摘要
试验拟通过构建A亚群禽白血病病毒(ALV-A) p27基因原核表达载体,从而制备抗p27蛋白鼠源多克隆抗体。将感染ALV-A毒株的DF-1细胞反复冻融后提取RNA并反转录为cDNA,利用PCR扩增获得ALV-A p27基因,将目的基因克隆至pColdⅠ载体,并转化入大肠杆菌BL21(DE3)菌中进行诱导表达,经镍柱纯化后免疫28～32日龄的雌性小鼠,采用间接ELISA法测定抗体效价,Western blot检测抗体特异性。试验经PCR扩增出750 bp左右的特异性条带,并成功构建pColdⅠ-ALV-A-p27原核表达载体,加入终浓度为1 mmol/L的IPTG于18℃诱导表达18～24 h成功得到重组蛋白,该蛋白主要以包涵体形式存在。间接ELISA检测显示,制备的鼠源多克隆抗体血清抗体效价为1∶3 200;Western blot检测结果表明,制备的多克隆抗体能特异性识别原核表达的p27蛋白。试验结果在ALV的抗原分析、血清学诊断等方面有着重要的应用价值。
The experiment was aimed to prepare polyclonal antibodies against p27 by constructing prokaryotic expression vector of avian leukosis virus subgroup A(ALV-A) p27 gene.The RNA was extracted from DF-1 cells infected with ALV-A virus strain after repeated freezing and thawing,and reversely transcribed into c DNA.The ALV-A p27 gene was amplified by PCR,and the target gene was cloned into pColdⅠ vector and transformed into E.coli BL21(DE3)strain for induction expression.The expression protein was purified by nickel column and inoculated into female mice at 28 to 32 days of age.And the antibody titer was determined by indirect ELISA and the antibody specificity was detected by Western blot.The specific band about 750 bp was amplified by PCR and the prokaryotic expression vector of pColdⅠ-ALV-A-p27 was successfully constructed.The recombinant protein was successfully obtained by inducing the expression of IPTG with the final concentration of 1 mmol/L at 18 ℃ for 18 to 24 hours,and the protein was mainly in the form of inclusion body.The indirect ELISA assay showed that the titer of murine multi-antibody serum antibody was 1∶3 200;the results of Western blot showed that the prepared polyclonal antibody could specifically recognize the prokaryotic expression of p27 protein.The test results had important application value in antigen analysis and serological diagnosis of ALV.
作者
徐丽
林汉卿
温贵兰
李昌红
张升波
田浪
杨佰启
陈彦希
文明
龚新勇
XU Li;LIN Hanqing;WEN Guilan;LI Changhong;ZHANG Shengbo;TIAN Lang;YANG Baiqi;CHEN Yanxi;WEN Ming;GONG Xinyong(College of Animal Science,Guizhou Uiversity,Guiyang,Guizhou 550025;Key Laboratory of Animal Disease and Veterinary Public Health in Guizhou Province,Guiyang,Guizhou 550025;Animal Disease Prevention and Control Center of Sinan County,Tongren,Guizhou 565100)
出处
《中国家禽》
北大核心
2019年第2期15-19,共5页
China Poultry
基金
贵州地方品种鸡ALV快速检测与中药控制关键技术研究(黔科合支撑[2019]2286)
贵州省科学技术基金(黔科合J字[2015]2046号)
国家自然科学基金项目(31460668)
贵州省高层次创新型人才培养(黔财教[2017]号)
贵州大学博士基金(贵大人基合字(2013)12号)
贵州省科技计划课题(联合基金)(黔科合LH字[2014]7666)
贵州省科技平台及人才团队计划(黔科合平台人才[2018]5253)
贵州省研究生工作站计划项目[GZZ2017002]
关键词
ALV-A
P27
原核表达
鼠源多克隆抗体
ALV-A
p27
prokaryotic expression
mouse-derived polyclonal antibodies
