腐败梭菌α毒素蛋白的原核表达及间接ELISA抗体检测方法的建立

Prokaryotic expression of Clostridium septicum alpha toxin protein and establishment of an indirect ELISA method for detection of anti-αtoxin antibody

为建立绵羊腐败梭菌α毒素抗体间接ELISA检测方法,将腐败梭菌α毒素基因进行密码子优化后,通过基因合成连接到pGEX-4T-1载体,构建重组质粒pGEX-4T-1-α。将重组质粒转入到大肠杆菌BL21(DE3)p LysS感受态细菌,用IPTG诱导重组α毒素蛋白表达并纯化表达蛋白,切除表达蛋白携带的GST标签后,得到重组腐败梭菌α毒素蛋白。以腐败梭菌α毒素蛋白为包被抗原,建立腐败梭菌α毒素抗体间接ELISA检测方法。结果表明,腐败梭菌α毒素蛋白以200 ng/孔,绵羊血清稀释度为1∶200,封闭液为10 g/L BSA,家兔抗山羊HRP酶标二抗稀释度为1︰8000,避光显色20 min为最佳的ELISA条件。检测50份阴性血清,计算平均值(x^(-))、标准差(s),依据(x^(-)+2s)确定建立的腐败梭菌α毒素抗体间接ELISA检测方法的阴阳性临界值为0.244。检测血清敏感度为1︰6400;批内、批间重复性试验结果显示,该方法的变异系数均小于10%;特异性试验结果显示,该方法与绵羊B型产气荚膜梭菌、C型产气荚膜梭菌、绵羊布鲁菌、鼠伤寒沙门菌及羊痘病毒阳性血清均无交叉反应。用建立的间接ELISA和商品化腐败梭菌α试剂盒分别检测100份临床血清样本,结果表明这2种方法的阳性符合率为94.87%,阴性符合率为81.97%,总符合率为87.00%。对三联四防疫苗免疫绵羊血清的检测结果表明,本方法的检测结果能够更好地反映出接种疫苗后免疫绵羊血清中抗α毒素抗体的消长情况。该检测方法具有很好的敏感性、重复性和特异性,适用于腐败梭菌感染临床血清样品的检测和腐败梭菌疫苗免疫效果的评价。

To establish an indirectELiSA method for the detection of the antibodies to Clostridium septicum(CS)α-toxin,the codon-optimizedαtoxin gene of C.septicum was synthesized and ligated into the pGEX-4T-1 vector and the recombinant plasmid pGEX-4T-1-αwas constructed,then the recombinant plasmid was transformed into Escherichia coli BL21(DE3)pLysS for IPTG induced protein expression.After the recombinant protein was purified and the GST tag was removed,the recombinant CSα-toxin protein was used as the coating antigen to establish an indirect ELISA for detection of anti-CSα-toxin antibody.It was found that the optimized ELISA condition was to coat the recombinant protein at the concentration of 200 ng/well,the dilution ratio of sheep serum was 1:200,the blocking solution was 10 g/L BSA,the dilution ratio of rabbit anti-goat HRP antibody was 1:8000,and the color solution was added and reacted in the dark for 20 min.Totally 50 CS negative serum samples were tested and the mean(x^(-))and standard deviation(s)were calculated.Based on(x^(-)+2s)value,the cut-off value of the established indirect ELISA was 0.244.The sensitivity of serum was 1:6400.Both the intra-assay and inter-assay reproducibility results showed that the coefficient of variation was less than 10%.The specificity testing results showed that there was no cross-react with positive sera for Clostridium perfringens type B,Clostridium perfringens type C,Brucella ovis,Salmonella typhimurium and sheeppox virus.A total of 100 clinical serum samples were tested with the established indirect ELISA and a commercial ELISA kits,the results showed that the rate of positive consistent,negative consistent and total consistent of our method to the commercial kit was 94.87%,81.97%,87.00%,respectively.The detection results of sheep sera immunized with commercial Clostridium vaccines showed our method is better reflected the fluctuation of anti-CSα-toxin antibody in immunized sheep sera.This method has good sensitivity,repeatability and specificity,and is suitable for the detection of clinical serum samples of C.septicum infection and for the evaluation of the immune effect of corresponding vaccines.