CN1296485C - 能够产生蛋白质及能够在无谷氨酰胺的培养基上生长的谷氨酰胺营养缺陷型人类细胞 - Google Patents
能够产生蛋白质及能够在无谷氨酰胺的培养基上生长的谷氨酰胺营养缺陷型人类细胞 Download PDFInfo
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Abstract
以可编码蛋白质的外源DNA序列或能够改变可编码蛋白质之内生基因表达的外源DNA序列及可编码谷氨酰胺合成酶的外源DNA序列转染谷氨酰胺营养缺陷型人类细胞,其中这些外源DNA序列位于一个或多个DNA构建物上,该转染的细胞能够产生该蛋白质及能够生长在无谷氨酰胺的培养基中。
Description
本发明是关于新颖的能够产生蛋白质和能够生长在无谷氨酰胺的培养基中的谷氨酰胺营养缺陷型人类细胞。此外,其是关于新颖的产生蛋白质的方法和关于在谷氨酰胺营养缺陷型人类细胞中把谷氨酰胺合成酶(GS)当作可选择标记的用途。
由哺乳动物细胞培养所产生的蛋白质是用来提供治疗和诊断应用的蛋白质。现今,哺乳动物细胞培养是用于人类和动物医药之许多重要蛋白质的较佳来源,尤其那些相当大的、复杂的和糖基化的(N.B.Finter等,在大规模哺乳动物细胞培养技术,1990,A.S.Lubiniecki编,Marcel Dekker,Inc.,纽约)。
例如,藉细胞培养产生人类蛋白质红血球生成素(EPO)已在WO 93/09222中描述。使用以可编码人类EPO外源基因转染的人类纤维组织母细胞,人类EPO已获得可观的比生成速率。在进一步产生人类EPO的方法(WO 94/12650),是使用以能够活化内生编码的EPO之DNA序列转染的人类纤维肉瘤细胞系HT1080。类似的方法已于WO99/09268中描述。永生的(Immortalised)人类细胞如Namalwa、Hela S3及HT 1080细胞用能够活化内生编码的EPO之DNA序列转染。
描述于WO 93/09222、WO 94/12650及WO 99/09268用来产生人类EPO的细胞系都在含有谷氨酰胺的培养基中培养。这是不利的,因为当谷氨酰胺被培养的细胞当作能量基质时,会产生氨这种代谢产物,其具有细胞毒性及会抑制细胞生长。再者,由于它在细胞高尔基体内对pH的影响,而会抑制蛋白质糖基化。
产生大量组织血浆素原活化剂(一种糖基化蛋白质)已在WO 87/04462中描述。GS在其中已被用来作为放大是统,在谷氨酰胺原养型中国仓鼠卵巢(CHO)细胞用于共放大可编码组织血浆素原活化剂(tPA)的基因,其通过以可编码GS的基因转染细胞。然而,如同在EP-A 148 605发现的,用CHO细胞来产生糖基化的人类蛋白质是不利的。由CHO细胞合成的蛋白质其平均碳水化合物成分会与自然生成的糖基化人类蛋白质有所不同,这是因为人类细胞含有α2.3唾液酸转移酶及α2.6唾液酸转移酶。CHO细胞只具有α2.3唾液酸转移酶,所以不能产生α2.6端唾液酸与寡糖部分的键结,CHO细胞缺乏硫化碳水化合物结构的酵素,CHO细胞虽然有α1-6岩藻糖基转移酶(附着中心的岩藻糖残基),亦缺乏α1-3岩藻糖基转移酶(附着末端的岩藻糖残基)。人类细胞则具有两者岩藻糖基转移酶(Cumming D.A.,1991,Glycobiology卷1,No.2,115-130,Jenkins N.及Curling E.M.A.,1994,enzyme and Micorbial Technology,卷16,354-364;Lee等,1989,Journal of Biological Chemistry,卷264,13848-13855)。所以,由CHO细胞合成之糖基化的蛋白质不会有希望得到的特征,例如如于人类细胞产生的活体内生物活性。
在WO 89/10404中,报告了制造骨髓瘤细胞例如鼠融合瘤、鼠浆细胞瘤细胞及大鼠融合瘤细胞谷氨酰胺独立的方法,其藉由以GS转化他们。其进一步证明GS可以用来共放大可编码免疫球蛋白分子轻及重链的基因及用来共放大在骨髓瘤细胞系中可编码纤维蛋白分解酵素的基因。然而,啮齿类细胞系具有缺点,即以N-乙醇酰基神经氨酸残基的附着代替N-乙酰神经氨酸、无法进行硫化及α1.3半乳糖转移酶的存在。在啮齿类细胞中合成之糖蛋白的寡糖结构因此可以在人类产生免疫性。
本发明之目的是提供一种改进的方法,其不具有用于产生蛋白质时的上述缺点(尤其当产生糖蛋白时),及可获得高蛋白质效价。
利用权利要求1所述的新颖的谷氨酰胺营养缺陷型人类细胞和权利要求7所述的新颖的方法,可以达成此目的。
根据本发明,可得到谷氨酰胺营养缺陷型人类细胞,所述细胞用(第一)可编码蛋白质之外源DNA序列或能够改变可编码蛋白质之内生基因表达的外源DNA序列转染,及进一步以(第二)可编码谷氨酰胺合成酶(GS)(较佳的为哺乳动物GS)之外源DNA序列转染,其中这些外源DNA序列位于一或一个以上DNA构建物,该转染的细胞能够产生该蛋白质及能够生长在无谷氨酰胺的培养基中。
图1显示细胞系R223悬浮培养于无血清培养基之适应作用的细胞浓度图,经由重复连续的次培养。
图2显示HT1080细胞悬浮培养于无血清培养基之适应作用概要。
图3显示细胞系HT1080悬浮培养于无血清培养基之适应作用的细胞浓度图,经由重复连续的次培养。
图4显示从GS转染子3E10及从无转染的R223细胞系中免疫纯化的EPO之IEF分析,其是生长在产业高密度生长培养基。泳道2:收集的(收集)3E10;泳道3:3E10峰;泳道4:无转染的R223细胞系峰;泳道5:收集的无转染的R223细胞系。
图5显示R223细胞系之GS-R223转染子3E10及从无转染的R223细胞系免疫纯化的EPO之IEF分析,生长在传统的Iscove培养基。泳道4:自添加谷氨酰胺的Iscove培养基收集的无转染的R223细胞系;泳道5:自无谷氨酰胺的Iscove培养基收集的GS-223细胞系3E10。
图6显示了图5所示凝胶泳道4从顶部到底部光密度扫描的色谱图。
图7显示了图5所示凝胶泳道5从顶部到底部光密度扫描的色谱图。
″可编码蛋白质的外源DNA序列″或″能够改变可编码蛋白质之内生基因表达的外源DNA序列″及″可编码GS的外源DNA序列″通常位于DNA构建物上,例如表达载体或感染性载体。质体可以用来作为表达载体。作为感染性载体则可以使用如反转录病毒、泡疹病毒、腺病毒、腺病毒伴随病毒、流行性腮腺炎病毒及脊髓灰质炎病毒载体。较佳的为表达载体,尤其使用质体。
″可编码蛋白质的外源DNA序列″可以包括额外序列,例如调控序列如启动子及/或增强子、多聚腺苷酸化位点及剪接接头,通常用于表达外源基因或可以包括额外一或多个分开的靶向性序列及视需要的可编码可选择标记之DNA(如WO 93/09222中所描述)。
″能够改变可编码蛋白质之内生基因表达的外源DNA序列″可以包括外源DNA序列,其不编码蛋白质的基因产物,但可编码部分该基因产物(如外显子),及可以包括额外序列,例如调控序列及剪接接头,通常用于表达外源DNA序列。他们可以进一步包括靶向性序列及视需要的可编码可选择标记之DNA(如WO 93/09222中所描述)。
通常,″能够改变可编码蛋白质之内生基因表达的外源DNA序列″是在转染进细胞后插入细胞的染色体DNA。同源重组或靶向性是在此用来取代或使与含有调控序列之内生基因相关的调控区域失去作用。作为调控序列可以使用如启动子及/或增强子,其造成基因表达量高于相对应之无转染的细胞(如WO 93/09222所描述),合适的启动子可以是可调控的或持续表达的启动子。合适的启动子可以是强启动子,其是取决于所使用的细胞系,如人类巨细胞病毒主要立即早期启动子(hCMV-MIE)、SV 40早期及晚期启动子、其他腺病毒启动子、任何多瘤病毒或乳多泡病毒早期及晚期启动子、干扰素α1启动子、鼠金属硫蛋白启动子、Rous肉瘤病毒长端重复启动子、β-珠蛋白启动子、伴白蛋白启动子、卵白蛋白启动子、鼠β-珠蛋白启动子及人类β-珠蛋白启动子。
根据本发明的″可编码GS之外源DNA序列″可以受强启动子及弱启动子的控制,如果外源DNA序列是单单需要表达可编码GS的基因,则使用强启动子,当外源DNA序列是作为可选择标记,以及若GS是用于放大作用时,则使用弱启动子。合适的启动子可以是可调控的或持续表达的启动子。启动子可以选择,例如,GS是在足够使转染的细胞生长的浓度中表达,但细胞培养时并不产生高量谷氨酰胺代谢产物氨,通常不大于4mM,较佳的不大于2mM,更佳的少于2mM氨。
″可选择标记″提供了可选择的表现型,使鉴别及分离受体细胞成为可能。GS可以在本发明用来当作可选择标记,以筛选出成功转染的谷氨酰胺营养缺陷型人类细胞,其并入和表达可编码GS的外源DNA序列。
根据所用的细胞系,强启动子可以是如hCMV-MIE、SV 40早期及晚期启动子、其他腺病毒启动子、任何多瘤病毒或乳多泡病毒早期及晚期启动子、干扰素α1启动子、鼠金属硫蛋白启动子、Rous肉瘤病毒长端重复启动子、β-珠蛋白启动子、伴白蛋白启动子、卵白蛋白启动子、鼠β-珠蛋白启动子及人类β-珠蛋白启动子。
根据所用的细胞系,弱启动子可以是如鼠白血病病毒长端重复、单纯疹病毒胸苷激酶及鼠乳房肿瘤病毒长端重复,较佳的,可编码GS的基因是在强启动子控制下,更佳的是在hCMV-MIE启动子控制下。一项可能的具体实例,一种来自仓鼠之可放大的哺乳动物GS序列及其在哺乳动物细胞中作为可选择标记的用途已为该项技术所熟知,且如WO 87/04462、WO 91/06657及WO 89/01036所描述;本发明实施例使用此仓鼠GS表达单元及个别的筛选方法如参考文献所提及。
″可编码蛋白质之外源DNA序列″或″能够改变可编码蛋白质之内生基因表达的外源DNA序列″及″可编码GS之外源DNA序列″位于一个或多个DNA构建物上,较佳的,这些外源DNA序列位于多于一个,更佳的位于两个DNA构建物上。若这些外源DNA序列位于一个DNA构建物上,他们可能功能上相组合,例如他们的表达会受同一个调控序列,如启动子及/或增强子(如WO 89/10404所描述)驱策。
谷氨酰胺营养缺陷型人类细胞系指所有不表达GS或表达GS很差的人类细胞,所以能够生长在含有谷氨酰胺的培养基,但在无谷氨酰胺的培养基不能生长或生长很差。用于本发明之谷氨酰胺营养缺陷型人类细胞系必死的谷氨酰胺营养缺陷型人类细胞或永生的谷氨酰胺营养缺陷型人类细胞。必死的谷氨酰胺营养缺陷型人类细胞系在培养基中具有有限寿命的谷氨酰胺营养缺陷型人类细胞,永生的(又称永久的或建立的)谷氨酰胺营养缺陷型人类细胞系在培养基中如习于该项技术者所熟知以适当地继代培养和次培养,具有明显无限寿命之谷氨酰胺营养缺陷细胞。
必死的谷氨酰胺营养缺陷型人类细胞的例子可以是人类纤维组织母细胞及人类胎肺组织细胞,永生的谷氨酰胺营养缺陷型人类细胞的例子可以是人类纤维肉瘤细胞,如HT1080细胞系(如DSMZ No.ACC-315或ATCC No.CCL 121)及B-淋巴母细胞人类细胞如HL60(DSMZ No.Acc-3)或Namalwa(DSMZ Acc-24)细胞系。用于本发明较佳的是永生的谷氨酰胺营养缺陷型人类细胞,更佳的,此永生的谷氨酰胺营养缺陷型人类细胞系用B-淋巴母细胞或纤维肉瘤细胞,更佳的是用人类纤维肉瘤细胞,最佳的是用HT1080细胞系(如ATCC No.CCL 121)。
谷氨酰胺营养缺陷型人类细胞可藉由已知的基因工程技术以外源DNA序列转染。
以外源DNA序列转染取决于序列是否位于一个或多个DNA构建物上,若序列位于多于一个DNA构建物上,转染可随各序列分别发生或共转染。当转染随各序列分别发生时,序列转染顺序通常是随意的,随各序列分别发生的转染较佳先以″可编码该蛋白质的外源DNA序列″或″能够改变可编码该蛋白质之内生基因表达的外源DNA序列″转染,然后再以″可编码GS的外源DNA序列″转染,转染的谷氨酰胺营养缺陷细胞可以在各别转染之后培养并评估蛋白质生成。
为了成功筛选转染的细胞,所述细胞嫩生长在无谷氨酰胺的培养基中。细胞可以直接生长在无谷氨酰胺的培养基或先在含有谷氨酰胺的培养基,然后逐步稀释成无谷氨酰胺的培养基,如先以谷氨酰胺浓度10mM,然后以2mM至0mM逐步稀释。合适的筛选方法可以根据所用的细胞系来选择。如前述显知,本发明能够产生蛋白质及能够生长在无谷氨酰胺的培养基中的谷氨酰胺营养缺陷型人类细胞系得自以可编码该蛋白质的外源DNA序列或能够改变可编码蛋白质之内生基因表达的外源DNA序列及可编码谷氨酰胺合成酶之外源DNA序列转染该细胞,其中这些外源DNA序列位于一个或多个DNA构建物上。
可编码蛋白质的外源DNA序列或能够改变可编码蛋白质之内生基因表达的外源DNA序列可以在转染后根据如WO 94/12650所描述之基因放大已知方法来放大。可放大的基因可编码酵素,如DHFR(二氢叶酸还原酶)、GS、腺苷脱氨酶、天冬酰胺合成酶、天冬氨酸氨甲酰基转移酶、金属硫蛋白-1、鸟氨酸脱羧酶、P-糖蛋白质、核苷酸还原酶、胸苷激酶或黄嘌呤鸟嘌呤磷酸核糖转移酶可以用于此目的。含有这些基因的放大复制的细胞系例如能够在没有酵素代谢产物的培养基或在含有相关的选择剂之培养基中存活,相关的选择剂是例如在DHFR情况下为甲氨喋呤(MTX)及在GS情况下为甲硫氨酸磺酰胺(methionine sulphoximine)(MSX)。
由本发明转染的谷氨酰胺营养缺陷型人类细胞所产生之蛋白质是无糖基化和糖基化蛋白质,糖基化蛋白质是指具有至少一个寡糖链的蛋白质。
无糖基化蛋白质的例子如无糖基化荷尔蒙,像黄体生成素释放激素、甲状腺素释放激素、胰岛素、生长抑素、催乳素、促肾上腺皮质激素、黑素细胞促黑激素、升压素、以及其衍生物如去氨加压素、催产素、降钙素、副甲状腺素(PTH)或其片段(如PTH(I-43))、促胃液素、胰泌素、肠促胰酶素、激胆囊素、血管紧张素、人类胎盘促黄体激素、人类纤毛膜促性腺激素(HCG)、雨蛙肽及胃动素;无糖基化的止痛剂物质如脑啡肽及其衍生物(参见US-A 4 277 394及EP-A 031567)、内啡肽、daynorphin及kyotorphin;无糖基化酵素如无糖基化神经传导物质例如蛙皮素、神经紧张素、缓激肽和物质P;神经生长因子(NGF)家族、上皮生长因子(EGF)及纤维组织母细胞生长因子(FGF)家族之无糖基化生长因子及无糖基化荷尔蒙及生长因子受体。
糖基化蛋白质的例子是荷尔蒙及荷尔蒙释放因子,如生长荷尔蒙,包括人类生长荷尔蒙、牛生长荷尔蒙、生长荷尔蒙释放因子、副甲状腺素、甲状腺刺激素、EPO、脂蛋白质、α-1-抗胰蛋白酶、卵泡刺激素、降钙素、黄体生成素、胰增血糖素、凝血因子如因子VIIIC、因子IX、组织因子及von Willebrand因子、抗凝血因子例如蛋白质C、心房利钠因子、肺表面活性物质、血浆素原活化剂例如尿激酶或人类尿或组织型血浆素原活化剂(t-PA)、凝血酶、造血生长因子、脑啡肽、RANTES(调控活化自然的T-细胞表达及分泌)、人类巨噬细胞发炎蛋白质(MIP-1-α)、血清蛋白素例如人类血清蛋白素、米勒抑制物、松弛素A链、松弛素B链、前松弛素、鼠促性腺激素相关肽、微生物蛋白质例如β-lactanase、DNase、抑制素、激活素、肾素、血管内皮生长因子(VEGF)、荷尔蒙或生长因子受体、整合素、蛋白质A或D、类风湿性因子、神经营养因子例如源自骨神经营养因子(BDNF)、神经营养蛋白-3、-4、-5或-6(NT-3、NT-4、NT-5或NT-6)、或神经生长因子例如NGF-β、源自血小板生长因子(PDGF)、纤维组织母细胞生长因子例如FGF及bFGF、表皮生长因子(EGF)、转化生长因子(TGF)例如TGF-α及TGF-β,包括TGF-β1、TGF-β2、TGF-β3、TGF-β4或TGF-β5、类胰岛素生长因子-I及-II(IGF-I及IGF-II)、des(1-3)-IGF-I(脑IGF-I)、类胰岛素生长因子结合蛋白、CD蛋白(一群分化蛋白质)例如CD-3、CD-4、CD-8及CD-19、骨诱导因子、免疫毒素、骨形态发生蛋白质(BMP)、细胞活素及其受体,还有嵌合蛋白,包含其受体的细胞活素,包括例如肿瘤坏死因子α及β、他们的受体(TNFR-1,EP 417 563及TNFR-2,EP 417 014)及其衍生物,一种干扰素例如干扰素-α、-β、及-γ,细胞族群刺激因子(CSFs),如M-CSF、GM-CSF及G-CSF,白介素(ILs)如IL-1到IL-10,超氧化物歧化酶,T-细胞受体、表面膜蛋白、降解加速因子、病毒抗原例如AIDS夹膜的一部份、运输蛋白、归巢受体、地址素、调控蛋白、抗体、嵌合蛋白如免疫粘附素,及任何上述所列片段之糖基化蛋白质。较佳的,本发明产生糖基化蛋白质,更佳的本发明产生N-糖基化蛋白质,最佳的糖基化荷尔蒙像EPO为N-糖基化及其生物活性是依赖其上,或尤其产生EPO。
任何于该项技术已知之合适的培养程序及培养设备均可用来生长本发明转染的人类细胞。添加约0.1至20%(较佳0.5至15%)血清之常见无谷氨酰胺的基础培养基以及无血清无谷氨酰胺的常见基础培养基都可作为培养基用,此外,也可用不含动物来源蛋白质的常见无谷氨酰胺的基础培养基,较佳的使用无血清无谷氨酰胺的常见基础培养基。
可以用的血清如胎牛血清或成牛血清,较佳是使用胎牛血清。常见之可用的无谷氨酰胺的基础培养基为,例如无谷氨酰胺的Eagle极限基础培养基(MEM)、无谷氨酰胺的Dulbecco改进的Eagle培养基(DMEM)、无谷氨酰胺的Iscove氏DMEM培养基(N.Iscove及F.Melchers,Journal of Experimental Methods,1978,147,923)、无谷氨酰胺的Ham氏F12培养基(R.G.Ham,Proceedings of National Academy ofScience,1965,53,288)、无谷氨酰胺的L-15培养基(A.Leibovitz,American Journalof Hygiene,1963,78,173)、无谷氨酰胺的RPMI 1640培养基(G.E.Morre等,TheJournal of the American Medical Association,1967,199,519)、无谷氨酰胺的专用培养基和其合适比例之混合物。用于高密度细胞培养的强化常见细胞培养生长培养基已在该项技术所熟知,且已描述于如GB 2251249,其也非常适合作为本发明之无谷氨酰胺的培养基。
常见的添加物可以加到常见的无谷氨酰胺的基础培养基。常加入的添加物包括血清中的蛋白质以及视需要可以对细胞生长及/或细胞生存有正面影响之进一步成分。血清中的蛋白质如牛血清白蛋白(BSA)、运铁蛋白及/或胰岛素;可以对细胞生长及/或细胞生存有正面影响之进一步成分如大豆脂、硒及乙醇胺。取代谷氨酰胺及/或核苷的胺基酸可以依所用的细胞系加到培养基中,胺基酸的例子如异亮氨酸、亮氨酸、缬氨酸、赖氨酸、天冬酰胺、天冬氨酸、谷氨酸、丝氨酸、丙氨酸。视需要,谷氨酰胺可以低浓度加到常见的无谷氨酰胺的基础培养基中,通常少于1mg/l,较佳的少于0.5mg/l,以支持其生物合成作用(如转胺反应)。
若可编码蛋白质的外源DNA序列或能够改变可编码蛋白质之内生基因表达的外源DNA序列在转染后以可放大的(amplifiable)基因放大,相关的选择剂可以加到常见的无谷氨酰胺的基础培养基中,选择剂施用的浓度范围依所用的细胞系而定,通常用10μM及更高的浓度。
谷氨酰胺营养缺陷型人类细胞可以是固着依赖或固着独立,其可以作为起始材料来获得根据本发明的转染的谷氨酰胺营养缺陷型人类细胞,根据本发明,该转染的细胞能够产生蛋白质及能够生长在无谷氨酰胺的培养基中。若使用固着依赖人类细胞,如HT1080细胞系(ATCC No.CCL 121),其可以适应成为固着独立HT1080细胞系,能够悬浮生长于无血清培养基,尚未于文献中描述。
适应作用可以发生在以可编码蛋白质的外源DNA序列或能够改变可编码蛋白质之内生基因表达的外源DNA序列及可编码GS的外源DNA序列转染之前或之后。较佳的,细胞先以可编码蛋白质的外源DNA序列或能够改变可编码蛋白质之内生基因表达的外源DNA序列转染,再使其适应悬浮生长于无血清培养基,然后进一步以可编码GS之外源DNA序列转染。若需要,转染的细胞可以再使其适应悬浮生长于无血清无谷氨酰胺的培养基。
本发明之转染的谷氨酰胺营养缺陷型人类细胞可以是固着依赖或固着独立,并能够悬浮生长于无血清无谷氨酰胺的培养基。较佳的转染的谷氨酰胺营养缺陷型人类细胞系固着独立,且能够悬浮生长在无血清无谷氨酰胺的培养基。
适应作用使其成为能够悬浮生长于无血清培养基之固着独立细胞,可以藉由在第一步用含有血清的培养基适应细胞而达成。这可以经由如胰蛋白酶处理细胞,然后接着摇动或藉摇动释放细胞来达成。细胞然后在第二步藉接着减少血清含量来适应无血清培养基,在适应时,若使用选择剂,可以减少其用量,以避免抑制细胞生长。然而,细胞也可以在第一步藉由接着减少血清含量适应生长于无血清培养基,第二步适应成为能够悬浮生长的固着独立细胞,藉由如胰蛋白酶处理细胞然后接着摇动或藉摇动释放细胞来达成,两个步骤也可以同时施行。
较佳的,细胞系在第一步含血清培养基适应成为固着独立细胞用,藉由摇动释放细胞然后第二步经由接着减少血清含量来适应于无血清培养基。
如上所描述的培养基可以做为含血清培养基的基本。在此所定义之选择剂可以加到培养基中,选择剂施用的浓度范围取决于所用的细胞系,通常使用10μM及更高浓度。含有血清的培养基通常添加约0.1到20%,较佳的0.2到10%,最佳的0.5到5%血清,可以使用的血清如上所提及。接着减少血清含量可以藉由逐步减少血清含量,例如从10%到1%到0%来达成。
本发明转染的谷氨酰胺营养缺陷型人类细胞系用在产生蛋白质的方法中,藉由在适合用来表达该蛋白质及回收此蛋白质的条件下之培养基培养该细胞,产生的蛋白质是描述如上。如上所描述之常见的无谷氨酰胺基础培养基及常见的添加物可以作为培养基,合适的培养条件是如WO 96/39488所描述之习知的用于哺乳动物细胞体外培养者。
蛋白质可以从细胞培养物中以习知的分离技术分离,例如免疫亲和或离子交换管柱分划、沉淀、逆相HPLC、色层分析、色层焦集法、SDS-PAGE、胶体过滤。习于该项技术者应了解,适合用于有兴趣的多肽之纯化方法,可能需要根据多肽在重组细胞培养中表达的特性来做修饰。
实施例
实施例1.准备人类纤维肉瘤细胞系HT1080-R223
含有多人类EPO基因拷贝的固着依赖人类HT1080-R223细胞系是产业上用来生产EPO的细胞,一开始是由Transkaryotic Therapies公司所创造(剑桥,MA 02139(US)),其是源自固着依赖人类纤维肉瘤HT 1080细胞系。亲本HT1080细胞系(ATTC No.CCL121)具有产生EPO的能力,其通过类似于DNA构建物pREPO18(描述于WO 95/31 560)的DNA构建物pREPO22转染,除了DHFR基因是在相反方向以及pREPO22含有比pREPO18少约600碱基对同源序列。以后将此细胞系称为R223细胞系。
实施例2.R223细胞系的适应作用以能够悬浮生长于无血清培养基中
藉由胰蛋白酶处理释放在静置瓶中以附着培养生长的细胞,悬浮于专用的含有谷氨酰胺之无血清培养基(进一步以“HM9”提及),添加10%透析的胎牛血清(dFBS)及500nM MTX,然后以和震荡瓶培养。生长在六天后开始,细胞次培养到同样的培养基中(图1)。一旦可信的生长模式建立了,就减少培养基的血清含量至1%,再一次,可信的生长可在添加的血清完全用尽之前被再建立。
使添加血清和无血清的培养物过度生长以评估产量,结果显示于表1,并附有附着培养的数据。足够的适应作用之后,悬浮培养的比生长速率(即使没有血清)与添加血清之附着培养一样。
在添加血清的培养基中从附着培养到悬浮培养时的适应作用,EPO合成比速率下降50%,从24到12EU/106细胞/小时,然而,此速率在适应无血清生长之后,大体上会恢复至18EU/106细胞/小时。
表1
在适应无血清培养基之前或之后,R223细胞在附着培养及悬浮培养的生长及产量。
最大细胞数/mL×10-6 | 比生长速率h-1 | 累积细胞h/m1×10-6 | EPOEU/ml | qEPOEU/106细胞/h | |
附着培养(DMEM+10%dFBS500nM MTX) | 不适用 | 0.0157 | 不适用 | 不适用 | 24 |
悬浮培养(HM9+10%dFBS500nM MTX) | 1.4 | 0.0185 | 334 | 4199 | 12 |
悬浮培养(HM9(无血清)500nM MTX) | 1.1 | 0.0166 | 210 | 3600 | 18 |
实施例3.HT1080(ATCC CCL121)的适应作用以能够悬浮生长在无血清培养基中
HT1080细胞系ATCC CCL121是从美国典型培养物保藏中心(Rockville,Maryland,USA)取得,一开始细胞附着培养生长于含有10%胎牛血清(FBS)的DMEM中。
悬浮和无血清的适应作用可以根据图2的程序进行。为了开始悬浮培养,细胞通过胰蛋白酶处理从附着培养中释放,而释放的细胞再悬浮于加了2%FBS的HM9,这些接着以震荡培养,一旦细胞在加了2%FBS的HM9的悬浮生长建立了,以相同的培养基稀释培养物作子代培养,可信的HT1080细胞系悬浮生长在30天后建立(图3)。在那之后培养基血清含量减少,最后完全不见。细胞持续在无血清中继续次培养生长。
实施例4.氨对R223细胞系生长及产量的影响
当以谷氨酰胺作为能量基质时,氨是培养的细胞产生的代谢产物,具有细胞毒性并会抑制细胞生长,除此之外,它会藉由对细胞高基氏体pH的影响,抑制蛋白质糖基化。在瓶中无供应之培养的R223细胞通常产生5mM氨,在有养分供应之发酵槽培养则产生10mM。
在一开始评估氨的影响时,R223细胞系生长在震荡瓶中,没有加氨或加氨(2、5或10mM),重复培养之各氨浓度是在不同的pH值下得到,藉改变施加气体之CO2含量。此处之主要目标是决定氨对生长抑制的程度,及测试减低的pH能不能克服此生长抑制。
氨被发现会抑制细胞生长(表2),而减低的pH并不能缓和氨所造成的抑制作用,虽然用来减少pH的升高之pCO2本身可能造成一些生长抑制。
培养在仅3天后结束,加长培养是无效的,因为不挥发性的酸代谢产物累积会造成所有培养物pH下降数个pH单位。
表2
在不同的培养pH值中,氨对R223细胞生长及产量的影响。pH用施加气体的CO2含量调整。
pCO2% | 加入的氨mM | 起始pH | 每毫升的最大细胞数×10-6 | 比生长速率h-1 | EPOEU/ml | q-EPOEU/106细胞/h |
5 | 0 | 7.26 | 0.9 | 0.020 | 1719 | 35 |
10 | 0 | 6.93 | 0.7 | 0.017 | 1580 | 40 |
15 | 0 | 6.75 | 0.4 | 0.012 | 1186 | 56 |
5 | 2 | 7.15 | 0.8 | 0.018 | 1796 | 38 |
10 | 2 | 6.88 | 0.7 | 0.017 | 1928 | 42 |
15 | 2 | 6.80 | 0.3 | 0.009 | 1404 | 78 |
5 | 5 | 7.18 | 0.6 | 0.018 | 2639 | 40 |
10 | 5 | 6.91 | 0.6 | 0.014 | 1663 | 51 |
15 | 5 | 6.75 | 0.3 | 0.016 | 1626 | 45 |
5 | 10 | 7.17 | 0.5 | 0.016 | 2222 | 43 |
10 | 10 | 6.90 | 0.6 | 0.014 | 1966 | 50 |
15 | 10 | 6.75 | 0.2 | 0.008 | 1891 | 86 |
在接下来的实验(表3)pH通过调整培养基NaHCO3的含量来调整,并维持施加气体的CO2含量在无抑制作用的浓度。测试的pH范围从pH 7.0到7.5(要强调的是,在瓶中培养的培养基pH不能被控制,而且会大大的下降,即使最先两天的培养,所指的pH值是各培养之起始pH)。比生长速率在pH 7.5(NaHCO3于3g/l)时最大,但在这个pH下,因为10mM氨的存在使生长速率及最大细胞浓度减半,EPO合成比速率减少四倍(表3)。
当pH 7.25(NaHCO3于1.5g/l)时,相较于pH 7.5的培养,比生长速率减少,EPO合成比速率增加,然而因为有10mM氨存在,生长速率较不受影响,EPO合成比速率不受影响。
在最低的研究pH,pH 7.0(NaHCO3于0.75g/l),比生长速率进一步减少但仍比pH 7.5时较不受氨影响。EPO合成比速率因为加入的氨之存在而增加(这可能是因为生长速率降低)。
表3
在不同的培养pH值下,氨对R223细胞生长及产量的影响。pH通过改变培养基中NaHCO3含量来调整。
起始pH | 最大细胞数/ml×10-6 | 比生长速率h-1 | EPOEU/ml | qEPOEU/106个细胞/h | |
NaHCO3 3g/l | 7.49 | 1.3 | 0.030 | 951 | 16 |
NaHCO3 3g/l10mM氨 | 7.51 | 0.5 | 0.016 | 256 | 4 |
NaHCO3 1.5g/l | 7.26 | 1.1 | 0.027 | 1151 | 24 |
NaHCO3 1.5g/l10mM氨 | 7.27 | 0.7 | 0.021 | 1191 | 26 |
NaHCO3 0.75g/l | 7.02 | 0.9 | 0.020 | 995 | 26 |
NaHCO3 0.75g/l10mM氨 | 7.01 | 0.4 | 0.016 | 1009 | 42 |
实施例5
R223细胞系在5公升规模发酵槽培养的产量
对于R223细胞系,实施了三个5公升的发酵,控制在介于6.95和7.15的不同pH值(参见表4),每一个培养得到浓缩的营养供应,其含有胺基酸和葡萄糖,设计来维持主要的消耗养分在足够浓度。培养物在pH 7.15及pH 7.05生长的很好,但在pH 6.95则无法生长,可能是因为用来控制该pH之高浓度CO2。
表4
发酵槽培养之R223细胞系的产量及代谢。
发酵条件 | 最大细胞数/ml×10-6 | 倍殖时间小时 | 累积的细胞小时/ml×10-6 | EPOEU/ml | qEPOEU/106细胞数/h | 最终氨mg/l |
pH 7.15NaHCO3 2g/l | 1.0 | 46 | 197 | 10795 | 58 | 163 |
pH 7.05NaHCO3 2g/l | 1.0 | 51 | 195 | 11414 | 64 | 157 |
pH 6.95NaHCO3 2g/l | 0.2 | 105 | 62 | 2995 | 70 | 未做 |
实施例6.以GS转染的R223及GS转染子在附着培养的产量。
用来转染的细胞系来自实施例2之R223细胞系悬浮适应的无血清储备溶液,当这些细胞长到像大的多细胞聚集时,再将他们以胰蛋白酶处理使其成为实质上的单细胞悬浮。
转染从实施例2获得的约107单悬浮适应R223细胞系分液(在不含钙或镁的磷酸缓冲盐)是混合以20μg含有GS基因之线状DNA(DNA序列pCMGS Bam H1描述于Bebbington等,1992,Biotechnology 10,169-175)及使用Biorad Gene Pulser(450伏特,250μF)施以电穿孔。作为控制组者,等量分液之细胞在不加DNA下作电穿孔。
细胞以HM9培养基稀释,不加MTX,含有0或10%dFBS,及分布在96孔培养盘或25cm2瓶,35.5至37℃培养。
HM9培养基一开始含有2mM谷氨酰胺,但1天后稀释成0.5mM,然后十天后换成无谷氨酰胺的HM9培养基,并且在第1天或第10天要再加入MTX(500nM)于培养物中。
至于在不加DNA下作电穿孔的控制组细胞培养,没有生长。
以DNA电穿孔的细胞,有15个GS转染子可以在96孔盘鉴别出来,不管起始培养基有没有MTX,都可获得GS转染子。15个GS转染子中,有7个成功的进展到瓶培养(表5),剩下的8个GS转染子呈现异常的细胞型态,或生长的不好,遗弃之。
分离出的每一个起始的七个GS转染子,设置一组复制静置瓶培养,使用10%添加血清之无谷氨酰胺的HM9培养基,每隔1到4天,牺牲培养物以计数细胞数,并以EPO ELISA测量EPO浓度。从这些数据集成可以估计每一个GS转染子的EPO合成比速率,数据整理于表5及(作为比较)数据也包括无转染的R223细胞系,所有GS转染子相较于无转染的R223细胞系呈现提高的EPO合成比速率,最佳的GS转染子,3E10,具有高于无转染R223细胞系五到六倍之合成速率。
表5
R223细胞系GS转染子及无转染的R223细胞系之EPO合成比速率。
数据来自生长在含有10%dFBS之附着培养,在制造转染子的培养基中不加甲氨喋呤,但在转染后第一天或第十天再加到培养物中。
GS转染子 | 添加MTX(500nM)到转染盘的日子 | qEPO(附着)EU/106细胞数/h |
3B3 | 10 | 59 |
3E10 | 10 | 139 |
3E11 | 10 | 45 |
4D9 | 10 | 35 |
8F11 | 1 | 47 |
8F12 | 1 | 79 |
8G3 | 1 | 52 |
评估附着培养中无转染的R223细胞系 | 不适用 | 24 |
实施例7.适应GS转染子于悬浮培养及无血清培养基。
由实施例6所得到的七个GS转染子,GS转染子#3E10及GS转染子#8G3进行悬浮培养。
细胞附着培养生长于静置瓶,内含无谷氨酰胺的HM9培养基,添加10%dFBS及500nM MTX,于35.5至37℃。细胞在五或六天后藉摇动释放,再悬浮于同样的培养基中,于相同温度以震荡瓶培养,生长在二或三天后开始,细胞于相同培养基次培养一次,然后过度生长以评估产量。效价及产物合成速率至少高于生长在10%dFBS及500nM MTX的无转染R223细胞系两倍(表6)。
表6
GS转染子3E10及8G3在震荡瓶悬浮培养的生长及产量。无谷氨酰胺的HM9培养基含有10%dFBS及500nM MTX。
GS转染子 | 最大细胞数/ml×10-6 | 最大EPO效价EU/ml | q-EPO(悬浮)EU/106个细胞/h |
3E10 | 0.7 | 11113 | 57 |
8G3 | 1.2 | 10171 | 33 |
评估在悬浮培养生长之无转染的R223细胞系,于10%dFBS及500nM MTX中 | 1.4 | 4199 | 12 |
GS转染子3E10及8G3的悬浮培养,无谷氨酰胺的HM9培养基中dFBS含量是逐步减少,3E10从10%至2%至1%至0.2%至0.1%至0%,8G3从10%至2%至1%,并在各dFBS浓度进一步减少前,建立可信的细胞生长,8G3没有进一步适应于1%dFBS。
实施例8.GS转染子3E10在无血清悬浮培养的产量及代谢。
实施例7中适应于无血清生长的GS转染子3E10悬浮培养培养于无血清无谷氨酰胺的HM9培养基中,35.5到37℃。对于GS转染子3E10细胞系,培养在相同条件于含有谷氨酰胺的HM9培养基,其具有比无转染的R223细胞系快2到3倍的EPO合成比速率(表7)。
表7
GS转染子3E10细胞在无血清悬浮培养的生长及产量。
最大细胞数/ml×10-6 | EPOEU/ml | q-EPOEU/106/h | |
3E10(无谷氨酰胺的HM9培养基) | 1.1-1.2 | 9731-14234 | 40-67 |
R223(HM9培养基加6mM谷氨酰胺) | 1.0-1.6 | 3075-5818 | 13-24 |
GS转染的细胞的高EPO产量伴随代谢氨释放的减少,不同于在含有6mM谷氨酰胺HM9培养基中瓶培养典型会产生5mM氨的无转染之R223细胞系,GS转染子3E10于无谷氨酰胺的HM9培养基只产生1.8mM氨(表8)。
表8
GS转染子3E10之氨合成减少。
最大细胞数/ml×10-6 | 氨mM | q氨微摩尔/106个细胞/h | |
R223(含有6mM谷氨酰胺之HM9培养基) | 0.91.21.0 | 4.85.05.5 | 302327 |
3E10(无谷氨酰胺的HM9培养基) | 0.8 | 1.8 | 11 |
实施例9
分析产物品质。
由GS转染子3E10在瓶培养产生的EPO被免疫纯化并分析其糖型分布,图4显示EPO之等电点聚焦法(IEF)胶体分析,其是得自最高细胞浓度时及收集生长于无谷氨酰胺的HM9培养基之3E10细胞培养物,如实施例8所描述,包括从生长在HM9培养基之无转染R223细胞系之可相较的样本。从GS转染子3E10产生的EPO(相较于无转染的R223细胞系)呈现更酸的异构物,分析IEF胶可以定量异构物的分布和计算理论上异构物的相对活性(IRA)。
表9
分析来自于无转染的R223细胞系及GS转染子3E10之EPO异构物相对活性。
条带编号 | 比活性* | |
更碱 | 1 | |
2 | ||
3 | ||
4 | ||
5 | ||
6 | 0.071 | |
7 | 0.194 | |
8 | 0.273 | |
9 | 0.373 | |
10 | 0.658 | |
11 | 0.989 | |
12 | 0.999 | |
13 | 1.000 | |
14 | 0.796 | |
15 | ||
16 | ||
更酸 | 17 | |
其他次要条带 |
无转染的R223 | |
各异构物百分比 | 活性(百分比×比活性) |
5.2 | |
5.2 | |
6.3 | |
11.4 | |
10.5 | |
9.2 | 0.7 |
8.8 | 1.7 |
8.7 | 2.4 |
5.5 | 2.1 |
4.4 | 2.9 |
4.3 | 4.3 |
4.5 | 4.5 |
1.4 | 1.4 |
0.2 | 0.2 |
14.4 | |
总合=100% | 总合=IRA=20.2 |
GS转染子3E10 | |
各异构物百分比 | 活性(百分比×比活性 |
2.5 | |
3.2 | |
3.6 | |
6.0 | |
6.2 | |
7.5 | 0.5 |
10.5 | 2.0 |
11.6 | 3.2 |
9.3 | 3.5 |
9.5 | 6.3 |
7.8 | 7.7 |
6.1 | 6.1 |
5.3 | 5.3 |
1.7 | 1.4 |
0.5 | |
8.7 | |
总合=100% | 总合=IRA=36.0 |
*活性数据来自EP-A 0428 267
数据指出了GS转染子3E10有相当大的产物品质提升(表9及10),IRA几乎两倍高于控制组无转染的R223细胞系培养。
也决定了来自GS转染子3E10之EPO的假设N-多醣电荷“Z”,表10没有决定无转染的R223细胞系之“Z”,然而,GS转染子3E10(273于最大细胞浓度及265于收集时)之“Z”值超过瓶培养(183-228)无转染R223细胞系的值。“Z”是根据Hermentin等于Glycobiology,1996,6,217-230决定;将个别%-唾液酸化的(sialyated)异构物之部分乘以对应的该异构物负电荷,可得Z,视其是否缺乏唾液酸基/中性、单涎、三-、四或五涎而定。该乘积之数学总合术语是Z。Z数字是关于活体内给予的治疗上糖蛋白质(Hermentin,ibd.)之清除速率。
表10
来自无转染R223细胞系及GS转染子3E10之EPO产物品质分析。
效价EU/mL | 异构物相对活性 | ’Z’ | |
R223亲本(加6mM谷氨酰胺之HM9培养基)峰收集 | 3518 | 25.2 | 未做 |
6150 | 20.2 | 未做 | |
3E10(无谷氨酰胺的HM9培养基) 峰收集 | 3921 | 43.6 | 273 |
6862 | 36.0 | 265 |
实施例10.在6公升发酵槽无血清悬浮培养之GS转染子3E10的产量、代谢及产物品质。
GS转染子3E10是生长在分批培养于气举式(airlift)发酵槽,培养基是无谷氨酰胺的HM9,不含血清,每一个培养有浓缩的养分供应,其含有胺基酸及葡萄糖,设计使主要消耗之养分维持在足够浓度。结果显示在表11,也包括无转染的亲本R223发酵数据,用于比较。
GS转染子呈现延长的生存能力,所以培养期间增加,产物合成的比速率相同于无转染的亲本细胞系,但较长培养寿命造成最大产物浓度增加,氨累积至少低于GS转染子四倍,GS转染子产物品质(以IRA测量)改善。
表11
GS转染子3E10细胞在无血清悬浮培养的生长及产量,产生的EPO产物品质用IRA定量。
发酵条件 | 最大细胞数/ml×10-6 | 最大CCH*/ml×10-6 | 收集日 | 氨mg/L | 收集效价EU/ml | 整体qEPOEU/106细胞数/h | 收集时的IRA |
无谷氨酰胺 | 1.03 | 306 | 21 | 41 | 20936 | 69 | 43.6 |
无谷氨酰胺 | 1.13 | 360 | 24 | 20 | 20150 | 52 | 42.6 |
无谷氨酰胺 | 0.94 | 319 | 24 | 15 | 18680 | 49 | 48.5 |
以标准方法用谷氨酰胺之亲本R223 | 1.11 | 216 | 13 | 165 | 12225 | 58 | 28 |
*累积细胞时数,细胞浓度的时间积分[Renard等,1988,Biotechnology Letters10(9):1-96]。
实施例11.在基于Iscove氏的培养基中无血清悬浮培养GS转染子的产量及代谢
实施例7中适应于无血清生长的GS转染3E10是培养在震荡瓶,在无血清无谷氨酰胺的Iscove培养基,亲本细胞系R223是对应生长在相等的添加谷氨酰胺之培养基。
所用的培养基是Iscove修饰的Dulbecco培养基,含有Iscove添加物(牛血清白蛋白0.4g/L、人类全(holo)运铁蛋白30mg/L)、重组人类胰岛素10mg/L、LutrolF68 1g/L及乙醇胺60μL/L。用于培养R223细胞系的培养基含有4mM谷氨酰胺,但用于GS转染的细胞系则不加谷氨酰胺,改以4mM谷氨酸钠加4mM天冬酰胺。
GS转染的细胞系3E10之产物合成比速率大约50%高于无转染的亲本是R223(表12),转染的细胞系3E10之氨生成比速率低七倍(表13)。
产物是从这些个别培养纯化而来,并在等电点聚焦法(IEF)胶体上分析,此分析结果显示于图5,其显示染色后的IEF胶。IEF胶在pH 2.5到6.5变性条件下跑,并以考马斯蓝染色。
图5解说 | |
泳道1、3、6和7 | 空白 |
泳道2和7 | pI标记 |
泳道4 | 从在含有谷氨酰胺的Iscove培养基中生长的R223免疫亲和纯化的产物 |
泳道5 | 免疫亲和纯化的产物,其是从从在无谷氨酰胺的Iscoves培养基中生长的GS转染的R223细胞系之转殖3E10 |
来自R223的产物至少有13条可见的条带,分布在整条胶体上;来自GS转染的细胞系3E10生成的产物,较碱的条带(胶体上方)强度比从细胞系R223产物条带弱,但细胞系3E10较酸的条带(胶体下方)则强度增加。有至少一条额外的酸性条带可在GS转染子3E10产物中可侦测到,但R223产物侦测不到亲本,这显示来自GS转染的3E10的产物的唾液酸化程度增加。
表12
在无血清悬浮培养于Iscove培养基中的GS转染子3E10细胞之生长及产量
最大存活细胞数/mL | EPOEU/mL | qepoEU/106细胞数/h | |
生长在无谷氨酰胺的Iscove培养基之3E10 | 0.55 | 1340 | 11.5 |
生长在含有谷氨酰胺Iscove培养基之R223 | 0.72 | 993 | 7.3 |
表13
在无血清悬浮培养于Iscove培养基中GS转染子3E10细胞之氨生成减少
最大存活细胞数/mL | 氨mM | q氨纳摩/106细胞数/h | |
生长在无谷氨酰胺的Iscove培养基之3E10 | 0.55 | 0.61 | 2.6 |
生长在含有谷氨酰胺Iscove培养基之R223 | 0.72 | 2.39 | 17.4 |
通过光密度扫描IEF-凝胶图象进一步量化图5的凝胶数据。计算出了每个条带代表的产物的相对比例。数据总结在表14中,每个条带的相对比例表示为占凝胶各个泳道上总产物的百分比。
酸性更强的条带(8-14)具有最高的生物学活性,而酸性较弱的条带活性相对较低或没有活性。从凝胶(图5)和光密度扫描图谱(见图6,凝胶泳道4和图7,凝胶泳道5)显见,GS转染的细胞系的产物,GSR223在酸性更强的同种型中富含。对于未转染的细胞系,R223,条带8-14占了总产物的44%,而GS转染的细胞系,GSR223,该比例达到了73%。在图6和7中,峰是根据图5中凝胶条带的编号进行辨别的。
R223 | GS转染的细胞系GSR223 | |||
条带编号 | 占总数的比例 | 占总数的比例 | ||
1 | 20.626 | 条带1-7的总和=55.735% | 1.185 | 条带1-7的总和=26.865% |
2 | 9.604 | 1.346 | ||
3 | 3.291 | 1.913 | ||
4 | 1.934 | 2.54 | ||
5 | 4.593 | 3.362 | ||
6 | 7.870 | 7.265 | ||
7 | 7.817 | 9.254 | ||
8 | 15.905 | 条带8-14的总和=44.263% | 18.347 | 条带8-14的总和=73.137% |
9 | 13.384 | 18.87 | ||
10 | 8.103 | 14.339 | ||
11 | 5.522 | 11.978 | ||
12 | 1.349 | 7.609 | ||
13 | 0 | 1.482 | ||
14 | 0 | 0.512 | ||
总计=99.998% | 总计=100.002% |
Claims (8)
1.一种谷氨酰胺营养缺陷型人类细胞,所述细胞用可编码蛋白质的外源DNA序列或能够改变可编码蛋白质之内生基因表达的外源DNA序列及可编码谷氨酰胺合成酶的外源DNA序列转染,其中,这些外源DNA序列位于一个或多个DNA构建物上,该转染的细胞能够产生该蛋白质并能够生长在无谷氨酰胺的培养基中,所述蛋白质是糖基化蛋白质。
2.如权利要求1所述的谷氨酰胺营养缺陷型人类细胞,其中,所述外源DNA序列位于一个以上DNA构建物上。
3.如权利要求1或2所述的谷氨酰胺营养缺陷型人类细胞,其中,所述谷氨酰胺营养缺陷型人类细胞系永生的谷氨酰胺营养缺陷型人类细胞。
4.如权利要求3所述的谷氨酰胺营养缺陷型人类细胞,其中,所述永生的谷氨酰胺营养缺陷型人类细胞系人类纤维肉瘤细胞。
5.如权利要求4所述的谷氨酰胺营养缺陷型人类细胞,其中,所述人类纤维肉瘤细胞系HT1080细胞系。
6.如权利要求1-5中任一项所述的谷氨酰胺营养缺陷型人类细胞,其中,所述转染的细胞被固着独立并能够悬浮生长在无血清无谷氨酰胺的培养基。
7.一种产生糖基化蛋白质的方法,其特征在于,所述方法包含步骤
a)将权利要求1所述的谷氨酰胺营养缺陷型人类细胞在适合所述糖基化蛋白质表达的条件下培养于培养基中,和
b)取得该糖基化蛋白质。
8.一种谷氨酰胺合成酶在谷氨酰胺营养缺陷型人类细胞中作为可选择标记的应用。
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CN2009100029634A Division CN101492656B (zh) | 2002-01-17 | 2003-01-17 | 能够产生蛋白质及能够在无谷氨酰胺的培养基上生长的谷氨酰胺营养缺陷型人类细胞 |
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GB8601597D0 (en) | 1986-01-23 | 1986-02-26 | Wilson R H | Nucleotide sequences |
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