CN109609533B - Construction and application of CAR lentiviral expression vector based on humanized CD276 antibody - Google Patents
- ️Fri Jul 10 2020
技术领域technical field
本发明涉及CAR慢病毒表达载体的技术领域，具体领域为基于人源化CD276抗体的CAR慢病毒表达载体构建。The present invention relates to the technical field of CAR lentiviral expression vectors, and the specific field is the construction of CAR lentiviral expression vectors based on humanized CD276 antibody.
背景技术Background technique
嵌合抗原受体(CAR)是一种人工合成的结构。利用转基因技术，将CAR序列转导入T细胞中，能够产生识别特异靶点、杀伤肿瘤细胞的CAR-T细胞。CAR-T细胞在血液系统肿瘤中取得了良好的治疗效果，但在实体瘤中效果不佳。A chimeric antigen receptor (CAR) is a man-made structure. Using transgenic technology, the CAR sequence is transferred into T cells, which can generate CAR-T cells that recognize specific targets and kill tumor cells. CAR-T cells have achieved good therapeutic effects in hematological tumors, but not in solid tumors.
CAR-T细胞治疗实体瘤面临的主要技术问题包括：The main technical issues facing CAR-T cell therapy for solid tumors include:
1、靶点抗原的表达是否广泛；2、CAR序列是否具有较强免疫原性，导致免疫排斥；3、CAR是否能将识别信号有效传递给T细胞。1. Whether the target antigen is widely expressed; 2. Whether the CAR sequence has strong immunogenicity, leading to immune rejection; 3. Whether the CAR can effectively transmit the recognition signal to T cells.
发明内容SUMMARY OF THE INVENTION
本发明的目的在于提供基于人源化CD276抗体的CAR慢病毒表达载体构建及其应用，以解决现有技术中CAR-T细胞治疗的问题。The purpose of the present invention is to provide the construction of CAR lentiviral expression vector based on humanized CD276 antibody and its application, so as to solve the problem of CAR-T cell therapy in the prior art.
CD276是在肿瘤中广泛表达，是较好的CAR-T细胞治疗靶点。本发明通过构建包含人源化CD276特异单链抗体(scFv)、CD8信号传导片段、以及CD28或41BB刺激信号分子的CAR序列，制备了靶向CD276的CAR(CD276-28z-CAR或CD276-BBz-CAR)表达质粒与CAR-T细胞。CD276 is widely expressed in tumors and is a good target for CAR-T cell therapy. The present invention prepares a CAR targeting CD276 (CD276-28z-CAR or CD276-BBz) by constructing a CAR sequence comprising a humanized CD276-specific single-chain antibody (scFv), a CD8 signal transduction fragment, and a CD28 or 41BB stimulation signal molecule -CAR) expression plasmid and CAR-T cells.
CAR主要由3部分构成：胞外识别区、信号转导区和胞内信号区。其中，胞外识别区决定了CAR-T细胞杀伤的特异性，往往由scFv序列构成，但很多scFv序列是小鼠来源的，会造成较强的免疫排斥反应，导致CAR-T细胞治疗无效。而本发明选择了高亲和性识别CD276的人源化改造的scFv序列(anti-CD276scFv)，既能够有效避免免疫排斥，又能够高效识别靶细胞。胞内信号区也是决定CAR-T细胞治疗效果的重要结构，通过加入共刺激信号分子(CD28或41BB)，保证CAR-T细胞具备较强的杀伤功能与扩增能力。此外，信号转导区的结构选择也影响CAR-T细胞功能。利用CD8铰链区保证胞外识别区与信号区之间有了较好结合，保证了CAR-T细胞的功能。CAR is mainly composed of three parts: extracellular recognition domain, signal transduction domain and intracellular signal domain. Among them, the extracellular recognition region determines the specificity of CAR-T cell killing and is often composed of scFv sequences. However, many scFv sequences are derived from mice, which will cause strong immune rejection and make CAR-T cell therapy ineffective. The present invention selects a humanized scFv sequence (anti-CD276 scFv) that recognizes CD276 with high affinity, which can effectively avoid immune rejection and efficiently recognize target cells. The intracellular signal region is also an important structure that determines the therapeutic effect of CAR-T cells. By adding costimulatory signal molecules (CD28 or 41BB), CAR-T cells have strong killing function and expansion ability. In addition, the structural choice of the signal transduction region also affects CAR-T cell function. The CD8 hinge region is used to ensure a good combination between the extracellular recognition region and the signal region, which ensures the function of CAR-T cells.
为提高CAR-T细胞治疗效果，针对现有CAR结构缺陷，本发明对3个主要结构进行了优化，其技术方案具体如下：In order to improve the therapeutic effect of CAR-T cells, in view of the structural defects of the existing CAR, the present invention optimizes three main structures, and the technical solutions are as follows:
基于人源化CD276抗体的CAR慢病毒表达载体构建方法，包括以下步骤：(1)CAR结构优化；(2)CAR序列合成及载体构建；The construction method of CAR lentiviral expression vector based on humanized CD276 antibody includes the following steps: (1) CAR structure optimization; (2) CAR sequence synthesis and vector construction;
其中，步骤(1)优化后的CAR结构为CD276-28z-CAR结构或者CD276-BBz-CAR结构；CD276-28z-CAR编码蛋白序列如SEQ ID No:1所示；CD276-BBz-CAR编码蛋白序列如SEQ IDNo:7所示。Wherein, the optimized CAR structure in step (1) is the CD276-28z-CAR structure or the CD276-BBz-CAR structure; the CD276-28z-CAR encoded protein sequence is shown in SEQ ID No: 1; the CD276-BBz-CAR encoded protein The sequence is shown in SEQ ID No:7.
本发明所述的基于人源化CD276抗体的CAR慢病毒表达载体构建方法，其特征在于：所述CD276-28z-CAR结构包括人CD8a分子信号肽、人源化CD276单链抗体、人CD8a分子柔性片段、人CD28分子跨膜区与胞内区和人CD3z分子胞内区；The method for constructing a humanized CD276 antibody-based CAR lentiviral expression vector according to the present invention is characterized in that: the CD276-28z-CAR structure includes human CD8a molecule signal peptide, humanized CD276 single-chain antibody, human CD8a molecule Flexible fragment, transmembrane region and intracellular region of human CD28 molecule and intracellular region of human CD3z molecule;
其中，人CD8a分子信号肽序列如SEQ ID No:2所示；人源化CD276单链抗体序列如SEQ ID No:3所示；人CD8a分子柔性片段序列如SEQ ID No:4所示；人CD28分子跨膜区与胞内区序列如SEQ ID No:5所示；人CD3z分子胞内区序列如SEQ ID No:6所示。Among them, the sequence of human CD8a molecule signal peptide is shown in SEQ ID No: 2; the sequence of humanized CD276 single chain antibody is shown in SEQ ID No: 3; the sequence of human CD8a molecule flexible fragment is shown in SEQ ID No: 4; The sequence of the transmembrane region and the intracellular region of the CD28 molecule is shown in SEQ ID No: 5; the sequence of the intracellular region of the human CD3z molecule is shown in SEQ ID No: 6.
本发明所述的基于人源化CD276抗体的CAR慢病毒表达载体构建方法，其特征在于：所述CD276-BBz-CAR结构包括人CD8a分子信号肽、人源化CD276单链抗体、人CD8a分子柔性片段与跨膜区、人41BB分子胞内区和人CD3z分子胞内区；The humanized CD276 antibody-based CAR lentiviral expression vector construction method of the present invention is characterized in that: the CD276-BBz-CAR structure includes human CD8a molecule signal peptide, humanized CD276 single-chain antibody, human CD8a molecule Flexible fragment and transmembrane region, intracellular region of human 41BB molecule and intracellular region of human CD3z molecule;
其中，人CD8a分子信号肽序列如SEQ ID No:8所示；人源化CD276单链抗体如SEQID No:9所示；人CD8a分子柔性片段与跨膜区序列如SEQ ID No:10所示；人41BB分子胞内区序列如SEQ ID No:11所示；人CD3z分子胞内区序列如SEQ ID No:12所示。Among them, the signal peptide sequence of human CD8a molecule is shown in SEQ ID No: 8; the humanized CD276 single-chain antibody is shown in SEQ ID No: 9; the flexible fragment and transmembrane region sequence of human CD8a molecule is shown in SEQ ID No: 10 ; Human 41BB molecule intracellular region sequence is shown in SEQ ID No: 11; Human CD3z molecule intracellular region sequence is shown in SEQ ID No: 12.
本发明所述的基于人源化CD276抗体的CAR慢病毒表达载体构建方法，其特征在于，所述步骤(2)具体包括：合成CAR编码序列，将获得的DNA序列通过酶切连接插入到pCDH-EF1-MSC质粒中，构建慢病毒表达质粒pCDH-EF1-CAR-CD276-28z或pCDH-EF1-CAR-CD276-BBz。The method for constructing a CAR lentiviral expression vector based on a humanized CD276 antibody according to the present invention is characterized in that, the step (2) specifically includes: synthesizing the CAR coding sequence, and inserting the obtained DNA sequence into pCDH by enzymatic ligation - EF1-MSC plasmid, construct lentiviral expression plasmid pCDH-EF1-CAR-CD276-28z or pCDH-EF1-CAR-CD276-BBz.
本发明所述的CAR慢病毒表达载体构建方法获得的基于人源化CD276抗体的CAR慢病毒表达载体。The CAR lentiviral expression vector based on the humanized CD276 antibody obtained by the method for constructing the CAR lentiviral expression vector of the present invention.
采用本发明所述的基于人源化CD276抗体的CAR慢病毒表达载体制备CAR-T细胞，其方法包括以下步骤：(1)慢病毒包装；(2)T细胞纯化与感染；(3)T细胞CAR表达效率检测；(4)CAR-T细胞体外扩增；Using the humanized CD276 antibody-based CAR lentiviral expression vector of the present invention to prepare CAR-T cells, the method includes the following steps: (1) lentiviral packaging; (2) T cell purification and infection; (3) T cells Cell CAR expression efficiency detection; (4) CAR-T cell expansion in vitro;
其中，步骤(1)具体为，包装细胞293T铺板24小时后，将pCDH-EF1-CAR-CD276-28z或pCDH-EF1-CAR-CD276-BBz表达质粒与包装质粒混合，利用磷酸钙转染试剂进行293T细胞转染；转染48小时后，收集上清，准备用于T细胞感染。Wherein, step (1) is as follows: 24 hours after the packaging cells are plated with 293T, the pCDH-EF1-CAR-CD276-28z or pCDH-EF1-CAR-CD276-BBz expression plasmid is mixed with the packaging plasmid, and calcium phosphate transfection reagent is used. 293T cell transfection was performed; 48 hours after transfection, the supernatant was collected and prepared for T cell infection.
本发明所述的制备CAR-T细胞的方法，其步骤(2)具体为，人外周血经密度梯度离心后，分离外周血单个核细胞；利用T细胞分离试剂盒获得纯化的CD3+T细胞，再按照2个细胞加入1个磁珠的比例，加入适量CD3/CD28磁珠活化2天；2天后，加入病毒上清与polybrene孵育过夜；次日，离心清洗T细胞3次后，加入含1000U IL-2与5％胎牛血清的RPMI1640培养基扩增T细胞。In the method for preparing CAR-T cells according to the present invention, step (2) is specifically as follows: after human peripheral blood is subjected to density gradient centrifugation, peripheral blood mononuclear cells are separated; purified CD3 + T cells are obtained by using a T cell separation kit , and then add an appropriate amount of CD3/CD28 magnetic beads according to the ratio of 2 cells to 1 magnetic bead for activation for 2 days; after 2 days, add the virus supernatant and incubate with polybrene overnight; the next day, after centrifuging and washing the T cells 3 times, add T cells were expanded in RPMI1640 medium with 1000 U IL-2 and 5% fetal bovine serum.
本发明所述的制备CAR-T细胞的方法，其步骤(3)具体为，T细胞感染3天后，利用流式细胞术对T细胞表面CAR的表达情况进行检测。In the method for preparing CAR-T cells according to the present invention, step (3) is specifically as follows: 3 days after the infection of the T cells, flow cytometry is used to detect the expression of CAR on the surface of the T cells.
本发明所述的制备CAR-T细胞的方法，其步骤(4)具体为，计数1×106个T细胞进行感染，并在感染后3，7，10和14天时进行计数，检测CAR-T细胞增殖情况。In the method for preparing CAR-T cells according to the present invention, the step (4) is specifically: counting 1×10 6 T cells for infection, and counting them at 3, 7, 10 and 14 days after infection, and detecting CAR-T cells. T cell proliferation.
与现有技术相比，本发明的有益效果是：抗原表达强度与CAR-T细胞治疗效果紧密相关。本发明以肿瘤广泛高表达的CD276为靶点进行CAR-T细胞治疗，结果显示该抗原是较好的肿瘤治疗靶点。另外，本发明采用了人源化的抗CD276scFv作为CAR-T细胞识别区，可以有效避免免疫排斥发生，有助于治疗效果改善。通过添加胞内刺激信号(CD28或41BB)，能够改善T细胞功能。此类转基因修饰的CAR-T细胞具备更强、更持久的杀伤能力，有望提高肿瘤治疗效果。Compared with the prior art, the beneficial effect of the present invention is that the intensity of antigen expression is closely related to the therapeutic effect of CAR-T cells. The present invention uses CD276, which is widely and highly expressed in tumors, as a target for CAR-T cell therapy, and the results show that the antigen is a better tumor therapy target. In addition, the present invention adopts the humanized anti-CD276 scFv as the CAR-T cell recognition region, which can effectively avoid the occurrence of immune rejection and help to improve the therapeutic effect. By adding intracellular stimulatory signals (CD28 or 41BB), T cell function can be improved. Such genetically modified CAR-T cells have stronger and longer-lasting killing ability, which is expected to improve the effect of tumor treatment.
附图说明Description of drawings
图1为CD276-28z-CAR结构示意图；Figure 1 is a schematic diagram of the structure of CD276-28z-CAR;
图2为CD276-BBz-CAR结构示意图；Figure 2 is a schematic diagram of the structure of CD276-BBz-CAR;
图3为流式细胞术检测T细胞表达CAR表达；Figure 3 is flow cytometry detection of T cells expressing CAR expression;
图4为CAR-T细胞增殖情况；Figure 4 shows the proliferation of CAR-T cells;
图5说明CAR-T细胞能够特异杀伤CD276阳性细胞；Figure 5 shows that CAR-T cells can specifically kill CD276 positive cells;
图6说明以CD276为靶点的CAR-T细胞的肿瘤杀伤效果更好；Figure 6 shows that the tumor killing effect of CAR-T cells targeting CD276 is better;
图7说明CAR-T细胞能够控制肿瘤生长。Figure 7 illustrates that CAR-T cells can control tumor growth.
具体实施方式Detailed ways
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.
1.为提高CAR-T细胞治疗效果，针对现有CAR结构缺陷，对3个主要结构进行了优化。具体包括CD276-28z-CAR结构和CD276-BBz-CAR结构。1. In order to improve the therapeutic effect of CAR-T cells, three main structures have been optimized for the existing structural defects of CAR. Specifically, it includes CD276-28z-CAR structure and CD276-BBz-CAR structure.
(1)CD276-28z-CAR结构(1) CD276-28z-CAR structure
如图1所示，CAR基本结构包括：人CD8a分子信号肽(Leading signal)、人源化CD276单链抗体(scFv)、人CD8a分子柔性片段(CD8Hinge)、人CD28分子跨膜区与胞内区(CD28)，人CD3z分子胞内区(CD3z)。As shown in Figure 1, the basic structure of CAR includes: human CD8a molecule signal peptide (Leading signal), humanized CD276 single chain antibody (scFv), human CD8a molecule flexible fragment (CD8Hinge), human CD28 molecule transmembrane region and intracellular domain (CD28), the intracellular domain (CD3z) of the human CD3z molecule.
CD276-28z-CAR编码蛋白序列如下：(SEQ ID No:1所示)The protein sequence encoded by CD276-28z-CAR is as follows: (SEQ ID No: 1)
MALPVTALLLPLALLLHAARPGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSDSSAIYYADTVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARGRENIYYGSRLDYWGQGTTVTVSSGSTSGSGKPGSGEGSTKGDIQLTQSPSFLSASVGDRVTITCKASQNVDTNVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNNYPFTFGQGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*MALPVTALLLPLALLLHAARPGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSDSSAIYYADTVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARGRENIYYGSRLDYWGQGTTVTVSSGSTSGSGKPGSGEGSTKGDIQLTQSPSFLSASVGDRVTITCKASQNVDTNVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNNYPFTFGQGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*
其中，in,
人CD8a分子信号肽(Leading signal)序列：(SEQ ID No:2所示)Human CD8a molecule signal peptide (Leading signal) sequence: (SEQ ID No: 2)
MALPVTALLLPLALLLHAARPGSMALPVTALLLPLALLLHAARPGS
人源化CD276单链抗体(Humanized scFv)序列：(SEQ ID No:3所示)Humanized CD276 single chain antibody (Humanized scFv) sequence: (shown in SEQ ID No: 3)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSDSSAIYYADTVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARGRENIYYGSRLDYWGQGTTVTVSSGSTSGSGKPGSGEGSTKGDIQLTQSPSFLSASVGDRVTITCKASQNVDTNVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNNYPFTFGQGTKLEIKEVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSDSSAIYYADTVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARGRENIYYGSRLDYWGQGTTVTVSSGSTSGSGKPGSGEGSTKGDIQLTQSPSFLSASVGDRVTITCKASQNVDTNVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGTTFTLGTEIQPEDFATKYYNNY
人CD8a分子柔性片段(CD8Hinge)序列：(SEQ ID No:4所示)Human CD8a molecule flexible fragment (CD8Hinge) sequence: (shown in SEQ ID No: 4)
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD
人CD28分子跨膜区与胞内区(CD28)序列：(SEQ ID No:5所示)Human CD28 molecule transmembrane region and intracellular region (CD28) sequence: (SEQ ID No: 5)
FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS
人CD3z分子胞内区(CD3z)序列：(SEQ ID No:6所示)Human CD3z molecular intracellular region (CD3z) sequence: (shown in SEQ ID No: 6)
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
(2)CD276-BBz-CAR结构(2) Structure of CD276-BBz-CAR
如图2所示，CAR基本结构包括：人CD8a分子信号肽(Leading signal)、人源化CD276单链抗体(scFv)、人CD8a分子柔性片段与跨膜区(CD8Hinge TM)、人41BB分子胞内区(41BB)，人CD3z分子胞内区(CD3z)。As shown in Figure 2, the basic structure of CAR includes: human CD8a molecule signal peptide (Leading signal), humanized CD276 single-chain antibody (scFv), human CD8a molecule flexible fragment and transmembrane region (CD8Hinge TM), human 41BB molecule cell Intracellular domain (41BB), human CD3z molecule intracellular domain (CD3z).
CD276-BBz-CAR编码蛋白序列如下：(SEQ ID No:7所示)The protein sequence encoded by CD276-BBz-CAR is as follows: (shown in SEQ ID No: 7)
MALPVTALLLPLALLLHAARPGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSDSSAIYYADTVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARGRENIYYGSRLDYWGQGTTVTVSSGSTSGSGKPGSGEGSTKGDIQLTQSPSFLSASVGDRVTITCKASQNVDTNVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNNYPFTFGQGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*MALPVTALLLPLALLLHAARPGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSDSSAIYYADTVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARGRENIYYGSRLDYWGQGTTVTVSSGSTSGSGKPGSGEGSTKGDIQLTQSPSFLSASVGDRVTITCKASQNVDTNVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNNYPFTFGQGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*
其中，in,
人CD8a分子信号肽(Leadingsignal)序列：(SEQ ID No:8所示)Human CD8a molecule signal peptide (Leadingsignal) sequence: (shown in SEQ ID No: 8)
MALPVTALLLPLALLLHAARPGSMALPVTALLLPLALLLHAARPGS
人源化CD276单链抗体(Humanized scFv)序列：(SEQ ID No:9所示)Humanized CD276 single chain antibody (Humanized scFv) sequence: (shown in SEQ ID No: 9)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSDSSAIYYADTVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARGRENIYYGSRLDYWGQGTTVTVSSGSTSGSGKPGSGEGSTKGDIQLTQSPSFLSASVGDRVTITCKASQNVDTNVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNNYPFTFGQGTKLEIKEVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSDSSAIYYADTVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARGRENIYYGSRLDYWGQGTTVTVSSGSTSGSGKPGSGEGSTKGDIQLTQSPSFLSASVGDRVTITCKASQNVDTNVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGTTFTLGTEIQPEDFATKYYNNY
人CD8a分子柔性片段与跨膜区(CD8Hinge TM)序列：(SEQ ID No:10所示)Human CD8a molecule flexible fragment and transmembrane region (CD8Hinge TM) sequence: (SEQ ID No: 10)
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVIT
人41BB分子胞内区(41BB)序列：(SEQ ID No:11所示)Human 41BB molecular intracellular region (41BB) sequence: (shown in SEQ ID No: 11)
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL
人CD3z分子胞内区(CD3z)序列：(SEQ ID No:12所示)Human CD3z molecule intracellular domain (CD3z) sequence: (shown in SEQ ID No: 12)
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
2.CAR序列合成及载体构建2. CAR sequence synthesis and vector construction
CAR编码序列由上海生工公司合成，获得的DNA序列通过酶切连接插入到pCDH-EF1-MSC质粒中，构建慢病毒表达质粒pCDH-EF1-CAR-CD276-28z或pCDH-EF1-CAR-CD276-BBz。The CAR coding sequence was synthesized by Shanghai Sangong Company, and the obtained DNA sequence was inserted into the pCDH-EF1-MSC plasmid by restriction enzyme digestion to construct the lentiviral expression plasmid pCDH-EF1-CAR-CD276-28z or pCDH-EF1-CAR-CD276 -BBz.
3.慢病毒包装3. Lentiviral Packaging
包装细胞293T铺板24小时后，将pCDH-EF1-CAR-CD276-28z或pCDH-EF1-CAR-CD276-BBz表达质粒与包装质粒(psPAX2与pMD2.G)混合，利用磷酸钙转染试剂进行293T细胞转染。转染48小时后，收集上清，准备用于T细胞感染。24 hours after the packaging cells were plated with 293T, the pCDH-EF1-CAR-CD276-28z or pCDH-EF1-CAR-CD276-BBz expression plasmids were mixed with the packaging plasmids (psPAX2 and pMD2.G), and 293T was transfected with calcium phosphate transfection reagent. Cell transfection. Forty-eight hours after transfection, supernatants were collected and prepared for T cell infection.
4.T细胞纯化与感染4. T Cell Purification and Infection
人外周血经密度梯度离心后，分离外周血单个核细胞。利用德国美天旎公司的T细胞分离试剂盒获得纯化的CD3+T细胞，再按照2个细胞加入1个磁珠的比例，加入适量CD3/CD28磁珠活化2天。2天后，加入病毒上清与polybrene(6μg/mL)孵育过夜。次日，离心清洗T细胞3次后，加入含1000U IL-2与5％胎牛血清的RPMI1640培养基扩增T细胞。Human peripheral blood was subjected to density gradient centrifugation to separate peripheral blood mononuclear cells. Purified CD3 + T cells were obtained using the T cell isolation kit from Miltenyi Company, Germany, and then an appropriate amount of CD3/CD28 magnetic beads was added to activate for 2 days according to the ratio of 2 cells to 1 magnetic bead. After 2 days, viral supernatants were added and incubated with polybrene (6 μg/mL) overnight. The next day, after washing the T cells by centrifugation for 3 times, the RPMI1640 medium containing 1000 U IL-2 and 5% fetal bovine serum was added to expand the T cells.
5.T细胞CAR表达效率5. T cell CAR expression efficiency
T细胞感染3天后，利用流式细胞术对T细胞表面CAR的表达情况进行检测。结果显示CAR表达阳性率达到～60％-70％(如图3)，证明CAR表达质粒构建及病毒包装成功。After 3 days of T cell infection, flow cytometry was used to detect the expression of CAR on the surface of T cells. The results showed that the positive rate of CAR expression reached ~60%-70% (as shown in Figure 3), which proved that the CAR expression plasmid was constructed and the virus packaging was successful.
6.CAR-T细胞体外扩增6. In vitro expansion of CAR-T cells
计数1×106个T细胞进行感染，并在感染后3，7，10和14天时进行计数，检测CAR-T细胞增殖情况。如图4所示，CAR-T细胞均能够高效增殖(14天内扩增约50-60倍)，其中CAR-CD276-BBz细胞的增殖能力更好。1×10 6 T cells were counted for infection, and counted at 3, 7, 10 and 14 days after infection to detect the proliferation of CAR-T cells. As shown in Figure 4, all CAR-T cells can proliferate efficiently (about 50-60 times in 14 days), and CAR-CD276-BBz cells have better proliferative ability.
7.CAR-T细胞杀伤效果7. CAR-T cell killing effect
T细胞感染14天后，计数T细胞与靶细胞，并利用细胞染料(eFluor670)对靶细胞进行标记。然后按照效靶比(效应细胞:靶细胞，E:T)1:1，1:5，1:20的比例，将T细胞(效应细胞)与CD276高表达靶细胞(Calu-3、KYSE70和SW620)，CD276低表达靶细胞(A549和H322)或CD276阴性靶细胞(95D)共孵育6小时。共孵育结束后，离心收集细胞，利用凋亡染色试剂盒标记细胞，然后流式细胞术分析靶细胞凋亡情况(如图5)。结果显示，CAR-T细胞对表达CD276的肿瘤细胞具有很强的杀伤能力，而对CD276阴性细胞的影响很小，说明该CAR-T细胞具有很强的特异性杀伤能力。After 14 days of T cell infection, T cells and target cells were counted, and target cells were labeled with a cell dye (eFluor670). Then according to the ratio of effector to target (effector cells:target cells, E:T) 1:1, 1:5, 1:20, T cells (effector cells) and CD276 high-expressing target cells (Calu-3, KYSE70 and SW620), CD276 low-expressing target cells (A549 and H322) or CD276-negative target cells (95D) were incubated for 6 hours. After the co-incubation, the cells were collected by centrifugation, and the cells were labeled with an apoptosis staining kit, and then the apoptosis of the target cells was analyzed by flow cytometry (Figure 5). The results showed that CAR-T cells had strong killing ability to tumor cells expressing CD276, but had little effect on CD276-negative cells, indicating that the CAR-T cells had strong specific killing ability.
8.CD276特异CAR-T细胞具有较强杀伤能力8. CD276-specific CAR-T cells have strong killing ability
为了进一步验证CD276特异CAR-T细胞的杀伤能力，我们利用细胞活性实时检测系统观察了CAR-T细胞对靶细胞A549(既表达CD276又表达Her2)的杀伤。CAR-T细胞(CAR-CD276-28z，CAR-CD276-BBz或CAR-Her2-28z)按照E:T＝1:10的比例分别加入到靶细胞中，然后对靶细胞的活性进行了5天的连续观察(如图6)。结果显示，与Her2特异CAR-T细胞相比，CD276靶向CAR-T细胞的肿瘤杀伤效果更好，说明以CD276为靶点能够提高CAR-T细胞治疗效果。To further verify the killing ability of CD276-specific CAR-T cells, we observed the killing of target cells A549 (expressing both CD276 and Her2) by CAR-T cells using a cell viability real-time detection system. CAR-T cells (CAR-CD276-28z, CAR-CD276-BBz or CAR-Her2-28z) were added to the target cells at the ratio of E:T=1:10, and then the activity of the target cells was measured for 5 days continuous observation (Figure 6). The results show that compared with Her2-specific CAR-T cells, CD276-targeted CAR-T cells have better tumor killing effect, indicating that targeting CD276 can improve the therapeutic effect of CAR-T cells.
9.动物实验验证CAR-T细胞功能9. Animal experiments to verify the function of CAR-T cells
免疫缺陷小鼠皮下接种5×106个Calu-3细胞10天后，经尾静脉分别注射5×106个CAR-T细胞或T细胞进行治疗。治疗0，3，7，14天时，利用小动物活体成像设备，观察肿瘤大小。如图7所示，在CAR-T细胞注射后7天(A)，肿瘤体积明显缩小，而且随着治疗时间的延长，CAR-T细胞治疗组的肿瘤体积越来越小(B)，提示CAR-T细胞具有良好的肿瘤杀伤能力。Immunodeficient mice were subcutaneously inoculated with 5 × 10 6 Calu-3 cells for 10 days, and then were injected with 5 × 10 6 CAR-T cells or T cells through the tail vein for treatment. At 0, 3, 7, and 14 days of treatment, the tumor size was observed using a small animal in vivo imaging device. As shown in Figure 7, 7 days after CAR-T cell injection (A), the tumor volume was significantly reduced, and with the prolongation of treatment time, the tumor volume in the CAR-T cell treatment group became smaller and smaller (B), suggesting that CAR-T cells have good tumor killing ability.
由以上实验结果可知，抗原表达强度与CAR-T细胞治疗效果紧密相关。本发明以肿瘤广泛高表达的CD276为靶点进行CAR-T细胞治疗，结果显示该抗原是较好的肿瘤治疗靶点。另外，本发明采用了人源化的抗CD276scFv作为CAR-T细胞识别区，可以有效避免免疫排斥发生，有助于治疗效果改善。通过添加胞内刺激信号(CD28或41BB)，能够改善T细胞功能。此类转基因修饰的CAR-T细胞具备更强、更持久的杀伤能力，有望提高肿瘤治疗效果。It can be seen from the above experimental results that the intensity of antigen expression is closely related to the therapeutic effect of CAR-T cells. The present invention uses CD276, which is widely and highly expressed in tumors, as a target for CAR-T cell therapy, and the results show that the antigen is a better tumor therapy target. In addition, the present invention adopts the humanized anti-CD276 scFv as the CAR-T cell recognition region, which can effectively avoid the occurrence of immune rejection and help to improve the therapeutic effect. By adding intracellular stimulatory signals (CD28 or 41BB), T cell function can be improved. Such genetically modified CAR-T cells have stronger and longer-lasting killing ability, which is expected to improve the effect of tumor treatment.
尽管已经示出和描述了本发明的实施例，对于本领域的普通技术人员而言，可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由所附权利要求及其等同物限定。Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, and substitutions can be made in these embodiments without departing from the principle and spirit of the invention and modifications, the scope of the present invention is defined by the appended claims and their equivalents.
序列表sequence listing
<110> 郑州大学第一附属医院<110> The First Affiliated Hospital of Zhengzhou University
<120> 基于人源化CD276抗体的CAR慢病毒表达载体构建及其应用<120> Construction and application of CAR lentiviral expression vector based on humanized CD276 antibody
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<170> SIPOSequenceListing 1.0<170> SIPOSequenceListing 1.0
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<212> PRT<212> PRT
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
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Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu LeuMet Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 151 5 10 15
His Ala Ala Arg Pro Gly Ser Glu Val Gln Leu Val Glu Ser Gly GlyHis Ala Ala Arg Pro Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly
20 25 30 20 25 30
Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala SerGly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser
35 40 45 35 40 45
Gly Phe Thr Phe Ser Ser Phe Gly Met His Trp Val Arg Gln Ala ProGly Phe Thr Phe Ser Ser Phe Gly Met His Trp Val Arg Gln Ala Pro
50 55 60 50 55 60
Gly Lys Gly Leu Glu Trp Val Ala Tyr Ile Ser Ser Asp Ser Ser AlaGly Lys Gly Leu Glu Trp Val Ala Tyr Ile Ser Ser Asp Ser Ser Ala
65 70 75 8065 70 75 80
Ile Tyr Tyr Ala Asp Thr Val Lys Gly Arg Phe Thr Ile Ser Arg AspIle Tyr Tyr Ala Asp Thr Val Lys Gly Arg Phe Thr Ile Ser Arg Asp
85 90 95 85 90 95
Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Asp GluAsn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Asp Glu
100 105 110 100 105 110
Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Arg Glu Asn Ile Tyr TyrAsp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Arg Glu Asn Ile Tyr Tyr
115 120 125 115 120 125
Gly Ser Arg Leu Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val SerGly Ser Arg Leu Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser
130 135 140 130 135 140
Ser Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly SerSer Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser
145 150 155 160145 150 155 160
Thr Lys Gly Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser AlaThr Lys Gly Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala
165 170 175 165 170 175
Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn ValSer Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val
180 185 190 180 185 190
Asp Thr Asn Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro LysAsp Thr Asn Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
195 200 205 195 200 205
Leu Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Ser ArgLeu Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Ser Arg
210 215 220 210 215 220
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser SerPhe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
225 230 235 240225 230 235 240
Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn AsnLeu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Asn
245 250 255 245 250 255
Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Thr ThrTyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Thr Thr
260 265 270 260 265 270
Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser GlnThr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln
275 280 285 275 280 285
Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly AlaPro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala
290 295 300 290 295 300
Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp Val Leu ValVal His Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp Val Leu Val
305 310 315 320305 310 315 320
Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val AlaVal Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala
325 330 335 325 330 335
Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His SerPhe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser
340 345 350 340 345 350
Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys HisAsp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His
355 360 365 355 360 365
Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser ArgTyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg
370 375 380 370 375 380
Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly GlnVal Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln
385 390 395 400385 390 395 400
Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr AspAsn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp
405 410 415 405 410 415
Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys ProVal Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro
420 425 430 420 425 430
Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln LysGln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
435 440 445 435 440 445
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu ArgAsp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
450 455 460 450 455 460
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr AlaArg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
465 470 475 480465 470 475 480
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro ArgThr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
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<212> PRT<212> PRT
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
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Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu LeuMet Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 151 5 10 15
His Ala Ala Arg Pro Gly SerHis Ala Ala Arg Pro Gly Ser
20 20
<210> 3<210> 3
<211> 247<211> 247
<212> PRT<212> PRT
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly GlyGlu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 151 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser PheSer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe
20 25 30 20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp ValGly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 35 40 45
Ala Tyr Ile Ser Ser Asp Ser Ser Ala Ile Tyr Tyr Ala Asp Thr ValAla Tyr Ile Ser Ser Asp Ser Ser Ala Ile Tyr Tyr Ala Asp Thr Val
50 55 60 50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu TyrLys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 8065 70 75 80
Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr CysLeu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 85 90 95
Ala Arg Gly Arg Glu Asn Ile Tyr Tyr Gly Ser Arg Leu Asp Tyr TrpAla Arg Gly Arg Glu Asn Ile Tyr Tyr Gly Ser Arg Leu Asp Tyr Trp
100 105 110 100 105 110
Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Ser Thr Ser Gly SerGly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Ser Thr Ser Gly Ser
115 120 125 115 120 125
Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Asp Ile Gln LeuGly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Asp Ile Gln Leu
130 135 140 130 135 140
Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly Asp Arg Val ThrThr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly Asp Arg Val Thr
145 150 155 160145 150 155 160
Ile Thr Cys Lys Ala Ser Gln Asn Val Asp Thr Asn Val Ala Trp TyrIle Thr Cys Lys Ala Ser Gln Asn Val Asp Thr Asn Val Ala Trp Tyr
165 170 175 165 170 175
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala SerGln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser
180 185 190 180 185 190
Tyr Arg Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser GlyTyr Arg Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly
195 200 205 195 200 205
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe AlaThr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala
210 215 220 210 215 220
Thr Tyr Tyr Cys Gln Gln Tyr Asn Asn Tyr Pro Phe Thr Phe Gly GlnThr Tyr Tyr Cys Gln Gln Tyr Asn Asn Tyr Pro Phe Thr Phe Gly Gln
225 230 235 240225 230 235 240
Gly Thr Lys Leu Glu Ile LysGly Thr Lys Leu Glu Ile Lys
245 245
<210> 4<210> 4
<211> 45<211> 45
<212> PRT<212> PRT
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
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Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile AlaThr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
1 5 10 151 5 10 15
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala GlySer Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
20 25 30 20 25 30
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys AspGly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
35 40 45 35 40 45
<210> 5<210> 5
<211> 68<211> 68
<212> PRT<212> PRT
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 5<400> 5
Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser LeuPhe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu
1 5 10 151 5 10 15
Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg SerLeu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser
20 25 30 20 25 30
Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro GlyArg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly
35 40 45 35 40 45
Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe AlaPro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala
50 55 60 50 55 60
Ala Tyr Arg SerAla Tyr Arg Ser
6565
<210> 6<210> 6
<211> 113<211> 113
<212> PRT<212> PRT
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 6<400> 6
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln GlyArg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly
1 5 10 151 5 10 15
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu TyrGln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
20 25 30 20 25 30
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly LysAsp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
35 40 45 35 40 45
Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu GlnPro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln
50 55 60 50 55 60
Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly GluLys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu
65 70 75 8065 70 75 80
Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser ThrArg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr
85 90 95 85 90 95
Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro ProAla Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro
100 105 110 100 105 110
ArgArg
<210> 7<210> 7
<211> 491<211> 491
<212> PRT<212> PRT
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 7<400> 7
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu LeuMet Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 151 5 10 15
His Ala Ala Arg Pro Gly Ser Glu Val Gln Leu Val Glu Ser Gly GlyHis Ala Ala Arg Pro Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly
20 25 30 20 25 30
Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala SerGly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser
35 40 45 35 40 45
Gly Phe Thr Phe Ser Ser Phe Gly Met His Trp Val Arg Gln Ala ProGly Phe Thr Phe Ser Ser Phe Gly Met His Trp Val Arg Gln Ala Pro
50 55 60 50 55 60
Gly Lys Gly Leu Glu Trp Val Ala Tyr Ile Ser Ser Asp Ser Ser AlaGly Lys Gly Leu Glu Trp Val Ala Tyr Ile Ser Ser Asp Ser Ser Ala
65 70 75 8065 70 75 80
Ile Tyr Tyr Ala Asp Thr Val Lys Gly Arg Phe Thr Ile Ser Arg AspIle Tyr Tyr Ala Asp Thr Val Lys Gly Arg Phe Thr Ile Ser Arg Asp
85 90 95 85 90 95
Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Asp GluAsn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Asp Glu
100 105 110 100 105 110
Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Arg Glu Asn Ile Tyr TyrAsp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Arg Glu Asn Ile Tyr Tyr
115 120 125 115 120 125
Gly Ser Arg Leu Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val SerGly Ser Arg Leu Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser
130 135 140 130 135 140
Ser Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly SerSer Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser
145 150 155 160145 150 155 160
Thr Lys Gly Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser AlaThr Lys Gly Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala
165 170 175 165 170 175
Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn ValSer Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val
180 185 190 180 185 190
Asp Thr Asn Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro LysAsp Thr Asn Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
195 200 205 195 200 205
Leu Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Ser ArgLeu Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Ser Arg
210 215 220 210 215 220
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser SerPhe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
225 230 235 240225 230 235 240
Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn AsnLeu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Asn
245 250 255 245 250 255
Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Thr ThrTyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Thr Thr
260 265 270 260 265 270
Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser GlnThr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln
275 280 285 275 280 285
Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly AlaPro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala
290 295 300 290 295 300
Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp AlaVal His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala
305 310 315 320305 310 315 320
Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile ThrPro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr
325 330 335 325 330 335
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe MetLys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
340 345 350 340 345 350
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg PheArg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
355 360 365 355 360 365
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser ArgPro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg
370 375 380 370 375 380
Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr AsnSer Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn
385 390 395 400385 390 395 400
Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys ArgGlu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg
405 410 415 405 410 415
Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys AsnArg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn
420 425 430 420 425 430
Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala GluPro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu
435 440 445 435 440 445
Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys GlyAla Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly
450 455 460 450 455 460
His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr TyrHis Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr
465 470 475 480465 470 475 480
Asp Ala Leu His Met Gln Ala Leu Pro Pro ArgAsp Ala Leu His Met Gln Ala Leu Pro Pro Arg
485 490 485 490
<210> 8<210> 8
<211> 23<211> 23
<212> PRT<212> PRT
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 8<400> 8
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu LeuMet Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 151 5 10 15
His Ala Ala Arg Pro Gly SerHis Ala Ala Arg Pro Gly Ser
20 20
<210> 9<210> 9
<211> 247<211> 247
<212> PRT<212> PRT
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 9<400> 9
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly GlyGlu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 151 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser PheSer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe
20 25 30 20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp ValGly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 35 40 45
Ala Tyr Ile Ser Ser Asp Ser Ser Ala Ile Tyr Tyr Ala Asp Thr ValAla Tyr Ile Ser Ser Asp Ser Ser Ala Ile Tyr Tyr Ala Asp Thr Val
50 55 60 50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu TyrLys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 8065 70 75 80
Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr CysLeu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 85 90 95
Ala Arg Gly Arg Glu Asn Ile Tyr Tyr Gly Ser Arg Leu Asp Tyr TrpAla Arg Gly Arg Glu Asn Ile Tyr Tyr Gly Ser Arg Leu Asp Tyr Trp
100 105 110 100 105 110
Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Ser Thr Ser Gly SerGly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Ser Thr Ser Gly Ser
115 120 125 115 120 125
Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Asp Ile Gln LeuGly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Asp Ile Gln Leu
130 135 140 130 135 140
Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly Asp Arg Val ThrThr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly Asp Arg Val Thr
145 150 155 160145 150 155 160
Ile Thr Cys Lys Ala Ser Gln Asn Val Asp Thr Asn Val Ala Trp TyrIle Thr Cys Lys Ala Ser Gln Asn Val Asp Thr Asn Val Ala Trp Tyr
165 170 175 165 170 175
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala SerGln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser
180 185 190 180 185 190
Tyr Arg Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser GlyTyr Arg Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly
195 200 205 195 200 205
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe AlaThr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala
210 215 220 210 215 220
Thr Tyr Tyr Cys Gln Gln Tyr Asn Asn Tyr Pro Phe Thr Phe Gly GlnThr Tyr Tyr Cys Gln Gln Tyr Asn Asn Tyr Pro Phe Thr Phe Gly Gln
225 230 235 240225 230 235 240
Gly Thr Lys Leu Glu Ile LysGly Thr Lys Leu Glu Ile Lys
245 245
<210> 10<210> 10
<211> 66<211> 66
<212> PRT<212> PRT
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 10<400> 10
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile AlaThr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
1 5 10 151 5 10 15
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala GlySer Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
20 25 30 20 25 30
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr IleGly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile
35 40 45 35 40 45
Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu ValTrp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val
50 55 60 50 55 60
Ile ThrIle Thr
6565
<210> 11<210> 11
<211> 42<211> 42
<212> PRT<212> PRT
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 11<400> 11
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe MetLys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
1 5 10 151 5 10 15
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg PheArg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
20 25 30 20 25 30
Pro Glu Glu Glu Glu Gly Gly Cys Glu LeuPro Glu Glu Glu Glu Gly Gly Cys Glu Leu
35 40 35 40
<210> 12<210> 12
<211> 113<211> 113
<212> PRT<212> PRT
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 12<400> 12
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln GlyArg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly
1 5 10 151 5 10 15
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu TyrGln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
20 25 30 20 25 30
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly LysAsp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
35 40 45 35 40 45
Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu GlnPro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln
50 55 60 50 55 60
Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly GluLys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu
65 70 75 8065 70 75 80
Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser ThrArg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr
85 90 95 85 90 95
Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro ProAla Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro
100 105 110 100 105 110
ArgArg