CN117949667A - 一种基于工程细胞系检测抗原肽段亲和力的方法 - Google Patents
一种基于工程细胞系检测抗原肽段亲和力的方法 Download PDFInfo
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Abstract
本发明属于基因工程领域、生物医药领域,具体涉及一种基于工程细胞系检测抗原肽段亲和力的方法。具体地,所述方法包括将目标肽与工程细胞相接触,所述工程细胞具有以下特征:1)野生型工程细胞的表面缺乏HLA,2)在工程细胞中过表达的HLA分子,所述HLA的型别与目标肽对应,3)工程细胞中的TAP基因被敲除或TAP的表达被抑制。所述方法丰富了对不同HLA型别的抗原肽免疫原性的验证方法。
Description
技术领域
本发明属于基因工程领域、生物医药领域,具体涉及一种基于工程细胞系检测抗原肽段亲和力的方法。
背景技术
HLA(human leucocyte antigen)复合体是人类多态性最丰富的遗传系统,定位于第6号染色体短臂6P21.31区,长3600kb。1999年已完成全部序列分析及基因定位,在此区域内共确认了224个基因座位中128个为功能性基因,其中39.8%和免疫功能相关。HLA基因根据其编码分子的分布与功能不同分为3个区,即Ⅰ类基因区,Ⅱ类基因区,Ⅲ类基因区。经典的HLAⅠ类抗原包括HLA-A、HLA-B、HLA-C;HLAⅡ类抗原包括HLA-DP、HLA-DQ、HLA-DR。非经典的HLAⅠ、Ⅱ类有HLA-F、E、H、X、DN、DO、DM等。HLAⅠ类几乎分布于身体全部细胞表面,Ⅱ类主要是定位于巨噬细胞和B淋巴细胞表面的糖蛋白。
HLA等位基因频率不仅在不同人群、不同地域存在明显差异,而且与某些疾病的发生存在一定的相关性。HLA分型不仅为了解人种来源和民族的迁徙提供重要的科学资料,而且在医学实践中有重要意义。
中国人群中常见的HLA-A基因有HLA-A*02:01、HLA-A*11:01,HLA-A*24:02,其中,HLA-A*11:01是中国人群最为常见的HLA-I类分子的型别。但是,对于多种多样的HLA型别缺乏验证其相应抗原肽的亲和力的工具细胞系。下一代测序技术和生物信息学分析能力的发展使得大量新生抗原肽被发现,如何验证这些抗原肽与HLA分子的亲和力及其免疫原性面临着巨大的挑战。
发明内容
为了丰富了对不同HLA型别的抗原肽免疫原性的验证方法,本发明构建了一种工程细胞系,基于该工程细胞系可以有效检测抗原肽段亲和力。具体地,本发明提供了以下技术方案:
第一方面,本发明提供了一种灵敏检测目标肽免疫原性的方法,所述方法包括将目标肽与工程细胞相接触,所述工程细胞具有以下特征:
1)野生型工程细胞的表面缺乏HLA,
2)在工程细胞中过表达的HLA分子,所述HLA的型别与目标肽对应,
3)工程细胞中的TAP基因被敲除或TAP的表达被抑制。
优选地,所述方法中还包括免疫原性检测的步骤,所述免疫原性检测的方法包括但不限于流式细胞技术、酶联免疫吸附测定(Enzyme-linked immunosorb ent assay,ELISA)、放射免疫分析法(RIA)、荧光免疫、化学发光免疫测定、电化学发光(Electrochemiluminescence immunoassay,ECLI)检测、表面等离子体共振检测法(SPR)、全自动纳升级免疫分析工作站Gyrolab、酶联免疫斑点法(enzyme linked immunospotassay,ELISPOT)、生物测定法等,所述ELI SA包括四种主要类型:直接法、间接法、竞争法和双抗夹心法。
优选地,本发明所述HLA的型别包括任意型别,在此引入HLA数据库https://hla.alleles.org/nomenclature/index.html中所有型别;示例性的,HLA I类包括HLA-A、HLA-B、HLA-C、HLA-E、HLA-F、HLA-G,其中,HLA-A中又包括A*01:01、A*01:02、A*01:03、A*01:06、A*01:07、A*01:08、A*01:09、A*01:10、A*01:12、A*01:13等,HLA-B中又包括B*07:02、B*07:03、B*07:04、B*07:05、B*07:06、B*07:07、B*07:08、B*07:09、B*07:10、B*07:11等;HLAII类包括HLA-DRA、HLA-DRB1、HLA-DRB2-9、HLA-DQA1、HLA-DQB1、HLA-DPA1、HLA-DPB1,各类中还包括多种分型。
优选地,本发明具体实施例中以A0201(A*02:01)、A1101(A*11:01)、A2402(A*24:02)、B4001(B*40:01)、B4601(B*46:01)、C0102(C*01:02)、C0702(C*07:02)进行了示例性的验证。
优选地,所述目标肽所对应的HLA型别是本领域技术人员根据常规技术可以确认的。所述目标肽包括具有任意序列的肽。
本发明所述术语“目标肽”、“多肽”、“多肽片段”和“蛋白质”在此可以互换使用,指的是氨基酸残基的聚合物及其变体和合成类似物。因此,这些术语适用于天然存在的氨基酸聚合物,以及其中一个或多个氨基酸残基是人工合成的非天然存在的氨基酸(如相应的天然存在氨基酸的化学类似物)的氨基酸聚合物。
本发明所述TAP也即抗原处理相关转运体(Antigen transfer associatedprocess protein)是由TAP1和TAP2两个亚基形成的异二聚体跨膜转运蛋白。
优选地,所述TAP基因被敲除是TAP1和/或TAP2被敲除,优选,TAP1和TAP2同时被敲除。
优选地,所述敲除的方法包括CRISPR技术、RNA干扰技术、反义寡核苷酸(ASO)技术、TALEN技术、ZFN技术、Cre-loxP基因重组技术等基因编辑技术所使用的试剂。
优选地,所述敲除的方法是CRISPR技术。
优选地,所述过表达是通过病毒载体转染实现的。
本发明所述原始细胞还可以是任意来源、任意类型的细胞,优选人类细胞,优选非免疫细胞,所述人类细胞例如:造血细胞、神经细胞、血液细胞、脂肪细胞、间充质细胞、肌肉细胞、心脏细胞、肝脏细胞、胰腺细胞、皮肤细胞等。所述人类细胞还可以是细胞系,例如:CHO细胞、293细胞、CHO-K1细胞、HEK293细胞、Caco2细胞、U2-OS细胞、NIH 3T3细胞、NSO细胞、SP2细胞、CHO-S细胞、DG44细胞、K-562细胞、U-937细胞、MRC5细胞、IMR90细胞、Jurkat细胞、HepG2细胞、HeLa细胞、HT-1080细胞、HCT-116细胞、Hu-h7细胞、Huvec细胞、Molt 4细胞等。
优选地,所述原始细胞是K562细胞。
具体地,所述野生型工程细胞也即未经任何人工处理的原始细胞,所述原始细胞表面缺乏的HLA分子包括HLA I类和/或HLA II类。在本发明具体实施例中以K562作为原始细胞为例。
另一方面,本发明提供了以上工程细胞的制备方法,所述方法包括过表达HLA和敲除(包括抑制)TAP基因的步骤。
在一种具体的实施方式中,前述一种灵敏检测目标肽免疫原性的方法通过该制备方法得到的工程细胞进行。
本发明所述过表达即提高所述HLA的瞬时表达量或稳定表达量。在一种实施例中,编码所述重组蛋白的核酸可以染色体性地整合入细胞的基因组中,并指导细胞中相应大蛋白的表达,稳定转染的细胞可反复使用,所述整合可以是随机或靶向的;在另一种实施例中,编码所述HLA的核酸可以位于染色体外,例如在质粒、粘粒、人工染色体、微型染色体中表达。
优选地,所述表达包括通过瞬时表达或稳定表达。
优选地,所述稳定表达是通过使用转座子、病毒载体、重组酶或基因编辑技术将目标序列整合到宿主基因组实现的。
优选地,所述病毒载体包括慢病毒载体、痘病毒载体、单纯疱疹病毒载体、腺病毒载体、腺相关病毒载体。
优选地,所述病毒载体是慢病毒载体。
优选地,所述瞬时表达是通过转染载体实现的。
优选地,所述转染的方法包括物理方法、化学方法。
优选地,所述物理方法包括磷酸钙沉淀、脂质转染法、粒子轰击、微注射、电穿孔(电转)。
优选地,所述化学方法包括胶体分散系统、基于脂质的系统。
优选地,所述敲除TAP基因的方法包括CRISPR技术、RNA干扰技术、反义寡核苷酸(ASO)技术、TALEN技术、ZFN技术、Cre-loxP基因重组技术等基因编辑技术所使用的试剂。
优选地,所述敲除TAP基因的方法是CRISPR技术。
优选地,所述CRISPR技术敲除TAP基因所使用的试剂包括Cas蛋白成分和sgRNA成分。
优选地,所述敲除TAP基因是通过Cas蛋白成分和sgRNA成分共同转入细胞实现的。
优选地,所述转入的方法包括电转(电穿孔)、磷酸钙沉淀、脂质转染法、粒子轰击、微注射。
优选地,所述转入的方法是电转。
优选地,所述敲除TAP是敲除TAP1和/或TAP2,优选,同时敲除TAP1和TAP2。
优选地,所述敲除TAP是同时靶向SEQ ID NO.1和SEQ ID NO.2所示位点进行CRISPR编辑,根据位点序列设计相应的sgRNA是本领域技术人员所熟知的。
优选地,所述敲除TAP基因是TAP的表达降低到原始水平的90%、80%、70%、60%、50%、40%、30%、20%、10%或更低。
本发明所述CRISPR编辑包括敲除,也包括突变,凡使TAP1和/或TAP2的表达受到影响皆纳入此范围内。
在另一种具体地实施例中,本领域技术人员熟知,将编码基因编辑工具(Cas蛋白成分和sgRNA成分)的核苷酸及其他调控元件构建于适宜的载体中,再转化细胞,也可以实现对细胞内基因组的编辑。
本发明所述的Cas蛋白可以是野生型或其突变体,所述的突变体的突变类型包括氨基酸的替换、取代或缺失,所述的突变体可以改变/不改变Cas蛋白的酶切活性。本领域技术人员所知,现有技术中已报到的多种具有核酸切割活性的Cas蛋白,该公知蛋白或其改造后的变体均可以实现本发明的功能,本文通过引用方式将其纳入保护范围。
优选地,所述的Cas蛋白是指蛋白家族,可以根据其来源不同而具有不同的结构,如来源于酿脓链球菌(Streptococcus pyogenes)的SpCas9、来源于葡萄球菌(Staphylococcus aureus)的SaCas9。
另一方面,本发明提供了上述制备方法所制备得到的工程细胞。
优选地,所述工程细胞不仅是指特定的个体细胞,并且也是指此一细胞的子代或潜在的子代。由于在后续世代中可能因突变或环境影响而发生某些改变,因此所述子代实际上可能与亲代细胞不同,但却仍涵盖于本文所用术语工程细胞的范围内。
另一方面,本发明还提供了以上工程细胞在检测目标肽免疫原性的应用。
优选地,所述目标肽与所述工程细胞中过表达HLA型别相对应。
本发明所述术语免疫原性(Immunogenicity)是指目标肽诱发对自身或相关蛋白的免疫应答或免疫相关事件的能力。
附图说明
图1是HLA慢病毒表达质粒图谱。
图2是不同K562过表达细胞系HLA表达水平的检测结果。
图3是TAP基因敲除前后的K562-HLA过表达细胞系中的HLA表达水平的检测结果。
图4是TAP基因敲除前后K562-HLA-A0201细胞负载抗原肽能力流式检测结果。图5是不同K562-HLA-A0201OE-TAPKO单克隆细胞负载抗原肽前后MFI比值检测结果。
图6不同K562-HLA-A1101OE-TAPKO单克隆细胞负载抗原肽前后MFI比值检测结果。
图7不同K562-HLA-A2402OE-TAPKO单克隆细胞负载抗原肽前后MFI比值检测结果。
图8其他HLA型别的K562-HLAOE-TAPKO细胞负载抗原肽前后MFI比值检测结果。
图9K562-HLA-A0201OE-TAPKO细胞与T2细胞对不同A2型抗原肽亲和力对比结果。
具体实施方式
下面结合具体实施例对本发明作更进一步的说明,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,根据本发明的技术方案及其发明构思加以等同替换或改变,都应涵盖在本发明的保护范围之内。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
通用方法
1、293FT与K562细胞复苏与培养
打开水浴锅,将温度设置为37℃,等待升温。将培养液置于水浴锅中预热至37℃备用。取一支15ml无菌离心管加入10ml已预热的培养液。分别取1支293FT和k562野生型冻存细胞,37℃水浴轻柔且快速使其融化,然后转入15mL离心管内,加入5mL新鲜培养基,混匀后350rcf常温低速离心5min,弃上清后,用适量完全培养基将细胞重悬,之后转移至T75培养瓶,于37℃、5%CO2培养箱培养。贴壁细胞待培养瓶底部细胞融合度达80~90%时,对细胞进行1:3传代培养;悬浮细胞一般2-3天观察一次培养基状态,溶液变黄后可进行传代或补液操作,调整细胞起始密度为5×105cells/mL,待细胞培养2-3代状态稳定后,可进行后续实验操作。
2、K562细胞的细胞冻存
K562细胞冻存密度为5×106cells/ml,冻存体积为1ml/支。根据培养体积预先计算细胞总数,准备一定数量的冻存管。将细胞悬液转移至无菌离心管中,350rcf常温低速离心5min。弃上清,反复拨弹离心管,将细胞弹至松散,取一定量的冻存液重悬细胞,轻柔吹吸混匀至均一细胞悬液。取20μl细胞悬液进行计数,记录活细胞密度、活率等,根据活细胞总数调整细胞密度为5×106cells/ml。将细胞悬液每管1ml,依次分装至贴好标签的冻存管中,拧紧冻存管管盖,迅速放入程序性降温盒置于-80℃冰箱,次日转移至液氮罐中。
3、HLA过表达慢病毒包装
步骤(1):取生长状态良好的293FT细胞,消化重悬后计数,取1.2×107个细胞加入T175培养瓶内,20mL完全培养基,摇晃混匀后放于37℃细胞培养箱过夜培养;
步骤(2):第二天观察细胞汇合度,当其达到50~60%时进行转染(过高或过低均会影响细胞转染效率),转染按照下列表格进行取样混合,Lipo3000与P3000分开进行配制,配制完成后室温静置5min,然后将两者混合到一起,轻弹混匀,室温静置30min,之后用移液器缓慢滴加到待包装病毒的细胞培养瓶内,做好标记,摇晃混匀,放置于CO2培养箱内持续培养;HLA慢病毒表达质粒图谱如图1所示;
表1、转染试剂配制表
步骤(3):质粒转染后48h,将培养基上清全部转移至50mL离心管内,然后向细胞培养瓶内添加20mL新鲜完全培养基,继续放回培养箱内培养,收集的上清放至4℃冰箱保存;
步骤(4):质粒转染后72h,收集培养基上清,并与48h的培养基上清合并至一起,4500rpm,4℃离心5min,去除细胞碎片等沉淀,然后将上清用0.22μM的真空过滤瓶除掉杂质,向过滤后的溶液中加入1/5总体积的PEG8000浓缩试剂,摇晃混匀后放置于4℃冰箱进行过夜浓缩沉降;
步骤(5):次日上午取出液体,在生物安全柜内转移至50mL离心管中,4500rpm,4℃离心50min,沉淀中包含我们所需的慢病毒。弃掉上清,将离心管在擦手纸上倒扣2~3min,尽量控干液体,然后每管加入200μL预冷的D-PBS将沉淀重悬,混匀后分装至1.5mL离心管内,50μL/管,-80℃保存或直接用于感染目的细胞。
实施例1、构建过表达不同型别HLA的K562稳转细胞系
首先利用293FT工具细胞将HLA表达基因包装成慢病毒,然后利用慢病毒感染的方式将HLA基因稳定整合到K562细胞的基因组内,使其能够稳定遗传和表达。按照通用方法准备细胞和HLA过表达慢病毒,慢病毒感染目的细胞K562并挑选阳性单克隆。
步骤(1):将培养的K562野生型细胞离心,完全培养基重悬后进行计数,取一部分细胞调整密度至3×105cells/mL,接种至12孔板内,1mL/孔,同时每孔加入5μL的polybrene促感染试剂和50μL纯化后的慢病毒,摇晃混匀后放入细胞培养箱内进行培养;
步骤(2):细胞正常传代与扩大培养,传两代后可取部分细胞流式检测HLA表达情况,PBS清洗1遍后加入2-3μL的BV510 Mouse Anti-Human HLA-ABC(BD公司)抗体,避光染色20min,之后加PBS清洗1遍,弃去上清,加入300μL的PBS重悬细胞(均无菌操作),之后取一部分上机流式检测HLA阳性率与表达量;
步骤(3):流式检测细胞HLA表达与野生型相比有明显差异的,可将剩余细胞进行流式分选单克隆,选择HLA表达强度前5%的细胞,至少分选1个96孔板;
步骤(4):单克隆细胞扩增培养起来后,可按照上述步骤(2)操作检测单克隆细胞的HLA表达量,比K562野生型显著升高的即为HLA过表达细胞系,选取表达量最好的单克隆进行冻存保种入库。
结果显示:
通过流式检测其HLA表达强度与阳性率,挑选表达效果最好的细胞,结果如图2所示。K562-HLA-A1101OE细胞HLA阳性率为98.68%;K562-HLA-A0201OE细胞HLA阳性率为98.61%;K562-HLA-A2402OE细胞HLA阳性率为98.15%;HLA-B/C基因过表达的细胞中HLA表达水平较野生型也均有明显提升。选用以上细胞进行后续实验。
实施例2、CRISPR/Cas9敲除K562-HLA过表达细胞系的TAP1/TAP2基因
利用实施例1构建K562-HLA过表达细胞系,通过Neon电转系统(Thermo公司)将Cas9蛋白与TAP1、TAP2的gRNA共同电转至细胞内,后续通过流式检测HLA的表达水平间接检测TAP基因的敲除效率。
所涉及的试剂:TrueCutTMCas9 Protein v2(Thermo,货号:A36498)、TrueGuideTMSynthetic sgRNA(Thermo,货号:A35533,NeonTM)、10μL电转试剂盒(Thermo,货号:MPK1096)
TAP1、2的靶点序列是:
TAP1靶点:5’-TAGAGCTAGCCATTGGCACT-3’(SEQ ID NO.1)
TAP2靶点:5’-GGGGGCTGCTAAAGCTAAGA-3’(SEQ ID NO.2)
操作步骤:
(1)取一个新的12孔板,根据实验选合适数目的孔,每孔加入1mL完全培养基后放入37℃培养箱进行预热;
(2)取1支干净的1.5mL离心管,加入Cas9 v2蛋白0.25μL/反应(1.25μg)、TAP1sgRNA 3.25pmol/反应、TAP2 sgRNA 3.25pmol/反应(1.5nmol干粉加入100μL无核酶水溶解,终浓度为15pmol/μL),电转R液补齐至5μL/反应,枪头吸打混匀后放置于室温下共孵育20min;
(3)孵育期间取生长状态良好的K562-HLA过表达细胞,离心收集细胞,加入适量的PBS重悬,计数后取2×106个细胞,离心后再次使用PBS清洗一遍,之后吸尽上清,加入50μL的电转R液重悬细胞,将其密度调整至4×107cells/mL;
(4)孵育完成后,取等体积重悬后的细胞加入Cas9与gRNA共同孵育的离心管内,吸打混匀,然后使用Neon电转仪进行电转,1700V、20ms、1pulse,电转完成后迅速加入提前预热的12孔板培养基内并放回培养箱恢复培养,同时取5uL重悬后的细胞加入12孔板培养基内,后续用作检测的对照组;
(5)细胞正常扩增培养,期间取部分细胞检测敲除效率。取细胞使用PBS清洗1遍后加入2μL的BV510 Mouse Anti-Human HLA-ABC抗体,避光染色20min,之后加PBS清洗1遍,弃去上清,加入300μL的PBS重悬细胞(均无菌操作),取一部分上机流式检测HLA阳性率与表达量;
(6)流式检测TAP基因敲除后细胞HLA表达与野生型相比有明显差异的,可将剩余细胞进行流式分选单克隆,选择HLA表达明显下降的细胞,至少分选2个96孔板;
(7)单克隆细胞扩增培养起来后(约2-3周),可按照上述步骤(5)操作检测单克隆细胞的HLA表达量,比未敲除的K562-HLA细胞明显低的即为TAP基因敲除的细胞系;
结果显示:
通过流式检测TAP基因敲除前后的K562-HLA过表达细胞系表面HLA的表达水平,确定TAP基因敲除的效果,结果如图3所示。
TAP基因敲除后,细胞不能正常递呈内源抗原肽至细胞表面,缺失抗原肽的HLA复合物不能稳定存在于细胞表面,经流式检测其HLA水平明显低于未敲除TAP基因的K562-HLA细胞。
实施例3、敲除TAP基因的K562-HLA细胞系对相应抗原肽的负载能力检测
利用构建好的K562-HLAOE-TAPKO细胞系,使用对应HLA型别的阳性抗原肽与细胞共同孵育,负载上抗原肽的HLA复合物则能够稳定存在于细胞表面,之后通过流式细胞术检测细胞表面的HLA表达水平,确定细胞对抗原肽的负载能力。
1、首先以K562-HLA-A0201OE-TAPKO细胞检测流程为例进行检测。
操作步骤:
(1)抗原肽溶解及稀释
根据抗原肽合成报告选择抗原肽溶解试剂(培养基/DMSO),计算有效肽含量和摩尔浓度。
计算公式:
有效肽质量=总含量×肽纯度×净氮含量
配制200μM抗原肽溶解体积简便计算公式:
若所用抗原肽质量较小,可将抗原肽加入溶剂(溶剂不能是DMSO)直接溶解至200μM。若使用DMSO溶解抗原肽,需加入少量DMSO溶解,再使用培养基将抗原肽稀释至200μM,且DMSO的终浓度需<10%(v/v)。
(2)β2M工作液配制
β2M原浓度为5mg/ml,用RPMI 1640培养基配制β2M工作液为50μg/ml。
(3)细胞负载抗原肽
细胞终浓度为1×106/ml,抗原肽终浓度为60μM,β2M终浓度为2.5μg/ml;取适量待检测细胞,350g室温离心5min,1ml RPMI 1640培养基重悬细胞并计数,调整细胞浓度为2×106/ml。
取一块48孔板,做好标记,分别设置实验组,对照组和Blank组。实验组和对照组分别加入待检测细胞,抗原肽和β2M,最后用培养基补至终体积。空载组加入待检测细胞和β2M,用培养基补至终体积。终体系200μl/孔接种于48孔板中。于二氧化碳培养箱中孵育24h。
阳性肽对照组:负载HLA-A*02:01型CMV病毒抗原肽G44(HLA-A*02:01阳性抗原肽G44,HCMV pp65 495-504:NLVPMVATV,SEQ ID NO.3);
阴性肽对照肽:负载HLA-A*1101型EBV病毒抗原肽G93(EBV EBNA-4416-424:IVTDFSVIK,SEQ ID NO.4);
Blank组:待测细胞空载组。
4)流式检测HLA-A2
收集待检测细胞到流式管中,350g离心5min,弃上清。2ml PBS清洗细胞2次,350g室温离心5min,弃上清,100μl PBS重悬细胞。每管中加入2μl PE anti-human HLA-A2,室温避光孵育20min。2ml PBS清洗细胞1次,350g室温离心5min,弃上清,200μl PBS重悬细胞。使用流式细胞仪检测细胞表面HLA-A2表达水平,记录检测的MFI值。
(5)数据处理
计算公式:
结果显示:
利用培养获得的K562-HLA-A0201OE-TAPKO单克隆细胞与未敲除的K562-HLA-A0201OE细胞负载抗原肽,比较其负载能力差异,两者负载肽后流式检测结果如图4所示。
敲除TAP基因后的单克隆细胞负载G44阳性肽后细胞群体明显右移,而负载阴性对照抗原肽G93的细胞与未负载肽的blank组细胞完全重合,表明抗原肽的结合是特异的。
未敲除的细胞负载G44阳性肽后细胞群体也有一些向右偏移,而负载G93后却无明显变化,表明未敲除TAP基因前K562-HLA-A0201OE细胞也有一定的抗原肽负载能力,但敲除TAP基因后其负载能力有明显的提升。
按照同样的方法对不同TAP敲除的单克隆细胞进行负载能力检测,计算MFI比值后的对比结果如图5所示。14号克隆的MFI比值最高,是未敲除TAP基因的对照组细胞的8倍,其他单克隆的MFI比值相较于对照组均有所提升,表明TAP基因敲除后不同单克隆细胞对抗原肽的亲和能力均有不同程度的提升。
2、K562-HLA-A1101OE-TAPKO和K562-HLA-A2402OE-TAPKO细胞检测
将以下抗原肽替换前述阳性抗原肽G44进行实验:
HLA-A*11:01阳性抗原肽G101(HCMV pp65:ATVQGQNLK,SEQ ID NO.5)
HLA-A*24:02阳性抗原肽G70(HCMV pp65 113-121:VYALPLKML,SEQ ID NO.6)
对K562-HLA-A1101OE-TAPKO和K562-HLA-A2402OE-TAPKO单克隆细胞的负载肽能力检测结果分别如图6与图7所示。相较于未敲除TAP基因的对照组细胞,敲除TAP基因后的单克隆负载相应阳性抗原肽后MFI比值均有提升,表明按照本发明所述方法制备HLAOE-TAPKO细胞均可挑选出对抗原肽检测能力显著提升的单克隆细胞,可更为灵敏的检测对相应HLA型别的抗原肽的免疫原性。
3、K562-HLA-B4001OE-TAPKO、K562-HLA-B4601OE-TAPKO、K562-HLA-C0102OE-TAPKO和K562-HLA-C0702OE-TAPKO细胞检测
将以下抗原肽替换前述阳性抗原肽G44进行实验:
HLA-B*40:01阳性抗原肽(pB4001:IEVDGKQVEL,SEQ ID NO.7)
HLA-B*46:01阳性抗原肽(pB4601:FIKDGSSTY,SEQ ID NO.8)
HLA-C*01:02阳性抗原肽(pC0102:VAPWNSLSL,SEQ ID NO.9)
HLA-C*07:02阳性抗原肽(pC0702:KYFDEHYEY,SEQ ID NO.10)
K562-HLA-B4001OE-TAPKO、K562-HLA-B4601OE-TAPKO、K562-HLA-C0102OE-TAPKO和K562-HLA-C0702OE-TAPKO细胞的负载肽能力检测结果如图8所示。B4001细胞敲除TAP基因后,单克隆负载肽能力较未敲除的细胞有明显提升,但混合克隆经检测与未敲除的细胞差异不大,表明挑取单克隆的效果更优异。B4601、C0102、C0702细胞敲除TAP基因后单克隆细胞生长困难,使用混合克隆进行负载肽能力检测,结果与未敲除的细胞相比MFI ratio无明显差异,表明该型别的HLA可能不适用于此种方法进行抗原肽亲和力检测。
实施例4、对比K562-HLA-A0201OE-TAPKO细胞与T2细胞对不同A2型抗原肽的亲和力
利用K562-HLA-A0201OE-TAPKO细胞与T2细胞(人淋巴母细胞T2)分别负载5条不同的A2型抗原肽,孵育与检测条件均相同,比较相同条件下两者对不同的抗原肽序列的亲和力差异。
涉及试剂:
抗原肽名称 | 来源基因名称 | 抗原肽序列 | SEQ ID NO. | 预测亲和力(nM) |
G53 | GPC3 | FVGEFFTDV | 11 | 43.4 |
G54 | MAGE-A1 | KVLEYVIKV | 12 | 14.9 |
G63 | MAGE-C2 | FLAKLNNTV | 13 | 13.0 |
G65 | SSX2 | KASEKIFYV | 14 | 28.0 |
G66 | TERT | ILAKFLHWL | 15 | 14.7 |
操作步骤:
(1)试剂准备。根据抗原肽合成报告选择抗原肽溶解试剂(培养基),计算有效肽含量,有效肽含量=总含量×纯度×净氮含量,多肽以摩尔浓度负载。β2m原浓度为5mg/ml。
(2)取用适量待检测细胞,1ml无血清RPMI 1640培养基重悬细胞并计数。调整细胞浓度为1×106/ml,100μl/孔接种于48孔板中,即每孔细胞数量为1×105。加入β2m使其终浓度为2.5μg/ml,多肽浓度为30μM,总体系200μl,于二氧化碳培养箱中过夜孵育。
(3)流式检测。收集细胞到流式管中,350g离心5min,弃上清。2ml PBS清洗细胞2次,350g室温离心5min,弃上清,100μl PBS重悬细胞。每管中加入1μl PE anti-human HLA-A2,室温避光孵育20min,2ml PBS清洗细胞1次,350g室温离心5min,弃上清,200μl PBS重悬细胞。使用流式细胞仪检测待检测细胞表面HLA-A2分子表达水平。
(4)数据处理。荧光指数(MFI ratio)=(MFI加肽组-MFI不加肽组)/MFI不加肽组,分别绘制荧光指数曲线。
结果显示:
两种细胞对不同A2型抗原肽的亲和力检测和对比结果如图9所示。
T2细胞天然缺失TAP基因,但构建的K562-HLA-A0201OE-TAPKO细胞对GPC3、MAGE-A1、MAGE-C2、SSX2和TERT抗原肽的亲和力比T2细胞更优异,表明该细胞对A2型抗原肽检测的灵敏度更高,能够替代T2细胞进行抗原免疫原性验证。
以上内容是结合具体的优选实施方式对本发明所作的进一步详细说明,不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干简单推演或替换,都应当视为属于本发明的保护范围。
Claims (10)
1.一种灵敏检测目标肽免疫原性的方法,所述方法包括将目标肽与工程细胞相接触,所述工程细胞具有以下特征:
1)野生型工程细胞的表面缺乏HLA,
2)在工程细胞中过表达的HLA分子,所述HLA的型别与目标肽对应,
3)工程细胞中的TAP基因被敲除或TAP的表达被抑制;
优选地,所述过表达的HLA分子包括A0201、A1101、A2402、B4001、B4601、C0102、C0702;
优选地,所述方法中还包括免疫原性检测的步骤,所述免疫原性检测的方法包括流式细胞技术、酶联免疫吸附测定、放射免疫分析法、荧光免疫、化学发光免疫测定、电化学发光检测、表面等离子体共振检测法、全自动纳升级免疫分析工作站、酶联免疫斑点法、生物测定法。
2.如权利要求1所述方法,所述原始细胞表面缺乏HLA I类和/或HLA II类;
优选地,所述原始细胞是人类细胞;
优选地,所述原始细胞是人类细胞的细胞系;
优选地,所述原始细胞包括293细胞、HEK293细胞、Caco2细胞、U2-OS细胞、NIH 3T3细胞、NSO细胞、SP2细胞、DG44细胞、K562细胞、U-937细胞、MRC5细胞、IMR90细胞、Jurkat细胞、HepG2细胞、HeLa细胞、HT-1080细胞、HCT-116细胞、Hu-h7细胞、Huvec细胞、Molt 4细胞;
优选地,所述原始细胞是K562细胞。
3.如权利要求1所述方法,所述TAP基因被敲除是TAP1和/或TAP2被敲除,优选,TAP1和TAP2同时被敲除;
优选地,所述敲除的方法包括CRISPR技术、RNA干扰技术、反义寡核苷酸技术、TALEN技术、ZFN技术、Cre-loxP基因重组技术等基因编辑技术所使用的试剂;
优选地,所述敲除的方法是CRISPR技术。
4.一种工程细胞的制备方法,所述方法包括过表达HLA和敲除TAP基因的步骤;
优选地,所述工程细胞是权利要求1所述工程细胞。
5.如权利要求4所述制备方法,所述过表达包括提高所述HLA的瞬时表达量或稳定表达量;
优选地,提高所述稳定表达量包括通过使用转座子、病毒载体、重组酶或基因编辑技术将目标序列整合到宿主基因组实现
优选地,所述病毒载体包括慢病毒载体、痘病毒载体、单纯疱疹病毒载体、腺病毒载体、腺相关病毒载体;
优选地,所述病毒载体是慢病毒载体;
优选地,所述瞬时表达是通过转染载体实现的;
优选地,所述转染的方法包括物理方法、化学方法;
优选地,所述物理方法包括磷酸钙沉淀、脂质转染法、粒子轰击、微注射、电穿孔;
优选地,所述化学方法包括胶体分散系统、基于脂质的系统。
6.如权利要求4所述制备方法,所述敲除TAP基因的方法包括CRISPR技术、RNA干扰技术、反义寡核苷酸技术、TALEN技术、ZFN技术、Cre-loxP基因重组技术等基因编辑技术所使用的试剂;
优选地,所述敲除TAP基因的方法是CRISPR技术;
优选地,所述CRISPR技术敲除TAP基因所使用的试剂包括Cas蛋白成分和sgRNA成分;
优选地,所述敲除TAP基因是通过Cas蛋白成分和sgRNA成分共同转入细胞实现的,或者,是通过将编码Cas蛋白成分和sgRNA成分的核苷酸构建于载体中并转化细胞实现的;
优选地,所述转入的方法包括电转、磷酸钙沉淀、脂质转染法、粒子轰击、微注射;
优选地,所述转入的方法是电转。
7.如权利要求4所述制备方法,所述敲除TAP是敲除TAP1和/或TAP2,优选,同时敲除TAP1和TAP2;
优选地,所述敲除TAP是同时靶向SEQ ID NO.1和SEQ ID NO.2所示位点进行CRISPR编辑;
优选地,所述敲除TAP基因是TAP的表达降低到原始水平的90%、80%、70%、60%、50%、40%、30%、20%、10%或更低。
8.如权利要求4所述制备方法,所述Cas蛋白包括SpCas9、SaCas9及其突变体。
9.权利要求4所述制备方法所制备得到的工程细胞。
10.权利要求9所述工程细胞在检测目标肽免疫原性的应用;
优选地,所述目标肽与其中过表达HLA型别相对应。
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WO2021251271A1 (ja) * | 2020-06-09 | 2021-12-16 | 帝人株式会社 | Mhc-クラスi発現が抑制された細胞 |
WO2023239736A1 (en) * | 2022-06-09 | 2023-12-14 | Amgen Inc. | Methods of screening for peptide-hla class i alloreactivity |
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WO2021251271A1 (ja) * | 2020-06-09 | 2021-12-16 | 帝人株式会社 | Mhc-クラスi発現が抑制された細胞 |
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