抗卡波济肉瘤相关疱疹病毒vIL-6蛋白合成肽多克隆抗体的制备及初步应用  

Generation and characterization of polyclonal antibody against synthetic Kaposi' s sarcoma-associated herpesvirus-encoding peptides of viral interleukin-6 and its application

作　　者：张晓洁 陈菲[3] 郭园园[3] 秦娣[3] 卢春[3] 严沁[3] 

机构地区：[1]南京医科大学第一附属医院 [2]江苏省妇幼保健院检验科,南京210036 [3]南京医科大学病原生物学系,南京210029

出　　处：《临床检验杂志》2015年第3期175-180,189,共7页Chinese Journal of Clinical Laboratory Science

基　　金：江苏省自然科学基金青年基金(BK20140908);南京医科大学科技发展基金(2013NJMU007)

摘　　要：目的制备与鉴定抗卡波济肉瘤相关疱疹病毒(Kaposi's sarcoma-associated herpesvirus,KSHV)病毒白细胞介素6(viral interleukin-6,v IL-6)多克隆抗体,并探讨将其初步应用于检测天然v IL-6蛋白的价值。方法设计并合成3条v IL-6候选肽,免疫新西兰兔,制备抗v IL-6多克隆抗体,用间接ELISA法鉴定该抗体的效价。构建重组质粒p HAGE-v IL-6并转染293T细胞和EA.hy926细胞,表达并纯化v IL-6-His融合蛋白,通过western blot鉴定抗v IL-6多克隆抗体对该蛋白质的识别。通过western blot和免疫荧光方法检测对KSHV阳性细胞中天然v IL-6蛋白的识别。结果由3号v IL-6候选合成肽制备的抗v IL-6多克隆抗体效价大于8 000,能特异性识别重组质粒p HAGE-v IL-6转染293T细胞和EA.hy926细胞产生的v IL-6-His融合蛋白,以及天然v IL-6蛋白。结论本实验采用合成肽作为半抗原制备的抗v IL-6多克隆抗体,可以应用于检测重组和天然v IL-6蛋白。Objective To generate rabbit polyclonal antibody against Kaposi＇s sarcoma-associated herpesvirus（ KSHV） encoding peptides of viral interleukin-6（ v IL-6）,identify the specificity of the prepared polyclonal v IL-6 antibody by constructing the recombinant plasmid,and detect the native viral protein using the prepared polyclonal v IL-6 antibody. Methods After the bioinformatics analysis for the conformational antigenic epitopes of the v IL-6,three candidate polypeptides of v IL-6 were designed and synthesized. New Zealand white rabbits were immunized with the synthetic peptides to generate polyclonal antibodies against v IL-6. Enzyme-linked immunosorbent assay（ ELISA） was performed to characterize the titers of the prepared polyclonal antibodies. The recombinant plasmid,p HAGE-v IL-6,was constructed to transfect 293 T and EA. hy926 cells to obtain v IL-6-His fusion protein,which was identified by western blot. The native viral protein in KSHV-infected primary effusion lymphoma（ PEL） cells was recognized using the polyclonal antibodies against v IL-6 by western blot and immunofluorescence assay（ IFA）. Results The titer of the polyclonal antibodies against v IL-6 prepared with the third antigenic polypeptide was greater than 8 000 based on ELISA and could react specifically with the v IL-6-His fusion protein in 293 T and EA. hy926 cells as well as the native viral protein in PEL cells. Conclusion We successfully prepared the polyclonal antibody against v IL-6 to react with fusion protein and native viral protein by using the synthesized peptides as hapten,which should be available for detecting recombinant and native v IL-6 protein.

关 键 词：卡波济肉瘤相关疱疹病毒 病毒白细胞介素6蛋白 合成肽 多克隆抗体 

分 类 号：R392.11[医药卫生—免疫学]