The Construction Of Ts87 Gene Recombinant Plasmid And The Expression Of Ts87 In Eukaryotic Cell In Vitro

Trichinosis is one of zoonoses in the world, which can bring numerous economic loss and serious health problems. Because of the difficulties on diagnosis and treatment ,it is very important for the prevention of trichinosis.Trichinella spiralis(T.s) has a complex life history and a variety of antigen components, so , despite of a lot of studies ,no prevention measures specific for T.s has been found until now .The method of making DNA vaccine is a new technology that did not come to being until 1990. It is that recombinant plasmid which contain a protective antigen gene was rejected into animal's muscle, then the protein that the recombinant plasmid expressed in vivo will directly induce immune response in the body. Because DNA vaccine can not only induce protective humoral immunity but also induce strong T cell toxic response, its application on parasital diseases prevention is becoming more and more popular and studies on this area is gradually deepening . DNA vaccine has been considered to be a potentially promising approach, which certainly throwed lights on T.s prevention.Ts87 gene fragment was picked out by screening adult T.s cDNA library using sera of rabbit raised against soluble antigen of T.s and using sera of rabbit infected artificially with T.s.The Ts87 fragment was ligated to vector pCDNAS.l,which contains a CMV promoter /enhancer.In order to construct the recombinant plasmid pCDNA3.1/Ts87,first,the Ts87gene fragment was rePCRed using the redesigned primers to introduce RE sites of HindlD and BamH I , and KOZAK sequence .Then the rebuilt Ts87 fragment was cloned into T-vector and transformed into E.Coli DH5 a . The positive clones were selected by blue-white screening and the insert fragment was sequenced. Then the rebuilt Ts87 fragment was ligated to pCDNA3.1. With the same method the positive clones were selected by Amp screening. After the recombinant plasmid pCDNA3.1/ Ts87 was identified by digestion of Hindllland BamH I, it transformed into COS7 by Lipofectamine.Expression product was identified by immunohistochemical method, SDS-PAGE and Western-blot .The immunocytochemistry result has shown that specific brown-staining grains were found in the cytoplasm of cells transformed by recombinant plasmid versus not seen in cells transformed by pCDNA3.1 or normal cells;The SDS-PAGE result has revealed that a band about 3 8KB was found in cell lysis transformed by recombinant plasmid versus not in cells transformed by pCDNAS.l or normal cells;The Western-blot result has showed that only the band about 38KD was recognized by sera from rabbit infected by T.s artificially and sera from rabbit immunized with soluble antigen of T.s and with protein expressed by Ts87 gene and by a monoclonal antibody of T.s .To sum up,a recombinant plasmid pCDNA3.1/Ts87 was constructed and expressed successfully in eukaryotic cells ,which has laid a strong basis on further studies of T.s vaccine.