Cloning And Expression Of The Gene Encoding Excretory-secretory Products P49 Of Thichinella Spiralis

Trichinella spiralis, a kind of parasite, can cause disease to more than 150 kinds of mammalian, including human. Trichinellosis can cause people severe immflamation, even to death. Since human firstly detected Trichinella spiralis in 1835, this disease hasn't been controlled efficiently and diffused to all over the world and has brought great loss to people in food industry and livestock husbandry. So it is necessary to develop a quick and easy way to detect Trichinella spiralis. According to a lot of research, excretory-secretory antigen of Trichinella spiralis has high specificity and sensitivity and can be used for detection of Trichinella spiralis.Primers were designed and synthesized. Using worm RNA as a template, the DNA encoding excretory-secretory antigen of Trichinella spiralis were amplified by reverse transcriptase-polymerase chain reaction (RT-PCR). The products from PCR were cloned into pGEM-T vector and sequenced. To subclone the P49 gene into vector SK(+) and then into vector pET-28b , and transform the recombinant plasmid into competent cells of E coli BL2i. The results showed that a specific band of about 0.96Kb was amplified. There was highly homology of fragment to be sequenced with that of P49 gene in Genbank. The expressed protein induced by IPTG was analyzed with SDS-PAGE and Western blot.Molecular weight of expressed protein was about 38kDa and content approximate 7 % ofwhole bacterial lysate. The expressed products could be recognized by positive sera ofswine infected with Trichinella spiralis. It is suggested that the expressed protein had antigenicity and provided the basis for detection of Trichinella spiralis basing on Colloidal Gold Dipstick Assay and development of vaccine for Trichinella spiralis.