HIV-1 Gag多表位嵌合基因的构建及表达蛋白的抗体制备

Construction and Expression of Chimeric Domains Derived from HIV-1 Gag and Its Antibody Preparation

旨在通过构建Gag的抗原多表位融合基因及在原核系统的高表达,为HIV诊断及可能的疫苗制备提供试验基础。选定HIV-1 Gag基因中3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3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 blotting测定融合蛋白的表达,并免疫动物制备相应抗体。结果显示,构建的HIV-1 Gag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576bp。在大肠杆菌BL21(DE3)中高效表达的重组蛋白分子量为27kD,以包涵体的形式存在。纯化目的蛋白免疫家兔,制备多克隆抗体IgG。ELISA和免疫荧光方法检测显示制备的多克隆抗体能具有特异性反应。成功构建和高表达了HIV-1 Gag多表位融合蛋白,纯化蛋白制备的抗体与HIV-1Gag有特异性结合。为进一步研究HIV-1奠定了试验基础。

Human immunodeficiency virus （HIV）is the etiologic agent of AIDS. For clinical diagnosis and developing vaccine of HIV-1,we prepared HIV-1 Gag fusion protein and its antibody. Three gene fragments of HIV-1 Gag containing highly immunogenic epitopes were amplified by PCR from plasmid of HIV-1 HXB2. The PCR products were ligated and cloned into a prokaryotic expression vector pET28a（＋） and the accuracy of the inserted fragments was identified by sequencing. To express the HIV-1 Gag fusion epitopes in E. coli cells and identify fusion protein,the induced protein was checked with SDS-PAGE and Western blotting （WB）. The purified protein was used to immunize rabbit for 3 times to prepare polyclonal antibody. Rabbit anti-serum specificity and its titer to HIV-1 Gag were assayed by ELISA and cell immunofluorescence（CIF）.Results indicated that the length of the chimeric fragment derived from HIV-1 Gag was 576 bp,and encoded 27 kD protein. Procaryotic expression plasmid was constructed successfully which can high effectively express Gag fusion protein. Recombinant fusion protein was expressed in Eserichia coli BL21（DE3） as an insoluble protein. A high titer antibody through immunizing rabbit with the purified protein was obtained.The data of CIF demonstrated that the anti-serum had high specificity for HIV-1 Gag expressed in B16 cells.Therefore,it proved that procaryotic expression plasmid which can highly express Gag fusion protein was successfully constructed. Prepared polyclonal antibody was high specific and affinitic by immunized rabbit. The founding and subjects were provided for the research of HIV-1 diagnosis kit and vaccine.