猪细小病毒7型VP2蛋白的原核表达及其多克隆抗体的制备  

Prokaryotic expression of Porcine parvovirus type 7 VP2 protein and preparation of its polyclonal antibody

作　　者：吕紫欣 张鑫杰 薛少华 黄喜荣 刘建奎[1,3,4] 戴爱玲 LYU Zixin;ZHANG Xinjie;XUE Shaohua;HUANG Xirong;LIU Jiankui;DAI Ailing(College of Life Sciences,Longyan University,Longyan 364012,China;College of Animal Science(College of Bee Science),Fujian Agriculture and Forestry University,Fuzhou 350002,China;Fujian Engineering Research Center for Swine Disease Control and Prevention,Longyan 364012,China;Fujian Provincial Key Laboratory for the Prevention and Control of Animal Infectious Diseases and Biotechnology,Longyan 364012,China)

机构地区：[1]龙岩学院生命科学学院,福建龙岩364012 [2]福建农林大学动物科学学院(蜂学学院),福州350002 [3]福建省生猪疫病防控工程技术研究中心,福建龙岩364012 [4]福建省家畜传染病防治与生物技术重点实验室,福建龙岩364012

出　　处：《黑龙江畜牧兽医》2024年第14期56-60,共5页Heilongjiang Animal Science And veterinary Medicine

基　　金：中央引导地方科技发展专项(2021L3028);福建省科技重大专项(2019NZ09005);龙岩市科技计划重大项目(2019LY1001)。

摘　　要：为了表达猪细小病毒7型(PPV7)VP2蛋白(由Cap基因编码)并制备其多克隆抗体,试验从GenBank数据库中下载PPV7 Cap基因序列,选择不同毒株的高频序列作为目的基因序列,使用T4 DNA连接酶将目的基因与pET-32a表达载体连接,将连接产物转化至DH5α感受态细胞中,并使用IPTG对重组菌进行诱导,对表达的重组蛋白进行可溶性分析与纯化并通过Western-blot检测重组蛋白的反应原性,用纯化后的重组蛋白免疫新西兰大白兔制备多克隆抗体并分别通过间接ELISA方法、Western-blot检测多克隆抗体的效价及其与重组蛋白的反应性。结果表明:诱导后的重组菌在56 ku处出现明显条带,重组蛋白主要以包涵体的形式表达;纯化后的重组蛋白为单一条带,质量浓度为0.46 mg/mL,能与His标签抗体特异性结合;制备的多克隆抗体的效价为1∶51200,能与重组蛋白特异性结合。说明PPV7 VP2蛋白获得了成功表达,该蛋白具有良好的反应原性和免疫原性,所制备的多克隆抗体效价较高。In order to express Porcine parvovirus type 7(PPV7) VP2 protein(encoded by Cap gene) and prepare its polyclonal antibody, in the experiment, PPV7 Cap gene sequences from GenBank database were downloaded and the high frequency sequences of different strains were selected as the target gene sequences;T4 DNA ligase was used to connect the target gene with pET-32a expression vector and the ligated product was transformed into DH5α competent cells;the recombinant bacteria were induced by IPTG, and the expressed recombinant protein was analyzed for solubility and purification, and the immunogenicity of the recombinant protein was detemined by Western-blot;the purified recombinant protein was used to immunize New Zealand White rabbits for the preparation of polyclonal antibody;the titer of the polyclonal antibody and the reactivity with the recombinant protein were detected by indirect ELISA and Western-blot, respectively. The results showed that the recombinant bacteria showed obvious bands at 56 ku after induction, and the recombinant protein was mainly expressed in the form of inclusion bodies;the purified recombinant protein was a single band with a mass concentration of 0.46 mg/mL, which could bind specifically to the His-tag antibody;the titer of the prepared polyclonal antibody was 1∶51 200. Polyclonal antibody could bind specifically to the recombinant protein. The results suggested that PPV7 VP2 protein was successfully expressed, which had good reactivity and immunogenicity, and the prepared polyclonal antibody had high titer.

关 键 词：猪细小病毒7型 VP2蛋白 Cap基因 原核表达 多克隆抗体 

分 类 号：S852.65[农业科学—基础兽医学]