hMSH2和hMSH6蛋白相互作用体系的建立与hMSH2基因错义突变的功能评估  

Establishment of a hMSH2/hMSH6 protein interaction system and functional evaluation of hMSH2 gene missense mutations

作　　者：朱明[1,2] 范怡梅[1] 朱妍蓓[1] 王亚平[1] 

机构地区：[1]南京大学医学院江苏省医学分子技术重点实验室,210093 [2]江苏省肿瘤防治研究所分子生物学研究室

出　　处：《中华医学遗传学杂志》2013年第5期559-564,共6页Chinese Journal of Medical Genetics

基　　金：国家自然科学基金（30972535,81070273）

摘　　要：目的建立hMSH2和hMSH6蛋白相互作用体系，以评估检出的hMSH2基因错义突变的功能意义。方法应用基因克隆技术构建重组质粒pGADT7-hMSH2、pGBKT7-hMSH6及7个不同hMSH6结构域的重组pGBKT7质粒。通过定点诱变技术构建10个pGADT7-hMSH2突变质粒。将重组质粒共转化酵母AH109菌株，观察转化子在组氨酸缺陷型培养基上的生长状况。结果hMSH6蛋白的MutSⅡ-V、MutSⅢ—V结构域分别与hMSH2蛋白在酵母AH109中具有双杂交作用。以pGBKT7-hMSH6-MutSⅡ—V和pGADT7-hMSH2在酵母AH109的相互作用为基础建立hMSH2／hMSH6相互作用平台。与野生型hMSH2相比较，c．505A〉G、C．1168C〉T、C．1255C〉A、C．126IC〉A突变体在组氨酸缺陷的培养基上生长正常；C．1223A〉G、C．1886A〉G、C．2108C〉A、c．2516A〉G突变体生长缓慢；C．518T〉G和c．1664delA突变体不生长。结合文献中病例对照、氨基酸改变分析，判定C．518T〉G为病理性突变；C．1223A〉G、C．1886A〉G、C．2108C〉A、C．2516A〉G为疑似病理性突变；C．505A〉G、C．1168C〉T、C．1255C〉A、C．1261C〉A为正常变异。结论初步建立了hMSH2、hMsH6蛋白相互作用功能分析平台，并将其用于hMSH2基因错义突变的功能评估。Objective To construct a hMSH2/hMSH6 protein interaction system, and to use it for evaluating missense mutations detected in hMSH2 gene. Methods Recombinant plasmids pGADT7- hMSH2, pGBKT7-bMSH6 and 7 recombinant pGBKT7 plasmids with different hMSH6 domains were constructed through genetic engineering. Subsequently, site-directed mutagenesis was used to construct 10 mutant pGADT7-hMSH2 plasmids, which were transformed into yeast AH109. The growth of transformants was observed on a histidinedeficient culture. Results Both hMSH6 -MutS II-V and MutS Ⅲ-Ⅴ could interact with hMSH2 in yeast AH109. Yeast two-hybrid transformants pGADTT-hMSH2 / pGBKT7-hMSH6-MutS II-V were used to construct a hMSH2/hMSH6 protein interaction system. Compared with wild-type hMSH2, yeast two-hybrid transformants c. 505A：〉G, c. 1168C：〉T, e. 1255C〉A, c. 1261C〉A could grow normally, c. 1223A〉G, c. 1886A〉G, c. 2108C〉A and c. 2516A〉G grew slowly, c. 518T〉 G and c. 1664 delA could not grow in a histidine-deficient medium in yeast AH109. Conclusion A hMSH2/hMSH6 protein interaction system has been constructed with yeast two-hybrid system, which has been used for functional evaluation of hMSH2 gene missense mutations, c. 518T〉G is a pathological mutation, c. 1223A〉G, c. 1886A〉G, c. 2108C〉A, c. 2516A〉G may in part affect the hMSH2 function. And c. 505A〉G, c. 1168C〉T, c. 1255C〉A, c. 1261C〉A were innocuous variants.

关 键 词：HMSH2基因 错义突变 酵母双杂交 结直肠癌 

分 类 号：R735.3[医药卫生—肿瘤]