CN116814697B - 一种适用于构建鲤科鱼稳转细胞系的慢病毒包装系统及应用 - Google Patents
一种适用于构建鲤科鱼稳转细胞系的慢病毒包装系统及应用 Download PDFInfo
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Abstract
本发明属于遗传育种领域中的分子遗传学研究领域,公开了一种适用于构建淡水鱼稳转细胞系的慢病毒包装系统及应用。该方法简单高效,适用于所有鲤科鱼类稳转细胞系的构建和性状相关基因的功能研究。采用这一方法,结合流式细胞筛选,我们建立了两个稳定转入绿色荧光蛋白(GFP)基因的草鱼肾脏(CIK)细胞系。本发明提供的优化后的慢病毒包装系统可用于草鱼经济性状相关基因的转基因功能研究和遗传机制解析,也可以解决鲤科鱼类的转基因家系高效构建的问题。
Description
技术领域
本发明属于遗传育种领域中的分子遗传学研究领域,具体涉及一种适用于构建鲤科鱼稳转细胞系的慢病毒包装系统及应用。
背景技术
传统构建稳转细胞株的方法主要是通过转染试剂和逆转录病毒介导,转染试剂中使用较多的是基于脂质体的转染试剂,如LIP2000和LIP3000等,逆转录病毒中使用较多的则是慢病毒系统。基于脂质体的转染试剂和慢病毒系统在哺乳动物细胞,特别是293T细胞中转染效率很高,是建立哺乳动物稳转细胞株的两种主流方法。
目前在鱼类细胞,特别是在淡水鱼细胞做转染时,主要使用的是基于脂质体的转染试剂。但由于鱼类细胞的生长模式和培养条件与哺乳动物细胞存在较大差异,基于脂质体的转染方法在鱼类细胞中转染效率低,整合入细胞基因组的效率更低,因此利用现有方法,构建鱼类稳转细胞系较为困难。原因有以下两个方面:①鱼类细胞培养环境温度较低,代谢较慢,脂质体转染效率低;②脂质体转染需要无血清环境,但无血清环境对鱼类细胞的毒性高,造成细胞存活率低。
慢病毒载体(Lentiviral vectors,LVs)是在HIV-1基础上去掉有害基因但保留其感染性,从而改造成的病毒载体系统,它能高效地将目的基因导入人的原代细胞或细胞系,对分裂细胞和非分裂细胞均具有感染能力。已有一些水产领域从事分子生物学研究的人员,试图将慢病毒系统应用于鱼类转基因细胞株的构建,但并未针对系统中的质粒进行增强感染性方面的改造,目前还没有构建高效鱼类慢病毒系统质粒的报道。推测主要原因是可购买的慢病毒包装系统,虽然其感染普广,但主要侵染范围还是来源于哺乳类的各类细胞,如神经细胞、肝细胞、心肌细胞、内皮细胞、干细胞、原代细胞等,并不能高效进入鱼的细胞。
针对上述问题,申请人提供了一种适用于构建草鱼或鲤鱼稳转细胞系的慢病毒包装系统,该系统可直接用于经济鱼类相关基因的转基因功能研究,也可以解决淡水鱼类的转基因家系高效构建的问题。
发明内容
本发明的目的在于提供一种适用于构建鲤科鱼稳转细胞系的慢病毒包装系统,所述的包装系统包括:改造的pMD2.G,改造的pLenti CMV/TO eGFP Puro和psPAX2质粒,所述的改造的pMD2.G质粒是将pMD2.G质粒中的水疱性口炎病毒糖蛋白(VSV-G)基因替换为密码子优化后的鲤春病毒糖蛋白(SVCV-G)基因或草鱼弹状病毒糖蛋白(GrCRV-G)基因;改造的pLenti CMV/TO eGFP Puro质粒是将pLenti CMV/TO eGFP Puro质粒中的两个LTR序列替换为斑马鱼内源性逆转录病毒(ZFERV)中的转座子序列(zLTR)。
本发明的另一个目的在于提供上述慢病毒包装系统在构建鲤科鱼稳转细胞系中的应用。
本发明还有一个目的在于提供上述慢病毒包装系统在构建转基因鲤科鱼家系中的应用。
本发明最后一个目的在于提供上述慢病毒包装系统在鲤科鱼相关基因功能研究中的应用。
为了达到上述目的,本发明采取以下技术措施:
鲤科鱼稳转细胞系的慢病毒包装系统的构建思路:
申请人在前期实验过程中发现,利用商品化的三质粒慢病毒包装系统,对淡水鱼,例如草鱼,鲤鱼,斑马鱼等的转染效率极低,甚至出现不转染的情况。因此,申请人考虑到鱼类细胞和哺乳动物细胞的差异,将三质粒系统中的pMD2.G质粒中的水疱性口炎病毒糖蛋白基因(VSV-G)替换为感染目标鱼病毒的糖蛋白基因。由于宿主差异,因此需要对替换后糖蛋白基因进行优化,由于软件推荐的优化后的基因导入后,并不能很好的实现转染,因此申请人利用生物信息学进行计算,进行基因的手动优化,同时对pLenti CMV/TO eGFP Puro质粒的转座子元件进行了改造,替换为鱼类逆转录病毒的转座子序列。最终,获得了鲤科鱼的稳转细胞系的慢病毒包装系统。
本发明所述的鲤科鱼的稳转细胞系的慢病毒包装系统,包括pMD-SVCV-G质粒或pMD-GrCRV-G质粒、pLenti CMV/TO eGFP Puro zLTR质粒,和psPAX2,所述的pMD-SVC V-G质粒是将pMD2.G质粒中的水疱性口炎病毒糖蛋白(VSV-G)基因替换为密码子优化后的鲤春病毒糖蛋白(SVCV-G)基因;所述的pMD-GrCRV-G质粒是将pMD2.G质粒中的水疱性口炎病毒糖蛋白(VSV-G)基因替换为密码子优化后的草鱼弹状病毒糖蛋白(GrCR V-G)基因;所述的pLenti CMV/TO eGFP Puro zLTR质粒是将pLenti CMV/TO eGFP Pu ro质粒中的两个LTR序列替换为斑马鱼内源性逆转录病毒(ZFERV)中的转座子序列(zL TR)。pMD-SVCV-G质粒的多核苷酸序列为SEQ ID NO.1所示,pMD-GrCRV-G质粒的多核苷酸序列为SEQ ID NO.2所示,pLenti CMV/TO eGFP Puro zLTR质粒的多核苷酸序列为SEQ ID NO.3所示。psPAX2是商业化质粒。
本发明的保护内容还包括:
上述慢病毒包装系统在构建鲤科鱼稳转细胞系中的应用。
上述慢病毒包装系统在构建转基因鲤科鱼家系中的应用。
上述慢病毒包装系统在鲤科鱼相关基因功能研究中的应用。
上述应用的应用方法,包括将pMD-SVCV-G质粒或pMD-GrCRV-G质粒、pLenti CM V/TO eGFP Puro zLTR质粒或改造的pLenti CMV/TO eGFP Puro zLTR质粒,和psPAX2形成的转染复合物转染293T细胞进行病毒包装后,再转染鲤科鱼细胞;
所述的改造的pLenti CMV/TO eGFP Puro zLTR质粒,是将目的基因与pLentiCMV/TO eGFP Puro zLTR中的GFP融合,或者替换。
本发明与现有技术相比,具有以下优点和效果:
本发明对已有的商业化慢病毒包装质粒进行多质粒元件优化,包装出可高效感染草鱼CIK细胞的慢病毒颗粒。本发明方法主要具有两个优点:
①进行独特的病毒糖蛋白密码子优化,可稳定感染哺乳类和鱼类细胞。与传统质粒转染相比,慢病毒感染无需转染试剂,且包装和感染的效率高;
②通过荧光蛋白标记,可发现,本发明的包装系统可将目的蛋白整合入宿主细胞并稳定表达。相比瞬时转染或普通质粒转染,慢病毒载体可以将外源基因有效地整合到宿主染色体上,并可稳定持续表达。
附图说明
图1为本发明提供的慢病毒包装系统稳转GFP基因后CIK细胞的发光情况:
其中:A为优化前SVCV-G作为替换方案的慢病毒感染CIK细胞的初始荧光(左),优化后SVCV-G作为替换方案的慢病毒感染CIK细胞的初始荧光(中)和传代后的荧光(右);
B为优化前GrCRV-G作为替换方案的慢病毒感染CIK细胞的初始荧光(左),优化后GrCRV-G作为替换方案的慢病毒感染CIK细胞的初始荧光(中)和传代后的荧光(右)。C图为未感染慢病毒的阴性对照CIK细胞所发荧光。
具体实施方式
本发明所述技术方案,如未特别说明,均为本领域的常规方案;所述试剂或材料,如未特别说明,均来源于商业渠道。
实施例1:
一种适用于构建鲤科鱼稳转细胞系的慢病毒包装系统:
1)从武汉淼灵生物科技有限公司购买pMD2.G(货号:P0262)、pLenti CMV/TO eGFPPu ro(货号:P5470)、psPAX2(货号:P0261)。
2)以pMD2.G质粒为骨架构建pMD-SVCV-G和pMD-GrCRV-G质粒。
(1)从NCBI下载鲤春病毒糖蛋白SVCV-G序列(AAB39501.1)和草鱼弹状病毒糖蛋白GrCRV-G序列(YP009094266.1)。
(2)依据人和草鱼基因组注释信息的CDS序列和蛋白序列,分别计算人和草鱼所有注释基因中各种氨基酸对应的密码子种类和频率,统计20种氨基酸对应频率最高的密码子。以人的密码子偏好为基准,如在人中出现同一个氨基酸,对应最高频率的密码子有多个,则从草鱼中选择频率较高的密码子,以此获得SVCV-G和GrCRV-G两种蛋白优化的氨基酸对应密码子及其排列顺序,将优化排列的核酸序列合成后,用EcoRI单酶切分别连接至pMD2.G载体上,获得pMD-SVCV-G和pMD-GrCRV-G质粒,所述的pMD-SVCV-G质粒的多核苷酸序列为SEQ ID NO.1所示,pMD-GrCRV-G质粒的多核苷酸序列为SEQ ID NO.2所示。
3)以pLenti CMV/TO eGFP Puro质粒为骨架构建pLenti CMV/TO eGFP Puro zLTR质粒。
(1)从NCBI下载ZFERV的基因组序列(AF503912),根据注释信息找出ZFERV基因组序列上游和下游的zLTR序列。
(2)分析pLenti CMV/TO eGFP Puro上LTR两侧序列(同源臂)和酶切位点,基于此合成ZFERV基因组序列上游和下游的zLTR序列(带同源臂上下游100bp和酶切位点EcoR I和XhoI),对pLenti CMV/TO eGFP Puro进行EcoRI和XhoI双酶切后,利用同源重组的方式,分别替换入ZFERV基因组序列上游和下游的zLTR序列,获得pLenti CMV/TO eGFP Puro zLTR质粒。所述的pLenti CMV/TO eGFP Puro zLTR质粒的多核苷酸序列为SEQ ID NO.3所示。
至此获得一种适用于构建鲤科鱼稳转细胞系的慢病毒包装系统,包括:pMD-SVCV-G或pMD-GrCRV-G、pLenti CMV/TO eGFP Puro zLTR;和psPAX2。
实施例2:
一种适用于构建鲤科鱼稳转细胞系的慢病毒包装系统转染效率的验证:
1)合成SVCV-G和GrCRV-G的未经密码子优化序列连接至pMD2.G载体,另外两个质粒同实验组相同,作为优化前对照组。
pMD-SVCV-G或pMD-GrCRV-G、pLenti CMV/TO eGFP Puro zLTR;和psPAX2慢病毒包装系统作为实验组。
2)慢病毒包装及纯化浓缩:
接种293T细胞于100mm培养皿中,37℃过夜培养至第二天细胞汇合度约为80%。转染前2h换新鲜10ml无血清培养基,将5.4ug pLenti CMV/TO eGFP Puro、5.4ug psPAX2和1.2ug pMD-SVCV-G或pMD-GrCRV-G加入到500ul Opti-MEM培养基中,轻微振荡混匀,然后加入10ul LIP3000 DNA转染试剂,混匀后室温孵育30min形成转染复合物。
3)将转染复合物逐滴均匀加入到293T细胞中,6h后加入1ml FBS,37℃过夜培养。24h后换10ml新鲜完全培养基,分别于48h和72h收集病毒悬液。
4)500g离心5min,收集上清过0.45um纤维素醋酸酯滤膜除去细胞碎片,滤液在4℃条件下80000g离心2h后,弃上清,加入0.5-1ml PBS重悬,-80℃保存,避免反复冻融。
5)慢病毒感染:
(1)感染前一天接种CIK细胞于6孔板中至第二天细胞汇合度约为80%,均匀滴加制备好的慢病毒悬液(MOI<0.5),28℃感染6h后换新鲜完全培养基,实验同时设置未感染慢病毒的阴性对照孔。
(2)24h/48h/72h各观察一次细胞荧光表达情况。感染细胞经过胰蛋白酶消化,离心后用PBS重悬,过0.75um滤网去除细胞团和杂质,使用Cytoflex S流式分析仪进行流式检测,收集发光细胞,计算未经密码子优化的序列GFP荧光率分别为1.29%(SVCV-G作为糖蛋白,图1中A左)和2.76%(GrCRV-G作为糖蛋白,图1中B左),经过密码子优化后的荧光率分别为23.56%和27.08%(图1中A中和图1中B中)。
(3)发光细胞经过3次传代和传代后的流式分析仪筛选,荧光率逐渐提高,第3次传代后分选细胞的GFP荧光率分别为87.31和93.24%(图1中A右和图1中B右)。未感染慢病毒的阴性对照仅见少许细胞碎片发光,见图1中C。结果说明慢病毒感染成功,GFP基因已稳定整合入CIK细胞表达,而非瞬时转染表达。
(4)同样在鲤鱼上皮细胞(EPC)进行感染试验,将纯化浓缩后的慢病毒感染EPC细胞,第一代转染后的EPC细胞发光率介于18.93-19.75%之间,远高于基于脂质体转染试剂的感染效率(约1%)。
Claims (5)
1. 一种适用于构建鲤科鱼稳转细胞系的慢病毒包装系统,包括pMD-SVCV-G质粒、pLenti CMV/TO eGFP Puro zLTR质粒和psPAX2质粒;或包括pMD-GrCRV-G质粒、pLentiCMV/TO eGFP Puro zLTR质粒和psPAX2质粒;
所述的pMD-SVCV-G质粒的多核苷酸序列为SEQ ID NO.1所示,pMD-GrCRV-G质粒的多核苷酸序列为SEQ ID NO.2所示,pLenti CMV/TO eGFP Puro zLTR质粒的多核苷酸序列为SEQID NO.3所示,所述的鲤科鱼为草鱼或鲤鱼。
2.权利要求1所述的慢病毒包装系统在构建鲤科鱼稳转细胞系中的应用,所述的鲤科鱼为草鱼或鲤鱼。
3.权利要求1所述的慢病毒包装系统在构建转基因鲤科鱼家系中的应用,所述的鲤科鱼为草鱼或鲤鱼。
4.权利要求1所述的慢病毒包装系统在鲤科鱼相关基因功能研究中的应用,所述的鲤科鱼为草鱼或鲤鱼。
5.根据权利要求2或3或4所述的应用,其应用过程包括:将质粒A、质粒B和质粒C形成的转染复合物转染293T细胞进行病毒包装后,再转染鲤科鱼细胞;
所述的质粒A为pMD-SVCV-G质粒或pMD-GrCRV-G质粒,质粒B为pLenti CMV/TO eGFPPuro zLTR质粒或改造的pLenti CMV/TO eGFP Puro zLTR质粒,质粒C为psPAX2质粒;
所述的改造的pLenti CMV/TO eGFP Puro zLTR质粒,是将目的基因与pLenti CMV/TOeGFP Puro zLTR中的GFP融合,或者替换。
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