克隆中国B、C、E亚型的HIV-1代表株建立适于我国流行株的异源双链泳动分析  

Cloning the representative strains of China HIV 1 subtypes B, C and E for heteroduplex mobility analysis (HMA)

作　　者：肖瑶[1,2] 姚均[1,2] 陈刚[1,2] 潘品良[1,2] 邵一鸣[1,2] 

机构地区：[1]卫生部艾滋病预防与控制中心参比室 [2]同济医科大学微生物教研室

出　　处：《中华实验和临床病毒学杂志》1999年第1期33-36,共4页Chinese Journal of Experimental and Clinical Virology

摘　　要：目的构建我国人免疫缺陷病毒１型（ＨＩＶ－１）Ｂ、Ｃ和Ｅ各亚型代表株ｅｎｖ基因质粒，用于对中国ＨＩＶ－１进行分型。方法将与我国ＨＩＶ－１Ｂ、Ｃ和Ｅ各亚型共享序列最接近的毒株用套式聚合酶链反应（ＰＣＲ）技术扩增包膜（ｅｎｖ）基因，克隆到ｐＧＥＭ－Ｔｅａｓｙ载体中，构建成用于异源双链泳动分析（ＨＭＡ）分型的中国标准亚型质粒，并进行了序列分析和分型的敏感性分析。结果构建的中国ＨＩＶ－１Ｂ、Ｃ和Ｅ亚型标准亚型质粒对我国已知亚型的样品进行分析，有清晰的异源双链带形成，形成的异源双链与国外同种亚型质粒与样品形成的异源双链带相比，距同源双链更为接近，分型结果更为精确。中国ＨＩＶ－１Ｂ、Ｃ和Ｅ亚型标准株的核苷酸序列与国际常用于ＨＭＡ分型的ＴＨ１４、ＩＮＤ８６８、ＭＡ９５９、ＴＨ２２、等序列比较，相应亚型的核苷酸同源性是：Ｂ亚型９０．２％，８８．３％，Ｃ亚型８３．７％，Ｅ亚型９２．３％。结论采用ＨＭＡ方法，用中国ＨＩＶ－１Ｂ、Ｃ和Ｅ各亚型标准质粒对中国ＨＩＶ－１进行分型，中国ＨＩＶ－１代表株的ＨＭＡ各参考亚型质粒提高了中国ＨＭＡ分型的敏感性，也为世界卫生组织（ＷＨＯ）艾滋病规划署（ＵＮＡＩＤＳ）的ＨＩＶ分离鉴定网增加了供分型的?Objective To clone the HIV 1 env gene and establish the HMA reagents suitable for use with the Chinese HIV strains.Methods Using Nest PCR technique, we amplified env gene from representative strains which contain the most proximate consensus of China HIV 1 subtypes B, C and E. The genes were then cloned into pGEM Teasy vector for HMA subtyping. Result The env gene of representative strains of China HIV 1 subtypes B, C and E were amplified and cloned into plasmids. The plasmids were used in HMA and compared with those standard env gene of other countries. The heteroduplex bands obtained with ours env genes were more proximate to homoduplex bands. The homogeneity of the corresponding China HIV 1 subtypes with standard TH14(B), IND868(C), MA959(C) and TH22(E) are 90.2%(B), 88.3%(C), 83.7(C)% and 92.3%(E) respectively. Conclusion The HMA reagents we established in this study have higher sensitivity in HMA analysis for China HIV 1 strains when compared with the international HMA reagents. The HMA reagents also provide new reference reagents for UNAIDS HIV isolation network.

关 键 词：亚型 质粒 序列分析 敏感性分析 HIV-1 HMA 

分 类 号：R373.9[医药卫生—病原生物学]