【作者】 刘长梅；

【导师】 庄文忠； 安利国；

【作者基本信息】 山东师范大学 ， 动物学， 2001， 硕士

【摘要】 新城疫（Newcastle Disease，ND）是一种由新城疫病毒（Newcastledisease virus，NDV）引起的具有高度传染性的病毒性传染病，能感染鸡、鸽、鸭和野生鹦鹉等多种鸟类，往往给养禽业造成极大的危害。目前用于防制该病的疫苗为传统的常规疫苗，其研制都是以大量培养病原微生物为基础的，这不仅给疫苗的制造带来局限性，同时存在潜在的不安全因素，如病原微生物灭活不彻底，导致强毒散播；致弱的疫苗存在毒力返强问题等。自70年代发展起来的分子生物学技术为解决传统疫苗的缺陷、研制高效优质的新型疫苗带来了希望。 随着分子生物学的快速发展，以分子操作为基础的基因工程疫苗研究方兴未艾，由于基因工程疫苗具有原料来源简单、安全性高、便于区分感染动物和免疫动物、易于制作多价疫苗等优点，使基因工程疫苗具有巨大的发展潜力和市场前景。 脂质体可将蛋白质抗原以细胞膜抗原的形式提呈给宿主免疫系统，既可诱发体液免疫，又可诱导细胞免疫，并且对所包被的物质具有缓释作用，为近十几年发展起来的新型抗原载体。 本课题将鸡新城疫病毒的重要保护抗原基因-血凝素-神经氨酸苷酶基因克隆到杆状病毒表达载体，并使其在昆虫细胞得到成功表达，利用表达的蛋白质抗原，对鸡新城疫脂质体-基因工程亚单位疫苗进行了系统的研究，取得了如下结果：1．利用血凝试验及血凝抑制试验对重组病毒进行了鉴定，证明重组 杆状病毒特异性地表达了鸡新城疫病毒的血凝素-神经氨酸苷酶 蛋白（Hemagglutinin-neuraminidase protein，HNP）。2．通过反复的摸索及实践，发现在该脂质体疫苗的制备中，卵磷脂 与胆固醇的最佳质量比为2:1，病毒液的最适pH值为7.2-7.6，成 功地建立了脂质体-鸡新城疫基因工程亚单位疫苗制备技术。 山东帅他人学攸I：毕业论义 鸡新城疫林H丁程亚平位疫苗的研九3．利用电子显微镜、紫外分光光度计、无菌检验、安全性检验等对 脂质体疫苗进行了质量检测，结果表明研制的脂质体疫苗符合常 规疫苗的质量要求。4．对脂质体疫苗进行了免疫剂量筛选试验和免疫效力检测，结果表 明脂质体疫苗免疫后的血凝抑制抗体效价可达 51 OgZ，攻毒保护 率为60％，免疫后IL－2（lute，1。。巾i n 2，IL－2）的表达量增加。 通过本研究，成功建立了脂质体－鸡新城疫基因工程亚单位疫苗制备技术，为脂质体－重组蛋白疫苗的研制提供了有重要参考价值的‘欠验十物学数折Z，为脂质休疫十穴’1；，。”中’文际应用打卜了良灯的尽川比。更多还原

【Abstract】 Newcastle disease(ND), caused by Newcastle diseasevirus(NDV), is a highly contagious disease,and often makes agreat economic loss to avian industries. In the study, fordeveloping a new type of vaccine for controlling the disease,a recombinant baculovirus containing a eDNA which encodeshemagglutinin膎euraminidase (HN) of Newcastle disease virus(NDV) was constructed, which could produce a large amount ofHN glveoprotein successfully by infecting Spodopterafrugiperda (Sf) cells. Using the expressed recombinant proteins,liposome vaccines were developed and characterized. The resultsobtained in this study are summarized as follows.1.The recombinant protein expressed by haculovirus wasidentified by hemagglutination test and hemagglutinationinhibition test. The i-塻ults demonstrated the recombinanthacu]ovirus expressed HN protein of NDV specifically.2.Using the expressed HN protein, liposome vaccines weredeveloped, of which optimal mass ratio between lecithin andcholesterol 鵤nd the optimal pH of virus liquid weredetermined.3.The liposome vaccine was detected by electron microscope,spectrophotometer, asepsis test etc.,which demonstratedthat the lipsome vaccine developed in this study had a goodquality.4.Immune dose of the vaccine was determined by immune doseselection test, O.5m1 dose group of which produced higherW~駘Mki~antibodies than other groups.3.Antibody production in the 1 iposome vaccine膇noculatedchickens was examined by I-lI test. The results showed thatthe inoculated chickens produced HI antibodies, and the HItiters of the sera reached to 32.6.Protective effect of the liposome vaccine was examined invivo. 60% of the chickens inoculated with the vaccinesurvived from the challenge by the virulent NDV withoutshowing any clinical signs of Newcastle disease. On theother hand, the uninocuiated control chickens showedtypical clinical signs after the challenge.7.Expression of IL? (Jnterleukin 2), IFN膟 (Interferon膇)and cMGF(chicken Myelomonocytic Growth Factor),weredetected by RT腜CR (Reverse transcript ion膒olymerase chainreact ion) . The results demonstrated that expression of iL?increased due to inoculation of Iiposome vaccines.The results of the study provide important basic data fordevelopment of liposome vaccines using recombinant baculovirusexpressed proteins.更多还原