土拨鼠肝炎病毒核心蛋白质粒的构建、原核表达及多克隆抗体的制备与鉴定  

Cloning and Prokaryotic Expression of Woodchuck Hepatitis Virus Core Gene,and Preparation and Identification of Polyclonal Antibodies against Woodchuck Hepatitis Virus Core Protein

作　　者：张振华[1] 田拥军[1] 李磊[1] 王宝菊[1] 孟忠吉[1] 陆蒙吉 杨东亮[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院临床免疫研究室 [2]德国Essen大学医学院病毒研究所,essen45149

出　　处：《华中科技大学学报（医学版）》2007年第5期567-570,577,F0002,共6页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

基　　金：国家"十五"科技攻关计划资助项目(No2001BA705B05)

摘　　要：目的构建土拨鼠肝炎病毒核心蛋白质粒并进行原核表达、抗体制备。方法应用基因工程技术将编码截短型土拨鼠肝炎病毒核心蛋白(WHcAg1～149aa)的基因片段装入原核表达载体pQE60上,在JM109菌内进行诱导表达,使用切胶回收及Ni-NTA柱两种方法纯化目的蛋白。将纯化蛋白免疫家兔制备多克隆抗体,并采用酶联免疫吸附实验、免疫组织化学及Western blot检测抗体的灵敏度和特异性。结果成功地构建了含截短型土拨鼠肝炎病毒核心区基因的质粒并获得表达,经纯化得到了分子量约为16.5kD的重组核心蛋白,免疫家兔获得了高效价的特异性多克隆抗体,而且与HBcAg有交叉反应。结论获得的重组截短型土拨鼠肝炎病毒核心抗原(1～149aa)纯度高,免疫原性强。获得的兔抗-WHc效价高,特异性好,与HBcAg有交叉反应。Objective To construct the prokaryotic expression plasmid expressing woodchuck hepatitis virus core antigen（WHcAg） and prepare polyclonal antibodies.Methods DNA fragment coding truncated WHcAg（1-149aa） was cloned into pQE60 vector using gene engineering technique.Recombinant plasmid was transformed into host strain JM109 and then protein expression was induced.Recombinant protein was purified by Ni-NTA purification column and acrylamide gel electrophoresis.Polyclonal antibodies were developed by immunizing rabbits with purified recombinant protein,and the sensitivity and specificity were tested by ELISA,immunohistochemistry and Western blot assays.Results Recombinant plasmid expressing truncated WHcAg was constructed successfully.A protein with the apparent molecular weight of 16.5 was successfully expressed and purified.High titer polyclonal antibody with a high specificity was obtained by immunizing rabbits with the purified recombinant protein.There was a cross reaction between the polyclonal antibody and HBcAg.Conclusion The truncated recombinant WHcAg obtained in this study was highly purified and had a strong antigenicity.The polyclonal antibodies developed had both high sensitivity and high specificity and had a cross reaction with HBcAg.

关 键 词：土拨鼠肝炎病毒 核心蛋白 原核表达 多克隆抗体 抗原抗体交叉反应 

分 类 号：R392[医药卫生—免疫学]