布鲁菌BAB基因的原核表达及间接ELISA检测方法的建立

doi:10.16431/j.cnki.1671-7236.2020.11.027

摘要/Abstract

摘要： 本研究以布鲁菌Rev.1株基因组为模板，扩增BAB基因序列，将其克隆至原核表达载体pET-30a（+），获得重组质粒pET-30a-BAB，对重组质粒进行原核表达，经Western blotting检测后，利用表达产物构建检测布鲁菌病的间接ELISA方法。结果表明，本试验成功克隆并表达了BAB基因，纯化表达产物经SDS-PAGE分析显示，本研究获得较纯的重组BAB蛋白；Western blotting试验表明，表达蛋白可与布鲁菌羊阳性血清发生特异性反应，具有良好的反应原性；以重组BAB蛋白作为包被抗原，建立并优化了检测BAB抗体的间接ELISA方法。确定最佳包被条件：BAB蛋白包被量0.25 μg/mL，血清稀释度为1：400；封闭液为3%猪源明胶；二抗稀释度为1：6 000；显色时间为10 min。应用建立方法对临床40份羊血清进行检测，计算得出临界值为0.607。即当待检血清的P/N ≥ 1.5，且D_{450 nm} ≥ 0.607时，判定为阳性，当D_{450 nm} ≤ 0.561时，判定为阴性，当0.607 < D_{450 nm} < 0.561时，判定为疑似值，需要进行复检。与虎红平板试验和试管凝集试验比较，阳性符合率为100%，阴性符合率为71.88%，总符合率为77.5%。

关键词: 布鲁菌病; Rev.1株; BAB基因; 原核表达; ELISA

Abstract: In this study,the genome of Brucella Rev.1 strain was used as a template to amplify the BAB gene sequence,clone it into the prokaryotic expression vector pET-30a(+),obtain the recombinant plasmid pET-30a-BAB,and perform prokaryotic expression on the recombinant plasmid.The indirect ELISA method was constructed by using the expression products detected by Western blotting.The results showed that the BAB gene was successfully cloned and expressed in this experiment,and the purified expression product was analyzed by SDS-PAGE.This study obtained a relatively pure recombinant BAB protein;Western blotting test showed that the expressed protein could react specifically with Brucella sheep positive serum and had good reactogenicity;Using the recombinant BAB protein as the coating antigen,an indirect ELISA method for detecting BAB antibodies was established and optimized.The best determined coating conditions were as follows BAB protein coating amount was 0.25 μg/mL,serum dilution was 1:400;blocking solution was 3% pig-derived gelatin;secondary antibody dilution was 1:6 000;color development time was 10 min.The established method was used to detect 40 clinical sheep serums,and the cut-off value was calculated to be 0.607.That was,when the serum tested had P/N ≥ 1.5 and D_{450 nm} ≥ 0.607,it was judged as positive,when D_{450 nm} ≤ 0.561,it was judged as negative,and when 0.607<D_{450 nm}<0.561,it was judged as a suspect value,and retest was required.Compared with the Huhong plate test and the test tube agglutination test,the positive coincidence rate was 100%,the negative coincidence rate was 71.88%,and the total coincidence rate was 77.5%.

Key words: brucellosis; strain Rev.1; BAB gene; prokaryotic expression; ELISA

中图分类号:

S885.3

杭天宇, 赵洪哲, 宋前进, 张静, 姬智, 温永俊, 关平原. 布鲁菌BAB基因的原核表达及间接ELISA检测方法的建立[J]. 中国畜牧兽医, 2020, 47(11): 3641-3650.

HANG Tianyu, ZHAO Hongzhe, SONG Qianjin, ZHANG Jing, JI Zhi, WEN Yongjun, GUAN Pingyuan. Prokaryotic Expression of Brucella BAB Gene and Establishment of iELISA[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(11): 3641-3650.

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