大肠杆菌K_(88)纤毛抗原的提纯和抗血清的制备  

Purification of K_(88) Pilus Antigen of the Escherichia coli and Preparation of the Antiserum

作　　者：李虹[1] 朱良全[1] 刘亚华 范书才[1] 

机构地区：[1]中国兽医药品监察所,北京100081 [2]乾元浩生物股份有限公司南京生物药厂,南京210012

出　　处：《中国兽药杂志》2006年第12期23-26,14,共5页Chinese Journal of Veterinary Drug

摘　　要：以5%绵羊血改良M inca琼脂筛选和克隆表达良好的K88+菌株,改良M inca琼脂培养获得K88菌液。用加热法及高速匀浆法使K88纤毛从菌体上脱下。饱和硫酸铵沉淀粗提,用Sepha-rose CL-4B凝胶过滤纯化K88抗原。提纯的抗原经SDS-PAGE电泳测定其分子量约为25 000,且仅此一条蛋白带;与K88抗血清进行琼脂扩散试验只出现一条沉淀线。用提纯的抗原经多次免疫家兔制备了K88抗血清。抗血清在玻板凝集试验中只凝集K88菌株,不凝集K99,987P或F41菌株;在琼扩试验中,只与K88粗提抗原出现一条沉淀线,且效价为1∶64,而不与K99,987P抗原出现沉淀线;在免疫电泳试验中,与无细胞K88纤毛液只出现一条沉淀线。鉴定结果表明,所制备的K88血清特异性强、效价高,可作为单因子血清用于凝集试验、琼扩试验等血清学试验,对K88菌株进行鉴定,对K88纤毛抗原进行定性和定量检测。The cultures of E. coli K88 strain were obtained by selecting and cloning well-expressed K88^ ＋ strains with improved Minca agar medium by adding 5% sheep blood and incubating the chosen strain with improved Minca agar medium. The K88 pilus were purified by Sepharose CL-4B gel filtration after separated from the bacteria by the heating and high-speed dispersing treatments and extracted by ammonium sulfate precipitation. The purified antigen was identified as a single protein band with a molecular weight of 25 000 through SDS-PAGE and a single precipitating line with K88 antiserum in the gel double diffusion test. The K88 antiserum was prepared by immunizing the rabbits for three times with the purified antigen. The prepared antiserum was identified as agglutinating with Ksa strains but not with K99, 987P or F41 in the slide agglutination test and a single precipitating line appearing with extracted K88 antigen but not with K99 or 987P in the gel double diffusion test and immunoelectro- phoresis test. The titer of antiserum was 1 ： 64 in the gel double diffusion test. The results showed the prepared K88 antiserum had the high specificity and titer and can be used for detection of K88 strains and identification of K88 pilus antigen in the immunity assays, such as the agglutination test and the gel double diffusion test.

关 键 词：大肠杆菌 K88纤毛抗原 K88抗血清 

分 类 号：S852.612[农业科学—基础兽医学]