气肿疽梭菌CctA基因的原核表达及其间接ELISA检测方法的建立

研究旨在原核表达气肿疽梭菌细胞毒素A （CctA）基因，用纯化的重组蛋白建立其间接ELISA检测方法，a。利用大肠杆菌密码子的偏爱性优化CctA基因序列，克隆至原核表达载体pET-28a （+），双酶切鉴定原核表达质粒pET28a-CctA并测序。将重组质粒转入大肠杆菌，经IPTG诱导得到高表达的重组CctA包涵体蛋白。包涵体蛋白变性后经镍（Ni）柱纯化，SDS-PAGE检测其纯化效果。以豚鼠抗气肿疽梭菌抗血清为一抗，用Western blotting方法检测重组CctA蛋白的反应原性。用棋盘滴定法建立间接ELISA检测方法，以复性后的重组CctA蛋白作为检测抗原，摸索抗原包被浓度、封闭液的种类及浓度、抗体的最适稀释度和反应条件等。选用3批气肿疽灭活疫苗免疫豚鼠后采集血清，同时用攻毒和间接ELISA两种方法验证疫苗的免疫效力。质粒双酶切结果显示，得到大小约853 bp的条带，与预期相符，且测序结果正确。SDS-PAGE结果表明，成功表达并纯化大小为35 ku的重组CctA蛋白。Western blotting结果显示，豚鼠抗气肿疽梭菌抗血清与重组CctA蛋白具有良好的反应原性。建立的间接ELISA法的最适条件为：抗原的包被浓度为0.5 μg/mL，于4 ℃包被过夜；封闭液选择10%胎牛血清，37 ℃孵育2 h；二抗的稀释度为1：8 000；室温避光显色10 min后终止反应，测定D_{450 nm}值。当P/N>4.6时，间接ELISA法检测结果与豚鼠攻毒试验结果拟合度较好。本研究成功表达CctA基因并纯化了重组CctA蛋白，建立的以重组CctA蛋白为检测抗原的间接ELISA检测方法，有望成为气肿疽灭活疫苗免疫效果验证的替代方法。

Prokaryotic Expression of Clostridium chauvoei CctA Gene and Establishment of Its Indirect ELISA

The aim of this study was to express Clostridium chauvoei toxin A (CctA) gene in prokaryotic system and establish indirect ELISA detection method using purified recombinant protein,and to verify the immune effect of gangraena emphysematosa inactivated vaccine.The sequence of CctA gene was cloned into prokaryotic expression vector pET-28a(+) according to the codon bias of Escherichia coli (E.coli).The prokaryotic expression plasmid pET28a-CctA was identified by double enzyme digestion and sequencing.The high expression of recombinant CctA inclusion body protein was induced by IPTG after recombinant plasmid was transferred into E.coli.The inclusion body protein was purified by Ni column after denaturation and its purification effect was tested by SDS-PAGE.Western blotting was used to detect the reactivity of recombinant CctA protein.Establishment of indirect ELISA was accompanied by the checkerboard titration method.The recombinant CctA protein was used as detected antigen,and the concentration of the coated antigen,the type and concentration of the blocking solution,the optimal dilution of the antibody and the reaction conditions were explored.After the guinea pigs were immunized with 3 batches of gangraena emphysematosa inactivated vaccine,the serum was collected.The immune effect of the vaccine was verified both by challenge and indirect ELISA.The results of double digestion of plasmid showed that the band about 853 bp was in line with expectation,and the sequencing results was correct.SDS-PAGE results indicated that 35 ku recombinant CctA protein was successfully expressed and purified.The results of Western blotting showed that the antiserum of guinea pigs against Clostridium chauvoei had good reactivity with recombinant CctA protein.The optimal conditions of indirect ELISA method were as follows.The coating concentration of antigen was 0.5 μg/mL overnight at 4 ℃,10% fetal bovine serum was selected for blocking solution and incubated at 37 ℃ for 2 h,the dilution of the secondary antibody was 1:8 000,the reaction was stopped after 10 min at room temperature and D_{450 nm} was determined.When P/N>4.6,the results of indirect ELISA fitted well with the guinea pig challenge test.In this study,CctA gene was successfully expressed and recombinant CctA protein was purified,and indirect ELISA was established with recombinant CctA protein as detected antigen,which was expected to be an alternative method to verify the immune effect of gangraena emphysematosa inactivated vaccine.