新城疫病毒F蛋白和禽致病性大肠杆菌OmpA嵌合蛋白的表达及双特异性抗体制备  

Expression of chimeric protein of Newcastle disease virus F and avian pathogenic Escherichia coli OmpA and preparation of bispecific antibody

作　　者：袁橙 王永娟 郭梦娇 刘思琪 周雨豪 YUAN Cheng;WANG Yongjuan;GUO Mengjiao;LIU Siqi;ZHOU Yuhao(The School of Veterinary Medicine,Jiangsu Agri-animal Husbandry Vocational College,Taizhou 225300,China;Veterinary Medicine,Yangzhou University,Yangzhou 225009,China)

机构地区：[1]江苏农牧科技职业学院动物医学院,江苏泰州225300 [2]扬州大学兽医学院,江苏扬州225009

出　　处：《黑龙江畜牧兽医》2024年第1期103-108,共6页Heilongjiang Animal Science And veterinary Medicine

基　　金：江苏省高等学校自然科学研究面上项目(21KJB230007);江苏高校“青蓝工程”项目(苏教师函〔2020〕10号);泰州市科技支撑计划(农业)项目(TN202118);泰州市“凤城英才”托举工程项目(泰科协发〔2021〕45号);江苏省大学生创新创业训练计划项目(202112806006Y)。

摘　　要：为获得Ⅶ型新城疫病毒(Newcastle disease virus,NDV)融合蛋白(fusion protein,F蛋白)和O18型禽致病性大肠杆菌(avian pathogenic Escherichia coli,APEC)外膜基因A(outer-membrane proteases,OmpA)嵌合蛋白和双特异性抗体,试验首先采用基因合成技术获得Ⅶ型NDV F基因和O18型APEC的OmpA基因片段,使用同源重组技术将F基因插入到OmpA基因中,再将重组基因片段插入到原核表达载体pGEX-4T-1中构建嵌合表达质粒,对重组质粒中的插入基因序列进行测序鉴定;将序列正确的嵌合表达质粒转入大肠杆菌BL21(Rosetta)感受态细胞中,利用IPTG诱导嵌合蛋白表达,使用SDS-PAGE和Western-blot检验嵌合蛋白的表达情况;纯化嵌合蛋白后免疫兔获得抗血清,抗血清再经抗原亲和纯化层析柱纯化获得多克隆抗体,采用间接酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)测定抗体效价。结果表明:成功合成了Fo和OmpA1、OmpA2基因片段,构建并表达了重组质粒pGEX-O1-Fo-O2,纯化后的嵌合蛋白O1FoO2在大肠杆菌中以包涵体形式存在,制备的多克隆抗体对O1FoO2的效价约为1.1×10^(6),对GST的效价约为4.1×10^(4)。说明制备的嵌合蛋白和双特异性抗体质量较好。In order to obtain the chimeric protein and bispecific antibody of the fusion protein(F)of Newcastle disease virus(NDV)typeⅦand outer membrane gene A(OmpA)of avian pathogenic Escherichia coli(APEC)type O18,firstly,the F gene of Newcastle disease virus typeⅦand OmpA gene fragment of APEC type O18 were obtained by gene synthesis technique.Homologous recombination technique was used to insert F gene into OmpA gene,and then the recombinant gene fragment was inserted into the prokaryotic expression vector pGEX-4T-1 to construct chimeric expression plasmid.The insertion gene sequence in the recombinant plasmid was sequenced and identified.The chimeric expression plasmid with correct sequence was transformed into BL21(Rosetta)Escherichia coli(E.coli),and inducing chimeric protein expression using IPTG.The expression of chimeric proteins was detected by SDS-PAGE and Western-blot.After purification of chimeric protein,rabbits were immunized to obtain antiserum,and antiserum was purified by antigen affinity purification chromatography column to obtain polyclonal antibodies.Enzyme linked immunosorbent assay(ELISA)was used to determine the antibody titer.The results showed that Fo,OmpA1 and OmpA2 gene fragments were synthesized successfully,and the recombinant plasmid pGEX-O1-Fo-O2 was constructed and expressed;the purified chimeric protein O1FoO2 existed in the form of inclusion body in Escherichia coli;the titer of the prepared polyclonal antibody against O1FoO2 was about 1.1×10^(6);the titer against GST was about 4.1×10^(4).The results indicated that the quality of chimeric protein and bispecific antibody was good.

关 键 词：新城疫病毒F蛋白 大肠杆菌OmpA蛋白 嵌合蛋白 原核表达 多克隆抗体 

分 类 号：S855[农业科学—临床兽医学] S831[农业科学—兽医学]