Mol Biol Rep (2014) 41:467–475 DOI 10.1007/s11033-013-2881-z

Construction and identification of a cDNA library for use in the yeast two-hybrid system from duck embryonic fibroblast cells post-infected with duck enteritis virus Xinghong Gao • Renyong Jia • Mingshu Wang • Dekang Zhu • Shun Chen Meng Lin • Zhongqiong Yin • Yin Wang • Xiaoyue Chen • Anchun Cheng

•

Received: 7 February 2013 / Accepted: 21 November 2013 / Published online: 30 November 2013 Ó Springer Science+Business Media Dordrecht 2013

Abstract To explore and isolate genes related to duck embryonic fibroblast cells (DEFs) post-infected with duck enteritis virus (DEV), a cDNA library was established using SMART (Switching Mechanism At 50 end of the RNA Transcript) technique coupling with a homologous recombination method. The cells were harvested and total RNA was extracted at 48 h post infection. Then the mRNAs were purified and reverse transcribed to firststrand cDNAs using oligo (dT) primers (CDS III). Subsequently, long distance-PCR was performed, the doublestranded cDNAs were purified, and a transformation assay was carried out in that order. Eventually, a high qualitative library was successfully established according to an

Xinghong Gao, Renyong Jia and Mingshu Wang contributed equally to the work.

Electronic supplementary material The online version of this article (doi:10.1007/s11033-013-2881-z) contains supplementary material, which is available to authorized users. X. Gao  R. Jia (&)  M. Wang  D. Zhu  S. Chen  M. Lin  A. Cheng (&) Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, Sichuan, China e-mail: [email protected] A. Cheng e-mail: [email protected] M. Wang e-mail: [email protected] D. Zhu e-mail: [email protected] S. Chen e-mail: [email protected]

evaluation on quality. The transformation efficiency was about 2.33 9 106 transformants/4.34 lg pGADT7-Rec ([1.0 9 106). The cell density of the library was 1.75 9 109 cells/mL ([29107 cells/mL). The titer of the primary cDNA library and amplified cDNA library was 6.75 9 105 and 2.33 9 107 CFU/mL respectively. The numbers for the primary cDNA library and amplified cDNA library were 1.01 9 107 and 1.14 9 109, respectively, and the recombinant rate was 97.14 %. The sequence results of 27 randomly picked independent clones revealed the insert ranged from 0.323 to 2.017 kb with an average insert size of 0.807 kb. Full-length transcripts of DEV-CHv LORF3, UL26 and UL35 genes were acquired through sequence similarity analysis from the non-redundant nucleic acid or protein database. Five polyA sites were identified in the DEV-CHv genome. Also, a new transcript of 668 bp was found between the IRS gene and US1 gene of the DEV-CHv genome. Thus, we concluded that the constructed cDNA library will be a useful tool in proteomic analysis of interactions between the DEV and host DEFs, X. Gao  R. Jia  M. Wang  D. Zhu  S. Chen  M. Lin  Z. Yin  Y. Wang  X. Chen  A. Cheng Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, 46 Xinkang Road, Ya’an 625014, Sichuan, China e-mail: [email protected] Y. Wang e-mail: [email protected] X. Chen e-mail: [email protected] R. Jia  M. Wang  D. Zhu  S. Chen  X. Chen  A. Cheng Key Laboratory of Animal Disease and Human Health of Sichuan Province, Chengdu 611130, Sichuan, China

M. Lin e-mail: [email protected]

123

468

and discovery of biomarkers studies on the mechanism of DEV and subsequently exploitation original vaccines and antiviral drugs to prevent or cure diseases. Keywords Duck enteritis virus (DEV)  Duck embryonic fibroblast cells (DEFs)  SMART technology  cDNA library

Introduction Since the first cDNA library was constructed by Maniatis [1], which made a significant contribution to the foundation of molecular and genomic research, the construction of cDNA libraries has become one of the most fundamental tools in genetics and serves as a physical resource for outer-to-outer clones in genomics research. Once a cDNA library is constructed, genes of interest can be acquired from it. Historically, in the previous 23 years, the process of cDNA library generation was labyrinthian and laborious, though there were tremendous benefits with the successful generation of a cDNA library. Since SMART technology appeared, substantive changes have taken place in founding a successful cDNA library. In the early years, because the primer was not added to the 50 -terminal cap structure of the mRNA, which lost whole or small fragments and was degraded partially by RNase in the 50 -terminal, a high percentage of 50 -truncated clones were not generated with traditional methods and protocols. Currently, SMART technology is employed to construct cDNA libraries. With the SMART technique [2, 3], in principle, all of the mRNAs can be reverse transcribed, especially for low-copy or low abundance genes that are used in special research, such as studying the interaction of proteins, discovery of biomarkers for the development of therapy for a disease and studies on the mechanisms of some pathogens. Duck enteritis virus (DEV), a linear and double stranded DNA virus, is about 158*162 kb [4–7]. It has been classified as belonging to the Alpha-Herpesvirinae subfamily of the herpesviridae family, which contains the Alpha, Beta, and Gamma Herpesvirinae subfamilies on the basis of the Ninth ICTV (International Committee on Taxonomy of Viruses, ICTV) reports. However, it has not been grouped into any genus [8]. Currently, DEV has received attention because it results in significant economic losses in domestic and wild waterfowls (e.g., ducks, geese and swans) due to high mortality and decreased egg production rates [9]. Genomic DNA or cDNA libraries have been constructed for many microorganisms [10–12], animals [13–16] and plant species [17–23] and genes related to biological processes have been determined. Also, genes related to the biology of animal pathogens can be discovered with a

123

Mol Biol Rep (2014) 41:467–475

cDNA library. For example, genes responsible for pathogen invasion, development, survival, pathogenicity and virulence have been found. Few reports have been published concerning the construction of a cDNA library from duck [24, 25]. In our previous study [25], a library that contains only the DEV-CHv strain genes was developed. In Zhang’s study [24], a duck liver resource cDNA library, which not only contained DEV-GZ strain genes but also duck liver genes, was constructed. And this cDNA library was used to study the mechanisms of DEV infected duck liver. They showed that DEV-GZ strain virulence was attenuated, but DEV-CHv strain virulence was strong enough to cause heavy economic losses. In our study, a cDNA library resource from the DEFs infected with DEVCHv strain was constructed. Though embryonic cell differentiation was not complete, these cells could still be infected. Most importantly, the genes had an integral organization source provided by the DEFs. Therefore, this cDNA library was constructed from the DEFs post-infected with the DEV-CHv strain, it should include expressed genes unique to this viral and DEFs. Eventually, according to the interaction proteins screened from every gene or, more importantly, a DEV gene by yeast two-hybrid system (Y2H) assay, researchers can determine the pathogenic molecular mechanisms of DEV-CHv strain and subsequently explore original vaccines and antiviral drugs to prevent or cure diseases.

Materials and methods Cells and virus DEFs, which were made by 10 days’ detersive and DEVfree duck embryo coming from the farm of Sichuan Agricultural University, were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10 % newborn calf serum (NBCS, Gibco), penicillin/ streptomycin and incubated at 37 °C for 24 h. DEFs were infected with DEV at a multiplicity of 5 PFU per cell, and harvested post-infection at 48 h while the percentage of cytopathic effect (CPE) attained 50 %. For virus infection, DMEM medium supplemented with 2*3 % NBCS was used. And approximately, there were one hundred million cells in one of 50 cm2 arrow-necked bottles. Total RNA extraction Cells were scraped by drawknife distinctively used for cells and collected by centrifugation. The total RNA was isolated from the samples with Trizol assay reference to the book of molecular cloning [26] and manufacturer’s protocol with some improvements. Approximately, total RNA

Mol Biol Rep (2014) 41:467–475

from every 107 cells could be obtained with 10 lL diethyl pyrocarbonate (DEPC) H2O dissolved, and there was 80 lL total RNA in total. The concentration and the integrity of RNAs were detected by nucleic acid and protein detection instrument and 1.2 % formaldehyde degeneration agarose gel respectively. Isolation of the mRNA from total RNA To obtain a high quality cDNA library, the mRNA was isolated from the total RNA with the mRNA purification kit (OligotexTM-dT30\super[mRNA Purification Kit, TAKARA) protocol as its manufacture. The concentration of the mRNA was also measured with a nucleic acid and protein detection instrument. First-strand cDNA synthesis In order to obtain the first-Strand cDNA, 2.5 lL mRNAs (about 0.1 lg) were transcribed by the kit of Make Your Own ‘Mate & PlateTM’ Library System (Clontech, USA) with a thermal cycler (Bio-Rad, USA), and they were synthesized as directed as the manufacturer. To identify the quality of single stranded cDNA (ss cDNA), 6.0 lL of the PCR product was analyzed with 1 % agarose gel, and then, stored at -80 °C provisionally. Simultaneously, total RNA and rat liver mRNA were the positive control and water was negative control. Amplification of double-stranded cDNA by long distance PCR (LD-PCR) The double-stranded cDNAs (ds cDNA) were obtained by LD-PCR which was specially used to the amplification of large fragment genes by the Advantage 2 PCR Kit (Clontech, USA). In a sterile microcentrifuge tube, 50 lL reaction volume was established like this: deionized H2O 35.0 lL, 109 advantage 2 PCR buffer 5.0 lL, 109 Melting solution 5.0 lL, 509 dNTP mix 1.0 lL, 509 advantage 2 polymerase mix 1.0 lL, 50 -PCR primer 1.0 lL, 30 PCR primer 1.0 lL, and first-strand cDNA 1.0 lL. The mixture was centrifuged briefly after being mixed gently, carried out its program as follows: pre-thermal denaturation, 95 °C for 30 s; followed by 22 cycles which contains two steps, the one was thermal denaturation at 95 °C for 10 s, the other was elongation at 68 °C for 6 min (extension/enhance 5 s after each cycle); at last, another 5 min was used to elongation once more at 68 °C. To keeping the rate of PCR-induced mutations in a minimum, the number of PCR cycles was optimized, and the rat liver source cDNA was used as a positive control. To identify the quality of ds cDNA, 7.0 lL of the PCR product was analyzed by 1 % agarose gel.

469

Double-stranded cDNA size fractionation by CHROMA SPIN-400 column To get rid of low-molecular weight ds cDNA fragments, small DNA contaminants, and unincorporated nucleotides efficiently from the ds cDNA, ds cDNA size fractionation was carried out using two CHROMA SPIN-400 columns (Clontech, USA) according to the protocol. Each 93 lL sample was added into one of the preprocessing CHROMA SPIN TE-400 columns. After centrifugation at 700 g for 5 min, appropriate amounts of 3 M sodium acetate and absolute ethanol (-20 °C) were added into a sterile 1.5 mL centrifuge tube combined with the collected samples. Then, the mixture was kept in -20 °C for 1 h, and it was centrifuged at 1,4000g for 30 min at room temperature (RT). The supernatant was carefully removed and abandoned, and the pellet was dried at RT for several minutes. Then, the pellet was resolved with 22 lL deionized H2O at RT, and kept at -20 °C. The quality of the purified ds cDNA was analyzed with a 1 % agarose gel and the concentration was measured with a nucleic acid and protein detection instrument. Construction and detection of the cDNA library For the construction of a high quality cDNA library, preparation of competent yeast cells Y187 and a transformation assay were carried out with a YeastmakerTM Yeast Transformation System 2 Kit (Clontech, USA) according to the manufacturer’s instructions. About 4.34 lg (10 lL) of purified ds cDNA were used in the transformation assay. To detect transformation efficiency, 100 lL was spread in dilutions of 1:10, 1:100 and 1:1,000 on 100 mm SD/-Leu plates, and others productions were spread on 150 mm plates for 150 lL each (a total of 100 plates). After being incubated at 30 °C for 4 days, the plates were harvested with 5 mL YPDA freezing medium for each. All washed liquids were harvested in a sterile flask and mixed well. Centrifugation was carried out at 1,000g for 5 min, the pellet was resuspended with YPDA freezing medium and 25 % glycerol, and the concentration of the mixture was made compatible. Then, they were aliquoted at 1.0 or 50 mL and stored at -80 °C. Simultaneously, the capacity, the transformation efficiency and the titer of the library were measured. Insert fragments detection The primers (forward primer: 50 -CTATTCGATGATGA AGATACCCCACCAAACCC-30 ; reverse primer: 50 -GT GAACTTGCGGGGTTTTTCAGTATCTACGAT-30 ) were used to amplify the insert fragment in pGADT7 vector. 35 recombinant clones were randomly picked and as templates

123

470

they were used to amplify cloned inserts. The reactions bulk volume was 20.0 lL, and as following: 109 advantage 2 PCR buffer 2.0 lL, 509 advantage 2 polymerase mix 0.4 lL, 509 dNTP 0.4 lL, 10 pM forward primer and reverse primer 0.1 lL respectively, and aseptic UP water 17.0 lL. The PCR amplification reactions were performed at 95 °C for 5 min, and coupled with 35 cycles at 94 °C for 30 s, 58 °C for 30 s and elongated at 72 °C for 3 min, then 72 °C for another 10 min. The PCR product was loaded on a 1 % agarose gel. Sequencing and sequence analysis Plasmid DNAs of 27 randomly picked independent clones were prepared by using an Easy Yeast Plasmid Isolation Kit (Clontech, USA), and were transformed into Escherichia coli DH5a to breeding. Sequencing was carried out with T7 primer and insert fragments detection primer as upstream primer and downstream primer respectively (Meiji, China). Sequence similarity of cDNA was tested against various sequence databases, including the GenBank non-redundant nucleotide library (bBLASTn), non-redundant protein sequences library (bBLASTx) and dbEST (All nonredundant GenBank CDS, by the BLAST program), accessed through the NCBI (National Center for Biotechnology Information, NCBI) at the following http server: http://www.ncbi.nlm.nih.gov. For the score of sequence similarities from GenBank, the BLASTn score was higher than 200, and the BLASTx score was higher than 100.

Results Isolation and analysis of total RNA The integrity of the total RNA, extracted with the Trzol reagent from the post-infected DEFs, was examined by 1.2 % formaldehyde degenerating agarose gel electrophoresis. It showed three bands corresponding to ribosomal 28 S, 18 S and 5 S RNA. The 28 S band was twofold more abundant than the 18 s band, and there was no visible degradation (Supporting Information-S1). The ratio of A260/A280 of total RNA was 2.033 and the concentration of total RNA was 2.315 lg/lL (total volume 10 lL) measured with a nucleic acid and protein detection instrument. These results indicated that the total RNA was of good quality and was used to isolate mRNA. Isolation of mRNA from total RNA High quality mRNA was acquired with the combination of both Trizol reagent and an OligotexTM-dT30\super[mRNA Purification Kit, according to Wang [11]. In this study, to

123

Mol Biol Rep (2014) 41:467–475

obtain a high quality cDNA library, the mRNA was also isolated from the total RNA. The concentration of the mRNA was 0.04 lg/lL (total volume 16 lL), and OD260/280 was 2.103 as measured with a nucleic acid and protein detection instrument. Thus, the mRNAs from the post-infected DEFs were of high quality and quantity and could be used to construct the cDNA library. First-strand cDNA synthesis According to the protocol of Make Your Own ‘‘Mate & PlateTM’’ Library System Kit (Clontech, USA), 2.0 lL (4.6 lg) of the total RNA and 2.0 lL (0.08 lg) of the high quality mRNAs were chosen to synthesize the first-strand cDNAs. As shown in Supporting Information S-2a and S-2b, the extent of molecular weight examination of the ss cDNA from the mRNAs and total RNA were both displayed as a distributed smear from 0.1 to 8 kb in 1 % agarose gels. Amplification and purification of ds cDNA To keep the rate of PCR-induced mutations at a minimum, the number of PCR cycles was optimized. And the rat liver source cDNA was used as a positive control, water used as a negative control. From Fig. 1a and b, 21*24 cycles and 27 cycles of amplification were accomplished respectively. The results showed that amplification production both at 24 and 27 cycles had more low fragments than that at 21 cycles, although there were much more fragments larger than 4 kb, indicated that mutations had taken place (Fig. 1a). So the step of optimized number of PCR cycles (21*23 cycles) was employed again (Fig. 1b). Figure 1b displays that the length of the ds cDNA fragments, sourced from the DEFs, was 0.1*4.0 kb, and this long distributed smear of ds cDNA indicated that the ds cDNA was successfully synthesized according to LD-PCR technique. Supporting Information-S3 exhibited that fragments smaller than 200 bp were eliminated by cDNA fractionation with the CHROMA SPIN-400 column, which could effectively avoid the very small inserts and non-recombinant clones in the library. After fractionation, the production concentration of resources from the DEFs cDNA was 0.434 lg/lL and that from the rat liver source cDNA was 0.274 lg/lL. Detection of the transformation and quality of the cDNA library Detection of the transformation efficiency and titer of the cDNA library After 4 days incubation at 30 °C, colonies appeared on the SD/-Leu plates and the mixture from the transformation

Mol Biol Rep (2014) 41:467–475

471

Fig. 1 The detection of the ds cDNA-fragments size from the LD-PCR. a The number of PCR cycles was optimized (21, 24, 27 cycles respectively); the rat liver source cDNA as a positive control (21#, 24#, 27# cycles respectively); M wide range DNA marker 500*15,000 bp. b The number of PCR cycles was further optimized (21, 22, 23 cycles respectively); M wide range DNA marker 500*12,000 bp

dilutions of 1:10, 1:100, 1:1,000 and 1:10,000 were counted. Also, the transformation efficiency, library titer and library quantity were calculated according to the following formulas [27]: transformation efficiency = the colonies/ plating volume 9 dilution factor 9 mixture volume/quality of pGADT7-Rec, the library titer = the colonies (CFU)/volume of plating 9 dilution factor, and the library quantity = the library titer 9 volume of library. Thus, the transformation efficiency of this library was about 2.33 9 106 ([1.0 9 106), the titer of the primary cDNA library was 6.75 9 105 CFU/mL, and the quantity of the primary cDNA library was 1.01 9 107. The concentration of liquid mixtures from the plates was uniform coupled with the measurement of the cell density using a hemacytometer, and the cell density of the library was 1.75 9 109 cells/mL ([29107 cells/mL). After the mixture was subpackaged, the library titer and library quantity were determined. The titer of the amplified cDNA library was 2.33 9 107 CFU/mL, and the quantity of the amplified cDNA library was 1.14 9 109. Identification of the inserts and the recombination rate of the cDNA library The library was generated by a homologous recombination assay in vivo and, in order to identify the length of the inserts and the recombination rate of the cDNA library, 35 randomly picked individual clones were amplified by PCR using the AD vector universal primers. In Fig. 2, all of the fragments were more than 200 bp except for sample 18, which did not have the insert. The results revealed the insert ranged from 0.35 to 2.1 kb with an average size of 0.79 kb. Three samples were 0.35 kb (8.82 %), 23 samples were between 0.5 and 1.0 kb (67.64 %), 6 samples were between 1 and 2.0 kb (17.65 %), and 1 sample (2.94 %) was more than 2.0 kb. The recombination rate was about 97.14 %, as obtained from the formula [27]: the

recombination rate = the number of clones with insert fragments/the total number of clones detected 9 100 %. The above information also indicated that the small fragment cDNAs were removed from the libraries. Sequencing and sequence analysis Further detection for the quality of this library, sequencing of twenty-seven randomly picked independent clones were accomplished. For the length of the inserts, all of them were more than 200 bp, and 4 samples were between 0.30 and 0.5 kb (14.82 %), 9 samples were between 0.5 and 0.8 kb (33.33 %), 9 samples were between 0.8 and 1.0 kb (33.33 %), 3 samples were between 1 and 2.0 kb (11.11 %), and 1 sample was more than 2.0 kb. The results also revealed the insert ranged from 0.323 to 2.017 kb, with an average insert size of 0.807 kb. The sequence similarity analysis (Supporting Information-S4) showed fourteen cDNAs were DEV genes, such as DEV-ORF3, DEV LORF3-like protein CDS, DEV-UL35 CDS and DEV-UL46 CDS. Three cDNAs were duck mitochondrial genes, which all were duck mitochondrial cytochrome c oxidase subunitIgenes. Three cDNAs were similar to the Gallus gallus genes, including the Gallus gallus cyclin Pas1/PHO80 domain containing 1 (CNPPD1) gene, Gallus gallus ring finger protein 130 gene, and Gallus gallus 40S ribosomal protein S17 gene. One cDNA was similar to the Rattus norvegicus rRNA promoter binding protein gene, and six cDNAs did not match any gene of the database. Because the genome of duck has not been published, and only the duck mitochondrial genome has been reported [28, 29], the cDNAs that were similar to the Gallus gallus genes, Rattus norvegicus gene, and not matching with any gene of the database were probably duck genes. Furthermore, the results were considered as highly credible in that the BLASTn scores were higher than 200 and the BLASTx scores were higher than 100.

123

472

Mol Biol Rep (2014) 41:467–475

Fig. 2 The identification of the inserts size. M1 DL 2,000 bp; M2 DNA Marker 200*5,000 bp. Lines 1*35 were 35 recombinant individual colonies, which were randomly picked and amplified by performing PCR with the AD vector universal primers. All of the fragments were more than 200 bp except for sample 18, which did not

have the insert. Three samples were about 0.35 kb, 23 samples were between 0.5*1.0 kb, 6 samples were between 1*2.0 kb, 1 sample was more than 2.0 kb, and the insert ranged from 0.35 to 2.1 kb. NC (negative control): templates of PCR application were water

Discussion

about 3 % of the total RNA. In this research, the abundance of the mRNA obtained from the total RNA was 2.77 % of the total RNA. The two numbers were contiguous, which indicated that the mRNAs from the DEV-infected DEFs were of high quality and quantity and could be used to construct the cDNA library. As shown in Supporting Information-S2a and S2b, the first strand cDNAs resourced from the mRNAs had a uniform distribution. However, that from the total RNAs were overdispersed, and most of them were smaller than 1,000 bp. And these results indicated that a high-quality cDNA library should be a resource from mRNA. In order to obtain high-quality ds cDNA and avoid the occurrence of selection bias favor the smaller cDNAs during PCR [30], the ss cDNAs resourced from the mRNAs were selected firstly as a template for ds cDNA synthesis (Supporting Information-S2); secondly, fewer PCR cycles were employed to minimize such bias as previously suggested [30] before performing LD-PCR. And the optimized number of LD-PCR cycles were 22 cycles for the test team and 21 cycles for the positive control team. And the quantity of both mRNAs during the first-strand cDNA synthesis leaded to the different of two optimal cycles. To obtain a high transformation and recombinant ratio, fragments smaller than 200 bp were eliminated effectively by CHROMA SPIN-400 purification columns, which

Based on the research of Wang [11], the results indicated that the combination method of both Trizol reagent and the mRNA isolation kit could overcome some limitations and yield mRNA extracts with good quality and quantity. We therefore used a similar procedure. The electrophoresis of the total RNA exhibited three bright bands corresponding to ribosomal 28 S, 18 S and 5 S RNA, and the 28 S band was twofold more abundant than the 18 S band. Thus, there was little or no visible RNA degradation or contamination (polysaccharides and proteins) occurring during isolation of the total RNA. The ratio of A260/A280 of the total RNA was 2.033, which was in the middle of the reference values (1.9*2.1). It was generally considered that the purity of the RNA was 100 % as acquired, and there was no degradation (RNA) or contamination. Thus, the total RNAs obtained from the cells were integral and pure, and the total RNA could be used to isolate mRNA or synthesize firststand cDNA. In addition, we known that the quantity and quality of cDNA was critical to establishing a high quality cDNA library, but both of them were affected by mRNA. The quantity of cDNA cloud be enhanced by increasing the quantity of mRNA. Therefore, the steps for isolating mRNAs were critical steps in establishing a cDNA library. It was also shown that the abundance of mRNA exists in

123

Mol Biol Rep (2014) 41:467–475

473

Table 1 The DEF Cells with post-infected DEV Y2H cDNA library transformation efficiency, insert size and the quality of library

The transformation efficiency

DEF cDNA library

Expected

2.33 9 106 transformants/4.34 lg pGADT7-Rec

C1 9 106 transformants/3ug pGADT7-Rec

Insert size (kb) Minimum

0.4 kb

Maximum

1.5 kb

Average

0.8 kb

% of positive recombinant clones

29/30

Cell density of frozen library (cells/mL)

1.75 9 109 cells/mL

1 [2 9 107 cells/mL

cDNA library titer (CFU/mL) The primary cDNA library

6.75 9 105 CFU/mL

The amplified cDNA library

2.33 9 107 CFU/mL

The cDNA library quantity The primary cDNA library The amplified cDNA library

1.01 9 107 1.14 9 109

Table 2 Insert fragments length by sequencing Insert size (kb)

[1 9 107 cells/mL

Number of fragments

Rate (%)

0.30*0.50

4

14.82

0.50*0.80 0.80*1.0

9 9

33.33 33.33

1.0*2.0

3

11.11

[2.0

1

3.70

avoided the very small inserts and non-recombinant clones in the cDNA library. In addition, the ds cDNA and pGADT7-Rep ligation ratio was 1:1, according to the Xiao Hong [31] and Naiara Rodrı’guez-Ezpeleta studies [32], and approximately 4.34 lg (10 lL) ds cDNA and 5 lg (10 lL) pGADT7-Rep were used for transformation. Eventually, the transformation efficiency of the library was 2.33 9 106 ([1.0 9 106), and the recombination rate was at about 97.14 %. Table 1 also showed that the transformation efficiency of the library was about 2.33 9 106, which was more than 1.0 9 106. According to Clareke-Carbon’s formula [26], a cDNA library should theoretically contain at least 1.7 9 105 independent clones to be sufficient for including the majority of mRNAs expressed [19], and for a clone derived from low-abundance mRNA would be screened out with 99 percent probability from the library [33]. In this article, the titer of the primary and amplified cDNA library was 6.75 9 105 and 2.33 9 107 CFU/mL, respectively. The capacity of the two cDNA libraries constructed in this work was 1.01 9 107 and 1.14 9 109 CFU/mL, respectively. The recombination rate of the library was 97.14 % with only 1 of the 35 clones not acquiring a PCR product. These results indicated that this cDNA library is a good

source for finding specific genes and should be able to contribute to the understanding of mechanisms of DEV invading duck embryonic fiber cells Table 2. Comparison the results of inserts detection with that by sequencing indicated that the average insert size of the amplified cDNA library was contiguous (0.79 and 0.807 kb, respectively). And the results also showed that most inserts were more than 0.5 kb and especially were between 0.5*1.0 kb. Interestingly, the result was similar to the size range of cDNAs from the Mouse ESCs Y2H cDNA libraries [27]. In summary, all these results displayed that this cDNA library was constructed successfully. Further analysis showed there were fourteen mRNAs sequences analogously to the DEV-CHv strain genome DNA by BLAST sequencing results. Five polyA sites were identified in the DEV-CHv genome, and they were located in the 116702nd, 54571st, 73662nd, 133526th and 25292nd sites of the DEV-CHv strain genome. Furthermore, they also corresponded to three full-length mRNAs of the LORF3, UL35 and UL26 genes (GenBank accession numbers: KC526636, KC526637, KC526638), a new virus transcript that is located in between the IRS and the US1 gene (GenBank accession number: KC526639) and a non-full-length mRNA (GenBank accession number: KC526640). The transcripts of the LORF3, UL35 and UL26 genes matched with the 118491*116698, 55110*54571 and 74800*73662 areas of the genome DNA with 100 % similarity. The transcript of LORF3 was 1,822 bp, which was comprised by 50 -UTR area of -23 bases apart from the upstream of the LORF3 gene initiation site, full-length LORF3 CDS (1,443 bp), the 30 UTR area of 328 bp non-coding region, and the polyA tail of the rest 28 bp bases. The full-length transcript of UL35 of 587 bp included part of the UL34 gene and the full-length of the UL35 CDS. The 50 -UTR area was -182 bases apart from

123

474

the upstream of the UL35 gene initiation site, the 30 -UTR area was ?21 bases apart from the downstream of the CDS region of UL35 gene. Long transcripts of the HSV-1 UL24 gene had two polyA sites in the UL26 gene, one was in the middledownstream of the UL26 gene, and the other was downstream of the UL26 gene [34, 35]. The 73662 site lay in the middledownstream of the UL26 gene. Accordingly, the polyA site located in the middle-downstream of the HSV-1UL26 gene, also existed in the DEV-CHv UL26 gene. This transcript contained a UL25 gene sequence (74845*76641) of 34 bp, and a UL26 gene sequence (72644*74767) of 1,105 bp. Presumably, this sequence was a short transcript of the DEVCHv UL26 gene whose full-length was 2,124 bp, and was 1,160 bp. Although it was also one part of the long-transcript of the UL24 gene, it coded a complete short transcript of the UL26 gene. Therefore, this sequence was considered a short transcript of the UL26 gene. The new transcript of 668 bp, matched with the 134166*133526th base of the DEV-CHv genome. Nevertheless, the 134166th and 133526th sites were both in between the IRS and the US1 gene. At the 76*78th and 268*270th of the 50 - terminal sequence, there was a ATG codon. Furthermore, there also was a ‘-10 sequence’ (TATAA, also called Pribrow box) on the upstream -9 to -13rd base of the ATG codon. Therefore, our conclusion was that a new promoter was located in the 134166th base of the DEV-CHv genome and there was a new and full-length transcript between the IRS gene and US1 gene. Another transcript, which was similar to the DEV-CHv genome in the 24958-25292 bases with 100 % similarity, was 359 bp. The 50 -termination was at the interior of UL46 gene, and there was no promoter, so it was concluded that the transcript was not a full-length mRNA sequence. However, the 30 -termination lay between the UL46 (22912*25131) gene and the UL45 (25353*26027) gene and was apart from the downstream of the UL46 gene coding region for 162 bp, indicating that the polyA site of UL46 gene transcription was at the 162nd position after the UL46 gene coding region. Some sequences of viral mRNA (DEV-ORF3, DEVUL35 CDS and DEV-UL46 CDS) and duck’s mRNA (duck mitochondrial cytochrome c oxidase subunitIgenes, similar to the Gallus gallus genes, and similar to the Rattus norvegicus rRNA promoter binding protein gene), obtained by sequencing, were included in this cDNA library using yeast two-hybrid system. This cDNA library, constructed from the DEFs post-infected with the DEV-CHv strain, included expressed genes unique to this viral and genes of DEFs, especially genes after DEV-infected DEFs. More importantly, duck genome has not been reported by far. So the construction of this library was much significant, which not only contributed to the research of the pathogenic molecular mechanisms of DEV-CHv strain, but also employed for studying on DEFs genes.

123

Mol Biol Rep (2014) 41:467–475

Experimentally, yeast two-hybrid (Y2H) technology is by far the most useful and widely used method to detect the protein–protein interactions in high-throughput screenings of proteomics. Take research of herpesvirus for an example [36], in order to be able to perform a comparative analysis of all three herpesvirus subfamilies, all genes of five herpesvirus, VZV (varicella-zoster virus), KSHV (Kaposi’s sarcoma-associated herpesvirus), HSV-1 (herpes simplex virus 1), mCMV (murine cytomegalovirus) and EBV (Epstein–Barr virus), were systematically tested by genome-wide yeast-two-hybrid screens (Y2H) both in Fossum’s work and others’ studies [37–40]. And a core set of highly conserved protein (41 conserved proteins) interactions in all five viruses was identified. From the core interactions map, UL33 protein not only interacted with the nuclear egress proteins UL31/UL34, but also interacted with many other core proteins, such as UL19, UL42, UL17 proteins, and so on. Accordingly, we concluded that UL33 protein, which was imperative during the replication, probably was one of biomarkers. Similarity, this library will be a useful tool in proteomic analysis of interactions between the DEV and host DEFs according to the yeast two-hybrid assay, discovery of biomarkers studies on the mechanism of DEV and subsequently exploitation original vaccines and antiviral drugs to prevent or cure diseases. Acknowledgments The research was supported by China Agricultural Research System (CARS-43-8), the Ministry of Education Program (20125103110013), Sichuan Province Research Programs (2013HH0042/2013TD0015/11ZA084/12TD005/2011ZO0034/ 2011JO0040) and China 973 program (2011CB111606).

References 1. Maniatis T, Hardison RC, Lacy E, Lauer J, O’Connell C, Quon D (1978) The isolation of structural genes from libraries of eucaryotic DNA. Cell 15:687–701 2. Suzuki Y, Yoshitomo-Nakagawa K, Maruyama K, Suyama A, Sugano S (1997) Construction and characterization of a full lengthenriched and a 50 -end-enriched cDNA library. Gene 200:149–156 3. Zhu Y, Machleder E, Chenchik A, Li R, Siebert P (2001) Reverse transcriptase template switching: a SMARTTM approach for fulllength cDNA library construction. Biotechniques 30:892–897 4. Li Y, Huang B, Ma X, Wu J, Li F, Ai W, Song M, Yang H (2009) Molecular characterization of the genome of duck enteritis virus. Virology 391:151–161 5. Wang J, Ho¨per D, Beer M, Osterrieder N (2011) Complete genome sequence of virulent duck enteritis virus (DEV) strain 2085 and comparison with genome sequences of virulent and attenuated DEV strains. Virus Res 160:316–325 6. Wu Y, Cheng A, Wang M, Yang Q, Zhu D, Jia R, Chen S, Zhou Y, Wang X, Chen X (2012) Complete genomic sequence of Chinese virulent duck enteritis virus. J Virol 86:5965 7. Wu Y, Cheng A, Wang M, Zhu D, Jia R, Chen S, Zhou Y, Chen X (2012) Comparative genomic analysis of duck enteritis virus strains. J Virol 86:13841–13842

Mol Biol Rep (2014) 41:467–475 8. King AMQ, Lefkowitz E, Adams MJ, Carstens EB (2011) Virus taxonomy: ninth report of the International Committee on Taxonomy of Viruses. Elsevier Academic Press, SanDiego 9. Fadly AM, Glisson JR, McDougald LR, Nolan L, Swayne DE (2008) Duck virus enteritis. Diseases of poultry, 12th edn. Blackwell, London, pp 384–393 10. Ling P, Wang M, Chen X, Campbell KG (2007) Construction and characterization of a full-length cDNA library for the wheat stripe rust pathogen (Puccinia striiformis f. sp. tritici). BMC genomics 8:145 11. Wang YY, Zhang T, Zhou QM, Wei JC (2011) Construction and characterization of a full-length cDNA library from mycobiont of Endocarpon pusillum (lichen-forming Ascomycota). World J Microbiol Biotechnol 27:2873–2884 12. Zhao WJ, Zhang H, Bo X, Li Y, Fu X (2009) Generation and analysis of expressed sequence tags from a cDNA library of moniezia expansa. Mol Biochem Parasitol 164:80–85 13. Degrado-Warren J, Dufford M, Chen J, Bartel PL, Shattuck D, Frech GC (2008) Construction and characterization of a normalized yeast two-hybrid library derived from a human proteincoding clone collection. Biotechniques 44:265–274 14. Gou D, Chow L, Chen N, Jiang D, Li W (2001) Construction and characterization of a cDNA library from 4-week-old human embryo. Gene 278:141–147 15. Han XF, Luo J, Wu N, Matand K, Yang BJ, Wu HJ, Zhang LJ, Wang HB (2008) Construction and characterization of a goat mammary gland cDNA library. Mol Biotechnol 38:187–193 16. Ma Y, Ruan Q, Ji Y, Wang N, Li M, Qi Y, He R, Sun Z, Ren G (2011) Novel transcripts of human cytomegalovirus clinical strain found by cDNA library screening. Genet Mol Res 10:566–575 17. Lee LY, Wu FH, Hsu CT, Shen SC, Yeh HY, Liao DC, Fang MJ, Liu NT, Yen YC, Dokla´dal L (2012) Screening a cDNA library for protein–protein interactions directly in planta. Plant Cell 24:1746–1759 18. Ma J, Huang X, Wang X, Chen X, Qu Z, Huang L, Kang Z (2009) Identification of expressed genes during compatible interaction between stripe rust (Puccinia striiformis) and wheat using a cDNA library. BMC genomics 10:586 19. Tao NG, Xu J, Cheng YJ, Deng XX (2006) Construction and characterization of a cDNA library from the pulp of Cara Cara navel orange (Citrus sinensis Osbeck). J Integr Plant Biol 48:315–319 20. Thanh T, Chi VTQ, Abdullah MP, Omar H, Noroozi M, Ky H, Napis S (2011) Construction of cDNA library and preliminary analysis of expressed sequence tags from green microalga Ankistrodesmus convolutus Corda. Mol Biol Rep 38:177–182 21. Umezawa T, Sakurai T, Totoki Y, Toyoda A, Seki M, Ishiwata A, Akiyama K, Kurotani A, Yoshida T, Mochida K (2008) Sequencing and analysis of approximately 40,000 soybean cDNA clones from a full-length-enriched cDNA library. DNA Res 15:333–346 22. Yang D, Tang Z, Zhang L, Zhao C, Zheng Y (2009) Construction, characterization, and expressed sequence tag (EST) analysis of normalized cDNA library of thermo-photoperiod-sensitive genic male sterile (TPGMS) wheat from spike developmental stages. Plant Mol Biol Rep 27:117–125 23. Yoshida S, Ishida JK, Kamal NM, Ali AM, Namba S, Shirasu K (2010) A full-length enriched cDNA library and expressed

475

24.

25.

26. 27.

28. 29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

sequence tag analysis of the parasitic weed, Striga hermonthica. BMC plant biol 10:55 Zhang H, Mao J, Yang Y, Wang K, Zhou B, Wen M (2011) Constructionof yeast two hybrid cDNA library of duck sliver inoculated with duck enteritis virus. Chin Vet Sci 41:470–473 Cheng A, Wang M, Wen M, Zhou W, Guo Y, Jia R, Xu C, Yuan G, Liu Y (2006) Construction of duck enteritis virus gene libraries and discovery, cloning and identification of viral nucleocapsid protein gene. Chin High Technol Lett 16:948–953 Green MR, Sambrook J (2012) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York Zheng Y, Tan X, Pyczek J, Nolte J, Pantakani DVK, Engel W (2013) Generation and characterization of yeast two-hybrid cDNA libraries derived from two distinct mouse pluripotent cell types. Mol Biotechnol. doi:10.1007/s12033-012-9561-4 Desjardins P, Ramirez V, Morais R (1990) Gene organization of the Peking duck mitochondrial genome. Curr Genet 17:515–518 Ramirez V, Savoie P, Morais R (1993) Molecular characterization and evolution of a duck mitochondrial genome. J Mol Evol 37:296–310 Wellenreuther R, Schupp I (2004) SMART amplification combined with cDNA size fractionation in order to obtain large fulllength clones. BMC Genomics 5:36 Xiao-hong C, Zhi C, Hang-ping Y, Feng C, Hai-hong Z, Hongjuan Z (2005) Construction and characterization of a cDNA library from human liver tissue with chronic hepatitis B. J Zhejiang Univ Sci B 6:288–294 Rodrıguez-Ezpeleta N, Teijeiro S, Forget L, Burger G, Lang BF (2009) Construction of cDNA Libraries: focus on protists and fungi. Methods Mol Biol 533:33–47 Shao Z, Cong X, Yuan J, Yang G, Chen Y, Pan J, An L (2009) Construction and characterization of a cDNA library from head kidney of Japanese sea bass (Lateolabrax japonicus). Mol Biol Rep 36:2031–2037 Hann LE, Cook WJ, Uprichard SL, Knipe DM, Coen DM (1998) The role of herpes simplex virus ICP27 in the regulation of UL24 gene expression by differential polyadenylation. J Virol 72:7709–7714 Liu S, Chen S, Li H, Kong X (2007) Molecular characterization of the herpes simplex virus 1 (HSV-1) homologues, UL25 to UL30, in duck enteritis virus (DEV). Gene 401:88 Fossum E, Friedel CC, Rajagopala SV, Titz B, Baiker A, Schmidt T (2009) Evolutionarily conserved herpesviral protein interaction networks. PLoS Pathog 5:e1000570 Calderwood MA, Venkatesan K, Xing L, Chase MR, Vazquez A, Holthaus AM (2007) Epstein–Barr virus and virus human protein interaction maps. Proc Natl Acad Sci 104:7606–7611 Lee JH, Vittone V, Diefenbach E, Cunningham AL, Diefenbach RJ (2008) Identification of structural protein–protein interactions of herpes simplex virus type 1. Virology 378:347–354 Rozen R, Sathish N, Li Y, Yuan Y (2008) Virion-wide protein interactions of Kaposi’s sarcoma-associated herpesvirus. J Virol 82:4742–4750 Uetz P, Dong Y-A, Zeretzke C, Atzler C, Baiker A, Berger B (2006) Herpesviral protein networks and their interaction with the human proteome. Science 311:239–242

123