酵母粉诱导重组人甲状旁腺激素表达研究
发布时间：2018-11-15 08:36
【摘要】：人甲状旁腺激素(Trx-hPTH)是人体内由甲状旁腺主细胞分泌的多肽类激素,具有调节钙磷平衡功能,由84个氨基酸组成,经研究发现它能够促进人体对Ca2+的重吸收和磷酸盐的排出,刺激软骨细胞和成骨细胞分裂,增加骨基质密度,是治疗骨质疏松症的蛋白药物。 本文以大肠杆菌E. coli BL21(DE3)为宿主,pET32a为表达载体,进行人甲状旁腺激素的发酵表达,首先研究了在不同培养基中菌体生长和融合蛋白表达的特性,筛选出TB培养基作为发酵培养基,研究重组菌在摇瓶培养时的最佳发酵条件,选用LB培养基作为活化培养基,TB培养基作为发酵培养基,研究在不同培养条件下菌体浓度和目的蛋白表达量,优化发酵结果如下：用自来水配制培养基,pH为7,37℃下培养,活化培养5.5h后加诱导剂IPTG, IPTG终浓度为0.2mM,诱导3.5h时,Trx-hPTH的胞内蛋白量达到最大。 同时在实验中发现不添加外源诱导剂时,目标蛋白也有表达,这与培养基中的酵母粉有关,本文对酵母粉的诱导规律进行研究,比较了三个品牌的酵母粉的诱导效果,三个品牌分别是BBI, Sangon, OXIOD,研究发现用不同的水源(蒸馏水和自来水)配制培养基,目标蛋白表达结果有差异,Sangon酵母粉在两种水源的培养基中都能表达,BBI在两种培养基中均不能表达,OXIOD酵母粉在蒸馏水的培养基中无明显表达,在自来水配制的培养基中表达显著。OXIOD酵母粉在浓度大于24g/1时能诱导大量目标蛋白表达。对OXIOD酵母粉进行分离,先后使用透析袋,不同的色谱柱进行分离。
[Abstract]:Human parathyroid hormone (Trx-hPTH) is a polypeptide hormone secreted by parathyroid gland main cells. It has the function of regulating calcium and phosphorus balance and is composed of 84 amino acids. It can promote the reabsorption of Ca2 and the excretion of phosphate, stimulate the division of chondrocytes and osteoblasts, and increase the density of bone matrix. It is a protein drug for the treatment of osteoporosis. In this paper, E. coli BL21 (DE3) was used as host and pET32a as expression vector to express human parathyroid hormone. Firstly, the characteristics of cell growth and expression of fusion protein in different media were studied. TB medium was selected as fermentation medium. The optimum fermentation conditions of recombinant bacteria in shaking flask culture were studied. LB medium was used as activation medium and TB medium as fermentation medium. The optimum fermentation results were as follows: prepared with tap water, pH was cultured at 737 鈩，
本文编号：2332774
[Abstract]:Human parathyroid hormone (Trx-hPTH) is a polypeptide hormone secreted by parathyroid gland main cells. It has the function of regulating calcium and phosphorus balance and is composed of 84 amino acids. It can promote the reabsorption of Ca2 and the excretion of phosphate, stimulate the division of chondrocytes and osteoblasts, and increase the density of bone matrix. It is a protein drug for the treatment of osteoporosis. In this paper, E. coli BL21 (DE3) was used as host and pET32a as expression vector to express human parathyroid hormone. Firstly, the characteristics of cell growth and expression of fusion protein in different media were studied. TB medium was selected as fermentation medium. The optimum fermentation conditions of recombinant bacteria in shaking flask culture were studied. LB medium was used as activation medium and TB medium as fermentation medium. The optimum fermentation results were as follows: prepared with tap water, pH was cultured at 737 鈩，
本文编号：2332774
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