【作者】 贺奋义；

【导师】 余四九；

【作者基本信息】 甘肃农业大学 ， 临床兽医学， 2012， 博士

【摘要】 鸽I型副粘病毒(Pigeon Paramyxovirus1，PPMV-Ⅰ)病又称鸽瘟或鸽新城疫，是一种由禽I型副粘病毒(Avian Paramyxovirus1， PMV-Ⅰ)引起的一种急性、热性、高度性传染病。PPMV-Ⅰ引起鸽群突然发病，迅速蔓延，临床症状上与鸡新城疫嗜神经速发株引起的十分类似，主要临床特点是肠炎、下痢和神经症状，尤以嗜神经速发型为多见，发病率和死亡率都很高。鸽新城疫病其传播迅速、发病率高，发病后死亡率较高，死亡率可达50-80%以上，严重的可高达95%以上，处于孵化期的种胚感染后可全部死亡。给养鸽业造成的影响和危害巨大，是养鸽业重点防控的传染病之一。鸽新城疫病毒编码两种囊膜糖蛋白，融合蛋白(F)和血凝素-神经氨酸酶(HN)，F蛋白位于囊膜外表面，形成纤突，对鸽新城疫的毒力及感染性起决定性作用，是鸽新城疫的主要免疫原性蛋白；F基因还具有较高的遗传稳定性，是新城疫基因分型的主要依据，也是决定鸽新城疫致病力强弱的关键因素；F基因的遗传变异与鸽新城疫的变异和流行密切相关，是鸽新城疫分子流行病学研究的首选基因，因此，F基因是研究鸽新城疫核酸疫苗的首选目的基因。本文通过鸽新城疫种毒PL株在非免疫健康鸡胚上增殖动态的试验研究和病毒的灭活时间、灭活温度以及甲醛浓度的试验研究，确定了鸽新城制苗用病毒最佳制备方法和最佳灭活条件，在此基础上，研制出鸽新城疫油乳剂灭活疫苗和蜂胶佐剂灭活疫苗，经物理性状检验、安全性检验、急性毒性试验、免疫持续期试验、最佳免疫剂量测定，效力试验以及田间应用试验表明，鸽新城疫蜂胶佐剂灭活疫苗不仅均匀度好，粘度低，通针性良好，注射后易被机体吸收，在接种部位不会形成硬结，而且免疫效果好，对鸽群免疫后能起到坚强的保护作用。此外，本文还用RT-PCR方法，从鸽新城疫病毒PL株基因组中克隆、鉴定了融合蛋白基因(F基因)，并在大肠杆菌中进行了高效表达，以纯化的F表达产物为抗原，建立了鸽新城疫病毒抗体间接ELISA检测方法；同时，把鸽新城疫F基因插入酵母表达载体pPic9k和真核表达载体PC-DNA3.1/V5-His中，构建了鸽新城疫F基因毕赤酵母表达系统和F基因真核表达体系，为鸽新城疫F基因生物活性的进一步研究及新型核酸疫苗的研制奠定基础。本项目研究的主要内容包括：1、鸽新城疫种毒增殖规律和灭活方法的研究：采用鸽新城疫PL株为种毒，在鸡胚上连续盲传5代，使其扩大增殖，收获尿囊液做为基础种子毒。通过种子毒在非免疫健康鸡胚上增殖动态的试验研究表明，病毒在羊水、尿囊膜(包含羊膜)、尿囊液中含量较高、增殖动态趋于一致，而在胚体及卵黄中含量较低，接种病毒后72h为最佳收毒时间。经病毒的最佳灭活时间、灭活温度以及甲醛浓度的试验研究，确定了0.05%甲醛在20℃条件下经过48h；70℃的温度条件下经过20min灭活病毒为最佳灭活方法，不仅能有效的完全灭活病毒，而且能使病毒的血凝活性保持在94%以上。2、鸽新城疫油乳剂灭活苗和蜂胶灭活疫苗的制备：用70℃，20min的灭活方法分别制备尿囊液蜂胶佐剂灭活苗和四元材料(鸡胚尿囊液、羊水、尿囊膜、羊膜)混合蜂胶佐剂灭活苗，用0.05%甲醛在20℃条件下经48h的灭活方法分别制备鸽新城疫尿囊液蜂胶佐剂灭活苗、尿囊液油乳剂灭活苗和四元材料(尿囊液、尿囊膜、羊膜、羊水)混合油乳剂灭活苗。制备的六种疫苗经物理性状检验、安全性检验、急性毒性试验、免疫持续期试验综合分析表明，均具有良好的抗原性和安全性。3.鸽新城疫油乳剂灭活苗和蜂胶佐剂灭活苗的性能比较试验：用制备的六种鸽新城疫油乳剂灭活苗和蜂胶佐剂灭活苗分别免疫注射乳鸽，结果表明四种蜂胶佐剂灭活苗免疫抗体消长水平和免疫期基本一致，二种油乳剂苗免疫抗体消长水平和免疫期基本一致，从而证明四元材料的疫苗与尿囊液毒疫苗的性能基本一致；但四元材料蜂胶佐剂灭活苗和油乳剂灭活苗在抗体产生时间及其达到峰值的时间和数值上有差异，蜂胶苗明显优于油苗。蜂胶佐剂灭活苗在免疫后5d抗体开始上升(平均为2.8log2～2.9log2)，14d抗体可达到高峰(平均为11.2log2～11.4log2)，而油乳剂灭活苗在免疫后9d抗体效价开始上升(平均为3.0log2～3.1log2)，21d才达到峰值(平均为9.0log2～9.2log2)。蜂胶佐剂灭活苗180d仍可维持较高水平(平均为6.6log2～6.7log2)；在免疫后30d、90d、180d用200ELD50强毒株攻击，其保护指数均为100%；对1日龄乳鸽接种1个使用剂量进行急性毒性试验观察，15d生长曲线表明，不影响鸽的生长，甚至有提高的趋势；室温保存3个月，4℃-8℃保存6个月，疫苗的物理性状和免疫保护性没有明显变化。4、鸽新城疫F基因的克隆及序列分析：通过设计一对引物，利用RT-PCR扩增以及亚克隆方法，从鸽新城疫病毒PL株中扩增出F基因，将扩增产物克隆进pMD18-T载体，转化DH5α感受态细胞，经菌落PCR鉴定筛选出阳性菌落并进行序列测定。结果表明：F基因为1662bp，编码553个氨基酸，与国内外报道的其他NDV毒株的F基因氨基酸数量相同；通过BLAST法与GenBank中国内外17个鸽源和鸡源部分新城疫毒株进行相似性比较及对照系统进化树的结果分析，表明我们的分离株（PL株）与17个毒株之间的相似性介于76.3-98.6%；对分离的鸽新城疫F融合蛋白的氨基酸序列与国内外8株新城疫氨基酸序列进行对比分析，结果氨基酸同源性为93.6%-96.7%；F0蛋白裂解位点的氨基酸组成为112R-R-R-K-R-F117，符合强毒株的序列。5、鸽新城疫F基因的原核表达及其生物活性研究：用PCR方法获得了缺失F蛋白信号肽、胞质区和跨膜区的F基因片段，将其亚克隆到pGEX-4T-1原核表达载体中，与GST蛋白融合表达，经IPTG诱导和SDS-APGE分析，表明目的蛋白在大肠杆菌BL21中得到高效表达，表达产物分子量约为60Kda；以表达的融合蛋白为抗原初步建立了间接酶联免疫吸附试验(ELISA)方法，用方阵法确定其最佳反应条件：抗原1∶128稀释即抗原包被量为0.6μg/孔，血清1∶400稀释，与一抗的最佳作用时间为60min，与酶标二抗的最佳作用时间为60min，底物显色的最佳作用时间为15min。6、鸽新城疫F基因在毕赤酵母中表达：根据鸽新城疫F基因序列，设计引物，将其亚克隆到真核表达载体pPIC9K，用SalI内切酶线性化后电击转化GS115毕赤酵母感受态细胞，用影印法在含G418的YPD平板上筛选高拷贝重组菌，用甲醇诱导表达并进行SDS-APGE分析。7、鸽新城疫F基因的真核表达及其生物活性研究：将鸽新城疫PL株F基因插入到真核表达载体pcDNA3.1V5-Hi中，构建出真核表达质粒PCDNA3.1-PPMV-1-F；将构建的真核表达重组质粒免疫接种非免疫新城疫的乳鸽，并在免疫后第28d进行攻毒保护试验。结果表明，在免疫后第7d产生抗体，14d抗体水平达到最高，之后抗体水平开始下降；攻毒保护试验结果显示，对鸽新城疫强毒攻击保护率为33.33%（5/15），具有一定的免疫保护作用。更多还原

【Abstract】 Pigeon Paramyxovirus1(PPMV-Ⅰ) disease, is also known as Pigeon plague or PigeonNewcastle disease, which is a kind of Avian Paramyxovirus1(PMV-Ⅰ) can cause an acute,febrile, highly infectious disease. PPMV-Ⅰis caused by the sudden onset of the flock, and therapid spread of addicted the nerve-speed hair strains are very similar with clinical symptoms ofNewcastle disease. The mainly clinical features are enteritis, diarrhea and neurologic symptoms,especially in the perineural fast hairstyle, morbidity and mortality are high. Pigeon Newcastledisease is spreaded rapidly, the incidence and mortality rate are high, what can reach more than50-80%or serious can be as high as95%and up to serious infection in the incubation period ofthe seed embryo all died. Huge impact and harm are caused to the Pigeon industry which is thePigeon industry’s focus on the prevention and control of infectious diseases.Pigeon Newcastle disease virus encoding two glycoprotein, fusion protein (F) and thehemagglutinin-neuraminidase (HN), F protein located in the sac membrane outer surface, forminga spike, the Pigeon Newcastle disease pathogenicity and infection play a decisive role in PigeonNewcastle disease, which is the main immunogenic protein; F gene also has high genetic stability,which is the main basis of the Newcastle disease genotyping, also is the key factor for deciding thePigeon Newcastle disease causative force; genetic variation of F gene and Pigeon Newcastledisease variation and prevalence are closely related, it is a preferred gene for Pigeon Newcastledisease molecular epidemiology research. Therefore, F gene is the preferred target gene vaccinefor the study of Pigeon Newcastle disease virus nucleic acid.In this paper, by the test study of the Pigeon Newcastle disease drug PL strains in chickenembryos non-immune healthy chicken embryos proliferation dynamic test, virus inactivation time,the best inactivated temperature and the concentration of formaldehyde, which was determinedthe preparation of pigeon Newcastle disease vaccine virus preparation method, and the bestinactivated condition. On the base of the above, the preparation of the different viral sources oilemulsion inactivated vaccines and propolis adjuvant inactivated vaccine were produced, and theprocess for the preparation of the vaccines to physical properties, safety, the acute toxicity,immune duration, the best immune dosimetry, the potency and field application were tested. Thefinalized Pigeon Newcastle disease propolis adjuvant inactivated vaccine had good uniformity,which contain low viscosity, good through needle after injection, easily absorbed by the body, andwould’t form the inoculation site induration. It could play a excellently protective effect for theflock after immunization. In addition, by using of RT-PCR method, pigeon Newcastle disease virus PL genomic wascloned, and the fusion protein gene (F gene) was identificated, and was highly expressed in E. coli.The antibody indirect ELISA of Pigeon Newcastle disease virus was established by the expressionproduct purified F antigen;at the same time, the pigeon Newcastle disease F gene was insertedinto the yeast expression carrier pPic9k and eukaryotic expression vector PC-DNA3.1/V5-His, inwhich built pigeon Newcastle disease F gene Completion Pichia expression system and F geneeukaryotic expression system, the foundation was established which for the further study of thebiological activity of the F gene of pigeon Newcastle disease and the development of novel DNAvaccine.The main research contents are as follows.:1. The study on Pigeon Newcastle Disease Viruses proliferation law and methods ofinactivation: using PL as a poison. Continuous on the chick embryo blinded fivegenerations to expand proliferation, collected allantoic fluid as foundation seed virus. Bythe study of seed poison dynamic proliferation in non-immune healthy chicken embryoswhich had been shown that the virus was on high levels in the amniotic fluid, allantoicmembrane (containing the amniotic membrane), and allantoic fluid. It was in theconsistent of dynamically proliferation. Though in the embryoid bodies and yolk were onlow levels, the best closing poison time was of the72hours after virus inoculation. On thestudy of the best time after virus inactivation, inactivation temperature, and theconcentration of formaldehyde, determined the condition of0.05%formaldehyde,20°Cwith48h,70°C with20min was the best. Not only was the effect to completely inactivatethe virus, but also the virus hemagglutination activity was maintained at above94%.2. The study on Pigeon Newcastle disease virus inactivated oil emulsion vaccine andpreparation of propolis inactivated vaccine: which were respectively prepared on the condition of70℃,20min’s inactivation methods for allantoic fluid of Propolis Adjuvant Inactivated Vaccineand four element (chick allantoic fluid, amniotic fluid, chorioallantoic membrane, amnion) mixedPropolis Adjuvant Inactivated Vaccine.0.05%formaldehyde in the condition of20℃after48hinactivation methods which were respectively prepared Pigeon Newcastle disease allantoic fluidof Propolis Adjuvant Inactivated Vaccine, allantoic fluid of oil emulsion inactivated vaccine andfour element (allantoic fluid, chorioallantoic membrane, amniotic membrane, amniotic fluid)mixed oil emulsion inactivated vaccine. Preparation of the six vaccines were tested by physicscharacter, safety, acute toxicity, immune duration comprehensive analysis, which indicated thatthe six kinds of vaccine had good antigenicity and safety. 3. The performance comparison test of Pigeon Newcastle disease virus inactivated oilemulsion vaccine and Inactivated propolis vaccine: preparing the six Pigeon Newcastle diseasevirus inactivated oil emulsion vaccine with propolis inactivated vaccine immunized pigeonsrespectively, the results showed that four kinds of propolis vaccine immune antibody level andimmune period were basically the same, two kinds of oil emulsion vaccine immune antibody leveland immune period basically was consistency, four proof material and allantoic fluid attenuatedvaccine was of similar performance; but the four element propolis inactivated vaccine andinactivated oil emulsion vaccine on antibody production time and peak time and numericaldifference was obviously better than that of oil, propolis vaccine. Propolis Adjuvant InactivatedVaccine on immune5D antibodies after start up (average2.8log2～2.9log2),14d antibodiescould reach a peak (average11.2log2～11.4log2), while the oil emulsion inactivated vaccineafter immunization in9D antibody titer begin to rise (average3.0log2~3.1log2,21d) not to peak(average9.0log2～9.2log2).180d could still maintain higher level (the average was6.6log2～6.7log2).Propolis inactivated vaccine after immunization in30d,90d,180d with200ELD50virulent attack, its protection index was100%.In1day-old Pigeon inoculated with1dose acutetoxicity test and observation,15d growth curves that did not affect the pigeons, growth, and evenhad a trend of increase. Propolis Adjuvant Inactivated Vaccine stored at room temperature for3months,4℃-8℃for6months, the vaccine’s physical traits and immune protection did notsignificantly change.4.Pigeon Newcastle disease virus F gene cloning and sequence analysis: through a pair ofprimers were designed, using RT-PCR amplification and clone method, from Pigeon Newcastledisease virus PL strain F gene was amplified by the PCR products were cloned into pMD18-T,vector, transformation of DH5α competent cells, colony PCR identification screened positivecolonies and sequence determination. The results showed that: F as1662bp, encoding a protein of553amino acids, and other domestic and foreign reports of NDV strain F gene amino acid number;by the method of BLAST and GenBank Chinese inside and outside17Pigeon and chickenNewcastle disease virus source part similarity comparison and contrast of phylogenetic treeanalysis results, show that our isolates (PL strain) and17strains between the similarity between76.3-98.6%; on the separation of Pigeon Newcastle disease virus F fusion protein amino acidsequence with domestic and international8strains of Newcastle disease amino acid sequenceswere compared and analyzed, the results of amino acid homology to93.6%-96.7%; F0proteincleavage site amino acid composition of112R-R-R-K-R-F117, consistent with the virulentsequence. 5.The study on the prokaryotic expression and biological activity of the F gene for pigeonNewcastle disease: using PCR method to obtain the deletion of F protein signal peptide,cytoplasmic and transmembrane region of the F gene fragment, subcloned into prokaryoticexpression vector pGEX-4T-1, and GST fusion protein, induced by IPTG and SDS-APGEanalysis showed that objective, protein in E. coli BL21was highly expressed, the molecularweight of the expressed product was about60Kda; to express the fusion protein as antigenpreliminary established the indirect enzyme linked immunosorbent assay (ELISA) method, usingmatrix method to determine the optimal reaction conditions: antigen was diluted on1∶128andthe coating antigen was0.6μ g/hole,1:400diluted serum, and one of the best time for60min,and enzyme labeled two of best action time is60min, chromogenic substrate the best time for15min.6.The expression of Pigeon Newcastle disease virus F gene in Pichia pastoris: according tothe sequence of F gene of Newcastle disease in pigeons, primers, subcloned into eukaryoticexpression vector pPIC9K, SalI enzyme after linearization by electroporation GS115Pichiacompetent cells, using photolithography in G418containing YPD panel on screening high copyexpression of recombinant bacteria, induced by methanol and SDS-APGE analysis.7.The study of the Pigeon Newcastle disease virus F gene’s eukaryotic expression andbiological activity: the F gene of Pigeon Newcastle disease virus strain PL was inserted intoeukaryotic expression vector pcDNA3.1V5-His, constructed eukaryotic expression plasmidPCDNA3.1-PPMV-1-F; the constructed eukaryotic expression recombinant plasmid vaccinationimmunity milk Pigeon Newcastle disease and after immunization in article28d in protective test.The results showed that, in the7d immune antibody, in immune14d antibodies levels reached amaximum, then antibody levels was began to decline; attack protection test results were beenshowed, the Pigeon Newcastle disease virus attack protection rate was of33.33%(5/15), whichhad a certain protective immunity in mice.更多还原