CAJ | 학술논문

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（ hu-man 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gp120BAL V1V2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293 F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。
목적：장서흉선기질림파세포생성소（ mouse thymic stromal lymphopoietin ，mTSLP）여인면역결함병독1형（ hu-man immunodeficiency virus type 1，HIV-1）B아형분리주BAL적gp120 V1/V2결구역재293F진핵세포표체체계중진행융합표체。방법：이진핵표체질립pCEP-Pu위재체，구건mTSLP여gp120BAL V1V2융합기인적중조단백표체질립pCTV1V2BAL。한제성매절전영여측서험증질립구건정학후，사용PEI순시전염293 F현부세포，표체산물이SDS-PAGE여Western blot진행분석，병대순화후중조단백적V1/V2항원표위진행료ELISA분석。결과：SDS-PAGE、Western blot분석현시，재표관분자량35 kD지55 kD적범위내존재일조불균일적당단백조대，병차능여항mTSLP다극륭항체화항His표첨단극륭항체발생반응。 HIV-1양성병인혈청능구식별mTSLP-V1/V2BAL상적HIV-1V1/V2항원표위。결론：재293F중성공표체료mTSLP-V1/V2BAL융합단백，해단백구유HIV-1gp120BALV1/V2구적항원표위，능구용우HIV-1 gp120 V1/V2아단위역묘적연구。
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 .