人F1F0-ATP合成酶β亚基的原核表达、纯化及抗体制备

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

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55000,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∶640000,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。

AIM:To express and purify the β 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 Ni2+ metal-chelating chromatograph,and refolded by...