【作者】 胡北侠；

【导师】 刘秀梵； 张如宽； 吴艳涛；

【作者基本信息】 扬州大学 ， 预防兽医学， 2001， 硕士

【摘要】 以国内新城疫病毒（Newcastle disease Virus，NDV）标准强毒株F₄₈E₈接种10日龄SPF鸡胚，收集死亡鸡胚尿囊液经蔗糖密度梯度离心后获得较纯的病毒。以纯化的NDV建立了间接ELISA方法，并用于单抗筛选的第一检测系统。应用蓝斑筛选技术对表达NDV融合蛋白（Fusion Protein，F）的重组鸡痘病毒（Recombinant Fowlpox Virus，rFPV）进行筛选后获得较纯的重组病毒，通过间接免疫荧光（Immunofluorescence，ⅡF）试验证实了NDV F基因在rFPV中的表达。用纯化的rFPV感染96孔细胞培养板上次代鸡胚成纤维细胞（Chicken Embryo Fibroblast，CEF），建立了检测单抗的ⅡF方法，作为单抗筛选的第二检测系统。 以纯化的NDV和经SDS-PAGE分离的NDV F蛋白免疫BALB/C小鼠，应用淋巴细胞杂交瘤技术，经间接ELISA和ⅡF试验反复筛选并三次亚克隆后共获得7株能够稳定分泌针对NDV F蛋白单克隆抗体的杂交瘤细胞，分别命名为C₅-2-A₇、F₇-G₅、F₉-2-G₅、B₂-C8-F₁₀、C₁₁-C₆-E₂、55-C₂-A₉，51-F₆-F₇。其中前5株是用NDV全病毒作免疫原，后2株是用SDS-PAGE分离的F蛋白作免疫原。单抗的生物学特性鉴定表明：7株单抗均无血凝抑制性（HI），而单抗中和性各有差异。此外，对单抗的免疫球蛋白 胡北侠：新城疫病毒融合蛋白单克隆抗体的研制 三 亚类、所针对的抗原表位，ELSA效价等进行了鉴定和研究。Western blot 结果显示：除F7－G，、Cll－C6－EZ外，其它单抗均能识别56KD的蛋白多肽； 其中 CS一2－A7、F7一GS、Fg一2一GS、BZ一CS一FI＊，C；；一q一EZ 5株单抗的 ELSA效 价明显比 55－CZ－Ag，sl－F6－F7 2株高。用这 7株单抗对国内夕不同时期分离 的NDV毒株进行排谱发现，单抗几乎可以与所有毒株反应，表明所得的单 抗都是针对 NDV F蛋白的抗原保守区。更多还原

【Abstract】 Chinese challenge strain F48E8 of newcastle disease virus(NDV) was papagated in the allantoic cavity of 10-day old embiyonated SPF chicken eggs. The allantoic fluid of NDV F48E8 was purified and concentrated by centrifugation with 10-60% continuous sucrose gradient. The indirect ELISA was developed by using the purified NDV as antigen and used to screen monoclonal antibodies (MAbs). Recombinant fowlpox vivus(rFPV) were selected and purified many times in chicken embryo fibroblast (CEF) cultures overlaid with agarose gel containing x-gal by blue plaques screening. The expression of the NDV F gene in recombinant FPV was confirmed by indirect immunofluorescence(IIF) with ployclonal chicken antiserum against NDV. The 1W with the CEF infected by the purified rFPV as the second screening system was used to select the clones which had been positive in indirect ELISA. The BALBIC mice were immunized by the intact NDV and the F protein of the sodium dodecyl sufate 梩reated NDV (SD SNDV). By using the hybridoma technique seven MAbs with specificity to the NDV F protein were established. They were named C5-2-A7, F7-G5, F9-2-G5, B2-C8-F10, Ci 1-C6-E2, 55-C2- A9, 51 -F6-F7 respectively. The first five MAbs were derived from fusions with the intact NDV as immunogen, and the other two were abtained from the F protein of the SDSNDV. All of the MAbs were negative in HI test while only three of them neutralized viral infectivity in embryonated eggs. The first five of them bound to the virus at higher titers than the others in indirect ELISA. These MAbs were able to recognize the protein about 56KD in western blot with the exception of the Mabs F7-G5 ,C11-C6-E2. The reacting patterns of MAbs with different NDV strains indicated that all of the MAbs were against the conserved antigenic epitopes of the F protein of NDV.更多还原