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[目的]克隆乳腺癌相关分子人乳腺珠蛋白(humanmammaglobin,hMAM)基因并诱导其表达,为后期研制乳腺癌早期诊断试剂盒奠定基础,从而为早期发现乳腺癌提供科学的监测方法。[方法]从乳腺癌组织提取总RNA,通过逆转录聚合酶链反应(RT-PCR)扩增hMAM基因编码序列,将扩增产物克隆至PGEM-T载体中,DNA测序,用限制性内切酶切下目的基因,与相同酶切的表达载体pQE40连接、筛选、鉴定,构建pQE40-hMAM融合表达质粒,以硫代半乳糖苷(IPTG)诱导在大肠杆菌M15中表达,聚丙烯酰胺凝胶电泳(SDS-PAGE)分析表达结果。[结果]琼脂糖凝胶电泳显示聚合酶链反应(PCR)扩增产物的分子量大小与预期相符。对重组质粒分析表明,插入片段的序列与文献报道hMAM基因编码序列一致。在IPTG的诱导下,M15重组菌高效表达出一个相对分子质量约为34kD的产物。[结论]重组表达质粒pQE40-hMAM表达成功。
[Objective] The study aimed to clone and induce the expression of hMAM gene in breast cancer, and lay the foundation for the early development of early diagnosis kit for breast cancer, so as to provide a scientific method for the early detection of breast cancer. [Methods] Total RNA was extracted from breast cancer tissues. The coding sequence of hMAM gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR). The amplified product was cloned into PGEM-T vector. The DNA was sequenced and sequenced with restriction endonuclease The target gene was excised and ligated with the same restriction enzyme pQE40. The pQE40-hMAM fusion expression plasmid was constructed and identified. The recombinant plasmid pQE40-hMAM was expressed in E. coli M15 induced by thiogalactoside (IPTG). Polyacrylamide gel The expression was analyzed by electrophoresis (SDS-PAGE). [Result] The results of agarose gel electrophoresis showed that the molecular weight of PCR product was in line with the expectation. The analysis of the recombinant plasmids showed that the sequence of the inserted fragment was consistent with that of the hMAM gene reported in the literature. Under the induction of IPTG, recombinant M15 efficiently expressed a product with a relative molecular mass of about 34 kD. [Conclusion] The recombinant plasmid pQE40-hMAM was successfully expressed.