牛结核病鉴别诊断的方法研究

Differential diagnosis of bovine tuberculosis by indirect ELISA assay

目的构建含结核分枝杆菌(Mycobacterium tuberculosis)tb10.4基因片断的原核表达载体,在E.coli中诱导表达带组氨酸标记的该融合蛋白。分别以TB10.4蛋白,PPD蛋白以及TB10.4与MPT83混合蛋白为抗原,用间接ELISA法鉴别牛结核病。方法PCR法扩增tb10.4基因片段,连接到pET-22b(+)原核表达载体中,将重组子转化到大肠杆菌蛋白酶缺陷型菌株BL21(DE3)/PolysS,用IPTG诱导,进行蛋白表达、纯化。之后将TB10.4,PPD,TB10.4及MPT83混合蛋白作为抗原用间接ELISA法检测牛结核病。结果使用结核杆菌早期分泌蛋白TB10.4作为抗原用间接ELISA方法快速检测牛结核病,并区分BCG免疫的健康牛和感染牛的结果显示灵敏度为23%,没有假阳性反应。结论用PPD作为抗原进行检测灵敏度达到48%,但假阳性率达到21%;用TB10.4与MPT83混合抗原检测时灵敏度下降到6%,假阳性率1%。

The TB10.4 gene fragment was firstly amplified and cloned to prokaryotic expression vector pET-22h（＋ ）, and then the recombinant plasmid pET-22b（＋）/tb10.4 was transfected to the expression vector strain E. coli BL21 （DE3）/ PolysS and induced with IPTG to express the fusion protein. This fusion protein was used as antigen in indirect ELISA assay for the differential diagnosis of bovine tuberculosis, and comparison was made in indirect ELISA assays using PPD, TB10.4 protein and TB10.4 mixed with MPT83 as antigen, for their sensitivity and specificity. It was found that when TB10.4 was used as antigen in the indirect ELISA assay, the sensitivity approached up to 23 % without false positive reaction. However, when PPD was used as antigen, although the sensitivity might reach to 48%, but the specificity dropped to 21%. When TB 10.4 mixed with MPT83 was used as antigen, the sensitivity declined to 6 % with 1 % of false positive reaction.