重组人Jagged2胞外区蛋白的真核表达与免疫原性鉴定  

Eukaryotic Expression and Immunogenicity Study of Human Jagged2 Extracellular Domain

作　　者：费文仪 王世静 冯宇琪 赵玉红 杨欣宇 沈乐 张万鹏 王旻[1] 吴旻[1] FEI Wen-yi;WANG Shi-jing;FENG Yu-qi;ZHAO Yu-hong;YANG Xin-yu;SHEN Le;ZHANG Wan-peng;WANG Min;WU Min(School of Life Science and Technology,China Pharmaceutical University,Nanjing 210009,China)

机构地区：[1]中国药科大学生命科学与技术学院,江苏南京210009

出　　处：《药物生物技术》2022年第2期111-115,共5页Pharmaceutical Biotechnology

基　　金：国家自然科学基金资助项目(No.NSFC81703404);江苏高校优势学科建设工程资助项目(PAPD);中国药科大学“双一流”学科创新团队建设项目(No.CPU2018GY14);大学生创新创业训练计划项目(No.202010316176)。

摘　　要：本研究目的在于使用哺乳动物真核表达系统获得具有高免疫原性重组人Jagged2胞外区C2-EGF2区段(aa27~309),为后续抗Jagged2抗体相关研究奠定基础。首先,合成JAG2胞外区基因并插入pcDNA3.1(+)质粒构建哺乳细胞真核表达载体。使用阳离子脂质体转染试剂瞬时转染HEK293F细胞,收集培养上清,使用镍离子亲和层析柱梯度洗脱纯化,纯化后蛋白样品经过12%胶浓度SDS-PAGE电泳,免疫Balb/c小鼠并利用间接ELISA法测试小鼠抗血清效价。结果显示,150 mmol/L咪唑浓度下洗脱出的Jagged2蛋白在相对分子质量35000左右出现单一电泳条带,ImageJ分析纯度达90%以上。3次免疫后小鼠产生的抗血清效价最高可达10^(6)以上。此结果表明真核表达得到的Jagged2胞外区蛋白具有较高的纯度和免疫原性,可以作为免疫原蛋白支持后续的抗体筛选。Jagged2 is one of the Notch ligands,which has been linked to metastasis of several epithelial-derived tumors.The purpose of this study was to produce recombinant human Jagged2 extracellular C2-EGF2 domain(aa 27-309)by mammalian expression system and evaluate its immunogenicity to lay a foundation for the preparation of anti-Jagged2 monoclonal antibodies.Extracellular region of JAG2 gene was synthesized and cloned into pcDNA3.1(+)plasmid to construct HEK293 F expression vector.The Jagged2 protein was purified by Nickel ion affinity chromatography and quantified using 12%SDS-PAGE.Five Balb/c mice was immunized by target protein and the serum titer was detected by ELISA.Purified protein which eluted by 150 mmol/L exceeded 95%purity by SDS-PAGE.Titer of mouse antiserum reached above 10^(6)after immuning for three times.These data showed that the Jagged2 expressed by mammalian cell which has high purity and immunogenicity is suitable as antigen for monoclonal antibodies preparation.

关 键 词：NOTCH Jagged2 哺乳细胞 真核细胞表达 亲和层析 免疫原性 

分 类 号：Q78[生物学—分子生物学]