Cloning of human fullª²length ALKª²1 in HEK293 cells and construction of eukaryotic expression vector

    SHU Maoª²Guo, GUO Shuª²Zhong, WANG Xinª²Hong, GUO Xiaoª²Tong, LI Liª²Wen

    1PLA Center of Plastic Surgery, 3Center of Oncology, 4PLA Institute of Orthopedics, Xijing Hospital, Fourth Military Medical University, Xi¬ðan 710033, China, 2Shaanxi Space Flight Hospital, Xi¬ðan 710032, China

    ¡¾Abstract¡¿ AIM: To clone and express human fullª²length ALKª²1 cDNA in HEK293 cells. METHODS: Human ALKª²1 specific primers were designed to obtain fullª²length cDNA through RTª²PCR from lung tissues of human embryos and it was then cloned into vector pcDNA3.1/mycª²His(-) A. The recombinant plasmid pcDNA3.1(-)/ALKª²1 was transfected into HEK293 cells after it was verified by restriction enzyme digestion and DNA sequencing. The expression of ALKª²1 gene was detected by Western Blot using mouse antiª²6XHis antibody. RESULTS: A 1.7 Kb fullª²length ALKª²1 cDNA was obtained by RTª²PCR and inserted into pcDNA3.1/mycª²His(-). DNA sequencing results confirmed that the sequence of ALKª²1 was consistent with that reported. Western Blot showed that a Mr 62¡Á103 ALKª²1 protein could be expressed after transfecting pcDNA3.1(-)/ALKª²1 into HEK293 cells. CONCLUSION: Human fullª²length ALKª²1 cDNA is successfully cloned and can be expressed in transfected HEK293 cells, which provides a solid basis for exploring the effects of ALKª²1 mediated TGF¦Â signal pathway in wound healing and the development of keloid. ......