Cloning, Expression And Identification Of Cystatin TsCystatin1 Of Trichinella Sprialis

Trichinella spiral is an intra celluar parasitic nematode and the life cycle can complete within one host. The host specificity of T. spiralis is very low and nearly all the mammalian can be infected. Trichinellosis caused by Trichinella spp. is a serious foodborne parasitic zoonosis. Cysteine protease inhibitor (Cystatin) is a high efficient endogenous inhibitor of cysteine proteases and is ubiquitious present in organism. Cystatin of parasites play a pivotal role in immune invasion and adaption to parasitism. Cystatins secreted by parasitic nematode have apparent immune modulation effect and could inhibit immune response when parasite penetrates into the host, and it's one of the main factors to induce immune supression of the host.In this study specific primers of T.spiralis cystatin gene (TsCystatin1) was designed from an EST sequence of newborn larval stage and RT-PCR was performed to amplify the cystatin gene full ORF from total RNA from T. spiralis newborn larvae. The results showed that TsCystatin1 gene full ORF consisted of 669 bp which encoding 221 amino acids with conserved amino acids in cystatin domain. Genome DNA amplification indicated this gene contains two extrons and one intron of 80 bp. RT-PCR amplification and real-time PCR indicated this gene is expressed in adult, newborn larvae and different development stages of muscle larvae and the mRNA can be detected as early as 14 dpi. TsCystatin1 gene fragment was cloned into the prokaryotes expression vector PGEX-4T-1 and recombinant pGEX-4T-TsCystatin1 was transformed into E.coli BL21 to induce expression. SDS-PAGE results showed the expression product is about 48.3 ku and mainly exists in soluable form when induced at low temperature. The immune serum from rabbit immunized with the recombinant protein could recognize a specific band of 44.0 ku in T.spiralis muscle larvae antigen.Protease inhibition assay indiceted that the recombiant protein could inhibit activity of cathepsin B. Western-blot analysis showed TsCystatin1 recombinant protein could not be recognized by T. spiralis infected swine serum and after immunization with recombinant protein and DNA vaccine of TsCystatin1 no significant protectin against infection was observed. These results will lay the foundation for the function study of TsCystatin1 of T. spiralis.