EHEC O157主要毒素基因克隆表达及单克隆抗体制备与鉴定

发布时间：2018-01-19 00:13

本文关键词： EHEC O157 Stx1B Stx2B eae 原核表达单克隆抗体　出处：《东北农业大学》2010年硕士论文　论文类型：学位论文

【摘要】： 肠出血性大肠杆菌(EHEC)是一种重要的食源性病原菌,自1975年被首次分离接着被确认为致病菌以来,世界各地包括中国在内都有不同规模的暴发流行,可引起出血性或非出血性腹泻、出血性结肠炎(HC)、溶血性尿毒综合征(HUS)、血栓性血小板减少性紫癜(TTP)等全身性并发症。O157是EHEC的代表菌株,其主要毒力特征之一是引起肠上皮细胞出现黏附与脱落(attaching and effacing, AE)损伤,编码AE损伤的基因都位于细菌染色体上LEE致病岛内,其中最重要的就是eae基因,编码一个被称为紧密素的外膜蛋白。EHEC O157另外一个毒力因子是由插入到染色体上的原噬菌体编码的志贺毒素(Stx),它能使感染机体器官发生病理变化,或者继发其他的临床症状甚至引起死亡。大量研究证明,紧密素和志贺毒素Stx是可以用于开发疫苗以预防EHEC感染的重要免疫原。本试验利用PCR技术从大肠杆菌EDL933全基因组中克隆了Stx1B、Stx2B、eae三段基因,经序列分析,结果与参考序列同源性达99%以上。将产物克隆到原核表达载体pGEX-6P-1,构建重组表达质粒pGEX-Stx1B、pGEX-Stx2B、pGEX-eae,转入大肠杆菌BL21中,经IPTG诱导,实现了重组融合蛋白的高效表达,表达量分别约占菌体总蛋白的27%、27.7%和19.5%,且均主要以包涵体形式表达,通过切胶的方法纯化目的蛋白。纯化蛋白免疫BABL/c小鼠,利用细胞融合技术,经间接ELISA筛选和3次细胞亚克隆,Stx1B、Stx2B、eae分别获得5、5、2株杂交瘤细胞,分别命名为1G11、2H8、1B10、3A6、4B1;1B3、4B4、2C3、3D1、1C2;2D4、3B4。经单克隆抗体亚类试剂盒鉴定,2D4为IgM,其他均为IgG类抗体。12株杂交瘤细胞染色体数为103±5,明显多于SP2/0骨髓瘤细胞65～75条。Western blotting表明1G11、2H8、1B10,1B3、4B4、2C3,2D4、3B4与相应的GST-Stx1B、GST-Stx2B、GST-eae融合蛋白反应均能产生特异性条带,而与空载体诱导后的GST标签蛋白不反应。制备了1G11、2H8腹水单抗,采用辛酸-饱和硫酸铵法纯化后,经SDS-PAGE电泳分析IgG重链和轻链区带明显,几乎没有杂带。ELISA叠加试验可知,1G11、2H8、1B10抗同一抗原表位,1B3、4B4、2C3抗同一抗原表位,2D4、3B4抗不同的抗原表位。12株杂交瘤细胞在体外连续传代3个月和冻存后再复苏均不影响抗体分泌的稳定性;与沙门菌、大肠杆菌、牛结核杆菌、李氏杆菌、链球菌、金黄色葡萄球菌均无交叉反应。亲和力分析表明,1G11、2H8、1B10,1B3、4B4、2C3,2D4、3B4 8株单抗的亲和常数均在107～109M-1之间,亲和力较高,为这些单抗敏感、稳定和快捷的检测应用提供了必要的保证。总之,12株单抗的成功制备为EHEC O157病原的快速、特异性诊断提供了诊断试剂,同时也为该病致病机理和特异性治疗的进一步研究提供物质基础,为成功研制O157疫苗积累了数据。
[Abstract]:Enterohemorrhagic Escherichia coli (EHEC) is an important foodborne pathogen, which has been isolated for the first time since 1975 and then confirmed as a pathogenic bacteria. All over the world, including China, there are outbreaks of different scales, which can cause hemorrhagic or non-hemorrhagic diarrhea, hemorrhagic colitis, hemorrhagic colitis, hemolytic uremic syndrome (HUSS). Thrombotic thrombocytopenic purpura (TTP) and other systemic complications. O157 is the representative strain of EHEC. One of its main virulence characteristics is to cause adhesion and abscission of and effusion (AEs) injury in intestinal epithelial cells. The genes encoding AE damage are located in the LEE pathogenicity island on the bacterial chromosome, the most important of which is the eae gene. Another virulence factor that encodes an outer membrane protein, EHEC O157, called compaction, is the Shiga toxin Stx encoded by the prophage inserted into the chromosome. It can cause pathological changes in infected organs, or other clinical symptoms or even death. A large number of studies prove that. Titin and Shigella toxin Stx are important immunogens that can be used to develop vaccines to prevent EHEC infection. In this study, three segments of Stx1BX Stx2BUE eae gene were cloned from the whole genome of Escherichia coli EDL933 by PCR technique and sequenced. Results the homology with the reference sequence was over 99%. The product was cloned into the prokaryotic expression vector pGEX-6P-1 and the recombinant expression plasmid pGEX-Stx1BnpGEX-Stx2B was constructed. PGEX-eaewas transferred into Escherichia coli BL21 and induced by IPTG, the recombinant fusion protein was highly expressed, which accounted for about 27% of the total bacterial protein. Both 27.7% and 19.5g were expressed in the form of inclusion body, and the target protein was purified by gumming method. The purified protein was used to immunize BABL/c mice. By indirect ELISA screening and three cell subclones Stx2Beae, 5 BABL/c mice were obtained. Two hybridoma cells were named as 1G11O2H8B103A6A6B1; 1B _ 3N _ 4B _ 4N _ 2C _ 3C _ 3N _ 3D _ 1C _ 2; The chromosome number of the hybridoma cells was 103 卤5. The chromosome number of the other hybridoma cells was 103 卤5, which was identified as IgM by monoclonal antibody subclass kit. There were more than 6575 SP2/0 myeloma cells. Western blotting showed that 1G112H8, 1B10, 1B3, 4B4, 2C3D4. 3B4 reacted with the corresponding GST-Stx1BU GST-Stx2BU GST-eae fusion protein to produce specific bands. The 1G112H8 ascites monoclonal antibody was prepared and purified by octanoic acid-saturated ammonium sulfate method. The IgG heavy chain and light chain bands were obviously analyzed by SDS-PAGE electrophoresis. There was almost no heteroband. Elisa superposition test showed that 1G112H8H8 1B10 could resist the same antigen epitope 1B3. The antigenic epitopes of 4B4O2C3 against different epitopes. 12 hybridoma cells were subcultured for 3 months in vitro and resuscitated after cryopreservation did not affect the stability of antibody secretion. There was no cross reaction with Salmonella, Escherichia coli, Mycobacterium bovis, Listeria, Streptococcus, Staphylococcus aureus. The affinity constants of the McAbs of 2C3D4D4B48 were between 107 and 109M-1, and they were sensitive to these McAbs because of their high affinity. The stable and rapid detection and application provided the necessary guarantee. In a word, the successful preparation of 12 McAbs provided a diagnostic reagent for the rapid and specific diagnosis of EHEC O157. It also provides material basis for further research on the pathogenesis and specific treatment of the disease, and accumulates data for the successful development of O157 vaccine.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

【参考文献】