表达鸡传染性支气管炎病毒S1蛋白重组腺病毒的构建及免疫原性的初步分析

畜牧兽医学报 ›› 2012, Vol. 43 ›› Issue (9): 1449-1454.doi:

表达鸡传染性支气管炎病毒S1蛋白重组腺病毒的构建及免疫原性的初步分析

宋菲菲^1,2，康震³，齐艳君³，袁颖烁^1,2，张守峰²，张乐萃^1*，扈荣良^2*

（1. 青岛农业大学动物科技学院, 青岛 266109; 2. 军事医学科学院军事兽医研究所,
长春 130122; 3. 寿光六和肉鸡养殖有限公司, 潍坊 262700）

收稿日期:2012-02-21 出版日期:2012-09-25 发布日期:2012-09-25
通讯作者: 张乐萃，教授，E-mail: lczhang@qau.edu.cn; 扈荣良，研究员，E-mail: Ronglianghu@hotmail.com
作者简介:宋菲菲（1987-），女，山东即墨人，硕士生，主要从事预防兽医学研究，E-mail: songfeifei125@126.com

Construction and lmmunogenicity Detection of Recombinant Adenovirus
Expressing S1 Gene of Infections Bronchitis Virus

SONG Fei-fei^1,2, KANG Zhen³, QI Yan-jun³, YUAN Ying-shuo^1,2,
ZHANG Shou-feng², ZHANG Le-cui^1*, HU Rong-liang^2*

(1. Qingdao Agricultural University, College of Animal Science and Technology, Qingdao 266109,
China; 2. Institute of Military Veterinary, Academy of Military Medical Sciences, Changchun
130122, China; 3. Liuhe Broilor Cultivation Limited Company, Weifang 262700, China)

Received:2012-02-21 Online:2012-09-25 Published:2012-09-25

摘要/Abstract

摘要： 拟构建表达鸡传染性支气管炎病毒（IBV）QS10株S1蛋白的重组复制缺陷型人5型腺病毒，并在本动物上进行初步的免疫学试验。通过RTPCR获得S1基因，插入腺病毒表达系统穿梭质粒，构建重组穿梭质粒pacAd5 CMV-S1，转染293AD细胞获得表达S1蛋白的重组腺病毒。PCR检测显示，S1基因已重组到腺病毒基因组；间接免疫荧光和Western blot检测证明该重组病毒在293AD细胞中真实表达了具有免疫反应性的IBV S1糖蛋白。重组病毒培养滴度可达到10⁷ TCID₅₀·mL^－1。免疫接种SPF鸡后，通过ELISA检测，免疫鸡产生了针对IBV的特异性抗体。作者成功构建了表达IBV S1蛋白的重组腺病毒，该重组病毒可在SPF鸡体内诱导产生IBV特异性抗体。

Abstract: To construct human adenovirus type 5 vectored recombinant virus expressing S1 glycoprotein gene of avian infectious bronchitis virus (IBV), S1 gene of IBV QS10 strain obtained through RT-PCR was inserted into shuttle plasmid of the adenoviral expression system, forming pacAd5 CMV-S1, as well as the backbone plasmid, which was linearized with PacⅠand transfected into 293AD cells. Detection with PCR revealed that the recombinant virus was successfully packaged with a titer up to 10⁷ TCID₅₀·mL^－1. Furthermore, The results of Western blotting and indirect immunofluorescence assay (IFA) indicated that the S1 gene was significantly expressed. Sera antibodies against IBV of the SPF chicken immunized with the recombinant virus were detected by enzyme-linked immunosorbent assay (ELISA). The recombinant adenovirus expressing S1 gene of IBV was successfully constructed with potent capacity to induce the specific antibodies in chickens and had the good immunogenicity, which laid the foundation for further development of recombinant avian infectious bronchitis vaccine.

中图分类号:

S852.659.6

宋菲菲, 康震, 齐艳君, 袁颖烁, 张守峰, 张乐萃, 扈荣良. 表达鸡传染性支气管炎病毒S1蛋白重组腺病毒的构建及免疫原性的初步分析[J]. 畜牧兽医学报, 2012, 43(9): 1449-1454.

SONG Fei-Fei, KANG Zhen, QI Yan-Jun, YUAN Ying-Shuo, ZHANG Shou-Feng, ZHANG Le-Cui, HU Rong-Liang. Construction and lmmunogenicity Detection of Recombinant Adenovirus
Expressing S1 Gene of Infections Bronchitis Virus[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2012, 43(9): 1449-1454.

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参考文献

［1］CHU V C, MCELORY L J, CHU V, et al. The avian coronavirus infectious bronchitis virus undergoes direct low-pH-dependent fusion activation during entry into host cells［J］. Virol, 2006, 80: 3180-3188.

［2］BIJLENGA G, COOK J K, GELB J, et al. Development and use of the H strain of avian infectious bronchitis virus from the Netherlands as a vaccine: a review［J］. Avian Pathol, 2004, 33: 550-557.

［3］CAVANGH D. Severe acute respiratory syndrome vaccine development: experiences of vaccination against avian infectious bronchitis coronavirus［J］. Avian Pathol, 2003, 32: 567-582.

［4］KUSTERS J G, NIESTERS H G M, BLEUMINK-PLUYM N M C, et al. Molecular epidemiology of infectious bronchitis virus in the Netherlands［J］. Gen Virol, 1987, 68: 343-352.

［5］WANG L, JUNKER D, COLLISSON E W. Evidence of natural recombination within the S1 gene of infectious bronchitis virus［J］. Virology, 1993, 192: 710-716.

［6］YANG Z Y, KONG W P, HUANG Y, et al. A DNA vaccine induces SARS coronavirus neutralization and protective immunity in mice［J］. Nature, 2004, 428: 561-564.

［7］STOHLMAN S A, KYUWA S, COHEN M, et al. Mouse hepatitis virus nucleocapsid protein-specific cytotoxic T lymphocytes are Ld restricted and specific for the carboxy terminus［J］. Virology, 1992, 189(1): 217-224.

［8］WEBSTER D P, DUNACHIE S, VUOLA J M, et al. Enhanced T cell mediated protection against malaria in human challenges by using the recombinant poxviruses FP9 and modified vaccinia virus Ankara［J］. PNAS, 2005, 102(13): 4836-4842.

［9］WEISS S R, NAVAS-MARTIN S. Coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus［J］. Microbiol Mol Biol Rev, 2005, 69: 635-664.

［10］BINNS M M, BOURSWELL M E, CAVANAGH D, et al. Cloning and sequencing of the gene encoding the spike protein of the coronavirus IBV［J］. Gen Virol, 1985, 66: 719-726.

［11］KWON H M, JACKWOOD M W. Molecular cloning and sequence comparison of the S1 glycoprotein of the Gray and JMK strains of avian infectious bronchitis virus［J］. Virus Genes, 1995, 9: 219-229.

［12］TOMLEY F M, MOCKETT A P, BOURSNELL M E, et al. Expression of the infectious bronchitis virus spike protein by recombinant vaccinia virus and induction of neutralizing antibodies in vaccinated mice［J］. Gen Virol, 1987, 68: 2291-2298.

［13］WANG X, SCHNITZLEIN W M, TRIPATHY D N, et al. Construction and immunogenicity studies of recombinant fowl poxvirus containing the S1 gene of Massachusetts 41 strain of infectious bronchitis virus［J］. Avian Dis, 2002, 46: 831-838.

［14］JOHNSON M A, POOLEY C, IGNJATOVIC J, et al. A recombinant fowl adenovirus expressing the S1 gene of infectious bronchitis virus protects against challenge with infectious bronchitis virus［J］. Vaccine, 2003, 21: 2730-2736.

［15］CAVANAGH D, NAQI S. Infectious bronchitis virus［M］//CALNEK B W, BARNES H J, BEARD C W, et al. Disease of Poultry. 9th ed. Ames, IA: Iowa State University Press, 1997: 511-526.

［16］RUSSELL W C. Update on adenovirus and its vectors［J］. Gen Virol, 2000, 81: 2573-2604.

［17］YAROSH O K, WANDEL A I, GRAHAM F L, et al. Human adenovirus types vectors expressing rabies glycoprotein［J］. Vaccine, 1996, 14(13): 1257-1264.

［18］XIANG Z Q, YANG Y P, WILSON J M, et al. A replication-defective human adenovirus recombinant serves as a highly efficacious vaccine carrier［J］. Virol, 1996, 219: 220-227.

［19］PREVEC L, COMPBELL J B, CHRISTIE B S, et al. A recombinant human adenovirus vaccine against rabies［J］. Infect Dis, 1990, 161: 27-33.

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