mTSLP-V1/V2融合蛋白的真核表达与鉴定

Eukaryotic expression and characterization of mouse TSLP and HIV-1 gp120BAL V1/V2 fusion protein

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 express fusion protein of mouse thymic stromal lymphopoietin （TSLP） and HIV-1 gp120BAL V1/V2 subdomain in 293F cell.Methods：Full length of the V1V2 sequence from BAL isolate was fused with the C-terminus of mouse thymic stromal lymphopoietin （TSLP） and sub-cloned into pCEP-Pu vector to construct the eukaryotic expression plasmid-pCTV1V2BAL.The recombinant plasmid was confirmed by enzyme digestion and sequencing , then transfected into 293 F cells using PEI as a transfection reagent .The fusion protein was purified by metal chelate affinity chromatography and characterized by SDS -PAGE and Western blot . The epitopes of V1/V2 in fusion protein were identified by ELISA .Results：The SDS-PAGE and Western blot results showed that there were highly heterogeneous glycoprotein bands at the site between 35 kD and 55 kD, which reacted with anti-mTSLP rabbit polyclonal antibody and anti-His tag mouse monoclonal antibody .The ELISA analysis showed that antibodies to V 1/V2BAL existed in the sera of HIV-1 positive patients.Conclusion：The mTSLP-V1/V2 fusion protein was successfully expressed in 293F cells, which may be useful for HIV-1 vaccine research .