CN1228119A - 用于病毒生长的固定化细胞系 - Google Patents
用于病毒生长的固定化细胞系 Download PDFInfo
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Abstract
本发明涉及来自原代鸡胚成纤维细胞的固定化细胞系的生产及其应用。该细胞可用作病毒增殖、重组蛋白表达和重组病毒生产的基质。
Description
发明领域
本发明涉及细胞生物学和病毒学领域。更具体而言,本发明涉及用于病毒增殖的固定化细胞系的应用。
发明背景
1931年,Alices Miles Woodruff和Goodpasture介绍了培养病毒的一种新方法。他们报导,禽痘病毒可以在发育鸡胚的尿囊绒膜上生长。接种病毒后,在膜上出现含病毒的病斑。与用作早期病毒研究基质的动物相比,禽蛋相对较便宜,易于获得。禽蛋具有多种细胞,膜易于为不同病毒感染,并可保持可控制的稳定状态。由于可以方便地提供多种病毒易感的多种细胞类型,鸡胚对病毒学的发展作出了重要贡献。
尽管禽蛋支持多种病毒株的复制,但感染禽蛋和维持病毒生长的方法既费时又费力。例如,尿囊绒膜接种时,首先在蛋壳和壳膜上打一个孔。在气囊处的蛋壳上打孔,让空气进入壳膜和尿囊绒膜之间,形成一个人工气囊,将样品置于此处。样品接触绒膜上皮,病毒在膜上生长成病斑。正如预料的那样,随着细胞培养技术的出现,使用禽蛋进行病毒复制已经减少。
多种细胞均可以在体外生长。与禽蛋相比,细胞培养物容易维持,并能保持在高度可控制的环境下。然而,仍有一些病毒株在含胚卵细胞上生长似乎较在培养细胞上好。此外,很多培养细胞系携带内源的感染因子,包括原虫,低水平细菌污染,内源病毒等。某些支持病毒复制最有效的细胞类型在病毒原种生产方面存在问题,即细胞含有内源病毒。内源病毒或以低水平复制或在细胞感染另一种病毒株后激活。例如,已知啮齿动物细胞携带内源病毒,培养中啮齿动物细胞的电镜照片经常可见细胞内存在着可识别的病毒粒子。污染的细胞系不能用作活的或灭活商用疫苗的基质。
对于某些病毒,选用的病毒复制方法是含胚鸡卵。例如,人流感病毒、狂犬病毒、犬温热病毒、禽疱疹病毒1型、呼肠病毒和禽痘病毒优先在含胚卵(因为卵支持高滴度病毒原种的生长)或来自含胚卵的原代细胞上生长。其它情况下,病毒在卵中生长,因为需要可靠的无病毒细胞基质。
原代细胞培养物是由完整组织新分离的细胞培养物。这些细胞通常为无病毒材料的良好来源,非常适合作为病毒复制的宿主细胞。原代细胞用来复制病毒并非总是有效,原代动物细胞在培养物中的寿命有限,最终会衰老。衰老细胞停止分裂,最终死亡。培养细胞的长期分裂能力取决于几个参数,包括细胞来源的种类,培养时组织的年龄。衰老的细胞无法长期培养,因而不能作为商用病毒原种生长的可增殖宿主。
某些原代细胞不会衰老,而获得了无限增殖的能力。啮齿动物细胞似乎非常容易发生自发的永生化(Curatolo等,In Vitro 20:597-601,1984),但普通人和鸟细胞即使能够自发永生化,也非常少见(Harvey等,Genes andDevelopment 5:2375-2385,1991;Pereira-Smith,J.Cell Physiol 144:546-9,1990;Smith等,Science 273:63-67,1996)。为何特定的细胞群体会永生化有多种原因。用已知诱导基因突变的试剂处理细胞可以诱导细胞永生化。有些人推测相对于衰老的生长停止主导着永生化,灭活生长抑制基因的事件可以导致永生化(Perira-Smith等,Proc.Natl.Acad.Sci.(USA)85:6042-6046,1988)。
获得永生化无病毒细胞可以无需或减少使用原代动物组织培养物。原代培养物通常是不定的细胞群体,并且经常被污染。这些培养物经常不能满足商用疫苗制品的强制性要求。原代细胞培养物可以被Cirodnaviredeae(如Chickenenima病毒)或Egg Drop综合征病毒污染。例如,Marek病疫苗(一种活病毒疫苗)可以在鸭蛋上作为病毒原种生长,用于家禽接种。1976年,接种了该疫苗的鸡群显示出Egg Drop综合征的迹象,这是由鸭腺病毒引起的,据信该病毒污染了疫苗原种,并已改变可在鸡中生长。
在疫苗工业中,对产品安全、稳定性和效力的强制性要求驱使各公司采用细胞系作为现在应用卵基和原代细胞的疫苗基质实践的最佳替代途径。在美国和欧洲,由于对疫苗基质的强制性环保要求日益严格,在人用和兽用疫苗制品的生产过程中均需考虑安全和稳定性。考虑到美国政府对试验、研究和驯养脊椎动物利用和保护的规定和动物保护法(7 U.S.C.ξ2131)规定,在所有情况下,应当考虑用体外生物系统等方法代替活体动物模型系统,鉴定适合病毒生长的细胞系代替含胚卵也是有利的。需求的细胞是无病毒并支持外源病毒生长从而产生动物疫苗制品的细胞。
发明概述
本发明涉及自发永生化的鸡成纤维细胞系的鉴定和获得该细胞系的方法。具体而言,本发明涉及来自原代鸡胚成纤维细胞的自发永生化细胞系,其具有自发永生化细胞系UMNSAH-DF1的特征,根据布达佩斯条约的规定保藏于ATCC。此外,本发明涉及这些细胞的培养物和这些永生化细胞系的永生化亚克隆,它们支持病毒复制。
一方面,本发明的永生化细胞含有病毒,本发明的另一种永生化细胞含有至少一种能够指导重组蛋白在细胞中表达的载体。在一个实施方案中,本发明的细胞表达重组蛋白,另一方面,本发明细胞中包含的载体编码重组病毒的至少一部分。在另一个实施方案中,该载体为逆转录病毒载体。
另一方面,本发明公开了一种由鸡胚成纤维细胞产生永生化细胞系的方法,包括以下步骤:培养原代鸡胚成纤维细胞;将培养物中的成纤维细胞传代直至它们开始细胞衰老;在细胞衰老时浓缩细胞以维持大约30%到60%的培养物汇合;鉴别非衰老细胞转化灶;培养非衰老细胞30代以上。
再一方面,本发明公开了一种在细胞中培养病毒的方法,包括以下步骤:培养来自原代鸡胚成纤维细胞的自发永生化细胞系;用病毒感染细胞;使病毒在细胞中复制;收集在细胞中复制的病毒。
优选实施方案详述
目前还没有基本无病毒、无病毒蛋白或非化学转化的禽细胞系供应。原代细胞系难以持续产生用于病毒原种生产,必需分别证实为无污染的基质才能用于病毒生长。本发明公开了鸡胚成纤维(CEF)细胞包括来自EastLansing Line(ELL-0)鸡胚的细胞的永生化。
本文中,永生化指能够连续培养30代以上的非啮齿动物细胞,培养物中倍增时间保持为约1-2天,已连续培养大约6个月以上。通常认为鸟细胞在传代大约20-25代后为永生化。永生化细胞与转化细胞的区别在于,不象转化细胞,永生化细胞是密度依赖的和/或生长停滞(如接触抑制)的。转化细胞能够在软琼脂中生长,注入试验动物后通常能够形成肿瘤。本发明的细胞可用作培养毒或表达重组蛋白或病毒的基质,特别是强调细胞不含污染病毒或病毒蛋白时。该细胞也可用于研究细胞衰老和永生化的内在机制。
来自10日龄ELL-0卵的鸡胚成纤维细胞(CEF)原代细胞通过以下方法获得:取10日龄胚胎躯干,将组织绞碎,将细胞置于培养物中。受精卵获自Hy-Vac(Adel,Iowa)。卵及产卵者由供应商证实无禽流感病毒(A型)、禽呼肠病毒、禽腺病毒(I-III群)、禽脑脊髓炎病毒、禽痘病毒、新城疫病毒、副粘病毒(2型)、支原体、沙门氏菌和其它已知感染家禽原种的感染因子。原代细胞的分离和永生化细胞的鉴定见实施例1。
进行细胞鉴定是因为发现永生化细胞时,要对细胞群体进行选择以研究细胞衰老的影响。已知人和禽细胞在组织培养条件下是最难永生化的细胞。不象啮齿动物细胞,未见有来自正常供体的人或鸡成纤维细胞永生化的方法报导(Smith等,Science 273:63-67,1996)。在禽成纤维细胞中,未处理细胞一般可维持20-25代。即,这些禽细胞的原代培养物传代30次后已死亡或将死亡。如本发明所述,传代至20次时,在大约12次到20次将细胞转移至较小的培养皿上并浓缩(见实施例1)。观察生长更快的转化灶,使用克隆环(cloning ring,Bellco Glass,Inc.Vineland,N.J.)分离这些转化灶并在培养物中扩增。
本文中衰老意指每日群体倍增为约0.5或更少。本发明中,永生化细胞指传代超过30次的细胞,每日群体倍增为约0.6-1.2(使用锥虫蓝排除每日计数总细胞和活细胞来确定),优选为约0.7-1.0,同时具有接触抑制、密度依赖和正常细胞形态。
如实施例1所述,获自原始鉴定的转化灶的细胞已经历超过400次(群体倍增)和超过160次传代。本文中转化灶指可与周围细胞形态区分的形态一致的细胞群。这些细胞转化灶可以移出和亚克隆,供进一步研究。本发明的细胞持续维持每22-24小时倍增一次。这些细胞表现为接触抑制,逆转录酶阴性(见实施例2),密度依赖停滞,非整倍体(染色体涂布分析:在油镜下,核型为二倍体/四倍体核型的混合物,某些细胞表现出明显的染色体1易位),长成1.1-1.9×105细胞/cm2的高密度。未见多核的巨大细胞。细胞表型一致。细胞也保持快速生长的特征,这对病毒增殖有重要意义。
细胞未被转化,这可通过它们不能在软琼脂上生长(实施例3)证实。此外,将细胞注入鸡翅不会产生肿瘤(见实施例4)。本发明例举的细胞命名为UMNSAH-DF1细胞,根据布达佩斯条约的规定,保藏于美国典型培养物保藏中心(ATCC),12301 Parklawn Drive,Rockville Maryland,20852,保藏号为CRL-12203,保藏日为1996年10月11日。
本发明也涉及培养中的本发明永生化鸡胚成纤维细胞和本发明永生化细胞的亚克隆。例如,本发明的细胞经鉴定为自发永生化细胞。细胞获自已知为无病毒、无化学污染的产卵者(产生作为本发明原料的胚胎组织的母鸡),用来产生本发明细胞的胚胎组织也是无化学污染(即未经已知的致癌剂或其它已知转化啮齿动物细胞的试剂处理)、无已知病毒的。本发明的永生化细胞在培养物中时,可以进一步亚克隆以选择其它在细胞群体中有可能不同的参数,这些细胞仍保持接触抑制和病毒感染敏感性。
测试细胞复制HVT(火鸡疱疹病毒)、禽疱疹病毒(血清型III)、禽痘病毒和呼肠病毒的能力。可以测试细胞为多种其它病毒复制Circodnavirideae,鸡HSV血清型II的能力,并已将细胞作为转染基质进行了测试。该细胞可用于增殖禽和非禽病毒。实施例5详述了增殖HVT,禽痘病毒和呼肠病毒的方法。这些细胞可用作病毒生产的基质,具体而言,这些细胞可用于逆转录病毒生产,因为这些细胞和产卵者(即其母本)未测定到逆转录病毒感染。这些细胞能够支持禽肉瘤白血病病毒和Rous肉瘤病毒的复制。
为生产病毒原种,本发明的细胞可以接种至组织培养瓶、滚瓶、搅拌培养物、中空纤维反应器或其它大规模培养系统。滚瓶病毒增殖时,细胞以大约2-5×104细胞/cm2表面接种。起始病毒生长的感染复数(感染性病毒粒子与细胞之比)取决于病毒株。病毒学领域的技术人员及特定病毒和病毒株培养的技术人员无需过多的试验,通过感染增殖、温度、基质变化等标准操作即可使病毒原种的产率最高。
感染后收获病毒获得感染性病毒原种的方法根据病毒株也有所不同。有包膜病毒进入培养基较无包膜病毒要慢。病毒原种可获自培养基或与条件培养基合并的细胞裂解物。对裂解病毒(在病毒侵入期间有效裂解细胞的病毒),低速离心除去细胞碎片后收获条件培养基(如含病毒的已用过(spent)培养基)即可。由多种病毒株收获和保存病毒的方法在本领域是公知的。
在本领域中也已知有多种方法可以对来自细胞培养物的病毒定量。例如,疱疹病毒科成员和多种在细胞单层表面产生细胞病理学转化灶的病毒的病毒原种滴度,通过噬斑试验(斑形成单位/毫升培养液或对疫苗接种物病毒定量为斑形成单位/剂)或以半数组织培养感染量(TCID50)可以方便地定量。快速裂解病毒通过TCID50(在限定时间内能感染50%培养物的病毒原种剂量或稀释度)可以更好地定量。培养和对病毒定量的方法在本领域是已知,病毒定量方法的教导可见Field等编,Fundamental Virology 1991,Raven Press,New York,或Mandell等编,Principles and Practice ofInfectious Diseases,1985,John Wiley & Sons,New York。
除支持病毒生长之外,本发明的细胞也可用作产生重组病毒包括逆转录病毒的包装细胞系。这些细胞还可用于产生重组蛋白包括病毒蛋白等。使编码重组蛋白的核酸整合进核酸载体的方法在本领域是公知的,所述核酸载体处于能够指导蛋白在真核细胞(如本发明的永生化细胞)中表达的调控因子控制之下。表达载体是能够指导重组蛋白表达的可复制核酸片段。通过本领域的杂志出版物和供应商可以得到多种表达载体包括逆转录表达载体。可复制的表达载体成分通常包括但不限于以下一种或多种:复制起点,一个或多个标记基因,增强子,启动子,可任选的信号序列和转录终止序列。选择或标记基因编码的蛋白用来鉴定转化或转染细胞群体。典型的选择基因编码的蛋白能赋予对抗生素或其它毒素的抗性,补充营养缺陷型的缺陷或提供不能由复合培养基获得的关键营养素。
带有编码重组蛋白的核酸的表达载体转染进细胞,用来指导重组蛋白在本发明的永生化细胞中表达。优选该载体能够编码任何能在鸡胚成纤维细胞中表达的重组蛋白,包括但不限于病毒蛋白,如逆转录酶和/或病毒结构蛋白。在细胞中产生重组蛋白的载体的实例包括产生肿瘤抑制蛋白或病毒结构蛋白的逆转录病毒载体,如以下文献中所报导的:Givol等,Oncogene 11(12):2609-2618,1995,Givol等,Cell Growth & Differentiation5(4):419-429,1994,Akiyama等,Virology 203(2):211-220,1994和Boyer等,Oncogene 20:457-66,1993。
本发明的细胞可用作表达重组病毒包括但不限于重组逆转录病毒的基质。本发明的细胞适合作为遗传工程病毒的包装细胞系,用于基因治疗等。使用特定细胞系作为包装细胞系的构建和方法在本领域中是已知的。例如,Boerkoel等(Virology 195(2):669-79,1993)报导了使用原代鸡胚成纤维细胞作为包装细胞系包装病毒的方法。这些方法可用于包装本发明永生化细胞中的病毒。
由于多数禽细胞系和所有转化禽细胞以及实际上所有的小鼠转化细胞系要么含有内源病毒等病毒污染物,要么产生病毒蛋白,它们不适合用来生产人或动物疫苗。这些细胞不能用来生产重组蛋白,因为内源污染物可以污染纯化的重组蛋白制品。有利的是,本发明的细胞为解决这些问题提供合适的替代途径。
本发明的细胞可以用作支持来自其它细胞的病毒生长的基质。这些其它细胞包括原代细胞,或在其它细胞或胶原、层粘连蛋白等细胞外基质蛋白存在时培养表现出生长更好或寿命更长的培养细胞。在一个实施方案中,这些细胞与病毒混合,然后与本发明的细胞混合,比例为所述细胞:本发明的细胞为约1∶5到1∶20,更优选为约1∶10(大约1个细胞与本发明的细胞10个)。混合细胞然后进行培养。在第二个实施方案中,这些细胞与病毒混合,并涂布于已贴附在组织培养表面的本发明的永生化细胞表面。本发明的细胞用来支持其它细胞,本发明的细胞可以提供生长因子等以及细胞外基质成分等(在此无意限制本发明的范围),以在其它细胞生产病毒时支持其它细胞。
本发明的具体实施方案将在以下详述,已对本发明范围之内的可能变化给出了提示。对本领域技术人员来说,有多种替代的技术和方法可以用来成功地实现本发明。
实施例1:自发鸡胚成纤维细胞系的建立
两打ELL-0蛋购自East Lansing USDA种禽场。将蛋在无菌的分离孵育箱中孵育10天,用来获得原代培养物。胚胎组织使用胰蛋白酶/EDTA溶液分离,涂布于含10%胎牛血清(Gibco),1%抗生素/抗霉菌素(Gibco)和2mM L-谷氨酰胺的DMEM培养基(Gibco)。分离的细胞悬液收集于50毫升离心管中,其中所含的10%胎牛血清用来灭活胰蛋白酶,700×g离心10分钟。
细胞重悬于10毫升Dulbecco改良Eagle培养基,其中添加了36μg/ml胰岛素(Sigma),1.6μg/ml转铁蛋白(Sigma,St.Louis,MO),2mM L-谷氨酰胺,10%胎牛血清,1%抗生素/抗霉菌素,转移至25cm2 corning组织培养瓶,40.5℃在5% CO2,95%空气中温育。温育24小时后,更换培养基,原代培养物含大量外植块,其中心为表皮样细胞,周围为成纤维细胞。
培养物长至汇合(5天),使用胰蛋白酶/EDTA溶液(0.05%胰蛋白酶和0.02%乙二胺四乙酸(EDTA),溶于PBS)由平皿中移出,重新涂布进行第2次传代。第2次传代时,某些细胞冰冻于含50%DMEM培养基,12%DMSO和38%胎牛血清的条件培养基中。这些细胞在液氮的气相冰冻24小时,然后转移至液氮的液相中长期保存。
第2次传代的细胞(P2)以2.7×104细胞/cm2的接种密度重新涂布。细胞传代培养数月。培养的成纤维细胞快速生长8-9代,然后生长开始变慢,有明显的细胞死亡。在转捩期间,细胞使用ATV溶液(8gm/l NaCl,0.4gm KCl,1gm葡萄糖,0.58gm NaHCO3,0.5gm胰蛋白酶(Difco 1:250),0.2gm versene(二钠盐),1000mL)传代。细胞在Dulbecco改良Eagle培养基中生长,其中添加了36μg/ml胰岛素(Sigma),1.6μg/ml转铁蛋白(Sigma),2mM L-谷氨酰胺,10%胎牛血清,1%抗生素/抗霉菌素。可以见到第11代细胞(P11)大部分死亡或将死亡;但是,有一小细胞亚群似乎保持着活力。P11细胞置于平皿上4周,每3天更换新鲜培养基。一些细胞被冰冻,其余细胞浓缩至较小的区域,再生长2周,直至它们汇合可以再次传代。P15细胞在细胞形态上似乎更为一致,生长速率为每日群体倍增0.32。P20时,群体倍增增加至每日大约0.7-0.8。此时,细胞似乎具有非常均一的形态。细胞被命名为UMNSAH/DF#1,已连续培养超过19个月。细胞已传代160次。P5细胞冰冻(如上述)并解冻。扩增亚克隆的细胞,该方法的重复性通过鉴定其它克隆来证实。另外一些亚克隆获自P11。
实施例2:测试细胞的病毒污染物
使用PCR鉴定污染核酸片段来测试本发明细胞的病毒污染物。有多种针对不同病毒的商用检测试剂盒可用来确定本发明的细胞是否含有污染病毒。同样,也有测定病毒抗原的商用试验(如商用的ELISA试验等),此处抗原来自多种不同病毒。这些试验可以使用常规的试验手段用于本发明的细胞,以证实培养物不含污染病毒。
在一个系列的试验中,测试细胞的逆转录酶活性。来自快速生长培养物的1×106细胞分离于4ml培养基中。培养基在-80℃冻融几次裂解细胞。带有裂解细胞的培养基用10%甘油梯度分层。该梯度使用SW40转头(Beckman Instruments,Palo Alto,CA)40000rpm离心60分钟。如果有病毒粒子的话就会沉淀。弃去培养基,沉淀重悬于20μl Nonidet P-40(SigmaChemical Co.,St.Louis,MO)。
将eppendorf管加热至41℃。将5μl样品加入至45μl逆转录酶混合物中,其中含有45mM Tris,pH7.8,2mM 2-β巯基乙醇,2mM乙酸锰,0.1%Triton X-100,dATP,dCTP,dGTP(Boehringer Mannheim Biochemical,Indianapolis,IN)各10μM,2.4μg polyA(Sigma),60ng引物dT 12-19(Pharmacia),0.4μCi/反应3H胸苷三磷酸(15,000-28,000cpm/pmole活性,Amersham)。
反应在41℃温育1小时。阴性对照包括5μl重蒸水和45μl混合物。在试验中包括两个已知的阳性对照。通过加入1毫升10%三氯乙酸(TCA,Columbus Chemical Industries,Inc.,Columbus,WI)终止反应。混合物过Whatman GF/C玻璃0.45微米预滤器。使用5%TCA洗涤几次。滤纸转移至Beckman Instruments Scintillation Counter,使用含5毫升液闪液的液闪瓶。样品在050-600窗设置(window setting)计数。较混合物背景(阴性对照)增加3倍以上视为阳性。
经测定原代培养物和本发明的永生化细胞均为逆转录酶阴性。逆转录酶试验的进一步信息见Crittenden等,Virology 57:128-138,1974。
实施例3:软琼脂集落形成试验评价细胞的致瘤潜能
为测定致瘤潜能,测试细胞在软琼脂中的生长。软琼脂的制备如下:在21.6ml加富McCoy 5A培养基〔Gibco,120ml胎牛血清(热灭活),5ml丙酮酸钠(2.2%储存液),1ml L-丝氨酸(21mg/ml储存液),5ml L-谷氨酰胺(200mM储存液),12.5ml Hepes(1M储存液)〕,5.9ml天冬酰胺(4.4mg/ml过滤除菌储存液)中混合12毫升2%琼脂糖。7毫升温热培养基/琼脂倒入100mm2组织培养皿,在室温下固化1小时。
通过胰蛋白酶处理由活跃生长的培养物中移出细胞,获得悬浮于含10%胎牛血清(含L-谷氨酰胺和抗生素-抗霉菌素)的新鲜DMEM培养基中的单细胞悬液。大约1×106细胞加入4.25ml含10%胎牛血清,0.75 ml 1%琼脂糖和2β巯基乙醇的DMEM培养基中。需要小心确定温热的培养基/琼脂糖在加入细胞时是42℃。将5毫升上述细胞悬液迅速覆盖在琼脂糖平板上。
细胞在培养箱中于37℃、5%CO2 95%空气中培养并观察35天。复制平板用3对-硝基苯-5-苯基亚氯酸四鎓(INT染色)染色,并在0,5,10,15,20,30,35天观察集落形成和生长。所有大于60μm的染色集落视为阳性。
测试的所有细胞均为阴性。有关软琼脂试验的进一步信息见Hamburger等,Prog.Clin.Biol.Res.48:p43,135,179,1980。
实施例4:永生化细胞的致瘤性
根据University of Minnesota Animal Usage Protocol(protocol#9503000-1,1995年3月-1996年12月)的原则,将细胞注入试验动物体内,以确定细胞是否具有致瘤性。
活跃生长的细胞由细胞培养物平板中移出,并注入6只SPAFAS系成年鸡(Hy-Vac,Adcl,Iowa)。4×106细胞注入鸡的翅蹼(wing webs)。注射部位每周观察,持续3.5个月。在注射部位未观察到任何转染细胞的肿瘤,所有动物至今健康存活。试验表明这些永生化细胞不具致瘤性。
实施例5:细胞支持病毒生长的能力
细胞以5×105细胞/cm2接种至滚瓶。让细胞贴附24小时,收获对照进行细胞计数。用于病毒感染的细胞在DMEM(4.5g/l葡萄糖),4%胎牛血清,2mM L-谷氨酰胺,50mg/L庆大霉素中培养。以0.0006 HVT病毒粒子/细胞的感染复数感染细胞。每日观察滚瓶的CPE进展。感染后46小时收获滚瓶,此时大约为50% CPE。HVT感染细胞在生长培养基中加入10% DMSO后冰冻,浓度为2.0×107细胞/ml。HVT滴度通过噬斑试验定量。病毒以生长培养基梯度稀释,铺在允许细胞的汇合单层上。培养物温育设定时间,将细胞固定并染色。计数在单层上的噬斑,病毒滴度表示为斑形成单位/剂。
也测定了这些细胞支持呼肠病毒生长的能力。2.5×108细胞用滴度为8.2 TCID50/ml的呼肠病毒WSS-Reo 1773株感染,感染复数为0.005,0.001或0.0005感染性病毒粒子/细胞。感染细胞在滚瓶中生长,在感染后48,64和72小时进行测定,以证实增殖性病毒生长。
试验6:转染皮肤细胞作为细胞基质的应用
本发明的细胞可用作支持原代细胞的病毒复制的基质。在这些试验中,永生化细胞与原代细胞混合。在一个试验中,原代细胞被感染,与永生化细胞混合,置于培养物中。在另一个试验中,原代细胞被感染,并铺在永生化细胞上,所述永生化细胞已经在组织培养瓶中定位作为底层。在一个实施例中,病毒是Egg Drop综合征病毒,原代细胞为原代鸡胚肝细胞。在第二个实施例中,原代细胞为内皮细胞,优选肾内皮细胞,病毒为感染性支气管炎病毒。优选原代细胞与永生化细胞的比例为大约1∶5到1∶20,更优选大约1∶10。来自生长在混合细胞群体中的原代细胞的病毒滴度高于来自培养中原代细胞的病毒滴度。永生化细胞允许原代细胞用于商品化的病毒增殖。
所有引用的参考文献全文引作参考。尽管本发明以特定实施方案的方式进行了叙述,但本专利的保护范围仅由下列权利要求限定。
Claims (9)
1.自发永生化的细胞系,来自原代鸡胚成纤维细胞,具有UMNSAH-DF1细胞系的特征,保藏于美国典型培养物保藏中心,保藏号为#CRL-12203。
2.支持病毒复制的权利要求1的永生化细胞系的培养物或永生化亚克隆。
3.含病毒的权利要求1或2的细胞。
4.含有至少一种能够指导重组蛋白在细胞中表达的载体的权利要求1或2的细胞。
5.表达重组蛋白的权利要求4的细胞。
6.权利要求4的细胞,其中载体编码重组病毒的至少一部分。
7.权利要求4的细胞,其中载体为逆转录病毒载体。
8.由鸡胚成纤维细胞产生永生化细胞系的方法,包括以下步骤:
培养原代鸡胚成纤维细胞;
将成纤维细胞传代直至它们开始衰老;
细胞衰老期间浓缩细胞以维持大约30%-60%培养物汇合;
鉴定培养物中的非衰老细胞转化灶;
分离非衰老细胞;和
将非衰老细胞传代30次以上。
9.在细胞中培养病毒的方法,包括以下步骤:
培养权利要求1的细胞;
用病毒感染细胞;
让病毒在细胞中复制;和
收集在细胞中复制的病毒。
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CN102740881A (zh) * | 2009-05-12 | 2012-10-17 | 特兰斯吉恩股份有限公司 | 正痘病毒产生和纯化方法 |
CN105408471A (zh) * | 2013-07-25 | 2016-03-16 | 国家农艺研究院 | 用于筛选对复制禽病毒受纳的细胞系的方法 |
CN105408471B (zh) * | 2013-07-25 | 2019-01-18 | 国家农艺研究院 | 用于筛选对复制禽病毒受纳的细胞系的方法 |
CN109234239A (zh) * | 2018-07-13 | 2019-01-18 | 广东永顺生物制药股份有限公司 | 一种鸭瘟病毒的培养方法 |
CN109234239B (zh) * | 2018-07-13 | 2021-09-14 | 广东永顺生物制药股份有限公司 | 一种鸭瘟病毒的培养方法 |
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