携带新城疫病毒F基因DNA疫苗的减毒鼠伤寒沙门氏菌的构建及其免疫原性  

Construction and immunogenicity of the attenuated Salmonella typhimurium harbouring Newcastle disease virus DNA vaccine

作　　者：潘志明[1] 焦新安[1] 唐丽华[1] 黄金林[1] 张辉[1] 张晓明[1] 张小荣[2] 刘秀梵[2] 

机构地区：[1]扬州大学生物科学与技术学院,江苏扬州225009 [2]扬州大学农业部畜禽传染病学重点开放实验室,江苏扬州225009

出　　处：《中国兽医学报》2007年第1期17-21,共5页Chinese Journal of Veterinary Science

基　　金：国家"863"计划资助项目(2002AA245051);国家自然科学基金资助项目(30170700);江苏省自然科学基金资助项目(BK2003043)

摘　　要：根据GenBank中发表的新城疫病毒(NDV)融合蛋白(F)基因序列,设计1对引物,通过RT-PCR扩增出鹅源NDV分离株JS5F基因(约1700bp),测序确认后,将其克隆入真核表达载体pVAX1,获得重组真核表达质粒pVAX1-F。pVAX1-F经脂质体转染COS-7细胞,间接免疫荧光试验检测出F基因在COS-7细胞中的表达产物。将pVAX1-F转化减毒鼠伤寒沙门氏菌SL7207,构建成功携带DNA疫苗的重组沙门氏菌SL7207(pVAX1-F)。重组菌以109CFU/只的剂量2次免疫BALB/c小鼠,免疫小鼠可以检测到特异性针对NDVF蛋白的血清抗体和小肠粘膜抗体应答,SL7207(pVAX1-F)免疫组抗体水平显著高于SL7207(pVAX1)组(P<0.05)。将SL7207(pVAX1-F)以109CFU/只剂量口服免疫1日龄雏鸡,免疫保护试验结果显示,SL7207(pVAX1-F)免疫组对鸡具有良好的保护率(77.27%),与空白对照组和SL7207(pVAX1)空载体组之间存在显著性差异(P<0.05)。结果表明,该运送DNA疫苗的减毒沙门氏菌系统在体内能成功释放所携带的质粒,并能刺激机体产生免疫应答,可对NDV强毒攻击提供良好的免疫保护作用,提示该疫苗候选株对新城疫的控制有重要应用前景。A pair of primers were designed and synthesized according to the previously published sequence of fusion protein（F） gene of Newcastle disease virus（NDV） and used to amplify F gene by reverse-transcrlption polymerase chain reaetion（RT PCR） from the genomie RNA of a NDV strain JS5 isolated from goose, The PCR prod- uct was identified by sequencing. Then recombinant eukaryotic expression vector pVAX1-F was constructed through inserting F gene into MCS of pVAX1. The recombinant plasmid pVAX1-F was transfected in COS-7 cells, and identified for the transient expression of F gene by indirect immunofluorescent assay. Finally,the recombinant plasmid was transformed into attenuated Salmonella typhimurium SL7207, The recombinant was screened and designated as SL7207 （pVAX1-F）. This recombinant bacterial could significantly elicit specific serum and intestinal mucosal antibody immune response in immunized BALB/c mice ,compared with SL7207 （pVAX1） immunized mice. 1-day-old commercial ISA brown chickens were immunized orally with SL7207（pVAX1-F） at dosage of 109 CFU at two weeks interval. The chickens immunized with SL7207（pVAX1-F） could be protected with the protective rate of 77.27% ,significantly higher than those with SL7207（pVAX1）（29.42%） or PBS（12.5% ） （P〈0.05）, These results demonstrated that the Salmonella delivering system could release haboured NDV DNA vaccines in viva and elicit spcific immune response. A novel mucosal DNA vaccine was developed and would be useful for controlling the infection and epidemic of ND in the poultry.

关 键 词：减毒沙门氏菌 DNA疫苗 新城疫病毒 免疫原性 

分 类 号：S852.65[农业科学—基础兽医学] Q78[农业科学—兽医学]