传染性法氏囊病病毒VP5缺失减毒株的构建

发布时间：2018-07-05 19:33

本文选题：传染性法氏囊病病毒 + 反向遗传　；参考：《浙江大学》2006年博士论文

【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
[Abstract]:Infectious bursal disease (IBD) is an acute contact infectious disease caused by infectious bursal disease virus (IBDV). The destruction of B lymphocytes with sIgM caused apoptosis of lymphocytes in spleen thymus and peripheral blood and resulted in immunodeficient and immunosuppressive diseases characterized by lymphocyte failure and necrosis. A novel infectious bursal disease virus (IBDV) genomic segment heterozygous virus was constructed from susceptible chickens. The silencing mutation NaeI was introduced into the A segment 2366-2371nt of IBDV wild strain ZJ2000 by PCR site-directed mutation. The pCI-AKZJ2000 and the eukaryotic expression vectors pCI-AKZJ2000 and pCI-mBHZ2 were cloned into the eukaryotic expression vector pCI-AKZJ2000 and the eukaryotic expression vectors pCI-AKZZ2 and pCI-mBHZ2 containing the full-length segments of A and B segments of the attenuated vaccine strain HZ2 were prepared, respectively. Different combinations of these plasmids (pCI-AKZJ2000 / pCI-mBHZ2 and pCI-AKHZ2 / pCI-mBHZ2) were injected into chicks by liposome mediated muscle pathway. The results of serum antibody, virus antigen and virus particle detection, chicken embryo passage test and molecular genetic marker identification showed that the hybrid virus rAKZJ / HZ and recombinant virus Xihu2002 strain were rescued from chicken. This study provides a new strategy for the reverse genetic system of IBDV, especially for the rescue of IBDV virulent strains that can not adapt to cells. The results also suggest that the B segment sequence of IBDV genome plays an important role in virulence of the virus and provides a way to study the role of VP1 protein in viral replication and pathogenicity. Rabbit anti IBDV VP5 polyclonal antibody was prepared. The VP5 gene was amplified by RT-PCR from chicken embryo allantoic fluid infected with TL2004 strain of IBDV, and then the N-terminal GST-Tag fusion expression plasmid pGEX-VP5 was constructed under the control of T7 promoter. Sequence analysis showed that the VP5 gene was 438 BP in length, encoding a 145 amino acid VP5 protein. PGEX-VP5 was transformed into Escherichia coli BL21, and GST-VP5 fusion protein (44kD) was highly expressed under the induction of IPTG. The high purity fusion protein was obtained by the method of inclusion body purification. Western blot and Elisa analysis showed that the antiserum titer of the fusion protein was over 1: 12800 and had good immunoreactivity. The results provide a good basis for the further study of the role of VP5 in the replication and pathogenesis of IBDV and the deletion of VP5 gene of IBDV. On the above basis, the effects of VP5 gene sequence and encoded protein on IBDV replication were explored. The ORF A2 (VP5) initiation codon of A segment of IBDV HZ2 strain was determined by PCR site-directed mutation.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R373

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