摘要目的 克隆人肺表面活性物质相关蛋白C(SP-C)基因,构建真核表达载体pcDNA3.1(+)/SP-C,并检测其在体外的表达,为SP-C的批量生产提供可靠的方法.方法 提取肺癌手术患者病灶周围正常肺组织总RNA,逆转录-聚合酶链反应(RT-PCR)技术获得SP-C cDNA序列.用NotI和XhoI内切酶双酶切SP-C cDNA序列和质粒pcDNA3.1(+),胶回收后体外连接.酶切和测序后,用脂质体包裹转染人乳腺癌细胞株MCF-7,采用RT-PCR和蛋白质免疫印迹法(Western blotting)检测SP-C的表达.结果 能够正确克隆人SP-C基因并插入至质粒pcDNA3.1(+)中;重组质粒体外转染MCF-7细胞后可以表达SP-C蛋白.结论 采用体外重组技术,成功构建了人SP-C真核表达载体 pcDNA3.1(+)/SP-C,并能在体外表达SP-C,为下一步构建人SP-C乳腺特异表达载体,利用乳腺生物反应器大量生产SP-C奠定了基础.

abstractsObjective To clone human pulmonary surfactant-associated protein C (SP-C) and construct an eukaryote expression vector pcDNA3.1(+)/SP-C, and then to examine the SP-C expression in vitro, which may provide a reliable way of massive production of SP-C. Methods The total RNA of normal lung tissue neighboring lung cancer from patients undergoing operation was extracted, and SP-C cDNA was then obtained by reverse transcription-polymerase chain reaction (RT-PCR). Both SP-C cDNA sequence and plasmid pcDNA3.1(+) were digested with NotI and XhoI, then connected by T4 DNA ligase after recycling agarose. After identification by restriction analysis and DNA sequencing, the recombinant was transfected into human breast cancer MCF-7 cells by using lipofectin reagent, and then the expression of SP-C was examined by RT-PCR and Western blotting. Results Human SP-C cDNA could be correctly cloned into the plasmid pcDNA3.1(+), and SP-C protein may be expressed in MCF-7 cells after transfection. Conclusion By in vitro recombination, the eukaryote expression vector pcDNA3.1(+)/SP-C was successfully constructed and expressed SP-C in vitro, which rendered preparation for the construction of specific expression vector of human SP-C, and it laid the foundation of massive production of SP-C through mammary gland bioreactor.