大片吸虫磷酸丙糖异构酶基因的克隆表达和免疫原性分析OACSTPCD

Triosephosphate isomerase(TPI)is a key glycolytic enzyme that exists in the organisms and plays an important role in obtaining energy for the life of Fasciola gigantica.In this study,the full-length cDNA encoding FgTPI was cloned according to the gene sequence of TPI from Fasciola gigantica,and the primer with Bam HⅠ and XhoⅠ as enzyme digestion sites was designed.The cDNA from Fasciola gigantica was used as a template.The results were as follows:The open reading frame(ORF)of FgTPI was 762 bp,encoding 253 amino acids with an isoelectric point of 8.07.The ORF of was sub-cloned into pET-28a(+)vectors and expressed in Escherichia coli with a mo-lecular weight of 32 kDa.The BALB/c mouse was injected with the purified recombinant of FgTPI,and the polyclonal antibodies were pre-pared.ELISA assay analysis showed that rFgTPI could induce high levels of anti-rFgTPI-specific IgG antibodies.The result of western blot-ting showed that recombinant FgTPI had good antigenicity by using the serum from the sheep infected with Fasciola gigantica.The recombi-nant protein showed the optimal activity of TPI at pH 7.5 and temperature of 35℃,according to α-glycerol phosphate dehydrogenase cou-pling method.The above results indicated that FgTPI could be a potential candidate for vaccine against Fascioliasis,which would provide ba-sis for further investigation into the biological function of FgTPI.