CN111518773B - 一种抗新型冠状病毒s蛋白的car-t细胞、制备方法及其应用 - Google Patents
一种抗新型冠状病毒s蛋白的car-t细胞、制备方法及其应用 Download PDFInfo
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- C12N2800/107—Plasmid DNA for vertebrates for mammalian
Abstract
本发明提供一种抗新型冠状病毒S蛋白的CAR‑T细胞,所述CAR‑T细胞,经抗新型冠状病毒S蛋白优化的单链抗体基因修饰,所述优化的单链抗体为序列表中SEQ ID NO.2所示的核苷酸;所述新型冠状病毒为COVID‑19;本发明还提供上述CAR‑T细胞的制备方法和应用。本发明提供一种免疫细胞疗法CAR‑T技术,可以精准清除体内感染的COVID‑19病毒细胞,安全高效的治疗新型冠状病毒,本发明构造的CAR‑T细胞杀伤效果显著,对表达COVID‑19病毒S蛋白抗原的细胞杀伤率高达90%以上;本发明首次利用单个B淋巴细胞克隆和筛选策略技术筛选出针对COVID‑19病毒的scFv(单链抗体)。
Description
技术领域
本发明涉及一种抗新型冠状病毒S蛋白的CAR-T细胞、制备方法及其应用,属于属于生物医药技术领域。
背景技术
COVID-19病毒引起的肺炎一般症状表现为发热、乏力、干咳、逐渐出现呼吸困难,严重者会出现急性呼吸窘迫综合征、脓毒症休克、难以纠正的代谢性酸中毒、出凝血功能障碍。基于目前的流行病学调查,该病的潜伏期1-14天,多为3-7天,潜伏期也具有传染性,危害极大。自流行病爆发以来,既有西药(α型干扰素、洛匹那韦、利托那韦)、中药(轻症)等多种疗法积极加入治疗的行列,又有精准医学和干细胞疗法等高科技疗法加入流行病的预防和控制。临床研究表明,对包括SARS-CoV-2在内的所有人类冠状病毒感染,除了对症治疗外,国内外尚没有治愈新冠病毒的靶向药物上市,也没有开发出成熟有效的疫苗。目前临床治疗方法主要是采用高效抗逆转录病毒药物和注射新冠患者康复者血浆疗法,这些疗法虽然也起到了一定的治疗作用,但对病人本身的机体副作用伤害也较大或者容易引起一些免疫反应,都达不到理想的治疗效果。康复期COVID-19患者的血清抗体对治疗危重的COVID-19患者有一定的疗效,因此研制抗COVID-19的抗体对于新型冠状病毒的特异性治疗具有重要意义。
冠状病毒是一种有包膜的、非节段的单股正链RNA球形病毒,直径为 120~160 nm(图1),分为α、β、γ 和 δ四个属,新型冠状病毒属于β属,病毒粒子的外周是整齐排列的纤突,被脂质膜所包裹,包括刺突糖蛋白(spikegly coprotein,S蛋白)、膜蛋白(membraneprotein,M蛋白)和包膜蛋白(envelope protein,E蛋白)。其中,S蛋白(含有S1和S2两个亚基)是最重要的致病蛋白,最新研究表明,在COVID-19与SARS病毒S蛋白的保守性最高,其在结合细胞受体和介导病毒与宿主细胞膜的融合过程中起着重要作用,在COVID-19感染过程中是中和病毒感染的主要靶标。因此,选取COVID-19病毒的S蛋白为靶点,使用国际上最先进的CAR-T技术治疗COVID-19病毒,可以有效地阻断病毒侵入宿主细胞,清除体内的新型冠状病毒,达到彻底治愈的目的。
对于急重症患者,多采用抗病毒药物和注射新冠患者康复者血浆疗法治疗COVID-19患者,新型冠状病毒感染的肺炎诊疗方案(试行第七版)推荐抗病毒治疗可试用α-干扰素(广谱抗病毒药物)雾化吸入、洛匹那韦/利托那韦或可加用利巴韦林(广谱抗病毒药物)。对于轻症患者,可以结合中医疗法进行治疗。
抗病毒药物副作用大,患者易出现药物耐受。中药治疗效果缓慢,对于重症患者治疗效果差。这两种方法只能降低体内的病毒含量,无法彻底清除体内的COVID-19病毒。
发明内容
针对现有技术存在的不足,本发明提供一种抗新型冠状病毒S蛋白的CAR-T细胞、制备方法及其应用,选取COVID-19病毒最保守的区域S蛋白为靶点,筛选出抗COVID-19病毒的特异性单链抗体,并进行密码子优化,得到抗COVID-19病毒的单链抗体,采用第三代CAR,使用COVID-19患者自体的外周血,体外进行基因编辑(将构建的CAR结构转入T细胞)之后,靶向治疗新型冠状病毒,无副作用,安全性高,治疗周期短,可以实现对新冠病毒感染的细胞的精准清除,杀伤效率高,从而抑制病毒在感染细胞内的复制。
本发明采取以下技术方案:
一种抗新型冠状病毒S蛋白的CAR-T细胞,所述CAR-T细胞,经抗新型冠状病毒S蛋白的优化的单链抗体基因修饰,所述优化的单链抗体为序列表中SEQ ID NO.2所示的核苷酸;所述新型冠状病毒为COVID-19。
一种抗新型冠状病毒S蛋白的CAR-T细胞的制备方法,所述制备方法包括抗COVID-19-CAR表达载体的构建,所述抗COVID-19-CAR表达载体,包括抗新型冠状病毒S蛋白的优化的单链抗体。
所述优化的单链抗体的制备方法,包括筛选记忆B细胞、中和抗体实验、单细胞抗体测序、抗S蛋白抗体靶点的确认、序列优化。
所述筛选记忆B细胞,从COVID-19康复者的静脉血中,分选出记忆性B细胞,然后用DMEM培养基进行悬浮得到细胞悬液,按照18-22个细胞/孔的密度接种到培养板中培养,每孔接种0.09-0.12ml细胞悬液,用CD40配体刺激活化并加入IL-2和IL-21因子培养10-12天,让记忆性B细胞活化成为抗体分泌细胞,收集上清备用。
所述CD40配体的终浓度为1.8-2.2μg/ml,所述IL-2的终浓度为1.9-2.1 ng/ml,所述IL-21因子的终浓度为1.9-2.1 ng/ml。
所述中和抗体实验,检测OD492值为1.1-1.3;选用的中和抗体的假病毒为VERO-S-LUC-eGFP细胞系;所述抗S蛋白抗体靶点的确认,得到单链抗体,为序列表中SEQ ID NO.7所示的核苷酸;
所述抗COVID-19-CAR表达载体包括CD8导引子- scFv- CD8 Hinge区- CD28跨膜区- CD28跨膜区-CD3ζ信号传导区。
所述制备方法还包括慢病毒包装及滴度检测;所述慢病毒包装及滴度检测,重组慢病毒的滴度为4.6-4.7×106pfu/mL。
所述制备方法还包括T细胞制备;所述T细胞制备,从COVID-19患者自体外周血中分离、诱导培养T细胞,得到的T细胞CD3+阳性率>80%,CD3+ CD56+双阳性率>20%。
一种抗新型冠状病毒S蛋白的CAR-T细胞的应用,所述抗新型冠状病毒S蛋白的CAR-T细胞用于治疗新型冠状病毒COVID-19。
与现有技术相比,本发明取得以下有益效果:
(1)本发明提供一种免疫细胞疗法CAR-T技术,可以精准清除体内感染的COVID-19病毒细胞,安全高效的治疗新型冠状病毒,本发明构造的CAR-T细胞杀伤效果显著,对表达COVID-19病毒S蛋白抗原的细胞杀伤率高达90%以上。
(2)本发明首次利用单个B淋巴细胞克隆和筛选策略技术筛选、优化出针对COVID-19病毒的scFv(单链抗体)。
(3)本发明构造的CAR-T细胞用于治疗COVID-19病毒,特异性强,安全性高,无副作用。
附图说明
图1为冠状病毒的结构示意图;
图2为抗COVID-19-CAR模块示意图;
图3为基因重组示意图;
图4为流式细胞术检测抗COCID-19-CAR载体在T细胞表面的表达效率的流式图;
图5为本发明的COVID-19 CAR-T细胞、pri COVID-19 CAR-T细胞和效应T细胞杀伤率的柱状图;
图6为本发明靶向S蛋白的单链抗体scFv的重链核苷酸序列的优化对比;
图7为本发明靶向S蛋白的单链抗体scFv的重链氨基酸序列的优化对比;
图8为本发明靶向S蛋白的单链抗体scFv的轻链核苷酸序列的优化对比;
图9为本发明靶向S蛋白的单链抗体scFv的轻链氨基酸序列的优化对比。
具体实施方式
实施例1 单个B淋巴细胞克隆和筛选特异性单链抗体
(1)筛选记忆B细胞
抽取10ml COVID-19康复者的静脉血,用TBD样本密度分离液(购自天津灏洋华科生物),分离获得外周血单个核细胞(PBMC),按照人源记忆性B细胞磁珠分选试剂盒分选出记忆性B细胞。将筛选出的记忆B细胞用DMEM培养基进行悬浮得到细胞悬液,按照20个/孔的密度接种U型底的96孔培养板中培养,每孔接种0.1ml细胞悬液,用CD40配体(终浓度2μg/ml)刺激活化并加入IL-2(终浓度2ng/ml)和IL-21因子(终浓度2ng/ml)在37℃、5% CO2条件下培养11天,让记忆性B细胞活化成为抗体分泌细胞,收集上清备用。
(2)中和抗体实验
向96孔平底培养板中加入待测上清25μL/孔(每孔约5×104个记忆B细胞),加入包含10% FBS、1% PBS的DMEM培养基稀释的假病毒(VERO-S-LUC-eGFP细胞系,约1×104个细胞,能表达S蛋白抗原的细胞,由山东大学药学院提供)25μL/孔,混匀,同时设细胞对照孔和病毒对照孔,37℃、5% CO2培养箱孵育1h,加VERO-E6细胞(购自通派生物科技有限公司,用于检测中和抗体的中和能力),加入量为:1×104个/(50μL/孔),加入终浓度为20μg/ml DE-AE-Dextran;37℃、5% CO2培养箱继续孵育48h。培养结束后裂解细胞,酶标仪测OD492值。中和抗体的效价判断以低于阳性的50%OD值为依据。当OD492数值在0.6-1.5之间,即确认为有效抗体。本发明涉假病毒组的OD492值分别为1.2,初步说明得到的中和抗体具有一定的中和病毒活性;上述1×104个细胞,是指25μL中含有的VERO-S-LUC-eGFP细胞的个数。
(3)单细胞抗体测序
将B细胞收集到EP管中,用RNAiso TM Plus将细胞裂解提取总RNA。将上述EP管放入离心机中4℃,12000rpm离心10min,上清转移到一个新的EP管中,弃沉淀。在上清中加入等体积的氯仿溶液,涡旋震荡,室温静置7min。4℃,12000rpm离心15min,此时EP管中的液体分3层,中间一层白色的为蛋白层,取上层的水相转移到新的EP管中(RNA存在于水相中);加入等体积的异丙醇到转移的上清中,温和的使其混匀,在光下能稍微看到絮状沉淀后室温静置7min;4℃,12000rpm离心10min,弃上清;加入750μL、75%的乙醇洗涤,4℃,12000rpm离心5min,重复一次;弃乙醇,真空干燥5min,加入50μL无RNAase水溶解,即获得总RNA,放在-20℃冰箱中保存备用。以提取的总RNA作模板,Prime ScriptTM RT reagent Kit withgDNA Eraser试剂盒合成第一链cDNA,放于-20℃冰箱保存备用。
RT-PCR反应体系:20μL反应体系中含10μLPCR Master Mix、0.5μmol/L上/下游引物、0.5μL cDNA、用ddH2O补足20μL。
引物:根据网站设计100对靶向S蛋白的抗体引物序列,从中筛选出10对效果较好的引物。
10对引物分别进行RT-PCR扩增,分别得到重链和轻链扩增产物,将获得的扩增产物进行一代测序,获得重链和轻链序列。
RT-PCR反应程序:95℃/5min,95℃/10s,58℃/10s,72℃/30s,共40个循环。将收集的PCR产物纯化后进行基因测序,获得抗体的重链和轻链序列。
(4)抗S蛋白抗体靶点的确认
要决定它在S蛋白抗原上的抗原表位,用ELISA的方法决定它是否为靶向S蛋白的抗体。方法如下:将获得scFv序列连接至表达载体并转化到大肠杆菌校正菌HB2151中表达形成可溶性的scFv,抗体纯化后与S蛋白(包括S1和S2蛋白)氨基酸构成的肽相结合,观察抗体与相应肽的结合特异性。使用ELISA试剂盒检测(购自福因德科技(武汉)有限公司),当获得的scFv与S蛋白反应,发生特异性结合时,表明筛选出的抗体为靶向S蛋白的单链抗体scFv,见序列表中的SEQ ID NO.7。
实施例2 靶向S蛋白的scFv的序列优化
已知COVID-19病毒靶向S蛋白中和抗体的中和位点位于S蛋白第315-523氨基酸之间,而COVID-19病毒与人细胞表面受体血管紧张肽转移酶(ACE2) 的结合位点也恰在这一段。依据这一已知位点,对获得scFv序列进行密码子优化,得到优化的单链抗体,增强单链抗体与S蛋白的亲和力,提高杀伤效果,scFv序列的重链和轻链的核苷酸、氨基酸的对比见图6-9。
序列优化前的单链抗体表述为单链抗体,序列优化后的单链抗体表述为优化的单链抗体。
实施例3 抗COVID-19-CAR表达载体的构建
抗COVID-19-CAR模块示意图2。Anti-SP(S蛋白)-CAR各模块核酸序列如下:
(1)CD8导引子Leader核酸人工序列(SEQ ID NO.1)
(2)Anti-SP single chain antibody fragment(scFv)核酸人工序列(SEQ IDNO.2)(优化后的核酸序列)
(3)CD8 Hinge区(CD8α)核酸人工序列(SEQ ID NO.3)
(4)CD28跨膜区(CD8 TM)核酸人工序列(SEQ ID NO.4)
(5)CD137共刺激区核酸人工序列(SEQ ID NO.5)
(6)CD3ζ信号传导区核酸人工序列(SEQ ID NO.6)
分别将SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、C SEQ ID NO.5、SEQ ID NO.6(基因重组图3)委托生工生物工程(上海)有限公司合成其整个表达框并插入标准载体pUC57载体上,得到pUC-COVID-19-CAR。将pUC-COVID-19-CAR和pLent-C-GFP载体进行Fast Digest AsiSI(购自Thermo Fisher公司)和Fast Digest NotI(购自ThermoFisher公司)双酶切后,16℃进行过夜连接形成pLent-COVID-19-CAR(结构示意图见图2)。将上述pLent-COVID-19-CAR转化到E.coli(DH5α)。提取质粒pLent-COVID-19-CAR并委托生工生物工程(上海)有限公司进行测序。经测序正确后备用。
实施例4慢病毒包装及滴度检测
将慢病毒包装细胞系293T接种于含有DMEM+10% FBS 10cm培养皿中,37℃、5%的CO2条件下培养,贴壁率为70%-80%时准备转染。取无菌的1.5ml EP管,按下列组分配制反应体系:无血清DMEM:3ml;pLent-COVID-19-CAR质粒:10μg;GM easyTM Lentiviral Mix:10μl(10μg);HG Transgene TM Reagent:60μl。混匀后,室温放置20min后,均匀滴加到含有293T细胞培养皿中,置于CO2培养箱中培养。转染24h后,小心吸掉细胞培养液弃于盛有消毒液的废液杯中,然后加15ml含有10%血清新鲜的培养基继续培养。换液48h后,吸取细胞上清液于50ml离心管,4℃,500g离心5min,上清液用0.45μm滤器过滤后转移到新的离心管中。收获病毒后进行浓缩,获得浓缩过的COVID-19-CAR病毒液,直接去检测滴度或-70℃低温冰箱中保存备用。将上述病毒采用TCID 50测定滴度,将处于对数生长期的293T细胞以1×104 Cells/孔的量接种于96孔细胞培养板,样品用5% FBS DMEM按10倍倍比稀释系列浓度,加样于96孔板中,每个浓度加样10孔,设2孔空白对照。于37℃、5% CO2培养,逐日观察细胞出现毒斑情况,一般需要观察5-7天,据出现毒斑的浓度和孔数计算样品的TCID 50结果。结果表明,重组慢病毒的滴度4.69×106pfu/mL。
实施例5 COVID-19 CAR-T细胞的制备
(1)T细胞制备
取50ml COVID-19患者自体外周血,用TBD样本密度分离液,分离获得PMBC。用含有1000 IU/ml重组干扰素α2a(购自沈阳三生制药)的培养基DMEM(购自CORNING公司,88-551-CM)诱导培养24小时后,加入1000IU/ml的重组IL-2(购自沈阳三生制药)、50ng/ml的OKT-3和5%的患者自体血浆诱导继续培养24小时。每隔两天倍比加液,培养至第14天,流式细胞术检测T细胞中的CD3+、CD56+的阳性表达率(CD3-FITC,CD16/CD56-PE抗体购自BECKMAN公司,A07735)。CD3+阳性率>80%,CD3+ CD56+双阳性率>20%,视为T细胞诱导成功,并留取该细胞待病毒感染。
(2)慢病毒感染T细胞
T细胞经活化后,取出1×106个细胞,加入浓缩过的COVID-19-CAR病毒液,混匀,MOI=8。分别在一周后对转染的T细胞进行相关检测。通过荧光显微镜检测嵌合抗原受体表达,由于GFP与CAR共表达,检测GFP的阳性细胞即为表达嵌合抗原受体的阳性细胞。
图4为流式细胞术检测的利用本发明构建的COVID-19-CAR载体在T细胞表面上的表达情况。结果表明有32.9%的细胞是GFP(慢病毒载体本身表达GFP蛋白)阳性,说明利用本发明构建的COVID-19-CAR能够在T细胞表面表达,且转染效率为32.9%。
实施例6 COVID-19 CAR细胞的体外杀伤实验
(1)构建表达COVID-19病毒S蛋白的细胞系
构建plvx-S-luc-eGFP重组质粒,包装重组慢病毒,感染Vero细胞后,通过嘌呤霉素筛选获得阳性的细胞系Vero-S-LUC-eGFP,使细胞表面即抗原表达S蛋白,又可以表达eGFP绿色荧光蛋白及荧光素酶。检测细胞表面COVID-19病毒S蛋白、EGFP绿色荧光蛋白表达情况。当ELSIA方法检测到S蛋白表达,流式细胞仪检测eGFP表达率达90%以上时,即为成功构建VERO-S-LUC-eGFP细胞系(该细胞系由山东大学药学院提供)。
(2)抗COVID-19 CAR-T细胞的体外杀伤实验
分别将抗COVID-19 CAR-T细胞(靶向S蛋白的优化的单链抗体scFv,以下简称COVID-19 CAR-T细胞)、抗pri COVID-19 CAR-T细胞CAR-T细胞(靶向S蛋白的scFv序列优化前,以下简称pri COVID-19 CAR-T细胞,制备方法同抗COVID-19 CAR-T细胞)、效应T细胞(未转染CAR)作为效应细胞,Vero-S-LUC-eGFP细胞系作为靶细胞(稳定表达S蛋白抗原),进行杀伤活性测定。取对数生长期的靶细胞,用含有10% FBS的DMEM以1×105个细胞重悬,接种到96孔板,每孔100μL,即1×104个/孔。37℃、5% CO2的培养箱中培养过夜。按照效应细胞数/靶细胞数(效靶比E:T)=5:1、10:1、20:1的比例,加入效应细胞至96孔板中,分组如下:
空白对照组(等量10% FBS+DMEM培养基);
靶细胞对照组(只加等量靶细胞);
效应T细胞实验组(未转染CAR的效应T细胞和靶细胞);
pri COVID-19 CAR-T细胞实验组(pri COVID-19 CAR-T细胞细胞和靶细胞)。
COVID-19 CAR-T细胞实验组(COVID-19 CAR-T细胞和靶细胞)。
每孔的最终体积加培养基固定至100μL,根据效靶比调整不同细胞的数量。
每组设置3个重复。放回37℃,5% CO2的培养箱继续培养24h。取出96孔板,按照Promega MTS试剂盒要求,使用酶标仪测定酶标仪检测450nm波长,读取OD值。实验组抗COVID-19 CAR-T细胞的细胞毒活性,以杀伤率表示,按照MTS试剂盒要求计算公式如下:杀伤率=[1-(实验组OD值-效应细胞对照组OD值)/靶细胞对照组OD值]×100%。
结果表明,效应细胞数/靶细胞数(效靶比E:T)=10:1时,效应细胞杀伤效果最好。效靶比过高空间有限,影响细胞生长,过低无法完全抑制病毒的复制。COVID-19 CAR-T细胞对Vero-S-LUC-eGFP细胞的杀伤效率高达90%,而pri COVID-19 CAR-T细胞实验组为60%,效应T细胞组(未转染CAR)杀伤率不足30%(见图5)。COVID-19 CAR-T细胞特异性杀伤活性显著高于pri COVID-19 CAR-T细胞组,其杀伤效果是pri COVID-19 CAR-T细胞组的1.5倍,表明优化后的scFv对靶细胞的杀伤性显著提高。因而,本发明制备的抗COVID-19 CAR-T细胞具有高效杀伤COVID-19病毒(靶向S蛋白抗原)的能力。
序列表
<110> 山东兴瑞生物科技有限公司
<120> 一种抗新型冠状病毒S蛋白的CAR-T细胞、制备方法及其应用
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gccgtatatt actgtgcgaa acgcagtctg agttttgact actggggcca gggaaccctg 180
gtcaccgtct cgctggagtg ggtctcacct attggtaggc ttggtcaggc tacaacctac 240
gcagactccg gcactctgac catcagcgcc ctgcagaagc actatactac tcctccgacg 300
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ggaggaggaa gcacggacat ccagcccatg acccagtctc catccggact gtctgcatct 420
gtaggagaca gagtcaccat cacttgccgg gcaagtcaga gcattcctga agattttgca 480
acttactact gtcaacagag ttacagtacc cctaatacgt tcggcgcaga acaaggtctc 540
atctcagaag aggatttaaa tggatatctg gtgcagggcc gtttcacttt ctatgctatg 600
gttcagctgg gggagtctgg cggtggcctc cagatgaaca gcctgcgtgc tgaggacacc 660
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Claims (7)
1.一种抗新型冠状病毒S蛋白的CAR-T细胞,其特征在于:所述CAR包括CD8导引子-scFv- CD8 Hinge区- CD28跨膜区- CD137共刺激区-CD3ζ信号传导区;所述scFv为抗新型冠状病毒S蛋白的优化的单链抗体;所述scFv的核苷酸序列如序列表中SEQ ID NO.2所示;所述新型冠状病毒为COVID-19。
2.根据权利要求1所述的一种抗新型冠状病毒S蛋白的CAR-T细胞的制备方法,其特征在于:所述制备方法包括抗COVID-19-CAR表达载体的构建,所述抗COVID-19-CAR表达载体,包括抗新型冠状病毒S蛋白的优化的单链抗体。
3.根据权利要求2所述的一种抗新型冠状病毒S蛋白的CAR-T细胞的制备方法,其特征在于:所述优化的单链抗体的制备方法,包括筛选记忆B细胞、中和抗体实验、单细胞抗体测序、抗S蛋白抗体靶点的确认、序列优化。
4.根据权利要求3所述的一种抗新型冠状病毒S蛋白的CAR-T细胞的制备方法,其特征在于:所述筛选记忆B细胞,从COVID-19康复者的静脉血中,分选出记忆性B细胞,然后用DMEM培养基进行悬浮得到细胞悬液,按照18-22个细胞/孔的密度接种到培养板中培养,每孔接种0.09-0.12ml细胞悬液,用CD40配体刺激活化并加入IL-2和IL-21因子培养10-12天,让记忆性B细胞活化成为抗体分泌细胞,收集上清备用。
5.根据权利要求4所述的一种抗新型冠状病毒S蛋白的CAR-T细胞的制备方法,其特征在于:所述CD40配体的终浓度为1.8-2.2μg/mL,所述IL-2的终浓度为1.9-2.1ng/mL,所述IL-21因子的终浓度为1.9-2.1ng/mL。
6.根据权利要求3所述的一种抗新型冠状病毒S蛋白的CAR-T细胞的制备方法,其特征在于:所述中和抗体实验,检测OD492值为1.1-1.3;选用的中和抗体的假病毒为VERO-S-LUC-eGFP细胞系;所述抗S蛋白抗体靶点的确认,得到单链抗体,所述单链抗体的核苷酸序列如序列表中SEQ ID NO.7所示。
7.根据权利要求2所述的一种抗新型冠状病毒S蛋白的CAR-T细胞的制备方法,其特征在于:所述制备方法还包括慢病毒包装及滴度检测;所述慢病毒包装及滴度检测,重组慢病毒的滴度为4.6-4.7×106pfu/mL。
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