【作者】 赵魁；

【导师】 高丰；

【作者基本信息】 吉林大学 ， 基础兽医学， 2010， 博士

【摘要】 养羊业是我国畜牧业的一个重要内容,然而,长期以来,养羊业并未象养猪业、养鸡业等一样受到人们的重视,因此仍处于较为落后的状况。此外,羊群密度的增大、频繁的从外地引种以及粗放的饲养管理等因素的存在导致羊痘、羊传染性脓疱病、蓝舌病、坏死杆菌病等一些严重危害养羊业发展的疾病的频繁发生。羊传染性脓疱病作为羊群中常见的病毒性传染病之一,严重威胁着养羊业的健康发展。近年来,该病的发生呈上升趋势,因此不断有该病发生和流行的报道。本研究对长春市郊养羊户饲养羊群中一起疑似羊传染性脓疱病例进行分析,根据流行病学特点、临床症状和系统的病理学观察以及分子生物学检测等实验室诊断结果,证实该病为羊传染性脓疱病。然后利用原代犊牛睾丸细胞从病羊唇部痂皮中成功分离获得1株病毒,并通过形态学观察、理化学测定、动物回归试验和PCR等方法对该病毒进行鉴定,证实其为羊传染性脓疱病毒(ORFV),被命名为ORFV/JL/08/CC。将经超速离心纯化的病毒免疫家兔制备出多抗,通过免疫组织化学检测病原在各组织脏器中的分布情况,结果显示,在感染唇部皮肤棘细胞的胞浆中表现出阳性反应,从而进一步证明了该病的发生原因为感染ORFV所致。而对ORFV/JL/08/CC分离株的分离鉴定为进行针对羊传染性脓疱病的疫苗研制提供物质条件。然后利用PCR扩增获得该病毒株的主要免疫原性基因ORFV 011(B2L)和ORFV 059(F1L),测序后提交Genbank收录(登录号分别为FJ808074和FJ808075)。此外,从氨基酸和核苷酸水平上对其同源性进行分析,同时对两基因进行了遗传进化分析。结果表明,该分离株的ORFV 011(B2L)和ORFV 059(F1L)基因高度保守,可作为核酸疫苗研制的目的基因。在获得ORFV 011(B2L)和ORFV 059(F1L)的基础上,利用一编码(G4S)2多肽的碱基linker连接并通过重叠PCR的方法扩增获得ORFV 011(B2L)-linker-ORFV(F1L)融合基因,在此基础上,利用pcDNA3.1(+)真核表达载体成功构建了相应的疫苗质粒,通过脂质体法将其分别转染MDBK细胞,并通过RT-PCR、间接免疫荧光、SDS-PAGE以及Western-blotting等方法从mRNA水平和蛋白水平上证实了目的基因在动物细胞的转录和表达,这将为我们后续进行的动物免疫试验提供理论依据。采用初免-加强免疫的策略,将制备纯化的疫苗质粒经肌肉注射免疫Balb/c小鼠,之后通过间接ELISA、淋巴细胞增殖试验、流式细胞术等方法对疫苗质粒引起机体特异性的细胞免疫应答和体液免疫应答水平进行检测,结果表明,构建的疫苗质粒能够诱导免疫小鼠产生特异性的细胞免疫应答和体液免疫免疫应答,且以Th1型免疫应答为主;攻毒试验结果表明所构建的疫苗质粒安全性好,且能提供有效的免疫保护,该研究结果将为后续进行羊只免疫试验及其在临床上的应用奠定基础。更多还原

【Abstract】 Contagious ecthyma, also known as Sore mouth, is an acute and high contagious disease of sheep, goat and humans caused by Orf virus. The virus mainly causes proliferative lesions of infected skin and mucosa of lambs aged 3-6 months old. The disease is characterized by the formation of papules, nodules, or vesicles that progress into thick crusts or heavy scabs on the lips, around the nostrils, oral cavity mucosa, gingival, breast and tail.The infectivity and morbidity of the disease is very high, and usually spread in a flock of sheep or goats. The mortality rates up to 90% have been reported in a susceptible flock of sheep. However, the mortality is rare and usually does not exceed 10%, and sometimes up to 50% in the condition of infected following by Bacillus necrophorus or other pathogens. The disease has an economic impact on sheep farmers due to decrease in performance and production. At present, the disease has a worldwide distribution and is found ubiquitously wherever sheep and goats are farmed in China. The disease has been reported in provinces of Xizang, Xinjiang, Gansu, Neimeng, Jilin, Shanxi, Sichuan and Yunnan.The natural outbreak of the Orf virus among sheep occurred in a farm with 60 lambs at the age of 1-6months old in the Jilin province of China during November 2008. Of the 60 lambs in the flock, 25 developed proliferative lesions around the mouth. In young kids, lesions appeared on the junctions of the lips, the muzzle, as well as the tongue. In the early days, papules were nodular, varying in size between approximately 3 and 8 mm. The nodules or vesicles appeared after 2-3 days. Finally, nodules or vesicles that progressed into thick crusts or heavy scabs. Among 25 infected animals, 23 recovered 2-3 weeks after clinical signs first appeared, and two of the affected 6-week-old lambs died due to inanition owing to sucking diffculty caused by the severe mouth lesions of cauliflower-like. According to the epidemiology feature and clinical symptom, the disease was diagnosed preliminarily as contagious ecthyma. In order to diagnose finally, we analyzed systematically by the observation of pathohistology, negative staining and PCR. Necropsies were performed on the the inner organs of heart, liver, spleen, lungs, kidney, and skin. The pathological changes of the samples were recorded, and were fixed in 10% buffered formalin for histological examination or un-fixed for electron microscopic evaluation, and for PCR analysis. Pathology of the organs of lambs appeared in skin of lips and liver. The skin of lips progressed into thick crusts or heavy scabs and the colour of liver appeared yellow. Microscopic lesions in the skin collected from lip lesions of infected animals included acanthosis, degeneration and karyorrhexis in spinous cells of the stratum spinosum and characteristic intracytoplasmic eosinophilic inclusion bodies in keratinocytes. Further, a morderate level of fatty degeneration was found in the liver, which could have been caused by the difficulty in consuming food and water. Electron microscopic examination revealed the presence of characteristic parapoxvirus virions. The virions presented an ovoid shape, and were approximately 220nm in length and 152nm in width. In addition, the PCR results confirmed the causative agent of the disease was ORFV. According to clinical symptoms, necrospies and results of agent, we confirmed the occurrence of the disease was caused by infection of ORFV.Virus isolation was performed on bovine testis cells after confirmed. The homogenate supernatant treated with antibiotics inoculated monolayer cells. The CPE was observed day by day. The result showed that the cells appeared typical CPE while were blind passaged to the 5th generation. The cell culture fluid was collected and frozen-thawed 3 times. After obtained ORFV isolate, the virus was identified systematically by morphology, physico-chemical property, animal regression experiment and PCR. Massive virions were observed in the supernatant of cells by electron microscopic. The typical parapoxvirus virions were observed in the cytoplasm by extra thin section. In addition, the morphology and physico-chemical property feathers of the virus were consistent with DNA viruses. The results of animal regression experiment indicated that the rabbit and lamb inoculated by hypodermic injection and scarification oral cavity mucosa, respectively, appeared the similar clinical symptom with natural infection case. The specific gene of ORFV was obtained by PCR. The results mentioned above confirmed the agent was ORFV. The anti-ORFV polyclonal antibody was prepared from rabbit immuned with purified virions by ultracentrifugation, and was used to the detection of antigen in organs collected from died lambs by immunohistochemistry. The result showed that the virus antigen was localized in the cytoplasm of spinous cells in the stratum spinosum. The result further confirmed the disease was caused by ORFV infection.In order to obtain a secure, effective and new generation vaccine, we carried on preparation of DNA vaccine against ORFV by using ORFV 011(B2L) gene and ORFV 059(F1L) gene. Primers for the specific amplification for the complete ORFV 011(B2L) and ORFV 059(F1L) gene sequence based on published genomic sequence available in Genbank, and Kozak sequence and linker structure encoding (G4S)2 were inserted. The ORFV 011(B2L) gene and ORFV 059(F1L) gene were obtained by cloning and sequencing, and the complete sequences of the ORFV 011(B2L) and ORFV 059(F1L) genes were submitted to the NCBI Genbank database and were assigned accession number FJ808074 and FJ 808075. Sequence identities of nucleotides, as well as those of animo acids, were analyzed using the Clustal W and Blast. A phylogenetic tree derived from deduced amino acid sequences was constructed for the parapoxviruses using N-J method of MEGA 4.0. Comparing with the corresponding sequences including ORFV, BPSV, PVNZ, and other parapoxviruses available in Genbank, the phylogenetic studies of the ORFV 011(B2L) and ORFV 059(F1L) genes showed that ORFV/JL/08/CC isolate clustered in different branches, but was closer to the ORFV- Mukteswar 67/04 (DQ263305) isolate and the ORFV-OV/C2 isolate (AY040083), respectively. We obtained similar results from sequence analyses of the nucleotide and deduced amino acid of the ORFV 011(B2L) and ORFV 059(F1L) genes. Sequence analyses of the nucleotide and deduced amino acid of the ORFV 011(B2L) gene showed that the ORFV/JL/08/CC isolate shared closer relationship with other ORFV isolates from different regions (96.5-98.9% and 95.3-97.4%), and shared the highest homology with ORFV-Mukteswar 67/04 isolate from India (98.9% and 97.4%) . In the ORFV 059 (F1L) gene, the ORFV/JL/08/CC isolate shared the highest homology (98.4% and 97.9%) with the ORFV-OV/C2. The result above indicated the ORFV 011(B2L) and ORFV 059(F1L) genes were conservative among viral genome. In addition, the ORFV 011(B2L) gene and ORFV 059(F1L) gene of the ORFV/JL/08/CC isolate shared closer relationship with ORFV-Mukteswar 67/04 and ORFV-OV/C2, respectively. Hence, we concluded the isolate probably entered in our country by introduction from abroad, and formed independent branches during evolution. It was demonstrated that the length of the ORFV 011(B2L) gene (1137 bp) was identical in all Orf virus isolates, but several random nucleotide and amino acid mutations were found. The deduced amino acid sequences revealed seven unique substitutions (L47, R89, S113, F118, K228, L263, and S319) in the ORFV/JL/08/CC isolate that differed from those (F47, K89, G113, L118, E228, R263, and N319) of the other Orf virus isolates (data not shown). The length of ORFV 059 (F1L) gene (1011 bp) of the ORFV/JL/08/CC isolate was different from other Orf virus isolates. By multiple alignment of deduced amino acid sequences, the deletions of some amino acids were found to be located at the 5’termini of the ORFV 059 (F1L) gene. In addition, the result also revealed three unique substitutions (I133, L143, and A164) in the ORFV/JL/08/CC isolate that differed from those (F133, F143, and T164) of other Orf virus isolates. The variation probably formed during evolution, and these findings may provide useful information concerning the genotype of the Jilin province isolate.The ORFV 011(B2L) gene and ORFV 059(F1L) gene were linked by a linker structure encoding (G4S)2, and expression plasmids of pcDNA3.1-ORFV 011(B2L)、pcDNA3.1-ORFV 059(F1L) and pcDNA3.1-ORFV 011(B2L)-linker-ORFV 059(F1L) were constructed by eukaryotic expression vector pcDNA3.1(+). Recombinant plasmids were identified by enzyme digestion and PCR. The result indicated the construction of three recombinant plasmids were correct. Expression plasmids were sterily extracted, and were transfected MDBK cells by liposome. The cell strains which could stably expression were screened by G418. The transcription and translation of exogenous gene in MDBK cells was identified from mRNA level and translational level by RT-PCR, IFA, SDS-PAGE and Western-blotting. The result of RT-PCR indicated that three recombinant plasmids were were transcribed successfully in transfected cells. In addition, the detection of IFA indicated that green fluorescence was found in the cytoplasm of transfected cells. While, the result of SDS-PAGE and Western-blotting of cellular protein showed that fragments were found at positions of 42 kDa, 39 kDa and 79 kDa, which were consistent with anticipation. This further confirmed the expression of exogenous gene.Female Balb/c mice aged 6-8 weeks old were immuned by intramuscular injection with recombinant plasmid which were large-scale preparation using alkaline lysis. The immuned trategy was Prime-boost. The level of cell immunity and humoral immunity of immuned mice was detected by indirect ELISA, MTT and FCM. In addition, the security and immunoprotection role were evaluated by challenge experiments. First, the specific antibody level and antibody subtype of of sera was detected by SN and ELISA. The result found that the specific antibody of sera was detected after 1 week. The antibody level reached the highest. The result of antibody subtype detection found that the ratio of IgG2a and IgG1 was more than one after the third immunion. The result showed that DNA vaccine plasmids evoked Th1 type immunity response. In addition, the reproductive activity of splenic lymphocyte stimulated respectively by specific ORFV and non-specific ConA was detected by MTT, and the obvious T lymphocyte reproductive activity was found. It indicated that the immune system of immuned mice occupied high activated state. The changed regularity of T cell subgroup of splenic lymphocyte was analyzed by FCM. The result indicated CD4+and CD8+ T cell of immuned groups had obviously increase compared with control groups. The further confirmed the DNA vaccine plasmids could evoked cell immunity response. According to the results mentioned above, ORFV DNA vaccine plasmids could evoked specific cell immunity and humoral immunity response of immuned mice. In addition, the security and immunoprotection effect were evaluated by challenge experiments at the tenth day after the third immunization. The results indicated that the ORFV DNA vaccine plasmids were secure and could provide efficient immunoprotection. The results mentioned above will provide experiment evidence for the immunity experiments of lambs, while will establish foundation for the clarity of ORFV immunity.更多还原