CN1041181A - 制备人/牛碱性成纤维细胞生长因子新衍生物的方法 - Google Patents
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Abstract
本发明涉及用DNA重组技术生产碱性成纤维细胞生长因子(bFGF)衍生物。这些bFGF衍生物可在血管形成过程中用作野生型分子的拮抗物和/或超级增效物。可使用已用携带编码人和牛bFGF及其衍生物之核苷酸顺序的质粒转化的大肠杆菌菌株制备这些衍生物以及野生型bFGF。
Description
本发明涉及制备碱性成纤维细胞生长因子(bFGF)之新的分子整体衍生物的重组DNA方法,以及有关的表达质粒和DNA编码顺序。本发明新的bFGF分子变体可作为血管生成过程中野生型分子的拮抗物或超增效物。
本发明所公开的生产这些新分子以及野生型bFGF的方法,是基于使用已用携带编码人和牛bFGF及其衍生物之核苷酸顺序的质粒转化了的大肠杆菌重组菌株。
在许多重要的生物学过程,如器官发育和伤口愈合等生理过程或肿瘤生长等病理过程中出现毛细血管生成(Denekamp J:Vascular endothelium as the vulnerable element in tumors.Acta Radiol.(oncol)23 p.217-225,1984;Hobson B.and Denekamp J.:Endothelial proliferation in tumours and normal tissues:continous labelling studies.Br.J.Cancer 49 p.405-413,1984;Folkman J:Tumor antiogenesis,Adv.Cancer Res.43 p.175-203,1985)。
虽然在形态上已确定了导致新血管形成的一系列变化,但有关这一过程的分子机制尚不甚明了。因为毛细血管内皮细胞在正常情况下形成一个静止的单层,而且其增殖是在血管形成过程中引起的,所以这些细胞的生长控制似乎是十分严谨的(Folkman J:Tumor angiogenesis,Adv.Cancer Res.43 p.175-203,1985;Joseph-Silverstein J.and Rifkin B.D.:Endothelial Cell growth factors and the vessel wall,Seminars in Thromb.and Hemost.13 p.504-513,1987)。
血浆中明显缺乏内皮细胞生长因子可部分地群释正常情况下内皮细胞的静止性质。
虽然在几乎所有组织的提取物以及许多正常和肿瘤细胞系中都存在内皮细胞分裂素,但事实上在血浆却没有见到(Joseph Silverstein J.and Rifkin B.D.:Endothelial cell growth factors and the vessel wall,Seminars in Thromb and Hemost.13 p.504-513,1987;Folkman J.and Klagsbrun M.:Angiogenic Factors,Science,235 p.442-447,1987)。
因此,定位诱导内皮细胞的迅速增殖可包括在对环境条件反应中由细胞内释放内皮细胞分裂素。
已明确定性的内皮细胞分裂素是一族多肽生长因子,包括碱性成纤维细胞生长因子(bFGF),因其对肝素有高度亲合性,故也称为肝素结合性生长因子(Thomas K.:Fibroblast growth factors,FASEB J.,1 p 434-440,1987;Gospodarowicz D.,Neufeld G.and Schweigerer L.:Fibroblast grwth factor:Structural and biological properties,J.Cell.Physiol.,5 p.15-26,1987)。
已经由大多数中胚层或神经外胚层分化的组织或细胞中纯化了碱性FGF。
结构研究表明,bFGF是由146个氨基酸构成的单链多肽,其也可以以缺失前10-20个氨基酸之氨基末端剪切的形式存在。
放射受体结合试验和生物学检验证明修剪形式的FGF与天然bFGF有同样的效力(Gospodarowicz D.,Neufeld G.and Schweigerer L,:Fibroblast growth factor,Mol.Cell.Endocrin.46 p.187-206,1986;Gospodarowicz D.,Neufeld G.and Schweigerer L,:Molecular and biogical characterization of fibroblast growth factor:an angiogenic factor which also controls the proliferation and differen tiation of mesoderm and neuroectoderm derived cells.,Cell.Differ.,19 p.1-17,1986;Thomas K.and Gimenez Gallego G.:Fibroblast growth factors:broad spectrum mitogens with potent angiogenic activity.Trends Biochem.Sci.11 p 81-84,1986)。
另外,将纯化程序中自匀浆组织内提取的方法由酸性提取改为中性提取并使用蛋白酶抑制物,已得到了一种含154个氨基酸残基较长形式的FGF(Veno N.,Baird A.,Esch F.,Ling N.and Guillemin R.:Isolation of anamino terminal extended form of basic fibroblast grwth factor.Biochrm.Biophys.Res.Commun.,138 p.580-588,1986;Story M.T.,Esch.F.,Shimasaki S.,Sasse J.,Jacobs S.C.and Lawson R.K.:Amino terminal Sequence of a large form of basic fibroblast growth factor isolated from human benign prostatic hyperplastic tissue.Biochem Biophys.Res.Commun.142 p.702-709,1987;Klagsbrun M.,Smith S.,Sullivan R.,SHing Y.,Davidson S.,Smith J.A. and Sasse J.:Multiple forms of basic fibroblast growth factor:amino-terminal cleavages by tumor cell-and brain cell-derived acid proteinases.Proc.Natl.Acad.Sci.USA 84 p.1839-1843,1987)。
已观察到FGF的这种微观不均一性可能是由于在细胞内或在纯化期间氨基末端附近部分水解造成的。至少有部分是这样的。但因为各种形式似乎具有同等活性,所以这种微观不均一性可能在生理上是无关紧要的。
在遗传进化中,碱性FGF似乎是极为保守的。例如,牛和人bFGF在其146个氨基酸中只有两个不同,整个氨基酸顺序同源性为98.7%(Gospodarowicz D.,Neufeld G.和Schweigerer L.:Molecular and biological characterization of fibroblast growth factor:anangiogenic factor which alss controls the proliferation and differentiation of mesoderm and neuroectoderm derived cells.,Cell.Differ.,19 p.1-17,1986)。
相对于bFGF的是酸性FGF(aFGF),它与bFGF有55%的总顺序同源性。酸性FGF是一种140个氨基酸的多肽,其也可以缺失前6个氨基酸之NH2末端剪切的形式存在(Gimenez-Gallego G.,Corn G.,Hatcher V.B.and Thomas K.A.:The complete amino acid sequence of human braimdericed acidic fibroblast growth factor.Biochem.Biophys.Res.Commun.138,p.611-617,1986)。
碱性FGF和酸性FGF具有两个潜在的肝素结合区域,一个位于它们的NH2末端附近,另一个位于COOH末端附近。两个区域可能都与FGF对肝素的强亲合性有关(Gospodarowicz D.,Neufeld G.and Schweigerer L.:Fibroblast growth factor,Mol.Cell.Endocrin.46 p.187-306,1986;Gospodarowicz D.,Neufeld G.and Schweigerer L.:Molecular and biological characterization of fibroblast growth factor:an angiogenic factor which also controls the proliferation and differentiation of mesoderm and neuroectoderm derived cells.,Cell.Differ.,19 p.1-17,1986);Baird A.,Schubert D.,Ling N.,and Guillemin R.,Receptor-and heparin-binding domains of basic fibroblast growth factor,Proc.Natl.Acad.Sci.USA,85,p.2324-2328,1988)。
aFGF和bFGF的高度同源性,提示它们是由一个原始基因衍生的。最近,已经克隆了FGF基因并已测定了bFGF和aFGF的互补DNA顺序(Abraham J.A.,Whang J.L.,Tumolo A.,Mergia A.,Friedman J.,Gospodarowicz D.and Fiddes J.C.:Human basic fibroblast growth factor:nucleotide seguence and genemic organization,EMBO J.,5 p.2523-2528,1986;Abraham J.A.,Mergia A.,Whang J.L.,Tumolo A.,Friedman J.,Hjerrild K.A.,Gospodarowicz D.and Fiddes J.C.:Nucleotide sequence of a bovine clone encoding the angiognic protein,basic fibroblast growth factor,Science 233,p.545-548,1986;Jaye M.,Howk R.,Brugess G.A..,Ricca W.,Chiu I.M.,Ravera M.W.,O′ Brien S.J.,Modi W.S.,Maciag T.and Drolian W.N.:Human endothelial cell grouwth factor:Cloning nucleotide sequence and chromosome localization,Science 233,p.541-545,1986)。
对人和牛中cDNA克隆之核苷酸顺序的分析提示,碱性FGF的初级转译产物包含155个氨基酸。然而,最近Sommer等人已分离出一种在NH2末端有两个多余氨基酸之新的157个氨基酸形式的人碱性FGF(Sommer A.,Brewer M.T.,Thompson R.C.,Moscatelli D.,Presta M.and Rifkin D.B.:A form of human basic fibroblast growth fator with an extended amino terminus,Biochem.Biophys.Res.Commun.144,p.543-550,1987)。
有趣的是,这两个氨基相应于以前描述的人cDNA克隆中的密码子。图1中总结了迄今分离的或根据cDNA顺序推测的碱性FGF的不同形式。图2则显示了155氨基酸形式之FGF的一级结构。
如上面提到的,本发明涉及人碱性FGF的分子变体。已通过对编码155氨基酸形式FGF之基因的位点特异性诱变获得了这些以前在自然界中从未见到的新的分子载体。
然而,碱性FGF氨基末端的微观不均一性及其生理学不相干性表明,本发明公开并描述的对155氨基酸形式的修饰等效于可能基于FGF的其他形式得到的同一种FGF(见下述)。
正如已经指出的,血管形成是一个严格控制的过程,其保证了它在实体肿瘤发展中发挥重要的病理学作用。从bFGF在血管形成中所起的重要作用看,如果这种与内源性FGF竞争的分子变体是生物学上无活性的,便可以在抗肿瘤治疗中作为一种有价值的工具。
另一方面,新毛细血管生长是在控制再生、生长和发育的自动调节机制的基础上进行的。因此,提高了生物学活性的bFGF类似物则可能具有许多潜在应用价值,如用于愈合烧伤、创伤(包括角膜创伤)和外科手术切口;治疗皮肤溃疡(包括褥疮);心脏病发作后通过加速损伤组织内血管生成而恢复心肌血流量;以及用于治疗某些肌肉骨骼损伤。
因此,本发明的目的在于设计并产生已改变了生物学活性之碱性FGF的重组类似物。
为了了解碱性FGF的氨基酸顺序中如何改变会影响其功能性质,可以考虑许多与碱性FGF有很高同源性的相关蛋白质,这个蛋白质家族包括酸性FGF以及hst和int-2这两个最近发现的致癌基因产物(Yoshida T.,Miyagawa K.,Odagiri H.,Sakamoto H.,Little P.F.R.,Terada M.and Sugomura T.:Geromic sequence of hst,a fibroblast growth factors and the int-2-encoded protein,Proc.Natl.Acad.Sci.USA84,p.7305-7309,1987;Delli Bovi P.,Curatola A.M.,Kern F.G.,Greco A.,Ittmann M.and Basilico C.:An oncogene isolated by fransfection of Kaposi′s sarcoma DNA encodes a growth factor that is a member of the FGF family,Cell 50,p.729-737,1987)。
包括bFGF在内的所有这些分子构成了一个参予细胞生长和调节的因子家族。图3中比较了这些蛋白质的一级结构顺序。
当考虑这些蛋白质之间的同源性时,可观察到一级结构中的高保守区域。这些区域的保守性不仅是结构上的,而且表现于这些蛋白质间的某些功能同等性。
这些蛋白质确实对肝素有很强的亲合力,而且似乎在血管形成过程(可能包括支持肿瘤生长的新的毛细血管增生)中起到重要作用。
如果保守区域是这些蛋白质具有共同特征的根源,则高度差异的顺序便是这些因子有不同生物学作用的原因。因此,改变任何高度差异区域即可显著地影响碱性FGF的生物学活性。
基于这些考虑,本发明人已使用基因工程技术构建了人和牛碱性FGF的新衍生物,其一种情况是在bFGF分子的不同区域内丢失了氨基酸顺序,第二种情况是在特定位置上有氨基酸取代。应根据几种已知生长因子间的同源性和差异选择修饰方法。有关分子变体的特征如下所述。可在选择的表达系统中获得以重组蛋白质形式的类似物。
当bFGF基因在适当生物体内表达之前,用基因工程技术对其进行修饰,即可达到预期的改变。bFGF基因即是指能够通过克隆cDNA文库或组装合成的寡核苷酸而得到的DNA顺序(Maniatis T.,Frisch E.F.and Sambrook J.:Molecular cloning,A laboratory manual,cold spring Harboun Laboratory,Cold Spring Harbour,NY,1982)。本发明还涉及生产bFGF及其衍生物的重组DNA方法。
分子变体的特征
本发明中,“类似物”、“变体”或“衍生物”是指改变了氨基酸顺序的bFGF分子。迄今已分离出的及在引言部分中描述的天然bFGF均可被改变成等价类似物。其中优选的类似物是含155个氨基酸的变体。本发明中人和牛bFGF氨基酸顺序都已经过了修饰。新的bFGF衍生物则是以寡核苷酸定向诱变方法构建的。
特别是本发明设计并合成了特定的寡核苷酸以造成人和牛bFGF基因内编码区的缺失。在“方法”部分中将详细描述用以获得变体的诱变技术。
可将突变的基因引入可指导新bFGF衍生物合成的大肠杆菌表达载体内。然后纯化、定性并最后大量产生重组分子。
下文中将详细描述构成本发明主题的新的bFGF衍生物,并在图4中作一般性的图解说明。其相应于155个残基形式氨基酸编号即蛋氨基是1号残基,丝氨酸是155号残基。但所有已述及的bFGF都可用于构建变体。
优选的bFGF的衍生物是:
M1-bFGF,为缺失氨基酸顺序中残基27至32(Lys-Asp-Pro-Lys-Arg-Leu)的bFGF衍生物;
M2-bFGF,为缺失氨基酸顺序中残基54到58(Glu-Lys-Ser-Asp-Pro)的bFGF衍生物;
M3-bFGF,为缺失氨基酸顺序中残基70至75(Gly-Val-Val-Ser-Ile-Lys)的bFGF衍生物;
M4-bFGF,为缺失氨基酸顺序中残基78到83(Cys-Ala-Asn-Arg-Tyr-Leu)的bFGF衍生物;
M5-bFGF,为缺失氨基酸顺序中残基110到120(Asn-Asn-Tyr-Asn-Thr-Tyr-Arg-Ser-Arg-Lys-Tyr)的bFGF衍生物;
M6a-bFGF,为分别在128和129位的赖氨酸和精氨酸残基被谷氨酰胺取代的bFGF衍生物;
M6b-bFGF,为119和128位的赖氨酸残基以及118和129位的精氨酸残基均被谷氨酰胺取代的bFGF衍生物。
分子变体的具体氨基酸顺序如图5所示。
在氨基或羧基末端加有或在两个末端都加有附加氨基酸残基的多肽被视为属于本发明范围内的。
从用DNA重组技术表达变体的技术原因看,这样的延伸可能是必要的(Courtney M.,Jallat S.,Tessier L.H.,Benavente A.and Crystal R.G.:Synthesis in E.Coli of alfal-anti-trypsin variants of therapeutic potential for emphysema and thrombosis Nature 313,p.149-151,1985;Nagai L.and Thogersen H.C.:Generation of β-globin by seguencespecific proteolysis of a hybrid protein produced in E.Coli.Nature,309,p.810-812,1984)。
另外,附加残基可用于提高变体的药理学效能,如通过延长其在血浆内的循环半寿期。
建立了生产变体的具体方法。
为了有效地产生新的bFGF衍生物,我们建立了在实验室和中试规模上制备为对不同分子进行生物学和临床估价所需量之变体的重组DNA方法。
该方法是基于发酵培养出经基因工程技术修饰,从而可在高水平上表达突变基因之大肠杆菌的菌株。
具体生产程序包括:
1)表达bFGF的合成DNA序列的构建
用合成方法重组构建用于表达野生型碱性FGF和其衍生物的所有顺序。此可通过使用自动DNA合成仪(如Applied Biosystem公司的380B型DNA合成仪)合成含交迭顺序的寡核苷酸来完成(Caruthers M.H.:Gene synthesis machines;DNA chemistry and its uses.Science,230,p.281-285,1985)。
连接交迭寡核苷酸以形成双股DNA链,并用DNA聚合酶和T4连接酶充填缺口。
直接在有意义链的FGF编码顺序之5′端提供了一个ATG起始信号。就155氨基酸形式的bFGF来说,我们可使用天然存在编码第一个氨基酸残基之蛋氨酸的ATG作为起始密码子,这样就没有额外的氨基酸加到被表达之多肽的N末端(Abraham J.A.,Whang J.L.,Tumolo A.,Mergia A.,Friedman J.,Gospodarowicz D.and Fiddes J.C.:Human basic fibroblast growth factor:nwcleotide sequence and genomic organization,EMBO J.,5,p.2523-2528,1986;Abraham J.A.,Mergia A.,Whang J.L.,Tumolo A.,Friedman J.,Hjerrild K.A.,Gospodarowicz D.and Fiddes J.C.:Nucleotide sequnence of a bovine clone encoding the angiogenic protein,basic fibroblast growth factor,Science 233,p.545-548,1986)。
另外,为提高FGF在大肠杆菌内的表达,可在不改变蛋白质一级结构的情况下改变其5′末端顺序。更具体地说,即是按图6所示修饰核苷酸顺序。
2)表达bFGF衍生物之突变顺序的构建
为得到本发明中所述的变体,可改变野生型bFGF,使之产生预期的缺失。为此可用位点特异性诱变方法进行基因修饰(Norris K.,Norris F.,Chrisiansen L.and Fiil N.:Efficient site-directed mutagenesis by simultaneous use of two primers,Nucleic Acid Research 11,5103-5112,1983)。
该方法的原理是将bFGF基因再克隆到能够以单链形式得到的载体(如噬菌体载体M13)内。该重组单链则与编码所需修饰部分的互补性合成寡脱氧核苷酸退火。然后使用DNA聚合酶和连接酶延伸新链并将其连接成环形。用新产生的异源双链DNA转化一个使之能在其中复制并产生子代的细胞系,在这里携带野生型基因或有所需缺失之基因的噬菌体将分离为两种不同的分子。
然后可用起始诱变寡核苷酸作探针去识别突变的基因。位点针对性诱变技术将在“方法”部分中详细描述。
具体地说,是将编码野生型bFGF(牛或人的)的合成顺序插入到M13载体中,并用所得单链作为诱变模板(Norris K.,Norris F.,Christiansen L.and Fiil N.:Efficient siite-directed mutagenesis by simultaneous use of two primers,Nucleic Acid Research 11,p.5103-5112,1983)。
3)野生型bFGF及其衍生物的表达
为在大肠杆菌中的重组体菌株中表达bFGF的野生型和突变顺序,可将这些顺序插入到携带负责转录和转译新基因之顺序的表达质粒中。更具体地说,我们使用了大肠杆菌的色氨酸启动子和λCⅡ蛋白的核糖体结合位点区域作为调节信号(Hendrix R.W.,Roberts J.W.,Stahl F.W.and Weisberg R.A.:Lambda Ⅱ,Cold Spring HarbourLaboratory,Cold Spring Harbour,N.Y,1983)。
启动子Ptrp得自产品质粒pDR720(Pharmacia)。Shine-Dalgarno CⅡ顺序则是根据已公布的顺序用化学合成方法制得的(Hendrix R.W.,Roberts J.W.,Stahl F.W.and Weisberg R.A.:Larnbda Ⅱ,Cold Spring Harbour Laboratory,Cold Spring Harbowr,N.Y,1983)。
图7中图群显示了用于野生型bFGF及其衍生物的典型表达载体。具体地说,它是经组装下列片段而构建的:
a)质粒pDS20的大的EcoRⅠ-BamHⅠ片段(Duester G.,Elford R.M.,Holmes W.N.:Fusion of the Escherichia coli leucyl transfer-RNA-I Promoter to the gal-K gene,Analysis of seguences necessary for growth rate dependent regulation cell 30,p.855-964,1982);
b)质粒pDR720的EcoRⅠ-SalⅠ片段(Pharmacia,Sweden),其携带色氨酸启动子;
c)合成的编码CⅡ核糖体结合位点的SalⅠ-NdeⅠ寡核苷酸;
d)合成的携带编码bFGF和其衍生物之野生型或突变顺序的NdeⅠ-XhoⅡ(BamHⅠ-可相容的)片段。
图6中给出了编码野生型bFGF的优选顺序。编码bFGF类似物的优选顺序是按图5所示突变情况对该顺序进行修饰得到的。
对新基因表达的诱导和对所得重组蛋白的分析如“方法”部分所述。
4)重组分子的纯化及生物学检测
可由细菌溶胞产物中纯化重组突变体,并在与野生型bFGF平行进行的比较实验中检测其生物学特征。
典型的纯化方法是:用超声波破碎细胞,离心并将上清液过S-Sepharose离子交换柱。
用氯化钠梯度洗脱蛋白质并直接加于肝素-Sepharose亲合柱上。
用氯化钠梯度洗脱蛋白质并用凝胶过滤法除盐,然后冻干。如此纯化的类似物即可用于生物学活性检测。
变体的性质、其实用性及使用方法。
本发明的新分子可用作野生型bFGF分子的拮抗物和/或增效物。
可参照Presta等人所述的方法(Molecular and Cellular Biol.6,p.4060,1986),基于增加内皮细胞增殖的能力来估计bFGF增效活性。
可参照Baird等所述的方法(Proc.Natl.Acad.Sci.USA 85,p.2324,1988),基于125I-bFGF的结合抑制率来估计bFGF拮抗活性。
例如,已发现当以1ng/ml的剂量给予本发明缩写为M6b的化合物时,内皮细胞增殖能力提高50%,此表明其具有特别明显的bFGF增效活性。
另如,当存在300ng/ml本发明缩写为M3-bFGF和M6a的变体化合物时,125I-bFGF(3ng/ml)的结合抑制率分别约为20%和70%,此即表明它们具有bFGF拮抗活性。
作为bFGF增效物,本发明的化合物可用作血管形成、细胞生长或细胞存活的促进剂,用于组织修复,如创伤、烧伤、骨折、表面损伤、溃疡的愈合,还包括心肌缺血和梗塞期间的组织修复。
作为bFGF的拮抗物,本发明的化合物可用作血管形成抑制剂,故可用于治疗以新血管生成为主要病理改变的疾病,如眼视网膜病、神经血管性青光眼、皮肤病如牛皮癣、慢性炎症、湿风湿性关节炎,以及如前所述用于治疗某些肿瘤,特别是可作为抑制肿瘤血管生成有价值工具用治疗血管生成性肿瘤。
本发明的化合物可与一种或多种医药上可接受的载体和/或稀释剂结合,制成一种药物组合物用于哺乳动物和人体。
活性物质的实用剂量将根据病人的年龄、体重和状态,以及给药途径和预期的疗程而定。
该医药组合物可经局部、眼、口、静脉内、皮下或肌肉内给药,可使用常用赋形剂以常规方法配制。
局部用药时,可将活性成分与常用的油脂或乳化佐剂混合制成乳剂、洗剂或糊剂。局部使用的洗剂可含有10-100mg/ml活性物质,并可每天在感染部位施用达7次。
用于滴眼时,可在缓冲液或生理盐水或其他适当赋形剂中配成药水。
口服给药时可配制成片剂或胶囊剂,其中可含有活性成分及稀释剂,如乳糖、右旋糖、蔗糖、纤维素、玉米淀粉或马铃薯淀粉;润滑剂如硅胶、滑石粉、硬脂酸、硬脂酸镁或钙,和/或聚乙二烯;粘合剂如淀粉、阿拉伯胶、明胶、甲基纤维素、羧甲基纤维素或聚乙烯吡咯烷酮;解聚剂剂淀粉、藻酸、藻酸盐或淀粉甘醇酸钠;发泡混合物;着色剂;甜味剂;润滑剂如卵磷脂、多乙氧基醚、十二烷基硫酸盐;以及一般用于医药配方中的非毒性、无药理学活性的物质。可按已知方法,经混合、造颗、压片、包糖衣或包膜等过程制造所说的药物制剂。
用于肌肉内注射的悬浮液或溶液可含有活性化合物及其药医上可接受的载体,如无菌水、橄榄油、油酸乙脂、二元醇如α-丙二醇,必要时还可含有适当量的盐酸利多卡因。
用于静脉内注射或输注的溶液可含有无菌水(最好是无菌等渗盐溶液)作为载体。
本发明的化合物可以作为其医药上可接受的盐或络合物使用。
作为盐,可以是与无机酸如盐酸、氢溴酸、硫酸和磷酸盐,以及有机酸如马来酸、柠檬酸、乙酸、苯甲酸、琥珀酸、抗坏血酸和酒石酸形成的酸加成盐。
络合物的例子如锌或铁络合物。
方法
1)质粒的构建
使用自动DNA合成仪(Applied Biosysten,380 B型)合成含有交迭顺序的寡核苷酸,得到合成的DNA片段。将片段再克隆到M13载体中之后用双脱氧法测定核苷酸顺序(Messing J.:Methods Enzymol,101,p.20-78,1983)。
使用限制性内切酶、连接酶和聚合酶(按制造商推荐的剂量和使用方法),依据标准程序转化大肠杆菌细胞(Maniatis T.,Frisch E.F.and Sambrook J.:Molecular Cloning,Alaboratry manual,Cold Spring Harbour Laboratory,Cold Speing Harbour,NY,1982)。
2)诱变
将编码野生型bFGF(人或牛形式的)的合成顺序再克隆到M13噬菌体载体中。按照已公开的方法(Messing J.:Methods Enzymol.101,p.20-78,1983)培养单链形式的重组噬菌体载体。取20ng这些M13单链DNA在10mM Tris-HCl(pH7.5)、0.1mM EDTA、50mM NaCl中于95℃下加热5分钟,并经逐步冷却至室温与合适的寡核苷酸一起退火。然后加入下列各组分:ATP加到0.4mM(终浓度);dCTP、dGTP、dTTP加到0.12mM;dATP加到0.04mM;1单位大肠杆菌DNA聚合酶I的Klenow片段及0.5单位T4 DNA连接酶(Boehringer Mannheim)。终体积为50μl的35mM Tris-HCl(pH7.5)、0.1mM EDTA、6mM MgCl2、0.006mM DTT。于15℃下保温12小时后,用该DNA按已公开的方法(Manniatis T.,Frisch E.F.and Sambrook J,:Molecular cloning,ALaboratory manual,Cold Spring Harbour Laboratory,Cold Spring Harbour,NY,1982)转染大肠杆菌JM101细胞。
在含有10mM MgCl2、5mM DTT和10单位T4多核苷酸激酶(Boehringer Mannheim)的70mM Tris-HCl缓冲液(pH8)中,于50μl于37℃保温30分钟的反应混合物内,使用150μCi(r-32P)ATP(New Emgland Nuclear,6000 Ci/mmol)放射标记用以引起预期突变的寡核苷酸。然后用标记的寡核苷酸与诱变的噬菌体DNA进行噬班杂文。
在含有3×SSC、0.1%SDS、10×Denhardt氏液和0.2mg/ml变性鲑鱼精子DNA的10mM Tris-HCl缓冲液(pH8)中于65℃下杂交过夜。
在几次变液的0.4×SSC、0.1%SDS中于65℃下将硝酸纤维素滤膜洗涤30分钟,并对X光胶片曝光过夜。使用AmershamM13顺序检测药盒,以Sanger氏双脱氧核苷酸顺序测定法检测显示阳性杂交之噬斑的核苷酸顺序。
3)基因表达的诱导和分析
用含有四环素(3μg/ml)的Luria核养液培养携带质粒的细胞。用添加于0.4%葡萄糖、0.4%酪蛋白氨基酸和10mg/ml硫胺素的M9培养基诱导处于色氨酸启动子控制下的基因表达。
在没有色氨酸的M9培养基中生长6小时后,离心收集细胞。沉淀细菌培养物的等分样品,用载样缓冲液重新悬浮并用Laemmli所述的SDS-PAGE法分析之(Laemmli U.K.:Cleavage of structural proteins during the assembly of the head of bacteriophage T4,Nature 227,p.680-685,1970)。
另外,也可用溶菌酶或超声波破碎细胞,并分别分析经离心分离的可溶性和不溶性部分。电泳后用考马氏亮兰染色凝胶中的总细胞蛋白质。
用抗合成肽(含bFGF衍生的顺序)的多克隆兔抗血清探查Western转移印迹。按生产商推荐的方法使用包含生物素化羊抗兔IgG(作为第二抗体)的Vectastain ABC药盒(Vector Laboratories,Ca.USA)进行检测。
附图说明
图1图示了迄今分离的不同天然形式的碱FGF。已根据cDNA核苷酸顺序推断出的含155个氨基酸的bFGF。
图2显示了人和牛FGF的氨基酸顺序。两顺序的差异在121和137位。相当于牛bFGF的氨基酸(不同于人形式者)用黑体符号示出。
图3为前已公开发表的资料(Yoshida T.,Miyagawa K.,Odagiri H.,Sakamoto H.,Little P.F.R.,Terada M.and Sugimura T.:Genomic sequence of hst,a transforming gene encoding a protein homologous to fibroblast growth factors and the int-2-encoded protein,Proc.Natl.Acad.Sci.USA,84 p.7305-7309,1987)。
hst蛋白、人碱性FGF(hbFGF)、人酸性FGF(haFGF)及小鼠int-2蛋白的完整氨基酸顺序对准排列并互相比较。短横线指示为有最适排列而插入的间隙。完全相同于hst顺序的残基括在方框内,顺序上方的编号以hst为准。
图4图示了碱性FGF衍生物。编号是指155氨基酸bFGF的顺序编号。涂黑的区域代表缺失的氨基酸顺序。黑点代表单个氨基酸取代。
图5并列显示了人bFGF及其变体的完整氨基酸顺序。短横线指示缺失的氨基酸。星号代表单个氨基酸置换。
图6为人和牛bFGF的核苷酸顺序。该顺序是用合成法重新构建的,并用于表达bFGF。用星号标示前20个氨基酸的密码子,它们已在没有改变相应氨基酸残基的情况得以修饰。编码牛顺序中两个不同氨基酸的密码子在相对之密码子下方并列示出。
图7中pDS20代表用于构建bFGF表达质粒的通用质粒本底物。用bFGF基因取代galk基因,并向其中插入得自λ噬菌体的表达信号Ptrp和Shine-Dalgarno顺序“CⅡ”以构建pEC80。
本发明涉及到分离新的碱性FGF分子衍生物。所说的碱性FGF分子可以是人或牛来源的。
使用DNA重组技术制得的这些新衍生物都是155氨基酸bFGF的缺失或置换变异体。据文献报导,可以分离到人或牛不同形式的碱性FGF,其不同仅在于氨基末端延伸的长度。具体地说,短到126个氨基酸或长到157个氨基酸的bFGF均具有等同的活性。
本发明人已在bFGF分子的完全不同于该异源氨基末端区的区域中完成了突变。例如使用了155氨基酸形式的bFGF。因此,很显然也可以对其他形式的bFGF,如含有126个到157个氨基酸的bFGF,进行代表了本发明之主题的修饰。
如前所述,本发明的新分子可以作为野生型bFGF分子的拮抗物和/或超级增效物。另如上面所指出的,bFGF的拮抗物可能是抑制肿瘤血管形成的十分有价值的治疗工具。而bFGF的超级增效物与天然顺序相比则表现有改善了的药理学性质。
对本发明化合物之生物学活性的估计,可明确鉴别bFGF分子中某些对bFGF的生物学和生物化学性质特别重要的区域。
经鉴定这些区域,特别是衍生物M1-bFGF、M2-bFGF和,M3-FGF中缺失的区域,指示在同样区域中进行进一步突变(如置换、缺失或修饰)可产生治疗上更为有效的bFGF衍生物。
这些进一步的突变也包括于本发明的范围内。
如前面已述及的,本发明还涉及生产这些新的分子整体的DNA重组方法。令人感兴趣的是,该方法也可成功地应用于生产野生型bFGF。另外,所有这些考虑对人和牛碱性FGF顺序都是有效的。
本发明说明书中公开的DNA重组方法,是基于使用用携带编码预期的FGF分子之基因的质粒DNA转化的大肠杆菌菌株而进行的。当然,本发明人也注意到已公开的其他DNA重组方法这一事实(Iwane M.,Kurokawa T.,Sasada R.,Seno M.,Nakagawa S.,Igarashi K.:Expression of cDNA encoding human basic fibroblast growth factor in E.Coli,Biochem.Biophis.Res.c Commun.146,p.470-477,1987)。但本文所公开的方法就其新颖性和重组bFGF的产量特征来说,此前却从未描述过。
该方法的主要特征之一是用作生产碱性FGF的宿主的大肠杆菌菌株的类型。事实上,菌株的类型是影响大肠杆菌中异源基因表达的主要参数之一(Harris T.J.R.and Emtage J.S.:Expression of heterologous gene in E.Coli Microbiological Sciences 3,p.28-31,1986)。
根据本发明,业已发现B型大肠杆菌菌株是用于生产重组bFGF及bFGF衍生物的十分有效的宿主。当将相同的bFGF表达载体转化到其他大肠杆菌(K-12型、C型等)内时,确实并不能产生同样多的bFGF。
本方法的第二个特征是与在编码不同bFGF分子的基因内导入某些“最适密码子”。事实上,发明人已发现,为了提高表达水平,在不改变氨基酸顺序的情况下修饰一定数目的DNA密码子是必要的。本发明说明书中公开的顺序(见图6)是优选的DNA编码顺序。
用作宿主的菌株类型和最适密码子这两个特征构成了本发明用于生产bFGF及其突变型之DNA重组方法的新颖性方面。适用于生产碱性FGF的这两方面的特征,在迄今为止的科学文献中从未提到或发表过。
Claims (12)
1、制备选自下列一组中之碱性成纤维细胞生长因子衍生物的DNA重组方法:
a)一种人或牛碱性成纤维细胞生长因子衍生物;其特征在于缺失氨基酸残基27至32(Lys-Asp-Pro-Lys-Arg-Leu),上述氨基酸残基的序号相当于155氨基酸形式的人或牛碱性FGF;
b)一种人或牛碱性成纤维细胞生长因子衍生物,其特征在于缺失氨基酸残基54至58(Glu-Lys-Ser-Asp-Pro-),上述氨基酸残基的序号相当于155氨基酸形式的人或牛碱性FGF;
c)一种人或牛碱性成纤维细胞生长因子衍生物,其特征在于缺失氨基酸残基70至75(Gly-Val-Val-Ser-Ile-Lys),上述氨基酸残基的序号相当于155氨基酸形式的人或牛碱性FGF;
d)一种人或牛碱性成纤维细胞生长因子衍生物,其特征在于缺失氨基酸残基78至83(Cys-Ala-Asn-Arg-Tyr-Leu),上述氨基酸残基的序号相当于155氨基酸形式的人或牛碱性FGF;
e)一种人或牛碱性成纤维细胞生长因子衍生物,其特征在于缺失氨基酸残基110至120(Asn-ASn-Tyr-Asn-Thr-Tyr-Arg-Ser-Arg-Lys-Tyr),上述氨基酸残基的序号相当于155氨基酸形式的人或牛碱性FGF;
f)一种人或牛碱性成纤维细胞生长因子衍生物,其特征在于用谷氨酰胺(Gln)残基取代氨基酸残基128(Lys)129(Arg),上述氨基酸残基的序号相当于155氨基酸形式的人或牛碱性FGF;以及
g)一种人或牛碱性成纤维细胞生长因子衍生物,其特征在于用谷氨酰胺(Gln)残基取代所有氨基酸残基118(Arg)、119(Lys)、128(Lys和129(Arg),上述氨基酸残基的序号相当于155氨基酸形式的人或牛碱性FGF。
2、根据权利要求1的制备人或牛碱性成纤维细胞生长因子衍生物的方法,所说的衍生物包括具有根据权利要求1之突变的其他天然形式的人或牛bFGF。
3、根据权利要求1的制备任何前述权利要求所述的bFGF衍生物的方法,其中所说的衍生物还在氨基和/或羧基末端包含附加的氨基酸残基。
4、根据权利要求1的制备前述权利要求所述之bFGF衍生物的方法,其中所说的衍生物有或没有糖苷侧链。
5、用于表达基因组DNA、cDNA、合成的或经修饰的基因之表达质粒pFC80,其中所说的基因编码天然或经修饰的人或牛bFGF氨基酸顺序。
6、根据权利要求5的表达质粒,其至少包含一个对一种抗生素具有抗性的基因或任何其他可选择的标志。
7、根据权利要求5或6的表达质粒,它用于表达编码权利要求1之衍生物的基因。
8、B型大肠杆菌菌株用于生产权利要求1之重组衍生物。
9、利用权利要求5的质粒和B型大肠杆菌菌株生产权利要求1的衍生物。
10、如图6所示的DNA编码顺序。
11、基于结合使用B形大肠杆菌菌株及图6所示的DNA顺序以生产人和牛bFGF的重组DNA方法。
12、基于结合使用B形大肠杆菌菌株和图6所示的DNA顺序以生产根据权利要求1之衍生物的DNA重组方法。
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GB8821795.5 | 1988-09-16 | ||
GB888821795A GB8821795D0 (en) | 1988-09-16 | 1988-09-16 | New derivatives of human/bovine basic fibroplast growth factor |
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WO2018040535A1 (zh) * | 2016-08-31 | 2018-03-08 | 黄志锋 | Fgf1突变体和给药方法 |
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US5143829A (en) * | 1990-03-29 | 1992-09-01 | California Biotechnology Inc. | High level expression of basic fibroblast growth factor having a homogeneous n-terminus |
US5478804A (en) * | 1990-09-19 | 1995-12-26 | The Salk Institute For Biological Studies | Treatment of tumorigenic pathophysiological conditions with FGF-cytoxic conjugates |
US6833354B1 (en) | 1990-12-19 | 2004-12-21 | Kaken Pharmaceutical Co., Ltd. | Agent for the treatment of bone diseases |
JP3135138B2 (ja) * | 1990-12-19 | 2001-02-13 | 科研製薬株式会社 | 骨疾患治療剤 |
EP0505108B1 (en) * | 1991-03-21 | 1995-10-18 | The Procter & Gamble Company | Compositions for regulating skin wrinkles comprising an Arg-Ser-Arg-Lys based peptide |
JP3303211B2 (ja) * | 1991-04-26 | 2002-07-15 | 武田薬品工業株式会社 | bFGFムテインおよびその製造法 |
TW275068B (zh) * | 1993-09-24 | 1996-05-01 | American Cyanamid Co | |
AUPO247496A0 (en) | 1996-09-23 | 1996-10-17 | Resmed Limited | Assisted ventilation to match patient respiratory need |
US6214795B1 (en) * | 1996-11-12 | 2001-04-10 | Praecis Pharmaceuticals, Inc. | Peptide compounds useful for modulating FGF receptor activity |
US6274712B1 (en) | 1997-12-23 | 2001-08-14 | 3-Dimensional Pharmaceuticals, Inc. | Analogs of human basic fibroblast growth factor mutated at one or more of the positions glutamute 89, aspartate 101 or leucine 137 |
FR2780416B1 (fr) * | 1998-06-10 | 2002-12-20 | Rhone Poulenc Nutrition Animal | Procede industriel de production de proteines heterologues chez e. coli et souches utiles pour le procede |
WO2001013031A2 (en) | 1999-08-13 | 2001-02-22 | Chiron Corporation | Dose of an angiogenic factor and method of administering to improve myocardial blood flow |
WO2001046416A1 (en) * | 1999-12-22 | 2001-06-28 | 3-Dimensional Pharmaceuticals, Inc. | Analogs of human basic fibroblast growth factor |
JP4212357B2 (ja) | 2000-11-27 | 2009-01-21 | 株式会社ディナベック研究所 | 血管新生遺伝子をコードするパラミクソウイルスベクターおよびその利用 |
US8772460B2 (en) * | 2011-12-16 | 2014-07-08 | Wisconsin Alumni Research Foundation | Thermostable FGF-2 mutant having enhanced stability |
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DK122687A (da) * | 1986-03-14 | 1987-09-15 | Takeda Chemical Industries Ltd | Polypeptid, dna og anvendelse deraf |
CA1323317C (en) * | 1987-01-16 | 1993-10-19 | Allen R. Banks | Production of fibroblast growth factor |
JP2526965B2 (ja) * | 1987-02-24 | 1996-08-21 | 武田薬品工業株式会社 | ムテイン,dnaおよびその用途 |
EP0377579A1 (en) * | 1987-07-07 | 1990-07-18 | California Biotechnology, Inc. | Recombinant fibroblast growth factors |
EP0326907A1 (en) * | 1988-01-26 | 1989-08-09 | Takeda Chemical Industries, Ltd. | Polypeptide, DNA and its use |
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AU4317189A (en) | 1990-04-02 |
MY104910A (en) | 1994-06-30 |
ES2061855T3 (es) | 1994-12-16 |
DE68911461T2 (de) | 1994-05-19 |
YU179489A (en) | 1991-10-31 |
DK120290D0 (da) | 1990-05-15 |
AU620925B2 (en) | 1992-02-27 |
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WO1990002800A1 (en) | 1990-03-22 |
ZA897020B (en) | 1990-08-29 |
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