【摘要】 目的:本项目将通过构建中国仓鼠卵巢细胞(Chinese hamster ovary,CHO)真核表达系统获取小鼠Vsig4膜外端和免疫球蛋白Ig G3a-Fc段的融合蛋白,鉴定Vsig4-Fc和Vsig4纳米抗体的相互作用。方法:采用重合延伸PCR法融合小鼠Ig G3a-Fc和Vsig4胞外段的基因序列,将该融合基因插入真核表达载体中并转染CHO细胞。Western blotting鉴定转染细胞上清中的目标蛋白,通过连续两次亚克隆筛选,获得高表达小鼠Vsig4-Fc融合蛋白的单克隆,之后大量培养增殖转染细胞并收集细胞培养上清,选择Protein A柱纯化方法纯化Vsig4-Fc蛋白,最后经ELISA法鉴定Vsig4-Fc和纳米抗体的结合能力。结果:在CHO细胞中成功构建了小鼠Vsig4-Fc真核表达稳转系,并且在真核表达体系中获得可表达15 mg/L的双分子结构Vsig4-Fc的稳定转染细胞系。经鉴定小鼠Vsig4-Fc融合蛋白能与Vsig4纳米抗体结合。结论:重合延伸PCR法使得Vsig4和Fc基因片段的融合更为高效,两次亚克隆筛选优势细胞系大幅提高了真核蛋白的表达量,为进一步研究Vsig4的生物学功能奠定重要基础。更多还原

【Abstract】 Objective: The current research aims to construct the extracellular domain of Vsig4 and the Fc domain of mouse immunoglobulin Ig G3 a fusion protein via mammalian expression system for Vsig4 functional assays. Methods: The gene of Fc region includes hinge-CH2-CH3 in mouse Ig G3 a. It was introduced to the C terminal of Vsig4 extracellular domain gene via overlapping extension PCR. Then, the chimeric plasmid was generated by inserting the fusion DNA fragment into p EGZ-Term/Vsig4-Fc plasmids.After transfection of CHO cells by chimeric plasmids using Lipofectamine, the CHO/Vsig4-Fc transfectants was selected by complete medium containing Geneticin. Furthermore, continuous cloning and medium ELISA were performed to gain higher yield of fusion protein. Protein A column was chosen for the fusion protein purification and expression of fusion protein were validated via ELISA using anti-Vsig4 nanobody. Results: The CHO transfectants which produce Vsig4-Fc fusion protein in yield of 15 mg/m L and with bivalent form were generated successfully. The Vsig4-Fc protein is able to bind with anti-Vsig4 Nanobody. Conclusions: The fusion DNA fragment of Vsig4 and Fc was generated by overlapping extension PCR method and the fusion protein was produced. The Vsig4 functional assays in the fields of T cells activity regulation and complement pathway inhibition will be benefited with the presented functional Vsig4-Fc fusion protein.更多还原