【作者】 万旺军；

【导师】 于涟；

【作者基本信息】 浙江大学 ， 生物医学工程， 2005， 硕士

【摘要】 传染性法氏囊病病毒（Infectious Bursal Disease Virus,IBDV）主要侵害雏鸡中枢免疫器官法氏囊,导致以免疫抑制为主的淋巴细胞衰竭综合症,造成对多种病原的易感性增加和其他疫苗的免疫失败。传统IBD人工免疫主要是弱毒苗和灭活苗免疫,但自80年代以来出现的IBDV变异株和超强毒株给该病的防治带来了困难。DNA疫苗因能诱导机体产生全面的免疫应答而成为疫苗学研究领域的焦点,但由于其诱导产生的免疫应答的效果还不够理想,因此如何增强其免疫效果成为目前DNA疫苗研究的关键问题。 本研究小组在前期工作中已证实了IBDV多聚蛋白（VP2/4/3）DNA疫苗具有良好的免疫原性,可诱发机体产生满意的免疫保护效果,而且ChIL-2（Chicken Interleukin 2）能够增强IBDV DNA疫苗免疫效果。在此基础上,本研究通过基因融合技术构建了IBDVVP2/4/3和ChIL-2的融合基因真核表达质粒,利用ChIL-2的分子免疫佐剂效应来增强IBDV DNA疫苗的免疫效果。由于目前对质粒DNA在真核细胞中的表达及影响其表达产物分泌的因素还不甚清楚,因而不能对融合基因表达质粒中基因连接位置进行优化,本研究将ChIL-2分别置于IBDV VP2/4/3的N端及C端,进行了两种方式的基因融合。应用重叠延伸剪切技术（splicing by overlapping extension）,分别经三次PCR获得融合基因片段VP2/4/3-IL-2、IL-2-VP2/4/3,定向插入真核表达载体pCI中,获得含有IBDV VP2/4/3和ChIL-2融合基因的真核表达载体pCI-VP2/4/3-IL-2、pCI-IL-2-VP2/4/3,从而使IBDVVP2/4/3与ChIL-2基因能够在同一细胞内得到表达,以避免两种质粒共同免疫时可能存在的细胞摄取不成比例,从而高效地发挥ChIL-2的分子免疫佐剂作用。同时在扩增VP2/4/3-IL-2和IL-2-VP2/4/3融合基因时,引物中设计了包含编码15个低疏水性及低电荷效应的氨基酸序列（G₄S）₃作为接头,甘氨酸结构简单,易于折叠,对融合蛋白的空间构象影响小,并保持其天然生物学活性。 在脂质体介导下,将融合基因真核表达质粒pCI-VP2/4/3-IL-2、pCI-IL-2-VP2/4/3分别转染Vero细胞进行表达。RT-PCR检测证实导入的外源基因在Vero细胞中得到了转录;FITC标记的山羊抗兔IgG进行间接免疫荧光试验（IPA）可检测到特异性的黄绿色荧光。通过SDS-PAGE检测转染72h后的细胞裂解液,pCI-IL-2-VP2/4/3的表达产物在60KD、56KD以及30KD处均可检测到特异性条带,pCI-VP2/4/3-IL-2的表达产物在48KD、44KD和42KD处也可检测到特异性的条带,但均未见与VP4蛋白分子量大小相对应的条带。而后相同的样品进行Western-blot鉴定,以免源IBDV抗血清为一抗,pCI-IL-2-VP2/4/3更多还原

【Abstract】 Infectious bursal disease virus(IBDV) can cause infectious disease with the character of immunosuppression and large scale of loss due to higher susceptibility to other pathogens and immunization defeat. Classic method of inactive vaccine and low-virulence vaccine have been confronted with the threat of variant strains and very virulent strains of IBDV isolated since 1980’s. DNA vaccine, which can induce all-round immunity, has been a focus of vaccine research. However, immune effect of DNA vaccine is not ideal, so how to enhance the Immunogenicity of DNA vaccine has been the key to recent studies.Chicken interleukin 2(ChIL-2), as a molecular immune adjuvant, can be used to enhance the immunogenicity of IBDV DNA vaccine. At the same time, ChIL-2 can cooperate with IBDV VP2/4/3 in biological activity. In prophase work, we have make sure that pCI-VP2/4/3 has good protection efficacy and chicken interleukin 2 can enhance the immunogenicity of IBDV DNA vaccine clearly. Recombinant eukaryotic expression vectors, which contain fusion genes of infectious bursal disease virus (IBDV) polyprotein (VP2/4/3) gene and chicken interleukin 2 (ChIL-2) gene, were constructed by using the technique of splicing by overlapping extension (SOEing) and three times PCR. We have used two modes of gene fusion of IBDV VP2/4/3 and ChIL-2 because the factors of expression and secretion of fusion genes in eukaryotic cell is indistinct. So ChIL-2 was placed at C end and N end of VP2/4/3 respectively. Fusion gene fragments were directly cloned into pCI, an eukaryotic expression vector, to get recombinant expression plasmids pCI-VP2/4/3-IL-2 and pCI-IL-2-VP2/4/3. In this way, ChIL-2 and IBDV VP2/4/3 genes can be expressed in the same cell, and ChIL-2 can exert adjuvant effection highly. Otherwise, we used the primers containing a linker which is composed of 15 low hydrophobe and low charge amino acids so that the expression products of fusion genes can be close to nature state.The recombinant plasmids were transfered into Vero cells introduced by Lipofectamine2000 reagent. Gene specific RT-PCR showed that the fusion genes were transcribted in Vero cells. Indirect immuno-fluorescent assay(IFA) showed that the expressed protein was immune-reactivity with chicken anti-IBDV serum and anti-ChIL-2 serum respectively. By SDS-PAGE and Western blotting, 60KD、 56KD and 30KD special bands ofexpression product of pCI-IL-2-VP2/4/3 can be identified respectively in cell fragmentation which have been thansferd after 72 hours. At the same time, 48KDn 44KD and 42KD special bands of expression product of pCI-VP2/4/3-IL-2 can be identified respectively in the same condition, but VP4 protein haven’t been detected at all. Incubating with rabbit anti-IBDV serum as primer antibody, western blotting showed that 60KD、 56KD and 30KD special bands of expression product of pCI-IL-2-VP2/4/3, corresponding to the molecular weight of IL-2/VP2 precursor protein、 mature protein and VP3 .This result suggests that the fusion genes are expressed correctly in Vero cells. Whereas incubating with rabbit anti-ChIL-2 serum as primer antibody, western blotting showed that 44KD and 48KD special bands of expression product of pCI-VP2/4/3-IL-2, corresponding to the molecular weight of VP3/IL-2 precursor protein and mature protein. This indicates that not only the fusion genes are expressed correctly in Vero cells but also the expression products have immune-activity. And the linker retains the nature state of fusion genes.Recombinant plasmids of pCI-VP2/4/3-IL-2 and pCI-IL-2-VP2/4/3 are prepared for DNA vaccine. Fourteen-day-old non-immunized chickens were vaccinated intramuscularly with DNA vaccines, and boosted two weeks later. Anti-IBDVserum ELISA antibody and neutralization antibody were detected in designated dates. All test groups were challenged with reference virulent IBDV strain BC6/85 at 7th week and all chickens were killed after 3 days. Protection rate and bursal/body were computed, and histopathology detection was performed. These results indicated: 1)Recombinant plasmids of pCI-更多还原