H9亚型禽流感病毒黏膜DNA疫苗与灭活油乳苗的联合免疫研究  

The prime-boost strategy with mucosal DNA vaccine and killed oil-emulsified vaccine against H9 subtype of avian influenza virus

作　　者：程宁宁[1] 潘志明[1] 刘蓓蓓[1] 孙林[1] 耿士忠[1] 贾自文[1] 黄金林[1] 焦新安[1] 

机构地区：[1]扬州大学江苏省人兽共患病学重点实验室,扬州225009

出　　处：《中国人兽共患病学报》2008年第5期425-429,共5页Chinese Journal of Zoonoses

基　　金：国家自然科学基金(30425031);全国优秀博士学位论文作者专项资金(FANEDD200358);国家863计划(2006AA10A206);江苏省自然科学基金项目(BK2007511)

摘　　要：目的构建减毒沙门氏菌介导的H9亚型禽流感病毒黏膜DNA疫苗,并探讨黏膜DNA疫苗与灭活油乳苗联合应用的免疫效力。方法以RT-PCR法扩增H9N2亚型禽流感病毒分离株(A/Chicken/China/N/2005)的血凝素(HA)基因,然后克隆入pmcDNA3.1+载体中,获得重组质粒pmcDNA3.1-HA。通过电穿孔法将重组质粒转入减毒鼠伤寒沙门氏菌SL7207,构建沙门氏菌介导的黏膜DNA疫苗SL7207(pmcDNA3.1-HA)。重组菌与灭活油乳苗联合免疫1日龄商品代伊莎褐蛋鸡,并设立重组菌SL7207(pmcDNA3.1-HA)单独免疫组、油苗免疫组、空载体免疫组及空白对照组。用HI或ELISA测定血清和小肠黏膜抗体效价,以H9N2亚型禽流感病毒攻击,计算免疫保护力。结果联合免疫组和重组菌SL7207(pmcD-NA3.1-HA)单独免疫组能激发机体产生黏膜免疫应答,且与其他试验组之间存在显著性差异(P<0.05)。联合免疫组和重组菌SL7207(pmcDNA3.1-HA)单独免疫组的免疫保护力均与空载体组、空白对照组有显著差异(P<0.05),且联合免疫组的免疫保护率最高,达100%。结论成功构建H9亚型禽流感病毒黏膜DNA疫苗,DNA疫苗与灭活油乳苗联合应用具有良好的免疫协同作用。In order to determine the synergistic immune efficacy of the DNA vaccine with the killed oil-emulsified vaccine of HgN2 subtype avian influenza virus（AIV）, the mucosal DNA vaccine named SL7207 （pmcDNA-HA） was construsted, and the immunogenicity of the prime-boost strategy with the DNA vaccine and killed oil-emulsified vaccine was evaluated in chicken model. The hemagglutinin （HA） gene from the HgN2 subtype AIV was amplified with the RT-PCR and sub-cloned into vector pmcDNA3.1 ＋. The recombinant plasmid was transformed into the attenuated Salmonella typhimurium SL7207, and the positive recombinant was designated as SL7207（pmcDNA3.1-HA）. One-day-old commercial ISA brown chickens were immunized with SL7207（pmcDNA3.1-HA） plus killed oil-emulsified vaccine of AIV,SL7207（pmcDNA3.1-HA） ,killed oil-emulsified vaccine of AIV,SL7207 （pmcDNA3.1）, and PBS. It was shown that the intestinal mucosal antibody titers in combined inoculation and SL7207（pmcDNA3.1-HA） groups were significantly higher than those of other groups （P〈0.05）. The result of protective immunity showed both the group of combined inoculation and SL7207 （pmcDNA3.1-HA） providing significantly protection compared with the negative control groups（P〈0.05）, and the group of combined inoculation obtaining 100% protection. In conclusion, the mucosal DNA vaccine of HgN2 subtype AIV was successfully constructed, and the mucosal DNA vaccine plus killed oil-emulsified vaccine has good synergistic effect of protection.

关 键 词：H9亚型禽流感 减毒沙门氏菌 DNA疫苗 灭活油乳苗 联合免疫 

分 类 号：S855.3[农业科学—临床兽医学]