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Derouazi M Flaction R Girard P de Jesus M Jordan M Wurm FM 《Biotechnology letters》2006,28(6):373-382
Microinjection is a gene transfer technique enabling partial control of plasmid delivery into the nucleus or cytoplasm of
cultured animal cells. Here this method was used to establish various recombinant mammalian cell lines. The injection volume
was estimated by fluorescence quantification of injected fluorescein isothyocynate (FITC)-dextran. The DNA concentration and
injection pressure were then optimized for microinjection into the nucleus or cytoplasm using a reporter plasmid encoding
the green fluorescent protein (GFP). Nuclear microinjection was more sensitive to changes in these two parameters than was
cytoplasmic microinjection. Under optimal conditions, 80–90% of the cells were GFP-positive 1 day after microinjection into
the nucleus or the cytoplasm. Recombinant cell lines were recovered following microinjection or calcium phosphate transfection
and analyzed for the level and stability of recombinant protein production. In general, the efficiency of recovery of recombinant
cell lines and the stability of reporter protein expression over time were higher following microinjection as compared to
CaPi transfection. The results demonstrate the feasibility of using microinjection as a method to generate recombinant cell
lines.
Revisions requested 27 October 2005; Revisions received 12 December 2005 相似文献
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中华仓鼠卵巢细胞(Chinese Hamster Ovary Cells,CHO)是科研及生产中用于蛋白表达的常用体系。与大肠杆菌相比,CHO获得高表达的细胞株所需时间更长,蛋白产量更低。因此,大规模培养细胞所需的成本较高,培养条件也不易掌握。但该体系产生的蛋白纯度高,因而被广泛用于工业生产中。本文对CHO细胞的培养方式、pH值测定、渗透压、溶氧及培养液成分的选择等多方面条件进行综述,为优化中华仓鼠卵巢细胞培养的策略及具体方法提供理论依据。 相似文献
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通过在中国仓鼠卵巢细胞(CHO)中过表达热休克蛋白70以提高其表达抗体的能力。首先从中国仓鼠基因组DNA中扩取HSP70基因,构建真核表达质粒pcDNA3.1-HSP70,再将重组质粒稳定转染到CHO/dhfr-细胞中,筛选获得稳定的细胞系,运用RT-qPCR检测和Western blot分析HSP70基因的过表达。在过表达HSP70的CHO细胞组和对照细胞组(转染空载体pcDNA3.1的CHO细胞组)中分别转染表达抗-HBs的质粒,应用ELISA检测两组细胞表达抗-HBs的能力。RT-qPCR结果显示实验组CHO细胞中HSP70基因的表达量明显高于对照组细胞;ELISA检测结果表明过表达HSP70的CHO细胞组抗-HBs表达量高于对照组细胞(P<0.05)。研究揭示HSP70能有效促进细胞内分泌性蛋白的表达。 相似文献
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外源蛋白在中国仓鼠卵巢细胞中高效表达的策略 总被引:10,自引:0,他引:10
高效表达外源蛋白,在生物制药中有重要意义.中国仓鼠卵巢细胞(Chinese hamster ovary cell)是表达外源蛋白的最佳真核表达系统之一.影响外源蛋白在CHO细胞表达的因素甚多,主要包括载体、宿主细胞和外源基因几方面.深入了解和灵活运用它们之间的关系,有助于获得外源基因在CHO细胞中的高效表达. 相似文献
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目的:通过悬浮适应,使中国仓鼠卵巢细胞(CHO细胞)获得悬浮生长的特性,并可在悬浮培养条件下较快地生长。方法:将CHO细胞以3×10^5/mL接种于100mL的三角瓶内,培养时加入1%小牛血清、1g/LPIuronic F-68、25μg/mL硫酸葡聚糖,培养体积35mL,摇床转速90r/min,每24h离心换液,当细胞增殖为2×10^6/mL时传代。结果:经过悬浮适应,细胞的平均比生长速率由适应最初的0.27/d提高为适应后的0.48/d,最大总细胞密度由适应初期的2.5×10^6/mL提高为适应后的6.3×10^6/mL,目的蛋白活性也由适应前的2781U/mL提高为适应后的8878U/mL,适应后细胞的葡萄糖平均比消耗率为1.42μmol/(10^6细胞·d),低于适应前的2.16μmol/(10^6细胞·d)。结论:贴壁生长的CHO细胞经过悬浮适应,不仅可以在悬浮培养条件下快速生长,而且细胞对葡萄糖的利用率也得到提高。 相似文献
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Thirteen vitamins, twenty amino acids, hormones, inorganic salts, and other chemical agents, which constitute typical serum-free
media, were evaluated for the development of fortified medium to enhance cell growth and productivity of recombinant antibody
in the cultures of the recombinant Chinese hamster ovary (rCHO) cells. Two different rCHO cell lines, rCHO-A producing recombinant
antibodies against the human platelet and rCHO-B secreting recombinant antibodies against the S surface antigen of Hepatitis
B, respectively, were cultivated in batch suspension mode. Concentration of interested component in the tested medium was
doubled to examine the fortification effect. Growth of rCHO-A cell and its antibody production were slightly improved with
addition of either choline chloride, folic acid, thiamine⋅HCl, or LongTMR3IGF-I. On the other hand, in the cultivation of rCHO-B cell which was more sensitive to its environmental changes, hormones
such as LongTMR3IGF-I and triiodothyronine (T3) as well as various vitamins involving choline chloride, i-inositol, niacinamide, pyridoxine HCl, and thiamine⋅HCl enhanced
the cell growth and antibody production. Particularly, when concentration of consuming amino acid was doubled, remarkable
increase in specific productivity was served, resulting in high final antibody concentration. These results were believed
to provide a fundamental strategy of medium fortification useful for improvement of recombinant antibody production in serum-free
medium.
These authors contributed equally to this work 相似文献
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人工转录因子研究进展 总被引:3,自引:0,他引:3
转录因子是真核表达调控中非常重要的一类反式作用因子,通常由DNA结合结构域与效应结构域两部分组成,研究发现这两个结构域可以各自独立发生作用。基于转录因子的这种结构特点,可以人为地选择针对特定序列的DNA结合结构域与具有特定作用的效应结构域构建人工转录因子。目前人工转录因子的DNA结合结构域多为C2H2 型锌指结构,每一个锌指单元由大约30个氨基酸组成,识别DNA双螺旋大沟中相连的3bp序列,并可通过氢键作用与相应的碱基结合;多个锌指可以串联成簇,从而识别并结合较长的DNA序列区域。常见的人工转录因子的效应结构域有激活结构域以及抑制结构域,不同的效应结构域赋予人工转录因子不同的功能。目前人工转录因子已经在基础研究、药物设计以及基因治疗等领域得到了广泛的应用。 相似文献
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克服位置效应提高外源基因在CHO细胞中稳定高效表达的策略 总被引:4,自引:0,他引:4
能够生产有功用的治疗性蛋白的一个重要前提是获得稳定的重组蛋白高表达细胞株,然而筛选一个能够持续稳定表达外源蛋白的重组细胞株是费时费力的过程。有多篇文献报道了重组蛋白细胞株表达的不稳定性。位置效应是高表达细胞株不稳定性的重要因素,克服或利用位置效应是当前获得稳定高表达重组蛋白细胞株的有效途径。为解决外源基因插入的随机性所带来的不可预知的后果,可以事先在CHO细胞基因组中筛选转录热点区域,再通过位点特异性或同源重组的方式,实现外源基因的定点整合。各种调节位置效应的DNA元件陆续被发现,可以利用它们去调控基因表达及增加细胞株的稳定性。 相似文献
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Kazuhiko Fukuda Takehiro Shoda Hitoshi Morikawa Shigehisa Kato Hiroyuki Mima Kenjiro Mori 《Journal of neurochemistry》1998,71(5):2186-2192
Abstract: To gain insight into the molecular mechanism for nociceptin function, functional coupling of the nociceptin receptor expressed in Chinese hamster ovary (CHO) cells with phospholipase A2 (PLA2 ) was examined. In the presence of A23187, a calcium ionophore, activation of the nociceptin receptor induced time- and dose-dependent release of arachidonate, which was abolished by pretreatment of the cells with pertussis toxin (PTX). Immunoblot analysis using anti-Ca2+ -dependent cytosolic PLA2 (cPLA2 ) monoclonal antibody demonstrates that activation of the nociceptin receptor induces a time- and dose-dependent electrophoretic mobility shift of cPLA2 , suggesting that phosphorylation of cPLA2 is induced by the nociceptin receptor. Pretreatment of the cells with PD98059, a specific mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 inhibitor, or staurosporine, a potent inhibitor of serine/threonine protein kinases and tyrosine protein kinases, partially inhibited the nociceptin-induced cPLA2 phosphorylation and arachidonate release. These results indicate that the nociceptin receptor expressed in CHO cells couples with cPLA2 through the action of PTX-sensitive G proteins and suggest that cPLA2 is activated by phosphorylation induced by the nociceptin receptor via mechanisms partially dependent on p44 and p42 mitogen-activated protein kinases. 相似文献
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Ly N. Nguyen Martina Baumann Heena Dhiman Nicolas Marx Valerie Schmieder Mohamed Hussein Peter Eisenhut Inmaculada Hernandez Jadranka Koehn Nicole Borth 《Biotechnology journal》2019,14(11)
For the industrial production of recombinant proteins in mammalian cell lines, a high rate of gene expression is desired. Therefore, strong viral promoters are commonly used. However, these have several drawbacks as they override cellular responses, are not integrated into the cellular network, and thus can induce stress and potentially epigenetic silencing. Endogenous promoters potentially have the advantage of a better response to cellular state and thus a lower stress level by uncontrolled overexpression of the transgene. Such fine‐tuning is typically achieved by endogenous enhancers and other regulatory elements, which are difficult to identify purely based on the genomic sequence. Here, Chinese hamster ovary (CHO) endogenous promoters and enhancers are identified using histone marks and chromatin states, ranked based on expression level and tested for normalized promoter strength. Successive truncation of these promoters at the 5′‐ and 3′‐end as well as the combination with enhancers are identified in the vicinity of the promoter sequence further enhance promoter activity up to threefold. In an initial screen within stable cell lines, the strongest CHO promoter appears to be more stable than the human cytomegalovirus promoter with enhancer, making it a promising candidate for recombinant protein production and cell engineering applications. A deeper understanding of promoter functionality and response elements will be required to take full advantage of such promoters for cell engineering, in particular, for multigene network engineering applications. 相似文献
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A designed angiopoietin-1 (Ang1) chimeric protein with nonleaky angiogenic activity, COMP-Ang1, is an effective alternative to native Ang1 for therapeutic angiogenesis in vivo. Recombinant Chinese hamster ovary (rCHO) cell lines expressing a high level (>20 mug/mL) of COMP-Ang1 and an amino-terminal FLAG-tag were constructed by transfecting the expression vector into dihydrofolate reductase-deficient CHO cells and the subsequent gene amplification in medium containing stepwise increments in methotrexate level such as 0.02, 0.08, 0.32, and 1 muM. The COMP-Ang1 secreted from rCHO cells was purified at a purification yield of 40.3% from the culture medium using an anti-FLAG M2 agarose affinity gel. SDS-PAGE and Western blot analyses showed that rCHO cells secrete COMP-Ang1 in homopentameric and homotetrameric glycoprotein forms. Furthermore, COMP-Ang1 binds to the Tie2 receptor and phosphorylates Tie2, indicating its potential for therapeutic angiogenesis. 相似文献
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干扰素α2b(interferonα2b,IFNα2b)是一种用于病毒性疾病和肿瘤性疾病治疗的多功能细胞因子,因其在体内的半衰期短限制了其在临床上的应用。将IFNα2b连接到人血清白蛋白(human serum albumin,HSA)的C端,构建融合蛋白HSA-IFNα2b。构建含融合蛋白的真核表达质粒p MH3/HSA-IFNa2b,经电转的方法转入中国仓鼠卵巢(Chinese hamster ovary,CHO)细胞中。经G418抗性压力筛选和目的蛋白的表达量筛选,最终获得一株高表达的稳定细胞株(CHO/p MH3/HSA-IFNa2b)。表达的目的蛋白经Western blot验证显示,产物具有IFNα2b和HSA的双抗原性。经悬浮驯化稳定后,通过批次筛选得到一株稳定的高克隆表达株,产量约为65mg/L,进一步选取高表达克隆株在悬浮驯化中不同代数进行批次培养,不同代数之间蛋白质的表达量和生长情况没有明显的差异,获得一株稳定遗传表达的单克隆细胞株,3L摇瓶的流加培养结果显示,最佳发酵时间为15天,蛋白质表达量为121mg/L。经离心获得的发酵液,经两步纯化后获得蛋白质纯度高达96.8%的目的蛋白,总回收率为22.3%。参照《中国药典》2015版对IFNα2b的检测方法,结果显示,CHO表达的HSA-IFNα2b比活性为4.16×106IU/mg。首次将HSA-IFNα2b在哺乳动物细胞CHO中构建表达,表达获得高活性的HSA-IFNα2b融合蛋白。 相似文献
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Yvette Torrens Jean-Claude Beaujouan Monique Saffroy Jacques Glowinski Martine Tencé 《Journal of neurochemistry》1998,70(5):2091-2098
Abstract: In [3H]myristic acid-prelabeled Chinese hamster ovary cells stably expressing the rat NK1 tachykinin receptor, the selective NK1 agonist [Pro9]substance P ([Pro9]SP) time and concentration dependently stimulated the formation of [3H]phosphatidylethanol in the presence of ethanol. This [Pro9]SP-induced activation of phospholipase D (PLD) was blocked by NK1 receptor antagonists and poorly or not mimicked by NK2 and NK3 agonists, respectively. In confirmation of previous observations, [Pro9]SP also stimulated the hydrolysis of phosphoinositides, the release of arachidonic acid, and the formation of cyclic AMP (cAMP). All these [Pro9]SP-evoked responses could be mimicked by aluminum fluoride, but they remained unaffected in cells pretreated with pertussis toxin, suggesting that a Gi/Go protein is not involved in these different signaling pathways. The activation of PLD by [Pro9]SP was sensitive to external calcium and required an active protein kinase C because the inhibition of this kinase (Ro 31-8220) or its down-regulation (long-term treatment with a phorbol ester) abolished the response. In contrast, a cAMP-dependent process was not involved in the activation of PLD because the [Pro9]SP-evoked response was neither affected by Rp-8-bromoadenosine 3′,5′-cyclic monophosphorothioate nor mimicked by cAMP-generating compounds (cholera toxin or forskolin) or by 8-bromo-cyclic AMP. A functional coupling of NK1 receptors to PLD was also demonstrated in the human astrocytoma cell line U 373 MG stimulated by SP or [Pro9]SP. These results suggest that PLD activation could be an additional signaling pathway involved in the mechanism of action of SP in target cells expressing NK1 receptors. 相似文献