人F1F0-ATP合成酶β亚基的原核表达、纯化及抗体制备

目的: 原核表达、纯化人F1F0-ATP合成酶β亚基(hATP5B)并制备其多克隆抗体.方法: 以人脐带静脉内皮细胞 (HUVEC) 总RNA为模板, 通过RT-PCR扩增hATP5B 成熟肽编码序列, 克隆入原核表达载体pET28a( ), 转化大肠杆菌E.coli BL21(DE3)并诱导表达, 表达产物用Ni2 螯合层析纯化, 纯化产物用透析复性, 产物用SDS-PAGE和 Western blot进行鉴定.复性产物免疫家兔, 制备多克隆抗体; 多抗的效价用间接ELISA法检测, 并用Western blot和细胞免疫荧光分析多抗的特异性和抗原结合性.结果: 测序证实获得hATP5B成熟肽编码序列.SDS-PAGE鉴定表明, 表达、纯化、复性产物的相对分子质量(Mr)均约55 000, 与理论值相符.灰度扫描分析重组hATP5B(recombinant hATP5B, rhATP5B)的表达量占菌体蛋白总量的36.8%, 纯化产物纯度达98.3%, 复性产物纯度达99.2%.Western blot反应阳性.多抗的平均抗体效价为1∶640 000, 可特异识别HUVEC中的天然抗原.结论: 原核表达的hATP5B具有良好的免疫原性, 其免疫家兔后获得的多抗具有高效价和特异性.hATP5B的原核高效表达和其抗体制备为进一步研究hATP5B的功能奠定了实验基础.

Prokaryotic expression, purification, and polyclonal antibody generation of β subunit of human F1F0-ATP synthase

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 of the expressed, purified, and refolded products were about relative molecular masses 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.