人ALK-1的克隆和真核表达载体的构建

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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. OBJECTIVE: To clone human ALK-1 gene and construct its eukaryotic expression vector.Methods: ALK-1 specific primers were designed and total RNA of human embryonic lung was extracted, and the cDNA of human ALK-1 was obtained by RT-PCR. The cDNA was cloned into pcDNA3.1 / myc-His (-) and identified by restriction enzyme digestion and sequencing, then transfected into HEK293 cells.The expression of target protein was detected by Western Blot.Results: The full length cDNA of human ALK- The results of sequencing confirmed that pcDNA3.1 (-) / ALK-1 was constructed successfully.The sequencing results showed that the full-length cDNA of ALK-1 was exactly the same as that of ALK-1 in GeneBank.After transfection into HEK293 cells, Mr = 62 × 103 Of the target protein.Conclusion: ALK-1 full-length cDNA was obtained and the ALK-1 eukaryotic expression vector was successfully constructed, which confirmed that ALK-1 protein was expressed after transfection of pcDNA3.1 (-) / ALK-1 into HEK293 cells In-depth study of ALK-1-mediated TGFβ signaling pathway in wound healing and keloid formation, the role of development and laid the foundation.
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