【作者】 洪琴；

【导师】 李广兴；

【作者基本信息】 东北农业大学 ， 基础兽医学， 2011， 硕士

【摘要】 本实验将疑似“混合型”鸡痘的病料进行病理学检查,发现皮肤、气管和喉头病料的细胞胞浆内嗜酸性染色的A型包涵体。病原分离鉴定表明,病毒能在10日龄鸡胚绒毛尿囊膜上形成典型的痘斑,并在CEF上形成鸡痘病毒典型病变。据GenBank发表的FPV美国标准强毒株基因序列,利用Oligo 6.0和Primer 5.0软件设计1对扩增4b core蛋白基因引物,通过PCR扩增测序及动物回归试验证明所分离的病毒是鸡痘病毒（FPV）,命名为HH2008。本实验根据Afonso等人已发表的FPV核苷酸全序列（AF198100）,针对FPV140上下游设计了1对引物,PCR扩增出FPV140完整的开放阅读框,进行亚克隆构建原核表达质粒pET30a-ENV,表达蛋白大小约为42 ku。制备兔抗ENV多克隆抗体,抗体效价可达到2¹⁸,Westernblot检测发现多抗血清特异性好,可通过ELISA鉴定鸡痘病毒。本试验将FPV ORF140基因亚克隆至pVAX1真核表达载体中,构建了重组真核表达质粒pVAX-ENV。利用一段编码（G₄S）₃多肽的碱基Linker将ChIFN-γ分别与FPV ENV基因进行连接,成功构建pVAX-ENV-IFN融合基因真核表达质粒。通过脂质体转染法将pVAX-ENV和pVAX-ENV-IFN转入BHK21细胞中并检测到目的蛋白表达。重组真核表达质粒免疫雏鸡进行体内表达检测,ENV基因和ENV-IFN融合基因能够在体内进行表达,同时表达后的融合蛋白可以被ChIFN-γ多克隆抗体和FPV ENV多克隆抗体检测出来,进一步证明了融合基因表达的蛋白保留了融合前两段基因各自的生物学活性,为后续动物试验提供了理论基础。动物免疫试验分为5个组,即E组（FPV ENV基因真核表达质粒免疫组）、E-I组（pVAX-ENV-IFN真核表达质粒免疫组）、V组（鸡痘商品化疫苗免疫组）、P组（pVAX1空质粒免疫组）和PBS对照组（B组）。利用淋巴细胞转化试验、酶联免疫吸附试验和流式细胞术对不同处理组雏鸡的各项免疫指标进行了检测,最后应用病毒攻击试验比较不同质粒免疫组间保护率的差异。结果表明,免疫组各日龄雏鸡免疫指标均比pVAX1空载体组和PBS组高,以ENV-IFN融合基因的结果尤为显著。其中T、B淋巴细胞增殖功能、CD4⁺、CD8⁺、T细胞数量和外周血液中抗FPV ENV特异性抗体与pVAX1空载体组和PBS组相比较都有明显的提高,且以融合基因组和疫苗组最为显著。综上所述,ChIFN-γ基因在配合FPV抗原基因免疫过程中,机体免疫应答能力增强,提高了机体抵抗病毒的攻击能力。本试验构建ChIFN-γ与病毒抗原基因融合基因质粒鸡免疫保护试验为进一步研究ChIFN-γ生物学活性提供了试验依据,同时为探讨研制新型免疫增强型抗病毒疫苗提供了理论基础。更多还原

【Abstract】 In this research, we firstly examined chickens suspected of“mixed type”fowl pox with pathological methods. Eosinophilic dying A type inclusion body could be observed in the skin, trachea and laryngeal. Then virus was isolated and passaged onto the chorioallantoic membrane of 10-day-old embryonated chicken eggs for several times, the compact variola could be formed on CAM. The CAM-adapted virus was propagated on primary chicken embryo fibroblast cell （CEF） until cytopathic effect was noted. According to American standards FPV genome sequence published in GenBank, a pair of primers for 4b core protein gene was designed with Oligo 6.0 and Primer 5.0 software, sequence analysis of PCR product and regression of chicken test proved that the isolated pathogen was fowl poxvirus, named HH2008.According to published gene sequence （AF198100） in GenBank, a pair of primers for ORF 140 gene was designed. After idenfication and sequence analysis of the PCR product, it was subcloned into prokaryotic expression vector and designed pET30a-ENV, then expressed in E. Coli Rosetta system and ENV protein is approximately 42 ku as expected. The recombinant ENV protein was purified and used as immunogen to generate rabbit polyclonal antibody. Western-blot and ELISA indicated that the antibody could detect FPV ENV protein. More importantly, an indirect ELISA was developed with this anti-FPV140 antibody was capable of distinguishing FPV from other common avian pathogens.FPV ORF140 gene was then modified and inserted into an eukaryotic expression vector, pVAX1, to construct pVAX1-ENV. Furthermore, a flexible linker, which express （G₄S）₃ polypeptide was used to link ChIFN-γgene with FPV ORF140 gene and got a fusion gene eukaryotic expression plasmid （pVAX1-ENV-Linker-IFN）. Transient transfection of these two plasmids in vitro showed that the pVAX-ENV and pVAX1-ENV-Linker-IFN could express in BHK21 cells demonstrated by their antibodies, respectively. Intramscular injection experiment of these two plasmids in vivo showed pVAX-ENV and pVAX-ENV-IFN were able to expressed too. It proved that the protein expressed by fusion gene could retain their bioactivities, and provided theoretical foundation for the following immunoprotection research.The experimental chicken were divided into 5 groups, E group inoculated with pVAX-ENV plasmid, E-I group inoculated with pVAX-ENV-IFN plasmid, V group inoculated with FP commericial vaccine, P group inoculated with empty pVAX vetor, and B group inoculate with PBS as control. Thereafter, lymphocyte proliferative assay, enzyme Linkered immunosorbent assay and flow cytometric technique were used to detect the immune response of different groups. Finally chickens in different groups were challenged with FPV HH2008 strain to compare their protection rate. The results showed that most of immune response of V, E and E-I groups were higher than those of B and P groups, especially the E-I groups. T, B lymphocyte proliferative function, the number of CD4⁺, CD8⁺ T cell, the special anti-FPV ENV antibody titer in immune groups were all higher than those of B, P groups. And the E-I and V groups were the most significant among these groups. The histopathological change of FPV target organs of each group were examined after challenge and the immunoprotection rates were calculated among every group. Above all, ChIFN-γwere inoculated with FPV antigen gene, it could improve the host immune response and higher protection rate could be observed against FPV chanllenge. Furthermore, FPV antigen genes and ChIFN-γfusion plasmid was firstly constructed and the results could provide solid scientific ground to the further study of ChIFN-γbioactivities, and the theoretical basis to the dicovery of new type immune-enhancing vaccine for virual diseases.更多还原