禽白血病病毒衣壳蛋白表达载体的构建及应用  

Construction and Application of Caspid Protein Eukaryotic Expression Vector of Avian Leukosis Virus

作　　者：孙舒 胡海 程晓薇[1,2] 秦爱建 钱琨[1,2,3] SUN Su HU Hai CHENG Xiaowei QIN Aijian QIAN Kun(Key Lab for Avian Preventive Medicine,Ministry of Education, Yangzhou University, Yangzhou, Jiangsu 225009 Key Laboratory of Jiangsu Preventive Veterinary Medieine, Yangzhou University, Yangzhou, Jiangsu 225009 Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu 225009)

机构地区：[1]扬州大学教育部禽类预防医学重点实验室,江苏扬州225009 [2]扬州大学江苏省动物预防医学重点实验室,江苏扬州225009 [3]江苏高校动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009

出　　处：《中国家禽》2017年第18期18-22,共5页China Poultry

基　　金：国家重点研发计划(2016YFD0500803;2017YFD0500700);国家自然科学基金(31772734);江苏省优势学科项目;公益性行业(农业)科研专项(201203055);扬州大学新世纪人才项目

摘　　要：为利用分段表达载体鉴定禽白血病病毒(ALV)衣壳蛋白单克隆抗体识别区域,构建ALV衣壳蛋白p27全长及分段真核表达载体。将实验室保存的pGEX-6p-1-p27质粒双酶切亚克隆到pcDNA3.1真核表达载体中,构建pcDNA3.1-p27全长表达质粒。同时设计引物,扩增p27基因1~480 bp的A片段以及241~720 bp的B片段,构建pcDNA3.1-p27A和pcDNA3.1-p27B分段表达载体,并在DF-1细胞中暂态表达,利用IFA和Western blot方法鉴定病毒衣壳蛋白单克隆抗体的识别区域。结果表明:重组质粒均能正确表达,并鉴定出两株单克隆抗体5D3和4F12的抗原识别区域均位于衣壳蛋白p27蛋白碳端161~240位氨基酸区域。研究成功构建并表达了ALV衣壳蛋白p27的全长和分段真核表达载体,并利用分段表达质粒鉴定了两株单克隆抗体的抗原识别区域,为深入研究衣壳蛋白p27的生物学功能提供了生物材料。In order to identify antigen recognition region of monoclnnal antibodies aganist capsid protein of avian leukosis virus, a full-length and segmented the eukaryotic expression vectors of capsid protein p27 of avian leukosis virus （ALV）were constructed in this study. The full-length p27 gene was digested by restricted enzyme from pGEX-6p-1-p27 and subcloned into the eukaryotic expression vector pcDNA-3.1. Primers were designed for the amplification of p27 fragment A （1 to 480 bp）and fragment B （241 to 720 bp）. And both fragments were cloned into pcDNA3.1-flag expression vector. The recombinant plasmids were transfected into DF-1 cells for transient expression. Indirect immunofluorescence （IFA）and Western blot （Western blot）assay were performed to identify antigen recognition region of monoclonal antibodies. The results of IFA and Western blot showed that the recombinant plasmids were expressing correctly. And the recognition regions of monoclonal antibodys 5D3 and 4F12 were idetified at 161st to 240th amino acid region in the carbon side of the p27 protein. In this study, the full-length and segmented eukaryotic expression vectors of capsid protein p27 of ALV were successfully constructed and expressed, and the antigen recognition region of two monoclonal antibodies were identified by segmented expression plasmids, which provided biological materials for the further biofunctional study of capsid protein p27.

关 键 词：衣壳蛋白p27 真核表达载体 单克隆抗体 抗原识别区域 

分 类 号：S855-3[农业科学—临床兽医学]