人自噬基因hAPG_(12)的克隆及其重组真核表达载体的构建

Cloning of hAPG_(12) and construction of its eukaryotic expression vector

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

Objective:To investigate the function of the hAPG_ 12 in the process of autophagy , the human autophagic gene hAPG_ 12 was cloned,and its eukaryotic expression vector pEGFP-C_2-hAPG_ 12 was constructed. Methods:Poly(A) mRNA obtained from human primary peripheral blood mononuclear cells was reverse-transcribed to cDNA with oligo(dT)primer,PCR was performed with a pair of hAPG_ 12 specific primer.The PCR products were inserted into pUC_ 18 ,and the resulted plasmid was transformed into DH5α. The positive clone was selected and sequenced.Then the gene that was comfirmed by DNA sequencing was subcloned into pEGFP-C_2 to construct eukaryotic expression vector. Results:The PCR amplification yielded a single band of about 225 base pairs,sequence analysis of the PCR products revealed that it is consistent with that of the references published(GenBank BC011033). Furthermore, a recombinant plasmid pEGFP-C_2-hAPG_ 12 for eukaryotic expression was also obtained after subcloning. Conclusion:The hAPG_ 12 gene has been cloned from human primary peripheral blood mononuclear cells,and its eukaryotic expression vector with reporter gene-a enhance green fluorescent protein (EGFP) gene has been obtained.The results have provided basis for the further studies of the roles of hAPG_ 12 in the process of autophagy.