CN116731953A - 弹状病毒阴性草地贪夜蛾昆虫细胞株及其筛选、鉴定和应用 - Google Patents
弹状病毒阴性草地贪夜蛾昆虫细胞株及其筛选、鉴定和应用 Download PDFInfo
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Abstract
本发明属于基因工程、细胞工程技术领域,具体涉及一种弹状病毒阴性草地贪夜蛾昆虫细胞株及其筛选、鉴定和应用。本发明通过筛选、鉴定获得弹状病毒阴性草地贪夜蛾昆虫细胞株WSK‑Sf9,保藏编号为CCTCC NO:C202246。该细胞株经巢式PCR、转录组二代测序、荧光定量PCR以及探针法定量PCR等多种不同的高灵敏性的检测方法验证,最终筛选得到Sf‑弹状病毒阴性草地贪夜蛾昆虫细胞株WSK‑Sf9,按照药典要求对该细胞的无菌、支原体、外源病毒、致瘤性等进行检测,结果显示各项指标均符合要求,能基于杆状病毒表达系统生产重组蛋白和用于重组蛋白疫苗生产。
Description
技术领域
本发明属于基因工程、细胞工程技术领域,涉及一种新的草地贪夜蛾昆虫细胞株,具体涉及一种弹状病毒阴性草地贪夜蛾昆虫细胞株及其筛选、鉴定和应用。
背景技术
昆虫细胞表达系统已被广泛用于重组蛋白的生产,与其它表达系统相比,其具有许多优势,如:昆虫杆状病毒专一寄生于无脊椎动物,安全性高;重组蛋白表达水平高;可对重组蛋白进行正确折叠和翻译后修饰,获得具有生物活性的蛋白;适应于多基因表达如病毒样颗粒的复杂设计;适用于大规模无血清培养等。
目前,全球已有多个昆虫细胞表达系统生产的重组蛋白疫苗获批上市,并展现出良好的效果和安全性,其中以GSK公司生产的Cervarix宫颈癌疫苗、Dendreon公司生产的Provenge前列腺癌疫苗以及ProteinSciences公司生产的FluBlok流感疫苗为代表,另外,还有许多处于临床前实验阶段的重组蛋白疫苗也表现出良好的应用前景。
Sf9细胞(Spodoptera frugiperda cell,草地贪夜蛾细胞)是昆虫杆状病毒表达系统中最为常用的昆虫细胞,用于表达生产外源蛋白,包括抗体、疫苗以及重组蛋白等。Sf9细胞衍生于IPLBSF-21细胞系(也称为Sf21),来源于1977年分离培养的秋蝇蠕虫(草地贪夜蛾)蛹的卵巢组织。草地贪夜蛾弹状病毒(Sf-rhabdovirus,Sf-弹状病毒)是由美国FDA研究人员于2014年在草地贪夜蛾昆虫细胞系Sf9及其亲本细胞系Sf21中发现的一种新的负链RNA病毒,它包含了编码N,P,M,G和L等5个结构蛋白的基因信息,另外还检测到在G和L之间多出来一段未知功能的基因序列X。虽然该Sf-弹状病毒未整合到宿主细胞的基因组,但具有完整的基因组,有可能包装成完整的病毒颗粒,给基于利用Sf9细胞杆状病毒表达系统来获得疫苗等重组蛋白的生产和使用带来了潜在的风险。
发明内容
本发明通过筛选、鉴定获得弹状病毒阴性草地贪夜蛾Sf9细胞衍生系WSK-Sf9,简称WSK-Sf9,该细胞株能基于杆状病毒表达系统生产重组蛋白和用于重组蛋白疫苗生产。
本发明的目的在于提供弹状病毒阴性草地贪夜蛾昆虫细胞株WSK-Sf9,保藏编号为CCTCC NO:C202246。保藏命名为草地贪夜蛾Sf9细胞衍生系WSK-Sf9,保藏日期为:2022年2月16日;保藏中心为中国典型培养物保藏中心(CCTCC),地址为湖北省武汉市武昌区八一路299号武汉大学保藏中心,邮编430072。
本发明弹状病毒阴性草地贪夜蛾昆虫细胞株WSK-Sf9是采用有限稀释法筛选单一克隆,经多种不同的高灵敏性的检测方法验证,包括巢式PCR、转录组二代测序、荧光定量PCR以及探针法定量PCR,筛选得到弹状病毒阴性草地贪夜蛾昆虫细胞株WSK-Sf9;同时按照药典要求,对其无菌、支原体、外源病毒、致瘤性进行检测,结果显示各项指标均符合要求,能够作为生产用细胞基质。
本发明的另一目的在于提供弹状病毒阴性草地贪夜蛾昆虫细胞株WSK-Sf9在基于杆状病毒表达系统生产重组蛋白中的应用。
其中,所述应用包括基于杆状病毒表达系统生产重组蛋白药物或疫苗的应用。
其中,所述重组蛋白药物包括细胞因子、激素、重组酶类或抗体。
其中,所述细胞因子包括重组人白细胞介素、重组人表皮生长因子、重组人干扰素、重组人成纤维细胞生长因子、重组人促红细胞生成素或重组人粒细胞巨噬细胞刺激因子。
其中,所述激素包括重组人生长激素、重组人胰岛素、胰岛素类似物或重组人促卵泡成熟激素。
其中,所述重组酶类包括重组人α葡萄糖苷酶或重组人尿激酶原。
其中,所述抗体包括单克隆抗体、Fab抗体、scFv抗体或纳米抗体。
其中,所述疫苗包括重组蛋白疫苗或病毒样颗粒疫苗。
其中,所述重组蛋白疫苗包括新型冠状病毒蛋白疫苗、乙型肝炎病毒蛋白疫苗或狂犬病毒蛋白疫苗;优选地,为新型冠状病毒蛋白疫苗。
其中,所述病毒样颗粒疫苗包括新型冠状病毒病毒样颗粒疫苗(SARS-CoV-2-VLP)、人乳头瘤病毒样颗粒疫苗(HPV-VLP)、流感病毒样颗粒疫苗(HA-VLP)、脊髓灰质炎病毒样颗粒疫苗(PV-VLP)、呼吸道合胞病毒样颗粒疫苗(RSV-VLP)或手足口病病毒样颗粒疫苗(EV71-VLP)。优选地,为新型冠状病毒病毒样颗粒疫苗。
实现本发明WSK-Sf9在基于杆状病毒表达系统生产重组蛋白中的应用或在疫苗生产过程中的应用的技术方案是:利用Bac-to-Bac昆虫杆状病毒表达系统,包装生产杆状病毒,感染WSK-Sf9细胞表达目的蛋白,通过亲和纯化技术手段获得目的重组蛋白。
有益效果:本发明采用有限稀释法筛选单一克隆,经巢式PCR、转录组二代测序、荧光定量PCR以及探针法定量PCR等多种不同的高灵敏性的检测方法验证,筛选得到弹状病毒阴性草地贪夜蛾昆虫细胞株WSK-Sf9;同时按照药典要求,对其无菌、支原体、外源病毒、致瘤性进行检测,结果显示各项指标均符合要求,能用于生产临床使用的蛋白疫苗等重组蛋白产品。而且,本发明利用Bac-to-Bac昆虫杆状病毒表达系统,包装生产杆状病毒,感染WSK-Sf9细胞表达目的蛋白,通过亲和纯化等技术手段获得目的重组蛋白。
本发明筛选、鉴定获得的弹状病毒阴性草地贪夜蛾昆虫细胞株WSK-Sf9,保藏编号为CCTCC NO:C202246。保藏日期为:2022年2月16日;保藏中心为中国典型培养物保藏中心(CCTCC),地址为湖北省武汉市武昌区八一路299号武汉大学保藏中心,邮编430072,保藏的培养物名称及鉴别特征为草地贪夜蛾Sf9细胞衍生系WSK-Sf9。
附图说明
图1为本发明鉴定WSK-Sf9细胞的巢式PCR琼脂糖凝胶电泳结果;
图2为本发明筛选得到的弹状病毒阴性草地贪夜蛾昆虫细胞株WSK-Sf9的形态特征;
图3为本发明筛选得到的弹状病毒阴性草地贪夜蛾昆虫细胞株WSK-Sf9的核型分析;
图4为本发明筛选得到的弹状病毒阴性草地贪夜蛾昆虫细胞株WSK-Sf9的生长曲线;
图5为本发明筛选得到的弹状病毒阴性草地贪夜蛾昆虫细胞株WSK-Sf9的连续传代培养曲线;
图6为本发明筛选得到的弹状病毒阴性草地贪夜蛾昆虫细胞株WSK-Sf9表达外源性重组蛋白的时间梯度检测。
具体实施方式
下面将结合具体实施例对本发明的方案进行解释。本领域技术人员将会理解,下面的实施例仅用于说明本发明,而不应视为限定本发明的范围。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。
以下实施中将进一步说明该Sf-弹状病毒阴性草地贪夜蛾昆虫细胞株WSK-Sf9的筛选、鉴定和应用,下面结合附图对本发明作进一步的描述说明。
实施例1:Sf-弹状病毒阴性草地贪夜蛾昆虫细胞株WSK-Sf9的筛选和鉴定
目前唯一市售的Sf-弹状病毒阴性的Sf-RVN细胞(Sf-rhabdovirus-negativeSf9),由Jarvis教授团队通过有限稀释法联合抗病毒药物处理从Sf9筛选得到(MaghodiaAB,et al.Protein expression and purification.2016;122:45-55.),并通过针对L基因的巢式PCR验证其为Sf-弹状病毒阴性,该细胞归属GlycoBac公司(http://www.glycobac.com/sf-rvn-cells),它已与Millipore/Sigma公司达成合作伙伴关系,委托Millipore/Sigma销售该细胞。
根据非专利参考文献:Ma H,Nandakumar S,Bae EH,Chin PJ,Khan AS.TheSpodoptera frugiperda Sf9 cell line is a heterogeneous population ofrhabdovirus-infected and virus-negative cells:Isolation and characterizationof cell clones containing rhabdovirus X-gene variants and virus-negative cellclones.Virology 2019;536:125-33.,草地贪夜蛾Sf9细胞系是一种异质细胞群,其包含了Sf-弹状病毒感染阳性和Sf-弹状病毒阴性的两类细胞群,可以通过有限稀释法获得单一的Sf-弹状病毒阴性的细胞群。本发明利用有限稀释法挑取单一细胞克隆,再通过巢式PCR、转录组测序、荧光定量PCR(Q-PCR)以及探针法定量PCR检测等方法筛选、鉴定Sf-弹状病毒阴性的细胞。
1)Sf9亲本细胞系购自ThermoFisher公司(Lot No:2043331),将购回的细胞定义为P0代次,本筛选实验所用代次为P4代,细胞培养基为SIM SF无血清培养基(北京义翘神州科技股份有限公司,MSF1)。将悬浮培养的Sf9细胞按10倍梯度有限稀释到孔板中,每隔几天观察一次细胞状态,当有较明显的单一细胞群形成时,将其转移到新的孔板中扩大培养。然后收集适量细胞,提取总RNA,利用针对Sf-弹状病毒特异M基因的巢式PCR引物(表1)对候选细胞做初步鉴定。
表1.针对Sf-弹状病特异M基因的巢式PCR引物序列信息
2)将候选Sf-弹状病毒阴性的细胞连续传代培养,并收集细胞样品冻存于-80℃备用。经过45代连续培养,利用巢式PCR技术进行检测,结果显示其中一株命名为WSK-Sf9的细胞,其P1~P45代细胞中Sf-弹状病毒M基因均为阴性(如图1)。
3)转录组测序:分别收取5×106的亲本Sf9细胞和WSK-Sf9细胞进行转录组测序,结果显示在亲本Sf9细胞的转录组中检测到Sf-弹状病毒的RNA基因信息(GenBank:KF947078.1),而在WSK-Sf9中未检测到。
4)荧光定量PCR:设计两对针对Sf-弹状病毒M基因的定量PCR引物(表2),利用Bio-Rad SsoFastEvaGreensupermix试剂盒进行荧光定量PCR检测,结果显示WSK-Sf9 P3和P28均未检测到荧光信号,证明该WSK-Sf9细胞为Sf-弹状病毒阴性(表3)。
表2.针对Sf-弹状病毒M基因的特异性Q-PCR引物
表3.Q-PCR实验数据
5)探针法定量PCR:设计针对Sf-弹状病毒M基因的taqMan探针(表4),利用定量PCR检测,结果显示WSK-Sf9主细胞库(MCB)和工作细胞库(WCB)均未检测到荧光信号,再次证明该WSK-Sf9细胞为Sf-弹状病毒阴性(表5)。
表4.针对Sf-弹状病毒M基因的特异性taqMan探针引物
表5.Q-PCR实验数据
实施例2:Sf-弹状病毒阴性草地贪夜蛾昆虫细胞株WSK-Sf9的生长特征
1)WSK-Sf9培养特性:该细胞可以在无血清培养基中贴壁或悬浮生长,培养温度为27℃,悬浮生长时平均直径为16.28±0.34mm,如图2WSK-Sf9的形态特征。
2)WSK-Sf9细胞核型分析:对亲本Sf9细胞进行核型分析,在统计的60个分裂中期细胞中,染色体数目主要分布在180~250条之间,每个分裂中期细胞染色体数目平均为215条;而对WSK-Sf9细胞进行核型分析,在统计的60个分裂中期细胞中,染色体数目主要分布在191~538条之间,每个分裂中期细胞染色体数目平均为299条(如图3)。这也说明该Sf-弹状病毒阴性WSK-Sf9细胞株不同于其亲本Sf9细胞。
3)WSK-Sf9细胞生长曲线:将处于对数生长期的WSK-Sf9稀释到1×106/ml左右,传代到250ml透气盖摇瓶中,培养体积为100ml,将其置于27℃恒温摇床中连续培养,每24小时计数一次细胞密度及活率,经过三个不同批次的培养,其生长曲线及活率如图4所示,该细胞于96小时后生长至1×107/ml左右,并持续6天处于该水平,最高细胞密度接近1.2×107/ml,从第11天开始,细胞活率逐渐降低,细胞倍增时间平均为23小时左右。
4)WSK-Sf9细胞连续传代培养曲线:将处于对数生长期的WSK-Sf9稀释到1×106/ml左右,传代到250ml透气盖摇瓶中,培养体积为100ml,将其置于27℃恒温摇床中连续培养,每3天计数细胞浓度和活率,并传代培养。一般培养3天后,细胞浓度均能达到6~8×106/ml,活率均大于98%以上,连续传代培养曲线如图5所示,培养100代次后,细胞生长特性稳定。
实施例3:Sf-弹状病毒阴性草地贪夜蛾昆虫细胞株WSK-Sf9的安全性检测
依据2020版《中国药典》三部,对WSK-Sf9细胞做如下一系列检定。
1)种属鉴别:利用DNA Barcoding法检测,结果显示该WSK-Sf9细胞为草地贪夜蛾来源;
2)无菌检查:利用薄膜过滤法检测,结果显示为无菌生长;
3)分枝杆菌检查:利用培养法检测,结果显示为分枝杆菌阴性;
4)支原体检查:利用培养法、指示细胞培养法和Touchdown PCR法,结果显示为支原体阴性;
5)螺原体检查:利用荧光PCR法,结果显示为螺原体阴性;
6)外源病毒检查:
(1)利用体外细胞培养观察法、红血球吸附试验和红血球吸附试验,对猴源Vero细胞、人源MRC-5细胞、Sf9细胞、BHK-21细胞、蚊子细胞Aedes以及果蝇细胞D.Mel等不同细胞传代培养进行检测,均形态正常,检测为阴性。
(2)利用体内法进行乳鼠,成鼠和鸡胚(5-6日龄鸡胚、9-11龄鸡胚)接种,结果显示均符合规定。
7)逆转录病毒检测:利用透射电镜、HEK293细胞接种传代感染性试验检测以及化学试剂诱导法病毒检查未见病毒样颗粒,结果符合规定;
8)牛源性病毒检查以及猪源性病毒检查均为阴性;
9)其他特定病毒检查,对包括杆状病毒(Baculoviruses)、T.ni兽棚病毒变异株(FHVvar)、弹状病毒(Rhabdoviruses)、虫媒病毒蓝舌病毒科(Reoviridae)、批膜病毒科(Togaviruses)、黄病毒科(Flaviviruses)、布尼亚病毒科(Bunyaviruses)、猪瘟病毒科(Asfaviruses)、无脊椎动物病毒囊泡病毒科(Ascoviridae)、虹彩病毒科(Iridoviridae)、痘病毒科(Poxviridae)、杆状病毒科(Baculoviridae)、多分DNA病毒科(Poldnaviridae)、细小病毒科(Parvoviridae)、双核糖核酸病毒科(Birnaviridae)、呼肠孤病毒科(Reoviridae)、微核糖核酸病毒科(Picornaviralses)、二顺反子病毒科(Dicistrovitidae)、罗达病毒科(Nodaviridae)、四病毒科(Tetraviridae)进行检测,结果显示均为阴性,符合规定。
10)致瘤性检查:利用WSK-Sf9细胞对实验鼠进行接种,结果显示该细胞无致瘤性,符合规定。
综上,该Sf-弹状病毒阴性草地贪夜蛾昆虫细胞株WSK-Sf9的各项安全性检查均符合规定,满足生物制品生产用细胞基质的各项要求。
实施例4:Sf-弹状病毒阴性草地贪夜蛾昆虫细胞株WSK-Sf9表达外源重组蛋白
构建包含新型冠状病毒SARS-CoV-2的RBD结构域的杆状病毒表达载体,利用WSK-Sf9包装用于重组蛋白表达的杆状病毒。分别培养Sf9和WSK-Sf9细胞,待其浓度为2.5×106/ml时,按照MOI=0.5的比例接种病毒分别感染Sf9和WSK-Sf9细胞,并收集感染前(0小时)和感染后24小时、48小时、72小时和96小时的上清液,通过western-blot,用针对His标签的抗体进行检测,结果显示,WSK-Sf9细胞中重组蛋白的表达水平较Sf9细胞上调。经GMP车间生产、纯化,与佐剂复合后,该重组蛋白可用于预防新型冠状病毒的感染。这说明该Sf-弹状病毒阴性草地贪夜蛾昆虫细胞株WSK-Sf9可以用来表达生产蛋白疫苗等外源性重组蛋白,其表达水平高于Sf9细胞。
序列表
<110> 成都威斯克生物医药有限公司
<120> 弹状病毒阴性草地贪夜蛾昆虫细胞株及其筛选、鉴定和应用
<130> A220064K(序)
<141> 2022-03-01
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
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<213> 人工序列(Artificial Sequence)
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<212> DNA
<213> 人工序列(Artificial Sequence)
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ccacatctcc gctatcacca 20
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<212> DNA
<213> 人工序列(Artificial Sequence)
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aggagaagga gcggttgga 19
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<212> DNA
<213> 人工序列(Artificial Sequence)
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<212> DNA
<213> 人工序列(Artificial Sequence)
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<212> DNA
<213> 人工序列(Artificial Sequence)
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<212> DNA
<213> 人工序列(Artificial Sequence)
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<213> 人工序列(Artificial Sequence)
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<212> DNA
<213> 人工序列(Artificial Sequence)
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gtatgcaggt ggttgaggct 20
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<212> DNA
<213> 人工序列(Artificial Sequence)
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ctccttctcc tccacccaca t 21
Claims (11)
1.弹状病毒阴性草地贪夜蛾昆虫细胞株WSK-Sf9,保藏编号为CCTCC NO:C202246。
2.权利要求1所述的细胞株WSK-Sf9在基于杆状病毒表达系统生产重组蛋白中的应用。
3.如权利要求2所述应用,其特征在于:包括基于杆状病毒表达系统生产重组蛋白药物或疫苗的应用。
4.如权利要求3所述应用,其特征在于:所述重组蛋白药物包括细胞因子、激素、重组酶类或抗体。
5.如权利要求4所述应用,其特征在于:所述细胞因子包括重组人白细胞介素、重组人表皮生长因子、重组人干扰素、重组人成纤维细胞生长因子、重组人促红细胞生成素或重组人粒细胞巨噬细胞刺激因子。
6.如权利要求4所述应用,其特征在于:所述激素包括重组人生长激素、重组人胰岛素、胰岛素类似物或重组人促卵泡成熟激素。
7.如权利要求4所述应用,其特征在于:所述重组酶类包括重组人α葡萄糖苷酶或重组人尿激酶原。
8.如权利要求4所述应用,其特征在于:所述抗体包括单克隆抗体、Fab抗体、scFv抗体或纳米抗体。
9.如权利要求3所述应用,其特征在于:所述疫苗包括重组蛋白疫苗或病毒样颗粒疫苗。
10.如权利要求9所述应用,其特征在于:所述重组蛋白疫苗包括新型冠状病毒蛋白疫苗、乙型肝炎病毒蛋白疫苗或狂犬病毒蛋白疫苗;优选地,为新型冠状病毒蛋白疫苗。
11.如权利要求9所述应用,其特征在于:所述病毒样颗粒疫苗包括新型冠状病毒病毒样颗粒疫苗、人乳头瘤病毒样颗粒疫苗、流感病毒样颗粒疫苗、脊髓灰质炎病毒样颗粒疫苗、呼吸道合胞病毒样颗粒疫苗或手足口病病毒样颗粒疫苗;优选地,为新型冠状病毒病毒样颗粒疫苗。
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