M122蛋白与宿主因子Myst4的相互作用位点  

Interaction Sites between M122 Protein and Host Factor Myst4

作　　者：王慧[1] 张菊[1] 舒赛男[1] 刘兴楼[1] 李革[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,武汉430030

出　　处：《实用儿科临床杂志》2011年第22期1702-1705,共4页Journal of Applied Clinical Pediatrics

基　　金：国家自然科学基金(30671859)

摘　　要：目的明确M122蛋白与宿主因子Myst4相互作用的位点。方法以诱饵载体pGBKT7-M122为模板,采用PCR方法扩增不同长度大小的M122基因片段,并将其分别插入至pMD-T simple载体,构建含有不同长度M122基因片段的重组质粒。将构建成功的各个重组质粒分别用限制性内切酶EcoRI和SalI进行双酶切鉴定,并送测序。将测序正确的重组质粒采用同样的内切酶进行双酶切,凝胶回收试剂盒回收不同长度大小的M122基因片段,并将其分别亚克隆至pGBKT7-BD载体构建含有不同长度M122基因片段的诱饵质粒。将构建成功的各个诱饵质粒分别用限制性内切酶EcoRI和SalI进行双酶切鉴定,并送测序。将测序正确的各个诱饵质粒分别与pGADT7-Myst4质粒共同转化至酵母菌株AH109感受态细胞,转化后的酵母细胞分别涂板于营养缺陷培养基SD/-Trp/-Leu和SD/-Trp/-Leu/-His/-Ade/X-α-Gal平板。M122与宿主因子Myst4相互作用的位点通过营养缺陷筛选确定。结果成功扩增了编码不同M122蛋白突变体的基因片段,并将其正确插入诱饵载体,构建了含有不同长度大小M122基因片段的诱饵质粒;通过酵母双杂交筛选明确了M122蛋白与宿主因子Myst4相互作用的结合位点位于M122蛋白的1～148氨基酸。结论 M122蛋白的1～148氨基酸是其与宿主因子Myst4相互作用所必需的,为研究M122蛋白在MCMV致神经系统损伤的分子机制中的作用提供了实验基础。Objective To find the domains of M122 involved in the interaction with Myst4. Methods The DNA fragments encoding various truncated M122 were amplified by polymerase chain reaction using pGBKT7 - M122 as a template, and then inserted into pMD - T smipie vector, respectively. After verified with restriction endonuclease digestion of EcoRI and SalI, the right fragments of various truncated M122 gene determined by sequencing were subcloned into pGBKT7 - BD vector, respectively. All the recombinant plasmids were verified by restriction enzyme digestion and DNA sequencing. Then recombinant plasmids were transformed respectively into yeast strain AH109 together with plasmid pGADT7 - Myst4. The transformed yeast cells were plated on nutrient deficiency medium SD/- Trp/- Leu and SD/- Trp/- Leo./- His/- Ade/X - α - Gal. The M122 domains involved in the interaction were identified by auxotrophic selection. Results DNA fragments encoding deletion mutants of the M122 protein were amplified and cloned into bait vector. The reconstructed bait plasmids containing various truncated M122 were successfully constructed. The binding site of M122 and Myst4 was mapped to the region （ residues l - 148 ） of M122. Conclusions The region （ residues 1 - 148 ） of M122 is responsible for the interactions of M122 with Myst4. It provides an experimental basis for further studying the actions of M122 in molecular neuropathogenesis of mouse cytomegalovirus.

关 键 词：巨细胞病毒 M122蛋白 Myst4 酵母双杂交分析 

分 类 号：R346[医药卫生—基础医学]