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目的:构建结核分枝杆菌(MTB)Rv1759c结构域(Rv1759cD domain,Rv1759cD)与人IL-2(hIL-2)融合基因,并在大肠杆菌中表达获得重组的融合蛋白Rv1759cD-IL-2。方法:用PCR方法从MTB H37Rv基因组扩增Rv1759cD基因片段,测序后与hIL-2基因构建融合基因,并克隆到表达载体pProEX HTa。融合基因在大肠杆菌DH5α中诱导表达,经SDS-PAGE分析后,分别与His mAb、IL-2mAb和结核病人血清进行Western-blot鉴定,采用Ni-NTA亲和层析纯化蛋白。结果:获得的Rv1759cD基因经测序与GenBank公布的序列完全一致,与hIL-2基因连接后,构建的融合基因在大肠杆菌中有效表达。表达蛋白相对分子量为30KDAa,与预测值相符;Western-blot结果显示,在相对分子量30KDAa处分别与His mAb和鼠抗IL-2 mAb形成结合带,并与结核病人血清出现特异性结合。通过Ni-NTA亲和层析,可得到纯化的目的蛋白。结论:成功表达、纯化和鉴定了Rv1759cD-IL-2融合蛋白,并有可能作为新型结核病疫苗的靶抗原。
Objective: To construct the fusion gene Rv1759c domain (Rv1759cD domain) and human IL-2 (hIL-2) of Mycobacterium tuberculosis (MTB) and express the recombinant fusion protein Rv1759cD-IL-2 in E. coli. METHODS: The Rv1759cD gene fragment was amplified from the MTB H37Rv genome by PCR. The fusion gene was constructed with the hIL-2 gene after sequencing and cloned into the expression vector pProEX HTa. The fusion gene was induced in E. coli DH5α. After SDS-PAGE analysis, the fusion protein was identified by Western-blot with His mAb, IL-2 mAb and tuberculosis patient serum respectively. The protein was purified by Ni-NTA affinity chromatography. Results: The obtained Rv1759cD gene was sequenced exactly in accordance with the sequence published in GenBank. After being ligated with hIL-2 gene, the constructed fusion gene was efficiently expressed in E. coli. The relative molecular weight of the expressed protein was 30KDAa, which was in good agreement with the predicted value. Western-blot results showed that the recombinant protein had a binding band with His mAb and murine anti-IL-2 mAb at the molecular weight of 30KDAa, respectively. By Ni-NTA affinity chromatography, the purified target protein can be obtained. CONCLUSION: The Rv1759cD-IL-2 fusion protein was successfully expressed, purified and identified, and it may be used as the target antigen of novel TB vaccine.