maspin/PCR2.1表达载体的构建及意义

The construction and significance of maspin/PCR2.1 expression vector

目的:构建maspin/PCR2.1表达载体,为深入研究maspin基因抑制肝癌生长、转移、浸润作用和机制奠定基础。方法:PCR扩增maspin基因全长,将表达载体PCR2.1用Hind111+XbaI进行双酶切后,T4DNA连接酶连接成重组质粒,转染到人肝癌高转移细胞株MHCC-97中进行表达,检测转染前后maspin表达的变化。结果:重组质粒在大肠杆菌株JM109内扩增;提纯、纯化后,用HindIII、XbaI酶切鉴定及测序鉴定证明maspin基因已完整、正确的插入到PCR2.1表达载体,并在人肝癌MHCC-97细胞中上调了maspin基因mRNA和蛋白水平表达。结论:成功构建了maspin/PCR2.1表达载体,能在真核细胞中。

Objective:To construct the expression vector maspin /Pcr2.1 in order to provide the evidence for the inhibitory effect of maspin gene on growth,metastasis and invasion of hepatocellular carcinoma(HCC) and to discuss its mechnism.Methods:The full length of maspin gene was cloned,and the expression vector PCR2.1 was digested by Hind111/XbaI,then the maspin gene and the PCR2.1 vector were ligated with T4 DNA ligase to be recombinant vector.The recombinant vector maspin/PCR2.1 was transfected into humman hcc MHCC-97 cell line,and the expressive changes of maspin gene were detected.Results:The recombinant plasmid was amplified in the E.coli.JM109.After the identification of redigesting by HindIII/XbaI and sequencing,the reconstructive plasmid was confirmed to contain the correct and full nucleotide sequence of maspin gene,and the mRNA and protein level of maspin gene were up-regulated in hcc MHCC-97 cell.Conclusion:The expression vector maspin/PCR2.1 is constructed successfully and can be expressed in eukaryotic cells.