摘要
目的:构建含结核分枝杆菌38kD蛋白基因的真核表达载体并转染HEK293T细胞,高效表达分泌性38kD蛋白。方法:设计合成的结核分枝杆菌38kD基因被克隆到T载体,然后亚克隆到真核表达载体pcDNA3.0,经酶切鉴定正确后,PEI转染法导入293T细胞,换不含血清的DMEM培养基培养3 d后收集细胞及上清,采用Western印迹检测38kD蛋白的表达。结果:酶切结果显示,获得正确的含38kD基因的重组表达载体;Western印迹结果显示表达载体导入293T细胞中后能在细胞及上清中检测到38kD蛋白表达。结论:构建了含重组结核分枝杆菌38kD蛋白基因的真核表达载体pcDNA3.0-38kD,该载体可在哺乳动物细胞HEK293T中高效分泌性表达38kD蛋白,为结核病诊断试剂盒研发奠定了基础。
Objective： To construct a robust expressing vector of Mycobacterium tuberculosis 38kD protein, which is able to induce high efficient and secretory expression of 38kD protein in transfected HEK293T cells. Methods： The 38kD gene of M.tuberculosis was synthesized and cloned to T-vector, and then subeoloned into the peDNA3.0 vector. The recombinant pcDNA3.0-38kD was verified by enzyme digestion, and then transfected into HEK293T cells by polyethylenemine（PEI）-based transfection. The transfected cells and supernatants were collected separately for the detection of 38kD protein by Western blot after the media were changed to serum-free DMEM media for 3 days. Results： We have obtained a robust M.tuberculosis 38kD protein expressing vector, pcDNA3.0-38kD, which can elicit the expression of high level of 38kD protein in both cell supernatants and lysates in transfeeted HEK293T cells. Conclusion： High level of secretory M.tuberculosis 38kD protein was expressed in mammalian cells, and it can be potentially used in the development of serological diagnostic kit for tuberculosis.
出处
《生物技术通讯》
CAS
2017年第4期451-454,533,共5页
Letters in Biotechnology
