人ALK-1的克隆和真核表达载体的构建

Cloning of human full-length ALK-1 in HEK293 cells and construction of eukaryotic expression vector

目的:克隆人ALK-1基因并构建其真核表达载体.方法:设计ALK-1特异性引物,提取人胚胎肺组织总RNA,用RT-PCR方法获取人ALK-1 cDNA.将ALK-1 cDNA克隆至pcDNA3.1/myc-His(-),双酶切鉴定并测序后,转染HEK293细胞.用Western Blot检测目的蛋白的表达.结果:成功获得人ALK-1全长cDNA,双酶切鉴定证实成功构建pcDNA3.1(-)/ALK-1.测序结果表明ALK-1全长cDNA与GeneBank中ALK-1序列完全一致.转染HEK293细胞后,可检测出Mr约为62×103的目的蛋白.结论:获得了ALK-1全长cDNA并成功构建了ALK-1真核表达载体,证实pcDNA3.1(-)/ALK-1转染HEK293细胞后可表达ALK-1蛋白,为深入研究ALK-1介导的TGFβ信号转导通路在创面愈合以及瘢痕疙瘩发生,发展中的作用奠定了基础.

AIM： To clone and express human full-length ALK-1 cDNA in HEK293 cells. METHODS. Human ALK-1 specific primers were designed to obtain full-length cDNA through RT-PCR from lung tissues of human embryos and it was then cloned into vector pcDNA3. 1/myc-His （-） A. The recombinant plasmid pcDNA3.1 （ - ）/ALK-1 was transfected into HEK293 cells after it was verified by restriction enzyme digestion and DNA sequencing. The expression of ALK-1 gene was detected by Western Blot using mouse anti-6XHis antibody. RESULTS： A 1.7 Kb full-length ALK-1 cDNA was obtained by RT-PCR and inserted into pcDNA 3.1/myc-His（ - ）. DNA sequencing results confirmed that the sequence of ALK-1 was consistent with that reported. Western Blot showed that a Mr 62 × 10^3 ALK- protein could be expressed after transfecting pcDNA3-1（ - ）/ALK-1 into HEK293 cells. CONCLUSION： Human full-length ALK-1 cDNA is successfully cloned and can be expressed in transfected HEK293 cells, which provides a solid basis for exploring the effects of ALK-1 mediated TGFβ signal pathway in wound healing and the development of keloid.