【作者】 张鹏；

【导师】 李和平； 廖玉才；

【作者基本信息】 华中农业大学 ， 植物学， 2008， 硕士

【摘要】 禽流感（Avian influenza,AI）是一种发生于家禽和野鸟中的烈性传染病,近年来禽流感疫情的频繁发生,给世界各国的家禽养殖业和人类公共卫生健康造成了极大的损失和威胁。因此,研究、发展高效、安全、生产工艺简单、价格低廉的禽流感疫苗生产技术体系具有重要的现实意义。随着植物高效表达载体系统和遗传转化技术的不断发展,植物已被用作生产包括疫苗及抗体等在内的一系列重要蛋白的生物反应器,成为植物基因工程中一个重要研究领域。植物作为生物反应器具有许多优点:可大规模生产,贮藏简单,使用方便,可对真核蛋白进行正确的翻译后加工、形成有活性的分子,生产成本低廉,不涉及公众极为关注的有关转基因动物的伦理道德问题,从而使转基因植物成为一种新型、高效的生物反应器。本研究选择我国H5N1亚型禽流感病毒表面抗原血凝素（HA）和神经氨酸酶（NA）基因,分别构建到原核表达载体、单子叶植物表达载体和双子叶植物表达载体中,研究了在细菌中经IPTG诱导表达、烟草叶片瞬时表达以及转基因拟南芥稳定表达,分析鉴定了目的蛋白的表达,还探索了小麦愈伤组织的遗传转化。结果表明,HA和NA可以在细菌细胞中正常表达,利用融合蛋白中的GST标签,经亲和层析纯化获得较高纯度的HA和NA蛋白;利用根癌农杆菌—真空过滤法介导的烟草瞬间表达法,从瞬间表达3天后的烟草叶片中提取烟草叶片总蛋白,Western分析证实,HA和NA蛋白均可在烟草叶片中表达积累,在瞬间表达的烟草叶片检测到预期大小的特异蛋白带;以农杆菌介导的Floral dip法,将重组的双子叶植物表达载体转化拟南芥,通过PCR对抗性筛选得到的转化植株进行鉴定,HA和NA转基因的阳性率分别为85.7%和86.7%;提取T₂代PCR鉴定阳性植株叶片总蛋白,通过ELISA筛选,分别得到2株表达量较高的转基因植株;对其进行RT-PCR鉴定和Western杂交检测,证实了HA和NA的正常转录和表达;进一步通过根癌农杆菌介导法转化小麦愈伤组织,筛选得到的转化植株经PCR初步鉴定,证实了目的基因整合到小麦的基因组中。因此,禽流感病毒表面抗原血凝素和神经氨酸酶基因可在植物细胞中瞬间和稳定表达。这些研究结果为利用植物作为生物反应器生产禽流感植物疫苗打下了基础。更多还原

【Abstract】 Bird flu （Avian influenza, AI） is an infectious disease that occurred in wild birds and poultry. In recent years, the frequent occurrence of bird flu cases caused great losses and threats to the poultry industry and the public human health. Therefore, the development of efficient and safe systems for bird flu vaccine production with simple process and low price has important practical significance.With the rapid development of efficient plant expression vector systems and plant genetic transformation technology, various important proteins including vaccines and antibodies have been successfully produced in different plants and plant cells. Plant bioreactor has become an important, fast growing field of plant genetic engineering. Plant bioreactor has many advantages over other systems such as easy large-scale production, simple storage, easy use and low cost of production for foreign proteins. Plants can process post-translational modifications and proper protein folding, generating highly active molecules. Also, it is not involved in any ethical issues that were observed in transgenic animals. Thus, transgenic plants have become a new, efficient system as a bioreactor.Surface antigen hemagglutinin （HA） and neuraminidase （NA） genes of H5N1 subtype of an avian influenza virus from China were cloned into bacterial expression vector pGEX-6p-1, monocotyledon expression vector and dicotyledon expression vector, respectively. And the quantities of proteins expressed in bacteria by induction of IPTG as well as in plant leaves were purified and analyzed. Genetic transformation of calli derivde from wheat was also investigated.The results indicated that HA and NA could be expressed well in the bacteria, and affinity-purified by chromatography with GST matrix. The two genes were constructed to dicotyledon expression vector, and then were transformed into Agrobacterium tumefaciens. After the vacuum filtration of tobacco leaves for transient expression, total protein of tobacco leaves was extracted after three days. Purified proteins of both HA and NA were detected by Western blotting, and there are specific protein bands from the transiently expressed tabacco leaves, confirming proper expression of these two genes in tabocco leaves.By Agrobacterium-mediated floral dip, HA and NA genes constructed dicotyledon expression vector were transformed into Arabidopsis thaliana. The transgenic plants were screened by PCR after selection on antibiotic media, and the positive rates were 85.7% and 86.7%, respectively. Total leaf proteins of T₂ transgenic plants were isolated and used for ELISA screening, from which two transgenic plants with high expression levels were identified. Through RT-PCR and Western blot anlysis, both HA and NA proteins with expected molecular sizes were detected in the two plants. The results show that it is feasible to produce bird flu vaccine by Arabidopsis plants. The two genes constructed into monocotyledon expression vector were transformed into wheat calli by Agrobacterium-mediated method. The transformed plants were identified by PCR, suggesting the integeration of the foreign genes into wheat genome. The current studies could be used as a basis for further expression of avian influenza vaccines in plants.更多还原