利用噬菌体展示技术筛选草鱼呼肠孤病毒VP39蛋白相互作用多肽  

SCREENING OF PEPTIDES INTERACTING WITH GRASS CARP REOVIRUS VP39 PROTEIN BY PHAGE DISPLAY TECHNOLOGY

作　　者：龙晨 徐宁 谢雅晴 吕利群[1] LONG Chen;XU Ning;XIE Ya-Qing;LÜLi-Qun(National Pathogen Collection Center for Aquatic Animals,Shanghai Ocean University,Shanghai 201306,China;Key Laboratory of Freshwater Aquatic Genetic Resources,Ministry of Agriculture and Rural Affairs,Shanghai Ocean University,Shanghai 201306,China)

机构地区：[1]上海海洋大学国家水生动物病原库,上海201306 [2]上海海洋大学农业农村部淡水水产种质资源与利用重点实验室,上海201306

出　　处：《水生生物学报》2023年第6期859-865,共7页Acta Hydrobiologica Sinica

基　　金：国家现代农业产业技术体系项目(CARS-45)资助。

摘　　要：VP39是草鱼呼肠孤Ⅲ型病毒(GCRV GenotypeⅢ,GCRV-Ⅲ)S9基因编码的蛋白,为研究VP39蛋白在GCRV-Ⅲ感染草鱼细胞过程中行使的生物学功能,将克隆VP39基因序列并构建原核表达载体pET32a-VP39,通过原核表达得到VP39-HIS融合蛋白;利用VP39蛋白溶液免疫小鼠,制备鼠抗VP39多克隆抗体,通过Western Blot对抗体进行评估;利用制备的多克隆抗体探究GCRV-Ⅲ感染细胞过程中VP39蛋白表达动力学;利用噬菌体展示技术筛选与VP39蛋白特异性结合的多肽序列并进行分析。SDS-PAGE电泳结果显示,VP39-HIS融合蛋白可良好溶于PBS中,蛋白大小约为39 kD;Western Blot检测表明实验所制备的VP39多克隆抗体在1﹕10000稀释比例下,既能识别原核表达的VP39-HIS融合蛋白,也能识别GCRV-Ⅲ感染CIK细胞后表达的VP39蛋白,具有良好的效价与特异性;在病毒侵染过程中,VP39前期表达量较少,在中后期大量表达;噬菌体展示技术筛选出两条多肽与VP39蛋白有高度亲和性,经过在NCBI上比对后发现草鱼基因组中有7个基因与筛出的多肽具有同源性,表明其可能与VP39存在相互作用。研究制备了鼠抗VP39多克隆抗体,为GCRV-Ⅲ的免疫学检测方法提供了新的路径;结合多肽的筛选也为VP39在GCRV-Ⅲ侵染过程中参与的生物学功能研究奠定了基础。VP39 is a protein encoded by S9 gene of typeⅢgrass carp reovirus(GCRV-Ⅲ).In order to study the biological function of VP39 protein in the process of GCRV infection of grass carp cells,the sequence of VP39 gene was cloned and the prokaryotic expression vector PET32A-VP39 was constructed.The fusion protein VP39-HIS was obtained by using prokaryotic expression method.Mouse anti-VP39 polyclonal antibody was prepared by immunizing mice with VP39 solution protein,and the antibody was evaluated by Western Blot.The polyclonal antibody was used to investigate the expression dynamics of VP39 protein in GCRV infected cells.The results of SDS-PAGE showed that the fusion protein of vp39-his was well soluble in PBS and the protein size was about 39 kDa.Western Blot analysis showed that the prepared VP39 polyclonal antibody could recognize both the prokaryotic expression of VP39-HIS fusion protein and the expression of VP39 protein after GCRV infection with CIK cells at the dilution ratio of 1﹕10000,showing good titer and specificity.In the process of virus infection,the expression of VP39 was low in the early stage and high in the middle and late stage.Two peptides were screened by phage display technology for specific binding to VP39 protein,and further bioinformatics analysis also found that 7 genes in grass carp genome had homology with the peptide,indicating that these genes may interact with VP39.In this study,mouse anti-VP39 polyclonal antibody was prepared,which provided a new immunological method for GCRV-Ⅲdetection.The screening of binding peptides also laid a foundation for the study of the biological function of VP39 in the process of GCRV infection.

关 键 词：Ⅲ型草鱼呼肠孤病毒 VP39蛋白 多克隆抗体 噬菌体展示技术 

分 类 号：S943[农业科学—水产养殖]