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建立9种临床常见的对非核苷类逆转录酶抑制剂(non-nucleoside reverse-transcriptase inhibitors,NNRTIs)耐药型HIV-1重组病毒药理评价模型。应用重叠PCR定点突变技术将耐药突变位点引入HIV-1核心基因(pNL4-3.Luc.R-E-),以水泡性口膜炎病毒的外壳糖蛋白(vesicular stomatitis virus glycoprotein,VSV-G)包装含耐药突变位点的HIV-1核心,形成耐药型HIV-1重组假病毒颗粒,简称VSVG/HIV-mut(包括VSVG/HIV-wt、VSVG/HIV-K103N、VSVG/HIV-Y181C、VSVG/HIV-L100I,K103N、VSVG/HIV-Y188L、VSVG/HIV-K103N,Y181C、VSVG/HIV-K103N,P225H、VSVG/HIV-K103N,Y188L、VSVG/HIV-K103N,G109A和VSVG/HIV-K103N,V108I)。经验证9种NNRTIs耐药型HIV-1重组病毒颗粒均具有高感染能力,对核苷类阳性药物不耐药,而对非核苷类阳性药物呈现不同程度的耐药性(17～10000倍),且耐药倍数与报道的数据基本一致。所建立的耐药型模型是针对NNRTIs耐药的HIV-1复制环节的细胞水平药效学评价体系,野生型和9种耐药型HIV-1模型的联合应用,可为新型非核苷类逆转录酶抑制剂的研发提供更全面药效学评价的安全平台。
To establish a pharmacological evaluation model of nine common clinical non-nucleoside reverse-transcriptase inhibitors (NNRTIs) resistant HIV-1 recombinant viruses. Overlapped PCR site-directed mutagenesis was used to introduce the drug-resistant mutation site into HIV-1 core gene (pNL4-3.Luc.RE-) and vesicular stomatitis virus glycoprotein (VSV-G) HIV-1 core containing drug-resistant mutation sites was packaged to form drug-resistant HIV-1 recombinant pseudovirions, referred to as VSVG / HIV-mut (including VSVG / HIV-wt, VSVG / HIV-K103N, VSVG / HIV-Y181C HIV-K103N, Y188L, VSVG / HIV-K103N, G109A and VSVG / HIV -K103N, V108I). Nine NNRTIs drug-resistant HIV-1 recombinant virus particles were proved to be highly infectious, resistant to nucleoside-positive drugs, and non-nucleoside-positive drugs to varying degrees (17 to 10,000 times) , And the resistance multiple was consistent with the reported data. The established drug resistance model is a cell-level pharmacodynamic evaluation system for HIV-1 replication resistant to NNRTIs. The combination of wild-type and 9 drug-resistant HIV-1 models can be used as a new non-nucleoside reverse Development of beta-lactase inhibitors provides a secure platform for more comprehensive pharmacodynamic assessment.