重组Rab8截短体蛋白的原核表达、纯化和抗体制备

目的 获得Rab8纯化蛋白,制备多克隆抗体,为深入研究Rab8在病毒感染宿主细胞过程中的作用奠定基础.方法 首先从HepG2细胞中克隆Rab基因,然后从Rab8质粒中亚克隆Rab8 C端编码122个氨基酸的基因片段,构建Rab8与6His的融合蛋白原核表达质粒.原核表达与蛋白纯化后,免疫动物,制备Rab8多克隆抗体.采用ELISA法检测其效价.Western检验抗体特异性,间接免疫荧光染色法验证其免疫组化特性.结果 克隆了人Rab8编码基因,构建了表达Rab8 C端编码122个氨基酸的原核表达质粒pQE31-Rab8-122,经过大肠杆菌表达、镍亲和层析柱纯化,获得相对分子量约15 000的融合蛋白,免疫Balb/c小鼠后收获抗血清.ELISA显示抗体效价达1/20 000.Western blot和免疫荧光染色结果显示此多克隆抗体能够与原核表达的Rab8蛋白发生特异性反应,并能够识别HepG2细胞中内源性Rab8蛋白.结论 本研究获得原核表达的Rab8纯化蛋白,成功的制备了Rab8多克隆抗体,为进一步研究Rab8参与病毒感染宿主细胞提供了重要的实验材料.

Prokaryotic expression and purification of truncated Rab8 recombinant protein and preparation of polyclonal antibody against Rab8

Objective To obtain purified Rab8 protein and prepare polyclonal antibody against Rab8. Methods Rab8 was TA-cloned from HepG2 cells by RT-PCR and the prokaryotic expression plasmid containing 6His was constructed with the gene segment consisting of C terminal 122 amino acids of Rab8. After expression in E. coil and purification with Ni affinity column, the purified Rab8 protein was used to immunize Balb/c mouse tO obtain the antiserum. The titers and specificity of the antibody were measured by ELISA, Western blotting, and indirect immunofluorescence staining. Results Human Rab8 gene was cloned and the plasmid pQE31-Rab8-122 expressing a 15000 Da fusion protein was constructed. The antibody against Rab8 showed high titer and specificity, and could recognize native Rab8 protein. Conclusion The purified Rab8 protein and anti-Rab8 polyclonal antibody are obtained and it might be available for studying mechanism of Rab8 in viral infection.