中华医学遗传学杂志
中華醫學遺傳學雜誌
중화의학유전학잡지
CHINESE JOURNAL OF MEDICAL GENETICS
2013年
5期
559-564
,共6页
朱明%范怡梅%朱妍蓓%王亚平
硃明%範怡梅%硃妍蓓%王亞平
주명%범이매%주연배%왕아평
hMSH2基因%错义突变%酵母双杂交%结直肠癌
hMSH2基因%錯義突變%酵母雙雜交%結直腸癌
hMSH2기인%착의돌변%효모쌍잡교%결직장암
hMSH2 gene%Missense mutation%Yeast two-hybrid%Colorectal cancer
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.1261C＞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.1664 delA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.
目的 建立hMSH2和hMSH6蛋白相互作用體繫,以評估檢齣的hMSH2基因錯義突變的功能意義.方法 應用基因剋隆技術構建重組質粒pGADT7-hMSH2、pGBKT7-hMSH6及7箇不同hMSH6結構域的重組pGBKT7質粒.通過定點誘變技術構建10箇pGADT7-hMSH2突變質粒.將重組質粒共轉化酵母AH109菌株,觀察轉化子在組氨痠缺陷型培養基上的生長狀況.結果 hMSH6蛋白的MutSⅡ-Ⅴ、MutSⅢ-Ⅴ結構域分彆與hMSH2蛋白在酵母AH109中具有雙雜交作用.以pGBKT7-hMSH6-MutSⅡ-Ⅴ和pGADT7-hMSH2在酵母AH109的相互作用為基礎建立hMSH2/hMSH6相互作用平檯.與野生型hMSH2相比較,c.505A＞G、c.1168C＞T、c.1255C＞A、c.1261C＞A突變體在組氨痠缺陷的培養基上生長正常；c.1223A＞G、c.1886A ＞G、c.2108C＞A、c.2516A＞G突變體生長緩慢；c.518T＞G和c.1664 delA突變體不生長.結閤文獻中病例對照、氨基痠改變分析,判定c.518T＞G為病理性突變；c.1223A＞G、c.1886A＞G、c.2108C＞A、c.2516A＞G為疑似病理性突變；c.505A＞G、c.1168C＞T、c.1255C＞A、c.1261C＞A為正常變異.結論 初步建立瞭hMSH2、hMSH6蛋白相互作用功能分析平檯,併將其用于hMSH2基因錯義突變的功能評估.
목적 건립hMSH2화hMSH6단백상호작용체계,이평고검출적hMSH2기인착의돌변적공능의의.방법 응용기인극륭기술구건중조질립pGADT7-hMSH2、pGBKT7-hMSH6급7개불동hMSH6결구역적중조pGBKT7질립.통과정점유변기술구건10개pGADT7-hMSH2돌변질립.장중조질립공전화효모AH109균주,관찰전화자재조안산결함형배양기상적생장상황.결과 hMSH6단백적MutSⅡ-Ⅴ、MutSⅢ-Ⅴ결구역분별여hMSH2단백재효모AH109중구유쌍잡교작용.이pGBKT7-hMSH6-MutSⅡ-Ⅴ화pGADT7-hMSH2재효모AH109적상호작용위기출건립hMSH2/hMSH6상호작용평태.여야생형hMSH2상비교,c.505A＞G、c.1168C＞T、c.1255C＞A、c.1261C＞A돌변체재조안산결함적배양기상생장정상；c.1223A＞G、c.1886A ＞G、c.2108C＞A、c.2516A＞G돌변체생장완만；c.518T＞G화c.1664 delA돌변체불생장.결합문헌중병례대조、안기산개변분석,판정c.518T＞G위병이성돌변；c.1223A＞G、c.1886A＞G、c.2108C＞A、c.2516A＞G위의사병이성돌변；c.505A＞G、c.1168C＞T、c.1255C＞A、c.1261C＞A위정상변이.결론 초보건립료hMSH2、hMSH6단백상호작용공능분석평태,병장기용우hMSH2기인착의돌변적공능평고.
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-hMSH6 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 histidine-deficient culture.Results Both hMSH6-MutSⅡ-Ⅴ and MutS Ⅲ-Ⅴ could interact with hMSH2 in yeast AH109.Yeast two hybrid transformants pGADT7-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,c.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.
