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应用单基因组扩增法分析HIV-1准种基因变异
作者:秦彦珉 王晓辉 孔东锋 张顺祥

摘要:

目的  探索应用单基因组扩增分析HIV-1准种基因变异。方法  将深圳2010年6个基因测序出现重叠峰的样本RNA稀释至单拷贝,采用一步法RT-PCR套式扩增,扩增产物纯化后进行基因测序,应用Mega 4.02多功能基因分析软件分析不同准种序列的基因距离,构建进化树模型。同时,测定样本的CD4T淋巴细胞浓度和病毒载量。结果  同一感染者体内HIV-1准种在进化树上形成了各自独立的簇,有些簇存在明显的亚簇,表明某些病毒株在复制中具有竞争优势。6个样本HIV准种的基因距离介于0.008与0.06之间,与感染持续时间和病毒载量有关。结论  单基因组扩增法可以很好地分辨出HIV-1准种的基因变异,对于HIV-1准种基因距离的分析可以判断感染时间和分析免疫逃逸机制,其相关影响因素尚需更大样本的分析。

关键词:HIV;基因扩增;准种;有限稀释法

Abstract:

Objective  To establish the single genome amplification (SGA) method and analyze the quasispecies in HIV-infected patients.Methods  All 6 sample RNA acquired in 2010 in Shenzhen and genetic sequenced as overlap peaks were extracted and diluted to a single copy, nest-PCR after one step RT-PCR was employed to amplify HIV-1 genome, and then PCR products was purified and sequenced. Mega 4.02 software was used to analyze the genetic distance among HIV-1 quasispecies, and phylogenetic tree was constructed.Results  Our data showed that viral sequences derived from different patients were grouped into different clusters. Subcluster was also observed in several clusters, indicating these existed competition and preferential replication of certain viral strains. The genetic distance within one cluster of 6 samples were between 0.008 and 0.06, it was likely to associate with the duration since infection and viral load.Conclusion  SGA is a useful approach to gain information on quasispecies, the genetic distance within one cluster may help to determine the infection time and immune escaping. The analysis of related affecting factors need more samples.

Key words: HIV;Gene amplification;Quasispecies;Limiting dilution method

发表日期:2014/9

引用本文:

图/表:

  • 10.3760/cma.j.issn.0253-9624.2014.09.015.F001:图1 6株深圳地区HIV-1毒株准种进化树模型

    10.3760/cma.j.issn.0253-9624.2014.09.015.F001:图1 6株深圳地区HIV-1毒株准种进化树模型

  • 10.3760/cma.j.issn.0253-9624.2014.09.015.T001:表1 HIV感染者相关检测指标及个体间准种基因距离分析

    10.3760/cma.j.issn.0253-9624.2014.09.015.T001:表1 HIV感染者相关检测指标及个体间准种基因距离分析

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