人Kai-1基因真核表达载体的构建及其体外表达  

Cloning and Expression Analysis of Kai-1 in Hela Cells

作　　者：张健[1] 吕飒美[1] 赵素平[1] 党珊[1] 何小勤[1] 陈倩[1] 易默[1] 史丽萍[1] 

机构地区：[1]陕西省人民医院消化内二科,西安710068

出　　处：《科学技术与工程》2014年第33期16-19,共4页Science Technology and Engineering

摘　　要：克隆人Kai-1基因,构建其真核表达载体,并在体外细胞系中进行表达验证。提取人正常肝细胞系HL7702的总RNA,通过RT-PCR其反转录为c DNA;以该c DNA为模板,通过PCR扩增出Kai-1基因的编码区,将该目的片段纯化后亚克隆入真核表达载体pc DNA3.1myc-his(-)中,利用菌落PCR及DNA测序分别对Kai-1基因编码区的大小及序列进行鉴定。将所构建的重组质粒通过脂质体瞬时转染Hela细胞,48 h后裂解细胞,利用Western blot检测有无目的蛋白的表达。测序证实所克隆的Kai-1编码区c DNA正确地插入pc DNA3.1myc-his(-)中,经Western blot检测证实其在Hela细胞中得到表达。成功克隆了人Kai-1 c DNA,构建了其真核表达载体,并在Hela细胞中得到有效表达,为进一步研究Kai-1的功能奠定了基础。To construct the recombinant expression vector for Kai-1 and detect its expression in Hela cells,total RNA was isolated from normal human liver cell line HL7702 and RT-PCR was conducted to acquire the c DNA. A1 200 bp fragment containing the coding region of Kai-1 was amplified by PCR and the resulting product was purified. Then this fragment was used as new template and the coding region of Kai-1（ 927 bp） was amplified,purified and inserted into the EcoRI and HindⅢ sites of the pc DNA3. 1myc-his（-） A expression vector. The sequence was confirmed by colony PCR and DNA sequencing. In the end,pc DNA3. 1 myc-his（-） A-Kai-1 was transiently transfected into Hela cells and the expression of this new construct was detected by western blot. The full length coding region of Kai-1 was obtained and confirmed,the expression of Kai-1 was detected successfully in Hela cells. The eukaryotic expression vector of Kai-1 has been successfully constructed,which will provide a useful tool for designing an in-depth investigation of the role of Kai-1 in tumorigenesis.

关 键 词：KAI-1 真核表达 克隆 CDNA 

分 类 号：R318.04[医药卫生—生物医学工程]