Expression Of Genes Of Schistosoma Japonicum And Studies On The Use Of Recombinant Rroducts In The Diagnosis Of Schistosomiasis

Schistosomiasis is a serious disease which can not only infect humans but also animals. At present, Schistosomiasis japonicum is still a serious public health problem in China. The repeated infection is very serious. The animals are the most important infectants of the disease. So the immunol-preventation research is required and diagnostic antigen which has high specificity and high sensitivity has to be studied. The 23kD integral membrane protein of S.japonicum is an antigen of some interests in terms of both anti-parasite vaccination and immuno-diagnosis. The studies focused on the clone and expression of the gene coding the Sj23 membrane protein in eukyotic expression system, Bombyx mori Baculoviridae expression system and assayed the effect of the recombinant protein in clinic diagnosis of Schistosomiasis. The eggs of Schistosoma are the key factors which arise the Schistosomiasis. Therefore, the studies on cathepthin L of S.japonicum, which has a link with the production of egg, are also carried out to estimate it's effect in diagnosis of Schistosomiasis at the same time.The gene coding the 23kD integral membrane protein of schistosome was subcloned from plasmid pET-28(c) to the transfer vector pBacPAK-His-1 with BamH I and Xhol I .The purified DNA was obtained by large-scale preparation of plasmid. The DNA of BmNPV(Bombyx mori Nucleopolyhedrovirus) which was purified with the method of preparation of virus DNA was spliced into linear DNA by the endorestrict enzyme, Bsu-1. Bombyx mori was cotransfected by the DNA of SJ23 together with the DNA of BmNPV.The recombinant virus was obtained through three cycles of screening. The target protein was shown by the SDS-PAGE and identified by Western-blot. The molecular weight of recombinant protein is 26kD. The preliminary experiment showed that it's useful in diagnosing the Schistosomiasis.The recombinant plasmid pET-28a(+)-SjCL was transferred into E.coli BL21 which was processed by the Cacl2 and then was identified through splice of endorestrict enzyme and PCR. SDS-PAGE showed that the highest expression happened 8 hours later after the induction by IPTG. The density of recombinant protein which was purified by Ni ion affinity chromatography is 0.95mg/ml. The positive rate was 67.31% when serum of cow infected with S.japonicum were diagnosed by the recombinant protein. The result showed that the recombinant SjCL had a good prosperity in clinic diagnosis of Schistosomiasis.