重组葡萄球菌肠毒素SEA和SEO单克隆抗体的研制

发布时间：2018-03-13 20:15

本文选题：葡萄球菌肠毒素　切入点：重组蛋白　出处：《天津大学》2010年硕士论文　论文类型：学位论文

【摘要】：葡萄球菌肠毒素(Staphylococcal enterotoxins,SEs)是由葡萄球菌产生的、已超过20种基因型报道的、氨基酸序列和结构相似,具有潜在超抗原活性的一组食源性致病毒素。目前对新发现肠毒素结构、功能活性的认识和不同型肠毒素的检测鉴别尚缺乏有效手段。制备型特异性单抗对认识不同型肠毒素的结构和功能、不同毒素的检测鉴别、分析肠毒素基因簇的表达调控机制等具有重要意义。本研究通过构建葡萄球菌肠毒素A(SEA)和O(SEO)的原核表达载体,应用纯化的重组葡萄球菌肠毒素制备了两种肠毒素单克隆抗体,并对单抗特性做了研究。应用克隆的肠毒素基因sea、selo构建pET28α原核表达载体,转化重组菌BL21(DE3)表达,经镍鳌合层析纯化获得可溶性重组蛋白SEA-His、SEO-His。应用已构建的pEGX-6P-SEA和pEGX-6P-SEO表达载体转化宿主菌BL21,经诱导表达、亲和层析纯化获得SEA-GST、SEO-GST重组蛋白,以此蛋白为免疫原免疫8周龄Balb/c小鼠,3次免疫后取其脾细胞与SP2/0骨髓瘤细胞融合,制备杂交瘤细胞。以SEA-His、SEO-His为包被蛋白建立间接ELISA方法筛选阳性克隆,阳性率分别为8.5%(34/400)、5.3%(24/450)。有限稀释法对SEA肠毒素阳性杂交瘤细胞4A2,SEO肠毒素阳性杂交瘤细胞5C12、8C7进一步亚克隆,获得稳定分泌抗重组肠毒素蛋白单克隆抗体的杂交瘤细胞株,分别命名为A型:4A2-C5、4A2-B10;O型:5C12-C3、5C12-B4、8C7-G2、8C7-H8。对亚克隆获得的杂交瘤细胞株分泌的单抗特性进行了研究。经间接ELISA检测,4A2-C5、5C12-C3、8C7-G2杂交瘤细胞培养上清效价分别为1:1600、1:1600、1:6400,腹水效价分别达1:6.4×10~3、1:5.12×10~4、1:1.024×10~5;3株单克隆抗体4A2-C5、8C7-G2、5C12-C3亲和常数分别为3.35×10~6、1.89×10~6、1.45×10~6;亚类鉴定结果显示,4A2-C5、5C12-C3为IgG2b亚类;8C7-G2为IgM亚类。竞争ELISA对单抗识别抗原位点的分析结果表明,5C12-C3和8C7-G2分别识别SEO蛋白的不同抗原表位;Western blotting表明获得的3株单克隆抗体均能识别相应重组蛋白的B细胞线性表位;应用SEB和SEC1肠毒素阳性血清的阻断ELISA结果表明,SEA、SEO、SEB和SEC1肠毒素间存在交叉抗原。通过单抗特异性鉴定,结合肠毒素成熟肽氨基酸比对、表位预测、易接近性预测、二级结构预测分析,推断获得的3株单抗为葡萄球菌肠毒素群特异性抗体。这为葡萄球菌肠毒素功能特性的分析、检测奠定了基础。
[Abstract]:Staphylococcal enterotoxin SES (Staphylococcal enterotoxins) is a group of foodborne pathogenic toxins produced by Staphylococcal enterotoxins, which have been reported by more than 20 genotypes and have similar amino acid sequences and structures and have potential superantigen activity. The recognition of functional activity and the detection and identification of different types of enterotoxins are still lack of effective means. The specific monoclonal antibody pairs recognize the structure and function of different types of enterotoxins, and the detection and identification of different types of toxins. It is important to analyze the expression regulation mechanism of enterotoxin gene cluster. In this study, two monoclonal antibodies against staphylococcal enterotoxin were prepared by constructing prokaryotic expression vectors of staphylococcal enterotoxin (Staphylococcal enterotoxin), and using purified recombinant staphylococcal enterotoxin (staphylococcal enterotoxin). The characteristics of McAb were also studied. PET28 伪 prokaryotic expression vector was constructed by using the cloned enterotoxin gene seaselo. The recombinant protein SEA-His-SEO-Hiswas purified by nickel chelate chromatography. The recombinant protein was transformed into the host strain BL21 by using the constructed pEGX-6P-SEA and pEGX-6P-SEO expression vectors, and the expression was induced. SEA-GST-SEO-GST recombinant protein was purified by affinity chromatography. The SEA-GST-SEO-GST recombinant protein was used as immunogen to immunize 8-week-old Balb/c mice. The spleen cells of SEA-GST-SEO-GST were fused with SP2/0 myeloma cells. Hybridoma cells were prepared. SEA-His-SEO-His was used as coating protein to establish indirect ELISA method to screen positive clones, the positive rates were 8.5N 34 / 400 / 5.30.The subclone of SEA enterotoxin positive hybridoma cell line 5C128C7 was further subcloned by finite dilution method. Hybridoma cell lines secreting monoclonal antibodies against recombinant enterotoxin protein were obtained and named as type A: type A: 4A2-C5, 4A2-B10O type: 5C12-C3, 5C12-B4, 8C7-G2C7-H8. The monoclonal antibody properties of hybridoma cell lines obtained by subcloning were studied. The titers of the supernatants of the culture supernatants of 4A2-C5C5-C12-C3O8C7-G2 hybridoma cells were 1: 16001: 1: 16001: 6400, and the ascites titers were 1: 6.4 脳 1031.12 脳 1035.12 脳 104w 1: 1.024 脳 105C 3 McAb 4A2-C5C5C5C7-G2O2C2C12-C3, respectively. The results of subclass identification showed that 4A2-C5C5C12-C3 was IgG2b subclass 8C7-G2 as IgM subclass. The results of competitive ELISA analysis of mAb recognition antigenic sites showed that the three McAbs identified by Western blotting could recognize different epitopes of SEO protein respectively. The B cell linear epitopes of the corresponding recombinant proteins can be recognized. The results of blocking ELISA with SEB and SEC1 enterotoxin positive serum showed that there were cross antigens between SEOSEB and SEC1 enterotoxin. The results of specific identification of monoclonal antibody, amino acid ratio of mature peptide of enterotoxin, epitope prediction, accessibility prediction, etc. It was deduced that the three monoclonal antibodies were specific antibodies to staphylococcal enterotoxin group, which laid a foundation for the analysis of the functional characteristics of staphylococcal enterotoxin.
【学位授予单位】：天津大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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