鸡传染性支气管炎和禽衣原体病的多表位DNA疫苗构建及其免疫原性研究  

Construction of Multi-epitope DNA Vaccine Against Infectious Bronchitis and Avian Chlamydiosis and Its Immunogenicity

作　　者：扈立伟 任君 卢宝平 刘青 朱凡杰 顾天越 包利霞 刘婷婷 王鑫源 朱晓琛 张东超 金天明 HU Liwei;REN Jun;LU Baoping;LIU Qing;ZHU Fanjie;GU Tianyue;BAO Lixia;LIU Tingting;WANG Xinyuan;ZHU Xiaochen;ZHANG Dongchao;JIN Tianming(Tianjin Key Laboratory of Agricultural Animal Breeding and Healthy Husbandry,College of Animal Science and Veterinary Medicine,Tianjin Agricultural University,Tianjin 300384;Tianjin Engineering Technology Center of Livestock Pathogen Detection and Genetic Engineering Vaccine,College of Animal Science and Veterinary Medicine,Tianjin Agricultural University,Tianjin 300392;Tianjin Academy of Agricultural Sciences,Tianjin Key Laboratory of Animal Molecular Breeding and Biotechnology,Tianjin 300192)

机构地区：[1]天津农学院动物科学与动物医学学院,天津市农业动物繁育与健康养殖重点实验室,天津300384 [2]天津农学院动物科学与动物医学学院,天津市畜禽病原检测与基因工程疫苗工程技术中心,天津300392 [3]天津市农业科学院,天津市畜禽分子育种与生物技术重点实验室,天津300192

出　　处：《中国家禽》2024年第5期62-69,共8页China Poultry

基　　金：天津市教委科技计划项目(2019KJ032);甘肃省科技计划项目(22CX8NL215);国家自然科学基金青年基金项目(32202760)。

摘　　要：试验旨在设计和构建基于鸡传染性支气管炎病毒(IBV)S1蛋白和鹦鹉热衣原体(Cps)主要外膜蛋白(MOMP)的抗原多表位DNA疫苗,并分析其免疫原性。研究利用免疫信息学方法筛选出IBV S1蛋白和Cps MOMP蛋白的优势抗原表位,设计多表位基因M,并利用多种生物信息学软件对其进行预测分析;将M基因、S1和MOMP串联基因分别连接至真核表达载体pEGFP-N1并转染至DF-1细胞,利用Western blot检测目标蛋白的表达情况。将上述重组质粒肌内注射7日龄雏鸡,四免后检测免疫鸡血清中特异性抗体IgG、细胞因子及T淋巴细胞含量。结果显示:构建的表位蛋白结构稳定、免疫原性较好且无副作用;重组质粒pEGFP-M和pEGFP-S1-MOMP与预期大小相符;与pEGFP-N1组和PBS组相比,四免后,pEGFP-M免疫组鸡血清中抗IBV IgG和抗Cps IgG抗体水平极显著升高(P<0.01),与pEGFP-S1-MOMP组相当;pEGFP-M组鸡血清中细胞因子γ-干扰素(IFN-γ)、白细胞介素-4(IL-4)水平,T淋巴细胞抗原CD3+、CD4+和CD8+含量均极显著高于pEGFP-N1组和PBS组(P<0.01)。结果表明,构建的多表位DNA疫苗pEGFP-M能够激发鸡的体液免疫和细胞免疫反应,为研制同时预防鸡传染性支气管炎和禽衣原体病的多表位疫苗奠定基础。The aim of this study was to design and construct an antigenic multi-epitope DNA vaccine based on the S1 protein of infectious bronchitis virus(IBV)and the major outer membrane protein(MOMP)of Chlamydia psittaci(Cps),and analyze the resulting immunogenicity.The dominant epitopes of the IBV S1 protein and the Cps MOMP were identified through immunoinformatics methods.Subsequently,the multi-epitope gene M was designed and predicted using various bioinformatics software.The M gene,along with the tandem genes S1 and MOMP,were fused to the eukaryotic expression vector pEGFP-N1 and transfected into DF-1 cells.The expression of the target protein was confirmed by Western blot.Intramuscular injections of the recombinant plasmids were administered to 7-day-age chickens,and the serum of immunized chickens was analyzed after four immunizations to measure specific antibody IgG,cytokines and T-lymphocyte levels.The results showed that the constructed epitope protein possessed a stable structure,exhibited good immunogenicity,and had no side effects.The recombinant plasmids pEGFP-M and pEGFP-S1-MOMP were successfully verified.Compared with pEGFP-N1 group and PBS group,the levels of anti-IBV IgG and anti-Cps IgG antibodies in the serum were significantly increased in pEGFP-M immunized group(P<0.01),which were comparable to pEGFP-S1-MOMP group.Furthermore,pEGFP-M group displayed significantly higher levels ofγ-interferon(IFN-γ),interleukin-4(IL-4),and thymus lymphocyte antigens CD3+,CD4+,and CD8+in serum compared to pEGFP-N1 group and PBS group(P<0.01).The results suggested that the established multi-epitope DNA vaccine pEGFP-M could effectively stimulate both humoral and cellular immune responses in chickens,providing a foundational basis for the development of multi-epitope vaccines to prevent infectious bronchitis and avian chlamydiosis.

关 键 词：鸡传染性支气管炎病毒 鹦鹉热衣原体 S1基因 MOMP基因 表位疫苗 

分 类 号：S855.3[农业科学—临床兽医学]