CN110687298A - 一种以Chitosan多糖为骨架制备MHC抗原肽多聚体检测试剂的新方法 - Google Patents
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Abstract
抗原特异T细胞是免疫应答中的主要参与细胞，它参与免疫效应和调节免疫反应的进程。临床上在自身免疫疾病，抗肿瘤免疫反应，过敏变态反应等疾病过程中，抗原特异T细胞是重要的病理指标，可以与自身抗体同时检测，在某些自身抗体不明确的特殊疾病中更是最直接相关的免疫反应指标。本专利设计通过以多糖chitosan为骨架，标记上NTA‑Ni镍离子，然后用基因重组方法合成带有Histidine的MHC class I重链，与β2m结合后，装上抗原表位肽(epitope peptide)形成复合体。将MHC/peptide复合体与骨架连接，由于一个NTA只结合2个位点，所以NTA‑Ni上有2个空的Histidine结合位置，一个骨架分子上可以连接20‑30个MHC/peptide复合体分子。定量快速检测抗原特异T细胞的数量，也可以用于体外分离抗原特异T细胞。具有检测灵敏度高，结合力强,背景低的特点。
Description
技术领域
生物医药技术领域。
背景技术
抗原特异T细胞是免疫应答中的主要参与细胞，它参与免疫效应和调节免疫反应的进程，检测抗原特异T细胞是反应特异性免疫应答的最有效指标方法。临床上在自身免疫疾病，抗肿瘤免疫反应，过敏变态反应等疾病过程中，抗原特异T细胞是重要的病理指标，可以与自身抗体同时检测，在某些自身抗体不明确的特殊疾病中更是最直接相关的免疫反应指标。抗原特异T细胞的检测不能用纯抗原分子，因为T细胞上面的T细胞受体（TCR）只识别MHC/peptide复合体，不能识别单个纯抗原。以前检测抗原特异T细胞的方法包括CTL杀伤试验，有限稀释法，细胞内细胞因子染色，ELISPOT, 这些方法都有局限性，都不是直接观察抗原特异T细胞，只是通过抗原特异T细胞间接的效应来评估。用MHC/peptide多聚体检测抗原特异T细胞是在1996年由美国斯坦福大学医学院首次描述，目前检测抗原特异T细胞的方法是用MHC/peptide多聚体，有四聚体（tetramer），五聚体(pentamer)和长链的dextramer多聚体。每一个四聚体和五聚体分子中分别含有四个和五个MHC/peptide复合体，可以分别结合四个和五个T细胞受体，对于检测血液中含量低的抗原特异T细胞，灵敏度受到局限。Dextramer含有8-10个MHC/peptide复合体提高了灵敏度，但是试剂稳定性也受到挑战，试剂有效期不是很长。由于单个TCR和MHC/peptide多聚体的结合比较弱，所以结合越多的TCR能够显著提高检测的灵敏度。进一步说，由于抗原特异T细胞一般在血液中数量很少，所以足够的检测灵敏度是检测成功的关键。
发明内容
本专利设计通过以多糖chitosan为骨架，标记上NTA-Ni镍离子，然后用基因重组方法合成带有Histidine的MHC class I重链，与β2m结合后，装上抗原表位肽(epitopepeptide)形成复合体。将MHC/peptide复合体与骨架连接，由于一个NTA只结合2个位点，所以NTA-Ni上有2个空的Histidine结合位置，一个骨架分子上可以连接20-30个MHC/peptide复合体分子，显著提高了检测灵敏度。Chitosan是稳定的糖分子，镍离子的螯合作用强，以此方法制备的试剂比较稳定。本专利与现有MHC/peptide多聚体试剂有根本的不同，它第一次利用chitosan为骨架，以金属镍离子螯合为原理，将带有His标记的hisMHC/peptide装上骨架，制备高效价的MHC/peptide多聚体检测试剂。
本专利设计利用天然chitin binding protein (CBP)为配体，在配体蛋白上标记各种荧光素（flurochrome）,使用荧光素标记配体用流式细胞仪来显示被hisMHC/peptide多聚体结合的抗原特异T细胞。CBP和chitosan的结合非常特异，与其它以biotin-streptavidin为原理的结合相比，非特异背景染色低。另外，两者的结合力强，不容易解离。
本专利以hisMHC/peptide多聚体和荧光素标记CBP组成抗原特异T细胞检测试剂，根据此基本原理可以制备各种MHC/peptide多聚体，比如各种不同MHC class I分子（如HLAA*0201，A*1101, A*2402，HLA B*5801, B*5201等），以及装配各种抗原多肽（如CMV表位多肽，EBV表位多肽，HCV表位多肽，HBV表位多肽等），此方法是一种制备检测抗原特异T细胞的MHC/peptide多聚体试剂的新方法。
图1是NTA-Ni和His-Tag结合示意图。NTA分子通过螯合结合镍离子后，一个二价镍离子分子还可以结合二个Histidine分子。
图2是Chitosan和N-Succinyl Chitosan分子结构图。N-Succinyl Chitosan的溶解度高于Chitosan，有着更广泛的应用。
图3是hisMHC/peptide多聚体结构示意图。利用基因重组蛋白表达含His-Tag的MHC Class I重链，His-Tag结合在C端。β2-microglobulin在纯化重组蛋白后通过蛋白复性过程加上。
图4是hisMHC/peptide多聚体结合在N-Succinyl Chitosan多糖骨架上，多糖通过NTA-Ni预处理过，荧光素标记CBP结合在Chitosan多糖骨架上的示意图。整个分子通过用在流式细胞染色，来检测血液中抗原特异T细胞。其中MHC Class I 分子和多肽分子可以根据所需要检测的抗原特异T细胞和个体MHC表型来替换。CBP分子是蛋白质，可以标记各种荧光素分子，例如FITC，PE， PE-Cy5.5, Percp, APC, APC-Cy7, Percp-Cy5.5, PE-Cy7等等。本发明，首次利用Chitosan多糖分子为骨架，通过二价镍离子介导，结合Histidine修饰的MHC分子，用荧光素标记CBP，制备检测抗原特异T细胞的MHC多聚体试剂，与现有MHC多聚体试剂相比，具有灵敏度高，结合力强，稳定性好等特点。
发明创造的优点或有益效果
与现有技术相比，本发明提供了一个新的MHC多聚体试剂，用于检测血液中抗原特异T细胞的数量。基于长链多糖骨架，每个骨架分子至少可以结合20-30个hisMHC/peptide多聚体，比传统四聚体多5-6倍，所以Chitosan-Ni-NTA-HisMHC/peptide多聚体具有检测灵敏度高，结合力强，因为使用了荧光素标记的CBP来着色，染色背景低，特异性高。而传统的生物素和链霉亲和素系统，由于内源性生物素的存在，总是存在一定的背景染色。用本发明原理制备的Chitosan-Ni-NTA-HisMHC/peptide多聚体可以用在临床检测中，定量快速检测抗原特异T细胞的数量，也可以用于体外分离抗原特异T细胞。
具体实施例及附图
取2例个体PBMC（3x106）分别用10% FCS 1640 悬浮在6孔细胞培养板，加入30ug/mlCMV多肽pp65(NLVPMVATV)刺激48小时后，用HLA A*0201/pp65(NLVPMVATV)和PE标记CBP染色，用流式细胞仪分析察看被染色的CMV pp65抗原特异T细胞的含量，图5是使用本发明和PE-CBP检测特异性抗原T细胞流式结果分析图。
结果显示，当取CD8+细胞gate分析PE的荧光强度，两个个体的抗原刺激后PBMC分别含有5.57%和4.8%的CMV pp65抗原特异T细胞，显示用这个发明原理制备的HLA A*0201/pp65(NLVPMVATV)和PE标记CBP可以检测抗原特异T细胞。
图6是使用本发明检测抗原特异性T细胞流程图。检测流程如下：
1)取静脉血1mL,加入1mL pbs 混匀后，加入等体积的淋巴细胞分离液。
2)400g/分钟，离心20分钟，取环状乳白色淋巴细胞层。
3)加入2mL pbs，清洗细胞，300g/分钟，离心5分钟。弃上清。
4)将细胞重悬于50μL pbs中，加入适量的Chitosan-Ni-NTA-HisMHC/peptide多聚体，轻轻混匀后，室温孵育30分钟。
5)加入2mL pbs了qu缓冲液，置于离心机中，300g/分钟，离心5分钟。弃上清。
6) 将细胞重悬于50μL pbs中，加入适量荧光标记的CBP和抗CD8荧光标记抗体，轻轻混匀，室温避光孵育10分钟。
7)加入2mL pbs了qu缓冲液，置于离心机中，300g/分钟，离心5分钟。弃上清。重复一次。
8)将细胞重悬于400μL 缓冲液中，流式上机。
Claims (4)
1.一种以Chitosan长糖链为骨架，用nitroltriacetic acid (NTA)处理后，螯合二价镍离子（Ni2+）形成用以结合Histidine标记的MHC Class I/多肽表位复合体Chitosan-Ni-NTA。
2.一种HisMHC/peptide多聚体通过上面的Hi-tag与Chitosan-Ni-NTA-HisMHC/peptide形成聚合体检测试剂。
3.一种将各种荧光素（例如FITC，PE，APC, Percp，PE-Cy7，APC-Cy7，PE-Cy5.5, Percp-Cy5.5等）标记来检测Chitosan-Ni-NTA-HisMHC/peptide聚合体的chitin bindingprotein（CBP）。
4.一种使用上述二种试剂（Chitosan-Ni-NTA-HisMHC/peptide和荧光素标记CBP）来检测人和动物血液中抗原特异T细胞的检测方法（包括流式细胞染色，免疫荧光染色等等）。
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