CN1246154A - Ob融合蛋白组合物及方法 - Google Patents
Ob融合蛋白组合物及方法 Download PDFInfo
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- CN1246154A CN1246154A CN97181817A CN97181817A CN1246154A CN 1246154 A CN1246154 A CN 1246154A CN 97181817 A CN97181817 A CN 97181817A CN 97181817 A CN97181817 A CN 97181817A CN 1246154 A CN1246154 A CN 1246154A
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Abstract
本发明涉及Fc-OB融合蛋白的组合物,及该组合物的制备方法及其应用。尤其是本发明涉及一种遗传学的或化学的融合蛋白,该融合蛋白包括与OB蛋白,其衍生物或类似物的N末端部分融合的Fc免疫球蛋白区,其衍生物或类似物。
Description
发明领域
本发明涉及Fc-OB融合蛋白组合物及其制备和应用的方法。
背景
尽管肥胖的分子基础大部分都未知,但“OB基因”及其编码蛋白(“OB蛋白”或“leptin”)的鉴定给机体调节脂肪沉积的机制带来一线光明。见,PCT公开,WO96/05309(12/22/96),Friedman等:Zhang等,自然372:425-432(1994);也见修订自然374:479(1995)。同在正常野生型小鼠体内一样,OB蛋白在ob/ob突变小鼠(由于OB基因产物缺乏而肥胖的小鼠)也活化。该生物活性表现为体重减轻。综合见,Barrinaga,“肥胖”蛋白使小鼠苗条,科学269:475-456(1995)OB蛋白及衍生物因此作为动物(包括哺乳类和人)控制体重和肥胖的调节分子,其详细情况公布于PCT出版物WO96/05309(12/22/96),在此收入参考文献及图。
OB蛋白的其它生物学作用未被很好描述。据知,例在ob/ob突变小鼠中,施用OB蛋白导致血清胰岛素水平及血清葡萄糖水平的降低。施用OB蛋白亦能造成躯体脂肪的减少。此现象在ob/ob突变小鼠及非肥胖正常小鼠中都被观察到。Pelleymounter等.,科学269:540-543(1995);Halaas等.,科学269:543-546(1995)。也见,Campfield等.,科学269:546-549(1995)(外周及中心施用微克剂量的OB蛋白在ob/ob和饮食诱导肥胖小鼠而非db/db肥胖小鼠中导致食物摄入减少和体重减轻。)以上报告中均未观察到毒性,即使为最高剂量也是如此。
尽管OB蛋白被允诺进行临床应用,其在体内活化的方式并未被清楚地阐明。OB受体的消息显示其被测出在大鼠下丘脑中与OB蛋白高亲和结合力,这暗示了OB受体的定位。Stephens等.,自然377:530-532。db/db鼠表现与ob/ob鼠相同的表型,即,极端肥胖和II型糖尿病;此表型被认为是由于OB受体的缺乏引起,尤其因为db/db小鼠不能对OB蛋白施用产生反应。见Stephens等.,上述。
随着重组DNA技术的进步,治疗用的重组蛋白的有效性造成蛋白设计和化学修饰的进步。上述修饰的一个目标是蛋白保护和减少降解。融合蛋白和化学附着物能有效阻断蛋白水解酶与蛋白主链的物理接触,从而阻止降解。在某些情况下,附加优点包括增加治疗蛋白的稳定性,循环时间及生物活性。综述文章Francis,生长因子评论3:4-10(1992.5)(Mediscript,Mountview Court,Friern Barnet Lane出版,伦敦N20,OLD,UK)描述了蛋白修饰及融合蛋白。
上述的一种修饰方法是应用免疫球蛋白的Fc区段。抗体包含两个功能独立的部分,一个称为“Fab”的可变区,与抗原结合,一个称为“Fc”的恒定区,提供对效应子功能的连接如补体或吞噬细胞。免疫球蛋白的Fc部分具有长原生质半衰期而Fab为短寿的。Capon等,自然337:525-531(1989)。
治疗用蛋白产品应用Fc区域进行构建以提供较长半衰期或加入功能如Fc受体结合,蛋白A结合,补体固定和胎盘转移,等这些功能位于免疫球蛋白的Fc蛋白中。例,将IgG1抗体的Fc区段与CD30-L的N-末端相融合。CD30-L是一种与在何杰金氏病肿瘤细胞,退行性淋巴细胞,T细胞白血病细胞和其它恶性细胞类型中表达的CD30受体结合的分子。见,美国专利,No.5,480,981。IL-10,一种抗炎症及抗排斥剂与鼠的Fcγ2a融合,以延长细胞因子的短循环半衰期。Zheng,X.等,免疫学杂志,154:5590-5600(1995)许多研究也评估了应用肿瘤坏死因子受体与人IgG1的Fc蛋白相联以治疗败血休克的病人。Fisher,C.等.,N.Eng1.医学杂志.334:1697-1702(1996);Van Zee.K.等.,免疫学杂志,156:2221-2230(1996)。Fc也被用于与CD4受体融合以产生治疗AIDS的治疗蛋白。见:Capon等,自然,337:525-531(1989)。此外,白细胞介素2的N-末端与IgG1或IgG3的Fc部分相融合,以克服白细胞介素2的短半衰期和其全身毒性。见,Harvill等.,免疫技术,1:95-105(1995)。
由于OB蛋白被鉴定为有前途的蛋白药物,因此需要发展OB类似物组合物在临床应用上结合或替代OB蛋白的施用。这些进步将包括OB类似物组合物,其中通过蛋白构成和化学修饰成功降低蛋白的降解,提高其稳定性和循环时间。本发明提供上述组合物。
发明概述
本发明涉及OB融合蛋白组合物,该组合物的制备方法及应用。本发明尤其涉及一种遗传性融合蛋白,该蛋白包括与OB蛋白或类似物的N端部分融合的免疫球蛋白的Fc区域或类似物。Fc-OB融合蛋白可通过Fc区域的半胱氨酸残基二聚化。但与预期不同的是,对OB蛋白的N端用Fc进行遗传融合修饰表现出稳定性,清除速率和减少降解的优点,这些优点在OB蛋白或以Fc与OB蛋白C末端融合中不可见。令人惊奇的也十分重要的是,与OB蛋白或对OB蛋白C末端的Fc修饰相比较,N-末端的修饰提供了预料之外的保护蛋白不降解的作用,增加了循环时间及稳定性。这些对OB蛋白进行Fc修饰带来的预料之外的优点给OB蛋白的使用者带来了好处,因为这些变化可降低用药剂量和用药频率。因此,在以下细节详述中,本发明有许多方面涉及通过将Fc区域与OB蛋白(或其类似物)融合对蛋白进行的遗传修饰以及特异性修饰,其制备及方法。
因此,一方面本发明提供Fc-OB融合蛋白其中Fc基因融合于OB蛋白(及其类似物)的N-末端。而且,Fc部分可通过本领域熟知的肽或化学连接物连于OB蛋白(或其类似物)的N-末端。如上所述且将在以下更多细节中描述的与OB蛋白及C端OB-Fc融合蛋白相比,Fc-OB融合蛋白具有预料之外的保护不被降解作用及延长的循环时间和增加的稳定性。本发明的其它方面,不仅包括Fc-OB融合蛋白组合物,还包括编码这些蛋白的DNA序列,相关载体以及包含这些载体的宿主细胞,载体和宿主细胞对于生产本发明的融合蛋白十分有用。
其二,本发明提供了制备Fc-OB融合蛋白的方法,该方法包括制备重组蛋白的重组DNA技术。此外,该方面还包括发酵和纯化的方法。
另一方面,本发明提供在人或动物中处理超重的方法,包括通过施用Fc-OB融合蛋白调节脂肪沉积。由于Fc-OB融合蛋白的特征,方法着重于通过应用Fc-OB减重剂而减少OB蛋白的用药量和用药次数。
另一方面,本发明为治疗过度肥胖引起的共发病,如糖尿病,血脂异常或高血脂症,动脉硬化,动脉噬斑,减轻或预防胆结石形成、中风,以及增加胰岛素敏感度,增加瘦肉组织重量。
另一方面,本发明还提供Fc-OB蛋白,类似物及其衍生物的相关药用组合物,用于以上疗法中。
附图的主要描述
图1重组鼠metOB(双链)DNA(SEQ.ID.NOs.:1和2)和氨基酸序列(SEQ.ID.NO.3)。
图2重组人metOB类似物(双链)DNA(SEQ.ID.NOs.:4和5)和氨基酸序列(SEQ.ID.NO.6)。
图3(A-C)重组人metFc-OB(双链)DNA(SEQ.ID.NOs.:7和8)和氨基酸序列(SEQ.ID.NO.9)。
图4(A-C)重组人metFc-OB变异的(双链)DNA(SEQ.ID.NOs.:10和11)和氨基酸序列(SEQ.ID.NO.13)。
图5(A-C)重组人metFc-OB变异的(双链)DNA(SEQ.ID.NOs.:13和14)和氨基酸序列(SEQ.ID.NO.15)。
图6(A-C)重组人metFc-OB变异的(双链)DNA(SEQ.ID.NOs.:16和17)和氨基酸序列(SEQ.ID.NO.18)。
详细说明
本发明涉及Fc-OB融合蛋白组合物,该组合物的制备方法及应用。本发明尤其涉及免疫球蛋白的Fc区遗传地或化学方法融合到OB蛋白的N-末端。预料之外的,与OB蛋白或Fc融合在OB蛋白C末端相比,Fc融合至OB蛋白N-末端显示出其优势。N-末端修饰的Fc-OB蛋白令人惊讶地提供了预期之外的保护蛋白不降解的作用,延长循环时间以及增加稳定性。因此,Fc-OB融合蛋白和其类似物及衍生物的相关制备及应用方法在以下详述中都有描述。
组合物
起始于SEQ.ID.NO.9(见图3)的重组人Fc-OB序列的Fc序列可从人免疫球蛋白IgG-1重链中选择,见Ellison,J.W.等.,核酸研究.10:4071-4079(82),或本领域任何已知Fc序列(例其它IgG类,包括但不局限于IgG-2,IgG-3和IgG-4,或其它免疫球蛋白)中选择。Fc部分的变异物,类似物或衍生物可通过如进行不同的残基或序列替代来构建。
半胱氨酸残基可被删除或由其它氨基酸取代以防止Fc序列的二硫化物交联的形成。位于SEQ.ID.NO.9的位置5的特殊氨基酸为半胱氨酸残基。SEQ.ID.NO.9的重组Fc-OB序列为378氨基酸的Fc-OB蛋白(甲硫氨酸残基不计)。图3的重组Fc-OB蛋白之首个氨基酸序列所示为在-1位置以甲硫氨酸为+1。
可在位置5移去半胱氨酸残基或用一个或多个氨基酸取代。丙氨酸可在位置6取代半胱氨酸形成图4(SEQ.ID.NO.12)的变异的氨基酸序列。图4的重组Fc-OB蛋白是一种378个氨基酸的Fc-OB蛋白(未包括甲硫氨酸残基。图4的重组Fc-OB蛋白的第一个氨基酸序列,在-1位的甲硫氨酸指定为+1。
同样地,位于SEQ.ID.NO.9位置5的半胱氨酸可被丝氨酸或其它氨基酸残基取代或去除。在SEQ.ID.NO.15的位置1,2,3,4和5去除氨基酸也可制备变异物或类似物。在这些位置也可取代属于本发明的范围之中。图5的重组Fc-OB蛋白为373氨基酸的Fc-OB蛋白(甲硫氨酸残基不计)图5中重组Fc-OB蛋白的第一个氨基酸序列从在-1位置的甲硫氨酸为+1。
也可引入4个氨基酸的取代进行修饰以去除Fc受体结合位点和补体(Clq)结合位点。在SEQ.ID.NO.15进行的不同修饰方法包括在位置15用谷氨酸取代亮氨酸,在位置98用丙氨酸取代谷氨酸、在位置100和102用丙氨酸取代赖氨酸。(见图6和SEQ.ID.NO.18)。图6中重组Fc-OB蛋白为373氨基酸的Fc-OB蛋白(不计甲硫氨酸残基)。图6所示的重组Fc-OB蛋白的第一个氨基酸序列从-1位的甲硫氨酸为+1。
同样地,一个或多个酪氨酸残基可被苯丙氨酸替代。而且也可考虑其它不同的氨基酸插入,删除和(或)取代,这些方法均属于本发明的范围之内。进一步的,选择方法也可以以改变的氨基酸的形式,如肽模拟物或D-氨基酸。Fc蛋白也可通过不同长度的化学的或氨基酸的连接子部分与Fc-OB蛋白的OB蛋白相联。这些化学连接子在本领域内是众所周知的。氨基酸连接子序列包括但不仅限于:
(a)ala,ala,ala;
(b)ala,ala,ala,ala;
(c)ala,ala,ala,ala,ala;
(d)gly,gly;
(e)gly,gly,gly;
(f)gly,gly,gly,gly,gly;
(g)gly,gly,gly,gly,gly,gly,gly;
(h)gly-pro-gly;
(i)gly,gly,pro,gly,gly;和
(j)(a)~(i)的任意组合。
Fc-OB融合蛋白中的OB部分可从重组鼠中选择SEQ.ID.NO.3(见图3)中阐明,或来自重组人蛋白中,如Zhang等.,自然,上述,在此引入仅作参考,或来自那些在28位缺失谷氨酰胺残基的蛋白。(见Zhang等,自然,上述428页。)也可应用如SEQ.ID.NO.6(见图2)所述重组人OB蛋白类似物,它包括:(1)在35位点精氨酸取代赖氨酸;和(2)74位点亮氨酸取代异亮氨酸。(此类似物简短缩写为重组人R->L35,I->L74)。重组人和重组鼠的蛋白或类似物在OB蛋白N-末端有或无融合Fc部分的氨基酸序列在以下阐明,-1位为甲硫氨酸残基。但在任何现有OB蛋白类似物中,甲硫氨酸残基可能缺失。
鼠蛋白基本上与人蛋白同源,尤其是成熟蛋白更是如此,更进一步尤其在N-末端同源性高。在重组的人的蛋白序列中,通过改变与鼠序列不同的氨基酸(如取代氨基酸残基,可制备重组人蛋白的类似物。因为重组人蛋白在小鼠中具有生物活性,此种类似物在人中也很可能有活性。例:根据SEQ.ID.NO.6的数字顺序,使用在35位残基为赖氨酸,74位残基为异亮氨酸的人蛋白,其中第一位氨基酸为缬氨酸,146氨基酸为半胱氨酸,在位置32,35,50,64,68,71,74,77,89,97,100,105,106,107,108,111,118,136,138,142和145可用其它一个或多个氨基酸取代。可选择小鼠蛋白(SEQ.ID.NO.3)相应位置的氨基酸或其它氨基酸。
可根据大鼠OB蛋白序列,可进一步制备“共有序列”分子。Murakami等,生物化学生物生理学研究通讯。
209:944-952(1995)在此引仅作参考。大鼠OB蛋白与人OB蛋白在以下位点不同(使用SEQ.ID.NO.6的编号):4,
32,33,
35,
50,68,
71,
74,
77,78,
89,
97,100,101,102,
105,
106,
107,
108,
111,
118,
136,
138和
145。在这些不同位点,可用另一个或多个氨基酸取代。下划线的位点为小鼠和大鼠OB蛋白均与人OB蛋白差异的部位,因此,尤其适合改变。可根据大鼠OB蛋白在一个和多个位点以氨基酸取代或应用其它氨基酸。
在大鼠和小鼠OB蛋白中与成熟人OB蛋白均有差异的位点为:4,32,33,35,50,64,68,71,74,77,78,89,97,100,102,105,106,107,108,111,118,136,138,142和145。根据SEQ.ID.NO.6 OB的蛋白可在以上一个或多个氨基酸位点由另一个氨基酸取代,如在相应大鼠或小鼠序列中找到的氨基酸取代仍具有活性。
此外,在猕猴OB蛋白氨基酸中发现与成熟人OB蛋白有差异的为(用圆括号中单字母氨基酸缩写形式区别):8(S),35(R),48(V),53(Q),60(I),66(I),67(N),68(L),89(L),100(L),108(E),112(D),和118(L)。因为重组人OB蛋白在Cynomolgus猴中具有活性,根据SEQ.ID.NO.6的人OB蛋白(35位赖氨酸和74位异亮氨酸),有一个或多个猕猴偏离氨基酸被其它氨基酸取代,如圆括号中的氨基酸,也可有效。应该提到的是,一些猕猴中的偏离氨基酸也可在以上小鼠种中找到(位35,68,89,100和112)。因此,可制备鼠/猕猴/人共有的分子(使用SEQ.ID.NO.6的号码,35位为赖氨酸,74位为异亮氨酸。),在一到多个如下位点可被其它氨基酸取代,这些位点为:4,8,32,33,
35,48,50,53,60,64,66,67,
68,71,74,77,78,
89,97,
100,102,105,106,107,108,111,
112,118,136,138,142和145。
其它类似物可通过删除一部分蛋白氨基酸序列制备。例如,缺少引导序列的成熟蛋白(-22至-1)。可制备如下截短形式的人OB蛋白分子(使用SEQ.ID.NO.6编号):
(a)氨基酸98-146
(b)氨基酸1-32
(c)氨基酸40-116
(d)氨基酸1-99和112-146相连
(e)氨基酸1-99连接到112-146其中100-111的一至多个氨基酸置于氨基酸99到112之间。
而且,截短形式也可改变与人OB蛋白不同的一至多个氨基酸(在大鼠、小鼠或猕猴OB蛋白)。而且,任何改变可以以改变的氨基酸形式出现,如肽模拟物或D-氨基酸。
因此,本发明包括Fc-OB融合蛋白,其中OB蛋白从以下选择。
(a)如SEQ.ID.NO.3(如下)或SEQ.ID.NO.6所示的氨基酸序列1-146;
(b)如SEQ.ID.NO.6阐明的氨基酸序列1-146,其中35位为赖氨酸残基,74位为异亮氨酸残基;
(c)(b)的部分氨基酸序列在以下1至多个位点发生不同氨基酸取代(根据SEQ.ID.NO.6的编码,即使28位谷氨酰胺残基缺失的情况下也保留相同的计数顺序):4,32,33,35,50,64,68,71,74,77,78,89,97,100,102,105,106,107,108,111,118,136,138,142和145;
(d)(a)(b)或(c)可以在28位缺失谷氨酰胺残基的氨基酸序列;
(e)(a)(b)(c)或(d)在N-末端具甲硫氨酸残基的氨基酸序列;
(f)来自以下(按SEQ.ID.NO.6号码)的截短的OB蛋白类似物:
(i) 氨基酸98-146
(ii) 氨基酸1-32
(iii) 氨基酸40-116
(iv) 氨基酸1-99和112-146
(v) 氨基酸1-99和112-146其中100-111的一至多个氨基酸置于氨基酸99-112之间。
(vi) (i)截短OB类似物,该类似物在100,102,105,106,107,108,111,118,136,138,142和145位有一至多个氨基酸被其它氨基酸取代;
(vii) (ii)截短的类似物在4,8和32位有一至多个氨基酸被其它氨基酸取代;
(viii) (iii)截短的类似物在50,53,60,64,66,67,68,71,74,77,78,89,97,100,102,105,106,107,108,111和112位有,一至多个氨基酸被其它氨基酸取代;
(vix) (iv)截短的类似物在4,8,32,33,35,48,50,53,60,64,66,67,68,71,74,77,78,89,97,112,118,136,138,142和145位有至多个氨基酸被其它氨基酸取代;
(x) (v)截短的类似物在4,32,33,35,50,64,68,71,74,77,78,89,97,100,102,105,106,107,108,111,118,136,138,142和145位有一至多个氨基酸被其它氨基酸取代;
(xi) (i)-(x)的任何具N-末端甲硫氨酸残基截短的类似物;
(g)(a)~(f)之任一的OB蛋白或类似物的衍生物形成一个化学部分与蛋白部分相连的结构;
(h)(g)的衍生物,其中所述化学部分为水溶性聚合物部分。
(i)(h)的衍生物,其中所述水溶性聚合物部分为聚乙二醇;
(j)(h)的衍生物,其中所述水溶性聚合物部分为多聚氨基酸部分;
(k)(h)部分以由(f)形成的衍生物,其中所述部分只与N-末端所述蛋白部分相连;
(l)在药用可接受的载体中的(a)至(k)任何一种OB蛋白,类似物或衍生物。
衍生物
本发明的Fc-OB融合蛋白(这里所指的“蛋白”不经另外说明,惯指包括“肽”,Fc,OB或类似物,如下所列举。)由一至多个化学部分连接至Fc-OB融合蛋白部分衍生而成。这些化学修饰的衍生物可进一步制成为动脉内,腹膜内,肌内皮下,静脉内,口的、鼻的、肺的、表面的或如以下讨论的其它途径的施用。对生物活性蛋白进行化学修饰在特定环境下提供更多优点,如提高稳定性,延长蛋白药物的循环时间,降低致免疫性。见,U.S.专利No.4,179,337,Davis等,授权于1979.12.18.综述见Abuchowski等关于作为药物的酶类。(J.S.Holcerberg和J.Roberts,eds.PP.367-383(1983);Francis等,上述。
合适于衍生用的化学部分可从多种水溶性聚合物中选择。所选的聚合物应可水溶,这样与聚合物连接的蛋白不会在水环境如生理环境下沉淀。优选地,为最终产品制备的治疗用途,聚合物应为可药学可接受的。本领域技术人员可根据考虑选择期待的聚合物,如聚合物/蛋白结合物是否用于治疗,如果是这样,还应考虑要求剂量,循环时间,抗蛋白酶降解以及其它一些问题。对于本发明蛋白和肽,通过用要求形式(即通过渗透泵,或更优选地,注射或输注,或进一步制成口服,肺或鼻传递)的施用衍生物,并如此处描述地观察生物效用,可确定衍生过程的有效性。
水溶性聚合物可从包括聚乙二醇,乙二醇/丙二醇共聚物,羧甲基纤维素、葡聚糖、聚乙烯醇、聚乙烯吡咯烷酮,聚-1,3-二氧戊环,聚-1,3,6-三氧杂环己烷,乙二醇/马来酐共聚物,多聚氨基酸(均聚物或随机共聚物),和葡聚糖或聚(n-乙烯吡咯烷酮)聚乙二醇,丙二醇同源聚合物,聚丙烯氧化物/乙烯氧化物共聚物、聚氧乙烯多元醇和聚乙烯基醇中选择。聚乙二醇丙醛因其在水中的稳定性在工业生产中更具优势。同样也可用琥珀酸盐和苯乙烯。
用以制成Fc-OB融合蛋白的OB或Fc蛋白可通过将多聚氨基酸或分支点氨基酸与Fc或OB蛋白(或类似物)部分相连而制备。例如,多聚氨基酸可以为一个额外的载体氨基酸,它同Fc融合至OB蛋白或本发明中OB类似物的作用一样,除Fc-OB融合蛋白的其它优点以外也可延长蛋白循环的半衰期。因本发明的治疗和美容目的,这些多聚氨基酸应不产生中和抗原反应或其它不良反应的种类。这类多聚氨基酸可从包括血清白蛋白(如人血清白蛋白),额外抗体或其部分(例Fc区),或其它多聚氨基酸,例赖氨酸。如F所示,与多聚氨基酸相连的位置可为Fc-OB蛋白部分的N-末端,或C-末端,或其它中间部分,也可通过化学“连接子”部分连到Fc-OB蛋白上。
聚合物可为任意分子量,也可以分支或不分支。对于聚乙二醇,优选的分子量大约在2kDa和100kDa之间(“大约”指在制备聚乙二醇过程中,一些分子比标示分子量更重,而另一些更轻),以便于处理或生产。根据要求的治疗特点(例,要求的稳定释放的持续时间,效用,如果有生物学活性,处理的难易,抗原性的程度或缺失以及其它聚乙二醇对于治疗用蛋白或类似物已知的作用),其它规格也可应用。
这样连接的聚合物分子数目可能不同,本领域的技术人员能确定其功能上的作用。聚合物分子可能是单来源衍生的,也可是用相同或不同化学部分(例,聚合物如不同分子量的聚乙二醇)在衍生过程中,双,三,四或组合来源衍生。聚合物分子对蛋白(或多肽)分子的比例可随其在反应混合物中浓度变化。一般地说,最佳比率(就反应效应而言即无过剩反应蛋白或聚合物)由以下因素决定,如要求的衍生程度(例单,双,三,四等),选择的聚合物的分子量,聚合物是否分支以及反应条件。
化学部分连到蛋白时应考虑到对蛋白的功能性或抗原性区的影响。本领域技术人员可用多种连接方法。例,EP0401384在此引入仅作参考。(连接PEG至G-CSF),也见Malik等,Exp.Hematol 20:1028-1035(1992)(用tresyl chloride报告GM-CSF的聚乙二醇化)。例,聚乙二醇可通过反应基团共价结合在氨基酸残基上,如游离氨基或羧基。反应基团为活化的聚乙二醇分子可与之结合的那些种类。具游离氨基的氨基酸残基包括赖氨酸残基和N-末端氨基酸残基。具游离羧基的氨基酸残基包括天冬氨酸残基,谷氨酸残基和C-末端氨基酸残基。巯基也可用作反应基团以连接聚乙二醇分子。为治疗目的,优选的连接是在氨基上,如在N-末端或赖氨酸基团的连接。如果要求受体结合,应避免对受体结合起重要作用的残基进行连接。
Fc-OB融合蛋白可特殊要求为N-末端化学修饰的。本组合物以聚乙二醇为例说明,可选择不同的聚乙二醇分子(根据分子量,分支等),在反应混合物中聚乙二醇分子对蛋白(或多肽)分子的比例,执行聚乙二醇化反应的类型,及获得所选N-末端聚乙二醇化蛋白的方法,获得N-末端聚乙二醇化制备物(即必要时将此部分与其它单聚乙二醇化部分分离)的方法可通过从聚乙二醇化蛋白分子群体中纯化N-末端聚乙二醇化物质。选择的N-末端化学修饰可伴随还原的烷基取代,该烷基化作用利用在衍生特殊蛋白中所用不同类型的原始氨基(赖氨酸在N-末端)的差异反应性。在合适反应条件下,基本上可得到与N-末端用含聚合物的羰基群选择性衍生化的蛋白。例,可在某pH进行反应以选择地N-末端聚乙二醇化蛋白,该反应允许利用蛋白赖氨酸残基ε-氨基和N-末端残基α-氨基pKa的差异进行。通过这样选择性地衍生,可控制将水溶性聚合物连接到一种蛋白上;与聚合物的结合优势地发生在蛋白N-末端,且对其它反应基团如赖氨酸侧链氨基无明显修饰发生。使用还原性烷基取代,水溶性聚合物可为上述的类型,且应具有单独反应醛基以同蛋白结合。可用含有独立反应醛基的聚乙二醇丙醛。
为容易生产治疗药品,优选的是N-末端单聚乙二醇化衍生物。N-末端聚乙二醇化保证为一种均质产物。相对于双-,三-或多聚乙二醇化产物,该产物的特征是简化处理方法。应用以上还原性烷基取代方法,制备N-末端产物,是易于商业生产的优选方法。
复合物
Fc-OB融合蛋白,类似物或其衍生物可复合为一种结合组合物施用。这种结合组合物具有延长循环时间的效用,此效用超过Fc-OB融合蛋白,类似物或衍生物的作用。此组合物可为一种蛋白(或同义的,肽)。结合蛋白的例子之一为OB蛋白受体或其部分,如其水溶性部分。其它结合蛋白可通过检查血清中Fc-OB蛋白或OB蛋白来确定,或通过经验筛选结合的存在确定。应用的结合蛋白一般不会干扰OB蛋白,Fc-OB融合蛋白或其类似物或衍生物结合到内源OB蛋白受体或作用信号转导的能力。
药物组合物
本发明也提供了应用Fc-OB融合蛋白和衍生物的药物组合物的方法,该药物组合物可进行注射,口服,肺,鼻,经皮肤或其它形式的施用。一般地说,该发明包括的药物组合物包括有效数量的蛋白和本发明的衍生产物以及药用可接受的稀释剂,防腐剂,溶剂,乳化剂,佐剂和/或载体。这些组合物包含不同缓冲液内容(例Tris-HCl,乙酸盐,磷酸盐)的稀释剂,pH和离子强度;添加剂如去污剂和溶剂(例Tween 80,聚氧乙烯山梨醇脂肪酸酯80),抗氧化剂(例,抗坏血酸,焦二硫酸钠);防腐剂(例,硫柳汞,苯甲醇)和大量物质(例,乳糖,甘露醇);结合形成质如聚乳酸,聚羟基乙酸等的多聚化合物,或结合形成脂质体,也可用Hylauronic酸,它具有在循环中促进持久的持续时间的作用。这些组合物会影响物理状态,稳定性及体内释放的速率,以及在体内本发明蛋白和衍生物的清除速率。见,例,Remington’s药学科学,18th Ed.(1990 Mack出版公司.,Easton,PA,18042)1435-1712页,在此引入仅作参考。组合物可制备成液体形式,或制成为干粉,如冻干的形式。也可考虑使用植入的持久释放制剂如透皮制剂。
在此考虑应用的是口服固体剂量形式,在Remington’s药物科学18thEd.,1990(Mack出版公司EastonPA 18042)89章中概述,在此引入仅作参考。固体制剂形式包括片剂,胶囊,药丸,锭剂或糖锭,扁囊剂,小糖丸。脂质体的或类蛋白包被也可用于形成本发明的组合物(例如美国专利号4,925,673所披露的类蛋白微球)。可用脂质体囊化法以及脂质体可由不同聚合物衍生而成(例,U.S.Patent No.5,013,556)。治疗中可能的固体剂量形式在Marshall,K.In:现代药物学由G.S.Banker和C.T.Rhodes第四章,1979中详细描述,在此引入仅作参考。一般地说,制剂将包括Fc-OB融合蛋白(类似物或衍生物)和惰性成分,惰性成分用来抵抗胃环境的,以及在肠中释放的生物活性物质。
特别要考虑的是以上衍生蛋白的口服剂量形式。Fc-OB融合蛋白也可被化学修饰,因此衍生物的口服传递是有效的。概括地讲,期待的化学修饰是至少一部分与蛋白(或多肽)分子连接,所述的部分允许(a)抑制蛋白水解,(b)在胃或肠可摄入到血流中。同样期待增加蛋白全面的稳定性及增加在体内的循环时间。这样部分的例子包括:聚乙二醇,乙二醇和丙二醇的共聚物,羧甲基纤维素,葡聚糖,聚乙烯基醇;聚乙烯吡咯烷酮和多聚脯氨酸,Abuchowski和Davis,可溶的多聚酶加合物。在“作为药物的酶类,Hocenberg和Roberts,eds.,Wiley-Interscience,New York,NY,(1981),pp.367-383;Newmark,等,J.应用生物化学杂志。4:185-189(1982)。其它能用的聚合物为多聚1,3-二氧戊烷和多聚-1,3,6-三氧杂环己烷。优选用于治疗的,在上述所披露的,为聚乙二醇部分。
对于Fc-OB融合蛋白,类似物或衍生物,其释放位置可能在胃,小肠(例,十二指肠,空肠,回肠)或大肠。本领域技术人员会有各种有效组方,这些组方不在胃溶解,而在十二指肠或肠其它地方释放物质。优选地,该释放将避免胃环境的有害作用,且由保护Fc-OB融合蛋白,类似物或衍生物,或在胃环境之外如在肠释放生物活性物质实现。
为保证对胃的抗性,至少在pH5.0不通透的包衣是必需的。更常见的无惰性成分的例子是那些用作肠道的包衣的醋酸纤维素偏苯三酸盐(CAT),羟丙基甲基纤维素邻苯二甲酸盐(HPMCP),HPMCP50,HPMCP55,聚乙酸乙烯酯邻苯二甲酸盐(PVAP),Eudragit L30D,Aquateric,醋酸纤维素,邻苯二甲酸盐(CAP),Eudragit L,Eudragit S,和Shellac。这些包衣可用作混合薄膜。
包衣或包衣的混合物也可用于片剂中,而其目的不为保护避免受胃环境破坏。这些包衣包括糖衣或使片剂更易吞咽的包衣。胶囊可包括一个坚硬的壳(如明胶)组成,以便传递干药物即粉末;可用对于液体形式药物柔软的明胶包被。扁囊剂的外壳物质可能为浓淀粉或其它可食纸。对于丸剂,可应用菱形,模型化片剂或片剂研磨剂,湿润团技术。
治疗物可被包括在制剂中如细多粒子的1mm颗粒大小的颗粒或药丸的形式。用于制成胶囊施用的物质也可为粉末,轻微压缩的填料,甚至片剂。治疗药物可通过压缩制备。
色素和调味剂也可包括在内。例蛋白(或衍生物)可先被制成制剂(如通过脂质体或微球体囊化体),然后纳入到可食产品中,如含有色素和调味剂的冷藏饮料。
可用惰性物质稀释或增加治疗物的体积。这些稀释物包括碳水化合物类,尤其是甘露糖,α-乳糖,无水乳糖,纤维素,蔗糖,修饰过的葡聚糖和淀粉。某些无机盐可用作填充物如三磷酸钙,碳酸镁和氯化钠。一些商业用烯释剂为Fast-Flo,Emdex,STA-Rx 1500,Emcompress和Avicell。
分解剂可以固体剂量形式加入到治疗用的制剂中。用做分解剂的物质包括但不仅限于淀粉,包括建立在淀粉基础上的商用分解剂,Explotab。乙酸淀粉钠,安伯来特。羧甲基纤维素钠,Ultramylopectin,藻酸钠,明胶,橙皮,羧甲基纤维素酸,天然海绵和皂土均可用。其它形式的分解剂为不溶的阳离子交换树脂。粉状树胶可用作分解剂和粘合剂,粉状树脂包括琼脂,Karaya或西黄蓍胶。藻酸和其钠盐作为分解剂亦十分有效。
粘合剂可用于保持治疗剂一起形成为坚硬的片剂,它包括来自天然产物的物质如阿拉伯胶,西黄蓍胶,淀粉和明胶。其它种类包括甲基纤维素(Mc),乙基纤维素(Ec)和羧甲基纤维素(CMC)。聚乙烯吡咯烷酮(PVP)和羟丙甲基纤素(CMC)。聚乙烯吡咯烷酮(PVP)和羟丙基甲基纤维素(HPMC)二者都可用于酒精溶剂中使治疗物颗粒化。
抗摩擦剂可加入到治疗用制剂中以阻止制剂过程中的粘着。润滑剂可用作治疗物和模型外壳之间的隔离层,它们包括而不仅限于;硬脂酸包括其镁盐和钙盐的酸,聚四氟乙烯(PTFE),液体石蜡,植物油和蜡。可溶的润滑剂也可用如十二烷基硫酸钠,十二烷基硫酸镁,不同分子量的聚乙二醇,Carbowax 4000和6000。
可提高制剂过程中药物的流动特性且在压缩过程中帮助重排的滑剂也可添加。这些滑剂包括淀粉,滑石,热源石英和水合硅铝酸盐。
为帮助治疗物溶解至水环境,可加入表面活性剂作为湿润剂,表面活性剂包括阴离子去污剂如十二烷基硫酸钠,十六烷磺基琥珀酸钠和十六烷磺酸钠。也可用阳离子去污剂,包括洁尔灭或氯化苄乙铵。可能的非离子型去垢剂可加入到制剂中作为表面活性剂的有lauromacrogol400,polyoxyl 40硬脂酸酯,聚乙烯加氢蓖麻油10,50和60,甘油单硬脂酸,聚山梨酸酯40,60,65和80,蔗糖脂肪酸酯,甲基纤维素和羟甲基纤维素。这些表面活性剂可在蛋白或衍生物制剂中单独或按不同比例混合掺入。
添加剂可能提高蛋白(衍生物)的摄入,例脂肪酸、油酸、亚油酸和亚麻酸。
制剂被控制地释放是所期待的。可掺入药物到无活性基质中,此无活性基质可允许以渗透或沥滤机制释放,例树胶,缓慢降解基质可加入到制剂中,例藻酸盐,多糖。另一种使治疗物被控释放的形式是通过建立在Oros治疗系统(Alza Corp)的一种方法,即药物包被在半透膜中,此半透膜允许水进入并推动药物因渗透作用而通过单个小孔。一些肠衣也具延迟释放作用。
其它还有些可于制剂的包衣。这包括能应用于包衣盘的不同的糖。治疗剂可为薄膜包被的片剂形式,这样应用的物质在本例中分为2类。第一类为非肠道物质包括甲基纤维素,乙基纤维素,羟乙基纤维素,甲基羟乙基纤维素,羟丙基纤维素,羟丙基甲基纤维素,羧基-甲基纤维素钠,providone和聚乙二醇。第二类包括肠道物质,这些物质为邻苯二甲酸的普通酯。
物质混合后可用于提供最佳的薄膜包衣。薄膜包衣可在包衣盘或液体床中制备或通过压缩包衣法。
在此也注意到本蛋白(或其衍生物)的肺传递。该蛋白(或衍生物)在吸气时传递到哺乳动物的肺并横越肺表皮至血流中(其它有关报告包括Adjei等药物学研究7565-569(1990);Adjei等,药物国际杂志63:135-144(1990)(醋酸亮丙瑞林);Braquet等,心血管药物学杂志13(增刊5):s.143-146(1989)(内皮素-1);Hubbard等;国内药物年刊3:206-212(1989)(α1-抗胰蛋白酶);Smith等.,临床研究杂志。84:1145-1146(1989)(α-1-蛋白酶);Oswein等.,“蛋白的雾状分散技术”,呼吸药物传递专题讨论会II进展,Keystone,Colorado,March,1990(重组人生长激素);Debs等,免疫学杂志140:3482-3488(1988)(干扰素γ和肿瘤坏死因子α)和Platz等.,U.S.专利No.5,284,656(粒细胞集落刺激因子)。
为本发明的实践应用,注意到广泛的用于治疗产品的肺传递的医学方法,包括但不仅限于雾化机,表控的剂量吸气器,粉末吸气器,这些方法都是本领域技术人员所熟悉的。
一些适于本发明实施的商用设备的具体实施例子有Ultravent雾化机,由Mallinckrodt,Inc.,生产,St.Louis,Missoun;Acorn II雾化机Marquest医药产品生产,Engleword,Colorado;Ventolin仪表控制的剂量吸气器,Glaxo Inc.生产.,Research Triangle Park,North Caroliha;Spinhaler粉末吸气器,Fisons Crop.生产,Bedford,Massachusetts。
所有这些装置需要应用合适的制剂来分散蛋白(类似物或衍生物)。典型的,每种制剂特殊对应使用的装置,除稀释剂,佐剂和/或对有用于治疗的载体外,还可加入合适的推进剂。
蛋白(或衍生物)最好制备为,颗粒形式,一般颗粒大小不超过10μm(或微米),最优选的为0.5至5μm,以便最有效传递到远端肺。
载体包括碳水化合物如海藻糖、甘露糖、木糖醇、蔗糖、乳糖、山梨糖醇。其它用于制剂的成分可包括DPPC,DOPE,DSPC和DOPC。天然或合成表面活性剂也可用。可应用聚乙二醇(即使离开其在衍生蛋白或类似物中的应用)。葡聚糖,如环化葡聚糖可用,胆盐和其它相关促进剂也可用。纤维素和纤维素衍生物也可用。氨基酸可用,如其在缓冲制剂中的应用。
同时也注意到脂质体,微囊体或微球体,包含物复合体或其它形式载体的应用。
适用于喷雾器,无论喷射器或超声发射器的制剂,一般包括Fc-OB蛋白,类似物或其衍生物。这些蛋白在水中以每毫升0.1至25mg生物活性蛋白溶液浓度溶解。制剂也可包括缓冲液或单糖(例,为蛋白的稳定性和调节渗透压。喷雾制剂还包括表面活性剂,用于减少或阻止表面诱导的蛋白聚集,这种聚集是形成气雾剂时溶液的原子化引起。
用于仪表控制的剂量吸气器的制剂一般构成在表面活性剂帮助下,重悬在推进剂中良好分配的含蛋白(或衍生物)的粉末。推进剂可为任何便于此目的常规物质,如氯氟烃,氢氯氟烃或氢氟烃或碳氢化合物,包括三′氯氟代甲烷,二氯二氟甲烷,二氯四氟乙醇,和1,1,1,2-四氟乙烷或其组合,合适的表面活性剂包括山梨聚糖三油酸酯和大豆卵磷脂,油酸也是有用的表面活性剂。
为粉末吸入设备准备的制剂主要包括良好分散的干粉,其中包含蛋白(或衍生物),也可包含大量试剂如乳糖,山梨糖醇、蔗糖,甘露糖,海藻糖、或木糖醇,其数量要使干粉末从设备分散的数量,例50至90%的制剂量。
也可考虑蛋白(或类似物或衍生物)的鼻传递。鼻传递允许蛋白在施用治疗产物到鼻后直接传到血流而无需产物在肺的沉淀。用于鼻递的制剂包括葡聚糖或环葡聚糖也可考虑通过粘膜的传递。
剂量
本领域的技术人员通过施用和观察期待的治疗作用可确定有效的剂量。由于OB蛋白N-末端的修饰,与OB蛋白和C-末端修饰的OB蛋白相比,本发明提供了意想不到的保护不受降解作用,且提高了循环时间和稳定性。本领域技术人员可通过这些变化确定可需要较少剂量或较小频率剂量的有效剂量。
优选地,该分子的制剂在10μg/kg/天和10mg/kg/天,将产生符合期待的治疗作用。有效剂量可通过超时运用诊断工具来决定。例,为测量血液中(血浆成分血清)中OB蛋白或Fc-OB融合蛋白的量的诊断可首先运用以决定蛋白的内源水平。这些诊断工具可以抗体分析形式,如抗体夹心分析。内源OB蛋白的量需首先确定,才决定基线。当内源的和外源的OB蛋白或Fc-OB融合蛋白(即体内找到的蛋白,类似物或衍生物,无论为自己产生的或施用的)的量在治疗的连续过程中被确定的,治疗剂量也被确定。剂量可能在治疗过程中变化,在起始阶段相对为高剂量,直到疗效可见,然后施以较低剂量以保持疗效。
理想地,在(X)下情况下,当期待单独的血脂水平降低,或期待保持血脂水平的降低,或期待自体物质变瘦的增加,剂量将不足以导致体重减轻。因此,在治疗肥胖病人伊始,剂量可被施用使体重减轻和伴随血脂水平降低或伴行脂肪组织减少/瘦物质增加,一旦成功减少足够体重,可施用剂量以阻止重获体重,更足够保持期待的血脂水平或瘦物质增加(或阻止瘦物质的消耗)。此剂量可凭经验确定,因OB或Fc-OB蛋白的作用是可逆的,(例,Campfield等科学269:546-549(1995)在547)。因此,假如在未期待体重减轻时某剂量造成观察到体重减少,就应施用较低剂量以达到期待的血脂水平或瘦组织物质的增加,且保持期待的体重。
为提高个体对胰岛素的敏感性,应考虑相似的剂量,瘦物质增加而无体重减轻,可能足够成功降低个体在施用胰岛素(或潜在的,支链淀粉,噻唑烷二酮(thiazolidinediones)或其他潜在的治疗糖尿病的药物)进行糖尿病治疗时的量。
为增加全面的强度,应考虑相似的剂量。可得到瘦物质增加同时伴行全面强度增加,而所用的剂量不足以导致体重减少的结果。其它优点,如红血细胞(和血液中氧合作用)增加和骨骼吸收作用或骨质疏松的下降可在非体重减轻下获得。
组合
本方法可应用于与其它药物结合,如那些处理糖尿病有效的(例胰岛素,可能地,噻唑烷二酮支链淀粉,或其拮抗剂),胆固醇和血压降低药物(如那些降低血脂水平或其它心血管药物),和增加活性药物(例安非他明)。食欲抑制剂也可用(如那些作用于5-羟色胺或神经肽水平的药物)。这样的施用可同时进行或依次地进行。
另外,本方法可用于与外科手术的步骤结合,如旨在改变身体的全面的美容手术外形(例旨在减身体物质的吸脂术或激光手术)。心脏手术的健康益处,如分流术手术或其它旨在缓解由于脂肪沉淀,血管阻塞而造成的有害病状,如动脉斑,可在同时使用本发明和方法下增加其益处。旨在消除胆结石的方法,如超声或激光方法,也可在本治疗方法之前,当中或以后应用。进一步地,本方法可用作外科手术或治疗骨折,肌肉损伤或其它可提高瘦组织物质而改善的疗法结合使用。
以下实施例用于进一步更详尽解释本发明,但不是限制本发明的范围。实施例1.通过皮下注射应用鼠Fc-OB蛋白
本实施例论证了皮下注射鼠Fc-OB蛋白导致正常鼠体重减轻。正常(非肥胖)CD1小鼠通过皮下注射施用鼠Fc-OB蛋白经过22天时间。注射到22天时10mg蛋白/kg体重/天的剂量导致体重从基础重量下降了14%(+/-1.1%)。加入PBS剂量导致到22天注射后体重从基础重量下降3.9%(+/-3.3%)。在肥胖CD1小鼠中,应用10mg蛋白/kg体重/天Fc-OB蛋白,经过22天注射后,导致体重从基础重量下降10%(+/-4.3%),而同样施用PBS则导致体重从基础重量下降8.7%(+/-1.3%)。
以下为CD1小鼠(8周龄)与基础体重相比的百分比变化。
表1
皮下注射体重减轻
时间(天) | 介导液(PBS) | 瘦/重组Fc-OB融合蛋白 | 肥胖/重组Fc-OB融合蛋白 |
1-2 | -.44+/1.1 | -3.6+/-.41 | -1.03+/-1.36 |
3-4 | -1.07+/-.13 | -6.8+/-1.5 | -2.7+/-1.1 |
56 | -.13+/-1.1 | -9.5+/-1.2 | -4.9+/-.95 |
7-8 | -.92+/-.29 | -12.5+/-1.6 | -7.7+/-2.9 |
9-10 | 1.6+/-1.3 | -12.6+/-1.9 | -8.2+/-2.9 |
11-12 | -1.98+/-1 | -13.6+/-1.96 | -8.6+/-2.9 |
13-14 | -5.2+/-1.3 | -14.6+/-1.7 | -10.1+/-3.6 |
15-16 | -8.6+/-0.1 | -14.5+/-2 | -9.4+/-2.2 |
17-18 | -8.5+/-.64 | -16.1+/-1.8 | -9.6+/-2.99 |
19-20 | -4.1+/-.99 | -16+/-1.5 | -10.4+/-3.3 |
21-22 | -3.9+/-3.3 | -14.1+/-1.1 | -10+/-4.3 |
可见,在22天皮下注射方案之后,接受Fc-OB蛋白的动物在瘦动物中失去14.1%的体重,在肥胖动物中失去10%体重,此值与仅接受PBS介导液的动物和与基础重量相比得到。
令人惊奇的是,接受Fc-OB蛋白动物在22天后继续减少体重至28天,即最后一次注射的4天之后。正常(非肥胖)CD1小鼠通过皮下注射施用10mg蛋白/kg体重/天的鼠Fc-OB蛋白在第22天停止,导致第28天体重比基础体重下降21%,与第22天相比,第22天体重下降14%。相似的,肥胖CD1小鼠施用10mg蛋白/kg体重/天的鼠Fc-OB蛋白在第22天停止导致第28天13%体重从基础重量下降,而第22天仅下降10%。在第34天,肥胖小鼠体重下降保持在10%,而瘦小鼠恢复到5%下降。每个体系的对照组从22天到34天平均增重,在肥胖小鼠中为4%而瘦鼠中为7%。实施例2:通过对C57小鼠的皮下注射使用人Fc-OB蛋白
本实施例论证了在正常小鼠中皮下注射施用人Fc-OB蛋白导致体重下降,正常(非肥胖)C57小鼠通过7天时间的皮下注射施用人Fc-OB蛋白。10mg蛋白/kg体重/天的剂量导致7天注射后,体重从基础体重下降12%(+/-1.3%)。1mg蛋白/kg体重/天的剂量7天注射后,导致体重从基础体重下降8.9%(+/-1.5%)。7天注射后在肥胖C57小鼠中应用10mg蛋白/kg体重/天人OB蛋白剂量导致体重从基础体重下降1.1%(+/-0.99%),而1mg蛋白/kg体重/天的剂量导致从基础体重下降2.5%(+/-1.1%)。
结果
以下为C57小鼠(8周龄)从基础体重的百分比差别:
表2皮下注射后体重减轻
时间(天) | 介导液(PBS) | 重组Fc-OB融合蛋白 | 重组OB蛋白 |
1-2 | .258+/-1.3 | -6.4+/-1.6 | -2.1+/-.91 |
3-4 | 2.2+/-1.1 | -12.1+/-1.5 | -7.8+/-.36 |
5-6 | 4.5+/-2 | -11.5+/-1.5 | -1.7+/-.6 |
7-8 | 7.0+/-2.1 | -11.9+/-1.6 | 0.1+/-1.2 |
9-10 | 9.0+/-1.9 | -11.5+/-1.3 | 7.2+/-2.7 |
11-12 | 10+/-3.8 | -9+/-1.4 | 10.9+/-2.9 |
13-14 | 12.5+/-4.4 | -9.5+/-1.6 | 12.3+/-6.4 |
15-16 | 11.1+/-1.0 | -3.0+/-1.5 | 10.3+/-3.3 |
17-18 | 17.2+/-3.6 | 8.0+/-1.3 | 13.3+/-3.4 |
可见,在以10mg/kg体重/天皮下注射方案7天后,到17天时,接受Fc-OB蛋白的动物恢复了它们8%体重,接受1mg蛋白/kg体重/天剂量的动物7天皮下注射方案后12天恢复6.4%的体重。
这些研究还显示在第7天至第22天的恢复阶段,在第7天最后注射之后,在以Fc-OB处理的C57小鼠中体重恢复比OB处理鼠缓慢。这说明Fc-OB蛋白不如OB蛋白清除快,因此造成延长的减轻体重效应。实施例3:以Fc-OB蛋白处理CF7小鼠的剂量反应
另一项研究证明持续施用Fc-OB蛋白具有剂量反应。在该研究中,肥胖CF7小鼠,重35-40g,被用与以上实施例相似的方法施以人Fc-OB蛋白。结果显示在以下表3(用%体重减少与基础体重相比,与以上同样方法测量)。
表3持续施用的剂量反应
剂量 | 时间 | %体重减少 |
0.25mg/kg/天 | 第5天 | 4 |
0.5mg/kg/天 | 第5天 | 12 |
1mg/kg/天 | 第5天 | 16 |
可见,剂量从0.25mg/kg/天到1mg/kg/天增加了体重减少从4%至16%。同样值得注意的是,第5天,1mg/kg/天剂量造成体重16%减少。这些研究也显示缓慢的体重恢复到0%说明Fc-OB蛋白不迅速清除,造成延长的体重减少效应。实施例4:重组人Fc-OB在CD1小鼠和狗中的药物动力学
本研究证明人重组met Fc-OB蛋白在CD1小鼠和狗中的药物动力学特性。按照静脉内或皮下加药1mg/kg/天,重组人met Fc-OB蛋白和人met OB蛋白在血清中的浓度由酶联免疫吸附分析(ELISA)确定。
在两物种中,当根据高峰血清浓度和该血清浓度以下曲线(AUC)的较大区域确定量时,与重组人met OB蛋白相比,这些数值在接触该蛋白后增加。Fc-OB具有比重组人met OB蛋白缓慢的系统清除。在Fc-OB与OB蛋白比较时可见较缓慢的清除率和较长的半衰期。体积的加大不仅造成蛋白稳定性增加还降低肾清除的效率。结果Fc-OB在循环系统中清除较慢。Fc-OB蛋白的峰值时间,峰值血清浓度和AUC的增加与较低清除率一致。Fc-OB蛋白与OB蛋白相比实际产出更高系统接触。结果在以下表4所示。
表4药物动力学特征
实施例5:
种 | CD-1小鼠 | CD-1小鼠 | 猎犬 | |||
施用途径 | 静脉内 | 皮下 | 皮下 | |||
OB蛋白 | Fc-OB蛋白 | OB蛋白 | Fc-OB蛋白 | OB蛋白 | Fc-OB蛋白 | |
剂量水平(mg/kg) | 1 | 1 | 1 | 1 | 0.5 | 0.5 |
峰值时间(h) | 0.14 | 6 | 2.8 | 8 | ||
峰值血清浓度(ng/mL) | 1520 | 7550 | 300 | 1120 | ||
AUC(ng·h/mL) | 1470 | 366000 | 1230 | 132000 | 2200 | 52500 |
半衰期(h) | 0.491 | 21.4 | 0.388 | 2.13 | 22.9 | |
清除率(mg/h/kg) | 681 | 2.73 |
本实施例论证了在既不肥胖且无升高的血脂水平的正常小鼠中,施用人重组Fc-OB蛋白导致胆固醇,葡萄糖和甘油三酯水平降低。另外,本实施例证明这些水平在经过3天恢复期后仍保持低水平。
正常CD1小鼠通过皮下注射施用重组人Fc-OB蛋白,在即最后一天注射23天后,取24小时的血样。如上所讨论,动物在所施剂量下体重减轻。如表5所示,与对照相比,小鼠以剂量依赖样式,持续降低其血清胆固醇,葡萄糖,甘油三酯:
表5
剂量 | 葡萄糖 | 胆固醇 | 甘油三酯 |
PBS | 232.6+/-15.1 | 67.8+/-3.6 | 52.6+/-3.7 |
1mg/kg/天 | 225.8+/-29.1 | 54+/-5.6 | 43+/-8.7 |
10mg/kg/天 | 193.2+/-21.4 | 53.4+/-5.7 | 38+/-11 |
1mg/kg每二天 | 242.0+/-9.3 | 52.6+/-4.4 | 40.8+/-7.2 |
10mg/kg每二天 | 197.4+/-27.9 | 51.4+/-5.9 | 29.8+/-6.3 |
1mg/kg每三天 | 244.8+/-19.5 | 60.8+/-7.3 | 54+/-7.1 |
10mg/kg每三天 | 188+/-31.2 | 52.2+/-6.9 | 26.2+/-10.7 |
这些数据说明Fc-OB蛋白或类似物或其衍生物,是有效的血脂降低剂。实施例6:
肥胖人患者施用人Fc-OB蛋白,或其类似物或衍生物为减轻体重之目的。这些肥胖患者也具升高的血脂水平,包括高水平胆固醇,在200mg/100ml以上。患者在Fc-OB治疗后获得满意的体重减少。Fc-OB蛋白或其类似物或衍生物的维持剂量施用至非肥胖患者以维持低血脂水平,包括低胆固醇水平,低于200mg/100ml。剂量施用不足以造成进一步体重减轻。施用是慢性的。Fc-OB蛋白或类似物或衍生物的循环水平可用诊断试剂盒检测,如抗OB蛋白(如果要求时,或其它可用的抗原来源)的抗体检测。实施例7
非肥胖患者经过冠状分流术手术或其它侵入处理以缓和发展阶段动脉斑形成期。手术后,患者施用维持剂量的Fc-OB蛋白或类似物或衍生物以阻止动脉斑的重形成。施用剂量不足以造成体重减轻。服法为慢性。Fc-OB蛋白或类似物或衍生物的循环水平用诊断试剂盒检测,如抗OB蛋白的抗体分析(如果要求时,或其它可用的抗原来源)。实施例8
非肥胖患者因限制血流经阻塞动脉而患高血压。该患者施以一定剂量Fc-OB蛋白或类似物或其衍生物,足以减少造成堵塞动脉的动脉斑。因此,该患者的进一步动脉斑的形成和高血压被控制。如病情重出现,该患者则被重新施用有效量的Fc-OB蛋白。类似物或衍生物,该量足够重新恢复血流,而不足以造成体重下降。Fc-OB蛋白或类似物或衍生物的循环水平用诊断试剂盒监测,如抗体抗Fc-OB分析蛋白(如果要求时,或其它可用抗原来源)。实施例9:
患者患胆结石。无论胆结石移除,希望避免其他的胆结石生成,或胆结石移除但保留胆囊(例使用激光或超声外科手术)希望避免其他的胆结石生成。患者被施以有效量的Fc-OB蛋白,类似物或其衍生物以导致阻止另外的胆结石积累或胆结石重积累。循环Fc-OB蛋白或类似物的水平用诊断试剂盒监测,如抗体抗Fc-OB分析蛋白(如果要求时,或其它可用的抗体来源)。实施例10:
一位糖尿病患者期待用较小剂量胰岛素治疗糖尿病。该患者被施以有效量的Fc-OB蛋白,类似物或其衍生物,导致瘦组织物质增加,该患者对胰岛素的敏感性增加,缓解糖尿病症状所需的剂量下降,无论以所需的胰岛素单位形式或以每天所需注射胰岛素的次数形式都下降。循环的Fc-OB蛋白或类似物或衍生物的水平可通过诊断试剂盒监测,如抗OB蛋白的抗体分析(如果要求时,或其它可用抗原来源)。实施例11:
一个非肥胖患者为治疗目的期待瘦组织物质增加,例如从瘦组织物质消耗的病症中恢复。该患者被施以有效剂量的Fc-OB蛋白或其类似物或衍生物,导致期待的瘦组织物质增加。瘦组织物质增加用DEXA扫描法监测。循环Fc-OB蛋白或类似物或衍生物水平可通过诊断试剂盒监测,如抗OB蛋白(如果要求时,或其它可用的抗原来源)的抗体分析。材料和方法
动物野生型CD1小鼠V和(+/+)C57B16小鼠应用于以上实施例。鼠龄在起始治疗时间点为8周,动物体重稳定。
饲养和体重测量
小鼠用在粉末食物进料器磨碎的啮齿动物食物(PMI Feeds.Inc.)(Allentow Caging and Equipment)饲养,该法比常规块状食物更准确和敏感的测量。每天同一时间(2:00pm)称量体重,持续期待的时期。注射前一天的体重定为基础体重,使用重达18-22克的小鼠。
笼养小鼠单独笼养,保持在人道条件下。
施用蛋白或介质蛋白(如下所述)或介质(磷酸缓冲盐,pH7.4)通过皮下注射或静脉内施用。
对照对照动物为仅注射媒介质,而未添加Fc-OB蛋白或OB蛋白至介质中。
蛋白序列ID.Nos.1,2和3阐明鼠重组OB DNA和蛋白(图1),序列ID.Nos.4,5和6阐明重组人OB DNA和蛋白的类似物(图2)。如上所指出,重组人OB蛋白如SEQ.ID.No.6,35位具赖氨酸残基而74位具异亮氨酸残基。进一步的,重组人蛋白在Zhang等,自然,上述,和PCT出版物WO96/05 309(12/22/96)(二者连图引入仅作参考)所阐述。图1和2的鼠和人类似物重组蛋白,为以本方法处理形成Fc-OB蛋白和药物生产的OB蛋白的例证。其它OB蛋白或Fc蛋白或其类似物或其衍生物也可用于形成Fc-OB融合蛋白。
在此,重组OB蛋白的氨基酸序列的(第一个氨基酸始为+1,为缬氨酸,在-1位的氨基酸为甲硫氨酸。C-末端氨基酸为146位(半胱氨酸)(见图1和2)。图3重组人Fc-OB蛋白的第一个氨基酸序列始为+1,为谷氨酸,-1位的氨基酸为甲硫氨酸。C末端氨基酸为378位(半胱氨酸)。图4重组人Fc-OB蛋白变体的首个氨基酸序列始为+1,为谷氨酸,-1位氨基酸为甲硫氨酸。C-末端氨基酸为378位(半胱氨酸)。图5的重组人Fc-OB蛋白氨基酸变体的第一个氨基酸序列始指为+1,为天冬氨酸,-1位氨基酸为甲硫氨酸。C-末端氨基酸为373位(半胱氨酸)。图6的重组人Fc-OB蛋白变体中第一个氨基酸始为+1,为天冬氨酸,-1位氨基酸为甲硫氨酸。C-末端氨基酸为373位(半胱氨酸)。
表达载体和宿主菌株
使用的表达载体质粒为pAMG21(ATCC)登记号98113),为pCFM1656(ATCC登记号69576)的衍生物,具合适限制性位点在lux PR启动子下游插入基因(见美国专利No.5,169,318描述lux表达系统)。Fc-OB DNA,如下所述在图3-6中所示,在限制性核酸内切酶NdeI和BamHI线性化作用下产生结合到表达载体pAMG21,并转化至大肠杆菌宿主菌株FM5。大肠杆菌FM5细胞由Amgen Inc.,Thousand Oaks,CA从大肠杆菌K-12菌株衍生。(Bachmann,等,细菌综述,40:116-167(1976)),包括结合的λ噬菌体抑制子基因。cI587(Sussman等,C.R.学术科学254:1517-1579(1962))。载体产生,细胞转化,克隆筛选按标准方法操作,(例Sambrook等,分子克隆:实验手册,第2版,冷泉港实验室出版,冷泉港,NY.)。宿主细胞在LB培养基中生长。
Fc-OB DNA构建
质粒pFc-A3(下述)作为人免疫球蛋白IgG-1重链从氨基酸99位(谷氨酸)至天然羧基端的序列来源。人IgG-1序列可从Genebank(P01857)获得。
人OB序列披露在上述Zhang等自然,同上PCT出版物WO96/05309包括图引入仅作参考。OB DNA连接到以限制性内切酶XbaI和BamHI连线性化的表达载体pCFM1656中,使用标准克隆方法,例Sambrook,等.,分子克隆:实验指南,第2版,冷泉港实验室出版,冷泉港,N.Y.。携带OB DNA序列质粒pCFM1656作为重组人OB基因序列的来源。
使PCR重叠扩增法使两序列融合(HO,S.N.,等多聚酶链式反应重叠扩增定点诱变,基因77:51-59(1989)),PCR产物用限制性内切酶NdeI切割,以产生5′-粘性末端,用限制性内切酶BamHI产生3′-粘性末端。载体pAMG21,以相似方法切割。连接反应用融合片段和线性载体实施。连接DNA通过电融合转化入大肠杆菌宿主菌中。检查卡那霉素(50μg/ml)选择琼脂皿上存活的克隆的Fc-OB分类蛋白的表达。分离来自各个克隆的质粒,检验基因编码区的序列。
当要求对Fc-OB蛋白基因作额外修饰时,再次应用PCR技术制造变化。在融合蛋白(SEQ.ID.No.9)的N-末端实施两种变化以产生变体SEQ.ID.NOS.12和15。另一变体构建以介导四个氨基酸取代以清除Fc-受体结合位点(15位亮氨酸以谷氨酸取代),补体(Clq)结合位点(98位谷氨酸以丙氨酸取代100位赖氨酸被丙氨酸替代,102位赖氨酸被丙氨酸替代。(见:Xin Xiao Zhang等免疫学杂志154:5590-5600(1995))。该构建体的模板为SEQ.ID.No.15,产生的变体为SEQ.ID.Nos.18。
pFC-A3载体构建
质粒pFC-A3,含有编码人免疫球蛋白IgG-1重链Fc部分(见Ellison,J.W.等,核酸研究,10:4071-4079(1982)),从铰链区首位氨基酸Glu-99至羧基末端添加5′-NotI融合位点和3′-SalI和XbaI位点,均由PCR扩增人脾cDNA文库得到。PCR反应终体积为(100μl),使用2单位Vent DNA聚合酶加在20mM Tris-HCl(pH8.8),10mM KCl,10mM(NH4)2SO4,2mM MgSO4,0.1%Triton X-100,每个dNTP 400mM和要扩增的1ng cDNA文库和1μM每种引物。反应起始为95℃变性2分钟,而后30个循环,循环为95℃30秒,55℃30秒,73℃2分钟。5′端引物掺入在NotI位点紧邻5′端至IgG-1铰链区的首个残基(Glu-99)。3′-端引物掺入SalI和XbaI位点。717碱基对PCR产物用NotI和SalI消化,产生的DNA片段1%琼脂糖电泳分离纯化并克隆至NotI,SalI消化的pBluescript II KS载体(Stratagene)中。在得到的质粒pFc-A3的插入,测序以确定PCR反应的忠实性。
制备方法
以下制备方法用于制备生物活性重组甲硫氨酸鼠或人类似OB蛋白和Fc-OB融合蛋白。相似方法可用于制备生物活性甲硫氨酸人OB蛋白。
发酵方法
应用批量发酵方法。培养基组成如下所述。
包括基本氮源的培养基部分进行灭菌(升温至120℃-123℃25-35分钟),在发酵容器中进行,冷却后,无菌加入碳,镁,磷酸和微量金属源。发酵罐中加入以上重组鼠蛋白制备菌500mL(LB液体培养基中生长)的过夜培养物。当培养至最佳密度(600nm测量作为细胞密度指示)。达到15-25吸光值时,加入(1mL/L)自发诱导物溶液(0.5mg/mL高丝氨酸内酯)入培养物中诱导重组基因表达。发酵过程允许另外进行10至16小时,而后离心收获液体培养基。
培养基组成:
一次操作需原料:
34g/L 酵母抽提物
78g/L 大豆蛋白
0.9g/L 氯化钾
5.0g/L Hexaphos
1.7g/L 柠檬酸
120g/L 甘油
0.5g/L MgSO4·7H2O
0.2mg/L 痕量金属溶液
0.5mL/L P2000消泡剂痕量金属溶性
氯化铁(FeCl3·6H2O) 27g/L
氯化锌(ZnCl2·4H2O) 2g/L
氯化钴(CoCl2·6H2O) 2g/L
钼酸钠(NaMoO4·2H2O) 2g/L
氯化钙(CaCl2·2H2O) 1g/L
硫酸铜(CuSO4·5H2O) 1.9g/L
硼酸(H3BO3): 0.5g/L
氯化锰(MnCl2·4H2O): 1.6g/L
柠檬酸钠二水合物: 73.5g/L
人Fc-OB融合蛋白纯化过程
纯化人Fc-OB融合蛋白按以下步骤进行(除非其它说明,以下步骤在4℃操作)。纯化鼠和人OB蛋白方法公开于PCT出版物WO96/05309,上述,在此引入仅作参考。
1.细胞糊。大肠杆菌细胞糊重悬在5倍体积蒸馏水中。水中的细胞进一步二次通过微流体器破裂。破裂细胞在Beckman JB-6离心机用J5-4.2转子4.2k rpm离心1小时。
2.包含体洗涤。来自上述的上清液去除,以5倍体积蒸馏水重悬沉淀。混合物如步骤1离心。
3.溶解。沉淀以10体积50mM Tris.pH 8.5,8M盐酸胍,10mM二硫苏糖醇溶解,室温搅拌1小时。溶液为40mM胱胺二氢氯化物,搅拌1小时。
4.步骤3的溶液加入20至30体积的以下重新拌入溶液:50mMTris。pH8.5,0.8M精氨酸,2M尿素和4mM半胱氨酸。重新拌入物8℃搅拌16小时。
5.缓冲液交换。步骤4的溶液浓缩,过滤到10mM Tris,pH8.5。
6.酸沉淀。步骤5的溶液用50%冰乙酸调至pH4.75,室温孵育30分钟,溶液过滤。
7.阳离子交换层析。步骤6的溶液调至pH7.0,10℃上样至CM,琼脂糖凝胶快速流动柱中。20柱体积梯度洗脱,洗脱液为10mM磷酸盐,pH7.0,0至0.1M NaCl。
8.阴离子交换层析。步骤7的CM洗脱集中液用5mM Tris,pH7.5稀释5倍,10℃上样至Q琼脂糖快速流动柱中,20柱体积洗脱液为10mM Tris,pH7.5,0至0.2M NaCl洗脱。
9.疏水相互层析。Q琼脂糖洗脱集中液溶到0.75硫酸铵,室温上样到甲基Macroprep疏水相互作用柱。20柱体积洗脱液10mM磷酸盐,pH7.0,0.75M至0M硫酸铵,洗脱。
10.缓冲液交换。步骤9的集中液如需要则进行浓缩再对PBS缓冲液透析。
当本发明以优选实施方案描述时,可以理解本领域技术人员可进行各种变化和修饰。因此,应该认为所附的权利要求书覆盖上述所有的等值变化方法,如权利所要求的,这些方法均在本发明的范围之内。
序列表(1)一般信息:
(i)申请人:Mann,Michael B.
Hecht,Randy I.
(ii)发明题目:OB融合蛋白组合物和方法
(iii)序列数:18
(iv)通信地址:
(A)收件人:Amgen Inc.
(B)大街:1840 DeHavilland Drive
(C)城市:Thousand Oaks
(D)州:CA
(E)国家:美国
(F)邮政编码:91320-1789
(v)可读计算机类型:
(A)介质类型:软盘
(B)计算机:IBM PC兼容机
(C)操作系统:PC-DOS/MS-DOS
(D)软件:Patent In Release#1.0,版本1.30
(vi)目前申请资料:
(A)申请号:US08/770,973
(B)申请提出日期:1996,12,20
(C)分类:
(viii)律师/代理人信息:
(A)姓名:Knight,Matthew W.
(B)登记号:36,846
(C)参考/摘要号:A-416(2)SEQ ID NO.1信息:
(i)序列特征:
(A)长度:491碱基对
(B)类型:核酸
(C)链型:双链
(D)拓扑结构:线状
(ii)分子类型:cDNA
(ix)特点:
(A)名称/关键:misc-feature
(B)定位:41
(D)其它信息:/注意=“Met=ATG”
(xi)序列描述:SEQ.ID.NO.1:TCTAGATTTG AGTTTTAACT TTTAGAAGGA GGAATAACAT ATGGTACCGA TCCAGAAAGT 60TCAGGACGAC ACCAAAACCT TAATTAAAAC GATCGTTACG CGTATCAACG ACATCAGTCA 120CACCCAGTCG GTCTCCGCTA AACAGCGTGT TACCGGTCTG GACTTCATCC CGGGTCTGCA 180CCCGATCCTA AGCTTGTCCA AAATGGACCA GACCCTGGCT GTATACCAGC AGGTGTTAAC 240CTCCCTGCCG TCCCAGAACG TTCTTCAGAT CGCTAACGAC CTCGAGAACC TTCGCGACCT 300GCTGCACCTG CTGGCATTCT CCAAATCCTG CTCCCTGCCG CAGACCTCAG GTCTTCAGAA 360ACCGGAATCC CTGGACGGGG TCCTGGAAGC ATCCCTGTAC AGCACCGAAG TTGTTGCTCT 420GTCCCGTCTG CAGGGTTCCC TTCAGGACAT CCTTCAGCAG CTGGACGTTT CTCCGGAATG 480TTAATGGATC C 491(2)SEQ ID NO.2信息:
(i)序列特征:
(A)长度:491碱基对
(B)类型:核酸
(C)链型:双链
(D)拓扑结构:线状
(ii)分子类型:cDNA
(xi)序列描述:SEQ.ID.NO.2:AGATCTAAAC TCAAAATTGA AAATCTTCCT CCTTATTGTA TACCATGGCT AGGTCTTTCA 60AGTCCTGCTG TGGTTTTGGA ATTAATTTTG CTAGCAATGC GCATAGTTGC TGTAGTCAGT 120GTGGGTCAGC CAGAGGCGAT TTGTCGCACA ATGGCCAGAC CTGAAGTAGG GCCCAGACGT 180GGGCTAGGAT TCGAACAGGT TTTACCTGGT CTGGGACCGA CATATGGTCG TCCACAATTG 240GAGGGACGGC AGGGTCTTGC AAGAAGTCTA GCGATTGCTG GAGCTCTTGG AAGCGCTGGA 300CGACGTGGAC GACCGTAAGA GGTTTAGGAC GAGGGACGGC GTCTGGAGTC CAGAAGTCTT 360TGGCCTTAGG GACCTGCCCC AGGACCTTCG TAGGGACATG TCGTGGCTTC AACAACGAGA 420CAGGGCAGAC GTCCCAAGGG AAGTCCTGTA GGAAGTCGTC GACCTGCAAA GAGGCCTTAC 480AATTACCTAC C 491(2)SEQ ID NO.3信息:
(i)序列特征:
(A)长度:147个氨基酸
(B)类型:氨基酸
(C)链型:未知
(D)拓扑结构:未知
(ii)分子类型:蛋白质
(ix)特点:
(A)名称/关键:蛋白质
(B)定位:1
(D)其它信息:/注意=Met(ATG)从1开始
(xi)序列描述:SEQ.D.NO.3:Met Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys Thr Lea Ile Lys1 5 10 15Thr Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr Gln Ser Val Ser
20 25 30Ala Lys Gln Arg Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro
35 40 45Ile Leu Ser Leu Ser Lys Met Asp Gln Thr Leu Ala Val Tyr Gln Gln
50 55 60Val Leu Thr Ser Leu Pro Ser Gln Asn Val Leu Gln Ile Ala Asn Asp65 70 75 80Leu Glu Asn Leu Arg Asp Leu Leu His Leu Leu Ala Phe Ser Lys Ser
85 90 95Cys Ser Leu Pro Gln Thr Ser Gly Leu Gln Lys Pro Glu Ser Leu Asp
100 105 110Gly Val Leu Glu Ala Ser Leu Tyr Ser Thr Glu Val Val Ala Leu Ser
115 120 125Arg Leu Gln Gly Ser Leu Gln Asp Ile Leu Gln Gln Leu Asp Val Ser
130 135 140Pro Glu Cys145(2)SEQ ID NO.4信息:
(i)序列特征:
(A)长度:454碱基对
(B)类型:核酸
(C)链型:双链
(D)拓扑结构:线状
(ii)分子类型:cDNA
(ix)特点:
(A)名称/关键:misc-feature
(B)定位:4
(D)其它信息:/注意=“Met=ATG”
(xi)序列描述:SEQ.ID.NO.4:CATATGGTAC CGATCCAGAA AGTTCAGGAC GACACCAAAA CCTTAATTAA AACGATCGTT 60ACGCGTATCA ACGACATCAG TCACACCCAG TCGGTGAGCT CTAAACAGCG TGTTACAGGC 120CTGGACTTCA TCCCGGGTCT GCACCCGATC CTGACCTTGT CCAAAATGGA CCAGACCCTG 180GCTGTATACC AGCAGATCTT AACCTCCATG CCGTCCCGTA ACGTTCTTCA GATCTCTAAC 240GACCTCGAGA ACCTTCGCGA CCTGCTGCAC GTGCTGGCAT TCTCCAAATC CTGCCACCTG 300CCATGGGCTT CAGGTCTTGA GACTCTGGAC TCTCTGGGCG GGGTCCTGGA AGCATCCGGT 360TACAGCACCG AAGTTGTTGC TCTGTCCCGT CTGCAGGGTT CCCTTCAGGA CATGCTTTGG 420CAGCTGGACC TGTCTCCGGG TTGTTAATGG ATCC 454(2)SEQ ID NO.5信息:
(i)序列特征:
(A)长度:454碱基对
(B)类型:核酸
(C)链型:双链
(D)拓扑结构:线状
(ii)分子类型:cDNA
(xi)序列描述:SEQ.ID.NO.5:GTATACCATG GCTAGGTCTT TCAAGTCCTG CTGTGGTTTT GGAATTAATT TTGCTAGCAA 60TGCGCATAGT TGCTGTAGTC AGTGTGGGTC AGCCACTCGA GATTTGTCGC ACAATGTCCG 120GACCTGAAGT AGGGCCCAGA CGTGGGCTAG GACTGGAACA GGTTTTACCT GGTCTGGGAC 180CGACATATGG TCGTCTAGAA TTGGAGGTAC GGCAGGGCAT TGCAAGAAGT CTAGAGATTG 240CTGGAGCTCT TGGAAGCGCT GGACGACGTG CACGACCGTA AGAGGTTTAG GACGGTGGAC 300GGTACCCGAA GTCCAGAACT CTGAGACCTG AGAGACCCGC CCCAGGACCT TCGTAGGCCA 360ATGTCGTGGC TTCAACAACG AGACAGGGCA GACGTCCCAA GGGAAGTCCT GTACGAAACC 420GTCGACCTGG ACAGAGGCCC AACAATTACC TAGG 454(2)SEQ ID NO.6信息:
(i)序列特征:
(A)长度:147氨基酸
(B)类型:氨基酸
(C)链型:未知
(D)拓扑结构:未知
(ii)分子类型:蛋白质
(ix)特点:
(A)名称/关键:蛋白质
(B)定位:1
(D)其它信息:/注意=“Met(ATG)从1开始”
(xi)序列描述:SEQ.D.NO.6:Met Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys1 5 10 15Thr Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr Gln Ser Val Ser
20 25 30Ser Lys Gln Arg Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro
35 40 45Ile Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala Val Tyr Gln Gln
50 55 60Ile Leu Thr Ser Met Pro Ser Arg Asn Val Leu Gln Ile Ser Asn Asp65 70 75 80Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser
85 90 95Cys His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly
100 105 110Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser
115 120 125Arg Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln Leu Asp Leu Ser
130 135 140Pro Gly Cys145(2)SEQ ID NO.7信息:
(i)序列特征:
(A)长度:1150碱基对
(B)类型:核酸
(C)链型:双链
(D)拓扑结构:线状
(ii)分子类型:cDNA
(ix)特点:
(A)名称/关键:misc-feature
(B)定位:4
(C)其它信息:/注意=“Met=ATG”
(xi)序列描述:SEQ.ID.NO.7:CATATGGAAC CCAAATCTTG TGACAAAACT CACACATGCC CACCGTGCCC AGCACCTGAA 60CTCCTGGGGG GACCGTCAGT CTTCCTCTTC CCCCCAAAAC CCAAGGACAC CCTCATGATC 120TCCCGGACCC CTGAGGTCAC ATGCGTGGTG GTGGACGTGA GCCACGAAGA CCCTGAGGTC 180AAGTTCAACT GGTACGTGGA CGGCGTGGAG GTGCATAATG CCAAGACAAA GCCGCGGGAG 240GAGCAGTACA ACAGCACGTA CCGTGTGGTC AGCGTCCTCA CCGTCCTGCA CCAGGACTGG 300CTGAATGGCA AGGAGTACAA GTGCAAGGTC TCCAACAAAG CCCTCCCAGC CCCCATCGAG 360AAAACCATCT CCAAAGCCAA AGGGCAGCCC CGAGAACCAC AGGTGTACAC CCTGCCCCCA 420TCCCGGGATG AGCTGACCAA GAACCAGGTC AGCCTGACCT GCCTGGTCAA AGGCTTCTAT 480CCCAGCGACA TCGCCGTGGA GTGGGAGAGC AATGGGCAGC CGGAGAACAA CTACAAGACC 540ACGCCTCCCG TGCTGGACTC CGACGGCTCC TTCTTCCTCT ACAGCAAGCT CACCGTGGAC 600AAGAGCAGGT GGCAGCAGGG GAACGTCTTC TCATGCTCCG TGATGCATGA GGCTCTGCAC 660AACCACTACA CGCAGAAGAG CCTCTCCCTG TCTCCGGGTA AAGTACCGAT CCAGAAAGTT 720CAGGACGACA CCAAAACCTT AATTAAAACG ATCGTTACGC GTATCAACGA CATCAGTCAC 780ACCCAGTCGG TGAGCTCTAA ACAGAAAGTT ACAGGCCTGG ACTTCATCCC GGGTCTGCAC 840CCGATCCTGA CCTTGTCCAA AATGGACCAG ACCCTGGCTG TATACCAGCA GATCTTAACC 900TCCATGCCGT CCCGTAACGT TATCCAGATC TCTAACGACC TCGAGAACCT TCGCGACCTG 960CTGCACGTGC TGGCATTCTC CAAATCCTGC CACCTGCCAT GGGCTTCAGG TCTTGAGACT 1020CTGGACTCTC TGGGCGGGGT CCTGGAAGCA TCCGGTTACA GCACCGAAGT TGTTGCTCTG 1080TCCCGTCTGC AGGGTTCCCT TCAGGACATG CTTTGGCAGC TGGACCTGTC TCCGGGTTGT 1140TAATGGATCC 1150(2)SEQ ID NO.8信息:
(i)序列特征:
(A)长度:1150碱基对
(B)类型:核酸
(C)链型:双链
(D)拓扑结构:线状
(ii)分子类型:cDNA
(xi)序列描述:SEQ.ID.NO:8:GTATACCTTG GGTTTAGAAC ACTGTTTTGA GTGTGTACGG GTGGCACGGG TCGTGGACTT 60GAGGACCCCC CTGGCAGTCA GAAGGAGAAG GGGGGTTTTG GGTTCCTGTG GGAGTACTAG 120AGGGCCTGGG GACTCCAGTG TACGCACCAC CACCTGCACT CGGTGCTTCT GGGACTCCAG 180TTCAAGTTGA CCATGCACCT GCCGCACCTC CACGTATTAC GGTTCTGTTT CGGCGCCCTC 240CTCGTCATGT TGTCGTGCAT GGCACACCAG TCGCAGGAGT GGCAGGACGT GGTCCTGACC 300GACTTACCGT TCCTCATGTT CACGTTCCAG AGGTTGTTTC GGGAGGGTCG GGGGTAGCTC 360TTTTGGTAGA GGTTTCGGTT TCCCGTCGGG GCTCTTGGTG TCCACATGTG GGACGGGGGT 420AGGGCCCTAC TCGACTGGTT CTTGGTCCAG TCGGACTGGA CGGACCAGTT TCCGAAGATA 480GGGTCGCTGT AGCGGCACCT CACCCTCTCG TTACCCGTCG GCCTCTTGTT GATGTTCTGG 540TGCGGAGGGC ACGACCTGAG GCTGCCGAGG AAGAAGGAGA TGTCGTTCGA GTGGCACCTG 600TTCTCGTCCA CCGTCGTCCC CTTGCAGAAG AGTACGAGGC ACTACGTACT CCGAGACGTG 660TTGGTGATGT GCGTCTTCTC GGAGAGGGAC AGAGGCCCAT TTCATGGCTA GGTCTTTCAA 720GTCCTGCTGT GGTTTTGGAA TTAATTTTGC TAGCAATGCG CATAGTTGCT GTAGTCAGTG 780TGGGTCAGCC ACTCGAGATT TGTCTTTCAA TGTCCGGACC TGAAGTAGGG CCCAGACGTG 840GGCTAGGACT GGAACAGGTT TTACCTGGTC TGGGACCGAC ATATGGTCGT CTAGAATTGG 900AGGTACGGCA GGGCATTGCA ATAGGTCTAG AGATTGCTGG AGCTCTTGGA AGCGCTGGAC 960GACGTGCACG ACCGTAAGAG GTTTAGGACG GTGGACGGTA CCCGAAGTCC AGAACTCTGA 1020GACCTGAGAG ACCCGCCCCA GGACCTTCGT AGGCCAATGT CGTGGCTTCA ACAACGAGAC 1080AGGGCAGACG TCCCAAGGGA AGTCCTGTAC GAAACCGTCG ACCTGGACAG AGGCCCAACA 1140ATTACCTAGG 1150(2)SEQ ID NO.9信息:
(i)序列特征:
(A)长度:379氨基酸
(B)类型:氨基酸
(C)链型:未知
(D)拓扑结构:未知
(ii)分子类型:蛋白质
(ix)特点:
(A)名称/关键:蛋白质
(B)定位:1
(D)其它信息:/注意=“Met(ATG)从1开始”
(xi)序列描述: SEQ ID.NO.9:Met Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro1 5 10 15Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
20 25 30Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
35 40 45Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
50 55 60Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu65 70 75 80Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
85 90 95Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
100 105 110Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
115 120 125Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
130 135 140Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro145 150 155 160Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
165 170 175Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
180 185 190Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
195 200 205Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
210 215 220Lys Ser Leu Ser Leu Ser Pro Gly Lys Val Pro Ile Gln Lys Val Gln225 230 235 240Asp Asp Thr Lys Thr Leu Ile Lys Thr Ile Val Thr Arg Ile Asn Asp
245 250 255Ile Ser His Thr Gln Ser Val Ser Ser Lys Gln Lys Val Thr Gly Leu
260 265 270Asp Phe Ile Pro Gly Leu His Pro Ile Leu Thr Leu Ser Lys Met Asp
275 280 285Gln Thr Leu Ala Val Tyr Gln Gln Ile Leu Thr Ser Met Pro Ser Arg
290 295 300Asn Val Ila Gln Ile Ser Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu305 310 315 320His Val Leu Ala Phe Ser Lys Ser Cys His Leu Pro Trp Ala Ser Gly
325 330 335Leu Glu Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr
340 345 350Ser Thr Glu Val Val Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp
355 360 365(2)SEQ ID NO.10信息:
(i)序列特征:
(A)长度:1150碱基对
(B)类型:核酸
(C)链型:双链
(D)拓扑结构:线状
(ii)分子类型:cDNA
(ix)特点:
(A)名称/关键:misc-feature
(B)定位:4
(D)其它信息:/注意=“Met=ATG”
(xi)序列描述:SEQ.ID.NO.10:CATATGGAAC CAAAATCTGC TGACAAAACT CACACATGTC CACCTTGTCC AGCTCCGGAA 60CTCCTGGGGG GTCCTTCAGT CTTCCTCTTC CCCCCAAAAC CCAAGGACAC CCTCATGATC 120TCCCGGACCC CTGAGGTCAC ATGCGTGGTG GTGGACGTGA GCCACGAAGA CCCTGAGGTC 180AAGTTCAACT GGTACGTGGA CGGCGTGGAG GTGCATAATG CCAAGACAAA GCCGCGGGAG 240GAGCAGTACA ACAGCACGTA CCGTGTGGTC AGCGTCCTCA CCGTCCTGCA CCAGGACTGG 300CTGAATGGCA AGGAGTACAA GTGCAAGGTC TCCAACAAAG CCCTCCCAGC CCCCATCGAG 360AAAACCATCT CCAAAGCCAA AGGGCAGCCC CGAGAACCAC AGGTGTACAC CCTGCCCCCA 420TCCCGGGATG AGCTGACCAA GAACCAGGTC AGCCTGACCT GCCTGGTCAA AGGCTTCTAT 480CCCAGCGACA TCGCCGTGGA GTGGGAGAGC AATGGGCAGC CGGAGAACAA CTACAAGACC 540ACGCCTCCCG TGCTGGACTC CGACGGCTCC TTCTTCCTCT ACAGCAAGCT CACCGTGGAC 600AAGAGCAGGT GGCAGCAGGG GAACGTCTTC TCATGCTCCG TGATGCATGA GGCTCTGCAC 660AACCACTACA CGCAGAAGAG CCTCTCCCTG TCTCCGGGTA AAGTACCGAT CCAGAAAGTT 720CAGGACGACA CCAAAACCTT AATTAAAACG ATCGTTACGC GTATCAACGA CATCAGTCAC 780ACCCAGTCGG TGAGCTCTAA ACAGAAAGTT ACAGGCCTGG ACTTCATCCC GGGTCTGCAC 840CCGATCCTGA CCTTGTCCAA AATGGACCAG ACCCTGGCTG TATACCAGCA GATCTTAACC 900TCCATGCCGT CCCGTAACGT TATCCAGATC TCTAACGACC TCGAGAACCT TCGCGACCTG 960CTGCACGTGC TGGCATTCTC CAAATCCTGC CACCTGCCAT GGGCTTCAGG TCTTGAGACT 1020CTGGACTCTC TGGGCGGGGT CCTGGAAGCA TCCGGTTACA GCACCGAAGT TGTTGCTCTG 1080TCCCGTCTGC AGGGTTCCCT TCAGGACATG CTTTGGCAGC TGGACCTGTC TCCGGGTTGT 1140TAATGGATCC 1150(2)SEQ ID NO.11信息:
(i)序列特征:
(A)长度:1150碱基对
(B)类型:核酸
(C)链型:双链
(D)拓扑结构:线状
(ii)分子类型:cDNA
(xi)序列描述:SEQ.ID.NO.11:GTATACCTTG GTTTTAGACG ACTGTTTTGA GTGTGTACAG GTGGAACAGG TCGAGGCCTT 60GAGGACCCCC CAGGAAGTCA GAAGGAGAAG GGGGGTTTTG GGTTCCTGTG GGAGTACTAG 120AGGGCCTGGG GACTCCAGTG TACGCACCAC CACCTGCACT CGGTGCTTCT GGGACTCCAG 180TTCAAGTTGA CCATGCACCT GCCGCACCTC CACGTATTAC GGTTCTGTTT CGGCGCCCTC 240CTCGTCATGT TGTCGTGCAT GGCACACCAG TCGCAGGAGT GGCAGGACGT GGTCCTGACC 300GACTTACCGT TCCTCATGTT CACGTTCCAG AGGTTGTTTC GGGAGGGTCG GGGGTAGCTC 360TTTTGGTAGA GGTTTCGGTT TCCCGTCGGG GCTCTTGGTG TCCACATGTG GGACGGGGGT 420AGGGCCCTAC TCGACTGGTT CTTGGTCCAG TCGGACTGGA CGGACCAGTT TCCGAAGATA 480GGGTCGCTGT AGCGGCACCT CACCCTCTCG TTACCCGTCG GCCTCTTGTT GATGTTCTGG 540TGCGGAGGGC ACGACCTGAG GCTGCCGAGG AAGAAGGAGA TGTCGTTCGA GTGGCACCTG 600TTCTCGTCCA CCGTCGTCCC CTTGCAGAAG AGTACGAGGC ACTACGTACT CCGAGACGTG 660TTGGTGATGT GCGTCTTCTC GGAGAGGGAC AGAGGCCCAT TTCATGGCTA GGTCTTTCAA 720GTCCTGCTGT GGTTTTGGAA TTAATTTTGC TAGCAATGCG CATAGTTGCT GTAGTCAGTG 780TGGGTCAGCC ACTCGAGATT TGTCTTTCAA TGTCCGGACC TGAAGTAGGG CCCAGACGTG 840GGCTAGGACT GGAACAGGTT TTACCTGGTC TGGGACCGAC ATATGGTCGT CTAGAATTGG 900AGGTACGGCA GGGCATTGCA ATAGGTCTAG AGATTGCTGG AGCTCTTGGA AGCGCTGGAC 960GACGTGCACG ACCGTAAGAG GTTTAGGACG GTGGACGGTA CCCGAAGTCC AGAACTCTGA 1020GACCTGAGAG ACCCGCCCCA GGACCTTCGT AGGCCAATGT CGTGGCTTCA ACAACGAGAC 1080AGGGCAGACG TCCCAAGGGA AGTCCTGTAC GAAACCGTCG ACCTGGACAG AGGCCCAACA 1140ATTACCTAGG 1150(2)SEQ ID NO.12信息:
(i)序列特征:
(A)长度:579氨基酸
(B)类型:氨基酸
(C)链型:未知
(D)拓扑结构:未知
(ii)分子类型:蛋白质
(ix)特点:
(A)名称/关键:蛋白质
(B)定位:1
(D)其它信息:/注意=“Met(ATG)从1开始”
(xi)序列描述:SEQ.ID.NO.12:Met Glu Pro Lys Ser Ala Asp Lys Thr His Thr Cys Pro Pro Cys Pro1 5 10 15Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
20 25 30Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
35 40 45Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
50 55 60Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu65 70 75 80Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
85 90 95Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
100 105 110Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
115 120 125Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu130 135 140Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro145 150 155 160Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
165 170 175Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
180 185 190Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
195 200 205Phe Ser Cys Sar Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
210 215 220Lys Ser Leu Ser Leu Ser Pro Gly Lys Val Pro Ile Gln Lys Val Gln225 230 235 240Asp Asp Thr Lys Thr Leu Ile Lys Thr Ile Val Thr Arg Ile Asn Asp
245 250 255Ile Ser His Thr Gln Ser Val Ser Ser Lys Gln Lys Val Thr Gly Leu
260 265 270Asp Phe Ile Pro Gly Leu His Pro Ile Leu Thr Leu Ser Lys Met Asp
275 280 285Gln Thr Leu Ala Val Tyr Gln Gln Ile Leu Thr Ser Met Pro Ser Arg
290 295 300Asn Val Ile Gln Ile Ser Asn Asp Leu Glu Asn Lau Arg Asp Leu Leu305 310 315 320His Val Leu Ala Phe Ser Lys Ser Cys His Leu Pro Trp Ala Ser Gly
325 330 335Leu Glu Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr
340 345 350Ser Thr Glu Val Val Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp
355 360 365Met Leu Trp Gln Leu Asp Leu Ser Pro Gly Cys
370 375(2)SEQ ID NO.13信息:
(i)序列特征:
(A)长度:1135碱基对
(B)类型:核酸
(C)链型:双链
(D)拓扑结构:线状
(ii)分子类型:cDNA
(ix)特点:
(A)名称/关键:misc-feature
(B)定位:4
(D)其它信息:/注意=“Met=ATG”
(xi)序列描述:SEQ.ID.NO.13:CATATGGACA AAACTCACAC ATGTCCACCT TGTCCAGCTC CGGAACTCCT GGGGGGTCCT 60TCAGTCTTCC TCTTCCCCCC AAAACCCAAG GACACCCTCA TGATCTCCCG GACCCCTGAG 120GTCACATGCG TGGTGGTGGA CGTGAGCCAC GAAGACCCTG AGGTCAAGTT CAACTGGTAC 180GTGGACGGCG TGGAGGTGCA TAATGCCAAG ACAAAGCCGC GGGAGGAGCA GTACAACAGC 240ACGTACCGTG TGGTCAGCGT CCTCACCGTC CTGCACCAGG ACTGGCTGAA TGGCAAGGAG 300TACAAGTGCA AGGTCTCCAA CAAAGCCCTC CCAGCCCCCA TCGAGAAAAC CATCTCCAAA 360GCCAAAGGGC AGCCCCGAGA ACCACAGGTG TACACCCTGC CCCCATCCCG GGATGAGCTG 420ACCAAGAACC AGGTCAGCCT GACCTGCCTG GTCAAAGGCT TCTATCCCAG CGACATCGCC 480GTGGAGTGGG AGAGCAATGG GCAGCCGGAG AACAACTACA AGACCACGCC TCCCGTGCTG 540GACTCCGACG GCTCCTTCTT CCTCTACAGC AAGCTCACCG TGGACAAGAG CAGGTGGCAG 600CAGGGGAACG TCTTCTCATG CTCCGTGATG CATGAGGCTC TGCACAACCA CTACACGCAG 660AAGAGCCTCT CCCTGTCTCC GGGTAAAGTA CCGATCCAGA AAGTTCAGGA CGACACCAAA 720ACCTTAATTA AAACGATCGT TACGCGTATC AACGACATCA GTCACACCCA GTCGGTGAGC 780TCTAAACAGA AAGTTACAGG CCTGGACTTC ATCCCGGGTC TGCACCCGAT CCTGACCTTG 840TCCAAAATGG ACCAGACCCT GGCTGTATAC CAGCAGATCT TAACCTCCAT GCCGTCCCGT 900AACGTTATCC AGATCTCTAA CGACCTCGAG AACCTTCGCG ACCTGCTGCA CGTGCTGGCA 960TTCTCCAAAT CCTGCCACCT GCCATGGGCT TCAGGTCTTG AGACTCTGGA CTCTCTGGGC 1020GGGGTCCTGG AAGCATCCGG TTACAGCACC GAAGTTGTTG CTCTGTCCCG TCTGCAGGGT 1080TCCCTTCAGG ACATGCTTTG GCAGCTGGAC CTGTCTCCGG GTTGTTAATG GATCC 1135(2)SEQ ID NO.14信息:
(i)序列特征:
(A)长度:1135碱基对
(B)类型:核酸
(C)链型:双链
(D)拓扑结构:线状
(ii)分子类型:cDNA
(xi)序列描述:SEQ.ID.NO.14:GTATACCTGT TTTGAGTGTG TACAGGTGGA ACAGGTCGAG GCCTTGAGGA CCCCCCAGGA 60AGTCAGAAGG AGAAGGGGGG TTTTGGGTTC CTGTGGGAGT ACTAGAGGGC CTGGGGACTC 120CAGTGTACGC ACCACCACCT GCACTCGGTG CTTCTGGGAC TCCAGTTCAA GTTGACCATG 180CACCTGCCGC ACCTCCACGT ATTACGGTTC TGTTTCGGCG CCCTCCTCGT CATGTTGTCG 240TGCATGGCAC ACCAGTCGCA GGAGTGGCAG GACGTGGTCC TGACCGACTT ACCGTTCCTC 300ATGTTCACGT TCCAGAGGTT GTTTCGGGAG GGTCGGGGGT AGCTCTTTTG GTAGAGGTTT 360CGGTTTCCCG TCGGGGCTCT TGGTGTCCAC ATGTGGGACG GGGGTAGGGC CCTACTCGAC 420TGGTTCTTGG TCCAGTCGGA CTGGACGGAC CAGTTTCCGA AGATAGGGTC GCTGTAGCGG 480CACCTCACCC TCTCGTTACC CGTCGGCCTC TTGTTGATGT TCTGGTGCGG AGGGCACGAC 540CTGAGGCTGC CGAGGAAGAA GGAGATGTCG TTCGAGTGGC ACCTGTTCTC GTCCACCGTC 600GTCCCCTTCC AGAAGAGTAC GAGGCACTAC GTACTCCGAG ACGTGTTGGT GATGTGCGTC 660TTCTCGGAGA GGGACAGAGG CCCATTTCAT GGCTAGGTCT TTCAAGTCCT GCTGTGGTTT 720TGGAATTAAT TTTGCTAGCA ATGCGCATAG TTGCTGTAGT CAGTGTGGGT CAGCCACTCG 780AGATTTGTCT TTCAATGTCC GGACCTGAAG TAGGGCCCAG ACGTGGGCTA GGACTGGAAC 840AGGTTTTACC TGGTCTGGGA CCGACATATG GTCGTCTAGA ATTGGAGGTA CGGCAGGGCA 900TTGCAATAGG TCTAGAGATT GCTGGAGCTC TTGGAAGCGC TGGACGACGT GCACGACCGT 960AAGAGGTTTA GGACGGTGGA CGGTACCCGA AGTCCAGAAC TCTGAGACCT GAGAGACCCG 1020CCCCAGGACC TTCGTAGGCC AATGTCGTGG CTTCAACAAC GAGACAGGGC AGACGTCCCA 1080AGGGAAGTCC TGTACGAAAC CGTCGACCTG GACAGAGGCC CAACAATTAC CTAGG 1135(2)SEQ ID NO.15信息:
(i)序列特征:
(A)长度:374氨基酸
(B)类型:氨基酸
(C)链型:双链
(D)拓扑结构:未知
(ii)分子类型:蛋白质
(ix)特点:
(A)名称/关键:蛋白质
(B)定位:1
(D)其它信息:/注意=“Met(ATG)从1开始”
(xi)序列描述:SEQ.ID.NO.15:Met Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu1 5 10 15Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
20 25 30Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
35 40 45His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
50 55 60Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr65 70 75 80Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
85 90 95Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
100 105 110Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
115 120 125Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
130 135 140Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val145 150 155 160Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
165 170 175Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
180 185 190Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
195 200 205Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
210 215 220Ser Pro Gly Lys Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys Thr225 230 235 240Leu Ile Lys Thr Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr Gln
245 250 255Ser Val Ser Ser Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro Gly
260 265 270Leu His Pro Ile Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala Val
275 280 285Tyr Gln Gln Ile Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gln Ile
290 295 300Ser Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe305 310 315 320Ser Lys Ser Cys His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp
325 330 335Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val
340 345 350Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln Leu
355 360 365Asp Leu Ser Pro Gly Cys
370(2)SEQ ID NO.16信息:
(i)序列特征:
(A)长度:1135碱基对
(B)类型:核酸
(C)链型:双链
(D)拓扑结构:线状
(ii)分子类型:cDNA
(ix)特点:
(A)名称/关键:misc-feature
(B)定位:4
(D)其它信息:/注意=“Met=ATG”
(xi)序列描述:SEQ.ID.NO.16:CATATGGACA AAACTCACAC ATGCCCACCG TGCCCAGCTC CGGAACTCGA AGGTGGTCCG 60TCAGTCTTCC TCTTCCCCCC AAAACCCAAG GACACCCTCA TGATCTCCCG GACCCCTGAG 120GTCACATGCG TGGTGGTGGA CGTGAGCCAC GAAGACCCTG AGGTCAAGTT CAACTGGTAC 180GTGGACGGCG TGGAGGTGCA TAATGCCAAG ACAAAGCCGC GGGAGGAGCA GTACAACAGC 240ACGTACCGTG TGGTCAGCGT CCTCACCGTC CTGCACCAGG ACTGGCTGAA TGGCAAAGCT 300TACGCATGCG CGGTCTCCAA CAAAGCCCTC CCAGCCCCCA TCGAGAAAAC CATCTCCAAA 360GCCAAAGGGC AGCCCCGAGA ACCACAGGTG TACACCCTGC CCCCATCCCG GGATGAGCTG 420ACCAAGAACC AGGTCAGCCT GACCTGCCTG GTCAAAGGCT TCTATCCCAG CGACATCGCC 480GTGGAGTGGG AGAGCAATGG GCAGCCGGAG AACAACTACA AGACCACGCC TCCCGTGCTG 540GACTCCGACG GCTCCTTCTT CCTCTACAGC AAGCTCACCG TGGACAAGAG CAGGTGGCAG 600CAGGGGAACG TCTTCTCATG CTCCGTGATG CATGAGGCTC TGCACAACCA CTACACGCAG 660AAGAGCCTCT CCCTGTCTCC GGGTAAAGTA CCGATCCAGA AAGTTCAGGA CGACACCAAA 720ACCTTAATTA AAACGATCGT TACGCGTATC AACGACATCA GTCACACCCA GTCGGTGAGC 780TCTAAACAGA AAGTTACAGG CCTGGACTTC ATCCCGGGTC TGCACCCGAT CCTGACCTTG 840TCCAAAATGG ACCAGACCCT GGCTGTATAC CAGCAGATCT TAACCTCCAT GCCGTCCCGT 900AACGTTATCC AGATCTCTAA CGACCTCGAG AACCTTCGCG ACCTGCTGCA CGTGCTGGCA 960TTCTCCAAAT CCTGCCACCT GCCATGGGCT TCAGGTCTTG AGACTCTGGA CTCTCTGGGC 1020GGGGTCCTGG AAGCATCCGG TTACAGCACC GAAGTTGTTG CTCTGTCCCG TCTGCAGGGT 1080TCCCTTCAGG ACATGCTTTG GCAGCTGGAC CTGTCTCCGG GTTGTTAATG GATCC 1135(2)SEQ ID NO.17信息:
(i)序列特征:
(A)长度:1135碱基对
(B)类型:核酸
(C)链型:双链
(D)拓扑结构:线状
(ii)分子类型:cDNA
(xi)序列描述:SEQ.D.NO.17:GTATACCTGT TTTGAGTGTG TACGGGTGGC ACGGGTCGAG GCCTTGAGCT TCCACCAGGC 60AGTCAGAAGG AGAAGGGGGG TTTTGGGTTC CTGTGGGAGT ACTAGAGGGC CTGGGGACTC 120CAGTGTACGC ACCACCACCT GCACTCGGTG CTTCTGGGAC TCCAGTTCAA GTTGACCATG 180CACCTGCCGC ACCTCCACGT ATTACGGTTC TGTTTCGGCG CCCTCCTCGT CATGTTGTCG 240TGCATGGCAC ACCAGTCGCA GGAGTGGCAG GACGTGGTCC TGACCGACTT ACCGTTTCGA 300ATGCGTACGC GCCAGAGGTT GTTTCGGGAG GGTCGGGGGT AGCTCTTTTG GTAGAGGTTT 360CGGTTTCCCG TCGGGGCTCT TGGTGTCCAC ATGTGGGACG GGGGTAGGGC CCTACTCGAC 420TGGTTCTTGG TCCAGTCGGA CTGGACGGAC CAGTTTCCGA AGATAGGGTC GCTGTAGCGG 480CACCTCACCC TCTCGTTACC CGTCGGCCTC TTGTTGATGT TCTGGTGCGG AGGGCACGAC 540CTGAGGCTGC CGAGGAAGAA GGAGATGTCG TTCGAGTGGC ACCTGTTCTC GTCCACCGTC 600GTCCCCTTGC AGAAGAGTAC GAGGCACTAC GTACTCCGAG ACGTGTTGGT GATGTGCGTC 660TTCTCGGAGA GGGACAGAGG CCCATTTCAT GGCTAGGTCT TTCAAGTCCT GCTGTGGTTT 720TGGAATTAAT TTTGCTAGCA ATGCGCATAG TTGCTGTAGT CAGTGTGGGT CAGCCACTCG 780AGATTTGTCT TTCAATGTCC GGACCTGAAG TAGGGCCCAG ACGTGGGCTA GGACTGGAAC 840AGGTTTTACC TGGTCTGGGA CCGACATATG GTCGTCTAGA ATTGGAGGTA CGGCAGGGCA 900TTGCAATAGG TCTAGAGATT GCTGGAGCTC TTGGAAGCGC TGGACGACGT GCACGACCGT 960AAGAGGTTTA GGACGGTGGA CGGTACCCGA AGTCCAGAAC TCTGAGACCT GAGAGACCCG 1020CCCCAGGACC TTCGTAGGCC AATGTCGTGG CTTCAACAAC GAGACAGGGC AGACGTCCCA 1080AGGGAAGTCC TGTACGAAAC CGTCGACCTG GACAGAGGCC CAACAATTAC CTAGG 1135(2)SEQ ID NO.1 8信息:
(i)序列特征:
(A)长度:374氨基酸
(B)类型:氨基酸
(C)链型:未知
(D)拓扑结构:未知
(ii)分子类型:蛋白质
(ix)特点:
(A)名称/关键:蛋白质
(B)定位:1
(D)其它信息:/注意=“Met(ATG)从1开始”
(xi)序列描述:SEQ.ID.NO.18:Met Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Glu1 5 10 15Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
20 25 30Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
35 40 45His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
50 55 60Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr65 70 75 80Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
85 90 95Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys Ala Leu Pro Ala Pro
100 105 110Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
115 120 125Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
130 135 140Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val145 150 155 160Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
165 170 175Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
180 185 190Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
195 200 205Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
210 215 220Ser Pro Gly Lys Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys Thr225 230 235 240Leu Ile Lys Thr Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr Gln
245 250 255Ser Val Ser Ser Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro Gly
260 265 270Leu His Pro Ile Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala Val
275 280 285Tyr Gln Gln Ile Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gln Ile
290 295 300Ser Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe305 310 315 320Ser Lys Ser Cys His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp
325 330 335Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val
340 345 350Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln Leu
355 360 365Asp Leu Ser Pro Gly Cys
370
Claims (19)
1.一种具有选自R1-R2和R1-L-R2的通式的蛋白质,其中R1为Fc蛋白或其类似物,R2为OB蛋白或其类似物,L为连接子。
2.根据权利要求1的蛋白,其中Fc,其类似物或衍生物选自下组:
(a)Fc氨基酸序列如SEQ.ID.NOS.:9,12,15和18所示;
(b)(a)的氨基酸序列,具有以下一个或多个位置(根据SEQ.ID.NOS.9使用编号),发生不同氨基酸取代或删除:
(i) 一个或多个半胱氨酸残基被丙氨酸或丝氨酸残基取代;
(ii) 一个或多个酪氨酸残基被苯丙氨酸残基取代;
(iii) 5位的氨基酸被丙氨酸取代;
(iv) 20位的氨基酸被谷氨酸取代;
(v) 103位的氨基酸被丙氨酸取代;
(vi) 105位的氨基酸被丙氨酸替代;
(vii) 107位的氨基酸被丙氨酸替代;
(viii)1,2,3,4或5的氨基酸被删除;
(ix) 一个或更多残基取代或删除,以切除Fc受体结合位点;
(x) 一个或更多残基取代或删除,以切除补体(Clq)结合位点;
(xi) i-x亚部分的组合;
(c)(a)或(b)氨基酸序列,其N-末端具甲硫氨酸残基,
(d)(a)至(c)的Fc蛋白、类似物或衍生物,包括与蛋白部分连接的一种化学部分;
(e)(d)的衍生物,其中所述化学部分为水溶性聚合物部分;
(f)(e)的衍生物,其中所述水溶性聚合物部分为聚乙二醇;
(g)(e)亚部分衍生物,其中所述水溶性聚合物部分为多聚氨基酸部分;
(h)(e)的衍生物,其中所述水溶性聚合物部分只与所述蛋白部分N-末端相连。
3.如权利要求1的蛋白,其中OB蛋白,其类似物或衍生物选自下组:
(a)SEQ.ID.NO.3或SEQ.ID.NO.6所示的氨基酸序列1-146;
(b)SEQ.ID.NO.6所示的氨基酸序列1-146,在35位为赖氨酸残基和74位为异亮氨酸残基;
(c)(b)的氨基酸序列,在以下一个或更多位点发生了不同的氨基酸取代(根据SEQ.ID.NO.6编号):4,8,32,33,35,48,50,53,60,64,66,67,68,71,74,77,78,89,97,100,102,105,106,107,108,111,112,118,136,138,142和145;
(d)(a),(b)或(c)的氨基酸序列,在28位可以缺失谷氨酰胺残基;
(e)(a),(b),(c)或(d)的氨基酸序列,在N-末端具甲硫氨酸残基;
(f)选自如下部分的截短的OB蛋白类似物(使用SEQ.ID.NO.6的号码,35位具赖氨酸残基,74位具异亮氨酸残基):
(i) 氨基酸98-146
(ii) 氨基酸1-32
(iii) 氨基酸40-116
(iv) 氨基酸1-99和112-146
(v) 氨基酸1-99和112-146有一个或多个氨基酸100-111顺序地置于氨基酸99和112之间;和,
(vi) (f)(i)截短的OB类似物,有一个或多个氨基酸在100,102,105,106,107,108,111,112,118,136,138,142和145被另一个氨基酸取代;
(vii) (f)(ii)截短的类似物,有一个或多个氨基酸在4,8和32被其它氨基酸取代;
(viii)(f)(iii)截短的类似物,有一个或多个氨基酸在50,53,60,64,66,67,68,71,74,77,78,89,97,100,102,105,106,107,108,111和112被其它氨基酸替代;
(vix) (f)(iv)截短的类似物,有一个或多个氨基酸在4,8,32,33,35,48,50,53,60,64,66,67,68,71,74,77,78,89,97,112,118,136,138,142和145被其它氨基酸替代;
(x) (f)(v)截短的类似物,有一个或多个氨基酸在4,8,32,33,35,48,50,53,60,64,66,67,68,71,74,77,78,89,97,100,102,105,106,107,108,111,112,118,136,138,142和145位被其它氨基酸替代;
(xi) (f)(i)-(x)之任一的截短的类似物,具N-末端甲硫氨酸残基;
(g)(a)~(f)之任一的OB蛋白或类似衍生物,包括一个与蛋白部分相连的化学部分;
(h)(g)的衍生物,其中所述化学部分为水溶性聚合物部分;
(i)(h)的衍生物,其中所述水溶性聚合物部分为聚乙二醇;
(j)(h)的衍生物,其中所述水溶性聚合物部分为多聚氨基酸部分,和
(k)(h)亚部分的衍生物,其中所述水溶性聚合物部分只与所述蛋白部分的N-末端相连。
4.如权利要求1的蛋白,其中连接子序列是一个或多个选自:甘氨酸、天冬酰胺、丝氨酸、苏氨酸和丙氨酸的氨基酸。
5.如权利要求1的蛋白,其中连接子选自:
(a)ala,ala,ala;
(b)ala,ala,ala,ala;
(c)ala,ala,ala,ala,ala;
(d)gly,gly;
(e)gly,gly,gly;
(f)gly,gly,gly,gly,gly;
(g)gly,gly,gly,gly,gly,gly,gly;
(h)gly-pro-gly;
(i)gly,gly,pro,gly,gly;
(j)化学部分;和
(k)(a)~(j)的任意组合。
6.一种融合蛋白,包括与OB蛋白,其类似物或衍生物相融合的Fc蛋白,其类似物或衍生物。
7.一种编码如下一蛋白的核酸序列,所述蛋白具有选自从R1-R2和R1-L-R2的通式,其中R1为Fc蛋白或其类似物,R2为OB蛋白或其类似物,L为连接子。
8.根据权利要求7的编码蛋白的核酸序列,该蛋白具有选自如下部分的Fc,类似物或衍生物部分;
(a)如SEQ.ID.NOS.:9,12,15和18所示的Fc氨基酸序列;
(b)(a)的氨基酸序列,在以下一个或多个位点发生不同氨基酸的取代或删除,(根据SEQ.ID.NO.9编号);
(i) 一个或多个半胱氨基酸被丙氨酸或丝氨酸残基取代;
(ii) 一个或多个酪氨酸残基被苯丙氨酸残基取代;
(iii) 5位的氨基酸被丙氨酸取代;
(iv) 20位的氨基酸被谷氨酸取代;
(v) 103位的氨基酸被丙氨酸取代;
(vi) 105位的氨基酸被丙氨酸替代;
(vii) 107位的氨基酸被丙氨酸替代;
(viii) 1,2,3,4或5位的氨基酸被删除;
(ix) 一个或多个残基取代或删除,以切除Fc受体结合位点;
(x) 一个或多个残基取代或删除,以切除补体(Clq)结合位点;
(xi) i-x的组合;
(c) (a)或(b)的氨基酸序列,其N-末端具有甲硫氨酸残基;
(d) 任何(a)~(c)的Fc蛋白、其类似物或衍生物,包括与蛋白部相连接的化学部分;
(e) (d)的衍生物,其中所述化学部分为水溶性聚合物部分;
(f) (e)的衍生物,其中所述水溶性聚合物部分为聚乙二醇;
(g) (e)的衍生物,其中所述水溶性聚合物部分为多聚氨基酸部分;和
(h) (e)的衍生物,其中所述水溶性聚合物部分单只与所述蛋白部分的N-末端连接。
9.根据权利要求7的编码一种蛋白的核酸序列,该蛋白具选自如下部分的OB蛋白,其类似物或衍生物部分:
(a)SEQ.ID.NO.3或SEQ.ID.NO.6所示的氨基酸序列1-146;
(b)如SEQ.ID.NO.6所示的氨基酸序列1-146,35位具赖氨酸残基,74位具异亮氨酸残基;
(c)(b)的氨基酸序列,在以下一个或多个位点发生不同氨基酸替代(根据SEQ.ID.NO.6编号):4,8,32,33,35,48,50,53,60,64,66,67,68,71,74,77,78,89,97,100,102,105,106,107,108,111,112,118,136,138,142和145;
(d)(a),(b)或(c)的氨基酸序列,在28位可以缺失谷氨酰胺残基;
(e)(a),(b),(c)或(d)的氨基酸序列,在N-末端具有甲硫氨酸残基;
(f)一种选自如下的截短的OB蛋白类似物(采用SEQ.ID.NO.6的编号,在35位具赖氨酸残基,74位具异亮氨酸残基):
(i) 氨基酸98-146
(ii) 氨基酸1-32
(iii) 氨基酸40-116
(iv) 氨基酸1-99和112-146
(v) 氨基酸1-99和112-146,有一个或多个氨基酸100-111顺序置于氨基酸99和112之间,
(vi) (f)(i)截短的OB类似物,有一个或多个氨基酸在100,102,105,106,107,108,111,112,118,136,138,142和145被其它氨基酸取代;
(vii) (f)(ii)截短的类似物,有一个或多个氨基酸在4,8和32被其它氨基酸所取代;
(viii) (f)(iii)截短的类似物,有一个或多个氨基酸在50,53,60,64,66,67,68,71,74,77,78,89,97,100,102,105,106,107,108,111和112被其它氨基酸取代;
(ix) (f)(iv)截短的类似物,具有一个或多个氨基酸在4,8,32,33,35,48,50,53,60,64,66,67,68,71,74,77,78,89,97,112,118,136,138,142和145被其它氨基酸所替代;
(x) (f)(v)截短的类似物,有一个或多个氨基酸在4,8,32,33,35,48,50,53,60,64,66,67,68,71,74,77,78,89,97,100,102,105,106,107,108,111,112,118,136,138,142和145被其它氨基酸所替代;
(xi) (f)(i)~(x)之任一的截短的类似物,N-末端具甲硫氨酸残基;
(g)任何(a)~(f)之任一的OB蛋白或类似衍生物,包括连接到蛋白部分的化学部分;
(h)(g)的衍生物,其中所述化学部分为水溶性聚合物部分。
(i)(h)的衍生物,其中所述水溶性聚合物部分为聚乙二醇;
(j)(h)的衍生物,其中所述水溶性聚合物部分为多聚氨基酸部分,和
(k)(h)的衍生物,其中所述水溶性聚合物部分单独与所述蛋白部分N-末端相连。
10.如权利要求7编码一种蛋白的核酸序列,该蛋白具有的连接子序列的氨基酸选自Gly、Asn、Ser、Thr和Ala。
11.如权利要求7的编码一种蛋白的核酸序列,该蛋白具有的连接子选自:
(a)ala,ala,ala;
(b)ala,ala,ala,ala;
(c)ala,ala,ala,ala,ala;
(d)gly,gly;
(e)gly,gly,gly;
(f)gly,gly,gly,gly,gly;
(g)gly,gly,gly,gly,gly,gly,gly;
(h)gly-pro-gly;
(i)gly,gly,pro,gly,gly;
(j)化学部分;和
(k)(a)~(j)的任何组合。
12.一种编码融合蛋白的核酸序列,该蛋白具Fc蛋白,其类似物或衍生物,它与OB蛋白,其类似物或衍生物的N-末端融合。
13.一种含有如权利要求7或12的核酸序列的载体。
14.如权利要求13的载体,其中该载体是载有权利要求7或12的核酸序列的pAMG21。
15.一种包含权利要求13的载体的原核或真核的宿主细胞。
16.一种产生如权利要求1或6蛋白的方法,该方法包括在合适的条件下培养权利要求15的宿主菌和分离所产生的蛋白的步骤。
17.权利要求16的方法,该方法进一步包括纯化产生的蛋白的步骤。
18.一种药用组合物,该组合物包括在药学可接受的稀释剂,佐剂或载体中的有效量的权利要求1或6的蛋白。
19.一种治疗以下疾病的方法,这些疾病包括肥胖,糖尿病,高血脂,动脉硬化,动脉斑,减少或阻止胆结石形成,瘦肉组织量不足,对胰岛素敏感性不足,和中风,其中所述的方法包括施用有效量的根据权利要求1或6的蛋白。
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US77097396A | 1996-12-20 | 1996-12-20 | |
US08/770,973 | 1996-12-20 | ||
US08/770973 | 1996-12-20 |
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JP (2) | JP4175668B2 (zh) |
KR (1) | KR100937550B1 (zh) |
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CN112618698A (zh) * | 2019-10-08 | 2021-04-09 | 北京东方百泰生物科技股份有限公司 | 一种人白细胞介素10-Fc融合蛋白的注射制剂 |
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Cited By (3)
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CN110183530A (zh) * | 2019-06-21 | 2019-08-30 | 深圳市亚辉龙生物科技股份有限公司 | 瘦素免疫原、杂交瘤细胞、单克隆抗体、多克隆抗体及应用 |
CN112618698A (zh) * | 2019-10-08 | 2021-04-09 | 北京东方百泰生物科技股份有限公司 | 一种人白细胞介素10-Fc融合蛋白的注射制剂 |
CN112618698B (zh) * | 2019-10-08 | 2021-10-08 | 北京东方百泰生物科技股份有限公司 | 一种人白细胞介素10-Fc融合蛋白的注射制剂 |
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