白念珠菌磷脂酶B的原核表达、纯化及功能验证

发布时间：2018-06-10 19:33

  本文选题：磷脂酶B + 白念珠菌　； 参考：《中国人民解放军军医进修学院》2011年博士论文

【摘要】：研究背景 白念珠菌是一种重要的人类致病真菌,可引起机体浅部和深部的广泛损害,毒力因子包括黏附素、利于侵入的酶、形态发生及表型转换、受体等几个方面。其中磷脂酶B是白念珠菌重要的毒力因子,与白念珠菌致病有直接关系即在白念珠菌入侵宿主的早期发挥作用,包括对上皮细胞的黏附、入侵和损伤。磷脂酶B与白念珠菌感染密切相关,其在感染过程中的作用逐渐受到重视,并逐渐成为研究热点。 目的 构建磷脂酶B1和B2的重组表达体,在大肠杆菌中进行诱导表达,纯化表达产物并进行生物活性检测,为探索磷脂酶B1和B2重组蛋白的致病和耐药机制奠定基础,也为研制新型抗真菌药物、诊断试剂及抗真菌疫苗等提供理论基础及科学依据。 方法 从白念珠菌中提取基因组DNA为模板,用PCR方法获取磷脂酶B1和B2基因。将原核表达载体pEGX-4T-1分别与磷脂酶B1和B2基因行双酶切,琼脂糖凝胶纯化,连接酶切产物,转化大肠杆菌TOP10感受态细菌,筛选菌落和测序鉴定。将重组原核表达载体pEGX-4T-1/PLB1和pEGX-4T-1/PLB2转化大肠杆菌BL21(DE3)感受态细胞,在37℃经IPTG诱导表达出包涵体融合蛋白。包涵体蛋白尿素中变性复性后经亲和层析、阴离子交换层析和反相高效液相层析纯化,并用凝血酶酶切标签,得到纯化的磷脂酶B1和B2蛋白,并通过凝胶扩散法检测其生物学活性。 结果 经PCR扩增获得的目的基因分子量与预计相同,并定向插入原核表达载体pEGX-4T-1中,双酶切后电泳获得预期的磷脂酶B1和B2基因条带,测序证实为正确序列。重组原核表达载体pEGX-4T-1/PLB1和pEGX-4T-1/PLB2的基因工程菌在37℃经IPTG诱导后均表达出包涵体融合蛋白,其在尿素中变性复性后经纯化、酶切后得到目的蛋白,并验证其具有生物学活性。 结论 1、成功构建了pEGX-4T-1/PLB1和pEGX-4T-1/PLB2原核表达载体,将其转化至大肠杆菌后表达出磷脂酶B1和B2的重组蛋白； 2、重组蛋白变性、复性后经亲和层析等纯化及凝血酶酶切后得到了目的蛋白； 3、重组蛋白磷脂酶B1和B2具有良好的生物学活性
[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，

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