人Gfil基因克隆及重组Gfil慢病毒表达载体的构建

目的 克隆人Gill基因的全长cDNA,构建用于真核细胞表达的含有Gfil基因ORF区的重组慢病毒表达载体pLOX-Gfil,为研究Gfil基因的功能打下基础.方法 应用RT-PCR从人K562细胞系中扩增出Gfil cDNA片段·经回收纯化与pGEM-T载体连接并转化感受态菌DH5a,通过蓝白筛选酶切鉴定出阳性菌落,质粒提取,BamH I限制性内切酶酶切获得目的 基因,插入慢病毒载体pLOX中并转化感受态细菌DH5a.质粒提取后进行酶切及PCR鉴定,对阳性克隆进行DNA序列分析.结果 RT-PCR扩增获得含有约1.2 kb的DNA片段,酶切及PCR鉴定证实pLOX-Gfil含有大小正确的正向Gfil cDNA,DNA序列分析的结果 与发表于GenBank上的序列(NM005263)完全一致.结论 成功克隆人Gfil基因并构建用于真核细胞表达的重组慢病毒载体pLOX-Gfil.

Cloning of Human Gfil cDNA and Construction of Recombinant Lentiviral Expressing Vector Gfil

Objective To clone the full-length of human Gill cDNA and construct the recombinant lentiviral expressing vector pLOX-Gfil for eukaryotic expression,providing a basis for further study on the biological functions of Gfil.Methods Total RNA was isolated from K562 cells,and the full-length Gfil cDNA was amplified by RT-PCR and then ligated with pGEM-T vector after retrieve and purification.The ligation product was transformed into competent cells DH5a.The positive recombinant clones were selected and identified by a complementation,restriction endonuclease digestion.The cloning vector and the lentiviral vector pLOX first digested with BarnH I were ligated and transformed.The enzyme and PCR analyses were performed to confirm the recombinant vector,and then DNA sequence analysis.Results A fragment of 1.2 kb was obtained by RT-PCR.The enzyme and PCR analyses revealed that the correct Gfil cDNA was cloned.The sequence of cloned cDNA was identical to the sequence deposited in GenBank (NM005263).Conclusion Gfil was cloned correctly and the recombinant lentiviral vector pLOX-Gfil for eukaryotic expression was constructed successfully.