CN107964045B - 一种人鼠嵌合抗CXCR2全分子IgG及其应用 - Google Patents
一种人鼠嵌合抗CXCR2全分子IgG及其应用 Download PDFInfo
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Abstract
本发明公开了一种人鼠嵌合抗CXCR2全分子IgG及其应用,属于生物制药领域。包括重链可变区和轻链可变区,其特征在于:所述轻链可变区的核酸序列为SEQ ID NO.1所示,所述重链可变区的核酸序列为SEQ ID NO.2所示。本发明使用特异性重组CXCR2蛋白免疫小鼠,采用杂交瘤技术制备鼠源抗CXCR2单克隆抗体,并采用基因工程技术和抗体工程技术,制备重组人鼠嵌合抗CXCR2全分子IgG。该嵌合抗体可有效识别CXCR2胞外区氨基酸片段,并抑制CXCR2蛋白与GROα蛋白的结合。
Description
技术领域
本发明属生物制药领域,涉及一种人鼠嵌合抗CXCR2的全分子IgG抗体,还涉及上述全分子IgG抗体的DNA分子、表达载体、宿主细胞和应用。
背景技术
趋化因子(chemokine)是能使细胞发生趋化作用的细胞因子总称,最早发现在哺乳动物、鸟类及鱼类动物体内,为一类功能结构相似,分子量在8~10kDa小分子量蛋白质。趋化因子由中性粒细胞,单核细胞等多种细胞分泌产生,主要表达于中性粒细胞,巨噬细胞等炎性细胞表面,亦可表达于上皮细胞、平滑肌细胞和成纤维细胞等细胞。根据多肽链一级结构特点不同,将趋化因子分为CXC亚家族和CC亚家族。趋化因子中的CXC家族具有ELR(谷氨酸-亮氨酸-精氨酸)结构,该结构可在多种肿瘤中促进肿瘤新生血管的形成,并参与介导炎症因子的化学趋化作用。
趋化因子受体是一类介导趋化因子行使功能的GTP蛋白耦联的跨膜受体(GPCR),通常表达于免疫细胞、内皮细胞等细胞膜上,在肿瘤新生血管的形成、肿瘤增殖以及侵袭和转移过程中发挥着多种作用。CXCR2属于趋化因子受体家族,为G蛋白偶联受体。CXCR2基因定位于染色体2q35,其分子由330个氨基酸组成,存在7个跨膜区,分成细胞外自由的N端、3个细胞外环和C端等部分,氨基端残基和第一个胞外环是其与配体结合的的关键部位。CXCR2表达于黑色素瘤细胞、细胞因子活化的嗜酸性粒细胞、嗜碱性粒细胞、T淋巴细胞、肥大细胞和树突状细胞等。研究表明,CXCR2的存在与多种肿瘤相关。在敲除CXCR2基因的Lewis肺癌小鼠模型,肿瘤的生长比对照组明显减缓。从病理组织学分析得到,敲除CXCR2小鼠模型中瘤体坏死面积增大,肿瘤区域的血管密度也相应降低,可见肿瘤的生长及血管生成均被抑制。在使用CXCR2拮抗剂后,食管癌细胞株WHCO1增殖下降40%。
目前已公布的以CXCR2为靶点的抗体中,多数以诊断和科学研究为主,尚无明确可以与CXCR2胞外区特定氨基酸区域结合的报道,且兼具抑制或阻断CXCR2结合功能的抗体。
发明内容
本发明旨在提供一种具有中和作用的人鼠嵌合抗CXCR2抗体IgG,以及该抗体CDR区域的核酸及氨基酸序列,抗体可变区核酸及氨基酸序列,抗体全分子氨基酸序列,抗原结合表位氨基酸序列,制备鼠源源抗CXCR2抗体的方法,人鼠嵌合抗CXCR2全分子IgG的方法,及其对抑制CXCR2与GROα蛋白的结合反应中的用途。
本发明所制备的抗体,已明确可与CXCR2蛋白特异性结合,且抗原结合表位氨基酸序列为CERRNHIDRALD,该抗体可有效阻断CXCR2蛋白与GROα蛋白的结合,具有明显的中和活性。目前国内外,以CXCR2为靶点的抗体,且明确抗原表位,以及对GROα的竞争性抑制的相关专利未见报道。
本发明提供一种人鼠嵌合抗CXCR2全分子IgG,包括重链可变区和轻链可变区,抗体轻链可变区的核酸序列为SEQ ID NO.1所示,抗体重链可变区的核酸序列为SEQ ID NO.2所示。抗体轻链可变区的氨基酸序列为SEQ ID NO.3所示,抗体重链可变区的氨基酸序列为SEQ ID NO.4所示。抗体轻链抗原互补区CDR的氨基酸序列为SEQ ID NO.5,SEQ ID NO.6,SEQ ID NO.7所示。抗体重链抗原互补区CDR的氨基酸序列为SEQ ID NO.8,SEQ ID NO.9,SEQ ID NO.10所示。抗体的轻链氨基酸序列为SEQ ID NO.11所示,重链氨基酸序列为SEQID NO.12所示。抗原结合表位氨基酸序列为SEQ ID NO.13所示。
一种含有上述编码单克隆抗体重、轻链的核苷酸编码的表达载体,该质粒载体含有所述的氨基酸序列。
该系统用于表达含有所述抗体的核酸及氨基酸序列。
所述的人鼠嵌合抗CXCR2全分子IgG,与CXCR2胞外区蛋白特异性结合,氨基酸序列为SEQ ID NO.13所示,并阻断CXCR2蛋白与GROα蛋白的结合。
所述的人鼠嵌合抗CXCR2全分子IgG在制备抑制CXCR2功能的肿瘤诊断或治疗药物中的应用。
一种抑制CXCR2生物学功能的药物,有效成分为人鼠嵌合抗CXCR2全分子IgG。
本发明使用特异性重组CXCR2蛋白免疫小鼠,采用杂交瘤技术制备鼠源抗CXCR2单克隆抗体,并采用基因工程技术和抗体工程技术,制备重组人鼠嵌合抗CXCR2全分子IgG。该嵌合抗体可有效识别CXCR2胞外区氨基酸片段,并抑制CXCR2蛋白与GROα蛋白的结合。
附图说明
图1酶联免疫吸附实验
图2SDS-PAGE检测
M:ProteinMarker 1∶293F细胞培养上清2:纯化洗脱液3:纯化流穿液
图3Western-blot检测
图4质谱检测
图5Biacore T100抗体亲和力检测KD=5.719e-8
图6GROα与人鼠嵌和抗CXCR2E4IgG竞争性结合CXCR2蛋白的检测
图7ELISA检测MV3细胞加入不同剂量重组GROα时抗CXCR2E4IgG竞争性结合CXCR2
图8CXCR2表达差异的肿瘤细胞中抗CXCR2E4IgG的抑制作用
具体实施方式
1.鼠源抗CXCR2杂交瘤细胞的制备
2.鼠源抗CXCR2抗体的筛选、制备和鉴定
3.人鼠嵌合抗CXCR2全分子IgG的制备、表达及纯化
4.人鼠嵌合抗CXCR2全分子抗体的特性分析
5.抗CXCR2全分子IgG对GROα蛋白的竞争性抑制作用检测
实施例1.鼠源抗CXCR2杂交瘤细胞的制备
根据人CXCR2基因(UniProtKB P25025)胞外区序列(A286-A297)设计CERRNHIDRALD-C为特异性表位的抗原,由南京金斯瑞生物有限公司合成,5mg(95%纯度),并分别偶联OVA和KLH。偶联OVA的短肽为免疫用抗原,偶联KLH的短肽为筛选检测用抗原。
用CXCR2胞外区重组蛋白作为免疫原在纯系BALB/c小鼠腹部皮下注射进行免疫接种,每次100μg/ml,共五次,在最后一次免疫接种前三天进行腹腔内加强免疫。融合当天取小鼠脾脏,用DMEM培养基(美国GIBCO公司)制备成单细胞悬液,在50%PEG(PH 8.0)存在下,将脾细胞和SP2/0小鼠骨髓瘤细胞进行融合,用HAT选择性培养基(DMEM培养基98ml,HT贮存液1ml,A贮存液1ml)培养7天,换用HT培养基(DMEM培养基99ml,HT 1ml)培养。
实施例2.鼠源抗CXCR2抗体的筛选、制备和鉴定
根据杂交瘤细胞生长情况行酶联免疫法(ELISA)检测筛选,具体方法见下述。取检测阳性孔的细胞再次克隆化培养,经过6次亚克隆后,待所有的孔内单克隆细胞检测都为CXCR2-KLH阳性、KLH阴性后,取数孔扩大培养并部分冻存。
ELISA方法筛选CXCR2阳性而正常鼠血清阴性的单克隆抗体,具体方法如下:
(1)化学合成的CXCR2-KLH多肽包被ELISA的96孔板,用包被液(0.1M碳酸盐缓冲液,pH9.6)稀释至20μg/ml,每孔加入100μl,4℃过夜;
(2)用PBST洗涤液(PBS含0.05%吐温)洗涤5次后加入10%BSA(200μl/孔)封闭,37℃孵育2h,洗涤5次后备用。
(3)每个孔中加入50μl杂交瘤细胞培养上清,以脾切除小鼠的血清为阳性对照(1:1000稀释),以正常小鼠的血清为阴性对照(1∶1000稀释)37℃孵育1h。
(4)将以1∶5000稀释的羊抗鼠Ig-HRP第二抗体(Thermo公司)100μl/孔加入到孔内,37℃孵育1h,PBST洗涤5次;
(5)加入过氧化物酶底物显色液100μl/孔,室温下15分钟后用2M硫酸中止反应。用全波长酶标仪检测(Thermo labsystems,USA),比色采用双波长450nm/630nm,检测结果大于正常鼠血清OD值2.5倍的为CXCR2阳性克隆,最终选取1株稳定分泌抗CXCR2抗体的杂交瘤细胞,命名为E4,其分泌的单抗命名为CXCR2E4。
使用美国Sigma公司的单克隆抗体亚型鉴定试剂盒(ISO-2KT)进行抗体的亚型,结果示抗CXCR2抗体E4的亚型为Ig2a。
实施例3.人鼠嵌合抗CXCR2全分子IgG的制备、表达及纯化
复苏CXCR2杂交瘤细胞E4,参照Trizol Reagent Kit说明书,提取细胞总RNA,RT-PCR方法获得cDNA。参照Phage Display和BLAST数据库的统计资料分别设计19条VHforward和17条Vκforward引物,4条VH reverse和3条Vκreverse引物,引物序例如下:
Vκforward primers
Vκ-1∶5’-GGGCCCAGGCGGCCGAGCTCGAYATCCAGCTGACTCAGCC-3’
Vκ-2∶5’-GGGCCCAGGCGGCCGAGCTCGAYATTGTTCTCWCCCAGTC-3’
Vκ-3∶5’-GGGCCCAGGCGGCCGAGCTCGAYATTGTGMTMACTCAGTC-3’
Vκ-4:5’-GGGCCCAGGCGGCCGAGCTCGAYATTGTGYTRACACAGTC-3’
Vκ-5∶5’-GGGCCCAGGCGGCCGAGCTCGAYATTGTRATGACMCAGTC-3’
Vκ-6∶5’-GGGCCCAGGCGGCCGAGCTCGAYATTMAGATRAMCCAGTC-3’
Vκ-7∶5’-GGGCCCAGGCGGCCGAGCTCGAYATTCAGATGAYDCAGTC-3’
Vκ-8:5’-GGGCCCAGGCGGCCGAGCTCGAYATYCAGATGACACAGAC-3’
Vκ-9:5’-GGGCCCAGGCGGCCGAGCTCGAYATTGTTCTCAWCCAGTC-3’
Vκ-10:5’-GGGCCCAGGCGGCCGAGCTCGAYATTGWGCTSACCCAATC-3’
Vκ-11:5’-GGGCCCAGGCGGCCGAGCTCGAYATTSTRATGACCCARTC-3’
Vκ-12∶5’-GGGCCCAGGCGGCCGAGCTCGAYATTKTGATGACCCARAC-3’
Vκ-13∶5’-GGGCCCAGGCGGCCGAGCTCGAYATTGTGATGACBCAGKC-3’
Vκ-14:5’-GGGCCCAGGCGGCCGAGCTCGAYATTGTGATAACYCAGGA-3’
Vκ-15:5’-GGGCCCAGGCGGCCGAGCTCGAYATTGTGATGACCCAGWT-3’
Vκ-16:5’-GGGCCCAGGCGGCCGAGCTCGAYATTGTGATGACACAACC-3’
Vκ-17:5’-GGGCCCAGGCGGCCGAGCTCGAYATTTTGCTGACTCAGTC-3’Vκ3’reverseprimers
VκR1:5’-AGATGGTGCAGCCACAGTTCGTTTKATTTCCAGYTTGGTCCC-3’
VκR2:5’-AGATGGTGCAGCCACAGTTCGTTTTATTTCCAACTTTGTCCC-3’
VκR3:5’-AGATGGTGCAGCCACAGTTCGTTTCAGCTCCAGCTTGGTCCC-3’VH 5’forwardprimers
VH 1:5’-GCTGCCCAACCAGCCATGGCCCTCGAGGTRMAGCTTCAGGAGTC-3’
VH 2∶5’-GCTGCCCAACCAGCCATGGCCCTCGAGGTBCAGCTCAGCAGTC-3’
VH 3∶5’-GCTGCCCAACCAGCCATGGCCCTCGAGGTGCAGCTGAAGSASTC-3’
VH 4∶5’-GCTGCCCAACCAGCCATGGCCCTCGAGGTCCARCTGCAACARTC-3’
VH 5∶5’-GCTGCCCAACCAGCCATGGCCCTCGAGGTYCAGCTBCAGCARTC-3’
VH 6∶5’-GCTGCCCAACCAGCCATGGCCCTCGAGGTYCARCTGCAGCAGTC-3’
VH 7∶5’-GCTGCCCAACCAGCCATGGCCCTCGAGGTCCACGTGAAGCAGTC-3’
VH 8∶5’-GCTGCCCAACCAGCCATGGCCCTCGAGGTGAASSTGGTGGAATC-3’
VH 9∶5’-GCTGCCCAACCAGCCATGGCCCTCGAGGTGAWGYTGGTGGAGTC-3’
VH 10∶5’-GCTGCCCAACCAGCCATGGCCCTCGAGGTGCAGSKGGTGGAGTC-3’
VH 11∶5’-GCTGCCCAACCAGCCATGGCCCTCGAGGTGCAMCTGGTGGAGTC-3’
VH 12∶5’-GCTGCCCAACCAGCCATGGCCCTCGAGGTGAAGCTGATGGARTC-3’
VH 13∶5’-GCTGCCCAACCAGCCATGGCCCTCGAGGTGCARCTTGTTGAGTC-3’
VH 14∶5’-GCTGCCCAACCAGCCATGGCCCTCGAGGTRAAGCTTCTCGAGTC-3’
VH 15∶5’-GCTGCCCAACCAGCCATGGCCCTCGAGGTGAARSTTGAGGAGTC-3’
VH 16∶5’-GCTGCCCAACCAGCCATGGCCCTCGAGGTTACTCTRAAAGWGTSTG-3’
VH 17∶5’-GCTGCCCAACCAGCCATGGCCCTCGAGGTCCAACTVCAGCARCC-3’
VH18:5’-GCTGCCCAACCAGCCATGGCCCTCGAGGTGAACTTGGAAGTGTC-3’
VH 19∶5’-GCTGCCCAACCAGCCATGGCCCTCGAGGTGAAGGTCATCGAGTC-3’
VH 3’reverse primers
VH R1:5’-CGATGGGCCCTTGGTGGAGGCTGAGGAGACGGTGACCGTGGT-3’
VH R2:5’-CGATGGGCCCTTGGTGGAGGCTGAGGAGACTGTGAGAGTGGT-3’
VH R3:5’-CGATGGGCCCTTGGTGGAGGCTGCAGAGACAGTGACCAGAGT-3’
VH R4:5’-CGATGGGCCCTTGGTGGAGGCTGAGGAGACGGTGACTGAGGT-3’
按一定比例将上述引物混匀后,分别扩增VH、VL基因,扩增条件为95℃4min,95℃30s,56℃30s,72℃30s,30个循环;最后72℃延伸10min,电泳回收、纯化扩增的基因片段,连至pMD-18T,转化大肠杆菌Top10F’,测序后获得轻链、重链可变区序列。
(1)PCR
反应体系如下:
反应条件如下:
(2)2%琼脂糖凝胶电泳,紫外下观察目的条带,切胶回收。
(3)胶回收试剂盒纯化目的DNA片段,去离子水洗脱。
(4)双酶切IgG表达质粒
IgG表达质粒pFUSE-CHIg-hG1、pFUSE-CLIg-hk(购自Invivogen公司)包含IgG1型人源的重链和轻链(Kappa)恒定区碱基编码序列。
a.pFUSE-CHIg-hG1、pFUSE-CLIg-hk模板载体的双酶切
反应体系如下:
反应条件为:37℃酶切过夜。
b.1%琼脂糖凝胶电泳,紫外下切胶回收。
c.胶回收试剂盒纯化目的DNA片段,去离子水洗脱。
(5)Infusion PCR重组表达质粒
重链IF-PCR扩增引物:
F:5’-TACAGGTGTCCACTCGCTAGACGTGCAGCTGCAGGAATCGGGA-3’
R:5’-TGGGCCCTTGGTGGATGCTGCAGAGACAGTGAC-3’
轻链IF-PCR扩增引物:
F:5’-CTTACAGACGCTCGCTGCCAGATTGTGCTCACTCAG-3’
R:5’-TGCAGCCACCGTACGTTTGATTTCCAGTTT-3’
反应体系如下:
反应条件为:50℃孵育15min。
取5μL反应液转化感受态细菌,铺于相应抗性的平板上,次日挑克隆送测序。将测序结果正确的克隆保存菌种并扩大培养,提取质粒。
(6)抗CXCR2全分子IgG的表达
a.取50μg重组后的重链质粒于1mL的Opti-MEM培养基中,取50μg的轻链质粒于1mL的Opti-MEM培养基中,取200μL的293Fectin于2.8mL的Opti-MEM培养基中,将上述三种混合液室温静置5min。
b.将两个质粒混合液混合均匀后,补加500μL的Opti-MEM培养基混合均匀后直接加入转染试剂293Fectin的混合液,混合均匀后静置20min。期间处理293F细胞,将293F细胞离心后用293F Expression Medium重悬,然后计数以及用台盼蓝计算细胞活力比,吸取1.00×108个细胞于培养瓶中,用293F Expression Medium定容至94mL。
c.20min结束后将6mL的DNA、293Fectin的复合物加入准备好的293F细胞中。
d.将细胞放在摇床培养箱中培养,培养条件8%CO2,120rmp,37℃,6天后收集细胞上清。
(7)抗CXCR2全分子IgG的纯化
将收集的细胞培养上清用0.45μm的滤膜过滤,同时过滤平衡液和洗脱液。使用用AKATA P100蛋白纯化系统按照Protein A纯化的标准步骤纯化,以1mL/min的流速上样,以1.5mL/min的流速洗脱。
实施例4.人鼠嵌合抗CXCR2全分子抗体的特性分析
1)酶联免疫吸附实验
用包被液(0.1M碳酸盐缓冲液,pH9.6)稀释CXCR2重组蛋白(南京军区军事医学研究所赠送)至2μg/mL包被ELISA96孔板,每孔加入100μL,4℃过夜;用PBST(PBS含0.5%Tween20)5%脱脂牛奶-洗涤缓冲液封闭,37℃孵育2h;PBST洗涤5次后,每孔加入100μl倍比稀释的抗CXCR2E4全分子IgG(2μg/mL起始浓度,14个浓度梯度稀释),37℃孵育2h;每孔加入100μl羊抗人二抗(1∶4000稀释),37℃孵育1h;PBST洗涤5次后,加过氧化物酶底物显色液,室温下15min后用2M硫酸中止反应,上机检测蛋白吸光度值。
结果如图1示,CXCR2E4抗体能与重组CXCR2蛋白起明显的抗原抗体反应。
2)免疫共沉淀
分别将人肝癌细胞株BEL7402、SMMC7721、HepG2、QYC7701,人肝细胞LO2,人黑色素瘤细胞株A375裂解蛋白与5μgCXCR2E4抗体混合,用PBS定容到300μl,放置于4℃环境共孵育;2h后加入Protein A免疫磁珠,继续孵育1h。离心10min去除上清,用PBST漂洗5遍后,加入50μl柠檬酸洗脱液,离心并收集上清,加入10μl Tris-base中和上清液。使用SDS-PAGE和Western-blot鉴定(结果见图2和图3)。
3)质谱检测
将SDS-PAGE胶中相应位置的胶切样后送质谱检测,结果见图4。
4)亲和力检测
根据等电点以及按照BiacoreX100 control soft的protocol优化偶联条件,斜率优化选择醋酸钠作为偶联稀释缓冲液。用此缓冲液稀释CXCR2E4抗体样品至25ug/ml后偶联到CM5芯片(GE#BR100012)上。预设偶联水平1500RU。用pH7.4的Running buffer稀释mAb系列样品,稀释一系列浓度至0uM、5nM、10nM 20nM、40nM、80nM。设置进样时间为180s,解离时间10min,再生缓冲液用50mMpH2.2Gly-HCl。按照BiacoreX100 control soft的protocol进行上机测试。经检测抗体的亲和力KD为5.719e(-8)(见图5)。
实施例5.抗CXCR2全分子IgG对GROα蛋白的竞争性抑制作用检测
以培养的人肝癌细胞株BEL7402、SMMC7721、SK-Hep-1做实验组;人黑色素瘤细胞株MV3做阳性对照组,成纤维细胞株NIH 3T3做阴性对照组。选取对数生长期的细胞,用0.25%胰蛋白酶消化并制备成单细胞悬液,按每孔1×103接种于96孔板,100μl/孔。置37℃5%CO2恒温保湿培养箱中培养24h。
1)用PBS冲洗3遍。
2)每孔加入固定液(50%丙酮,50%异丙醇)100μl,室温放置15min。
3)用PBS冲洗5遍后再加入5%脱脂牛奶(200μl/孔)封闭,37℃孵育2h,洗涤5次后备用。
4)将重组GROα蛋白分别按照0,0.1,0.2,0.4,0.8,1.6,3.2μg/ml的浓度加入,同时每孔加入等量的人鼠嵌合抗CXCR2抗体E4(5μg/ml)。每孔中最终加入样品体积为100μl,反应设3个复孔。以免疫小鼠的血清为阳性对照(1∶200稀释)37℃孵育1.5h;另一组将抗CXCR2抗体E4分别按照0,1.25,2.5,5,7.5,10μg/ml的浓度加入,同时每孔加入等量的重组GROα蛋白(1μg/ml)。每孔中最终加入样品体积为100μl,反应设3个复孔。以PBS代替抗CXCR2抗体E4为阴性对照,37℃孵育1.5h。
5)用PBST洗涤5~6次,5min/次。
6)1∶2000稀释的HRP标记羊抗人IgG,100μl/孔,37℃孵育1.5h。
7)PBST洗涤5次;使用Pierce TMB显色试剂盒,每孔加入TMB 50μl、H2O2
50μl,室温下显色15min,用2M硫酸中止反应。
8)用酶标仪检测,比色采用双波长450nm/630nm。
分别检测等量抗体与倍比增加的重组GROα蛋白的竞争作用,以及等量GROα蛋白与倍比增加的抗体的竞争作用。实验结果均提示人鼠嵌合抗CXCR2抗体E4可以与GROα和CXCR2的结合位点结合,发挥竞争性抑制作用(见图6~8)。
SEQUENCE LISTING
<110> 南京医科大学
<120> 一种人鼠嵌合抗CXCR2全分子IgG及其应用
<130> 20171119
<160> 13
<170> PatentIn version 3.5
<210> SEQ ID NO.1
<211> 324
<212> DNA
<213> 人工序列
<400> SEQ ID NO.1
gatattttgc tcactcagtc tccagcaatc atgtctgcat ctctagggga acgggtcacc 60
atgacctgca ctgccagctc aagtgtaagt tccagttact tgcactggta ccagcagaag 120
ccaggatcct cccccaaact ctggatttat agtacatcca acctggcttc tggagtccca 180
gctcgcttca gtggcagtgg gtctgggacc tcttactctc tcacaatcag cagcatggag 240
gctgaagatg ctgccactta ttactgccac cagtatcatc gttccccgtg gacgtttggt 300
ggagggacca aactggaaat gaaa 324
<210> SEQ ID NO.2
<211> 360
<212> DNA
<213> 人工序列
<400> SEQ ID NO.2
gaggtgcagc tggtggaatc gggacctggc ctggtgaaac cttctcagtc tctgtccctc 60
acctgcactg tcactggcta ctcaatcacc agtgattatg cctggaactg gatccggcag 120
tttccaggaa acaaactgga gtggatgggc tacataagct acagtggtag cactagctac 180
aacccatctc tcaaaagtcg aatctctatc actcgagaca catccaagaa ccagttcttc 240
ctgcagttga attctgtgac tactgaggac acagccacat attactgtgc aagacgggac 300
tacggctacc tcacctggtt tgcttactgg ggccaaggga ctctggtcac tgtctctgca 360
<210> SEQ ID NO.3
<211> 108
<212> PRT
<213> 人工序列
<400> SEQ ID NO.3
Asp Ile Leu Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly
1 5 10 15
Glu Arg Val Thr Met Thr Cys Thr Ala Ser Ser Ser Val Ser Ser Ser
20 25 30
Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Leu Trp
35 40 45
Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser
50 55 60
Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu
65 70 75 80
Ala Glu Asp Ala Ala Thr Tyr Tyr Cys His Gln Tyr His Arg Ser Pro
85 90 95
Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Met Lys
100 105
<210> SEQ ID NO.4
<211> 120
<212> PRT
<213> 人工序列
<400> SEQ ID NO.4
Glu Val Gln Leu Val Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp
20 25 30
Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp
35 40 45
Met Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe
65 70 75 80
Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Ala Arg Arg Asp Tyr Gly Tyr Leu Thr Trp Phe Ala Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ala
115 120
<210> SEQ ID NO.5
<211> 7
<212> PRT
<213> 人工序列
<400> SEQ ID NO.5
Ser Ser Val Ser Ser Ser Tyr
1 5
<210> SEQ ID NO.6
<211> 3
<212> PRT
<213> 人工序列
<400> SEQ ID NO.6
Ser Thr Ser
1
<210> SEQ ID NO.7
<211> 9
<212> PRT
<213> 人工序列
<400> SEQ ID NO.7
His Gln Tyr His Arg Ser Pro Trp Thr
1 5
<210> SEQ ID NO.8
<211> 9
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<213> 人工序列
<400> SEQ ID NO.8
Gly Tyr Ser Ile Thr Ser Asp Tyr Ala
1 5
<210> SEQ ID NO.9
<211> 7
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<213> 人工序列
<400> SEQ ID NO.9
Ile Ser Tyr Ser Gly Ser Thr
1 5
<210> SEQ ID NO.10
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<213> 人工序列
<400> SEQ ID NO.10
Ala Arg Arg Asp Tyr Gly Tyr Leu Thr Trp Phe Ala Tyr
1 5 10
<210> SEQ ID NO.11
<211> 230
<212> PRT
<213> 人工序列
<400> SEQ ID NO.11
Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr
1 5 10 15
Asp Ala Arg Cys Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser
20 25 30
Ala Ser Leu Gly Glu Arg Val Thr Met Thr Cys Thr Ala Ser Ser Ser
35 40 45
Val Ser Ser Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Ser Ser
50 55 60
Pro Lys Leu Trp Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro
65 70 75 80
Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile
85 90 95
Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys His Gln Tyr
100 105 110
His Arg Ser Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
115 120 125
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
130 135 140
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
145 150 155 160
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
165 170 175
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
180 185 190
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
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Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
210 215 220
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<213> 人工序列
<400> SEQ ID NO.12
Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly
1 5 10 15
Val His Ser Leu Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val
20 25 30
Lys Pro Ser Gln Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser
35 40 45
Ile Thr Ser Asp Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn
50 55 60
Lys Leu Glu Trp Met Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ser Tyr
65 70 75 80
Asn Pro Ser Leu Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys
85 90 95
Asn Gln Phe Phe Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala
100 105 110
Thr Tyr Tyr Cys Ala Arg Arg Asp Tyr Gly Tyr Leu Thr Trp Phe Ala
115 120 125
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys
130 135 140
Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
145 150 155 160
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
165 170 175
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
180 185 190
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
195 200 205
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
210 215 220
Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg
225 230 235
<210> SEQ ID NO.13
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<212> PRT
<213> 人工序列
<400> SEQ ID NO.13
Cys Glu Arg Arg Asn His Ile Asp Arg Ala Leu Asp
1 5 10
Claims (7)
1.一种人鼠嵌合抗CXCR2全分子IgG,包括重链可变区和轻链可变区,其特征在于:
抗体轻链抗原互补区CDR1的氨基酸序列为SEQ ID NO.5,抗体轻链抗原互补区CDR2的氨基酸序列为SEQ ID NO.6,抗体轻链抗原互补区CDR3的氨基酸序列为SEQ ID NO.7所示;
抗体重链抗原互补区CDR1的氨基酸序列为SEQ ID NO.8,抗体重链抗原互补区CDR2的氨基酸序列为SEQ ID NO.9,抗体重链抗原互补区CDR3的氨基酸序列为SEQ ID NO.10所示。
2.如权利要求1所述的人鼠嵌合抗CXCR2全分子IgG,其特征在于:轻链可变区的氨基酸序列为SEQ ID NO.3所示,重链可变区的氨基酸序列为SEQ ID NO.4所示。
3.如权利要求1所述的一种人鼠嵌合抗CXCR2全分子IgG,其特征在于:轻链氨基酸序列为SEQ ID NO.11所示,重链氨基酸序列为SEQ ID NO.12所示。
4.编码如权利要求1所述的人鼠嵌合抗CXCR2全分子IgG的核酸分子,其特征在于:编码所述轻链可变区的编码核酸序列为SEQ ID NO.1所示,编码所述重链可变区的编码核酸序列为SEQ ID NO.2所示。
5.一种含有编码权利要求1-4 中任一项的人鼠嵌合抗CXCR2全分子IgG的重链和轻链的核苷酸的表达载体。
6.一种在真核细胞中表达权利要求1~4中任一项所述的人鼠嵌合抗CXCR2全分子IgG的方法。
7.权利要求1-4中任一项所述的人鼠嵌合抗CXCR2全分子IgG在制备阻断CXCR2蛋白与GROα蛋白结合的制剂中的应用,其特征在于:所述的人鼠嵌合抗CXCR2全分子IgG与CXCR2胞外区蛋白特异性结合的氨基酸序列为SEQ ID NO.13所示。
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