布鲁氏菌S2疫苗株间接ELISA检测方法的建立

Establishment of an indirect ELISA detection method for Brucella S2 vaccine strain

为了区分布鲁氏菌S2疫苗株和布鲁氏菌野毒株,试验通过比对NCBI上公布的布鲁氏菌S2疫苗株和M28野毒株全基因组序列,筛选出S2疫苗株特有基因GL_0002189,对该基因进行克隆、原核表达及蛋白质纯化,以纯化的重组蛋白作为抗原建立间接ELISA检测方法,对该方法的反应条件进行优化后确定判定点,同时对建立的间接ELISA检测方法进行重复性验证、特异性和敏感性试验,并对部分曾接种过布鲁氏菌S2疫苗的83份牛血清进行检测。结果表明:重组蛋白GL_0002189同时以可溶形式和包涵体形式表达,经纯化得到大小为29.2 ku的重组蛋白;在建立的间接ELISA检测方法中,最适重组蛋白包被浓度为2.5 ng/μL,最适血清稀释度为1∶100,最佳酶标二抗稀释度为1∶5 000,判定点为0.586,该方法的特异性为95.0%,敏感性为93.3%,且具有较好的重复性。利用建立的间接ELISA检测方法对83份牛血清进行检测,检出率为7.2%。说明试验建立的间接ELISA检测方法可区分布鲁氏菌S2疫苗免疫牛血清和布鲁氏菌野毒感染牛血清。

In order to distinguish Brucella S2 vaccine strain from Brucella wild strains,in this experiment,by comparing the whole genome sequences of Brucella S2 vaccine strain and M28 wild strain published in NCBI,the unique gene GL_0002189 of S2 vaccine strain was screened.Cloning of the gene,prokaryotic expression and protein purification were carried out.Indirect ELISA detection method was established using purified recombinant protein as antigen;the reaction conditions of the method were optimized to determine the decision points,and the reproducibility,specificity and sensitivity tests of the established indirect ELISA detection method were performed.83 positive bovine sera that had been vaccinated against Brucella S2 were tested.The results showed that the recombinant protein GL_0002189 expressed simultaneously in soluble form and inclusion body form,and the recombinant protein with a size of 29.2 ku was obtained by purification.Among the established indirect ELISA detection method,the optimal recombinant protein coating concentration was 2.5 ng/μL;the optimal serum dilution was 1∶100,and the optimal secondary antibody dilution was 1∶5000.The decision point was 0.586;the specificity of the method was 95.0%;the sensitivity was 93.3%,and it had good reproducibility.The established indirect ELISA method was used to detect 83 positive bovine sera,and the detection rate was 7.2%.The results suggested that the indirect ELISA detection method established by the trial could distinguish between Brucella S2 vaccine immunized bovine sera and Brucella wild virus infected bovine sera.