HIV-1 gp160多表位嵌合基因的构建及表达蛋白的免疫原性分析

Construction and Expression of Fusion Domains Derived from HIV-1 gp160 and Immunogenicity Analysis of Its Purified Protein

目的：HIV感染引起的AIDS已经成为严重影响人类健康和社会发展的全球性疾病。酶联免疫吸附试验和免疫印迹检验组合则被认为是HIV检测的“金标准”。因此本实验构建gp160的抗原多表位融合基因及在原核系统的高表达,为HIV抗体测定提供特异、价廉的抗原。方法：选定HIV-1 gp160基因中三个片段包含较多抗原表位的区域,设计带有酶切位点的引物,用PCR的方法从HIV-1HXB2全基因扩增编码这三个片段的基因序列,通过质粒提取、酶切、测序方法鉴定基因片段的正确性,SDS-PAGE和Western Blot测定融合蛋白的抗原特异性。结果：构建的HIV-1 gp160多表位嵌合基因的原核表达质粒,酶切和测序结果表明基因序列正确,基因全长969bp。在大肠杆菌BL21（DE3）中高效表达的重组蛋白分子量为37kDa,以包涵体的形式存在。结论：应用western blot测定10例正常人和12例HIV/AIDS病人血浆显示HIV-1 gp160多表位融合蛋白具有良好的抗原特异性。成功构建了高表达HIV-1 gp160多表位蛋白的原核表达系统,纯化的融合蛋白有较强的抗原特异性。

Objective： Human immunodeficiency virus （HIV） is the etiologic agent of AIDS. Immunogenic epitopes of HIV resides on virus-encoded envelope glycoproteins. To prepare HIV-1 gp160 protein and used it for clinical diagnosis, three gene fragments of HIV-1 env containing highly immunogenic epitopes were amplified by PCR from plasmid of HIV-1 HXB2. The resulting PCR products were linked and cloned into a prokaryotic expression vector pET28a（ ＋ ） and the accuracy of the inserted fragments was identified by sequencing. To express the HIV-1 gp160 fusion epitopes in E. coli cells and identify fusion protein, the induced protein was checked with SDS-PAGE and Western Blot（WB）. Methods： The identified HIV positive serum samples were tested by Western Blot （WB） to analysis immunogenicity of the purified protein. The length of the chimeric fragment derived from HIV-1 gp160 was 969bp, and encoded 37kDa amino acid residues. Resuits： Procaryotic expression plasmid was constructed successfully which can highly effectively express gp160 fusion protein. Recombinant fusion protein was expressed in Eserichia coli BL21 （DE3） as an insoluble protein. Conclusions： Procaryotic expression plasmid which can highly effectively express gp160 fusion protein was constructed successfully. The founding and subjects were provided for the research of diagnosis kit.