Cloning,Expression And Immunoprotection Of Ubiquitin Conjugating Enzyme From Mother Sporocyst Of Schistosoma Japonicum

Objective:Schistosoma japonicum ubiquitin conjugating enzyme(SjE2G)gene is a up-regulated gene in the co-culture mother sporocyst of S.japonicum with protein extraction from O.hupensis head-foot,and which may be a vaccine candidate for schistosomiasis.E2G mRNA expression in co-culture mother sporocyst,will further investigated in this study,and SjE2G will be cloned and transferred E.coli BL21 to express E2G protein,which will be used to explore the expression localization of SjE2G in mother sporocyst and its immune protection for anti-shistosomiasis.Method:Firstly,according to the open reading frame of SjE2G,specific primers for real-time PCR was designed to detect the SjE2G mRNA expression level in two groups of mother sporocyst(mother sporocyst in normal cultured condition and mother sporocyst in co-culture system with protein extraction from O.hupensis head-foot).Secondly,specific full-length primers of SjE2G was designed and used to amplified the full-length of SjE2G with the cDNA template from mother sporocyst of S.japonicum.The PET2 8 a(+)-SjE2 G recombinant expression plasmid was subsequently constructed and transformed into E.coli BL21 bacterial.SjE2G recombinant protein which was induced expression in BL21 with IPTG,was collected and purified to immune animals.The SjE2G-immunized rabbits were done to obtain immune anti-SjE2G sera,which was used to make sure expression distribution of SjE2G in mother sporocyst with immunohistochemical staining method.Moreover,the SjE2G-immunized mice were infected with S.japonicum cercariae on the sixth week.After 42 days later,the infection worm burden and egg burden in liver of each mice were counted,then the relativity worm and egg reduction rate of SjE2G-immunized mice compared with nonimmunized control mice were statisticed to evaluate immunoprotecive efficiency of E2G.Results:Real-time PCR results showed that S.jE2G mRNA expression in co-culture mother sporocyst was up-regulated significantly compared with normal culture mother sporocyst(p<0.01).A 672bp length PCR amplified fragment of E2G was obtained and successfully cloned in expression plasmid to construct recombinant PET28a(+)-SjE2G plasmid.A recombinant SjE2G protein which molecular weight was 23kDa was induced and purified with IPTG induction.The results of immunohistochemical staining showed that SjE2G was mainly expressed and distributed on the wall thickness and cytoplasm of mother sporocyst of Sjaponicum.After immunized with SjE2G protein,the infection burdon and egg burdon of liver were very similar among all three test groups.Consequently,for the E2G-immunized groups,worm reduction rate of was 13%and eggs reduction rate was 6%;for adjuvant control group,the former was 7%and the latter was 2%.There were no statistically signification among groups.Conclusion:SjE2G gene was an up-regulated gene in the co-culture mother sporocyst of Sjaponicum with oncomelania head-foot tissue extraction.SjE2G was mainly expressed on the wall thickness and cytoplasm of mother sporocyst of S.japonicum,and SjE2G had no protective immunity against S.japonicum infection.