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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。
The aim of this study is to provide experimental basis for HIV diagnosis and possible vaccine preparation by constructing Gag antigen multi-epitope fusion gene and its high expression in prokaryotic system. The three regions of HIV-1 Gag gene were selected to contain the regions with more epitopes. The primers with restriction sites were designed and the gene sequences encoding these three fragments were amplified by PCR from the HIV-1 HXB2 whole genome The correctness of the gene fragments was identified by plasmid extraction, restriction enzyme digestion and sequencing. The expression of the fusion protein was detected by SDS-PAGE and Western blotting, and the corresponding antibodies were prepared from the animals. The results showed that the constructed prokaryotic expression plasmid of HIV-1 Gag multi-epitope chimeric gene, digestion and sequencing results showed that the gene sequence was correct and the gene was 576 bp in length. The recombinant protein that is highly expressed in E. coli BL21 (DE3) has a molecular weight of 27 kD and exists as an inclusion body. The target protein was purified to immunize rabbits to prepare polyclonal antibody IgG. ELISA and immunofluorescence assay showed that the prepared polyclonal antibody could react specifically. The HIV-1 Gag multi-epitope fusion protein was successfully constructed and highly expressed. The purified protein was specifically bound to HIV-1 Gag. It laid the experimental foundation for further research on HIV-1.