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目的:探讨克隆人自噬基因h APG12 ,构建重组真核表达载体p EGFP- C2 -h APG12 ,为进一步研究h APG12 的功能奠定基础。方法:从正常人外周血单个核细胞中提取总RNA,采用两步法RT- PCR,首先把RNA逆转录为c DNA,而后通过一对h APG12 的特异性引物扩增出目的基因,PCR产物与测序载体p UC18连接后转化到大肠杆菌DH5 α,而后对单菌落进行菌落PCR、酶切和测序鉴定。将测序正确的h APG12 亚克隆入真核表达载体p EGFP-C2 ,该重组载体转化到大肠杆菌DH5 α后进行菌落PCR和酶切鉴定。结果:测序结果表明从正常人外周血单个核细胞中所获得的h APG12 c DNAs含有2 2 5个碱基,与Genbank( BC0 1 1 0 33)序列完全一致。构建的重组真核表达载体经鉴定证实h APG12 基因已完全正确亚克隆到p EGFP- C2 。结论:成功克隆了人自噬基因h APG12 并构建了具有报告基因-增强绿色荧光蛋白( EGFP)基因的重组真核表达载体p EGFP- C2 - h APG12 ,为以后研究h APG12 的功能奠定基础
OBJECTIVE: To investigate the cloning of human autophagy gene h APG12 and to construct recombinant eukaryotic expression vector p EGFP-C2 -h APG12, which may lay the foundation for further study on the function of h APG12. Methods: Total RNA was extracted from normal human peripheral blood mononuclear cells. The two-step RT-PCR method was used to reverse transcribe RNA into c DNA. The target gene was amplified by a pair of specific primers of h APG12. PCR products After being ligated with the sequencing vector pUC18, the recombinant plasmid was transformed into E. coli DH5α, and single colony PCR, digestion and sequencing were performed. The cloned hAPG12 was subcloned into the eukaryotic expression vector pEGFP-C2. The recombinant vector was transformed into E. coli DH5α and then identified by colony PCR and restriction enzyme digestion. Results: The sequencing results showed that h APG12 c DNAs obtained from normal human peripheral blood mononuclear cells contained 225 bases, which was completely consistent with the sequence of Genbank (BC0 1 1 0 33). The constructed recombinant eukaryotic expression vector was identified and confirmed h APG12 gene has been completely correctly subcloned into p EGFP-C2. CONCLUSION: The human apyrase gene h APG12 was successfully cloned and a recombinant eukaryotic expression vector p EGFP-C2 - h APG12 with reporter gene - enhanced green fluorescent protein (EGFP) gene was constructed, which laid the foundation for further study on the function of h APG12