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| 结核分枝杆菌Ag85B与IL-2基因双顺反子真核表达质粒的构建及体外表达 | |
| 引用本文： | 刘焰,彭剑虹,马立新,刘君炎,曹晓春.结核分枝杆菌Ag85B与IL-2基因双顺反子真核表达质粒的构建及体外表达[J].武汉大学学报(医学版),2006,27(6):691-694. |
| 作者姓名： | 刘焰 彭剑虹 马立新 刘君炎 曹晓春 |
| 作者单位： | 1. 武汉大学医学院免疫学系,武汉,4300712 2. 武汉大学中南医院基因诊断中心,武汉,430071 3. 湖北大学生命科学院微生物研究室,武汉,430064 |
| 摘 要： | 目的:构建结核分枝杆菌Ag85B与白细胞介素2(IL-2)双顺反子真核表达质粒。方法:以结核杆菌H37Rv基因组DNA为模板,通过PCR扩增出Ag85B基因。采用亚克隆的技术从pcDNA3.1-IL-2中获得小鼠IL-2基因的cDNA片段。将两者分别定向插入真核双表达载体pIRES,构建表达两个目基因的双顺反子重组质粒,然后用脂质体包裹体外转染A549细胞,RT-PCR检测Ag85B及IL-2的表达。结果:酶切分析及序列测定证实重组质粒构建正确;该重组质粒能在体外表达Ag85B及IL-2 mRNA。结论:成功构建了结核杆菌Ag85B及IL-2基因双顺反子真核表达质粒,为进一步在整体动物水平的实验研究奠定了基础。 |
| 关 键 词： | 结核杆菌 Ag85B 白细胞介素2 双顺反子 |
| 文章编号： | 1671-8852(2006)06-0691-04 |
| 收稿时间： | 2006-05-21 |
| 修稿时间： | 2006年6月21日 |
Construction and Expression of Bicistronic Eukaryotic Plasmid Containing Mycobacterium Tuberculosis Ag85B Gene and IL-2 Gene |
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| LIU Yan,PENG Jianhong,MA Lixin,LIU Junyan,CAO Xiaochun.Construction and Expression of Bicistronic Eukaryotic Plasmid Containing Mycobacterium Tuberculosis Ag85B Gene and IL-2 Gene[J].Medical Journal of Wuhan University,2006,27(6):691-694. | |
| Authors: | LIU Yan PENG Jianhong MA Lixin LIU Junyan CAO Xiaochun |
| Affiliation: | 1.Dept. of Immunology, School of Medcine , Wuhan University, Wuhan 430071 , China; 2.Genetic Diagnosis Center, Zhongnan Hospital of Wuhan University, Wuhan 430071 , China; 3.Dept. of Microbiology, Faculty of Life Science, Hubei University, Wuhan 430064 , China |
| Abstract: | Objective: To construct the bicistronic eukaryotic plasmid containing Mycobacterium tuberculosis Ag85B gene and IL-2 gene.Methods: The DNA fragments of Ag85B gene were amplified from the genome DNA of Mycobacterium tuberculosis by polymerase chain reaction(PCR) and the cDNA encoding murine IL-2 was obtained by subcloning.The two gene fragments were inserted into eukaryotic coexpression plasmid vector pIRES to construct recombinant plasmid.Then A_(549)cells were transfected with this recombinant plasmid using lipofectamine reagent and the expression of Ag85B and IL-2 were detected by RTPCR.Results: The bicistronic eukaryotic plasmid pIRES-85B-IL-2 was proved to be correct by restriction enezyme digestion and nucleotide sequencing and it could express Ag85B and IL-2 mRNA in vitro.Conclusion: The bicistronic eukaryotic plasmid containing Mycobacterium tuberculosis Ag85B gene and IL-2 gene was constructed successfully,which lays a foundation for the study in animals. |
| Keywords: | Ag85B |
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