Molecular Identification Of Trichinella SPP And Cloning, Expression And Characterization Of Cystatin Gene Family Of Trichinella Sprialis

Trichinella spiralis is an intracellular parasitic nematode and can complete the life cycle within one host. The host spectrum of T. spiralis is very wide and nearly all the mammalian can be infected. Trichinellosis caused by Trichinella spp. is a serious foodborne parasitic zoonosis. Trichinellosis pathogen is Trichinella genus nematodes including8species and4genotypes. Because of the significant differences in geographical distribution, host range, infectivity, pathogenicity, and host immune response, the identification of Trichinella isolates is important to etiology, immunology, epidemiology, clinical and prevention of trichinellosis. Cysteine protease inhibitor (Cystatin) is a high efficient endogenous inhibitor of cysteine protease and is ubiquitous present in organism. Parasite cystatins play a pivotal role in immune invasion and adaption to parasitism. Cystatins secreted by parasitic nematode have immune modulation effect and could inhibit immune response during parasite invasion, and it’s one of the main factors to induce host immune suppression.In this study, with Trichinella mitochondrial genome ribosomal RNA gene as a molecular marker, a multiplex PCR-based genotyping method was established to identify the four Trichinella spp in Eurasia including T. spiralis, T. nativa, T. britovi and T. pseudospiralis. With T. spiralis and T. nativa standard isolates as control, mitochondrial ribosomal RNA genes multiple PCR was used to identify11Chinese Trichinella isolates, moreover with5S rRNA gene spacer region as a molecular marker, the phylogenetic clustering analysis was further performed. The results confirmed that the out of11Chinese Trichinella isolates, those9isolates collected from pigs in Deng county Henan province, Xi’an county Shanxi province, Harbin city Heilongjiang province, Tianjin city, Baoshan county Yunnan province, Dali city Yunnan province, Tibet Nagqu, as well as Inner Mongolia, UlanHot dog isolate Trichinella sp-Tyl, were grouped into T. spiralis. The dog isolates of Changchun city Jilin province and Inner Mongolia, UlanHot Trichinella sp-Ty5isolates were grouped into T. nativa.5S rRNA gene spacer sequence analysis showed that the sequence is highly conserved between different isolates of the same species. Three members of Trichinella cysteine protease inhibitor (TsCystatin) gene family, TsCystatin1, TsCystatin2and TsStefin were successfully cloned. The open reading frame was666bp,423bp and294bp, encoding221,140and97amino acids with typical cystatin conserved domain. TsCystatin gene family can be transcribed in various stages of T spiralis. TsCystatin genes were expressed in prokaryotic systems and the purified TsCystatin recombinant protein showed protease inhibitory activity, these laid the foundation for further study on biological function.