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Identification of antigenic genes in Trichinella spiralis by immunoscreening of cDNA libraries
Veterinary Parasitology 159 (2009) 272–275
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Identification of antigenic genes in Trichinella spiralis by immunoscreening of cDNA libraries§ X.P. Wu a, B.Q. Fu a,b, X.L. Wang a, L. Yu a, S.Y. Yu a, H.K. Deng a, X.Y. Liu a, P. Boireau a,b, F. Wang a, M.Y. Liu a,* a b
Key Laboratory for Zoonosis Research, Ministry of Education, Institute of Zoonosis, Jilin University, 5333 Xian Road, 130062 Changchun, PR China JRU BIPAR INRA AFSSA ENVA UPVM, 94703 Maisons-Alfort Cedex, France
A R T I C L E I N F O
A B S T R A C T
Keywords: Trichinella spiralis Antigenic genes Immunoscreening cDNA libraries
Genes encoding antigens of adult worm, newborn larvae and muscle larvae of Trichinella spiralis were identified by immunoscreening their corresponding cDNA libraries. The cDNA libraries of T. spiralis adult (3 day old, Ad3) and newborn larvae (NBL) were screened using the serum of a pig infected with 20,000 muscle larvae (ML) at 26 days post-infection (dpi). Fifty-two positive clones representing 18 unique genes were obtained from the Ad3 cDNA library. The deduced amino acid sequences of 8 cDNAs were sequence homologues of the serine protease-like protein family. In the screening of NBL cDNA library, 81 positive clones representing 8 unique genes were isolated and of these, 70 clones corresponded to an NBL stage-specific serine protease gene. The ML cDNA library was screened using pig anti-Trichinella serum obtained at 60 dpi and 18 positive clones representing 8 unique genes were identified. Ten clones encoded a protein that is identical to a T. spiralis serine protease inhibitor. In general, our results revealed that antigenic serine protease-like proteins were found during the two invasive stages, Ad and NBL when these libraries were screened using 26 dpi antiserum, whereas a serine protease inhibitor was found in abundance in the ML library when it is screened using 60 dpi antiserum. These data are consistent with serine proteases playing an important role during the invasive stages of Trichinella infections, but become inhibited or internally controlled when the parasite enters a more stable, non-developing environment. ß 2008 Elsevier B.V. All rights reserved.
1. Introduction Infection of animals and humans by Trichinella spiralis remains a serous public threat in both developed and developing countries (Murrell and Pozio, 2000; Liu and Boireau, 2002; Dupouy-Camet, 2006). The economic importance and the serious public health problems caused
§ The nucleotide sequences identified in this study (Tables 1–3) have been deposited in the GenBankTM, EMBL and DDBJ databases under the Accession numbers EU263308–EU263333, EU331363, DQ864973. * Corresponding author. Tel.: +86 431 87836713; fax: +86 431 87983442. E-mail address: [email protected] (M.Y. Liu).
0304-4017/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2008.10.035
by trichinellosis have stimulated research on new molecular diagnostic methods and immunoprophylaxis for livestock and wild animals. The life cycle of T. spiralis is completed within a single host species and muscle larvae (ML), adult worms (Ad) and newborn larvae (NBL) are three major antigenic stages. Stage specificity has been observed in a subset of antigens (Boireau et al., 1997) and can evoke stage-specific host immune responses. Conventional ELISA using ES antigens from ML often cannot detect a light or moderate infection in pigs for three weeks or longer after larvae become infective, owing in part to the uniqueness of these antigenic stages. In recent years, a number of antigens have been identified from T. spiralis by immunoscreening cDNA expression libraries, including the serine proteinase inhibitor gene
X.P. Wu et al. / Veterinary Parasitology 159 (2009) 272–275
(Nagano et al., 2001), the gene family encoding glutamic and serine rich proteins (Zarlenga et al., 2002), the serine proteinase gene (Nagano et al., 2003) and tropomyosin (Nakada et al., 2003). In the present work, we have isolated genes encoding highly antigenic proteins from cDNA libraries of T. spiralis at different stages by immunological screening. These data may form a foundation for identifying recombinant antigens that can be used in the diagnosis or vaccination against trichinellosis. 2. Materials and methods
273
2.4. Sequence analysis Selected clones were sequenced from both the 50 and 3 ends using the dideoxy chain-termination method on an automated DNA sequencer. After removal of flanking vector sequences, DNA sequences were analyzed with DNASIS software. Comparison against GenBank nucleotide and protein databases was performed using the NCBI-BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) network server at the National Center for Biotechnology Information. 0
2.1. Parasite and cDNA libraries
3. Results
T. spiralis (ISS534) ML were recovered from mice 35 days post-infection by artificial digestion with pepsin–HCl (1% pepsin, 1% HCl at 42 8C for 45 min). cDNA libraries of ML, Ad3 and NBL were previously constructed (Liu et al., 2001; Zhu et al., 2005).
3.1. Sequence analysis of positive clones from Ad3 cDNA library
2.2. Absorption of swine anti-Trichinella sera A domestic pig was infected orally with 20,000 ML of T. spiralis. Pre-infection serum was collected as negative control and infection sera were collected at 26 dpi and 60 dpi. Cross-reactive antibodies were removed from the sera through pre-absorption according to Sambrook and Russell (2001) using non-recombinant vector lambda ZAP II phage. 2.3. Immunoscreening of cDNA libraries For each cDNA library, 100,000 plaque forming units (pfu) were screened with absorbed antisera obtained at 26 dpi for the Ad3 and NBL cDNA libraries, and at 60 dpi for the ML cDNA library according to conventional methods. After the second screening, positive plaques were picked and the phagemids were prepared by in vivo excision. The plasmids were transformed into E. coli SOLR strain (E. coli XLOLR strain for plasmids from NBL cDNA library).
The Ad3 cDNA library screened with 26 dpi pig antiTrichinella serum identified 52 positive clones representing 18 unique genes (Table 1). Clone Ad5 showed high sequence identity with a previously identified T. spiralis serine proteinase inhibitor gene (Nagano et al., 2001). Twenty-three clones encoded the same gene, where clone WN10 contained the full open reading frame (ORF) of 406 amino acid (AA) residues. Sequence homology searches indicated that clone WN10 was 99% similar to the T. spiralis hypothetical ORF 9.10 mRNA, and was highly similar to the cDNA encoding p46 kDa antigen from ML ES products (Sugane and Matsuura, 1990) and the multi-domain cystatin-like protein (Robinson et al., 2007). Clone Ad147 showed some similarity to T. spiralis hypothetical ORF 9.10 mRNA as did clone Ad53 to T. spiralis 53 kDa ES antigen. Interestingly, 8 novel cDNAs derived from 16 clones showed high sequence identity to each other and to the T. spiralis ESTs from different stages. All of them encoded serine protease-like proteins. Six of these cDNAs contained the full ORF encoding a peptide of 429 AA. The first 18 AA of the deduced polypeptide were predicted to be a signal peptide, and SMART analysis revealed a putative trypsin-like serine protease domain from position 37 to
Table 1 Analysis of clones from Ad3 cDNA library by immunoscreening with T. spiralis infected pig serum. Clone name (identical clones)
Accession number
Size (bp/aa)
Sequence similarity
Accession number
BLASTX E value
Ad5 (2) WN10 (3) Ad147 (4) Ad53 Ad67 Ts Adsp-1 Ts Adsp-2 (3) Ts Adsp-3 Ts Adsp-4 Ts Adsp-5 (5) Ts Adsp-6 Ts Adsp-7 (3) Ts Adsp-8 Ad42 Ad71 Ad76 Ad62 Ad2
DQ864973 EU263325 EU331362 EU263318 EU263319 EU263326 EU263327 EU263328 EU263329 EU263330 EU263331 EU263332 EU263333 EU263322 EU331363 EU263309 EU263308 EU263321
1307/373 1352/406 950/237 1453/451 1095/312 589/193 1594/429 1375/429 1539/429 1534/429 1205/370 1372/429 1399/429 907/296 751/243 886/209 1165/388 735/174
T. spiralis serine proteinase inhibitor T. spiralis hypothetical ORF 9.10 T. spiralis hypothetical ORF 9.10 T. spiralis 53 kDa excretory/secretory antigen Trichuris trichiura putative porin precursor Trypsin-like serine protease family
AAF63473 AAB48491 AAB48491 AAA97512 AAC04763 AAK16516
Caenorhabditis elegans paramyosin Plasmodium yoelii Ran-binding protein Caenorhabditis elegans calcium-sensing receptor No sequence similarity No sequence similarity
NP_492085 EAA19671 NP_501400 – –
0.0 2e135 8e12 7e11 2e11 2e24 3e44 3e46 5e46 2e46 4e35 3e44 3e44 1e69 3e07 1e21 – –
X.P. Wu et al. / Veterinary Parasitology 159 (2009) 272–275
274
Table 2 Analysis of clones from NBL cDNA library by immunoscreening with T. spiralis infected pig serum. Clone name (identical clones)
Accession number
Size (bp/aa)
Sequence similarity
Accession number
BLASTX E value
NBL84 (2)
EF041139
1379/459
T. spiralis glutamic acid rich protein cNBL 1500 T. spiralis glutamic acid rich protein cNBL 1700
AAM19759 AAM19760
0.0 0.0
NBL122 (2) NBL39 (2) NBLWN18 (70) NBL50 (2) NBL43 NBLWN5 NBLWN11
DQ864973 EU263320 – EU263319 EU263324 EU263316 EU263317
1307/373 474/117 1587/465 1095/312 1276/425 1552/475 1540/389
T. spiralis serine proteinase inhibitor T. spiralis hypothetical ORF 11.30 precursor T. spiralis newborn larvae-specific serine protease SS2 Trichuris trichiura putative porin precursor Homo sapiens heat shock protein HSP 90-alpha 2 Plasmodium yoelii glutamine–asparagine rich protein Caenorhabditis elegans putative RNA binding G-patch domain protein
AAF63473 AAB48488 AAK16520 AAC04763 CAI64495 EAA18721 NP_497893
0.0 3e45 1e58 2e11 e166 9e13 7e24
Table 3 Analysis of clones from ML cDNA library by immunoscreening with T. spiralis infected pig serum. Clone name (identical clones)
Accession number
Size (bp/aa)
Sequence similarity
Accession number
BLASTX E value
MLWM5 (10)
DQ864973
1307/373
T. spiralis serine proteinase inhibitor
AAF63473
0.0
ML42 (2)
EU263312
678/170
T. spiralis hypothetical ORF 17.20 T. pseudospiralis 21 kDa ES protein
AAB48489 AAF79206
3e85 2e63
ML3 ML59 ML41 ML63 ML1 ML64
EU263323 EU263313 EU263311 EU263314 EU263310 EU263315
671/158 447/113 563/153 1170/192 1311/437 767/199
Ostertagia ostertagi globin-like ES protein F6 Arabidopsis thaliana 60s acidic ribosomal protein P1 Dunaliella tertiolecta nucleoside diphosphate kinase Homo sapiens putative G-protein coupled receptor SH120 Mus musculus nuclear matrix protein SNEV No sequence similarity
CAD20463 BAB11203 AAK38732 CAI13224 NP_598890 –
2e07 9e12 2e56 9e43 e135 –
277. Clones Ad67, Ad42, Ad71 and Ad76 encoded proteins with similarities to a putative porin precursor, paramyosin, Ran-binding protein and calcium-sensing receptor, respectively. Clones Ad62 and Ad2 showed no significant matches with known sequences. 3.2. Sequence analysis of positive clones from NBL cDNA library The cDNA library of NBL was also screened with 26 dpi pig anti-Trichinella serum and 81 positive clones were selected, which represented 8 different genes (Table 2). Clone NBL84 shared high identity with members of NBL specifically expressed glutamic acid-rich antigenic protein family (Zarlenga et al., 2002). Clone NBL122 aligned with a T. spiralis serine proteinase inhibitor gene. Clone NBL39 showed high similarity to T. spiralis hypothetical ORF 11.30 precursor. Sequence analysis revealed 70 of the positive clones were identical to the NBL stage-specific serine protease gene, previously identified by Liu et al. (2007). The putative porin precursor was also identified (clone NBL50). Clones NBL43, NBLWN5 and NBLWN11 encoded proteins which showed sequence similarity to heat shock protein HSP 90, glutamine–asparagine rich protein, and a putative RNA binding G-patch domain protein, respectively. 3.3. Sequence analysis of positive clones from ML cDNA library The 60 dpi pig anti-Trichinella serum was used to screen the T. spiralis ML cDNA library and 18 positive
clones corresponding 8 unique genes were obtained (Table 3). Sequence analysis showed that 10 of 18 clones represented the same gene, where clone MLWM5 contained the full ORF. These clones showed the strongest reactivity on the hybridization membranes. Clone MLWM5 was similar in sequence to a T. spiralis serine protease inhibitor gene (Nagano et al., 2001) where the two differed by 3 bp and 2 deduced AA. Clone ML42 encoded a protein with high identity to T. spiralis hypothetical ORF 17.20. The other sequences coincided with genes coding for a globin-like ES protein F6, the 60 s acidic ribosomal protein P1, a nucleoside diphosphate kinase, the G-protein coupled receptor SH120 and a nuclear matrix protein SNEV, respectively. 4. Discussion Recombinant DNA techniques have been used to produce large amounts of recombinant antigens of T. spiralis; however, most of the recombinant antigens come from T. spiralis ML and only a few from Ad and NBL such as glutamic rich proteins (Zarlenga et al., 2002) and T. spiralis tropomyosin (Nakada et al., 2003). By immunoscreening multiple cDNA libraries, we succeeded in identifying a series of genes encoding antigens from the three different development stages of T. spiralis, including the known glutamic rich proteins and serine protease inhibitor, p46 kDa, along with other novel candidates. This research suggests that a large portion of the antigenic proteins produced by both Ad and NBL, represent serine proteases including a polymorphic multigene family of serine protease in Ad3 and a stage specific serine
X.P. Wu et al. / Veterinary Parasitology 159 (2009) 272–275
protease gene in NBL. Parasite proteases are likely involved in varied aspects of host–parasite interactions, such as host tissue invasion, parasite nutrition, anti-coagulation and the evasion of host immune responses (Nagano et al., 2003). However, results presented herein suggest that serine proteases play important roles in Trichinella development related to invasion and migration of the parasite, because Ad and NBL stages are where most of these classes of antigens were found. A highly antigenic serine protease inhibitor gene was identified from Ad3, NBL and ML cDNA libraries. The 3 bp differences in the ORF of cloneWM5 and the serpin gene (Nagano et al., 2001) may represent single nucleotide polymorphisms; however, the differences among these two sequences are unlikely to affect antigenicity because both were obtained by immunoscreening. The serpin gene was transcribed largely in ML, consistent with T. spiralis not requiring serine protease activity during the more stable ML stage. Parasite serpins have essential extracellular functions such as modulation and inhibition of host immune responses, fibrinolysis, coagulation, and inflammation (Zang and Maizels, 2001). The ability to inhibit coagulation during the early stages of parasitism provides an important mechanisms for some parasites to survive in the host. Proteinase inhibitors are known to provoke strong immune responses (Frank et al., 1998) and may provide a new targets for immunodiagnostics and vaccine production. In conclusion, immunoscreening is a useful technique for the isolation of highly antigenic genes. From this, we have successfully obtained a series of genes encoding highly antigenic proteins from the three different stages of T. spiralis, which include both known and novel sequences. The true stage specificity of each gene and gene product requires further elucidation inasmuch as the Ad and NBL libraries were screened only with sera presumed to be enriched with antibodies to Ad and NBL antigens (26 dpi serum), and the ML library was screened only with serum presumed enriched with antibodies to ML antigens (60 dpi). Cross-reactivity of these antiserum is still required to validate any stage specificity. Nonetheless, these results are a foundation for future studies on advancing methodology for developing more specific and sensitive diagnostic tests and better targets for efficient vaccine development. Acknowledgements This research was financially supported by grants of China 863 program 2006AA02Z451, National Natural
275
Science Foundation of China 30771885 and 30825033, and EUTRICHIPORSE QLRT-2000-01156. Conflict of interest None of the authors has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the work presented herein. References Boireau, P., Vayssier, M., Fabien, J.F., Perret, C., Calamel, M., Soule´, C., 1997. Antigenic analysis of L1M of Trichinella T1 and T5 using 40 monoclonal antibodies: characterization of eleven antigenic groups and identification of new species markers. Parasitology 115, 641–651. Dupouy-Camet, J., 2006. Trichinellosis: still a concern for Europe. Euro. Surveill. 11, 5. Frank, G.R., Mondesire, R.R., Brandt, K.S., Wisnewski, N., 1998. Antibody to the Dirofilaria immitis aspartyl protease inhibitor homologue is a diagnostic marker for feline heartworm infections. J. Parasitol. 84, 1231–1236. Liu, M.Y., Boireau, P., 2002. Trichinellosis in China: epidemiology and control. Trends Parasitol. 18, 553–556. Liu, M.Y., Zhu, X.P., Xu, K.C., Lu, Q., Boireau, P., 2001. Biological and genetic characteristics of two Trichinella isolates in China; comparison with European species. Parasite 8 (2 Suppl.), S34–S38. Liu, M.Y., Wang, X.L., Fu, B.Q., Li, C.Y., Wu, X.P., Rhun, D., Chen, Q.J., Boireau, P., 2007. Identification of stage-specifically expressed genes of Trichinella spiralis by suppression subtractive hybridization. Parasitology 135, 77–83. Murrell, K.D., Pozio, E., 2000. Trichinellosis: the zoonoses that won’t go quietly. Int. J. Parasitol. 30, 1339–1449. Nagano, I., Wu, Z., Nakada, T., Matsuo, A., Takahashi, Y., 2001. Molecular cloning and characterization of a serine proteinase inhibitor from Trichinella spiralis. Parasitology 123, 77–83. Nagano, I., Wu, Z., Nakada, T., Boonmars, T., Takahashi, Y., 2003. Molecular cloning and characterization of a serine proteinase gene of Trichinella spiralis. J. Parasitol. 89, 92–98. Nakada, T., Nagano, I., Wu, Z., Takahashi, Y., 2003. Molecular cloning and expression of the full-length tropomyosin gene from Trichinella spiralis. J. Helminthol. 77, 57–63. Robinson, M.W., Massie, D.H., Connolly, B., 2007. Secretion and processing of a novel multi-domain cystatin-like protein by intracellular stages of Trichinella spiralis. Mol. Biochem. Parasitol. 151, 9–17. Sambrook, J., Russell, D., 2001. Screening expression libraries. In: Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp. 14.1–14.44. Sugane, K., Matsuura, T., 1990. Molecular analysis of the gene encoding an antigenic polypeptide of Trichinella spiralis infective larvae. J. Helminthol. 64, 1–8. Zang, X., Maizels, R.M., 2001. Serine proteinase inhibitors from nematodes and the arms race between host and pathogen. Trends Biochem. Sci. 26, 191–197. Zarlenga, D.S., Boyd, P., Lichtenfels, J.R., Hill, D., Gamble, H.R., 2002. Identification and characterisation of a cDNA sequence encoding a glutamic acid-rich protein specifically transcribed in Trichinella spiralis newborn larvae and recognised by infected swine serum. Int. J. Parasitol. 32, 1361–1370. Zhu, X.P., Garcia-Reyna, P., Fu, B.Q., Yang, J., Li, C.V., Yang, Y.P., Liu, M.Y., Ortega-Pierres, G., Boireau, P., 2005. A stage-specific open reading frame from three-day old adult worms of Trichinella spiralis encodes zinc-finger motifs. Parasite 12, 151–157.
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Identification of antigenic genes in Trichinella spiralis by immunoscreening of cDNA libraries§ X.P. Wu a, B.Q. Fu a,b, X.L. Wang a, L. Yu a, S.Y. Yu a, H.K. Deng a, X.Y. Liu a, P. Boireau a,b, F. Wang a, M.Y. Liu a,* a b
Key Laboratory for Zoonosis Research, Ministry of Education, Institute of Zoonosis, Jilin University, 5333 Xian Road, 130062 Changchun, PR China JRU BIPAR INRA AFSSA ENVA UPVM, 94703 Maisons-Alfort Cedex, France
A R T I C L E I N F O
A B S T R A C T
Keywords: Trichinella spiralis Antigenic genes Immunoscreening cDNA libraries
Genes encoding antigens of adult worm, newborn larvae and muscle larvae of Trichinella spiralis were identified by immunoscreening their corresponding cDNA libraries. The cDNA libraries of T. spiralis adult (3 day old, Ad3) and newborn larvae (NBL) were screened using the serum of a pig infected with 20,000 muscle larvae (ML) at 26 days post-infection (dpi). Fifty-two positive clones representing 18 unique genes were obtained from the Ad3 cDNA library. The deduced amino acid sequences of 8 cDNAs were sequence homologues of the serine protease-like protein family. In the screening of NBL cDNA library, 81 positive clones representing 8 unique genes were isolated and of these, 70 clones corresponded to an NBL stage-specific serine protease gene. The ML cDNA library was screened using pig anti-Trichinella serum obtained at 60 dpi and 18 positive clones representing 8 unique genes were identified. Ten clones encoded a protein that is identical to a T. spiralis serine protease inhibitor. In general, our results revealed that antigenic serine protease-like proteins were found during the two invasive stages, Ad and NBL when these libraries were screened using 26 dpi antiserum, whereas a serine protease inhibitor was found in abundance in the ML library when it is screened using 60 dpi antiserum. These data are consistent with serine proteases playing an important role during the invasive stages of Trichinella infections, but become inhibited or internally controlled when the parasite enters a more stable, non-developing environment. ß 2008 Elsevier B.V. All rights reserved.
1. Introduction Infection of animals and humans by Trichinella spiralis remains a serous public threat in both developed and developing countries (Murrell and Pozio, 2000; Liu and Boireau, 2002; Dupouy-Camet, 2006). The economic importance and the serious public health problems caused
§ The nucleotide sequences identified in this study (Tables 1–3) have been deposited in the GenBankTM, EMBL and DDBJ databases under the Accession numbers EU263308–EU263333, EU331363, DQ864973. * Corresponding author. Tel.: +86 431 87836713; fax: +86 431 87983442. E-mail address: [email protected] (M.Y. Liu).
0304-4017/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2008.10.035
by trichinellosis have stimulated research on new molecular diagnostic methods and immunoprophylaxis for livestock and wild animals. The life cycle of T. spiralis is completed within a single host species and muscle larvae (ML), adult worms (Ad) and newborn larvae (NBL) are three major antigenic stages. Stage specificity has been observed in a subset of antigens (Boireau et al., 1997) and can evoke stage-specific host immune responses. Conventional ELISA using ES antigens from ML often cannot detect a light or moderate infection in pigs for three weeks or longer after larvae become infective, owing in part to the uniqueness of these antigenic stages. In recent years, a number of antigens have been identified from T. spiralis by immunoscreening cDNA expression libraries, including the serine proteinase inhibitor gene
X.P. Wu et al. / Veterinary Parasitology 159 (2009) 272–275
(Nagano et al., 2001), the gene family encoding glutamic and serine rich proteins (Zarlenga et al., 2002), the serine proteinase gene (Nagano et al., 2003) and tropomyosin (Nakada et al., 2003). In the present work, we have isolated genes encoding highly antigenic proteins from cDNA libraries of T. spiralis at different stages by immunological screening. These data may form a foundation for identifying recombinant antigens that can be used in the diagnosis or vaccination against trichinellosis. 2. Materials and methods
273
2.4. Sequence analysis Selected clones were sequenced from both the 50 and 3 ends using the dideoxy chain-termination method on an automated DNA sequencer. After removal of flanking vector sequences, DNA sequences were analyzed with DNASIS software. Comparison against GenBank nucleotide and protein databases was performed using the NCBI-BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) network server at the National Center for Biotechnology Information. 0
2.1. Parasite and cDNA libraries
3. Results
T. spiralis (ISS534) ML were recovered from mice 35 days post-infection by artificial digestion with pepsin–HCl (1% pepsin, 1% HCl at 42 8C for 45 min). cDNA libraries of ML, Ad3 and NBL were previously constructed (Liu et al., 2001; Zhu et al., 2005).
3.1. Sequence analysis of positive clones from Ad3 cDNA library
2.2. Absorption of swine anti-Trichinella sera A domestic pig was infected orally with 20,000 ML of T. spiralis. Pre-infection serum was collected as negative control and infection sera were collected at 26 dpi and 60 dpi. Cross-reactive antibodies were removed from the sera through pre-absorption according to Sambrook and Russell (2001) using non-recombinant vector lambda ZAP II phage. 2.3. Immunoscreening of cDNA libraries For each cDNA library, 100,000 plaque forming units (pfu) were screened with absorbed antisera obtained at 26 dpi for the Ad3 and NBL cDNA libraries, and at 60 dpi for the ML cDNA library according to conventional methods. After the second screening, positive plaques were picked and the phagemids were prepared by in vivo excision. The plasmids were transformed into E. coli SOLR strain (E. coli XLOLR strain for plasmids from NBL cDNA library).
The Ad3 cDNA library screened with 26 dpi pig antiTrichinella serum identified 52 positive clones representing 18 unique genes (Table 1). Clone Ad5 showed high sequence identity with a previously identified T. spiralis serine proteinase inhibitor gene (Nagano et al., 2001). Twenty-three clones encoded the same gene, where clone WN10 contained the full open reading frame (ORF) of 406 amino acid (AA) residues. Sequence homology searches indicated that clone WN10 was 99% similar to the T. spiralis hypothetical ORF 9.10 mRNA, and was highly similar to the cDNA encoding p46 kDa antigen from ML ES products (Sugane and Matsuura, 1990) and the multi-domain cystatin-like protein (Robinson et al., 2007). Clone Ad147 showed some similarity to T. spiralis hypothetical ORF 9.10 mRNA as did clone Ad53 to T. spiralis 53 kDa ES antigen. Interestingly, 8 novel cDNAs derived from 16 clones showed high sequence identity to each other and to the T. spiralis ESTs from different stages. All of them encoded serine protease-like proteins. Six of these cDNAs contained the full ORF encoding a peptide of 429 AA. The first 18 AA of the deduced polypeptide were predicted to be a signal peptide, and SMART analysis revealed a putative trypsin-like serine protease domain from position 37 to
Table 1 Analysis of clones from Ad3 cDNA library by immunoscreening with T. spiralis infected pig serum. Clone name (identical clones)
Accession number
Size (bp/aa)
Sequence similarity
Accession number
BLASTX E value
Ad5 (2) WN10 (3) Ad147 (4) Ad53 Ad67 Ts Adsp-1 Ts Adsp-2 (3) Ts Adsp-3 Ts Adsp-4 Ts Adsp-5 (5) Ts Adsp-6 Ts Adsp-7 (3) Ts Adsp-8 Ad42 Ad71 Ad76 Ad62 Ad2
DQ864973 EU263325 EU331362 EU263318 EU263319 EU263326 EU263327 EU263328 EU263329 EU263330 EU263331 EU263332 EU263333 EU263322 EU331363 EU263309 EU263308 EU263321
1307/373 1352/406 950/237 1453/451 1095/312 589/193 1594/429 1375/429 1539/429 1534/429 1205/370 1372/429 1399/429 907/296 751/243 886/209 1165/388 735/174
T. spiralis serine proteinase inhibitor T. spiralis hypothetical ORF 9.10 T. spiralis hypothetical ORF 9.10 T. spiralis 53 kDa excretory/secretory antigen Trichuris trichiura putative porin precursor Trypsin-like serine protease family
AAF63473 AAB48491 AAB48491 AAA97512 AAC04763 AAK16516
Caenorhabditis elegans paramyosin Plasmodium yoelii Ran-binding protein Caenorhabditis elegans calcium-sensing receptor No sequence similarity No sequence similarity
NP_492085 EAA19671 NP_501400 – –
0.0 2e135 8e12 7e11 2e11 2e24 3e44 3e46 5e46 2e46 4e35 3e44 3e44 1e69 3e07 1e21 – –
X.P. Wu et al. / Veterinary Parasitology 159 (2009) 272–275
274
Table 2 Analysis of clones from NBL cDNA library by immunoscreening with T. spiralis infected pig serum. Clone name (identical clones)
Accession number
Size (bp/aa)
Sequence similarity
Accession number
BLASTX E value
NBL84 (2)
EF041139
1379/459
T. spiralis glutamic acid rich protein cNBL 1500 T. spiralis glutamic acid rich protein cNBL 1700
AAM19759 AAM19760
0.0 0.0
NBL122 (2) NBL39 (2) NBLWN18 (70) NBL50 (2) NBL43 NBLWN5 NBLWN11
DQ864973 EU263320 – EU263319 EU263324 EU263316 EU263317
1307/373 474/117 1587/465 1095/312 1276/425 1552/475 1540/389
T. spiralis serine proteinase inhibitor T. spiralis hypothetical ORF 11.30 precursor T. spiralis newborn larvae-specific serine protease SS2 Trichuris trichiura putative porin precursor Homo sapiens heat shock protein HSP 90-alpha 2 Plasmodium yoelii glutamine–asparagine rich protein Caenorhabditis elegans putative RNA binding G-patch domain protein
AAF63473 AAB48488 AAK16520 AAC04763 CAI64495 EAA18721 NP_497893
0.0 3e45 1e58 2e11 e166 9e13 7e24
Table 3 Analysis of clones from ML cDNA library by immunoscreening with T. spiralis infected pig serum. Clone name (identical clones)
Accession number
Size (bp/aa)
Sequence similarity
Accession number
BLASTX E value
MLWM5 (10)
DQ864973
1307/373
T. spiralis serine proteinase inhibitor
AAF63473
0.0
ML42 (2)
EU263312
678/170
T. spiralis hypothetical ORF 17.20 T. pseudospiralis 21 kDa ES protein
AAB48489 AAF79206
3e85 2e63
ML3 ML59 ML41 ML63 ML1 ML64
EU263323 EU263313 EU263311 EU263314 EU263310 EU263315
671/158 447/113 563/153 1170/192 1311/437 767/199
Ostertagia ostertagi globin-like ES protein F6 Arabidopsis thaliana 60s acidic ribosomal protein P1 Dunaliella tertiolecta nucleoside diphosphate kinase Homo sapiens putative G-protein coupled receptor SH120 Mus musculus nuclear matrix protein SNEV No sequence similarity
CAD20463 BAB11203 AAK38732 CAI13224 NP_598890 –
2e07 9e12 2e56 9e43 e135 –
277. Clones Ad67, Ad42, Ad71 and Ad76 encoded proteins with similarities to a putative porin precursor, paramyosin, Ran-binding protein and calcium-sensing receptor, respectively. Clones Ad62 and Ad2 showed no significant matches with known sequences. 3.2. Sequence analysis of positive clones from NBL cDNA library The cDNA library of NBL was also screened with 26 dpi pig anti-Trichinella serum and 81 positive clones were selected, which represented 8 different genes (Table 2). Clone NBL84 shared high identity with members of NBL specifically expressed glutamic acid-rich antigenic protein family (Zarlenga et al., 2002). Clone NBL122 aligned with a T. spiralis serine proteinase inhibitor gene. Clone NBL39 showed high similarity to T. spiralis hypothetical ORF 11.30 precursor. Sequence analysis revealed 70 of the positive clones were identical to the NBL stage-specific serine protease gene, previously identified by Liu et al. (2007). The putative porin precursor was also identified (clone NBL50). Clones NBL43, NBLWN5 and NBLWN11 encoded proteins which showed sequence similarity to heat shock protein HSP 90, glutamine–asparagine rich protein, and a putative RNA binding G-patch domain protein, respectively. 3.3. Sequence analysis of positive clones from ML cDNA library The 60 dpi pig anti-Trichinella serum was used to screen the T. spiralis ML cDNA library and 18 positive
clones corresponding 8 unique genes were obtained (Table 3). Sequence analysis showed that 10 of 18 clones represented the same gene, where clone MLWM5 contained the full ORF. These clones showed the strongest reactivity on the hybridization membranes. Clone MLWM5 was similar in sequence to a T. spiralis serine protease inhibitor gene (Nagano et al., 2001) where the two differed by 3 bp and 2 deduced AA. Clone ML42 encoded a protein with high identity to T. spiralis hypothetical ORF 17.20. The other sequences coincided with genes coding for a globin-like ES protein F6, the 60 s acidic ribosomal protein P1, a nucleoside diphosphate kinase, the G-protein coupled receptor SH120 and a nuclear matrix protein SNEV, respectively. 4. Discussion Recombinant DNA techniques have been used to produce large amounts of recombinant antigens of T. spiralis; however, most of the recombinant antigens come from T. spiralis ML and only a few from Ad and NBL such as glutamic rich proteins (Zarlenga et al., 2002) and T. spiralis tropomyosin (Nakada et al., 2003). By immunoscreening multiple cDNA libraries, we succeeded in identifying a series of genes encoding antigens from the three different development stages of T. spiralis, including the known glutamic rich proteins and serine protease inhibitor, p46 kDa, along with other novel candidates. This research suggests that a large portion of the antigenic proteins produced by both Ad and NBL, represent serine proteases including a polymorphic multigene family of serine protease in Ad3 and a stage specific serine
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protease gene in NBL. Parasite proteases are likely involved in varied aspects of host–parasite interactions, such as host tissue invasion, parasite nutrition, anti-coagulation and the evasion of host immune responses (Nagano et al., 2003). However, results presented herein suggest that serine proteases play important roles in Trichinella development related to invasion and migration of the parasite, because Ad and NBL stages are where most of these classes of antigens were found. A highly antigenic serine protease inhibitor gene was identified from Ad3, NBL and ML cDNA libraries. The 3 bp differences in the ORF of cloneWM5 and the serpin gene (Nagano et al., 2001) may represent single nucleotide polymorphisms; however, the differences among these two sequences are unlikely to affect antigenicity because both were obtained by immunoscreening. The serpin gene was transcribed largely in ML, consistent with T. spiralis not requiring serine protease activity during the more stable ML stage. Parasite serpins have essential extracellular functions such as modulation and inhibition of host immune responses, fibrinolysis, coagulation, and inflammation (Zang and Maizels, 2001). The ability to inhibit coagulation during the early stages of parasitism provides an important mechanisms for some parasites to survive in the host. Proteinase inhibitors are known to provoke strong immune responses (Frank et al., 1998) and may provide a new targets for immunodiagnostics and vaccine production. In conclusion, immunoscreening is a useful technique for the isolation of highly antigenic genes. From this, we have successfully obtained a series of genes encoding highly antigenic proteins from the three different stages of T. spiralis, which include both known and novel sequences. The true stage specificity of each gene and gene product requires further elucidation inasmuch as the Ad and NBL libraries were screened only with sera presumed to be enriched with antibodies to Ad and NBL antigens (26 dpi serum), and the ML library was screened only with serum presumed enriched with antibodies to ML antigens (60 dpi). Cross-reactivity of these antiserum is still required to validate any stage specificity. Nonetheless, these results are a foundation for future studies on advancing methodology for developing more specific and sensitive diagnostic tests and better targets for efficient vaccine development. Acknowledgements This research was financially supported by grants of China 863 program 2006AA02Z451, National Natural
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