甲状旁腺素相关蛋白1-141和1-86的表达与生物活性测定
发布时间：2018-11-09 21:13
【摘要】：甲状旁腺素相关蛋白(parathyroid hormone-related protein,PTHrP)最初作为一种引起恶性肿瘤患者发生高钙血症的蛋白被发现。随着对其研究的深入,证实其在大多数组织器官都有所表达,提示其在正常细胞生长分化中的重要性。PTHrP主要通过自分泌、旁分泌和胞内分泌的作用方式发挥作用。PTHrP被蛋白酶作用后,产生一组具有不同生物活性的多肽,它的作用靶组织与靶器官涉及非常广泛。PTHrP的这些生理功能,预示着PTHrP未来在临床上广泛的应用前景。 本实验采用TKM法自人血细胞中提取基因组DNA,并以之为模板PCR扩增hPTHrP1-141编码基因。将该基因克隆入pGEM-T easy载体,并进行DNA序列分析。在此基础上,将编码hPTHrP1-141和hPTHrP1-86的基因片段分别克隆到表达载体pQE-30Xa上,并转化入E. coli M15[pREP4],进行融合表达。表达条件经优化后,rhPTHrP1-141和rhPTHrP1-86的表达量分别达到60mg/L培养基和4mg/L培养基。重组蛋白rhPTHrP1-141和rhPTHrP1-86分别占菌体可溶性蛋白的34.64%和6.38%。目的蛋白含6×His纯化标签,可以经金属螯合亲和层析吸附于Ni-NTA树脂层析柱,梯度增加冲洗液中咪唑浓度以除去非特异吸附的杂蛋白后,将目的蛋白洗脱,超滤离心法浓缩。体外培养大鼠骨肉瘤细胞系UMR 106,以rhPTH1-34标准品作为阳性对照,将PTHrP1-141和PTHrP1-86加入细胞培养液,使其终浓度分别为10~(10)mol/L、10~(-9)mol/L、10~(-8)mol/L、10~(-7)mol/L、10~(-6)mol/L和10~(-5)mol/L。PTHrP能与存在于UMR 106细胞膜上的与G蛋白相偶连的PTH1型受体(PTH1R)结合,激活
[Abstract]:Parathyroid hormone-associated protein (parathyroid hormone-related protein,PTHrP) was originally identified as a protein that causes hypercalcemia in patients with malignant tumors. With the further study of PTHrP, it has been confirmed that it is expressed in most tissues and organs, suggesting its importance in normal cell growth and differentiation. PTHrP is mainly expressed through autocrine. Paracrine and cytosolic endocrine function. PTHrP is treated by protease to produce a group of polypeptides with different biological activities. Its target tissues and target organs are involved in a wide range of physiological functions of PTHrP. It indicates that PTHrP will be widely used in clinic in the future. In this study, genomic DNA, was extracted from human blood cells by TKM method and used as template PCR to amplify hPTHrP1-141 coding gene. The gene was cloned into pGEM-T easy vector and sequenced by DNA. On this basis, the gene fragments encoding hPTHrP1-141 and hPTHrP1-86 were cloned into the expression vector pQE-30Xa, and transformed into E. coli M15 [pREP4] for fusion expression. After optimized expression conditions, the expression of rhPTHrP1-141 and rhPTHrP1-86 reached 60mg/L medium and 4mg/L medium respectively. The recombinant protein rhPTHrP1-141 and rhPTHrP1-86 accounted for 34.64% and 6.38% of the soluble protein, respectively. Objective the protein contains 6 脳 His purified label and can be adsorbed on Ni-NTA resin column by metal chelating affinity chromatography. The target protein is eluted and concentrated by ultrafiltration centrifugation by gradient increasing the concentration of imidazole in the flushing solution to remove the non-specific adsorbed impurity. Rat osteosarcoma cell line UMR 106 was cultured in vitro. PTHrP1-141 and PTHrP1-86 were added into cell culture medium with rhPTH1-34 standard as positive control. The final concentration of PTHrP1-141 and PTHrP1-86 were 10 ~ (10) mol/L,10~ (-9) mol/L, respectively. 10 ~ (-8) mol/L,10~ (-7) mol/L,10~ (-6) mol/L and 10 ~ (-5) mol/L.PTHrP can bind and activate PTH1 type receptor (PTH1R) which is coupled to G protein on UMR 106 cell membrane.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：Q51
本文编号：2321577
[Abstract]:Parathyroid hormone-associated protein (parathyroid hormone-related protein,PTHrP) was originally identified as a protein that causes hypercalcemia in patients with malignant tumors. With the further study of PTHrP, it has been confirmed that it is expressed in most tissues and organs, suggesting its importance in normal cell growth and differentiation. PTHrP is mainly expressed through autocrine. Paracrine and cytosolic endocrine function. PTHrP is treated by protease to produce a group of polypeptides with different biological activities. Its target tissues and target organs are involved in a wide range of physiological functions of PTHrP. It indicates that PTHrP will be widely used in clinic in the future. In this study, genomic DNA, was extracted from human blood cells by TKM method and used as template PCR to amplify hPTHrP1-141 coding gene. The gene was cloned into pGEM-T easy vector and sequenced by DNA. On this basis, the gene fragments encoding hPTHrP1-141 and hPTHrP1-86 were cloned into the expression vector pQE-30Xa, and transformed into E. coli M15 [pREP4] for fusion expression. After optimized expression conditions, the expression of rhPTHrP1-141 and rhPTHrP1-86 reached 60mg/L medium and 4mg/L medium respectively. The recombinant protein rhPTHrP1-141 and rhPTHrP1-86 accounted for 34.64% and 6.38% of the soluble protein, respectively. Objective the protein contains 6 脳 His purified label and can be adsorbed on Ni-NTA resin column by metal chelating affinity chromatography. The target protein is eluted and concentrated by ultrafiltration centrifugation by gradient increasing the concentration of imidazole in the flushing solution to remove the non-specific adsorbed impurity. Rat osteosarcoma cell line UMR 106 was cultured in vitro. PTHrP1-141 and PTHrP1-86 were added into cell culture medium with rhPTH1-34 standard as positive control. The final concentration of PTHrP1-141 and PTHrP1-86 were 10 ~ (10) mol/L,10~ (-9) mol/L, respectively. 10 ~ (-8) mol/L,10~ (-7) mol/L,10~ (-6) mol/L and 10 ~ (-5) mol/L.PTHrP can bind and activate PTH1 type receptor (PTH1R) which is coupled to G protein on UMR 106 cell membrane.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：Q51
【引证文献】
相关硕士学位论文 前4条
1 张明新;HLA-B寡核苷酸分型芯片制备方法的建立及初步应用[D];天津医科大学;2006年
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3 张瑞;HLA寡核苷酸分型芯片的制备及其初步应用[D];天津医科大学;2007年
4 王春雨;大鼠DJ-1基因的克隆和表达[D];天津医科大学;2008年
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