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构建pMT/BiP/V5-HisA-HIVgp120真核表达载体,通过稳定转染获得稳定表达gp120蛋白的果蝇Schnei-der2(S2)细胞系,并对gp120蛋白进行大量表达和纯化。应用聚合酶链式反应技术从重组载体PVR1012-gp120中扩增出中国流行株CRF07-BCgp120全长基因后,将其插入载体pMT/BiP/V5-HisA。应用脂质体转染技术将真核表达载体和抗性筛选质粒共转染果蝇S2细胞,通过杀稻瘟菌素反复筛选,建立稳定高表达gp120蛋白的果蝇S2细胞系,并通过镍柱亲和层析和分子筛法纯化目的蛋白。用SDS-PAGE对重组蛋白的分子量和纯度进行分析,并通过Western blot和酶联免疫吸附技术(ELISA)对其进行鉴定。成功构建了gp120真核表达载体并获得了稳定转染该蛋白的果蝇S2细胞系,成功的表达了具有良好抗体反应性的gp120全长蛋白。此结果为深入研究HIV包膜蛋白gp120的生物学特性及进行HIV疫苗的研究提供了良好的物质基础。
The eukaryotic expression vector pMT / BiP / V5-HisA-HIV-gp120 was constructed. The stable cell line Schnei-der2 (S2) expressing gp120 protein was obtained by stable transfection. The gp120 protein was expressed and purified in large quantities. Polymerase chain reaction (PCR) was used to amplify the full-length Chinese CRF07-BCgp120 gene from the recombinant vector PVR1012-gp120 and inserted into vector pMT / BiP / V5-HisA. Liposome transfection technique was used to co-transfect Drosophila S2 cells with the eukaryotic expression vector and the resistant screening plasmid. The Drosophila S2 cell line stably expressing high gp120 protein was established by repeated screening with blasticidin, Purification of target protein by column affinity chromatography and molecular sieve. The molecular weight and purity of the recombinant protein were analyzed by SDS-PAGE and identified by Western blot and enzyme-linked immunosorbent assay (ELISA). The gp120 eukaryotic expression vector was successfully constructed and the Drosophila S2 cell line stably transfected with the protein was successfully obtained. The gp120 full-length protein with good antibody reactivity was successfully expressed. The results provide a good material basis for further study of the biological characteristics of HIV envelope protein gp120 and the research of HIV vaccine.