中国组织工程研究与临床康复2007，Vol.11Issue(29)：5836-5839,4.
结核分枝杆菌融合基因Hsp65-Esat6荧光表达载体的构建及表达
Construction and expression of the fusional genes Hsp65-Esat6 expressing plasmid of mycobacterium tuberculosis
李君武 1黄泽棋 1姚翠婵 1周曙光 1李晓栋 1宋东 1黄清华1
作者信息
- 1. 暨南大学医学院微生物免疫学教研室,广东省广州市,510632
- 折叠
摘要
Abstract
BACKGROUND:There is a linker between Hsp65 and Esat6 coding a segment of water repellent polypeptide.It is very gentle and easy to be folded,which is profitable to translate the keno-folding correctly between the two proteins and to make the space structure of fusional proteins consistent with those two native ones.Then,a correct structure is formed and the immunogenicity of the fusional proteins is improved.OBJECTIVE:This study was to clone the fusional genes Hsp65-Esat6 from mycobacterium tuberculosis(MTB)H37Rv, then to construct into a eukaryotic expressing vector which contains an enhanced green fluorescent protein(EGFP) reporter gene,and finally to identify the expression of Hsp65 protein and Esat6 protein by IHC methods.DESIGN:Single sample experiment.SETTING:Department of Microbiology ＆ Immunology,Medical College of Jinan University.MATERIALS:Plasmid pVAE was donated by Professor Cao from South China University of Technology.MTB H37Rv,E.coil DH5 α,expressing plasmid pEGFP-C1 and Hela cells were donated by Doctor Hu Ping.METHODS:The experiment was conducted at the Department of Microbiology ＆ Immunology and the Medical Experimental Center of Jinan University between September 2005 and June 2006.The whole-genome was extracted from MTB H37Rv by molecular cloning technique,and used it as template to amplify Hsp65 (no terminator) gene by polyrnerase chain reaction (PCR),to recombine it with pEGFP-C1 vector after purification to construct pEGHsp65(no terminator)recombination vector.pVAE vector was used as template to amplify Linker-Esat6 gene(with terminator)by PCR, and then to recombine it with pEGHSP65(no terminator)to construct an eukaryotic expressing vector pEGHsp65-Esat6 with the fusional genes Hsp65-Esat6 inside.Finally,genes Hsp65-Esat was checked by molecule biology methods such as PCR, restriction endonuclease and DNA sequencing.Hela cells were transfected with pEGHsp65-Esat6 and the expression of EGFP and the efficiency of transfection were observed.The expressions of Hsp65 protein and Esat6 protein were detected by IHC methods.MAIN OUTCOME MEASURES:①The size of pEGHsp65-Esat6.②The DNA sequencing result of the pEGFP-C1.③The expression of EGFP in the transfected Hela cells.④The IHC results of Hsp65 protein and Esat6 protein in Hela cells.RESULTS:①The size of pEGFP-C1 was 4.7 kb,that of pEGHsp65 was 6.4 kb,and that of pEGHsp65-Esat6 was 6.7 kb.There were differences between their speeds In agarose electrophoresis.②The results showed that it was the same as reported Hsp65 sequence and Esat6 sequence of MTB H37Rv.③After transfecting pEGHsp65-Esat6 for 24 hours,EGFP was found in 30% of Hela through Laser scanning confocal microscope. But there was no EGFP in non-transfected Hela.④Hsp65 protein and Esat6 protein with biological activities were detected in transfected Hela cells by IHC methods.CONCLUSION: Using Hsp65 and Esat6 as immunogens,we have successfully cloned and constructed a eukaryotic expressing vector which contain fusionaI genes Hsp65-Esat6 of MTB H37Rv and EGFP.关键词
结核分枝杆菌/Hsp65/Esat6/融合基因/DNA疫苗分类
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李君武,黄泽棋,姚翠婵,周曙光,李晓栋,宋东,黄清华..结核分枝杆菌融合基因Hsp65-Esat6荧光表达载体的构建及表达[J].中国组织工程研究与临床康复,2007,11(29):5836-5839,4.
