【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。更多还原

【Abstract】 In order to determine the synergistic immune efficacy of the DNA vaccine with the killed oil-emulsified vaccine of H9N2 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 H9N2 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 H9N2 subtype AIV was successfully constructed,and the mucosal DNA vaccine plus killed oil-emulsified vaccine has good synergistic effect of protection.更多还原