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目的:构建含人类免疫缺陷病毒1型(HIV-1)编码病毒蛋白R(Vpr)基因的重组真核表达质粒并初步探索Vpr基因编码蛋白对卡波济肉瘤相关疱疹病毒(KSHV)溶解性周期复制的影响。方法:构建pVpr-Flag重组质粒并进行酶切鉴定和序列测定;将pVpr-Flag重组质粒临时转染BCBL-1和NIH3T3细胞,采用RT-PCR、IFA和Western blot分别从mRNA和蛋白水平检测Vpr基因的表达情况;提取临时转染pVpr-Flag重组质粒的BCBL-1细胞总RNA,进行RT-PCR检测KSHV次要衣壳蛋白编码基因ORF26(仅在病毒裂解期表达)mRNA转录水平,初步探索HIV-1Vpr基因编码蛋白对KSHV复制的影响。结果:克隆的Vpr基因序列与GenBank中已登记的Vpr序列100%同源。RT-PCR、IFA和Western blot结果显示Vpr基因mRNA及其编码蛋白在真核细胞中得到了转录和表达。RT-PCR结果进一步显示,Vpr基因编码蛋白能够降低KSHV ORF26 mRNA转录水平。结论:成功构建含Vpr基因序列的重组质粒并在真核细胞中获得正确表达;初步探索表明Vpr蛋白能够抑制KSHV溶解性周期复制。
OBJECTIVE: To construct a recombinant eukaryotic expression vector containing the gene encoding human viral immunodeficiency virus type 1 (HIV-1) V (Vpr) and to explore the effect of Vpr gene encoding protein on the solubility of Kaposi’s sarcoma-associated herpes virus (KSHV) The effect of copy. Methods: The recombinant plasmid pVpr-Flag was constructed and digested with restriction endonucleases and sequenced. The recombinant plasmid pVpr-Flag was transiently transfected into BCBL-1 and NIH3T3 cells. RT-PCR, IFA and Western blot were used to detect the expression of Vpr The total RNA of BCBL-1 cells transfected with recombinant plasmid pVpr-Flag was extracted and the transcription level of KSHV secondary capsid protein encoding gene ORF26 (only during virus lysis) was detected by RT-PCR Effect of HIV-1 Vpr gene-encoded protein on KSHV replication. RESULTS: The cloned Vpr gene sequence was 100% homologous to the registered Vpr sequence in GenBank. RT-PCR, IFA and Western blot showed that the mRNA and protein of Vpr gene were transcribed and expressed in eukaryotic cells. The results of RT-PCR further showed that the protein encoded by Vpr gene can reduce the transcriptional level of KSHV ORF26 mRNA. CONCLUSION: The recombinant plasmid containing Vpr gene sequence was successfully constructed and correctly expressed in eukaryotic cells. Preliminary studies showed that Vpr protein can inhibit KSHV lytic cycle replication.