The trimeric envelope glycoprotein (gp120/gp41)₃ of human immunodeficiency virus-1 (HIV-1) mediates viral and host cell membrane fusion, initiated by binding of viral envelope gp120 protein to the CD4 receptor on host immune cells. Functional env genes from infected individuals have been widely used as templates for vaccine design, for setting up viral neutralization assays and to study the viral evolution and pathogenesis. Traditional topoisomerase or T4 DNA polymerase mediated approaches for cloning single genome amplified (SGA) env genes are labor-intensive, cost-ineffective with low-throughput, thereby enabling functional analysis of only a limited number of env genes from the diverse circulating quasispecies in infected individuals. Herein, we report an efficient, easy to optimize and high-throughput approach for cloning diverse HIV-1 env genes. Multiple env/rev gene cassettes, derived from infected infants, were subjected to SGA using Phusion polymerase and utilized as megaprimers in overlap extension PCR mediated cloning (OEC), circumventing the requirement for novel enzymes. Furthermore, utilization of Phusion polymerase for both the amplification of env/rev cassettes and OEC allows convenient monitoring and optimization, thereby providing much greater flexibility and versatility for analysis of env genes from HIV-1 infected individuals.

中文翻译：

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克隆HIV-1 env基因的通用高通量策略

三聚体包膜糖蛋白（gp120 / gp41）₃人类免疫缺陷病毒1（HIV-1）介导的病毒和宿主细胞膜融合是由病毒包膜gp120蛋白与宿主免疫细胞上的CD4受体结合而引发的。来自感染个体的功能性env基因已被广泛用作疫苗设计，建立病毒中和试验以及研究病毒进化和发病机理的模板。传统的拓扑异构酶或T4 DNA聚合酶介导的克隆单基因组扩增（SGA）env基因的方法是劳动密集型，成本低，低通量的方法，因此仅能对感染的多种循环准种中有限数量的env基因进行功能分析个人。在本文中，我们报告了一种高效，易于优化和高通量的克隆多种HIV-1 env基因的方法。多个env / rev基因盒，来源于感染婴儿的婴儿，使用Phusion聚合酶进行SGA处理，并在重叠延伸PCR介导的克隆（OEC）中用作巨型引物，从而避免了对新酶的需求。此外，利用Phusion聚合酶扩增env / rev盒和OEC都可以方便地进行监测和优化，从而为分析HIV-1感染者的env基因提供了更大的灵活性和多功能性。