ICAP1α基因及其突变体T38A、I138A真核表达载体的构建和鉴定  

Construction and identification of ICAP1α and the eukaryotic expression vector of its mutants T38A and I138A

作　　者：程忠良[1] 秦瑾[1] 刘正湘[1] 林敬阳[1] 刘毓[1] 左后娟[1] 汪道文[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院心血管内科,湖北武汉430030

出　　处：《医学研究生学报》2009年第12期1243-1247,I0009,共6页Journal of Medical Postgraduates

基　　金：国家自然科学基金资助项目(批准号:30670856)

摘　　要：目的:整合素β1-ICAP1α复合体在细胞黏附等过程中介导双向跨膜信号转导,并与囊泡特异性标记物共定位。文中构建并鉴定ICAP1α基因及其突变体T38A、I138A真核表达载体。方法:以pCMV-SPORT6/ICAP1α质粒为模板,PCR扩增ICAP1α基因片段,双酶切后与AAV载体重组连接。同时采用重叠PCR定点突变法构建其突变体pAAV-T38A、pAAV-I138A表达载体。重组质粒经酶切鉴定和DNA序列测定,筛选出重组pAAV-ICAP1α、pAAV-T38A和pAAV-I138A真核表达载体。结果:阳性重组质粒酶切后测序比对鉴定,与预期序列完全相符。结论:成功构建pAAV-ICAP1α、pAAV-T38A和pAAV-I138A真核表达载体,可为ICAP1α基因及其突变体T38A、I138A的生物学功能研究提供基因材料。Objective： The complex Integrin β1-ICAP1α mediates the bidirectional transmembrane signal transduction and is colocalized with the specific markers of vesicles in cell adhesion and other processes.This study aimed to construct and identify the gene ICAP1α and the eukaryotic expression vector of its mutants T38A and I138A.Methods： Based on the plasmid DNA pCMV-SPORT6/ICAP1α,ICAP1α fragments were amplified by PCR,and the products were digested with NotⅠ and BamHⅠ and inserted into the AAV vector.The eukaryotic expression vectors of the mutants pAAV-T38A and pAAV-I138A were constructed by the overlap PCR site-directed mutagenesis method.The recombinant plasmids were identified by digestion and DNA sequencing,and the eukaryotic expression vectors of pAAV-ICAP1α,pAAV-T38A and pAAV-I138A were screened.Results： DNA sequencing showed the outcome of the restriction endonuclease digestion of the positive recombinant plasmids to be in full consistence with what had been expected.Conclusion： The eukaryotic expression vector of pAAV-ICAP1α,pAAV-T38A and pAAV-I138A was successfully constructed,which can be used for the studies of the biological function of the ICAP1α gene and its mutants T38A and I138A.

关 键 词：ICAP1α T38A I138A 整合素Β1 

分 类 号：Q782[生物学—分子生物学]