MMP-7基因真核表达载体的构建及其在HeLa细胞中的表达与鉴定

目的：构建MMP-7基因真核重组质粒,检测并鉴定MMP-7在人宫颈癌HeLa细胞中的表达。方法：提取宫颈癌组织总RNA,通过基因克隆构建MMP-7基因真核表达重组质粒pcDNA3.1（＋）/MMP-7,酶切、PCR及基因测序鉴定,用阳离子脂质体介导采用基因转染技术转染人宫颈癌Hela细胞,RT-PCR检测外源基因的表达、间接免疫荧光法检测对表达产物进行鉴定。结果：成功构建了重组表达质粒pcDNA3.1（＋）/MMP-7并转染了人宫颈癌Hela细胞,通过RT-PCR可以检测到MMP-7 mRNA在Hela细胞中的表达,经间接免疫荧光反应可检测到明显的阳性反应,而转染空载体组表达阴性。结论：构建的重组质粒pcDNA3.1（＋）/MMP-7能在Hela细胞中表达,为该蛋白在人子宫癌后续的功能研究奠定了基础。

The Construction of MMP-7 Eukaryotic Expression Vector and it＇s Expression in Human Cervical Carcinoma Hela Cell

Objective： To construct the MMP-7 eukaryotic expression vector and detect the expression of MMP-7 gene in human cervical carcinoma Hela cell. Methods： Total RNA was extracted from human cervical carcinoma tissue,constructed the eukaryotic expression vector of pcDNA3.1 （＋）/MMP-7 through gene clone technology, the recombinant plasmid was identificated by restriction en- zyme, PCR and gene sequence analysis. Then, we carried out RT-PCR to detect the expression of exogenous gene after it was transfected into human cervical carcinoma Hela cell by lipofectamine and the expression products was qualificated by indirect immunofluorescence. Results： Successfully constructed the eukaryotic expression vector ofpcDNA3.1（＋）/MMP-7 and the recombinant plasmid had transfect- ed into the human cervical carcinoma Hela cell, through RT-PCR we have detected the expression of MMP-7 mRNA in Hela cell, the vector beating fusion gene could be expressed in eukaryotic cells and was detected by indirect immunofluotescence technique and the vector pcDNA3.1 （＋）could not be expressed in the Hela cell. Conclusions： The successful construction of the recombinant pasmid pcDNA3.1 （＋）/MMP-7 can be expressed in human cervical carcinoma Hela cell and will be benefit to the further study in human cervical carcinoma.