Ⅰ型鸭肝炎病毒VP1基因的克隆表达及结合肽的筛选  

Expression of recombinant VP1 protein of duck hepatitis virus 1 and screening of specific binding peptides

作　　者：王臣[1] 杨静[1] 张巫凡 郭香玲[1] 吴庭才[1] 陈溥言[2] 

机构地区：[1]河南科技大学兽医肿瘤免疫学重点实验室,河南洛阳471003 [2]南京农业大学农业部动物细菌学重点实验室,江苏南京210095

出　　处：《中国兽医科学》2016年第4期473-478,共6页Chinese Veterinary Science

基　　金：国家自然科学基金项目(31101792);河南省高校青年骨干教师项目(2012GGJS-077)

摘　　要：为鉴定与I型鸭肝炎病毒(DHV-1)特异性结合的多肽,通过基因工程方法获得DHV-1 VP1蛋白,并以DHV-1 VP1蛋白作为靶标,利用噬菌体随机12肽库进行3轮亲和筛选。ELISA鉴定结果显示,10个噬菌体克隆与VP1蛋白有较强的体外结合特性;竞争抑制试验结果表明,VP1蛋白对噬菌体克隆P4与P6结合具有阻断作用。对P4和P6克隆的序列测定结果显示,插入的12肽序列分别为P4-12:LLSPGTHHSPWP;P6-12:LLADTTHHRPWT,其保守序列为THHXPW。体外试验结果显示,多肽P4-12和P6-12本身对鸭胚成纤维细胞的增殖无明显影响,多肽P4-12和P6-12在0.02、0.2、2、20 g/m L浓度时均能显著降低DHV-1在鸭胚成纤维细胞中的病毒效价和病毒拷贝数。以上结果表明,多肽P4-12和P6-12均能抑制DHV-1在鸭胚成纤维细胞中的增殖,这一研究结果为进一步探明DHV-1与宿主细胞的相互作用及抗病毒制剂的开发提供了试验线索。To identify the peptides that specifically bind to duck hepatitis virus 1（DHV-1）,we cloned DHV-1 VP1 gene and provided evidence that the gene in a recombinant prokaryotic expression plas mid was successfully expressed in E.coli BL21.To identify the peptides that specifically binding to DHV-1 VP1 protein,VP1 protein was used to screen out binding peptides from the 12-mer random phage display peptide library.After three rounds of biopanning,ELISA and competitive inhibition test, two positive phage clones（P4 and P6） were found and the sequencing results showed that they were LLSPGTHHSPWP（P4-12）,and LLADTTHHRPWT（P6-12）,respectively,both of which shared a conserved sequence THHXPW.Furthermore,TCID50 and quantitative RT-PCR were used to detect the effect of VP1 protein binding peptide on the proliferation of DHV-1 in duck embryo fibroblast（DEF）.The results indicated that P4-12 and P6-12 peptide had no significant effect on the proliferation of DEF.However,P4-12 and P6-12 peptides（0.02,0.2,2 and 20 g /m L,respectively）significantly reduced the virus titer and virus copes of DHV-1 in DEF.Taken together,this study result provides novel insights on the interaction mechanism of DHV-1 and host cells.

关 键 词：I型鸭肝炎病毒 VP1蛋白 鸭胚成纤维细胞 噬菌体展示 结合肽 

分 类 号：S852.659.6[农业科学—基础兽医学]