小鼠CD40胞外段的原核表达及多克隆抗体的制备

doi:10.14088/j.cnki.issn0439-8114.2020.06.026

湖北农业科学 ›› 2020, Vol. 59 ›› Issue (6): 130-134.doi: 10.14088/j.cnki.issn0439-8114.2020.06.026

小鼠CD40胞外段的原核表达及多克隆抗体的制备

曹旗, 潘悦, 郑宇, 王菡, 任洪林, 胡盼, 李岩松, 周玉, 柳增善, 卢士英

吉林大学动物医学学院/人兽共患病研究所/人兽共患病教育部重点实验室,长春 130062

收稿日期:2020-01-10 出版日期:2020-03-25 发布日期:2020-06-13
通讯作者: 卢士英（1973-）,女,教授,博士,博士生导师,主要从事动物性食品安全研究,（电子信箱）lushiying1129@163.com。
作者简介:曹旗（1992-）,男,湖北丹江口人,在读硕士研究生,研究方向为动物性食品安全,（电话）18844199729（电子信箱）1332719329@qq.com;
基金资助:
吉林大学省校共建项目（SXGJSF2017-6）

Prokaryotic expression of mouse extracellular domain of CD40 and preparation of polyclonal antibody

CAO Qi, PAN Yue, ZHENG Yu, WANG Han, REN Hong-lin, HU Pan, LI Yan-song, ZHOU Yu, LIU Zeng-shan, LU Shi-ying

College of Veterinary Medicine,Jilin University/Institute of Zoonosis/Key Laboratory of Zoonosis Research Ministry of Education,Changchun 130062,China

Received:2020-01-10 Online:2020-03-25 Published:2020-06-13

摘要/Abstract

摘要： 为了探讨CD40抗体与CD40结合对B细胞应答小分子半抗原的增强作用,利用TRIzol法提取BALB/c小鼠脾脏总RNA,并反转录成cDNA;根据CD40的胞外段CDS设计引物,通过PCR扩增目的基因,构建pGEX4T-1-CD40表达载体,进行原核表达及多克隆抗体制备。结果表明,成功扩增了522 bp的目的基因,表达并纯化获得了45 ku的CD40重组蛋白,多克隆抗体抗体不但可与重组蛋白结合,与小鼠脾脏天然CD40蛋白也可以特异性结合。说明CD40重组蛋白表达成功,且制备的抗体有望模拟CD40L为B细胞活化提供第二信号。

关键词: CD40, 原核表达, 多克隆抗体, 半抗原, 免疫增强

Abstract: To investigate the enhanced effect of CD40 antibody binding to CD40 on B-cell response to small molecule haptens,in this study, the total RNA of BALB/c mouse spleen was extracted by TRIzol method and reverse transcribed into cDNA; Primers were designed based on the CDS region of the extracellular domain of CD40, and the target gene was amplified by PCR to construct the pGEX4T-1-CD40 expression vector for prokaryotic expression;The polyclonal antibody was prepared. The results showed that the 522 bp target gene was successfully amplified, CD40 recombinant protein of 45 ku was expressed and purified. The antibody obtained by Western-blot detection not only binds to recombinant protein, but also can specifically bind to mouse spleen natural CD40 protein. Those indicated that the expression of CD40 recombinant protein is successful, and the prepared antibody was expected to mimic CD40L to provide the second signal for B cell activation.

Key words: CD40, prokaryotic expression, polyclonal antibody, hapten, immune enhancement

中图分类号:

Q786

曹旗, 潘悦, 郑宇, 王菡, 任洪林, 胡盼, 李岩松, 周玉, 柳增善, 卢士英. 小鼠CD40胞外段的原核表达及多克隆抗体的制备[J]. 湖北农业科学, 2020, 59(6): 130-134.

CAO Qi, PAN Yue, ZHENG Yu, WANG Han, REN Hong-lin, HU Pan, LI Yan-song, ZHOU Yu, LIU Zeng-shan, LU Shi-ying. Prokaryotic expression of mouse extracellular domain of CD40 and preparation of polyclonal antibody[J]. HUBEI AGRICULTURAL SCIENCES, 2020, 59(6): 130-134.

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小鼠CD40胞外段的原核表达及多克隆抗体的制备

Prokaryotic expression of mouse extracellular domain of CD40 and preparation of polyclonal antibody

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