【作者】 韦栋平；

【导师】 刘秀梵；

【作者基本信息】 扬州大学 ， 预防兽医学， 2004， 博士

【摘要】 新城疫是由副粘病毒科的新城疫病毒（Newcastle diseae virus，NDV）引起的一种烈性家禽传染病，它对我国养禽业的危害最为严重。低致病性H9亚型禽流感病毒（Avian Influenza virus，AIV）是目前影响我国养禽业的主要AIV亚型，它对我国养禽业也已造成了严重的经济损失。当前我国对ND和H9亚型AI的控制主要应用常规疫苗的免疫接种。但是作为我国目前控制ND和H9亚型AI流行的重要手段，常规疫苗的免疫接种会干扰现有技术条件下的免疫监测和流行病学调查。而且油乳剂灭活疫苗使用后会引起严重应激，并且生产成本偏高等。为了研制一种可以克服上述缺点的新型载体联合疫苗，本研究用鸡痘病毒（Fowlpox viros，FPV）作为表达载体，构建了能够同时表达NDV融合蛋白（Fusion protein，F）基因和H9亚型AIV血凝素（Hemagglutinin，HA）基因的重组鸡痘病毒，并对其免疫效力进行了评估。 1．共表达NDV F基因和H9亚型AIV HA基因的重组鸡痘病毒的构建及其生物学特性鉴定 以酚-氯仿提纯NDV F48E8株的基因组RNA作为反转录聚合酶链反应（RT-PCR）模板，扩增出长度约为1.7Kb的NDV F基因片段，将其克隆入pGEM-Teasy载体，构建重组质粒pGEM-TF并进行测序确证；以质粒pUCHA为模板，用PCR扩增出约为1.7Kb的H9亚型AIV HA基因片段，将其克隆入pGEM-T easy载体，构建重组质粒pGEM-THA并进行测序确证。分别从pGEM-TF和pGEM-THA切下F基因和HA基因，通过一系列分子生物学操作步骤插入到质粒pFPV7S中的FPV基因组复制非必需片段，并使分别调控F基因和HA基因转录的鸡痘病毒启动子P_E／L和合成启动子P_S以反向方式连接，重组质粒命名为p7SHF。最后将P11-LacZ报告基因表达盒插入到质粒p7SHF中，获得转移载体pFPVHF，用以转扬州大学博士学位论文染经预先感染FPV 282E4疫苗株的鸡胚成纤维细胞（CEF）.通过在含有X.Gal的营养琼脂上挑选蓝色病毒蚀斑，经过多次蓝斑筛选和克隆纯化后，获得稳定生长的重组鸡痘病毒（rFPv一F一HA）。PCR和Southem一blot检测证实了F基因和HA基因已成功插入到FPV的基因组:间接免疫荧光试验结果表明重组病毒能够同时正确表达HA和F蛋白。 比较rFPv一F一HA和wt一FPv在CEF上的繁殖滴度，结果发现两者在CEF中的繁殖滴度无显性著差异，提示载体病毒的复制能力并未因外源基因的插入而下降。将rFPV一F一HA在CEF上连续传代培养20代次后，蓝色空斑试验和PCR检测结果表明插入的F基因和HA基因均能够在病毒的传代过程中稳定遗传;间接免疫荧光试验证实第20代次的重组鸡痘病毒仍然能够正确表达F和HA蛋白，进一步表明了rFPv一F一HA具有良好的遗传稳定性。2.NOV「蛋白抗体的间接ELISA检测方法的建立及评估 用经过充分灭活的NDV F48E8株作为包被抗原，建立了检测由FPV载体介导表达的NDVF蛋白的抗体的间接ELJSA方法，并用动物试验进行了初步评估。以方阵滴定法确定抗原的最佳包被浓度为355n留孔，待检血清最佳稀释度为1:40。通过交叉试验、阻断试验和重复性试验证实所建立的间接ELisA方法具有高度的特异性和良好的可重复性。用该间接EUSA方法测定了20份阳性血清样品的P加值，并通过对该20份阳性血清的P/N值与ELisA抗体效价（ELISA titer， ET）的自然对数值（InET）的线性回归分析，获得了回归方程1 nET=0 .201 XP/N+4 .632，通过测定待检血清在l:40稀释度的P/N值，用方程直接求出血清的ELISA抗体滴度，可以实现定量检测。应用该间接ELISA方法对表达NDVF基因的重组鸡痘病毒免疫鸡群进行了血清抗体检测，结果表明，血清的间接ELISA抗体效价与中和试验所测定相应血清的中和抗体效价高度相关（P<0 .01）。攻毒保护试验结果显示，间接ELISA抗体效价和机体保护力之间表现一定程度的相关性。该方法的建立为后续试验评估表达NDVF基因的重组鸡痘病毒免疫鸡群的免疫效果提供了一种可靠的血清学方法。3.共表达NDVF基因和H9亚型AIVHA基因的重组鸡痘病毒的免疫效 力试验3.1试验鸡接种rFPv一F一HA后抵抗H9亚型Alv感染的免疫保护作用 7日龄SPF雏鸡随机分为4组，分别于颈部皮下接种rFPv一F一HA（104PFU/羽）、韦栋平:共表达NDVF基因和H9亚型AlvHA基因的重组鸡痘病毒及其免疫效力iiirFPv一队（lo4PFu/羽）、wt一FPv（一o4PFu/羽）和Hg亚型Al油乳剂灭活疫苗（0.ZmL/羽）。免疫后7²1天，除wt一FPv外，各种疫苗均可诱生针对H9亚型Alv的特异性HI抗体。其中以油乳剂灭活疫苗诱生的HI抗体效价最高，而rFPv一F一HA诱生的Hl抗体效价与单表达队基因的rFPV一队诱生的HI抗体效价差异不显著。免疫后21天，各组试验鸡用剂量为107ELDS丫羽的Hg亚型川vF株滴鼻攻毒，并于攻毒后5天采集试验鸡的口腔棉拭子，按照常规方法处理后，接种10日龄SPF鸡胚作病毒分离试验。结果显示各组试验鸡的口腔排毒率为:wt一FPV免疫组（100%）、rFPV一F一HA免疫组（16.7%）、rFPV一队免疫组（16.7%）和油乳剂灭活疫苗免疫组 （16.7%）。 用含有残余抗NDV和H9亚型AIV母源抗体的26日龄商品蛋鸡进行免疫效力试验，结果发现，尽管由于母源抗体的干扰，rFPV一F一HA及rFPV一HA在商品蛋鸡体内诱生的HI抗体效价远低于?更多还原

【Abstract】 Both Newcastle disease (ND) and H9 subtype avian influenza (AI) have been serious disease problems and caused considerable economic losses in poultry industry in China. So far, vaccination with conventional vaccines including the attenuated live vaccines and inactivated vaccines in oil emulsion to protect birds against ND and H9 subtype AI has been a routine procedure in China. However, the use of these vaccines is restricted because of the disadvantages they inherit including their interference of routine serological surveillance, higher cost and side effect of greater streos in birds post-vaccination. Here, we described the construction of recombinant fowlpox virus co-expressing the fusion protein (F) gene of Newcastle disease virus (NDV) and the HA gene of H9 subtype avian influenza virus (AIV) and the evaluation of its protective efficacy against either NDV or AIV challenge in chickens for the development of better vaccine.1. Construction of recombinant fowlpox virus co-expressing the F gene of NDV and the HA gene of H9 subtype AIV and its biological propertiesThe genomic RNA of NDV strain F48E8 was prepared by phenol-chloroform extraction and used as template for RT-PCR to obtain the fusion protein (F) gene, which was cloned into pGEM-T easy vector to form pGEM-TF and its sequence was determined. The hemagglutinin (HA) gene of H9 subtype ATV in pUCHA was modified by using PCR. Its amplified product was then inserted into pGEM-T easy vector and the resulting plasmid pGEM-THA was determined by sequencing. In order to construct recombinant plasmid p7SHF, the F gene in pGEM-TF and the HA gene in pGEM-THA were excised, respectively, and inserted into the randomly selectednonessential region of the fowlpox virus (FPV) under the transcriptional control of fowlpox virus promoter Pe/L. and the synthetic promoter Ps, respectively, which were ligated in opposite direction. The P11-LacZ reporter gene from pppG18 was cloned into p7SHF resulting in recombinant transfer vector pFPVHF. Pre-infected with Chinese vaccine strain 282E4 of FPV (wt-FPV), chicken embryo fibroblast (CEF) monolayers was transfected with pFPVHF to generate recombinant fowlpox virus co-expressing F and HA, designated rFPV-F-HA. By selection of blue plaques on the CEF overlaid with agar containing X-Gal, rFPV-F-HA was screened and subjected to further rounds of plaque purification until a pure population was achieved. PCR and Southern-blot assay indicated that the F and HA gene had been inserted into the genomic DNA of FPV. Subsequently, F and HA expressed in CEF infected with rFPV-F-HA were confirmed by indirect immunofluorescence assay (IFA).Comparison of the viral titer between rFPV-F-HA and wt-FPV grown in CEF demonstrated that the ability of rFPV-F-HA to replicate in CEF was not impaired for insertion of the target DNA fragments. The rFPV-F-HA was further detected by blue plaque assay and PCR after it was passaged for 20 times on CEF monolayers. These results showed that the inserted F gene and HA gene were stably integrated. Expression of F and HA in rFPV- F-HA confirmed by IFA suggested that it be genetically stable. 2. Detection of antibody in chickens to Newcastle disease virus fusion protein by indirect ELISAAn indirect ELISA for detecting antibody in chickens induced by the fusion protein of NDV expressed by recombinant fowlpox viruses (rFPVs) was developed with great sensitivity, high specificity and strong reproducibility, and subsequently evaluated with sera from SPF chickens vaccinated with or without rFPVs. The optimum coating concentration of NDV antigen was determined by checkerboard titration using pre-immune chicken antiserum to recombinant fowlpox virus expressing the F gene of NDV and amounted to 355ng of NDV antigen per well. The serum sample for detection was diluted to 1:40. A near-linear relationship existed between positive /negative ratio(P/N) of anti-sera at a single working dilution (1:40) and the In of the corresponding observed serum ELISA titers (ET) as determined by a standard serial-dilution method. Regres更多还原