HSPb1真核表达载体构建及其在乳腺癌细胞中表达探究.docVIP

HSPb1真核表达载体构建及其在乳腺癌细胞中表达探究 　 作者：时鹏　王明明　孙靖中　冯进波 【摘要】 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。 【关键词】 乳腺肿瘤 热休克蛋白质类 因表达 基因重组 克隆 分子 Construction of HSPb1 expression vector and its function in breast cancer cells 【ABSTRACT】 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 HSPb1 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 ampicillin 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-HSPb1 was transfected into breast cancer cell line MDA-MB-231. The HSPb1 expression was detected by RT-PCR and Western blot. Results: Recombinant pcDNA3.1-HSPb1 contained the correct recombinant human HSPb1 sequences. Conclusions: The pcDNA3.1-HSPb1 plasmids might express the target gene in high level. 【KEY WORDS】 Breast neoplasms·Heat-shock protain·Gene expression·Gene rearrangement·Cloning molecular 　　HSPb1是小分子量热休克蛋白（small heat shock protein，sHSP）亚家族中的重要一员。作为一种应激蛋白，具有保护细胞免受应激因素损伤、维持细胞内稳态的作用。近年来的研究发现它还参与肌动蛋白的聚合和解聚过程［１，２］，影响细胞的增殖、分化及细胞凋亡的信号传导［3-6］。国外文献报道，HSPb1在乳腺癌和前列腺癌等肿瘤的发生、临床预后及化疗耐药等方