摘要
目的获得带FLAG标签的结核分枝杆菌PE35的真核表达载体,并在293T细胞中检测其表达。方法用PCR方法从质粒上获得PE35基因全长,克隆到pCDNA3-FLAG真核表达载体上;用脂质体介导的基因瞬时转染法,将构建正确的表达载体转染293T细胞;Western-blot法检测细胞中的FLAG融合蛋白的表达。结果酶切鉴定和DNA序列分析显示构建了正确的FLAG-PE35真核表达载体,并能在真核细胞中表达相对分子量大小相符的重组蛋白。结论成功构建了FLAG-PE35真核表达载体,并在真核细胞中得到表达。为研究PE35蛋白在结核分枝杆菌中的功能研究奠定了基础。
Objective To construct the eukaryotic expression vector of pcDNA-FLAG-PE35 and evaluate Mycobacterium Tuberculosis（TB） PE35 expression in SV40-transformed embryonic kidney cell line 293T.Methods The full-length coding gene of Mycobacterium Tuberculosis PE35 was amplified from a plasmid with standard PCR method.The expression construct of pcDNA-FLAG-PE35 was generated by inserting the amplified gene of TB PE35 into the eukaryotic expression vector of pcDNA3-FLAG.The expressed protein of TB PE35 was evaluated by Western blot in transient transfected 293T cells.Results The pattern of restriction enzyme digestion and result of nucleotide sequencing confirmed that the gene of TB PE35 was accurately inserted into eukaryotic expression vector pcDNA-FLAG-PE35.The expression of recombinant protein was detected by Western blot.Conclusion The production of recombinant protein will facilitate the understanding of molecular mechanism of TB PE35 related pathogenesis in TB infection.
出处
《中国预防医学杂志》
CAS
2011年第12期995-997,共3页
Chinese Preventive Medicine
基金
国家重大传染病专项基金资助项目(基金编号:2008ZX10003-012)
关键词
结核分枝杆菌
PE35
真核表达
Mycobacterium tuberculosis（TB）
PE35
Eukaryotic expression
