本文選題：艱難梭菌 + 細(xì)胞毒素B膜受體結(jié)合區(qū)��； 參考：《第一軍醫(yī)大學(xué)》2005年碩士論文

【摘要】：目的: 艱難梭菌(Clostridium difficile)是造成抗生素相關(guān)性結(jié)腸炎及偽膜性結(jié)腸炎(PMC)的最常見(jiàn)致病菌。該菌造成老年人和免疫功能低下的病人抗生素相關(guān)性腹瀉及腸炎的發(fā)病率及死亡率明顯升高。Cdifficile產(chǎn)毒株準(zhǔn)確快速的診斷、鑒定,對(duì)確定病源、控制其傳播、有效治療患者是極其重要的。國(guó)外建立的酶聯(lián)免疫吸附實(shí)驗(yàn)(ELISA)檢測(cè)C difficile毒素,其優(yōu)點(diǎn)是靈敏度和特異度高,結(jié)果可在2-4小時(shí)內(nèi)得出,從而更適合指導(dǎo)臨床實(shí)際工作,故被大力推廣,但國(guó)內(nèi)對(duì)艱難梭菌診斷試劑研究較少,尤其是對(duì)Cdifficile細(xì)胞毒素B(cdtB)的檢測(cè)。本課題利用cdtB特點(diǎn),以基因工程技術(shù)對(duì)其羧基末端功能區(qū)進(jìn)行克隆、表達(dá)并純化出該蛋白,制備多克隆抗體,主要目的在于對(duì)該重組蛋白生物學(xué)特性進(jìn)行研究和探索,以期建立一種可快速、穩(wěn)定的檢測(cè)出Cdifficile細(xì)胞毒素B的ELISA診斷試劑。 方法: 1、基因工程技術(shù)制備cdtB膜受體結(jié)合區(qū)域的重組蛋白。以C difficile標(biāo)準(zhǔn)株VPI10463的全長(zhǎng)DNA序列為模板,利用PCR技術(shù)擴(kuò)增出編碼cdtB膜受體結(jié)合區(qū)域的功能基因,經(jīng)EcoRI和XhoI雙酶切后,定向插入同樣經(jīng)這兩種酶雙酶切的原核表達(dá)載體pET-22b(+)中,在DNAT_4連接酶作用下進(jìn)行連接以獲得重組質(zhì)粒。重組質(zhì)粒經(jīng)測(cè)序,結(jié)果與GenBank記錄的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.
【學(xué)位授予單位】：第一軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類(lèi)號(hào)】：R378.8

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本文編號(hào)：1802099