本文選題：表達(dá)載體 + 重組蛋白　； 參考：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2005年碩士論文

【摘要】：利用大腸桿菌生產(chǎn)多肽類藥物具有成本低,周期短且不存在病毒致癌基因污染等優(yōu)點(diǎn),所以大腸桿菌是目前基因工程中細(xì)胞因子和多肽類藥物生產(chǎn)的主要工程菌。但大多外源基因在大腸桿菌中表達(dá)時(shí)往往以包含體形式存在,這就造成復(fù)性的困難和成本的提高。原核生物表達(dá)系統(tǒng)缺乏真核細(xì)胞特有的加工后處理;高度還原環(huán)境大腸桿菌胞質(zhì)使表達(dá)的重組蛋白往往不能形成正確的二硫鍵與完整的四級(jí)結(jié)構(gòu)都使得重組蛋白的活性較低。因此如何獲得高效可溶性并具有類似天然生物活性的重組蛋白是利用大腸桿菌進(jìn)行基因工程技術(shù)的關(guān)鍵。大腸桿菌胞間質(zhì)內(nèi)的Dsb(二硫鍵氧化還原酶)家族使得胞間質(zhì)內(nèi)氧化電勢(shì)較高為形成二硫鍵提供了有利條件。利用Dsb家族和目的蛋白融合表達(dá)具有讓外源蛋白周質(zhì)腔定位并輔助其正確形成二硫鍵和空間結(jié)構(gòu)的功能。 本課題基于以往的研究基礎(chǔ),利用Dsb蛋白家族的功能和特性構(gòu)建了串聯(lián)表達(dá)載體pET-SWG1-SWG2-NGF(神經(jīng)生長因子β亞基)和pET-SWG1-SWG2-C_7+Fc(環(huán)七肽和Fc融合蛋白),并通過優(yōu)化表達(dá)條件實(shí)現(xiàn)了融合蛋白SWG1-SWG2-NGF和SWG1-SWG2-C_7+Fc在大腸桿菌中高效可溶性表達(dá)。伴侶蛋白和目的蛋白之間凝血酶位點(diǎn)酶切和再次分離純化后,獲得了具有較高生物活性的目的蛋白NGFβ亞基和C_7+Fc。 通過本課題研究,構(gòu)建了高效可溶性表達(dá)載體,實(shí)現(xiàn)NGFβ亞基和C_7+Fc在原核高效可溶表達(dá)并具有良好生物活性。這為實(shí)現(xiàn)其它細(xì)胞因子或多肽類藥物利用原核表達(dá)提供了模式。也為探討原核生物的表達(dá)機(jī)制提供參考。
[Abstract]:The use of Escherichia coli to produce polypeptide drugs has the advantages of low cost, short period and no contamination of viral oncogenes, so Escherichia coli is the main engineering bacteria in the production of cytokines and polypeptide drugs in gene engineering. However, most of the foreign genes expressed in E. coli often exist in the form of inclusions, which leads to the difficulty of renaturation and the increase of cost. The expression system of prokaryotes lacks the unique post-processing of eukaryotic cells, and the cytoplasm of highly reduced Escherichia coli makes the expressed recombinant proteins often unable to form the correct disulfide bond and complete quaternary structure, which results in the low activity of the recombinant proteins. Therefore, how to obtain highly soluble recombinant protein with similar natural biological activity is the key to the genetic engineering of Escherichia coli. The Dsb (disulfide redox reductase) family in the interstitial of Escherichia coli provides favorable conditions for the formation of disulfide bonds. The fusion expression of DSB family and target protein has the function of localizing and assisting the formation of disulfide bonds and spatial structures of exogenous proteins. This subject is based on the previous research. A series expression vector pET-SWG1-SWG2-NGF (nerve growth factor 尾 subunit) and pET-SWG1-SWG2-C7Fc (cyclic heptapeptide and FC fusion protein) were constructed by using the function and characteristics of DSB protein family. The fusion proteins SWG1-SWG2-NGF and SWG1-SWG2-C7Fc were obtained by optimizing expression conditions. High efficient and soluble expression in bacteria. The target protein NGF 尾 subunit and C7Fc with high biological activity were obtained after digestion and purification of the thrombin site between chaperone protein and target protein. Through this study, a high efficient soluble expression vector was constructed to realize the expression of NGF 尾 subunit and C7Fc in prokaryotic cells, and the expression of NGF 尾 subunit and C7Fc in prokaryotic cells had good biological activity. This provides a model for the prokaryotic expression of other cytokines or polypeptide drugs. It also provides a reference for exploring the expression mechanism of prokaryotes.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類號(hào)】：Q782

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 丁鳴,余建法,丁仁瑞;融合表達(dá)載體的研究進(jìn)展[J];生物技術(shù);1998年04期

2 楊U，

本文編號(hào)：2023205