CN108794629A - 一种vegf-d单克隆抗体及试剂盒 - Google Patents

一种vegf-d单克隆抗体及试剂盒 Download PDF

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CN108794629A
CN108794629A CN201810711880.1A CN201810711880A CN108794629A CN 108794629 A CN108794629 A CN 108794629A CN 201810711880 A CN201810711880 A CN 201810711880A CN 108794629 A CN108794629 A CN 108794629A
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吕鹏辉
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Abstract

本发明公开了一种VEGF‑D单克隆抗体，其重链的氨基酸序列如VH：BAA24264所示，其轻链的氨基酸序列如VL：XP_009304362所示。所述VEGF‑D单克隆抗体具有较高的亲和力，特异性强，在体外具有良好的生物学活性。将本发明VEGF‑D单抗与VEGF‑D多抗建立的VEGF‑D蛋白检测试剂盒可作为评价肿瘤淋巴管生成和淋巴转移指标。

Description

一种VEGF-D单克隆抗体及试剂盒
技术领域
本发明属于生物技术领域，具体涉及一种VEGF-D单克隆抗体及VEGF-D蛋白检测试剂盒。
背景技术
在肿瘤转移机制的研究中，淋巴系统作为肿瘤尤其是实体瘤转移通道的重要意义不断被认识。临床病理学研究显示实体瘤播散的最早途径是经淋巴道的区域性淋巴结播散。目前大多数研究都认为，VEGF-D/VEGFR-3信号系统是肿瘤淋巴管生成最重要的调控机制，并在肿瘤淋巴转移过程中发挥了重要作用。
VEGF-D是1998年Achen等[1]用计算机进行同源搜索时发现的，与VEGF-C结构相似，定位于染色体Xp22.31。VEGF-D在人体结合内皮细胞受体，蛋白水解程序可以调控VEGF-D的生物活性。VEGFR-3最早从人胎盘和红白血病细胞基因库中克隆出来，定位于染色体5q33-5q35，与VEGFR-1、VEGFR-2同属于受体型酪氨酸蛋白激酶家族。VEGFR1和VEGFR2主要表达于血管内皮细胞，VEGFR-3主要表达于淋巴管内皮细胞。所有VEGFR都具有相同的7个细胞外免疫球蛋白同源区域，其中第2和第3个区域是配体结合的关键部位，并且前3个区域是建立完整结合的必须条件。肿瘤组织中淋巴管生成增加，表现为淋巴管内皮细胞增殖，肿瘤组织淋巴管密度明显增高，并与淋巴结转移相关。VEGF-D可以诱导肿瘤组织内部和肿瘤组织周围淋巴管生成，并与显著增加的淋巴脉管浸润和淋巴结转移率相关。对人类许多肿瘤的临床研究也证实，高表达的VEGF-C或D与肿瘤患者淋巴结转移、淋巴管浸润以及不良预后相关。
临床研究的结果证实VEGF-C、VEGF-D/VEGFR-3信号系统在肿瘤淋巴转移过程中发挥了重要作用。目前研究认为其机制可能是：肿瘤细胞分泌淋巴管内皮生长因子VEGF-C、D以旁分泌的形式作用于淋巴管内皮细胞上的酪氨酸激酶受体VEGFR-3，经过信号传导，最终引起淋巴管内皮细胞的增殖、分化、迁移和管腔形成，肿瘤组织淋巴管密度增加。这些肿瘤组织内增多的淋巴管与生理状态下的淋巴管结构相似，但数量增多，管壁薄，多位于肿瘤实质周围，因此这就为肿瘤细胞浸润转移提供了更多的通道，有利于肿瘤细胞转移扩散和淋巴结转移。除此以外，VEGF-C、D与VEGFR-3结合后能够促进淋巴管内皮细胞释放蛋白水解酶类，促进肿瘤细胞的基质浸润，同时还可改变淋巴管内皮细胞的黏附特性、表面趋化因子及其受体的表达，影响肿瘤细胞进入淋巴管的过程，从而主动促进肿瘤淋巴管转移。
关于淋巴管生成因子及其受体对肿瘤淋巴转移和预后的关系和意义，近年来颇受重视。淋巴管生成不仅发生在主要的位点,而且发生在滤过的淋巴结,在肿瘤细胞之前到达淋巴结。肿瘤内VEGF-D的高表达,已被证实能引起肿瘤内部淋巴管的形成,并且能够促进淋巴结的转移。研究发现VEGF-D表达增高可促进肿瘤淋巴管生成及增加淋巴管密度，从而促进淋巴转移，这与诸多瘤体的预后密切相关。在NSCLC组织中VEGF-D的表达量明显高于肺良性病变组织，其表达与NSCLC的发生发展有关。表明，在正常组织中淋巴管生成因子及其受体处于失活或封闭状态，在肿瘤病理状态下NSCLC细胞可通过自分泌VEGF-D诱导淋巴管新生，促进肿瘤发展。VEGF-D的表达与淋巴管密度和淋巴结转移密切相关,表明VEGF-D在促进肿瘤的淋巴管生成和淋巴结转移中起重要的作用,阻断VEGF-D的作用后,有可能抑制肿瘤淋巴管生成和降低淋巴结转移的能力。研究发现血清VEGF-D的水平在淋巴管平滑肌增生症中明显升高,但在管瘤病、肺朗格汉斯组织细胞增多症及肺气肿的患者中是正常的,而淋巴管平滑肌增生症有和肿瘤相似的特点。VEGF-C、D/VEGFR-3一方面通过旁分泌作用机制促进肿瘤淋巴管生成而为肿瘤细胞转移提供通道，另一方面通过自分泌作用机制促进肿瘤细胞生长、增殖、迁移，从而在肿瘤淋巴管生成和淋巴结过程中起到了重要的调控作用。这也就为抗肿瘤淋巴管生成和淋巴转移的基因治疗提供了可能的靶点，监测VEGF-C、D的含量对评价肿瘤淋巴管生成和淋巴转移为肿瘤淋巴转移的治疗开辟一条新的途径。
近十年来标记免疫分析技术的研究和应用发展迅速，已广泛应用于生物医学基础理论研究及临床疾病诊断各领域。用于检测血清学指标的方法主要包括放射性同位素免疫分析、酶联免疫吸附法、和化学发光免疫分析。这些方法既可以作为初筛试验也可以作为确认试验，其中化学发光法具有检测线性范围宽、检测仪器简单、操作方便等优点。
现有技术中针对肿瘤淋巴转移的生物标记物产品目前国内尚属空白，同类产品其特异性和亲和力等方面还存在进一步的提升可能，基于此，我们开发了VEGF-C测定试剂盒(化学发光法)及其制备方法。
发明内容
为了克服现有中的上述问题，本发明提供了一种VEGF-D单克隆抗体，具有较高的亲和力，在体外具有良好的生物学活性；开发前景广阔。
一种VEGF-D单克隆抗体，其特征在于，所述抗体的重链可变区氨基酸序列为：MetTyr Arg Glu Trp Val Val Val Asn Val Phe Met Met Leu Tyr Val Gln Leu Val GlnGly Ser Ser Asn Glu His Gly Pro Val Lys Arg Ser Ser Gln Ser Thr Leu Glu ArgSer Glu Gln Gln Ile Arg Ala Ala Ser Ser Leu Glu Glu Leu Leu Arg Ile Thr HisSer Glu Asp Trp Lys Leu Trp Arg Cys Arg Leu Arg Leu Lys Ser Phe Thr Ser MetAsp Ser Arg Ser Ala Ser His Arg Ser Thr Arg Phe Ala Ala Thr Phe Tyr Asp IleGlu Thr Leu Lys Val Ile Asp Glu Glu Trp Gln Arg Thr Gln Cys Ser Pro Arg GluThr Cys Val Glu Val Ala Ser Glu Leu Gly Lys Ser Thr Asn Thr Phe Phe Lys ProPro Cys Val Asn Val Phe Arg Cys Gly Gly Cys Cys Asn Glu Glu Ser Leu Ile CysMet Asn Thr Ser Thr Ser Tyr Ile Ser Lys Gln Leu Phe Glu Ile Ser Val Pro LeuThr Ser Val Pro Glu Leu Val Pro Val Lys Val Ala Asn His Thr Gly Cys Lys CysLeu Pro Thr Ala Pro Arg His Pro Tyr Ser Ile Ile Arg Arg Ser Ile Gln Ile ProGlu Glu Asp Arg Cys Ser His Ser Lys Lys Leu Cys Pro Ile Asp Met Leu Trp AspSer Asn Lys Cys Lys Cys Val Leu Gln Glu Glu Asn Pro Leu Ala Gly Thr Glu AspHis Ser His Leu Gln Glu Pro Ala Leu Cys Gly Pro His Met Met Phe Asp Glu AspArg Cys Glu Cys Val Cys Lys Thr Pro Cys Pro Lys Asp Leu Ile Gln His Pro LysAsn Cys Ser Cys Phe Glu Cys Lys Glu Ser Leu Glu Thr Cys Cys Gln Lys His LysLeu Phe His Pro Asp Thr Cys Ser Cys Glu Asp Arg Cys Pro Phe His Thr Arg ProCys Ala Ser Gly Lys Thr Ala Cys Ala Lys His Cys Arg Phe Pro Lys Glu Lys ArgAla Ala Gln Gly Pro His Ser Arg Lys Asn Pro；
轻链可变区氨基酸序列为：Met Lys Lys Gln Lys Cys Ala Gly Leu His MetLeu Leu Leu Leu Tyr Val Arg Leu Met Leu Ala Val Asp Ala Tyr Arg Pro Gln ArgAsp Thr Asn Gln Glu Lys Trp Glu Gln Glu Leu Arg Glu Ala Gly Ser Leu Asp GluLeu Leu Met Leu Thr Glu Tyr Pro Asp Trp Lys Leu Trp Arg Cys Arg Leu Lys LeuLys His Phe Asp Asp Val Thr Pro Pro Glu Asn Arg Arg Ser Thr Arg Tyr Ala AlaAla Ser Phe Ser Pro Glu Met Leu Lys Asp Ile Asp Asp Glu Trp Gln Lys Thr GlnCys Met Pro Arg Glu Thr Cys Val Asp Val Ala Lys Glu Leu Gly Thr Asn Thr AlaVal Phe Phe Lys Pro Pro Cys Val Ser Val Phe Arg Cys Gly Gly Cys Cys Asn LysGlu Gly Val Thr Cys Arg Asn Thr Ser Met Thr Tyr Val Asn Lys Thr Val Ser ThrThr His Cys Ile Ala Phe。
进一步地，本发明还提供了编码上述轻链可变区和重链可变区氨基酸序列的多核苷酸。
进一步地，本发明还提供了包含上述多核苷酸的重组DNA表达载体，所述重组DNA表达载体中包含编码VEGF-D单克隆抗体重链可变区、轻链可变区和和抗体恒定区的DNA序列。
进一步地，本发明提供了包含上述VEGF-D单克隆抗体的检测试剂盒。该试剂盒制备方法是：以Nunc化学发光反应板为反应支持物，将VEGF-D单克隆抗体用0.5mPBS稀释至2.5ug/ml，以100ul/孔包被至化学发光空白板中，4℃下24小时后，用0.9％Nacl洗板3次，加入含有1％BSA的0.5mol PBS封闭，4℃下12小时后甩干。
本发明具有以下优点：
(1)本发明的VEGF-D单克隆抗体，具有较高的亲和力，特异性强，在体外具有良好的生物学活性。
(2)包含有VEGF-D单克隆抗体的检测试剂盒检测方法简便、准确性强、成本低。
(3)将VEGFd354与VEGF-D多抗建立VEGF-D蛋白检测试剂盒可作为评价肿瘤淋巴管生成和淋巴转移手段。
附图说明
图1是VEGFd354抗体剂量-反应体系示意图。
图2是VEGFd354抗体质控血清检测散点图。
具体实施方式
借助于下述具体实施方式可更好地理解本发明，然而，这些具体实施方式仅用于举例说明本发明，不应被解释为对本发明的限制。
实施例1
一、VEGF-D抗原的制备：
(1)根据GenBank数据库中人VEGF-D(D89630.1)的序列设计一对引物，上游引物为vP：5’-ATATGTACAGAGAGTGGGTAGTGGTGAATG-3’，引入XhoI酶切位点；下游引物为vP：5’-TTAACAGCATGCTGCTTTGC-3’，引入EcoRI酶切位点，通过RT-PCR方法从人肝细胞中提取总RNA，然后反转录成cDNA，以cDNA为模板，用Pyrobest DNA聚合酶进行VEGF-D基因特异性扩增。扩增后的PCR产物进行凝胶回收，回收的VEGF-D基因与pPIC9K质粒分别进行Xho I和EcoR I酶切，T4连接酶连接回收后的目的片段，连接产物转化DH5α感受态细胞，通过氨苄(1mg/mL)抗体筛选出阳性克隆，体取质粒并经酶切和测序鉴定。
(2)pPIC9K-VEGF-D表达载体经Stu I线性化、电转化至感受态的毕赤酵母GS115(his4)，涂布含G418的MD平板筛选高拷贝的转化子，待菌落长出，挑选单克隆菌株进行PCR鉴定，将PCR结果为阳性的克隆以质粒pPIC9-Kal/GS115(His4)转化至毕赤酵母细胞，在7.5L培养罐内高密度细胞3B5发酵，以1％-2％的甲醛诱导表达VEGF-D。发酵上清液经Phenyl Sephadex G-25凝胶过滤层析，Heparin Sepharose FF肝素亲和层析，SephacrylS-100凝胶过滤层析，得到充足人VEGF-D蛋白，表达水平为50mg/L，进行SDS-PAGE电泳鉴定，纯化后的VEGF-D蛋白纯度98％。
二、VEGF-D多抗的制备及标记
(1)用雄性新西兰白兔作为免疫动物，先用10mg卡介苗注射刺激动物，一周后开始免疫。使用VEGF-D蛋白作为免疫原；免疫模式采用足下注射及背部4-6点皮下注射，免疫佐剂使用弗氏完全佐剂及弗氏不完全佐剂，分四次免疫动物，每次取VEGF-D抗原1mg将等量的弗氏完全佐剂和抗原溶液分别吸入两个注射器内充分乳化30-60分钟后后足皮下注射，每次间隔两周。取耳静脉血检测效价，CLIA达到1:50000进行劲动脉放血，离心取血清。利用DEAE离子交换纯化，所得抗体溶液达98％以上。
(2)VEGF-D多抗用0.5mol PBSPH7.5稀释1mg/ml，配置EZ-LinkTMSulfo-NHS-LC-LC-Biotin货号21338生物素试剂10mM，标记多抗与生物素，摩尔比1:20；室温反应1小时，PD10脱盐柱过柱，收集并保存。
三、VEGF-D单克隆抗体的制备
(1)小鼠免疫：培养稳定表达的3B5细胞，离心后用PBS(pH7.4)重悬，免疫3只雌性BALA/c小鼠，每只BALB/c小鼠皮下注射5×106细胞，连续3次，每次间隔2周，第4次免疫后7d，用CLIA法检测抗血清效价，取效价最高的小鼠，脾内注射1×105个细胞加强免疫，3d后取小鼠脾脏，研磨，并对脾细胞计数待用。
(2)细胞融合与抗体制备：取脾细胞与骨髓细胞Sp2/0按细胞计数6:1的比例融合，PEG1200诱导，融合后加到铺好的饲养层的96孔板培养一周后用HAT培养基半量换液，CLIA方法筛选阳性细胞株。用间接CLIA法筛选阳性杂交瘤细胞株。经3次有效稀释法克隆化后，选择I株VEGF-DAb持续分泌阳性率98％以上的杂交瘤细胞5B6并扩大培养准备腹腔注射小鼠。接种杂交瘤细胞前I周，小鼠腹腔注射500μL液体石蜡。接种时离心收集杂交瘤细胞，用不完全培养液悬浮并混匀，将细胞数调至1×109/L，每只小鼠腹腔注射500μL，1周后小鼠腹部明显肿胀，消毒下腹部皮肤后，抽取腹水，所得腹水3000r/min，收集上清。
(3)选用GE公司proteinG纯化柱纯化腹水；用20mMPB缓冲液平衡纯化柱，加入腹水上样，用PH2.70.1mol甘氨酸盐酸缓冲液进行洗脱，用含有PH8.71moltris缓冲液的EP管收集，0.05mMPB透析，浓缩冻存。
四、单克隆抗体应用筛选
(1)包被前述方法制备好的单克隆抗体，以0.5mPBS稀释抗体至1-5ug/ml。100ul每孔包被至化学发光空白板中，4℃过夜后，0.9％Nacl洗3次，加入含有1％BSA每孔150ul封闭，4℃过夜后甩干备用。
(2)筛选最优单抗：稀释制备的抗原按比例稀释8个梯度(0、10、50、100、200、400、1000、2000pg/m)，依次加入前述制备好的化学发光板中，每孔50ul，再加入生物素化多抗及标记有辣根过氧化物酶的链霉亲和素，37℃温育1h，PBST洗涤5次，加入化学发光底物液，避光反应检测发光值；对比不同单抗包被化学发光板下校准品的线性相关系数R值，以VEGF-D浓度为横坐标X值，RLU值为纵坐标Y值。最终挑选具有良好线性，评价指标最好的单克隆抗体VEGFd354。单抗隆抗体作为夹心法CLIA检测方法的评价标准应S0RLU值＜100，S5/S0(P/N)最大，30例质控血清检出率大于90％等条件。
VEGFd354抗体剂量-反应体系示意图如图1所示。
VEGFd354抗体质控血清检测散点图如图2所示。
五、杂交瘤中单克隆抗体基因调取与制备
(1)用Trizol试剂提取5×106的杂交瘤细胞5B6的总RNA，再以OligodT为引物，AMV逆转录酶逆转录温度37℃15min，合成得到cDNA。以合成cDNA为模板，针对RNA序列的引物和基因特异性引物GSP进行巢式PCR扩增，PCR的条件均为：95℃变性5min，94℃30s，55℃30s，72℃1min，共35个循环，最后再于72℃延伸10min。
(2)将PCR产物经1％的琼脂糖凝胶电泳鉴定，切取并回收目的凝胶条带，调取目的基因，连接到pGEM-T载体中，EcoRI酶切鉴定阳性的重组质粒，进行DNA序列的测定分别获得VL和VH基因序列。
VH(重链可变区序列)
LOCUS BAA24264 354aa
Met Tyr Arg Glu Trp Val Val Val Asn Val Phe Met Met Leu Tyr Val GlnLeu Val Gln Gly Ser Ser Asn Glu His Gly Pro Val Lys Arg Ser Ser Gln Ser ThrLeu Glu Arg Ser Glu Gln Gln Ile Arg Ala Ala Ser Ser Leu Glu Glu Leu Leu ArgIle Thr His Ser Glu Asp Trp Lys Leu Trp Arg Cys Arg Leu Arg Leu Lys Ser PheThr Ser Met Asp Ser Arg Ser Ala Ser His Arg Ser Thr Arg Phe Ala Ala Thr PheTyr Asp Ile Glu Thr Leu Lys Val Ile Asp Glu Glu Trp Gln Arg Thr Gln Cys SerPro Arg Glu Thr Cys Val Glu Val Ala Ser Glu Leu Gly Lys Ser Thr Asn Thr PhePhe Lys Pro Pro Cys Val Asn Val Phe Arg Cys Gly Gly Cys Cys Asn Glu Glu SerLeu Ile Cys Met Asn Thr Ser Thr Ser Tyr Ile Ser Lys Gln Leu Phe Glu Ile SerVal Pro Leu Thr Ser Val Pro Glu Leu Val Pro Val Lys Val Ala Asn His Thr GlyCys Lys Cys Leu Pro Thr Ala Pro Arg His Pro Tyr Ser Ile Ile Arg Arg Ser IleGln Ile Pro Glu Glu Asp Arg Cys Ser His Ser Lys Lys Leu Cys Pro Ile Asp MetLeu Trp Asp Ser Asn Lys Cys Lys Cys Val Leu Gln Glu Glu Asn Pro Leu Ala GlyThr Glu Asp His Ser His Leu Gln Glu Pro Ala Leu Cys Gly Pro His Met Met PheAsp Glu Asp Arg Cys Glu Cys Val Cys Lys Thr Pro Cys Pro Lys Asp Leu Ile GlnHis Pro Lys Asn Cys Ser Cys Phe Glu Cys Lys Glu Ser Leu Glu Thr Cys Cys GlnLys His Lys Leu Phe His Pro Asp Thr Cys Ser Cys Glu Asp Arg Cys Pro Phe HisThr Arg Pro Cys Ala Ser Gly Lys Thr Ala Cys Ala Lys His Cys Arg Phe Pro LysGlu Lys Arg Ala Ala Gln Gly Pro His Ser Arg Lys Asn Pro；
VL(轻链可变区序列)
LOCUS XP_009304362 169aa
Met Lys Lys Gln Lys Cys Ala Gly Leu His Met Leu Leu Leu Leu Tyr ValArg Leu Met Leu Ala Val Asp Ala Tyr Arg Pro Gln Arg Asp Thr Asn Gln Glu LysTrp Glu Gln Glu Leu Arg Glu Ala Gly Ser Leu Asp Glu Leu Leu Met Leu Thr GluTyr Pro Asp Trp Lys Leu Trp Arg Cys Arg Leu Lys Leu Lys His Phe Asp Asp ValThr Pro Pro Glu Asn Arg Arg Ser Thr Arg Tyr Ala Ala Ala Ser Phe Ser Pro GluMet Leu Lys Asp Ile Asp Asp Glu Trp Gln Lys Thr Gln Cys Met Pro Arg Glu ThrCys Val Asp Val Ala Lys Glu Leu Gly Thr Asn Thr Ala Val Phe Phe Lys Pro ProCys Val Ser Val Phe Arg Cys Gly Gly Cys Cys Asn Lys Glu Gly Val Thr Cys ArgAsn Thr Ser Met Thr Tyr Val Asn Lys Thr Val Ser Thr Thr His Cys Ile Ala Phe。
将获得重链和轻链可变区基因序列和抗体的恒定区一起进行密码子优化并合成，分别获得完整抗体基因的重链和轻链，将获得的轻链和重链基因分别克隆到pMD18-T表达载体中。质粒分别提取100-150ug的量，并去内毒素(＜1EU/mg)，将获得的2个质粒1:1共转染到50ml体积的悬浮的感受态TG1细胞中，转染试剂采用PEI，质粒和PEI的比值为1:2。转染3-7天后，收集细胞上清，SDS-PAGE检测抗体是否正确表达，ProteinA柱纯化，浓缩后获得合格的VEGF-D抗体VEGFd354，纯度大于98％。
六、VEGF-D检测CLIA试剂盒的制备
(1)单抗包被：以Nunc化学发光反应板为反应支持物，将筛选出的最优单抗VEGFd354，用0.5mPBS稀释至2.5ug/ml。以100ul/孔包被至化学发光空白板中，4度24小时后，用0.9％Nacl 300ul/孔洗板3次，加入含有1％BSA的0.5molPBS/孔150ul封闭，4度12小时甩干备用。
(2)加样：稀释制备的VEGF-D抗原按比例稀释6个梯度(0、10、50、100、200、400、800pg/m)及待测血清样本，各50ul/孔加入包被VEGFd354的包被板中，50ul/孔再加入生物素化VEGF-D多抗及标记有辣根过氧化物酶的链霉亲和素(0.5mPBS，1:10000稀释)，37℃温育1h，PBST洗涤5次，除去未结合的VEGF多抗及链霉亲和素-辣根过氧化物酶。
(3)加入鲁米诺-过氧化物化学发光底物50ul/孔，利用TZD-CL-200S化学发光免疫分析仪检测化学发光强度(RLU)。
七、VEGF-D检测试剂盒的性能
(1)VEGF-D检测试剂盒的分析性能
重复性：VEGF-D检测试剂盒的批内变异系数(CV)不大于10％；批间变异系数(CV)不大于15％；
分析灵敏度：试剂盒最低检出限不大于30pg/mL；
分析特异性：一定浓度特异性物质(FGF2、EGF)检测结果不大于50pg/mL；
线性范围：在0-800pg/mL浓度范围内，线性相关系数r不小于0.9900。
(2)VEGF-D检测试剂盒的临床性能
开展266例临床样本检测，133例正常人，133例肺癌淋巴转移患者。试剂盒正常参考值0-110pg/ml，试剂盒检测结果如下：
符合率及其置信区间
交叉表
计数
符合率及其置信区间
对称度量值
a.没有假定空假设。
b.使用渐近标准错误假定空假设。
一致性系数Kappa(K)值为：0.962。
血管内皮生长因子D(VEGF-D)化学发光检测试剂盒肺癌淋巴结转移符合率为99％，能够作为监测肺癌淋巴转移的有效临床指标。
SEQUENCE LISTING
<110> 浙江众意生物科技有限公司
<120> 一种VEGF-D单克隆抗体及试剂盒
<130>
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 354
<212> PRT
<213> VEGF-D单克隆抗体重链可变区
<400> 1
Met Tyr Arg Glu Trp Val Val Val Asn Val Phe Met Met Leu Tyr Val
1 5 10 15
Gln Leu Val Gln Gly Ser Ser Asn Glu His Gly Pro Val Lys Arg Ser
20 25 30
Ser Gln Ser Thr Leu Glu Arg Ser Glu Gln Gln Ile Arg Ala Ala Ser
35 40 45
Ser Leu Glu Glu Leu Leu Arg Ile Thr His Ser Glu Asp Trp Lys Leu
50 55 60
Trp Arg Cys Arg Leu Arg Leu Lys Ser Phe Thr Ser Met Asp Ser Arg
65 70 75 80
Ser Ala Ser His Arg Ser Thr Arg Phe Ala Ala Thr Phe Tyr Asp Ile
85 90 95
Glu Thr Leu Lys Val Ile Asp Glu Glu Trp Gln Arg Thr Gln Cys Ser
100 105 110
Pro Arg Glu Thr Cys Val Glu Val Ala Ser Glu Leu Gly Lys Ser Thr
115 120 125
Asn Thr Phe Phe Lys Pro Pro Cys Val Asn Val Phe Arg Cys Gly Gly
130 135 140
Cys Cys Asn Glu Glu Ser Leu Ile Cys Met Asn Thr Ser Thr Ser Tyr
145 150 155 160
Ile Ser Lys Gln Leu Phe Glu Ile Ser Val Pro Leu Thr Ser Val Pro
165 170 175
Glu Leu Val Pro Val Lys Val Ala Asn His Thr Gly Cys Lys Cys Leu
180 185 190
Pro Thr Ala Pro Arg His Pro Tyr Ser Ile Ile Arg Arg Ser Ile Gln
195 200 205
Ile Pro Glu Glu Asp Arg Cys Ser His Ser Lys Lys Leu Cys Pro Ile
210 215 220
Asp Met Leu Trp Asp Ser Asn Lys Cys Lys Cys Val Leu Gln Glu Glu
225 230 235 240
Asn Pro Leu Ala Gly Thr Glu Asp His Ser His Leu Gln Glu Pro Ala
245 250 255
Leu Cys Gly Pro His Met Met Phe Asp Glu Asp Arg Cys Glu Cys Val
260 265 270
Cys Lys Thr Pro Cys Pro Lys Asp Leu Ile Gln His Pro Lys Asn Cys
275 280 285
Ser Cys Phe Glu Cys Lys Glu Ser Leu Glu Thr Cys Cys Gln Lys His
290 295 300
Lys Leu Phe His Pro Asp Thr Cys Ser Cys Glu Asp Arg Cys Pro Phe
305 310 315 320
His Thr Arg Pro Cys Ala Ser Gly Lys Thr Ala Cys Ala Lys His Cys
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Arg Phe Pro Lys Glu Lys Arg Ala Ala Gln Gly Pro His Ser Arg Lys
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Asn Pro
<210> 2
<211> 169
<212> PRT
<213> VEGF-D单克隆抗体轻链可变区
<400> 2
Met Lys Lys Gln Lys Cys Ala Gly Leu His Met Leu Leu Leu Leu Tyr
1 5 10 15
Val Arg Leu Met Leu Ala Val Asp Ala Tyr Arg Pro Gln Arg Asp Thr
20 25 30
Asn Gln Glu Lys Trp Glu Gln Glu Leu Arg Glu Ala Gly Ser Leu Asp
35 40 45
Glu Leu Leu Met Leu Thr Glu Tyr Pro Asp Trp Lys Leu Trp Arg Cys
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Arg Leu Lys Leu Lys His Phe Asp Asp Val Thr Pro Pro Glu Asn Arg
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Arg Ser Thr Arg Tyr Ala Ala Ala Ser Phe Ser Pro Glu Met Leu Lys
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Asp Ile Asp Asp Glu Trp Gln Lys Thr Gln Cys Met Pro Arg Glu Thr
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Lys Pro Pro Cys Val Ser Val Phe Arg Cys Gly Gly Cys Cys Asn Lys
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Glu Gly Val Thr Cys Arg Asn Thr Ser Met Thr Tyr Val Asn Lys Thr
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<210> 3
<211> 30
<212> DNA
<213> 人工序列
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atatgtacag agagtgggta gtggtgaatg 30
<210> 4
<211> 20
<212> DNA
<213> 人工序列
<400> 4
ttaacagcat gctgctttgc 20

Claims (5)

1.一种VEGF-D单克隆抗体，其特征在于，所述抗体的重链可变区氨基酸序列为：MetTyr Arg Glu Trp Val Val Val Asn Val Phe Met Met Leu Tyr Val Gln Leu Val GlnGly Ser Ser Asn Glu His Gly Pro Val Lys Arg Ser Ser Gln Ser Thr Leu Glu ArgSer Glu Gln Gln Ile Arg Ala Ala Ser Ser Leu Glu Glu Leu Leu Arg Ile Thr HisSer Glu Asp Trp Lys Leu Trp Arg Cys Arg Leu Arg Leu Lys Ser Phe Thr Ser MetAsp Ser Arg Ser Ala Ser His Arg Ser Thr Arg Phe Ala Ala Thr Phe Tyr Asp IleGlu Thr Leu Lys Val Ile Asp Glu Glu Trp Gln Arg Thr Gln Cys Ser Pro Arg GluThr Cys Val Glu Val Ala Ser Glu Leu Gly Lys Ser Thr Asn Thr Phe Phe Lys ProPro Cys Val Asn Val Phe Arg Cys Gly Gly Cys Cys Asn Glu Glu Ser Leu Ile CysMet Asn Thr Ser Thr Ser Tyr Ile Ser Lys Gln Leu Phe Glu Ile Ser Val Pro LeuThr Ser Val Pro Glu Leu Val Pro Val Lys Val Ala Asn His Thr Gly Cys Lys CysLeu Pro Thr Ala Pro Arg His Pro Tyr Ser Ile Ile Arg Arg Ser Ile Gln Ile ProGlu Glu Asp Arg Cys Ser His Ser Lys Lys Leu Cys Pro Ile Asp Met Leu Trp AspSer Asn Lys Cys Lys Cys Val Leu Gln Glu Glu Asn Pro Leu Ala Gly Thr Glu AspHis Ser His Leu Gln Glu Pro Ala Leu Cys Gly Pro His Met Met Phe Asp Glu AspArg Cys Glu Cys Val Cys Lys Thr Pro Cys Pro Lys Asp Leu Ile Gln His Pro LysAsn Cys Ser Cys Phe Glu Cys Lys Glu Ser Leu Glu Thr Cys Cys Gln Lys His LysLeu Phe His Pro Asp Thr Cys Ser Cys Glu Asp Arg Cys Pro Phe His Thr Arg ProCys Ala Ser Gly Lys Thr Ala Cys Ala Lys His Cys Arg Phe Pro Lys Glu Lys ArgAla Ala Gln Gly Pro His Ser Arg Lys Asn Pro；
轻链可变区氨基酸序列为：Met Lys Lys Gln Lys Cys Ala Gly Leu His Met LeuLeu Leu Leu Tyr Val Arg Leu Met Leu Ala Val Asp Ala Tyr Arg Pro Gln Arg AspThr Asn Gln Glu Lys Trp Glu Gln Glu Leu Arg Glu Ala Gly Ser Leu Asp Glu LeuLeu Met Leu Thr Glu Tyr Pro Asp Trp Lys Leu Trp Arg Cys Arg Leu Lys Leu LysHis Phe Asp Asp Val Thr Pro Pro Glu Asn Arg Arg Ser Thr Arg Tyr Ala Ala AlaSer Phe Ser Pro Glu Met Leu Lys Asp Ile Asp Asp Glu Trp Gln Lys Thr Gln CysMet Pro Arg Glu Thr Cys Val Asp Val Ala Lys Glu Leu Gly Thr Asn Thr Ala ValPhe Phe Lys Pro Pro Cys Val Ser Val Phe Arg Cys Gly Gly Cys Cys Asn Lys GluGly Val Thr Cys Arg Asn Thr Ser Met Thr Tyr Val Asn Lys Thr Val Ser Thr ThrHis Cys Ile Ala Phe。
2.一种编码权利要求1所述的轻链可变区和重链可变区氨基酸序列的多核苷酸。
3.一种包含如权利要求2所述的多核苷酸的重组DNA表达载体，所述重组DNA表达载体中包含编码VEGF-D单克隆抗体重链可变区、轻链可变区和和抗体恒定区的DNA序列。
4.一种包含权利要求1所述VEGF-D单克隆抗体的检测试剂盒。
5.权利要求4所述检测试剂盒的制备方法，其特征在于，以Nunc化学发光反应板为反应支持物，将VEGF-D单克隆抗体用0.5mPBS稀释至2.5ug/ml，以100ul/孔包被至化学发光空白板中，4℃下24小时后，用0.9％Nacl洗板3次，加入含有1％BSA的0.5mol PBS封闭，4℃下12小时后甩干。
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WO2000037025A2 (en) * 1998-12-21 2000-06-29 Ludwig Institute For Cancer Research Antibodies to truncated vegf-d and uses thereof
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WO2000037025A2 (en) * 1998-12-21 2000-06-29 Ludwig Institute For Cancer Research Antibodies to truncated vegf-d and uses thereof
CN1684706A (zh) * 2002-07-23 2005-10-19 路德维格癌症研究所 活化或抑制vegf－d和vegf－c的方法和组合物

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