HIV包膜蛋白Gp120在果蝇Schneider 2细胞内的表达纯化及初步性质鉴定

Establishment of Stable Cell Line and Expression and Purification of HIV gp120 in Drosophila S2 Cells

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。

Constructing eukaryotic expressing vector of pMT/Bip/V5-His A-HIV gp120 and transfecting S2 cells to establish stable gp120-expressing S2 cell line.The gp120 gene of HIV CRF07-BC epidemic in China was amplified by polymerase chain reaction from the recombinant vector PRSV-gp120 and confirmed by DNA sequence analysis.The DNA fragment of HIV gp120 was digested with the restriction endonucleases NcoI and XhoI,then inserted into eukaryotic expressing vector pMT/BiP/V5-His A.A selection vector containing the streptomyces griseochromogenes bsd gene conferring blasticidin resistance was cotransfected into drosophila S2 cells by CellfectinⅡreagent.The stable cell line was established following repeated screening by blasticidin.HIV gp120 was purified by a Ni-NTA affinity column followed by molecular sieve chromatography.Recombinant protein was characterized by SDS-PAGE,Western blot and ELISA.The eukaryotic expressing vector pMT/BiP/V5-His A-HIV gp120 was constructed,stable expressing cell line was established,and protein was expressed and purified successfully.This result will contribute to functional study of gp120 and vaccine design against AIDS.