CN116790504A - 培养gbf1敲除st细胞的培养基、方法及其用途 - Google Patents
培养gbf1敲除st细胞的培养基、方法及其用途 Download PDFInfo
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Abstract
本发明提出了一种ST细胞系,所述ST细胞系中的GBF1基因被沉默。根据本发明的实施例,将GBF1敲除的ST细胞系进行培养,接种猪瘟病毒后,通过检测猪瘟病毒的增殖情况,发现猪瘟病毒复制速率明显加快,最终病毒滴度有明显提高,说明有望用于猪瘟疫苗生产。
Description
技术领域
本发明涉及基因工程领域,具体地,本发明涉及培养GBF1敲除ST细胞的培养基、方法及其用途。
背景技术
猪瘟病毒(classical Swine fever virus,CSFV)是猪流行性腹泻(ClassicalSwine fever,CSF)的病原体,猪瘟病毒感染后会抑制病猪的免疫功能,常引发与其他疾病的混合感染及母猪的隐形感染,作为危害我国生猪养殖业的一类主要的传染病,每年造成巨大的经济损失。
目前针对此病尚无有效的治疗方法,预防猪瘟主要以接种有效的疫苗为主,现如今市场上使用最多、效果最好是弱毒活细胞苗,其是以猪瘟病毒在ST细胞中增殖后经收集、浓缩、过滤等工艺制备的。
因此,开发出一种新型的治疗猪瘟的方法刻不容缓。
发明内容
本发明旨在至少在一定程度上解决现有技术中存在的技术问题至少之一。
CRISPR/Cas9技术是近些年迅速发展起来的能够用于哺乳动物细胞的新型基因编辑技术,在很多领域均具有非常好的应用前景。利用该技术对ST细胞中调控细胞转运蛋白进行缺失,提高猪瘟病毒在ST细胞中的增殖与释放,将有助于提高猪瘟疫苗的产量或质量。
外被体蛋白Ⅰ(COPⅠ)是一种由7个不同亚基组成的胞质溶胶蛋白质复合物,COPⅠ囊泡可以携带各类分子从高尔基体到内质网的逆向转运,介导高尔基体内物质的逆向和正向运输过程,并维持高尔基体结构与膜囊的成熟。有研究表明,细胞内的囊泡运送系统与病毒的侵染、定位、组装增殖与释放密切相关,而高尔基体特异性BFA抗性因子1(GBF1)相关蛋白是COP I重要的下游调控因子,其可影响病毒在细胞中的增殖、组装与释放。发明发现,通过CRISPR基因编辑技术对ST细胞中GBF1基因进行缺失,可实现猪瘟病毒在ST细胞中的高效组装与复制,以提高猪瘟疫苗的产量与质量。
因此,发明人研发了一种GBF1基因敲除的ST细胞系的构建方法,利用CRISPR/Cas9技术对猪瘟弱毒疫苗生产用ST细胞系中的GBF1基因进行敲除,将用于GBF1基因的sgRNA和ssDNA共转染至ST细胞后,利用嘌呤霉素筛选结合梯度稀释,用克隆环法结合亚克隆技术分离到多个细胞克隆。提取基因组DNA后进行PCR扩增、测序,结合Western-Blot验证,并成功鉴定到了1个GBF1基因敲除的ST细胞克隆。将GBF1敲除的ST细胞系中扩大培养,接种猪瘟病毒后,通过检测猪瘟病毒的增殖情况,发现猪瘟病毒复制速率明显加快,最终病毒滴度有明显提高,说明有用于猪瘟疫苗生产的前景。
为此,在本发明的一个方面,本发明提出了一种ST细胞系。根据本发明的实施例,所述ST细胞系中的GBF1基因被沉默。
需要说明的是,本申请所述的“被沉默”是指基因的活性被抑制或基因的表达量下调,既可包括基因的活性被部分抑制或基因的表达量被部分下调,也可包括基因的活性被全部抑制或基因的表达量被全部下调。
根据本发明的实施例,所述GBF1基因被沉默是通过CRISPR/Cas9技术实现的。
在本发明的又一个方面,本发明提出了前面所述的ST细胞系在制备猪瘟疫苗中的用途。发明人发现采用CRISPR基因编辑技术对ST细胞中GBF1基因进行缺失,可实现猪瘟病毒在ST细胞中的高效组装与复制,从而提高猪瘟疫苗的产量与质量。
在本发明的另一个方面,本发明提出了一种培养用于制备疫苗的ST细胞的方法。根据本发明的实施例,所述方法包括:将前面所述的细胞系在经过优化的ST-SLM培养基中进行培养,以便获得用于制备疫苗的ST细胞,其中,所述经过优化的ST-SLM培养基是在基础DMEM培养基中额外添加下列成分:L-丙氨酸、L-盐酸精氨酸、L-天门冬氨酸、L-门冬酰胺、L-半胱氨酸盐酸盐一水、L-胱氨酸二盐酸盐、L-谷氨酸、L-谷氨酰胺、甘氨酸、L-盐酸组氨酸一水、L-羟脯氨酸、L-异亮氨酸、L-亮氨酸、L-盐酸赖氨酸、L-甲硫氨酸、L-苯丙氨酸、L-脯氨酸、L-丝氨酸、丙氨酰谷氨酰胺、丙酮酸钠、HEPES、无水磷酸氢二钠、D-无水葡萄糖、还原型谷胱甘肽、七水硫酸亚铁、七水硫酸锌、维生素C、氯化胆碱、肌醇、九水硝酸铁、氯化锂、重组人胰岛素、柠檬酸铁、维生素B12、五水硫酸铜、六水氯化镁、D-生物素、次黄嘌呤、亚油酸、硫辛酸、1,4-丁二胺二盐酸盐、维生素B2、胸苷、对氨基苯甲酸、氯化铝、乙酸钡、六水氯化钴、四水氯化锰、氟化钠、氯化亚锡、氯化镉、二氧化锗、亚硒酸钠、九水偏硅酸钠、氯化镍、偏钒酸铵、胆固醇、维生素E、维生素A醋酸酯、烟酸、2D-脱氧核糖、氢化可的松、EGF、IGF、钼酸铵、乙醇胺、胰岛素、维生素E醋酸酯、肉豆蔻酸、油酸、棕榈酸、硬脂酸、吐温80。ST细胞在经过优化的ST-SLM培养基能够大量增殖,在降低血清使用量的同时解决细胞结团率高、生长速度慢的问题。
根据本发明的实施例,额外添加成分在所述经过优化的ST-SLM培养基中的终浓度为:
根据本发明的实施例,所述基础DMEM培养基包括:
根据本发明的实施例,ST细胞系中的GBF1基因被沉默是通过CRISPR/Cas9技术实现的;其中,sg RNA包括SEQ ID NO:1或2所示的核苷酸序列:
CACCGCCATCAAACGAAATGCCCGA(SEQ ID NO:1);
AAACTCGGGCATTTCGTTTGATGGC(SEQ ID NO:2)。
在本发明的又一个方面,本发明提出了一种制备病毒疫苗的方法。根据本发明的实施例,所述方法包括:根据前面所述的方法培养所述ST细胞;和在所述培养过程中接种病毒,以利用所述ST细胞对所述病毒进行扩增;和收集所述扩增的病毒并进行对所述病毒进行减毒或者灭活处理,以便获得所述病毒疫苗。
根据本发明的实施例,所述病毒包括选自猪瘟病毒、狂犬病毒、流感病毒、冠状病毒、法氏囊病毒、登革热病毒、肠道病毒、脑炎病毒、痘病毒、正粘病毒、副粘病毒、反向病毒、披膜病毒、黄病毒、肠道病毒、细小核糖核酸病毒、嵌沙样病毒、疱疹病毒、腺病毒、牛痘病毒以及SARS病毒的至少之一。
根据本发明的具体实施例,所述病毒选自猪瘟病毒。
在本发明的又一个方面,本发明提出了一种用于培养ST细胞的经过优化的ST-SLM培养基。根据本发明的实施例,所述ST细胞携带经过基因改造的GBF1基因,所述经过优化的ST-SLM培养基是在DMEM培养基中额外添加下列成分:L-丙氨酸、L-盐酸精氨酸、L-天门冬氨酸、L-门冬酰胺、L-半胱氨酸盐酸盐一水、L-胱氨酸二盐酸盐、L-谷氨酸、L-谷氨酰胺、甘氨酸、L-盐酸组氨酸一水、L-羟脯氨酸、L-异亮氨酸、L-亮氨酸、L-盐酸赖氨酸、L-甲硫氨酸、L-苯丙氨酸、L-脯氨酸、L-丝氨酸、丙氨酰谷氨酰胺、丙酮酸钠、HEPES、无水磷酸氢二钠、D-无水葡萄糖、还原型谷胱甘肽、七水硫酸亚铁、七水硫酸锌、维生素C、氯化胆碱、肌醇、九水硝酸铁、氯化锂、重组人胰岛素、柠檬酸铁、维生素B12、五水硫酸铜、六水氯化镁、D-生物素、次黄嘌呤、亚油酸、硫辛酸、1,4-丁二胺二盐酸盐、维生素B2、胸苷、对氨基苯甲酸、氯化铝、乙酸钡、六水氯化钴、四水氯化锰、氟化钠、氯化亚锡、氯化镉、二氧化锗、亚硒酸钠、九水偏硅酸钠、氯化镍、偏钒酸铵、胆固醇、维生素E、维生素A醋酸酯、烟酸、2D-脱氧核糖、氢化可的松、EGF、IGF、钼酸铵、乙醇胺、胰岛素、维生素E醋酸酯、肉豆蔻酸、油酸、棕榈酸、硬脂酸、吐温80。ST细胞在经过优化的ST-SLM培养基能够大量增殖,在降低血清使用量的同时解决细胞结团率高、生长速度慢的问题。
根据本发明的实施例,额外添加成分在所述经过改造的ST-SLM培养基中的终浓度为:
根据本发明的实施例,所述基础DMEM培养基包括:
在本发明又一个方面,本发明提出了一种疫苗。根据本发明的实施例,所述疫苗是根据前面所述的方法制备的。通过该方法制备的疫苗能够加快猪瘟病毒增殖速率,提高猪瘟疫苗的产量与质量。
在本发明又一个方面,本发明提出了前面所述的疫苗在制备药物中的用途,所述药物用于治疗或者预防病毒相关疾病。
根据本发明的实施例,所述病毒包括选自猪瘟病毒、狂犬病毒、流感病毒、冠状病毒、法氏囊病毒、登革热病毒、肠道病毒、脑炎病毒、痘病毒、正粘病毒、副粘病毒、反向病毒、披膜病毒、黄病毒、肠道病毒、细小核糖核酸病毒、嵌沙样病毒、疱疹病毒、腺病毒、牛痘病毒以及SARS病毒的至少之一。
本发明的附加方面和优点将在下面的描述中部分给出,部分将从下面的描述中变得明显,或通过本发明的实践了解到。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解,其中:
图1是根据本发明实施例的ST细胞基因敲除过程图1;
图2是根据本发明实施例的ST细胞基因敲除过程图2;
图3是根据本发明实施例的Western blot结果图;
图4是根据本发明实施例的ST细胞生长曲线图;
图5是根据本发明实施例的RT-PCR结果图
图6是根据本发明实施例的ST细胞倍增时间;
图7是根据本发明实施例的ST细胞感染猪瘟病毒后病毒效价;
具体实施方式
下面详细描述本发明的实施例。下面描述的实施例是示例性的,仅用于解释本发明,而不能理解为对本发明的限制。实施例中未注明具体技术或条件的,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
实施例1 GBF1基因敲除的ST细胞系的构建
根据NCBI中猪的GBF1基因序列,利用张锋实验室网站在线设计针对GBF1基因的第2个外显子区域设计sgRNA序列,如表1所示,合成、退火并连接至pX0-PG中,如图1和2所示,获得重组质粒,扩增后提取备用;根据Invitrogen Lipofectamine 3000的标准转染程序,CRISPR质粒转染至ST细胞中,转染48h后加入终浓度10μg/mL的嘌呤霉素筛选5-7天后,进行细胞计数,分别铺100个和300个细胞,一周后单个细胞克隆形成后用克隆环挑取单克隆,转移至24孔板,在5%CO2培养箱中,温度为37℃,DMEM培养基中添加有10%NBS和1%抗生素的条件下培养;收集不同细胞克隆分别在DNA水平和蛋白水平进行鉴定。
表1
基因(gene) | 引物序列(Primer sequences)(5’-3’) |
sg RNA1-Exon2-F | CACCG CCATCAAACGAAATGCCCGA |
sg RNA1-Exon2-R | AAAC TCGGGCATTTCGTTTGATGG C |
实施例2 Western blot检测GBF1基因敲除效果
将猪瘟病毒按照相同的感染复数MOI感染GBF1基因敲除的ST细胞和对照ST细胞。48h后用PBS漂洗2次,加入预冷的RIPA裂解液裂解;在冰上裂解细胞,用移液器吹打细胞至完全裂解,将裂解产物转移至离心管中;4℃10000rpm离心10分钟;对蛋白样品进行煮样处理,进行蛋白电泳;电泳结束后根据电泳分离胶的尺寸,裁剪PVDF膜和四块滤纸,将PVDF膜浸泡于无水甲醇中激活30s,将激活后的PVDF膜和四块滤纸浸泡于转膜缓冲液中备用;按三明治夹心方式将胶和膜夹在滤纸中间,并将胶、膜和滤纸放置于半干转仪正负极之间,叠放顺序为:仪器负极-2层滤纸-胶-PVDF膜-2层滤纸-仪器正极,其中,将电泳胶面与半干转仪的负极相连,PVDF膜与正极相连。盖上半干转仪的上盖,接上电源,准备转膜;
以恒流(50mA)进行转膜,室温下转膜时间约为1小时15分钟,具体时间按照蛋白大小,以及每次转膜的效果可进行略微调整;取出PVDF膜封闭过夜或在水平摇床上封闭3h,封闭后用1×TBST洗涤1次,时间5min;按照实验所需稀释一抗,在水平摇床上孵育60min或4℃静置过夜,吸净一抗稀释液,用1×TBST洗涤5次,每次5min;按照二抗工作浓度稀释二抗,摇床上孵育30min,吸净二抗稀释液,用1×TBST洗涤5次,每次5min;最后显影,用化学发光成像系统曝光拍照。
如图3所示,与对照组相比,sgRNA缺失实验组ST细胞GBF1蛋白表达量显著减少,表明该基因被敲除。
实施例3 CCK8法绘制细胞生长曲线
本试验用于测定GBF1基因敲除ST细胞和对照ST细胞生长速率,简言之,该方法包括:将上述GBF1基因敲除ST细胞和对照ST细胞分别消化后计数,以每孔10000个细胞,接种至96孔板,置细胞培养箱正常培养;为了测定特定时间的细胞活力,每孔培养基中加入10μL的CCK8溶液,放置培养箱继续孵育培养1h,在多功能酶标仪上测定450nm处的吸光值;根据测定的吸光值制作细胞的生长曲线。
试验结果如图4所示,与对照ST细胞相比,敲除ST细胞生长无显著变化,表明敲除GBF1基因对ST细胞生长无显著影响。
实施例4 GBF1基因敲除细胞系接毒验证
ST细胞在96孔微量培养板中生长到汇合度大约为70%时备用;首先在1.5mL离心管中将待检测猪瘟兔化弱毒病毒液分别作连续10倍的梯度稀释,细胞用100μl PBS洗两遍之后,将稀释好的病毒液接种到96孔板中,每一稀释度接种一列,共8孔,每孔接种100μl;在培养箱中孵育1h,吸掉病毒液,用100μl PBS洗两次细胞后吸干净PBS,加入含有10%NBS的DMEM培养基,培养箱中继续培养;逐日观察并记录细胞病变结果,连续观察5-7天后根据Reed-Muench法计算TCID50值,结果如表2、表3所示,敲除ST细胞系培养的猪瘟病毒滴度更高。
表2GBF1基因敲除ST细胞感染猪瘟病毒的病毒滴度
经计算,GBF1基因敲除ST细胞系感染猪瘟病毒的lgTCID50=10-6.3/0.1mL
表3对照ST细胞感染猪瘟病毒的病毒滴度
经计算,对照ST细胞系感染猪瘟病毒的lgTCID50=10-5.0/0.1mL
实施例5RT-PCR检测病毒RNA的相对表达量
收集的病毒样品利用病毒RNA快速提取试剂盒提取病毒RNA后,利用TaKaRa反转录试剂盒进行反转录得到cDNA,以cDNA为模板进行PCR扩增,扩增产物通过凝胶电泳成像进行鉴定,结果如图5所示,GBF1敲除ST细胞系组目标条带更亮,表明有更多的病毒粒子。
实施例6配制培养基
1.配置DMEM培养基,并添加10%NBS,作为对照血清培养基,DMEM如下:
原料名称 | mg/L |
L-盐酸精氨酸 | 84.00 |
L-谷氨酰胺 | 584.00 |
L-盐酸组氨酸一水 | 42.00 |
L-异亮氨酸 | 105.00 |
L-亮氨酸 | 105.00 |
L-盐酸赖氨酸 | 146.00 |
L-甲硫氨酸 | 30.00 |
L-苯丙氨酸 | 66.00 |
L-丝氨酸 | 42.00 |
L-苏氨酸 | 95.00 |
L-色氨酸 | 16.00 |
L-胱氨酸二盐酸盐 | 63.00 |
甘氨酸 | 30.00 |
L-缬氨酸 | 94.00 |
苯酚红 | 15.00 |
二水氯化钙 | 265.00 |
氯化钾 | 400.00 |
无水硫酸镁 | 97.67 |
氯化钠 | 6400.00 |
一水磷酸二氢钠 | 125.00 |
九水硝酸铁 | 0.10 |
D-无水葡萄糖 | 4500.00 |
丙酮酸钠 | 110.00 |
D-泛酸钙 | 4.00 |
叶酸 | 4.00 |
氯化胆碱 | 4.00 |
肌醇 | 7.20 |
烟酰胺 | 4.00 |
维生素B2 | 0.40 |
维生素B1 | 4.00 |
维生素B6 | 4.00 |
L-酪氨酸二钠盐 | 104.00 |
2.配置优化后的ST-SLM培养基,在DMEM基础上添加1%NBS及下表物质:
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实施例7改良培养基ST-SLM培养效果
分别使用DMEM+10%NBS、ST-SLM培养基,按照3.0×106cells的量将改良ST细胞接种于T75方瓶,置于37℃、5%CO2培养箱进行培养,培养72h后,按上述细胞量分种至新培养瓶进行连续传代,检测细胞活率、细胞密度,并计算细胞倍增时间。
结果如图6所示,结果表明改良培养基ST-SLM的培养效果明显优于基础培养基,细胞生长速度快,平均倍增时间比基础培养基缩短了28%。
实施例8改良ST-SLM培养基及细胞系生产应用
取上述用DMEM+10%NBS、ST-SLM培养基培养的基因敲除ST细胞,细胞铺满后倒掉培养基,更换病毒维持液,按1%的体积比接种猪瘟病毒进行培养,每4天收获一次抗原液,采用免疫荧光法对病毒半数组织感染量进行测定。结果如图7所示,使用ST-SLM培养基培养的基因敲除ST细胞,在低血清条件下能符合猪瘟疫苗质量标准,表明该改造细胞系及优化的ST-SLM培养基在猪瘟疫苗生产中具有良好的应用前景。
本说明书的描述中,参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中,对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且,描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外,在不相互矛盾的情况下,本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例,可以理解的是,上述实施例是示例性的,不能理解为对本发明的限制,本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。
Claims (15)
1.一种ST细胞系,其特征在于,所述ST细胞系中的GBF1基因被沉默。
2.根据权利要求1所述的ST细胞系,其特征在于,所述GBF1基因被沉默是通过CRISPR/Cas9技术实现的。
3.权利要求1或2所述的ST细胞系在制备猪瘟疫苗中的用途。
4.一种培养用于制备疫苗的ST细胞的方法,其特征在于,包括:
将权利要求1或2所述的细胞系在经过优化的ST-SLM培养基中进行无血清培养,以便获得用于制备疫苗的ST细胞,
其中,
所述经过优化的ST-SLM培养基是在基础DMEM培养基中额外添加下列成分:
L-丙氨酸、L-盐酸精氨酸、L-天门冬氨酸、L-门冬酰胺、L-半胱氨酸盐酸盐一水、L-胱氨酸二盐酸盐、L-谷氨酸、L-谷氨酰胺、甘氨酸、L-盐酸组氨酸一水、L-羟脯氨酸、L-异亮氨酸、L-亮氨酸、L-盐酸赖氨酸、L-甲硫氨酸、L-苯丙氨酸、L-脯氨酸、L-丝氨酸、丙氨酰谷氨酰胺、丙酮酸钠、HEPES、无水磷酸氢二钠、D-无水葡萄糖、还原型谷胱甘肽、七水硫酸亚铁、七水硫酸锌、维生素C、氯化胆碱、肌醇、九水硝酸铁、氯化锂、重组人胰岛素、柠檬酸铁、维生素B12、五水硫酸铜、六水氯化镁、D-生物素、次黄嘌呤、亚油酸、硫辛酸、1,4-丁二胺二盐酸盐、维生素B2、胸苷、对氨基苯甲酸、氯化铝、乙酸钡、六水氯化钴、四水氯化锰、氟化钠、氯化亚锡、氯化镉、二氧化锗、亚硒酸钠、九水偏硅酸钠、氯化镍、偏钒酸铵、胆固醇、维生素E、维生素A醋酸酯、烟酸、2D-脱氧核糖、氢化可的松、EGF、IGF、钼酸铵、乙醇胺、胰岛素、维生素E醋酸酯、肉豆蔻酸、油酸、棕榈酸、硬脂酸、吐温80。
5.根据权利要求4所述的方法,其特征在于,额外添加成分在所述经过优化的ST-SLM培养基中的终浓度为:
终浓度为48mg/L的L-丙氨酸,
终浓度为40mg/L的L-盐酸精氨酸,
终浓度为16mg/L的L-天门冬氨酸,
终浓度为24mg/L的L-门冬酰胺,
终浓度为16mg/L的L-半胱氨酸盐酸盐一水,
终浓度为12.8mg/L的L-胱氨酸二盐酸盐,
终浓度为80mg/L的L-谷氨酸,
终浓度为160mg/L的L-谷氨酰胺,
终浓度为48mg/L的甘氨酸,
终浓度为8mg/L的L-盐酸组氨酸一水,
终浓度为32mg/L的L-羟脯氨酸,
终浓度为16mg/L的L-异亮氨酸,
终浓度为24mg/L的L-亮氨酸,
终浓度为24mg/L的L-盐酸赖氨酸,
终浓度为16mg/L的L-甲硫氨酸,
终浓度为8mg/L的L-苯丙氨酸,
终浓度为48mg/L的L-脯氨酸,
终浓度为72mg/L的L-丝氨酸,
终浓度为560mg/L的丙氨酰谷氨酰胺,
终浓度为80mg/L的丙酮酸钠,
终浓度为960mg/L的HEPES,
终浓度为116mg/L的无水磷酸氢二钠,
终浓度为2000mg/L的D-无水葡萄糖,
终浓度为0.32mg/L的还原型谷胱甘肽,
终浓度为0.48mg/L的七水硫酸亚铁,
终浓度为0.48mg/L的七水硫酸锌,
终浓度为1.2mg/L的维生素C,
终浓度为16mg/L的氯化胆碱,
终浓度为16mg/L的肌醇,
终浓度为0.02mg/L的九水硝酸铁,
终浓度为0.5mg/L的氯化锂,
终浓度为4mg/L的重组人胰岛素,
终浓度为4mg/L的柠檬酸铁,
终浓度为0.5104mg/L的维生素B12,
终浓度为0.000208mg/L的五水硫酸铜,
终浓度为6.704mg/L的六水氯化镁,
终浓度为0.0144mg/L的D-生物素,
终浓度为0.2864mg/L的次黄嘌呤,
终浓度为0.00992mg/L的亚油酸,
终浓度为0.0336mg/L的硫辛酸,
终浓度为0.0096mg/L的1,4-丁二胺二盐酸盐,
终浓度为0.0368mg/L的维生素B2,
终浓度为0.0624mg/L的胸苷,
终浓度为0.0416mg/L的对氨基苯甲酸,
终浓度为0.000032mg/L的氯化铝,
终浓度为0.000092mg/L的乙酸钡,
终浓度为0.00004mg/L的六水氯化钴,
终浓度为0.0000032mg/L的四水氯化锰,
终浓度为0.00012mg/L的氟化钠,
终浓度为0.000028mg/L的氯化亚锡,
终浓度为0.000028mg/L的氯化镉,
终浓度为0.000012mg/L的二氧化锗,
终浓度为0.0008mg/L的亚硒酸钠,
终浓度为0.00016mg/L的九水偏硅酸钠,
终浓度为0.000004mg/L的氯化镍,
终浓度为0.000008mg/L的偏钒酸铵,
终浓度为0.07472mg/L的胆固醇,
终浓度为0.02656mg/L的维生素E,
终浓度为0.0032mg/L的维生素A醋酸酯,
终浓度为0.0008mg/L的烟酸,
终浓度为0.012mg/L的2D-脱氧核糖,
终浓度为0.00016mg/L的氢化可的松,
终浓度为0.00096mg/L的EGF,
终浓度为0.00096mg/L的IGF,
终浓度为0.006mg/L的钼酸铵,
浓度为0.00439mg/L的乙醇胺,
终浓度为1.31841mg/L的胰岛素,
终浓度为0.1552795mg/L的维生素E醋酸酯,
终浓度为0.0221828mg/L的肉豆蔻酸,终浓度为0.0221828mg/L的油酸,
终浓度为0.0221828mg/L的棕榈酸,
终浓度为0.0221828mg/L的硬脂酸,
终浓度为4.88mg/L的吐温80。
6.根据权利要求3所述的方法,其特征在于,所述基础DMEM培养基包括:终浓度为84.00mg/L的L-盐酸精氨酸;
终浓度为584.00mg/L的L-谷氨酰胺;
终浓度为42.00mg/L的L-盐酸组氨酸一水;
终浓度为105.00mg/L的L-异亮氨酸;
终浓度为105.00mg/L的L-亮氨酸;
终浓度为146.00mg/L的L-盐酸赖氨酸;
终浓度为30.00mg/L的L-甲硫氨酸;
终浓度为66.00mg/L的L-苯丙氨酸;
终浓度为42.00mg/L的L-丝氨酸;
终浓度为95.00mg/L的L-苏氨酸;
终浓度为的16.00mg/L L-色氨酸;
终浓度为63.00mg/L的L-胱氨酸二盐酸盐;
终浓度为30.00mg/L的甘氨酸;
终浓度为94.00mg/L的L-缬氨酸;
终浓度为15.00mg/L的苯酚红;
终浓度为265.00mg/L的二水氯化钙;
终浓度为400.00mg/L的氯化钾;
终浓度为97.67mg/L的无水硫酸镁;
终浓度为6400.00mg/L的氯化钠;
终浓度为125.00mg/L的一水磷酸二氢钠;
终浓度为0.10mg/L的九水硝酸铁;
终浓度为4500.00mg/L的D-无水葡萄糖;
终浓度为110.00mg/L的丙酮酸钠;
终浓度为4.00mg/L的D-泛酸钙;
终浓度为4.00mg/L的叶酸;
终浓度为4.00mg/L的氯化胆碱;
终浓度为7.20mg/L的肌醇;
终浓度为4.00mg/L的烟酰胺;
终浓度为0.40mg/L的维生素B2;
终浓度为4.00mg/L的维生素B1;
终浓度为4.00mg/L的维生素B6;
终浓度为104.00mg/L的L-酪氨酸二钠盐。
7.根据权利要求4所述的方法,其特征在于,ST细胞系中的GBF1基因被沉默是通过CRISPR/Cas9技术实现的;
其中,sg RNA包括SEQ ID NO:1或SEQ ID NO:2所示的核苷酸序列。
8.一种制备病毒疫苗的方法,其特征在于,包括:
根据权利要求4~7任一项所述的方法培养所述ST细胞;和
在所述培养过程中接种病毒,以利用所述ST细胞对所述病毒进行扩增;和
收集所述扩增的病毒,并对所述病毒进行减毒或者灭活处理,以便获得所述病毒疫苗。
9.根据权利要求8所述的方法,其特征在于,所述病毒包括选自猪瘟病毒、狂犬病毒、流感病毒、冠状病毒、法氏囊病毒、登革热病毒、肠道病毒、脑炎病毒、痘病毒、正粘病毒、副粘病毒、反向病毒、披膜病毒、黄病毒、肠道病毒、细小核糖核酸病毒、嵌沙样病毒、疱疹病毒、腺病毒、牛痘病毒以及SARS病毒的至少之一。
10.一种用于培养ST细胞的经过优化的ST-SLM培养基,其特征在于,所述ST细胞的GBF1基因被沉默,
所述经过优化的ST-SLM培养基是在基础DMEM培养基中额外添加下列成分:
L-丙氨酸、L-盐酸精氨酸、L-天门冬氨酸、L-门冬酰胺、L-半胱氨酸盐酸盐一水、L-胱氨酸二盐酸盐、L-谷氨酸、L-谷氨酰胺、甘氨酸、L-盐酸组氨酸一水、L-羟脯氨酸、L-异亮氨酸、L-亮氨酸、L-盐酸赖氨酸、L-甲硫氨酸、L-苯丙氨酸、L-脯氨酸、L-丝氨酸、丙氨酰谷氨酰胺、丙酮酸钠、HEPES、无水磷酸氢二钠、D-无水葡萄糖、还原型谷胱甘肽、七水硫酸亚铁、七水硫酸锌、维生素C、氯化胆碱、肌醇、九水硝酸铁、氯化锂、重组人胰岛素、柠檬酸铁、维生素B12、五水硫酸铜、六水氯化镁、D-生物素、次黄嘌呤、亚油酸、硫辛酸、1,4-丁二胺二盐酸盐、维生素B2、胸苷、对氨基苯甲酸、氯化铝、乙酸钡、六水氯化钴、四水氯化锰、氟化钠、氯化亚锡、氯化镉、二氧化锗、亚硒酸钠、九水偏硅酸钠、氯化镍、偏钒酸铵、胆固醇、维生素E、维生素A醋酸酯、烟酸、2D-脱氧核糖、氢化可的松、EGF、IGF、钼酸铵、乙醇胺、胰岛素、维生素E醋酸酯、肉豆蔻酸、油酸、棕榈酸、硬脂酸、吐温80。
11.根据权利要求10所述的用于培养ST细胞的经过优化的ST-SLM培养基,其特征在于,额外添加成分在所述经过改造的ST-SLM培养基中的终浓度为:
终浓度为48mg/L的L-丙氨酸,
终浓度为40mg/L的L-盐酸精氨酸,
终浓度为16mg/L的L-天门冬氨酸,
终浓度为24mg/L的L-门冬酰胺,
终浓度为16mg/L的L-半胱氨酸盐酸盐一水,
终浓度为12.8mg/L的L-胱氨酸二盐酸盐,
终浓度为80mg/L的L-谷氨酸,
终浓度为160mg/L的L-谷氨酰胺,
终浓度为48mg/L的甘氨酸,
终浓度为8mg/L的L-盐酸组氨酸一水,
终浓度为32mg/L的L-羟脯氨酸,
终浓度为16mg/L的L-异亮氨酸,
终浓度为24mg/L的L-亮氨酸,
终浓度为24mg/L的L-盐酸赖氨酸,
终浓度为16mg/L的L-甲硫氨酸,
终浓度为8mg/L的L-苯丙氨酸,
终浓度为48mg/L的L-脯氨酸,
终浓度为72mg/L的L-丝氨酸,
终浓度为560mg/L的丙氨酰谷氨酰胺,
终浓度为80mg/L的丙酮酸钠,
终浓度为960mg/L的HEPES,
终浓度为116mg/L的无水磷酸氢二钠,
终浓度为2000mg/L的D-无水葡萄糖,
终浓度为0.32mg/L的还原型谷胱甘肽,
终浓度为0.48mg/L的七水硫酸亚铁,
终浓度为0.48mg/L的七水硫酸锌,
终浓度为1.2mg/L的维生素C,
终浓度为16mg/L的氯化胆碱,
终浓度为16mg/L的肌醇,
终浓度为0.02mg/L的九水硝酸铁,
终浓度为0.5mg/L的氯化锂,
终浓度为4mg/L的重组人胰岛素,
终浓度为4mg/L的柠檬酸铁,
终浓度为0.5104mg/L的维生素B12,
终浓度为0.000208mg/L的五水硫酸铜,
终浓度为6.704mg/L的六水氯化镁,
终浓度为0.0144mg/L的D-生物素,
终浓度为0.2864mg/L的次黄嘌呤,
终浓度为0.00992mg/L的亚油酸,
终浓度为0.0336mg/L的硫辛酸,
终浓度为0.0096mg/L的1,4-丁二胺二盐酸盐,
终浓度为0.0368mg/L的维生素B2,
终浓度为0.0624mg/L的胸苷,
终浓度为0.0416mg/L的对氨基苯甲酸,
终浓度为0.000032mg/L的氯化铝,
终浓度为0.000092mg/L的乙酸钡,
终浓度为0.00004mg/L的六水氯化钴,
终浓度为0.0000032mg/L的四水氯化锰,
终浓度为0.00012mg/L的氟化钠,
终浓度为0.000028mg/L的氯化亚锡,
终浓度为0.000028mg/L的氯化镉,
终浓度为0.000012mg/L的二氧化锗,
终浓度为0.0008mg/L的亚硒酸钠,
终浓度为0.00016mg/L的九水偏硅酸钠,
终浓度为0.000004mg/L的氯化镍,
终浓度为0.000008mg/L的偏钒酸铵,
终浓度为0.07472mg/L的胆固醇,
终浓度为0.02656mg/L的维生素E,
终浓度为0.0032mg/L的维生素A醋酸酯,
终浓度为0.0008mg/L的烟酸,终浓度为0.012mg/L的2D-脱氧核糖,
终浓度为0.00016mg/L的氢化可的松,
终浓度为0.00096mg/L的EGF,
终浓度为0.00096mg/L的IGF,
终浓度为0.006mg/L的钼酸铵,
浓度为0.00439mg/L的乙醇胺,
终浓度为1.31841mg/L的胰岛素,
终浓度为0.1552795mg/L的维生素E醋酸酯,
终浓度为0.0221828mg/L的肉豆蔻酸,
终浓度为0.0221828mg/L的油酸,
终浓度为0.0221828mg/L的棕榈酸,
终浓度为0.0221828mg/L的硬脂酸,
终浓度为4.88mg/L的吐温80。
12.根据权利要求10所述的方法,其特征在于,所述基础DMEM培养基包括:终浓度为84.00mg/L的L-盐酸精氨酸;
终浓度为584.00mg/L的L-谷氨酰胺;
终浓度为42.00mg/L的L-盐酸组氨酸一水;
终浓度为105.00mg/L的L-异亮氨酸;
终浓度为105.00mg/L的L-亮氨酸;
终浓度为146.00mg/L的L-盐酸赖氨酸;
终浓度为30.00mg/L的L-甲硫氨酸;
终浓度为66.00mg/L的L-苯丙氨酸;
终浓度为42.00mg/L的L-丝氨酸;
终浓度为95.00mg/L的L-苏氨酸;
终浓度为的16.00mg/L L-色氨酸;
终浓度为63.00mg/L的L-胱氨酸二盐酸盐;
终浓度为30.00mg/L的甘氨酸;
终浓度为94.00mg/L的L-缬氨酸;
终浓度为15.00mg/L的苯酚红;
终浓度为265.00mg/L的二水氯化钙;
终浓度为400.00mg/L的氯化钾;
终浓度为97.67mg/L的无水硫酸镁;
终浓度为6400.00mg/L的氯化钠;
终浓度为125.00mg/L的一水磷酸二氢钠;
终浓度为0.10mg/L的九水硝酸铁;
终浓度为4500.00mg/L的D-无水葡萄糖;
终浓度为110.00mg/L的丙酮酸钠;
终浓度为4.00mg/L的D-泛酸钙;
终浓度为4.00mg/L的叶酸;
终浓度为4.00mg/L的氯化胆碱;
终浓度为7.20mg/L的肌醇;
终浓度为4.00mg/L的烟酰胺;
终浓度为0.40mg/L的维生素B2;
终浓度为4.00mg/L的维生素B1;
终浓度为4.00mg/L的维生素B6;
终浓度为104.00mg/L的L-酪氨酸二钠盐。
13.一种疫苗,其特征在于,所述疫苗是根据权利要求8或9所述的方法制备的。
14.根据权利要求10所述的疫苗在制备药物中的用途,所述药物用于治疗或者预防病毒相关疾病。
15.根据权利要求14所述的方法,其特征在于,所述病毒包括选自猪瘟病毒、狂犬病毒、流感病毒、冠状病毒、法氏囊病毒、登革热病毒、肠道病毒、脑炎病毒、痘病毒、正粘病毒、副粘病毒、反向病毒、披膜病毒、黄病毒、肠道病毒、细小核糖核酸病毒、嵌沙样病毒、疱疹病毒、腺病毒、牛痘病毒以及SARS病毒的至少之一。
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