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

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 constructed, and the recombinant protein of S-ScFv4F10 has a fairly high affinity with CTLA-4 antigen.