小鼠巨细胞病毒即刻早期基因M122酵母双杂交诱饵载体的构建及自激活作用的检测  

Construction and Self-Activation Detection of Bait Vector Containing of Mouse Cytomegalovirus Immediate-Early Gene M122 in Yeast Two-Hybrid System

作　　者：王慧[1] 田佳[1] 罗丹[1] 刘兴楼[1] 舒赛男[1] 张慧娟[1] 方峰[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院儿科学系,武汉430030

出　　处：《实用儿科临床杂志》2010年第4期289-292,共4页Journal of Applied Clinical Pediatrics

基　　金：国家自然科学基金(30671859)

摘　　要：目的构建小鼠巨细胞病毒(MCMV)即刻早期基因M122的酵母双杂交诱饵载体pGBKT7-M122,并检测其自激活作用及对酵母AH109菌株有无毒性作用,用于筛选小鼠胚胎脑cDNA文库。方法采用反转录(RT)-PCR的方法扩增MCMV的M122基因片段,并将其插入到pMD18-Tsimple vector,构建重组质粒pMD18-T-M122。用限制性内切酶EcoRⅠ和SalⅠ将重组质粒酶切鉴定,并测序。测序正确的pMD18-T-M122重组质粒和载体pGBKT7-BD分别用限制性内切酶EcoRⅠ和SalⅠ进行双酶切,凝胶回收M122基因片段及pGBKT7-BD载体片段。将回收的M122基因片段和pGBKT7-BD载体片段,用T4 DNA连接酶16℃连接过夜,构建诱饵质粒pGBKT7-M122。对pGBKTT-M122进行酶切鉴定,测序。将测序正确的pGBKT7-M122转化AH109酵母感受态细胞,转化菌液涂布于营养缺陷培养基SD/-Trp平板和SD/-Trp/X-α-Gal平板。空载体质粒pGBKT7-BD和阳性质粒pCL作为对照亦被转入AH109酵母感受态细胞,转化菌液分别涂布于SD/-Trp平板和SD/-Leu/X-α-Gal平板。观察平板上菌落的颜色、数量及大小。结果1.成功构建了用于酵母双杂交筛选的诱饵载体pGBKT7-M122,其在SD/-Trp/X-α-Gal平板上的菌落为白色。2.转化有重组质粒pGBKT7-M122和空载体质粒pGBKT7的酵母菌株AH109,在2个SD/-Trp平板上长出的菌落大小一致数量相当。结论构建的诱饵质粒pG-BKT7-M122无自激活作用,对酵母菌株AH109亦无毒性。能够用于酵母双杂交系统以筛选与诱饵蛋白相互作用的蛋白分子。Objective To construct a bait vector containing mouse cytomegalovirus （MCMV） M122 gene in yeast two - hybrid system and detect the self- activation of this recombinated plasmid in order to screen the cDNA library of mouse fetal brain. Methods The DNA fragment of coding M122 was amplified by reverse transcriptase polymerase chain reaction, and then inserted into pMD18 -T simple vector. After verified with restriction endonuclease digestion of EcoR Ⅰ and Sal Ⅰ ,the right fragment of M122 gene determined by sequencing was subcloned into pGBKT7 - BD vector. Then right fragment of recombinant plasmid pGBKT7 - M122 detected by both the same endonuclease and sequence analysis was transformed into the yeast cell AH109. The transformed yeast cells were plated on nutrient deficiency medium SD/- Trp and SD/- Trp containing X - α - Gal. Positive plasmid pCL and empty pGBKT7 vector as controls were also transformed into the yeast strain AH109, and the transformed yeast cells were respectively plated on nutrient deficiency medium SD/- Trp and SD/- Leu containing X - α - Gal. The color,number and size of colonies on the plates were observed. Results The pGBKT7 - M122 plasmid was successfully constructed. The colonies containing pGBKT7 -M122 were white,while the colonies of the positive control were blue. The colonies containing pGBKT7 - M122 and empty pGBKT7 vector were similar on the number and size. Conclusion The recombinated plasmid pGBKT7 - M122 had no autoactivation and toxicity,and was proved to be suitable for the further research on the interactors with M122 in the yeast two- hybrid system.

关 键 词：M122基因 诱饵载体 酵母双杂交系统 自激活作用 小鼠巨细胞病毒 

分 类 号：R346[医药卫生—基础医学]