新城疫病毒F蛋白抗原表位串联基因的构建及其原核表达  

Construction of prokaryotic expression vector for F protein epitope gene cascade of Newcastle disease virus

作　　者：程宝艳[1] 彭明义[2] 余为一[2] 张丹俊[1] 詹凯[1] 

机构地区：[1]安徽省农业科学院畜牧兽医研究所,安徽合肥230031 [2]安徽农业大学动物科技学院,安徽合肥230036

出　　处：《中国兽医学报》2010年第6期734-737,共4页Chinese Journal of Veterinary Science

基　　金：国家自然科学资金资助项目(30671537);农业部蛋鸡行业专项(NYHYZX07-039);蛋鸡产业技术体系资助项目

摘　　要：用自行设计的3对引物,通过RT-PCR从接毒的SPF鸡胚的尿囊液中克隆了新城疫病毒F基因3段抗原表位区段,大小分别为81、108、105bp。应用添加互补性酶切位点的基因工程方法,将3段抗原表位基因片段拼接成多表位串联基因,大小为306bp。将此基因片段插入含组氨酸(His)基因的质粒pET-32-a中,构建了重组质粒pET-32-a-F306。经诱导表达,获得相对分子质量约为31000的融合蛋白,其中F蛋白抗原表位串联基因片段表达产物约为11000。经亲和层析,获得纯化His-F306融合蛋白,进一步用该蛋白免疫小鼠,制备了鼠源抗新城疫病毒F蛋白抗体。经过琼扩、ELISA及Western-blot检测,表明该融合蛋白中的抗原表位串联基因所表达的蛋白具有良好的抗原性。Three epitope configuration regions of Newcastle disease virus F gene from SPF chicken embryo about 81,108,105 bp were cloned by RT-PCR using three pair of primers.The tandem-arranged multiple epitope gene of the F gene about 306 bp was obtained by appending complementary endonuclease of genetic engineering means.The fragment of Newcastle disease virus F gene was cloned into pET-32-a.The recombinant plasmid,namely pET-32-a-F306,was transformed into Escherichia coli BL21 and His-F306 fusion protein was induced to express,A MW of the fusion protein was about 31 000 as analyzed by SDS-PAGE,the partial segment size in F gene was 11 000.The purified fusion protein was used to immunized mice and the specific antibody was observed,which was identified further in immunodifusion,ELISA and Westtern-blot.The results indicated that this expressed fusion protein had a favorable antigenieity.

关 键 词：新城疫病毒 F基因 抗原表位 原核表达 

分 类 号：S852.65[农业科学—基础兽医学]