摘要:
目的 分析昆明市静脉吸毒（intravenous drug user，IDU）感染者艾滋病病毒1型（human immunodeficiency virus type 1，HIV-1） env基因C2-V4区序列特征及代表性广谱单克隆中和抗体（broadly monoclonal neutralizing antibodies，bmNAbs）的表位变异。方法 在昆明市收集165份IDUHIV-1感染者血浆样品，提取RNA，通过逆转录得到cDNA，巢式聚合酶链反应（nested-polymerase chain reaction，nested-PCR）扩增HIV-1膜蛋白基因C2-V4区，应用BioEdit和MEGA等生物信息软件分析所获得序列的特征，并分析2G12、PGT135、PGT128、b12和VRC01等bmNAbs的表位变异。结果 165条序列主要为CRF08_BC （74.55%）和CRF07_BC （23.03%）重组毒株，各可变环变异程度由小到大为V3区 < C3区 < V4区，并且CRF08_BC样本的C2-C4、V3区、C3区和V4区基因离散率分别大于CRF_07BC的相应区域；V3环顶端四肽存在5种类型：GPGQ （95.76%）、GPGR （1.82%）、GPRQ （1.21%）、GPGL （0.61%）和RPGQ （0.61%）；辅助受体主要为CCR5（97.58%）。90%以上的CRF08_BC和CRF07_BC重组毒株存在与2G12识别相关的糖基化位点的缺失；而b12、VRC01、PGT135和PGT128等抗体识别的位点在CRF08_BC和CRF07_BC重组毒株中的变异率低。结论 CRF08_BC和CRF07_BC为昆明市IDU感染者的主要流行毒株；CRF08_BC基因离散率高于CRF07_BC毒株；V3区序列变异程度小于C3区及V4区；2G12、PGT135、PGT128、b12和VRC01等代表性bmNAbs表位在CRF08_BC和CRF07_BC毒株存在不同程度的变异。
Abstract:
Objective To investigate the sequence characteristics of C2-V4 region of HIV-1 env genes and to analyze the epitope variation of representative broadly monoclonal neutralizing antibodies (bmNAbs) in intravenous drug users in Kunming City. Methods 165 HIV-1 infected plasma samples were collected from intravenous drug users in Kunming. RNA was extracted and the cDNA was obtained by reverse transcription. The C2-V4 region of the HIV-1 env genes was amplified by nested PCR and analyzed by bioinformation software such as BioEdit and MEGA, etc. And the epitope variation of bmNAbs such as 2G12, PGT135, PGT128, b12 and VRC01 were analyzed. Results The 165 sequences of C2-V4 region were mainly CRF08_BC (74.55%) and CRF07_BC (23.03%) recombinant strains. The diversity degree of the variable loops was as follows:V3 region < C3 region < V4 region;And the gene divergence of C2-C4, V3, C3 and V4 regions of CRF08_BC strains was greater than that of CRF07_BC, respectively. The crown tetrapeptide of V3 loop existed in 5 types:GPGQ (95.76%), GPGR(1.82%), GPRQ(1.21%), GPGL(0.61%) and RPGQ(0.61%), and the majority of strains utilize CCR5 as co-receptor (97.58%). Over 90% CRF08_BC and CRF07_BC recombinant strains had deletions of glycosylation sites associated with 2G12 recognition, while mutations at the sites recognized by b12, VRC01, PGT135 and PGT128 were low. Conclusions CRF08_BC and CRF07_BC are the main circulating strains in intravenous drug users in Kunming, and the gene divergence of CRF08_BC is higher than that of CRF07_BC. Sequence analysis demonstrate that V3 region is more conservative than C3 and V4 regions. Varying degrees of epitope variation among representative bmNAbs such as 2G12, PGT135, PGT128, b12 and VRC01 exist in CRF08_BC and CRF07_BC strains.
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