新城疫病毒F蛋白免疫活性片段的原核表达  

Expression of nucleotide fractions encoding immunogenic proteins of the fusion protein of NDV in Escherichia coli

作　　者：敖艳华[1] 陈晓春[1] 廖明[1] 蒋文泓[2] 聂飞[2] 任涛[1] 

机构地区：[1]华南农业大学兽医学院农业部养禽与禽病防治重点开放实验室,广东广州510642 [2]广东省农业厅,广东广州510500

出　　处：《中国兽医科学》2008年第5期374-379,共6页Chinese Veterinary Science

基　　金：广东省自然科学基金项目(5500-E07106);广东省科技攻关计划项目(2005A20401003,2004B20201030);国家高技术研究发展计划(863)项目(2006AA10A205)

摘　　要：利用已构建的新城疫病毒F基因重组质粒,通过氨基酸序列分析和InsightⅡ基因工作站的MODELER程序和同源模建技术,预测了试验株F蛋白的表面抗原分布位置。设计引物扩增了2段含有多个抗原位点的多肽片段,通过双酶切定向克隆到原核表达载体pET32a,获得了重组质粒pET32a-F1和pET32a-F2以及2个片段串联起来后克隆的重组质粒pET32a-F3。重组质粒经诱导表达并取产物进行分析,结果显示,F1、F2、F3片段均获得了融合表达。Western-blotting证实,表达产物F1、F2和F3与NDV阳性血清均具有免疫反应性。用表达的重组蛋白加免疫佐剂经皮下接种免疫鸡,免疫2次后产生较高的抗体水平,用100 LD50的超强毒株GD-05-2攻击,重组蛋白免疫组鸡可达到约70%的保护率,并可显著抑制排毒。Distribution of surface antigen of Newcastle disease virus（NDV） strain GD-05-2 fusion protein was predicted by using amino acid sequence analysis,MODELER program in Insight Ⅱ gene station and homologous modeling technology. The gene fragments F1 and F2 encoding the antigenic structural domains of NDV fusion protein were amplified from the recombinant plasmid pMD18-F containing the full length sequence of NDV F gene encoding the fusion protein by PCR with specific primers,and then F3 gene fragment was amplified by ligating F1 with F2 using PCR. To obtain recombinant plasmids pET32a-F1, pET32a-F2 and pET32a-F3,the three genes were inserted into pET32a vector after cleavage by corresponding enzymes. All the three recombinant plasmids were transformed into Escherichia coli Rossetta and induced with IPTG. The expressed products F1 ,F2,and F3 proteins were recognized by NDV positive serum in Western-blotting test. One-day-old chickens in protein immunization groups were inoculated subcutaneously with the F1,F2 and F3 proteins emulsified with two kinds of Freund＇s adjuvants for 2 times,respectively. The same day-old chickens in two control groups were inoculated respectively with normal saline solution or NDV La Sota vaccine. All the chickens were challenged with 100 LD50 of NDV strain GD-05-2. The three recombinant proteins could induce protection rate of approximately 70% against the challenge with a virulent NDV to a some degree,and could reduce the viral shed from such a challenge.

关 键 词：新城疫病毒 F蛋白 抗原表位 原核表达 

分 类 号：S852.659.5[农业科学—基础兽医学] Q786[农业科学—兽医学]