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.
出处
《中华医学遗传学杂志》
CAS
CSCD
北大核心
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）
