结核分枝杆菌重组蛋白CFP-10-ESAT-6-PPE68的表达、纯化与多克隆抗体的制备

Expression,purification and preparation of the polyclonal antibody of the recombinant protein CFP-10-ESAT-6-PPE68 of tuberculosis

目的构建重组质粒pET32a(+)-cfp-10-esat-6-ppe68,得到重组结核分枝杆菌CFP-10-ESAT-6-PPE68蛋白及兔抗结核分枝杆菌CFP-10-ESAT-6-PPE68蛋白的多克隆抗体,为下一步应用于临床结核病的检测奠定基础。方法将结核分枝杆菌cfp-10-esat-6-ppe68基因转入质粒pET32a,将经PCR、酶切、序列测定鉴定为阳性的质粒转入大肠杆菌DE3,IPTG诱导表达重组蛋白,经亲和层析法纯化重组蛋白后用凝血酶切除载体蛋白,免疫家兔制备多克隆抗体并进行纯化。结果成功构建重组质粒表达体系pET32a(+)-cfp-10-esat-6-ppe68,制得去除载体蛋白的高纯度重组蛋白CFP-10-ESAT-6-PPE68,并成功得到高纯度抗结核分枝杆菌CFP-10-ESAT-6-PPE68蛋白的抗体。结论成功表达结核重组蛋白并制得高纯度多克隆抗体。

Objective Based on supporting the clinical detection for tuberculosis,this subject aims to construct a recombinant plasmid pET32a（＋）-cfp-10-esat-6-ppe68 in order to obtain purified recombinant proten CFP-10-ESAT-6-PPE68 and the rabbit polyclonal antibody of CFP-10-ESAT-6-PPE68. Methods The recombinant plasmid pET32a-cfp-lO-esat- 6-ppe68 was constructed and confirmed by PCR, double enzyme digestion and sequence analysis, and then transferred to E.Coli DE3. The expression of recombinant protein cfp-lO-esat-ppe68 was induced by IPTG and purified by affinity chromatography before separation of the target protein and carrier protein by Thrombin. Polyclonal antibody of cfp-10-esat- 6-ppe68 was obtained from immunized domestic rabbits. Results The recombinant plasmid pET32a-cfp-10-esat-6-ppe68 was constructed successfully.Moreover, both the highly purified recombinant protein CFP-10-ESAT-6-PPE68 and the anti- CFP-10-ESAT-6-PPE68 antibody were obtained. Conclusion The recombinant tuberculosis protein CFP-10-ESAT-6- PPE68 and the antibody against CFP-lO-ESAT-6-PPE68 were obtained successfully.