人F1F0-ATP合酶β亚基的原核表达、纯化及多克隆抗体制备

[Prokaryotic expression, purification, and polyclonal antibody generation of beta subunit of human F1F0-ATP synthase].

作者信息

Yu Li-li, Zhang Yuan-jun, Zhang Xia, Wang Yi-bo, Lin Jian-bo, Ni Jian, Peng Yan

机构信息

Department of Pharmacy, Shanghai Jiaotong University, Shanghai 200240, China.

出版信息

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2008 Mar;24(3):253-5.

PMID:18328186

Abstract

AIM

To express and purify the beta subunit of human F1F0-ATP synthase (hATP5B) in prokaryotic system, and generate its polyclonal antibody in rabbits.

METHODS

The coding sequence of hATP5B mature peptide was amplified by RT-PCR from human umbilical vein endothelial cells (HUVEC) and then cloned into prokaryotic expression vector pET28a(+). The plasmid was transformed into E.coli BL21(DE3) to express hATP5B in reponse to IPTG induction. Expressed protein was purified by Ni(2+) metal-chelating chromatograph, and refolded by dialysis. The products were analyzed by SDS-PAGE and Western blot. Refolded protein was injected into rabbits to generate polyclonal antibody. The titer of the polyclonal antibody was determined by indirect ELISA. The specificity and the binding ability were detected by Western blot and cell immunofluorescence analysis.

RESULTS

DNA sequencing confirmed that the coding sequence of hATP5B mature peptide was completely concordant with the original sequence (NM_001686, hATP5B's GenBank accession number). SDS-PAGE showed that the relative molecular masses (M(r)) of the expressed, purified, and refolded products were about relative molecular masses M(r) 55,000, which was in accordance with the predicted. Grayscale scanning showed that the expressed recombinant hATP5B (rhATP5B) accounted for 36.8% of the total bacteria protein, and the purity of purified product was 98.3%, of refolded 99.1%. The result of Western blot is positive. The titer of the polyclonal antibody was 1:640,000, and it specifically recognized the native antigen in HUVEC.

CONCLUSION

hATP5B expressed in prokaryotic system has strong immunogenicity, and the polyclonal antibody with high titer and specificity was obtained from immunization of rabbits. The high level prokaryotic expression of hATP5B and the preparation of its antibody lay the foundation for further function research of hATP5B.

摘要

目的

在原核系统中表达并纯化人F1F0 - ATP合酶β亚基（hATP5B），并在兔体内制备其多克隆抗体。

方法

通过RT - PCR从人脐静脉内皮细胞（HUVEC）中扩增hATP5B成熟肽的编码序列，然后克隆到原核表达载体pET28a(+)中。将质粒转化到大肠杆菌BL21(DE3)中，经IPTG诱导表达hATP5B。表达的蛋白通过Ni(2+)金属螯合层析纯化，经透析复性。产物经SDS - PAGE和Western印迹分析。将复性后的蛋白注射到兔体内制备多克隆抗体。通过间接ELISA测定多克隆抗体的效价。通过Western印迹和细胞免疫荧光分析检测其特异性和结合能力。

结果

DNA测序证实hATP5B成熟肽的编码序列与原始序列（NM_001686，hATP5B的GenBank登录号）完全一致。SDS - PAGE显示表达、纯化和复性产物的相对分子质量（M(r)）约为55,000，与预测值相符。灰度扫描显示表达的重组hATP5B（rhATP5B）占总细菌蛋白的36.8%，纯化产物纯度为98.3%，复性后为99.1%。Western印迹结果为阳性。多克隆抗体效价为1:640,000，能特异性识别HUVEC中的天然抗原。