CN112062818B - 一种提高转导效率的慢病毒载体及其应用 - Google Patents
一种提高转导效率的慢病毒载体及其应用 Download PDFInfo
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Abstract
本发明提供一种提高转导效率的慢病毒载体及其应用,慢病毒载体是以HIV‑1为基础发展起来的基因治疗载体,可以将外源基因导入动物和人的多种原代细胞或细胞系中,具有广阔的应用前景。本发明建立了使用VSVG突变体从而不受MARCH8影响的慢病毒载体系统,提高了利用正常293T细胞制备的慢病毒的转导效率。同时,本申请建立了MARCH8缺失的293T细胞,利用此缺失MARCH8的293T细胞进行慢病毒的制备,提高了产生慢病毒的转导效率,进一步优化了慢病毒载体系统。
Description
技术领域
本发明涉及生物技术领域,具体涉及一种VSVG突变体及其不受MARCH8影响的慢病毒载体系统,以及其制备与应用。
背景技术
慢病毒载体是以HIV-1为基础发展起来的基因治疗载体。慢病毒载体可以将外源基因(或shRNA等)导入动物和人的原代细胞或细胞系中,有效整合到宿主染色体上,进而可以持久表达。同时,慢病毒载体具有广阔的宿主范围,可以感染多种类型细胞,如淋巴细胞、肝细胞、神经元细胞等,能够达到良好的基因治疗效果,具有广阔的应用前景。慢病毒载体介导的基因表达或RNA干扰作用持续且稳定,主要原因是目的基因整合到宿主的基因组中,随细胞基因组的分裂而分裂,而且慢病毒能有效感染非分裂细胞。文献报道表明,慢病毒载体介导的目的基因可持续表达于多种组织或细胞中,如脑、肝脏、视网膜、干细胞等。
慢病毒表达载体包含了包装、稳定整合所需的遗传信息。为产生高滴度的病毒颗粒,常利用表达载体和包装质粒共转染293T细胞,在293T细胞中进行病毒的包装,成熟的慢病毒颗粒被分泌到培养基中,离心所得上清可以直接感染靶细胞,慢病毒携带目的基因在靶细胞中通过反转录整合到基因组,进而持续表达。现今常用的慢病毒包装系统主要使用VSVG蛋白(水疱性口炎病毒G蛋白)替代Env包膜蛋白,扩大了病毒载体的靶细胞范围,而且增加了载体的稳定性,可以通过高速离心浓缩病毒载体,从而提高滴度,VSVG包膜蛋白在慢病毒载体构建及使用中发挥着不可替代的作用。
VSV具有广泛的宿主范围,可以感染多种细胞,表明它可以通过某种普遍存在的受体进入宿主细胞。研究发现,低密度脂蛋白受体LDLR可以作为VSV、VSVG包装的假病毒的进入所需受体,普遍存在的LDLR家族成员为VSV、VSVG包装的假病毒的广泛应用提供可能。据此,在制备慢病毒的293T细胞中缺陷LDLR蛋白,可以减少产生的慢病毒再次进入细胞,从而提高慢病毒的产量。
近几年,在HIV-1与宿主限制因子的相互作用研究中发现E3泛素连接酶MARCH8可以抑制HIV-1的复制,通过减少病毒颗粒中包膜糖蛋白的插入,降低病毒颗粒的感染性。研究同时发现,MARCH8可以导致VSVG降解,从而降低VSVG在细胞中的表达。MARCH8属于一类膜结合的E3泛素连接酶MARCHs蛋白家族,MARCHs蛋白N端胞内区均含有一个C4HC3 RING-finger结构域。MARCHs蛋白的发现最早源于对病毒免疫逃逸机制的研究,病毒来源的MARCHs蛋白的主要功能是下调细胞表面主要组织相容性复合物(Majorhistocompatibility complex,MHC)Ⅰ类分子表达,帮助病毒实现免疫逃逸。目前已知胞内MARCHs蛋白共有十一个成员,其中,除了MARCH7和MARCH10没有预测的跨膜结构域(Transmembrane domain, TM)外,其它MARCHs家族成员均有两个或以上的跨膜区。含有两个跨膜区的MARCHs蛋白共有七个,分别是MARCH1, 2, 3, 4, 8, 9和11,根据氨基酸序列相似性,将这七个蛋白分为三个亚家族,分别是:MARCH1和8,MARCH2和3,MARCH4, 9和11。对于MARCHs的功能研究集中于通过泛素化调节胞内蛋白的降解和运输。已经发现,MARCH1和MARCH8能够下调MHCⅠ,MHCⅡ,CD86和白介素1受体辅助蛋白(IL-1 Receptor AccessoryProtein,IL1RAP)等多种细胞膜蛋白的表达,是一种重要的免疫调节蛋白。
最近研究发现,胞内MARCHs作为限制因子抑制HIV-1复制。通过siRNA文库筛选,发现MARCH8是抑制HIV-1复制的宿主限制因子。随后Takuya等发现MARCH8能够抑制多种类型HIV复制。MARCH8的抗病毒作用依赖其E3酶活性,但不依赖细胞蛋白酶体作用。过表达实验发现,MARCH8不影响病毒的产量,而通过减少病毒颗粒中包膜糖蛋白的插入,降低病毒颗粒的感染性。同时,MARCH8可以通过泛素化VSVG蛋白,导致VSVG蛋白的降解。
在上述研究背景的基础上,本申请建立了使用VSVG突变体从而不受MARCH8影响的慢病毒载体系统,提高了利用正常293T细胞制备的慢病毒的转导效率。同时,本申请建立了MARCH8缺失的293T细胞,利用此缺失MARCH8的293T细胞进行慢病毒的制备,提高了产生的慢病毒的转导效率,进一步优化了慢病毒载体系统。利用本申请建立的优化的慢病毒载体系统,可以为基因缺陷性疾病,如输血依赖型β地中海贫血、脑肾上腺脑白质营养不良、X连锁重症联合免疫缺陷病等的基因治疗提供更高效的慢病毒载体,还可以为CAR-T等利用慢病毒载体治疗肿瘤的技术服务。
发明内容
本发明对慢病毒载体系统进行改造,建立可增加转导效率的慢病毒载体系统。
本发明首先提供了一种VSVG突变体,其特征在于突变包括491或493或496或497或511位或其组合。
更优选的,本发明提供的VSVG突变体为V4R或者V5R。
进一步的,本发明提供一种上述VSVG突变体序列在制备慢病毒载体中的应用。
其中所述的应用为将上述VSVG蛋白突变体蛋白替代Env包膜蛋白应用在慢病毒载体的制备过程中。
进一步的,所述慢病毒载体包括:VSVG(pMD2.G)(Addgene,#12259),或其突变体V4R、V5R,慢病毒包装质粒表达Gag-Pol 的psPAX2(Addgene,#12260),带有GFP的plenti-GFP(pBMN(CMV-copGFP-Luc2-Puro))(Addgene,#80389)组成的三质粒系统。
另外的,本发明提供了一种包装细胞系,其包含MARCH8缺失的细胞。
进一步的,本发明提供上述细胞系的获得方法,具体为:
(1)Lenti-gRNA-Cas9质粒的构建
针对MARCH8基因设计gRNA序列,包括gRNA1和gRNA2。
gRNA1的序列为:GTAAGACCAAAGAAAAGGAG(SEQ ID NO.1) ;
gRNA2的序列为:GAGCTCGCAGCAGCGCGTGT(SEQ ID NO.2)。
通过分子克隆方法将这两条靶向MARCH8的gRNA序列导入lentiCRISPR v2(Addgene,#52961)中,获得靶向MARCH8的gRNA质粒gRNA-Cas9。
(2)缺失MARCH8的293T细胞系的建立
将293T细胞接种在六孔板无抗生素 DMEM 完全培养基中,培养过夜,细胞生长平铺 60%~70%时,转染gRNA-Cas9质粒。24h后加嘌呤霉素进行筛选。两周后获得缺失MARCH8的293T细胞系。
另外的,本发明提供了包含MARCH8缺失的细胞在制备慢病毒载体中的应用。
优选的,慢病毒体系包括VSVG或其突变体V4R、V5R,慢病毒包装质粒表达Gag-Pol的psPAX2,带有GFP的plenti-GFP。
进一步的,本发明提供一种生产重组慢病毒的方法,包括培养如上所示的细胞系,转染包含如上所述VSV-G突变体质粒,慢病毒包装质粒表达Gag-Pol 的psPAX2,带有GFP的plenti-GFP。转染48h后,收取上清。
进一步的,本发明提供一种生产重组慢病毒的方法,包括缺失MARCH8的293T及对照细胞接种无抗生素DMEM 完全培养基中,培养过夜,细胞生长平铺 60%~70%时,转染VSVG、psPAX2、lenti-GFP质粒,48h后,收取上清。
进一步的,本发明提供一种利用上述的方法产生的慢病毒。
进一步的,本发明提供一种包含VSVG突变的慢病毒载体,其特征在于包含突变491或493或496或497或511位,或其组合,包括本发明所用VSVG以及所有表达VSVG的质粒。
进一步的,所述突变为VSVG的491、493、496、497位赖氨酸突变为精氨酸(V4R)。
进一步的,所述突变为VSVG的491、493、496、497、511位赖氨酸突变为精氨酸(V5R)。
进一步的,本发明提供一种药物制剂,其包含药学上可接受的载体以及根据上述的慢病毒颗粒或上述方法生产的慢病毒颗粒。
进一步的,本发明提供一种重组慢病毒载体表达系统,其包含上述的慢病毒颗粒或权利要求9-11所述方法生产的慢病毒颗粒。
进一步的,本发明提供一种将异源基因递送至靶细胞的方法,包括用上述的方法中收集的慢病毒颗粒,感染所述靶细胞。
优选的,其中所述靶目标细胞包括293T、SupT1、THP1、PBMC、活化T细胞等,以及各种传代和原代的肿瘤细胞、上皮细胞、成纤维细胞、肌细胞、神经细胞、干细胞等。
进一步的,本发明提供一种如上所述的慢病毒颗粒或方法生产的慢病毒颗粒在基因治疗中的应用。
优选的,所述应用在于体外过继免疫治疗。
优选的,所述应用如CAR-T,TCR-T等。
附图说明
为了更清楚地说明本申请实施例或现有技术中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍。
图1为MARCH8或突变体W114A降解VSVG的情况。Con为空载体对照,hMAR8为野生型MARCH8,W114A为突变体W114A,Flag指示MARCH8或突变体W114A的标签抗体,Actin为内参。
图2为 VSVG的泛素化情况。Con为空载体对照,hMAR8为野生型MARCH8,W114A为突变体W114A,HA指示泛素Ub的标签。
图3为VSVG的定位情况。hMAR8为野生型MARCH8,W114A为突变体W114A,Flag指示MARCH8或突变体W114A的标签抗体,DAPI为核染料。
图4为VSVG胞质区赖氨酸位置及突变体示意图。
图5为MARCH8降解VSVG或突变体的情况。
图6为利用VSVG突变体V4R、V5R制备慢病毒,并检测转导效率的结果。
图7为MARCH8缺失的293T细胞中MARCH8的表达情况。V2为对照细胞,g1、g2分别为利用MARCH8两条gRNA构建的MARCH8敲除细胞系。
图8为利用MARCH8缺失的293T细胞制备慢病毒,并检测转导效率的结果。V2为对照细胞,g1、g2分别为利用MARCH8两条gRNA构建的MARCH8敲除细胞系。
具体实施方式
为了使本领域的技术人员更好地理解本发明的技术方案,下面将结合附图对本发明作进一步的详细介绍。
293T、HeLa细胞用含10%胎牛血清、100U/mL青霉素、100μg/mL链霉素的DMEM培养液,在37℃,5%CO2培养箱中培养。SupT1、THP1、PBMC细胞用含10%胎牛血清、100U/mL青霉素、100μg/mL链霉素的RPMI-1640培养液,在37℃,5%CO2培养箱中培养。
实施例1 建立使用VSV-G突变体从而不受MARCH8影响的慢病毒载体
1、方法
1.1、MARCH8及VSVG蛋白的表达及定位的检测
1.1.1、蛋白的提取
在293T(5×105个)细胞中转染MARCH8(SEQ ID NO.3)或其突变体W14A(SEQ IDNO. 4, MARCH8第114位氨基酸由W突变为A)、VSVG(SEQ ID NO.5)或其赖氨酸突变体(V491,SEQ ID NO.6,VSVG第491位氨基酸由K突变为R; V493,SEQ ID NO.7、VSVG第493位氨基酸由K突变为R;V496,SEQ ID NO.8、VSVG第496位氨基酸由K突变为R; V497,SEQ ID NO.9、VSVG第497位氨基酸由K突变为R;V511,SEQ ID NO.10、VSVG第511位氨基酸由K突变为R;V4R,SEQID NO.11、VSVG第491、493、496、497位氨基酸同时由K突变为R;V5R,SEQ ID NO.12,VSVG第491、493、496、497、511位氨基酸同时由K突变为R),48h后收取细胞。3000 rpm离心3min,PBS洗一次,加入100μL细胞裂解液RIPA,-20℃保存备用。
1.1.2、Western Blot检测蛋白的表达
(l)首先制备12%的分离胶 (4mL 30%丙烯酰胺溶液,2.5mL Tris/HCl pH8.8,100μL 10%SDS,100μL 10%过硫酸氨,5μL TEMED),灌胶,液面顶端加入水,室温下聚合60min。
(2)倒干净上层水,再制备5%的积层胶(0.5mL 30%丙烯酰胺溶液,0.5mL Tris/HCl pH6.8,40μL 10%SDS,50μL 10%过硫酸氨,5μL TEMED),在积层胶上插入梳齿,室温下聚合50min。待胶完全凝聚后,小心拔下梳齿。
(3)收获的裂解液12000rpm,4℃离心10min 。取60 μL,加入4×上样缓冲液20μL,98℃ 煮 10min。
(4)制备的电泳胶装进电泳槽,在内槽加满新鲜配制的电泳缓冲液(没过制胶板),将上步处理好的样品取20μL缓缓加入积层胶梳齿孔中,再在电泳槽外槽内加入适量电泳缓冲液,进行电泳,约1.5h,结束电泳。
(5)切掉多余胶块,按照从负极到正极分别为滤纸、胶、硝酸纤维素膜(NC膜)、滤纸的顺序叠放在转膜夹板中,放入转膜装置,加满新配的转膜液,冰上或 4℃, 80V 120min,将胶上的蛋白转移到 NC膜上。
(6)转好的膜用5%脱脂奶粉封闭,水平摇床上25 rpm,室温孵育 l h。
(7)将膜浸在含适当稀释抗体(Flag 抗体或VSVG抗体 1:1000 稀释)的 5%脱脂奶粉中,水平摇床上 25 rpm,4℃过夜。
(8)50 rpm水平摇床上,用 TBST 洗膜 4 次,5min/次。
(9)弃掉洗液,加入 1:10000 稀释的Alexa Fluor®488goat anti-mouse或goatanti-rabbit二抗,水平摇床上 25rpm,室温避光孵育 lh。
(10)弃掉二抗,25rpm水平摇床上用 TBST 洗膜 4 次,5min/次。
(11)取出膜,利用LI-COR Odyssey仪器扫膜。
1.1.3、通过免疫荧光实验检测VSVG蛋白的定位
在HeLa细胞中转染MARCH8或其突变体W114A、VSVG或其赖氨酸突变体(具体序列如上),24h后PBS洗2遍,用4%的多聚甲醛固定15min,再用含0.1%Triton的多聚甲醛室温打孔10min。PBS洗4次,再用封闭液室温封闭2h。上一抗Flag、VSVG抗体,室温2h或者4℃过夜。0.1%Triton 的PBS洗4次,上二抗 Alexa Fluor®488 goat anti-mouse、Alexa Fluor®594 goat anti-rabbit,避光室温1h。PBS洗1遍,DAPI染色5min,PBS洗2遍,通过激光共聚焦显微镜观察。
1.2、免疫共沉淀Co-IP检测VSVG的泛素化
293T细胞(4×106个)接种在10cm碟子无抗生素DMEM 完全培养基中,培养过夜,细胞生长平铺 60%~70%时,转染Flag-MARCH8或其突变体W114A、VSVG及HA-Ub质粒。48h后,收获细胞,加入RIPA裂解,离心后取裂解液上清加入VSVG抗体及磁珠4℃混4h,取出后离心,加入RIPA洗4次,最后加入2Xloading buffer,98℃煮10min,取上清进行SDS-PAGE及WesternBlot实验。
1.3、Gibco™Dynabeads CD3/CD28 CTS磁珠分离活化T细胞
使用CTS Dynabeads CD3/CD28(磁珠:细胞比例为3:1)在细胞培养袋中分离并激活来自健康供体的PBMC的T细胞 (不完全培养基)。在移除磁珠后,在细胞培养板中扩增T细胞。将分离的和活化的T细胞在完全培养基(不完全培养基加100U/mL IL-2)中扩增7–10天;在刺激后第2,3或5天磁性移除CTS Dynabeads CD3/CD28,随后进行慢病毒转导。
1.4、利用VSVG突变体制备慢病毒,转导靶细胞,并检测转导效率
293T细胞(4×106个)接种在10cm碟子无抗生素DMEM 完全培养基中,培养过夜,细胞生长平铺 60%~70%时,转染VSVG或其491、493、496、497及511位突变或其组合(V491、V493、V496、V497、V511、V4R、V5R)、psPAX2、lenti-GFP质粒。48h后,收取病毒上清,进行p24ELISA定量及超离后Western Blot检测p24等。同时收获细胞,加入RIPA裂解,进行SDS-PAGE及Western Blot实验。
p24 ELISA定量上清的病毒量后,取等量病毒加入293T、SupT1、THP1 、PBMC、以及PBMC经CD3/CD28免疫磁珠分离活化的T细胞中进行转导实验,72h后收取细胞流式细胞仪检测GFP的表达情况,从而指示其转导效率。
2、结果
2.1 MARCH8利用其E3泛素连接酶活性诱导VSVG降解
本实验室将VSVG与MARCH8或MARCH8E3酶活性突变体W114A (Flag标签)共同表达于 293T细胞,通过Western Blot检测,发现野生型MARCH8可以明显降低VSVG的表达,而MARCH8的E3酶活性突变体W114A丧失了降解VSVG的能力,表明MARCH8的E3酶活性是其降解VSVG所必需的(如图1所示)。
2.2 MARCH8通过其E3酶活性泛素化修饰VSVG
我们将VSVG与MARCH8或MARCH8酶活性突变体W114A共同表达于 293T细胞,利用Co-IP检测VSVG的泛素化,结果表明MARCH8可以泛素化VSVG,而MARCH8的酶活性突变W114A丧失了泛素化VSVG的能力(如图2所示)。
2.3 MARCH8导致VSVG在细胞膜上的表达降低
将VSVG与MARCH8或MARCH8酶活性突变体W114A (Flag标签)共同表达于 HeLa细胞,利用免疫荧光实验检测VSVG的定位及表达情况,结果表明MARCH8可以降低VSVG在细胞膜上的表达,而MARCH8的酶活性突变W114A则不影响VSVG的定位及表达(如图3所示)。
2.4 寻找抵抗MARCH8降解的VSVG突变体
泛素化修饰主要发生在细胞质中,而VSVG的胞内区主要包括489-511位氨基酸,分析此区域,可能被泛素化的位点主要包括491、493、496、497及511位的赖氨酸,据此我们构建了这五个位点分别突变为精氨酸、前四个位点均突变的V4R以及5个位点同时突变的突变体V5R(如图4所示)。将VSVG或这些突变体与Flag-MARCH8共同表达于 293T细胞,通过Western Blot检测,结果显示5个赖氨酸位点同时突变的VSVG突变体V5R不会被MARCH8所降解(如图5所示),据此,找到可以抵抗MARCH8降解的VSVG突变体V5R。
2.5 利用VSVG突变体V4R、V5R制备慢病毒,提高其转导效率。
将VSVG(pMD2.G)(Addgene,#12259))或其491、493、496、497及511位的突变(V491、V493、V496、V497及V511),或其组合V4R、V5R(包括所有表达VSVG的质粒以及携带491、493、496、497及511位突变的质粒)等,与慢病毒包装质粒表达Gag-Pol 的psPAX2、带有GFP的plenti-GFP共同转染293T细胞,48h后收取上清获得慢病毒,经Western Blot(如图6A所示)或p24 ELISA试剂盒(如图6B所示)检测,利用VSVG突变体V4R、V5R制备慢病毒,上清及细胞中p24的量不受影响。取等量的慢病毒进一步转导293T、SupT1、THP1、PBMC、以及PBMC经CD3/CD28免疫磁珠磁珠分离活化的T细胞,72h后通过流式细胞仪检测转导后靶细胞中GFP的表达情况,从而指示利用VSVG突变体制备的慢病毒的转导效率。结果显示,利用VSVG突变体V4R、V5R制备的慢病毒的转导效率均高于野生型VSVG(如图6C、D、E、F、G所示),而V5R制备的慢病毒的转导效率略高于V4R,表明建立了使用VSV-G突变体从而不受MARCH8影响转导效率提高的慢病毒载体系统。
实施例2 建立MARCH8缺失的293T细胞,利用此细胞制备慢病毒,提高转导效率
1、方法
1.1 、缺失MARCH8的293T细胞系的建立
1.1.1、Lenti-gRNA-Cas9质粒的构建
针对MARCH8基因,通过网站http://www.e-crisp.org/E-CRISP/设计gRNA序列,包括gRNA1和gRNA2。
gRNA1的序列为:GTAAGACCAAAGAAAAGGAG(SEQ ID NO.1);
gRNA2的序列为:GAGCTCGCAGCAGCGCGTGT(SEQ ID NO.2)。
本实验室通过分子克隆方法将这两条靶向MARCH8的gRNA序列导入lentiCRISPRv2 (Addgene,#52961)中,获得靶向MARCH8的gRNA质粒。
1.1.2、缺失MARCH8的293T细胞系的建立
293T细胞(5×105个)接种在六孔板无抗生素 DMEM 完全培养基中,培养过夜,细胞生长平铺 60%~70%时,转染gRNA-Cas9质粒。24h后加入0.6ug/ml嘌呤霉素(puromycin)进行筛选。两周后获得缺失MARCH8的293T细胞系。
1.2、在缺失MARCH8的293T细胞中制备慢病毒,并检测转导活性
缺失MARCH8的293T(g1、g2)及对照细胞V2(4×106)接种在10cm碟子无抗生素DMEM完全培养基中,培养过夜,细胞生长平铺 60%~70%时,转染VSVG、psPAX2、lenti-GFP质粒。48h后,收取上清,进行p24 ELISA定量及超离后Western Blot检测p24等。同时收获细胞,加入RIPA裂解,进行SDS-PAGE及Western Blot实验。
p24 ELISA定量上清的病毒量后,取等量病毒加入293T、SupT1、THP1、PBMC、以及PBMC经CD3/CD28免疫磁珠分离活化的T细胞中进行转导实验,72h后收取细胞流式细胞仪检测GFP的表达情况从而指示其转导效率。具体方法同实施例一。
2、结果
2.1 建立MARCH8缺陷的293T细胞系
经过嘌呤霉素筛选,最终获得的缺失MARCH8细胞系中,利用Western Blot检测,细胞系内源MARCH8蛋白的表达与对照V2相比明显降低(如图7所示)。
2.2、利用MARCH8缺陷的293T细胞系制备慢病毒,提高其转导效率。
将VSVG与慢病毒包装质粒表达Gag-Pol 的psPAX2、带有GFP的plenti-GFP共同转染MARCH8缺陷的293T细胞系,48h后收取上清获得慢病毒,经Western Blot(如图8A所示)或p24 ELISA试剂盒(如图8B所示)检测,上清及细胞中p24的量不受影响。取等量的慢病毒进一步转导293T、SupT1、THP1、PBMC、以及PBMC经CD3/CD28免疫磁珠磁珠分离活化的T细胞,72h后通过流式细胞仪检测转导后靶细胞中GFP的表达情况,从而指示利用缺陷细胞系制备的慢病毒的转导效率。结果显示,利用MARCH8缺陷细胞(g1、g2)制备的慢病毒的转导效率均高于对照V2细胞(如图8C、D、E、F、G所示),表明建立了使用MARCH8缺陷的293T细胞系提高转导效率的慢病毒载体系统。
本说明书中提到的所有出版物均通过引用以其整体并入本文。本领域技术人员将会理解,可以对具体实施例中所示的本公开进行许多变化和/或修改,而不脱离如广泛描述的本公开的精神或范围。因此,本实施例在所有方面都被认为是说明性的而不是限制性的。
尽管已经参照特定实施例描述了本公开,但是应该理解的是,这些实施例仅仅是本公开的原理和应用的说明。因此可以理解的是,可以对说明性实施例作出许多修改,并且在不脱离由所附权利要求限定的本公开的精神和范围的情况下可以设计出其它布置。
〈110〉 青岛宁逸生物科技有限公司
〈120〉一种提高转导效率的慢病毒载体及其应用
〈160〉 12
〈170〉 PatentIn version 3.3
〈210〉 1
〈211〉 20
〈212〉 nt
〈213〉gRNA1
〈400〉 GTAAGACCAAAGAAAAGGAG
〈210〉 2
〈211〉 20
〈212〉 nt
〈213〉gRNA2
〈400〉 GAGCTCGCAGCAGCGCGTGT
〈210〉 3
〈211〉 291
〈212〉 aa
〈213〉 MARCH8
〈400〉MSMPLHQISAIPSQDAISARVYRSKTKEKEREEQNEKTLGHFMSHSSNISKAGSPPSASAPAPVSSFSRTSITPSSQDICRICHCEGDDESPLITPCHCTGSLHFVHQACLQQWIKSSDTRCCELCKYEFIMETKLKPLRKWEKLQMTSSERRKIMCSVTFHVIAITCVVWSLYVLIDRTAEEIKQGQATGILEWPFWTKLVVVAIGFTGGLLFMYVQCKVYVQLWKRLKAYNRVIYVQNCPETSKKNIFEKSPLTEPNFENKHGHGICHSDTNSSCCTEPEDTGAEIIHV
〈210〉 4
〈211〉 291
〈212〉 aa
〈213〉 W114A
〈400〉MSMPLHQISAIPSQDAISARVYRSKTKEKEREEQNEKTLGHFMSHSSNISKAGSPPSASAPAPVSSFSRTSITPSSQDICRICHCEGDDESPLITPCHCTGSLHFVHQACLQQAIKSSDTRCCELCKYEFIMETKLKPLRKWEKLQMTSSERRKIMCSVTFHVIAITCVVWSLYVLIDRTAEEIKQGQATGILEWPFWTKLVVVAIGFTGGLLFMYVQCKVYVQLWKRLKAYNRVIYVQNCPETSKKNIFEKSPLTEPNFENKHGHGICHSDTNSSCCTEPEDTGAEIIHV
〈210〉 5
〈211〉 511
〈212〉 aa
〈213〉 VSVG
〈400〉MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK
〈210〉 6
〈211〉 511
〈212〉 aa
〈213〉 V491
〈400〉MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIRLKHTKKRQIYTDIEMNRLGK
〈210〉 7
〈211〉 511
〈212〉 aa
〈213〉 V493
〈400〉MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLRHTKKRQIYTDIEMNRLGK
〈210〉 8
〈211〉 511
〈212〉 aa
〈213〉 V496
〈400〉MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTRKRQIYTDIEMNRLGK
〈210〉 9
〈211〉 511
〈212〉 aa
〈213〉 V497
〈400〉MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKRRQIYTDIEMNRLGK
〈210〉 10
〈211〉 511
〈212〉 aa
〈213〉 V511
〈400〉MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGR
〈210〉 11
〈211〉 511
〈212〉 aa
〈213〉 V4R
〈400〉MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIRLRHTRRRQIYTDIEMNRLGK
〈210〉 12
〈211〉 511
〈212〉 aa
〈213〉 V5R
〈400〉MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIRLRHTRRRQIYTDIEMNRLGR
Claims (9)
1.一种VSVG突变体,其特征在于所述VSVG突变体的氨基酸序列如SEQ ID NO:11或SEQID NO:12所示。
2.如权利要求1所述的VSVG突变体在制备慢病毒载体中的应用。
3.一种包装细胞系在制备慢病毒体系中的应用,其特征在于,所述包装细胞系包含MARCH8缺失的细胞,慢病毒体系包括含有权利要求1所示的VSVG突变体的载体,慢病毒包装质粒表达Gag-Pol 的psPAX2,带有GFP的plenti-GFP。
4.一种生产重组慢病毒的方法,包括培养包含MARCH8缺失的细胞系,转染包含如权利要求1所述VSVG突变体的质粒、慢病毒包装质粒表达Gag-Pol 的psPAX2、带有GFP的plenti-GFP,转染48h后,收取上清。
5.根据权利要求4所述的方法产生的慢病毒。
6.一种包含如权利要求1所述的VSVG突变体的慢病毒载体。
7.一种非治疗目的的将异源基因递送至靶细胞的方法,包括用根据权利要求4所述方法制备携带异源基因的慢病毒,感染所述靶细胞。
8.如权利要求7所述的方法,其中所述靶细胞选自293T、SupT1、THP1、PBMC、活化T细胞。
9.如权利要求7所述的方法,其中所述靶细胞选自传代和原代的肿瘤细胞、上皮细胞、成纤维细胞、肌细胞、神经细胞、干细胞。
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