本文選題：磷脂酶B + 白念珠菌　； 參考：《中國(guó)人民解放軍軍醫(yī)進(jìn)修學(xué)院》2011年博士論文

【摘要】：研究背景 白念珠菌是一種重要的人類致病真菌,可引起機(jī)體淺部和深部的廣泛損害,毒力因子包括黏附素、利于侵入的酶、形態(tài)發(fā)生及表型轉(zhuǎn)換、受體等幾個(gè)方面。其中磷脂酶B是白念珠菌重要的毒力因子,與白念珠菌致病有直接關(guān)系即在白念珠菌入侵宿主的早期發(fā)揮作用,包括對(duì)上皮細(xì)胞的黏附、入侵和損傷。磷脂酶B與白念珠菌感染密切相關(guān),其在感染過(guò)程中的作用逐漸受到重視,并逐漸成為研究熱點(diǎn)。 目的 構(gòu)建磷脂酶B1和B2的重組表達(dá)體,在大腸桿菌中進(jìn)行誘導(dǎo)表達(dá),純化表達(dá)產(chǎn)物并進(jìn)行生物活性檢測(cè),為探索磷脂酶B1和B2重組蛋白的致病和耐藥機(jī)制奠定基礎(chǔ),也為研制新型抗真菌藥物、診斷試劑及抗真菌疫苗等提供理論基礎(chǔ)及科學(xué)依據(jù)。 方法 從白念珠菌中提取基因組DNA為模板,用PCR方法獲取磷脂酶B1和B2基因。將原核表達(dá)載體pEGX-4T-1分別與磷脂酶B1和B2基因行雙酶切,瓊脂糖凝膠純化,連接酶切產(chǎn)物,轉(zhuǎn)化大腸桿菌TOP10感受態(tài)細(xì)菌,篩選菌落和測(cè)序鑒定。將重組原核表達(dá)載體pEGX-4T-1/PLB1和pEGX-4T-1/PLB2轉(zhuǎn)化大腸桿菌BL21(DE3)感受態(tài)細(xì)胞,在37℃經(jīng)IPTG誘導(dǎo)表達(dá)出包涵體融合蛋白。包涵體蛋白尿素中變性復(fù)性后經(jīng)親和層析、陰離子交換層析和反相高效液相層析純化,并用凝血酶酶切標(biāo)簽,得到純化的磷脂酶B1和B2蛋白,并通過(guò)凝膠擴(kuò)散法檢測(cè)其生物學(xué)活性。 結(jié)果 經(jīng)PCR擴(kuò)增獲得的目的基因分子量與預(yù)計(jì)相同,并定向插入原核表達(dá)載體pEGX-4T-1中,雙酶切后電泳獲得預(yù)期的磷脂酶B1和B2基因條帶,測(cè)序證實(shí)為正確序列。重組原核表達(dá)載體pEGX-4T-1/PLB1和pEGX-4T-1/PLB2的基因工程菌在37℃經(jīng)IPTG誘導(dǎo)后均表達(dá)出包涵體融合蛋白,其在尿素中變性復(fù)性后經(jīng)純化、酶切后得到目的蛋白,并驗(yàn)證其具有生物學(xué)活性。 結(jié)論 1、成功構(gòu)建了pEGX-4T-1/PLB1和pEGX-4T-1/PLB2原核表達(dá)載體,將其轉(zhuǎn)化至大腸桿菌后表達(dá)出磷脂酶B1和B2的重組蛋白； 2、重組蛋白變性、復(fù)性后經(jīng)親和層析等純化及凝血酶酶切后得到了目的蛋白； 3、重組蛋白磷脂酶B1和B2具有良好的生物學(xué)活性
[Abstract]:Background Candida albicans is an important human pathogenic fungus, which can cause extensive damage to the superficial and deep part of organism. Virulence factors include adhesion, invasive enzyme, morphogenesis and phenotypic transformation, receptor and so on. Phospholipase B is an important virulence factor of Candida albicans, which is directly related to the pathogenicity of Candida albicans, that is, it plays an early role in the host invasion of Candida albicans, including adhesion, invasion and injury to epithelial cells. Phospholipase B is closely related to the infection of Candida albicans, and its role in the process of infection has gradually been paid attention to, and gradually become a research hotspot. Objective to construct the recombinant expression of phospholipase B1 and B2 and express them in Escherichia coli. The purification of the expressed product and the detection of its biological activity lay a foundation for exploring the pathogenicity and drug resistance mechanism of phospholipase B1 and B2 recombinant proteins, and also for the development of new antifungal drugs. Methods Genomic DNA was extracted from Candida albicans as template and phospholipase B1 and B2 genes were obtained by PCR. The prokaryotic expression vector pEGX-4T-1 was digested with phospholipase B1 and B2 genes respectively, then purified by agarose gel, ligated with enzyme digestion product, transformed into E. coli TOP10 competent bacteria, screened colonies and identified by sequencing. The recombinant prokaryotic expression vectors pEGX-4T-1 / PLB1 and pEGX-4T-1 / PLB2 were transformed into Escherichia coli BL21DE-3) competent cells, and the inclusion body fusion protein was induced by IPTG at 37 鈩，

本文編號(hào)：2004352