Molecular Cloning, Expression And Antigenicity Analysis Of Serine Protease Inhibitor Gene From Trichinella Spiralis

Trichinellosis caused by Trichinella spp. is a serious foodborne parasitic zoonosis and wide spread among more than 150 species of animals including human. Human can obtain this infection through ingestion of raw meat infected by Trichinella spp. and serious infection can be fatal. Trichinellosis is an important public health issue which not only threatens human health but also causes great economic losses to livestock production.In this study the specific primers derived from T. spiralis serine proteinase inhibitor gene in GenBank database was designed and used to amplify the TsSerPIN gene from total RNA isolated from T. spiralis muscle larvae. The RT-PCR product was purified, digested and ligated into the expression vector pET30a. After transformation to E. coli DH5αand identification by PCR, restriction digestion and sequencing, pET30a-TsSerPIN1 plasmid was transformed to E. coli BL21 (DE3) and induced by IPTG for expression. The results that pET30a-TsSerPIN1 recombinant expression plasmid was successfully constructed and contained a insert of 1122 bp encoding 373 amino acids which showed 99% identity to Trichinella spp.serine proteinase inhibitor gene in GenBank database. The TsSerPIN1 recombinant protein was highly expressed in the form of inclusion body with the molecular weight of about 48.5 kDa. In Western-blot analysis the purified TsSerPIN1 recombinant protein could be specifically recognized by T. spiralis infected swine serum. Different fragments of the TsSerPIN gene were further cloned and all of the four fragments were expressed. Western-blots indicated that only TsSerPIN1 and TsSerPIN5 recombinat proteins could be recognized by T. spiralis infected swine serum. Indirect ELISA showed that TsSerPIN1 and TsSerPIN5 recombinant proteins were similar in reactivity, indicating that TsSerPIN5 had the main epitopes in the C terminal of this protein. ELISA on experimentally infected swine serum, field swine serum and human serum revealed good reactivity of TsSerPIN5 recombinant protein. After immunization of mice with TsSerPIN1 and TsSerPIN5 recombinant proteins and challenge with T. spiralis larvae, the immunized mice could get parties protection with muscle larvae reduction of 40.7% and 54.5%, respectively. and produced specific antibody response. TsSerPIN5 could be a candidate for immunodignosis and vaccines of Trichinellosis.