| 郑芳 罗思羽 韩燕 张富军 温玉荣.Vsig4 和免疫球蛋白Fc段融合蛋白的真核表达纯化[J].现代生物医学进展英文版,2016,16(26):5001-5005. |
| 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 SystemExpression |
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| DOI： |
| 中文关键词: Vsig4-Fc 融合蛋白 真核蛋白表达 蛋白质纯化 重合延伸PCR |
| 英文关键词: Vsig4-Fc Fusion protein Mammalian protein expression Purification Overlapping extension PCR |
| 基金项目:国家自然科学基金项目（81501527）；陕西省自然科学基础研究计划-青年人才项目（2016JQ3024）；
西安交通大学基本科研业务费（xjj2015048） |
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| 中文摘要: |
| 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IgG3a-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IgG3a-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。 |
| 英文摘要: |
| 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.Conclusion: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. |
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