本文選題：CHO細(xì)胞　切入點(diǎn)：TRAIL-Fc融合蛋白　出處：《重慶理工大學(xué)》2011年碩士論文

【摘要】：腫瘤壞死因子相關(guān)凋亡誘導(dǎo)配體(tumor necrosis factor related apoptosis inducing ligand,TRAIL)是由Wiley于1995年首次克隆并鑒定的一個能夠誘導(dǎo)腫瘤細(xì)胞凋亡的TNF家族新成員。TRAIL對正常細(xì)胞無毒害作用,能夠選擇性殺傷腫瘤細(xì)胞,因此成為腫瘤治療研究的熱點(diǎn),也有望成為一種新型的抗腫瘤藥物。 目的: 本論文通過高密度培養(yǎng)CHO工程細(xì)胞株獲得重組TRAIL-Fc融合蛋白,建立TRAIL-Fc純化中試工藝并對其理化性質(zhì)和生物學(xué)活性進(jìn)行研究,為TRAIL-Fc最終應(yīng)用于臨床進(jìn)行腫瘤治療奠定實(shí)驗(yàn)基礎(chǔ)。 方法: (1) CHO工程菌無血清馴化:通過逐步降低血清和直接降血清兩種馴化方法獲得可在無血清培養(yǎng)基中穩(wěn)定生長的細(xì)胞株。為提高細(xì)胞株生長密度和活率對無血清培養(yǎng)基進(jìn)行添加物優(yōu)化。 (2) 5L反應(yīng)器培養(yǎng):采用分批補(bǔ)料式培養(yǎng),研究溫度、pH、溶氧控制及細(xì)胞生長代謝情況,探索CHO-TFc在5L反應(yīng)器中的發(fā)酵工藝。TRAIL-Fc融合蛋白純化工藝:利用Protein A親和層析柱、CM離子交換層析柱純化目的蛋白,并對目的蛋白進(jìn)行理化性質(zhì)研究。 (3) TRAIL-Fc融合蛋白生物學(xué)活性:采用MTT法檢測TRAIL-Fc融合蛋白體外對多株腫瘤細(xì)胞的生長抑制作用。建立H22小鼠肝癌模型,檢測TRAIL-Fc融合蛋白對體內(nèi)腫瘤細(xì)胞生長抑制作用。 結(jié)果: (1)最終確定以逐步降血清法對CHO-TFc細(xì)胞株進(jìn)行懸浮馴化,獲得能在無血清培養(yǎng)基中穩(wěn)定懸浮生長的細(xì)胞株。 (2)對無血清培養(yǎng)基進(jìn)行優(yōu)化,選擇在無血清培養(yǎng)基中添加20ng/mlIGF-1。 (3) 5L反應(yīng)器發(fā)酵工藝研究,以葡萄糖濃度及細(xì)胞密度為參數(shù)來確定補(bǔ)料策略。細(xì)胞以2.0×10~6cell/ml的密度接種,生長期葡萄糖濃度控制為2g/L,當(dāng)細(xì)胞密度達(dá)到6-8×10~6cell/ml時,降溫至32℃進(jìn)行表達(dá),控制殘?zhí)橇繛?.5g/L,最終發(fā)酵表達(dá)量為80-100mg/L。 (4)建立穩(wěn)定的純化工藝,純度可達(dá)90%以上。質(zhì)譜及Western blotting結(jié)果顯示,TRAIL-Fc融合蛋白結(jié)構(gòu)正確。體內(nèi)外活性測定結(jié)果顯示,TRAIL-Fc融合蛋白具有較強(qiáng)的腫瘤生長抑制的作用,且其在體內(nèi)的半衰期明顯延長。 結(jié)論: 成功完成CHO-TFc工程菌株懸浮馴化,建立5L發(fā)酵及純化工藝,獲得高純度的TRAIL-Fc融合蛋白,并對制備的目的蛋白進(jìn)行了體內(nèi)外活性檢測,證實(shí)了其潛在的腫瘤治療價值。
[Abstract]:Tumor necrosis factor related apoptosis inducing trail, a novel member of the TNF family, which was first cloned and identified by Wiley in 1995, has no toxic effect on normal cells.It can selectively kill tumor cells, so it has become a hot spot in tumor therapy, and it is also expected to be a new anti-tumor drug.Objective:In this paper, the recombinant TRAIL-Fc fusion protein was obtained by high density culture of CHO engineering cell lines, and a pilot process of TRAIL-Fc purification was established, and its physicochemical properties and biological activities were studied, which laid the experimental foundation for the final application of TRAIL-Fc in clinical tumor therapy.Methods:(1) serum-free acclimation of CHO engineering bacteria: two acclimation methods: gradually reducing serum and direct lowering serum were used to obtain stable cell lines which could grow in serum-free medium.In order to improve cell growth density and viability, the serum-free medium was optimized.(2) 5L reactor culture: batch feeding culture was used to study temperature and pH, dissolved oxygen control and cell growth and metabolism.To explore the fermentation process of CHO-TFc in 5L reactor. The purification process of TRAIL-Fc fusion protein. The aim protein was purified by Protein A affinity chromatography column CM ion exchange chromatography, and the physicochemical properties of the target protein were studied.Biological activity of TRAIL-Fc fusion protein: MTT assay was used to detect the inhibitory effect of TRAIL-Fc fusion protein on the growth of many tumor cells in vitro.The H 22 mouse hepatoma model was established to detect the inhibitory effect of TRAIL-Fc fusion protein on the growth of tumor cells in vivo.Results:Finally, the suspension acclimation of CHO-TFc cell line was carried out by the method of decreasing serum gradually, and the cell line which could grow stably in serum-free medium was obtained.The serum-free medium was optimized by adding 20ng / ml IGF-1 to the serum-free medium.The fermentation technology of 5 L reactor was studied. The feeding strategy was determined by the parameters of glucose concentration and cell density.The cells were inoculated with 2.0 脳 10~6cell/ml density and glucose concentration was controlled to 2 g / L in the growing period. When the cell density reached 6-8 脳 10~6cell/ml, the cells were expressed at 32 鈩，

本文編號：1721695