中国仓鼠卵巢细胞表达外源蛋白研究进展

中国仓鼠卵巢细胞(Chinese hamster ovary,CHO)是一种生物制药技术的重要表达系统之一,在生物制药过程中应用较为广泛,但由于其应用成本较高,限制了发展,随年研究的深入,研究者们对于此系统进行了深入的探索,提高了该系统的表达效率,降低了应用成本。     

重组蛋白在中国仓鼠卵巢细胞中高效表达的影响因素

高效表达重组蛋白 ,对于生物制药意义重大。大多数药用蛋白是糖蛋白 ,中国仓鼠卵巢细胞 (Chinesehamsterovarycell,CHO)是目前重组糖基蛋白生产的首选体系。影响外源蛋白在CHO细胞中表达的因素很多 ,从CHO细胞表达体系、表达载体系统、外源基因、表达细胞株的加压扩增与筛选、细胞大规模培养等方面对CHO高效表达加以阐述 ,同时提出存在的问题和未来的发展方向。     

牛病毒性腹泻病毒Erns蛋白的中华仓鼠卵巢细胞表达及免疫原性分析

本研究利用中华仓鼠卵巢(Chinese hamster ovary,CHO)细胞表达系统制备牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV) E^rns蛋白,并分析其免疫原性。以BVDV-1 NADL标准毒株基因序列为基础,构建BVDV E^rns蛋白重组真核表达质粒pcDNA3.1-BVDV-E^rns,转染悬浮培养的CHO细胞,进行上清分泌表达。SDS-PAGE分析E^rns蛋白的表达和纯化,并用抗His单克隆抗体和BVDV阳性血清进行Western blotting鉴定纯化蛋白;进一步使用纯化的E^rns蛋白免疫新西兰大白兔,通过间接酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)和细胞间接免疫荧光(indirect immunofluorescence,IFA)实验检测血清抗体水平及其免疫反应活性,用病毒中和实验测定免疫兔血清的中和抗体滴度。BCA蛋白定量试剂盒检测纯化的E^rns     

中华仓鼠卵巢(CHO)工程细胞无血清培养的研究

以DMEM：F12(1：1)为基础培养基,通过观察细胞生长状态和检测乙肝表面抗原的表达量作为评价指标,筛选适合于CHO工程细胞生长的生长因子,如：胰岛素、转铁蛋白、氢化可的松、硒酸钠,丁二胺等。并且建立了J5SFM培养基。该培养基与商品化的无血清培养基比较,能够使细胞生长维持较长的时间,表达产物分泌量也相对较高。     

中国仓鼠卵巢细胞表达新技术

中国仓鼠卵巢细胞（CHO细胞）是基因工程药物生产的最佳表达系统之一,在生物制药中被广泛应用。传统的获得高表达CHO细胞株的方法费时、费力。近年来出现了一些CHO细胞高效表达新技术,它们从克服位置效应,提高基因转录效率、mRNA翻译效率及稳定性、筛选高表达细胞的效率等不同层次调控外源基因在CHO细胞中的高效表达。与MTX加压扩增基因获得高效表达外源基因的方法比较,能够节约时间、减少工作量,不易丢失高表达细胞株。     

人骨形态发生蛋白7在中国仓鼠卵巢细胞中的表达研究

目的:构建重组人骨形态发生蛋白-7(rhBMP7)表达质粒,并研究其在中国仓鼠卵巢细胞中的表达。方法:将hBMP7重组表达质粒电转到中国仓鼠卵巢细胞(CHO)中,并用DOT-BLOT和ELISA方法分析检测rhBMP7在重组CHO细胞中的表达。结果:hBMP7 cDNA整合到CHO细胞基因组中并被转录。点杂交和ELISA检测证实rhBMP7在CHO细胞中得到表达。结论:hBMP7成功在CHO表达系统中得到表达。     

CHO细胞表达糖蛋白的研究进展*

中国仓鼠卵巢细胞(Chinese hamster ovary cells,CHO)表达系统因具有较高密度培养、高表达和相对完整的蛋白质糖基化修饰系统等特点,成为生产糖蛋白广泛应用的宿主表达细胞之一。目前已产生不同的CHO细胞系和各种功能细胞株以满足对糖蛋白的大量生产和其他实验需求。近年来,随着基因工程、蛋白质工程、细胞工程和发酵调控等技术的发展应用,由CHO细胞生产糖蛋白的产量和糖基化修饰程度取得了突破。然而,随着生物制品市场对于糖蛋白的需求增加,如何获得大量、均质的糖蛋白也成为急需解决的问题。综述了不同工程CHO表达系统的研究、应用、糖基化修饰系统,以及影响外源糖蛋白在CHO系统表达和糖基化修饰的理化因素,结合文献总结并预测了未来CHO细胞表达系统研究的四个具有重大意义的研究方向,以期在未来可以改善由CHO细胞表达糖蛋白的产量和质量。     

中国仓鼠卵巢细胞的悬浮适应及代谢特征

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。     

利用CRISPR/Cas9技术构建稳定表达人白蛋白基因的中国仓鼠卵巢细胞系

通过目的基因的随机整合方式,构建得到的CHO表达细胞系,常会因为目的基因插入到染色体内不稳定区域,而在长期传代过程中出现表达不稳定的现象,这主要是由于位点效应所导致的。为了解决这个问题,现利用CRISPR/Cas9技术介导的同源重组,将目的基因直接整合于CHO细胞染色体上的稳定表达区域,以克服位点效应带来的CHO表达细胞系的长期表达不稳定。利用该技术总共获得了2株外源基因(人白蛋白基因)定点整合细胞系; Western blot结果显示,细胞上清液的产物具有人白蛋白抗原性;细胞在贴壁状态下,两株细胞系在第3、12、23、35、50代,细胞每日表达的HSA质量接近,且均维持在0. 5pg cell/d;选取一株表达细胞系,经悬浮驯化后,在批次条件培养下,第1、25、50代的悬浮细胞,在摇瓶内的表达浓度均稳定在13～14mg/L。显示了将外源基因定点整合于CHO细胞基因组内的可行性;且外源基因整合在稳定表达区域后,重组细胞系具有对外源基因长期表达的稳定性。     

血管内皮细胞生长因子受体Flt-1胞外区基因的克隆及其在中国仓鼠卵巢细胞中的表达

从原代培养的人脐静脉血管内皮细胞(HUVEC)提取细胞总RNA,采用逆转录PCR(RT-PCR)方法得到VEGF受体Flt-1胞外区前3个IgG样区域cDNA片段(Flt-1n3).将获得的受体基因克隆到真核表达载体pcDNA3.1中,得到重组质粒pcDNA3.1/Flt-1n3,通过脂质体转染方法将其转入中国仓鼠卵巢细胞(CHO),用G418筛选得到稳定表达目的蛋白的细胞克隆.经固相结合实验筛选得到表达与VEGF特异结合的目的蛋白的细胞克隆,细胞测活结果表明,表达产物具有抑制人脐静脉内皮细胞(HUVEC)增殖的活性.鸡胚测活结果表明,CHO细胞表达的r Flt-1n3能抑制鸡胚绒毛尿囊膜的血管形成.     

过表达热休克蛋白70提高CHO细胞表达抗体的能力

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。     

Generation of Recombinant Chinese Hamster Ovary Cell Lines by Microinjection

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     

Activation of Phospholipase A2 by the Nociceptin Receptor Expressed in Chinese Hamster Ovary Cells

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.     

中国仓鼠卵巢细胞及其表达载体的改造

哺乳动物细胞因其表达的外源蛋白最接近天然构象,已成为生产重组蛋白药物的理想系统。其中,中国仓鼠卵巢细胞（CHO）是目前最为常用的表达系统,但这种系统也存在很多缺点,如大规模培养中表达量低、生产成本高、细胞无限度增殖及细胞凋亡等。目前,通过优化培养基配方和培养条件很难从根本上解决上述问题,必须从整个表达系统着手进行改造,其中CHO细胞本身和表达载体的改造最为关键。     

Functional Coupling of the NK1 Tachykinin Receptor to Phospholipase D in Chinese Hamster Ovary Cells and Astrocytoma Cells

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 G_i/G_o 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.     

Effects of Supplementation of Various Medium Components on Chinese Hamster Ovary Cell Cultures Producing Recombinant Antibody

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 Long^TMR³IGF-I. On the other hand, in the cultivation of rCHO-B cell which was more sensitive to its environmental changes, hormones such as Long^TMR³IGF-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