摘要
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(K15P_(ex8))序列做为模板,构建K15P_(ex8)的表达载体p QE-80L-K15P_(ex8)。诱导重组菌蛋白表达,免疫新西兰兔,制备抗K15P_(ex8)多克隆抗体,并用间接ELISA法测定抗体效价。将p FJ和p FJ-K15P两种重组质粒分别转入HEK 293T细胞,用Western blot法检测抗K15P_(ex8)多克隆抗体对K15P-Flag蛋白的识别。结果由K15P_(ex8)制备的抗K15P_(ex8)多克隆抗体效价大于1∶6 400,能特异性识别重组质粒p FJ-K15P转染293T细胞产生的K15P-Flag融合蛋白。结论初步证明K15P_(ex8)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。
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 transfeeted HEK 293T cells to produce K15P-Flag fusion protein,which was identified by Western blot. Re- sults The titer of the polyclonal antibody against K15P 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 K15P could react with the K15P-Flag fusion protein.
出处
《安徽医科大学学报》
CAS
北大核心
2016年第1期1-4,共4页
Acta Universitatis Medicinalis Anhui
基金
国家自然科学基金(编号:81271837)
安徽高校省级自然科学研究项目(编号:KJ2012A161)
安徽医科大学博士科研经费资助项目(编号:XJ200914)
