【作者】 许凌峰；

【导师】 俞炜源；

【作者基本信息】 中国人民解放军军事医学科学院 ， 遗传学， 2005， 博士

【摘要】 CHO-dhfr^-细胞表达体系是重要的药用蛋白表达体系,但其在培养过程中存在着一系列的问题,为了使其具有更适应大规模培养的特性,我们利用Flp定点整合系统,通过在CHO-dhfr^-细胞中稳定整合调控基因对CHO-dhfr^-细胞进行了的改造。首先在CHO-dhfr^-细胞中定点整合并过表达人抗凋亡基因Bc1-2,获得的重组细胞对诱导凋亡因素的耐受能力增强,凋亡水平降低,增加了对无血清和高氨离子浓度不利培养条件。乳酸是细胞的重要代谢产物,其积累可明显影响细胞的生长和对外源蛋白的表达。为了减少细胞内乳酸的积累,选择了四个CHO-dhfr^-细胞LDH-A基因中RNAi位点,设计了可转录产生相应shRNA片段的DNA序列,克隆于表达载体pAV/U6的下游。通过将shRNA表达载体在CHO-dhfr^-细胞中稳定整合使之持续表达针对CHO-dhfr细胞LDH-A基因的shRNA,在建立的四株重组细胞中,筛选到一株具有较低的乳酸脱氢酶的mRNA的细胞株,获得的重组细胞株CHO-shRNA能降低乳酸的产生和在培养基中的积累,使细胞的凋亡水平降低,提高了细胞贴壁培养密度11.4%。我们的第三方面工作是实现对细胞增殖进行调控,因为细胞在培养过程中的过度增殖不利于外源蛋白的产生,当细胞达到最佳密度后,生长的停滞将有利于提高外源蛋白的生产。为了实现对细胞生长调控蛋白P27表达的调控以实现细胞生长的停滞,利用T-REx^TM（四环素诱导）诱导表达系统,通过两步重组来达到建立稳定整合并可诱导表达p27基因的重组细胞株的目的,首先筛选了高表达TetR的重组细胞株,在此基础上,定点整合了受TetO₂调控之下的p27基因,以实现对p27基因的诱导表达和对细胞周期的控制,在p27诱导表达的情况下,有效的抑制细胞周期停滞于G1期,通过对EGFP检测,表明在细胞生长停滞条件下,细胞对外源蛋白的表达水平可提高70%。更多还原

【Abstract】 CHO-dhfr~- cell is the most important host cell for biopharmaceutical protein, however, there are some problems still existing in CHO-dhfr~- cell culturing.In order to make CHO-dhfr~- cell more suitable for culturing on a large scale, we modified the cell by steady integrating the anti-apoptosis gene Bcl-2 in genome of CHO-dhfr~- used the Flp target integrating system.Firstly, human anti-apoptosis gene Bcl-2 was target integrated into CHO-dhfr~- cell and overexpressed. The viability of the reconstructing cell increased to face the inductive apoptosis factors such as culturing in serum-free and in high-concentration ammonia ion.As accumulation of Lactic acid which is a kind of side products can obviously influence the growth of the cell. In order to reduce lactate formation in the cell culture by genetically manipulation of the pathway of lactate synthesis, 4 RNAi targets were selected in the LDH-A gene of CHO-dhfr~-cell. Four DNA sequence to produce corresponding shRNA is designed and cloned in the lower position of the expressing vector pAV/U6. shRNA toward LDH-A gene of CHO-dhfr~- can be expressed continuously by steady integrating expressing vector within genome of CHO-dhfr~- cell. One cell line was screened out from the 4 reconstructing cells lines, the result of analysis showed that the LDH-A mRNA , the accumulation of lactate , cell apoptosis in medium all decreased, the density of cell culturing clinging to walls increased by 11. 4%.The third part of the job was to control the cell proliferation bioprocesses. Because the over proliferation of the cell in culturing process was unfavorable for the heterologous protein production, growth stagnation is更多还原