共表达H5亚型AIV HA基因和IBDV VP2基因重组DEV的构建及免疫效力评价  

Construction of Recombinant DEV Co-expressing HA Gene of H5 Type AIV and VP2 Gene of IBDV and Evaluation of Protection Efficacy

作　　者：李爱心[1] 丁蕾蕾[1] 刘璐[2] 高立[1] 胡玉珍[1] 陈普成[1] 姜永萍[1] 柳金雄[1] 王笑梅[1] 陈化兰[1] 

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/农业部动物流感重点开放实验室,哈尔滨150069 [2]甘肃农业大学动物医学学院,兰州730070

出　　处：《农业生物技术学报》2017年第7期1197-1206,共10页Journal of Agricultural Biotechnology

基　　金：哈尔滨市应用技术研究与开发项目(2013AA6BN001)

摘　　要：禽流感(Avian influenza,AI)和传染性法氏囊病(Infectious bursal disease,IBD)严重危害家禽养殖业,疫苗免疫是防治的主要手段之一。为探索以鸭瘟病毒(Duck enteritis virus,DEV)(又称鸭病毒性肠炎病毒)为载体,研制在鸡(Gallus gallus)中使用的AI-IBD二联疫苗的可行性,本研究利用overlap PCR及Gateway LR克隆技术,扩增出血凝素(hemagglutinin,HA)-内部核糖体进入位点(internal ribosome entry site,IRES)-病毒蛋白2(virus protein 2,VP2)和VP2-IRES-HA基因片段,并将其克隆入DEV多片段拯救系统T-us78Kan ccd B粘粒短独特区7(unique short region 7,US7)和US8位点之间,利用该系统成功拯救出两株共表达H5亚型禽流感病毒(Avian influenza virus,AIV)HA基因和传染性法氏囊病病毒(Infectious bursal disease virus,IBDV)VP2基因的重组病毒r DEV-HA/VP2和r DEV-VP2/HA,并对其进行了初步免疫效果评价。经系统体外实验表明,病毒传至20代,外源基因均能在重组病毒中稳定遗传和表达,且不影响载体病毒的增殖。但免疫效力实验显示,重组病毒以103半数组织培养感染剂量(tissue culture infective dose,TCID50)、104TCID50、105TCID50免疫鸡后14 d,r DEV-HA/VP2最高仅诱导对同源AIV强毒70%的免疫保护和对超强IBDV(very virulent IBDV,vv IBDV)30%的免疫保护,部分鸡中能检测到血凝抑制(hemagglutination inhibition,HI)抗体,但整体水平较低,IBD抗体未检测到;r DEV-VP2/HA最高仅诱导对vv IBDV 40%的免疫保护,不能诱导对同源AIV强毒的免疫保护,HI抗体和IBD抗体均未检测到。结果显示,以IRES串联基因构建鸭瘟载体禽流感传染性法氏囊病二联疫苗的策略不可行。本研究为以DEV为载体构建在鸡中使用的多联疫苗的研究提供参考资料。Avain influenza （AI） and Infectious bursal disease （IBD） are harmful to poultry industry, and vaccination is one of the most effective method of controling these two diseases. In order to explore the feasibility of Duck enteritis virus （DEV） vector combined vaccine against AI and IBD that were used in chicken （Gallus gaUus）, two recombinant viruses that co-expressed hemagglutinin （HA） gene of H5 type Avian influenza virus （AIV） and virus protein 2 （VP2） gene of Infectious bursal disease virus （IBDV） were constructed, and experiments were done to evaluate the immune efficacy of the recombinant viruses. HA gene from A/ Chicken/Guizhou/4/2013 （HSN1） （GZ/4） and VP2 gene from IBDV （HLJ-0504） were connected by internal ribosome entry site （IRES） sequence in different orders, HA-IRES-VP2 and VP2-IRES-HA. The two gene fragments were then inserted between unique short region 7 （US7） and US8 genes of DEV genome in fosmid T-us78Kan ccdB which is overlapping fosmid DNAs rescue system. Using this system, the target gene between the US7 and US8 genes of the DEV genome were successfully inserted, and the two recombinant viruses rDEV-HA/VP2 and rDEV-VP2/HA were successfully constructed. The in vitro experiments showed that the inserted gene could be stably inherited and express without affecting replication of virus vector in cells. In order to evaluate the protective efficacy induced by recombinant virus in specific pathogen free （SPF） chicken, groups of ten 3-week-old SPF chicken were immunized with l03 tissue culture infective dose （TCIDs0）, 104 TCIDs0, 10s TCIDs0 recombinant virus, and challenged with 100 median lethal dose （LDs0） GZ/4 or 100 LD,0 very virulent IBDV （vvIBDV） at 14 days post vaccination. Recombinant DEV with only HA gene of GZ/4 inserting between US7 and US8 genes （rDEV H5-8） and commercial IBDV vaccine Gt strain were immunized as control. The result showed that chicken which were immunized with 103 TCIDs0, l04 TCID,0, 105 TCIDs0 rDEV-HA/

关 键 词：禽流感(AI) 传染性法氏囊病(IBD) 鸭瘟病毒(DEV) 活载体疫苗 

分 类 号：S852.65[农业科学—基础兽医学]