人肠道来源大肠杆菌β-半乳糖苷酶基因突变株的构建  

Construction of β-galactosidase Gene Mutant from Human Intestinal Escherichia Coli

作　　者：洪伟[1] 赵行行 吴昌学[1] 饶凤琴 程玉梅[2] 张婷[1] 齐晓岚[1] 禹文峰[1] 官志忠[1] HONG Wei;ZHAO Xingxing;WU Changxue;RAO Fengqin;CHEN Yumei;ZHANG Ting;QI Xiaolan;YU Wenfeng;GUAN Zhizhong(Key Laboratory of Ministry of Education for Epidemic and Ethnic Diseases in Guizhou Medical University&Key Laboratory of Molecular Biology in Guizhou,Guiyang 550004,Guizhou,China;ICU of Hospital Affiliated to Guizhou Medical University,Guiyang 550004,Guizhou,China)

机构地区：[1]贵州医科大学地方病与少数民族疾病教育部重点实验室&贵州省分子生物学重点实验室,贵州贵阳550004 [2]贵州医科大学附院ICU,贵州贵阳550004

出　　处：《贵州医科大学学报》2019年第12期1370-1376,共7页Journal of Guizhou Medical University

基　　金：国家自然科学基金(31560318,31601012,31760318);贵州省自然科学基金资助项目[黔科合基础(2019)1441,(2018)1132,(2018)5779-17]

摘　　要：目的:使用嗜热二型内含子基因失活系统(Thermotargetron)失活人肠道来源大肠杆菌(HSM174 DE3)β-半乳糖苷酶基因(LacZ),构建lacZ失活突变株。方法:使用分子克隆方法,构建lacZ基因失活质粒pHK-TT1A-lacZ426a、pHK-TT1A-lacZ1879a、pHK-TT1A-lacZ1880a及pHK-TT1A-lacZ1155s,将基因失活质粒转化大肠杆菌HSM174菌株(E.coli HSM174),转化子培养时提高温度至48℃,诱导TeI3c/4c二型内含子“归巢”活性,采用蓝白斑及PCR筛选lacZ基因失活突变株。结果:随机挑选20株克隆菌株的PCR结果显示,lacZ426a、lacZ1879a、lacZ1880a及lacZ1155s基因失活效率分别为60%、75%、60%及75%;白斑克隆基因失活效率为100%。结论:Thermotargetron系统可高效的实现E.coli HSM 174菌株lacZ基因失活,并可能具有在中温菌中广泛应用的潜力。Objective:To construct lacZ inactivated mutant by the thermophile groupⅡintron gene inactivation system for the inactivation of a human intestinal Escherichia coliβ-galactosidase gene(LacZ).Methods:The inactivated plasmid pHK-TT1A-lacZ426a,pHK-TT1A-lacZ1879a,pHK-TT1A-lacZ1880a and pHK-TT1A-lacZ1155s were constructed by molecular cloning.The gene-inactivated plasmid was transformed into Escherichia coli HSM174 strain(E.coli HSM174),and the temperature was raised to 48℃when the transformant was transformed.The"retrohoming"activity of the two-type intron of TeI3c/4c was induced by the raising temperature and the mutant strain of lacZ gene was screened by blue and white spot and PCR.Results:The PCR results of 20 cloned strains showed that the inactivation efficiency of lacZ426a,lacZ1879a,lacZ1880a and lacZ1155s gene were 60%,75%,60%and 75%,respectively.The inactivation efficiency of white colonies gene was 100%.Conclusion:Thermotargetron system can efficiently inactivate lacZ gene of Escherichia coli HSM 174 strain,and have the potential to be widely used in mesophilic bacteria.

关 键 词：基因失活系统 二型内含子 大肠杆菌 Β-半乳糖苷酶基因 基因失活 质粒 分子克隆 

分 类 号：R378.21[医药卫生—病原生物学]