禽巴氏杆菌ptfa基因原核表达载体的构建..doc

禽巴氏杆菌ptfa基因原核表达载体的构建 *** 指导教师 摘　要 多杀性巴氏杆菌（Pasteurella multocida P.m）是有荚膜的革兰氏阴性杆菌，属于巴氏杆菌科巴氏杆菌属。禽巴氏杆菌病（又称禽霍乱）是由巴氏杆菌所引起的家禽和野禽的一种急性、接触性、败血性疾病总称。近年来国内外学者的研究表明，菌毛是巴氏杆菌表面存在着一种比鞭毛更细、更短而直硬的丝状物，其化学组成是菌毛蛋白（Pilin）。它是禽多杀性巴氏杆菌的重要致病因子。菌毛在禽巴氏杆菌感染过程中与粘附有关。ptfa基因编码的蛋白是禽多杀性巴氏杆菌的菌毛蛋白，在机体抵抗禽多杀性巴氏杆菌攻击中具有重要作用。 本文通过已公布的菌毛蛋白ptfa基因的序列，设计了一对引物，以提取的巴氏杆菌基因组为模板，进行PCR特异性扩增出ptfa基因，将扩增得到的产物克隆到载体ET30a中，得到重组质粒ET30a＋ptfa 转化到大肠杆菌DH5ɑ菌株的感受态细胞中，将转化形成的重组细胞进行涂布（含卡那霉素的LB平板）筛选，挑取平板上的单菌落进行扩培，对扩培液再次进行纯化，提取纯化细胞的质粒进行PCR扩增检验，将呈阳性的提取质粒进行测序，成功获取ptfa基因同时表明原核表达载体构建成功，为巴氏杆菌疫苗的研究奠定了基础。 关 键 词：多杀性巴氏杆菌ptfa基因基因组提取原核表达载体 Prokaryotic construction of expression vector ptf gene from avian pasteurella multocida College of Food ＆ Bioengineering *** Tutor *** ABSTRACT Fowl cholera,which pathogen is pasteurella mutocida,is a kind of acute、contagiousand septicaemia infection disease in domestic poultry and wild birds.Many studies have shown that, Fimbriae on the Pasteurella surface is more detailed, straight , hard and shorter than the flagellum, its chemical composition is the pili protein (Pilin).It it is an important pathogenic factor.of Pasteurella multocida.Fimbriae has infections on the process of poultry fimbriae and adhesion.the protein which ptfa encoded is the pilus of Pasteurella multocida. it has an important role in the body against avian Pasteurella multocida . In this paper, a pair of primers were designed according to the ptfa gene sequence published . The Pasteurella multocida P.m genome was extracted as template for PCR amplified specific ptfa gene. The PCR product was recovered and cloned into the clone vector pET30a, and was transformed into E.coli strain DH5ɑ competent cells. The recombinant plasmid pET30a-ptfa was selected by Kanamycin. Plasmid was extracted and tested by PCR amplification. The results show that the prokaryotic expression vector was constructed successfully. KEY WORDS：Pasteurella multocida, ptfa gene, genomic extraction, Prokaryotic expression vector目　录前 言 1 第1章 文献综述 3 1.1