中文摘要：

目的构建整合酶区标准突变株真核表达载体，并进行初步功能验证，为HIV-1表型耐药研究提供技术方法。方法采用点突变的方法，以质粒pNL4-3．Luc．R-E为模板，扩增整合酶区片段（含突变位点Y143、Q148），经过BamHI和XhoI双酶切，通过T4DNA连接酶将纯化的PCR产物与经过BglⅡ和XhoI双酶切的pcDNA6．2-LTRgagpolSl连接，命名为pcDNA6．2-LTRgagpol，经过GatewayTM技术的BP／LR反应，将LTRgagpol片段重组到pNL4-3-attR，构成整合酶区标准突变质粒。在聚乙烯亚胺（PEI）转染试剂介导下，与水泡性口膜炎病毒的外壳糖蛋白质粒共转染HEK293细胞，得到HIV-1假病毒，应用整合酶抑制剂拉替拉韦与野生株假病毒进行表型耐药比较，将制备的病毒效价按4：1的比例感染HEK293细胞，同时加入拉替拉韦，终浓度为1μmol／L，感染48h后测荧光素酶报告基因。样本间抑制率比较采用单因素方差分析。结果带有整合酶区主要突变位点的HIV-l标准耐药株（pNL4-3-Q148R、pNL4-3-Y143H）构建成功。应用1μmol／L拉替拉韦进行表型耐药检测时，野生型病毒对拉替拉韦最为敏感，报告基因数值平均为1．72×10^3，Y143H耐药株（1．18×10。）和Q148R耐药株（2．82×10。）对拉替拉韦表现为耐药，对三者相对荧光素酶单位取对数值做统计学分析，野生株与Y143H耐药株相比差异有统计学意义（F=38．800，P=0．025），野生株与Q148R耐药株相比差异有统计学意义（F=174．355，P=0．006）。Y143H耐药株与Q148R耐药株相比，Q148R耐药株对拉替拉韦敏感性更低，差异具有统计学意义（F=22．551，P=0．042）。结论成功构建了带有整合酶区主要突变位点的HIV一1标准耐药株载体，并成功表达，为研究临床HIV-1标本表型耐药典定了基础。

英文摘要：

Objective To construct eukaryotic expression vector of human immunodefieiency virus （HIV）-I integrase standard mutant and conduct preliminary functional verification, and to provide study method for HIV-1 phenotypic drug resistance. Methods In this study, point mutation was used. The integrase sequences （containing Y143 and Q148 mutations） were amplified by polymerase chain reaction （PCR） with pNL4-3. Luc. R-E plasmid as template. Purified PCR product was digested by restriction enzymes Bgl Ⅱ and Xho I and linked with pcDNA6. 2 LTRgagpolS1 vector digested by the same restriction enzymes via T4 DNA ligase. The product was named as pcDNA 6.2-LTRgagpol. LTRgagpol fragment was cloned into pNL4-3 attR plasmid by GatewayTM BP/LR reaction to form the standard plasmid containing integrase mutations. The standard plasmid, together with VSV-G, was transfected into HEK293 cells using PEI-mediated transfection reagent to obtain HIV-1 pseudotyped virus. Then integrase inhibitor raltegravir was used to examine the phenotypic sensitivity of HIV-1 variants and wild-type virus strain relatively. Then phenotypic drug resistance was compared between integrase inhibitor raltegravir and wild-type pseudovirus strain. HEK 293 cell line was infected with the prepared virus strain of 4 ： 1 ratio and raltegravir was added simultaneously with the final concentration of 1 μmol/L. The luciferase reporter gene was detected after 48 h of infection. The comparison of inhibition rate between samples was done using one-way analysis of variance. Results HIV-1 standard drug resistant strains with the integrase enzyme mutation sites were constructed successfully, the phenotypic drug resistance assay using raltegravir （1μmol/L） showed that wild-type strain was sensitive to raltegravir the the mean reporter gene copy of 1.72 × 103 , while Y143H resistant strain （1.18×10^4 ） and Q148R resistant strain （2. 82 × 10^4 ） were resistant against raltegravir. The analysis of log values of relative Iuciferase units showed that