H7N9禽流感DNA疫苗的免疫保护效力研究  

Protective efficacy of DNA vaccine against H7N9 avian influenza

作　　者：梁真洁 潘俊慧 于晓菲 陈普成[1] 曾显营[1] 柳金雄[1] 施建忠[1] 邓国华[1] 姜永萍[1] 陈化兰[1] LIANG Zhen-jie;PAN Jun-hui;YU Xiao-fei;CHEN Pu-cheng;ZENG Xian-ying;LIU Jin-xiong;SHI Jian-zhong;DENG Guo-hua;JIANG Yong-ping;CHEN Hua-lan(National Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute,CAAS,Harbin 150069,China)

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室

出　　处：《中国预防兽医学报》2019年第9期935-939,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：“十二五”农村领域国家科技计划(（2015BAD12B03/001)

摘　　要：为检测H7N9禽流感病毒(AIV) DNA疫苗的免疫保护效力,本研究将H7N9禽流感疫苗株A/Chicken/Guangxi/SD098/2017 (H7N9)[CK/GX/SD098/17 (H7N9)]的HA基因密码子优化后,定向克隆至载体pCAGGS中构建重组质粒pCA-SD098。将pCA-SD098转染至293T细胞后,采用间接免疫荧光(IFA)和western blot检测,结果显示,HA蛋白可以在293T细胞中正确表达。分别利用15μg和20μg重组质粒pCA-SD098免疫3周龄SPF鸡,3周后以相同的剂量和方式加强免疫,加强免疫一周后通过鼻腔分别感染105EID50的同源高致病性AIV (HPAIV)CK/GX/SD098/17 (H7N9)和异源低致病性AIV (LPAIV) A/Chicken/Chongqing/SD057/2017 (H7N9)[CK/CQ/SD057/17 (H7N9)],攻毒10 d内记录免疫鸡发病和死亡情况,检测免疫鸡HI抗体水平,并于攻毒后第3 d、第5 d和第7 d无菌采集所有鸡的喉头和泄殖腔拭子,采用红细胞凝集试验(HA)检测病毒的滴度。结果显示,重组质粒pCA-SD098免疫后可以诱导SPF鸡产生较高水平的HI抗体,15μg pCA-SD098剂量免疫鸡后能够完全抵御H7N9 HPAIV的致死性攻击,20μg pCA-SD098剂量可以有效阻断H7N9 LPAIV的感染。攻毒后,所有免疫鸡无发病、无死亡、无排毒,本研究为质粒pCA-SD098作为防控H7N9禽流感的候选DNA疫苗提供了实验依据。In order to evaluate the immune protective efficacy of DNA vaccine against H7N9 avian influenza virus(AIV),We inserted the codon-optimized HA gene of the vaccine strain, A/Chicken/Guangxi/SD098/2017(CK/GX/SD098/17, H7N9) into pCAGGs vector to generate a recombinant vector pCA-SD098. Indirect immunofluorescence assay and western blot analysis showed that the HA protein was correctly expressed in 293 T cells transfected with the pCA-SD098 construct. Groups of 3-week-old SPF chickens were inoculated intramuscularly with 15μg or 20μg of pCA-SD098, and were boosted with the same dose and manner three weeks after the primary immunization. One week post-booster, the immunized chickens were challenged intranasally with 105 EID50 of homologous highly pathogenic avian influenza virus(HPAIV) CK/GX/SD098/17(H7N9) or heterologous low pathogenic avian influenza virus(LPAIV) A/Chicken/Chongqing/SD057/2017(CK/CQ/SD057/17, H7N9). The signs of disease and death of experimental chickens were recorded for ten days, and the level of the HI antibodies was detected at different time post-challenge. Oropharyngeal and cloacal swabs were collected on day 3, 5, and 7 post-challenge for virus titration by hemagglutination assay(HA). The results showed that recombinant plasmid pCA-SD098 induced high level of HI antibodies,15 μg pCA-SD098 protected chickens from lethal challenge by the H7N9 HPAIV, and 20 μg pCA-SD098 completely blocked the infection by the H7N9 LPAIV. No signs of disease, death, or virus shedding were observed in the immunized chickens. These data indicate that the recombinant plasmid pCA-SD098 has the potential to be a DNA vaccine candidate against H7N9 AIV. Our study provides an experimental basis for the prevention and control of H7N9 avian influenza.

关 键 词：H7N9禽流感 DNA疫苗 免疫 保护效力 

分 类 号：S852.65[农业科学—基础兽医学]