【作者】 曾伟伟；

【导师】 王云峰；

【作者基本信息】 中国农业科学院 ， 预防兽医学， 2009， 硕士

【摘要】 新城疫是由新城疫病毒引起的禽的一种急性、高度接触性传染病,一直是危害养禽业的主要疫病,是世界公认的两大重要禽病之一。传统全病毒灭活疫苗和弱化疫苗在有效预防和控制新城疫的实践中发挥了重要作用,但也存在干扰常规血清学流行病监测、难于区分疫苗免疫与自然感染免疫反应的缺陷。为此,开展新城疫基因工程疫苗的研究,为新城疫的防制提供新的防疫手段具有重要意义。DNA疫苗的抗原合成和提呈过程与病原的自然感染相似等诸多优点,被认为是最具潜力的基因工程疫苗之一。本研究对新城疫病毒F基因进行了优化,并用IL-2和不同拷贝数的C3d作为分子佐剂,构建了F基因真核表达质粒,对其体外表达水平及体内免疫保护效果进行了评估,以期遴选出免疫剂量小、经济成本低、有商品化潜力的NDV DNA疫苗。首先,对NDV F48E9株的F基因进行优化,将F基因ORF的密码子全部替换为鸡体内偏嗜的密码子,在上游引入Kozak序列,改造后的基因通过人工合成后命名为optiF,插入到真核表达载体pVAX1获得重组质粒pVAX1-optiF。间接免疫荧光和Western-blot检测结果显示,与未经修饰的F基因(pVAX1-F)相比,optiF基因体外瞬时表达水平明显提高;2周龄SPF鸡接种试验(200μg/只剂量,间隔2周加强免疫,二免2周后攻毒)表明,F基因密码子的优化可显著提高NDV F48E9株DNA疫苗诱导的保护性体液免疫和细胞免疫应答水平,增强免疫保护效果,一免后2周,pVAX1-optiF质粒免疫鸡的特异性抗体水平显著高于pVAX1-F质粒免疫鸡(P<0.05),一免后27天,pVAX1-optiF质粒免疫鸡的CD4+、CD8+及TCRγδT淋巴细胞百分含量和淋巴细胞增殖反应OD570值均显著高于pVAX1-F质粒免疫鸡(P<0.01),所有pVAX1-optiF免疫鸡均获得完全保护,其保护率(12/12)显著高于pVAX1-F免疫组(2/12)。其次,构建含有密码子优化的NDV F48E9株F基因(optiF)的表达质粒pV-optiF、表达鸡源IL-2的真核表达质粒pV-IL-2和融合表达1-6个拷贝C3d(鸡源)与optiF基因的质粒pV-C3dn-optiF(n=1-6),以进一步探讨分子佐剂C3d和IL-2对低剂量NDV F基因DNA疫苗的免疫增强作用。结果表明,融合单拷贝分子佐剂C3d的重组质粒pV-C3d-optiF以及pV-optiF共免疫表达IL-2的质粒所诱导的保护性体液免疫、细胞免疫以及对强毒的攻击保护效果比相应的其他质粒好。其中,以免疫20μg pV-C3d-optiF和200μg pV-optiF共免疫表达IL-2的免疫效果最佳。总之,通过密码子优化和引入分子佐剂(C3d和IL-2)可以显著提高新城疫病毒F基因DNA疫苗的免疫应答水平,使其有效免疫保护剂量降低至20μg水平,具备推广应用的经济可行性。pVAX1-C3d-optiF和pVAX1-optiF+IL-2作为免疫效果良好、成本低廉的优化DNA疫苗,有望成为新城疫的高效、安全新型基因工程疫苗。更多还原

【Abstract】 Newcastle disease (ND) is a highly contagious viral disease of poultry and other bird species caused by Newcastle disease virus (NDV), its effects are most notable in domestic poultry due to their high susceptibility and the potential for severe impacts of an epidemic on the poultry industries. the conventional vaccine have played an important role to prevent and to control ND in practice,and the most widely used was NDV live vaccine, but both attenuated vaccine and inactivated vaccine can result in latent infection and exist serious defects that can not distinguish vaccine immunization and natural infection . so developing ND engineering vaccine is of great importance to prevent this disease.DNA vaccine which has various advantages is considered as one of the most potential engineering vaccine . this study two approaches were used to enhance the immunogenicity:codon-optimization of NDV F48E9 strain F gene inserts and C3d conjugation to F or combined immunization with IL-2 in order to select low dosage, economic and have commercial potential NDV DNA vaccine.First, we optimized the F gene of NDV F48E9 strain according to the chicken bias codon usages and adding Kozak sequence at the upstream . the optimized F (optiF) and the wild-type F genes were inserted into the pVAX1vector, respectively. The F protein transient expressed in COS 7 cells could be detectable by indirect immunofluorescence and Western-blot at 72 h pos-transfection. The results showed that the expression levels of optiF in vitro was considerably increased in comparison to that of the respective wide-type sequence in the same vector. Two-week-old SPF chickens were inoculated by multi-point injection of plasmids pVAX1, pVAX1-F and pVAX1-opti F, respectively, 200μg for each chichen, and the control group was injected with PBS alone. followed by booster immunization with the same dose at the two week after primary immunization. Two weeks later, the chickens were challenged by NDV F48E9 velogen strain with a dose of 105EID50, then calculating their morbidity and mortality. Blood samples were collected weekly for detecting the antibody directly against the F protein; peripheral blood lymphocytes were isolated and probed for the composition of CD4+,CD8+ and TCRγδT cells, and splenic lymphocytes proliferation assay for measuring cell-mediated immunity was done. The chickens vaccinated by pVAX1-optiF were found to be earlier F protein antibody production, significantly enhanced stimulation index and marked increase in percentage of CD4+,CD8+ and TCRγδT cells, comparing to that vaccinated by pVAX1-F. And the chickens of optiF vaccine group were completely protected from virus challenge. The results indicated that codon optimization of F gene could observably enhance the protection efficiency of NDV F48E9 strain DNA vaccine.we further investigate the immunopotentiation effects of molecular adjuvant C3d and IL-2 to NDV F-DNA vaccine at low dosage.so this study we construct various expression vectors: pV-optiF expressing F protein alone, pV-C3dn-optiF(n=1-6)fused to one to six copies of chicken C3d gene and pV-IL-2 expressing chicken IL-2. pV-optiF,pV-C3d-optiF and pV-C3d5-optiF were selected for this research based on the result of in vitro transient expression and pre-exprement .chicken were inoculated with decreasing doses (200μg, 50μg or 20μg)of these plamids respectively,another three groups inoculated with decreasing doses (200μg, 50μg or 20μg)of pV-optiF and co-immunization with 100μg plamid pV-IL-2 at the same time. Chicken vaccinated with the highest dose (200μg) of DNA had high anti-F protein specific antibody titers regardless of the addition of C3d and IL-2. At lower doses (50μg and 20μg) of DNA, chicken vaccinated with optiF fused C3d or co-immunization with IL-2 had enhanced immune responses compared to chicken vaccinated with DNA expressing F only. The protective humoral immunity, cellular immunity and immune protective effect against virulent virus of chicken vaccinated with pV-C3d-optiF or pV-optiF co-immunization with IL-2 are better than other plamids.In addition, chicken vaccinated with pVAX1-C3d-optiF at 20μg of DNA or vaccinated with pVAX1-optiF at 200μg and co-immunization with 100μg pVAX1- IL-2 obtain the optimum efficiencyIn conclusion,codon optimization and fusion one copy of C3d or co-immunization with plamid which express IL-2 can remarkably improve the immune response of NDV F-DNA vaccine, these approaches could function in a synergistic manner result in protect chickens from lethal NDV virus challenge, even at the low dose of 20μg, implied the potential possibility for the commercialization of ND DNA vaccine in the future. pVAX1-C3d-optiF and pVAX1-optiF+IL-2 have good immune effect and with low costs hoping to become an efficient and safe new ND engineering vaccine.更多还原