【作者】 李龙；

【导师】 于涟；

【作者基本信息】 浙江大学 ， 生物医学工程， 2006， 博士

【摘要】 传染性法氏囊病(IBD)是由传染性法氏囊病病毒(IBDV)引起的急性接触性传染病,是危害世界养禽业的主要传染病之一。IBDV主要侵害3-8周龄雏鸡的中枢免疫器官法氏囊,破坏带有sIgM的B淋巴细胞,引起脾脏、胸腺及外周血液淋巴细胞的凋亡,导致以淋巴细胞衰竭坏死为主要特征的免疫缺陷性和免疫抑制性疾病。 通过易感动物雏鸡,构建了一株传染性法氏囊病病毒基因组节段杂合病毒。应用PCR定点突变方法,在IBDV野毒株ZJ2000株A节段2366-2371nt引入沉默突变NaeI后,克隆入真核表达载体pCI,获得pCI-AKZJ2000,分别制备pCI-AKZJ2000、以及含有细胞致弱疫苗株HZ2株A和B节段全长的真核表达载体pCI-AKHZ2和pCI-mBHZ2的超纯质粒。不同组合的上述质粒(pCI-AKZJ2000/pCI-mBHZ2和pCI-AKHZ2/pCI-mBHZ2),在脂质体介导下,肌肉途径分别注射雏鸡。通过血清抗体、病毒抗原和病毒粒子检测、鸡胚传代试验、以及分子遗传标记鉴定,表明从鸡体内拯救出了杂合病毒rAKZJ/HZ和重组病毒Xihu2002株。本试验为IBDV的反向遗传系统提供了一个新策略,尤其是对于不能适应细胞的IBDV强毒株的拯救。结果还提示IBDV基因组B节段序列对病毒毒力具有重要作用,为研究VP1蛋白在病毒复制和致病中的作用提供了思路。 制备了兔抗IBDV VP5多克隆抗体。利用RT-PCR技术从IBDV地方分离株TL2004株感染鸡胚尿囊液中扩增到VP5基因,进而构建了T7启动子控制下的N端GST-Tag融合表达质粒pGEX-VP5。序列测定表明VP5基因全长438bp,编码一个由145个氨基酸组成的VP5蛋白。将pGEX-VP5转化大肠杆菌BL21,在IPTG的诱导下高效表达了GST-VP5融合蛋白(44kD)。通过包涵体纯化的方法,获得的较高纯度的融合蛋白,免疫新西兰兔,Western blot和ELISA分析表明,制备的融合蛋白抗血清效价在1:12800以上,并具有良好的免疫反应性,为进一步研究VP5在IBDV复制与致病中的作用,以及IBDV VP5基因缺失病毒打下了良好的基础。 在上述基础上,进而探索了VP5基因序列及编码蛋白对IBDV复制的影响。通过PCR定点突变方法,将IBDV HZ2株A节段ORF A2(VP5)起始密码子更多还原

【Abstract】 The infectious bursal disease virus (IBDV), a member of the Birnaviridae family, containing a bisegmented double-stranded RNA genome, encodes four structural viral proteins, VP1, VP2, VP3, and VP4, as well as a non-structural protein, VP5. IBDV is the causative agent of infectious bursal disease (IBD) in chickens. It causes high mortality in young chickens and establishes an immunosuppression state by destroying the precursors of B lymphocytes in the bursa of Fabricius and the postbursal B lymphocytes, leading to vaccination failure against other pathogens.To further evaluate the efficiency and optimize the procedure of previously constructed IBDV reverse genetic system based on plasmids cDNA co-transfection, in the present part, the segment A from two IBDV strains, field isolate ZJ2000 and attenuated strain HZ2, were inserted into one NaeI by site-directed silent mutagenesis, respetively. The two resulting mutants were subcloned into expression plasmid pCI under the control of the human cytomegalovirus (hCMV) immediate early enhancer and promoter to construct the recombinant plasmids pCI-AKZJ2000 and pCI-AKHZ2, respectively. Each of the two recombinants was combined with another recombinant pCI plasmid containing the marked segment B of strain HZ2 (pCI-mBHZ2), and injected intramuscularly into specific-pathogen-free (SPF) chickens. Two chimeric IBDV strains were recovered from the chickens post injection. Two out of eight chickens in each of two groups showed the bursal histopathological change. The reassortant virus derived from pCI-AKZJ2000/pCI-mBHZ2 can infect chicken embryos and shows relatively low virulence. We have developed a novel virus reverse genetic approach for the study of IBDV. The results also form the basis for investigating the role of VP1 in viral replication and pathogenecity.In order to characterize the role of VP5 in IBDV replication, IBDV VP5 gene was amplified and cloned into an N-terminal GST-Tag fusion expression vector, pGEX-4T-2, which was controlled by T7 promoter. The sequencing result showed thatthe VP5 gene was composed of 438 base pairs, and coded 145 amino acids. High VP5 product was expressed in E. coli BL21 induced by IPTG, and the GST-VP5 fusion protein existed in inclusion. High titer anti-VP5 serum was also prepared in New Zealand rabbit immunized with purified fusion protein inclusion.The nonstructural protein VP5 of IBDV has been reported to be associated with virus cell apoptosis and pathogenicity, but its regulator role in viral replication has not been unequivocally identified. In this part, three VP5 deficient IBDV strains were constructed with or without base pairs deletion by a plasmid-based infectious cDNA system. Indirect immuno-fluorescence assay showed that VP5 was not expressed by VP5 deficient strains. The effect of 5’ terminal base pairs of VP5 gene on viral replication kinetics was analyzed on chicken embryo fibroblast (CEF) cells. The results showed that VP5 and 5’ terminus of its gene is not essential for replication of IBDV. However, the deletion delayed the virus replication on CEF cells and the final titers decreased with increasing of deletion length. Thus, it is indicated that the 5’ terminus of VP5 gene contains an expression regulator for virus genome. The regulation mode was further characterized by comparison between virus replication and protein expression. The results showed that the putative factor affects protein translation rather than RNA replication.To characterize the effect of VP5 on IBDV replication, pathogenicity and immunogenicity, cell-derived attenuated strain HZ2 and VP5 deficient strain ANhe2 were oral given to 14-day-old SPF chickens respectively. The bursa/body weight ratios and bursal histopathological lesion were detected at certain intervals post inoculation. Viruses were re-isolated form bursas and titration at the same time intervals for replication characterization. It is showed that mild and transient bursal lesion was occurred post virus inoculation. Both strains could replicate in bursas within a short period after inoculation, although strain ANhe2 replicated a litter slower to peak at 7-d post inoculation.Both strains HZ2 and ANhe2 induced similar antibody response, which could neutralize IBDV strain HZ2. Three weeks after boosting at 4-week-old, all test chickens were challenged with IBDV reference strain BC6/85. Although both strainsprovided immune protection against challenge, the protection rate induced by strain ANhe2 (87.5%) is better than that induced by strain HZ2 (62.5%). Combined with gross and histopathological bursa change, it is showed that the deficiency of VP5 enhanced the immunogenicity of strain HZ2.更多还原