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

• 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
• 2:贵州医科大学附院ICU

摘要(Abstract)：

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 group Ⅱ intron gene inactivation system for the inactivation of a human intestinal Escherichia coli β-galactosidase gene(LacZ). Methods: The inactivated plasmid pHK-TT1 A-lacZ426 a, pHK-TT1 A-lacZ1879 a, pHK-TT1 A-lacZ1880 a and pHK-TT1 A-lacZ1155 s 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 TeI3 c/4 c 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 lacZ426 a, lacZ1879 a, lacZ1880 a and lacZ1155 s 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(KeyWords)： 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
gene inactivation system;type Ⅱ intron;Escherichia coli;β-galactomase gene;gene inactivation;plasmid;molecular cloning

Abstract:

Keywords:

基金项目(Foundation): 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]

作者(Author): 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

DOI: 10.19367/j.cnki.1000-2707.2019.12.002

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