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.
作者
洪伟
赵行行
吴昌学
饶凤琴
程玉梅
张婷
齐晓岚
禹文峰
官志忠
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)
出处
《贵州医科大学学报》
CAS
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]
