论文标题:转基因鱼腥藻7120质粒稳定性研究及rhTNF-α表达的发酵条件优化
Studies on Plasmid Stability of Recombinant Anabaena sp. PCC7120 and Optimization of Fermentation Conditions of rhTNF-α Expression
论文作者
论文导师 郭勇,论文学位 博士,论文专业 发酵工程
论文单位 华南理工大学,点击次数 100,论文页数 126页File Size6154K
2000-06-01论文网 http://www.lw23.com/lunwen_1697987/
recombinant Anabeane sp. PCC7120; rhTNF-α; plasmid stability; photoheterotophic culture; environmental factor
α型肿瘤坏死因子(α-TNF)是巨噬细胞产生的一种细胞因子，因具有抗肿瘤、抗病毒和免疫调节等多种生物学活性及功能，而具有广阔的临床应用前景。由于自然来源有限，重组TNF和其衍生物的研究迅速发展，其中由于蓝藻具有的诸多优点，如不含毒蛋白，本身富含多种保健成分，易培养等，使开发其成为TNF的新型宿主具有很好的商业价值。本研究在前人的基础上，得到已转入hTNF-α基因的鱼腥藻7120，为了产生更好的经济效益，研究了重组质粒的稳定性及其光异养和各种环境因子对其生长及表达的影响，旨在提高表达产量，推进其产业化进程。所得结果如下： 首先探索了鱼腥藻的短期保存方法：保护剂可以增强藻细胞对4℃低温的耐受力，其中4.0％的二甲亚砜加上4.0％的甘油效果最好；接种时，藻液滴加到斜面上均匀铺开利于后一步的保存；向斜面定期补充少许无菌水以避免干燥可保存较长时间。 外源质粒的转入会减慢鱼腥藻7120的生长速率：最佳的生物量收获时间与最大的TNF活性时间不同，大约在第16天，TNF活性达到最大值6.735×10~4U／mg，然后活性开始逐渐下降；添加新霉素对转基因鱼腥藻7120生长会造成一定的影响；建立了野生型鱼腥藻7120的敏感动力学方程，1ug／m(?)新霉素已到致死剂量；外源质粒PDC-TNF具有相当高的分裂稳定性和结构稳定性；在无选择压力新霉素存在下的传代培养过程中，TNF表达量会降低，这种降低是由于PDC-TNF质粒拷贝数减少而产生的，在无新霉素存在下，外源质粒拷贝数会大幅度降低，这是转基因鱼腥藻7120有益于自身生长和生理的一种调节机制。 光异养条件下可以显著提高转基因鱼腥藻7120的生长和TNF的表达。鱼腥藻7120只能吸收利用少数几种碳源，其中葡萄糖和蔗糖是不错的碳源。对于生长，最佳的碳氮源为6g／L葡萄糖、1.5g／L硝酸钠，高压灭菌方式灭菌；对于TNF的表达，最佳的碳氮源为6g／L蔗糖、1.5g／L硝酸钠。 有机氮源中，所试的几种氨基酸，只有精氨酸在光自养条件下可以促进藻细胞的生长，其它的氨基酸对藻细胞的生长有抑制作用；对TNF的表达，所试的几种氨基酸都没有促进作用。自养条件下，蛋白胨不仅可以促进藻细胞的生长，对
Human tumor necrosis factor alpha(hTNF- α ) is a cytokine produced by macrophages. With varied bio-activities and functions like anti-tumor, anti-virus and immunoregulation, hTNF- α has wide and significant clinical application prospects for cancer treatment Because of limited sources from nature, the studies on recombinant TNF and its derivatives have been developing very quickly. Cyanobacteria have many advantages, which make them a very potentially ideal host cell system, e.g. containing abundantly various health ingredients, free from toxic proteins, easy cultivation and commercialization.Former researchers have transferred the hTNF- α gene into Anabaena sp. PCC7120. In order to get better economic benefits, the dissertation focused on recombinant plasmid stability as well as effect of photoheterotrophic culture and environmental factors on growth and TNF expression, with the aim of improving TNF expression yield and advancing its industrialization process. The results are as fellows:Short-term preservation method for Anabaena sp. PCC7120 was investigated, cryoprotective agents could enhance low temperature(4℃) resistance capability of Anabaena sp. PCC7120 cells. Among these agents, 4.0% dimethyl sulfoxide with 4.0% glycerol had the best effect It"s good for the next preservation step by dripping homogeneously the Anabaena sp. PCC7120 solution in slats during inoculation. Adding little aseptic water regularly to slats could prolong preservation time.The growth rate of Anabaena sp. PCC7120 would slow down upon transfer of foreign plasmid. At the growth curve, the time of reaching the maximum TNF activity (6.735 ×10~4U per mg cells) was at around the sixteenth day, which was different from the optimum biomass harvest time. TNF activity decreased gradually afterwards. Adding neomycin would bring certain effect on growth of recombinant Anabaena sp. PCC7120. A sensitive kinetic equation for neomycin in wild type Anabaena sp. PCC7120 was set up, even if 1μg/m(?) was enough to kill the cells of wild type Anabaena sp. PCC7120.The segregative and structural stability of recombinant plasmid PDC-TNF was
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