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本研究旨在构建结核分枝杆菌H37Rv尿嘧啶糖苷酶UNG的原核表达载体及酶学分析,为结核分枝杆菌的碱基切除修复系统的研究奠定基础。提取结核分枝杆菌H37Rv基因组DNA,PCR法扩增ung基因,构建pQE-30-ung重组质粒,在表达宿主菌E.coli M15中诱导表达蛋白,经Ni 2+-NTA亲和层析柱和凝胶过滤层析纯化,通过表面等离子共振试验观察该蛋白与错配DNA结合的情况。结果显示,成功构建了原核表达载体pQE-30-ung,并能在宿主菌E.coli M15中表达,纯化后的酶具有识别异常DNA活性,为进一步研究奠定了基础。
The purpose of this study was to construct a prokaryotic expression vector and enzyme analysis of mycobacterium tuberculosis H37Rv uracil glucoamylase UNG, which laid the foundation for the study of the base excision repair system of Mycobacterium tuberculosis. The genomic DNA of Mycobacterium tuberculosis H37Rv was extracted and the ung gene was amplified by PCR. The recombinant plasmid pQE-30-ung was constructed and expressed in E.coli M15. The recombinant protein was induced by Ni 2 + -NTA affinity chromatography and Purification by gel filtration chromatography was performed to observe the binding of the protein to mismatched DNA by surface plasmon resonance assay. The results showed that prokaryotic expression vector pQE-30-ung was successfully constructed and expressed in E.coli M15. The purified enzyme could recognize abnormal DNA and lay the foundation for further study.