c-Src结构域真核表达载体的构建及鉴定  

Construction and Identification of the Eukaryotic Expression Vector of c-Src Domains

作　　者：张瑶楠[1] 石冲[1] 马永超[1] 王泽生[1] 张玉祥[1] 

机构地区：[1]首都医科大学基础医学院生物化学与分子生物学系,北京100069

出　　处：《河南大学学报（自然科学版）》2011年第5期500-504,共5页Journal of Henan University:Natural Science

基　　金：国家自然科学基金资助项目(30873033)

摘　　要：通过聚合酶链式反应(PCR),将c-Src的三个结构域SH3、SH2和KD分别定向克隆至C端带有HA蛋白标签序列的真核表达载体pcDNA3中.经HindIII和XbaI双酶切分析和测序鉴定正确后,应用脂质体法转染HeLa细胞48 h,蛋白免疫印迹法可以检测到细胞内SH3、SH2和KD的表达,证明重组表达质粒pcDNA3-SH2、pcDNA3-SH3和pcDNA3-KD构建成功,为进一步研究Src激酶在肿瘤发生发展中的作用奠定了基础.Polymerase chain reaction（PCR） was performed to obtain the DNA fragments encoding the three domains of c-Src named SH2,SH3 and KD respectively.The DNA fragments were inserted into the pcDNA3 vectors in-frame with the HA sequence.And the new constructs were confirmed by restriction enzyme digestion and DNA sequencing.HeLa cells were transfected with the pcDNA3-SH3,pcDNA3-SH2,pcDNA3-KD vectors and pcDNA3 vector respectively.The expressions of SH3,SH2 and KD were detected by Western blotting.The eukaryotic expression vectors pcDNA3-SH3,pcDNA3-SH2 and pcDNA3-KD were constructed successfully.This study laid a foundation for the research on the mechanism of Src protein tyrosine kinase in tumorigenesis and development of human cancers.

关 键 词：SRC SH3 SH2 KD 真核表达载体 

分 类 号：Q782[生物学—分子生物学]