HIV1 Tat蛋白对HCV RNA复制蛋白表达的影响.docxVIP

中南大学硕士学位论文 中南大学硕士学位论文 中文摘要 结论：成功构建了HIV．1 Tat基因的真核表达质粒载体，并在 huh．7中表达；HIV．1 Tat蛋白能促进HCV RNA的复制和蛋白表达。 实验结果为进一步探索HIV促进HCV致病性的分子机制奠定了基 础，同时也为HIV阳性的HCV感染者的疾病预防和治疗提供了新的 实验依据。 关键词：人类免疫缺陷病毒I型；Tat蛋白；丙型肝炎病毒；基 因表达；共感染 H 中南大学硕士学位论文 中南大学硕士学位论文 英文摘要 ABSTRACT Objective：Both human immunodeficiency virus(HIV)and hepatitis C virus(HCV)infections are distributed worldwide．Because of the same transmission routes，there is a high rate of HIV／HCV CO-infection，and they exert the mutual impact on their clinical outcomes， but the mechanism remains unclear．HIV Tat protein is an important regulatory protein，which can not only trans—activate the transcription of HIV，but also stimulates the replication and transcription of other virus．This study was designed to construct a eukaryotic expression plasmid of HIV-1 Tat protein and then to explore the influence of HIV一1 Tat protein on HCV RNA replication and protein expression． Methods：DNA fragment encoding HIV-1 Tat protein was amplified by PCR from the plasmid pNL4—3．Then the PCR product was digested and inserted into pcDNA3．1(+)，an eukaryotic expression plasmid，to generate a recombinant plasmid，designated as pcDNA3．1一tat．The resulting plasmid was verified by restriction enzyme mapping and direct DNA sequencing，and then was transfected into huh-7 cells by lipo-fectamine．RT-PCR and Western blot were used to identify whether the object protein was correctly expressed in huh-7 cells．The full-length HCV replicon pcDNA6／TR—Tight／JFH 1／AR and pcDNA3．1—tat were CO—transfected into eukaryotic huh-7 cells by lipo-fectamine．The replication and expression of HCV RNA were detected by RT—PCR and Western blot respectively． Results：The results showed that the 240 bp DNA fragment of tat was correctly amplified and the recombinant plasmid pcDNA3．1-tat Was successfully constructed，which could be correctly expressed in the huh一7 1II 中南大学硕士学位论文