CN112469733B - Mart-1(27-35)表位特异性t细胞受体 - Google Patents
Mart-1(27-35)表位特异性t细胞受体 Download PDFInfo
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Abstract
提供一种MART‑1(27‑35)表位特异性T细胞受体,该T细胞受体包含α链和β链;所述α链包含三个互补决定区,序列分别为SEQ ID No.3的第61‑66位、第84‑89位以及第124‑136位;所述β链包含三个互补决定区,氨基酸序列分别为SEQ ID No.4的第46‑50位、第68‑73位以及第112‑125位。提供表达TCR的T细胞可以有效的识别T2细胞负载的MART‑1(27‑35)表位多肽,并分泌IFN‑γ,证明其具有功能。该TCR与相关药物靶标联用,可以有效的进行药物开发。
Description
技术领域
本发明涉及一种MART-1(27-35)表位特异性T细胞受体。
背景技术
黑色素瘤,又称恶性黑色素瘤,是来源于黑色素细胞的一类恶性肿瘤,常见于皮肤,亦见于粘膜、眼脉络膜等部位。黑色素瘤是皮肤恶性程度最高的瘤种,容易出现远处转移。在亚洲人和有色人种中,原发于皮肤的黑色素瘤占50%~70%,欧美白种人中过度的紫外线照射是明确病因之一。紫外线可致使皮肤灼伤,并诱导DNA突变,进而诱导黑色素瘤发生。此外,光敏型皮肤、存在大量普通痣或发育异常痣以及皮肤癌家族史者均为高危人群。多发于亚洲和非洲地区的肢端型黑色素瘤所受紫外线照射极少,病因仍不明确。
近年来,人类黑色素瘤相关抗原已被陆续鉴定。MART-1为黑色素瘤相关抗原性较强的一种,其基因已被克隆,抗原的性质及呈递MART-1的HLA分子已得到部分阐明。研究发现,在MART-1上有几个免疫优势表位,它们能在体内外诱导产生CTL免疫应答。其中最常见的是HLA-A*02限制性的表位27-35,针对该表位刺激特异性的T细胞,可以获得特异性杀伤T细胞(CTL),这种CTL可以有效的杀伤MART-1阳性表达的肿瘤细胞。然而,目前针对MART-1(27-35)表位的临床治疗效果仍然是十分有限的,现有MART-1特异性的CD8+T细胞克隆的体外研究显示,尽管表达HLA-A*02与MART-1基因,然而却只能相对较少的被特异性T细胞识别。其中很重要的一个原因是,现有的MART-1对应的特异性T细胞的TCR不能高亲和力的识别MART-1(27-35)表位的靶细胞。因此,获得高亲和力MART-1(27-35)表位特异性的T细胞受体将具有非常重要的意义。
发明公开
本发明的目的是提供一种MART-1(27-35)表位特异性T细胞受体。其中,所述MART-1(27-35)表位的序列如SEQ ID No.5所示。
本发明所提供的MART-1(27-35)表位特异性T细胞受体,包含α链和β链。其中,所述α链包含三个互补决定区,氨基酸序列分别为SEQ ID No.3的第61-66位、第84-89位以及第124-136位;或这些序列的具有至多3个、2个或1个氨基酸改变的变体。所述β链包含三个互补决定区,氨基酸序列分别为SEQ ID No.4的第46-50位、第68-73位以及第112-125位;或这些序列的具有至多3个、2个或1个氨基酸改变的变体。
进一步地,所述α链的可变区的氨基酸序列为SEQ ID No.3的第35-136位;或这些序列的具有至多3个、2个或1个氨基酸改变的变体。所述β链的可变区的氨基酸序列为SEQID No.4的第20-125位;或这些序列的具有至多3个、2个或1个氨基酸改变的变体。
所述α链的恒定区的氨基酸序列为SEQ ID No.3的第148-288位;所述β链的恒定区的氨基酸序列为SEQ ID No.4的第136-314位。
更进一步地,所述α链的氨基酸序列具体为SEQ ID No.3;所述β链的氨基酸序列具体为SEQ ID No.4。
编码所述T细胞受体的核酸分子也属于本发明的保护范围。
编码所述T细胞受体的核酸分子包含编码所述T细胞受体的α链的核酸分子和编码所述T细胞受体的β链的核酸分子。
其中,编码所述T细胞受体的α链中三个互补决定区的核酸分子的序列分别为SEQID No.1的第181-198位、第250-267位以及第370-408位;或与这些序列具有99%以上、95%以上、90%以上、85%以上或者80%以上同一性且编码相同氨基酸残基的序列。编码所述T细胞受体的β链中三个互补决定区的核酸分子的序列分别为SEQ ID No.2的第136-150位、第202-219位以及第334-375位;或与这些序列具有99%以上、95%以上、90%以上、85%以上或者80%以上同一性且编码相同氨基酸残基的序列。
进一步地,编码所述α链的可变区的核酸分子的序列为SEQ ID No.1的第103-408位;或与这些序列具有99%以上、95%以上、90%以上、85%以上或者80%以上同一性且编码相同氨基酸残基的序列。编码所述β链的可变区的核酸分子的序列为SEQ ID No.2的第58-375位;或与这些序列具有99%以上、95%以上、90%以上、85%以上或者80%以上同一性且编码相同氨基酸残基的序列。
更进一步地,编码所述α链的核酸分子的序列具体为SEQ ID No.1;编码所述β链的核酸分子的序列具体为SEQ ID No.2。
含有所述核酸分子的表达盒、载体或细胞也属于本发明的保护范围。
进一步地,所述载体可为逆转录病毒载体,如慢病毒载体。
在本发明的一个实施例中,所述载体具体是通过将编码所述α链的核酸分子和编码所述β链的核酸分子用连接肽的编码序列连接后插入到慢病毒载体pRRLSIN.cPPT.PGK-GFP.WPRE的BamHI与SalI限制性内切酶切位点之间得到的。
进一步地,所述细胞可为T细胞。
含有所述载体或所述细胞的药物组合物也属于本发明的保护范围。
其中,所述药物组合物可用于预防和/或治疗黑色素瘤。
所述T细胞受体或所述核酸分子或所述载体或细胞在制备预防和/或治疗黑色素瘤的药物中的应用也属于本发明的保护范围。
所述T细胞受体或所述核酸分子或所述载体或细胞在预防和/或治疗黑色素瘤中的应用也属于本发明的保护范围。
本发明还要求保护一种预防和/或治疗黑色素瘤的方法。该方法可包括如下步骤:以前文所述的T细胞受体或所述核酸分子或所述载体或细胞来预防和/或治疗黑色素瘤。
附图说明
图1为MART-1(27-35)表位多肽第一轮刺激后的流式检测结果。左图为未经过刺激CD8-T细胞群体;右图为MART-1抗原刺激后四聚体检测流式细胞群体。
图2为MART-1(27-35)表位多肽第二轮刺激后的流式检测结果。左图为未经过刺激CD8-T细胞群体;右图为MART-1抗原刺激后四聚体检测流式细胞群体。
图3为MART-1(27-35)表位多肽特异性T细胞Elispot检测结果。从左到右7个孔,依次代表T+T2+有效多肽、T+T2+无关多肽、T+T2、T+有效多肽、T+无关多肽、T,T+OKT3。
图4为单细胞TCR扩增电泳图。框标记的条带进行酶切胶回收。
图5为TA克隆的菌落PCR电泳图。1-1代表一个克隆进行的TA克隆,1-2代表一个克隆进行的TA克隆,1-6代表一个克隆进行的TA克隆。
图6为重组病毒载体的部分结构示意图。
图7为clone#4序列对应的MART-1(27-35)表位多肽特异性T细胞的Elispot检测结果。
实施发明的最佳方式
以下的实施例便于更好地理解本发明,但并不限定本发明。下述实施例中的实验方法,如无特殊说明,均为常规方法。下述实施例中所用的试验材料,如无特殊说明,均为自常规生化试剂商店购买得到的。以下实施例中的定量试验,均设置三次重复实验,结果取平均值。
以下的实施例中所用到的实验试剂物品具体如下:
实验物品:采血管(含ACD抗凝剂),注射器,离心管,0.2μm滤膜,MS分选柱,磁力架,低吸附六孔板;0.2μm滤膜,达优冻存试剂盒,PCR管。
实验试剂:无菌盐溶液(DPBS),RPMI1640培养基,Ficoll,AIM-V培养基,无菌超纯水(0.1um滤膜过滤),MACS running buffer;AIM-V培养基,GM-CSF,IL-4,IFN-γ,LPS,IL-7。抗原呈递beads,AIM-V培养基,IL-21,IL-2,IL-15。无菌盐溶液(PBS),Human IFN-γELISpot试剂盒(MABTECH),四聚体(MART-1抗原)。
实施例1、高亲和力MART-1(27-35)表位特异性T细胞受体序列的获得
一、MART-1(27-35)表位特异性T细胞刺激
1、健康人外周血PBMC分离
血样收集于50ml,室温100g,15min;分别收上层血浆和下层血细胞;层血浆室温1100g,20min离心,弃沉淀;56℃(30min)灭活后,放于-20℃冷冻层静置15min;室温3800rpm,20min离心,离心后上清即为人血清,取之备用;取DPBS补足下层血细胞加至50ml,上下颠倒混匀;吸取20mL加入到50mL离心管中;在Ficoll上方小心加入混匀后的血样25mL,室温离心;离心结束后,液体分为四层,从上至下分别为血浆层、白膜层、Ficoll层和血细胞层,用巴氏滴管将白膜层小心的吸出,并转移至一个无菌的离心管中;加入3倍体积的1640培养基洗涤吸出的白膜层,并轻轻的吹打数次,室温500g,10min离心,小心的吸去上清,沉淀即为PBMC;加入DNAase消化成团细胞,肉眼判断成单细胞悬液加入5-6ml 4℃MACSrunning buffer终止。将终止后的单细胞悬液加到70μm的细胞筛网上,1-2ml的MACSrunning buffer洗三遍管子和筛网,室温300g,10min离心,重悬后计数。
2、CD8+T细胞分选
计数后PBMC按80μl buffer/107细胞加入MACS running buffer重悬,并加入20μlCD8磁珠/107cells,混匀,于4℃孵育15min;孵育结束后加入1-2mL buffer/107cells洗涤;完全吸去上清,PBMC弹散,并加入500μl buffer(0-108total cells)重悬;分选柱置于磁力架上,加入MACS running buffer平衡分选柱。(MS:500μl,LS:3mL)加入细胞悬液,用MACSrunning buffer洗涤管子和分选柱三次,每次体积同上;将柱子取下,加入1ml MACSrunning buffer后猛推柱子活塞,推出的液体即为CD8+T细胞,计数后按107/ml冻存;CD8-T细胞(阴性细胞)贴壁取DC细胞(1.5-2h或过夜)。
3、DC负载MART-1多肽
阴性细胞重悬于5%人血清AIM-V中,铺板;摇晃培养皿使未贴壁细胞重悬于上清中,吸出上清,再加入AIM-V培养基滴洗;贴壁细胞加入DC培养基,48h后补加半量5%人血清AIM-V培养基,24h后用冷的DPBS将细胞吹下来(原培养基与后续加入DPBS吹洗下来的细胞液分开在不同的管子),并按照12孔板5×105cells,1ml培养基每孔,培养基为5%人血清AIM-V,并加入细胞因子诱导DC细胞成熟,同时负载多肽(MART-1(27-35)表位多肽,SEQ IDNo.5),培养的DC细胞中加入多肽,37℃孵育16h。
4、负载多肽(MART-1(27-35)表位多肽的DC细胞与CD8+T细胞共培养
16h后将负载多肽(MART-1(27-35)表位多肽的DC细胞用冷的DPBS吹打下来与复苏的CD8+T共培养;将CD8+吹洗下来,并用AIM-V培养基吹洗孔板至少3遍,室温400g,5min离心;1ml AIM-V重悬,加入DNAase消化至单细胞悬液0-5min后加入5ml AIM-V终止,室温400g,5min离心;T细胞用5%人血清AIM-V重悬,按6.25×105细胞/cm2铺板。按照比例加入负载多肽(MART-1(27-35)表位多肽的DC细胞,并加入IL-21;72h后加入,每隔2-3天补液或半量换液,并补加细胞因子总体积的IL2,IL-7和IL-15;每隔2-3天,补加细胞因子或换液;培养至第5天时,制备第二轮刺激所用抗原呈递beads:day1计算所需磁珠总数,取出所需体积,磁铁吸附去上清,加入等体积硼酸溶液洗两次后等体积重悬,加入CD28和HLA-A2:Ig,4℃摇床过夜。培养至第10天,重悬细胞,取出用于检测和流式分选的细胞数,余下细胞用于进行第二轮共培养。
二、MART-1(27-35)表位特异性T细胞的单细胞TCR测序
1、加载了MART-1(27-35)表位多肽的HLA-A*02四聚体的合成
四聚体有四个单体连接而成,单个HLA分子蛋白与多肽形成的复合物称为单体,通过生物素-亲和链霉素连接四个单体,形成的四聚体。单体置换指的是单体上的多肽交换的过程,因不同实验需要,需要针对不同的抗原构建四聚体,不同抗原(多肽序列)的置换,称为单体置换。
取5μl 10mM MART-1(27-35)表位多肽(SEQ ID No.5),加入120μl PBS,放置冰上;向U型底部96孔板中加入20μl已稀释目的多肽和HLA-A*02单体,混匀;用锡箔纸封板,反应溶液至板底;UV灯管365nm交联30min,37℃避光孵育30min;取30μl MART-1(27-35)表位多肽置换HLA-A*02单体于新的平板中,加入3.3μl荧光偶联链霉亲和素,冰上放置30min,冰上孵育期间,配制终止液。4℃过夜或冰上避光30min,之后4℃保存备用。
2、MART-1(27-35)表位特异性T细胞流式检测
重悬步骤一4中经过刺激后的细胞并计数,取出用于流式分选的细胞数2×105/管,加入1ml PBS重悬,4℃500g,5min离心后,小心弃去上清,加入200μl PBS重悬;向tube中加入步骤1制备的四聚体(10μl/ml),混匀并4℃反应30min;反应时间结束后,加入1ml PBS重悬,4℃500g,5min离心,小心弃去上清,加入200μl PBS重悬,置于冰上用于流式检测;流式上样后,选择阳性群体用于分选单细胞。
MART-1(27-35)表位多肽第一轮刺激后的流式检测结果如图1所示,可见:相比于对照组,对照组为相同条件下培养,未经过刺激的T细胞,用MART-1(27-35)表位多肽抗原刺激后的T细胞可以用四聚体检测出0.668%的阳性肿瘤特异性T细胞。
MART-1(27-35)表位多肽第二轮刺激后的流式检测结果如图2所示,可见:相比于对照组,对照组为相同条件下培养,但未经过第一、第二轮刺激的T细胞,用MART-1(27-35)表位多肽抗原刺激后的T细胞可以用四聚体检测出的阳性肿瘤特异性T细胞比例有很大的提升,达到39.4%。
3、MART-1(27-35)表位多肽特异性T细胞Elispot检测
准备靶细胞:T2细胞计数,取出所需细胞数量,室温400g,5min离心后,用无血清IMDM培养基重悬。
靶细胞负载MART-1(27-35)表位多肽:根据取出的细胞数量确定合适的孔板和负载体积,MART-1(27-35)表位多肽配制成10μg/μl,按1000X加入负载体积中,重悬混匀,37℃,5%的CO2孵箱培养4h。同时设置负载无关多肽的对照组。
准备效应细胞(第二轮刺激后的流式筛选出的MART-1(27-35)表位多肽特异性T细胞):效应细胞计数,取出所需细胞,室温300g,10min离心后,用5%人血清AIM-V培养基重悬,置于冰上。
洗板:至抗原负载还剩45min时,在超净台中取出Human IFN-γELISpot试剂盒中的反应孔板,加入PBS,静置30s后拍掉孔中液体,该动作重复五次后,加入10%FBSRPMI1640培养基100μl/孔,37℃,5%的CO2孵箱孵育30min。
加样:抗原负载时间结束后,用5%人血清AIM-V培养基将孔板中的T2细胞吹洗下来,室温400g,5min离心后,用5%人血清AIM-V培养基重悬。将效应细胞50μl/孔加到反应孔板中后,加入靶细胞悬液50μl/孔,放入37℃,5%的CO2孵箱培养16-48h;培养时间结束后,加入PBS 150μl/孔,静置30s后拍掉孔中液体,该动作重复五次后,用0.5%FBS的PBS(0.2μm滤膜过滤)配制抗人IFN-γ检测抗体溶液(7-b6-1-ALP)。200X加入7-b6-1-ALP抗体并充分混匀0.5%FBS的PBS后,100μl/孔加到反应孔板中,反应板放入37℃,5%的CO2孵箱反应2h;反应时间结束后,加入PBS 150μl/孔,静置30s后拍掉孔中液体,该动作重复五次后,避光加入NBT/BCIP(0.2μm滤膜过滤)100μl/孔到反应孔中,避光显色30s-5min(以观察到阳性对照斑点明显为反应终点)后以大量自来水冲洗,晾干后观察结果。如图3所示,相比于对照组(相同的条件下的T+T2+无关多肽、T+T2、T+有效多肽、T+无关多肽、T为五个阴性对照组,T+OKT3为阳性对照组。其中,OKT3为CD3抗体,与T细胞反应会促使T细胞分泌IFN-γ,在显色反应中产生斑点,作为阳性对照使用),MART-1(27-35)表位多肽特异性T细胞可以有效的识别T2负载的多肽(靶细胞),并分泌IFN-γ,证明这群细胞是具有功能的。
4、单细胞TCR测序
所用试剂如表1所示。
表1单细胞TCR全长测序所需试剂
M0314L | Rnase Inhibitor,Murine | NEB |
T8787 | Triton x-100 | SIGMA |
4030 | dNTP Mixture | TAKARA |
18064071 | SuperScript II Reverse Transcriptase | Invitrogen |
B0300-1VL | Betaine solution | SIGMA |
20-303 | MgCl2 | MILLIPORE |
KK2602 | KAPA HiFi HotStart ReadyMix | KAPA BIOSYSTEMS |
AM9938 | Nuclease-free Water | AMBION |
B7022S | Gel Loading dye,Orange | NEB |
MD109-2 | 100bp DNA ladder | TIANGEN |
所用引物序列如表2。
表2MART-1(27-35)表位特异性T细胞的单细胞TCR全长测序所需引物
(1)细胞裂解
按照表3配制细胞裂解混合液。
表3细胞裂解混合液
细胞裂解混合液 | 体积μl | 终浓度 |
RNase/DNase-free water | 1.86 | |
10μM Oligo-dT Primer | 1 | 2.5μM |
10mM dNTP | 1 | 2.5mM |
40U/μl RNase Inhibitor | 0.1 | 2U/μl |
10%Triton X-100 | 0.04 | 0.2% |
总体积 | 4 |
配制时,按样品数110%配制(如有10个细胞样品,则配制11管的量)。配制好的裂解液吹打混匀后分装到洁净PCR管中,4℃14000rpm,30s离心(将液滴离心到管底并去除气泡),冰盒放置,待后续分入细胞;选择阳性群体(即步骤7所得的MART-1(27-35)表位特异性T细胞)并向装有裂解液的PCR管分入单细胞;分选完毕后,盖好管盖,短时离心,并调试好PCR仪准备进行单细胞裂解。
将0.2ml PCR管置于PCR仪内,72℃,3min孵育(细胞为bulk样本增至5min),热盖温度为75℃,裂解完成后立即置于冰上1min;10000rpm 4℃离心30s,后立即转至冰上;此步后,所有mRNAs都从单细胞中释放,并且Oligo-dT引物也已与mRNAs结合。
(2)按表4配制逆转录体系。
表4逆转录体系
成分 | 体积μl | 终浓度 |
5×SuperScript II | 2 | 1X |
5M Betaine | 2 | 1M |
100mM MgCl<sub>2</sub> | 0.9 | 9mM |
100mM DTT | 0.25 | 2.5mM |
100μM TSO | 0.1 | 1μM |
40U/ul RNAse inhibitor | 0.25 | 1U/μL |
200U/μl SSII | 0.5 | 10U/μL |
总体积 | 6 |
配制时,按样品数+0.5个配制(如有9个细胞样品,则配制9.5管的量)。配制好的Mix充分混匀后,依次加入到上步离心管中;
(3)吹打混匀、瞬时离心后,按如表5所示的条件进行逆转录反应(75℃热盖)。
表5逆转录反应条件
此步后,所有mRNAs的第一链cDNA合成完毕;
(4)按表6配制第一轮PCR Mix。
表6第一轮PCR Mix
成分 | 体积μl | 终浓度 |
2×KAPA HiFi HotStart ReadyMix | 12.5 | 1X |
IS PCR Primer(10μM) | 1 | 0.4μM |
TCRA-out Primer(10μM) | 0.5 | 0.2μM |
TCRB-out Primer(10μM) | 0.5 | 0.2μM |
NF-water | 0.5 | |
总体积 | 15 |
配制时,按样品数+0.5配制(如有9个细胞样品,则配制9.5管的量)。配制好的Mix充分混匀后,依次取15μl加入到上步离心管中,吹打混匀、瞬时离心后,按表7所示条件预扩增。
表7第一轮PCR预扩增条件
(5)按表8配制第二轮PCR Mix。
表8第二轮PCR Mix
成分 | 体积μl | 终浓度 |
2×KAPA HiFi HotStart ReadyMix | 12.5 | 1X |
IS PCR Primer(10μM) | 1 | 0.4μM |
TCRA-middle Primer(10μM) | 0.5 | 0.2μM |
TCRB-middle Primer(10μM) | 0.5 | 0.2μM |
NF-water | 9.5 | |
总体积 | 24 |
配制时,按样品数+0.5配制(如有9个细胞样品,则配制9.5管的量)。配制好的Mix充分混匀后,依次取24μl加入到上步离心管中,吹打混匀、瞬时离心后,按表9所示条件预扩增。
表9第二轮PCR预扩增条件
(6)按表10配制第三轮PCR Mix。
表10第三轮PCR Mix
成分 | 体积μl | 终浓度 |
2×KAPA HiFi HotStart ReadyMix | 12.5 | 1X |
IS PCR Primer(10μM) | 1 | 0.4μM |
TCRA-in Primer(10μM) | 0.5 | 0.2μM |
TCRB-in Primer(10μM) | 0.5 | 0.2μM |
NF-water | 9.5 | |
总体积 | 24 |
配制时,按样品数+0.5配制(如有9个细胞样品,则配制9.5管的量)。配制好的Mix充分混匀后,依次取24μl加入到上步离心管中,吹打混匀、瞬时离心后,按表11所示条件预扩增。
表11第三轮PCR预扩增条件
(7)电泳检测:PCR完成后电泳检测,采用2%琼脂糖凝胶,取15μL产物,加3ulLoading buffer混匀,130V电泳45min,目的条带切胶回收。之后与T载体连接,然后再进行菌落PCR鉴定。
图4为单细胞TCR扩增电泳图;图5为TA克隆的菌落PCR电泳图。图中结果显示,扩增出来的TCR片段与T载体连接成功,通过菌落PCR可以有效的检测出成功构建的插入片段载体。
(8)将测序的片段在IMGT网站上进行blast,分别与TCRα与TCRβ链的C区拼接,形成完整的TCR序列集合。
三、MART-1(27-35)表位特异性TCR序列的筛选及功能验证
将步骤二获得的MART-1(27-35)表位特异性T细胞的单细胞TCR序列合集按照丰度由高到低的顺序进行排序,选择丰度高的,按照丰度由高到低排序后,排名前5%的序列进行初步的功能验证,以确定最终的用于治疗的TCR全长序列。其中,一个丰度达到1.5%的功能性配对的TCRα/β序列(标记为clone#4),找到起始密码子后,根据恒定区(TRAC/TRBC)实际序列进行拼接,形成新序列,α链的完整编码基因的序列为SEQ ID No.1(编码SEQ IDNo.3所示α链),β链的完整编码基因的序列为SEQ ID No.2(编码SEQ ID No.4所示β链)。其中,SEQ ID No.1的第103-408位为α链可变区的编码基因(第181-198位、第250-267位和第370-408位分别为三个CDR的编码基因),SEQ ID No.2的第58-375位为β链可变区的编码基因(第136-150位、第202-219位和第334-375位分别为三个CDR的编码基因)。
按照图6所示示意图将SEQ ID No.1和SEQ ID No.2所示的α链和β链的编码基因通过P2A肽的基因序列连接后构建到慢病毒载体pRRLSIN.cPPT.PGK-GFP.WPR中,得到重组病毒载体。该重组病毒载体的结构描述为:将SEQ ID No.6所示的DNA片段插入到pRRLSIN.cPPT.PGK-GFP.WPRE载体的BamHI和SalI双限制性内切酶中间位置后得到的重组质粒。
将构建好的重组病毒载体感染T细胞,然后参照步骤一5中的方法进行Elispot检测,验证其功能。结果如图7所示,clone#4序列构建的病毒感染的T细胞相比GFP对照实验组,可以有效的识别T2负载的MART-1(27-35)表位多肽,并分泌IFN-γ,即可以有效的与靶细胞反应。
工业应用
本发明通过MART-1(27-35)表位体外刺激特异性T细胞,获得特异性T细胞群体,利用单细胞配对TCR测序技术,获得MART-1(27-35)表位对应的有效T淋巴细胞的TCR序列集合,再将这些序列集合通过丰度排序,进行体外的功能验证,最终获得了本发明请求保护的TCR。实验证明,本发明所提供的表达TCR的T细胞可以有效的识别T2细胞负载的MART-1(27-35)表位多肽(靶细胞),并分泌IFN-γ,证明这群T细胞是具有功能的。有效的TCR可以用于过继细胞的免疫细胞治疗。另外,这些序列集和进行亲和力提升或与相关药物靶标连用,可以有效的进行药物开发,市场前景广阔。
Claims (17)
1.一种MART-1(27-35)表位特异性T细胞受体,包含α链和β链;
所述α链包含三个互补决定区,氨基酸序列分别为SEQ ID No. 3的第61-66位、第84-89位以及第124-136位;
所述β链包含三个互补决定区,氨基酸序列分别为SEQ ID No. 4的第46-50位、第68-73位以及第112-125位。
2.根据权利要求1所述的T细胞受体,其特征在于:所述α链的可变区的氨基酸序列为SEQ ID No. 3的第35-136位,或与SEQ ID No. 3的第35-136位具有至多3个氨基酸的改变;
所述β链的可变区的氨基酸序列为SEQ ID No. 4的第20-125位,或与SEQ ID No. 4的第20-125位具有至多3个氨基酸的改变。
3.根据权利要求1所述的T细胞受体,其特征在于:所述α链的可变区的氨基酸序列与SEQ ID No. 3的第35-136位具有至多2个氨基酸的改变;
所述β链的可变区的氨基酸序列与SEQ ID No. 4的第20-125位具有至多2个氨基酸的改变。
4.根据权利要求1所述的T细胞受体,其特征在于:所述α链的可变区的氨基酸序列与SEQ ID No. 3的第35-136位具有至多1个氨基酸的改变;
所述β链的可变区的氨基酸序列与SEQ ID No. 4的第20-125位具有至多1个氨基酸的改变。
5.根据权利要求1-4中任一所述的T细胞受体,其特征在于:所述α链的恒定区的氨基酸序列为SEQ ID No. 3的第148-288位;所述β链的恒定区的氨基酸序列为SEQ ID No. 4的第136-314位。
6.根据权利要求1-4中任一所述的T细胞受体,其特征在于:所述α链的氨基酸序列为SEQ ID No. 3;所述β链的氨基酸序列为SEQ ID No. 4。
7.编码权利要求1-6中任一所述T细胞受体的核酸分子。
8.根据权利要求7所述的核酸分子,其特征在于:编码所述T细胞受体的核酸分子包含编码所述T细胞受体的α链的核酸分子和编码所述T细胞受体的β链的核酸分子;
编码所述T细胞受体的α链中三个互补决定区的核酸分子的序列分别为SEQ ID No. 1的第181-198位、第250-267位以及第370-408位;或与这些序列具有80%以上同一性且编码相同氨基酸残基的序列;
编码所述T细胞受体的β链中三个互补决定区的核酸分子的序列分别为SEQ ID No. 2的第136-150位、第202-219位以及第334-375位;或与这些序列具有80%以上同一性且编码相同氨基酸残基的序列。
9.根据权利要求7或8所述的核酸分子,其特征在于:编码所述α链的可变区的核酸分子的序列为SEQ ID No. 1的第103-408位;或与这些序列具有80%以上同一性且编码相同氨基酸残基的序列;
编码所述β链的可变区的核酸分子的序列为SEQ ID No. 2的第58-375位;或与这些序列具有80%以上同一性且编码相同氨基酸残基的序列。
10.根据权利要求7或8所述的核酸分子,其特征在于:编码所述α链的核酸分子的序列为SEQ ID No.1;编码所述β链的核酸分子的序列为SEQ ID No. 2。
11.含有权利要求7-10中任一所述核酸分子的表达盒。
12.含有权利要求7-10中任一所述核酸分子的载体。
13.含有权利要求7-10中任一所述核酸分子的细胞。
14.根据权利要求12所述的载体,其特征在于:所述载体是通过将编码所述α链的核酸分子和编码所述β链的核酸分子用连接肽的编码序列连接后插入到慢病毒载体pRRLSIN.cPPT.PGK-GFP.WPRE的BamHI和SalI之间后得到的。
15.根据权利要求13所述的细胞,其特征在于:所述细胞为T细胞。
16.含有权利要求12或14所述载体或权利要求13或15所述细胞的药物组合物。
17.权利要求1-6中任一所述的T细胞受体或权利要求7-10中任一所述的核酸分子或权利要求12或14所述的载体或权利要求13或15所述细胞在制备预防和/或治疗黑色素瘤的药物中的应用。
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