羊布鲁菌BCSP31蛋白的表达、纯化及其单克隆抗体的制备和鉴定

发布时间：2018-01-01 06:36

本文关键词：羊布鲁菌BCSP31蛋白的表达、纯化及其单克隆抗体的制备和鉴定　出处：《第四军医大学》2008年硕士论文　论文类型：学位论文

【摘要】： 布鲁菌是引起人兽共患布鲁菌病的病原菌。布鲁菌有7个生物种型,其中羊、牛、猪三个种既可感染家畜也可以感染人类。布鲁菌可通过多种途径感染人和动物,致病性强,流行范围广,危害十分严重。此外,布鲁菌还是一种潜在的生物战剂。布鲁菌病临床症状轻重不一,个体表现差异大,容易误诊,治疗不当会导致病情迁延,影响人畜健康并导致经济损失。此外,现有的布鲁菌检测方法仍不理想,存在特异性不高,不能区分疫苗免疫和自然感染,容易误诊和漏诊。随着近年来布鲁菌病日益增高的流行趋势,发展快速敏感特异的检测方法对控制布鲁菌病疫情,及时处置病畜、治疗患者有重要意义。布鲁菌细菌表面31kDa蛋白BCSP31具有抗原性强、在不同种间同源性高的特点。本研究拟通过纯化BCSP31蛋白,制备其单克隆抗体(mAb),为建立布鲁菌病ELISA检测方法奠定基础。 1主要方法将质粒pGEX-4T-1-BCSP31转化至大肠杆菌并诱导表达,采用GST亲和层析纯化的方法,分别获得GST-BCSP31融合蛋白和BCSP31纯化蛋白。用纯化的BCSP31蛋白免疫小鼠,将小鼠脾细胞与骨髓瘤Sp2/0细胞融合,ELISA间接法筛选阳性克隆,3次克隆化后建立杂交瘤细胞系。采用小鼠腹腔接种杂交瘤细胞制备腹水,正辛酸-硫酸铵法纯化mAb。采用Western-blot、ELISA以及表位相加试验等方法鉴定mAb特性。建立用于检测布鲁菌感染血清的ELISA间接夹心法。 2主要结果 GST-BCSP31融合蛋白在25℃诱导时为可溶性表达,经亲和层析纯化,分别获得GST-BCSP31融合蛋白2.48 mg/mL和BCSP31纯化蛋白0.5 mg/mL;Western-blot检测结果显示所获蛋白可与兔抗布鲁菌血清特异性反应。获得2株杂交瘤细胞系,1F1和1E5,均为IgG1亚类;2株mAb均可与羊布鲁菌BCSP31蛋白特异性反应,而不与HCV AS3蛋白、结核分枝杆菌DAF 44a蛋白和RPF蛋白、汉坦病毒GP和NP以及人IgG1等无关蛋白反应,也不与大肠杆菌裂解液反应,表明这两株mAb具有较高的特异性。并且这两株mAb在BCSP31蛋白上的抗原位点不同;mAb 1F1与BCSP31蛋白的相对亲和力高于1E5。ELISA间接夹心法的工作条件是以10μg/mL包被mAb,包被量为100μl/孔;抗原最佳浓度为20μg/mL,加入量为100μl/孔;HRP-羊抗兔最佳工作浓度为1:50000,加入量为100μl/孔。对相应参数进行评估,特异性、敏感性及重复性均良好。 BCSP31的纯化及其mAb的获得以及ELISA检测方法的建立,不仅有助于进一步了解与布鲁菌感染和免疫相关的抗原成分及其作用机制,也为进一步研制布鲁菌检测试剂盒提供了基础。
[Abstract]:Brucella is causing pathogen zoonotic brucellosis. Brucella has 7 species, including sheep, cattle, three pigs can be infected animal can also infect humans. Infection through a variety of ways and animal Brucella, highly pathogenic, wide, harm is very serious. In addition, Brucella is a potential biological warfare agents.
Brucellosis is an important clinical symptoms, individual differences, easily misdiagnosed, improper treatment can lead to persistent disease, affect human and animal health and lead to economic losses. In addition, Brucella and the existing detection methods is still not ideal, specificity is not high, can not distinguish between natural infection and vaccination, misdiagnosis and missed with the popular trend in recent years. The diagnosis of brucellosis is increasing, the rapid development of sensitive and specific detection method to control the brucellosis epidemic, timely disposal of animal disease, it has important significance for treatment of patients.
Brucella bacterial surface 31kDa protein BCSP31 has strong antigenicity and high homology among different species. In this study, monoclonal antibody (mAb) was prepared by purification of BCSP31 protein, which lay the foundation for establishing ELISA detection method of brucellosis.
1 main methods
The plasmid was transformed into Escherichia coli pGEX-4T-1-BCSP31 and induced by GST affinity chromatography method, the obtained GST-BCSP31 fusion protein and BCSP31 protein were purified by BCSP31. The purified protein to immunize mouse, the mouse spleen cells and myeloma cell fusion of Sp2/0, the positive clones were screened by indirect ELISA, the establishment of hybridoma cell lines were cloned 3 times after the mice were inoculated with ascites. Preparation of hybridoma cells were positive by caprylic acid - ammonium sulfate purification of mAb. by Western-blot, ELISA and epitope additivity test method for the identification of mAb characteristics. Established for the detection of serum Shandong bacteria infection cloth ELISA indirect sandwich method.
2 main results
The expression of GST-BCSP31 fusion protein is soluble at 25 DEG C during induction, purified by affinity chromatography, respectively GST-BCSP31 fusion protein and purified BCSP31 protein 2.48 mg/mL 0.5 mg/mL; Western-blot showed that the bacteria protein serum specific reaction with Rabbit anti Brucella. 2 hybridoma cell lines, 1F1 and 1E5, are IgG1 sub class; 2 strains of mAb can be used with B.melitensis BCSP31 protein specific protein reaction, but not with HCV AS3, DAF of Mycobacterium tuberculosis 44a protein and RPF protein, Hantaan virus GP and NP and IgG1 related protein reaction does not react with Escherichia coli lysate, indicating the specificity of the two strains with mAb high. And the two strains of mAb antigenic sites on the BCSP31 protein of mAb and 1F1; the relative affinity of BCSP31 protein was higher than that of indirect sandwich 1E5.ELISA working conditions are based on 10 g/mL coated mAb, coating amount of 100 hole l/ antigen; The best concentration is 20 micron g/mL, the dosage is 100 micron l/ hole, the best working concentration of HRP- Goat anti rabbit is 1:50000, and the dosage is 100 HRP- l/ hole. The corresponding parameters are evaluated, the specificity, sensibility and reproducibility are good.
The purification of BCSP31, the acquisition of mAb and the establishment of ELISA detection method are not only helpful to further understand the antigen components and their action mechanisms associated with brucellosis and immunity, but also provide a basis for further developing Brucella detection kit.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

【相似文献】