兔抗鼠MORF4L1 a多克隆抗体的制备与鉴定

doi:10.16352/j.issn.1001-6325.2022.05.025

基础医学与临床 ›› 2022, Vol. 42 ›› Issue (5): 726-731.doi: 10.16352/j.issn.1001-6325.2022.05.025

兔抗鼠MORF4L1 a多克隆抗体的制备与鉴定

李芳¹, 章元², 张业^1*

1.中国医学科学院基础医学研究所北京协和医学院基础学院生物化学与分子生物学系, 北京 100005;
2.中国医学科学院北京协和医学院北京协和医院神经外科,北京 100730

收稿日期:2022-02-07 修回日期:2022-03-22 出版日期:2022-05-05 发布日期:2022-04-28
通讯作者: * yezhang@pumc.edu.cn
基金资助:
国家自然科学基金(31871310)

Polyclonal antibody preparation and identification of rabbit anti-mouse MORF4L1 isoform a

LI Fang¹, ZHANG Yuan², ZHANG Ye^1*

1. Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences CAMS, School of Basic Medicine PUMC, Beijing 100005;
2. Department of Neurosurgery, Peking Union Medical College Hospital, CAMS & PUMC, Beijing 100730, China

Received:2022-02-07 Revised:2022-03-22 Online:2022-05-05 Published:2022-04-28
Contact: * yezhang@pumc.edu.cn

摘要/Abstract

摘要： 目的制备兔抗鼠MORF4L1 a变体的多克隆抗体以区分MORF4L1的a、b变体。方法根据对小鼠MORF4L1 a、b变体差异序列的抗原性、同源性的分析结果,合成MORF4L1 a的N端第52~66位的序列作为抗原肽(15肽,KSAVRPRRSEKSLKT),偶联KLH载体作为抗原免疫家兔,用Dot blot鉴定抗血清的特异性。对特异性良好的1号家兔抗血清进行抗原亲和层析纯化,用间接ELISA测定抗体效价。此外,构建MORF4L1 a、b变体的原核表达质粒和真核表达质粒。利用原核表达系统表达纯化后和在细胞中表达的MORF4L1 a、b蛋白进行Western blot鉴定抗体的特异性。结果 1号免疫兔抗血清能特异性识别多肽;纯化后抗体的效价达到1:512 000;成功构建pGEX-4T-2-M4S、pGEX-4T-2-M4L原核表达质粒和pCMV-3Tag-6-M4S、pCMV-3Tag-6-M4L真核表达质粒,并在原核系统和真核系统中成功表达。该抗体能与MORF4L1 a特异结合。结论成功制备了效价高、特异性较强的兔抗鼠MORF4L1 a抗体,为进一步揭示MORF4L1可变剪接的生物学意义和不同变体的生物学功能奠定了实验基础。

关键词: MORF4L1, 多克隆抗体, 变体

Abstract: Objective To prepare rabbit anti-mouse polyclonal antibody against MORF4L1 isoform to differentiate MORF4L1 isoform a and isoform b. Methods According to the antigenicity and autoploidy analysis of amino acid sequence of mouse MORF4L1 isoform a and isoform b,the N-terminal 52-66 amino acids peptide sequence (a 15 peptide,KSAVRPRRSEKSLKT) of mouse MORF4L1 isoform a was synthesized. Then the peptide was linked to a carrier protein KLH (keyhole limpet hemocyanin) as an antigen to prepare the antibody against MORF4L1 isoform a. The antiserum of rabbit 1# with good specificity was purified by antigen affinity chromatography and the antibody was titered by ELISA. In addition, the prokaryotic and eukaryotic expression plasmids of MORF4L1 isoform a and b were constructed. Western blot was used to identify the specificity of the antibody by using the MORF4L1 isoform a and b. Results The antiserum of rabbit 1# recognized polypeptide specifically. The titer of purified antibodywas 1:512 000.pGEX-4T-2-M4S,pGEX-4T-2-M4L prokaryotic expression plasmids and pCMV-3Tag-6-M4S,pCMV-3Tag-6-M4L eukaryotic expression plasmids were successfully constructed and expressed by prokaryotic and eukaryotic expression system. The antibody binds specifically to MORF4L1 isoform a. Conclusions Rabbit anti-mouse MORF4L1 a antibody with high titer and high specificity is successfully prepared, which lay an experimental foundation for further revealing the biological significance of MORF4L1 alternative splicing and the biological functions of different MORF4L1 isoforms.

Key words: MORF4L1, polyclonal antibody, isoform

中图分类号:

R392-33

李芳, 章元, 张业. 兔抗鼠MORF4L1 a多克隆抗体的制备与鉴定[J]. 基础医学与临床, 2022, 42(5): 726-731.

LI Fang, ZHANG Yuan, ZHANG Ye. Polyclonal antibody preparation and identification of rabbit anti-mouse MORF4L1 isoform a[J]. Basic & Clinical Medicine, 2022, 42(5): 726-731.

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兔抗鼠MORF4L1 a多克隆抗体的制备与鉴定

Polyclonal antibody preparation and identification of rabbit anti-mouse MORF4L1 isoform a

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