大肠埃希菌JM109表面抗原Ag43基因敲除与鉴定  

Knockout and identification of the surface antigen 43 gene in escherichia Coli JM109

作　　者：黄用豪[1] 赵焕阁[1] 周松森 林映莹[1] 谭光宏[1] 黄风迎[1] 

机构地区：[1]海南医学院海南省热带病重点实验室,海口571199

出　　处：《重庆医学》2015年第31期4330-4332,共3页Chongqing medicine

基　　金：国家自然科学基金资助项目(81272477);海南省自然科学基金资助项目(812198)

摘　　要：目的敲除大肠埃希菌JM109表面抗原43(Ag43)基因并对其进行鉴定。方法采用Sigma公司的TargeTron基因敲除系统和Ag43基因特异设计的PCR引物扩增获得突变Ⅱ组内含子RNA蛋白复合体(RNP)基因序列,然后将这段基因序插入表达RNP的质粒pACD4K-C中,获得Ag43特异的重组RNP质粒pACD4K-Ag43。最后将pACD4K-Ag43转化JM109,经过IPTG诱导表达将Ⅱ组内含子插入Ag43特异的部位。结果通过软件分析发现插入Ⅱ组内含子的最佳位点位于碱基1 812和1 913之间,琼脂糖凝胶电泳发现PCR扩增的突变Ⅱ组内含子RNP基因序列分子量大小和预期值(350bp)相一致,用NheⅠ和HindⅢ二种酶切分析重组质粒pACD4K-Ag43结果和预期值相符,PCR扩增相应产物和基因测序显示Ⅱ组内含子特异地插入Ag43基因碱基序列的1 812/1 913位点。结论成功地将大肠埃希菌JM109中的基因敲除,为更进一步深入研究Ag43基因的功能和将其作为制备重组Ag43嵌合蛋白的宿主菌奠定了基础。Objective To knockout and identify the Antigen 43 （Ag43） in the Escherichia Coli JM109. Methods Mutation group Ⅱ introns RNA protein complexes （RNP） gene sequence was obtained by Sigma Companyrs TargeTron Gene Knockout System and Ag43 gene specific designed PCR primers amplification, then, to acquired Ag43 specific recombinant RNP plasmid pACD4K-Ag4, this gene sequence was inserted into the plasmid pACD4K-C of RNA＇s expression. Finally, pEGFP-Ag43 was transformed into JM109 and inserted the group Ⅱ intron into the Ag43＇s locus by IPTG inducing expression. Results The best insertion locus was between 1 812 and 1 913. Through the agarose electrophoresis gel,the RNP gene sequence was consistent with the expected value （350 bp）. The pEGFP-Ag43 vector was correctly constructed which was proofed by endonuclease Nhe I and Hind Ⅱ digestion as predicted products （3 646 and 4 029 bp; 7 000 and 550 bp, respectively）, The PCR and gene sequence results indicated that the group Ⅱ intron was inserted into the locus between 1 812 and 1 913 in the Ag43 gene. Conclusion Successful knockout of the Ag43 in Escherichia Coli JM109 found basis to further study the Ag43Ps function and regard the coli as host bacteria of Ag43 chimeric protein recombinant.

关 键 词：抗原Ag43 基因敲除 细菌表面表达 Ⅱ组内含子 表达系统 

分 类 号：R378[医药卫生—病原生物学]