专利名称：可溶性血管内皮生长因子受体重组融合蛋白及用途的制作方法
技术领域：
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
CN 101781370 A
1 61 121 181 241 301 361 421 481 541 601 661 721 781 841 901 961 1021 1081 1141 1201 1261 1321 1381 1441 1501 1561 1621 1681 1741 1801
tctgtgggtt cttacaatta tggctttggc gatggcctct tacaagtgct tacagatctc
ctttgtgcaa
3gC33g皿gg
g皿gca皿^ tataggattt aagcttgtct gaataccctt tctgggagtg
tttgtcaggg g皿gccacgg
catgtactga
3CC^tCCC3
ccccagattg
C皿3CgCtg3 C3gttgg鄉
ccttgtgaag
gcggca皿tg
cccactgagc ctcacatggt cctgtttgca
aggtgctgct tgcctagtgt
tctgtaagac tctaccggga catttattgc 皿actgtggt gatecccag3 gctttactat
3tg3tgtggt teaattgtac cttcgaagca agatg^g^ tgtacacctg
tgggggagcg
gtgaga皿tc catgtacggt 皿gagtgcgc 皿tggag^g ttgctcteat tgtcagcttt ccttccacgt 郷卿gcgt
ggccgtcgcc ttctcttgat aactcttcaa gagtggcagt actcacaatt aactgacttg ttctgttagt gattccatgt
tcccagctac
tctgagtccg agc^g^ct tcagc3teag atttttgagc tgcagcatcc accttttgtt tgtcagaatc tggaateccc 3gtgagtg^ g^gcagagc tctaatctct ctatgccatt caacgagccc tgtgg郷ac
gacc郷ggt gtctttgtgg cccacagcct tectctttgg
ctgtggctct ctgcccaggc attacttgca gag固鄉g
gcctcggtca gacc皿catg ctcgggtcca gttcctgatg atgatcagct tctattatgt tctcatggaa
acctteacta
3gtgggCtg3
gcttttggaa cctgcgaagt cttgagtcca
catgtggtct cctgtggatt cctcccccgc agcc皿gctg ttcc郷gag
g33gCggtC3
cctgaaatta tgcactgcag ctgccaatcc
gcgtgg卿c tcagcataca ggggac卿g tgg郷tgac tcgg皿atg3 tttatgtcta gagtcgtgte tttcaaatct
atgctggcat acatagttgt ttgaactatc tggggattga accgagacct t卿tggtgt tgacc^g^ gtggratgga accttggtta
ctctggttgt cctaccagta atcacatcca tctcagtgac
taagteccct
3C^3gtCgg
ctttgcaacc acagatctac "gtggg卿 ccaccatgtt
ccgggccgcc ■■gacate ggacttggac tgagtgcagc cactggagcc tgttcaagat cattectgag caacgtgtca ttcctgggac ggtcttctgt cgttgtaggg tgttgg卿a cttcaactgg
■cccggagt
g^C3gC3C3
atctctggtg
CCC3CCCCC3
te卿cgggg tgtcatcctt gtatgtccca cggcaccact ctggtettgg
tgttetccaa g卿gg卿g
gtttgag皿c gttgcccaca ctcteatagc
7
ttttgatcatggagctt卿aatgcatccttgC3gg3CC3
gtctgccttgctc^gacagg^gacc^ggcgtggtcaggcagctcaca
gtcctagagcgtgtggcacccacgatcacagg333CCtgg
gggg^3gC3tcgaagtctc3tgC3CggC3tctgggaatcCCCCtCC3C3gatratgtgg
atgagacccttcaggcattgtggg肌ccgg
aacctcactatccgcagagtgacg皿ggcctctacacctgccaggcatgc
agtgttcttggctgtgc^atttttcataaCC3gg^^g
cagatatccagC3C3gtggCggccgctcgagtgttactgc
caggacccatatttteatgcaggtcattca
gatgtegcggtcttttcttaggcattttga卿gg卿gt
ccaaattgtctccttttacttcaaactttt
皿卿gtgtgg^tgtc^g
tttttcaatagc^ca皿^gacttcg3^ttattcggte
actgacttgaatgtccaacgcatgaactcatccaagtgatggctg咖tg
tcgccagcagg^gcg^^tgctgtttcg
gcatcccagt
1861192119812041210121612221228123412401246125212581264127012761〈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
Glu Ser Lys Val Leu Leu Ala Val Ala Leu6
Ala Ala Ser21
Leu Ser lie36
Leu Gin lie51
Pro Asn Asn66Asp81Asn96
Met1
Glu
16
Leu
31
Asn
46
Trp
61
Val
76
Pro
91
Thr
Pro
Thr
Leu
Thr
ArgArgThrTrpGlu
CysLys Val lie
Ser
Gly
ValGinThrGinGlyAsp
Gly LeuLys AspCys ArgSer GlyLeu PheThr Gly
ProlieGlySerCysAla
11
Ser
26
Leu
41
Gin
56
Glu
71
Lys
86
Tyr
101
Trp Leu Cys ValVal Ser Leu AspThr lie Lys AlaArg Asp Leu AspGin Arg Val GluThr Leu Thr lieLys Cys Phe Tyr
8[o川]ArgGluThrAspLeuAlaSerVallieTyrValTyrValGinAsp106111116TyrArgSerProPhelieAlaSerValSerAspGinHisGlyVal121126131ValTyrlieThrGluAsnLysAsnLysThrValVallieProCys136141146LeuGlySerlieSerAsnLeuAsnValSerLeuCysAlaArgTyr151156161ProGluLysArgPheValProAspGlyAsnArglieSerTrpAsp166171176SerLysLysGlyPheThrlieProSerTyrMetlieSerTyrAla181186191GlyMetValPheCysGluAlaLyslieAsnAspGluSerTyrGin196201206SerlieMetTyrlieValValValValGlyTyrArglieTyrAsp211216221ValValLeuSerProSerHisGlylieGluLeuSerValGlyGlu226231236LysLeuValLeuAsnCysThrAlaArgThrGluLeuAsnValGly241246251lieAspPheAsnTrpGluTyrProSerSerLysHisGinHisLys256261266LysLeuValAsnArgAspLeuLysThrGinSerGlySerGluMet271276281LysLysPheLeuSerThrLeuThrlieAspGlyValThrArgSer286291296AspGinGlyLeuTyrThrCysAlaAlaSerSerGlyLeuMetThr301306311LysLysAsnSerThrPheValArgValHisGluLysProPheVal316321326AlaPheGlySerGlyMetGluSerLeuValGluAlaThrValGly331336341GluArgValArglieProAlaLysTyrLeuGlyTyrProProPro346351356GlulieLysTrpTyrLysAsnGlylieProLeuGluSerAsnHis361366371ThrlieLysAlaGlyHisValLeuThrlieMetGluValSerGlu376381386ArgAspThrGlyAsnTyrThrVallieLeuThrAsnProlieSer
9
391396401LysGluLysGinSerHisValValSerLeuValValTyrValPro406411416ProGinlieGlyGluLysSerLeulieSerProValAspSerTyr421426431GinTyrGlyThrThrGinThrLeuThrCysThrValTyrAlalie436441446ProProProHisHislieHisTrpTyrTrpGinLeuGluGluGlu451456461CysAlaAsnGluProSerGinAlaValSerValThrAsnProTyr466471476ProCysGluGluTrpArgSerValGluAspPheGinGlyGlyAsn481486491LyslieGluValAsnLysAsnGinPheAlaLeulieGluGlyLys496501506AsnLysThrValSerThrLeuVallieGinAlaAlaAsnValSer511516521AlaLeuTyrLysCysGluAlaValAsnLysValGlyArgGlyGlu526531536ArgVallieSerPheHisValThrArgGlyProGlulieThrLeu541546551GinProAspMetGinProThrGluGinGluSerValSerLeuTrp
556561566CysThrAlaAspArgSerThrPheGluAsnLeuThrTrpTyrLys571576581LeuGlyProGinProLeuProlieHisValGlyGluLeuProThr586591596ProValCysLysAsnLeuAspThrLeuTrpLysLeuAsnAlaThr601606611MetPheSerAsnSerThrAsnAsplieLeulieMetGluLeuLys616621626AsnAlaSerLeuGinAspGinGlyAspTyrValCysLeuAlaGin631636641AspArgLysThrLysLysArgHisCysValValArgGinLeuThr646651656ValLeuGluArgValAlaProThrlieThrGlyAsnLeuGluAsn661666671GinThrThrSerlieGlyGluSerlieGluValSerCysThrAla676681686
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0189]SerGlyAsnPro Pro Pro
0190]691696
0191]ThrLeuValGlu Asp Ser
0192]706711
0193]AsnLeuThrlie Arg Arg
0194]721726
0195]ThrCysGinAla Cys Ser
0196]736741
0197]PhePhelielie Glu Gly
0198]751756
0199]PheCysArgTyr Pro Ala
0200]766771
0201]GinAspProTyr Val Lys
0202]781786
0203]AsnAlaGlyHis Ser Asp
0204]796801
0205]GlylieLeuLys Asn Trp
0206]811816
0207]GinSerGinlie Val Ser
0208]826831
0209]LysAspAspGin Ser lie
0210]841846
0211]AspMetAsnVal Lys Phe
0212]856861
0213]AspPheGluLys Leu Thr
0214]871876
0215]GinArgLysAla lie His
0216]886891
0217]SerProAlaAla Lys Thr
0218]901906
0219]PheArgGlyArg Arg Ala
0220]916921
0221]〈210>3
0222]〈211>23
0223]〈212>DNA
0224]〈213〉人工序列0225]〈220〉
0226]〈221>CDS
0227]〈22:》(1) (23)
Gin lie Met Trp Phe Lys Asp Asn Glu701
Gly lie Val Leu Lys Asp Gly Asn Arg716
Val Arg Lys Glu Asp Glu Gly Leu Tyr731
Val Leu Gly Cys Ala Lys Val Glu Ala746
Ala Gin Glu Lys Thr Asn Leu Glu Glu761
Gin Trp Arg Pro Leu Glu Cys Tyr Cys776
Glu Ala Glu Asn Leu Lys Lys Tyr Phe791
Val Ala Asp Asn Gly Thr Leu Phe Leu806
Lys Glu Glu Ser Asp Arg Lys lie Met821
Phe Tyr Phe Lys Leu Phe Lys Asn Phe836
Gin Lys Ser Val Glu Thr lie Lys Glu851
Phe Asn Ser Asn Lys Lys Lys Arg Asp866
Asn Tyr Ser Val Thr Asp Leu Asn Val881
Glu Leu lie Gin Val Met Ala Glu Leu896
Gly Lys Arg Lys Arg Ser Gin Met Leu911
Ser Gin
〈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-Y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-Y反义链PCR引物序 列 〈400>6 1 gctctagatt actgggatgc tcttcg
权利要求
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。
全文摘要
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。
文档编号A61K38/17GK101781370SQ20091009868
公开日2010年7月21日 申请日期2009年5月21日 优先权日2009年5月21日
发明者姚航平, 张立煌, 潘建平, 潘观根 申请人:浙江大学