HBsAg基因与细胞毒性T淋巴细胞相关抗原-4单链抗体的真核质粒载体构建及表达蛋白活性鉴定  

The construction, expression and immunological activity study of eukaryotic vector expressing cytotoxic T lymphocyte-associated antigen-4 ScFv and hepatitis B virus-S gene

作　　者：彭国平[1] 赵冬久[1] 周承[1] 周林福[2] 陈智[1] 

机构地区：[1]浙江大学医学院附属第一医院传染病研究所传染病诊治国家重点实验室,杭州310003 [2]浙江大学医学院细胞与分子生物学实验室

出　　处：《中华传染病杂志》2012年第6期324-329,共6页Chinese Journal of Infectious Diseases

基　　金：国家自然科学基金资助项目（30901267）;国家“十一五”重大专项资助项目（0082X10002-007）

摘　　要：目的构建HBsAg基因（HBV5）与抗鼠细胞毒性T淋巴细胞相关抗原4（CTLA-4）单链抗体（ScFv）的真核质粒载体，并检测融合表达蛋白特异性抗原的结合活性。方法将CTI。A-4单链抗体的真核表达质粒pSect2／ScFv4F10及含HBVS基因的质粒pSect2／S分别经SfiI和HindⅢ双酶切后，S基因片段进-步克隆至质粒pSect2／ScFv4F10中。将质粒pSect2／ScFv4F10、pSect2／ScFv4F10。转染中华仓鼠卵巢细胞，Western印迹法检测目的蛋白的表达。将表达的蛋白经超滤浓缩与亲和层析纯化后，分别通过竞争抑制ELISA和表面等离子共振（sPR）技术，检测其与重组小鼠CTLA-4抗原的结合活性及亲合常数。采用One-wayANOVA比较组间差异。结果成功构建可稳定表达ScFv4F10蛋白的真核质粒载体pSect2／ScFv4F10。十二烷基磺酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和Western印迹法检测表明，其表达蛋白的相对分子质量约为52×10^3。当将抗鼠CTLA-4的亲本单链抗体4F10浓度固定时，其与CTLA-4纯化抗原混合反应的吸光度（A）570值随ScFv融合蛋白比例的减少而逐步增大；且当ScFv、ScFv4F10与4F10单链抗体以摩尔比为2：1混合时，其对4F10结合抗原的竞争抑制率分别可达到72．6％和64．5％；亲本单链抗体、ScFv4F10、ScFv4F10与重组CTLA-4抗原的结合平衡常数KA值分别为7．29×10^8mol／L、9．52×10^6mol／L和2．04×10^6mol／L，解离平衡常数KD分别为1．40×10^-9mol／L、1．05×10^-7mol／L和4．91×10^-7mol／L。结论成功构建了HBVS基因与CTLA-4单链抗体ScFv4F10的真核表达质粒载体pSect2／ScFv4F10，其表达的蛋白与CTLA-4抗原具有一定的亲和活性。Objective To construct the eukaryotic vector that expressing hepatitis B virus （HBV） S and the single fragment of variety chain （ScFv） of monoclonal antiboy against cytotoxic T lymphocyte-associated antigen-4 （CTLA-4） and to analyze the immunological activity of recombinant S-ScFv protein. Methods The oringially constructed pSeet2/ScFv4F10 and pSect2/S were doubleenzyme digested by Sfi Ⅰ and Hind Ⅲ, respectively. Then the HBV S gene was cloned into the pSect2/ScFv4F10 vector. The pSect2/ScFv4F10 and pSect2/S-ScFV4F10 were expressed in Chinese hamster ovary （CHO） cells, and the expressed proteins were verified through sodium dodecyl sulfate- polyacrylamide gel electrophoresis （ SDS-PAGE ） and Western blotting. After ultrafiltration concentration and affinity chromatography, the biological affinity of the expressed ScFv4F10 and S- ScFv4F10 proteins were examined by competitive enzyme-linked immunosorbent assay （ELISA） and surface plasmon resonance （SPR） technology. The comparison between groups was done by One way ANOVA. Results The eukaryotic expression vector of pSect2/S-ScFv4F10 was successfully constructed, and relative molecular mass of the expressed protein of S-ScFv4F10 was about 52 000 that analyzed by SDS-PAGE and Western blotting. With the fixed concentration of 4F10-mAb against CTLA-4, the A570 value of the mixed reaction with purified CTLA-4 antigen gradually increased with the decrease of ScFc fusion protein proportion; when the molar ratio of ScFv, S-ScFv4F10：4F10-2： 1, the competitive inhibition rates against 4F10 conjugated antigen were 72.6% and 64.5%, respectively. The affinity constants of association kinetics for CTLA4 mAb, ScFvaF10 and S-SeFv4F10 with CTLA-4 antigen were 7.29 ×10^8 mol/L, 9.52 × 10^6 mol/L and 2.04 × 10^6 mol/L, respectively, and the dissociation constants of KD were 1. 40 × 10^-9 mol/L, 1. 05 × 10^-7 mol/L and 4. 91 × 10^-7 mol/L, respectively. Conclusions The eukaryotic expression vector of pSect2/S-ScFv4F10 is successfully constructe

关 键 词：基因 病毒 肝炎表面抗原 乙型 细胞毒性T淋巴细胞相关抗原 基因表达 质 粒 免疫活性 

分 类 号：R392[医药卫生—免疫学]