【作者】 隋兆峰；

【导师】 赵宏坤； 范伟兴；

【作者基本信息】 山东农业大学 ， 预防兽医学， 2003， 硕士

【摘要】 从山东省分离到了107株鸡大肠杆菌，攻毒试验后，对其中16株高致病性大肠杆菌进行了血清型鉴定，主要血清型为O78。通过血凝试验及PCR鉴定得知，这16株大肠杆菌主要表达Ⅰ型菌毛。菌毛的表达受许多因素的影响，本研究从培养时间，培养方式等条件对菌毛表达的影响进行了探索，实验结果表明：培养72小时，菌毛可良好表达，振摇培养优于静止培养。菌毛提取方法的研究结果表明，保温法优于搅拌法。提取的菌毛蛋白经SDS-PAGE测得其分子量约为17KDa。选择3株表达Ⅰ型菌毛的优势致病血清型鸡大肠杆菌作为试验株，分别为JX（O24）、QD5（ O78）、 QD2（O2）。经增菌培养后提取菌毛制备成单价和多价2种菌毛油乳苗。分别于1日龄免疫一次和1日龄、14日龄免疫二次对鸡只进行免疫试验，并于4周龄攻毒。结果表明：用血清型O78（QD5）制备的单价菌毛亚单位油乳苗能够有效保护其同源菌株的攻击，且接种免疫两次的效果明显优于一次免疫。交叉保护试验表明QD5（O78）、QD9（O2）和JX（O24）之间存在着一定的交叉保护作用。多价菌毛亚单位油乳苗免疫试验结果表明，由QD5（O78）、QD9（O2）和JX（O24）菌株提取的菌毛制成的多价菌毛亚单位油乳苗能有效保护鸡分别抵抗3种同源菌株经气囊的攻击。参照Genbank收录的禽源大肠杆菌Ⅰ型菌毛FimA基因序列，应用DNAStar软件分析，在其保守区设计了一对特异性引物。以煮沸法提取的16株鸡大肠杆菌基因组DNA为模板，进行PCR扩增。16个分离株中有14株（除QD6与FN外）扩增出特异的目的片段。选择其中2株（QD5与JX）的PCR产物进行了克隆和序列测定，并用DNAStar等软件分析测序结果，证明所扩增的产物为FimA片段，其核苷酸序列同源性较高，为99.8％。分析抗原性图谱发现，二者的抗原峰图基本相同，抗原变异主要集中在135～145位氨基酸之间。不同禽源大肠杆菌菌毛FimA基因的同源性在92.9％～100％之间，变异较大。<WP=8>参照Genbank收录的禽源大肠杆菌Ⅰ型菌毛FimH基因序列，应用DNAStar软件分析，在其保守区设计了一对特异性引物。对3株表达Ⅰ型菌毛的鸡致病性大肠杆菌的FimH基因进行了克隆并经测序得知，所扩增的产物为FimH片段，三者核苷酸序列之间虽有变异，但推导的氨基酸序列完全相同。三者的FimH基因序列与已发表的禽源大肠杆菌的进行比较发现，其同源性在97.9～100％之间，表明FimH基因高度保守。更多还原

【Abstract】 107 avian pathogenic E.coli strains were isolated from Shandong province, of which 16 high pathogenic E.coli strains were identified for serum type ,and most strains of serum type was O78.By hemagglutination and PCR analyze, 16 isolates mainly expressed type 1 pili. The results showed that after 72 hours of incubation,pili can be expressed well.Shaker cultures were observed to have an increased growth of pili compared with the stationary cultures.The molecular weight of pili was about 17KDa.Three high pathogenic avian E.coli(O2,O78,O24) isolates ,which had been identified type Ⅰpili by MSHA and PCR,were used to prepare fimbriae vaccines.Some chickens were vaccinated once on one day-old , another chickens vaccinated twice on one day-old and 14 day-old,respectively.Then challenged with E.coli(O2,O78,O24) via the air sac on 4 week-old. The results was that the chickens can efficiently resist attack of homologous E.coli via the air sac,and vaccinated twice with pili vaccine was good at vaccinated once.Cross-protection experiment showed that the pili of the three isolates had certain cross protection.Chickens vaccinated with multivalent pili oil-emulsified vaccine can efficiently resist attack of three homologous E.coli(O2,O78,O24) via the air sac.According to the published type 1 pili FimA nucleotide sequences of avian E.coli,two pairs of primers were designed and synthesized.Fourteen 549bp products were amplificated by polymerse chain reaction(PCR) Two of<WP=10>the which were cloned into pMD-18-T vectors, respectively.By analysis of the nuleotide sequences,the homologies of the two strains was 99.8％.The homologies of the FimA gene of two isolates with published type 1 nucleotide sequences of avian E.coli was 92.9％~100％. Using two pairs of primers designed according to the published sequences,the FimH genes of three isolates which had been identified type Ⅰpili by MSHA and PCR were obtained through PCR method.The amplified products of 839bp in length were cloned into pMD-18-T vectors, respectively.The analysis of the nuleotide sequences and its deduced amino acid sequences revealed that the homologies of the three isolates’ FimH genes was 99.6％ ~99.9％; the homologies of duced amino sequences of the three was the same; the homologies of the three isolates’ FimH with published type 1 pili FimH gene nucleotide sequences of avian E.coli was 97.9％ ~ 100％.更多还原