艰难梭菌细胞毒素B羧基末端功能区的克

发布时间：2018-04-25 16:28

本文选题：艰难梭菌 + 细胞毒素B膜受体结合区　；参考：《第一军医大学》2005年硕士论文

【摘要】：目的: 艰难梭菌(Clostridium difficile)是造成抗生素相关性结肠炎及伪膜性结肠炎(PMC)的最常见致病菌。该菌造成老年人和免疫功能低下的病人抗生素相关性腹泻及肠炎的发病率及死亡率明显升高。Cdifficile产毒株准确快速的诊断、鉴定,对确定病源、控制其传播、有效治疗患者是极其重要的。国外建立的酶联免疫吸附实验(ELISA)检测C difficile毒素,其优点是灵敏度和特异度高,结果可在2-4小时内得出,从而更适合指导临床实际工作,故被大力推广,但国内对艰难梭菌诊断试剂研究较少,尤其是对Cdifficile细胞毒素B(cdtB)的检测。本课题利用cdtB特点,以基因工程技术对其羧基末端功能区进行克隆、表达并纯化出该蛋白,制备多克隆抗体,主要目的在于对该重组蛋白生物学特性进行研究和探索,以期建立一种可快速、稳定的检测出Cdifficile细胞毒素B的ELISA诊断试剂。方法: 1、基因工程技术制备cdtB膜受体结合区域的重组蛋白。以C difficile标准株VPI10463的全长DNA序列为模板,利用PCR技术扩增出编码cdtB膜受体结合区域的功能基因,经EcoRI和XhoI双酶切后,定向插入同样经这两种酶双酶切的原核表达载体pET-22b(+)中,在DNAT_4连接酶作用下进行连接以获得重组质粒。重组质粒经测序,结果与GenBank记录的Cdifficile VPI10463的cdtB
[Abstract]:Objective: Clostridium traffic is the most common pathogen causing antibiotic associated colitis and pseudomembranous colitis. The incidence and mortality of antibiotic associated diarrhea and enteritis in the elderly and the patients with low immune function were significantly increased. The accurate and rapid diagnosis and identification of the strains produced by the strain were helpful in determining the source of the disease and controlling its transmission. Effective treatment of patients is extremely important. Enzyme linked immunosorbent assay (Elisa) established abroad for the detection of C difficile toxin has the advantages of high sensitivity and specificity, and the result can be obtained within 2-4 hours, which is more suitable for guiding clinical practice, so it has been popularized. However, there are few studies on the diagnostic reagents of Clostridium diffracta in China, especially the detection of Cdifficile cytotoxin BncdtB. In this paper, cdtB was used to clone the carboxyl terminal functional region of the recombinant protein, express and purify the protein, and prepare polyclonal antibody. The main purpose of this study was to study and explore the biological characteristics of the recombinant protein. In order to establish a rapid and stable detection of Cdifficile cytotoxin B ELISA diagnostic reagent. Methods: 1. The recombinant protein of cdtB membrane receptor binding region was prepared by genetic engineering. Using the full-length DNA sequence of C difficile standard strain VPI10463 as template, the functional gene encoding the binding region of cdtB membrane receptor was amplified by PCR technique. After being digested by EcoRI and XhoI, the functional gene was inserted into the prokaryotic expression vector pET-22b (), which was also digested by these two enzymes. The recombinant plasmid was obtained by ligation with DNAT_4 ligase. The recombinant plasmid was sequenced and compared with the cdtB of Cdifficile VPI10463 recorded by GenBank.
【学位授予单位】：第一军医大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R378.8

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