Accepted Manuscript Title: Preparation and Identification of a Single-chain Variable Fragment Antibody against Newcastle Diseases Virus F48E9 Author: Benqiang Li Jiaxin Ye Yuan Lin Man Wang Jianguo Zhu PII: DOI: Reference:
S0165-2427(14)00182-2 http://dx.doi.org/doi:10.1016/j.vetimm.2014.08.009 VETIMM 9242
To appear in:
VETIMM
Received date: Revised date: Accepted date:
7-3-2014 15-7-2014 12-8-2014
Please cite this article as: Li, B., Ye, J., Lin, Y., Wang, M., Zhu, J.,Preparation and Identification of a Single-chain Variable Fragment Antibody against Newcastle Diseases Virus F48E9, Veterinary Immunology and Immunopathology (2014), http://dx.doi.org/10.1016/j.vetimm.2014.08.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Preparation and Identification of a Single‐chain Variable
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Fragment Antibody against Newcastle Diseases Virus F48E9
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Benqiang Lia, Jiaxin Yea, Yuan Linb, Man Wanga, Jianguo Zhua* a
Shanghai Key Laboratory of Veterinary Biotechnology, College of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China b Department of Pathogenic Biology and Immunology, School of Basic Medical Sciences, Ningxia Medical University, Yinchuan750004, China
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* Corresponding author: Jianguo Zhu
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Correspondence address: Shanghai Key Laboratory of Veterinary Biotechnology, School of
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Agriculture and Biology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai,
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People’s Republic of China
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Tel: 86-21-34204273; Fax: 86-21-34206971
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Email-address: [email protected]
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ABSTRACT：This article describes a proposed method for convenient and efficient detection
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of Newcastle diseases virus (NDV) that uses the fusion of single-chain variable fragment (scFv)
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and pOPE101 vector. In order to select the single chain variable fragment (scFv) against NDV
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F48E9, the total RNA was extracted from the spleen of immunized chicken, and then was
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converted into cDNA via the reverse transcription. The scFv was spliced by using splice-overlap
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extension polymerase chain reaction (SOE-PCR). The scFv gene was cloned into a pOPE101
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vector and expressed in E.coli. Under the optimized conditions, antibody affinity was studied by
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indirect ELISA. One positive clone was selected by ELISA screening, named ZL.6. Based on the
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positive clone and the germline sequence, the results of sequence analysis showed that there are
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more variation in CDR of VH and VL. In addition, BHK21 cell culture was conducted to examine
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the potential antiviral activity of ZL.6. The experimental result demonstrated that ZL.6 was able to
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neutralize NDV F48E9 which infected BHK21 cells. So ZL.6 will be proved useful for further
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characterization of NDV as potential diagnostic tool and therapeutic agent.
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Key words:
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Newcastle diseases virus; scFv; Sequence comparison; Antiviral activity
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1. Introduction
Newcastle disease (ND), caused by Newcastle disease virus (NDV), is supposed to be one of
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the most seriously and highly contiguous poultry diseases, particularly, ND poses a great threat to
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the field of fowl aquaculture (Abi-Ghanem et al., 2008; Alexander, 2011). Although the
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traditional inactivated vaccine and attenuated vaccine contribute to controlling the disease, some
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problems arose, such as a strong virulence or too short a period of validity, which might reduce
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their effect on controlling or eliminating this disease (Corte-Real et al., 2005). Therefore, it is
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necessary to develop a new type method in order to enhance the chicken immunity and to
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effectively control this disease.
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Functional single-chain antibodies are the smallest functional fragments of antibodies that
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maintain the affinity and specificity of the whole antibody. Compared with intact IgG or Fab
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fragments, Single-chain antibody has such properties as low molecular weight, powerful
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penetrability, short half-life period, more rapid blood clearance, fine specificity and low
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immunogenicity (Feng et al., 2011; Parker et al., 2013). Furthermore, the chicken scFv antibodies
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were conveniently amplified by using only a single set of PCR primers (van Wyngaardt et al.,
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2004).It has a great potential for the development of therapeutic or diagnostic reagents (Ahmad
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et al., 2012).
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At this point, the selected positive scFv can specifically bind to the protein, that play
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important role in virus replications, so to block the viral DNA replication and inhibit the
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assembly and release of infectious viral particles (Foord et al., 2007). Under this circumstance,
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we attempt to explore the construction and antiviral activity of scFv specifically targeting the
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NDV, analyze the scFv gene sequence and the fluctuations of amino acid, and investigate the
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potential antiviral activity. The aim of this study was to assess the effect of scFv on Newcastle
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disease and thus lay a foundation for the prevention against and control of Newcastle disease. 2
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2. Materials and Methods
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2.1. Virus stains, vector and vaccine NDV F48E9 strain was provided by Shanghai Veterinary Research Institute of Chinese
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Academy of Agricultural Sciences; the pOPE101 Vector was kindly donated by Raybiotech Inc,
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USA(Schmiedl et al., 2000), with a C-terminal peptide containing a polyhistidine metal-binding
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tag and the MAb myc epitope. Vaccine of Bio ND VG/GA type was purchased from Beijing
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KangMu Animal Health Products ＆Veterinary Apparatus Supply Center. Newcaslte disease
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virus standard antiserum (positive serum) and negative serum were purchased from changzhou DI
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MENO Biothch CO., LTD. His tag
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2.2. Chicken immunization
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Ten ten-week-old female white leghorns were immunized with ND VG/GA type vaccines
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according to the manufacturer's instructions. The concentration of serum antibody was tested by
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an indirect Elisa method (Wang et al., 2012). When the antibody titer reached 32,000, the chickens
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were killed and the spleen were harvested immediately, and then stored at -80°C. Meanwhile,
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peripheral blood lymphocytes (PBL) were isolated from the chicken blood by lymphocyte
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separation medium (YanJin Biotech, China).
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2.3. cDNA synthesis, PCR and Cloning
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Total RNA was extracted from the chicken spleen and PBL by using Trizol reagent
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(Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions, and cDNA
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synthesized based on PrimeScript RT Master Mix’s instructions (Takara, Japan). The heavy (VH)
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and light (VL) chains of the immunoglobulin were amplified from cDNA using the primer pair
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VH1 back, VH1 for and VL1 back, VL1 for respectively. VH-linker and linker-VL were amplified
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though the gel-purified VH and VL PCR products that have been used in the previous section,
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including the primer pair of VH2 back, VH1 for and VL1 back, VL2 for respectively. VH2 for and
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VL2 back were created with built-in Not I restriction sites and built-in Nco I restriction sites
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(Table 1). The PCR conditions include a hot start followed by 30 cycles of denaturation at 95 ℃
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for 1 min, annealing at 94 ℃ for 40 s and extension at 50 ℃ 40 s with a final extension of 72
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℃ 1 min. The purified VH-linker and linker-VL were mixed and reamplified by using only the 3
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VH2 back and VL2 for primers and then scFv gene was spliced by overlap extension PCR
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(SOE-PCR). The PCR condition was performed as follows: denaturation at 55 ℃ for 1 min, 35
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cycle of 94 ℃ for 1 min, annealing at 63 ℃ for 1 min, extension at 72 ℃ for 2 min, followed
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by a final extension at 72 ℃ for 10 min. The scFv gene was then cloned into pOPE101 vector
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and the recombinant plasmid was used to transform E. coli JM109 stain (TransGen Biotech, China)
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for expression. The correct transformants were identified by PCR, restriction enzyme digestion
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sequencing.
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2.4. Screening specific binding clones by Indirect ELISA
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The correct clones were picked, and then the soluble antibody of the periplasm of the clones
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was extracted by using an osmotic shock method (Hagemeyer et al., 2009). In order to examine
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the affinity of scFv to NDV, the periplasm extractions of the correct clones were analyzed by the
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indirect ELISA. The method was performed according to the published protocols with minor
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modifications (Hoogenboom, 2005; Krah, 1991). Firstly, antigen NDV was coated on ELISA plate
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wells at 200 ng/well in sodium bicarbonate buffer (pH 9.6) overnight. Subsequently, the wells
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were blocked with 5 % milk/TBST for 1h at 37 ℃. 100 µl of periplasm extracts of each clones
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were added and reacted at 37 ℃ for 2 h. After extensive washes with corresponding wash buffers,
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anti-Myc tag mAb conjugated with HRP at 1:2000 was incubated with wells for 1 h at 37 ℃. The
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wells were washed 3 times with PBST. 100 µl of TMB (Tiangen, China) was used for coloration
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and 50 µl H2SO4 (2M) was used to stop the reaction. Signals were read at 490 nm wavelength.
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All samples were repeated in quadruplicate wells. Absorbances above 0.2 were considered
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positive(Sapats et al., 2003). The positive clones, identified as described above, were respectively
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analyzed by SDS-PAGE and sequenced (Sangon, Shanghai, China). Sequences were identified by
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using the Blast search program. The genome sequence of the scFv was analyzed by using the
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MegAlign software (DNAStar Inc., Madison, EI, USA), which was then compared to the
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published sequences from other members of the family germline by using the Clustal X v1.82
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program (USA).
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2.5. Expression and Western blotting analysis of recombinant protein
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The positive clones were cultured in LB medium containing ampicillin with shaking at 37 4
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℃ until the OD reached 0.6-0.8. Then production was induced by the addition of 1 mM IPTG and
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the cells were allowed to incubate at 37 ℃ for 6 h with shaking. Then the recombinant protein
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was further purified by Ni-NTA affinity chromatography and the concentration of purified protein
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was determined according to the published literature (Raitses Gurevich and Fishelson, 2013). For
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western blotting, the purified scFv fragment was separated on a 12 % SDS-PAGE and then
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electroblotted onto a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked in
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5 % non-fat dry milk power and then incubated with mouse anti-His-tag mAb for 1.5 h at room
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temperature. After washing, the membrane was incubated with HRP goat anti-mouse IgG (1/5000)
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for 1 h. The immunoreactive bands were visualized with DAB colorimetric western blot kit
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(Rockland).
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2.6. Virus-neutralization assay
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Firstly, the TCID50 of NDV F48E9 on BHK21 cells was calculated in accordance with the
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Reed-Muench method (Magari et al., 2010). The purified scFv protein was diluted with DMEM
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medium. And then each diluted protein 1µg with 100 TCID50 of NDV 50 µl were incubated for 1
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h at 37 ℃. Then antibody-virus mixture for one hole was added into 6-well culture monolayer
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plate containing the BHK21 cell at 37 ℃ in 5%(V/V) CO2 for 1 h, and then virus was replaced
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with the fresh complete medium. While the negative control without NDV and the positive control
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without scFv were added. The cytopathic effect (CPE) was observed for 48 h in succession
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(Pasman et al., 2012; Sapats et al., 2003). 12 h, 24 h, 36 h and 48 h, supernatants of cell cultures
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were harvested, serially diluted, and assayed for virus titer using the HA test.
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3. Results
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3.1. Construction and expression of recombinant scFv
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The product of RT-PCR was coincided with what we preconceived. Primers were designed
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for PCR amplification of the VH and VL gene from the spleen. A desired PCR product of 417 bp
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and 378 bp were obtained after agarose gel electrophoresis analysis (Fig 1A). And they were
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connected to scFv via a linker (45 bp) sequence by using SOE-PCR (Fig 1B). The ScFv gene was
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cut by the double restriction enzyme and further cloned into pOPE101 vector. The recombinant 5
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plasmid pOPE101-scFv was successfully constructed (Fig 1C), and the enzyme digestion analysis
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is identical with expected results. In order to obtain the anti-NDV scFv antibody, a constructed prokaryotic expression
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plasmid of pOPE101-scFv was transformed into E.coli JM109 and inducted by IPTG. The
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high-level expression of scFv is obtained in 6 hours after the induction (Fig 2A). The expressed
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fusion protein with molecular weight of about 30KD is detected by western blotting (Fig 2B).
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3.2. Indirect ELISA assay of the reaction between scFv and NDV
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158 correct clones were randomly picked up. Under optimized conditions, the affinity and
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specificity of antibodies were examined after being extracted from periplasmic of each clone by
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the indirect ELISA. Among the clones tested, one positive clone from the panning of total clones
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was obtained for binding to NDV F48E9 (OD490 above 0.2), named ZL.6; however, the other
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reacted clones were weak (OD490 below 0.2) (Fig 3).
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3.3 DNA sequence analysis of the positive ScFv clones
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The result of sequencing showed that ZL.6 gene amplified was 726 bp in length. The
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deduced amino acid sequence of ZL.6 was aligned with the germline gene of chicken and the
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other sequences which have been reported (McCormack et al., 1993). The amino acid-changed
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frequency of these clones is similar and complementary determining regions (CDR) are the mutant
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cluster region (Fig 4). The mutant amino acid rate of CDR is over 40 % and the CDR3 of VH and
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VL are approximately 90 % and 60 %. The mutant amino acid rate of FR is mostly within 12 %,
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the mutant amino acid rate of FR4 of VH and VL are very low. Therefore, the FR of VH and VL
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are relatively stable.
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3.4 Virus-neutralization assay
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In accordance with the Reed-Muench calculation of the activity, TCID50 tested calculated
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was 10-5.32/0.1ml. The CPE on BHK21 cells, characterized by syncytia owing to coalescing with
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adjacent to infected cells, occurred at 24 h post-infection. The neutralize test suggested that the
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recombinant antibody ZL.6 can inhibit the replication of NDV, some syncytia can be observed on
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cells at 48 h post-infection, while immune serum can effectively inhibit the infection of virus for
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only a few syncytia observed at 48 h post-infection. However, the negative control showed no
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neutralizing reactivity and the negative serum was ineffective [Fig 5]. Twelve, twenty-four, 6
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thirty-six, and forty-eight hour post-infection, supernatants of cell cultures were harvested, serially
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diluted, and assayed for virus titer using the HA test. Results showed that virus production was
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potently inhibited by the positive serum, ZL.6, negative-scFv, PBS [Fig 6].
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4. Discussion Newcastle disease is an acute and highly contagious poultry disease, which poses a serious
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threat to the development of poultry industry because of its acute occurrence and high lethality.
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The emergence of new genotypes and subgenotypes could contribute to the outbreak of ND (Palya
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et al., 2012; Panda et al., 2004). At present, the main prevention and therapeutic measures of ND
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is vaccination and high immune serum.
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The emergence of the specific antibody for B cells has increased enormously following the
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antigen selection and affinity maturation, so the antibodies with high affinity and specific would
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have been obtained by an antibody library, which has been constructed from immunized chicken
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(Refega et al., 2004). According to this principle, the specific scFv antibodies were screened by
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purified viral particles, which reacted with antibody by displaying the real space. The method of
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indirect ELISA for detecting the binding activity of antibody has been established successfully.
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After screening of abundant transformed clone in vitro, we got one clone reacted to NDV in
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ELISA. The OD490 of ZL.6 could reach to 0.34. However, OD490 of other clones is lower than
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0.1, means lower affinity of them. We successfully selected one positive antibody ZL.6 with
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specificity against NDV from immunized chickens.
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The variable region sequence of the antibody consists of the FR and CDR and the diversity
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of antibody mainly depends on gene conversion with upstream pseudogene and somatic mutation
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induced by antigen (van Wyngaardt et al., 2004). The amino acid sequences and the numbers of
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FR are not easily changed, however, the amino acid sequences and the number of CDR are highly
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variable, which determines the antibody specificity (Verma et al., 1998; Yamanaka et al., 1996).
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The results of sequence analysis reveals that sequence differences are predominantly localized in
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the CDRs and the sequences and numbers of CDRs are prone to varying. And the results obtained
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here are in agreement with what has been previously reported (Yang et al., 1998).
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The mechanisms of action of neutralizing antibody are to change the virus surface
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configuration, prevent the virus adsorption in susceptible cells, mask of receptor attachment sites, 7
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and inhibit virus uncoating (Yuan and Parrish, 2000). The results of neutralization in vitro showed
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that ZL.6 had an inhibitory activity against NDV F48E9, while the other negative clones as well as
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negative control had no inhibitory effect on the cell. Thus we presume that ZL.6 may bind to
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different structure protein or epitope of NDV, which plays an important role in infected cells. Above all, our results demonstrated that we got one chicken scFv ZL.6 with high affinity
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and specific against NDV by antibody library technology (Zhang et al., 2011). Although the
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neutralization capacity of the ZL.6 antibody still needs to be tested in vivo, the scFv antibody
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ZL.6 was potential for producing diagnostic or therapeutic reagent of Newcastle disease. It
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provided a theoretic basis and opened a new avenue for the prevention and treatment of Newcastle
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disease virus infection.
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Conflict of interest statement
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The authors declare that they have no competing interests.
Acknowledgements
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The authors thank the members of our groups for helpful discussions, especially Dr Ying Zhang for advice on the excellent technical assistance.
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Funding
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This work was funded by the project “Development of chicken antibody library and screening
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single-chain variable fragment derived from immune against main pathogen” supported by
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Shanghai Key Lab of Veterinary Biology.
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Fig.1. Overlapping PCR amplification of VH and VL recombinants from cDNA isolated from
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spleen of immunized chicken. (a)Lane 1, the PCR product of VH gene; Lane 2, the PCR product
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of VL gene. (b)Lane 1, the PCR product of VH-Linker gene; Lane 2, the PCR product of
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Linker-VL gene. (c) Lane 1, the PCR product of plasmid pOPE101 vector; Lane 2, the PCR
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product of scFv gene
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Fig.2. Expression analysis of recombinant protein ZL 6 and Western blot analysis of scFv
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fragment. (A)M, protein molecular weight Maker; Lane 1, the expression product of recombinant
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scFv without being induced; Lane 2-7: bacteria lysate induced by 1 mM IPTG at 37 ℃ for 1-6 h,
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respectively. (B)12 % SDS-PAGE of purified sample of scFv ZL.6 was recognized with mouse
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anti-His-tag mAb and goat anti-mouse polyclonal antibody.
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Fig.3. Detection of anti-NDV scFv by indirect ELISA. Microplates were coated with a serial
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dilution of NDV, followed by incubation with purified scFv and dilution of serum. The graph
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shows the optimal OD values and the data represents the average of three experiments
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Fig.4. The clustal W result of VH and VL of scFv in five clones shade residues that differ from
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Germline. Sequence gaps have been introduced to maximize alignment. Framework regions1-4
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and complementarity-determining regions for both the VH and VL are indicated above each
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germline sequence. Shadows represent the amino acid differ from the germline gene and dashe
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represent gaps.
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Fig.5. Inhibition of cytopathic effect by ZL 6. (a), Normal BHK21 cell before treated (b), BHK21
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cell 48 h post-infected with NDV pre-treated with immune serum for 1h (c), BHK21 cell 48 h
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post-infected with NDV pre-treated with ZL.6 for 1h (d), BHK21 cell 48 h post-infected with
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NDV pre-treated with negative scFv for 1h (e), BHK21 cell 48 h post-treated with PBS
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Fig.6. The determination of the effects of anti-NDV antibody on the virus production. Virus titer
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in the culture supernatants was measured at different time points by HA assay. Data shown are
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from one of the three experiments.
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Table 1 Primers employed in PCRs Polarity
Nucleotide sequence（5’-3’）
Size（bp）
VH1 back VH1 for VL1 back VL1 for VH2 back
Sense Antisense Sense Antisense Sense
VH2 for
Antisense
VL2 back
Sense
VL2 for
Antisense
GCCGTGACGTTGGACGAGTCCGG GGAGGAGACGATGACTTCGGT CTGACTCAGCCGTCCTCGGTGTC TAGGACGGTCAGGGTTGTCCC GGCCATGGCCGTGACGTTGGACGAGTCCGGG GCCAGAGCCACCTCCGCCTGAACCGCCTCCAC CGGAGGAGACGATGACTTCGGT GGCGGAGGTGGCTCTGGCGGTGGCGGATCGC TGACTCAGCCGTCCTCGGTGTC CTGCGGCCGCTAGGACGGTCAGGGTTGTCCC
366bp 312bp
417bp
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Primer name
342bp
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Heavy chain forward primer contains the restriction site Nco I (bold). At the light chain reverse primer was
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inserted the restriction site Not I (bold/italic).Primer VH1 for and VH1 back were used for amplication of the
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variable heavy (VH) region. VL1 for and VL1 back were used for amplication of the variable light (VL) region.
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S0165-2427(14)00182-2 http://dx.doi.org/doi:10.1016/j.vetimm.2014.08.009 VETIMM 9242
To appear in:
VETIMM
Received date: Revised date: Accepted date:
7-3-2014 15-7-2014 12-8-2014
Please cite this article as: Li, B., Ye, J., Lin, Y., Wang, M., Zhu, J.,Preparation and Identification of a Single-chain Variable Fragment Antibody against Newcastle Diseases Virus F48E9, Veterinary Immunology and Immunopathology (2014), http://dx.doi.org/10.1016/j.vetimm.2014.08.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Preparation and Identification of a Single‐chain Variable
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Fragment Antibody against Newcastle Diseases Virus F48E9
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Benqiang Lia, Jiaxin Yea, Yuan Linb, Man Wanga, Jianguo Zhua* a
Shanghai Key Laboratory of Veterinary Biotechnology, College of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China b Department of Pathogenic Biology and Immunology, School of Basic Medical Sciences, Ningxia Medical University, Yinchuan750004, China
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* Corresponding author: Jianguo Zhu
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Correspondence address: Shanghai Key Laboratory of Veterinary Biotechnology, School of
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Agriculture and Biology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai,
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People’s Republic of China
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Tel: 86-21-34204273; Fax: 86-21-34206971
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Email-address: [email protected]
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ABSTRACT：This article describes a proposed method for convenient and efficient detection
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of Newcastle diseases virus (NDV) that uses the fusion of single-chain variable fragment (scFv)
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and pOPE101 vector. In order to select the single chain variable fragment (scFv) against NDV
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F48E9, the total RNA was extracted from the spleen of immunized chicken, and then was
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converted into cDNA via the reverse transcription. The scFv was spliced by using splice-overlap
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extension polymerase chain reaction (SOE-PCR). The scFv gene was cloned into a pOPE101
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vector and expressed in E.coli. Under the optimized conditions, antibody affinity was studied by
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indirect ELISA. One positive clone was selected by ELISA screening, named ZL.6. Based on the
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positive clone and the germline sequence, the results of sequence analysis showed that there are
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more variation in CDR of VH and VL. In addition, BHK21 cell culture was conducted to examine
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the potential antiviral activity of ZL.6. The experimental result demonstrated that ZL.6 was able to
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neutralize NDV F48E9 which infected BHK21 cells. So ZL.6 will be proved useful for further
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characterization of NDV as potential diagnostic tool and therapeutic agent.
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Key words:
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Newcastle diseases virus; scFv; Sequence comparison; Antiviral activity
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1. Introduction
Newcastle disease (ND), caused by Newcastle disease virus (NDV), is supposed to be one of
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the most seriously and highly contiguous poultry diseases, particularly, ND poses a great threat to
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the field of fowl aquaculture (Abi-Ghanem et al., 2008; Alexander, 2011). Although the
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traditional inactivated vaccine and attenuated vaccine contribute to controlling the disease, some
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problems arose, such as a strong virulence or too short a period of validity, which might reduce
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their effect on controlling or eliminating this disease (Corte-Real et al., 2005). Therefore, it is
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necessary to develop a new type method in order to enhance the chicken immunity and to
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effectively control this disease.
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Functional single-chain antibodies are the smallest functional fragments of antibodies that
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maintain the affinity and specificity of the whole antibody. Compared with intact IgG or Fab
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fragments, Single-chain antibody has such properties as low molecular weight, powerful
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penetrability, short half-life period, more rapid blood clearance, fine specificity and low
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immunogenicity (Feng et al., 2011; Parker et al., 2013). Furthermore, the chicken scFv antibodies
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were conveniently amplified by using only a single set of PCR primers (van Wyngaardt et al.,
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2004).It has a great potential for the development of therapeutic or diagnostic reagents (Ahmad
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et al., 2012).
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At this point, the selected positive scFv can specifically bind to the protein, that play
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important role in virus replications, so to block the viral DNA replication and inhibit the
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assembly and release of infectious viral particles (Foord et al., 2007). Under this circumstance,
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we attempt to explore the construction and antiviral activity of scFv specifically targeting the
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NDV, analyze the scFv gene sequence and the fluctuations of amino acid, and investigate the
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potential antiviral activity. The aim of this study was to assess the effect of scFv on Newcastle
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disease and thus lay a foundation for the prevention against and control of Newcastle disease. 2
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2. Materials and Methods
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2.1. Virus stains, vector and vaccine NDV F48E9 strain was provided by Shanghai Veterinary Research Institute of Chinese
56
Academy of Agricultural Sciences; the pOPE101 Vector was kindly donated by Raybiotech Inc,
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USA(Schmiedl et al., 2000), with a C-terminal peptide containing a polyhistidine metal-binding
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tag and the MAb myc epitope. Vaccine of Bio ND VG/GA type was purchased from Beijing
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KangMu Animal Health Products ＆Veterinary Apparatus Supply Center. Newcaslte disease
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virus standard antiserum (positive serum) and negative serum were purchased from changzhou DI
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MENO Biothch CO., LTD. His tag
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2.2. Chicken immunization
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Ten ten-week-old female white leghorns were immunized with ND VG/GA type vaccines
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according to the manufacturer's instructions. The concentration of serum antibody was tested by
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an indirect Elisa method (Wang et al., 2012). When the antibody titer reached 32,000, the chickens
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were killed and the spleen were harvested immediately, and then stored at -80°C. Meanwhile,
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peripheral blood lymphocytes (PBL) were isolated from the chicken blood by lymphocyte
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separation medium (YanJin Biotech, China).
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2.3. cDNA synthesis, PCR and Cloning
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Total RNA was extracted from the chicken spleen and PBL by using Trizol reagent
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(Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions, and cDNA
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synthesized based on PrimeScript RT Master Mix’s instructions (Takara, Japan). The heavy (VH)
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and light (VL) chains of the immunoglobulin were amplified from cDNA using the primer pair
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VH1 back, VH1 for and VL1 back, VL1 for respectively. VH-linker and linker-VL were amplified
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though the gel-purified VH and VL PCR products that have been used in the previous section,
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including the primer pair of VH2 back, VH1 for and VL1 back, VL2 for respectively. VH2 for and
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VL2 back were created with built-in Not I restriction sites and built-in Nco I restriction sites
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(Table 1). The PCR conditions include a hot start followed by 30 cycles of denaturation at 95 ℃
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for 1 min, annealing at 94 ℃ for 40 s and extension at 50 ℃ 40 s with a final extension of 72
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℃ 1 min. The purified VH-linker and linker-VL were mixed and reamplified by using only the 3
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VH2 back and VL2 for primers and then scFv gene was spliced by overlap extension PCR
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(SOE-PCR). The PCR condition was performed as follows: denaturation at 55 ℃ for 1 min, 35
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cycle of 94 ℃ for 1 min, annealing at 63 ℃ for 1 min, extension at 72 ℃ for 2 min, followed
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by a final extension at 72 ℃ for 10 min. The scFv gene was then cloned into pOPE101 vector
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and the recombinant plasmid was used to transform E. coli JM109 stain (TransGen Biotech, China)
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for expression. The correct transformants were identified by PCR, restriction enzyme digestion
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sequencing.
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2.4. Screening specific binding clones by Indirect ELISA
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The correct clones were picked, and then the soluble antibody of the periplasm of the clones
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was extracted by using an osmotic shock method (Hagemeyer et al., 2009). In order to examine
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the affinity of scFv to NDV, the periplasm extractions of the correct clones were analyzed by the
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indirect ELISA. The method was performed according to the published protocols with minor
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modifications (Hoogenboom, 2005; Krah, 1991). Firstly, antigen NDV was coated on ELISA plate
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wells at 200 ng/well in sodium bicarbonate buffer (pH 9.6) overnight. Subsequently, the wells
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were blocked with 5 % milk/TBST for 1h at 37 ℃. 100 µl of periplasm extracts of each clones
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were added and reacted at 37 ℃ for 2 h. After extensive washes with corresponding wash buffers,
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anti-Myc tag mAb conjugated with HRP at 1:2000 was incubated with wells for 1 h at 37 ℃. The
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wells were washed 3 times with PBST. 100 µl of TMB (Tiangen, China) was used for coloration
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and 50 µl H2SO4 (2M) was used to stop the reaction. Signals were read at 490 nm wavelength.
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All samples were repeated in quadruplicate wells. Absorbances above 0.2 were considered
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positive(Sapats et al., 2003). The positive clones, identified as described above, were respectively
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analyzed by SDS-PAGE and sequenced (Sangon, Shanghai, China). Sequences were identified by
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using the Blast search program. The genome sequence of the scFv was analyzed by using the
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MegAlign software (DNAStar Inc., Madison, EI, USA), which was then compared to the
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published sequences from other members of the family germline by using the Clustal X v1.82
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program (USA).
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2.5. Expression and Western blotting analysis of recombinant protein
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The positive clones were cultured in LB medium containing ampicillin with shaking at 37 4
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℃ until the OD reached 0.6-0.8. Then production was induced by the addition of 1 mM IPTG and
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the cells were allowed to incubate at 37 ℃ for 6 h with shaking. Then the recombinant protein
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was further purified by Ni-NTA affinity chromatography and the concentration of purified protein
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was determined according to the published literature (Raitses Gurevich and Fishelson, 2013). For
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western blotting, the purified scFv fragment was separated on a 12 % SDS-PAGE and then
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electroblotted onto a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked in
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5 % non-fat dry milk power and then incubated with mouse anti-His-tag mAb for 1.5 h at room
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temperature. After washing, the membrane was incubated with HRP goat anti-mouse IgG (1/5000)
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for 1 h. The immunoreactive bands were visualized with DAB colorimetric western blot kit
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(Rockland).
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2.6. Virus-neutralization assay
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Firstly, the TCID50 of NDV F48E9 on BHK21 cells was calculated in accordance with the
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Reed-Muench method (Magari et al., 2010). The purified scFv protein was diluted with DMEM
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medium. And then each diluted protein 1µg with 100 TCID50 of NDV 50 µl were incubated for 1
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h at 37 ℃. Then antibody-virus mixture for one hole was added into 6-well culture monolayer
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plate containing the BHK21 cell at 37 ℃ in 5%(V/V) CO2 for 1 h, and then virus was replaced
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with the fresh complete medium. While the negative control without NDV and the positive control
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without scFv were added. The cytopathic effect (CPE) was observed for 48 h in succession
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(Pasman et al., 2012; Sapats et al., 2003). 12 h, 24 h, 36 h and 48 h, supernatants of cell cultures
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were harvested, serially diluted, and assayed for virus titer using the HA test.
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3. Results
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3.1. Construction and expression of recombinant scFv
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The product of RT-PCR was coincided with what we preconceived. Primers were designed
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for PCR amplification of the VH and VL gene from the spleen. A desired PCR product of 417 bp
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and 378 bp were obtained after agarose gel electrophoresis analysis (Fig 1A). And they were
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connected to scFv via a linker (45 bp) sequence by using SOE-PCR (Fig 1B). The ScFv gene was
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cut by the double restriction enzyme and further cloned into pOPE101 vector. The recombinant 5
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plasmid pOPE101-scFv was successfully constructed (Fig 1C), and the enzyme digestion analysis
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is identical with expected results. In order to obtain the anti-NDV scFv antibody, a constructed prokaryotic expression
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plasmid of pOPE101-scFv was transformed into E.coli JM109 and inducted by IPTG. The
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high-level expression of scFv is obtained in 6 hours after the induction (Fig 2A). The expressed
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fusion protein with molecular weight of about 30KD is detected by western blotting (Fig 2B).
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3.2. Indirect ELISA assay of the reaction between scFv and NDV
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158 correct clones were randomly picked up. Under optimized conditions, the affinity and
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specificity of antibodies were examined after being extracted from periplasmic of each clone by
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the indirect ELISA. Among the clones tested, one positive clone from the panning of total clones
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was obtained for binding to NDV F48E9 (OD490 above 0.2), named ZL.6; however, the other
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reacted clones were weak (OD490 below 0.2) (Fig 3).
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3.3 DNA sequence analysis of the positive ScFv clones
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The result of sequencing showed that ZL.6 gene amplified was 726 bp in length. The
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deduced amino acid sequence of ZL.6 was aligned with the germline gene of chicken and the
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other sequences which have been reported (McCormack et al., 1993). The amino acid-changed
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frequency of these clones is similar and complementary determining regions (CDR) are the mutant
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cluster region (Fig 4). The mutant amino acid rate of CDR is over 40 % and the CDR3 of VH and
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VL are approximately 90 % and 60 %. The mutant amino acid rate of FR is mostly within 12 %,
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the mutant amino acid rate of FR4 of VH and VL are very low. Therefore, the FR of VH and VL
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are relatively stable.
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3.4 Virus-neutralization assay
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In accordance with the Reed-Muench calculation of the activity, TCID50 tested calculated
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was 10-5.32/0.1ml. The CPE on BHK21 cells, characterized by syncytia owing to coalescing with
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adjacent to infected cells, occurred at 24 h post-infection. The neutralize test suggested that the
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recombinant antibody ZL.6 can inhibit the replication of NDV, some syncytia can be observed on
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cells at 48 h post-infection, while immune serum can effectively inhibit the infection of virus for
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only a few syncytia observed at 48 h post-infection. However, the negative control showed no
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neutralizing reactivity and the negative serum was ineffective [Fig 5]. Twelve, twenty-four, 6
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thirty-six, and forty-eight hour post-infection, supernatants of cell cultures were harvested, serially
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diluted, and assayed for virus titer using the HA test. Results showed that virus production was
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potently inhibited by the positive serum, ZL.6, negative-scFv, PBS [Fig 6].
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4. Discussion Newcastle disease is an acute and highly contagious poultry disease, which poses a serious
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threat to the development of poultry industry because of its acute occurrence and high lethality.
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The emergence of new genotypes and subgenotypes could contribute to the outbreak of ND (Palya
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et al., 2012; Panda et al., 2004). At present, the main prevention and therapeutic measures of ND
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is vaccination and high immune serum.
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The emergence of the specific antibody for B cells has increased enormously following the
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antigen selection and affinity maturation, so the antibodies with high affinity and specific would
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have been obtained by an antibody library, which has been constructed from immunized chicken
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(Refega et al., 2004). According to this principle, the specific scFv antibodies were screened by
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purified viral particles, which reacted with antibody by displaying the real space. The method of
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indirect ELISA for detecting the binding activity of antibody has been established successfully.
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After screening of abundant transformed clone in vitro, we got one clone reacted to NDV in
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ELISA. The OD490 of ZL.6 could reach to 0.34. However, OD490 of other clones is lower than
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0.1, means lower affinity of them. We successfully selected one positive antibody ZL.6 with
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specificity against NDV from immunized chickens.
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The variable region sequence of the antibody consists of the FR and CDR and the diversity
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of antibody mainly depends on gene conversion with upstream pseudogene and somatic mutation
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induced by antigen (van Wyngaardt et al., 2004). The amino acid sequences and the numbers of
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FR are not easily changed, however, the amino acid sequences and the number of CDR are highly
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variable, which determines the antibody specificity (Verma et al., 1998; Yamanaka et al., 1996).
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The results of sequence analysis reveals that sequence differences are predominantly localized in
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the CDRs and the sequences and numbers of CDRs are prone to varying. And the results obtained
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here are in agreement with what has been previously reported (Yang et al., 1998).
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The mechanisms of action of neutralizing antibody are to change the virus surface
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configuration, prevent the virus adsorption in susceptible cells, mask of receptor attachment sites, 7
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and inhibit virus uncoating (Yuan and Parrish, 2000). The results of neutralization in vitro showed
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that ZL.6 had an inhibitory activity against NDV F48E9, while the other negative clones as well as
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negative control had no inhibitory effect on the cell. Thus we presume that ZL.6 may bind to
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different structure protein or epitope of NDV, which plays an important role in infected cells. Above all, our results demonstrated that we got one chicken scFv ZL.6 with high affinity
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and specific against NDV by antibody library technology (Zhang et al., 2011). Although the
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neutralization capacity of the ZL.6 antibody still needs to be tested in vivo, the scFv antibody
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ZL.6 was potential for producing diagnostic or therapeutic reagent of Newcastle disease. It
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provided a theoretic basis and opened a new avenue for the prevention and treatment of Newcastle
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disease virus infection.
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Conflict of interest statement
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The authors declare that they have no competing interests.
Acknowledgements
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The authors thank the members of our groups for helpful discussions, especially Dr Ying Zhang for advice on the excellent technical assistance.
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Funding
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This work was funded by the project “Development of chicken antibody library and screening
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single-chain variable fragment derived from immune against main pathogen” supported by
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Shanghai Key Lab of Veterinary Biology.
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Fig.1. Overlapping PCR amplification of VH and VL recombinants from cDNA isolated from
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spleen of immunized chicken. (a)Lane 1, the PCR product of VH gene; Lane 2, the PCR product
289
of VL gene. (b)Lane 1, the PCR product of VH-Linker gene; Lane 2, the PCR product of
290
Linker-VL gene. (c) Lane 1, the PCR product of plasmid pOPE101 vector; Lane 2, the PCR
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product of scFv gene
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Fig.2. Expression analysis of recombinant protein ZL 6 and Western blot analysis of scFv
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fragment. (A)M, protein molecular weight Maker; Lane 1, the expression product of recombinant
294
scFv without being induced; Lane 2-7: bacteria lysate induced by 1 mM IPTG at 37 ℃ for 1-6 h,
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respectively. (B)12 % SDS-PAGE of purified sample of scFv ZL.6 was recognized with mouse
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anti-His-tag mAb and goat anti-mouse polyclonal antibody.
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Fig.3. Detection of anti-NDV scFv by indirect ELISA. Microplates were coated with a serial
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dilution of NDV, followed by incubation with purified scFv and dilution of serum. The graph
299
shows the optimal OD values and the data represents the average of three experiments
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Fig.4. The clustal W result of VH and VL of scFv in five clones shade residues that differ from
301
Germline. Sequence gaps have been introduced to maximize alignment. Framework regions1-4
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and complementarity-determining regions for both the VH and VL are indicated above each
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germline sequence. Shadows represent the amino acid differ from the germline gene and dashe
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represent gaps.
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Fig.5. Inhibition of cytopathic effect by ZL 6. (a), Normal BHK21 cell before treated (b), BHK21
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cell 48 h post-infected with NDV pre-treated with immune serum for 1h (c), BHK21 cell 48 h
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post-infected with NDV pre-treated with ZL.6 for 1h (d), BHK21 cell 48 h post-infected with
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NDV pre-treated with negative scFv for 1h (e), BHK21 cell 48 h post-treated with PBS
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Fig.6. The determination of the effects of anti-NDV antibody on the virus production. Virus titer
310
in the culture supernatants was measured at different time points by HA assay. Data shown are
311
from one of the three experiments.
ip t
305
cr
312
Ac ce pt e
d
M
an
us
313
11
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Table 1 Primers employed in PCRs Polarity
Nucleotide sequence（5’-3’）
Size（bp）
VH1 back VH1 for VL1 back VL1 for VH2 back
Sense Antisense Sense Antisense Sense
VH2 for
Antisense
VL2 back
Sense
VL2 for
Antisense
GCCGTGACGTTGGACGAGTCCGG GGAGGAGACGATGACTTCGGT CTGACTCAGCCGTCCTCGGTGTC TAGGACGGTCAGGGTTGTCCC GGCCATGGCCGTGACGTTGGACGAGTCCGGG GCCAGAGCCACCTCCGCCTGAACCGCCTCCAC CGGAGGAGACGATGACTTCGGT GGCGGAGGTGGCTCTGGCGGTGGCGGATCGC TGACTCAGCCGTCCTCGGTGTC CTGCGGCCGCTAGGACGGTCAGGGTTGTCCC
366bp 312bp
417bp
ip t
Primer name
342bp
cr
313
Heavy chain forward primer contains the restriction site Nco I (bold). At the light chain reverse primer was
315
inserted the restriction site Not I (bold/italic).Primer VH1 for and VH1 back were used for amplication of the
316
variable heavy (VH) region. VL1 for and VL1 back were used for amplication of the variable light (VL) region.
an
us
314
317
Ac ce pt e
d
M
318
12
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Ac
ce
pt
ed
M
an
us
cr
i
Figure
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Ac
ce
pt
ed
M
an
us
cr
i
Figure
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Ac
ce
pt
ed
M
an
us
cr
i
Figure
Page 15 of 18
Ac ce p
te
d
M
an
us
cr
ip t
Figure
Page 16 of 18
Ac
ce
pt
ed
M
an
us
cr
i
Figure
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Ac
ce
pt
ed
M
an
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cr
i
Figure
Page 18 of 18
