表达H9亚型禽流感病毒HA基因重组鸭肠炎病毒的构建  

Construction of a Recombinant Duck Enteritis Virus Expressing Hemagglutinin of H9N2 Avian Influenza Virus

作　　者：孙莹[1] 张兵[1] 李岭[1] 黄小洁[1] 侯力丹[1] 刘丹[1] 李启红[1] 李俊平[1] 王乐元[1] 李慧姣[1] 杨承槐[1] SUN Ying;ZHANG Bing;LI Ling;HUANG XiaoJie;HOU LiDan;LIU Dan;LI QiHong;LI JunPing;WANG LeYuan;LI HuiJiao;YANG ChengHuai(China Institute of Veterinary Drug Control,Beijing 100081)

机构地区：[1]中国兽医药品监察所

出　　处：《中国农业科学》2019年第23期4398-4405,共8页Scientia Agricultura Sinica

基　　金：国家重点研发计划(2017YFD0500800);北京市自然科学基金项目(6162025)

摘　　要：【背景】H9亚型禽流感病毒(AIV)存在宿主范围扩大、毒力增强的趋势,并为其他亚型AIV重排提供基因,给养禽业和公共卫生造成极大威胁。水禽不仅是流感病毒的宿主,更是其天然储存库,在禽流感病毒的传播和变异中发挥着重要作用。因此有效控制水禽感染对养禽业健康发展、公共卫生安全具有重要意义。鸭肠炎病毒(DEV)属于疱疹病毒,能感染鸭、鹅等雁形目禽类,可引起产蛋下降及高死亡率。DEV基因组大,免疫原性好,具有开发成活疫苗载体的潜力。【目的】构建缺失gE基因、表达H9亚型AIV HA基因的重组病毒rDEV-△gE-HA,探讨重组病毒rDEV-△gE-HA作为防治DEV-AIV的二联重组活载体疫苗的可行性。【方法】以H9N2亚型禽流感病毒HA基因作为靶基因,构建含有HA基因表达盒的转移载体pT-gE-HA,将其与携带绿色荧光蛋白标记的重组rDEV-△gE-GFP共转染CEF细胞后,进行蚀斑筛选、纯化表达HA基因的重组病毒rDEV-△gE-HA;利用PCR、基因测序鉴定重组病毒;在CEF中连续传代重组病毒20次,测定外源基因传代稳定性。以10^3 TCID50免疫易感鸭,分析重组病毒rDEV-ΔgE-HA对致死性DEV强毒攻毒保护效果;将不同剂量(10^3-10^6TCID50)rDEV-△gE-HA免疫鸭,免疫后14、21、28 d分别采集血清,测定H9血凝抑制(HI)抗体,并在免疫后28 d,以108EID50的剂量静脉注射H9N2AIV(A/duck/GD/08),攻毒后2 d,采集喉拭子,进行病毒分离试验。【结果】将构建的转移质粒载体pT-gE-HA与rDEV-△gE-GFP共转染CEF细胞,经过3轮蚀斑筛选,获得纯化的重组病毒rDEV-△gE-HA。PCR鉴定及基因测序结果显示,HA基因成功地插入到DEV基因组中,替换了绿色荧光蛋白。重组病毒在CEF中至少能稳定传代20代。重组病毒rDEV-ΔgE-HA以10^3 TCID50免疫易感鸭,能抵抗致死性DEV强毒攻击。重组病毒rDEV-Δg E-HA免疫易感鸭后14 d,各剂量免疫组均能检测到H9 HI抗体效价;免疫后21日,各组抗体�【Background】The H9N2 avian influenza virus(AIV) pathogenicity and transmissibility have recently showed an increasing trend. Moreover, it donates partial or even whole cassette of internal genes to generate novel reassortants, which is serious threat to poultry industry and public health. Waterfowls are considered as the natural host and reservoirs of AIVs and play an important role in the spread and mutation of AIV. Therefore, successful control of the spread of H9N2 in waterfowls contributes significantly to poultry industry and public health. Duck enteritis virus(DEV) taxonomically belongs to family Herpesviridae and infects ducks, geese, and swans, which results in high mortality and decreased egg production in domestic and wild waterfowl. DEV may be a promising candidate viral vector for aquatic poultry vaccination because it has a large genome and good immunogenicity. 【Objective】 In this study, we constructed a recombinant DEV expressing the hemagglutinin(HA) gene of a H9N2 virus that was inserted into the deleted viral g E gene, and then its characterization to explore the feasibility of the recombinant DEV as a live vectored vaccine was studied.【Method】 The HA gene of H9N2 was cloned to construct the transfer vector pT-gE-HA. Plasmid pT-gE-HA and rDEV-△gE-GFP were co-transfected into CEF cells. After plaque-purification, we obtained a pure recombinant virus which expressed H9N2 AIV HA protein, and named as rDEV-△gE-HA;PCR and sequencing assay were used to identify the recombinant virus. The recombinant virus was passaged in primary CEF 20 times to evaluate the genetic stability of the foreign gene in the recombinant virus. Ducks were inoculated with 10^3 EID50 rDEV-△gE-HA, then challenged with lethal DEV. Ducks were vaccinated intramuscularly with 10^3–10^6 TCID50 of rDEV-△gE-HA. At 14, 21, and 28 days post-vaccination(d.p.v.), sera were obtained from all ducks to monitor HI antibody against H9N2 AIV. At 28 d.p.v. all ducks were challenged with 10^8 EID50 H9N2(A/duck/GD/08) by intrav

关 键 词：鸭肠炎病毒 H9亚型禽流感病毒 HA 重组病毒 

分 类 号：S85[农业科学—兽医学]