截短型人骨保护素哺乳类载体的构建、优化及表达

发布时间：2018-08-28 14:47

【摘要】：骨保护素(osteoprotegerin,OPG)是破骨细胞分化因子(receptor activator of nuclear factor-κ B ligand,RANKL)的可溶性“诱骗”受体,能抑制破骨细胞的分化、成熟、活化及存活。它是一种分泌性糖蛋白,含21个信号肽和5个潜在的糖基化位点。成熟人OPG由7个结构域组成,其中N-端有4个半胱氨酸富集结构,也是其生物活性所在部位。最初OPG以单体形式在细胞内生成,再在第400个半胱氨酸(Cys400)的位置以二硫键连成二聚体。目前,虽有原核表达OPG的报道,但仍存在一些不足。例如,在大肠杆菌中表达真核蛋白,不能进行翻译后修饰,诸如不能完成蛋白的糖基化、磷酸化和二硫键的形成等,所以很难制得具备天然活性的蛋白质。真核细胞能够表达具有生物功能的OPG,但存在的普遍问题是表达量不高。近年本室成功构建了OPG的真核表达载体pcDNA3.1/DHFR-OPG,转染CHO-dhfr~-细胞后获得了表达,但表达量不高,不能满足后续实验的需要。本次研究在于进一步优化已建立好的表达系统,并进一步对表达产物进行免疫原性鉴定和生物活性测定。本次研究将通过对目的基因和载体结构进行改进,以达到优化表达的目的。在已有全长型人OPG重组哺乳类表达载体pcDNA3.1/DHFR-OPG的基础上,利用PCR技术扩增截短型人OPGcDNA(含有21个信号肽序列、D1-D4结构区和形成二硫键必需的Cys400),利用限制性内切酶EcoR Ⅰ将其克隆入构建好的带有二氢叶酸还原酶筛选标记的哺乳类表达载体pcDNA3.1/DHFR-GFP的相应克隆位点,构建成pcDNA3.1/DHFR-tOPG。并尝试对载体进行优化,以截短型OPG为模板,运用两次PCR技术,在目的基因的5'端非翻译区添加了一条长35bp的翻译增强序列和一条长37bp的内含子序列,构建了表达载体pcDNA3.1/DHFR-netOPG。按照Lipofectmine~(TM)2000 Reagent说明书将上述已线性化载体分别转入二氢叶酸还原酶(Dihydrofolate Reductase,DHFR)基因缺陷的CHO细胞中,首先在
[Abstract]:Osteoprotegerin (osteoprotegerin, OPG) is a osteoclast differentiation factor (receptor activator of).
Nuclear factor-kappa B ligand (RANKL), a soluble "decoy" receptor, inhibits the differentiation, maturation, activation and survival of osteoclasts. It is a secretory glycoprotein containing 21 signal peptides and 5 potential glycosylation sites. Mature human OPG consists of seven domains, of which the N-terminal has four cysteine-rich structures and is also bioactive. Location. OPG is initially formed as a monomer in the cell and then linked to a dimer at the 400 th cysteine (Cys400) site by disulfide bonding.
For example, the expression of eukaryotic proteins in E. coli can not be modified after translation, such as glycosylation, phosphorylation and disulfide bond formation, so it is difficult to produce naturally active proteins. In recent years, we successfully constructed the eukaryotic expression vector pcDNA3.1/DHFR-OPG of OPG and transfected CHO-dhfr~ - cells to express OPG, but the expression level was not high enough to meet the needs of subsequent experiments. The immunogenicity and biological activity of the substance were determined.
Based on the existing full-length human OPG recombinant mammalian expression vector pcDNA3.1/DHFR-OPG, the truncated human OPG cDNA (containing 21 signal peptide sequences, D1-D4 domain and Cys400 essential for disulfide bond formation) was amplified by PCR. The restriction endonuclease EcoR I was cloned into the corresponding cloning site of the mammalian expression vector pcDNA3.1/DHFR-GFP with dihydrofolate reductase screening marker and constructed into pcDNA3.1/DHFR-tOPG. The expression vector pcDNA3.1/DHFR-netOPG was constructed by adding a 35 BP translation enhancement sequence and a 37 BP intron sequence.
According to the Lipofectmine ~ (TM) 2000 Reagent specification, these linearized vectors were transferred into CHO cells with dihydrofolate reductase (DHFR) gene defect, first in
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R346

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