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目的 :从能感染HCV的人肝癌细胞HepG2中克隆出人LDLR基因 ,构建其真核表达质粒 ,并在小鼠肝癌细胞Hepa1 6中进行表达。方法 :从HepG2中提取总RNA ,采取逆转录PCR(RT PCR)方法得到人LDLR的cDNA。将获得的人LDLR基因克隆到真核表达载体pcDNA3中 ,进行酶切和序列鉴定。将重组质粒pcDNA3 hLDLR和空载体转入Hepa1 6 ,G4 18筛选 ,并用RT PCR和FACS检测人LDLR基因转录和蛋白的表达。结果 :人LDLR的cDNA已插入载体pcDNA3构建成真核表达质粒pcDNA3 hLDLR ,双酶切和测序表明 ,克隆的人LDLR与已登录GenBank的序列存在核苷酸多态性 ,但编码的氨基酸序列完全一致 ,该序列的GenBank登录号为gi|2 16 2 96 4 7|gb|AY114 15 5 .1,并且真核表达质粒的构建正确。用脂质体介导的方法转入Hepa1 6后 ,用RT PCR和FACS检测表明 ,细胞可表达人LDLR。结论 :成功地构建了真核表达质粒pcDNA3 hLDLR ,为进一步研究HCV和人LDLR的相互作用 ,以及建立可能的HCV细胞感染模型奠定了基础。
OBJECTIVE: To clone human LDLR gene from human hepatoma HepG2 infected with HCV and construct its eukaryotic expression plasmid, and to express it in HepG6 mouse hepatoma cells. Methods: Total RNA was extracted from HepG2 and cDNA of human LDLR was obtained by reverse transcription PCR (RT PCR). The obtained human LDLR gene was cloned into the eukaryotic expression vector pcDNA3, and digested and sequenced. The recombinant plasmid pcDNA3 hLDLR and empty vector were transfected into Hepa1 6 and G4 18 for screening, and the transcription and protein expression of human LDLR gene were detected by RT PCR and FACS. RESULTS: cDNA of human LDLR was inserted into pcDNA3 vector and constructed into eukaryotic expression plasmid pcDNA3 hLDLR. Double enzyme digestion and sequencing showed that there was nucleotide polymorphism in the cloned human LDLR and its registered GenBank, but the encoded amino acid sequence was completely The sequence of GenBank accession number was gi | 2 16 2 96 4 7 | gb | AY114 15 5 .1, and the eukaryotic expression plasmid was constructed correctly. After transfected into Hepa16 by liposome-mediated method, RT PCR and FACS showed that the cells could express human LDLR. CONCLUSION: The eukaryotic expression plasmid pcDNA3 hLDLR was successfully constructed, which laid the foundation for the further study of the interaction between HCV and human LDLR and the establishment of a possible HCV infection model.