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ScienceNews 2 0 0 3年 16 3卷 7期 10 9页报道 :已知人类肾脏中制造的天然蛋白红细胞生成素 (EPO)可用作调控人体内红细胞生成的激素。但目前大部分EPO是利用基因工程技术生产的。近据美国加利福尼亚州南圣弗郎西斯科市GryphonTherapeutis公司的科学家GerdKochendoerfer宣称 : 相似文献
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用无血清培养基在填充床生物反应生产rHuEPO 总被引:2,自引:0,他引:2
在填充床生物反应器用含5%FBS的DMEM:F12培养基培养产重组人促红细胞生成素(rHuEPO)的细胞C28 ̄10d后,使用自制的无血清生产培养基(SFM-p)生产rHuEPO。SFM-p培养基既能维持细胞生长,又能生产EPO,也便于纯化分离rHuEPO。使用填充床生物反应器培养细胞,能维持培养20 ̄25d,rHuEPO表达水平达12 ̄28.4mg/L之间,反应器的产率达到71.0mg/L/d, 相似文献
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利用COS7细胞暂时表达系统,研究转译起始序列对EPO-cDNA表达的影响。通过DNA重组技术,构建了原EPO-cDNA表达载体pCSV-EPO(1),其转译起始序列为5'AATTCATGG3'。同时通过定点突变技术,将起始序列改变成5'CCACCATGG3',而构建了另一表达载体PCSV-EPO(2)。后经序列分析证明无误后和前均通过DEAE-dextran法转染COS7细胞上清,测定结果为 相似文献
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用EPO基因组基因构建了腺病毒质粒型载体psp1B/hEPO,该质粒含有以RSV-LTR为启动子的完整的EPO基因表达盒.单独转染CHO细胞,经暂态表达检测到EPO的表达。用psp1B/hEPO与腺病毒拯救型载体pBHG11共转染293细胞,获得了表达EPO的重组腺病毒AdhEPO.经Southern杂交证实AdhEPO中有EPO表达盒,ELISA检测到了EPO阳性表达.用5×108pfu的AdhEPO给大鼠作一次性肌肉注射,观察到了其促进大鼠红细胞生成的短期效应。在注射后第1,3,5,7,10d分别检测了大鼠的红细胞压积、血红蛋白含量和红细胞计数等指标,发现大鼠的红细胞数量显著提高。在第10d红细胞压积从46±4%上升至65±6%。证实了重组腺病毒AdhEPO具有潜在的临床应用价值,可用于贫血症的基因治疗。 相似文献
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本实验从蛋鸡输卵管基因组DNA中扩增出约1.3kb的卵清蛋白5'端调控区(OV),并从质粒扩增出人促红细胞生成素(hEPO)基因组DNA。将hEPO亚克隆入pEGFP-C1载体的多克隆位点,命名为pEGFP-hEPO,然后将OV片段亚克隆入经Ase I和Vsp I双酶切的切口处,替换掉CMV启动子,经测序和酶切鉴定正确,成功构建了鸡卵清蛋白5'端调控区调控人促红细胞生成素的共表达载体pOV-GFP-hEPO。并利用脂质体转染法转染鸡输卵管上皮细胞,用荧光倒置显微镜观测绿色荧光蛋白的表达,结果显示共表达载体pOV-GFP-hEPO能够在鸡输卵管上皮细胞定位表达。为制备生产人促红细胞生成素的鸡输卵管生物反应器奠定了基础。 相似文献
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丁酸钠对CHO-EPO工程细胞株rhEPO表达量的影响 总被引:1,自引:0,他引:1
以稳定整合有pEDEPO的CHOEPO工程细胞株为研究对象,在无血清条件下,系统观察了05、10、25和50mmol/L4个浓度的丁酸钠作用于该细胞株的情况,结果表明:丁酸钠对CHOEPO工程细胞的生长有明显的抑制作用;影响CHOEPO工程细胞EPO表达,浓度10mmol/L可提高EPO表达量25倍左右,并可持续较长的一段时间;延缓CHOEPO工程细胞在无血清培养时的细胞脱落;提高CHOEPO工程细胞EPOmRNA水平 相似文献
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利用COS7细胞暂时表达系统,研究转译起始序列对EPO-cDNA表达的影响。通过DNA重组技术,构建了原EP0-cDNA表达载体pCSV-EP0(1).其转译起始序列为5’AATTCATGG3’.同时通过定点突变技术,将起始序列改变成5’CCACCATGG3’,而构建了另一表达载体pCSv—EPO(2)。后者经序列分析证明无误后和前者均通过DEAE-dextran法转染COS7细胞,用ELlSA法定量洲量EP0表达水平.转染后48小时及72小时收集含pCSV—EP0(1)的COS7细胞上清.测定结果为1580pg/mI及954pg/mI,而含pCSV—EPO(2)的上清则为25 300pg/m1和17 450pg/ml。这表明经优化起始序列的基因表达远高于未经优化的。 相似文献
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用无血清培养基在填充床生物反应器生产 rHuEPO 总被引:2,自引:0,他引:2
在填充床生物反应器用含5%FBS的DMEM:F12培养基培养产重组人促红细胞生成素(rHuEPO)的细胞C28~10d后,使用自制的无血清生产培养基(SFMp)生产rHuEPO。SFMp培养基既能维持细胞生长,又能生产EPO,也便于纯化分离rHuEPO。使用填充床生物反应器培养细胞,能维持培养20~25d,rHuEPO表达水平达12~28.4mg/L之间,反应器的产率达到71.0mg/L/d,比滚瓶的产率增加12~14倍。葡萄糖最高消耗量达到21g/L/d,细胞培养密度最高达到3.0×107/ml以上,每次可收无血清培养上清80~87L。由于细胞被固定在聚酯片上,培养上清中脱落细胞很少。观察了反应器的乳酸和氨的含量,其结果表明乳酸和氨含量分别低于3.5g/L和5mmol/L,不影响产物的表达。经过多批培养和生长rHuEPO的结果表明,自行配制的SFMp培养基在该反应器能有效地维持细胞生长和生产rHuEPO。 相似文献
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通过高表达Igf1/Bcl-2或Bcl-2/Cyclin E基因组合使CHO细胞适于在无蛋白培养基中抗凋亡培养 总被引:3,自引:0,他引:3
哺乳动物细胞表达系统是生产重组蛋白药物最常用的表达系统。但在无蛋白培养基中,哺乳动物细胞生长活力差,且容易发生细胞凋亡,因而难以大规模培养。为解决此问题,应用双顺反子表达载体在CHO-dhfr^-细胞中同时表达Igf-1/Bcl-2或Bcl-2/CyclinE基因组合,通过Bcl-2使细胞获得抗凋亡能力;通过1gf-1或CyclinE促进细胞生长分裂,使细胞获得在无蛋白培养基中生长的能力。以上述基因组合转染CHO-dhfr^-细胞,应用Western blot从G418抗性克隆中分别筛选到Bcl-2高表达克隆若干个,对其中表达Bcl-2最高的CHO-IB3和CHO-Bcl做进一步Western blot和流式细胞分析,确认此两个细胞株分别高表达Igf-1/Bcl-2和Bcl-2/CyclinE基因组合。分别通过撤去血清和加入放线菌素D诱导细胞凋亡,并以流式细胞术和DNA Ladder法检测细胞凋亡,证明CHO-IB3和CHO一BCl均具有较强的抗细胞凋亡能力。MTT法证明两个细胞株在不含血清的IMDM培养基中的增殖活力显著高于CHO-dhfr^-对照细胞。在细胞培养瓶中的连续培养实验表明,CHO-IB3和CHO-BCl在本实验室设计的IMEM无蛋白培养基中的生长速度和活细胞数显著高于CHO-dhfr^-对照细胞。提示此两个细胞系能够在无血清培养基中抗凋亡高活力生长,适于作为生物工程宿主细胞。 相似文献
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体外培养和冷冻保存对微囊化细胞生长和内皮抑素表达的影响 总被引:1,自引:0,他引:1
微囊化基因工程细胞移植治疗肿瘤是一种新兴的肿瘤治疗方法,如果将此技术应用到临床研究,就需要制备大量的细胞活性良好、重组蛋白表达量高的生物微胶囊。体外培养和冷冻保存是生物微胶囊制备过程中两个重要的环节,因此需要考察体外培养和冷冻保存对微囊化重组基因细胞生长和蛋白表达的影响。以重组CHO细胞为模型,考察了体外培养时间和冷冻保存对微囊化细胞在动物体内生长和内皮抑素表达的影响及体外培养时间对微囊化细胞冷冻保存的影响。结果表明:体外培养时间对微囊化细胞在动物体内生长、内皮抑素表达和微囊稳定性具有较大的影响,体外不培养和培养4d的微囊化细胞在小鼠腹腔内生长良好、内皮抑素表达量高,并且微囊稳定性好,而体外培养8d的微囊化细胞在移植后的第26天破裂。体外培养时间对微囊化细胞冷冻保存也具有较大的影响,体外培养4d和8d的微囊化细胞在液氮中冷冻保存40d,复苏后细胞生长良好、内皮抑素表达量高,而冻存前未经过体外培养的微囊化细胞,复苏后细胞几乎全部死亡。综上所述,生物微胶囊在体外比较适宜的培养时间为4d。并且冷冻保存对微囊化细胞在动物体内生长、内皮抑素表达和微囊稳定性没有显著的影响。 相似文献
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葡萄糖对重组CHO细胞生长代谢及EPO表达的影响 总被引:3,自引:0,他引:3
在CHO细胞批培养中 ,葡萄糖浓度从8.9增加到49.6mmol/L ,最大活细胞密度没有明显的差异 ,乳酸对葡萄糖的得率系数首先随着葡萄糖浓度的增加而增加 ,葡萄糖浓度达到 17.9mmol/L后 ,乳酸对葡萄糖的得率系数基本上维持恒定。在本实验中 ,葡萄糖浓度对谷氨酰胺代谢没有明显的影响。EPO的累积浓度首先随着起始葡萄糖浓度的增加 ( 8.9~ 17.9mmol L)而增加 ,进而又随着葡萄糖浓度的增加 (17.9~ 49.6mmol L)而下降 ,表明存在一最适浓度 ,在此浓度下重组CHO细胞的EPO表达最大。 相似文献
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目的:通过对贴壁培养CHO细胞筛选驯化,得到高表达的细胞后进行悬浮培养生产重组人促红细胞生成素(rHuEPO)。方法:利用96孔板和24孔板对CHO细胞进行筛选,得到高表达细胞株后进行驯化,使其适合悬浮培养,经过摇瓶扩增后接种到生物反应器中无血清培养,每天监测葡萄糖含量,测rHuEPO表达量。结果:悬浮培养CHO细胞生产rHuEPO,生产周期短,表达量比贴壁培养高出很多,操作方便,减少污染,易于放大,并建立了适合悬浮培养的CHO细胞株,为工业化悬浮培养CHO细胞生产rHuEPO提供了技术基础。结论:经过工艺优化后利用无血清悬浮培养生产促红细胞生成素平均表达量较贴壁培养高,生产周期短,有利于降低生产成本。 相似文献
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François LeFloch Bertrand Tessier Sébastien Chenuet Jean-Marc Guillaume Pierre Cans Jean-Louis Goergen Annie Marc 《Cytotechnology》2006,52(1):39-53
The necessity to perform serum-free cultures to produce recombinant glycoproteins generally requires an adaptation procedure
of the cell line to new environmental conditions, which may therefore induce quantitative and qualitative effects on the product,
particularly on its glycosylation. In previous studies, desialylation of EPO produced by CHO cells was shown to be dependent
on the presence of serum in the medium. In this paper, to discriminate between the effects of the adaptation procedure to
serum-free medium and the effects of the absence of serum on EPO production and glycosylation, adapted and non-adapted CHO
cells were grown in serum-free and serum-containing media. The main kinetics of CHO cells were determined over batch processes
as well as the glycosylation patterns of produced EPO by HPCE-LIF. A reversible decrease in EPO production was observed when
cells were adapted to SFX-CHOTM medium, as the same cells partially recovered their production capacity when cultivated in serum-containing medium or in
the enriched SFMTM serum-free medium. More interestingly, EPO desialylation that was not observed in both serum-free media was restored if the
serum-independent cells were recultured in presence of serum. In the same way, while the serum-independent cells did not release
a sialidase activity in both serum-free media, a significant activity was recovered when serum was added. In fact, the cell
adaptation process to serum-free conditions did not specifically affect the sialidase release and the cellular mechanism of
protein desialylation, which appeared to be mainly related to the presence of serum for both adapted and non-adapted cells. 相似文献
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To investigate the effect of culture pH in the range of 6.85-7.80 on cell growth and erythropoietin (EPO) production at 32.5 and 37.0 degrees C, serum-free suspension cultures of recombinant CHO cells (rCHO) were performed in a bioreactor with pH control. Lowering culture temperature from 37.0 to 32.5 degrees C suppressed cell growth, but cell viability remained high for a longer culture period. Regardless of culture temperature, the highest specific growth rate (mu) and maximum viable cell concentration were obtained at pH values of 7.00 and 7.20, respectively. Like mu, the specific consumption rates of glucose and glutamine decreased at 32.5 degrees C compared to 37.0 degrees C. In addition, they increased with increasing culture pH. Culture pH at 32.5 degrees C affected specific EPO productivity (q(EPO)) in a different fashion from that at 37 degrees C. At 37 degrees C, the q(EPO) was fairly constant in the pH range of 6.85-7.80, while at 32.5 degrees C, the q(EPO) was significantly influenced by culture pH. The highest q(EPO) was obtained at pH 7.00 and 32.5 degrees C, and its value was approximately 1.5-fold higher than that at pH 7.00 and 37.0 degrees C. The proportion of acidic EPO isoforms, which is a critical factor for high in vivo biological activity of EPO, was highest in the stationary phase of growth, regardless of culture temperature and pH. Although cell viability rapidly decreased in death phase at both 32.5 and 37.0 degrees C, the significant degradation of produced EPO, probably by the action of proteases released from lysed cells, was observed only at 37.0 degrees C. Taken together, through the optimization of culture temperature and pH, a 3-fold increase in maximum EPO concentration and a 1.4-fold increase in volumetric productivity were obtained at pH 7.00 and 32.5 degrees C when compared with those at 37.0 degrees C. These results demonstrate the importance of optimization of culture temperature and pH for enhancing EPO production in serum-free, suspension culture of rCHO cells. 相似文献
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Productivity and sialylation are two important factors for the production of recombinant glycoproteins in mammalian cell culture. In our previous study, we found that silkworm hemolymph increased the sialylation of recombinant secreted human placental alkaline phosphatase in the insect cells, promoted the transfer of sialic acids onto the glycoprotein oligosaccharides in an in vitro asialofetuin sialylation system, and enhanced recombinant protein production in the Chinese hamster ovary (CHO) cells. These beneficial effects were mainly due to the 30K proteins, which consist of five isoforms. Among the 30K proteins, 30Kc19 was determined to be the major component. In this study, the 30Kc19 gene was introduced into a CHO cell line producing recombinant human erythropoietin, and its effects on productivity and sialylation were investigated. The transient expression of 30Kc19 significantly improved the production and sialylation of EPO. A stable cell line containing 30Kc19 was also established to investigate the effect of 30Kc19 gene expression. The stable expression of 30Kc19 increased the production and sialylation by 102.6% and 87.1%, respectively. The enhanced productivity from 30Kc19 expression is believed to occur because the 30Kc19 protein suppresses the loss of mitochondrial membrane potential and consequently improves the generation of intracellular ATP. In addition, the positive effect of 30Kc19 expression on sialylation is believed to be due to its ability to maintain sialyltransferase activity. In conclusion, 30Kc19 expression is a novel approach to improve the production and sialylation of recombinant glycoproteins in CHO cells. 相似文献
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目的:建立一种从废弃胚胎中提高囊胚形成率和质量的培养体系,寻找多种促进内细胞团(ICM)数目增多、贴壁、增值的方法,提高人胚胎干细胞(human embryonic stem cell,hESC)建系效率,建立人胚胎干细胞库。方法:将179枚IVFDay3废弃的胚胎放入优选培养体系中培养(G2.5培养液中添加10%人血清蛋白,人白细胞抑制生长因子(hLIF),碱性成纤维细胞生长因子(bFGF))。到Day7将形成的囊胚全部用机械法分离ICM,接种于丝裂霉素C灭活处理的原代小鼠胚胎成纤维细胞(MEF)上,培养8-9天,每4-5天传代1次。结果:优选培养体系的囊胚形成率为29.1%(52/179),其中A级囊胚形成率为11.2%(20/179),50个ICM贴壁生长,20个出现克隆形态,成功建立11株hESC(FY-hES-11至FY-hES-21)。11株hESC均具有共同的多能性生物学特性。结论:优选培养体系可以明显提高囊胚形成的质量,促进ICM的增值,纯熟的机械切割法可以避免损伤ICM并提高其贴壁率,原代灭活的MEF饲养层可以明显促进细胞增殖。 相似文献
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Cultivation of Marine Sponges 总被引:9,自引:0,他引:9
There is increasing interest in biotechnological production of marine sponge biomass owing to the discovery of many commercially
important secondary metabolites in this group of animals. In this article, different approaches to producing sponge biomass
are reviewed, and several factors that possibly influence culture success are evaluated. In situ sponge aquacultures, based
on old methods for producing commercial bath sponges, are still the easiest and least expensive way to obtain sponge biomass
in bulk. However, success of cultivation with this method strongly depends on the unpredictable and often suboptimal natural
environment. Hence, a better-defined production system would be desirable. Some progress has been made with culturing sponges
in semicontrolled systems, but these still use unfiltered natural seawater. Cultivation of sponges under completely controlled
conditions has remained a problem. When designing an in vitro cultivation method, it is important to determine both qualitatively
and quantitatively the nutritional demands of the species that is to be cultured. An adequate supply of food seems to be the
key to successful sponge culture. Recently, some progress has been made with sponge cell cultures. The advantage of cell cultures
is that they are completely controlled and can easily be manipulated for optimal production of the target metabolites. However,
this technique is still in its infancy: a continuous cell line has yet to be established. Axenic cultures of sponge aggregates
(primmorphs) may provide an alternative to cell culture. Some sponge metabolites are, in fact, produced by endosymbiotic bacteria
or algae that live in the sponge tissue. Only a few of these endosymbionts have been cultivated so far. The biotechnology
for the production of sponge metabolites needs further development. Research efforts should be continued to enable commercial
exploitation of this valuable natural resource in the near future.
Received November 5, 1998; accepted June 20, 1999. 相似文献