PCV2 Cap蛋白原核表达、纯化及多克隆抗体的制备  

Prokaryotic Expression and Purification of PCV2 Cap Protein and Preparation of Polyclonal Antibody

作　　者：李钰昌 郑亮[1,2] 张华 吴志军[1,2,3] 肖莉杰 曹宏伟[1,2,3] Li Yuchang;Zheng Liang;Zhang Hua;Wu Zhijun;Xiao Lijie;Cao Hongwei(College of Life Science and Technology,Heilongjiang Bayi Agricultural University,Daqing163319;College of Animal Science and Technology,Heilongjiang Bayi Agricultural University;Biotechnology Center,Heilongjiang Bayi Agricultural University)

机构地区：[1]黑龙江八一农垦大学生命科学技术学院,大庆163319 [2]黑龙江八一农垦大学动物科技学院 [3]黑龙江八一农垦大学生物技术中心

出　　处：《黑龙江八一农垦大学学报》2023年第1期78-83,130,共7页journal of heilongjiang bayi agricultural university

基　　金：黑龙江省自然科学基金联合引导项目(LH2019C046)。

摘　　要：猪圆环病毒2型是一种常见的猪感染性病毒,Cap蛋白为猪圆环病毒2型的核衣壳蛋白,也是该病毒最主要的结构蛋白,是目前猪圆环病毒2型基因工程疫苗研制的关键蛋白。研究构建了Cap蛋白原核表达质粒pET28a-His-Cap,转化Transetta(DE3)表达菌株,IPTG诱导表达,经SDS-PAGE及蛋白免疫印迹试验验证,重组蛋白主要以可溶性形式表达,少数为包涵体蛋白。以Ni-NTA树脂进行蛋白纯化,纯化后蛋白浓度为629μg·mL^(-1),采用透析袋浓缩将重组蛋白浓缩,浓缩后蛋白终浓度为1.8 mg·mL^(-1)。将重组蛋白与弗氏佐剂等体积混合,充分乳化后制备抗原,免疫兔子采集全血,分离血清。经蛋白免疫印迹试验,该多抗与重组蛋白存在免疫反应性,ELISA检测抗体效价可达1∶32000,IFA试验证明该多抗能够特异性识别猪圆环病毒2型。Cap蛋白多克隆抗体的成功制备,为后续猪圆环病毒2型基因工程疫苗制备过程中验证Cap体外蛋白表达提供了材料。Porcine circovirus type 2 was a common porcine infectious virus.The Cap protein was the nucleocapsid protein of porcine circovirus type 2,and also the most important structural protein of the virus.It was currently the genetic engineering of porcine circovirus type 2 key protein for vaccine development.The Cap protein prokaryotic expression plasmid pET28a-His-Cap was constructed,transformed into Transetta(DE3)expression strains,and induced by IPTG.It was verified by SDS-PAGE and Western blot experiments that the recombinant protein was mainly expressed in a soluble form,with a few inclusion bodies.protein.The protein was purified with Ni-NTA resin,and the purified protein concentration was 629μg·mL^(-1).The recombinant protein was concentrated by dialysis bag concentration,and the final concentration of the concentrated protein was 1.8 mg·mL^(-1).The recombinant protein was mixed with Freund’s adjuvant in equal volumes,and the antigen was prepared after fμLl emμLsification.The rabbits were immunized to collect whole blood and separate the serum.Western blotting test showed that the polyclonal antibody was immunoreactive with the recombinant protein.The antibody titer detected by ELISA could reach 1∶32000.The IFA test proved that the polyclonal antibody can specifically recognize porcine circovirus type 2.The successful preparation of Cap protein polyclonal antibody provided material for the verification of Cap protein expression in vitro during the subsequent preparation of porcine circovirus type 2 genetically engineered vaccine.

关 键 词：PCV2 Cap蛋白 原核表达 多克隆抗体 

分 类 号：S828[农业科学—畜牧学] S852.651[农业科学—畜牧兽医]