新城疫病毒F蛋白结构域基因原核表达与抗原表位分析

Prokaryotic expression of structural domains of the Newcastle disease virus fusion protein and analyses of their antigenic epitopes.

以含新城疫病毒(NDV)融合蛋白(F)基因的重组质粒为模板,设计特异引物进行重叠-延伸PCR扩增获得F基因的抗原结构域基因片段Fa、Fb和Fa-l-Fb,经SalⅠ和NotⅠ酶切后,定向插入原核表达载体pGEX-6P-1,获得重组质粒pGEX-Fa、pGEX-Fb和pGEX-Fa-l-Fb;用定点突变方法获得重组质粒pGEX-Fa-mut.将重组质粒转化大肠杆菌BL21,转化菌经IPTG诱导、提取表达产物进行蛋白电泳和免疫印迹分析.结果表明:Fa-mut、Fb和Fa-l-Fb结构域基因均获得了融合表达,表达产物与NDV阳性血清具有免疫反应性;与pGEX-Fa-mut不同,pGEX-Fa重组质粒在大肠杆菌中未能表达,且转化菌在IPTG诱导过程中未见生长,提示未经突变的Fa片段可能为毒性蛋白.三维结构预测结果显示,目的蛋白片段中均保留F蛋白中原有相应的抗原表位.

The gene fragments Fa, Fb and Fa-l-Fb coding for the antigenic structural domains of the Newcastle disease virus （NDV） fusin protein were amplified from the recombinant plasmid containing full length NDV F gene by overlap extension PCR with specific primers. The genes were inserted between SalⅠ and NotⅠ sites of pGEX-6P-1 after cleavage by corresponding enzymes. The recombinant plasmids were named pGEX-Fa, pGEX- Fb and pGEX-Fa-l-Fb. The recombinant plasrnid pGEX-Fa-mut was obtained from pGEX-Fa by site-directed mutagenesis. Glutathione S-transferase （GST） fusion proteins containing the antigenic structural domains of the NDV F protein were expressed in E. coli BL21 containing pGEX-Fa-mut, pGEX-Fb and pGEX-Fa-l-Fb. The expressed fusion proteins were recognized by NDV positive serum from specific pathogen free （SPF） chickens. No target protein band of Fa was seen from E. coli BL21 transfected by pGEX-Fa, nor did E. coli strain harboring the plasmid show any growth upon IPTG induction, suggesting that the expression product of Fa fragment might be toxic to the host strain. The 3-D structure prediction reveals that all the antigenic epitopes in full-length F protein were retained in the corresponding expression products.