【摘要】 本实验室前期制备了1株分泌针对猪瘟病毒(CSFV)E2蛋白鼠源单克隆抗体的杂交瘤细胞株6E10,并验证其可特异性识别CSFV E2蛋白。由于杂交瘤细胞不稳定且不易储存，本研究将6E10抗体的轻、重链可变区基因和猪源抗体的恒定区基因融合并克隆至慢病毒表达载体pFUGW,分别构建了携带Strep标签的重组猪源化抗体轻、重链基因的慢病毒质粒pFU-p6E10-LC-Strep和pFU-p6E10-HC-Strep,进一步制备了慢病毒Lenti-p6E10-LC-Strep和Lenti-p6E10-HC-Strep,将二者共转导至HEK293S悬浮细胞，成功表达并纯化了重组猪源单克隆抗体6E10(p6E10)。经ELISA试验及Western blot证实，p6E10抗体能特异性识别E2蛋白。中和试验结果显示，p6E10抗体可以中和CSFV石门株。结果表明，本研究成功获得了针对CSFV E2蛋白的重组猪源化单克隆抗体p6E10,为深入研究CSFV E2蛋白的结构和功能以及开发新型诊断制剂奠定了基础。更多还原

【Abstract】 A hybridoma cell line secreting mouse monoclonal antibody(mAb) 6E10 against E2 protein of classical swine fever virus(CSFV) was generated in our laboratory.It has been shown that the mAb can specifically recognize the CSFV E2 protein.Since the hybridoma cells are unstable and difficult to store for long time, the chimeric antibody genes of the chain variable region genes from antibody 6E10 and the constant region genes of porcine antibody were amplified by PCR and cloned into the lentivirus vector pFUGW,giving rise to the lentivirus plasmids pFU-p6E10-LC-Strep and pFU-p6E10-HC-Strep.The plasmids were co-transfected with pSPAX2 and pMD2.G into HEK293T cells to generate the lentiviruses, termed Lenti-p6E10-LC-Strep and Lenti-p6E10-HC-Strep.The lentiviruses were co-transduced into HEK293S suspension cells to generate recombinant porcinized monoclonal antibody(p6E10).The p6E10 antibody expressed in supernatants of HEK293S suspension cells was successfully purified by AKTA system.Using ELISA and Western blot analysis, we demonstrated that p6E10 had high reactivity with the CSFV E2 protein.In conclusion, the recombinant p6E10 against the CSFV E2 protein was successfully generated in this study, which can be used to develop new diagnostic methods or to study the structure and function of E2 protein.更多还原