【作者】 张涛；

【导师】 徐恒；

【作者基本信息】 四川大学 ， 微生物学， 2007， 硕士

【摘要】 长期以来，对水产疫病的控制主要依靠使用各种抗生素和化学药物，但大量使用药物产生的药物残留问题与对食品品质与安全日益严格的要求这对矛盾越发突出。近年来，我国水产品因为药物残留超标而多次出口受阻就是惨痛的教训。不仅造成了巨大的经济损失，而且严重打击了养殖户的生产积极性，影响水产养殖业进一步发展。1991年，Hansen等首先报道了外源基因能够在鲤鱼肌肉组织中表达。1996年，Anderson等第一次报告了带有传染性造血组织坏死症病毒(Infectious haematopoietic necrosis virus，IHNV)糖蛋白基因的质粒能够在虹鳟中表达，并且能够保护IHNV病毒对受免疫鱼的感染。随后的一些实验也证明，在多种鱼体内，外源基因不仅能够高效，持久地表达，而且能够引起宿主鱼类对外源蛋白的免疫应答。这些为构建鱼类DNA疫苗提供了理论基础。同时对鱼病防治指出了一个新的途径。通过有计划的对养殖鱼类的主要疾病进行疫苗接种可以大大减少抗生素等化学药物在养殖鱼类上的使用量。提高水产品品质和食用安全性，促进我国水产品出口创汇。普通变形杆菌(Proteus.vulgaris)广泛存在于自然界，是一种条件致病菌。由该菌引发的人类疾病国内有较多报道，此类菌对虾、蟹、赤点石斑鱼、鳄鱼等水生生物均具有感染性甚至致死，其中不少品种是水产经济动物。本实验室成功从大口鲇病鱼体内分离出普通变形杆菌TWN-3株，经研究发现其pH和盐的耐受度强，在适宜生长温度范围内生长十分迅速，因此十分容易造成爆发性疾病，对水产养殖生产是一种潜在的威胁。同时研究还发现，该菌株对许多抗生素都不敏感，因此一旦发病，目前的常规鱼病防治手段难以取得明显的效果。尝试用DNA疫苗的方法对其进行预防不失为一个有益的尝试。成功构建DNA疫苗的必须获得相应抗原的主要保护性抗原基因。本研究用带有普通变形杆菌TWN-3株总DNA片段的重组pUC18质粒转化大肠杆菌BL21感受态细胞，构建普通变形杆菌TWN-3株DNA文库，获得了3.6×10~3个带有插入片段的重组子。根据Clarke-Carbon公式，该文库对普通变形杆菌TWN-3株所有基因的覆盖率在99％以上。然后用雄性新西兰大白兔制备普通变形杆菌TWN-3株的多克隆抗血清，采用免疫印迹筛选技术对组成文库的所有重组子进行初筛并对初筛呈阳性反应的重组子进行3次复筛，以排除假阳性的干扰，最终获得了8个稳定呈抗原阳性反应的重组子，为获得普通变形杆菌主要保护性抗原基因及DNA疫苗的研究奠定基础和提供依据。更多还原

【Abstract】 Ordinary, controlling the disease of the aquatic animals depends on the antibiotic and chemical drugs. But the conflict, which between the drug residues and the food quality and safety, was more and more serious. Because of the drug residues, China’s aquatic products were been forbidden import many times. This was not only cause a huge economic losses, but also badly hurt the production initiative of the fishermen, slow down the speed of the fishery’s development. In 1991, Hansen et al firstly reported that the foreign gene can be expressed in carp’s muscle tissue. In 1996, Anderson et al firstly report that the plasmid which carried the Infectious haematopoietic necrosis virus’s glycoprotein gene can be expressed in the rainbow trout, and after injected the plasmid, the fish show a immune ability to the IHNV. Many other research also prove the foreign gene can not only been expressed well in fish last a long time, but also can inspired the fish’s immune response to the foreign protein. All those researches provided a theory base for constructing the fish DNA vaccine. The vaccine can provide a new method to prevent fish disease. By widelv inoculating the major fish disease vaccines, the quantity of the antibiotic and chemical drugs can be decreased a lot, and the quality and safety of the fish can be increased.The Proteus.vulgaris widely distributed in the environment. It is a kind of opportunistic which can cause human disease and many kinds of aquatic animal’s disease, such as shrimp, crab, fish and crocodilc. A strain of Proteus.vulgaris was isolated from the disease catfishe around Chengdu, named TWN-3. It’s growth pH and salinity ranges are very wide, and it grows very fast at the optimum temperature. So when the environment is optimum, it can cause disease quickly and seriously. It is a potential threat to the fishery. The research also indicated that this strain is notsensitive to many antibiotics. So the disease which caused by the TWN-3 is hard to treat by conventional means. Try to construct the DNA vaccine to defend the disease which caused by TWN-3, is a hopeful attempt.The key to construct the DNA vaccine is to gain the major protective antigen gene. This research transformed E. coli BL21 strain by the pUC18 plasmid which carried the TWN-3’s genomic DNA fragments, and constructed a genomic library of TWN-3, which obtained 3.6×10~3 recombinants. According to the Clarke-Carbon formula, this gene library is covered more than 99％of all the genes. Then screen the gene library by rabbit anti- Proteus.vutgaris TWN-3 strain differential proteins serum. At last, gain 8 positive recombinants from the gene library.更多还原