酵母双杂交pGADT7-C3b重组质粒的构建与鉴定  

Construction and identification of pGADT7-C3b prey plasmid of yeast Two-Hybrid

作　　者：凌小雅 贾瑞璞 范阔海[2] 孙娜[1] 孙盼盼 孙耀贵[1] 杨惠珍 李宏全[1] 尹伟[1] LING Xiaoya;JIA Ruipu;FAN Kuohai;SUN Na;SUN Panpan;SUN Yaogui;YANG Huizhen;LI Hongquan;YIN Wei(College of Veterinary Medicine,Shanxi Agricultural University,Jinzhong 030600,China;Laboratory Animal Center,Shanxi Agricultural University,Jinzhong 030600,China)

机构地区：[1]山西农业大学动物医学学院,山西晋中030600 [2]山西农业大学实验动物管理中心,山西晋中030600

出　　处：《黑龙江畜牧兽医》2021年第19期5-9,共5页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家自然科学基金青年科学基金项目(31702221);山西省高等学校科技创新项目(2019L0364)。

摘　　要：为了研究猪红细胞CR1-like与补体片段C3b的互作关系,试验构建符合酵母双杂交系统要求的重组质粒pGADT7-C3b,对合成的含C3b基因片段的质粒进行双酶切鉴定,运用基因重组技术将鉴定正确的C3b基因与表达载体pGADT7连接并转化至大肠杆菌DH5α感受态细胞中筛选阳性克隆,将双酶切鉴定正确的重组质粒pGADT7-C3b转化至感受态酵母菌Y2HGold中并进行毒性检测和自激活检测。结果表明:合成的含C3b基因片段的质粒经双酶切鉴定得到的目的基因条带大小为1914 bp,将C3b基因与表达载体pGADT7连接并转化至大肠杆菌DH5α感受态细胞中,然后在含Amp的LB固体培养基中长出阳性菌落,重组质粒pGADT7-C3b经双酶切鉴定得到与预期大小相符的条带,经毒性检测发现转化有重组质粒pGADT7-C3b的Y2HGold酵母菌在SD/-Leu固体培养基上正常生长,在SD/-Leu、SD/-Leu/X-a-Gal固体培养基上长出白色菌落,在SD/-Leu/X-a-Gal/Aba固体培养基上无菌落生长。说明试验成功构建了pGADT7-C3b重组质粒,对Y2HGold酵母菌无毒性和自激活作用,符合酵母双杂交系统要求。In order to study the interaction between porcine erythrocytes CR1-like protein and complement fragment C3 b, a recombinant plasmid pGADT7-C3 b was constructed to meet the requirements of Yeast Two-Hybrid system, in this study, the synthetic plasmid containing C3 b gene fragment was identified by double enzyme digestion, and the correctly identified C3 b gene was connected with the vector pGADT7 by gene recombination technology and transformed into E.coli DH5α competent cells to screen the positive clones. The recombinant plasmid pGADT7-C3 b with double enzyme digestion was transformed into competent yeast Y2 HGold for toxicity and self-activation detection. The results showed that the size of target gene band was 1 914 bp by double enzyme digestion of the plasmid containing C3 b gene fragment. The C3 b gene was linked to the vector pGADT7 and transformed into E.coli DH5α competent cells, and positive colonies grew in the LB(+Amp) solid medium. The recombinant plasmid pGADT7-C3 b was identified by double enzyme digestion and the bands were consistent with the expected size. Toxicity test found that Y2 HGold yeast transformed with recombinant pGADT7-C3 b grew normally on SD/-Leu solid medium, and it had grown white colonies normally on SD/-Leu and SD/-Leu/X-a-Gal solid medium, but could not grow on SD/-Leu/X-a-Gal/Aba solid medium. It indicated that the pGADT7-C3 b recombinant plasmid was successfully constructed, and had no cytotoxicity and self-activation to Y2 HGold yeast, which met the requirements of the Yeast Two-Hybrid system.

关 键 词：酵母双杂交 C3B 猪CR1-like 重组质粒 毒性检测 自激活检测 

分 类 号：Q782[生物学—分子生物学]