靶向巨噬细胞膜蛋白Vsig4特异性纳米抗体的构建和筛选
发布时间：2019-04-04 17:44
【摘要】：目的构建V-set and immunoglobulin domain containing 4(Vsig4)特异性纳米抗体,以期作为巨噬细胞的分子探针。方法用Vsig4重组蛋白对羊驼进行免疫,分离血液中的淋巴细胞,利用噬菌体展示技术,构建噬菌体展示文库,经过连续3次生物淘筛获得与Vsig4蛋白结合的噬菌体,经测序和基因比对所得VHH序列,用ELISA法筛选出抗Vsig4的高亲和力纳米抗体,并用Vsig4稳定表达细胞系验证纳米抗体的结合能力。结果成功构建了插入率为70%、库容量为7.27×107的噬菌体表达文库,经过克隆筛选获得136个Vsig4阳性单克隆,经测序获得15个不同的VHH基因,将这些基因克隆至原核表达体系,表达和纯化后获得了高纯度的Vsig4纳米抗体,其中Nb119的亲和力最高,并且可以与Vsig4稳定表达细胞系结合。结论成功构建并筛选了特异性、高亲和力的Vsig4纳米抗体,以期用于检测巨噬细胞表面Vsig4的表达和构建特异性分子探针。
[Abstract]:Objective to construct V-set and immunoglobulin domain containing-4 (Vsig4)-specific nano-antibody to serve as a molecular probe for macrophages. Methods Vsig4 recombinant protein was used to immunize alpaca, and lymphocytes were isolated from the blood. The phage display library was constructed by phage display technique, and the phage binding to Vsig4 protein was obtained by three successive biological screening. The VHH sequence was obtained by sequencing and gene alignment, and the high affinity nano-antibodies against Vsig4 were screened by ELISA method. The binding ability of nano-antibodies was verified by Vsig4 stable expression cell line. Results A bacteriophage expression library with 70% insertion rate and 7.27 脳 10 ~ 7 library capacity was successfully constructed. After cloning and screening, a total of 136 Vsig4 positive clones were obtained. 15 different VHH genes were obtained by sequencing, and these genes were cloned into prokaryotic expression system. After expression and purification, high purity Vsig4 nano-antibodies were obtained, among which Nb119 had the highest affinity and could bind to the stable expression cell line Vsig4. Conclusion the specific and high affinity Vsig4 nano-antibodies were successfully constructed and screened in order to detect the expression of Vsig4 on the surface of macrophages and construct specific molecular probes.
【作者单位】： 西安交通大学医学部基础医学院 西安交通大学环境与疾病相关基因教育部重点实验室;西安交通大学生命科学与技术学院;
【基金】：国家自然科学基金资助项目(No.81501527) 陕西省自然科学基础研究计划项目(No.XJJ2015048)~~
【分类号】：R392.11
，
本文编号：2454027
[Abstract]:Objective to construct V-set and immunoglobulin domain containing-4 (Vsig4)-specific nano-antibody to serve as a molecular probe for macrophages. Methods Vsig4 recombinant protein was used to immunize alpaca, and lymphocytes were isolated from the blood. The phage display library was constructed by phage display technique, and the phage binding to Vsig4 protein was obtained by three successive biological screening. The VHH sequence was obtained by sequencing and gene alignment, and the high affinity nano-antibodies against Vsig4 were screened by ELISA method. The binding ability of nano-antibodies was verified by Vsig4 stable expression cell line. Results A bacteriophage expression library with 70% insertion rate and 7.27 脳 10 ~ 7 library capacity was successfully constructed. After cloning and screening, a total of 136 Vsig4 positive clones were obtained. 15 different VHH genes were obtained by sequencing, and these genes were cloned into prokaryotic expression system. After expression and purification, high purity Vsig4 nano-antibodies were obtained, among which Nb119 had the highest affinity and could bind to the stable expression cell line Vsig4. Conclusion the specific and high affinity Vsig4 nano-antibodies were successfully constructed and screened in order to detect the expression of Vsig4 on the surface of macrophages and construct specific molecular probes.
【作者单位】： 西安交通大学医学部基础医学院 西安交通大学环境与疾病相关基因教育部重点实验室;西安交通大学生命科学与技术学院;
【基金】：国家自然科学基金资助项目(No.81501527) 陕西省自然科学基础研究计划项目(No.XJJ2015048)~~
【分类号】：R392.11
，
本文编号：2454027
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