CN113234151A - 一种基于靶向SARS-CoV2病毒S蛋白的三聚体纳米抗体的CAR-NK的研制 - Google Patents

一种基于靶向SARS-CoV2病毒S蛋白的三聚体纳米抗体的CAR-NK的研制 Download PDF

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CN113234151A
CN113234151A CN202110635538.XA CN202110635538A CN113234151A CN 113234151 A CN113234151 A CN 113234151A CN 202110635538 A CN202110635538 A CN 202110635538A CN 113234151 A CN113234151 A CN 113234151A
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徐振宇
何连君
黄后宝
何银梅
徐新徽
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Anhui Tuoji Saiao Biotechnology Co.,Ltd.
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Abstract

目前，新冠病毒SARS‑CoV2(简称SC‑2)在世界各地迅速传播，已成为全球流行病。导致疫情严重的原因之一是缺乏有效的干预手段，虽然疫苗有望延缓甚至遏制病毒传播，但对于已感染的重症患者治疗却无效。SC‑2通过其表面的S蛋白与宿主细胞上的ACE2结合进入细胞而发挥作用。NK细胞作为人体免疫系统的第一道防线，可以主动识别和杀伤入侵机体的SC‑2病毒。对此我们提出研制种靶向SC‑2的S蛋白的通用型新型嵌合抗原受体NK细胞(T‑CAR‑NK)，利用筛选针对SC‑2的S蛋白的纳米抗体作为CAR中单链抗体部分，同时优选铰链及胞内信号区，组装成新型CAR；并联合分选自杀(RQR8)系统；最终将上述基因通过改进的高效慢病毒整合到健康成人脐血提取的NK细胞中，即制备出T‑CAR‑NK，此产品除具有清除SC‑2病毒及其感染的靶细胞潜力外，还实现通用性、并具低风险、安全性高等特点，为临床治疗新冠病毒感染奠定基础。

Description

一种基于靶向SARS-CoV2病毒S蛋白的三聚体纳米抗体的CAR- NK的研制
技术领域
本发明涉及生物技术领域，具体涉及一种基于靶向SARS-CoV2病毒S蛋白的三聚体纳米抗体的CAR-NK细胞及其制备与应用。
背景技术
新型冠状病毒传染性强，存在自然变异，科学家们对此进行了深入的研究，开展了一些列的临床研究，但目前尚未研发出有效的治疗手段。新冠病毒S蛋白在宿主细胞上结合ACE2，并与宿主蛋白酶(主要是TMPRSS2)协同作用，促进其进入宿主细胞中。基于ACE2在肺，小肠，肾等多个器官中均有表达，一旦感染后可引起全身炎症反应，严重者可能导致器官衰竭和死亡。虽然疫苗有望延缓甚至遏制病毒的传播，但是其时效性和有效性有待进一步验证，且对于已感染者无效。同时由于病毒存在高突变率，现有疫苗可能会失效。现有报道发现部分康复患者出现二次接触后感染的现象；甚至部分康复患者无接触再次转阳。因此新冠病毒不能过分依赖疫苗这一预防手段，研制出一种有效的治疗手段势在必行。
NK细胞，即自然杀伤细胞，作为人体免疫系统的第一道防线，可以主动识别和杀伤入侵机体的病毒以及被病毒感染的细胞。在病毒感染期间，NK细胞通过多种机制来感知炎症信号，包括有干扰素α，白介素12，15及182在病毒感染后的早期阶段大量表达，同时NK细胞表达的多种促炎因子的持续表达，如干扰素γ等，介导机体的抗病毒保护作用。除此之外NK细胞起杀伤作用的一个重要途径是抗体依赖的细胞毒性由CD16结合被抗体包被的病毒感染细胞，激活NK细胞，从而杀伤被病毒感染的细胞。现如今面对新冠病毒在全球的肆虐，多项关于NK细胞的临床研究项目正在同步开展。如美国Celularity和 SorrentoTherapeutics制药公司：应用脐带血来源的NK细胞治疗和预防新型冠状病毒的临床试验。NK细胞治疗是目前提升患者机体免疫力方面极为可行的预防和治疗方法。
近年来免疫治疗在肿瘤的治疗上得到突破性的进展，嵌合抗原受体 (chimericantigen receptor，CAR)得到越来越多的关注并取得了良好的治疗效果，以CD19 CAR-T为典型代表，在血液瘤的治疗中取得了巨大成就。随着 CAR-T在临床应用越来越广泛，也显现出一定弊端，其中最主要的就是细胞因子释放综合征和神经毒性，极大的影响了CAR-T的治疗效果。此外自体细胞获取给临床应用CAR-T细胞带了挑战，一方面一些病人由于疾病影响无法获得足够的T细胞和细胞质量差无发获取足量和活性好的CAR-T细胞；另一方面制备细胞需要一定的时间，使得一些病人无法及时得到治疗。
在癌症免疫治疗中的最新临床前研究表明，在临床安全性方面，CAR-NK 细胞优于CAR-T细胞，CAR-NK细胞不会产生严重的移植物抗宿主病 (GVHD)。更重要的是，与CAR-T细胞相比，NK细胞诱导细胞因子释放综合征(CRS)可能性更低。而这种综合征可能会加重患者的症状，根据前期临床数据显示导致SC-2感染如此严重的原因就是感染患者释放了大量细胞因子。与T细胞性比NK细胞不需要严格的HLA匹配，因此不需要用病人的自身的细胞来制备CAR-NK，由于病毒感染的病人往往淋巴系统损伤，淋巴细胞质量差数量缺乏，因此给临床制备有效足量的细胞带来巨大挑战，CAR-NK就很好地解决的了这一问题。此外NK细胞来源较为广泛，除了外周血之外还有脐带来源的NK细胞均已成功的进行临床转化，因此NK细胞极大地解决制备方面的难题，方便随时取用。且脐带血来源的NK细胞更加年轻，增殖能力更强，靶向杀伤癌细胞的效率也更高，收集和冷冻保存更加便捷。
目前较为成熟的CAR结构就是一段单链抗体，加上跨膜区再加上胞内刺激信号。本研究采用纳米抗体替换传统的单链抗体，纳米抗体是在羊驼外周血中存在的一种天然缺失轻链的抗体，只包含一个重链可变区(VHH)和两个常规的CH2与CH3区，是已知的可结合目标抗原的最小单位。相较于传统抗体来说纳米抗体具有分子量更小，特异性更强，亲和力更高等特点，同时对人的免疫原性弱，有研究报道纳米抗体能引起更小的副作用，安全性更高。本研究前期确定了新冠病毒S蛋白的三聚体结构，具有三个ACE2识别序列，根据该空间结构，我们针对该三聚体结构设计出新型CAR结构，以增强对于S蛋白的亲和力。
鉴于以上背景，对此我们提出研制出一种靶向SC-2的S蛋白的通用型新型嵌合抗原受体NK细胞(T-CAR-NK)，其借鉴CAR-NK技术，利用筛选针对 SC-2的S蛋白的新型纳米抗体作为CAR中单链抗体部分，同时优选铰链及胞内信号区，组装成新型CAR，最终将上述基因通过改进的高效慢病毒整合到脐血来源的NK细胞中，即制备出T-CAR-NK，此研究除了具有清除SC-2病毒及其感染的靶细胞潜力外，还实现通用性、并具低风险、安全性高等特点，为临床治疗新冠感染奠定基础。
发明内容
鉴于目前对SARS-CoV-2尚无有效治疗的手段，本发明提供一种T-CAR- NK细胞及其制备方法，利用该细胞能够有效清除SC-2病毒及其感染的靶细胞。
本发明提供一种T-CAR-NK细胞，所述T-CAR-NK细胞能够表达嵌合抗原受体T-CAR，其中，所述嵌合抗原受体包含抗原结合结构域，跨膜结构域，共刺激信号传导区和自杀基因。所述的抗原结构域为靶向S蛋白的三聚体纳米抗体，通过一段优化的linker序列串联，能够特异性的结合SARS-CoV-2的S蛋白，通过跨膜结构域和共刺激信号传导区激活该NK细胞，并且在必要时通过自杀基因输注利妥昔单抗及时清除T-CAR-NK细胞。该T-CAR-NK细胞具有如下特点：①靶向性强，具有清除SC-2及其感染的靶细胞潜力；②副作用小，相较于CAR-T，CAR-NK能降低CRS等风险；③安通用性好，利用脐血来源NK，有效降低GvHD3，并降低制备时间和成本；④理念创新，选用SC-2的S蛋白亲和纳米抗体作为CAR的靶向识别区，通过技术升级实现功能突破，不仅为 SC-2治疗提供一种治疗手段，也为其他病毒治疗提供一种思路。
本发明的另一方面提供一种T-CAR-NK的制备方法，具体包括如下步骤：
(1)构建T-CAR慢病毒表达载体质粒；
(2)利用第三代慢病毒包装系统和步骤(1)得到的质粒转染293T细胞，包装和制备慢病毒；
(3)分离提取健康成人脐血PBMC细胞，利用NK分选试剂盒分选出NK细胞，进一步刺激培养48小时
(4)取步骤(3)中培养的NK细胞，加入步骤(2)所制备的慢病毒进行转导，转导后24换成新鲜培养基并继续培养48小时检测转导效率。
(5)继续扩增培养15天收获T-CAR-NK细胞。
附图说明
图1靶向SC-2S蛋白的纳米抗体对于SC-2S蛋白的亲和力曲线
图2显示了表达T-CAR的DNA载体
图3外周血分离的NK细胞表面标志物检测
图4显示了表达T-CAR的慢病毒感染NK细胞分选后的T-CAR的表达效率
图5显示了T-CAR-NK对于假型SARS-Cov2慢病毒感染靶细胞的阻断效果
图6显示了表达S蛋白的慢病毒感染293T细胞后，细胞表面S蛋白的表达
图7显示了T-CAR-NK对于表达S蛋白的靶细胞的杀伤性
具体实施方式
下面对本发明实例中的技术方案进行清楚，完整地描述。显然，所描述的实例是本发明一部分实施，而不是全部的实施列。以下提供的本发明的实施列的详细描述并非旨在限制要求保护的本发明的范围，而是仅仅表示本发明的选定实施例。
实施例1靶向S蛋白的纳米抗体筛选
该纳米抗体利用His Bind Resin结合抗原蛋白，从噬菌体展示的抗体库中筛选抗体。此步委托第三方公司进行，最终筛选出靶向S蛋白的纳米抗体ANSP- 1，ANSP-2，分别如SEQ ID NO:1，SEQ ID NO:20所示序列。
实施例2靶向S蛋白纳米抗体对于SC-2S蛋白的亲和力验证
运用间接ELISA法测定靶向纳米抗体的亲和力，首先用SC-2S蛋白用包被缓冲液稀释至适合的浓度，在96孔板中，每孔加入100μl的包被液，4℃冰箱包被过夜。包被完成后，用洗涤液连续洗涤3次。每孔加入用0.5％BSA，放入 4℃封闭过夜。取出96孔板，洗涤液洗涤3次。将系列稀释的抗体依次加入酶标板中，同时做阴性和阳性对照，放于37℃孵育1h。取出96孔板，洗涤液洗涤3次。每孔加入二抗100μl，置于37℃孵育1h。取出96孔板，洗涤液洗涤 3次，每孔加入100μlTMB显色液，37℃10min，加入50μl的终止液(2M的浓硫酸)，在450nm处测定OD值。将OD值拟合出亲和力曲线，如图1所示，计算出EC50，EC50[ANSP-1]＝175.2nM，EC50[ANSP-2]＝823.4nM，ANSP-1纳米抗体的亲和力高于ANSP-2抗体的亲和力。
实施例3 T-CAR慢病毒表达载体质粒的构建
由于SRAS-Cov2 S蛋白的三聚体结构,将上述得到的纳米抗体构建成三聚体形式，linker序列为SEQ ID NO:8所示序列，构建成三聚体结构如SEQ ID NO：13和SEQ ID NO:21所示序列。与CD28-CD3ζ共刺激域如SEQ ID NO： 14所示序列串联，构建成靶向S蛋白的T-CAR[ANSP-1]及T-CAR[ANSP-2]，如SEQ ID NO：22和SEQ ID NO：23所示序列。连接到载体质粒pCDH-EF1- FHC(购自Invitrogen公司)的EcoRI和SalI酶切位点之间，该质粒委托委托安徽通用生物合成，命名为pCDH-spTCAR，载体如图2所示。
实例4 T-CAR-NK细胞的制备
1.慢病毒制备
(1)将8×106个293T细胞接种至10cm的培养皿中，24小时后待细胞融合度达到80％左右，将目的质粒pCDH-spTCAR和辅助质粒PMD2G， PSPAX2转染至293T细胞中。
(2)转染后48小时收取第一次上清液培养基，加入新鲜培养基，72小时收取第二次培养上清液，将两次收集到的上清培养基混合3000rpm， 4℃离心10min，去上清液用0.45μm的滤膜过滤。
(3)将上述病毒上清液加入到50ml离心管中，使用超高速离心机进行病毒浓缩，收集病毒浓缩液，-80℃保存备用。
(4)利用293-T细胞检测病毒滴度大约为5×108TU/ML。
2.NK的细胞分离提取
NK细胞为脐血来源，采取健康成人脐血100ml，按照1:1的稀释比,将全血用PBS缓冲液进行稀释，并转移到50ml离心管中。在新的50ml离心管中先加入15ml人外周血淋巴细胞分离液(TBD),之后沿壁小心加入25ml稀释后的全血,不可破坏两种液体之间的分界面。750g速度离心25-30分，设置离心机慢升慢降。离心后,大部分红细跑部被离心到管底,自下往上依次为红细胞、ficoll层、白细胞层、血清层。将白细胞层吸取到新的50ml离心管中，加PBS洗涤后弃上清，用RPMI 1640培养基进行重悬，使用NK细胞分选试剂盒(biolegend) 分选出NK细胞，分选后细胞表型检测如图3所示。利用细胞因子IL-18，IL- 15，IL-2刺激培养3天后进行慢病毒感染。
3.表达T-CAR的慢病毒感染NK细胞及效率检测
将上述分离培养的NK细胞移入到1.5ml离心管中，按MOI值100，1500g， 32℃，90min进行离心感染，感染结束后继续培养72小时流式检测感染效率，并进一步做磁珠分选后流式细胞术检测感染效率。如图4所示，分选之后T- CAR的表达效率达到96.9％，而未分选之前T-CAR的表达仅有10％，明显提高了T-CAR的表达效率。
实例5 T-CAR-NK阻断病毒感染效果检测
1.利用第三代慢病毒表达系统将表达S蛋白的质粒和表达组装病毒颗粒所需的质粒转染至293T细胞，收集病毒上清并进行浓缩收获病毒颗粒，此为假性 SARS-Cov2病毒。
2.将上述制备的T-CAR-NK细胞与假型病毒以及表达ACE2的293T细胞共孵育，设置T-CAR-NK：SARS-Cov2：293T为1:1:1培养72小时后，流式检测293T细胞感染SARS-coV-2假型病毒的情况，结果如图5所示，当加了T- CAR-NK后能明显阻断假型SARS-Cov2对靶细胞的感染，且T-CAR[ANSP-1]对于假性病毒感染的阻断效果强于T-CAR[ANSP-2]。
实例6 T-CAR-NK对表达S蛋白的靶细胞的杀伤性检测
1.表达S蛋白的293T细胞制备
1)将8×106个293T细胞接种至10cm的培养皿中，24小时后待细胞融合度达到80％左右，将目的质粒pCDH-spTCAR和包装质粒PMD2G， PSPAX2共转染至293T细胞中。
2)转染后48小时收取第一次上清液培养基，加入新鲜培养基，72小时收取第二次培养上清液，将两次收集到的上清培养基混合3000rpm，4℃离心10min，去上清液用0.45μm的滤膜过滤。
3)将上述病毒上清液加入到50ml离心管中，使用超高速离心机进行病毒浓缩。
4)将上述收集的慢病毒感染293T细胞，72小时检测293T细胞表面S蛋白表达情况，如图6所示，超过90％细胞均表达蛋白。
2.将上述制备的T-CAR-NK细胞以0.5:1，1:1，2:1，5:1与表达S蛋白的293T 细胞进行共培养，培养24小时后检测293T细胞裂解情况，并检测细胞因子的表达情况。杀伤效果如图7所示，与对照组NK相比T-CAR-NK细胞对靶细胞的杀伤性增强，并且T-CAR[ANSP-1]对于靶细胞的杀伤性明显强于T- CAR[ANSP-2]。
序列表
<110> 皖南医学院第一附属医院（皖南医学院弋矶山医院）
<120> 一种基于靶向SARS-CoV2病毒S蛋白的三聚体纳米抗体的CAR-NK的研制
<141> 2021-05-19
<160> 23
<170> SIPOSequenceListing 1.0
<210> 1
<211> 120
<212> PRT
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Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Asn Ser Arg Tyr
20 25 30
Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Ile Ser Met Ile Gly Arg Thr Tyr Asp Ala Asp Ser Val Lys
50 55 60
Asn Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Ala Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Thr Cys Ala
85 90 95
Ala Gly Ser Ser Arg Ser Ser Leu Leu Arg Tyr Gly Tyr Ala Gly Gln
100 105 110
Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 2
<211> 5
<212> PRT
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Arg Tyr Tyr Met Gly
1 5
<210> 3
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Ala Ile Ser Met Ile Gly Arg Thr Tyr Asp Ala Asp Ser Val Lys Asn
1 5 10 15
<210> 4
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Gly Ser Ser Arg Ser Ser Leu Leu Arg Tyr Gly Tyr
1 5 10
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Cys Ala Gly Gly Thr Gly Cys Ala Gly Cys Thr Gly Cys Ala Gly Gly
1 5 10 15
Ala Gly Ala Gly Cys Gly Gly Cys Gly Gly Cys Gly Gly Cys Cys Thr
20 25 30
Gly Gly Thr Gly Cys Ala Gly Gly Cys Cys Gly Gly Cys Gly Gly Cys
35 40 45
Ala Gly Cys Cys Thr Gly Cys Gly Cys Cys Thr Gly Ala Gly Cys Thr
50 55 60
Gly Cys Gly Cys Cys Gly Cys Cys Ala Gly Cys Gly Gly Cys Thr Ala
65 70 75 80
Cys Ala Cys Cys Ala Ala Cys Ala Gly Cys Cys Gly Cys Thr Ala Cys
85 90 95
Thr Ala Cys Ala Thr Gly Gly Gly Cys Thr Gly Gly Thr Thr Cys Cys
100 105 110
Gly Cys Cys Ala Gly Gly Cys Cys Cys Cys Cys Gly Gly Cys Ala Ala
115 120 125
Gly Gly Ala Gly Cys Gly Cys Gly Ala Gly Thr Thr Cys Gly Thr Gly
130 135 140
Gly Cys Cys Gly Cys Cys Ala Thr Cys Ala Gly Cys Ala Thr Gly Ala
145 150 155 160
Thr Cys Gly Gly Cys Cys Gly Cys Ala Cys Cys Thr Ala Cys Gly Ala
165 170 175
Cys Gly Cys Cys Gly Ala Cys Ala Gly Cys Gly Thr Gly Ala Ala Gly
180 185 190
Ala Ala Cys Cys Gly Cys Thr Thr Cys Ala Cys Cys Ala Thr Cys Ala
195 200 205
Gly Cys Cys Gly Cys Gly Ala Cys Ala Ala Cys Gly Cys Cys Ala Ala
210 215 220
Gly Ala Ala Cys Ala Cys Cys Gly Thr Gly Thr Ala Cys Cys Thr Gly
225 230 235 240
Cys Ala Gly Gly Cys Cys Ala Ala Cys Ala Gly Cys Cys Thr Gly Ala
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Ala Gly Cys Cys Cys Gly Ala Gly Gly Ala Cys Ala Cys Cys Gly Cys
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Cys Gly Thr Gly Thr Ala Cys Ala Cys Cys Thr Gly Cys Gly Cys Cys
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Gly Cys Cys Gly Gly Cys Ala Gly Cys Ala Gly Cys Cys Gly Cys Ala
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Gly Cys Ala Gly Cys Cys Thr Gly Cys Thr Gly Cys Gly Cys Thr Ala
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Cys Gly Gly Cys Thr Ala Cys Gly Cys Cys Gly Gly Cys Cys Ala Gly
325 330 335
Gly Gly Cys Ala Cys Cys Cys Ala Gly Gly Thr Gly Ala Cys Cys Gly
340 345 350
Thr Gly Ala Gly Cys Ala Gly Cys
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Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
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Gly Gly Gly Ser Gly Gly Gly Ser Leu Gln
20 25
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Gly Gly Gly Gly Ser
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Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
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Gly Gly Gly Ser
20
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Glu Ala Ala Ala Lys
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Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys
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Gly Ser Ala Asp Asp Ala Lys Lys Asp Ala Ala Lys Lys Asp Gly Lys
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Ser
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Ser Ser Ala Asp Asp Ala Lys Lys Asp Ala Ala Lys Lys Asp Asp Ala
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Lys Lys Asp Asp Ala Lys Lys Asp Ala
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Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Asn Ser Arg Tyr
20 25 30
Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Ile Ser Met Ile Gly Arg Thr Tyr Asp Ala Asp Ser Val Lys
50 55 60
Asn Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Ala Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Thr Cys Ala
85 90 95
Ala Gly Ser Ser Arg Ser Ser Leu Leu Arg Tyr Gly Tyr Ala Gly Gln
100 105 110
Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu
130 135 140
Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu
145 150 155 160
Ser Cys Ala Ala Ser Gly Tyr Thr Asn Ser Arg Tyr Tyr Met Gly Trp
165 170 175
Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Ala Ile Ser
180 185 190
Met Ile Gly Arg Thr Tyr Asp Ala Asp Ser Val Lys Asn Arg Phe Thr
195 200 205
Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Ala Asn Ser
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Leu Lys Pro Glu Asp Thr Ala Val Tyr Thr Cys Ala Ala Gly Ser Ser
225 230 235 240
Arg Ser Ser Leu Leu Arg Tyr Gly Tyr Ala Gly Gln Gly Thr Gln Val
245 250 255
Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
260 265 270
Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly
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Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala
290 295 300
Ser Gly Tyr Thr Asn Ser Arg Tyr Tyr Met Gly Trp Phe Arg Gln Ala
305 310 315 320
Pro Gly Lys Glu Arg Glu Phe Val Ala Ala Ile Ser Met Ile Gly Arg
325 330 335
Thr Tyr Asp Ala Asp Ser Val Lys Asn Arg Phe Thr Ile Ser Arg Asp
340 345 350
Asn Ala Lys Asn Thr Val Tyr Leu Gln Ala Asn Ser Leu Lys Pro Glu
355 360 365
Asp Thr Ala Val Tyr Thr Cys Ala Ala Gly Ser Ser Arg Ser Ser Leu
370 375 380
Leu Arg Tyr Gly Tyr Ala Gly Gln Gly Thr Gln Val Thr Val Ser Ser
385 390 395 400
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Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
1 5 10 15
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
20 25 30
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg
35 40 45
Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn
50 55 60
Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg
65 70 75 80
Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn
85 90 95
Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu
100 105 110
Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly
115 120 125
His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr
130 135 140
Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
145 150 155
<210> 15
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Met Gly Asn Ser Cys Tyr Asn Ile Val Ala Thr Leu Leu Leu Val Leu
1 5 10 15
Asn Phe Glu Arg Thr Arg Ser Leu Gln Asp Pro Cys Ser Asn Cys Pro
20 25 30
Ala Gly Thr Phe Cys Asp Asn Asn Arg Asn Gln Ile Cys Ser Pro Cys
35 40 45
Pro Pro Asn Ser Phe Ser Ser Ala Gly Gly Gln Arg Thr Cys Asp Ile
50 55 60
Cys Arg Gln Cys Lys Gly Val Phe Arg Thr Arg Lys Glu Cys Ser Ser
65 70 75 80
Thr Ser Asn Ala Glu Cys Asp Cys Thr Pro Gly Phe His Cys Leu Gly
85 90 95
Ala Gly Cys Ser Met Cys Glu Gln Asp Cys Lys Gln Gly Gln Glu Leu
100 105 110
Thr Lys Lys Gly Cys Lys Asp Cys Cys Phe Gly Thr Phe Asn Asp Gln
115 120 125
Lys Arg Gly Ile Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly Lys
130 135 140
Ser Val Leu Val Asn Gly Thr Lys Glu Arg Asp Val Val Cys Gly Pro
145 150 155 160
Ser Pro Ala Asp Leu Ser Pro Gly Ala Ser Ser Val Thr Pro Pro Ala
165 170 175
Pro Ala Arg Glu Pro Gly His Ser Pro Gln Ile Ile Ser Phe Phe Leu
180 185 190
Ala Leu Thr Ser Thr Ala Leu Leu Phe Leu Leu Phe Phe Leu Thr Leu
195 200 205
Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe
210 215 220
Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly
225 230 235 240
Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg
245 250 255
Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln
260 265 270
Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp
275 280 285
Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro
290 295 300
Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
305 310 315 320
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
325 330 335
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
340 345 350
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
355 360 365
<210> 16
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Met Ile His Leu Gly His Ile Leu Phe Leu Leu Leu Leu Pro Val Ala
1 5 10 15
Ala Ala Gln Thr Thr Pro Gly Glu Arg Ser Ser Leu Pro Ala Phe Tyr
20 25 30
Pro Gly Thr Ser Gly Ser Cys Ser Gly Cys Gly Ser Leu Ser Leu Pro
35 40 45
Leu Leu Ala Gly Leu Val Ala Ala Asp Ala Val Ala Ser Leu Leu Ile
50 55 60
Val Gly Ala Val Phe Leu Cys Ala Arg Pro Arg Arg Ser Pro Ala Gln
65 70 75 80
Asp Gly Lys Val Tyr Ile Asn Met Pro Gly Arg Gly Arg Val Lys Phe
85 90 95
Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu
100 105 110
Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp
115 120 125
Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg
130 135 140
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
145 150 155 160
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
165 170 175
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
180 185 190
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
195 200 205
<210> 17
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<400> 17
Met Gly Gly Leu Glu Pro Cys Ser Arg Leu Leu Leu Leu Pro Leu Leu
1 5 10 15
Leu Ala Val Ser Asp Cys Ser Cys Ser Thr Val Ser Pro Gly Val Leu
20 25 30
Ala Gly Ile Val Met Gly Asp Leu Val Leu Thr Val Leu Ile Ala Leu
35 40 45
Ala Val Tyr Phe Leu Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala
50 55 60
Glu Ala Ala Thr Arg Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr
65 70 75 80
Gln Glu Leu Gln Gly Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr
85 90 95
Gln Arg Pro Tyr Tyr Lys Arg Val Lys Phe Ser Arg Ser Ala Asp Ala
100 105 110
Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu
115 120 125
Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp
130 135 140
Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly
145 150 155 160
Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu
165 170 175
Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu
180 185 190
Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His
195 200 205
Met Gln Ala Leu Pro Pro Arg
210 215
<210> 18
<211> 472
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<400> 18
Met Ala Trp Arg Ala Leu His Pro Leu Leu Leu Leu Leu Leu Leu Phe
1 5 10 15
Pro Gly Ser Gln Ala Gln Ser Lys Ala Gln Val Leu Gln Ser Val Ala
20 25 30
Gly Gln Thr Leu Thr Val Arg Cys Gln Tyr Pro Pro Thr Gly Ser Leu
35 40 45
Tyr Glu Lys Lys Gly Trp Cys Lys Glu Ala Ser Ala Leu Val Cys Ile
50 55 60
Arg Leu Val Thr Ser Ser Lys Pro Arg Thr Met Ala Trp Thr Ser Arg
65 70 75 80
Phe Thr Ile Trp Asp Asp Pro Asp Ala Gly Phe Phe Thr Val Thr Met
85 90 95
Thr Asp Leu Arg Glu Glu Asp Ser Gly His Tyr Trp Cys Arg Ser Thr
100 105 110
Ala Leu Leu Thr Thr Leu Ser Leu Ser Pro Ser Asp Ser Ile Trp Trp
115 120 125
Tyr Leu Gln Pro Leu Pro Pro His Arg Pro Pro Gly Leu Pro Ala Thr
130 135 140
Trp Ser Leu His Arg Pro Arg Pro Arg Ala Val Cys Leu Pro Leu Gln
145 150 155 160
Glu Pro Asp Lys Pro Leu Ser Leu His Leu Pro Ser Leu Ser Leu His
165 170 175
Thr Arg Pro Leu Pro Phe Leu Ser Leu Cys Leu Pro Gly His Arg Thr
180 185 190
Pro Arg Ser Ala Leu Ala Leu Gln Pro Pro Leu Pro Trp Cys Leu Cys
195 200 205
Ser Val Asp Ser Ser Pro Arg Ala Trp Cys Cys Gln Pro Cys Ser Ser
210 215 220
Gly Gly Phe Gly Ile Gly Thr Cys Ser Ile Lys Gly Gly Leu Cys Cys
225 230 235 240
Thr Gln Leu Ser Pro Gly Pro Arg Pro Ile Asp Thr Ser His Ala Thr
245 250 255
Gly His Gln Gly Gly His Met Val Glu Asn His Met Ile His Leu Gly
260 265 270
His Ile Leu Phe Leu Leu Leu Leu Pro Val Ala Ala Ala Gln Thr Thr
275 280 285
Pro Gly Glu Arg Ser Ser Leu Pro Ala Phe Tyr Pro Gly Thr Ser Gly
290 295 300
Ser Cys Ser Gly Cys Gly Ser Leu Ser Leu Pro Leu Leu Ala Gly Leu
305 310 315 320
Val Ala Ala Asp Ala Val Ala Ser Leu Leu Ile Val Gly Ala Val Phe
325 330 335
Leu Cys Ala Arg Pro Arg Arg Ser Pro Ala Gln Asp Gly Lys Val Tyr
340 345 350
Ile Asn Met Pro Gly Arg Gly Arg Val Lys Phe Ser Arg Ser Ala Asp
355 360 365
Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn
370 375 380
Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg
385 390 395 400
Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu
405 410 415
Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser
420 425 430
Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly
435 440 445
Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu
450 455 460
His Met Gln Ala Leu Pro Pro Arg
465 470
<210> 19
<211> 431
<212> PRT
<213> Artificial sequence
<400> 19
Met Gly Trp Ile Arg Gly Arg Arg Pro Arg His Asn Leu Glu Met Ser
1 5 10 15
Glu Phe His Asn Tyr Lys Leu Gly Leu Ala Lys Ser Asp Phe Ser Thr
20 25 30
Arg Cys Gln Lys Gln Arg Cys Pro Val Ile Lys Ser Lys Cys Arg Glu
35 40 45
Asn Ala Ser Pro Leu Phe Phe Cys Cys Phe Ile Ala Val Ala Met Gly
50 55 60
Ile Arg Phe Ile Ile Met Val Thr Ile Trp Ser Ala Val Phe Leu Asn
65 70 75 80
Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys
85 90 95
Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln
100 105 110
Phe Phe Asn Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met
115 120 125
Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp
130 135 140
Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile
145 150 155 160
Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro
165 170 175
Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr
180 185 190
Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Ile Pro Asn Thr
195 200 205
Tyr Ile Cys Met Gln Arg Thr Val Met Gly Gly Leu Glu Pro Cys Ser
210 215 220
Arg Leu Leu Leu Leu Pro Leu Leu Leu Ala Val Ser Asp Cys Ser Cys
225 230 235 240
Ser Thr Val Ser Pro Gly Val Leu Ala Gly Ile Val Met Gly Asp Leu
245 250 255
Val Leu Thr Val Leu Ile Ala Leu Ala Val Tyr Phe Leu Gly Arg Leu
260 265 270
Val Pro Arg Gly Arg Gly Ala Ala Glu Ala Ala Thr Arg Lys Gln Arg
275 280 285
Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu Leu Gln Gly Gln Arg Ser
290 295 300
Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg Pro Tyr Tyr Lys Arg Val
305 310 315 320
Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn
325 330 335
Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val
340 345 350
Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln
355 360 365
Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp
370 375 380
Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg
385 390 395 400
Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr
405 410 415
Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
420 425 430
<210> 20
<211> 121
<212> PRT
<213> Artificial sequence
<400> 20
Gln Val Gln Leu Val Glu Thr Gly Gly Gly Leu Val Gln Pro Phe Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met Gly Trp Ala Arg Gln Val Pro Gly Tyr Gly Leu Glu Trp Val
35 40 45
Ser Tyr Gln Tyr Ser Asp Gly Ser Thr Glu Tyr Gln Asp Ser Val Ser
50 55 60
Gly Arg Phe Thr Ile Ser Ala Asp Asn Ala Lys Ser Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Ser Tyr Cys Ala
85 90 95
Thr Glu Gly Ser Leu Gly Gly Trp Gly Arg Asp Phe Gly Ser Ala Gly
100 105 110
Gln Gly Thr Gln Val Trp Val Ser Ser
115 120
<210> 21
<211> 403
<212> PRT
<213> Artificial sequence
<400> 21
Gln Val Gln Leu Val Glu Thr Gly Gly Gly Leu Val Gln Pro Phe Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met Gly Trp Ala Arg Gln Val Pro Gly Tyr Gly Leu Glu Trp Val
35 40 45
Ser Tyr Gln Tyr Ser Asp Gly Ser Thr Glu Tyr Gln Asp Ser Val Ser
50 55 60
Gly Arg Phe Thr Ile Ser Ala Asp Asn Ala Lys Ser Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Ser Tyr Cys Ala
85 90 95
Thr Glu Gly Ser Leu Gly Gly Trp Gly Arg Asp Phe Gly Ser Ala Gly
100 105 110
Gln Gly Thr Gln Val Trp Val Ser Ser Gly Gly Gly Gly Ser Gly Gly
115 120 125
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln
130 135 140
Leu Val Glu Thr Gly Gly Gly Leu Val Gln Pro Phe Gly Ser Leu Arg
145 150 155 160
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met Gly
165 170 175
Trp Ala Arg Gln Val Pro Gly Tyr Gly Leu Glu Trp Val Ser Tyr Gln
180 185 190
Tyr Ser Asp Gly Ser Thr Glu Tyr Gln Asp Ser Val Ser Gly Arg Phe
195 200 205
Thr Ile Ser Ala Asp Asn Ala Lys Ser Thr Val Tyr Leu Gln Met Asn
210 215 220
Ser Leu Lys Pro Glu Asp Thr Ala Val Ser Tyr Cys Ala Thr Glu Gly
225 230 235 240
Ser Leu Gly Gly Trp Gly Arg Asp Phe Gly Ser Ala Gly Gln Gly Thr
245 250 255
Gln Val Trp Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
260 265 270
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Glu
275 280 285
Thr Gly Gly Gly Leu Val Gln Pro Phe Gly Ser Leu Arg Leu Ser Cys
290 295 300
Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met Gly Trp Ala Arg
305 310 315 320
Gln Val Pro Gly Tyr Gly Leu Glu Trp Val Ser Tyr Gln Tyr Ser Asp
325 330 335
Gly Ser Thr Glu Tyr Gln Asp Ser Val Ser Gly Arg Phe Thr Ile Ser
340 345 350
Ala Asp Asn Ala Lys Ser Thr Val Tyr Leu Gln Met Asn Ser Leu Lys
355 360 365
Pro Glu Asp Thr Ala Val Ser Tyr Cys Ala Thr Glu Gly Ser Leu Gly
370 375 380
Gly Trp Gly Arg Asp Phe Gly Ser Ala Gly Gln Gly Thr Gln Val Trp
385 390 395 400
Val Ser Ser
<210> 22
<211> 642
<212> PRT
<213> Artificial sequence
<400> 22
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu
20 25 30
Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr
35 40 45
Thr Asn Ser Arg Tyr Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys
50 55 60
Glu Arg Glu Phe Val Ala Ala Ile Ser Met Ile Gly Arg Thr Tyr Asp
65 70 75 80
Ala Asp Ser Val Lys Asn Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
85 90 95
Asn Thr Val Tyr Leu Gln Ala Asn Ser Leu Lys Pro Glu Asp Thr Ala
100 105 110
Val Tyr Thr Cys Ala Ala Gly Ser Ser Arg Ser Ser Leu Leu Arg Tyr
115 120 125
Gly Tyr Ala Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly
130 135 140
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
145 150 155 160
Ser Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly
165 170 175
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Asn Ser Arg
180 185 190
Tyr Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe
195 200 205
Val Ala Ala Ile Ser Met Ile Gly Arg Thr Tyr Asp Ala Asp Ser Val
210 215 220
Lys Asn Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
225 230 235 240
Leu Gln Ala Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Thr Cys
245 250 255
Ala Ala Gly Ser Ser Arg Ser Ser Leu Leu Arg Tyr Gly Tyr Ala Gly
260 265 270
Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly
275 280 285
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln
290 295 300
Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg
305 310 315 320
Leu Ser Cys Ala Ala Ser Gly Tyr Thr Asn Ser Arg Tyr Tyr Met Gly
325 330 335
Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Ala Ile
340 345 350
Ser Met Ile Gly Arg Thr Tyr Asp Ala Asp Ser Val Lys Asn Arg Phe
355 360 365
Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Ala Asn
370 375 380
Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Thr Cys Ala Ala Gly Ser
385 390 395 400
Ser Arg Ser Ser Leu Leu Arg Tyr Gly Tyr Ala Gly Gln Gly Thr Gln
405 410 415
Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro
420 425 430
Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys
435 440 445
Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala
450 455 460
Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu
465 470 475 480
Leu Leu Ser Leu Val Ile Thr Lys Arg Gly Arg Lys Lys Leu Leu Tyr
485 490 495
Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu
500 505 510
Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu
515 520 525
Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln
530 535 540
Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu
545 550 555 560
Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly
565 570 575
Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu
580 585 590
Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly
595 600 605
Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser
610 615 620
Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro
625 630 635 640
Pro Arg
<210> 23
<211> 645
<212> PRT
<213> Artificial sequence
<400> 23
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gln Val Gln Leu Val Glu Thr Gly Gly Gly Leu
20 25 30
Val Gln Pro Phe Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
35 40 45
Thr Phe Ser Ser Tyr Ala Met Gly Trp Ala Arg Gln Val Pro Gly Tyr
50 55 60
Gly Leu Glu Trp Val Ser Tyr Gln Tyr Ser Asp Gly Ser Thr Glu Tyr
65 70 75 80
Gln Asp Ser Val Ser Gly Arg Phe Thr Ile Ser Ala Asp Asn Ala Lys
85 90 95
Ser Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala
100 105 110
Val Ser Tyr Cys Ala Thr Glu Gly Ser Leu Gly Gly Trp Gly Arg Asp
115 120 125
Phe Gly Ser Ala Gly Gln Gly Thr Gln Val Trp Val Ser Ser Gly Gly
130 135 140
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
145 150 155 160
Gly Ser Gln Val Gln Leu Val Glu Thr Gly Gly Gly Leu Val Gln Pro
165 170 175
Phe Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
180 185 190
Ser Tyr Ala Met Gly Trp Ala Arg Gln Val Pro Gly Tyr Gly Leu Glu
195 200 205
Trp Val Ser Tyr Gln Tyr Ser Asp Gly Ser Thr Glu Tyr Gln Asp Ser
210 215 220
Val Ser Gly Arg Phe Thr Ile Ser Ala Asp Asn Ala Lys Ser Thr Val
225 230 235 240
Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Ser Tyr
245 250 255
Cys Ala Thr Glu Gly Ser Leu Gly Gly Trp Gly Arg Asp Phe Gly Ser
260 265 270
Ala Gly Gln Gly Thr Gln Val Trp Val Ser Ser Gly Gly Gly Gly Ser
275 280 285
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln
290 295 300
Val Gln Leu Val Glu Thr Gly Gly Gly Leu Val Gln Pro Phe Gly Ser
305 310 315 320
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala
325 330 335
Met Gly Trp Ala Arg Gln Val Pro Gly Tyr Gly Leu Glu Trp Val Ser
340 345 350
Tyr Gln Tyr Ser Asp Gly Ser Thr Glu Tyr Gln Asp Ser Val Ser Gly
355 360 365
Arg Phe Thr Ile Ser Ala Asp Asn Ala Lys Ser Thr Val Tyr Leu Gln
370 375 380
Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Ser Tyr Cys Ala Thr
385 390 395 400
Glu Gly Ser Leu Gly Gly Trp Gly Arg Asp Phe Gly Ser Ala Gly Gln
405 410 415
Gly Thr Gln Val Trp Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro
420 425 430
Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro
435 440 445
Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu
450 455 460
Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys
465 470 475 480
Gly Val Leu Leu Leu Ser Leu Val Ile Thr Lys Arg Gly Arg Lys Lys
485 490 495
Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr
500 505 510
Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly
515 520 525
Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala
530 535 540
Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg
545 550 555 560
Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu
565 570 575
Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr
580 585 590
Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly
595 600 605
Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln
610 615 620
Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln
625 630 635 640
Ala Leu Pro Pro Arg
645

Claims (10)

1.靶向SARS-CoV2病毒S蛋白的纳米抗体ANSP-1，其特征在于，其三个互补决定区由CDR1、CDR2、CDR3组成，其中CDR1氨基酸序列如SEQ ID NO:2所示、CDR2的氨基酸序列如SEQID NO:3所示、CDR3的氨基酸序列如SEQ ID NO:4所示。
2.权利要求1所述的纳米抗体，其特征在于，所述纳米抗体氨基酸序列如SEQ ID NO:1所示。
3.编码权利要求1-2所述纳米抗体的基因，进一步，所述基因序列如SEQ ID NO:5所示。
4.靶向SARS-CoV2病毒S蛋白的三聚体纳米抗体，其特征在于，所述三聚体纳米抗体是将权利要求1所述纳米抗体与Linker相连，所述linker选自：SEQ ID NO：6，SEQ ID NO：7，SEQ ID NO：8，SEQ ID NO：9，SEQ ID NO：10，SEQ ID NO：11，SEQ ID NO：12所示的一种；进一步的，所述三聚体纳米抗体如SEQ ID NO:13所示。
5.靶向SARS-CoV2病毒S蛋白的T-CAR[ANSP-1]，其特征在于，如SEQ ID NO：22所示。
6.靶向SARS-CoV2病毒S蛋白的三聚体纳米抗体的T-CAR-NK细胞，其特征在于，所述纳米抗体如权利要求1所述，所述T-CAR-NK细胞是在NK细胞中表达T-CAR结构，所述NK细胞来自于健康成人脐血提取的NK细胞。
7.根据权利要求6所述的T-CAR-NK细胞，其特征在于所述的T-CAR结构包括抗原结合结构域，跨膜结构域，共刺激信号传导区，优选的，所述共刺激结构域选自以下任意组合中的一种:CD28-CD3ζ，4-1BB-CD3ζ，DAP10-CD3ζ，DAP12-CD3ζ，NKp44-DAP10-CD3ζ，NKG2D-DAP12-CD3ζ。
8.根据权利要求7所述的T-CAR-NK细胞，其特征在于，所述共刺激结构域分别为如SEQID NO:14，SEQ ID NO:15，SEQ ID NO:16，SEQ ID NO:17，SEQ ID NO:18，SEQ ID NO:19所示序列。
9.一种如权利要求6-8所述的靶向S蛋白的T-CAR-NK细胞的制备方法，其特征在于所述T-CAR-NK细胞制备方法包括如下步骤：
利用第三代慢病包装系统包装和制备表达T-CAR的慢病毒；
分离提取健康成人脐血NK细胞；
将制备的慢病毒感染NK细胞，并进一步扩大培养。
10.一种如权利要求1-3所述纳米抗体、权利要求4所述三聚体纳米抗体、权利要求5所述T-CAR、权利要求6-8所述的靶向S蛋白的T-CAR-NK细胞在制备抗病毒感染的药物中应用。
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11732030B2 (en) 2020-04-02 2023-08-22 Regeneron Pharmaceuticals, Inc. Anti-SARS-CoV-2-spike glycoprotein antibodies and antigen-binding fragments
WO2023217286A1 (zh) * 2022-05-13 2023-11-16 山西锦波生物医药股份有限公司 融合蛋白以及其应用
CN117511885A (zh) * 2024-01-08 2024-02-06 青岛华赛伯曼医学细胞生物有限公司 提高识别和杀伤肿瘤能力的工程化til细胞及其应用
US11999777B2 (en) 2020-06-03 2024-06-04 Regeneron Pharmaceuticals, Inc. Methods for treating or preventing SARS-CoV-2 infections and COVID-19 with anti-SARS-CoV-2 spike glycoprotein antibodies

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100150939A1 (en) * 2006-12-19 2010-06-17 Ablynx N.V. Amino acid sequences directed against a metalloproteinase from the adam family and polypeptides comprising the same for the treatment of adam-related diseases and disorders
US20200123233A1 (en) * 2008-06-05 2020-04-23 Ablynx N.V. Amino acid sequences directed against envelope proteins of a virus and polypeptides comprising the same for the treatment of viral diseases
CN111825762A (zh) * 2020-06-17 2020-10-27 武汉华美生物工程有限公司 抗sars-cov-2病毒s蛋白rbd结构域的纳米抗体及其用途
CN112062839A (zh) * 2020-09-22 2020-12-11 石河子大学 一种基于新型冠状病毒s蛋白s1亚基的纳米抗体及其应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100150939A1 (en) * 2006-12-19 2010-06-17 Ablynx N.V. Amino acid sequences directed against a metalloproteinase from the adam family and polypeptides comprising the same for the treatment of adam-related diseases and disorders
US20200123233A1 (en) * 2008-06-05 2020-04-23 Ablynx N.V. Amino acid sequences directed against envelope proteins of a virus and polypeptides comprising the same for the treatment of viral diseases
CN111825762A (zh) * 2020-06-17 2020-10-27 武汉华美生物工程有限公司 抗sars-cov-2病毒s蛋白rbd结构域的纳米抗体及其用途
CN112062839A (zh) * 2020-09-22 2020-12-11 石河子大学 一种基于新型冠状病毒s蛋白s1亚基的纳米抗体及其应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
MA,MINH等: "CAR-NK Cells Effectively Target the D614 and G614 SARS-CoV-2-infected Cells", 《BIORXIV》 *
MA,MINH等: "Efficacy of Targeting SARS-CoV-2 by CAR-NK Cells", 《BIORXIV》 *
史瑞芳等: "CAR-NK:肿瘤免疫细胞治疗新策略", 《中国肿瘤生物治疗杂志》 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11732030B2 (en) 2020-04-02 2023-08-22 Regeneron Pharmaceuticals, Inc. Anti-SARS-CoV-2-spike glycoprotein antibodies and antigen-binding fragments
US11999777B2 (en) 2020-06-03 2024-06-04 Regeneron Pharmaceuticals, Inc. Methods for treating or preventing SARS-CoV-2 infections and COVID-19 with anti-SARS-CoV-2 spike glycoprotein antibodies
WO2023217286A1 (zh) * 2022-05-13 2023-11-16 山西锦波生物医药股份有限公司 融合蛋白以及其应用
CN117511885A (zh) * 2024-01-08 2024-02-06 青岛华赛伯曼医学细胞生物有限公司 提高识别和杀伤肿瘤能力的工程化til细胞及其应用
CN117511885B (zh) * 2024-01-08 2024-06-11 青岛华赛伯曼医学细胞生物有限公司 提高识别和杀伤肿瘤能力的工程化til细胞及其应用

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