摘要目的 探讨Ⅰ型人类免疫缺陷病毒(HIV-1)包膜糖蛋白120 V4区氨基酸位点发生突变对CCR5及CXCR4嗜性毒株感染靶细胞能力的影响.方法 根据ADA株为CCR5嗜性毒株,只具有感染CCR5细胞的能力;HXB2株为CXCR4嗜性毒株,只具有感染CXCR4细胞的能力,通过重叠延伸剪接的方法,构建CCR5嗜性及CXCR4嗜性毒株V4区丙氨酸替换突变体.将突变体表达载体与带有荧光报告基因的HIV骨架基因表达载体共同转染真核细胞,制备假病毒颗粒.采用酶联免疫吸附(ELISA)法检测假病毒中HIV-1 P24抗原,对假病毒进行定量.将假病毒接20、40 ng感染U87.CD4.CCR5和U87.CD4.CXCR4细胞,以野生株ADA和HXB2毒株为对照,通过荧光素酶(RLU)测定,检测HIV-1 V4区氨基酸386-417位点各突变体假病毒对细胞的感染能力.结果 成功构建HIV-1 ADA和HXB2株V4区丙氨酸替换突变体,各获得10株突变体.在ADA株和HXB2株,389-391及414-417位点均发生丙氨酸替换突变体,无论是用20ng,还是40 ng假病毒感染,对CCR5及CXCR4细胞的感染能力均完全丧失[(0±0)％]；在ADA株,400-403和408-410位点发生丙氨酸替换突变体,当假病毒在20 ng时,感染细胞的能力达到了(124±35)％和(182±29)％；在40ng时,感染能力达到了(127±8)％和(134±16)％；在HXB2株,395-397位点发生丙氨酸替换突变体,当假病毒在20 ng时,感染能力达到了(144±42)％；在40 ng时,感染能力达到了(121±18)％；两株在其他位点发生丙氨酸替换突变体,但仅保留部分感染细胞能力( 15％ ～ 84％).结论 HIV-1包膜糖蛋白V4区389-391及414-417位点氯基酸发生丙氨酸突变,使病毒完全丧失感染靶细胞能力.

abstractsObjective To clarify the influence of human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein 120 V4 region with mutations at amino acid locuses on its abilities to enter target cells.Methods Based on the facts that ADA strains was a CCR5-tropic strain,only had the ability to infect CCR5 cells; that HXB2 strains was a CXCR4-tropic strain,only had the ability to infect CXCR4 cells,serial glycoprotein 120 mutants with alanine substitution in V4 region of ADA and HXB2 strains,were constructed by overlaping PCR.Eukaryotic expression vectors of mutants and expression vectors of HIV framework gene with luciferase reporter gene were cotransfected into eukaryotic cells to produce pseudoviruse.Concentration of HIV-1 gag P24 in pseudoviruses was detected by enzyme-linked immunosorbent assay(ELISA).U87.CD4.CCR5 and U87.CD4.CXCR4 cells were infected with 20 and 40 ng pseudoviruses,with wild ADA and HXB2 strains as control groups,respectively.The ability to infect cells of pseudovirus of each mutant with HIV-1 V4- region mutated at amine acid locuses 386-417 was measured by detecting the luciferase activity (relative light unit,RLU).Results Ten mutants with alanine substitution in V4 region of HIV-1 ADA and HXB2 strains were successfully constructed,respectively.Mutants of pseudoviruse with 20 ng and 40 ng at locuses 389-391 and 414-417 with alanine substitution of V4 region in both ADA and HXB2 strains lost completely the abilities to enter CCR5 and CXCR4 expressing cells[ (0 ± 0)％].It was found that introduction of alanine to ADAs 400-403 and ADAs 408-410 increased the ability to infect cells to (124 ± 35)％,(182 ± 29)％ and (127 ± 8)％,( 134 ± 16)％ with pseudoviruse of 20 ng and 40 ng,respectively.Likewise,the ability to infect CXCR4 expressing cells also increased to (144 ± 42 )％ and (121 ± 18 )％ with pseudoviruse of 20 ng and 40 ng,respectively by introduction of alanine to HXB2s 395-397.However,other mutants in V4 region of ADA and HXB2 only maintained partial entry abilities( 15％- 84％).Conclusions Mutants of V4 region of HIV-1 envelope glycoprotein 120 with alanine substitution at locuses 389-391 and 414-417 in both ADA and HXB2 strains have been constructed successfully.They completely lost the ability to enter target cells.