金黄色葡萄球菌肠毒素B适配子的筛选及初步应用

发布时间：2018-06-15 00:35

本文选题：指数富集配体系统进化 + 金黄色葡萄球菌肠毒素B　；参考：《福建医科大学》2010年硕士论文

【摘要】： 【目的】以金黄色葡萄球菌肠毒素B为靶物质筛选获得的富集单链DNA文库为起始文库,从中筛选获得能高亲和力和高特异性结合金黄色葡萄球菌肠毒素B的单个DNA适配子,并利用适配子建立的夹心法检测患者血清标本中SEB的含量,辅助诊断金黄色葡萄球菌感染;同时研究适配子在大鼠体内对SEB超抗原活性的抑制作用。【方法】 1.将富集文库的筛选产物分别进行扩增、纯化、胶回收和TA克隆转化至大肠杆菌DH5α中,LB液体增菌保存。挑取单个菌落培养,作为菌种保存,抽提质粒,利用相应引物进行PCR扩增,并以羧基磁珠作为分离介质获得单个适配子。 2.分别利用地高辛、荧光素和生物素标记的适配子直接测试法,对适配子的结合率和特异性等性质进行鉴定。 3.分别采用抗体一适配子夹心法和文库一适配子夹心法,用于SEB的定量检测;同时对两种检测方法进行比对。利用文库一适配子夹心法检测临床患者血清中SEB的含量。 4.将大鼠随机分成4组,分别进行不同的干预和处理;随后检测各组大鼠血清中IL-4, IL-6, ALT, AST,肌酐和尿酸的含量,并对各组检测结果进行分析。【结果】 1.筛选产物进行克隆和增菌培养后,选择其中40个克隆子进行了检测,共获得了30个条带位置正确的适配子,并从中筛选得到了一条结合力和特异性均较好的适配子。 2.文库一适配子夹心法和抗体一适配子夹心法均可用于SEB的检测,且两种检测方法的结果无统计学差异。而健康对照组与细菌感染组血清标本中SEB的含量具有统计学差异。 3.适配子在大鼠体内对SEB超抗原活性具有一定的抑制作用,可有效抑制IL-4和IL-6的分泌,并能降低血清中AST, ALT,尿酸和肌酐的含量。【结论】本实验成功筛选到了针对SEB的高亲和力和高特异性的ssDNA适配子,建立了文库一适配子夹心法可以对血清标本中SEB的含量进行检测。适配子的作用类似于抗体,能够与SEB特异性的结合,它可以作为金黄色葡萄球菌感染的检测分子和治疗药物,具有潜在的使用价值。
[Abstract]:[objective] to select the enriched single strand DNA library from Staphylococcus aureus enterotoxin B as the target material. A single DNA aptamer with high affinity and specificity binding to Staphylococcus aureus enterotoxin B was obtained. The content of SEB in serum samples was detected by the sandwich method established by the aptamer to assist the diagnosis of Staphylococcus aureus infection. At the same time, the inhibitory effect of aptamer on SEB superantigen activity in rats was studied. [methods] 1. The screening products of the enriched library were amplified, purified, reclaimed and transformed into Escherichia coli DH5 伪. A single aptamer was obtained from a single colony culture as a strain preservation, plasmid extraction, PCR amplification with the corresponding primers, and carboxyl magnetic beads as the separation medium. 2. The binding rate and specificity of aptamers were identified by direct assay of aptamers labeled with digoxin, fluorescein and biotin respectively. 3. The antibody-aptamer sandwich method and library-aptamer sandwich method were used for the quantitative detection of SEB, and the two detection methods were compared. The content of SEB in serum of clinical patients was detected by library-aptamer sandwich method. 4. 4. The rats were randomly divided into 4 groups for different intervention and treatment. The serum levels of IL-4, IL-6, alt, AST, creatinine and uric acid in each group were determined. The results of each group were analyzed. [results] 1. After the screening products were cloned and cultured, 40 clones were selected for detection, and 30 aptamers with the correct position were obtained. An aptamer with good binding ability and specificity was obtained. Library-aptamer sandwich method and antibody-aptamer sandwich method can be used to detect SEB, and there is no statistical difference between the two methods. The SEB content in serum samples of healthy control group and bacterial infection group was significantly different from that of bacterial infection group. The aptamer can inhibit the activity of SEB superantigen and inhibit the secretion of IL-4 and IL-6 in rats. It can reduce the content of AST, alt, uric acid and creatinine in serum. [conclusion] the high affinity and specificity of ssDNA adaptor for SEB were successfully screened in this experiment. A library-aptamer sandwich method was established to detect the content of SEB in serum samples. Aptamers have similar effects to antibodies and can bind to SEB specifically. They can be used as detection molecules and therapeutic drugs for Staphylococcus aureus infection.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R378

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