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2025年8月18日 星期一
  2017, Vol. 25 Issue (7): 1197-1206    
  专题:不同亚型低致病性禽流感病毒生物学特性研究 本期目录 | 过刊浏览 | 高级检索 |
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
李爱心1,丁蕾蕾2,刘璐3,高立4,胡玉珍2,陈普成2,姜永萍2,柳金雄2,王笑梅1,陈化兰1
1. 中国农业科学院哈尔滨兽医研究所
2.
3. 甘肃农业大学
4. 哈尔滨兽医研究所
Construction of Recombinant DEV Co-expressing HA Gene of H5 Type AIV and VP2 Gene of IBDV and Evaluation of Protection Efficacy
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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 ccdB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rDEV-HA/VP2和 rDEV-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)、104 TCID50、105 TCID50免疫鸡后14 d,rDEV-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, vvIBDV) 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;rDEV-VP2/HA最高仅诱导对vvIBDV 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。
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李爱心
丁蕾蕾
刘璐
高立
胡玉珍
陈普成
姜永萍
柳金雄
王笑梅
陈化兰
关键词 禽流感鸡传染性法氏囊病(IBD)鸭瘟病毒 (DEV)活载体疫苗    
Abstract: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 gallus), 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 (H5N1) (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 103 tissue culture infective dose (TCID50), 104 TCID50, 105 TCID50 recombinant virus, and challenged with 100 median lethal dose (LD50) GZ/4 or 100 LD50 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 TCID50, 104 TCID50, 105 TCID50 rDEV-HA/VP2 induced low level of hemagglutination inhibition (HI) antibody, and no IBD antibody could be detected. The protective efficiency against H5 AIV was lower than 70%, and the protective efficiency against vvIBDV was lower than 30%, respectively. While, chicken which were immunized with 103 TCID50, 104 TCID50, 105 TCID50 rDEV-VP2/HA could not induce HI or IBD antibody. The protective efficiency against IBDV was lower than 40%, and the protective efficiency against H5 AIV was 0. However, chicken which were immunized with rDEV H5-8 or Gt strain could induce 100% protection against H5 AIV or vvIBDV. In conclusion, the two recombinant viruses rDEV-HA/VP2 and rDEV-VP2/HA could not induce complete protection against AIV or IBDV, and the strategy of using IRES to connect HA and VP2 genes to construct the DEV vector vaccine against AI and IBD was not feasible. This study provides a reference for the construction of DEV vectored multi vaccine in chicken
Key wordsAvian influenza (AI)    Infectious bursal disease (IBD)    Duck enteritis virus (DEV)    Vectored live vaccine
收稿日期: 2017-03-13      出版日期: 2017-06-16
基金资助:H7N9亚型禽流感防制技术研发
通讯作者: 陈化兰     E-mail: chenhualan@caas.cn
引用本文:   
李爱心 丁蕾蕾 刘璐 高立 胡玉珍 陈普成 姜永萍 柳金雄 王笑梅 陈化兰. 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[J]. , 2017, 25(7): 1197-1206.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2017/V25/I7/1197
 
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