[Prokaryotic expression of mouse LRP16, preparation and identification of rabbit anti-mouse LRP16 polyclonal antibody].

细胞与分子免疫学杂志 Pub Date : 2025-06-01
Feifei Zhang, Jian Li, Xiangying Xu, Meiling Han, Zhe Zhang
{"title":"[Prokaryotic expression of mouse LRP16, preparation and identification of rabbit anti-mouse LRP16 polyclonal antibody].","authors":"Feifei Zhang, Jian Li, Xiangying Xu, Meiling Han, Zhe Zhang","doi":"","DOIUrl":null,"url":null,"abstract":"<p><p>Objective To investigate prokaryotic expression of the antigen sequence (amino acids 59-145) of mouse leukemia-related protein 16 (LRP16) protein and preparation of rabbit anti-mouse LRP16 polyclonal antibody. Methods The prokaryotic expression plasmid pLS962-LRP16 was constructed by the molecular cloning method and transferred into E.coli Rosetta to express LRP16 protein induced by IPTG. The recombinant protein was purified using Ni-NTA affinity columns followed by gel filtration chromatography. New Zealand white rabbits were immunized with the purified antigen to generate polyclonal antiserum, with antibody titer quantified by ELISA. Antigen-specific IgG was affinity-purified using Sepharose-coupled LRP16 and validated through Western blot and immunofluorescence assays. Results SDS-PAGE analysis confirmed insoluble expression of the LRP16 fusion protein as inclusion bodies. ELISA demonstrated exceptional antiserum titer (1:256 000). Western blot and immunofluorescence verified that the polyclonal antibody could specifically recognize endogenous LRP16 in murine tissues. Conclusion The prokaryotic expression of the LRP16 gene is successfully achieved, and the rabbit anti-mouse LRP16 polyclonal antibody exhibiting high specificity is prepared. This lays the foundation for further studies on the function of the LRP16 gene.</p>","PeriodicalId":61378,"journal":{"name":"细胞与分子免疫学杂志","volume":"41 6","pages":"544-551"},"PeriodicalIF":0.0000,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"细胞与分子免疫学杂志","FirstCategoryId":"3","ListUrlMain":"","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 0

Abstract

Objective To investigate prokaryotic expression of the antigen sequence (amino acids 59-145) of mouse leukemia-related protein 16 (LRP16) protein and preparation of rabbit anti-mouse LRP16 polyclonal antibody. Methods The prokaryotic expression plasmid pLS962-LRP16 was constructed by the molecular cloning method and transferred into E.coli Rosetta to express LRP16 protein induced by IPTG. The recombinant protein was purified using Ni-NTA affinity columns followed by gel filtration chromatography. New Zealand white rabbits were immunized with the purified antigen to generate polyclonal antiserum, with antibody titer quantified by ELISA. Antigen-specific IgG was affinity-purified using Sepharose-coupled LRP16 and validated through Western blot and immunofluorescence assays. Results SDS-PAGE analysis confirmed insoluble expression of the LRP16 fusion protein as inclusion bodies. ELISA demonstrated exceptional antiserum titer (1:256 000). Western blot and immunofluorescence verified that the polyclonal antibody could specifically recognize endogenous LRP16 in murine tissues. Conclusion The prokaryotic expression of the LRP16 gene is successfully achieved, and the rabbit anti-mouse LRP16 polyclonal antibody exhibiting high specificity is prepared. This lays the foundation for further studies on the function of the LRP16 gene.

[小鼠LRP16的原核表达及兔抗小鼠LRP16多克隆抗体的制备与鉴定]。
目的研究小鼠白血病相关蛋白16 (LRP16)抗原序列(59 ~ 145个氨基酸)的原核表达及兔抗小鼠LRP16多克隆抗体的制备。方法采用分子克隆法构建LRP16原核表达质粒pLS962-LRP16,转染大肠杆菌Rosetta,表达IPTG诱导的LRP16蛋白。采用Ni-NTA亲和柱和凝胶过滤层析纯化重组蛋白。用纯化后的抗原免疫新西兰大白兔制备多克隆抗血清,用ELISA法测定抗体滴度。使用sepharose偶联LRP16亲和纯化抗原特异性IgG,并通过Western blot和免疫荧光检测进行验证。结果SDS-PAGE分析证实LRP16融合蛋白不溶性表达为包涵体。ELISA检测出异常的抗血清效价(1:25 6000)。Western blot和免疫荧光验证该多克隆抗体能特异性识别小鼠组织中内源性LRP16。结论成功地实现了LRP16基因的原核表达,制备了高特异性的兔抗小鼠LRP16多克隆抗体。为进一步研究LRP16基因的功能奠定了基础。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
求助全文
约1分钟内获得全文 求助全文
来源期刊
自引率
0.00%
发文量
9567
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
确定
请完成安全验证×
copy
已复制链接
快去分享给好友吧!
我知道了
右上角分享
点击右上角分享
0
联系我们:info@booksci.cn Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。 Copyright © 2023 布克学术 All rights reserved.
京ICP备2023020795号-1
ghs 京公网安备 11010802042870号
Book学术文献互助
Book学术文献互助群
群 号:604180095
Book学术官方微信