【作者】 姜永厚；

【导师】 刘忠贵；

【作者基本信息】 东北农业大学 ， 基础兽医学， 2000， 博士

【摘要】 本文在成功构建鸡新城疫F基因疫苗，并观察了免疫SPF雏鸡后诱导的免疫应答及免疫保护作用基础上，首次系统研究了鸡IL-2在鸡新城疫F基因疫苗免疫中的作用，证实了国内首次克隆成功的鸡IL-2基因的免疫增强作用，及作为免疫佐剂应用于禽类基因免疫的可行性；在此基础上，为进一步提高F基因疫苗免疫效率，又成功地构建了鸡新城疫病毒F基因与鸡IL-2重组DNA疫苗，研究了其接种SPF雏鸡后免疫鸡只T、B淋巴细胞增殖反应和抗体变化规律，以及强毒攻击后的免疫保护作用。研究结果如下：1．新城疫病毒F基因疫苗研究 用新城疫病毒D26株F基因的cDNA与含人巨细胞病毒CMV启动子的真核表达载体pcDNA3重组，构建了新城疫F基因疫苗（pcDNA-F）。直接免疫接种SPF鸡后免疫鸡产生了针对新城疫病毒的抗体，免疫接种后前2周，免疫鸡的抗体产生并不明显，第3周后出现稍为明显的抗体反应。攻毒后，免疫鸡的抗体回忆反应十分迅速和强烈，出现一个较陡而又较高的峰值。而非免疫鸡在攻毒后，抗体反应相当弱。免疫组有部分试验鸡（30％）耐过强毒攻击。说明重组质粒的F基因已在体内得到表达，并诱导了机体的免疫应答反应和免疫保护作用。证明以F基因做为新城疫基因免疫的免疫原基因是可行的。2．鸡IL-2在新城疫病毒F基因疫苗免疫中的作用研究 用含鸡IL-2基因真核质粒与新城疫F基因疫苗联合免疫SPF雏鸡。抗体监测结果显示添加鸡IL-2质粒试验鸡的抗体滴度高于单独接种F基因疫苗试验鸡的抗体滴度；其中两种质粒混合接种组（pcDNA-F+IL2）抗体滴度又明显高于这两种质粒的分别接种组（pcDNA-F/IL-2）抗体滴度。 MTT法检测免疫鸡T、B淋巴细胞增殖反应：免疫后不同周龄，各免疫组试验鸡胸腺T淋巴细胞对ConA、法氏囊B淋巴细胞对PMA均有明显的反应性。接种鸡IL-2质粒试验鸡胸腺T淋巴细胞对ConA的反应性、法氏囊B淋巴细胞对PMA的反应性又显著高于单独接种F基因疫苗组，差异极显著（P＜0.01）。说明接种鸡IL-2表达质粒后，F基因疫苗诱导的体液免疫和细胞免疫应答均得到了加强。在添加IL-2质粒的两组中，混和接种IL-2质粒组的T、B淋巴细胞增殖反应又明显高于分别接种组，差异极显著（P＜0.01）。表明鸡IL-2质粒免疫增强作用的发挥有赖于同抗原基因的共同接种。 攻毒保护试验显示：混合接种组（pcDNA-F+IL2）中有50％的试验鸡耐过；分别接种组（pcDNA-F/IL-2）有40％试验鸡耐过。 添加IL-2质粒组试验鸡体液免疫、细胞免疫的增强及免疫保护率（50％）的提高，说明IL-2真核质粒在鸡体内得到表达，并且具有免疫增强作用。一方面刺激T淋巴细 东北农业大学 中文摘要 博士学位论文 胞的增殖、分化，增强细胞免疫应答；另一方面作用B细胞，促进其活化分泌Ig，加 速抗体生成，进而加强体液免疫。 3．新城疫病毒F基因与鸡thZ重组DNA疫苗的研究 利用国内首次克隆得到的鸡IL2与新城疫弱毒D26株的F基因，经载体改建，将 二者以非融合表达形式共同克隆于真核表达质粒pCDNA3上，经酶切分析、PCR鉴定 证实成功构建了共表达鸡 IL－2与新城疫 F蛋白的重组质粒（pCDNA－F－ILZ）。采用胸肌 多点注射方法接种 14日龄 SPF雏鸡，免疫后每周采集试验鸡胸腺、法氏囊、血液，无 菌分离T、B淋巴细胞和血清，以 MTT法和 ELISA分别检测免疫鸡 T。B淋巴细胞增 殖反应和抗体变化规律。结果显示：与单独接种F基因疫苗试验组相比，接种重组质 粒试验组鸡只的体液免疫和细胞免疫均明显增强；且免疫效果优于两种质粒的混合注 射。免疫后以新城疫标准强毒F。。巳攻击，接种重组质粒试验鸡有70％存活；混合接种 F基因和几－2质粒试验鸡有 5（）％存活；单独接种 F基因疫苗试验鸡只有 3（）％存活。试 验结果表明共表达IL．2可极大提高新城疫DNA疫亩的效力。 新城疫F基因与鸡IL。二重组质粒的成功构建在国内外尚未见报道，不仅首次证实 了鸡IL－2在新城疫基因免疫中的免疫增强作用，而且为鸡IL2这一新型佐剂在禽类基 因免疫中的应用提供了试验依据。同时山为探讨禽类重组基因疫苗的构建奠定了基础。 4．新城疫基因疫苗、鸡IL．2质粒免疫SPF雏鸡免疫器官的组织学变化 新城疫基因疫苗免疫后．试验鸡胸腺皮质增厚，其中淋巴细胞数目增客。法氏囊 淋巴滤泡明显增大，淋巳细胞数目增多。脾脏白髓区增大，淋巴小结数量增多，体积增 大，其中淋巴细胞数量增多。以上主要免疫器官在免疫后的组织学变化再次证实了基因 疫苗免疫可以诱导体液免疫和细胞免疫的结论。 另外，将几．2免疫鸡与空白对照鸡、F基固疫苗免疫鸡进行了形态学和组织学的 比较，接种IL．2质粒试验鸡剖检末见病理变化：整个免疫过程中，接种几－2质粒试验 鸡的更多还原

【Abstract】 Newcastle disease(ND)is a severe respiratory, neurological, or enteric disease and is characterized with a high frequency of death among infected chickens. The study investigated the protection of chickens against the ND by a recombinant DNA vaccine. A plasmid vector encoding NDV F protein, which is required for virus cell fusion and is important for vaccine induced immunity, was used as a model to study how DNA may be modulated by the simultaneous expression of chicken IL-2.Firstly, cDNA encoding NDV F protein of avirulent NDV D26 strain was cloned into pcDNA3 which contains CMV promoter. Two-week-old SPF chickens were intramuscularlly vaccinated with the eukaryotic plasmid. Antibody response showed that the specific antibody to NDV could be detected after vaccination, the titer of antibody was very high when chickens survived from challenge. 30% vaccinated chickens survived.Vaccination of SPF chickens with F protein expression plasmid and chicken IL-2 expression plasmid was set in two ways, one is coinjection, the other is separate injection. Antibody and Th B cell responses showed that the humoral and cellular immunity of F and IL-2 plasmid vaccinated chickens significantly enhanced compared with those vaccinated with F protein expression plasmid alone. Six weeks later, All chickens were challenged with a lethal dose of NDV F48E9 strain, 40% chickens vaccinated by separate injection of two plasmids survived, 50% chickens vaccinated by coinjection survived. The enhanced immunity by adding IL-2 plasmid showed that IL-2 was expressed, and it really has adjuvant effect, the effect of IL-2 expressing plasmid was dependent on coinjection with the plasmid expressing antigen.To ensure that IL-2 was delivered at the sites of immune interaction, we constructed a coexpression plasmid. The NDV D26 strain F gene with CMV promoter and BGH polyA signal sequence was amplified by PCR from eukaryotic plasmid pcDNA-F, which contains the full-length NDV F gene, and cloned into reconstructed eukaryotic plasmid pcDNA-1L2, which contains chicken IL-2 gene. Restriction endonuclease cleavage and PCR amplification showed that a bicistronic plasmid encoding NDV F gene and chicken JL-2 separately was successfully constructed. Two-week-old SPF chickens were intramuscularly inoculated with the recombinant plasmid. Antibody andlymphocyte proliferative responses showed that the humoral and cellular immunity of chickens vaccinated with the recombinant plasmid greatly increased compared with those inoculated only plasmid expressing NDV F protein. After challenge with a lethal dose of NDV F48E9 strain, 70% chickens vaccinated recombinant plasmid survived, only 30% chickens vaccinated with plasmid expressing F protein survived. These results proved the adjuvant effect of chicken IL-2, and showed that the efficacy of a DNA vaccine can be greatly improved by simultaneous expression of IL-2.The tissue sections of thymus bursa of Fabricius and spleen of experimental chickens showed that the cortex of thymus of chickens immunized with DNA vaccine broadened, the white pulp of spleen of chickens immunized with DNA vaccine enlarged, the number of lymphocytes in these immune organs of immunized chickens increased significantly compared with control chickens. In the meantime, there were no gross and histologic lesion found in immune organs of chickens vaccinated with chicken IL-2 plasmid. These results demonstrated that inoculating IL-2 plasmid is not only effective, but also safe.Doctoral Student: Jiang YonghouAdvisors:Prof. Liu Zhonggui Prof. Tong GuangzhiMajor:Basic Veterinary更多还原