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目的:将鼠胸腺基质淋巴细胞生成素(mouse thymic stromal lymphopoietin,mTSLP)与人免疫缺陷病毒1型(human immunodeficiency virus type 1,HIV-1)B亚型分离株BAL的gp120 V1/V2结构域在293F真核细胞表达体系中进行融合表达。方法:以真核表达质粒pCEP-Pu为载体,构建mTSLP与gp120BALV1V2融合基因的重组蛋白表达质粒pCTV1V2BAL。限制性酶切电泳与测序验证质粒构建正确后,使用PEI瞬时转染293F悬浮细胞,表达产物以SDS-PAGE与Western blot进行分析,并对纯化后重组蛋白的V1/V2抗原表位进行了ELISA分析。结果:SDS-PAGE、Western blot分析显示,在表观分子量35 kD至55 kD的范围内存在一条不均一的糖蛋白条带,并且能与抗mTSLP多克隆抗体和抗His标签单克隆抗体发生反应。HIV-1阳性病人血清能够识别mTSLP-V1/V2BAL上的HIV-1V1/V2抗原表位。结论:在293F中成功表达了mTSLP-V1/V2BAL融合蛋白,该蛋白具有HIV-1gp120BALV1/V2区的抗原表位,能够用于HIV-1 gp120 V1/V2亚单位疫苗的研究。
OBJECTIVE: To determine whether the gp120 V1 / V2 domain of mouse thymic stromal lymphopoietin (mTSLP) and human immunodeficiency virus type 1 (HIV-1) subtype B BAL isolates 293F eukaryotic expression system fusion expression. Methods: The eukaryotic expression plasmid pCEP-Pu was used as the vector to construct the recombinant protein expression plasmid pCTV1V2BAL of mTSLP and gp120BALV1V2 fusion gene. After restriction endonuclease electrophoresis and sequencing verified that the plasmid was correctly constructed, the 293F suspension cells were transiently transfected with PEI, and the expressed products were analyzed by SDS-PAGE and Western blot. The purified V1 / V2 antigen epitopes of the recombinant protein were subjected to ELISA analysis. Results: SDS-PAGE and Western blot showed that there was a heterogeneous glycoprotein band in the range of apparent molecular weight 35 kD to 55 kD and could react with anti-mTSLP polyclonal antibody and anti-His tag monoclonal antibody . HIV-1-positive patient sera recognize the HIV-1V1 / V2 epitope on mTSLP-V1 / V2BAL. CONCLUSION: The mTSLP-V1 / V2BAL fusion protein was successfully expressed in 293F and has the epitope of HIV-1 gp120BALV1 / V2 region and could be used in the study of HIV-1 gp120 V1 / V2 subunit vaccine.