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目的构建含结核分枝杆菌(MTB)脂蛋白G(Lpr G)基因的真核表达载体pc DNA3.1-His-Lpr G-FLAG,并在HEK293T细胞中表达和纯化Lpr G融合蛋白。方法以MTB H37Rv基因组DNA为模板,用PCR法扩增Lpr G基因,并通过DNA重组构建真核表达载体pc DNA3.1-His-Lpr G-FLAG,酶切鉴定并测序。将重组质粒转染HEK293T细胞后,应用Western blot法检测His-Lpr G-FLAG融合蛋白的表达。利用Ni-NTA金属亲和层析柱从细胞上清和细胞裂解液中纯化融合蛋白His-Lpr G-FLAG,并用SDS-PAGE和Western blot法检测纯化后的蛋白。结果成功构建了pc DNA3.1-His-Lpr G-FLAG的真核表达载体,Western blot结果表明转染质粒后,HEK293T细胞裂解液中检测到目的蛋白,相对分子量(Mr)为27 000。经Ni-NTA金属亲和层析柱纯化后得到高纯度的融合蛋白His-Lpr G-FLAG。结论成功构建并在HEK293T细胞中表达和纯化了His-Lpr G-FLAG融合蛋白。
Objective To construct an eukaryotic expression vector pcDNA3.1-His-Lpr G-FLAG containing Lp G gene of Mycobacterium tuberculosis (MTB) and express and purify Lpr G fusion protein in HEK293T cells. Methods The genomic DNA of MTB H37Rv was used as a template to amplify Lpr G gene by PCR. The eukaryotic expression vector pcDNA3.1-His-Lpr G-FLAG was constructed by DNA recombination, identified and sequenced. The recombinant plasmids were transfected into HEK293T cells and the expression of His-Lpr G-FLAG fusion protein was detected by Western blot. The fusion protein His-Lpr G-FLAG was purified from the cell supernatant and cell lysate by Ni-NTA metal affinity chromatography. The purified protein was detected by SDS-PAGE and Western blot. Results The eukaryotic expression vector pcDNA3.1-His-Lpr G-FLAG was successfully constructed. Western blot analysis showed that the target protein was detected in HEK293T cell lysate after transfection with a relative molecular weight of 27,000. Purified by Ni-NTA metal affinity chromatography to obtain high-purity fusion protein His-Lpr G-FLAG. Conclusion The His-Lpr G-FLAG fusion protein was successfully constructed and expressed in HEK293T cells.