摘要
目的:克隆结核杆菌含信号肽的Mtb8·4(MS)基因,导入真核表达载体pcDNA3·1(+),构建成重组质粒pcDNA3·1(+)-MS,并在真核细胞中进行表达。方法:提取人结核杆菌H37Rv株的基因组作为模板,进行PCR圹增,获得含信号肽的Mtb8·4(MS)基因后,与pcDNA3·1(+)载体进行连接重组,用限制性内切酶消化、PCR及DNA序列分析等多种方法进行鉴定;重组质粒转染COS-7细胞48h后,用RT-PCR方法鉴定MS在转录水平的表达情况。结果:pcD-NA3·1(+)-MS真核表达载体构建成功;转染COS-7细胞后,MS在转录水平成功表达。结论:pcDNA3·1(+)-MS真核表达质粒的构建以及MS在COS-7细胞中的成功表达,为进一步研究该真核表达质粒的免疫保护效果及制备结核病pcD-NA3·1(+)-MSDNA疫苗奠定了基础。
Objecticve:To construct and express the pcDNA3.1(+)-MS eukaryotic expression plasmid.Methods:Extracted DNA from M.Tuberculosis was amplified by PCR and the target gene we got was cloned into the unique HindⅢ and EcoR ⅰcloning sites of pcDNA3.1(+).Correct pcDNA3.1(+)-MS recombinant plasmid was identified by PCR,RE digestion and DNA sequencing .COS-7 cells were transfected with pcDNA3.1(+)-MS constructs by cationic liposom 48 hours later,mRNA of targets gene were detected by RT-PCR.Results:The accuracy of pcDNA3.1(+)-MS plasmid constructs was confirmed by a series of molecular biology techniques.Transfection of COS-7 cells with plasmid pcDNA3.1(+)-MS led to transient expression of MS proteins.Conclusion:The construction and expression of pcDNA3.1(+)-MS provided the possibility for investigating immunogenicity of the recombinant plasmid and preparing a new tuberculosis vaccine .
出处
《现代预防医学》
CAS
北大核心
2005年第7期711-713,750,共4页
Modern Preventive Medicine
基金
四川省青年科技基金资助(批准号:川青科基[2002]1号)
