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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 the yeast two-hybrid bait vector pGBKT7-M122 of mouse cytomegalovirus (MCMV) immediate early gene M122 and test its self-activation and toxicity to yeast AH109 strain for screening mouse embryonic brain cDNA library. Methods The M122 gene fragment of MCMV was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and inserted into pMD18-Tsimple vector to construct recombinant plasmid pMD18-T-M122. The recombinant plasmids were identified by restriction endonucleases EcoRI and SalI and sequenced. The correct sequencing pMD18-T-M122 recombinant plasmid and vector pGBKT7-BD were digested with restriction endonucleases EcoRI and SalI respectively, and the M122 gene fragment and the pGBKT7-BD vector fragment were recovered by gel filtration. The recovered M122 gene fragment and the pGBKT7-BD vector fragment were ligated overnight with T4 DNA ligase at 16 ° C to construct bait plasmid pGBKT7-M122. The pGBKTT-M122 digestion identification, sequencing. The recombinant plasmid pGBKT7-M122 was transformed into AH109 yeast competent cells. The transformed bacterial broth was plated on SD / -Trp and SD / -Trp / X-α-Gal plates. The empty plasmid pGBKT7-BD and the positive plasmid pCL were also transferred into AH109 yeast competent cells. The transformed bacterial broth was plated on SD / -Trp and SD / -Leu / X-α-Gal plates, respectively. Observe the color, number and size of the colonies on the plate. Results 1. The bait vector pGBKT7-M122 for yeast two-hybrid screening was successfully constructed and its colonies on the SD / -Trp / X-α-Gal plates were white. 2. The yeast strain AH109 transformed with the recombinant plasmids pGBKT7-M122 and empty vector plasmid pGBKT7 had the same number of colonies growing on the two SD / -Trp plates. Conclusion The constructed bait plasmid pG-BKT7-M122 has no self-activation and is non-toxic to yeast strain AH109. Can be used in yeast two-hybrid systems to screen for protein molecules that interact with bait proteins.