Vsig4和免疫球蛋白Fc段融合蛋白的真核表达纯化

A Fusion Protein of Vsig4 and Immunoglobulin Fc Domain Expression and Purification via Mammalian System Expression

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。

Objective： The current research aims to construct the extracellular domain of Vsig4 and the Fc domain of mouse immunoglobulin IgG3a fusion protein via mammalian expression system for Vsig4 functional assays. Methods： The gene of Fc region includes hinge-CH2-CH3 in mouse IgG3a. 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 pEGZ-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/mL 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 fimctional Vsig4-Fc fusion protein.