Objective To generate rabbit polyclonal antibody against Kaposi 's sarcoma-associated herpesvirus (KSHV) encoding peptides of K15, identify the detection of K15P protein in cell. Methods After selecting the eighth exon (EX8) sequence of K15P gene as a template, the recombinant plasmid named pQE-80L-K15Pex8 was constructed. Recombinant protein was induced and expressed. New Zealand white rabbits were immunized with the K15Pex8 to generate polyclonal antibodies against K15P. Indirect enzyme-linked immunosorbent assay (ELISA) was applied to characterize the titers of the polyclonal antibody. The two recombinant plasmids, pFJ and pFJ-K15P, were transfected HEK 293T cells to produce K15P-Flag fusion protein,which was identified by Western blot. Re-sults The titer of the polyclonal antibody against K15 P was more than 1: 6 400 with indirect ELISA and could re-act specifically with the K15P-Flag fusion protein in 293T cells. Conclusion We preliminarily find K15Pex8 pro-tein has immunoreactivity and the rabbit polyclonal antibody against K15 P could react with the K15 P-Flag fusion protein.%目的 制备卡波氏肉瘤相关疱疹病毒( KSHV) K15蛋白多克隆抗体,并将其应用于细胞内K15蛋白的检测. 方法选择K15基因第8 个外显子( K15Pex8 )序列做为模板,构建 K15Pex8的表达载体 pQE-80L-K15Pex8. 诱导重组菌蛋白表达,免疫新西兰兔,制备抗K15Pex8多克隆抗体,并用间接ELISA法测定抗体效价. 将pFJ和pFJ-K15P两种重组质粒分别转入HEK 293T细胞,用Western blot法检测抗K15Pex8多克隆抗体对K15P-Flag蛋白的识别. 结果 由K15Pex8制备的抗K15Pex8多克隆抗体效价大于1 : 6 400,能特异性识别重组质粒pFJ-K15P转染293T细胞产生的K15P-Flag融合蛋白. 结论 初步证明K15Pex8蛋白有免疫反应性,该多克隆抗体可用于重组的K15P-Flag融合蛋白的检测.
展开▼
