羊种布鲁氏菌wzt基因的原核表达及间接ELISA方法的建立

doi:10.16431/j.cnki.1671-7236.2019.12.030

摘要/Abstract

摘要： 为建立检测血清中布鲁氏菌抗体的间接ELISA方法，本试验采用PCR技术从羊种布鲁氏菌QY1菌株中扩增得到wzt基因片段，连接到pET-30a载体上，构建质粒pET-30a-wzt，将鉴定正确的质粒转化E.coli BL21（DE3）感受态细胞，经原核表达系统对其进行表达，表达产物用SDS-PAGE和Western blotting进行分析后，用亲和层析镍柱纯化wzt重组蛋白备用。以wzt重组蛋白为检测抗原，逐步优化条件后建立布鲁氏菌间接ELISA检测方法。结果显示，试验成功构建了pET-30a-wzt原核表达载体，并在BL21（DE3）宿主菌中表达；SDS-PAGE和Western blotting结果表明，重组蛋白约为35 ku，表达形式为上清，条带单一、无杂带，有很好的反应原性和特异性。ELISA优化试验确定了最佳包被浓度为15 μg/mL，血清最佳稀释度为1：80，酶标抗体的最佳稀释度为1：5 000；通过检测24份阴性样品确定临界值，当样品D_{450 nm}值≥ 0.30为阳性，样品D_{450 nm}值<0.30时为阴性；特异性试验表明，该方法不与小肠耶尔森菌、大肠杆菌发生交叉反应；批内及批间变异系数均<10%；用该方法对120份血清样本进行检测，并与虎红凝集试验进行相符性验证，符合率为96%。本试验建立的间接ELISA方法为布鲁氏菌病的检测提供了可靠的技术手段。

关键词: 布鲁氏菌; wzt基因; 原核表达; 间接ELISA

Abstract: In order to establish an indirect ELISA method for detection of Brucella antibody in serum,the wzt gene fragment was amplified from the strain of Brucella melitensis QY1 by PCR and ligated into the pET-30a vector to construct the plasmid pET-30a-wzt.The correct plasmid was transferred to E.coli BL21(DE3) competent cells were expressed by prokaryotic expression system,and the expressed products were analyzed by SDS-PAGE and Western blotting,and then the wzt recombinant protein was purified.The indirect ELISA assay for Brucella was established using wzt recombinant protein as the detection antigen and gradually optimizing the conditions.The results showed that the prokaryotic expression vector of pET-30a-wzt was successfully constructed and expressed in BL21(DE3) host bacteria.SDS-PAGE and Western blotting results showed that the recombinant protein was about 35 ku,which had good reactogenicity.The optimal coating concentration was determined to be 15 μg/mL,the optimal dilution of serum was 1:80,and the optimal dilution of the enzyme-labeled antibody was 1:5 000.The critical value was determined by detecting 24 negative samples,the sample was positive when the D_{450 nm} value was ≥ 0.30,and the sample was negative when the D_{450 nm} value was <0.30.The specificity test results showed that the method did not cross-react;The intra-and inter-assay coefficients of variation were both <10%;120 samples of serum were used in this method,the test was carried out and verified by the Rose-Bengal agglutination test,and the coincidence rate was 96%.This results indicated that the indirect ELISA method established in this experiment provided a reliable technical means for the detection of brucellosis.

Key words: Brucella; wzt gene; prokaryotic expression; indirect ELISA

中图分类号:

S852.61⁺4

赵鹭, 葛志毅, 刘永生, 李学瑞, 曹小安. 羊种布鲁氏菌wzt基因的原核表达及间接ELISA方法的建立[J]. 中国畜牧兽医, 2019, 46(12): 3707-3714.

ZHAO Lu, GE Zhiyi, LIU Yongsheng, LI Xuerui, CAO Xiaoan. Prokaryotic Expression of wzt Gene in Brucella melitensis and the Establishment of Indirect ELISA[J]. China Animal Husbandry and Veterinary Medicine, 2019, 46(12): 3707-3714.

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