布鲁氏菌多克隆抗体和单克隆抗体的制备与鉴定

发布时间：2018-01-09 20:09

本文关键词：布鲁氏菌多克隆抗体和单克隆抗体的制备与鉴定　出处：《河北农业大学》2009年硕士论文　论文类型：学位论文

【摘要】： 布鲁氏菌是一种革兰氏阴性、兼性细胞内寄生菌,它可以感染人和多种动物,导致人和动物的布鲁氏菌病。对于反刍动物,通常引起流产及不孕不育。人患布鲁氏菌病通常是由于接触患病动物所引起,人与人之间不能传播。过去,布鲁氏菌病曾一度在全世界范围内流行,造成严重的经济损失。现在布鲁氏菌病在欧、美等一些发达国家,已得到了有效的控制,但在广大发展中国家仍普遍流行。在我国,布鲁氏菌病曾得到过有效的控制,但九十年代后,布鲁氏菌病有回升的趋势,特别是近几年来,人和动物布鲁氏菌病的患病数迅速升高,这也使得对于布鲁氏菌的研究和布鲁氏菌病的诊断显得尤为重要。用牛种布鲁氏菌A19疫苗皮下多点注射免疫两只新西兰兔,两周一次。在第二次免疫两周后用试管凝集试验测定其血清效价为1:1600,达到所需标准,然后兔心脏采血并分离血清,用硫酸铵沉淀法粗提IgG,再用Sephadex-G25层析柱脱盐,Bradford法测定蛋白含量,最后SDS-PAGE电泳试验鉴定。结果显示成功得到了初步纯化的兔抗牛布鲁氏菌A19多克隆IgG。采用杂交瘤细胞技术,以布鲁氏菌全菌为抗原,以不同剂量和不同免疫方法免疫8周龄BALB/c小鼠,每次免疫间隔时间为3周,在融合前3d进行最后一次加强免疫。取小鼠脾细胞和SP2/0细胞经PEG-4000按10:1比例进行融合。采用布鲁氏菌细胞壁抗原作为筛选抗原,用自己建立的间接酶联免疫吸附法(Enzyme-Linked Immunosorbent Assay, ELISA)检测杂交瘤细胞培养液上清,筛选出的强阳性孔杂交瘤细胞用有限稀释法克隆培养,直到阳性率达到100%.经过3～4次亚克隆后,得到了2株能稳定分泌抗布鲁氏菌抗体的杂交瘤细胞株,其产生的单抗分别命名为3E7和1C6。采用ELISA方法检测细胞上清和腹水的效价,其中3E7细胞上清和腹水的效价分别为1:1600和1:20480,而1C6分别为1:800和1:10240。通过叠加试验对这两种单抗进行抗原位点分析,结果表明2株单抗分别针对不同的抗原位点。在单抗的稳定性试验中,两株细胞在四次克隆化培养的细胞上清的OD450均有上升趋势,四代腹水的抗体效价均在1:104～1:105之间,说明有较好的稳定性。对硫酸铵纯化后的3E7和1C6单抗进行SDS-PAGE电泳实验,结果显示,2株单抗的重链分子质量为50kD左右,轻链分子质量为25kD左右。用这两种单抗分别与布鲁氏菌细胞壁抗原进行Western-blot分析,结果显示两株单抗都能够和布鲁氏菌细胞壁抗原结合,且抗原抗体反应条带分别出现在分子质量为31kD和55kD的位置,提示这两种单抗可能是由分子质量为31kD和55kD的抗原产生。
[Abstract]:brucella is a gram - negative , and is also a parasitic fungus in a sex cell . It can infect humans and various animals . It can infect humans and animals . It usually causes abortion and infertility . In the past , the disease of brucella has been effectively controlled due to contact with diseased animals . In China , the disease of brucella has been effectively controlled . In China , the disease of brucella has recovered rapidly , especially in recent years . Two New Zealand rabbits were immunized subcutaneously with an A19 vaccine . Two weeks after the second immunization , the serum titer was 1 : 1600 , the required standard was reached , then the serum was isolated from the rabbit heart . The serum was extracted with ammonium sulfate precipitation method . The protein content was determined by using Sephadex - G25 chromatography column , and the protein content was determined by SDS - PAGE electrophoresis . The results showed that the purified rabbit anti - bovine brucella abortus A19 polyclonal IgG was successfully obtained . A hybridoma cell line with monoclonal antibody of 3E7 and 1C6 was prepared by ELISA . The results showed that both monoclonal antibodies were able to bind to the cell wall antigen of brucella . The results showed that the two monoclonal antibodies were able to bind to the cell wall antigen of brucella . The results showed that the two monoclonal antibodies were able to bind to the cell wall antigen of brucella . The results showed that the two monoclonal antibodies could be produced by the antigen of 31kD and 55kD respectively .

【学位授予单位】：河北农业大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

【引证文献】