CAJ | 학술논문

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.
목적 극륭인Gill기인적전장cDNA,구건용우진핵세포표체적함유Gfil기인ORF구적중조만병독표체재체pLOX-Gfil,위연구Gfil기인적공능타하기출.방법 응용RT-PCR종인K562세포계중확증출Gfil cDNA편단·경회수순화여pGEM-T재체련접병전화감수태균DH5a,통과람백사선매절감정출양성균락,질립제취,BamH I한제성내절매매절획득목적 기인,삽입만병독재체pLOX중병전화감수태세균DH5a.질립제취후진행매절급PCR감정,대양성극륭진행DNA서렬분석.결과 RT-PCR확증획득함유약1.2 kb적DNA편단,매절급PCR감정증실pLOX-Gfil함유대소정학적정향Gfil cDNA,DNA서렬분석적결과 여발표우GenBank상적서렬(NM005263)완전일치.결론 성공극륭인Gfil기인병구건용우진핵세포표체적중조만병독재체pLOX-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.