亲环素论文：解脂耶氏酵母亲环素的原核表达与纯化.docVIP

亲环素论文：解脂耶氏酵母亲环素的原核表达与纯化 【中文摘要】亲环素(Cyclophilins, CyPs)是一类广泛存在于各种具细胞结构的生物体内且结构高度保守的蛋白家族。它们是一种分子伴侣,类属肽脯氨酰顺反异构酶超家族,能催化含脯氨酸亚氨基肽键异构化,协助蛋白质正确折叠、组装和运输。此外,亲环素还与环孢素A结合参与对机体的免疫抑制,并在细胞凋亡、氧化应激、胆固醇代谢等生命活动中发挥的重要的作用。解脂耶氏酵母(Yarrow ia lipolylica)属于子囊菌纲真菌,它能在较广的pH范围内生存,并能以葡萄糖,脂肪酸和烃类等常见而廉价的物质作为代谢底物,是一种重要的工业酵母菌。解脂耶氏酵母是非致病菌,因此能用于食品和药物生产。此外,由于解脂耶氏酵母代谢分泌大量的有机酸和蛋白质到胞外培养基,所以也被人们开发成为—种优良的酵母表达系统。迄今发现并克隆的亲环素家族成员已经超过一百多种,研究者也从解脂耶氏酵母基因组中找到了13种亲环素基因。本文以解脂耶氏酵母亲环素基因YALIOC15653g (Gene ID:2909417)为研究对象,以解脂耶氏酵母总DNA为模板,PCR扩增该亲环素基因；产物经T-A克隆后进行测序鉴定；构建亲环素原核表达载体pET32a-CyP,转化大肠杆菌BL21(DE3)plysS;阳性克隆经IPTG诱导表达亲环素,经Ni-NTA金属螯合亲和层析纯化亲环素,纯化效果通过SDS电泳进行检验。现将结果小结如下：●测序结果证实成功扩增得到全长解脂耶氏酵母亲环素基因。●菌落PCR检验证实成功构建原核表达质粒pET32a-CyP。·SDS结果证实,成功在大肠杆菌中诱导表达出亲环素。以上结果表明,本研究成功克隆出解脂耶氏酵母亲环素基因,并能在原核细胞中正确表达,为进一步研究解脂耶氏酵母亲环素的结构和功能奠定了基础。 【英文摘要】Cyclophilins comprise a protein family of highly conservative structure which exist in almost all organisms. As the peptidyl-prolyl cis-trans isomerase (PPIase), cyclophilins can catalyze the cis-trans isomerization of the X-Pro peptide bonds. In the same time, cyclophilins work as molecular chaperones in helping proteins flod, assemble and transport. They are also playing pivotal roles in cyclosporin A-mediated immunosuppression and other events like programmed cell deaths, oxidative stress, cholesterol metabolism. The yeast Yarrowia lipolylica belongs to the ascomycetous yeast. It is an important industrial yeast which can survive wild range of pH conditions and take ordinary and cheap substances like glucose, fatty acids, hydrocarbons as substrates. Since Yarrowia lipolytica is nonpathogenic, it can be used in the food and medicine production. Yarrowia lipolylica secrets large amounts of metabolites like organic acids and proteins, so it was developed into a new yeast expression system. More than one hundred of cyclophilins have been identified, and there are also 13 putative cyclophilins through out the Yarrowia lipolytica genome.In this study, we picked one of the putative Yarrowia lipolytica cy