CN101781370A - 可溶性血管内皮生长因子受体重组融合蛋白及用途 - Google Patents
可溶性血管内皮生长因子受体重组融合蛋白及用途 Download PDFInfo
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Abstract
本发明提供一种可溶性血管内皮生长因子受体重组融合蛋白,涉及可溶性VEGFR2与γ-干扰素基因重组融合蛋白(sVEGFR2-IFN-γ),具有SEQ ID №:1的DNA序列和SEQ ID №:2的氨基酸序列。本发明以RT-PCR法,构建具有sVEGFR2和IFN-γ双功能的融合蛋白。本发明将抗肿瘤血管生成治疗和抗肿瘤细胞因子免疫治疗相结合,联合起来以提高抗肿瘤效果,可在制备抗肿瘤血管生成药物中的应用。
Description
技术领域
本发明属于生物技术领域,具体地说,涉及编码可溶性血管内皮生长因子受体2(sVEGFR2,又称sflk1或sKDR2)与γ-干扰素(IFN-γ)基因重组融合蛋白的多核苷酸,以及此多核苷酸编码的蛋白的制备和用途。
背景技术
恶性肿瘤是严重危害人类健康的重大疾病之一。恶性肿瘤的生长、浸润和转移必须依靠新生血管提供足够的营养才能实现。因此,近年来以抑制或破坏肿瘤血管生成为目的的肿瘤治疗新方法,已成为各国肿瘤学研究的热点。该治疗策略有其独特的优点:①血管内皮细胞具有遗传稳定性,不易产生耐药性;②药物最易到达作用部位,即血管表面;③对具有新血管生成的肿瘤都有效,具有通用性;④新生血管内皮细胞的有限破坏可使肿瘤细胞进入休眠状态并被诱导凋亡从而达到治疗肿瘤的目的。
迄今,已发现30多种与肿瘤血管生成有关的血管生成促进因子和抑制因子。主要的血管生成促进因子有:血管内皮生长因子(vascular endothelial growthfactor,VEGF)、血小板源性生长因子(platelet-derived growth factor,PDGF)、血管生成素(angiogenin)、转化生长因子(transforming growth factor,TGF)、表皮生长因子(epidermal growth factor,EGF)、成纤维细胞生长因子(fibroblastgrowth factor,FGF)等。主要的血管生成抑制因子包括血管抑素(angiostatin)、内皮抑素(endostatin)和血小板因子-4等。血管生成促进因子必须与相应的受体结合,才能发挥作用。其中VEGF与表达在活化的血管内皮细胞上的相应受体VEGFR2(VEGFR2又称flk1或KDR)结合所引起的信号转导是血管生成中的限速步骤,在整个血管生成过程中发挥着最为关键的作用。业已证实,VEGF和VEGFR2过度表达与人类肿瘤的侵袭和转移呈正相关。国外有人用优势阴性变异的VEGFR2(即VEGFR2胞外区,也称可溶性VEGFR2——sVEGFR2)与血管内皮细胞上VEGFR2竞争结合VEGF、或者用VEGF的单克隆抗体中和VEGF的活性、或者用VEGFR2的单克隆抗体结合VEGFR2,以阻断VEGF与VEGFR2的信号转导途径,可显著抑制肿瘤细胞诱导的血管生成和肿瘤的生长,具有显著的抗肿瘤作用。但是鼠源性的单克隆抗体的应用会诱导人体产生抗小鼠抗体(HAMA),从而限制其应用。近年来,血管生成抑制因子的基础和临床研究也有了可喜的成果,实验表明内皮抑素、血管抑素和TNP-470在动物体内均能抑制肿瘤诱导的血管生成,抑制肿瘤的生长。基因重组的血管内皮抑素已经上市,被用于肿瘤治疗。但是血管生成抑制因子治疗肿瘤必须长期用药,一旦停止,肿瘤又会继续生长,并且血管生成抑制因子的合成和提取费时、费事、成本高,因此治疗费用昂贵,一般人难于承受。因此,寻找新的简单有效而廉价的抗肿瘤血管生成制剂是当务之急,只有获得新的药物,才能使肿瘤的抗血管生成治疗有更大的应用前景,才能真正造福于广大肿瘤患者。
如前所述,VEGF与表达在血管内皮细胞上的相应受体VEGFR2结合所引起的信号转导是血管生成中的限速步骤,在整个血管生成过程中发挥着最为关键的作用。因此,VEGFR2是抗肿瘤血管生成治疗的关键靶点之一,若能通过某种途径打破对VEGFR2的自身免疫耐受,诱导产生针对VEGFR2的免疫应答,破坏表达VEGFR2的血管内皮细胞,无疑是抗肿瘤血管生成治疗最经济、最有效的方法。本申请人曾研究γ干扰素(IFN-γ)对体内最重要的抗原提呈细胞--树突状细胞(DC)分化发育的调控,发现IFN-γ是DC分化发育和功能成熟的自分泌因子,IFN-γ基因转染能促进DC的分化发育和功能成熟,IFN-γ基因修饰的DC其诱导Th1型免疫应答的能力显著增强;瘤内注射IFN-γ基因修饰的DC能增强DC的抗肿瘤作用。IFN-γ尚能直接抑制多种肿瘤的生长,同时它本身也有抑制肿瘤血管生成的作用。因此,为了更好地治疗肿瘤,本领域迫切需要开发一种新的制剂,将抗肿瘤血管生成治疗和抗肿瘤细胞因子免疫治疗联合起来。
发明内容
本发明的目的是提供一种可溶性血管内皮生长因子受体重组融合蛋白,涉及可溶性VEGFR2与γ-干扰素基因重组融合蛋白(sVEGFR2-IFN-γ),具有SEQID №:1的DNA序列和SEQ ID №:2的氨基酸序列。
本发明提供的重组融合蛋白通过以下方法制备:以RT-PCR法,分别从血管内皮细胞和外周血单个核细胞克隆sVEGFR2和IFN-γ的cDNA,构建携带sVEGFR2-IFN-γ融合基因的真核表达载体,以甲氨蝶啉浓度梯度筛选稳定表达sVEGFR2-IFN-γ融合蛋白的CHO细胞株,建立大规模无血清培养体系,表达并分离纯化sVEGFR2-IFN-γ融合蛋白。
本发明的另一个目的是提供该可溶性血管内皮生长因子受体重组融合蛋白在制备抗肿瘤血管生成药物中的应用。
本发明的特点是构建具有sVEGFR2和IFN-γ双功能的融合蛋白,制备新的有效的抗肿瘤制剂,将抗肿瘤血管生成治疗和抗肿瘤细胞因子免疫治疗相结合,联合起来以提高抗肿瘤效果。
附图说明
图1是本发明的人sVEGFR2-IFN-γ对VEGF165诱导的血管内皮细胞增殖的抑制作用。
图2是本发明的人sVEGFR2-IFN-γ对人外周血单个核细胞NK活性的增强作用。
具体实施方式
本发明结合附图和实施例作进一步的说明。
实施例1:用RT-PCR法克隆人sVEGFR2和IFN-γ的cDNA
1.RT-PCR克隆人sVEGFR2cDNA
用Trizol(Invitrogen)提取脐静脉内皮细胞总RNA。以First Strand cDNASynthesis Kit(Fermentas)将2μg总RNA逆转录成cDNA,逆转录体系为20μl。PCR扩增人sVEGFR2cDNA所用的引物如下:有义引物:5’-cg gga tcc atg gagagc aag gtg-3’,反义引物:5-cg gaa ttcttc caa gtt cgt ctt ttc c-3’。PCR反应体积为50μl,其中含逆转录模板2μl、0.4mM引物、0.2mM dNTP和1U Blend Taq DNA聚合酶(Takara)。扩增参数为94℃30秒、47℃30秒、72℃150秒,30个循环后PCR产物行1%琼脂糖凝胶电泳初步确认。DNA序列测定结果表明该PCR产物的编码DNA序列与GenBank中的VEGFR2(AF063658)胞外区序列完全相同。
2.RT-PCR克隆无信号肽编码序列的人IFN-γcDNA
用密度梯度离心法分离外周血单个核细胞,以含10%FCS的RPMI 1640完全培养液(Invitrogen)调整细胞浓度为1×106/ml,铺于24孔板,以5μg/ml的PHA刺激,在37℃、5%CO2条件下培养48小时。用Trizol(Invitrogen)提取经PHA刺激的外周血单个核细胞总RNA。以First Strand cDNA SynthesisKit(Fermentas)将2μg总RNA逆转录成cDNA,逆转录体系为20μl。PCR扩增无信号肽编码序列的人IFN-γcDNA所用的引物如下:有义引物:5’-ccg ctc gag tgt tac tgc cag gac-3’;反义链:5’-gctct agatta ctg gga tgc tct tcg-3’。PCR反应体积为50μl,其中含逆转录模板2μl、0.4mM引物、0.2mM dNTP和1U BlendTaq DNA聚合酶(Takara)。扩增参数为94℃30秒、42℃30秒、72℃30秒,30个循环后PCR产物行1%琼脂糖凝胶电泳初步确认。DNA序列测定结果表明该PCR产物的编码DNA序列与GenBank中的IFN-γ(NM00619)cDNA序列完全相同。
实施例2:人sVEGFR2-IFN-γ融合蛋白真核表达载体的构建
所用的真核表达载体为pcDNA3.1(+)(Invitrogen)。它含有CMV启动子、SV40复制起始点、新霉素抗性基因,可进行目的基因的短暂表达和稳定表达。外源基因克隆位点的两端分别是含有T7启动子和牛生长激素BGH的polyA信号,可用T7和BGH通用引物对目的外源基因进行直接测序。以实施例1同样的方法,以PCR分别扩增人sVEGFR2的cDNA和无信号肽编码序列的IFN-γcDNA。用BamHI和EcoRI双酶切人sVEGFR2的PCR产物,并将其直接插入同样酶切的真核表达载体pcDNA3.1(+);用XhoI和XbaI双酶切无信号肽编码序列的IFN-γPCR产物,然后将其插入到带有人sVEGFR2cDNA的pcDNA3.1(+)的XhoI和XbaI克隆位点之间,构建成表达人sVEGFR2和IFN-γ融合蛋白的真核表达载体——pcDNA3.1(+)/sVEGFR2-IFN-γ,其基因序列经测序验证。
实施例3:人sVEGFR2-IFN-γ融合蛋白真核重组表达与活性检测
将COS-7细胞按1×105/孔接种于24孔板,培养24小时后,按照LipofectamineTM2000(Invitrogen)说明书操作,将pcDNA3.1(+)/sVEGFR2-IFN-γ重组质粒与Lipofectamine按一定比例混合,转染COS-7细胞。于48小时后收集细胞培养上清进行生物活性检测。
1.检测融合蛋白sVEGFR2活性的血管内皮细胞增殖抑制试验:将100μl细胞培养上清与重组人VEGF165(BioVision,1ng/μl)10μl混合1h。将脐静脉内皮细胞按每孔5×103接种于96孔板,以含10%FCS的DMEM培养,2h后加入上述混合液为实验组,同时设VEGF165和培养液对照组。在培养结束前4h每孔加MTT(5mg/ml)10μl,继续培养至24h,弃上清,加入二甲基亚砜(DMSO)150μl,酶标仪检测OD570nm。此实验重复三次,每组设3个复孔。结果见附图1。
2.融合蛋白IFN-γ活性的检测:用密度梯度离心法分离外周血单个核细胞,以含10%FCS的RPMI1640完全培养液调整细胞浓度为1×107/ml,每孔1ml接种于24孔板,加入700μl的pcDNA3.1(+)/sVEGFR2-IFN-γ转染的COS-7细胞培养上清,并以RPMI1640完全培养液补足体积到每孔2ml;同时设RPMI1640对照孔,培养48h后,收集细胞,调节细胞浓度为1×106/ml作为效应细胞;将处于对数生长期的K562细胞作为靶细胞,调节细胞浓度为1×105/ml。将100μl效应细胞和100μl靶细胞(效靶比为10∶1)加入96孔板作为实验组,同时设靶细胞和效应细胞对照组。37℃孵育4h后,每孔加入MTT(5mg/ml)10μl,继续培养4h,弃上清,加入DMSO150μl,酶标仪读取OD570nm。此实验重复3次,每组设3个复孔。按下式计算NK活性:
结果见附图2。
实施例4:稳定表达人sVEGFR2-IFN-γ的真核细胞系的建立
所用的真核细胞系为dhfr-的CHO细胞系——DG44。将5×105的DG44细胞接种于6cm培养皿,在HAM’S/F-12培养基(Hyclone,含7%胎牛血清,30μM胸腺嘧啶,150ng/ml脯氨酸,4mM L-谷氨酰胺)中培养24h,将pcDNA3.1(+)/sVEGFR2-IFN-γ与pSV2-dhfr+载体按摩尔数之比9∶1共转染DG44,具体操作按LipofectamineTM2000(Invitrogen)说明书进行。转染后24h,加入G418终浓度为800μg/ml维持2周,每2-3天换液一次。2周后,收集细胞培养上清,ELISA检测sVEGFR2和IFN-γ的表达情况,并降低G418终浓度至300μg/ml继续筛选1周。
G418筛选完成后,在DG44细胞中以甲氨蝶呤(MTX,Invitrogen)1nM、10nM、20nM、50nM、100nM、200nM、500nM、1000nM的浓度梯度加压筛选,每一浓度梯度维持2周。每一浓度梯度时都对细胞培养上清中目的蛋白浓度进行检测,将表达目的蛋白最高者用于下一浓度梯度的筛选。将表达目的蛋白最高的DG44细胞以每孔0.5-1个细胞接种到96孔板,以含有500nM MTX的DMEM/F-12(Hyclone)培养基培养。2周左右能看到克隆形成,检测单克隆细胞上清目的蛋白的表达,将表达最高的6孔传到6孔板扩大培养一周左右,再行目的蛋白检测,选取表达最高孔的细胞继续下一轮有效稀释。经过三轮有限稀释后得到高效稳定表达sVEGFR2-IFN-γ融合蛋白的CHO细胞系(CHO/sVEGFR2-IFN-γ)。
实施例5:人sVEGFR2-IFN-γ的分离纯化
将1×105的CHO/sVEGFR2-IFN-γ细胞接种于WHEATON-33滚瓶,待细胞贴壁生长到70%左右时,换成50ml无血清DMEM/F 12培养基继续滚动培养(每分钟2转)至培养液变黄,收集细胞培养上清,12000rpm离心30min,用Labscale TFF超滤装置纯化浓缩上清液,先后用200KD和50KD的膜包去除杂蛋白并浓缩上清液。超滤后上清用explorer蛋白纯化仪进行纯化,先用HiTrap Desalting脱盐,脱盐后用DEAE Sepharose Fast Flow填料装柱进行阴离子交换层析。用pH 6.0,20mM的磷酸盐缓冲液平衡,加样品,用平衡缓冲液充分洗脱以除去未吸附蛋白,再用含1M的NaCl的磷酸盐缓冲液进行洗脱,收集融合蛋白所在的峰。经HIC Phenyl Sepharose 6FF(high sub)柱疏水层析,以pH6.0、50mM磷酸盐缓冲液梯度洗脱,收集目的蛋白所在峰。SDS-PAGE电泳,考马斯亮蓝R250染色后用凝胶扫描仪扫描检测样品纯度。收集的融合蛋白于Beckman DU640蛋白核酸分析系统进行定量,-80℃备用。融合蛋白于Savant真空低温干燥系统冻干后保存。
本发明涉及的序列
<160>6
<210>1
<211>2772
<212>DNA
<213>人工序列
<220>1
<221>CDS
<222>(1)...(2772)
<223>编码人sVEGFR2-IFN-gamma融合蛋白的基因序列
<400>1
1 atggagagca aggtgctgct ggccgtcgcc ctgtggctct gcgtggagac ccgggccgcc
61 tctgtgggtt tgcctagtgt ttctcttgat ctgcccaggc tcagcataca aaaagacata
121 cttacaatta aggctaatac aactcttcaa attacttgca ggggacagag ggacttggac
181 tggctttggc ccaataatca gagtggcagt gagcaaaggg tggaggtgac tgagtgcagc
241 gatggcctct tctgtaagac actcacaatt ccaaaagtga tcggaaatga cactggagcc
301 tacaagtgct tctaccggga aactgacttg gcctcggtca tttatgtcta tgttcaagat
361 tacagatctc catttattgc ttctgttagt gaccaacatg gagtcgtgta cattactgag
421 aacaaaaaca aaactgtggt gattccatgt ctcgggtcca tttcaaatct caacgtgtca
481 ctttgtgcaa gatacccaga aaagagattt gttcctgatg gtaacagaat ttcctgggac
541 agcaagaagg gctttactat tcccagctac atgatcagct atgctggcat ggtcttctgt
601 gaagcaaaaa ttaatgatga aagttaccag tctattatgt acatagttgt cgttgtaggg
661 tataggattt atgatgtggt tctgagtccg tctcatggaa ttgaactatc tgttggagaa
721 aagcttgtct taaattgtac agcaagaact gaactaaatg tggggattga cttcaactgg
781 gaataccctt cttcgaagca tcagcataag aaacttgtaa accgagacct aaaaacccag
841 tctgggagtg agatgaagaa atttttgagc accttaacta tagatggtgt aacccggagt
901 gaccaaggat tgtacacctg tgcagcatcc agtgggctga tgaccaagaa gaacagcaca
961 tttgtcaggg tccatgaaaa accttttgtt gcttttggaa gtggcatgga atctctggtg
1021 gaagccacgg tgggggagcg tgtcagaatc cctgcgaagt accttggtta cccaccccca
1081 gaaataaaat ggtataaaaa tggaataccc cttgagtcca atcacacaat taaagcgggg
1141 catgtactga cgattatgga agtgagtgaa agagacacag gaaattacac tgtcatcctt
1201 accaatccca tttcaaagga gaagcagagc catgtggtct ctctggttgt gtatgtccca
1261 ccccagattg gtgagaaatc tctaatctct cctgtggatt cctaccagta cggcaccact
1321 caaacgctga catgtacggt ctatgccatt cctcccccgc atcacatcca ctggtattgg
1381 cagttggagg aagagtgcgc caacgagccc agccaagctg tctcagtgac aaacccatac
1441 ccttgtgaag aatggagaag tgtggaggac ttccagggag gaaataaaat tgaagttaat
1501 aaaaatcaat ttgctctaat tgaaggaaaa aacaaaactg taagtaccct tgttatccaa
1561 gcggcaaatg tgtcagcttt gtacaaatgt gaagcggtca acaaagtcgg gagaggagag
1621 agggtgatct ccttccacgt gaccaggggt cctgaaatta ctttgcaacc tgacatgcag
1681 cccactgagc aggagagcgt gtctttgtgg tgcactgcag acagatctac gtttgagaac
1741 ctcacatggt acaagcttgg cccacagcct ctgccaatcc atgtgggaga gttgcccaca
1801 cctgtttgca agaacttgga tactctttgg aaattgaatg ccaccatgtt ctctaatagc
1861 acaaatgaca ttttgatcat ggagcttaag aatgcatcct tgcaggacca aggagactat
1921 gtctgccttg ctcaagacag gaagaccaag aaaagacatt gcgtggtcag gcagctcaca
1981 gtcctagagc gtgtggcacc cacgatcaca ggaaacctgg agaatcagac gacaagtatt
2041 ggggaaagca tcgaagtctc atgcacggca tctgggaatc cccctccaca gatcatgtgg
2101 tttaaagata atgagaccct tgtagaagac tcaggcattg tattgaagga tgggaaccgg
2161 aacctcacta tccgcagagt gaggaaggag gacgaaggcc tctacacctg ccaggcatgc
2221 agtgttcttg gctgtgcaaa agtggaggca tttttcataa tagaaggtgc ccaggaaaag
2281 acgaacttgg aagaattctg cagatatcca gcacagtggc ggccgctcga gtgttactgc
2341 caggacccat atgtaaaaga agcagaaaac cttaagaaat attttaatgc aggtcattca
2401 gatgtagcgg ataatggaac tcttttctta ggcattttga agaattggaa agaggagagt
2461 gacagaaaaa taatgcagag ccaaattgtc tccttttact tcaaactttt taaaaacttt
2521 aaagatgacc agagcatcca aaagagtgtg gagaccatca aggaagacat gaatgtcaag
2581 tttttcaata gcaacaaaaa gaaacgagat gacttcgaaa agctgactaa ttattcggta
2641 actgacttga atgtccaacg caaagcaata catgaactca tccaagtgat ggctgaactg
2701 tcgccagcag ctaaaacagg gaagcgaaaa aggagtcaga tgctgtttcg aggtcgaaga
2761 gcatcccagt aa
<210>2
<211>923
<212>PRT
<213>人工序列
<220>
<221>CDS
<222>(1)...(923)
<223>sVEGFR2-IFN-gamma融合蛋白的氨基酸序列
<400>2
Met Glu Ser Lys Val Leu Leu Ala Val Ala Leu Trp Leu Cys Val
1 6 11
Glu Thr Arg Ala Ala Ser Val Gly Leu Pro Ser Val Ser Leu Asp
16 21 26
Leu Pro Arg Leu Ser Ile Gln Lys Asp Ile Leu Thr Ile Lys Ala
31 36 41
Asn Thr Thr Leu Gln Ile Thr Cys Arg Gly Gln Arg Asp Leu Asp
46 51 56
Trp Leu Trp Pro Asn Asn Gln Ser Gly Ser Glu Gln Arg Val Glu
61 66 71
Val Thr Glu Cys Ser Asp Gly Leu Phe Cys Lys Thr Leu Thr Ile
76 81 86
Pro Lys Val Ile Gly Asn Asp Thr Gly Ala Tyr Lys Cys Phe Tyr
91 96 101
Arg Glu Thr Asp Leu Ala Ser Val Ile Tyr Val Tyr Val Gln Asp
106 111 116
Tyr Arg Ser Pro Phe Ile Ala Ser Val Ser Asp Gln His Gly Val
121 126 131
Val Tyr Ile Thr Glu Asn Lys Asn Lys Thr Val Val Ile Pro Cys
136 141 146
Leu Gly Ser Ile Ser Asn Leu Asn Val Ser Leu Cys Ala Arg Tyr
151 156 161
Pro Glu Lys Arg Phe Val Pro Asp Gly Asn Arg Ile Ser Trp Asp
166 171 176
Ser Lys Lys Gly Phe Thr Ile Pro Ser Tyr Met Ile Ser Tyr Ala
181 186 191
Gly Met Val Phe Cys Glu Ala Lys Ile Asn Asp Glu Ser Tyr Gln
196 201 206
Ser Ile Met Tyr Ile Val Val Val Val Gly Tyr Arg Ile Tyr Asp
211 216 221
Val Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu
226 231 236
Lys Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly
241 246 251
Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys
256 261 266
Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met
271 276 281
Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser
286 291 296
Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr
301 306 311
Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Pro Phe Val
316 321 326
Ala Phe Gly Ser Gly Met Glu Ser Leu Val Glu Ala Thr Val Gly
33 1336 341
Glu Arg Val Arg Ile Pro Ala Lys Tyr Leu Gly Tyr Pro Pro Pro
346 351 356
Glu Ile Lys Trp Tyr Lys Asn Gly Ile Pro Leu Glu Ser Asn His
361 366 371
Thr Ile Lys Ala Gly His Val Leu Thr Ile Met Glu Val Ser Glu
376 381 386
Arg Asp Thr Gly Asn Tyr Thr Val Ile Leu Thr Asn Pro Ile Ser
391 396 401
Lys Glu Lys Gln Ser His Val Val Ser Leu Val Val Tyr Val Pro
406 411 416
Pro Gln Ile Gly Glu Lys Ser Leu Ile Ser Pro Val Asp Ser Tyr
421 426 431
Gln Tyr Gly Thr Thr Gln Thr Leu Thr Cys Thr Val Tyr Ala Ile
436 441 446
Pro Pro Pro His His Ile His Trp Tyr Trp Gln Leu Glu Glu Glu
451 456 461
Cys Ala Asn Glu Pro Ser Gln Ala Val Ser Val Thr Asn Pro Tyr
466 471 476
Pro Cys Glu Glu Trp Arg Ser Val Glu Asp Phe Gln Gly Gly Asn
481 486 491
Lys Ile Glu Val Asn Lys Asn Gln Phe Ala Leu Ile Glu Gly Lys
496 501 506
Asn Lys Thr Val Ser Thr Leu Val Ile Gln Ala Ala Asn Val Ser
511 516 521
Ala Leu Tyr Lys Cys Glu Ala Val Asn Lys Val Gly Arg Gly Glu
526 531 536
Arg Val Ile Ser Phe His Val Thr Arg Gly Pro Glu Ile Thr Leu
541 546 551
Gln Pro Asp Met Gln Pro Thr Glu Gln Glu Ser Val Ser LeuTrp
556 561 566
Cys Thr Ala Asp Arg Ser Thr Phe Glu Asn Leu Thr Trp Tyr Lys
571 576 581
Leu Gly Pro Gln Pro Leu Pro Ile His Val Gly Glu Leu Pro Thr
586 591 596
Pro Val Cys Lys Asn Leu Asp Thr Leu Trp Lys Leu Asn Ala Thr
601 606 611
Met Phe Ser Asn Ser Thr Asn Asp Ile Leu Ile Met Glu Leu Lys
616 621 626
Asn Ala Ser Leu Gln Asp Gln Gly Asp Tyr Val Cys Leu Ala Gln
631 636 641
Asp Arg Lys Thr Lys Lys Arg His Cys Val Val Arg Gln Leu Thr
646 651 656
Val Leu Glu Arg Val Ala Pro Thr Ile Thr Gly Asn Leu Glu Asn
661 666 671
Gln Thr Thr Ser Ile Gly Glu Ser Ile Glu Val Ser Cys Thr Ala
676 681 686
Ser Gly Asn Pro Pro Pro Gln Ile Met Trp Phe Lys Asp Asn Glu
691 696 701
Thr Leu Val Glu Asp Ser Gly Ile Val Leu Lys Asp Gly Asn Arg
706 711 716
Asn Leu Thr Ile Arg Arg Val Arg Lys Glu Asp Glu Gly Leu Tyr
721 726 731
Thr Cys Gln Ala Cys Ser Val Leu Gly Cys Ala Lys Val Glu Ala
736 741 746
Phe Phe Ile Ile Glu Gly Ala Gln Glu Lys Thr Asn Leu Glu Glu
751 756 761
Phe Cys Arg Tyr Pro Ala Gln Trp Arg Pro Leu Glu Cys Tyr Cys
766 771 776
Gln Asp Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr Phe
781 786 791
Asn Ala Gly His Ser Asp Val Ala Asp Asn Gly Thr Leu Phe Leu
796 801 806
Gly Ile Leu Lys Asn Trp Lys Glu Glu Ser Asp Arg Lys Ile Met
811 816 821
Gln Ser Gln Ile Val Ser Phe Tyr Phe Lys Leu Phe Lys Asn Phe
826 831 836
Lys Asp Asp Gln Ser Ile Gln Lys Ser Val Glu Thr Ile Lys Glu
841 846 851
Asp Met Asn Val Lys Phe Phe Asn Ser Asn Lys Lys Lys Arg Asp
856 861 866
Asp Phe Glu Lys Leu Thr Asn Tyr Ser Val Thr Asp Leu Asn Val
871 876 881
Gln Arg Lys Ala Ile His Glu Leu Ile Gln Val Met Ala Glu Leu
886 891 896
Ser Pro Ala Ala Lys Thr Gly Lys Arg Lys Arg Ser Gln Met Leu
901 906 911
Phe Arg Gly Arg Arg Ala Ser Gln
916 921
<210>3
<211>23
<212>DNA
<213>人工序列
<220>
<221>CDS
<222>(1)...(23)
<223>按照大肠杆菌偏爱的密码子,设计了酶切位点的sVEGFR2有义链PCR引物序列
<400>3
1 cgggatccat ggagagcaag gtg
<210>4
<211>27
<212>DNA
<213>人工序列
<220>
<221>CDS
<222>(1)...(27)
<223>按照大肠杆菌偏爱的密码子,设计了酶切位点的sVEGFR2反义链PCR引物序列
<400>4
1 cggaattctt ccaagttcgt cttttcc
<210>5
<211>24
<212>DNA
<213>人工序列
<220>
<221>CDS
<222>(1)...(24)
<223>按照大肠杆菌偏爱的密码子,设计了酶切位点的IFN-γ有义链PCR引物序列
<400>5
1 ccgctcgagt gttactgcca ggac
<210>6
<211>26
<212>DNA
<213>人工序列
<220>
<221>CDS
<222>(1)...(26)
<223>按照大肠杆菌偏爱的密码子,设计了酶切位点的IFN-γ反义链PCR引物序列
<400>6
1 gctctagatt actgggatgc tcttcg
Claims (2)
1.一种可溶性血管内皮生长因子受体重组融合蛋白,涉及可溶性VEGFR2与γ-干扰素基因重组融合蛋白,具有SEQ ID №:1的DNA序列和SEQ ID №:2的氨基酸序列。
2.根据权利要求1所述的可溶性血管内皮生长因子受体重组融合蛋白在制备抗肿瘤血管生成药物中的应用。
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104531735A (zh) * | 2014-10-30 | 2015-04-22 | 浙江大学城市学院 | 血管内皮生长因子受体2抗原肽-mhc i类分子单链三聚体dna疫苗及用途 |
CN108671229A (zh) * | 2018-05-08 | 2018-10-19 | 华博生物医药技术(上海)有限公司 | 一种重组人血管内皮生长因子受体-抗体融合蛋白的药物组合制剂 |
CN117003878A (zh) * | 2023-09-28 | 2023-11-07 | 南京佰抗生物科技有限公司 | 抗sFLT-1蛋白的单克隆抗体组合物及应用 |
-
2009
- 2009-05-21 CN CN200910098685A patent/CN101781370A/zh active Pending
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104531735A (zh) * | 2014-10-30 | 2015-04-22 | 浙江大学城市学院 | 血管内皮生长因子受体2抗原肽-mhc i类分子单链三聚体dna疫苗及用途 |
CN108671229A (zh) * | 2018-05-08 | 2018-10-19 | 华博生物医药技术(上海)有限公司 | 一种重组人血管内皮生长因子受体-抗体融合蛋白的药物组合制剂 |
CN117003878A (zh) * | 2023-09-28 | 2023-11-07 | 南京佰抗生物科技有限公司 | 抗sFLT-1蛋白的单克隆抗体组合物及应用 |
CN117003878B (zh) * | 2023-09-28 | 2023-12-05 | 南京佰抗生物科技有限公司 | 抗sFLT-1蛋白的单克隆抗体及应用 |
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