摘要
食品中的肠道致病菌是引起人类食源性疾病的主要原因之一，临床症状主要表现为腹泻、呕吐，也是食物中毒的主要因素，是人类健康的一大杀手。其中，金黄色葡萄球菌(Saphylococcus aureus)是人类和某些动物的病原菌之一，常引起食物中毒。其致病力强弱主要取决于产生毒素和侵袭性酶的能力，其中金黄色葡萄球菌耐热性肠毒素SEB，是引起人类食物中毒和葡萄球菌胃肠炎的主要原因。
快速和自动化检验方法在应用微生物学领域还处于快速发展时期，目前用于微生物检验的检测方法有很多，出现了一些快速检测致病微生物的方法，到目前为止只有生物芯片技术能较好地满足人们对检测致病微生物的快速、准确、简易等要求。由于基因与其所表达的毒素蛋白之间有表达水平不对应等原因，单凭基因芯片检测结果是不能完全反应出生物体内的蛋白质水平，因此，要想得到完整的生物信息，其解决办法之一就是直接研究基因的表达产物一蛋白质。
本课题旨在制作出能应用于蛋白质芯片点印的SEB的单克隆抗体，为蛋白质芯片在致病微生物的应用上做一些前期的工作。所做实验课题主要内容包括金黄色葡萄球菌B型肠毒素的分离纯化及其单克隆抗体的制备。选用金黄色葡萄球菌产毒标准菌株CMCC(B)26002，采取玻璃纸覆盖琼脂平板的膜培养法培养产毒，毒素经预处理后，首先利用羧甲基阳离子交换纤维素CM—32柱层析，对B型肠毒素进行初步分离富集，然后依据分子筛原理按分子量大小经葡聚糖凝胶G—75进一步纯化，只经两步细分级分离就获得了纯化的能应用于蛋白质芯片的SEB。用分离纯化的SEB免疫BALB／C小鼠，然后取其脾细胞与骨髓瘤细胞Sp2／0融合，再经过选育、检测、鉴定等步骤，成功获得了SEB的单克隆抗体细胞，抗体特异性经酶联免疫吸附测定法检验，单克隆抗体细胞特异性强。本课题的研究成果为蛋白质芯片技术快速检测产肠毒素金黄色葡萄球菌奠定了基础。
Enteropathogenic microorganism in food is the main factor of food poisoning disease, and its symptom is mainly diarrhea, vomit. It is harmful to human's health. Staphylococcus aureus is one of the pathogens for human being and some animals and it can bring food poisoning. Its pathogenicity rest with the ability to secrete pathotoxin and zymins, and heat-resistant Staphylococcus aureus enterotoxin B is the main causation that results in food poisoning and Staphylococcus gastritis and enteritis.
Fast and automatic test ways is on the quick developing period. At present, there are many test ways in pathogenic microbiology test and some new quick test ways appear, but, at present, only biology chip can meet well human being's quick, veracious and simple demand for testing pathogenic microbiology. Moreover, gene doesn't correspond to its expressive toxin protein; gene chip's analytic result can not reflect protein's level. So, one of the methods is to study gene's expressive substance—protein.
This thesis is to make SEB monoclonal antibody that is used by protein chip and do some anterior work for protein chip's application in pathogenic microbiology. The main process includes the separation and purification of SEB and the making of SEB monoclonal antibody. As to producing toxin S. aureus standard bacterium CMCC (B) 26002, enterotoxin cultivation method use velum cultivation that agar culture dish are covered with dialytic cellophane paper. Enterotoxin is pretreated. The following work is to use positive ion-exchange carboxy methyl cellulose (CMC) to separate SEB, and then use Sephadex G-75 to purify SEB according to filter theory. Thus, we can gain purified SEB only by two steps. In the experiment, Purified SEB was used to immunize BALB/C mouse, then, let spleen cell and myeloma cells (Sp2/0) fuse. After selective cultivation, test and identification, The experiment gain successfully myeloma cells that can secrete SEB monoclonal antibody and its idiosyncrasy is good through enzyme linked immunosorbent assay (ELISA). The results of this experiment have established good foundation for protein chips in testing pathogenic microbiology.
引文
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