【摘要】：人甲狀旁腺激素(Trx-hPTH)是人體內(nèi)由甲狀旁腺主細(xì)胞分泌的多肽類激素,具有調(diào)節(jié)鈣磷平衡功能,由84個(gè)氨基酸組成,經(jīng)研究發(fā)現(xiàn)它能夠促進(jìn)人體對(duì)Ca2+的重吸收和磷酸鹽的排出,刺激軟骨細(xì)胞和成骨細(xì)胞分裂,增加骨基質(zhì)密度,是治療骨質(zhì)疏松癥的蛋白藥物。 本文以大腸桿菌E. coli BL21(DE3)為宿主,pET32a為表達(dá)載體,進(jìn)行人甲狀旁腺激素的發(fā)酵表達(dá),首先研究了在不同培養(yǎng)基中菌體生長(zhǎng)和融合蛋白表達(dá)的特性,篩選出TB培養(yǎng)基作為發(fā)酵培養(yǎng)基,研究重組菌在搖瓶培養(yǎng)時(shí)的最佳發(fā)酵條件,選用LB培養(yǎng)基作為活化培養(yǎng)基,TB培養(yǎng)基作為發(fā)酵培養(yǎng)基,研究在不同培養(yǎng)條件下菌體濃度和目的蛋白表達(dá)量,優(yōu)化發(fā)酵結(jié)果如下：用自來(lái)水配制培養(yǎng)基,pH為7,37℃下培養(yǎng),活化培養(yǎng)5.5h后加誘導(dǎo)劑IPTG, IPTG終濃度為0.2mM,誘導(dǎo)3.5h時(shí),Trx-hPTH的胞內(nèi)蛋白量達(dá)到最大。 同時(shí)在實(shí)驗(yàn)中發(fā)現(xiàn)不添加外源誘導(dǎo)劑時(shí),目標(biāo)蛋白也有表達(dá),這與培養(yǎng)基中的酵母粉有關(guān),本文對(duì)酵母粉的誘導(dǎo)規(guī)律進(jìn)行研究,比較了三個(gè)品牌的酵母粉的誘導(dǎo)效果,三個(gè)品牌分別是BBI, Sangon, OXIOD,研究發(fā)現(xiàn)用不同的水源(蒸餾水和自來(lái)水)配制培養(yǎng)基,目標(biāo)蛋白表達(dá)結(jié)果有差異,Sangon酵母粉在兩種水源的培養(yǎng)基中都能表達(dá),BBI在兩種培養(yǎng)基中均不能表達(dá),OXIOD酵母粉在蒸餾水的培養(yǎng)基中無(wú)明顯表達(dá),在自來(lái)水配制的培養(yǎng)基中表達(dá)顯著。OXIOD酵母粉在濃度大于24g/1時(shí)能誘導(dǎo)大量目標(biāo)蛋白表達(dá)。對(duì)OXIOD酵母粉進(jìn)行分離,先后使用透析袋,不同的色譜柱進(jìn)行分離。
[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，

本文編號(hào)：2332774