本文選題：大腸桿菌 + 藥物蛋白　； 參考：《華東理工大學(xué)》2017年碩士論文

【摘要】：大腸桿菌表達系統(tǒng)是目前應(yīng)用最為廣泛的重組蛋白表達系統(tǒng)。由于蛋白表達速度過快、活性二硫鍵來不及正確配對等等原因,外源蛋白在大腸桿菌胞內(nèi)往往會以無活性的包涵體形式表達。由于包涵體復(fù)性折疊機制不明確,把無活性的包涵體復(fù)性為完全的天然活性狀態(tài)是一件很難的事情。較低的復(fù)性效率也仍然是大腸桿菌體系表達的許多外源蛋白造成損失或者難以有效制備的主要原因。本課題以重組人表皮生長因子(hEGF)和新型HIV疫苗蛋白為研究對象,研究在包涵體藥物蛋白的復(fù)性純化過程中如何根據(jù)具體目的和要求,決定復(fù)性與純化的先后順序、選擇復(fù)性方法和純化方法、合理設(shè)計純化方案(合理銜接不同的純化方法)等等,以期為其他包涵體藥物蛋白的復(fù)性純化提供一定的參考價值。非融合表達hEGF蛋白的復(fù)性純化研究:采用的是"先復(fù)性后純化"的策略。經(jīng)過不斷探索,最終確定hEGF蛋白的復(fù)性純化工藝為:包涵體洗滌→包涵體變、復(fù)性→50%硫銨沉淀→凝膠過濾層析→DEAE陰離子交換層析。hEGF純化樣品的SDS-PAGE電泳純度為97%,HPLC液相色譜純度為96%;蛋白純化回收率為18%;且此純化樣品對Balb/c3T3細胞(小鼠胚胎成纖維細胞)具有明顯的生長促進作用。新型的藥物蛋白—HIV輔助T細胞疫苗的復(fù)性純化研究:(1)HIV疫苗包涵體蛋白的變性條件優(yōu)化。包涵體蛋白先用7M鹽酸胍變性緩沖液變性溶解,接著用8M尿素緩沖液對其進行稀釋,使鹽酸胍濃度降至1M。如此變性處理后的包涵體蛋白結(jié)構(gòu)更趨于穩(wěn)定,后期純化及復(fù)性過程中都不會發(fā)生蛋白沉聚現(xiàn)象。(2)HIV疫苗包涵體蛋白的純化與復(fù)性。選擇"先純化后復(fù)性"的策略,研究出一套成熟的復(fù)性純化工藝:包涵體洗滌→包涵體變性→鎳柱親和層析(HisTrapFF)→ 30%硫銨沉淀→CMFF陽離子交換層析→鎳柱親和層析(HisTrap HP)。最后目的蛋白的電泳純度提高到95%。HIV疫苗包涵體蛋白的純化總回收率為13%。純化后的變性狀態(tài)的包涵體蛋白復(fù)性方法選擇透析復(fù)性,復(fù)性過程中沒有產(chǎn)生明顯的蛋白沉淀。HIV疫苗蛋白的成功復(fù)性分離純化為進一步研究HIV輔助T細胞疫苗蛋白的理化性質(zhì)以及生物學(xué)功能提供了保障。
[Abstract]:E. coli expression system is the most widely used recombinant protein expression system. Due to the rapid expression of proteins and the late matching of active disulfide bonds, foreign proteins are often expressed in the form of inactive inclusion bodies in E. coli cells. Because the folding mechanism of inclusion body is not clear, it is difficult to renaturation the inactive inclusion body into a completely natural active state. Low renaturation efficiency is still the main reason for the loss of many foreign proteins expressed in Escherichia coli system or the difficulty of effective preparation. In this paper, recombinant human epidermal growth factor hEGF) and novel HIV vaccine proteins were used to study how to determine the sequence of renaturation and purification according to specific objectives and requirements in the renaturation and purification of inclusion body drug proteins. The methods of renaturation and purification were selected, and the purification schemes were designed reasonably (linking up with different purification methods) so as to provide some reference value for the renaturation and purification of other inclusion body drug proteins. Renaturation and purification of non-fusion expressed hEGF protein: the strategy of renaturation and purification was adopted. After continuous exploration, the final process of renaturation and purification of hEGF protein was as follows: inclusion body washing, inclusion body transformation, DEAE anion exchange chromatography. The purity of SDS-PAGE electrophoresis was 97%. The purity of HPLC was 96. The recovery rate of protein purification was 180.The purified sample was used to purify Balb/c3T3 cells (mouse embryogenesis). Fibroblasts) have obvious growth-promoting effects. Study on renaturation and purification of novel Drug protein-HIV-assisted T Cell Vaccine the denaturation conditions of inclusion body proteins of the 1: 1 HIV vaccine were optimized. The inclusion body protein was first denatured and dissolved with 7m guanidine hydrochloride denaturation buffer, then diluted with 8m urea buffer to reduce the concentration of guanidine hydrochloride to 1M. The structure of inclusion body protein after denaturation is more stable, and no protein deposition will occur during the later purification and renaturation. The purification and renaturation of inclusion body protein of HIV vaccine. By selecting the strategy of "first purification and then renaturation", a set of mature renaturation and purification techniques were developed: inclusion body washing body denatured nickel column affinity chromatography (HisTrapFF) with 30% ammonium sulfide precipitation and CMFF cation exchange chromatography and nickel column affinity chromatography (HisTrap HP). Finally, the purity of the protein was improved to 13% after purification of the inclusion body protein of 95%.HIV vaccine. The method of renaturation of inclusion body protein in purified denatured state was selected for dialysis renaturation. The successful renaturation and purification of HIV vaccine proteins without obvious protein precipitation during renaturation provided a guarantee for further study on the physicochemical properties and biological functions of HIV assisted T cell vaccine proteins.
【學(xué)位授予單位】：華東理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：TQ460.1

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本文編號：1991515