中华传染病杂志
中華傳染病雜誌
중화전염병잡지
CHINESE JOURNAL OF INFECTIOUS DISEASES
2014年
3期
129-133
,共5页
徐树莹%乔录新%徐萌%袁霖%李庆%丁渭%石英%陈德喜
徐樹瑩%喬錄新%徐萌%袁霖%李慶%丁渭%石英%陳德喜
서수형%교록신%서맹%원림%리경%정위%석영%진덕희
HIV-1%整合酶类%HIV整合酶抑制剂%点突变%质粒%表型%抗药性,病毒
HIV-1%整閤酶類%HIV整閤酶抑製劑%點突變%質粒%錶型%抗藥性,病毒
HIV-1%정합매류%HIV정합매억제제%점돌변%질립%표형%항약성,병독
HIV-1%Integrases%HIV integrase inhibitors%Point mutation%Plasmids%Phenotype%Drug resistance,viral
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BamH Ⅰ和XhoⅠ双酶切,通过T4 DNA连接酶将纯化的PCR产物与经过BglⅡ和Xho Ⅰ双酶切的pcDNA6.2-LTRgagpolS1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48 h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-1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1.72×103,Y143H耐药株(1.18×10 4)和Q148R耐药株(2.82×104)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-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.
目的 構建整閤酶區標準突變株真覈錶達載體,併進行初步功能驗證,為HIV-1錶型耐藥研究提供技術方法.方法 採用點突變的方法,以質粒pNL4-3.Luc.R-E為模闆,擴增整閤酶區片段(含突變位點Y143、Q148),經過BamH Ⅰ和XhoⅠ雙酶切,通過T4 DNA連接酶將純化的PCR產物與經過BglⅡ和Xho Ⅰ雙酶切的pcDNA6.2-LTRgagpolS1連接,命名為pcDNA6.2-LTRgagpol,經過GatewayTM技術的BP/LR反應,將LTRgagpol片段重組到pNL4-3-attR,構成整閤酶區標準突變質粒.在聚乙烯亞胺(PEI)轉染試劑介導下,與水泡性口膜炎病毒的外殼糖蛋白質粒共轉染HEK293細胞,得到HIV-1假病毒,應用整閤酶抑製劑拉替拉韋與野生株假病毒進行錶型耐藥比較,將製備的病毒效價按4∶1的比例感染HEK293細胞,同時加入拉替拉韋,終濃度為1μmol/L,感染48 h後測熒光素酶報告基因.樣本間抑製率比較採用單因素方差分析.結果 帶有整閤酶區主要突變位點的HIV-1標準耐藥株(pNL4-3-Q148R、pNL4-3-Y143H)構建成功.應用1μmol/L拉替拉韋進行錶型耐藥檢測時,野生型病毒對拉替拉韋最為敏感,報告基因數值平均為1.72×103,Y143H耐藥株(1.18×10 4)和Q148R耐藥株(2.82×104)對拉替拉韋錶現為耐藥,對三者相對熒光素酶單位取對數值做統計學分析,野生株與Y143H耐藥株相比差異有統計學意義(F=38.800,P=0.025),野生株與Q148R耐藥株相比差異有統計學意義(F=174.355,P=0.006).Y143H耐藥株與Q148R耐藥株相比,Q148R耐藥株對拉替拉韋敏感性更低,差異具有統計學意義(F=22.551,P=0.042).結論 成功構建瞭帶有整閤酶區主要突變位點的HIV-1標準耐藥株載體,併成功錶達,為研究臨床HIV-1標本錶型耐藥典定瞭基礎.
목적 구건정합매구표준돌변주진핵표체재체,병진행초보공능험증,위HIV-1표형내약연구제공기술방법.방법 채용점돌변적방법,이질립pNL4-3.Luc.R-E위모판,확증정합매구편단(함돌변위점Y143、Q148),경과BamH Ⅰ화XhoⅠ쌍매절,통과T4 DNA련접매장순화적PCR산물여경과BglⅡ화Xho Ⅰ쌍매절적pcDNA6.2-LTRgagpolS1련접,명명위pcDNA6.2-LTRgagpol,경과GatewayTM기술적BP/LR반응,장LTRgagpol편단중조도pNL4-3-attR,구성정합매구표준돌변질립.재취을희아알(PEI)전염시제개도하,여수포성구막염병독적외각당단백질립공전염HEK293세포,득도HIV-1가병독,응용정합매억제제랍체랍위여야생주가병독진행표형내약비교,장제비적병독효개안4∶1적비례감염HEK293세포,동시가입랍체랍위,종농도위1μmol/L,감염48 h후측형광소매보고기인.양본간억제솔비교채용단인소방차분석.결과 대유정합매구주요돌변위점적HIV-1표준내약주(pNL4-3-Q148R、pNL4-3-Y143H)구건성공.응용1μmol/L랍체랍위진행표형내약검측시,야생형병독대랍체랍위최위민감,보고기인수치평균위1.72×103,Y143H내약주(1.18×10 4)화Q148R내약주(2.82×104)대랍체랍위표현위내약,대삼자상대형광소매단위취대수치주통계학분석,야생주여Y143H내약주상비차이유통계학의의(F=38.800,P=0.025),야생주여Q148R내약주상비차이유통계학의의(F=174.355,P=0.006).Y143H내약주여Q148R내약주상비,Q148R내약주대랍체랍위민감성경저,차이구유통계학의의(F=22.551,P=0.042).결론 성공구건료대유정합매구주요돌변위점적HIV-1표준내약주재체,병성공표체,위연구림상HIV-1표본표형내약전정료기출.
Objective To construct eukaryotic expression vector of human immunodeficiency virus (HIV)-1 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 wildtype 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 (1 μmol/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× 104) and Q148R resistant strain (2.82 × 104) were resistant against raltegravir.The analysis of log values of relative luciferase units showed that the sensitivity decreased significantly compared with the wild-type strain (wild-type strain vs pNL4-3-Y143H,F=38.800,P=0.025; wild-type strain vs pNL4-3-Y143R,F=174.355,P=0.006).In addition,compared with pNL4-3-Y143H,the sensitivity of pNL4-3-Q148R mutant also decreased significantly (F=22.551,P=0.042).Conclusion HIV-1 standard resistant strains with the integrase enzyme mutation sites (Y143H,Q148R) have been successfully constructed and expressed,which could be the basis of HIV-1 phenotypic drug resistance research.
