摘要
目的构建M IG(CXCL 9)真核表达载体,为利用M IG研究肿瘤基因治疗奠定基础。方法从pBLA ST-M IG上切下含M IG全长的cDNA序列,定向插入pORF-m cs真核表达质粒,构建pORF-M IG重组质粒;然后经酶切分析和测序鉴定后,通过脂质体介导转染CO S-7细胞;再进一步用免疫印迹法鉴定重组质粒的表达活性,用趋化试验鉴定生物学活性。结果pORF-M IG重组质粒经酶切分析和测序证实含有M IG的全长cDNA序列,转染CO S-7细胞后高效表达M IG蛋白,对活化的淋巴细胞有趋化作用。结论成功构建了M IG(CXCL 9)真核表达质粒,为进一步利用pORF-M IG研究恶性肿瘤基因治疗奠定基础。
Objective To establish a transformant of monokine induced by interferon-γ （MIG） with the eukaryotic expression vector for further investigating the efficacy of its use in antitumor gene therapy. Methods The MIG full-length cDNA was amplified from pBLAST-MIG and was cloned into the eukaryote expression vector pORF-mcs, and the resulted recombinant plasmid was named pORF-MIG. The recombinant pORF-MIG was determined with restriction enzyme and sequencing, and then it was transfected into COS-7 cells by Lipfectamin. Expression of the transformant was detected by immunoblot assay, and the bioactivity was assessed by chemotaxis assay. Results The restriction analysis and the sequence determination confirmed that the recombinant pORF-MIG contained the MIG full-length cDNA. And the transformants of pORF-MIG expressed the MIG protein which could apparently attract the activated lymphocytes. Conclusion The recombinant pORF-MIG was constructed successfully, and this recombinant eukaryotic expression vector could be used in additional studies on the biological effect of MIG and its use in anti-tumor gene therapy.
出处
《四川大学学报（医学版）》
CAS
CSCD
北大核心
2006年第4期502-505,共4页
Journal of Sichuan University(Medical Sciences)
基金
国家973规划(资助号2001CB510001)资助
