CN114908053A - 孔雀成纤维永生化细胞系的制备方法及其在病毒扩增中的应用 - Google Patents
孔雀成纤维永生化细胞系的制备方法及其在病毒扩增中的应用 Download PDFInfo
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Abstract
一种孔雀成纤维永生化细胞系的制备方法及其在病毒扩增中的应用，通过将编码hTERT基因的真核表达质粒pCI‑neo‑hTERT转入原代培养的孔雀成纤维细胞，经筛选后连续培养至50代，得到永生化的细胞系PEF‑1，用于感染新城疫病毒(NDV)、禽流感病毒(AIV)及水泡性口炎病毒(VSV)，实现高效增殖。本发明采用基因重组技术，将端粒酶逆转录酶基因(hTERT)转染孔雀原代成纤维细胞，首次建立制备永生化的孔雀细胞系的方法。制备出永生化的孔雀成纤维细胞，生长旺盛、速度快。禽流感和新城疫等禽类病毒可以在该细胞中高效复制，且复制能力显著高于目前在用的MDCK细胞，使用方便，可以用来生产禽用病毒疫苗。
Description
技术领域
本发明涉及的是一种生物医药领域的技术，具体是一种孔雀成纤维永生化细胞系的制备方法及其在病毒扩增中的应用。
背景技术
新城疫病毒(NDV)属于副粘病毒科，副粘病毒科亚科。被世界卫生组织定为A类传染病的负链RNA病毒能够感染多种畜禽而且死亡率很高。尽管感染NDV后，孔雀的死亡率低，但临床研究发现，高致病性新城疫病毒能够感染蓝孔雀。
禽流感病毒(AIV)属甲型流感病毒，正黏病毒科RNA病毒。被国际兽疫局定为甲类传染病，又称真性鸡瘟或欧洲鸡瘟。按病原体类型的不同，禽流感可分为高致病性、低致病性和非致病性禽流感三大类。H9亚型禽流感属于低致病性禽流感，发病率高，但死亡率低。主要表现为轻度的呼吸道症状。但H9禽流感病毒可以感染人，一旦蓝孔雀感染H9亚型禽流感会对人类健康存在严重的威胁。
孔雀是公认的抗病能力强的禽类，禽流感(AIV)或新城疫病毒(NDV)等禽类强致病性病毒也能感染孔雀，然而，AIV和NDV对孔雀的致病性显著低于其它禽类。尽管AIV和NDV对孔雀致病性偏低，但孔雀感染AIV和NDV后，却可作为中间宿主将病毒传播给人类，引起人类疾病。目前主要应用的禽类商品化细胞系为鸡成纤维细胞系DF-1，然而，DF-1细胞系感染新城疫、禽流感等病毒后极易死亡，导致病毒产量极低，限制了其在疫苗生产中的应用。
发明内容
本发明针对现有技术制备得到的成纤维永生化细胞系经病毒感染细胞后上清液病毒含量低、病毒感染细胞后关键的细胞因子表达很低，无法满足生成疫苗要求的不足，提出一种孔雀成纤维永生化细胞系的制备方法及其在病毒扩增中的应用，采用基因重组技术，将端粒酶逆转录酶基因(hTERT)转染孔雀原代成纤维细胞，首次建立制备永生化的孔雀细胞系的方法。制备出永生化的孔雀成纤维细胞，生长旺盛、速度快。禽流感和新城疫等禽类病毒可以在该细胞中高效复制，且复制能力显著高于目前在用的MDCK细胞，使用方便，可以用来生产禽用病毒疫苗。
本发明是通过以下技术方案实现的：
本发明涉及一种基于孔雀成纤维永生化细胞系的应用，通过将编码hTERT基因的真核表达质粒pCI-neo-hTERT转入原代培养的孔雀成纤维细胞，经筛选后连续培养至50代，得到永生化的细胞系PEF-1，用于感染新城疫病毒(NDV)、禽流感病毒(AIV)及水泡性口炎病毒 (VSV)，实现高效增殖。
所述的原代培养的孔雀成纤维细胞，采用但不限于分离孔雀胚胎成纤维组织，剪碎至 1mm3大小后，经胰蛋白酶消化接种至细胞培养皿得到。
所述的真核表达质粒pCI-neo-hTERT具有G418抗性。
所述的筛选，优选在孔雀成纤维细胞转染24h后用1000ng/mL G418筛选阳性克隆，连续培养具有G418抗性的阳性克隆。
所述的连续培养，优选将筛选得到的阳性克隆连续培养超过50代次，得到永生化的细胞系PEF-1。
所述的永生化的细胞系PEF-1经梯度降温后，可置于液氮中长期保存，复苏后可继续正常培养。
所述的转入，具体为：采用脂质体转染方法，将编码hTERT和Neo基因的真核表达质粒pCI-neo-hTERT与脂质体预混后转入原代培养的孔雀成纤维细胞。
所述Neo为Marker基因。
所述的病毒水泡性口炎病毒为VSV-GFP包含绿色荧光蛋白。
所述的新城疫病毒为LaSota株包含绿色荧光蛋白。
所述的禽流感病毒为A/Chicken/Shanghai/010/2008(H9N2)-SH010株。
本发明涉及一种基于上述应用得到的孔雀成纤维细胞系生产病毒疫苗，通过在制备得到的永生化的孔雀成纤维细胞系与MDCK细胞系中接种禽流感病毒(AIV)，新城疫病毒(NDV- GFP)以及水泡性口炎病毒(VSV-GFP)，培养增殖并收获病毒后，通过TCID50检测病毒滴度。
所述的检测病毒滴度是指：将的孔雀成纤维细胞系接种于细胞瓶，待长满单层后，传代至96孔板，继续培养；将病毒上清10倍梯度稀释，从10-1-10-10；将稀释好的病毒接种至96孔板长成致密单层后的孔雀成纤维细胞系中，每一稀释度接种8个孔，同时设阴性对照，至于37℃CO2培养箱中继续培养；接种4-5天，记录每个稀释度出现细胞病变(CEP)的孔数，计算病毒滴度。
技术效果
本发明通过分离培养了孔雀原代成纤维细胞，并利用真核表达质粒pCI-neo-hTERT探索并构建了孔雀成纤维细胞系，该细胞系易感染病毒，且病毒产量高，能够作为生产禽源病毒疫苗的工具。
附图说明
图1为G418药筛之后传连续传代培养的孔雀成纤维细胞示意图；
图2为经冻存复苏后的孔雀成纤维细胞系示意图；
图3为Western Blot检测细胞hTERT基因的蛋白表达示意图；
图4为永生化的孔雀成纤维细胞系与MDCK细胞系接种新城疫病毒(NDV-GDP)，禽流感病毒(AIV)，和水泡性口炎病毒(VSV-GFP)后，细胞病变以及荧光图示意图；
图5为永生化的孔雀成纤维细胞系与MDCK细胞系感染病毒后，病毒滴度检测示意图。
具体实施方式
本实施例涉及一种孔雀成纤维细胞系的构建方法，所涉及的pCI-neo-hTERT真核表达系统购自Addgene公司；所采用的蓝孔雀胚购自芜湖如意生态农业有限公司。
本实施例具体包括以下步骤：
步骤1、孔雀成纤维细胞的分离与培养：将0日龄的孔雀胚放置37℃，50％恒温恒湿孵化箱，孵化10天后无菌取孔雀胚，去掉胚胎头，四肢，内脏和骨骼后用加1％双抗的PBS 洗三次，将剩余组织剪成约1mm3大小的组织块，转移至50mL离心管中，加适量胰蛋白酶 (含胰酶0.05％，含EDTA 0.02％)，37℃水浴摇床消化15min，直到无明显组织块后，加等体积DMEM+10％FBS的培养基终止消化，用70μm和100μm滤筛过滤。将滤液1000g离心5 分钟后用DMEM+10％FBS的培养基重悬后接种于10cm细胞培养皿，放入CO2培养箱中， 37℃培养24h，可长成单层。
步骤2、pCI-neo-hTERT真核表达系统的转染与筛选，具体包括：
2.1)待原代培养的孔雀成纤维细胞汇合度达到80％左右时，用胰蛋白酶消化，传代至 12孔板，每孔传1.0x 106个细胞，待12孔板细胞度汇合达到80％左右转染细胞。用OPTI 将pCI-neo-hTERT质粒稀释成500ng/100μL/孔，轻轻混匀。将3μL/孔的脂质体(NulenPlusTransTM)加入100μL OPTI中，轻轻混匀，将混合的质粒与脂质体稀释液混合，轻轻吹打均匀，室温孵育20min。
2.2)将孵育完成的质粒脂质体混合液逐滴均匀的加入12孔板，每孔200μL，留四个孔做对照。将转染完成的细胞置于37℃，5％CO2细胞培养箱，继续培养。
2.3)转染24小时后，加入(1000ug/mL)G418进行筛选，筛选3天后将G418浓度降至500ug/mL继续筛选，直到对照组细胞全部死完，用胰酶消化G418筛选后的阳性克隆，传代至24孔板继续培养，每隔2天传代一次。培养超过50代后，即为永生化的孔雀成纤维细胞系。
步骤3、永生化的孔雀成纤维细胞系的鉴定，具体包括：
3.1)细胞形态学特征和生长曲线的测定：孔雀成纤维细胞系具有典型的成纤维细胞特点，长条梭形，细胞核明显，细胞间存在接触抑制，如图2所示，该细胞系经过多次传代仍然具有典型的成纤维细胞的形态。
3.2)永生化的孔雀成纤维细胞系冻存与复苏：i)用胰蛋白酶消化永生化的孔雀成纤维细胞系，待细胞出现皱缩之后，加等体积全完培养基终止消化，用移液器轻轻吹打下剩下的贴壁细胞，直至全部贴壁细胞脱落，1500g离心5min，弃掉剩余培养基。按照： DMSO:DMEM:FBS＝1:5:4比例配置冻存液，重悬细胞后，转移至2mL细胞冻存管。将细胞放于程序冻存盒后，转移至-80℃冰箱。24h后转移至液氮长期保存；ii)永生化的孔雀成纤维细胞系的复苏。从液氮中取出细胞，放于37℃恒温水浴锅中解冻。解冻后1500g离心5min，用完全培养基重悬后，接种至T25细胞培养瓶，如图3所示，该永生化的孔雀成纤维细胞 PEF-1经冻存复苏后，依然有很好的活力。
3.3)Western Blot检测转染细胞中外源性hTERT蛋白表达：i)将孔雀原代成纤维细胞与培养至60代的孔雀成纤维细胞系接种至6孔板，分别接种2个孔，放入细胞培养箱直至细胞长满；ii)蛋白样品的制备：弃掉培养液，用PBS轻轻冲洗3遍，每孔加入100μL RIPA细胞裂解液(碧云天)，其中包含1μL PMSF。用细胞刮刀将细胞刮下后转移至1.5mL离心管，冰上裂解30min。12000g离心10min后吸取上清至新的1.5mL离心管，加入1×蛋白LoadingBuffer，100℃煮沸10min,放于-80℃冰箱待用；iii)SDS-PAGE电泳：根据SDS-PAGE试剂盒说明(雅酶)说配置10％SDS-PAGE胶，待SDS-PAGE胶凝固后，安装好电泳装置，每孔上样 10μL,80V电压跑出浓缩胶后，调整电压至120V,直到条带跑至最底层；iv)转膜：拆下电泳装置，取出PAGE胶，用快速转转膜仪器完成转膜(Genscript)。具体方法：PVDF膜用甲醇活化 30s后用转膜缓冲液清洗2min，按照海绵垫-PVDF膜-胶-海绵垫模式，放置于转膜装置中， 15V20min进行转膜；v)封闭及杂交：转膜结束后将PVDF膜，放置于5％脱脂奶粉室温摇床孵育，封闭1小时。TBST漂洗5min，重复3次。将稀释好的TERT和β-tubulin与膜共同孵育，4℃摇床过夜。一抗孵育完成后取出膜，TBST漂洗5min，重复3次，常温摇床孵育二抗 1小时。1小时后取出膜TBST漂洗5min，重复3次，准备显色；vi)显色：将膜放入显色液中孵育30s后，置于显色仪显色，如图4所示，转染hTERT基因存在于培养60代的孔雀细胞系中。
步骤4、用孔雀成纤维细胞系用于扩增病毒，具体包括：
4.1)孔雀成纤维细胞系与MDCK细胞系感染病毒：按照常规方法，对孔雀成纤维细胞系和MDCK细胞系接种禽流感病毒(AIV)，新城疫病毒(NDV-GFP)以及水泡性口炎病毒(VSV- GFP)，显微镜观察所述细胞感染病毒情况以及细胞病变情况，具体包括：
a)将孔雀成纤维细胞系与MDCK细胞系按照每孔1.0x 106个细胞传代至12孔板，放置于37℃，5％CO2细胞培养箱，继续培养。
b)待细胞两种细胞长满后，吸出培养基用PBS洗两次，更换为无血清DMEM培养基。将禽流感病毒(AIV)，新城疫病毒(NDV-GFP)以及水泡性口炎病毒(VSV-GFP)分别按照MOI＝1.0 接种与所述细胞中。
c)接种病毒12h和24h后，在显微镜下观察细胞病变情况，用荧光显微镜观察新城疫病毒(NDV-GFP)和水泡性口炎病毒(VSV-GFP)荧光强度。如图5所示，孔雀成纤维细胞系和MDCK细胞系均能感染禽流感病毒(AIV)，新城疫病毒(NDV-GFP)以及水泡性口炎病毒(VSV-GFP)，而且在感染12h，已经可以观察到新城疫病毒(NDV-GFP)和水泡性口炎病毒(VSV-GFP)荧光。
4.2)孔雀成纤维细胞系与MDCK细胞系病毒滴度测定：将孔雀成纤维细胞系按照每孔 5.0x 104个细胞传代至96孔板，放置于37℃，5％CO2细胞培养箱，继续培养；收集所述细胞系感染禽流感病毒(AIV)，新城疫病毒(NDV-GFP)以及水泡性口炎病毒(VSV-GFP)12h和24h 后的培养基上清；将所述病毒上清10倍梯度稀释于DMEM培养基中，从10-1-10-10，待96孔板成纤维细胞长满后，弃掉培养基，用PBS洗3次。将稀释好的病毒接种至所述96孔板长成致密单层后的孔雀成纤维细胞系中每一稀释度接种8个孔，同时设阴性对照，至于37℃ CO2培养箱中继续培养；接种4-5天，记录每个稀释度出现细胞病变(CEP)的孔数，计算所述病毒滴度。
结果显示孔雀成纤维细胞系接种禽流感病毒(AIV)，新城疫病毒(NDV-GFP)以及水泡性口炎病毒(VSV-GFP)可以在孔雀成纤维细胞系上大量增殖，而且病毒滴度高于MDCK细胞系。
与现有技术相比，本方法制备得到的孔雀成纤维细胞系接种病毒后增殖效率更高、产量更高，同时本方法减少因长期使用哺乳动物细胞生产禽病毒疫苗而诱发的病毒跨物种适应性突变，降低禽病毒对包括人类在内的哺乳动物的潜在威胁、降低哺乳动物源DNA、蛋白质等异源物质注射禽后引起禽不良反应。
上述具体实施可由本领域技术人员在不背离本发明原理和宗旨的前提下以不同的方式对其进行局部调整，本发明的保护范围以权利要求书为准且不由上述具体实施所限，在其范围内的各个实现方案均受本发明之约束。
Claims (10)
1.一种基于孔雀成纤维永生化细胞系的应用，其特征在于，通过将编码hTERT基因的真核表达质粒pCI-neo-hTERT转入原代培养的孔雀成纤维细胞，经筛选后连续培养，得到永生化的细胞系PEF-1，用于感染新城疫病毒(NDV)、禽流感病毒(AIV)及水泡性口炎病毒(VSV)，实现高效增殖。
2.根据权利要求1所述的基于孔雀成纤维永生化细胞系的应用，其特征是，所述的原代培养的孔雀成纤维细胞，通过分离孔雀胚胎成纤维组织，剪碎至1mm3大小后，经胰蛋白酶消化接种至细胞培养皿得到。
3.根据权利要求1所述的基于孔雀成纤维永生化细胞系的应用，其特征是，所述的真核表达质粒pCI-neo-hTERT具有G418抗性。
4.根据权利要求1或3所述的基于孔雀成纤维永生化细胞系的应用，其特征是，所述的筛选，在孔雀成纤维细胞转染24h后用1000ng/mL G418筛选阳性克隆，连续培养具有G418抗性的阳性克隆。
5.根据权利要求1所述的基于孔雀成纤维永生化细胞系的应用，其特征是，所述的连续培养，将筛选得到的阳性克隆连续培养超过50代次，得到永生化的细胞系PEF-1。
6.根据权利要求1所述的基于孔雀成纤维永生化细胞系的应用，其特征是，所述的永生化的细胞系PEF-1经梯度降温后，可置于液氮中长期保存，复苏后可继续正常培养。
7.根据权利要求1所述的基于孔雀成纤维永生化细胞系的应用，其特征是，所述的转入，具体为：采用脂质体转染方法，将编码hTERT和Neo基因的真核表达质粒pCI-neo-hTERT与脂质体预混后转入原代培养的孔雀成纤维细胞；
所述Neo为Marker基因。
8.根据权利要求1所述的基于孔雀成纤维永生化细胞系的应用，其特征是，所述的病毒水泡性口炎病毒为VSV-GFP包含绿色荧光蛋白；所述的新城疫病毒为LaSota株包含绿色荧光蛋白；所述的禽流感病毒为A/Chicken/Shanghai/010/2008(H9N2)-SH010株。
9.一种基于权利要求1～8中任一所述应用得到的孔雀成纤维细胞系生产病毒疫苗，其特征在于，通过在制备得到的永生化的孔雀成纤维细胞系与MDCK细胞系中接种禽流感病毒(AIV)，新城疫病毒(NDV-GFP)以及水泡性口炎病毒(VSV-GFP)，培养增殖并收获病毒后，通过TCID50检测病毒滴度。
10.根据权利要求9所述的孔雀成纤维细胞系生产病毒疫苗，其特征是，所述的检测病毒滴度是指：将的孔雀成纤维细胞系接种于细胞瓶，待长满单层后，传代至96孔板，继续培养；将病毒上清10倍梯度稀释，从10-1-10-10；将稀释好的病毒接种至96孔板长成致密单层后的孔雀成纤维细胞系中，每一稀释度接种8个孔，同时设阴性对照，至于37℃CO2培养箱中继续培养；接种4-5天，记录每个稀释度出现细胞病变(CEP)的孔数，计算病毒滴度。
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