牛分枝杆菌FixB、HspX和MPB83基因的原核表达及鉴定

发布时间：2018-12-11 22:44

【摘要】：为了给原核表达牛分枝杆菌Fix B、Hsp X和MPB83基因并纯化Fix B、Hsp X和MPB83蛋白,进一步研究它们在牛结核病诊断中作为特异性抗原的诊断价值及应用奠定基础,试验用PCR方法从牛结核分枝杆菌AF2122/97基因组中扩增出Fix B、Hsp X和MPB83基因片段,构建p ET-28a-Fix B、p ET-28a-Hsp X和p ET-28a-MPB83重组质粒,克隆到DH5ɑ感受态细胞,提取测序正确的阳性重组质粒,转化BL21(DE3)感受态细胞,用1 mmol/L IPTG于37℃诱导表达,SDS-PAGE分析目的蛋白的表达形式,Western-blot分析鉴定并用Ni-NTA层析柱纯化蛋白。结果表明:成功扩增了大小依次约为957 bp、435 bp和663 bp的Fix B、Hsp X和MPB83基因,将其连接p ET-28a原核表达载体,原核表达的Fix B、Hsp X和MPB83重组蛋白均以包涵体形式存在,分子质量依次约为37 ku、21 ku和28 ku。经Western-blot鉴定表达产物为Fix B、Hsp X和MPB83重组蛋白,Ni-NTA成功洗脱出目的蛋白。说明试验成功构建了牛分枝杆菌Fix B、Hsp X和MPB83基因的原核表达载体,并纯化了Fix B、Hsp X和MPB83重组蛋白,可用于进一步的应用研究。
[Abstract]:In order to express the genes of Fix BHsp X and MPB83 in prokaryotic expression and purify the proteins of Fix Bhsp X and MPB83, we further study their diagnostic value and application as specific antigens in the diagnosis of bovine tuberculosis. The recombinant plasmids of Fix Bu Hsp X and MPB83 were amplified from the AF2122/97 genome of Mycobacterium bovis by PCR method. The recombinant plasmids of p ET-28a-Fix BnP ET-28a-Hsp X and p ET-28a-MPB83 were constructed and cloned into DH5 receptive cells. The positive recombinant plasmid was extracted and transformed into BL21 (DE3) competent cells. The expression was induced by 1 mmol/L IPTG at 37 鈩，

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