5株不同亚型HIV-1毒株gp120序列特征分析及其蛋白的表达  

The analysis of 5 HIV-1 gp120 sequences from different clades and expression of their corresponding proteins in vitro

作　　者：王铮[1] 李敬云[1] 鲍作义[1] 李韩平[1] 庄道民[1] 刘思杨[1] 李林[1] 

机构地区：[1]军事医学科学院微生物流行病研究所全军艾滋病检测中心,病原微生物生物安全国家重点实验室,北京100071

出　　处：《中华微生物学和免疫学杂志》2009年第5期424-430,共7页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金资助项目（30700706）

摘　　要：目的分析我国HIV-1主要亚型毒株的gp120区域的序列特征，并表达出gp120糖基化蛋白，为研究gp120的致病机制和设计疫苗奠定基础。方法利用巢式PCR从来自于不同省份的5名HIV-1感染者外周血单个核细胞DNA中扩增全长gp120基因并测序，对序列特征进行分析，并将获得的gp120基因克隆入真核表达载体，体外表达获得gp120糖基化蛋白。结果序列分析显示，此5条gp120序列分属HIV-1 Thai-B、BC重组和AE重组亚型，虽然亚型不同，但这些gp120序列在相同的位置都存在着一些保守的糖基化位点，且都具备相同的Furin蛋白酶第一切割位点，与参考序列比较发现，不同的HIV亚型序列中，除V3序列长度较为保守外，V1、V2、V4、V5各区域都有不同程度的缺失现象，与BC重组和AE重组亚型相比，Thai-B亚型V3的顶端环呈现出多种组合。最终将这5条gp120序列都克隆入真核表达载体并表达出糖基化的gp120蛋白。结论在设计疫苗和检测试剂时应考虑到膜区的高变性，gp120糖蛋白的体外表达有利于进一步开展针对我国主要HIV流行毒株膜蛋白致病机制和疫苗学的研究。Objective To characterize 5 gp120 sequences from mainly circulating clades in China and expression of their gp120 glycoproteins. Methods gp120 genes were amplified from the PBMCs of 5 HIV-1 infected individuals in different provinces using nest PCR and their DNA sequences were determined. Sequence characteristics were analyzed and gp120 genes were sub-cloned into the mammalian expression vector to produce gp120 glycoproteins. Results Sequence characteristics indicated these sequences belong to the elade Thai-B, CRFBC and CRF_AE, respectively. There were some conservative N-linked glycosylation sites and primary Furin protease cleavage motifs in the same positions within gp120 amino acid sequences although these gp120 sequences were categorized into different clades. In comparison with referential strains, amino acids deletions were found in the V1, V2, V4, V5 regions except for the V3 loop; above all, V3 tip motifs of Thai-B exhibited the more polymorphic forms than those of CRF BC and CRF_AE. These 5 gp120 sequences were cloned into the eukaryotic expression vector and gp120 glycoproteins were produced successfully. Conclusion Hyper-variable nature of Env should be considered while designing HIV-1 vaccine or test reagent, and gp120 expression in vitro is helpful to further research on the Env pathogenesis and vaccine development against the mainly circulating HIV-1 isolates in China.

关 键 词：HIV-1 GP120 糖基化蛋白 

分 类 号：R373[医药卫生—病原生物学]