Journal of Helminlhology (1992) 66, 25-32
Molecular cloning of the cDNA encoding a 42 kDa antigenic polypeptide of Anisakis simplex larvae K. SUGANE, S. SUN and T. MATSUURA Department of Parasitology, Shinshu University School of Medicine, Asahi 3-1-1, Matsumoto City, Nagano Prefecture 390, Japan
ABSTRACT The gene encoding an antigenic polypeptide of Anisakis simplex larvae was studied using recombinant DNA techniques. cDNA synthesized from poly(A)-rich mRNA from A. simplex larvae was ligated into phage vector X gtll DNA and packaged in vitro. The phages were propagated on Escherichia coli and a X gtll expression library was constructed. A cDNA clone encoding a 42 kDa antigenic polypeptide was selected by immunoscreening of the library and identified by the epitope selection method. A clone containing cDNA for a 42 kDa protein was isolated. The gene encoding this 42 kDa antigenic polypeptide was characterized by DNA and RNA blot analysis using the cDNA as a probe. The gene was transcribed to mRNA with approximately 1400 nucleotides and translated to 42 kDa polypeptide. The antigenic p"-galactosidase fusion protein synthesized by bacteria had no cross-reactivity with other parasite-infected sera. KEY WORDS: Anisakis simplex, fusion protein, epitope selection. X gtll
INTRODUCTION
Anisakiasis is caused by infection with Anisakis larvae and may be acquired by eating raw fish (sushi or sashimi) (ASAISHI etal., 1989; OSHIMA, 1987). The clinical manifestations of this disease are thought to be caused by the invasion of larva into the mucosal and muscular layer of the stomach and intestine (KLIKS, 1983). Although some cases of gastric anisakiasis are diagnosed by endoscopic examination, clinical diagnosis is often difficult in intestinal anisakiasis which is inaccessible to diagnostic endoscopy (PINKUS & COOLIDGE, 1975). Immunodiagnosis using parasite antigen is important when larvae can not be found in the stomach or small intestine (AKAO et al., 1990), but cross-reactivity with other ascarids has been noted (TAKAHASHI et al., 1986; KENNEDY et al. 1988). In this study, antigenic polypeptides of A. simplex larvae were examined using recombinant DNA techniques and the gene encoding the 42 kDa antigenic polypeptide was characterized at DNA and RNA levels. MATERIALS AND METHODS Parasite
Third stage larvae of A. simplex were recovered from codfishes which had been obtained from a fish shop in Matsumoto City. Larvae were washed several times with sterile 0-85% saline solution, immediately frozen in liquid nitrogen and stored at -80°C until used. Sera
Five gastric anisakiasis patient sera were supplied by Dr. M. Tsuji (Hiroshima University, Hiroshima). All the patients had an epigastric pain within 12 h after eating raw mackerel and Anisakis larva could not be found in the stomach of patients by gastroendoscopic examination. All the sera were drawn between 3
26
K. SUGANE el al.
and 7 days after the onset of epigastric pain and were positive in the Ouchterlony test. Negative sera in the Ouchterlony test from five healthy volunteers who had no past history of gastrointestinal disorders were used as controls. Toxocara canis infected human sera were also received from Dr. M. Tsuji. Schistosoma japonicum or Paragonimus westermani infected human sera were provided by Dr. M. Ito (Nagoya City University, Nagoya). Human sera infected with Dirofilaria immitis or Taenia solium cysts (Cysticercus cellulosae) were given by Dr. K. Ando (Mie University, Tsu) and Dr. T. Uno (Nara University, Nara), respectively. These sera were demonstrated to have high titres of antibodies to respective parasites when examined by ELISA. Isolation and in vitro translation of mRNA from A. simplex larvae Frozen larvae were ground into powder with a Freezer/Mill pulverizer (Spex Industries Inc, USA). Total RNA was extracted using the hot phenol method (FERAMISCO et ai, 1982). In vitro translation of mRNA was performed using rabbit reticulocyte lysate (Radiochemical Centre, Amersham, UK) as described by 35 PELHAM & JACKSON (1976). S-methionine labelled translation products were immunoprecipitated with the appropriate amount of antibody and analysed directly by fluorography of SDS-PAGE gels (IRVING & HOWELL, 1981). Construction of cDNA library and immunoscreening of positive clones Double-stranded cDNA was synthesized from mRNA according to the method described by GUBLER & HOFFMAN (1983). cDNA was treated by EcoR I methylase, (New England Biolab., USA) and EcoR I linker (Takara Co., Japan) was added according to the manufacturer's instructions. The linked cDNA was ligated to dephosphorylated \ gtll arms and packagd in vitro. The recombinant >.-phage particles were propagated on E. coli (Y1090) and plaques formed were transferred to the nitrocellulose filter which was previously soaked in IPTG to induce the synthesis of (3-galactosidase fusion protein on the filter. Immunoscreening of positive clones was done as follows: fusion protein composed of |3-galactosidase-/L simplex protein on the filter was reacted with A. simplex infected human serum, and then with anti-human IgG antibody conjugated horseradish peroxidase (BioRad Lab., USA). Subsequently, 4-chloro-naphthal was added as a substrate, and coloured plaques selected (HUYNH et al., 1984). Epitope selection In order to extract the monospecific antibody to the antigenic fusion protein synthesized by each positive clone from infected serum, epitope selection of the A. simplex infected human serum was carried out according to the method of WEINBERGER et al. (1985) with some modifications. After the fusion protein on the nitrocellulose filter was reacted with antiserum in TBS (50 mM Tris-HCl (pH 7-4), 150 mM NaCl) and 0-5% skimmed milk, antigen-antibody complexes on the filter were washed with 005% Tween-20 in TE solution (10 mM Tris-HCl (pH 7-4), 1 mM EDTA), and monospecific antibody was eluted by 5 mM glycine-HCl (pH 2-3). The antibody solution was adjusted to pH 7-5 by adding 1 M Tris and used as a monospecific antibody solution after suspending in 0-5% skimmed milk. DNA and RNA blot hybridization Southern and Northern blot analyses were performed. For Southern blot analysis, genomic DNA isolated from larvae by the method of KEULEN et al. (1985) was digested with restriction endonucleases and electrophoresed on 0-8% agarose
cDNA encoding an antigen of A. simplex larvae
27
gel. RNA was also electrophoresed on 1-2% agarose gel containing 6-6% formaldehyde for Northern blot hybridization. After electrophoresis, nucleic acids were electrophoretically transferred to Hybond-N filter (Amersham, UK), hybridized with 32P-cDNA probe prepared as described by FEINBERG & VOGELSTEIN (1983) and autoradiographed. Western blotting E. coli (Y1089) cells lysogenized with X.Ca42 were grown and lysate containing the 42 kDa-antigenic fusion protein was prepared as reported by HUYNH et al. (1984). Somatic extracts of A simplex larvae prepared by our method (SUN et al., 1991) were electrophoresed on SDS-polyacrylamide gel, electrophoretically transferred to Hybond-C membrane (Amersham, UK), and reacted with the monospecific antibody which had been epitope-selected by ^Ca42 fusion protein or parasiteinfected serum. Immunocomplex was reacted with HRP-conjugated anti-IgG antibody followed by staining from peroxidase activity as reported by TOWBIN et al. (1979).
B 12
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3 4 5
12
3
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452920-
FIG. 1. A. Immunoprecipitation of in vitro translation products of mRNA from A. simplex larvae with infected human serum or monospecific antibody purified by epitope selection. Lane 1 in vitro translation products; lane 2 in vitro translation products immunoprecipitafed with normal human serum; lane 3 in vitro translation products immunoprecipitated with A. simplex infected human serum; lane 4 in vitro translation products immunoprecipitated with monospecific antibody X. gtll fi-galactosidase; lane 5 in vitro translation products immunoprecipitated with monospecific antibody to XCa42 fusion protein. The positions of markers are indicated, expressed in kilodaltons. *Antigenic polypeptide. FIG. l.B. Immunoprecipitation of in vitro translation products reacted with A. simplex infected human serum absorbed by X.Ca42 fusion protein. Lane 1 in vitro translation products immunoprecipitated with A. simplex infected human serum; lane 2 in vitro translation products immunoprecipitated with A. simplex infected human serum preabsorbed by XCa42 fusion protein; lane 3 in vitro translation products- immunoprecipitated with A. simplex infected human serum preabsorbed by X gtll fS-galactosidase. The positions of markers are indicated, expressed in kilodaltons. *Antigenic polypeptide.
K. SUGANE et at.
28
Sequence analysis of cDNA The cDNA insert of k gtll was subcloned into pTZ18U. The nucleotide sequence of the cDNA was determined by the dideoxynucleotide chain termination method (SANGER et al., 1977). RESULTS Identification of cDNA clone encoding 42 kDA-antigenic protein In vitro translation products of mRNA from A. simplex larvae (Fig. 1A, lane 1) were reacted with the A. simplex infected human serum and immunoprecipitated to detect antigenic polypeptides by SDS-PAGE autoradiography. The 42 kDa antigenic polypeptides reacted strongly with IgG antibody in infected serum (Fig. 1A lane 3*). Then, we tried to clone cDNA encoding the 42 kDa antigenic polypeptide. A X. gtll cDNA expression library derived from poly(A)-rich mRNA from A. simplex larvae was constructed and 28 positive clones were obtained from
C
1 2 3 4
5
B 6645-1.6 -1.4
29-
-09
FIG. 2. A. Southern blot analysis of the genomic DNA from A. simplex larvae. 1 \ig DNA was digested with EcoR I (lane 1), Hind III (lane 2) and Hae III (lane 3), electrophoresed on 0-8% agarose gel and transferred to Hybond N filter. Hybridization of DNA fragments with 12P-ACa42 cDNA was performed as described in Materials and Methods. The positions of markers are indicated, expressed in kilobase pairs. FIG. 2. B. Northern blot analysis of mRNA from A. simplex larvae. 1-5 ng poly(A) rich mRNA was electrophoresed on 1-2% agarose gel, transferred to Hybond N filter and hybridized with 32P-A.Ca42 cDNA. The positions of markers are indicated, expressed in kilobase pairs. FIG. 2. C. Western blot assay of somatic extracts of A. simplex larvae. 50 (ig somatic extracts of A. simplex larvae were electrophoresed on 12-5% SDS-polyacrylanide gel, electrophoretically transferred to Hybond-C membrane and reacted with the monospecific antibody which had been epitope-selected by XCa42 fusion protein. Immunocomplex was reacted with HRP-conjugated anti-IgG antibody. Lane 1 normal human serum; lane 2 A. simplex infected human serum; lane 3 A. simplex infected human serum preabsorbed by X.Ca42 fusion protein; lane 4 monospecific antibody to XCa42 fusion protein purified by epitope selection; lane 5 A. simplex infected human serum epitope-selected by X gtll fi-galactosidase. The positions of markers are indicated, expressed in kilodaltons.
cDNA encoding an antigen of A. simplex larvae
29
3-4x 105 recombinant clones by immunoscreening. In order to isolate cDNA clones encoding 42 kDa-antigenic polypeptide from positive clones, monospecific antibody purified by the epitode selection method was reacted with the in vitro translation products and antigenic protein in the translation products was examined by SDS-PAGE autoradiography. XCa42 was found to be a candidate clone encoding 42 kDa-antigenic polypeptide (Fig. 1A lane 5*). When the A. gtll lysate was used instead of the XCa42 lysate as a control, no antigenic polypeptide reactive with the epitope-selected antibody was detected (Fig. 1A lane 4). Furthermore, infected serum absorbed by the fusion protein derived from XCa42 lysogen did not react with 42 kDa-antigenic polypeptide in the in vitro translation products (Fig. IB, lane 2). The same results were obtained when the other four gastric anisakiasis patient sera were used. The result demonstrates the X.Ca42 is a clone encoding a 42 kDa-antigenic polypeptide. Structure and expression of the gene encoding 42 kDa-antigenic protein Southern and Northern blot analysis were carried out using >.Ca42 cDNA as a probe. Southern blot analysis showed that the cDNA probe hybridized with DNA fragments with more than 5 1 kb after digestion of genomic DNA by EcoR I. Also, two or three DNA fragments were hybridized with the probe after digestion of genomic DNA by Hind III and Hae III. Northern blot assay also demonstrated that the gene encoding 42 kDa antigen was transcribed to mRNA with 1400 nucleotides
205116—
66-
45-
i". 4-
FIG. 3. Antigen specificity of XCa42 fusion protein. 15 ng lysate of A.Ca42 lysogen was electrophoresed on 10% SDS-polyacrylamide gel and Western blot analysis was done using other parasite-infected human sera. Lane 1 A. simplex infected serum; lane 2 Toxocara canis infected serum; lane 3 Dirofilaria immitis infected serum; lane 4 Schistosoma japonicum infected serum; lane 5 Paragonimus westermani infected serum; lane 6 Taenia solium infected serum; lane 7 normal human serum. The positions of markers are indicated, expressed in kilodaltons. *XCa42 antigenic fusion protein.
30
K. SUGANE et al.
(Fig. 2B). However, Western blot analysis showed that the 40 kDa native polypeptide is somatic extracts of A. simplex larvae reacted with the epitopeselected monospecific antibody to 42 kDa antigenic polypeptide (Fig. 2C lane 4
Molecular cloning of the cDNA encoding a 42 kDa antigenic polypeptide of Anisakis simplex larvae K. SUGANE, S. SUN and T. MATSUURA Department of Parasitology, Shinshu University School of Medicine, Asahi 3-1-1, Matsumoto City, Nagano Prefecture 390, Japan
ABSTRACT The gene encoding an antigenic polypeptide of Anisakis simplex larvae was studied using recombinant DNA techniques. cDNA synthesized from poly(A)-rich mRNA from A. simplex larvae was ligated into phage vector X gtll DNA and packaged in vitro. The phages were propagated on Escherichia coli and a X gtll expression library was constructed. A cDNA clone encoding a 42 kDa antigenic polypeptide was selected by immunoscreening of the library and identified by the epitope selection method. A clone containing cDNA for a 42 kDa protein was isolated. The gene encoding this 42 kDa antigenic polypeptide was characterized by DNA and RNA blot analysis using the cDNA as a probe. The gene was transcribed to mRNA with approximately 1400 nucleotides and translated to 42 kDa polypeptide. The antigenic p"-galactosidase fusion protein synthesized by bacteria had no cross-reactivity with other parasite-infected sera. KEY WORDS: Anisakis simplex, fusion protein, epitope selection. X gtll
INTRODUCTION
Anisakiasis is caused by infection with Anisakis larvae and may be acquired by eating raw fish (sushi or sashimi) (ASAISHI etal., 1989; OSHIMA, 1987). The clinical manifestations of this disease are thought to be caused by the invasion of larva into the mucosal and muscular layer of the stomach and intestine (KLIKS, 1983). Although some cases of gastric anisakiasis are diagnosed by endoscopic examination, clinical diagnosis is often difficult in intestinal anisakiasis which is inaccessible to diagnostic endoscopy (PINKUS & COOLIDGE, 1975). Immunodiagnosis using parasite antigen is important when larvae can not be found in the stomach or small intestine (AKAO et al., 1990), but cross-reactivity with other ascarids has been noted (TAKAHASHI et al., 1986; KENNEDY et al. 1988). In this study, antigenic polypeptides of A. simplex larvae were examined using recombinant DNA techniques and the gene encoding the 42 kDa antigenic polypeptide was characterized at DNA and RNA levels. MATERIALS AND METHODS Parasite
Third stage larvae of A. simplex were recovered from codfishes which had been obtained from a fish shop in Matsumoto City. Larvae were washed several times with sterile 0-85% saline solution, immediately frozen in liquid nitrogen and stored at -80°C until used. Sera
Five gastric anisakiasis patient sera were supplied by Dr. M. Tsuji (Hiroshima University, Hiroshima). All the patients had an epigastric pain within 12 h after eating raw mackerel and Anisakis larva could not be found in the stomach of patients by gastroendoscopic examination. All the sera were drawn between 3
26
K. SUGANE el al.
and 7 days after the onset of epigastric pain and were positive in the Ouchterlony test. Negative sera in the Ouchterlony test from five healthy volunteers who had no past history of gastrointestinal disorders were used as controls. Toxocara canis infected human sera were also received from Dr. M. Tsuji. Schistosoma japonicum or Paragonimus westermani infected human sera were provided by Dr. M. Ito (Nagoya City University, Nagoya). Human sera infected with Dirofilaria immitis or Taenia solium cysts (Cysticercus cellulosae) were given by Dr. K. Ando (Mie University, Tsu) and Dr. T. Uno (Nara University, Nara), respectively. These sera were demonstrated to have high titres of antibodies to respective parasites when examined by ELISA. Isolation and in vitro translation of mRNA from A. simplex larvae Frozen larvae were ground into powder with a Freezer/Mill pulverizer (Spex Industries Inc, USA). Total RNA was extracted using the hot phenol method (FERAMISCO et ai, 1982). In vitro translation of mRNA was performed using rabbit reticulocyte lysate (Radiochemical Centre, Amersham, UK) as described by 35 PELHAM & JACKSON (1976). S-methionine labelled translation products were immunoprecipitated with the appropriate amount of antibody and analysed directly by fluorography of SDS-PAGE gels (IRVING & HOWELL, 1981). Construction of cDNA library and immunoscreening of positive clones Double-stranded cDNA was synthesized from mRNA according to the method described by GUBLER & HOFFMAN (1983). cDNA was treated by EcoR I methylase, (New England Biolab., USA) and EcoR I linker (Takara Co., Japan) was added according to the manufacturer's instructions. The linked cDNA was ligated to dephosphorylated \ gtll arms and packagd in vitro. The recombinant >.-phage particles were propagated on E. coli (Y1090) and plaques formed were transferred to the nitrocellulose filter which was previously soaked in IPTG to induce the synthesis of (3-galactosidase fusion protein on the filter. Immunoscreening of positive clones was done as follows: fusion protein composed of |3-galactosidase-/L simplex protein on the filter was reacted with A. simplex infected human serum, and then with anti-human IgG antibody conjugated horseradish peroxidase (BioRad Lab., USA). Subsequently, 4-chloro-naphthal was added as a substrate, and coloured plaques selected (HUYNH et al., 1984). Epitope selection In order to extract the monospecific antibody to the antigenic fusion protein synthesized by each positive clone from infected serum, epitope selection of the A. simplex infected human serum was carried out according to the method of WEINBERGER et al. (1985) with some modifications. After the fusion protein on the nitrocellulose filter was reacted with antiserum in TBS (50 mM Tris-HCl (pH 7-4), 150 mM NaCl) and 0-5% skimmed milk, antigen-antibody complexes on the filter were washed with 005% Tween-20 in TE solution (10 mM Tris-HCl (pH 7-4), 1 mM EDTA), and monospecific antibody was eluted by 5 mM glycine-HCl (pH 2-3). The antibody solution was adjusted to pH 7-5 by adding 1 M Tris and used as a monospecific antibody solution after suspending in 0-5% skimmed milk. DNA and RNA blot hybridization Southern and Northern blot analyses were performed. For Southern blot analysis, genomic DNA isolated from larvae by the method of KEULEN et al. (1985) was digested with restriction endonucleases and electrophoresed on 0-8% agarose
cDNA encoding an antigen of A. simplex larvae
27
gel. RNA was also electrophoresed on 1-2% agarose gel containing 6-6% formaldehyde for Northern blot hybridization. After electrophoresis, nucleic acids were electrophoretically transferred to Hybond-N filter (Amersham, UK), hybridized with 32P-cDNA probe prepared as described by FEINBERG & VOGELSTEIN (1983) and autoradiographed. Western blotting E. coli (Y1089) cells lysogenized with X.Ca42 were grown and lysate containing the 42 kDa-antigenic fusion protein was prepared as reported by HUYNH et al. (1984). Somatic extracts of A simplex larvae prepared by our method (SUN et al., 1991) were electrophoresed on SDS-polyacrylamide gel, electrophoretically transferred to Hybond-C membrane (Amersham, UK), and reacted with the monospecific antibody which had been epitope-selected by ^Ca42 fusion protein or parasiteinfected serum. Immunocomplex was reacted with HRP-conjugated anti-IgG antibody followed by staining from peroxidase activity as reported by TOWBIN et al. (1979).
B 12
66- •
3 4 5
12
3
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452920-
FIG. 1. A. Immunoprecipitation of in vitro translation products of mRNA from A. simplex larvae with infected human serum or monospecific antibody purified by epitope selection. Lane 1 in vitro translation products; lane 2 in vitro translation products immunoprecipitafed with normal human serum; lane 3 in vitro translation products immunoprecipitated with A. simplex infected human serum; lane 4 in vitro translation products immunoprecipitated with monospecific antibody X. gtll fi-galactosidase; lane 5 in vitro translation products immunoprecipitated with monospecific antibody to XCa42 fusion protein. The positions of markers are indicated, expressed in kilodaltons. *Antigenic polypeptide. FIG. l.B. Immunoprecipitation of in vitro translation products reacted with A. simplex infected human serum absorbed by X.Ca42 fusion protein. Lane 1 in vitro translation products immunoprecipitated with A. simplex infected human serum; lane 2 in vitro translation products immunoprecipitated with A. simplex infected human serum preabsorbed by XCa42 fusion protein; lane 3 in vitro translation products- immunoprecipitated with A. simplex infected human serum preabsorbed by X gtll fS-galactosidase. The positions of markers are indicated, expressed in kilodaltons. *Antigenic polypeptide.
K. SUGANE et at.
28
Sequence analysis of cDNA The cDNA insert of k gtll was subcloned into pTZ18U. The nucleotide sequence of the cDNA was determined by the dideoxynucleotide chain termination method (SANGER et al., 1977). RESULTS Identification of cDNA clone encoding 42 kDA-antigenic protein In vitro translation products of mRNA from A. simplex larvae (Fig. 1A, lane 1) were reacted with the A. simplex infected human serum and immunoprecipitated to detect antigenic polypeptides by SDS-PAGE autoradiography. The 42 kDa antigenic polypeptides reacted strongly with IgG antibody in infected serum (Fig. 1A lane 3*). Then, we tried to clone cDNA encoding the 42 kDa antigenic polypeptide. A X. gtll cDNA expression library derived from poly(A)-rich mRNA from A. simplex larvae was constructed and 28 positive clones were obtained from
C
1 2 3 4
5
B 6645-1.6 -1.4
29-
-09
FIG. 2. A. Southern blot analysis of the genomic DNA from A. simplex larvae. 1 \ig DNA was digested with EcoR I (lane 1), Hind III (lane 2) and Hae III (lane 3), electrophoresed on 0-8% agarose gel and transferred to Hybond N filter. Hybridization of DNA fragments with 12P-ACa42 cDNA was performed as described in Materials and Methods. The positions of markers are indicated, expressed in kilobase pairs. FIG. 2. B. Northern blot analysis of mRNA from A. simplex larvae. 1-5 ng poly(A) rich mRNA was electrophoresed on 1-2% agarose gel, transferred to Hybond N filter and hybridized with 32P-A.Ca42 cDNA. The positions of markers are indicated, expressed in kilobase pairs. FIG. 2. C. Western blot assay of somatic extracts of A. simplex larvae. 50 (ig somatic extracts of A. simplex larvae were electrophoresed on 12-5% SDS-polyacrylanide gel, electrophoretically transferred to Hybond-C membrane and reacted with the monospecific antibody which had been epitope-selected by XCa42 fusion protein. Immunocomplex was reacted with HRP-conjugated anti-IgG antibody. Lane 1 normal human serum; lane 2 A. simplex infected human serum; lane 3 A. simplex infected human serum preabsorbed by X.Ca42 fusion protein; lane 4 monospecific antibody to XCa42 fusion protein purified by epitope selection; lane 5 A. simplex infected human serum epitope-selected by X gtll fi-galactosidase. The positions of markers are indicated, expressed in kilodaltons.
cDNA encoding an antigen of A. simplex larvae
29
3-4x 105 recombinant clones by immunoscreening. In order to isolate cDNA clones encoding 42 kDa-antigenic polypeptide from positive clones, monospecific antibody purified by the epitode selection method was reacted with the in vitro translation products and antigenic protein in the translation products was examined by SDS-PAGE autoradiography. XCa42 was found to be a candidate clone encoding 42 kDa-antigenic polypeptide (Fig. 1A lane 5*). When the A. gtll lysate was used instead of the XCa42 lysate as a control, no antigenic polypeptide reactive with the epitope-selected antibody was detected (Fig. 1A lane 4). Furthermore, infected serum absorbed by the fusion protein derived from XCa42 lysogen did not react with 42 kDa-antigenic polypeptide in the in vitro translation products (Fig. IB, lane 2). The same results were obtained when the other four gastric anisakiasis patient sera were used. The result demonstrates the X.Ca42 is a clone encoding a 42 kDa-antigenic polypeptide. Structure and expression of the gene encoding 42 kDa-antigenic protein Southern and Northern blot analysis were carried out using >.Ca42 cDNA as a probe. Southern blot analysis showed that the cDNA probe hybridized with DNA fragments with more than 5 1 kb after digestion of genomic DNA by EcoR I. Also, two or three DNA fragments were hybridized with the probe after digestion of genomic DNA by Hind III and Hae III. Northern blot assay also demonstrated that the gene encoding 42 kDa antigen was transcribed to mRNA with 1400 nucleotides
205116—
66-
45-
i". 4-
FIG. 3. Antigen specificity of XCa42 fusion protein. 15 ng lysate of A.Ca42 lysogen was electrophoresed on 10% SDS-polyacrylamide gel and Western blot analysis was done using other parasite-infected human sera. Lane 1 A. simplex infected serum; lane 2 Toxocara canis infected serum; lane 3 Dirofilaria immitis infected serum; lane 4 Schistosoma japonicum infected serum; lane 5 Paragonimus westermani infected serum; lane 6 Taenia solium infected serum; lane 7 normal human serum. The positions of markers are indicated, expressed in kilodaltons. *XCa42 antigenic fusion protein.
30
K. SUGANE et al.
(Fig. 2B). However, Western blot analysis showed that the 40 kDa native polypeptide is somatic extracts of A. simplex larvae reacted with the epitopeselected monospecific antibody to 42 kDa antigenic polypeptide (Fig. 2C lane 4
