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

发布时间：2018-05-22 15:18

本文选题：禽流感(AI) + 传染性法氏囊病(IBD)　；参考：《农业生物技术学报》2017年07期

【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。
[Abstract]:Avian influenza (Avian influenza, AI) and infectious bursal disease (Infectious bursal disease, IBD) seriously harm the poultry breeding industry. Vaccine immunity is one of the main means of prevention and control. In order to explore the duck plague virus (Duck enteritis virus, DEV) (also known as duck viral enteritis virus) as the carrier, the two couplet used in chicken (Gallus) is developed. The feasibility of the vaccine was amplified by overlap PCR and Gateway LR cloning technology to amplify the hemagglutinin (HA) - internal ribosome entry site (internal ribosome entry site, IRES) - virus protein 2 (virus 2) and gene fragment Between the unique region 7 (unique short region 7, US7) and the US8 site, the system successfully rescued two strains of H5 subtype avian influenza virus (Avian influenza virus, AIV) HA gene and the recombinant virus of the infectious bursal disease virus. Preliminary immune evaluation. In vitro experiments showed that the virus was transmitted to 20 generations, and the exogenous genes were stable and expressed in the recombinant virus without affecting the proliferation of the virus, but the immunological efficacy experiment showed that the recombinant virus was cultured in 103 half of the tissues (tissue culture infective dose, TCID50), 104TCID50105TCID50 After immunizing chickens, 14 d, R DEV-HA/VP2 only induced immune protection to 70% of homologous AIV and 30% of ultra strong IBDV (very virulent IBDV, VV IBDV). In some chickens, the blood coagulation inhibition (hemagglutination inhibition) antibody was detected, but the whole level was low and the antibody was not detected. 40% immune protection did not induce immunity protection against the homologous AIV, and both HI and IBD antibodies were not detected. The results showed that the strategy of constructing the two joint vaccine of avian influenza infectious bursal disease by using IRES tandem gene was not feasible. This study provides a reference for the study of the multi joint vaccine used in chicken with DEV as the carrier. Information.
【作者单位】：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;甘肃农业大学动物医学学院;
【基金】：哈尔滨市应用技术研究与开发项目(2013AA6BN001)
【分类号】：S852.65

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