CN1291230A - 可结合生物素的受体分子 - Google Patents
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Abstract
一种新的跨膜蛋白能够结合生物素化分子,该蛋白质含有胞质结构域、跨膜结构域和胞外结构域,其中胞外结构域有生物素结合活性。
Description
发明领域
本发明涉及有结合生物素活性的跨膜蛋白。
发明背景
生物素(维生素H)是血液和组织里的一种浓度较低的水溶性物质。生物素的生物学作用是作为活化的CO2的一种载体,它在不需要额外自由能的情况下将CO2转移到受体。活化的羧基生物素通常附着在一种羧基生物素形成所必需的酶上。例如,生物素可以附着在丙酮酸羧化酶上,在乙酰CoA存在情况下,丙酮酸羧化酶催化羧基生物素的形成及随后将活化的羧基转移到丙酮酸形成草酰乙酸。
生物素也与来自鸡卵清的一种63kDa糖蛋白-亲和素及来自阿维丁链霉菌(Streptomyces avidinii)的一种非糖基化蛋白-链霉亲和素结合,它们的亲和力是自然界中已知最高的亲和力之一。这种结合的特点是几乎不可逆(Ka 1015 mol-1)。已证明亲和素和生物素间的亲和力在各种生物分析应用中非常有用。例如,亲和素-生物素复合物已被成功地用于各种检测系统,其中靶分子通过其羧基末端与生物素结合形成生物素化的分子,这种分子能比较容易地检测或从溶液里分离。生物素化不改变许多分子的生物学或物理化学特性,也不影响生物素辅基与亲和素结合的结合能力。
发明概述
现在已经认识到亲和素和链霉亲和素的生物素结合活性可以用来生产能结合生物素化分子的跨膜蛋白。
本发明中的蛋白质可以包含胞质结构域、跨膜结构域和胞外结构域,其中胞外结构域含有生物素结合活性。胞外结构域可以有亲和素或链霉亲和素的功能活性。
使用本发明中的蛋白或核酸分子有可能将生物素化的分子定位在组织中的特定位点。用这种方法定向的分子可通过内吞作用被组织或细胞吸收,使得这些分子可以在细胞内或细胞上发挥它们的作用。
附图简述
图1是本发明中融合蛋白的结构示意图,A代表亲和素,B代表内吞受体的跨膜结构域(C代表生物素);
图2是用穿梭载体进行克隆的策略图;
图3是用逆转录病毒载体进行克隆的策略图。
发明详述
本发明中的蛋白质可以用常规的重组DNA技术生产。通常,将编码生物素结合蛋白如亲和素、链霉亲和素或相关蛋白的功能结构域的DNA序列插入一种含有编码具有跨膜特性之蛋白质的DNA序列的基因工程构建体中。亲和素和链霉亲和素相关蛋白的实例包括AVR-1-AVR-5,AVR-X-AVR-V,Stv1和Stv2。
融合蛋白的各个结构域可通过聚合酶链式反应进行扩增,或用限制性酶消化,例如用凝胶电泳进行分离和纯化,随后用DNA连接酶进行连接,从而从亲本cDNA中分离出来。融合蛋白的构建体可以转染到任何适合的宿主细胞中,用标准的蛋白纯化方法进行培养和分离。
构建体也可以作为裸DNA或作为质粒/脂质体、质粒/聚乙烯亚胺、质粒/树枝状聚合物(dendrimer)或质粒/肽复合物使用。
或者,可将构建体导入复制缺陷型病毒中,它能使构建体定位在体内的特定位点。例如,构建体可以是含有编码融合蛋白的适当cDNA的逆转录病毒载体。一种复制缺陷型逆转录病毒,如莫洛尼鼠逆转录病毒,可以用于稳定转染靶细胞和组织。也可以使用其他病毒如复制缺陷型腺病毒、腺伴随病毒、肝病毒、乳头瘤病毒和Sinibis病毒。其他病毒也是本领域技术人员所熟知的。
除了亲和素、链霉亲和素或相关蛋白的功能结构域外,融合蛋白一般还包括内吞型受体的跨膜结构域。这些受体的应用使得能够将生物素化分子摄入靶细胞。可用于本发明的适当受体包括A类清除剂受体、低密度脂蛋白受体、极低密度脂蛋白受体、转铁蛋白受体和LOX-1受体。融合蛋白还可以含有位于受体蛋白和亲和素肽之间的连接子。只要不影响融合蛋白不同组成部分的功能活性,连接子可以任意长。
通常,亲和素或链霉亲和素肽序列与受体肽序列之间的融合是受体蛋白的胞外结构域与亲和素或链霉亲和素的生物素结合位点以外的任何位点的融合。
下面的实施例说明了本发明。
实施例
构建牛A类清除剂受体(ScR)(Kodama et al.(1990)Nature343:531-535)和生物素(Green(1975)Adv.Prot.Chem.29:85-133)之间的DNA构建体,该构建体编码的蛋白质有ScR胞质结构域、跨膜结构域和α-螺旋卷曲结构域,并连接着生物素结合结构域。融合蛋白完整的氨基酸序列显示在SQE ID No:2中,其中1-53位氨基酸是胞质结构域;55-79位氨基酸是跨膜结构域;81-111位氨基酸是间隔区;113-272位氨基酸是α-螺旋卷曲结构域。273-400位氨基酸是来源于亲和素cDNA(Gope et al.(1987)Nucleic Acid Res.15:3595-3606)的缺乏分泌信号的成熟亲和素肽序列。
简要地,从已转染质粒(pLScRNL)的培养细胞中得到ScR的cDNA,该质粒含有ScR cDNA以及内部劳氏肉瘤病毒启动子和HindⅢ位点。然后将分离的cDNA插入逆转录病毒载体pLSlARNL的HindⅢ位点。由聚合酶链式反应产生亲和素cDNA,将其插入逆转录病毒载体中ScR cDNA内的StyⅠ位点处。本发明的cDNA显示在SEQ ID No 1,其中1-989位核苷酸是Mo-MuSV的长末端重复区;1071-2270位核苷酸是融合蛋白的编码区;2376-3101位核苷酸是牛清除剂受体Ⅰ cDNA的非翻译区;3107-3376位核苷酸是RSV启动子区;3727-4522位核苷酸是neoR基因,4540-5177位核苷酸是Mo-MuLV的长末端重复区。
图2和图3表示本实施例中所用的方法。更具体地,图2显示怎样利用HindⅢ将含有内部RSV启动子的ScR cDNA从质粒pLScRNL切出,并克隆到穿梭载体的HindⅢ位点。图3显示如何将ScR-亲和素-RSVcDNA克隆到逆转录病毒载体pLRNL的HindⅢ位点。
转染了该载体的细胞中融合蛋白的表达可通过用抗亲和素抗体进行Northern杂交和免疫细胞组化来确证。
试验结果揭示全长的mRNA转录体被翻译成55 kDa的单体,该单体能在非还原条件下形成由S-S键连接的110kDa二聚体二级结构。在变性条件下检测到约110kDa的二聚体和55kDa的单体肽。这个结果与用计算机计算的45kDa单体融合蛋白类似。在非变性条件下(即在免疫印迹前进行乙酰化),最强的信号是约220kDa,其可以变性成110kDa的二聚体和55kDa的单体,表明形成了四聚体。220kDa蛋白的存在也可以用化学交联剂如NHS酯类证明。结果表明,亲和素即使附着在内吞受体的跨膜结构域上也仍保持可溶,并能形成四聚体。
通过共焦显微术和原子力显微术分析,融合蛋白表现为能结合FITC-生物素的一种功能蛋白。未转导的细胞和转染了含有LacZ基因的逆转录病毒的细胞作对照。用原子力显微术没有检测到生物素探针与LacZ转导的对照细胞的非特异结合。正如预计,转染细胞显示有特异结合,并且在未固定样品中可重复测定。测定的结合力是平均值149±19pN(平均值±sd)的倍数,该平均值如预计也在以前报道的生物素-链霉亲和素结合力160pN(Florin等(1994),科学,264:415-417)的范围之内。
通过用FACS分析法显示荧光标记的生物素分子与含有融合蛋白构建体的细胞的结合,也可以在体内证实构建体的功能。
融合蛋白在体内的功能活性用大鼠恶性神经胶质瘤模型进行分析。将BT4C野生型神经胶质瘤细胞经颅内移植到近交的BDIX雌性大鼠脑内的右侧胼胝体,深度为2.5mm。用高分辨率的MRI(磁共振成像)经常地监测肿瘤的生长。在接种肿瘤细胞三个星期后,将携有融合蛋白的cDNA、滴度为2x106cfu/ml的假型逆转录病毒或携有LacZ基因、滴度为1.3x106cofu/ml的假型逆转录病毒转移进肿瘤,首先注射到深度为2.5mm处,间隔十分钟注射到深度为1.5mm处。生长两天后重复进行基因转移。在最后一次注射的3天后,将老鼠处死,并用4%多聚甲醛(PFA)进行灌注固定。将脑取出,在注射部位纵切分成两半,在冰上切片,用针对抗亲和素的抗体作免疫反应分析。结果显示融合蛋白在大鼠恶性神经胶质瘤内有表达。在神经胶质瘤细胞中和肿瘤血管内类似血管内皮细胞的环样结构中检测到蛋白质。
序列表(1)一般资料:
(ⅰ)申请人:
(A)姓名:Eurogene Limited
(B)街道:Marquis House,57/68 Jermyn Street
(C)城市:London
(E)国家:英国
(F)邮政编码:SW1Y 6NY
(ⅱ)发明题目:可结合生物素的受体分子
(ⅲ)序列数:2
(ⅴ)计算机可读形式:
(A)介质类型:软盘
(B)计算机:IBM PC可兼容机
(C)操作系统:PC-DOS/MS-DOS
(D)软件:PatentIn Release #1.0,Version #1.30(EPO)
(2)SEQ ID NO:1的资料:
(ⅰ)序列特征:
(A)长度:5177个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ⅱ)分子类型:cDNA
(ⅸ)特征:
(A)名称/关键词:CDS
(B)位置:1071..2270
(ⅹⅰ)序列描述:SEQ ID NO:1:TTTGAAAGAC CCCACCCGTA GGTGGCAAGC TAGCTTAAGT AACGCCACTT TGCAAGGCAT 60GGAAAAATAC ATAACTGAGA ATAGAAAAGT TCAGATCAAG GTCAGGAACA AAGAAACAGC 120TGAATACCAA ACAGGATATC TGTGGTAAGC GGTTCCTGCC CCGGCTCAGG GCCAAGAACA 180GATGAGACAG CTGAGTGATG GGCCAAACAG GATATCTGTG GTAAGCAGTT CCTGCCCCGG 240CTCGGGGCCA AGAACAGATG GTCCCCAGAT GCGGTCCAGC CCTCAGCAGT TTCTAGTGAA 300TCATCAGATG TTTCCAGGGT GCCCCAAGGA CCTGAAAATG ACCCTGTACC TTATTTGAAC 360TAACCAATCA GTTCGCTTCT CGCTTCTGTT CGCGCGCTTC CGCTCTCCGA GCTCAATAAA 420AGAGCCCACA ACCCCTCACT CGGCGCGCCA GTCTTCCGAT AGACTGCGTC GCCCGGGTAC 480CCGTATTCCC AATAAAGCCT CTTGCTGTTT GCATCCGAAT CGTGGTCTCG CTGTTCCTTG 540GGAGGGTCTC CTCTGAGTGA TTGACTACCC ACGACGGGGG TCTTTCATTT GGGGGCTCGT 600CCGGGATTTG GAGACCCCTG CCCAGGGACC ACCGACCCAC CACCGGGAGG TAAGCTGGCC 660AGCAACTTAT CTGTGTCTGT CCGATTGTCT AGTGTCTATG TTTGATGTTA TGCGCCTGCG 720TCTGTACTAG TTAGCTAACT AGCTCTGTAT CTGGCGGACC CGTGGTGGAA CTGACGAGTT 780CTGAACACCC GGCCGCAACC CTGGGAGACG TCCCAGGGAC TTTGGGGGCC GTTTTTGTGG 840CCCGACCTGA GGAAGGGAGT CGATGTGGAA TCCGACCCCG TCAGGATATG TGGTTCTGGT 900AGGAGACGAG AACCTAAAAC AGTTCCCGCC TCCGTCTGAA TTTTTGCTTT CGGTTTGGAA 960CCGAAGCCGC GCGTCTTGTC TGCTGCAGCC AAGCTTGGGC TGCAGGTCGA CTCTAGAGGA 1020TCAATTCGGC ACGAGTAAAT CGGTGCTGCC GTCTTTAGGA CATATGAAGT ATG GCA 1076Met Ala1CAG TGG GAT GAC TTT CCT GAT CAG CAA GAG GAC ACT GAC AGC TGT ACA 1124Gln Trp Asp Asp Phe Pro Asp Gln Gln Glu Asp Thr Asp Ser Cys Thr5 10 15GAG TCT GTG AAG TTC GAT GCT CGC TCA GTG ACA GCT TTG CTT CCT CCC 1172Glu Ser Val Lys Phe Asp Ala Arg Ser Val Thr Ala Leu Leu Pro Pro20 25 30CAT CCT AAA AAT GGC CCA ACT CTT CAA GAG AGG ATG AAG TCT TAT AAA 1220His Pro Lys Asn Gly Pro Thr Leu Gln Glu Arg Met Lys Ser Tyr Lys35 40 45 50ACT GCA CTG ATC ACC CTT TAT CTC ATT GTG TTT GTA GTT CTC GTG CCC 1268Thr Ala Leu Ile Thr Leu Tyr Leu Ile Val Phe Val Val Leu Val Pro55 60 65ATC ATT GGC ATA GTG GCA GCT CAG CTC CTG AAA TGG GAA ACG AAG AAT 1316Ile Ile Gly Ile Val Ala Ala Gln Leu Leu Lys Trp Glu Thr Lys Asn70 75 80TGC ACG GTT GGC TCA GTT AAT GCA GAT ATA TCT CCA AGT CCG GAA GGC 1364Cys Thr val Gly Ser Val Asn Ala Asp Ile Ser Pro Ser Pro Glu Gly85 90 95AAA GGA AAT GGC AGT GAA GAT GAA ATG AGA TTT CGA GAA GCT GTG ATG 1412Lys Gly Asn Gly Ser Glu Asp Glu Met Arg Phe Arg Glu Ala Val Met100 105 110GAA CGC ATG AGC AAC ATG GAA AGC AGA ATC CAG TAT CTT TCA GAT AAT 1460Glu Arg Met Ser Asn Met Glu Ser Arg Ile Gln Tyr Leu Ser Asp Asn115 120 125 130GAA GCC AAT CTC CTA GAT GCT AAG AAT TTC CAA AAT TTC AGC ATA ACA 1508Glu Ala Asn Leu Leu Asp Ala Lys Asn Phe Gln Asn Phe Ser Ile Thr135 140 145ACT GAT CAA AGA TTT AAT GAT GTT CTT TTC CAG CTA AAT TCC TTA CTT 1556Thr Asp Gln Arg Phe Asn Asp Val Leu Phe Gln Leu Asn Ser Leu Leu150 155 160TCC TCC ATC CAG GAA CAT GAG AAT ATC ATA GGG GAT ATC TCC AAG TCA 1604Ser Ser Ile Gln Glu His Glu Asn Ile Ile Gly Asp Ile Ser Lys Ser165 170 175TTA GTA GGT CTG AAC ACC ACA GTA CTT GAT TTG CAG TTC AGT ATT GAA 1652Leu Val Gly Leu Asn Thr Thr Val Leu Asp Leu Gln Phe Ser Ile Glu180 185 190ACA CTG AAT GGC AGA GTC CAA GAG AAT GCA TTT AAA CAA CAA GAG GAG 1700Thr Leu Asn Gly Arg Val Gln Glu Asn Ala Phe Lys Gln Gln Glu Glu195 200 205 210ATG CGT AAA TTA GAG GAG CGT ATA TAC AAT GCA TCA GCA GAA ATT AAG 1748Met Arg Lys Leu Glu Glu Arg Ile Tyr Asn Ala Ser Ala Glu Ile Lys215 220 225TCT CTA GAT GAA AAA CAA GTA TAT TTG GAA CAG GAA ATA AAA GGG GAA 1796Ser Leu Asp Glu Lys Gln Val Tyr Leu Glu Gln Glu Ile Lys Gly Glu230 235 240ATG AAA CTG TTG AAT AAT ATC ACT AAT GAT CTG AGG CTG AAG GAT TGG 1844Met Lys Leu Leu Asn Asn Ile Thr Asn Asp Leu Arg Leu Lys Asp Trp245 250 255GAA CAT TCT CAG ACA TTG AAA AAT ATC ACT TTA CTC CAA GGT GCC AGA 1892Glu His Ser Gln Thr Leu Lys Asn Ile Thr Leu Leu Gln Gly Ala Arg260 265 270AAG TGC TCG CTG ACT GGG AAA TGG ACC AAC GAT CTG GGC TCC AAC ATG 1940Lys Cys Ser Leu Thr Gly Lys Trp Thr Asn Asp Leu Gly Ser Asn Met275 280 285 290ACC ATC GGG GCT GTG AAC AGC AGA GGT GAA TTC ACA GGC ACC TAC ATC 1988Thr Ile Gly Ala Val Asn Ser Arg Gly Glu Phe Thr Gly Thr Tyr Ile295 300 305ACA GCC GTA ACA GCC ACA TCA AAT GAG ATC AAA GAG TCA CCA CTG CAT 2036Thr Ala Val Thr Ala Thr Ser Asn Glu Ile Lys Glu Ser Pro Leu His310 315 320GGG ACA CAA AAC ACC ATC AAC AAG AGG ACC CAG CCC ACC TTT GGC TTC 2084Gly Thr Gln Asn Thr Ile Asn Lys Arg Thr Gln Pro Thr Phe Gly Phe325 330 335ACC GTC AAT TGG AAG TTT TCA GAG TCC ACC ACT GTC TTC ACG GGC CAG 2132Thr Val Asn Trp Lys Phe Ser Glu Ser Thr Thr Val Phe Thr Gly Gln340 345 350TGC TTC ATA GAC AGG AAT GGG AAG GAG GTC CTG AAG ACC ATG TGG CTG 2180Cys Phe Ile Asp Arg Asn Gly Lys Glu Val Leu Lys Thr Met Trp Leu355 360 365 370CTG CGG TCA AGT GTT AAT GAC ATT GGT GAT GAC TGG AAA GCT ACC AGG 2228Leu Arg Ser Ser Val Asn Asp Ile Gly Asp Asp Trp Lys Ala Thr Arg375 380 385GTC GGC ATC AAC ATC TTC ACT CGC CTG CGC ACA CAG AAG GAG 2270Val Gly Ile Asn Ile Phe Thr Arg Leu Arg Thr Gln Lys Glu390 395 400TGAGTGAGTG ACCAAGGTCC TCCTGGACTC CAGGTGAAAA AGGAGATAGA GGCCCTCCTG 2330GACAAAATGG TATACCAGGC TTTCCAGGTC TAATAGGTAC TCCAGGTCTT AAAGGTGATC 2390GGGGGGATCT CTGGTTTACC TGGAGTTCGA GGATTCCCAG GACCAATGGG GAAGACCGGG 2450AAGCCAGGAC TTAATGGACA AAAAGGCCAG AAGGGAGAAA AAGGGAGTGG AAGCATGCAA 2510AGACAATCTA ATACAGTCCG ACTGGTGGGT GGCAGCGGCC CTCACGAAGG CAGAGTGGAG 2570ATTTTTCACG AAGGCCAGTG GGGTACGGTG TGTGACGACC GCTGGGAACT GCGTGGAGGA 2630CTGGTCGTCT GCAGGAGCTT GGGATACAAA GGTGTTCAAA GTGTGCATAA GCGAGCTTAT 2690TTTGGAAAAG GTACGGGTCC AATATGGCTG AATGAAGTAT TTTGTTTCGG GAAAGAGTCA 2750TCCATTGAAG AGTGCAGAAT TAGACAGTGG GGTGTGAGAG CCTGTTCGCA CGACGAAGAT 2810GCTGGGGGTC ACTTTGCACC TACATAATGC ATCATATTTT CATTCACATT TTTTAAACTG 2870TTATAAAGTG ATTTTTTTCC TTTGCTTCAC TAAAATCAGC TTAATTAATA TTTAAGAAAC 2930TAAGAATTTT ATCCACAGAA AAGGAATATT TAAAAATCAC TGGATAAACA TATAAAATAG 2990CTTCATATTT GCTTCAAATA CCAGAACCAT TTCAACTTCT CTAGGTTTTT AAGTGGCTCG 3050TGCCGAATTG ATCCCCTCAG GATATAGTAG TTTCGCTTTT GCATAGGGAG GGGGAAATGT 3110AGTCTTATGC AATACTCTTG TAGTCTTGCA ACATGGTAAC GATGAGTTAG CAACATGCCT 3170TACAAGGAGA GAAAAAGCAC CGTGCATGCC GATTGGTGGA AGTAAGGTGG TACGATCGTG 3230CCTTATTAGG AAGGCAACAG ACGGGTCTGA CATGGATTGG ACGAACCACT GAATTCCGCA 3290TTGCAGAGAT ATTGTATTTA AGTGCCTAGC TCGATACAGC AAACGCCATT TGACCATTCA 3350CCACATTGGT GTGCACCTCC AAGCTTCACG CTGCCGCAAG CACTCAGGGC GCAAGGGCTG 3410CTAAAGGAAG CGGAACACGT AGAAAGCCAG TCCGCAGAAA CGGTGCTGAC CCCGGATGAA 3470TGTCAGCTAC TGGGCTATCT GGACAAGGGA AAACGCAAGC GCAAAGAGAA AGCAGGTAGC 3530TTGCAGTGGG CTTACATGGC GATAGCTAGA CTGGGCGGTT TTATGGACAG CAAGCGAACC 3590GGAATTGCCA GCTGGGGCGC CCTCTGGTAA GGTTGGGAAG CCCTGCAAAG TAAACTGGAT 3650GGCTTTCTTG CCGCCAAGGA TCTGATGGCG CAGGGGATCA AGATCTGATC AAGAGACAGG 3710ATGAGGATCG TTTCGCATGA TTGAACAAGA TGGATTGCAC GCAGGTTCTC CGGCCGCTTG 3770GGTGGAGAGG CTATTCGGCT ATGACTGGGC ACAACAGACA ATCGGCTGCT CTGATGCCGC 3830CGTGTTCCGG CTGTCAGCGC AGGGGCGCCC GGTTCTTTTT GTCAAGACCG ACCTGTCCGG 3890TGCCCTGAAT GAACTGCAGG ACGAGGCAGC GCGGCTATCG TGGCTGGCCA CGACGGGCGT 3950TCCTTGCGCA GCTGTGCTCG ACGTTGTCAC TGAAGCGGGA AGGGACTGGC TGCTATTGGG 4010CGAAGTGCCG GGGCAGGATC TCCTGTCATC TCACCTTGCT CCTGCCGAGA AAGTATCCAT 4070CATGGCTGAT GCAATGCGGC GGCTGCATAC GCTTGATCCG GCTACCTGCC CATTCGACCA 4130CCAAGCGAAA CATCGCATCG AGCGAGCACG TACTCGGATG GAAGCCGGTC TTGTCGATCA 4190GGATGATCTG GACGAAGAGC ATCAGGGGCT CGCGCCAGCC GAACTGTTCG CCAGGCTCAA 4250GGCGCGCATG CCCGACGGCG AGGATCTCGT CGTGACCCAT GGCGATGCCT GCTTGCCGAA 4310TATCATGGTG GAAAATGGCC GCTTTTCTGG ATTCATCGAC TGTGGCCGGC TGGGTGTGGC 4370GGACCGCTAT CAGGACATAG CGTTGGCTAC CCGTGATATT GCTGAAGAGC TTGGCGGCGA 4430ATGGGCTGAC CGCTTCCTCG TGCTTTACGG TATCGCCGCT CCCGATTCGC AGCGCATCGC 4490CTTCTATCGC CTTCTTGACG AGTTCTTCTG AGCGGGACTC TGGGGTTCGA TAAAATAAAA 4550GATTTTATTT AGTCTCCAGA AAAAGGGGGG AATGAAAGAC CCCACCTGTA GGTTTGGCAA 4610GCTAGCTTAA GTAACGCCAT TTTGCAAGGC ATGGAAAAAT ACATAACTGA GAATAGAGAA 4670GTTCAGATCA AGGTCAGGAA CAGATGGAAC AGCTGAATAT GGGCCAAACA GGATATCTGT 4730GGTAAGCAGT TCCTGCCCCG GCTCAGGGCC AAGAACAGAT GGAACAGCTG AATATGGGCC 4790AAACAGGATA TCTGTGGTAA GCAGTTCCTG CCCCGGCTCA GGGCCAAGAA CAGATGGTCC 4850CCAGATGCGG TCCAGCCCTC AGCAGTTTCT AGAGAACCAT CAGATGTTTC CAGGGTGCCC 4910CAAGGACCTG AAATGACCCT GTGCCTTATT TGAACTAACC AATCAGTTCG CTTCTCGCTT 4970CTGTTCGCGC GCTTCTGCTC CCCGAGCTCA ATAAAAGAGC CCACAACCCC TCACTCGGGG 5030CGCCAGTCCT CCGATTGACT GAGTCGCCCG GGTACCCGTG TATCCAATAA ACCCTCTTGC 5090AGTTGCATCC GACTTGTGGT CTCGCTGTTC CTTGGGAGGG TCTCCTCTGA GTGATTGACT 5150ACCCGTCAGC GGGGGTCTTT CATTTGG 5177
(2)SEQ ID NO:2的资料:
(ⅰ)序列特征:
(A)长度:400个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ⅱ)分子类型:蛋白质
(ⅹⅰ)序列描述:SEQ ID No:2:Met Ala Gln Trp Asp Asp Phe Pro Asp Gln Gln Glu Asp Thr Asp Ser1 5 10 15Cys Thr Glu Ser Val Lys Phe Asp Ala Arg Ser Val Thr Ala Leu Leu20 25 30Pro Pro His Pro Lys Asn Gly Pro Thr Leu Gln Glu Arg Met Lys Ser35 40 45Tyr Lys Thr Ala Leu Ile Thr Leu Tyr Leu Ile Val Phe Val Val Leu50 55 60Val Pro Ile Ile Gly Ile Val Ala Ala Gln Leu Leu Lys Trp Glu Thr65 70 75 80Lys Asn Cys Thr Val Gly Ser Val Asn Ala Asp Ile Ser Pro Ser Pro85 90 95Glu Gly Lys Gly Asn Gly Ser Glu Asp Glu Met Arg Phe Arg Glu Ala100 105 110Val Met Glu Arg Met Ser Asn Met Glu Ser Arg Ile Gln Tyr Leu Ser115 120 125Asp Asn Glu Ala Asn Leu Leu Asp Ala Lys Asn Phe Gln Asn Phe Ser130 135 140Ile Thr Thr Asp Gln Arg Phe Asn Asp Val Leu Phe Gln Leu Asn Ser145 150 155 160Leu Leu Ser Ser Ile Gln Glu His Glu Asn Ile Ile Gly Asp Ile Ser165 170 175Lys Ser Leu Val Gly Leu Asn Thr Thr Val Leu Asp Leu Gln Phe Ser180 185 190Ile Glu Thr Leu Asn Gly Arg Val Gln Glu Asn Ala Phe Lys Gln Gln195 200 205Glu Glu Met Arg Lys Leu Glu Glu Arg Ile Tyr Asn Ala Ser Ala Glu210 215 220Ile Lys Ser Leu Asp Glu Lys Gln Val Tyr Leu Glu Gln Glu Ile Lys225 230 235 240Gly Glu Met Lys Leu Leu Asn Asn Ile Thr Asn Asp Leu Arg Leu Lys245 250 255Asp Trp Glu His Ser Gln Thr Leu Lys Asn Ile Thr Leu Leu Gln Gly260 265 270Ala Arg Lys Cys Ser Leu Thr Gly Lys Trp Thr Asn Asp Leu Gly Ser275 280 285Asn Met Thr Ile Gly Ala Val Asn Ser Arg Gly Glu Phe Thr Gly Thr290 295 300Tyr Ile Thr Ala Val Thr Ala Thr Ser Asn Glu Ile Lys Glu Ser Pro305 310 315 320Leu His Gly Thr Gln Asn Thr Ile Asn Lys Arg Thr Gln Pro Thr Phe325 330 335Gly Phe Thr Val Asn Trp Lys Phe Ser Glu Ser Thr Thr Val Phe Thr340 345 350Gly Gln Cys Phe Ile Asp Arg Asn Gly Lys Glu Val Leu Lys Thr Met355 360 365Trp Leu Leu Arg Ser Ser Val Asn Asp Ile Gly Asp Asp Trp Lys Ala370 375 380Thr Arg Val Gly Ile Asn Ile Phe Thr Arg Leu Arg Thr Gln Lys Glu385 390 395 400
Claims (11)
1.一种含有跨膜结构域和胞外结构域的蛋白质,其中所述的胞外结构域包含有生物素结合活性。
2.权利要求1的蛋白质,该蛋白质进一步含有胞质结构域。
3.权利要求1或2的蛋白质,其中所述的胞外结构域包含有亲和素或链霉亲和素的功能活性。
4.根据上述任一权利要求的蛋白质,该蛋白质含有来自A类清除剂受体的氨基酸序列。
5.根据权利要求1至3中任一项所述的蛋白质,其中所述蛋白质包括SEQ ID No 2所示的氨基酸序列。
6.编码上述任一权利要求所述的蛋白质的核酸分子。
7.包含权利要求6所述的核酸分子的重组表达载体。
8.生产权利要求1至5中任一项所述蛋白质的方法,该方法包括用权利要求7所述的重组表达载体转染一种细胞系,以及在转染细胞内表达该蛋白质。
9.将分子体外运送到靶位点的方法,该方法包括将所述分子添加入含有该靶的溶液中,其中所述分子已被生物素化,并且所述靶包含权利要求1至5中任一项所述的蛋白质。
10.权利要求6的核酸分子在制备可用于对靶位点给药的药物中的应用,其中所述核酸分子编码的蛋白质在靶位点表达。
11.生物素化分子在制备可用于对靶位点给药的药物中的应用,其中所述靶位点含有权利要求1至5中任一项所述的蛋白质。
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB9803757.5 | 1998-02-23 | ||
GBGB9803757.5A GB9803757D0 (en) | 1998-02-23 | 1998-02-23 | Fusion proteins |
GBGB9813653.4A GB9813653D0 (en) | 1998-06-24 | 1998-06-24 | Fusion proteins |
GB9813653.4 | 1998-06-24 |
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CN1291230A true CN1291230A (zh) | 2001-04-11 |
CN1195852C CN1195852C (zh) | 2005-04-06 |
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CNB998032174A Expired - Fee Related CN1195852C (zh) | 1998-02-23 | 1999-02-23 | 可结合生物素的受体分子 |
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US (1) | US7208291B2 (zh) |
EP (1) | EP1056850B1 (zh) |
JP (1) | JP2002504328A (zh) |
KR (1) | KR100722578B1 (zh) |
CN (1) | CN1195852C (zh) |
AT (1) | ATE274058T1 (zh) |
AU (1) | AU750444B2 (zh) |
CA (1) | CA2319039A1 (zh) |
DE (1) | DE69919515T2 (zh) |
DK (1) | DK1056850T3 (zh) |
ES (1) | ES2226340T3 (zh) |
HU (1) | HUP0101614A3 (zh) |
NO (1) | NO328139B1 (zh) |
PL (1) | PL195990B1 (zh) |
PT (1) | PT1056850E (zh) |
WO (1) | WO1999042577A2 (zh) |
Cited By (1)
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CN105980402A (zh) * | 2013-12-20 | 2016-09-28 | 弗雷德哈钦森癌症研究中心 | 带标签的嵌合效应分子及其受体 |
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US20040209294A1 (en) * | 2002-09-02 | 2004-10-21 | National Food Research Institute | Method to produce a receptor chip using biotinylated protein |
US9623117B2 (en) * | 2011-04-04 | 2017-04-18 | Wisconsin Alumni Research Foundation | Method for selective targeting and entry of bacterial toxins to cells |
US11827904B2 (en) | 2015-04-29 | 2023-11-28 | Fred Hutchinson Cancer Center | Modified stem cells and uses thereof |
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US4839293A (en) * | 1986-02-24 | 1989-06-13 | The Trustees Of Columbia University In The City Of New York | DNA encoding streptavidin, streptavidin produced therefrom, fused polypeptides which include amino acid sequences present in streptavidin and uses thereof |
US5135736A (en) * | 1988-08-15 | 1992-08-04 | Neorx Corporation | Covalently-linked complexes and methods for enhanced cytotoxicity and imaging |
CA1340977C (en) | 1988-11-15 | 2000-04-25 | Monty Krieger | Scavenger receptor protein and antibody thereto |
US5457035A (en) | 1993-07-23 | 1995-10-10 | Immunex Corporation | Cytokine which is a ligand for OX40 |
US5846537A (en) | 1995-06-07 | 1998-12-08 | University Of Rochester | Modified avidin and streptavidin and methods of use thereof |
US6497881B1 (en) * | 1995-11-30 | 2002-12-24 | New York University | High efficiency tissue specific compound delivery system using streptavidin-protein a fusion protein |
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CN105980402A (zh) * | 2013-12-20 | 2016-09-28 | 弗雷德哈钦森癌症研究中心 | 带标签的嵌合效应分子及其受体 |
CN105980402B (zh) * | 2013-12-20 | 2021-06-15 | 弗雷德哈钦森癌症研究中心 | 带标签的嵌合效应分子及其受体 |
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KR100722578B1 (ko) | 2007-05-28 |
ATE274058T1 (de) | 2004-09-15 |
WO1999042577A3 (en) | 1999-10-21 |
HUP0101614A2 (hu) | 2001-09-28 |
AU750444B2 (en) | 2002-07-18 |
AU2631299A (en) | 1999-09-06 |
WO1999042577A2 (en) | 1999-08-26 |
JP2002504328A (ja) | 2002-02-12 |
ES2226340T3 (es) | 2005-03-16 |
CA2319039A1 (en) | 1999-08-26 |
NO20004195L (no) | 2000-08-22 |
US20040185059A1 (en) | 2004-09-23 |
CN1195852C (zh) | 2005-04-06 |
PT1056850E (pt) | 2004-12-31 |
DK1056850T3 (da) | 2004-11-29 |
EP1056850A2 (en) | 2000-12-06 |
NO328139B1 (no) | 2009-12-14 |
PL343079A1 (en) | 2001-07-30 |
DE69919515D1 (de) | 2004-09-23 |
KR20010085196A (ko) | 2001-09-07 |
US7208291B2 (en) | 2007-04-24 |
HUP0101614A3 (en) | 2006-04-28 |
EP1056850B1 (en) | 2004-08-18 |
PL195990B1 (pl) | 2007-11-30 |
NO20004195D0 (no) | 2000-08-22 |
DE69919515T2 (de) | 2005-04-07 |
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