人pcDNA3.1(+)-Cav-1真核表达载体的构建及鉴定

目的 构建pcDNA3.1 (+)-Cav-1真核表达载体,建立稳定转染pcCav-1的6-10B细胞系(6-10B-Cav-1).方法 提取5-8F细胞总RNA,利用RT-PCR获取caveolin-1(Cav-1)开放读码框(Open reading frame,ORF)序列构建Cav- 1/TA亚克隆载体,再将双酶切后的目的片段连入pcDNA3.1(+)真核表达载体并转染6-10B细胞.结果 RT-PCR扩增Cav-1基因ORF序列在783 bp附近见目的条带；质粒酶切鉴定及菌落PCR鉴定分别在783 bp及346 bp附近见目的条带；转染6-10B细胞后,转染组与对照组相比,Cav-1在mRNA水平表达差异具有统计学意义(P =0.027),在蛋白质水平前者比后者高出2倍以上.结论 人Cav-1重组真核表达载体pcDNA3.1( +)-Cav-1构建成功并获得稳定表达Cav -1的6-10B细胞,为进一步检测C av -1在鼻咽癌转移过程的作用奠定基础.

Construction and identification of Human pcDNA3.1(+)-Cav-1 eukaryotic expression vector

【Objective】 To construct the eukaryotic expression vector of pcDNA3.1(+)-Cav-1 and establish the 6-10B cell line which stably expressing Cav-1.【Methods】 Firstly,the open reading frame(ORF) sequence of Cav-1 was amplified by RT-PCR from 5-8F cells.Subsequently,the Cav-1/TA subcloning vector was constructed and then the double-digested fragment was connected to the pcDNA3.1(+) eukaryotic expression vector and which was transferred into 6-10B cells at last.【Results】 The ORF sequences of Cav-1 amplificated by RT-PCR was seem nearby 783 bp.The target bands were also detected near the 783 bp and 346 bp respectively through plasmid restriction enzyme digestion and PCR identification of colonies.The difference of expression of Cav-1 between the tranfected group and control group was statistically significant(P =0.027) at the mRNA level.And the expression of Cav-1 in the tranfected group was more than 2 times of the control group at the protein level.【Conclusion】 The human Cav-1 eukaryotic expression vector of pcDNA3.1(+)-Cav-1 is successfully constructed and the stably transfected 6-10B cell lines with pcDNA3.1(+)-Cav-1 has been established,which may lay the fundation for further studying the role of Cav-1 in nasopharyngeal carcinoma metastasis.