發(fā)布時(shí)間：2018-12-11 22:44

【摘要】：為了給原核表達(dá)牛分枝桿菌Fix B、Hsp X和MPB83基因并純化Fix B、Hsp X和MPB83蛋白,進(jìn)一步研究它們?cè)谂＝Y(jié)核病診斷中作為特異性抗原的診斷價(jià)值及應(yīng)用奠定基礎(chǔ),試驗(yàn)用PCR方法從牛結(jié)核分枝桿菌AF2122/97基因組中擴(kuò)增出Fix B、Hsp X和MPB83基因片段,構(gòu)建p ET-28a-Fix B、p ET-28a-Hsp X和p ET-28a-MPB83重組質(zhì)粒,克隆到DH5ɑ感受態(tài)細(xì)胞,提取測(cè)序正確的陽(yáng)性重組質(zhì)粒,轉(zhuǎn)化BL21(DE3)感受態(tài)細(xì)胞,用1 mmol/L IPTG于37℃誘導(dǎo)表達(dá),SDS-PAGE分析目的蛋白的表達(dá)形式,Western-blot分析鑒定并用Ni-NTA層析柱純化蛋白。結(jié)果表明:成功擴(kuò)增了大小依次約為957 bp、435 bp和663 bp的Fix B、Hsp X和MPB83基因,將其連接p ET-28a原核表達(dá)載體,原核表達(dá)的Fix B、Hsp X和MPB83重組蛋白均以包涵體形式存在,分子質(zhì)量依次約為37 ku、21 ku和28 ku。經(jīng)Western-blot鑒定表達(dá)產(chǎn)物為Fix B、Hsp X和MPB83重組蛋白,Ni-NTA成功洗脫出目的蛋白。說(shuō)明試驗(yàn)成功構(gòu)建了牛分枝桿菌Fix B、Hsp X和MPB83基因的原核表達(dá)載體,并純化了Fix B、Hsp X和MPB83重組蛋白,可用于進(jìn)一步的應(yīng)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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