本文關(guān)鍵詞： 鹽析萃取 血漿蛋白 重組人胰島素 離子交換層析 疏水層析　出處：《大連理工大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：IgG和白蛋白是血漿中主要的功能蛋白質(zhì),臨床上用于抗病毒、治療燒傷和失血過多引起的血流動(dòng)力學(xué)疾病等。胰島素是體內(nèi)唯一一個(gè)降低血糖的激素,臨床上多用來治療糖尿病。當(dāng)前,工業(yè)上蛋白質(zhì)的分離純化主要采用有機(jī)溶劑沉淀、多步離心等初分離的方法以及柱層析的方法進(jìn)行分離純化,這些方法存在著操作繁瑣、周期長(zhǎng)、蛋白的回收率低和易失活等問題,本論文開展了親水性有機(jī)溶劑鹽析萃取偶聯(lián)柱層析分離純化血漿中IgG和白蛋白的研究和鹽析萃取分離純化重組人胰島素的研究。首先,在前人的結(jié)果基礎(chǔ)上,驗(yàn)證了500g體系中17.5%乙醇/20% K2HPO4體系和14%乙醇/20% K2HPO4鹽析萃取分離效果,發(fā)現(xiàn)14%乙醇和20% K2HPO4體系放大穩(wěn)定,在pH7.0下,白蛋白和IgG分布在上相且得率分別為90%和65%。17.5%乙醇/20% K2HPO4體系則易使IgG沉淀。對(duì)14%乙醇/20% K2HPO4體系的萃取相,進(jìn)行了疏水層析和離子交換層析條件的優(yōu)化,在優(yōu)化的條件下,可以得到電泳純的IgG,但白蛋白的純度較低。其次考察了血漿蛋白在叔丁醇/鹽體系中的分配情況。結(jié)果表明,在15%叔丁醇/14% K2HPO4,pH 7.0和添加7.5% NaCl的條件下,血漿中的白蛋白主要分布在下相,收率為98%,而IgG以沉淀的方式分布在上下相界面中間,收率為77%,有利于兩種蛋白的分離。將叔丁醇／K2HPO4體系的的萃取相和沉淀相,使用優(yōu)化的疏水層析和離子交換層析進(jìn)行純化,可以得到電泳純度為100%的IgG和純度為87%的白蛋白,該方法操作簡(jiǎn)便、收率高,具有較好的應(yīng)用前景。最后研究了重組人胰島素在鹽析萃取體系中的分配情況,并通過對(duì)鹽析萃取體系的初篩及條件的優(yōu)化,篩選出了最優(yōu)的鹽析萃取體系。在18%乙醇/22% K2HPO4, pH9.0條件下,重組人胰島素分布在上相,回收率在98%以上。
[Abstract]:IgG and albumin are the main functional proteins in plasma, which are used clinically to treat hemodynamic diseases caused by burn and blood loss, etc. Insulin is the only hormone that lowers blood sugar. At present, the separation and purification of proteins in industry are mainly carried out by organic solvent precipitation, multi-step centrifugation and column chromatography. These methods have some problems, such as complicated operation, long period, low protein recovery and easy inactivation. In this paper, the separation and purification of IgG and albumin in plasma by hydrophilic organic solvent salt-out extraction coupled column chromatography and salt-out extraction and purification of recombinant human insulin were carried out. Based on the previous results, the extraction and separation effects of 17.5% ethanol / 20% K _ 2HPO _ 4 and 14% ethanol / 20% K _ 2HPO _ 4 were verified. It was found that the system of 14% ethanol and 20% K2HPO4 was stable and amplified under pH7.0. Albumin and IgG were distributed in the upper phase and the yields were 90% and 65.17.5% ethanol / 20% K2HPO4 system and IgG precipitated easily. For 14% ethanol / 20%. Extraction phase of K2HPO4 system. The conditions of hydrophobic chromatography and ion exchange chromatography were optimized. Under the optimized conditions, pure IgG could be obtained. However, the purity of albumin was low. Secondly, the distribution of plasma protein in tert-butanol / salt system was investigated. The results showed that 15% tert butanol / 14% K2HPO4 was obtained. Under the conditions of pH 7.0 and 7.5% NaCl, the albumin in plasma was mainly distributed in the lower phase, and the yield was 98. The IgG was distributed in the middle of the upper and lower phase interface by precipitation. The extraction and precipitation phases of tert-butanol / K2HPO4 system were purified by optimized hydrophobic chromatography and ion exchange chromatography. IgG with a purity of 100% and albumin with a purity of 87% can be obtained. The method is easy to operate and has a high yield. Finally, the distribution of recombinant human insulin in salting-out extraction system was studied, and the initial screening and conditions of salt-out extraction system were optimized. Under the conditions of 18% ethanol / 22% K2HPO4 and pH9.0, the recombinant human insulin was distributed in the upper phase and the recovery rate was over 98%.
【學(xué)位授予單位】：大連理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R446.6

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 趙媛媛;呂茂民;楊梅;馮晶晶;章金剛;;人內(nèi)皮抑素-白蛋白融合蛋白在畢赤酵母中的表達(dá)與鑒定[J];中國(guó)輸血雜志;2010年04期

2 裴曉言;顧名夏;黃鶴;徐蘭;周博達(dá);吳栗洋;康佳蕊;王寧;王月丹;;牛乳中的免疫球蛋白與健康的研究[J];現(xiàn)代生物醫(yī)學(xué)進(jìn)展;2007年03期

3 邱家山;黃曉軍;;人血漿蛋白連續(xù)沉淀分離技術(shù)試驗(yàn)[J];生物技術(shù)通訊;2006年05期

相關(guān)碩士學(xué)位論文 前1條

1 李玲玲;鹽析萃取偶聯(lián)柱層析分離純化血漿蛋白[D];大連理工大學(xué);2013年

，

本文編號(hào)：1447725