HSPb1真核表达载体的构建及其在乳腺癌细胞中表达的研究

Construction of HSPb1 expression vector and its function in breast cancer cells

目的:构建重组人HSPb1真核表达载体质粒,为研究HSPb1与乳腺癌发生和化疗耐药的关系做准备。方法:从人乳腺癌细胞MCF-7中提取总RNA,RT-PCR法反转录合成cDNA。设计引物调取目的基因片段,与T载体连接后转化大肠埃希菌DH-5α,筛选菌落并抽提质粒。测序正确后双酶切克隆进入真核表达载体pcDNA3.1中,得到重组质粒pcDNA3.1-HSPb1。用pcDNA3.1-HSPb1转染人乳腺癌细胞MDA-MA-231,RT-PCR法和Western blot法鉴定重组质粒在其中的表达。结果:经酶切证实重组质粒pcDNA3.1-HSPb1含有目的基因HSPb1。RT-PCR和Western blot检测表明,mRNA和蛋白水平,转染细胞HSPb1的表达均明显增强。结论:pcDNA3.1-HSPb1真核表达载体构建成功,它在乳腺癌细胞中可高表达目的基因,为进一步研究HSPb1在乳腺癌发生及化疗耐药中的作用奠定了基础。

Objective： To construct the eukaryotic expression plasmid of recombinant human heat shock protein b1 （HSPb1） and study its expression in breast cancer cells. Methods： Total RNA was isolated from human breast cancer cells MCF-7, and the full-length coding sequence of HSPbl was constructed by standard reverse transcriptional PCR method. The fragment was inserted into the shuttle vector T. Transformation of E.coli DH-5α with recombinant plasmids and identification of bacterial colonies containing recombinant plasmids by LB-agar plate containing 100μg/ml of ampi cillin were conducted, and recombinant plasmids were extracted and purified. All sequences amplified by PCR were confirmed by complete sequencing. Correct sequences were cloned into the pcDNA3.1 vector, pcDNA3.1-HSPbl was transfected into breast cancer cell line MDA-MB-231. The HSPbl expression was detected by RT-PCR and Western blot. Results： Recombinant pcDNA3.1-HSPbl contained the correct recombinant human HSPbl sequences. Conclusionus： The pcDNA3.1-HSPbl plasmids might express the target gene in high level.