CN1416348A - 修饰的凝血因子ⅷ - Google Patents
修饰的凝血因子ⅷ Download PDFInfo
- Publication number
- CN1416348A CN1416348A CN01806317A CN01806317A CN1416348A CN 1416348 A CN1416348 A CN 1416348A CN 01806317 A CN01806317 A CN 01806317A CN 01806317 A CN01806317 A CN 01806317A CN 1416348 A CN1416348 A CN 1416348A
- Authority
- CN
- China
- Prior art keywords
- leu
- ser
- glu
- thr
- lys
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/745—Blood coagulation or fibrinolysis factors
- C07K14/755—Factors VIII, e.g. factor VIII C (AHF), factor VIII Ag (VWF)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S930/00—Peptide or protein sequence
- Y10S930/01—Peptide or protein sequence
- Y10S930/10—Factor VIII, AHF; related peptides
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Hematology (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Physics & Mathematics (AREA)
- Diabetes (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Plant Pathology (AREA)
- Microbiology (AREA)
- Pharmacology & Pharmacy (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Glass Compositions (AREA)
- Fish Paste Products (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
本发明涉及一种修饰的猪凝血因子VIII的无B结构域的形式,和编码其的DNA以及其治疗血友病的用途。
Description
对相关申请的交叉参考
本申请要求美国专利申请号09/037,601,(1998年3月10日提交)的优先权,该申请是1996年6月26日提交的美国专利申请号08/670,707和1997年6月26日提交的国际申请号PCT/US97/11155的后续申请,08/670,707被授予美国专利号5,859,204。
联邦研究支持的认可
政府对导致本发明的研究通过国立卫生研究院基金编号HL46215给予过部分资助,因而对本发明具有权利。
发明背景
血液凝结从血小板粘附到损伤区域受伤血管的伤口壁上开始。然后,在一系列酶调控的级联反应中,可溶性纤维蛋白原分子被凝血酶转化成不溶性的纤维束,而固定血小板形成血栓。在级联反应的每一个步骤中都有一个蛋白前体转化成的蛋白酶,切割此系列中的下一个蛋白前体。在大多数步骤中需要辅助因子。
凝血因子VIII作为非活性前体在血液中循环,和von Willebrand因子非共价紧密结合。凝血因子VIII被凝血酶或Xa因子蛋白水解而活化,从而使它和vonWillebrand因子分离,并在级联反应中激活其促凝血功能。蛋白凝血因子VIIIa的活化形式是一种辅助因子,该因子能增强凝血因子IXa对凝血因子X活化催化效率达几个数量级。
具有凝血因子VIII缺陷或未用凝血因子VIII治疗就具有抗凝血因子VIII抗体的人,会患失控性内出血,从而可能导致一系列严重症状,从关节炎直到早逝。严重血友病病人(在美国大约为10,000),可用输入人凝血因子VIII来治疗,如果以足够多的次数和浓度施用该因子可恢复血液正常凝血能力。凝血因子VIII的经典定义,实际上是指存在于正常血浆中的能够纠正血友病A型患者的血浆凝血缺陷的物质。
在血友病病人治疗中,抑制凝血因子VIII活性的抗体(“抑制剂”或“抑制性抗体”)的产生是一种严重并发症。对凝血因子治疗性输入的反应中,有大约20%血友病A型患者产生了自身抗体。在以前未治疗过的、患有血友病A型的病人中产生了抑制性抗体,该抑制性抗体通常在治疗一年中产生。另外,使凝血因子VIII失活的自身抗体偶尔也可在以前凝血因子VIII水平正常的人体内产生。如果抑制抗体的滴度足够低,病人可通过增加凝血因子VIII的剂量进行治疗。然而,往往抑制抗体的滴度很高,以致于不能被(大量输入)凝血因子VIII所克服。另一治疗方案是在正常止血时,用凝血因子IX复合制剂(例如,KONYNE,Proplex)或重组人凝血因子VIIIa来绕过对凝血因子VIII的需要。另外,由于猪凝血因子VIII通常比人凝血因子VIII与抑制抗体的反应性要小得多,可使用部分纯化的猪凝血因子VIII制剂(HYATE:C)。许多对人凝血因子VIII产生抗体的病人已成功的用猪凝血因子VIII治疗,并长时间耐受了这种治疗。然而,猪凝血因子VIII的施用不是一种完全的解决方法,因为一些病人在一次或多次输入后,会对猪凝血因子VIII产生抑制抗体。
商品化的人血浆衍生凝血因子VIII的几种不同纯度的制品可用于血友病A型的治疗。这些制品包括:从许多献血者的合并血液中提取的部分纯化的凝血因子VIII,它经过对病毒加热和洗涤剂处理,但含有显著水平的抗原性蛋白;用单克隆抗体纯化的凝血因子VIII,具有更低水平的抗原性杂质和病毒污染;以及重组人凝血因子VIII,它的临床试验正在进行。不幸的是,人凝血因子VIII在生理浓度和pH时不稳定,在血液中以极低的浓度存在(0.2μg/ml血浆),特异性凝血活性很低。人们对于病毒或其它血液携带的污染物危险的关注,限制了从猪血纯化的猪凝血因子VIII的使用。
血友病病人需要每天更新凝血因子VIII来防止出血和所导致的变形性血友病关节病。然而,由于分离和纯化的困难、免疫原性、以及必须排除AIDS和肝炎感染的危险等,因子VIII的供给一直不足,并在治疗使用中产生问题。使用重组人凝血因子VIII或部分纯化的猪凝血因子VIII也不能解决所有这些问题。
和常用的、商品可得的、血浆衍生凝血因子VIII有关的问题激发了研制更好的凝血因子VIII制品的显著兴趣。需要以下更强的凝血因子VIII分子:每个分子能传递更多的凝血活性单位;在所选的pH和生理浓度下稳定的凝血因子VIII分子;更不易于导致抑制抗体产生的凝血因子VIII;在已产生抗人凝血因子VIII抗体的病人体内能避开免疫检测的凝血因子VIII分子。
因此,本发明的一个目的是提供一种凝血因子VIII,它能在凝血因子VIII缺陷或具有抗凝血因子VIII抑制抗体的病人体内纠治血友病。
本发明的另一个目的是提供治疗血友病的方法。
本发明还有一个目的是提供在所选pH和生理浓度下稳定的凝血因子VIII。
本发明还有另一个目的是提供比人凝血因子VIII具有更强凝血活性的凝血因子VIII。
本发明的还有一个目的是提供产生更少的针对它的抗体的凝血因子VIII。
本发明的还有一个目的是提供一种产生重组猪凝血因子VIII和特别修饰的猪凝血因子VIII的方法。
发明简述
由于确定了本文所列出的编码猪凝血因子VIII的全长DNA序列,使得第一次能够通过在合适的宿主细胞中表达编码猪凝血因子VIII的DNA合成了全长的猪凝血因子VIII。因此,纯化的重组猪凝血因子VIII是本发明的一个方面。编码猪凝血因子VIII的每个结构域及其任一片段的DNA,可以用相似方法表达。另外,全部或部分B结构域缺失的猪凝血因子VIII(无B-结构域的猪凝血因子VIII),作为本发明的一部分,已通过表达编码具有B结构域一个或多个密码子缺失的猪凝血因子VIII的DNA而制得。
本发明还提供了治疗凝血因子VIII缺陷病人的药物组合物和方法,包括施用重组猪凝血因子VIII或修饰的重组猪凝血因子VII,特别是无B结构域的猪凝血因子VIII。
附图简述
附图1A-1H提供了人、猪以及小鼠凝血因子VIII氨基酸序列的序列比较。
发明详述
除非另有指定或说明,本文所用的“凝血因子VIII”指来自任何动物的功能性凝血因子VIII蛋白分子。
除非另有指定,本文所用的“哺乳动物凝血因子VIII”包括具有任何非人哺乳动物氨基酸序列的凝血因子VIII。“动物”,如本文所用,指猪和其它非人哺乳动物。
“融合蛋白”或“融合性凝血因子VIII或其片段”,如本文所用,是一杂交基因的产物,其中一蛋白的编码序列被改变,例如,通过将它的一部分和另一不同基因的第二个蛋白编码序列在正确的阅读框中连接,使得发生连接区段的不间断转录和翻译,以产生编码该融合蛋白的杂交基因。
“对应的”核酸或氨基酸或两者的序列,如本文所用,是存在于凝血因子VIII或杂交凝血因子VIII分子或其片段中任一位点上的序列,和其它动物的凝血因子VIII分子一位点的序列有相同结构和/或功能,虽然核酸或氨基酸编号可能不相同。和另一凝血因子VIII序列“对应”的DNA序列和这样的序列实质上相对应,并能和指定SEQ ID NO的序列在严格条件下杂交。和另一凝血因子VIII序列“对应”的DNA序列还包括导致凝血因子VIII或其片段表达的序列,以及除非基因密码子的冗余,能与指定SEQ ID NO杂交的序列。
“独特的”氨基酸残基或序列,如本文所用,指在一种动物凝血因子VIII分子中,和其它动物的凝血因子VIII分子中的同源残基或序列不同的氨基酸序列或残基。
“比活性”,如本文所用,指能纠正人凝血因子VIII缺陷血浆的凝血缺陷的活性。比活性通过标准试验中每毫克总凝血因子VIII蛋白的凝血活性单位来衡量,其中将人凝血因子VIII缺陷血浆的凝血时间和正常人血浆的作比较。一单位凝血因子VIII活性等于一毫升正常人血浆中存在的活性。在该试验中,血块形成的时间越短,被测定的凝血因子VIII的活性越大。猪凝血因子VIII在人凝血因子试验中具有凝血活性。
“表达”意味着发生的一组过程,通过其利用遗传信息获得产物。编码猪凝血因子VIII的氨基酸序列的DNA可以在哺乳动物宿主细胞中“表达”,获得猪凝血因子VIII蛋白。材料、基因结构、宿主细胞和允许给定的DNA序列发生表达的条件是本领域熟知的,并可操纵,影响表达的时间和量,以及表达蛋白在胞内或胞外的位置。例如,通过在编码猪凝血因子VIII的DNA5’末端加上信号肽(常规上5’末端是编码蛋白质的NH2的末端),使表达的蛋白被运出宿主细胞,进入培养基。提供信号肽编码DNA联合猪凝血因子VIII编码DNA是有利的,因为表达的凝血因子VIII被运出细胞,简化了纯化过程。优选的信号肽是哺乳动物凝血因子VIII信号肽。
人凝血因子VIII cDNA核苷酸和预计的氨基酸序列分别显示于SEQ ID Nos:1和2。合成的凝血因子VIII大约为300kDa的单链蛋白,具有确定“结构域”序列NH2-A1-A2-B-A3-C1-C2-COOH的内部序列同源性。在凝血因子VIII分子中,“结构域”,如本文中所用,是由内部氨基酸序列相同性和凝血酶蛋白水解切割的位点所界定的一段连续氨基酸序列。除非另有指定,当该序列和人氨基酸序列(SEQ IDNO:2)排比对齐时,凝血因子VIII结构域包括以下氨基酸残基:A1,残基Ala1-Arg372;A2,残基Ser373-Arg740;B,残基Ser741-Arg1648;A3,残基Ser1690-Ile2032;C1,残基Arg2033-Asn2172;C2,残基Ser2173-Tyr2332。A3-C1-C2序列包括残基Ser1690-Tyr2332。剩余序列,残基Glu1649-Arg1689,常被称为凝血因子VIII活化肽。凝血因子VIII被凝血酶或凝血因子Xa水解而活化,它们使凝血因子VIII与von Willebrand因子分离,形成凝血因子VIIIa,它具有促凝血功能。凝血因子VIIIa的生物功能是增强凝血因子IXa对因子X活化的催化效率达几个数量级。凝血酶所活化的凝血因子VIIIa是一种160kDa的A1/A2/A3-C1-C2异质性三聚体,即与因子IXa和因子X在血小板或单核细胞表面形成的一种复合物。本文所用的“部分结构域”是形成结构域一部分的连续氨基酸序列。
人或动物凝血因子VIII的“亚基”,如本文所用,是该蛋白的重链或轻链。凝血因子VIII重链含有3个结构域,A1,A2,和B。凝血因子VIII轻链也含有3个结构域,A3,C1和C2。
术语“表位”、“抗原性位点”和“抗原决定簇”,如本文所用是同义的,定义为被抗体特异性鉴定的人、动物、杂交、或杂交等价凝血因子VIII或其片段的一部分。它可由任一编号的氨基酸残基组成,而且依赖于该蛋白的一级、二级、或三级结构。
术语“免疫原性位点”,如本文所用,定义为人或动物凝血因子VIII、或其片段中的一个区域,当用常规方法例如免疫试验(例如本文所述的ELISA或Bethesda试验)测定时,它在人或动物中能特异性诱导产生抗凝血因子VIII、其杂交、杂交等价物、或片段的抗体。它可由任一编号的氨基酸残基组成,而且它依赖于蛋白的一级、二级、和三级结构。在一些实施例中,杂交或杂交的等价凝血因子VIII或其片段在动物或人中不具免疫原性或比人或猪的凝血因子VIII免疫原性低。
“凝血因子VIII缺陷”。如本文所用,包括产生有缺陷的凝血因子VIII,或凝血因子VIII产生不足或不产生,或抑制抗体部分或完全抑制了凝血因子VIII的产生等引起的凝血活性缺陷。血友病A型是一类由X-连锁基因中的缺陷和其编码的凝血因子VIII蛋白的缺失或缺陷造成的凝血因子VIII缺陷。
术语“编码蛋白质,例如猪凝血因子VIII的DNA”意味着一种多脱氧核酸,其核苷酸序列对于宿主细胞来说含有蛋白质,例如猪凝血因子VIII的氨基酸序列的编码信息,根据已知的遗传密码的关系。
编码人或动物的凝血因子VIII或修饰的凝血因子VIII的DNA的“表达产物”是通过在合适的宿主细胞中表达所提到的DNA获得的产物,包括特征:引用的DNA编码的蛋白质的翻译前后的修饰,包括但不限于糖基化、蛋白酶解切割等。本领域已知这些修饰视宿主细胞类型和其它因素可发生或不同,并可导致产物的分子同工型,保留促凝血活性。见例如Lind,P.等,Eur.J.Biochem.232:1927(1995),在此引入以供参考。
“表达载体”是一种DNA元件,通常是环形结构,具有在理想的宿主细胞中自主复制,或整合到宿主细胞基因组中,并具有一些熟知的特征,允许在正确的位点和以正确方向插入载体序列的编码DNA表达。这些特征可以包括但不限于一种或多种指导编码的DNA转录起始的启动子序列,和其它的DNA元件,例如增强子、聚腺苷酸位点等,全部都是本领域熟知的。术语“表达载体”用于指在其序列中插入了要表达的DNA编码序列,和必要的表达控制元件,和一个插入位点,可以起到表达任何插入该位点的编码DNA的载体,所有都是本领域熟知的。因此例如,缺乏启动子的载体可以通过插入与编码DNA联合的启动子变成表达载体。
方法概述
美国专利号5,364,771描述了具有凝血活性的杂交人/猪凝血因子VIII的发现,其中用人或猪凝血因子VIII分子的元件取代了其它动物凝血因子VIII分子的对应元件。美国专利号5,663,060描述了促凝血的杂交人/动物和杂交等价凝血因子VIII分子,其中一种动物的凝血因子VIII分子元件取代了其它动物对应的凝血因子VIII分子元件。
目前的信息表明B结构域没有限制性表位,对凝血因子VIII功能没有已知的影响,在一些实施例中在用任何本文所述的方法制备的活性杂交或杂交等价的凝血因子VIII分子或其片段中除去了整个或部分B结构域(“B(-)因子VIII”)。
人凝血因子VIII基因按Toole,J.J.等(1984)Nature
312:342-347(GeneticsInstitute);Gitschier,J.等(1984)Nature
312:326-330(GenenTech);Wood,W.I.等(1984)Nature
312:330-337(Genentech);Vehar,G.A.等(1984)Nature312:337-342(Genentech);WO 87/04187;WO 88/08035;WO 88/03558;美国专利号4,757,006,所报道进行分离,并在哺乳动物细胞中表达,而氨基酸序列从cDNA推导。美国专利号4,965,199,(Capon等)公开了用于在哺乳动物宿主细胞中产生凝血因子VIII并纯化人凝血因子VIII的重组DNA方法。已报道了在CHO(中国仓鼠卵巢)细胞和BHKC(乳仓鼠肾细胞)中人凝血因子VIII的表达。修饰人凝血因子VIII除去部分或所有的B结构域(美国专利号4,868,112),并尝试了用人凝血因子V的B结构域替代凝血因子VIII的B结构域(美国专利号5,004,803)。编码人凝血因子VIII的cDNA序列和预计的氨基酸序列分别显示于SEQ ID NO:1和2。在SEQ ID NO:1中,编码区始于核苷酸位置208,三联体GCC是SEQ ID NO:2给出的成熟蛋白质的氨基酸号1(Ala)的密码子。
从血浆中分离并纯化了猪凝血因子VIII[Fass,D.N.等(1982)Blood59:594]。与N-末端轻链序列部分对应的猪凝血因子VIII的部分氨基酸序列,和血浆铜蓝蛋白及凝血因子V具有同源性,由Church等(1984)Proc.Natl.Acad.Sci.USA
81:6934所述。TooleJ.J.等(1984)Nature312:342-347描述了猪凝血因子VIII的4个氨基酸片段的N-末端的部分测序,但关于它们在凝血因子VIII分子中的位置未作特征分析。猪凝血因子VIII的B结构域和部分A2结构域的氨基酸序列见Toole,J.J.等(1986)Proc.Natl.Acad.Sci,USA
83:5939-5942报道。编码猪凝血因子VIII整个A2结构域的cDNA序列和预测的氨基酸序列,以及具有所有结构域、所有亚基、和特异性氨基酸序列被取代的杂交人/猪凝血因子VIII公开于1994年11月15日公告的美国专利号5,364,771(题为“杂交的人/猪凝血因子VIII”)和1993年10月14日出版的WO 93/20093中。编码和成熟的人凝血因子VIII的残基373-740(如SEQ ID NO:1中所示),具有序列相同性的猪凝血因子VIII A2结构域的cDNA序列,以及预测的氨基酸序列分别显示于SEQ ID NO:3和4中。最近,1994年5月26日出版的WO 94/11503中报道了猪凝血因子VIII缺乏开始的198个氨基酸的A1结构域部分和A2结构域的核苷酸和对应的氨基酸序列。Lollar等最后获得了整个编码猪凝血因子VIII,包括完整的A1结构域、活化肽、A3、C1和C2结构域的完整核苷酸序列以及编码的氨基酸序列,如1999年1月12日公开的美国专利5,859,204和1997年12月31日出版的WO 97/49725所公开的,两者在此引入以供参考。
从血浆中分离的猪和人凝血因子VIII都为二亚基蛋白质。该二亚基,即重链和轻链,通过非共价键结合,该键需要钙或其它二价金属离子。凝血因子VIII重链含有3个结构域,A1,A2,和B,它们共价连接。凝血因子VIII的轻链也含有3个结构域,命名为A3,C1,和C2。B结构域生物学功能不知道,故可通过蛋白水解或通过重组DNA技术方法从分子中除去,而不显著改变凝血因子VIII的任一可测定参数。人重组凝血因子VIII和血浆衍生的凝血因子VIII有相似的结构和功能,虽然除非在哺乳动物细胞中表达,否则不会糖基化。
人和猪的已活化凝血因子VIII(“凝血因子VIIIa”)由于重链在A1和A2结构域之间切开,具有3个亚基。这个结构被命名为A1/A2/A3-C1-C2。人凝血因子VIIIa在能稳定猪凝血因子VIIIa的条件下不稳定,可能是因为人凝血因子VIIIaA2亚基的较弱联合。人和猪凝血因子VIIIa的A2亚基的分离(丢失)和凝血因子VIIIa分子活性的丧失相关联。Yakhyoev,A.等(1997)Blood 90:增刊1,摘要#126,报道了A2结构域与低密度脂蛋白受体连接的蛋白质结合,提示这种结合介导的A2的细胞吸收起到下调凝血因子VIII活性的作用。
“无B结构域的凝血因子VIII”的表达通过加入B结构域增强。加入这些称为“SQ”的B结构域部分报道导致理想的表达[Lind,P.等(1995)见上]。“SQ”构建物缺乏全部人B结构域,除了B结构域N-末端的5个氨基酸和B结构域C末端的9个氨基酸。
可通过标准试验,例如,无血浆凝血因子VIII试验,一级凝血试验,和采用纯化的重组人凝血因子VIII作为标准的酶联免疫吸附试验,测定已纯化的杂交凝血因子VIII或其片段的免疫反应性和凝血活性。
根据熟练技术人员的偏爱和判断,可用其它载体包括质粒和真核病毒载体在真核细胞中表达重组基因构建物(见,例如,Sambrook等,16章)。可用其它载体和表达系统,包括细菌,酵母菌,和昆虫细胞系统,但因为糖基化的不同或缺少,不是优选的。
可在各种常用于培养和重组哺乳动物蛋白表达的细胞中表达重组杂交凝血因子VIII蛋白。特别是,已发现一定数量的啮齿类细胞系是表达大型蛋白的特别有用的宿主。优选的细胞系,可得自美国典型培养物保藏中心,Rockville,MD,包括乳仓鼠肾细胞,和中国仓鼠卵巢(CHO)细胞,它们可用常规程序和培养基培养。
猪凝血因子VIII中更强的凝血活性的基础,看来是由于人凝血因子VIIIa的A2亚基比猪凝血因子VIIIa的猪A2亚基更快的自发解离。A2亚基的解离导致活性的丧失[Lollar,P.等(1990)生物化学杂志
265:1688-1692;Lollar,P.等(1992)生物化学杂志
267:23652-23657;Lollar,P.等(1992)生物化学杂志267:13246-13250]。
具有减弱的免疫反应性的凝血因子VIII:
根据已知的凝血因子VIII的结构-功能关系,已确定了与抑制凝血因子VIII凝血活性的抗体(“抑制剂”或“抑制性抗体”)具有免疫反应性的表位特征。假定抑制性抗体可能是通过破坏与凝血因子VIII结构域结构有关的大分子相互反应,或它与von Willebrand凝血因子,凝血酶,凝血因子Xa,凝血因子Ixa,或凝血因子结合而来产生效果。然而,大多数抗人凝血因子VIII抑制性抗体是通过与位于凝血因子VIII的40kDa A2结构域或20kDa C2结构域的表位结合而起作用,导致破坏和这些结构域有关的特异性功能,如Fulchel等(1995)Proc.Natl.Acad.Sci USA
82:7728-7732;和Scandella等,(1988)Proc.Natl.Acad.Sci USA
85:6152-6156所述。除了A2和C2表位,可能在凝血因子VIII轻链的A3或C1结构域中有第三个表位,参见Scandella等(1993)Blood82:1767-1775。该假定的第三表位的意义未知,但看来它在凝血因子VIII中占据了表位反应性的一个微小部分。
抗-A2抗体阻断了凝血因子X活化,如Lollar等(1994)J.Cli.Invest.93:2497-2504所示。Ware等(1992)Blood Coagul.Fibrinolysis
3:703-716所述的先前用缺失诱变进行的作图研究,将A2表位定位于40kDa A2结构域的NH2-末端20kDa区域内。竞争性免疫放射试验表明A2抑制性抗体识别一个共有表位或紧密成簇表位,如Scandell等(1992)Throm.Haemostas
67:665-671所述,以及如美国专利5,859,204中所显示的。
可在临床试验中,在动物中测试动物或修饰的动物凝血因子VIII分子减弱的抗原性和/或免疫原性。在一类试验中,设计成测试凝血因子VIII是否对抑制抗体具有免疫反应性,对约25个具有凝血因子VIII缺陷,并具有已知治疗性人凝血因子VIII的凝血活性的抗体的病人施用凝血因子VIII,优选通过静脉灌注。动物或修饰的动物凝血因子VIII的剂量是5到50单位/公斤体重,优选10-50单位/公斤,最优选为40单位/公斤体重。在每次给药后约1个小时,在一步凝血试验中测定血样中凝血因子VIII的恢复。在灌注约5小时后再次取样,测定恢复。样品的总恢复和凝血因子VIII的消失速率可以预测抗体滴度和已知活性。如果抗体滴度高,凝血因子VIII的恢复通常不能测定。将恢复结果与用血浆衍生的人凝血因子VIII、重组人凝血因子VIII、血浆衍生的猪凝血因子VIII和其它常用的凝血因子的治疗形式或凝血因子替代品治疗的病人的恢复结果比较。
在鉴定了临床上重要的表位后,可表达具有与血浆衍生的猪凝血因子VIII比较,当在体外测试时对于广泛调查抑制剂血浆时,具有较低或相等的交叉反应性的重组凝血因子VIII。可以进行表位区域中的其它诱变,来减少交叉反应性。虽然理想,不需要降低交叉反应性来产生可能比现存的血浆衍生的猪凝血因子浓缩物有利的产品,现有的猪凝血因子浓缩物可能产生副作用,由于污染了猪蛋白质或污染了感染因子,例如病毒或朊病毒。重组猪或修饰的猪凝血因子VIII分子将不含外源猪蛋白。
诊断试验
凝血因子VIII cDNA和/或由它表达的蛋白,可全部或部分作为诊断试剂用于检测抗人或动物凝血因子VIII的抑制性抗体或检测抗杂交人/动物凝血因子或基质(包括例如,患凝血因子VIII缺陷的病人血清和体液样品)中的等价VIII的抑制性抗体。这些抗体试验包括例如ELISA试验,免疫印迹法,放射性免疫试验,免疫扩散试验,和凝血因子VIII生物活性试验(例如,用凝血试验)。制备这些试剂的技术和使用它们的方法为本领域熟练技术人员公知。例如,检测病人血清样品中抑制性抗体的免疫试验可包括使测试样品和足量的要测试的凝血因子VIII反应,在测试凝血因子VIII是抗原性的样品中,可以与抑制抗体形成可检测的复合物。
核酸和氨基酸探针可根据杂交凝血因子VIII cDNA或蛋白分子或其片段的序列来制备。在一些实施例中,这些探针可用染料或酶,荧光,化学发光,或商业可得的放射性标记物标记。氨基酸探针可用于,例如,筛选其中怀疑存在抗人,动物,或杂交人/动物凝血因子VIII抑制抗体的血清或其它体液。可定量测定病人中的抑制抗体水平,和健康对照比较,并可用来,例如,确定凝血因子VIII缺陷的病人是否能用动物或修饰的动物凝血因子VIII治疗。cDNA探针可用于,例如,筛选DNA文库的研究目的。
药物组合物
含有重组猪或修饰的猪凝血因子VIII的药物组合物,单用或和合适的医药学上稳定的化合物,传递载体,以及/或运输载体合用,参照已知方法,如Reminton’s药物科学,(E.W.Martin著)所述制备。
在一优选例中,用于静脉注射的优选运输或传递载体是生理盐水或磷酸盐缓冲液。
在另一优选例中,合适的稳定化合物,传递载体,和运输载体,包括但不限于其它人或动物蛋白质例如清蛋白。
磷脂载体和脂质体悬液也优选为药物学上可接受的运输或传递载体。这些载体可按照本领域技术人员已知的方法制备,而且可以含有例如磷脂酰丝氨酸/-磷脂酰胆碱或其它磷脂或去污剂组合物,因为凝血因子VIII和带负电的磷脂膜结合,它们一起向表面传递负电荷。脂质体可通过将合适的脂质,(例如硬脂酸磷脂酰乙醇胺,硬脂酸磷脂酰胆碱,花生酸磷脂酰胆碱,和胆固醇)溶于无机溶剂,然后蒸发,在容器表面留下一薄层干燥脂来制备。然后在容器中加入杂交凝血因子VIII的水溶液。用手旋摇容器,以从容器壁上释放脂材料,分散脂聚集物,从而形成脂质体悬液。
重组猪或修饰的猪凝血因子VIII可和其它合适的稳定化合物,传递载体,以及/或运输载体合用,包括维他命K依赖性凝结因子,组织凝血因子,和vorWillebrand因子(vWF)或vWF的片段(其含有凝血因子VIII结合位点),以及多糖例如蔗糖。
重组猪或修饰的猪凝血因子VIII可用基因治疗法传递,可用人凝血因子VIII传递的相同方法,使用的传递工具有例如逆转录病毒载体。该方法包括将凝血因子VIII cDNA掺入人细胞,细胞被直接移植入凝血因子VIII缺陷病人,或置于可植入装置中(可让凝血因子VIII渗透通过,但细胞不能通过),然后移植。优选方法是逆转录病毒介导的基因转移。在该方法中,一外源基因(例如凝血因子VIIIcDNA)被克隆入修饰的逆转录病毒的基因组中。该基因用病毒机制插入宿主细胞的基因组中,在其中通过细胞表达。逆转录病毒经过修饰,因而不会产生病毒,防止了宿主受病毒感染。该类型治疗的一般原理为本领域技术人员公知,在文献中已有综述[例如,Kohn,D.B.等(1989)Transfusion
29:812-820]。
重组猪或修饰的猪凝血因子VIII可与vWf结合储藏,以增加该杂交分子的半衰期和储藏寿命。另外,冻干凝血因子VIII可改进存在vWF时活性分子的产量。可采用商品供应商储藏人和动物凝血因子VIII的现有方法来储藏杂交凝血因子VIII。这些方法包括:(1)以部分纯化状态冻干凝血因子VIII(作为凝血因子VIII“浓缩液”,无须进一步纯化注射);(2)用Zimmerman方法免疫亲和纯化凝血因子VIII并在存在稳定凝血因子VIII的清蛋白时冻干;(3)在存在清蛋白时冻干重组凝血因子VIII。
另外,杂交凝血因子VIII于4℃时在0.6M NaCl,20mM MBS,和5mMCaCl2,pH6.0中无限稳定,而且可冰冻储藏于这些缓冲液中,融化时活性损失最小。
治疗方法
重组猪或修饰的猪凝血因子VIII可用于治疗有或无抑制性抗体的血友病病人,以及由于产生抑制性抗体而患获得性凝血因子VIII缺陷的病人由于凝血因子VIII缺陷引起的失控性出血(例如,关节内,颅内,或胃肠出血)。这些活性材料优选静脉内施用。
另外,重组猪或修饰的猪凝血因子VIII可通过移植基因工程改造的细胞来产生杂交物或通过植入含这种细胞的装置(如上所述)施用。
在一优选例中,重组猪或修饰的猪凝血因子VIII的药物学组合物单独或和稳定剂,传递载体,以及/或运载体合用,静脉注射入病人体内,按照注射人或动物凝血因子VIII所用的相同程序。
必须给予需要治疗的病人的重组猪或修饰的猪凝血因子VIII组合物的治疗剂量视凝血因子VIII缺陷的严重程度而变化。通常,剂量水平在频率,持续时间,以及单位上,与每个病人出血症状的严重程度和持续时间保持一致。因此,杂交凝血因子VIII包含于药物学上可接受的载体,传递载体,或稳定剂中,以足够量传递给病人,如标准凝血试验测定的那样,给予治疗有效量的杂交物停止了出血。
凝血因子VIII在经典上定义为存在于正常血浆中的能纠正患血友病A个体的血浆中凝血缺陷的物质。纯化或部分纯化形式的凝血因子VIII体外凝血活性是用于计算给病人注射的凝血因子VIII剂量,和病人血浆恢复了活性和纠正了内出血缺陷的可靠指标。在新凝血因子VIII分子体外标准试验和它们在狗注射模型中或在病人中的表现之间尚没有报道过差异,这是按照Lusher,J.M.等328 NewEngl.J.Med.
328:453-459;Pittman,D.D.等(1992)Blood
79:389-397;以及Brinkhous等,(1995)Proc.Natl.Acad.Sci.
82:8752-8755的报道。
通常,通过施用重组猪或修饰的猪凝血因子VIII使病人达到的理想血浆凝血因子VIII水平在正常的30-100%范围内。在施用重组猪或修饰的猪凝血因子VIII的优选模式中,组合物优选剂量范围5到50单位/公斤体重,更优选范围10-50单位/公斤体重,最优选剂量为20-40单位/公斤体重,静脉施用;间隔频率范围是8到24小时(患严重血友病病人);治疗持续天数是从1到10天,或直到出血症状被解决。见,例如,Roberts,H.R.,和M.R.Jones,″血友病和相关病症-凝血酶原(凝血因子II,凝血因子V,和凝血因子VII到XII)的先天缺陷,″Ch.153,1453-1474,1460,于
hematology Williams,W.J.,等编。(1990)。具有抑制抗体的病人可能需要更多的重组猪或修饰的猪凝血因子VIII,或因为它比人凝血因子VIII更高的比活性或减弱的抗体反应性或免疫原性,病人可能需要较少的重组猪或修饰的猪凝血因子VIII。如在用人或血浆衍生的猪凝血因子VIII治疗中,重组猪或修饰的猪凝血因子VIII量用一级凝血因子VIII凝血试验来确定,而且在所选定实例中,体外恢复情况可通过测定注射后病人血浆中的凝血因子VIII来确定。可以理解,对于任一特定的个体,具体的剂量方案应按照个体需要和施用或监控组合物者的职业医师的判断,根据时间而调整,而且本文上述的浓度范围仅是举例说明,并不限制权利要求所述的组合物的范围或实施。
治疗可按需要采用一次静脉施用组合物,或定期性或一段时间持续施用。另外,杂交或杂交等价凝血因子VIII可用脂质体在不同的时间间隔以一剂或数剂皮下或口腔内施用。
重组猪或修饰的猪凝血因子VIII也可用来治疗产生抗人凝血因子VIII抗体的血友病病人因凝血因子VIII缺陷而引起的失控性出血。在这种情况下,不需要比单独用人或动物凝血因子VIII更高的凝血活性。假如活性不被病人血浆中的抗体中和,凝血活性比人凝血因子VIII差的(亦即小于3,000单位/毫克)将会有用。
本文证明了重组猪和修饰的猪凝血因子VIII的比活力与人凝血因子VIII可能不同。比人凝血因子VIII具有更高促凝血活性的凝血因子VIII蛋白可用于治疗血友病,因为需要较低剂量来纠正病人的凝血因子VIII缺陷。具有比人凝血因子VIII具有较低促凝血活性的凝血因子VIII也适用于治疗用途,只要它们具有与正常人凝血因子VIII相比至少1%的比活力。本发明具有促凝血活性的凝血因子VIII因此被定义成至少具有人凝血因子VIII比活力的1%。
重组猪或修饰的猪凝血因子VIII分子和其分离,定性,制备,和使用的方法总的如上所述,参考下列非限制性实施例将会更好被理解。
实施例1:猪凝血因子VIII和杂交人/猪凝血因子VIII的试验
根据该分子的比活性,猪凝血因子VIII比人凝血因子VIII有更强的凝血活性。该结果基于使用合适的标准曲线从而使人和猪凝血因子VIII能公正的进行比较。凝血试验是根据凝血因子VIII缩短血友病A病人血浆凝血时间的能力。使用了两种试验:一级和二级试验。
在一级试验中,将0.1毫升血友病A血浆(George King Biomedical,Inc.)和0.1毫升活化的部分促凝血酶原激酶(APTT)(Organon Teknika)以及0.01毫升样品或含有稀释的柠檬酸化的正常人血浆标准品,一起在37℃水浴中培育5分钟。然后加入0.1毫升20mM CaCl2,产生血纤蛋白凝块的时间通过目测确定。
凝血因子VIII一单位定义为1毫升柠檬酸化的正常人血浆中存在的凝血因子VIII量。用人血浆作为标准,直接比较了猪和人凝血因子VIII的活性。血浆标准或纯化蛋白的稀释液制备于0.15M NaCl,0.02M HEPES,pH7.4中。根据3或4个血浆稀释度构建了标准曲线,最高的稀释度是1/50,以及根据log10凝结时间对log10血浆浓度作图,得到一张线性图。用内插法从标准曲线确定未知样品中的凝血因子VIII单位。
一级试验依赖于血友病A血浆中形成的活化剂内源性活化凝血因子VIII,而二级试验测定预活化的凝血因子VIII的促凝血活性。在二级试验中,将含有曾与凝血酶反应的凝血因子VIII的样品加到活化的部分促凝血激酶和人血友病A血浆的混合物中,该血浆已在37℃预培育了5分钟。得到的凝血时间根据上述同一人标准曲线换算成单位/毫升。二级试验的相对活性比一级试验的更高,因为凝血因子VIII已预活化。
实施例2:人和猪凝血因子VIII之间功能性差异的特性分析
猪和人血浆衍生凝血因子VIII和人重组凝血因子VIII的分离在文献中如Fulcher,C.A.等(1982)Proc.Natl.Acad.Sci.USA
79:1648-1652;Toole等(1984)Nature
312:342-347(Genetics Institute);Gitschier等(1984)Nature312:326-330(Genentech);Wood等(1984)Nature
312:330-337(Genentech);Vehar等,Nature
312:337-342(Genentech);Fass等(1982)Blood
59:594;Toole等(1986)Proc.Natl.Acad.Sci.USA
83:5939-5942所述。这可通过数种方法来完成。所有这些制备物在亚基组合物上都相似,尽管人和猪凝血因子VIII之间稳定性上有功能性差异。
为了比较人重组和猪凝血因子VIII,制备高度纯化的人重组凝血因子VIII(Cutter Laboratories,Berkeley,CA)和猪凝血因子VIII[免疫纯化,如Fass等(1982)Blood
59:594所述],将它们加到Mono QTM(Pharmacia-LKB,Piscataway,NJ)阴离子交换柱(Pharmacia,Inc.)上,通过高效液相层析(HPLC)。Mono QTM HPLC步骤的目的是除去人和猪凝血因子VIII中的少量杂质,将它们交换到常用缓冲液中,以实现比较目的。含有1000-2000单位凝血因子VIII的各小管加入5毫升H2O重建。然后加入Hepes(2M,pH7.4)至最终浓度0.02M。将凝血因子VIII加到Mono QTM HR 5/5柱上,该柱以0.15M NaCl,0.02M Hepes,5mMCaCl2,pH7.4(缓冲液A加0.15M NaCl)平衡,用10ml缓冲液A加0.15M NaCl洗涤;然后用20毫升线性梯度,(0.15M到0.90M NaCl)的缓冲液A以流速1ml/min洗脱。
为了比较人血浆衍生凝血因子VIII(用Mono QTM HPLC纯化)和猪凝血因子VIII,将免疫亲和纯化的猪血浆衍生凝血因子VIII用0.04M Hepes 5mM CaCl2、0.01%Tween-80,(pH7.4)1∶4稀释,以前文所述用于人凝血因子VIII的相同条件加到Mono QTM HPLC上。分离人和猪凝血因子VIII的这些方法对本领域的技术人员来说是标准的。
通过一级凝血试验测试柱组分的凝血因子VIII活性。测试的平均结果,表示为材料的每A280单位活性,给出于表II,表明当使用一级试验时,猪凝血因子VIII比人凝血因子VIII活性至少大6倍。
表II:人和猪凝血因子VIII凝血活性的比较
活性(U/A280)
猪 21,300
人血浆提取 3,600
人重组 2,400
实施例3:人和猪凝血因子VIII的稳定性比较
凝血因子VIII一级试验的结果反映样品中凝血因子VIII活化为凝血因子VIIIa,和可能丧失已形成的凝血因子VIIIa的活性。进行了人和猪凝血因子VIII稳定性的直接比较。将Mono QTM HPLC(Pharmacia,Inc.,Piscataway,N.J.)获得的样品稀释至相同浓度的缓冲液成分中,和凝血酶反应。在不同时间,取出样品用于二级凝血试验。通常,峰值活性(2分钟时)猪的比人的凝血因子VIIIa大10倍,然后猪和人凝血因子VIIIa的活性减弱,人的凝血因子VIIIa活性减弱得更快。
一般,即使采用能产生稳定的猪凝血因子VIIIa的条件,分离稳定的人凝血因子VIIIa的尝试也不成功。为说明这一点,用凝血酶活化Mono QTM HPLC纯化的人凝血因子VIII,并将它在产生稳定的猪凝血因子VIIIa的条件下加到Mono STM阳离子交换(Pharmacia,Inc.)HPLC上,如Lollar等(1989)生物化学
28:666所述。
人凝血因子VIII,43μg/ml(0.2μM)溶于0.2M NaCl,0.01M Hepes,2.5mMCaCl2,pH7.4,体积为10ml,和凝血酶(0.036μM))反应10分钟,在此时,加入FPR-CH2Cl D-苯基-丙基-精氨酰基-氯甲基酮至浓度为0.2μM,用以不可逆灭活凝血酶。然后混合物用40mM2-(N-吗啉代)乙磺酸(MES),5mM CaCl2,pH6.0作1∶1稀释,以2ml/min加到用5mM MES,5mM CaCl2,pH6.0(缓冲液B)和0.1M NaCl平衡的MonoSTM HR 5/5 HPLC柱上。凝血因子VIIIa不经柱洗涤,用20毫升线性梯度(0.1M到0.9M NaCl)以流速1ml/min洗脱。
二级试验中在这些条件下将具有凝血活性的组分单峰洗脱出来。峰组分的比活性是大约7,500U/A280。对Mono STM凝血因子VIIIa峰进行十二烷基磺酸钠凝胶电泳(SDS-PAGE),然后银染色蛋白,显示两条对应于凝血因子VIII的异二聚体(A3-Cl-C2/A1)衍生物。虽然在这些条件下由于A2片段浓度低银染色不能鉴定,但可用125I-标记法作为痕迹组分被鉴定。
和人凝血因子VIII结果相反,在相同条件下用MonoSTM HPLC分离的猪凝血因子VIIIa具有1.6×106U/A280的比活性。用SDS-PAGE分析猪凝血因子VIIIa,发现3条对应A1,A2和A3-C1-C2亚基的片段,说明猪凝血因子VIIIa有三个亚基。
人凝血酶活化的凝血因子VIII制备物在pH6.0时MonoSTM HPLC结果表明,人凝血因子VIIIa在产生稳定的猪凝血因子VIIIa的条件下是不稳定的。然而,尽管在峰组分中鉴定出痕迹量的A2片段,要确定凝血活性是由小量异三聚体凝血因子VIIIa还是由异二聚体凝血因子VIIIa所导致,仅靠该方法是不可能的。
需要在人凝血因子VIIIa丧失它的A2亚基前分离它的方法来解决这个问题。要达到该目的,涉及将MonoSTM缓冲液pH降低到pH5的一个过程来完成分离。MonoQTM纯化的人凝血因子VIII(0.5mg)用H2O稀释到最终组分为0.25mg/ml(1μm)凝血因子VIII(溶于0.25M NaCl,0.01M Hepes,2.5mM CaCl2,0.005%Tween-80,pH7.4)(总体积7.0毫升)。加入最终浓度为0.072μM的凝血酶,反应3分钟。然后用FPR-CH2Cl(0.2μM)灭活凝血酶。混合物用40mM醋酸钠,5mM CaCl2,0.01%Tween-80,pH5.0作1∶1稀释,以2ml/min加到用0.01M醋酸钠,5mM CaCl2,0.01%Tween-80,pH5.0平衡的MonoSTM HR 5/5 HPLC柱上。凝血因子VIIIa不经柱洗涤,用20毫升线性梯度,0.1M到1.0M NaCl的相同缓冲液以流速1ml/min洗脱。这导致峰组分中凝血活性的恢复,该峰含有可检测量的A2片段,如SDS-PAGE和银染色法所示。峰组分的比活性比在pH6.0时恢复的要大10倍(75,000U/A280比7,500U/A280)。然而,和pH6.0时分离的猪凝血因子VIIIa(其在4℃时无限稳定)相反,人凝血因子VIIIa活性在Mono STM洗脱后的几小时中稳步下降。另外,pH5.0时纯化并立即测试的凝血因子VIIIa的比活性只有猪凝血因子VIII的5%,表明在测试前发生了实质性解离。
这些结果说明人和猪凝血因子VIIIa由三个亚基组成(A1,A2,和A3-C1-C2)。A2亚基的解离在一定条件下例如生理性离子强度,pH和浓度下导致人和猪凝血因子VIIIa活性的丧失。在一定条件下猪凝血因子VIIIa的相对稳定性是由于A2亚基更强联合而致。
实施例4:编码猪凝血因子VIII A2结构域的DNA分离和测序
以前仅测序过编码猪凝血因子VIII的B结构域和部分A2结构域的核苷酸序列[Toole等(1986)Proc.Natl.Acad.Sci,USA
83:5939-5942]。本文公开了
全长 的猪凝血因子VIII A2结构域的cDNA和预测的氨基酸序列(分别为SEQ ID Nos:3和4)包括在本文中。
用逆转录猪脾脏总RNA克隆猪凝血因子VIII A2结构域,并PCR扩增;采用了基于已知的人凝血因子VIII cDNA序列的简并引物和基于一部分猪凝血因子VIII序列的精确猪引物。分离获得的1kb PCR产物,插入BluescriptTM(Stratagene)噬菌粒载体扩增。
猪A2结构域用双脱氧测序法完整测序。cDNA和预测的氨基酸序列分别在SEQID Nos:3和4中描述。
实施例5编码猪凝血因子VIII的完整DNA序列
Klenow片段,磷酸化ClaI接头,NotI接头,T4连接酶,和Taq DNA聚合酶购自Promega(Madison,Wisconsin)。聚核苷酸激酶购自LifeTechnologies,Inc.,Gaithersburg,Maryland。γ32P-ATP(Redivue,>5000Ci/mmol)购自Amerham。pBluescript II KS-和大肠杆菌Epicurean XLl-Blue细胞购自Stratagene(La Jolla,California)。合成寡核苷酸购自Life Technologies,Inc.,或Cruachem,Inc。当为克隆目的产生PCR产物时使用了5′-磷酸化引物。用作聚合酶链式反应(PCR)扩增猪fVIII cDNA或基因组DNA引物的寡核苷酸的核苷酸(nt)计数,使用了人fVIII cDNA作参照(Wood等(1984)同上)。
猪脾细胞总RNA用酸性硫氰酸胍-苯-氯仿抽提[Chomczynski等(1987)Anal.Biochem.
162:156-159]分离。除非另外说明,从总脾RNA制备猪cDNA用Moloney鼠白血病病毒逆转录酶(RT)和随机六聚物来引发反应(cDNA第一链合成试剂盒,Pharmacia Biotech)。RT反应包括45mM Tris-Cl,pH8.3,68mMKCl,15mM DTT,9mMMgCl2,0.08mg/ml牛血清白蛋白和1.8mM脱氧核苷酸三磷酸(dNTP)。猪基因组DNA从脾脏用标准方法分离(Strauss,W.M.(1995)在
分子生物学现有技术,F.M.Ausubet等编辑,John Wiley & Sons,pp.2.2.1-2.2.3)。从琼脂糖凝胶中分离DNA用Geneclean II(Bio 101)或Quiex II凝胶抽提试剂盒(Qiagen)。
PCR反应用Hybaid OmniGene热循环器进行。对于PCR反应用Taq DNA聚合酶,反应包括0.6mM MgCl2,0.2mM dNTP,0.5μM寡核苷酸引物,50U/ml聚合酶和0.1体积cDNA第一链反应混合物。除了另外说明,PCR产物经凝胶纯化,用Klenow片段补成平端,乙醇沉淀,并和去磷脂酰化的pBluescript II KS-的EcoRV位点连接,或用T4连接酶与磷酸化ClaI接头连接,再用ClaI消化,Sephacryl S400层析纯化,和ClaI-切口连接,除非另外说明,去磷酸化pBluescript II KS-连接用T4连接酶(快速DNA连接试剂盒,Boehringer Mannheim)进行。含有插入物的pBluscript II KS-质粒用来转化大肠杆菌Epicurean XL-1-Blue细胞。
质粒DNA测序用Applied Bisystems 373a自动DNA测序仪和PRISM染色终止试剂盒或手工使用Sequenase v.2.0测序试剂盒(Amersham Coporation)进行。PCR产物的直接测序,包括寡核苷酸32P-末端标记用循环测序法(dsDNA循环测序系统,Life Technologies)进行。
含有5′UTR序列,信号肽和A1结构域密码子的猪fVIII cDNA克隆的分离
猪fVIII cDNA 5′端至A2结构域段通过母猪脾总RNA嵌套式RT-PCR,用5′快速扩增cDNA末端(5′-RAGE)方案(Marathon cDNA扩增,Clontech,版本PR55453)扩增。这包括cDNA第一链合成,使用锁式对接寡聚胸腺嘧啶引物[Borson,N.D.等(1992)PCR方法应用
2:144-148],cDNA第二链合成使用大肠杆菌DNA聚合酶I,并与5′延伸双链衔接头:SEQ ID NO:5 5′CTA ATA CGA CTC ACT ATA GGGCTC GAG CGG CCG CCC GGG CAG GT-3′
3′-H2N-CCC GTC CA-PO4-5′连接。其短链3′末端用一氨基封闭,以减少非特异性PCR引导,而且它和3′端的8个核苷酸互补(Siebert,P.D.,等(1995)核酸研究
23:1087-1088)。PCR第一循环用衔接头特异的寡核苷酸,SEQ ID NO:6 5′-CCA TCC TAA TAC GAC TCA CTA TAG GGC-3′(命名为AP1)作为有义引物,猪fVIII A2结构域特异性寡核苷酸SEQ ID NO:7 5′-CCA TTG ACA TGA AGA CCG TTT CTC-3′(NT 2081-2104)作为反义引物进行。PCR第二循环用嵌套的衔接头特异的寡核苷酸,SEQ ID NO:8 5′-ACT CAC TATAGG GCT CGA GCG GC-3′(命名为AP2)作为有义引物,以及嵌套的猪A2结构域特异的寡核苷酸SEQ ID NO:9 5′-GGG TGC AAA GCG CTG ACA TCA GTG-3′(NT 1497-1520)作为反义引物。PCR用商品试剂盒(Advantage cDNA PCR核心试剂盒),使用抗体介导的热起始方案[Kellogg,D.E.等(1994)生物技术
16:1134-1137]进行。PCR条件包括采用试管温控,94℃变性60秒,然后进行30轮(第一PCR)或25轮(第二PCR)94℃变性30秒,60℃退火30秒,68℃延伸4分钟。该方法得到一个主要的约1.6kb产物,该产物与一个延伸入5′UTR中的约150bp片段的扩增相符。将该PCR产物用ClaI接头克隆入pBluescript中。对4个克隆的插入物进行双向测序。
这些克隆的序列包括和5′UTR137bp对应的区域、信号肽、A1结构域和部分A2结构域。在至少4个位点中的3个有共有序列。然而,诸克隆含有平均4个明显的PCR产生的突变子,推测是由于要产生可克隆的产物要进行多轮PCR而致。因此,我们用获自信号肽区域的序列设计了一个有义链磷酸化PCR引物,SEQ IDNO:10 5′-CCT
CTC GAG CCA CCA TGT CGA GCC ACC
CAG CTA GAGCTC TCC ACC TG-3′,命名为RENEOPIGSP,来合成另一个PCR产物以确认此序列并克隆入表达载体。粗体字序列代表起始密码子。该序列5′至此代表了与用于表达fVIII的哺乳动物表达载体ReNeo的插入位点5’相同的序列(Lubin等(1994)同上)。该位点包括一个XhoI切割位点(下划线)。RENEOPIGSP和nt1497-1520寡核苷酸用于引发Taq DNA聚合酶介导的PCR反应,使用母猪脾cDNA作为模板。购自几个其它制造商的DNA聚合酶不能得到可检测产物。PCR条件包括94℃变性4分钟,然后进行35轮94℃变性1分钟,55℃退火2分钟,72℃延伸2分钟,最后进行72℃延伸5分钟的步骤。PCR产物用ClaI接头克隆入pBluescript。对这些克隆的两个插入物进行双向测序,并匹配共有序列。
含有A3,C1和C2结构域密码子5′端一半的猪fVIII cDNA克隆的分离
首先克隆了对应于B-A3结构域片段(nt 4519-5571)和C1-C2结构域片段(nt6405-6990)的两种猪脾RT-PCR产物。获得的C2结构域的3′末端延伸入外显子26区域,它是fVIII的终止外显子。B-A3产物用猪特异性B结构域引物,SEQ ID NO:11 5′CGC GCG GCC GCG CAT CTG G
CA AAG CTG AGT T 3′,制备,其中下划线区域对应于猪fVIII中和人fVIII中nt4519-4530匹配的一个区域。该寡核苷酸的5′区域包括一个NotI位点,它原来用于克隆目的。用来产生B-A3产物的反义引物,SEQ ID NO:12 5′-GAA ATA AGC CCA GGC TTT GCA GTC RAA-3′是基于人fVIII cDNA序列nt 5545-5571的反向互补。PCR反应含有50mM KCl,10mMTris-Cl,pH9.0,0.1%Triton X-100,1.5mM MgCl2,2.5mM dNTPs,20μM引物,25单位/ml Taq DNA聚合酶和1/20体积RT反应混合物。PCR条件是94℃变性3分钟,然后进行30轮94℃变性1分钟,50℃退火2分钟,72℃延伸2分钟。PCR产物用T4 DNA激酶磷酸化,并加入NotI接头。用NotI切断后,将PCR片段克隆入BlueScript II KS-的NotI位点,转化入XL1-Blue细胞。
C1-C2产物用已知人序列分别合成有义和反义引物,SEQ ID NO:13 5′-AGGAAA TTC CAC TGG AAC CTT N-3'(nt 6405-6426)和SEQ ID NO:14 5′-CTG GGGGTG AAT TCG AAG GTA GCG N-3'(nt 6966-6990的反向互补)而制备。PCR条件和用于产生B-A2产物的条件相同。将所得片段用引物PCR ClonerCloning系统(5Prime-3 Prime,Inc.,Boulder,Colorado)连接到pNOT克隆载体,在JM109细胞中生长。
部分测序B-A3和C1-C2质粒,以分别制备猪特异性有义和反义寡核苷酸,SEQ ID NO:15 5′-GAG TTC ATC GGG AAG ACC TGT TG-3'(nt 4551-4573)和SEQID NO:16:5′-ACA GCC CAT CAA CTC CAT GCG AAG-3'(nt 6541-6564)。这些寡核苷酸用作引物,采用Clontech Advantange cDNA PCR试剂盒产生2013bp RT-PCR产物。该产物,和人nt 4551-6564对应,包括了对应于轻链活化肽(nt 5002-5124),A3结构域(nt 5125-6114)和大部分C1结构域(nt 6115-6573)的区域。C1-C2克隆的序列已证实人和猪cDNA从nt 6565到C1结构域3′末端是相同的。将PCR产物克隆入pBluescript II KS-的EcoRv位点。4个克隆作了完整双向测序。4位点中至少3个有共有序列。
含有C2结构域3′一半密码子的猪fVIII cDNA克隆的分离
人fVIIIC2结构域(核苷酸6574-7053)包含于外显子24-26[Gitschier J.等(1984)Nature
312:326-330]中。人外显子26含1958bp,对应核苷酸6901-8858。它含有1478bp的3′非翻译序列。通过3'RACE[Siebert等(1995)同上],反向PCR[Ochman,H.等(1990)生物技术(N.Y.).
8:759-760],限制性位点PCR[Sarkar,G.等(1993)PCR方法应用
2:318-322],“不可预测引导的”PCR[Dominguez,O.等(1994)核酸研究
22:3247-3248]以及筛选猪肝cDNA文库来克隆对应于C2结构域3′末端和3′UTR的外显子26 cDNA的尝试都失败了。用以前成功克隆了猪fVIII cDNA5′末端的相同衔接头连接的双链cDNA文库尝试了3′RACE。因此,该方法的失败不是因为对应于外显子26的cDNA不存在。
用靶向基因漫步(Walking)PCR方法[Parker,J.D.等(1991)核酸研究
19:3055-3060]克隆了C2结构域的3′半部分。猪特异性有义引物,SEQ ID NO:17 5′-TCAGGGCAATCAGGACTCC-3'(NT 6904-6924)根据原来的C2结构域序列合成,并与选自文库中可得到的寡核苷酸非特异性″漫步″引物一起用于PCR反应。然后PCR产物通过引物延伸分析[Parker等(1991)生物技术
10:94-101]导向,使用32P末端标记的猪特异性内部引物,SEQ ID NO:18 5′-CCGTGGTGAACGCTCTGGACC-3′(nt 6932-6952)。有趣的是,在测试的40个非特异性引物中,只有2个在引物延伸分析时得到阳性产物,而这两个对应于精确和简并的C2结构域3′末端的人序列:SEQ ID NO:19 5′-GTAGAGGTCCTGTGCCT CGCAGCC-3′(nt 7030-7053)和SEQ ID NO:20 5′-GTAGAGSTSCTGKGCCT CRCAKCCYAG-3′,(nt 7027-7053)。最初设计这些引物用于常规RT-PCR来产生产物,但未能产生足够的可用溴乙锭染料结合法显示的产物。然而,PCR产物可以通过更灵敏的引物延伸方法来鉴定。凝胶纯化该产物并直接测序。这将猪fVIII 3′序列延伸到nt7026。
其它序列通过前述5′-RACE方案所用衔接头连接的双链cDNA文库产生的嵌套PCR产物进行引物延伸分析而获得。第一轮反应用猪精确引物SEQ ID NO:215′-CTTCGCATGGAGTTGATGGGCTGT-3'(nt 6541-6564)和AP1引物。第二轮反应用SEQ ID NO:22 5′-AATCAGGACTCCTCCACCCCCG-3'(nt 6913-6934)和AP2引物。直接PCR测序将该序列3′延伸到C2结构域末端(nt 7053)。C2结构域序列除了靠近C2结构域3′末端的nt7045外是独一无二的。重复PCR反应的分析在该位点产生了A,G,或A/G双读。
用两个额外引物使测序延伸进入3′UTR,SEQ ID NO:23 5′-GGA TCC ACCCCA CGA GCT GG-3'(nt 6977-6996)和SEQ ID NO:24 5′CGC CCT GAG GCT CGAGGT TCT AGG-3'(nt 7008-7031)。获得大约15bp的3'UTR序列,虽然该序列在几个位点不清楚。然后根据对3′非翻译区的最好估计,合成几个反义引物。这些引物包括它们3′末端TGA终止密码子的反向互补序列。PCR产物获自猪脾基因组DNA和猪脾cDNA,它们用琼脂糖凝胶电泳和溴乙锭染色变为可见,使用了特异性引物SEQ ID NO:25 5′-AAT CAG GAC TCC TCC ACC CCC G-3'(nt 6913-6934)和3′UTR反义引物,SEQ ID NO:26 5′-CCTTGCAGGAATT CGATTCA-3'。要得到克隆目的所需的足够数量的材料,进行第二轮PCR,使用嵌套有义引物,SEQ IDNO:27 5′-CCGTGGTGAACGCTCTGGACC-3'(nt 6932-6952)和相同的反义引物。将141bp的PCR产物克隆入EcoRV-切开的pBluescript II KS-。双向测序获得衍生自基因组DNA的3个克隆和衍生自cDNA的3个克隆的序列。该序列是明确的,除了nt 7045(该位点基因组DNA总是A而cDNA总是G)。
与人,猪,和小鼠fVIII匹配的多个DNA序列(图1A-1H)。
信号肽,A1,A2,A3,C1和C2区域的匹配用CLUSTALW程序[Thompson,J.D.等(1994)核酸研究
22:4673-4680]进行。缺口打开和缺口延伸补偿分别是10和0.05。人,小鼠,和猪B结构域的排列对比先前已有描述[Elder等,(1993)同上]。人A2序列对应于SEQ ID NO:2中氨基酸373-740。猪A2氨基酸序列给出于SEQID NO:4,而小鼠A2结构域氨基酸序列给出于SEQ ID NO:28,氨基酸392-759。
实施例6:重组的无B结构域的猪凝血因子VIII(PB-)的活性表达
柠檬酸化血友病A型和正常合并人血浆购自Geoge King Biomedical,Inc。胎牛血清,遗传霉素,青霉素,链霉素,DMEM/F12培养基和AIM-V培养基购自Life Technologies,Inc。Taq DNA聚合酶购自Promega。Vent DNA聚合酶购自NewEngland Biolabs。Pfu DNA聚合酶和噬菌粒pBluescript II KS-购自Stratagene。合成寡核苷酸购自Life Technogies或Cruachem,Inc.限制性酶购自New EnglandBiolabs或Promega。当PCR产物被用作克隆目的制备时使用了5′-磷酸化引物。用作聚合酶链式反应(PCR)扩增猪fVIII cDNA或基因组DNA的引物的寡核苷酸的核苷酸(nt)编号用人fVIII cDNA(的编号)作为参照[Wood等,(1984)Nature312:330-337]。命名为HB-/ReNeo的fVIII表达载体获自Biogen,Inc。HB-/ReNeo含有氨苄青霉素和遗传霉素抗性基因,以及缺少整个B结构域的人fVIII cDNA,定义为凝血酶产生的Ser741-Arg1648切割片段。为了简化fVIII C2结构域cDNA的诱变,它在ReNeo中fVIII插入物的3′末端,通过重叠延伸剪接(SOE)诱变将NotI位点引入到HB-/ReNeo终止密码子3′的两个碱基处,[Horton,R.M.等(1993)MethodEnzylmol.
217:270-279]。该构建物命名为HB-ReNeo/NotI。
用酸性硫氰酸胍-苯-氯仿抽提总RNA[Chomczynski等(1987)Anal.Biochem.162:156-159]分离。从mRNA用Moloney鼠白血病病毒逆转录酶(RT)和随机六聚物按照制造商提供的说明(cDNA第一链合成试剂盒,Pharmacia Biotech)来合成cDNA。PCR反应采用Hybaid OmniGene热循环器,使用Taq,Vent,或Pfu DNA聚合酶进行。凝胶纯化PCR产物,乙醇沉淀,用T4 DNA连接酶(快速DNAl连接试剂盒,Boehringer Mannheim)连接入质粒DNA。用含有插入物的质粒转化大肠杆菌Epicurean XL-1-Blue细胞。PCR产生的所有新fVIII DNA通过双脱氧测序,使用Applied Bisystems 373a自动DNA测序仪和PRISM染色末端试剂盒得以确认。
含猪C2结构域的杂交fVIII表达载体,HP20的构建
将对应C1结构域3′末端和C2全部结构域的猪fVIII cDNA克隆入pBluescript,方法是通过脾总RNA的RT-PCR,使用根据已知的猪fVIII cDNA序列的引物[Healy,J.F.等(1996)Blood
88:4209-4214]。该构建物和HB-/ReNeo被用作模板在pBluescript中用SOE诱变构建人C1-猪C2融合产物。用ApaI和NotI除去该质粒中的C1-C2片段,并连接入ApaI/NotI-切开的HB-/ReNeo/NotI中以产生HP20/ReNeo/NotI。
含有猪轻链的B结构域除去的人/猪fVIII(HP18)-的构建
人fVIII轻链由氨基酸残基Asp1649-Tyr2332组成。以猪fVIII cDNA中的相应残基替代HB-的该区域产生了杂交的人/猪fVIII分子,命名为HP18。这时通过以相应于猪A2区、A3结构域、C1结构域和部分C2结构域的PCR产物取代HP20中的相应区域而完成。为了便于构建,通过SOE诱变在A2和A3结构域连接处nt2273引入了一个同义AvrII位点。
含有猪信号肽,A1结构域和A2结构域的B结构域除去的杂交人/猪
fVIII(HP22)的构建
人fVIII信号肽,A1结构域和A2结构域由氨基酸残基Met(-19)-Arg740组成。以猪fVIII cDNA中的相应残基取代HB-的该区域,产生了命名为HP22的分子。另外,通过SOE诱变将一个同义AvrI位点引入HP22的A2和A3结构域连接处的nt2273。通过将pBluescript中的猪信号肽-A1-部分A2片段[Healy等(1996)同上]和含有猪A2结构域的无B结构域杂交人/猪fVIII,命名为HP1[Lubin等(1994)见上]融合构建了HP22。
猪无B结构域fVIII-(PB
-
)的构建
用AvrI/NotI消化HP18/BS(+AvrII)的SpeI/NotI片段,并连接入AvrI/NotI-消化的HP22/BS(+AvrII)中,产生了构建物PB-/BS(+AvrII),它含有缺少整个B结构域的猪fVIII。通过将PB-/BS(+AvrII)的Xba/NotI片段连接入HP22/ReNeo/NotI(+AvrII),将PB-克隆入ReNeo。
重组fVIII分子的表达
将PB-/ReNeo/NotI(+AvrII)和HP22/ReNeo/NotI(+AvrII)瞬时转染入COS细胞,并如前所述表达[Lubin,I.M.等,(1994)生物化学杂志
269:8639-8641]。转染HB-/ReNeo/NotI和无DNA(模拟)作为对照。
PB-,HP22,和HB-的fVIII活性用如下的显色试验测定。COS细胞培养上清液中的fVIII样品用溶于0.15M NaCl,20mM HEPES,5Mm CaCl2,0.01%Tween-80,pH7.4的40nM凝血酶在存在10nM凝血因子IXa,425nM凝血因子X,和50μM单层磷脂酰丝氨酸-[磷脂酰胆碱(25/75w/w)运载体的情况下活化。5分钟后,用0.05MEDTA和100nM重组脱硫水蛭素终止反应,产生的凝血因子Xa用显色底物试验测定。在显色底物试验中,加入0.4mM Spectrozyme Xa,而对-硝基酰替苯胺释放率用溶液405nm吸光值来测定。
单个转染复制细胞培养上清液的结果(每分钟405nm吸光值)
HB-: 13.9
PB-: 139
HP22: 100
模拟: <0.2
这些结果表明无B结构域的猪fVIII和含有猪A1和A2亚基的无B结构域的fVIII具有活性,并提示它们具有比无B结构域的人fVIII更高的活性。
用肝素-Sepharose层析从生长培养基中部分纯化并浓缩PB-。肝素-Sepharose(10ml)已用0.075M NaCl,10mM HEPES,2.5mM CaCl2,0.005%Tween-80,0.02%叠氮化钠,pH7.4液平衡。将表达细胞的培养基(100-200毫升)加到肝素-Sepharose上,然后用30毫升不含叠氮化钠的平衡缓冲液洗涤。PB-用0.65MNaCl,20mM HEPES,5mM CaCl2,0.01%Tween-80,pH7.40洗脱,并储藏在-80℃。fVIII凝血活性产量通常为50-75%。
无B结构域的猪fVIII(PB
-
)的稳定表达
将转染细胞株保存于含10%胎牛血清,50U/ml青霉素,50μg/ml链霉素的Dulbecco′s改良Eagle′s培养基F-12中。胎牛血清在使用前50℃加热灭活1小时。将HB-/ReNeo和PB-/ReNeo/NotI(+AvrII)稳定转染入BHK细胞,用前述通用方案选择遗传霉素抗性株[Lubin等(1994)生物化学
269:8639-8641],除了保留在含有600μg/ml遗传霉素的生长培养基中的表达细胞外。将Corning T-75瓶中长成单层的细胞转入含有600微克/毫升遗传霉素培养基的Nunc三角瓶中,并长成单层。除去培养基,用无血清,无遗传霉素的AIM-V培养基(Life Technologies,Inc.)代替。用一级凝血因子VIII凝血活性试验(见上)监控凝血因子VIII的表达,每日收集一次100-150毫升培养基,进行4到5天。HB-和PB-在培养液中的最大表达水平分别为1到2单位/毫升,10-12单位/毫升凝血因子VIII凝血活性。
PB
-
纯化
用60%饱和硫酸铵从培养上清液中沉淀PB-,然后用W3-3免疫亲和层析和mono Q高效液相层析如前述纯化血浆衍生猪凝血因子VIII的方法纯化[Lollar等(1993)凝血因子VIII/凝血因子VIIIa。Method Enzymol.
222:128-143]。PB-的比凝血活性用一级凝血试验测定[Lollar等(1993)如上],和血浆提取猪凝血因子VIII相似。
当用SDS-聚丙烯酰胺凝胶电泳分析时,PB-制备物含有三条条带,表观分子量为160kDa,82kDa和76kDa。82kDa和72kDa条带如前所述是异二聚体,含有A1-A2和ap-A3-C1-C2结构域(其中ap指活化肽)[Toole等(1984)Nature
312:342-347]。将160kDa条带转到聚二氟乙烯膜上,进行NH2-末端测序,得到Arg-Ile-Xx-Xx-Tyr(其中Xx代表未测定),它是单链凝血因子VIII的NH2-末端顺序[Toole等(1984)见上]。因此,PB-通过A2和A3结构域之间切开而受到部分加工,这样它含有两种形式,单链A1-A2-ap-A3-C1-C2蛋白和A1-A2/ap-A3-C1-C2异二聚体。重组HB-的相似加工也已有报道[Lind等(1995)Eur.J.Biochem.
232:19-27]。
猪凝血因子VIII的特性分析
我们已测定了猪凝血因子fVIII对应于5′UTR的137bp,信号肽编码区域(57bp)和A1(1119bp),A3(990bp),C1(456bp),和C2(483bp)结构域的cDNA序列。与如前出版的B结构域序列,轻链活化肽区域[Toole等(1986)见上],和A2结构域[Lubin等(1994),见上]一起,本文所报道的序列完成了对应该翻译产物的猪fVIII cDNA的测定。含有5′UTR区域,信号肽,和A1区域cDNA的片段用5′-RACE RT-PCR方案克隆。根据人C2序列的引物在产生RT-PCR产物上是成功的。该产物克隆A3,C1,和C2结构域5′部分。对应于C2结构域3′半的cDNA和3′UTR cDNA被证实难以克隆。C2结构域的剩余部分最终用靶向基因漫步PCR方法克隆[Parker等,(1991)见上]。
本文报道的序列SEQ ID NO:29除了在靠近C2结构域3′末端的nt 7045,(它如本文上文所述,是A或G)是明确的。对应的密码子是GAC(Asp)或AAC(Asn)。人和小鼠密码子分别为GAC和CAG(Gln)。这是否代表多态性或可再现PCR假象未知。含有和GAC和AAC密码子对应的猪C2结构域取代的重组杂交人/猪无B结构域fVIII cDNA已被稳定表达,在促凝血活性中无可检测的差异。这表明在这两个C2结构域变体中无功能性差异。
全长猪fVIII SEQ ID NO:30的与公布的人[Wood等(1984)见上]和小鼠[Elder等(1993)见上]的预测氨基酸序列的排列对比,与翻译后修饰,蛋白酶解切割,以及其它大分子识别的位点一起显示于图1A-1H。对比序列的相同性程度显示于表VII。如前所示,这些动物的B结构域比A或C结构域更趋异。这和观察到B结构域尽管体积大但无已知功能是一致的[Elder等(1993)见上;Toole等(1986)见上]。本发明的结果确定了B结构域或猪fVIII不是活性所必须的。根据本文所示的序列数据,具有全部或部分B结构域缺失的猪fVIII可通过表达猪fVIII的编码DNA(去掉全部或部分猪B结构域)来合成。对应于A1结构域APC/凝血因子IXa切割肽(残基337-372)和轻链活化肽(表VII)的序列也具有更大的趋异性。位点336的凝血酶切割位点(产生337-372肽)在小鼠中明显缺失,因为该残基时谷氨酰胺而不是精氨酸[Elder等,(1993)见上]。凝血酶切割肽的相对快速趋异性(或在小鼠fVIII中可能是退化的337-372活化肽)先前已因纤维肽而受到注意[Creighton,T.E.(1993)于
蛋白质:结构和分子性质,W.H.Freeman,New York,pp.105-138]。这些肽一旦被切割引起的生物功能缺少已被引用为迅速趋异的可能原因。已提出人fVIII中的Arg562在fVIII和fVIIIa失活中是活化蛋白C的更重要的切割位点[Fay,P.J.等(1991)生物化杂志
266:20139-20145]。该位点在人,猪和小鼠fVIII中是保守的。
潜在的N-连接糖基化位点也以粗体字显示于图1A-1H。有8个保守的N-连接糖基化位点:一个在A1结构域中,一个在A2结构域中,4个在B结构域中,一个在A3结构域中,以及一个在C1结构域中。19个A和C结构域半胱氨酸是保守的,而B结构域半胱氨酸是趋异的。fVIII中的7个二硫键中的6个在凝血因子V和血清铜蓝蛋白的同源位点中也被找到,两个C结构域二硫键在凝血因子V中找到[Mcmullen,B.A.等(1995)Protein Sci.
4:740-746]。人fVIII在位点346,718,719,723,1664,和1680含有硫酸酪氨酸[Pittman,D.D.等(1992)生物化学
31:3315-3325;Michnick,D.A.等(1994)生物化学杂志
269:20095-20102]。这些残基在小鼠fVIII和猪fVIII中是保守的(图1),尽管CLUSTALM程序不能对比和人fVIIITyr346对应的小鼠酪氨酸。
小鼠和猪血浆能修正人血友病A型血浆的凝血缺陷,和这些动物A和C结构域中残基的保守水平相一致。猪fVIII的促凝血活性比人fVIII的要高[Lollar,P.等(1992)生物化学杂志
267:23652-23657]。如本文所述表达和纯化的重组猪凝血因子VIII(除去B结构域)也显示比人fVIII更高,和猪血浆衍生fVIII相同的比凝血活性。这可能因为活性A1/A2/A3-C1-C2 fVIIIa异二聚体的A2亚基自发解离率降低而致。促凝血活性中的这种差异是否反映了作为动物适应性例子的功能进化性变化[Perutz,M.F.(1996)高等蛋白化学
36:213-244]尚不清楚。现在对应于该翻译产物的猪fVIII DNA测序已经完成,同源扫描诱变[Cunningham,B.C.等(1989)Science
243:1330-1336]可提供鉴定人和猪fVIII之间引起后者更高活性的结构差异的方法。
猪fVIII通常和抑制性抗体反应较弱,该抑制性抗体产生于输注fVIII的血友病病人,或在普通人群中作为自身抗体而产生。这是用猪fVIII浓缩液治疗具抑制性抗体的病人的基础[Hay和Loizer(1995)见上]。大多数抑制抗体针对位于A2结构域或C2结构域中的表位[Fulcher,C.A.等(1985)Proc.Natl.Acad.Sci.USA82:7728-7732;Scadella,D.等(1988)Proc.Natl.Acad.Sci.USA
85:6152-6156;Scandella,D.等(1989)Blood
74:1618-1626]。另外,已在A3或C1结构域中鉴定出一个意义未明的表位[Scandella等(1989)见上;Scandella,D.等(1993)Blood82:1767-1775;Nakai,H.等(1994)Blood
84:224a]。A2表位已通过同源扫描诱变[Healey等(1995)见上]作图位于残基484-508之间。在这25个残基的片断中,有相对低比率的相同序列(16/25或64%)。有趣的是,根据针对它的抗体是抑制性抗体的事实,该区域看来功能上很重要,显然已经历了相对更迅速的遗传漂移。猪A2结构域和A3结构域的排列对比表明A2表位和A3结构域中对应区域的同源性不可检测到。
通过缺失作图已提出人fVIII的C2抑制抗体表位定位于残基2248-2312内[Scandella,D.等(1995)Blood
86:1811-1819]。人和猪fVIII在这65残基的片断中有83%相同性。然而,对C2表位定性的该区域同源扫描诱变揭示,C2表位的主要决定簇出乎意料的位于对应于人氨基酸2181-2243(SEQ ID NO:2)和图1H的区域内。
制备了人-猪杂交凝血因子VIII蛋白,其中人凝血因子VIII的C2结构域各部分用相应的猪凝血因子VIII部分,通过本文所述的策略予以替换(实施例5)。各种C2-杂交凝血因子VIII的合成通过构建编码杂交物的DNA完成,采用了SEQID NO:30中所示的编码猪C2区域的核苷酸序列,在转染的细胞中表达了各杂交DNA。这样可从生长培养基中部分纯化得到杂交凝血因子VIII。在不存在任何抑制抗体的情况下通过一级凝血试验测定活性。
用一组5种人抑制抗体来测试每种杂交凝血因子VIII。根据重组人C2结构域中和抑制性抗体的能力,先前曾显示含有抗凝血因子VIII抗体的抑制血浆直接针对人C2结构域。在所有测试的血浆中,C2结构域或轻链所中和的抑制抗体滴度大于79%,但重组人A2结构域中和的小于10%。另外,测定了抗C2杂交凝血因子VIII的小鼠单克隆抗体,该抗体和C2结构域结合,而且和人C2抑制抗体一样,它抑制了凝血因子VIII和磷脂以及和von Willebrand因子的结合。
通过比较针对C2杂交凝血因子VIII的抑制性抗体滴度,显示人C2抑制抗体(所识别)表位的主要决定簇为残基2181-2243(SEQ ID NO:2,也见图1H)区域。针对该区域COOH-末端残基2253的抗-C2抗体在五个病人血清的4个中没有鉴定到。比较了具有对应于人氨基酸残基号2181-2199和2207-2243的猪序列杂交物,显然这两个区域都对抗体结合有贡献。对应于人残基2181-2243的猪氨基酸序列是SEQ ID NO:30中的1982-2044。编码猪氨基酸编号1982-2044的猪DNA序列是SEQ ID NO:29中编号为5944-6132的核苷酸。
参照图1H,可见在区域2181-2243中,人和猪序列之间有16个氨基酸差异。这些差异见残基2181,2182,2188,2195-2197,2199,2207,2216,2222,2224-2227,2234,2238和2243。可进行这些编号残基中的一个或多个氨基酸置换以制备修饰的人凝血因子VIII,它和人抗-C2抑制性抗体不反应。如上所述,丙氨酸扫描诱变提供了天然存在残基用丙氨酸替代的便利方法。除了丙氨酸,如本文所述,其它氨基酸也一样可用于取代。用丙氨酸取代单个氨基酸,尤其是取代人/猪或人/小鼠之间不相同的氨基酸,或最有可能对抗体结合有贡献的氨基酸,可得到与抑制性抗体反应性减弱的修饰的凝血因子VIII。
图1A-1H一起提供了人、猪和小鼠凝血因子VIII氨基酸序列的排列序列比较。图1A比较信号肽区域(人,SEQ ID NO:31;猪,SEQ ID NO:30,氨基酸1-19;小鼠,SEQ ID NO:28,氨基酸1-19)。注意图1A-1H中的氨基酸将成熟蛋白的第一个丙氨酸编号为1,信号肽氨基酸分派为负数。SEQ ID NO:2中的人fVIII也从成熟蛋白的第一个氨基酸开始标为1。在小鼠fVIII(SEQ ID NO:28)和猪fVIII(SEQ ID NO:30)的氨基酸序列中,成熟序列的第一个氨基酸(丙氨酸)是编号20的氨基酸。图1A-1H显示了人、鼠和猪fVIII相应序列的排列对比,氨基酸相同性最大的区域被并列。图1A-1H中的氨基酸编号仅应用于人fVIII。图1B提供了人(SEQ ID NO:2,氨基酸1-372),猪(SEQ ID NO:30,氨基酸20-391),和小鼠(SEQ ID NO:28,氨基酸20-391)的A1结构域氨基酸序列。图1C提供了凝血因子VIII A2结构域氨基酸序列,来自人(SEQ ID NO:2,氨基酸373-740),猪(SEQID NO:30,氨基酸392-759)和小鼠(SEQ ID NO:28,氨基酸392-759)。图1D提供B结构域氨基酸序列,来自人凝血因子VIII(SEQ ID NO:2,氨基酸741-1648),猪(SEQ ID NO:30,氨基酸760-1449)和小鼠(SEQ ID NO:28,氨基酸760-1640)。图1E比较了人,猪和小鼠的凝血因子VIII轻链活化肽的氨基酸序列(分别为SEQ IDNO:2,氨基酸1649-1689;SEQ ID NO:30,氨基酸1450-1490;和SEQ ID NO:28,氨基酸1641-1678)。图1F提供了人,猪和小鼠凝血因子VIII A3结构域的序列比较(分别为SEQ ID NO:2,氨基酸1690-2019;SEQ ID NO:30,氨基酸1491-1820;和SEQ ID NO:28,氨基酸1679-2006)。图1G提供了人,猪和小鼠凝血因子VIII的C1结构域的氨基酸序列(分别为SEQ ID NO:2,氨基酸2020-2172;SEQ ID NO:30,氨基酸1821-1973;和SEQ ID NO:28,氨基酸2007-2159)。图1H提供了人,猪和小鼠凝血因子VIII的C2结构域序列数据(分别为SEQ IDNO:2,氨基酸2173-2332;SEQ ID NO:30,氨基酸1974-2133;和SEQ ID NO:28,氨基酸2160-2319)。
菱形代表酪氨酸硫化位点,可能的糖基化位点用粗体,提出它是凝血因子IXa的结合位点,磷脂和蛋白C用下划双线,结合抗A2和抗C2抑制性抗体的有关区域用斜体。星号突出的氨基酸序列是保守的。也见SEQ ID NO:29(猪凝血因子VIII cDNA)和SEQ ID NO:30(猪凝血因子VIII的推测氨基酸序列)。在此用人编号系统作为参照[Wood等(1984)见上]。A1,A2,和B结构域用位于位点372和740的凝血酶切割位点和1648处的未知蛋白酶切割位点界定分别为残基1-372,373-740,和741-1648[Eaton,D.L.等(1986)生物化学
25:8343-8347]。A3,C1,和C2结构域的界定分别为残基1690-2019,2020-2172,和2173-2332[Vehar等(1984)见上]。凝血酶(凝血因子IIa),凝血因子IXa,凝血因子Xa和APC的切割位点[Fay等(1991)见上;Eaton,D.等(1986)生物化学
25:505-512;Lamphear,B.J.等(1992)Blood
80:3120-3128]通过将酶名放在反应精氨酸上来显示。酸性肽用凝血酶或凝血因子Xa在位点1689从fVIII轻链上切下。凝血因子IXa的假设结合位点[Fay,P.J.等(1994)生物化学杂志
269:20522-20527;Lenting,P.J.等(1994)生物化学杂志269:7150-7155),磷脂(Foster,P.A.等(1990)Blood
75:1999-2004)和蛋白C(Walker,F.J.等(1990)生物化学杂志
265:1484-1489)用下划双线。涉及结合抗A2的区域[Lubin等(1994)见上; Healey等(1995)见上];以及前面提出的结合抗-A2的抑制性抗体的区域用斜体字。如本文所述鉴定出C2抑制性抗体表位(人氨基酸2181-243)显示于图IH中用下划单线。酪氨酸硫化位点[Pittman等(1992)见上]用◆表示。潜在的N-连接糖基化的识别序列(NXS/T,其中X不是脯氨酸)显示为斜体。
实施例7:POLl212的构建和在幼仓鼠肾细胞中的表达
POL1212是部分B结构域缺失的猪凝血因子VIII,缺少B结构域,仅留下B结构域的NH2端的12个氨基酸和-COOH端的12个氨基酸。
编码猪fVIII结构域A1、A2、ap-A3-C1和C2的cDNA如实施例5所述获得。猪凝血因子VIII的DNA核苷酸序列和衍生的氨基酸序列如SEQ ID NO:29和SEQ ID NO:30分别所示。扩增的片段分别克隆到质粒pBluescript II KS-(pBS)中。
POL1212指编码缺乏大部分B结构域,但含有编码A2和ap结构域之间24个氨基酸接头的DNA序列的猪fVIII的cDNA序列。POL1212在哺乳动物表达载体ReNeo中构建,它获自Biogen。ReNeo可以在细菌中复制,在COS细胞中作为附加体复制,瞬时表达凝血因子VIII,或稳定整合到各种哺乳动物细胞中。它含有1)衍生自pBR322的序列,包括复制起始点和氨苄青霉素抗性基因,2)在SV40启动子/增强子、SV40小t内含子和SV40聚腺苷酸信号调节元件控制下表达的新霉素抗性基因,3)插入fVIII及其信号肽的位点,其表达在SV40增强子、腺病毒2型主要晚期启动子和腺病毒2型三联前导序列控制下。可用任何具有相似功能成分的载体代替ReNeo载体。
分几步制备PL1212/ReNeo。首先,分别将编码猪fVIII重链(A1-A2)的cDNA和编码猪fVIII轻链(ap-A3-C1-C2)的cDNA装配到pBS中。从这些构建物,编码无B结构域的猪fVIII的DNA装配在pBS(PB-/pBS)中。猪fVIII的该形式缺乏整个B结构域,定义为对应人fVIII中残基741-1648的氨基酸(人核苷酸2278-5001)。接着,编码猪A2的DNA取代人无B结构域的fVIII表达载体ReNeo(HB-ReNeo)中的A2结构域。用先前制备的猪重链/pBS和PB-/pBS构建物取代编码猪重链的DNA和编码猪轻链的DNA,在两个额外步骤中取代人结构域。在A2和A3结构域之间插入编码5个C-末端和9个N-末端氨基酸的人B结构域的片段,产生称为PSQ/ReNeo的构建物[Healey等(1998),92:3701-3709]。残基Glu2181-Va12243含有人凝血因子VIII的C2结构域中的抑制表位的主要决定簇。该构建物作为模板,产生编码12个C-末端和12个N-末端氨基酸的猪B结构域的片段。该片段插入A2和A3结构域,得到最终构建物POL1212/ReNeo。
POL1212的24个氨基酸的接头由猪fVIII B结构域的开始12个和最后12个残基组成。POl1212接头具有下列序列:
SFAQNSRPPSASAPKPPVLRRHQR(SEQ ID NO:32)
对应于1212接头和周围的氨基酸的核苷酸序列是:
GTC ATT GAA CCT AGG AGC TTT GCC CAG AAT TCA AGA CCC CCT AGT GCG
(SEQ ID NO:33)
V I E P R S F A Q N S R P P S A
AGC GCT CCA AAG CCT CCG GTC CTG CGA CGG CAT CAG AGG GAC ATA
S A P K P P V L R R H Q R D I
AGC CTT CCT ACT
S L P T
POL1212接头是通过剪接-重叠延伸(SOE)诱变合成的,如下:
用于产生SOE产物的PCR反应如下:
反应#1
外侧引物:Rev4,它是猪A2引物,核苷酸1742-1761。(SEQ ID NO:29)序列为:5′-GAGGAAAACCAGATGATGTCA-3′(SEQ ID NO:34)
内侧引物:OL12,它是猪反向引物,覆盖OL1212的开始(5′)15个氨基酸和猪A2的最后(3′)5个氨基酸。序列是:5′-CTTTGGAGCGCTCGCACTAGGGGGTCTTGAATTCTGGGCAAAGCTCCTAGGTTCAATGAC-3′(SEQ ID NO:35)
模板:PSQ/ReNeo
产物:OL1212中A2结构域的核苷酸1742-2322的猪DNA,580bp
反应#2
外侧引物:P1949,它是猪A3引物,核苷酸2998-3021(SEQ ID NO:29)。序列为:5′-GGTCACTTGTCTACCGTGAGCAGC-3′(SEQ ID NO:29)
内侧引物:OL12+,它是猪引物,覆盖OL1212的最后(3′)16个氨基酸和活化肽(SEQ ID NO:29)的核苷酸2302-2367的开始(5′)6个氨基酸。序列是:5′-CCTAGTGCGAGCGCTCCAAAGCCTCCGGTCCTGCGACGGCATCAGAGGGACATAAGCCTTCCTACT-3′(SEQ ID NO:36)
模板:PSQ/ReNeo
产物:OL1212中A3结构域的核苷酸2302-3021的猪DNA,719bp
SOE反应
引物:Rev4,P2949-
模板:rxn#1(bp)和rxn#2(bp)低解链温度片段
产物:从A2结构域的核苷酸1742-A3结构域中的核苷酸3021(SEQ ID NO:29)的猪DNA,包括OL1212,1279bp。该反应产物用乙醇沉淀。
通过用BsaB I切割SOE产物(插入物)和PSQ/ReNeo(载体),将1212接头插入PSQ/ReNeo。用T4连接酶连接载体和插入物,产物用于转化大肠杆菌XL1-Blue细胞。从几个集落制备质粒DNA,用DNA序列分析证实1212接头的序列和其它PCR产生的序列。
幼仓鼠肾(BHK)CRL-1632细胞的培养
从ATCC,登录命名为CRL-1632获得BHK细胞系,并冷冻储藏在-20℃,直到进一步使用。在37℃融化细胞,置于10毫升完全培养基中,该培养基称为DMEM/F12,含有50U/ml青霉素、50微克/毫升链霉素+10%胎牛血清(FBS)。FBS购自Hyclone,Logan Utah。细胞在300RPM离心2分钟。吸去培养基,将细胞重新悬浮在2毫升培养基中,加到含有20毫升完全培养基的T-75瓶中。
POL1212同时在幼仓鼠肾(BHK)细胞和中国仓鼠卵巢(CHO)细胞中表达。使用了两种BHK谱系,来自ATCC的CRL-1632和另一种从R.Mcgillivray,Universityof British Columbia,[Funk等(1990),Biochemistry 29:1654-1660]。后者在发明人的实验室中亚培养,不经筛选,命名为BHK1632(Emory)。CHO细胞系是CHO-K1,ATCC登录号CCL-61。从Emory细胞系和从CHO-K1细胞获得的平均克隆的表达用显色试验活性判断比CRL-1632高。
在T-75瓶中生长的细胞形成汇合的单层。制备了携带POL1212/ReNeo质粒的大肠杆菌XL1-Blue细胞的LB/氨苄青霉素(50mg/ml)的60毫升培养物。
用POL1212/ReNeo转染CRL-1632 BHK细胞
用Qiagen,Valencia,CA Spin Miniprep试剂盒制备POL1212/ReNeo XL1-Blue的过夜培养物的DNA。将一瓶CRL-1632细胞分成0.2毫升的储藏瓶和用于转染的瓶中,用总共2毫升中的0.3毫升转染。另一个烧瓶装有新鲜培养基。培养基是DMEM/F12+10%Hyclone FBS+50U/ml青霉素、50微克/毫升链霉素。用新鲜DMEM/F12+10%Hyclone FBS+50U/ml青霉素、50微克/毫升链霉素将CRL-1632细胞分到6孔板中,目标是达到50-90%汇合,用于转染(各孔来自T-75瓶的0.3毫升细胞,于2毫升1∶5000Versene[Life TEchnologies,Gaithersburg,MD])。
在无菌的1-2毫升试管中制备了下列溶液:
A)48微升(10微克)Miniprep POL1212/ReNeo DNA+微升不含血清的培养基(DMEM/F12)+10微升LipofectinTM(Life Technologies Inc.,Gaithersburg,MD)。
B)将10微升Lipofectin+190微升培养基(模拟转染)温和混合,使DNA和Lipofectin在室温下反应15分钟。在这段过程中,细胞用2毫升DMEM/F12洗涤两次。然后将1.8毫升DMEM/F12加到细胞中。将DNA/Lipofectin复合物滴加到细胞中,温和旋转混合。细胞留在温箱中过夜。除去DNA/Lipofectin,并在细胞中加入3毫升含血清培养基。培养细胞30-48小时。遗传霉素购自LifeTechnologies,Gaithersburg,MD。将细胞培养物在10厘米培养皿上,在10毫升含有血清和535微克/毫升的遗传霉素的培养基中分成1∶20、1∶50和1∶100、1∶250、1∶500。在接下来的几天,不吸收POL1212/ReNeo质粒的细胞由于存在遗传霉素被杀死。剩余的细胞在遗传霉素中继续复制,在培养皿上形成可见的单层集落。
从BHK CRL-1632细胞表达和分析POL1212
将小塑料柱状圆环套在集落外。用完全培养基单个吸出集落,转移到试管中。这些集落称为环克隆的集落。环克隆的集落分别置于24孔板中,在完全培养基中生长。
转染的CRL-1632细胞凝血因子VIII表达的显色底物试验
将细胞培养上清液中的POL1212样品与50nM纯化的猪凝血因子IXa、0.05mM磷酯酰胆碱/磷酯酰丝氨酸(PCPS)小囊在0.15M NaCl、20mM HEPES、5mM CaCl2、0.01%Tween 80,pH7.4中混合。作为对照,使用来自模拟转染的细胞的细胞培养基。同时分别加入凝血酶和凝血因子X至最终浓度为40和425nM。凝血酶激活凝血因子VIII,然后与PCPS一起在凝血因子X的活化过程中作为凝血因子IXa的辅助因子。
在5分钟后,加入最终浓度为50mM的EDTA停止凝血因子IXa/凝血因子VIIIa/PCPS对凝血因子X的活化。同时,凝血酶对凝血因子VIII的活化由于加入最终浓度为100nM的凝血酶抑制剂,重组脱硫水蛭素。将反应混合物的25微升样品转移到微量滴定孔中,在其中加入74微升Spectrozyme Xa(AmericaDiagnostica,Greenwich,CT),它是凝血因子Xa的显色底物。Spectrozyme Xa的最终浓度是0.6mM。用Vmax动态平板读数器(Molecular Devices,Inc.,Menlopark,CA)连续检测5分钟由于凝血因子Xa切开Spectrozyme Xa在405nm处的吸光度。结果表示成A405/分钟。
10个环克隆的集落的凝血因子VIII显色试验
集落数 | A405/分钟(×103) |
缓冲液 | 0.2 |
1 | 2.1 |
2 | 8.4 |
3 | 6.4 |
4 | 10.7 |
5 | 12.5 |
6 | 7.6 |
7 | 51.3 |
8 | 139.5 |
9 | 3.8 |
10 | 8.4 |
这些结果显示全部10个所选集落表达至少比背景高10倍的凝血因子VIII活性。
一步凝血因子VIII凝血试验检测了集落8的培养基的活性它是最高的表达集落。在该试验中,将50毫升凝血因子VIII缺陷血浆(George King BiomedicalOverland Park,KA)、5毫升样品或标准和50毫升活化的颗粒凝血激酶时间试剂(Organon Teknika,Durham,NC)在37℃保温3分钟。样品含有在0.15M NaCl中稀释的集落8的培养基,mM肝素,pH7.4(HBS)或作为对照的完全培养基。加入50ml 20mM的CaCl2开始凝血。用ST$ BIO凝血仪(Diagnostica Stago,Parsippany,NJ)测定凝血时间。通过制备合并的、柠檬酸化的正常人血浆,批号0641(George KingBiomedical,Overland Park PA)的稀释物获得了标准曲线。标准的凝血因子VIII浓度是0.9单位/ml。
标准曲线:
稀释 | U/ml | 凝血时间 |
1)未稀释 | 0.96 | 45.2 |
2)1/3(HBS) | 0.32 | 53.7 |
3)1/11(HBS) | 0.087 | 62.5 |
4)1/21(HBS) | 0.046 | 68.9 |
凝血时间的线性回归对标准的浓度对数得到相关系数为0.997。
测试物质得到下列凝血时间,用标准曲线转换成单位/ml:
样品 | 凝血时间(秒) | 单位/ml |
1)集落8(24小时),1/10HBS | 40.6 | 1.74×10=17.4 |
2)集落8(24小时),1/10HBS | 41.1 | 1.63×10=16.3 |
3)集落8(24小时),1/20HBS | 47.7 | 0.69×20=13.8 |
4)集落8(24小时),1/20HBS | 47.2 | 0.73×20=14.6 |
5)完全培养基 | 82.9 | 0.007 |
6)完全培养基 | 83.3 | 0.006 |
这些结果显示集落8凝血活性比对照样品大约高2000倍。
编码POL1212的DNA序列如SEQ ID NO:37。POL1212的编码氨基酸序列如SEQ ID NO:38所示。POL1212的进一步纯化可以用各种已知方法,例如免疫亲和层析和HPLC层析进行-见实施例2和3。
总体评价
应理解可以在氨基酸序列或编码这些序列的DNA中引入微小改变,而不影响功能的主要性质。例如,在本领域已知的允许范围内可以增加或减少作为接头/保留在A2结构域之间的B-结构域序列的长度。可以在接头区内引入序列变化,而保留本文所指明的POL1212和本文所指明的和本领域已知的猪无B-结构域的凝血因子VIII的相等功能性质。基于与在人血中具有凝血活性的已知凝血因子VIII氨基酸序列的比较,可在基础POL1212氨基酸序列中产生例如单个氨基酸取代的序列变体或具有功能变化的肽区段的取代,同时保留等价的功能性质。上述类型的改变不是穷尽的,而只是举例说明可以由本领域普通技术人员制造的序列变化,而不改变蛋白质的功能性质。所有这些变化和修饰认为是在本发明要求的范围内或是其等价物。
序列ID表:
SEQ ID NO: | 命名: |
1 | 人凝血因子VIIIcDNA。编码从核苷酸号208开始的成熟蛋白的氨基酸号1。 |
2 | 人凝血因子氨基酸序列 |
3 | 猪凝血因子VIII A2结构域cDNA |
4 | 猪凝血因子VIII A2结构域氨基酸序列 |
5-27 | 寡核苷酸引物序列(实施例5) |
28 | 小鼠凝血因子VIII氨基酸序列 |
29 | 猪凝血因子VIIIcDNA |
30 | 猪凝血因子VIII氨基酸序列 |
31 | 人凝血因子VIII信号肽氨基酸序列 |
32-36 | 寡核苷酸引物(实施例7) |
37 | POL1212编码DNA |
38 | POL1212氨基酸序列 |
序列表
序列表<110>埃默里大学(Emory University)<120>修饰的凝血因子VIII<130>75-95I WO<140>PCT/US01/05076<141>2001-02-16<150>US 09/523,656<151>2000-03-10<150>US 09/037,601<151>1998-03-10<150>US 08/670,707<151>1996-06-26<160>38<170>PatentIn Ver.2.0<210>1<211>9009<212>DNA<213>人(Homo sapiens)<220><221>CDS<222>(208)..(7203)<400>1cagtgggtaa gttccttaaa tgctctgcaa agaaattggg acttttcatt aaatcagaaa 60ttttactttt ttcccctcct gggagctaaa gatattttag agaagaatta accttttgct 120tctccagttg aacatttgta gcaataagtc atgcaaatag agctctccac ctgcttcttt 180ctgtgccttt tgcgattctg ctttagt gcc acc aga aga tac tac ctg ggt gca 234
Ala Thr Arg Arg Tyr Tyr Leu Gly Ala
1 5gtg gaa ctg tca tgg gac tat atg caa agt gat ctc ggt gag ctg cct 282Val Glu Leu Ser Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu Leu Pro10 15 20 25gtg gac gca aga ttt cct cct aga gtg cca aaa tct ttt cca ttc aac 330Val Asp Ala Arg Phe Pro Pro Arg Val Pro Lys Ser Phe Pro Phe Asn
30 35 40acc tca gtc gtg tac aaa aag act ctg ttt gta gaa ttc acg gtt cac 378Thr Ser Val Val Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr Val His
45 50 55ctt ttc aac atc gct aag cca agg cca ccc tgg atg ggt ctg cta ggt 426Leu Phe Asn Ile Ala Lys Pro Arg Pro Pro Trp Met Gly Leu Leu Gly
60 65 70cct acc atc cag gct gag gtt tat gat aca gtg gtc att aca ctt aag 474Pro Thr Ile Gln Ala Glu Val Tyr Asp Thr Val Val Ile Thr Leu Lys
75 80 85aac atg gct tcc cat cct gtc agt ctt cat gct gtt ggt gta tcc tac 522Asn Met Ala Ser His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr90 95 100 105tgg aaa gct tct gag gga gct gaa tat gat gat cag acc agt caa agg 570Trp Lys Ala Ser Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg
110 115 120gag aaa gaa gat gat aaa gtc ttc cct ggt gga agc cat aca tat gtc 618Glu Lys Glu Asp Asp Lys Val Phe Pro Gly Gly Ser His Thr Tyr Val
125 130 135tgg cag gtc ctg aaa gag aat ggt cca atg gcc tct gac cca ctg tgc 666Trp Gln Val Leu Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Leu Cys
140 145 150ctt acc tac tca tat ctt tct cat gtg gac ctg gta aaa gac ttg aat 714Leu Thr Tyr Ser Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn
155 160 165tca ggc ctc att gga gcc cta cta gta tgt aga gaa ggg agt ctg gcc 762Ser Gly Leu Ile Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Ala170 175 180 185aag gaa aag aca cag acc ttg cac aaa ttt ata cta ctt ttt gct gta 810Lys Glu Lys Thr Gln Thr Leu His Lys Phe Ile Leu Leu Phe Ala Val
190 195 200ttt gat gaa ggg aaa agt tgg cac tca gaa aca aag aac tcc ttg atg 858Phe Asp Glu Gly Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met
205 210 215cag gat agg gat gct gca tct gct cgg gcc tgg cct aaa atg cac aca 906Gln Asp Arg Asp Ala Ala Ser Ala Arg Ala Trp Pro Lys Met His Thr
220 225 230gtc aat ggt tat gta aac agg tct ctg cca ggt ctg att gga tgc cac 954Val Asn Gly Tyr Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His
235 240 245agg aaa tca gtc tat tgg cat gtg att gga atg ggc acc act cct gaa 1002Arg Lys Ser Val Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu250 255 260 265gtg cac tca ata ttc ctc gaa ggt cac aca ttt ctt gtg agg aac cat 1050Val His Ser Ile Phe Leu Glu Gly His Thr Phe Leu Val Arg Asn His
270 275 280cgc cag gcg tcc ttg gaa atc tcg cca ata act ttc ctt act gct caa 1098Arg Gln Ala Ser Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln
285 290 295aca ctc ttg atg gac ctt gga cag ttt cta ctg ttt tgt cat atc tct 1146Thr Leu Leu Met Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser
300 305 310tcc cac caa cat gat ggc atg gaa gct tat gtc aaa gta gac agc tgt 1194Ser His Gln His Asp Gly Met Glu Ala Tyr Val Lys Val Asp Ser Cys
315 320 325cca gag gaa ccc caa cta cga atg aaa aat aat gaa gaa gcg gaa gac 1242Pro Glu Glu Pro Gln Leu Arg Met Lys Asn Asn Glu Glu Ala Glu Asp330 335 340 345tat gat gat gat ctt act gat tct gaa atg gat gtg gtc agg ttt gat 1290Tyr Asp Asp Asp Leu Thr Asp Ser Glu Met Asp Val Val Arg Phe Asp
350 355 360gat gac aac tct cct tcc ttt atc caa att cgc tca gtt gcc aag aag 1338Asp Asp Asn Ser Pro Ser Phe Ile Gln Ile Arg Ser Val Ala Lys Lys
365 370 375cat cct aaa act tgg gta cat tac att gct gct gaa gag gag gac tgg 1386His Pro Lys Thr Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp
380 385 390gac tat gct ccc tta gtc ctc gcc ccc gat gac aga agt tat aaa agt 1434Asp Tyr Ala Pro Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser
395 400 405caa tat ttg aac aat ggc cct cag cgg att ggt agg aag tac aaa aaa 1482Gln Tyr Leu Asn Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys410 415 420 425gtc cga ttt atg gca tac aca gat gaa acc ttt aag act cgt gaa gct 1530Val Arg Phe Met Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala
430 435 440att cag cat gaa tca gga atc ttg gga cct tta ctt tat ggg gaa gtt 1578Ile Gln His Glu Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val
445 450 455gga gac aca ctg ttg att ata ttt aag aat caa gca agc aga cca tat 1626Gly Asp Thr Leu Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr
460 465 470aac atc tac cct cac gga atc act gat gtc cgt cct ttg tat tca agg 1674Asn Ile Tyr Pro His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg
475 480 485aga tta cca aaa ggt gta aaa cat ttg aag gat ttt cca att ctg cca 1722Arg Leu Pro Lys Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro490 495 500 505gga gaa ata ttc aaa tat aaa tgg aca gtg act gta gaa gat ggg cca 1770Gly Glu Ile Phe Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro
510 515 520act aaa tca gat cct cgg tgc ctg acc cgc tat tac tct agt ttc gtt 1818Thr Lys Ser Asp Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val
525 530 535aat atg gag aga gat cta gct tca gga ctc att ggc cct ctc ctc atc 1866Asn Met Glu Arg Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile
540 545 550tgc tac aaa gaa tct gta gat caa aga gga aac cag ata atg tca gac 1914Cys Tyr Lys Glu Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp
555 560 565aag agg aat gtc atc ctg ttt tct gta ttt gat gag aac cga agc tgg 1962Lys Arg Asn Val Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp570 575 580 585tac ctc aca gag aat ata caa cgc ttt ctc ccc aat cca gct gga gtg 2010Tyr Leu Thr Glu Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val
590 595 600cag ctt gag gat cca gag ttc caa gcc tcc aac atc atg cac agc atc 2058Gln Leu Glu Asp Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile
605 610 615aat ggc tat gtt ttt gat agt ttg cag ttg tca gtt tgt ttg cat gag 2106Asn Gly Tyr Val Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu
620 625 630gtg gca tac tgg tac att cta agc att gga gca cag act gac ttc ctt 2154Val Ala Tyr Trp Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu
635 640 645tct gtc ttc ttc tct gga tat acc ttc aaa cac aaa atg gtc tat gaa 2202Ser Val Phe Phe Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu650 655 660 665gac aca ctc acc cta ttc cca ttc tca gga gaa act gtc ttc atg tcg 2250Asp Thr Leu Thr Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser
670 675 680atg gaa aac cca ggt cta tgg att ctg ggg tgc cac aac tca gac ttt 2298Met Glu Asn Pro Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe
685 690 695cgg aac aga ggc atg acc gcc tta ctg aag gtt tct agt tgt gac aag 2346Arg Asn Arg Gly Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys
700 705 710aac act ggt gat tat tac gag gac agt tat gaa gat att tca gca tac 2394Asn Thr Gly Asp Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr
715 720 725ttg ctg agt aaa aac aat gcc att gaa cca aga agc ttc tcc cag aat 2442Leu Leu Ser Lys Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn730 735 740 745tca aga cac cct agc act agg caa aag caa ttt aat gcc acc aca att 2490Ser Arg His Pro Ser Thr Arg Gln Lys Gln Phe Asn Ala Thr Thr Ile
750 755 760cca gaa aat gac ata gag aag act gac cct tgg ttt gca cac aga aca 2538Pro Glu Asn Asp Ile Glu Lys Thr Asp Pro Trp Phe Ala His Arg Thr
765 770 775cct atg cct aaa ata caa aat gtc tcc tct agt gat ttg ttg atg ctc 2586Pro Met Pro Lys Ile Gln Asn Val Ser Ser Ser Asp Leu Leu Met Leu
780 785 790ttg cga cag agt cct act cca cat ggg cta tcc tta tct gat ctc caa 2634Leu Arg Gln Ser Pro Thr Pro His Gly Leu Ser Leu Ser Asp Leu Gln
795 800 805gaa gcc aaa tat gag act ttt tct gat gat cca tca cct gga gca ata 2682Glu Ala Lys Tyr Glu Thr Phe Ser Asp Asp Pro Ser Pro Gly Ala Ile810 815 820 825gac agt aat aac agc ctg tct gaa atg aca cac ttc agg cca cag ctc 2730Asp Ser Asn Asn Ser Leu Ser Glu Met Thr His Phe Arg Pro Gln Leu
830 835 840cat cac agt ggg gac atg gta ttt acc cct gag tca ggc ctc caa tta 2778His His Ser Gly Asp Met Val Phe Thr Pro Glu Ser Gly Leu Gln Leu
845 850 855aga tta aat gag aaa ctg ggg aca act gca gca aca gag ttg aag aaa 2826Arg Leu Asn Glu Lys Leu Gly Thr Thr Ala Ala Thr Glu Leu Lys Lys
860 865 870ctt gat ttc aaa gtt tct agt aca tca aat aat ctg att tca aca att 2874Leu Asp Phe Lys Val Ser Ser Thr Ser Asn Asn Leu Ile Ser Thr Ile
875 880 885cca tca gac aat ttg gca gca ggt act gat aat aca agt tcc tta gga 2922Pro Ser Asp Asn Leu Ala Ala Gly Thr Asp Asn Thr Ser Ser Leu Gly890 895 900 905ccc cca agt atg cca gtt cat tat gat agt caa tta gat acc act cta 2970Pro Pro Ser Met Pro Val His Tyr Asp Ser Gln Leu Asp Thr Thr Leu
910 915 920ttt ggc aaa aag tca tct ccc ctt act gag tct ggt gga cct ctg agc 3018Phe Gly Lys Lys Ser Ser Pro Leu Thr Glu Ser Gly Gly Pro Leu Ser
925 930 935ttg agt gaa gaa aat aat gat tca aag ttg tta gaa tca ggt tta atg 3066Leu Ser Glu Glu Asn Asn Asp Ser Lys Leu Leu Glu Ser Gly Leu Met
940 945 950aat agc caa gaa agt tca tgg gga aaa aat gta tcg tca aca gag agt 3114Asn Ser Gln Glu Ser Ser Trp Gly Lys Asn Val Ser Ser Thr Glu Ser
955 960 965ggt agg tta ttt aaa ggg aaa aga gct cat gga cct gct ttg ttg act 3162Gly Arg Leu Phe Lys Gly Lys Arg Ala His Gly Pro Ala Leu Leu Thr970 975 980 985aaa gat aat gcc tta ttc aaa gtt agc atc tct ttg tta aag aca aac 3210Lys Asp Asn Ala Leu Phe Lys Val Ser Ile Ser Leu Leu Lys Thr Asn
990 995 1000aaa act tcc aat aat tca gca act aat aga aag act cac att gat ggc 3258Lys Thr Ser Asn Asn Ser Ala Thr Asn Arg Lys Thr His Ile Asp Gly
1005 1010 1015cca tca tta tta att gag aat agt cca tca gtc tgg caa aat ata tta 3306Pro Ser Leu Leu Ile Glu Asn Ser Pro Ser Val Trp Gln Asn Ile Leu
1020 1025 1030gaa agt gac act gag ttt aaa aaa gtg aca cct ttg att cat gac aga 3354Glu Ser Asp Thr Glu Phe Lys Lys Val Thr Pro Leu Ile His Asp Arg1035 1040 1045atg ctt atg gac aaa aat gct aca gct ttg agg cta aat cat atg tca 3402Met Leu Met Asp Lys Asn Ala Thr Ala Leu Arg Leu Asn His Met Ser1050 1055 1060 1065aat aaa act act tca tca aaa aac atg gaa atg gtc caa cag aaa aaa 3450Asn Lys Thr Thr Ser Ser Lys Asn Met Glu Met Val Gln Gln Lys Lys
1070 1075 1080gag ggc ccc att cca cca gat gca caa aat cca gat atg tcg ttc ttt 3498Glu Gly Pro Ile Pro Pro Asp Ala Gln Asn Pro Asp Met Ser Phe Phe
1085 1090 1095aag atg cta ttc ttg cca gaa tca gca agg tgg ata caa agg act cat 3546Lys Met Leu Phe Leu Pro Glu Ser Ala Arg Trp Ile Gln Arg Thr His
1100 1105 1110gga aag aac tct ctg aac tct ggg caa ggc ccc agt cca aag caa tta 3594Gly Lys Asn Ser Leu Asn Ser Gly Gln Gly Pro Ser Pro Lys Gln Leu1115 1120 1125gta tcc tta gga cca gaa aaa tct gtg gaa ggt cag aat ttc ttg tct 3642Val Ser Leu Gly Pro Glu Lys Ser Val Glu Gly Gln Asn Phe Leu Ser1130 1135 1140 1145gag aaa aac aaa gtg gta gta gga aag ggt gaa ttt aca aag gac gta 3690Glu Lys Asn Lys Val Val Val Gly Lys Gly Glu Phe Thr Lys Asp Val
1150 1155 1160gga ctc aaa gag atg gtt ttt cca agc agc aga aac cta ttt ctt act 3738Gly Leu Lys Glu Met Val Phe Pro Ser Ser Arg Asn Leu Phe Leu Thr
1165 1170 1175aac ttg gat aat tta cat gaa aat aat aca cac aat caa gaa aaa aaa 3786Asn Leu Asp Asn Leu His Glu Asn Asn Thr His Asn Gln Glu Lys Lys
1180 1185 1190att cag gaa gaa ata gaa aag aag gaa aca tta atc caa gag aat gta 3834Ile Gln Glu Glu Ile Glu Lys Lys Glu Thr Leu Ile Gln Glu Asn Val1195 1200 1205gtt ttg cct cag ata cat aca gtg act ggc act aag aat ttc atg aag 3882Val Leu Pro Gln Ile His Thr Val Thr Gly Thr Lys Asn Phe Met Lys1210 1215 1220 1225aac ctt ttc tta ctg agc act agg caa aat gta gaa ggt tca tat gag 3930Asn Leu Phe Leu Leu Ser Thr Arg Gln Asn Val Glu Gly Ser Tyr Glu
1230 1235 1240ggg gca tat gct cca gta ctt caa gat ttt agg tca tta aat gat tca 3978Gly Ala Tyr Ala Pro Val Leu Gln Asp Phe Arg Ser Leu Asn Asp Ser
1245 1250 1255aca aat aga aca aag aaa cac aca gct cat ttc tca aaa aaa ggg gag 4026Thr Asn Arg Thr Lys Lys His Thr Ala His Phe Ser Lys Lys Gly Glu
1260 1265 1270gaa gaa aac ttg gaa ggc ttg gga aat caa acc aag caa att gta gag 4074Glu Glu Asn Leu Glu Gly Leu Gly Asn Gln Thr Lys Gln Ile Val Glu1275 1280 1285aaa tat gca tgc acc aca agg ata tct cct aat aca agc cag cag aat 4122Lys Tyr Ala Cys Thr Thr Arg Ile Ser Pro Asn Thr Ser Gln Gln Asn1290 1295 1300 1305ttt gtc acg caa cgt agt aag aga gct ttg aaa caa ttc aga ctc cca 4170Phe Val Thr Gln Arg Ser Lys Arg Ala Leu Lys Gln Phe Arg Leu Pro
1310 1315 1320cta gaa gaa aca gaa ctt gaa aaa agg ata att gtg gat gac acc tca 4218Leu Glu Glu Thr Glu Leu Glu Lys Arg Ile Ile Val Asp Asp Thr Ser
1325 1330 1335acc cag tgg tcc aaa aac atg aaa cat ttg acc ccg agc acc ctc aca 4266Thr Gln Trp Ser Lys Asn Met Lys His Leu Thr Pro Ser Thr Leu Thr
1340 1345 1350cag ata gac tac aat gag aag gag aaa ggg gcc att act cag tct ccc 4314Gln Ile Asp Tyr Asn Glu Lys Glu Lys Gly Ala Ile Thr Gln Ser Pro1355 1360 1365tta tca gat tgc ctt acg agg agt cat agc atc cct caa gca aat aga 4362Leu Ser Asp Cys Leu Thr Arg Ser His Ser Ile Pro Gln Ala Asn Arg1370 1375 1380 1385tct cca tta ccc att gca aag gta tca tca ttt cca tct att aga cct 4410Ser Pro Leu Pro Ile Ala Lys Val Ser Ser Phe Pro Ser Ile Arg Pro
1390 1395 1400ata tat ctg acc agg gtc cta ttc caa gac aac tct tct cat ctt cca 4458Ile Tyr Leu Thr Arg Val Leu Phe Gln Asp Asn Ser Ser His Leu Pro
1405 1410 1415gca gca tct tat aga aag aaa gat tct ggg gtc caa gaa agc agt cat 4506Ala Ala Ser Tyr Arg Lys Lys Asp Ser Gly Val Gln Glu Ser Ser His
1420 1425 1430ttc tta caa gga gcc aaa aaa aat aac ctt tct tta gcc att cta acc 4554Phe Leu Gln Gly Ala Lys Lys Asn Asn Leu Ser Leu Ala Ile Leu Thr1435 1440 1445ttg gag atg act ggt gat caa aga gag gtt ggc tcc ctg ggg aca agt 4602Leu Glu Met Thr Gly Asp Gln Arg Glu Val Gly Ser Leu Gly Thr Ser1450 1455 1460 1465gcc aca aat tca gtc aca tac aag aaa gtt gag aac act gtt ctc ccg 4650Ala Thr Asn Ser Val Thr Tyr Lys Lys Val Glu Asn Thr Val Leu Pro
1470 1475 1480aaa cca gac ttg ccc aaa aca tct ggc aaa gtt gaa ttg ctt cca aaa 4698Lys Pro Asp Leu Pro Lys Thr Ser Gly Lys Val Glu Leu Leu Pro Lys
1485 1490 1495gtt cac att tat cag aag gac cta ttc cct acg gaa act agc aat ggg 4746Val His Ile Tyr Gln Lys Asp Leu Phe Pro Thr Glu Thr Ser Asn Gly
1500 1505 1510tct cct ggc cat ctg gat ctc gtg gaa ggg agc ctt ctt cag gga aca 4794Ser Pro Gly His Leu Asp Leu Val Glu Gly Ser Leu Leu Gln Gly Thr1515 1520 1525gag gga gcg att aag tgg aat gaa gca aac aga cct gga aaa gtt ccc 4842Glu Gly Ala Ile Lys Trp Asn Glu Ala Asn Arg Pro Gly Lys Val Pro1530 1535 1540 1545ttt ctg aga gta gca aca gaa agc tct gca aag act ccc tcc aag cta 4890Phe Leu Arg Val Ala Thr Glu Ser Ser Ala Lys Thr Pro Ser Lys Leu
1550 1555 1560ttg gat cct ctt gct tgg gat aac cac tat ggt act cag ata cca aaa 4938Leu Asp Pro Leu Ala Trp Asp Asn His Tyr Gly Thr Gln Ile Pro Lys
1565 1570 1575gaa gag tgg aaa tcc caa gag aag tca cca gaa aaa aca gct ttt aag 4986Glu Glu Trp Lys Ser Gln Glu Lys Ser Pro Glu Lys Thr Ala Phe Lys
1580 1585 1590aaa aag gat acc att ttg tcc ctg aac gct tgt gaa agc aat cat gca 5034Lys Lys Asp Thr Ile Leu Ser Leu Asn Ala Cys Glu Ser Asn His Ala1595 1600 1605ata gca gca ata aat gag gga caa aat aag ccc gaa ata gaa gtc acc 5082Ile Ala Ala Ile Asn Glu Gly Gln Asn Lys Pro Glu Ile Glu Val Thr1610 1615 1620 1625tgg gca aag caa ggt agg act gaa agg ctg tgc tct caa aac cca cca 5130Trp Ala Lys Gln Gly Arg Thr Glu Arg Leu Cys Ser Gln Asn Pro Pro
1630 1635 1640gtc ttg aaa cgc cat caa cgg gaa ata act cgt act act ctt cag tca 5178Val Leu Lys Arg His Gln Arg Glu Ile Thr Arg Thr Thr Leu Gln Ser
1645 1650 1655gat caa gag gaa att gac tat gat gat acc ata tca gtt gaa atg aag 5226Asp Gln Glu Glu Ile Asp Tyr Asp Asp Thr Ile Ser Val Glu Met Lys
1660 1665 1670aag gaa gat ttt gac att tat gat gag gat gaa aat cag agc ccc cgc 5274Lys Glu Asp Phe Asp Ile Tyr Asp Glu Asp Glu Asn Gln Ser Pro Arg1675 1680 1685agc ttt caa aag aaa aca cga cac tat ttt att gct gca gtg gag agg 5322Ser Phe Gln Lys Lys Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg1690 1695 1700 1705ctc tgg gat tat ggg atg agt agc tcc cca cat gtt cta aga aac agg 5370Leu Trp Asp Tyr Gly Met Ser Ser Ser Pro His Val Leu Arg Asn Arg
1710 1715 1720gct cag agt ggc agt gtc cct cag ttc aag aaa gtt gtt ttc cag gaa 5418Ala Gln Ser Gly Ser Val Pro Gln Phe Lys Lys Val Val Phe Gln Glu
1725 1730 1735ttt act gat ggc tcc ttt act cag ccc tta tac cgt gga gaa cta aat 5466Phe Thr Asp Gly Ser Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn
1740 1745 1750gaa cat ttg gga ctc ctg ggg cca tat ata aga gca gaa gtt gaa gat 5514Glu His Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp1755 1760 1765aat atc atg gta act ttc aga aat cag gcc tct cgt ccc tat tcc ttc 5562Asn Ile Met Val Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser Phe1770 1775 1780 1785tat tct agc ctt att tct tat gag gaa gat cag agg caa gga gca gaa 5610Tyr Ser Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu
1790 1795 1800cct aga aaa aac ttt gtc aag cct aat gaa acc aaa act tac ttt tgg 5658Pro Arg Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp
1805 1810 1815aaa gtg caa cat cat atg gca ccc act aaa gat gag ttt gac tgc aaa 5706Lys Val Gln His His Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys
1820 1825 1830gcc tgg gct tat ttc tct gat gtt gac ctg gaa aaa gat gtg cac tca 5754Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val His Ser1835 1840 1845ggc ctg att gga ccc ctt ctg gtc tgc cac act aac aca ctg aac cct 5802Gly Leu Ile Gly Pro Leu Leu Val Cys His Thr Asn Thr Leu Asn Pro1850 1855 1860 1865gct cat ggg aga caa gtg aca gta cag gaa ttt gct ctg ttt ttc acc 5850Ala His Gly Arg Gln Val Thr Val Gln Glu Phe Ala Leu Phe Phe Thr
1870 1875 1880atc ttt gat gag acc aaa agc tgg tac ttc act gaa aat atg gaa aga 5898Ile Phe Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu Asn Met Glu Arg
1885 1890 1895aac tgc agg gct ccc tgc aat atc cag atg gaa gat ccc act ttt aaa 5946Asn Cys Arg Ala Pro Cys Asn Ile Gln Met Glu Asp Pro Thr Phe Lys
1900 1905 1910gag aat tat cgc ttc cat gca atc aat ggc tac ata atg gat aca cta 5994Glu Asn Tyr Arg Phe His Ala Ile Asn Gly Tyr Ile Met Asp Thr Leu1915 1920 1925cct ggc tta gta atg gct cag gat caa agg att cga tgg tat ctg ctc 6042Pro Gly Leu Val Met Ala Gln Asp Gln Arg Ile Arg Trp Tyr Leu Leu1930 1935 1940 1945agc atg ggc agc aat gaa aac atc cat tct att cat ttc agt gga cat 6090Ser Met Gly Ser Asn Glu Asn Ile His Ser Ile His Phe Ser Gly His
1950 1955 1960gtg ttc act gta cga aaa aaa gag gag tat aaa atg gca ctg tac aat 6138Val Phe Thr Val Arg Lys Lys Glu Glu Tyr Lys Met Ala Leu Tyr Asn
1965 1970 1975ctc tat cca ggt gtt ttt gag aca gtg gaa atg tta cca tcc aaa gct 6186Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met Leu Pro Ser Lys Ala
1980 1985 1990gga att tgg cgg gtg gaa tgc ctt att ggc gag cat cta cat gct ggg 6234Gly Ile Trp Arg Val Glu Cys Leu Ile Gly Glu His Leu His Ala Gly1995 2000 2005atg agc aca ctt ttt ctg gtg tac agc aat aag tgt cag act ccc ctg 6282Met Ser Thr Leu Phe Leu Val Tyr Ser Asn Lys Cys Gln Thr Pro Leu2010 2015 2020 2025gga atg gct tct gga cac att aga gat ttt cag att aca gct tca gga 6330Gly Met Ala Ser Gly His Ile Arg Asp Phe Gln Ile Thr Ala Ser Gly
2030 2035 2040caa tat gga cag tgg gcc cca aag ctg gcc aga ctt cat tat tcc gga 6378Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His Tyr Ser Gly
2045 2050 2055tca atc aat gcc tgg agc acc aag gag ccc ttt tct tgg atc aag gtg 6426Ser Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser Trp Ile Lys Val
2060 2065 2070gat ctg ttg gca cca atg att att cac ggc atc aag acc cag ggt gcc 6474Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile Lys Thr Gln Gly Ala2075 2080 2085cgt cag aag ttc tcc agc ctc tac atc tct cag ttt atc atc atg tat 6522Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile Met Tyr2090 2095 2100 2105agt ctt gat ggg aag aag tgg cag act tat cga gga aat tcc act gga 6570Ser Leu Asp Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn Ser Thr Gly
2110 2115 2120acc tta atg gtc ttc ttt ggc aat gtg gat tca tct ggg ata aaa cac 6618Thr Leu Met Val Phe Phe Gly Asn Val Asp Ser Ser Gly Ile Lys His
2125 2130 2135aat att ttt aac cct cca att att gct cga tac atc cgt ttg cac cca 6666Asn Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr Ile Arg Leu His Pro
2140 2145 2150act cat tat agc att cgc agc act ctt cgc atg gag ttg atg ggc tgt 6714Thr His Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu Leu Met Gly Cys2155 2160 2165gat tta aat agt tgc agc atg cca ttg gga atg gag agt aaa gca ata 6762Asp Leu Asn Ser Cys Ser Met Pro Leu Gly Met Glu Ser Lys Ala Ile2170 2175 2180 2185tca gat gca cag att act gct tca tcc tac ttt acc aat atg ttt gcc 6810Ser Asp Ala Gln Ile Thr Ala Ser Ser Tyr Phe Thr Asn Met Phe Ala
2190 2195 2200acc tgg tct cct tca aaa gct cga ctt cac ctc caa ggg agg agt aat 6858Thr Trp Ser Pro Ser Lys Ala Arg Leu His Leu Gln Gly Arg Ser Asn
2205 2210 2215gcc tgg aga cct cag gtg aat aat cca aaa gag tgg ctg caa gtg gac 6906Ala Trp Arg Pro Gln Val Asn Asn Pro Lys Glu Trp Leu Gln Val Asp
2220 2225 2230ttc cag aag aca atg aaa gtc aca gga gta act act cag gga gta aaa 6954Phe Gln Lys Thr Met Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys2235 2240 2245tct ctg ctt acc agc atg tat gtg aag gag ttc ctc atc tcc agc agt 7002Ser Leu Leu Thr Ser Met Tyr Val Lys Glu Phe Leu Ile Ser Ser Ser2250 2255 2260 2265caa gat ggc cat cag tgg act ctc ttt ttt cag aat ggc aaa gta aag 7050Gln Asp Gly His Gln Trp Thr Leu Phe Phe Gln Asn Gly Lys Val Lys
2270 2275 2280gtt ttt cag gga aat caa gac tcc ttc aca cct gtg gtg aac tct cta 7098Val Phe Gln Gly Asn Gln Asp Ser Phe Thr Pro Val Val Asn Ser Leu
2285 2290 2295gac cca ccg tta ctg act cgc tac ctt cga att cac ccc cag agt tgg 7146Asp Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile His Pro Gln Ser Trp
2300 2305 2310gtg cac cag att gcc ctg agg atg gag gtt ctg ggc tgc gag gca cag 7194Val His Gln Ile Ala Leu Arg Met Glu Val Leu Gly Cys Glu Ala Gln2315 2320 2325gac ctc tac tgagggtggc cactgcagca cctgccactg ccgtcacctc 7243Asp Leu Tyr2330tccctcctca gctccagggc agtgtccctc cctggcttgc cttctacctt tgtgctaaat 7303cctagcagac actgccttga agcctcctga attaactatc atcagtcctg catttctttg 7363gtggggggcc aggagggtgc atccaattta acttaactct tacctatttt ctgcagctgc 7423tcccagatta ctccttcctt ccaatataac taggcaaaaa gaagtgagga gaaacctgca 7483tgaaagcatt cttccctgaa aagttaggcc tctcagagtc accacttcct ctgttgtaga 7543aaaactatgt gatgaaactt tgaaaaagat atttatgatg ttaacatttc aggttaagcc 7603tcatacgttt aaaataaaac tctcagttgt ttattatcct gatcaagcat ggaacaaagc 7663atgtttcagg atcagatcaa tacaatcttg gagtcaaaag gcaaatcatt tggacaatct 7723gcaaaatgga gagaatacaa taactactac agtaaagtct gtttctgctt ccttacacat 7783agatataatt atgttattta gtcattatga ggggcacatt cttatctcca aaactagcat 7843tcttaaactg agaattatag atggggttca agaatcccta agtcccctga aattatataa 7903ggcattctgt ataaatgcaa atgtgcattt ttctgacgag tgtccataga tataaagcca 7963ttggtcttaa ttctgaccaa taaaaaaata agtcaggagg atgcaattgt tgaaagcttt 8023gaaataaaat aacatgtctt cttgaaattt gtgatggcca agaaagaaaa tgatgatgac 8083attaggcttc taaaggacat acatttaata tttctgtgga aatatgagga aaatccatgg 8143ttatctgaga taggagatac aaactttgta attctaataa tgcactcagt ttactctctc 8203cctctactaa tttcctgctg aaaataacac aacaaaaatg taacagggga aattatatac 8283cgtgactgaa aactagagtc ctacttacat agttgaaata tcaaggaggt cagaagaaaa 8323ttggactggt gaaaacagaa aaaacactcc agtctgccat atcaccacac aataggatcc 8383cccttcttgc cctccacccc cataagattg tgaagggttt actgctcctt ccatctgcct 8443gcaccccttc actatgacta cacagaactc tcctgatagt aaagggggct ggaggcaagg 8503ataagttata gagcagttgg aggaagcatc caaagactgc aacccagggc aaatggaaaa 8563caggagatcc taatatgaaa gaaaaatgga tcccaatctg agaaaaggca aaagaatggc 8623tacttttttc tatgctggag tattttctaa taatcctgct tgacccttat ctgacctctt 8683tggaaactat aacatagctg tcacagtata gtcacaatcc acaaatgatg caggtgcaaa 8743tggtttatag ccctgtgaag ttcttaaagt ttagaggcta acttacagaa atgaataagt 8803tgttttgttt tatagcccgg tagaggagtt aaccccaaag gtgatatggt tttatttcct 8863gttatgttta acttgataat cttattttgg cattcttttc ccattgacta tatacatctc 8923tatttctcaa atgttcatgg aactagctct tttattttcc tgctggtttc ttcagtaatg 8983agttaaataa aacattgaca cataca 9009<210>2<211>2332<212>PRT<213>人(Homo sapiens)<400>2Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser Trp Asp Tyr1 5 10 15Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg Phe Pro Pro
20 25 30Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val Tyr Lys Lys
35 40 45Thr Leu Phe Val Glu Phe Thr Val His Leu Phe Asn Ile Ala Lys Pro
50 55 60Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln Ala Glu Val65 70 75 80Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser His Pro Val
85 90 95Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser Glu Gly Ala
100 105 110Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp Asp Lys Val
115 120 125Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu Lys Glu Asn
130 135 140Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser Tyr Leu Ser145 150 155 160His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile Gly Ala Leu
165 170 175Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr Gln Thr Leu
180 185 190His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly Lys Ser Trp
195 200 205His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp Ala Ala Ser
210 215 220Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr Val Asn Arg225 230 235 240Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val Tyr Trp His
245 250 255Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile Phe Leu Glu
260 265 270Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser Leu Glu Ile
275 280 285Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met Asp Leu Gly
290 295 300Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His Asp Gly Met305 310 315 320Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro Gln Leu Arg
325 330 335Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp Leu Thr Asp
340 345 350Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser Pro Ser Phe
355 360 365Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr Trp Val His
370 375 380Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro Leu Val Leu385 390 395 400Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn Asn Gly Pro
405 410 415Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met Ala Tyr Thr
420 425 430Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu Ser Gly Ile
435 440 445Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu Leu Ile Ile
450 455 460Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly Ile465 470 475 480Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys Gly Val Lys
485 490 495His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe Lys Tyr Lys
500 505 510Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp Pro Arg Cys
515 520 525Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg Asp Leu Ala
530 535 540Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu Ser Val Asp545 550 555 560Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val Ile Leu Phe
565 570 575Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu Asn Ile Gln
580 585 590Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp Pro Glu Phe
595 600 605Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val Phe Asp Ser
610 615 620Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp Tyr Ile Leu625 630 635 640Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe Ser Gly Tyr
645 650 655Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr Leu Phe Pro
660 665 670Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro Gly Leu Trp
675 680 685Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly Met Thr Ala
690 695 700Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp Tyr Tyr Glu705 710 715 720Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys Asn Asn Ala
725 730 735Ile Glu Pro Arg Ser Phe Ser Gln Asn Ser Arg His Pro Ser Thr Arg
740 745 750Gln Lys Gln Phe Asn Ala Thr Thr Ile Pro Glu Asn Asp Ile Glu Lys
755 760 765Thr Asp Pro Trp Phe Ala His Arg Thr Pro Met Pro Lys Ile Gln Asn
770 775 780Val Ser Ser Ser Asp Leu Leu Met Leu Leu Arg Gln Ser Pro Thr Pro785 790 795 800His Gly Leu Ser Leu Ser Asp Leu Gln Glu Ala Lys Tyr Glu Thr Phe
805 810 815Ser Asp Asp Pro Ser Pro Gly Ala Ile Asp Ser Asn Asn Ser Leu Ser
820 825 830Glu Met Thr His Phe Arg Pro Gln Leu His His Ser Gly Asp Met Val
835 840 845Phe Thr Pro Glu Ser Gly Leu Gln Leu Arg Leu Asn Glu Lys Leu Gly
850 855 860Thr Thr Ala Ala Thr Glu Leu Lys Lys Leu Asp Phe Lys Val Ser Ser865 870 875 880Thr Ser Asn Asn Leu Ile Ser Thr Ile Pro Ser Asp Asn Leu Ala Ala
885 890 895Gly Thr Asp Asa Thr Ser Ser Leu Gly Pro Pro Ser Met Pro Val His
900 905 910Tyr Asp Ser Gln Leu Asp Thr Thr Leu Phe Gly Lys Lys Ser Ser Pro
915 920 925Leu Thr Glu Ser Gly Gly Pro Leu Ser Leu Ser Glu Glu Asn Asn Asp
930 935 940Ser Lys Leu Leu Glu Ser Gly Leu Met Asn Ser Gln Glu Ser Ser Trp945 950 955 960Gly Lys Asn Val Ser Ser Thr Glu Ser Gly Arg Leu Phe Lys Gly Lys
965 970 975Arg Ala His Gly Pro Ala Leu Leu Thr Lys Asp Asn Ala Leu Phe Lys
980 985 990Val Ser Ile Ser Leu Leu Lys Thr Asn Lys Thr Ser Asn Asn Ser Ala
995 1000 1005Thr Asn Arg Lys Thr His Ile Asp Gly Pro Ser Leu Leu Ile Glu Asn1010 1015 1020Ser Pro Ser Val Trp Gln Asn Ile Leu Glu Ser Asp Thr Glu Phe Lys1025 1030 1035 1040Lys Val Thr Pro Leu Ile His Asp Arg Met Leu Met Asp Lys Asn Ala
1045 1050 1055Thr Ala Leu Arg Leu Asn His Met Ser Asn Lys Thr Thr Ser Ser Lys
1060 1065 1070Asn Met Glu Met Val Gln Gln Lys Lys Glu Gly Pro Ile Pro Pro Asp
1075 1080 1085Ala Gln Asn Pro Asp Met Ser Phe Phe Lys Met Leu Phe Leu Pro Glu1090 1095 1100Ser Ala Arg Trp Ile Gln Arg Thr His Gly Lys Asn Ser Leu Asn Ser1105 1110 1115 ll20Gly Gln Gly Pro Ser Pro Lys Gln Leu Val Ser Leu Gly Pro Glu Lys
1125 1130 1135Ser Val Glu Gly Gln Asn Phe Leu Ser Glu Lys Asn Lys Val Val Val
1140 1145 1150Gly Lys Gly Glu Phe Thr Lys Asp Val Gly Leu Lys Glu Met Val Phe
1155 1160 1165Pro Ser Ser Arg Asn Leu Phe Leu Thr Asn Leu Asp Asn Leu His Glu1170 1175 1180Asn Asn Thr His Asn Gln Glu Lys Lys Ile Gln Glu Glu Ile Glu Lys1185 1190 1195 1200Lys Glu Thr Leu Ile Gln Glu Asn Val Val Leu Pro Gln Ile His Thr
1205 1210 1215Val Thr Gly Thr Lys Asn Phe Met Lys Asn Leu Phe Leu Leu Ser Thr
1220 1225 1230Arg Gln Asn Val Glu Gly Ser Tyr Glu Gly Ala Tyr Ala Pro Val Leu
1235 1240 1245Gln Asp Phe Arg Ser Leu Asn Asp Ser Thr Asn Arg Thr Lys Lys His1250 1255 1260Thr Ala His Phe Ser Lys Lys Gly Glu Glu Glu Asn Leu Glu Gly Leu1265 1270 1275 1280Gly Asn Gln Thr Lys Gln Ile Val Glu Lys Tyr Ala Cys Thr Thr Arg
1285 1290 1295Ile Ser Pro Asn Thr Ser Gln Gln Asn Phe Val Thr Gln Arg Ser Lys
1300 1305 1310Arg Ala Leu Lys Gln Phe Arg Leu Pro Leu Glu Glu Thr Glu Leu Glu
1315 1320 1325Lys Arg Ile Ile Val Asp Asp Thr Ser Thr Gln Trp Ser Lys Asn Met1330 1335 1340Lys His Leu Thr Pro Ser Thr Leu Thr Gln Ile Asp Tyr Asn Glu Lys1345 1350 1355 1360Glu Lys Gly Ala Ile Thr Gln Ser Pro Leu Ser Asp Cys Leu Thr Arg
1365 1370 1375Ser His Ser Ile Pro Gln Ala Asn Arg Ser Pro Leu Pro Ile Ala Lys
1380 1385 1390Val Ser Ser Phe Pro Ser Ile Arg Pro Ile Tyr Leu Thr Arg Val Leu
1395 1400 1405Phe Gln Asp Asn Ser Ser His Leu Pro Ala Ala Ser Tyr Arg Lys Lys1410 1415 1420Asp Ser Gly Val Gln Glu Ser Ser His Phe Leu Gln Gly Ala Lys Lys1425 1430 1435 1440Asn Asn Leu Ser Leu Ala Ile Leu Thr Leu Glu Met Thr Gly Asp Gln
1445 1450 1455Arg Glu Val Gly Ser Leu Gly Thr Ser Ala Thr Asn Ser Val Thr Tyr
1460 1465 1470Lys Lys Val Glu Asn Thr Val Leu Pro Lys Pro Asp Leu Pro Lys Thr
1475 1480 1485Ser Gly Lys Val Glu Leu Leu Pro Lys Val His Ile Tyr Gln Lys Asp1490 1495 1500Leu Phe Pro Thr Glu Thr Ser Asn Gly Ser Pro Gly His Leu Asp Leu1505 1510 1515 1520Val Glu Gly Ser Leu Leu Gln Gly Thr Glu Gly Ala Ile Lys Trp Asn
1525 1530 1535Glu Ala Asn Arg Pro Gly Lys Val Pro Phe Leu Arg Val Ala Thr Glu
1540 1545 1550Ser Ser Ala Lys Thr Pro Ser Lys Leu Leu Asp Pro Leu Ala Trp Asp
1555 1560 1565Asn His Tyr Gly Thr Gln Ile Pro Lys Glu Glu Trp Lys Ser Gln Glu1570 1575 1580Lys Ser Pro Glu Lys Thr Ala Phe Lys Lys Lys Asp Thr Ile Leu Ser1585 1590 1595 1600Leu Asn Ala Cys Glu Ser Asn His Ala Ile Ala Ala Ile Asn Glu Gly
1605 1610 1615Gln Asn Lys Pro Glu Ile Glu Val Thr Trp Ala Lys Gln Gly Arg Thr
1620 1625 1630Glu Arg Leu Cys Ser Gln Asn Pro Pro Val Leu Lys Arg His Gln Arg
1635 1640 1645Glu Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr1650 1655 1660Asp Asp Thr Ile Ser Val Glu Met Lys Lys Glu Asp Phe Asp Ile Tyr1665 1670 1675 1680Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys Thr Arg
1685 1690 1695His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr Gly Met Ser
1700 1705 1710Ser Ser Pro His Val Leu Arg Asn Arg Ala Gln Ser Gly Ser Val Pro
1715 1720 1725Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr Asp Gly Ser Phe Thr1730 1735 1740Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His Leu Gly Leu Leu Gly1745 1750 1755 1760Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val Thr Phe Arg
1765 1770 1775Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser Leu Ile Ser Tyr
1780 1785 1790Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg Lys Asn Phe Val Lys
1795 1800 1805Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys Val Gln His His Met Ala1810 1815 1820Pro Thr Lys Asp Glu Phe Asp Cys Lys Ala Trp Ala Tyr Phe Ser Asp1825 1830 1835 1840Val Asp Leu Glu Lys Asp Val His Ser Gly Leu Ile Gly Pro Leu Leu
1845 1850 1855Val Cys His Thr Asn Thr Leu Asn Pro Ala His Gly Arg Gln Val Thr
1860 1865 1870Val Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser
1875 1880 1885Trp Tyr Phe Thr Glu Asn Met Glu Arg Asn Cys Arg Ala Pro Cys Asn1890 1895 1900Ile Gln Met Glu Asp Pro Thr Phe Lys Glu Asn Tyr Arg Phe His Ala1905 1910 1915 1920Ile Asn Gly Tyr Ile Met Asp Thr Leu Pro Gly Leu Val Met Ala Gln
1925 1930 1935Asp Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn
1940 1945 1950Ile His Ser Ile His Phe Ser Gly His Val Phe Thr Val Arg Lys Lys
1955 1960 1965Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr Pro Gly Val Phe Glu1970 1975 1980Thr Val Glu Met Leu Pro Ser Lys Ala Gly Ile Trp Arg Val Glu Cys1985 1990 1995 2000Leu Ile Gly Glu His Leu His Ala Gly Met Ser Thr Leu Phe Leu Val
2005 2010 2015Tyr Ser Asn Lys Cys Gln Thr Pro Leu Gly Met Ala Ser Gly His Ile
2020 2025 2030Arg Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro
2035 2040 2045Lys Leu Ala Arg Leu His Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr2050 2055 2060Lys Glu Pro Phe Ser Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile2065 2070 2075 2080Ile His Gly Ile Lys Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu
2085 2090 2095Tyr Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp Gly Lys Lys Trp
2100 2105 2110Gln Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu Met Val Phe Phe Gly
2115 2120 2125Asn Val Asp Ser Ser Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile2130 2135 2140Ile Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser2145 2150 2155 2160Thr Leu Arg Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys Ser Met
2165 2170 2175Pro Leu Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr Ala
2180 2185 2190Ser Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser Pro Ser Lys Ala
2195 2200 2205Arg Leu His Leu Gln Gly Arg Ser Asn Ala Trp Arg Pro Gln Val Asn2210 2215 2220Asn Pro Lys Glu Trp Leu Gln Val Asp Phe Gln Lys Thr Met Lys Val2225 2230 2235 2240Thr Gly Val Thr Thr Gln Gly Val Lys Ser Leu Leu Thr Ser Met Tyr
2245 2250 2255Val Lys Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly His Gln Trp Thr
2260 2265 2270Leu Phe Phe Gln Asn Gly Lys Val Lys Val Phe Gln Gly Asn Gln Asp
2275 2280 2285Ser Phe Thr Pro Val Val Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg2290 2295 2300Tyr Leu Arg Ile His Pro Gln Ser Trp Val His Gln Ile Ala Leu Arg2305 2310 2315 2320Met Glu Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr
2325 2330<210>3<211>1130<212>DNA<213>猪(Porcine)<400>3taagcaccct aagacgtggg tgcactacat ctctgcagag gaggaggact gggactacgc 60ccccgcggtc cccagcccca gtgacagaag ttataaaagt ctctacttga acagtggtcc 120tcagcgaatt ggtaggaaat acaaaaaagc tcgattcgtc gcttacacgg atgtaacatt 180taagactcgt aaagctattc cgtatgaatc aggaatcctg ggacctttac tttatggaga 240agttggagac acacttttga ttatatttaa gaataaagcg agccgaccat ataacatcta 300ccctcatgga atcactgatg tcagcgcttt gcacccaggg agacttctaa aaggttggaa 360acatttgaaa gacatgccaa ttctgccagg agagactttc aagtataaat ggacagtgac 420tgtggaagat gggccaacca agtccgatcc tcggtgcctg acccgctact actcgagctc 480cattaatcta gagaaagatc tggcttcggg actcattggc cctctcctca tctgctacaa 540agaatctgta gaccaaagag gaaaccagat gatgtcagac aagagaaacg tcatcctgtt 600ttctgtattc gatgagaatc aaagctggta cctcgcagag aatattcagc gcttcctccc 660caatccggat ggattacagc cccaggatcc agagttccaa gcttctaaca tcatgcacag 720catcaatggc tatgtttttg atagcttgca gctgtcggtt tgtttgcacg aggtggcata 780ctggtacatt ctaagtgttg gagcacagac ggacttcctc tccgtcttct tctctggcta 840caccttcaaa cacaaaatgg tctatgaaga cacactcacc ctgttcccct tctcaggaga 900aacggtcttc atgtcaatgg aaaacccagg tctctgggtc ctagggtgcc acaactcaga 960cttgcggaac agagggatga cagccttact gaaggtgtat agttgtgaca gggacattgg 1020tgattattat gacaacactt atgaagatat tccaggcttc ttgctgagtg gaaagaatgt 1080cattgaaccc agaagctttg cccagaattc aagaccccct agtgcgagca 1130<210>4<211>368<212>PRT<213>猪(Porcine)<400>4Ser Val Ala Lys Lys His Pro Lys Thr Trp Val His Tyr Ile Ser Ala1 5 10 15Glu Glu Glu Asp Trp Asp Tyr Ala Pro Ala Val Pro Ser Pro Ser Asp
20 25 30Arg Ser Tyr Lys Ser Leu Tyr Leu Asn Ser Gly Pro Gln Arg Ile Gly
35 40 45Arg Lys Tyr Lys Lys Ala Arg Phe Val Ala Tyr Thr Asp Val Thr Phe
50 55 60Lys Thr Arg Lys Ala Ile Pro Tyr Glu Ser Gly Ile Leu Gly Pro Leu65 70 75 80Leu Tyr Gly Glu Val Gly Asp Thr Leu Leu Ile Ile Phe Lys Asn Lys
85 90 95Ala Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly Ile Thr Asp Val Ser
100 105 110Ala Leu His Pro Gly Arg Leu Leu Lys Gly Trp Lys His Leu Lys Asp
115 120 125Met Pro Ile Leu Pro Gly Glu Thr Phe Lys Tyr Lys Trp Thr Val Thr
130 135 140Val Glu Asp Gly Pro Thr Lys Ser Asp Pro Arg Cys Leu Thr Arg Tyr145 150 155 160Tyr Ser Ser Ser Ile Asn Leu Glu Lys Asp Leu Ala Ser Gly Leu Ile
165 170 175Gly Pro Leu Leu Ile Cys Tyr Lys Glu Ser Val Asp Gln Arg Gly Asn
180 185 190Gln Met Met Ser Asp Lys Arg Asn Val Ile Leu Phe Ser Val Phe Asp
195 200 205Glu Asn Gln Ser Trp Tyr Leu Ala Glu Asn Ile Gln Arg Phe Leu Pro
210 215 220Asn Pro Asp Gly Leu Gln Pro Gln Asp Pro Glu Phe Gln Ala Ser Asn225 230 235 240Ile Met His Ser Ile Asn Gly Tyr Val Phe Asp Ser Leu Gln Leu Ser
245 250 255Val Cys Leu His Glu Val Ala Tyr Trp Tyr Ile Leu Ser Val Gly Ala
260 265 270Gln Thr Asp Phe Leu Ser Val Phe Phe Ser Gly Tyr Thr Phe Lys His
275 280 285Lys Met Val Tyr Glu Asp Thr Leu Thr Leu Phe Pro Phe Ser Gly Glu
290 295 300Thr Val Phe Met Ser Met Glu Asn Pro Gly Leu Trp Val Leu Gly Cys305 310 315 320His Asn Ser Asp Leu Arg Asn Arg Gly Met Thr Ala Leu Leu Lys Val
325 330 335Tyr Ser Cys Asp Arg Asp Ile Gly Asp Tyr Tyr Asp Asn Thr Tyr Glu
340 345 350Asp Ile Pro Gly Phe Leu Leu Ser Gly Lys Asn Val Ile Glu Pro Arg
355 360 365<210>5<211>44<212>DNA<213>人工序列<220><223>人工序列的描述:寡核苷酸引物<400>5ctaatacgac tcactatagg gctcgagcgg ccgcccgggc aggt 44<210>6<211>27<212>DNA<213>人工序列<220><223>人工序列的描述:寡核苷酸引物<400>6ccatcctaat acgactcact atagggc 27<210>7<211>24<212>DNA<213>人工序列<220><223>人工序列的描述:寡核苷酸引物<400>7ccattgacat gaagaccgtt tctc 24<210>8<211>23<212>DNA<213>人工序列<220><223>人工序列的描述:寡核苷酸引物<400>8actcactata gggctcgagc ggc 23<210>9<211>24<212>DNA<213>人工序列<220><223>人工序列的描述:寡核苷酸引物<400>9gggtgcaaag cgctgacatc agtg 24<210>10<211>50<212>DNA<213>人工序列<220><223>人工序列的描述:寡核苷酸引物<400>10cctctcgagc caccatgtcg agccaccatg cagctagagc tctccacctg 50<210>11<211>31<212>DNA<213>人工序列<220><223>人工序列的描述:寡核苷酸引物<400>11cgcgcggccg cgcatctggc aaagctgagt t 31<210>12<211>27<212>DNA<213>人工序列<220><223>人工序列的描述:寡核苷酸引物<400>12gaaataagcc caggctttgc agtcraa 27<210>13<211>22<212>DNA<213>人工序列<220><223>人工序列的描述:寡核苷酸引物<220><221>misc feature<222>(22)<223>22位的N是A,T,G或C。<400>13aggaaattcc actggaacct tn 22<210>14<211>25<212>DNA<213>人工序列<220><223>人工序列的描述:寡核苷酸引物<220><221>misc feature<222>(25)<223>25位的N是A,T,G或C。<400>14ctgggggtga attcgaaggt agcgn 25<210>15<211>23<212>DNA<213>人工序列<220><223>人工序列的描述:寡核苷酸引物<400>15gagttcatcg ggaagacctg ttg 23<210>16<211>24<212>DNA<213>人工序列<220><223>人工序列的描述:寡核苷酸引物<400>16acagcccatc aactccatgc gaag 24<210>17<211>19<212>DNA<213>人工序列<220><223>人工序列的描述:寡核苷酸引物<400>17tcagggcaat caggactcc 19<210>18<211>21<212>DNA<213>人工序列<220><223>人工序列的描述:寡核苷酸引物<400>18ccgtggtgaa cgctctggac c 21<210>19<211>24<212>DNA<213>人工序列<220><223>人工序列的描述:寡核苷酸引物<400>19gtagaggtcc tgtgcctcgc agcc 24<210>20<211>27<212>DNA<213>人工序列<220><223>人工序列的描述:寡核苷酸引物<400>20gtagagstsc tgkgcctcrc akccyag 27<210>21<211>24<212>DNA<213>人工序列<220><223>人工序列的描述:寡核苷酸引物<400>21cttcgcatgg agttgatggg ctgt 24<210>22<211>21<212>DNA<213>人工序列<220><223>人工序列的描述:寡核苷酸引物<400>22aatcaggact cctccacccc g 21<210>23<211>20<212>DNA<213>人工序列<220><223>人工序列的描述:寡核苷酸引物<400>23ggatccaccc cacgagctgg 20<210>24<211>24<212>DNA<213>人工序列<220><223>人工序列的描述:寡核苷酸引物<400>24cgccctgagg ctcgaggttc tagg 24<210>25<211>22<212>DNA<213>人工序列<220><223>人工序列的描述:寡核苷酸引物<400>25aatcaggact cctccacccc cg 22<210>26<211>20<212>DNA<213>人工序列<220><223>人工序列的描述:寡核苷酸引物<400>26ccttgcagga attcgattca 20<210>27<211>21<212>DNA<213>人工序列<220><223>人工序列的描述:寡核苷酸引物<400>27ccgtggtgaa cgctctggac c 21<210>28<211>2319<212>PRT<213>小鼠(Mus musculus)<400>28Met Gln Ile Ala Leu Phe Ala Cys Phe Phe Leu Ser Leu Phe Asn Phe1 5 10 15Cys Ser Ser Ala Ile Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser
20 25 30Trp Asn Tyr Ile Gln Ser Asp Leu Leu Ser Val Leu His Thr Asp Ser
35 40 45Arg Phe Leu Pro Arg Met Ser Thr Ser Phe Pro Phe Asn Thr Ser Ile
50 55 60Met Tyr Lys Lys Thr Val Phe Val Glu Tyr Lys Asp Gln Leu Phe Asn65 70 75 80Ile Ala Lys Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile
85 90 95Trp Thr Glu Val His Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala
100 105 110Ser His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala
115 120 125Ser Glu Gly Asp Glu Tyr Glu Asp Gln Thr Ser Gln Met Glu Lys Glu
130 135 140Asp Asp Lys Val Phe Pro Gly Glu Ser His Thr Tyr Val Trp Gln Val145 150 155 160Leu Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Pro Cys Leu Thr Tyr
165 170 175Ser Tyr Met Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu
180 185 190Ile Gly Ala Leu Leu Val Cys Lys Glu Gly Ser Leu Ser Lys Glu Arg
195 200 205Thr Gln Met Leu Tyr Gln Phe Val Leu Leu Phe Ala Val Phe Asp Glu
210 215 220Gly Lys Ser Trp His Ser Glu Thr Asn Asp Ser Tyr Thr Gln Ser Met225 230 235 240Asp Ser Ala Ser Ala Arg Asp Trp Pro Lys Met His Thr Val Asn Gly
245 250 255Tyr Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser
260 265 270Val Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu Ile His Ser
275 280 285Ile Phe Leu Glu Gly His Thr Phe Phe Val Arg Asn His Arg Gln Ala
290 295 300Ser Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu305 310 315 320Ile Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Lys
325 330 335His Asp Gly Met Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu
340 345 350Ser Gln Trp Gln Lys Lys Asn Asn Asn Glu Glu Met Glu Asp Tyr Asp
355 360 365Asp Asp Leu Tyr Ser Glu Met Asp Met Phe Thr Leu Asp Tyr Asp Ser
370 375 380Ser Pro Phe Ile Gln Ile Arg Ser Val Ala Lys Lys Tyr Pro Lys Thr385 390 395 400Trp Ile His Tyr Ile Ser Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro
405 410 415Ser Val Pro Thr Ser Asp Asn Gly Ser Tyr Lys Ser Gln Tyr Leu Ser
420 425 430Asn Gly Pro His Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Ile
435 440 445Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Thr Ile Gln His Glu
450 455 460Ser Gly Leu Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu465 470 475 480Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro
485 490 495His Gly Ile Thr Asp Val Ser Pro Leu His Ala Arg Arg Leu Pro Arg
500 505 510Gly Ile Lys His Val Lys Asp Leu Pro Ile His Pro Gly Glu Ile Phe
515 520 525Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp530 535 540Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Ile Asn Pro Glu Arg545 550 555 560Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu
565 570 575Ser Val Asp Gln Arg Gly Asn Gln Met Met Ser Asp Lys Arg Asn Val
580 585 590Ile Leu Phe Ser Ile Phe Asp Glu Asn Gln Ser Trp Tyr Ile Thr Glu
595 600 605Asn Met Gln Arg Phe Leu Pro Asn Ala Ala Lys Thr Gln Pro Gln Asp
610 615 620Pro Gly Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val625 630 635 640Phe Asp Ser Leu Glu Leu Thr Val Cys Leu His Glu Val Ala Tyr Trp
645 650 655His Ile Leu Ser Val Gly Ala Gln Thr Asp Phe Leu Ser Ile Phe Phe
660 665 670Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr
675 680 685Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro
690 695 700Gly Leu Trp Val Leu Gly Cys His Asn Ser Asp Phe Arg Lys Arg Gly705 710 715 720Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Ser Thr Ser Asp
725 730 735Tyr Tyr Glu Glu Ile Tyr Glu Asp Ile Pro Thr Gln Leu Val Asn Glu
740 745 750Asn Asn Val Ile Asp Pro Arg Ser Phe Phe Gln Asn Thr Asn His Pro
755 760 765Asn Thr Arg Lys Lys Lys Phe Lys Asp Ser Thr Ile Pro Lys Asn Asp
770 775 780Met Glu Lys Ile Glu Pro Gln Phe Glu Glu Ile Ala Glu Met Leu Lys785 790 795 800Val Gln Ser Val Ser Val Ser Asp Met Leu Met Leu Leu Gly Gln Ser
805 810 815His Pro Thr Pro His Gly Leu Phe Leu Ser Asp Gly Gln Glu Ala Ile
820 825 830Tyr Glu Ala Ile His Asp Asp His Ser Pro Asn Ala Ile Asp Ser Asn
835 840 845Glu Gly Pro Ser Lys Val Thr Gln Leu Arg Pro Glu Ser His His Ser
850 855 860Glu Lys Ile Val Phe Thr Pro Gln Pro Gly Leu Gln Leu Arg Ser Asn865 870 875 880Lys Ser Leu Glu Thr Thr Ile Glu Val Lys Trp Lys Lys Leu Gly Leu
885 890 895Gln Val Ser Ser Leu Pro Ser Asn Leu Met Thr Thr Thr Ile Leu Ser
900 905 910Asp Asn Leu Lys Ala Thr Phe Glu Lys Thr Asp Ser Ser Gly Phe Pro
915 920 925Asp Met Pro Val His Ser Ser Ser Lys Leu Ser Thr Thr Ala Phe Gly
930 935 940Lys Lys Ala Tyr Ser Leu Val Gly Ser His Val Pro Leu Asn Ala Ser945 950 955 960Glu Glu Asn Ser Asp Ser Asn Ile Leu Asp Ser Thr Leu Met Tyr Ser
965 970 975Gln Glu Ser Leu Pro Arg Asp Asn Ile Leu Ser Ile Glu Asn Asp Arg
980 985 990Leu Leu Arg Glu Lys Arg Phe His Gly Ile Ala Leu Leu Thr Lys Asp
995 1000 1005Asn Thr Leu Phe Lys Asp Asn Val Ser Leu Met Lys Thr Asn Lys Thr1010 1015 1020Tyr Asn His Ser Thr Thr Asn Glu Lys Leu His Thr Glu Ser Pro Thr1025 1030 1035 1040Ser Ile Glu Asn Ser Thr Thr Asp Leu Gln Asp Ala Ile Leu Lys Val
1045 1050 1055Asn Ser Glu Ile Gln Glu Val Thr Ala Leu Ile His Asp Gly Thr Leu
1060 1065 1070Leu Gly Lys Asn Ser Thr Tyr Leu Arg Leu Asn His Met Leu Asn Arg
1075 1080 1085Thr Thr Ser Thr Lys Asn Lys Asp Ile Phe His Arg Lys Asp Glu Asp1090 1095 1100Pro Ile Pro Gln Asp Glu Glu Asn Thr Ile Met Pro Phe Ser Lys Met1105 1110 1115 1120Leu Phe Leu Ser Glu Ser Ser Asn Trp Phe Lys Lys Thr Asn Gly Asn
1125 1130 1135Asn Ser Leu Asn Ser Glu Gln Glu His Ser Pro Lys Gln Leu Val Tyr
1140 1145 1150Leu Met Phe Lys Lys Tyr Val Lys Asn Gln Ser Phe Leu Ser Glu Lys
1155 1160 1165Asn Lys Val Thr Val Glu Gln Asp Gly Phe Thr Lys Asn Ile Gly Leu1170 1175 1180Lys Asp Met Ala Phe Pro His Asn Met Ser Ile Phe Leu Thr Thr Leu1185 1190 1195 1200Ser Asn Val His Glu Asn Gly Arg His Asn Gln Glu Lys Asn Ile Gln
1205 1210 1215Glu Glu Ile Glu Lys Glu Ala Leu Ile Glu Glu Lys Val Val Leu Pro
1220 1225 1230Gln Val His Glu Ala Thr Gly Ser Lys Asn Phe Leu Lys Asp Ile Leu
1235 1240 1245Ile Leu Gly Thr Arg Gln Asn Ile Ser Leu Tyr Glu Val His Val Pro1250 1255 1260Val Leu Gln Asn Ile Thr Ser Ile Asn Asn Ser Thr Asn Thr Val Gln1265 1270 1275 1280Ile His Met Glu His Phe Phe Lys Arg Arg Lys Asp Lys Glu Thr Asn
1285 1290 1295Ser Glu Gly Leu Val Asn Lys Thr Arg Glu Met Val Lys Asn Tyr Pro
1300 1305 1310Ser Gln Lys Asn Ile Thr Thr Gln Arg Ser Lys Arg Ala Leu Gly Gln
1315 1320 1325Phe Arg Leu Ser Thr Gln Trp Leu Lys Thr Ile Asn Cys Ser Thr Gln1330 1335 1340Cys Ile Ile Lys Gln Ile Asp His Ser Lys Glu Met Lys Lys Phe Ile1345 1350 1355 1360Thr Lys Ser Ser Leu Ser Asp Ser Ser Val Ile Lys Ser Thr Thr Gln
1365 1370 1375Thr Asn Ser Ser Asp Ser His Ile Val Lys Thr Ser Ala Phe Pro Pro
1380 1385 1390Ile Asp Leu Lys Arg Ser Pro Phe Gln Asn Lys Phe Ser His Val Gln
1395 1400 1405Ala Ser Ser Tyr Ile Tyr Asp Phe Lys Thr Lys Ser Ser Arg Ile Gln1410 1415 1420Glu Ser Asn Asn Phe Leu Lys Glu Thr Lys Ile Asn Asn Pro Ser Leu1425 1430 1435 1440Ala Ile Leu Pro Trp Asn Met Phe Ile Asp Gln Gly Lys Phe Thr Ser
1445 1450 1455Pro Gly Lys Ser Asn Thr Asn Ser Val Thr Tyr Lys Lys Arg Glu Asn
1460 1465 1470Ile Ile Phe Leu Lys Pro Thr Leu Pro Glu Glu Ser Gly Lys Ile Glu
1475 1480 1485Leu Leu Pro Gln Val Ser Ile Gln Glu Glu Glu Ile Leu Pro Thr Glu1490 1495 1500Thr Ser His Gly Ser Pro Gly His Leu Asn Leu Met Lys Glu Val Phe1505 1510 1515 1520Leu Gln Lys Ile Gln Gly Pro Thr Lys Trp Asn Lys Ala Lys Arg His
1525 1530 1535Gly Glu Ser Ile Lys Gly Lys Thr Glu Ser Ser Lys Asn Thr Arg Ser
1540 1545 1550Lys Leu Leu Asn His His Ala Trp Asp Tyr His Tyr Ala Ala Gln Ile
1555 1560 1565Pro Lys Asp Met Trp Lys Ser Lys Glu Lys Ser Pro Glu Ile Ile Ser1570 1575 1580Ile Lys Gln Glu Asp Thr Ile Leu Ser Leu Arg Pro His Gly Asn Ser1585 1590 1595 1600His Ser Ile Gly Ala Asn Glu Lys Gln Asn Trp Pro Gln Arg Glu Thr
1605 1610 1615Thr Trp Val Lys Gln Gly Gln Thr Gln Arg Thr Cys Ser Gln Ile Pro
1620 1625 1630Pro Val Leu Lys Arg His Gln Arg Glu Leu Ser Ala Phe Gln Ser Glu
1635 1640 1645Gln Glu Ala Thr Asp Tyr Asp Asp Ala Ile Thr Ile Glu Thr Ile Glu1650 1655 1660Asp Phe Asp Ile Tyr Ser Glu Asp Ile Lys Gln Gly Pro Arg Ser Phe1665 1670 1675 1680Gln Gln Lys Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp
1685 1690 1695Asp Tyr Gly Met Ser Thr Ser His Val Leu Arg Asn Arg Tyr Gln Ser
1700 1705 1710Asp Asn Val Pro Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr Asp
1715 1720 1725Gly Ser Phe Ser Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His Leu1730 1735 1740Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met1745 1750 1755 1760Val Thr Phe Lys Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser
1765 1770 1775Leu Ile Ser Tyr Lys Glu Asp Gln Arg Gly Glu Glu Pro Arg Arg Asn
1780 1785 1790Phe Val Lys Pro Asn Glu Thr Lys Ile Tyr Phe Trp Lys Val Gln His
1795 1800 1805His Met Ala Pro Thr Glu Asp Glu Phe Asp Cys Lys Ala Trp Ala Tyr1810 1815 1820Phe Ser Asp Val Asp Leu Glu Arg Asp Met His Ser Gly Leu Ile Gly1825 1830 1835 1840Pro Leu Leu Ile Cys His Ala Asn Thr Leu Asn Pro Ala His Gly Arg
1845 1850 1855Gln Val Ser Val Gln Glu Phe Ala Leu Leu Phe Thr Ile Phe Asp Glu
1860 1865 1870Thr Lys Ser Trp Tyr Phe Thr Glu Asn Val Lys Arg Asn Cys Lys Thr
1875 1880 1885Pro Cys Asn Phe Gln Met Glu Asp Pro Thr Leu Lys Glu Asn Tyr Arg1890 1895 1900Phe His Ala Ile Asn Gly Tyr Val Met Asp Thr Leu Pro Gly Leu Val1905 1910 1915 1920Met Ala Gln Asp Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Asn
1925 1930 1935Asn Glu Asn Ile Gln Ser Ile His Phe Ser Gly His Val Phe Thr Val
1940 1945 1950Arg Lys Lys Glu Glu Tyr Lys Met Ala Val Tyr Asn Leu Tyr Pro Gly
1955 1960 1965Val Phe Glu Thr Leu Glu Met Ile Pro Ser Arg Ala Gly Ile Trp Arg
1970 1975 1980Val Glu Cys Leu Ile Gly Glu His Leu Gln Ala Gly Met Ser Thr Leu1985 1990 1995 2000Phe Leu Val Tyr Ser Lys Gln Cys Gln Ile Pro Leu Gly Met Ala Ser
2005 2010 2015Gly Ser Ile Arg Asp Phe Gln Ile Thr Ala Ser Gly His Tyr Gly Gln
2020 2025 2030Trp Ala Pro Asn Leu Ala Arg Leu His Tyr Ser Gly Ser Ile Asn Ala
2035 2040 2045Trp Ser Thr Lys Glu Pro Phe Ser Trp Ile Lys Val Asp Leu Leu Ala
2050 2055 2060Pro Met Ile Val His Gly Ile Lys Thr Gln Gly Ala Arg Gln Lys Phe2065 2070 2075 2080Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp Gly
2085 2090 2095Lys Lys Trp Leu Ser Tyr Gln Gly Asn Ser Thr Gly Thr Leu Met Val
2100 2105 2110Phe Phe Gly Asn Val Asp Ser Ser Gly Ile Lys His Asn Ser Phe Asn
2115 2120 2125Pro Pro Ile Ile Ala Arg Tyr Ile Arg Leu His Pro Thr His Ser Ser
2130 2135 2140Ile Arg Ser Thr Leu Arg Met Glu Leu Met Gly Cys Asp Leu Asn Ser2145 2150 2155 2160Cys Ser Ile Pro Leu Gly Met Glu Ser Lys Val Ile Ser Asp Thr Gln
2165 2170 2175Ile Thr Ala Ser Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser Pro
2180 2185 2190Ser Gln Ala Arg Leu His Leu Gln Gly Arg Thr Asn Ala Trp Arg Pro
2195 2200 2205Gln Val Asn Asp Pro Lys Gln Trp Leu Gln Val Asp Leu Gln Lys Thr2210 2215 2220Met Lys Val Thr Gly Ile Ile Thr Gln Gly Val Lys Ser Leu Phe Thr2225 2230 2235 2240Ser Met Phe Val Lys Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly His
2245 2250 2255His Trp Thr Gln Ile Leu Tyr Asn Gly Lys Val Lys Val Phe Gln Gly
2260 2265 2270Asn Gln Asp Ser Ser Thr Pro Met Met Asn Ser Leu Asp Pro Pro Leu
2275 2280 2285Leu Thr Arg Tyr Leu Arg Ile His Pro Gln Ile Trp Glu His Gln Ile2290 2295 2300Ala Leu Arg Leu Glu Ile Leu Gly Cys Glu Ala Gln Gln Gln Tyr2305 2310 2315<210>29<211>6402<212>DNA<213>猪(Porcine)<220><221>CDS<222>(1)..(6399)<400>29atg cag cta gag ctc tcc acc tgt gtc ttt ctg tgt ctc ttg cca ctc 48Met Gln Leu Glu Leu Ser Thr Cys Val Phe Leu Cys Leu Leu Pro Leu1 5 10 15ggc ttt agt gcc ate agg aga tac tac ctg ggc gca gtg gaa ctg tcc 96Gly Phe Ser Ala Ile Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser
20 25 30tgg gac tac cgg caa agt gaa ctc ctc cgt gag ctg cac gtg gac acc 144Trp Asp Tyr Arg Gln Ser Glu Leu Leu Arg Glu Leu His Val Asp Thr
35 40 45aga ttt cct gct aca gcg cca gga gct ctt ccg ttg ggc ccg tca gtc 192Arg Phe Pro Ala Thr Ala Pro Gly Ala Leu Pro Leu Gly Pro Ser Val
50 55 60ctg tac aaa aag act gtg ttc gta gag ttc acg gat caa ctt ttc agc 240Leu Tyr Lys Lys Thr Val Phe Val Glu Phe Thr Asp Gln Leu Phe Ser65 70 75 80gtt gcc agg ccc agg cca cca tgg atg ggt ctg ctg ggt cct acc atc 288Val Ala Arg Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile
85 90 95cag gct gag gtt tac gac acg gtg gtc gtt acc ctg aag aac atg gct 336Gln Ala Glu Val Tyr Asp Thr Val Val Val Thr Leu Lys Asn Met Ala
100 105 110tct cat ccc gtt agt ctt cac gct gtc ggc gtc tcc ttc tgg aaa tct 384Ser His Pro Val Ser Leu His Ala Val Gly Val Ser Phe Trp Lys Ser
115 120 125tcc gaa ggc gct gaa tat gag gat cac acc agc caa agg gag aag gaa 432Ser Glu Gly Ala Glu Tyr Glu Asp His Thr Ser Gln Arg Glu Lys Glu
130 135 140gac gat aaa gtc ctt ccc ggt aaa agc caa acc tac gtc tgg cag gtc 480Asp Asp Lys Val Leu Pro Gly Lys Ser Gln Thr Tyr Val Trp Gln Val145 150 155 160ctg aaa gaa aat ggt cca aca gcc tct gac cca cca tgt ctc acc tac 528Leu Lys Glu Asn Gly Pro Thr Ala Ser Asp Pro Pro Cys Leu Thr Tyr
165 170 175tca tac ctg tct cac gtg gac ctg gtg aaa gac ctg aat tcg ggc ctc 576Ser Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu
180 185 190att gga gcc ctg ctg gtt tgt aga gaa ggg agt ctg acc aga gaa agg 624Ile Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Thr Arg Glu Arg
195 200 205acc cag aac ctg cac gaa ttt gta cta ctt ttt gct gtc ttt gat gaa 672Thr Gln Asn Leu His Glu Phe Val Leu Leu Phe Ala Val Phe Asp Glu
210 215 220ggg aaa agt tgg cac tca gca aga aat gac tcc tgg aca cgg gcc atg 720Gly Lys Ser Trp His Ser Ala Arg Asn Asp Ser Trp Thr Arg Ala Met225 230 235 240gat ccc gca cct gcc agg gcc cag cct gca atg cac aca gtc aat ggc 768Asp Pro Ala Pro Ala Arg Ala Gln Pro Ala Met His Thr Val Asn Gly
245 250 255tat gtc aac agg tct ctg cca ggt ctg atc gga tgt cat aag aaa tca 816Tyr Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Lys Lys Ser
260 265 270gtc tac tgg cac gtg att gga atg ggc acc agc ccg gaa gtg cac tcc 864Val Tyr Trp His Val Ile Gly Met Gly Thr Ser Pro Glu Val His Ser
275 280 285att ttt ctt gaa ggc cac acg ttt ctc gtg agg cac cat cgc cag gct 912Ile Phe Leu Glu Gly His Thr Phe Leu Val Arg His His Arg Gln Ala
290 295 300tcc ttg gag atc tcg cca cta act ttc ctc act gct cag aca ttc ctg 960Ser Leu Glu Ile Ser Pro Leu Thr Phe Leu Thr Ala Gln Thr Phe Leu305 310 315 320atg gac ctt ggc cag ttc cta ctg ttt tgt cat atc tct tcc cac cac 1008Met Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His His
325 330 335cat ggt ggc atg gag gct cac gtc aga gta gaa agc tgc gcc gag gag 1056His Gly Gly Met Glu Ala His Val Arg Val Glu Ser Cys Ala Glu Glu
340 345 350ccc cag ctg cgg agg aaa gct gat gaa gag gaa gat tat gat gac aat 1104Pro Gln Leu Arg Arg Lys Ala Asp Glu Glu Glu Asp Tyr Asp Asp Asn
355 360 365ttg tac gac tcg gac atg gac gtg gtc cgg ctc gat ggt gac gac gtg 1152Leu Tyr Asp Ser Asp Met Asp Val Val Arg Leu Asp Gly Asp Asp Val
370 375 380tct ccc ttt atc caa atc cgc tcg gtt gcc aag aag cat ccc aaa acc 1200Ser Pro Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr385 390 395 400tgg gtg cac tac atc tct gca gag gag gag gac tgg gac tac gcc ccc 1248Trp Val His Tyr Ile Ser Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro
405 410 415gcg gtc ccc agc ccc agt gac aga agt tat aaa agt ctc tac ttg aac 1296Ala Val Pro Ser Pro Ser Asp Arg Ser Tyr Lys Ser Leu Tyr Leu Asn
420 425 430agt ggt cct cag cga att ggt agg aaa tac aaa aaa gct cga ttc gtc 1344Ser Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Ala Arg Phe Val
435 440 445gct tac acg gat gta aca ttt aag act cgt aaa gct att ccg tat gaa 1392Ala Tyr Thr Asp Val Thr Phe Lys Thr Arg Lys Ala Ile Pro Tyr Glu
450 455 460tca gga atc ctg gga cct tta ctt tat gga gaa gtt gga gac aca ctt 1440Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu465 470 475 480ttg att ata ttt aag aat aaa gcg agc cga cca tat aac atc tac cct 1488Leu Ile Ile Phe Lys Asn Lys Ala Ser Arg Pro Tyr Asn Ile Tyr Pro
485 490 495cat gga atc act gat gtc agc gct ttg cac cca ggg aga ctt cta aaa 1536His Gly Ile Thr Asp Val Ser Ala Leu His Pro Gly Arg Leu Leu Lys
500 505 510ggt tgg aaa cat ttg aaa gac atg cca att ctg cca gga gag act ttc 1584Gly Trp Lys His Leu Lys Asp Met Pro Ile Leu Pro Gly Glu Thr Phe
515 520 525aag tat aaa tgg aca gtg act gtg gaa gat ggg cca acc aag tcc gat 1632Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp
530 535 540cct cgg tgc ctg acc cgc tac tac tcg agc tcc att aat cta gag aaa 1680Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Ser Ile Asn Leu Glu Lys545 550 555 560gat ctg gct tcg gga ctc att ggc cct ctc ctc atc tgc tac aaa gaa 1728Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu
565 570 575tct gta gac caa aga gga aac cag atg atg tca gac aag aga aac gtc 1776Ser Val Asp Gln Arg Gly Asn Gln Met Met Ser Asp Lys Arg Asn Val
580 585 590atc ctg ttt tct gta ttc gat gag aat caa agc tgg tac ctc gca gag 1824Ile Leu Phe Ser Val Phe Asp Glu Asn Gln Ser Trp Tyr Leu Ala Glu
595 600 605aat att cag cgc ttc ctc ccc aat ccg gat gga tta cag ccc cag gat 1872Asn Ile Gln Arg Phe Leu Pro Asn Pro Asp Gly Leu Gln Pro Gln Asp
610 615 620cca gag ttc caa gct tct aac atc atg cac agc atc aat ggc tat gtt 1920Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val625 630 635 640ttt gat agc ttg cag ctg tcg gtt tgt ttg cac gag gtg gca tac tgg 1968Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp
645 650 655tac att cta agt gtt gga gca cag acg gac ttc ctc tcc gtc ttc ttc 2016Tyr Ile Leu Ser Val Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe
660 665 670tct ggc tac acc ttc aaa cac aaa atg gtc tat gaa gac aca ctc acc 2064Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr
675 680 685ctg ttc ccc ttc tca gga gaa acg gtc ttc atg tca atg gaa aac cca 2112Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro
690 695 700ggt ctc tgg gtc cta ggg tgc cac aac tca gac ttg cgg aac aga ggg 2160Gly Leu Trp Val Leu Gly Cys His Asn Ser Asp Leu Arg Asn Arg Gly705 710 715 720atg aca gcc tta ctg aag gtg tat agt tgt gac agg gac att ggt gat 2208Met Thr Ala Leu Leu Lys Val Tyr Ser Cys Asp Arg Asp Ile Gly Asp
725 730 735tat tat gac aac act tat gaa gat att cca ggc ttc ttg ctg agt gga 2256Tyr Tyr Asp Asn Thr Tyr Glu Asp Ile Pro Gly Phe Leu Leu Ser Gly
740 745 750aag aat gtc att gaa ccc aga agc ttt gcc cag aat tca aga ccc cct 2304Lys Asn Val Ile Glu Pro Arg Ser Phe Ala Gln Asn Ser Arg Pro Pro
755 760 765agt gcg agc caa aag caa ttc caa acc atc aca agt cca gaa gat gac 2352Ser Ala Ser Gln Lys Gln Phe Gln Thr Ile Thr Ser Pro Glu Asp Asp
770 775 780gtg gag ctt gac ccg cag tct gga gag aga acc caa gca ctg gaa gaa 2400Val Glu Leu Asp Pro Gln Ser Gly Glu Arg Thr Gln Ala Leu Glu Glu785 790 795 800cta agt gtc ccc tct ggt gat ggg tcg atg ctc ttg gga cag aat cct 2448Leu Ser Val Pro Ser Gly Asp Gly Ser Met Leu Leu Gly Gln Asn Pro
805 810 815gct cca cat ggc tca tcc tca tct gat ctt caa gaa gcc agg aat gag 2496Ala Pro His Gly Ser Ser Ser Ser Asp Leu Gln Glu Ala Arg Asn Glu
820 825 830gct gat gat tat tta cct gga gca aga gaa aga ggc acg gcc cca tcc 2544Ala Asp Asp Tyr Leu Pro Gly Ala Arg Glu Arg Gly Thr Ala Pro Ser
835 840 845gca gcg gca cgt ctc aga cca gag ctg cat cac agt gcc gaa aga gta 2592Ala Ala Ala Arg Leu Arg Pro Glu Leu His His Ser Ala Glu Arg Val
850 855 860ctt act cct gag cca gag aaa gag ttg aag aaa ctt gat tca aaa atg 2640Leu Thr Pro Glu Pro Glu Lys Glu Leu Lys Lys Leu Asp Ser Lys Met865 870 875 880tct agt tca tca gac ctt cta aag act tcg cca aca att cca tca gac 2688Ser Ser Ser Ser Asp Leu Leu Lys Thr Ser Pro Thr Ile Pro Ser Asp
885 890 895acg ttg tca gcg gag act gaa agg aca cat tcc tta ggc ccc cca cac 2736Thr Leu Ser Ala Glu Thr Glu Arg Thr His Ser Leu Gly Pro Pro His
900 905 910ccg cag gtt aat ttc agg agt caa tta ggt gcc att gta ctt ggc aaa 2784Pro Gln Val Asn Phe Arg Ser Gln Leu Gly Ala Ile Val Leu Gly Lys
915 920 925aat tca tct cac ttt att ggg gct ggt gtc cct ttg ggc tcg act gag 2832Asn Ser Ser His Phe Ile Gly Ala GIy Val Pro Leu Gly Ser Thr Glu
930 935 940gag gat cat gaa agc tcc ctg gga gaa aat gta tca cca gtg gag agt 2880Glu Asp His Glu Ser Ser Leu Gly Glu Asn Val Ser Pro Val Glu Ser945 950 955 960gac ggg ata ttt gaa aag gaa aga gct cat gga cct gct tca ctg acc 2928Asp Gly Ile Phe Glu Lys Glu Arg Ala His Gly Pro Ala Ser Leu Thr
965 970 975aaa gac gat gtt tta ttt aaa gtt aat atc tct ttg gta aag aca aac 2976Lys Asp Asp Val Leu Phe Lys Val Asn Ile Ser Leu Val Lys Thr Asn
980 985 990aag gca cga gtt tac tta aaa act aat aga aag att cac att gat gac 3024Lys Ala Arg Val Tyr Leu Lys Thr Asn Arg Lys Ile His Ile Asp Asp
995 1000 1005gca gct tta tta act gag aat agg gca tct gca acg ttt atg gac aaa 3072Ala Ala Leu Leu Thr Glu Asn Arg Ala Ser Ala Thr Phe Met Asp Lys
1010 1015 1020aat act aca gct tcg gga tta aat cat gtg tca aat tgg ata aaa ggg 3120Asn Thr Thr Ala Ser Gly Leu Asn His Val Ser Asn Trp Ile Lys Gly1025 1030 1035 1040ccc ctt ggc aag aac ccc cta agc tcg gag cga ggc ccc agt cca gag 3168Pro Leu Gly Lys Asn Pro Leu Ser Ser Glu Arg Gly Pro Ser Pro Glu
1045 1050 1055ctt ctg aca tct tca gga tca gga aaa tct gtg aaa ggt cag agt tct 3216Leu Leu Thr Ser Ser Gly Ser Gly Lys Ser Val Lys Gly Gln Ser Ser
1060 1065 1070ggg cag ggg aga ata cgg gtg gca gtg gaa gag gaa gaa ctg agc aaa 3264Gly Gln Gly Arg Ile Arg Val Ala Val Glu Glu Glu Glu Leu Ser Lys
1075 1080 1085ggc aaa gag atg atg ctt ccc aac agc gag ctc acc ttt ctc act aac 3312Gly Lys Glu Met Met Leu Pro Asn Ser Glu Leu Thr Phe Leu Thr Asn 1090 1095 1100tcg gct gat gtc caa gga aac gat aca cac agt caa gga aaa aag tct 3360Ser Ala Asp Val Gln Gly Asn Asp Thr His Ser Gln Gly Lys Lys Ser1105 1110 1115 1120cgg gaa gag atg gaa agg aga gaa aaa tta gtc caa gaa aaa gtc gac 3408Arg Glu Glu Met Glu Arg Arg Glu Lys Leu Val Gln Glu Lys Val Asp
1125 1130 1135ttg cct cag gtg tat aca gcg act gga act aag aat ttc ctg aga aac 3456Leu Pro Gln Val Tyr Thr Ala Thr Gly Thr Lys Asn Phe Leu Arg Asn
1140 1145 1150att ttt cac caa agc act gag ccc agt gta gaa ggg ttt gat ggg ggg 3504Ile Phe His Gln Ser Thr Glu Pro Ser Val Glu Gly Phe Asp Gly Gly
1155 1160 1165tca cat gcg ccg gtg cct caa gac agc agg tca tta aat gat tcg gca 3552Ser His Ala Pro Val Pro Gln Asp Ser Arg Ser Leu Asn Asp Scr Ala1170 1175 1180gag aga gca gag act cac ata gcc cat ttc tca gca att agg gaa gag 3600Glu Arg Ala Glu Thr His Ile Ala His Phe Ser Ala Ile Arg Glu Glu1185 1190 1195 1200gca ccc ttg gaa gcc ccg gga aat cga aca ggt cca ggt ccg agg agt 3648Ala Pro Leu Glu Ala Pro Gly Asn Arg Thr Gly Pro Gly Pro Arg Ser
1205 1210 1215gcg gtt ccc cgc cgc gtt aag cag agc ttg aaa cag atc aga ctc ccg 3696Ala Val Pro Arg Arg Val Lys Gln Ser Leu Lys Gln Ile Arg Leu Pro
1220 1225 1230cta gaa gaa ata aag cct gaa agg ggg gtg gtt ctg aat gcc acc tca 3744Leu Glu Glu Ile Lys Pro Glu Arg Gly Val Val Leu Asn Ala Thr Ser
1235 1240 1245acc cgg tgg tct gaa agc agt cct atc tta caa gga gcc aaa aga aat 3792Thr Arg Trp Ser Glu Ser Ser Pro Ile Leu Gln Gly Ala Lys Arg Asn1250 1255 1260aac ctt tct tta cct ttc ctg acc ttg gaa atg gcc gga ggt caa gga 3840Asn Leu Ser Leu Pro Phe Leu Thr Leu Glu Met Ala Gly Gly Gln Gly1265 1270 1275 1280aag atc agc gcc ctg ggg aaa agt gcc gca ggc ccg ctg gcg tcc ggg 3888Lys Ile Ser Ala Leu Gly Lys Ser Ala Ala Gly Pro Leu Ala Ser GIy
1285 1290 1295aag ctg gag aag gct gtt ctc tct tca gca ggc ttg tct gaa gca tct 3936Lys Leu Glu Lys Ala Val Leu Ser Ser Ala Gly Leu Ser Glu Ala Ser
1300 1305 1310ggc aaa gct gag ttt ctt cct aaa gtt cga gtt cat cgg gaa gac ctg 3984Gly Lys Ala Glu Phe Leu Pro Lys Val Arg Val His Arg Glu Asp Leu
1315 1320 1325ttg cct caa aaa acc agc aat gtt tct tgc gca cac ggg gat ctc ggc 4032Leu Pro Gln Lys Thr Ser Asn Val Ser Cys Ala His Gly Asp Leu Gly1330 1335 1340cag gag atc ttc ctg cag aaa aca cgg gga cct gtt aac ctg aac aaa 4080Gln Glu Ile Phe Leu Gln Lys Thr Arg Gly Pro Val Asn Leu Asn Lys1345 1350 1355 1360gta aat aga cct gga agg act ccc tcc aag ctt ctg ggt ccc ccg atg 4128Val Asn Arg Pro Gly Arg Thr Pro Ser Lys Leu Leu Gly Pro Pro Met
1365 1370 1375ccc aaa gag tgg gaa tcc cta gag aag tca cca aaa agc aca gct ctc 4176Pro Lys Glu Trp Glu Ser Leu Glu Lys Ser Pro Lys Ser Thr Ala Leu
1380 1385 1390agg acg aaa gac atc atc agt tta ccc ctg gac cgt cac gaa agc aat 4224Arg Thr Lys Asp Ile Ile Ser Leu Pro Leu Asp Arg His Glu Ser Asn
1395 1400 1405cat tca ata gca gca aaa aat gaa gga caa gcc gag acc caa aga gaa 4272His Ser Ile Ala Ala Lys Asn Glu Gly Gln Ala Glu Thr Gln Arg Glu1410 1415 1420gcc gcc tgg acg aag cag gga ggg cct gga agg ctg tgc gct cca aag 4320Ala Ala Trp Thr Lys Gln Gly Gly Pro Gly Arg Leu Cys Ala Pro Lys1425 1430 1435 1440cct ccg gtc ctg cga cgg cat cag agg gac ata agc ctt cct act ttt 4368Pro Pro Val Leu Arg Arg His Gln Arg Asp Ile Ser Leu Pro Thr Phe
1445 1450 1455cag ccg gag gaa gac aaa atg gac tat gat gat atc ttc tca act gaa 4416Gln Pro Glu Glu Asp Lys Met Asp Tyr Asp Asp Ile Phe Ser Thr Glu
1460 1465 1470acg aag gga gaa gat ttt gac att tac ggt gag gat gaa aat cag gac 4464Thr Lys Gly Glu Asp Phe Asp Ile Tyr Gly Glu Asp Glu Asn Gln Asp
1475 1480 1485cct cgc agc ttt cag aag aga acc cga cac tat ttc att gct gcg gtg 4512Pro Arg Ser Phe Gln Lys Arg Thr Arg His Tyr Phe Ile Ala Ala Val
1490 1495 1500gag cag ctc tgg gat tac ggg atg agc gaa tcc ccc cgg gcg cta aga 4560Glu Gln Leu Trp Asp Tyr Gly Met Ser Glu Ser Pro Arg Ala Leu Arg1505 1510 1515 1520aac agg gct cag aac gga gag gtg cct cgg ttc aag aag gtg gtc ttc 4608Asn Arg Ala Gln Asn Gly Glu Val Pro Arg Phe Lys Lys Val Val Phe
1525 1530 1535cgg gaa ttt gct gac ggc tcc ttc acg cag ccg tcg tac cgc ggg gaa 4656Arg Glu Phe Ala Asp Gly Ser Phe Thr Gln Pro Ser Tyr Arg Gly Glu
1540 1545 1550ctc aac aaa cac ttg ggg ctc ttg gga ccc tac atc aga gcg gaa gtt 4704Leu Asn Lys His Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val
1555 1560 1565gaa gac aac atc atg gta act ttc aaa aac cag gcg tct cgt ccc tat 4752Glu Asp Asn Ile Met Val Thr Phe Lys Asn Gln Ala Ser Arg Pro Tyr1570 1575 1580tcc ttc tac tcg agc ctt att tct tat ccg gat gat cag gag caa ggg 4800Ser Phe Tyr Ser Ser Leu Ile Ser Tyr Pro Asp Asp Gln Glu Gln Gly1585 1590 1595 1600gca gaa cct cga cac aac ttc gtc cag cca aat gaa acc aga act tac 4848Ala Glu Pro Arg His Asn Phe Val Gln Pro Asn Glu Thr Arg Thr Tyr
1605 1610 1615ttt tgg aaa gtg cag cat cac atg gca ccc aca gaa gac gag ttt gac 4896Phe Trp Lys Val Gln His His Met Ala Pro Thr Glu Asp Glu Phe Asp
1620 1625 1630tgc aaa gcc tgg gcc tac ttt tct gat gtt gac ctg gaa aaa gat gtg 4944Cys Lys Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val
1635 1640 1645cac tca ggc ttg atc ggc ccc ctt ctg atc tgc cgc gcc aac acc ctg 4992His Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Arg Ala Asn Thr Leu1650 1655 1660aac gct gct cac ggt aga caa gtg acc gtg caa gaa ttt gct ctg ttt 5040Asn Ala Ala His Gly Arg Gln Val Thr Val Gln Glu Phe Ala Leu Phe1665 1670 1675 1680ttc act att ttt gat gag aca aag agc tgg tac ttc act gaa aat gtg 5088Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu Asn Val
1685 1690 1695gaa agg aac tgc cgg gcc ccc tgc cac ctg cag atg gag gac ccc act 5136Glu Arg Asn Cys Arg Ala Pro Cys His Leu Gln Met Glu Asp Pro Thr
1700 1705 1710ctg aaa gaa aac tat cgc ttc cat gca atc aat ggc tat gtg atg gat 5184Leu Lys Glu Asn Tyr Arg Phe His Ala Ile Asn Gly Tyr Val Met Asp
1715 1720 1725aca ctc cct ggc tta gta atg gct cag aat caa agg atc cga tgg tat 5232Thr Leu Pro Gly Leu Val Met Ala Gln Asn Gln Arg Ile Arg Trp Tyr1730 1735 1740ctg ctc agc atg ggc agc aat gaa aat atc cat tcg att cat ttt agc 5280Leu Leu Ser Met Gly Ser Asn Glu Asn Ile His Ser Ile His Phe Ser1745 1750 1755 1760gga cac gtg ttc agt gta cgg aaa aag gag gag tat aaa atg gcc gtg 5328Gly His Val Phe Ser Val Arg Lys Lys Glu Glu Tyr Lys Met Ala Val
1765 1770 1775tac aat ctc tat ccg ggt gtc ttt gag aca gtg gaa atg cta ccg tcc 5376Tyr Asn Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met Leu Pro Ser
1780 1785 1790aaa gtt gga att tgg cga ata gaa tgc ctg att ggc gag cac ctg caa 5424Lys Val Gly Ile Trp Arg Ile Glu Cys Leu Ile Gly Glu His Leu Gln
1795 1800 1805gct ggg atg agc acg act ttc ctg gtg tac agc aag gag tgt cag gct 5472Ala Gly Met Ser Thr Thr Phe Leu Val Tyr Ser Lys Glu Cys Gln Ala
1810 1815 1820cca ctg gga atg gct tct gga cgc att aga gat ttt cag atc aca gct 5520Pro Leu Gly Met Ala Ser Gly Arg Ile Arg Asp Phe Gln Ile Thr Ala1825 1830 1835 1840tca gga cag tat gga cag tgg gcc cca aag ctg gcc aga ctt cat tat 5568Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His Tyr
1845 1850 1855tcc gga tca atc aat gcc tgg agc acc aag gat ccc cac tcc tgg atc 5616Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys Asp Pro His Ser Trp Ile
1860 1865 1870aag gtg gat ctg ttg gca cca atg atc att cac ggc atc atg acc cag 5664Lys Val Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile Met Thr Gln
1875 1880 1885ggt gcc cgt cag aag ttt tcc agc ctc tac atc tcc cag ttt atc atc 5712Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile1890 1895 1900atg tac agt ctt gac ggg agg aac tgg cag agt tac cga ggg aat tcc 5760Met Tyr Ser Leu Asp Gly Arg Asn Trp Gln Ser Tyr Arg Gly Asn Ser1905 1910 1915 1920acg ggc acc tta atg gtc ttc ttt ggc aat gtg gac gca tct ggg att 5808Thr Gly Thr Leu Met Val Phe Phe Gly Asn Val Asp Ala Ser Gly Ile
1925 1930 1935aaa cac aat att ttt aac cct ccg att gtg gct cgg tac atc cgt ttg 5856Lys His Asn Ile Phe Asn Pro Pro Ile Val Ala Arg Tyr Ile Arg Leu
1940 1945 1950cac cca aca cat tac agc atc cgc agc act ctt cgc atg gag ttg atg 5904His Pro Thr His Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu Leu Met
1955 1960 1965ggc tgt gat tta aac agt tgc agc atg ccc ctg gga atg cag aat aaa 5952Gly Cys Asp Leu Asn Ser Cys Ser Met Pro Leu Gly Met Gln Asn Lys1970 1975 1980gcg ata tca gac tca cag atc acg gcc tcc tcc cac cta agc aat ata 6000Ala Ile Ser Asp Ser Gln Ile Thr Ala Ser Ser His Leu Ser Asn Ile1985 1990 1995 2000ttt gcc acc tgg tct cct tca caa gcc cga ctt cac ctc cag ggg cgg 6048Phe Ala Thr Trp Ser Pro Ser Gln Ala Arg Leu His Leu Gln Gly Arg
2005 2010 2015acg aat gcc tgg cga ccc cgg gtg agc agc gca gag gag tgg ctg cag 6096Thr Asn Ala Trp Arg Pro Arg Val Ser Ser Ala Glu Glu Trp Leu Gln
2020 2025 2030gtg gac ctg cag aag acg gtg aag gtc aca ggc atc acc acc cag ggc 6144Val Asp Leu Gln Lys Thr Val Lys Val Thr Gly Ile Thr Thr Gln Gly
2035 2040 2045gtg aag tcc ctg ctc agc agc atg tat gtg aag gag ttc ctc gtg tcc 6192Val Lys Ser Leu Leu Ser Ser Met Tyr Val Lys Glu Phe Leu Val Ser2050 2055 2060agt agt cag gac ggc cgc cgc tgg acc ctg ttt ctt cag gac ggc cac 6240Ser Ser Gln Asp Gly Arg Arg Trp Thr Leu Phe Leu Gln Asp Gly His2065 2070 2075 2080acg aag gtt ttt cag ggc aat cag gac tcc tcc acc ccc gtg gtg aac 6288Thr Lys Val Phe Gln Gly Asn Gln Asp Ser Ser Thr Pro Val Val Asn
2085 2090 2095gct ctg gac ccc ccg ctg ttc acg cgc tac ctg agg atc cac ccc acg 6336Ala Leu Asp Pro Pro Leu Phe Thr Arg Tyr Leu Arg Ile His Pro Thr
2100 2105 2110agc tgg gcg cag cac atc gcc ctg agg ctc gag gtt cta gga tgt gag 6384Ser Trp Ala Gln His Ile Ala Leu Arg Leu Glu Val Leu Gly Cys Glu
2115 2120 2125gca cag gat ctc tac tga 6402Ala Gln Asp Leu Tyr
2130<210>30<211>2133<212>PRT<213>猪(Porcine)<400>30Met Gln Leu Glu Leu Ser Thr Cys Val Phe Leu Cys Leu Leu Pro Leu1 5 10 15Gly Phe Ser Ala Ile Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser
20 25 30Trp Asp Tyr Arg Gln Ser Glu Leu Leu Arg Glu Leu His Val Asp Thr
35 40 45Arg Phe Pro Ala Thr Ala Pro Gly Ala Leu Pro Leu Gly Pro Ser Val
50 55 60Leu Tyr Lys Lys Thr Val Phe Val Glu Phe Thr Asp Gln Leu Phe Ser65 70 75 80Val Ala Arg Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile
85 90 95Gln Ala Glu Val Tyr Asp Thr Val Val Val Thr Leu Lys Asn Met Ala
100 105 110Ser His Pro Val Ser Leu His Ala Val Gly Val Ser Phe Trp Lys Ser
115 ` 120 125Ser Glu Gly Ala Glu Tyr Glu Asp His Thr Ser Gln Arg Glu Lys Glu
130 135 140Asp Asp Lys Val Leu Pro Gly Lys Ser Gln Thr Tyr Val Trp Gln Val145 150 155 160Leu Lys Glu Asn Gly Pro Thr Ala Ser Asp Pro Pro Cys Leu Thr Tyr
165 170 175Ser Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu
180 185 190Ile Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Thr Arg Glu Arg
195 200 205Thr Gln Asn Leu His Glu Phe Val Leu Leu Phe Ala Val Phe Asp Glu
210 215 220Gly Lys Ser Trp His Ser Ala Arg Asn Asp Ser Trp Thr Arg Ala Met225 230 235 240Asp Pro Ala Pro Ala Arg Ala Gln Pro Ala Met His Thr Val Asn Gly
245 250 255Tyr Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Lys Lys Ser
260 265 270Val Tyr Trp His Val Ile Gly Met Gly Thr Ser Pro Glu Val His Ser
275 280 285Ile Phe Leu Glu Gly His Thr Phe Leu Val Arg His His Arg Gln Ala
290 295 300Ser Leu Glu Ile Ser Pro Leu Thr Phe Leu Thr Ala Gln Thr Phe Leu305 310 315 320Met Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His His
325 330 335His Gly Gly Met Glu Ala His Val Arg Val Glu Ser Cys Ala Glu Glu
340 345 350Pro Gln Leu Arg Arg Lys Ala Asp Glu Glu Glu Asp Tyr Asp Asp Asn
355 360 365Leu Tyr Asp Ser Asp Met Asp Val Val Arg Leu Asp Gly Asp Asp Val
370 375 380Ser Pro Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr385 390 395 400Trp Val His Tyr Ile Ser Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro
405 410 415Ala Val Pro Ser Pro Ser Asp Arg Ser Tyr Lys Ser Leu Tyr Leu Asn
420 425 430Ser Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Ala Arg Phe Val
435 440 445Ala Tyr Thr Asp Val Thr Phe Lys Thr Arg Lys Ala Ile Pro Tyr Glu
450 455 460Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu465 470 475 480Leu Ile Ile Phe Lys Asn Lys Ala Ser Arg Pro Tyr Asn Ile Tyr Pro
485 490 495His Gly Ile Thr Asp Val Ser Ala Leu His Pro Gly Arg Leu Leu Lys
500 505 510Gly Trp Lys His Leu Lys Asp Met Pro Ile Leu Pro Gly Glu Thr Phe
515 520 525Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp
530 535 540Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Ser Ile Asn Leu Glu Lys545 550 555 560Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu
565 570 575Ser Val Asp Gln Arg Gly Asn Gln Met Met Ser Asp Lys Arg Asn Val
580 585 590Ile Leu Phe Ser Val Phe Asp Glu Asn Gln Ser Trp Tyr Leu Ala Glu
595 600 605Asn Ile Gln Arg Phe Leu Pro Asn Pro Asp Gly Leu Gln Pro Gln Asp
610 615 620Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val625 630 635 640Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp
645 650 655Tyr Ile Leu Ser Val Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe
660 665 670Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr
675 680 685Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro
690 695 700Gly Leu Trp Val Leu Gly Cys His Asn Ser Asp Leu Arg Asn Arg Gly705 710 715 720Met Thr Ala Leu Leu Lys Val Tyr Ser Cys Asp Arg Asp Ile Gly Asp
725 730 735Tyr Tyr Asp Asn Thr Tyr Glu Asp Ile Pro Gly Phe Leu Leu Ser Gly
740 745 750Lys Asn Val Ile Glu Pro Arg Ser Phe Ala Gln Asn Ser Arg Pro Pro
755 760 765Ser Ala Ser Gln Lys Gln Phe Gln Thr Ile Thr Ser Pro Glu Asp Asp
770 775 780Val Glu Leu Asp Pro Gln Ser Gly Glu Arg Thr Gln Ala Leu Glu Glu785 790 795 800Leu Ser Val Pro Ser Gly Asp Gly Ser Met Leu Leu Gly Gln Asn Pro
805 810 815Ala Pro His Gly Ser Ser Ser Ser Asp Leu Gln Glu Ala Arg Asn Glu
820 825 830Ala Asp Asp Tyr Leu Pro Gly Ala Arg Glu Arg Gly Thr Ala Pro Ser
835 840 845Ala Ala Ala Arg Leu Arg Pro Glu Leu His His Ser Ala Glu Arg Val
850 855 860Leu Thr Pro Glu Pro Glu Lys Glu Leu Lys Lys Leu Asp Ser Lys Met865 870 875 880Ser Ser Ser Ser Asp Leu Leu Lys Thr Ser Pro Thr Ile Pro Ser Asp
885 890 895Thr Leu Ser Ala Glu Thr Glu Arg Thr His Ser Leu Gly Pro Pro His
900 905 910Pro Gln Val Asn Phe Arg Ser Gln Leu Gly Ala Ile Val Leu Gly Lys
915 920 925Asn Ser Ser His Phe Ile Gly Ala Gly Val Pro Leu Gly Ser Thr Glu
930 935 940Glu Asp His Glu Ser Ser Leu Gly Glu Asn Val Ser Pro Val Glu Ser945 950 955 960Asp Gly Ile Phe Glu Lys Glu Arg Ala His Gly Pro Ala Ser Leu Thr
965 970 975Lys Asp Asp Val Leu Phe Lys Val Asn Ile Ser Leu Val Lys Thr Asn
980 985 990Lys Ala Arg Val Tyr Leu Lys Thr Asn Arg Lys Ile His Ile Asp Asp
995 1000 1005Ala Ala Leu Leu Thr Glu Asn Arg Ala Ser Ala Thr Phe Met Asp Lys
1010 1015 1020Asn Thr Thr Ala Ser Gly Leu Asn His Val Ser Asn Trp Ile Lys Gly1025 1030 1035 1040Pro Leu Gly Lys Asn Pro Leu Ser Ser Glu Arg Gly Pro Ser Pro Glu
1045 1050 1055Leu Leu Thr Ser Ser Gly Ser Gly Lys Ser Val Lys Gly Gln Ser Ser
1060 1065 1070Gly Gln Gly Arg Ile Arg Val Ala Val Glu Glu Glu Glu Leu Ser Lys
1075 1080 1085Gly Lys Glu Met Met Leu Pro Asn Ser Glu Leu Thr Phe Leu Thr Asn1090 1095 1100Ser Ala Asp Val Gln Gly Asn Asp Thr His Ser Gln Gly Lys Lys Ser1105 lll0 lll5 1120Arg Glu Glu Met Glu Arg Arg Glu Lys Leu Val Gln Glu Lys Val Asp
1125 1130 1135Leu Pro Gln Val Tyr Thr Ala Thr Gly Thr Lys Asn Phe Leu Arg Asn
1140 1145 1150Ile Phe His Gln Ser Thr Glu Pro Ser Val Glu Gly Phe Asp Gly Gly
1155 1160 1165Ser His Ala Pro Val Pro Gln Asp Ser Arg Ser Leu Asn Asp Ser Ala1170 1175 1180Glu Arg Ala Glu Thr His Ile Ala His Phe Ser Ala Ile Arg Glu Glu1185 1190 1195 1200Ala Pro Leu Glu Ala Pro Gly Asn Arg Thr Gly Pro Gly Pro Arg Ser
1205 1210 1215Ala Val Pro Arg Arg Val Lys Gln Ser Leu Lys Gln Ile Arg Leu Pro
1220 1225 1230Leu Glu Glu Ile Lys Pro Glu Arg Gly Val Val Leu Asn Ala Thr Ser
1235 1240 1245Thr Arg Trp Ser Glu Ser Ser Pro Ile Leu Gln Gly Ala Lys Arg Asn1250 1255 1260Asn Leu Ser Leu Pro Phe Leu Thr Leu Glu Met Ala Gly Gly Gln Gly1265 1270 1275 1280Lys Ile Ser Ala Leu Gly Lys Ser Ala Ala Gly Pro Leu Ala Ser Gly
1285 1290 1295Lys Leu Glu Lys Ala Val Leu Ser Ser Ala Gly Leu Ser Glu Ala Ser
1300 1305 1310Gly Lys Ala Glu Phe Leu Pro Lys Val Arg Val His Arg Glu Asp Leu
1315 1320 1325Leu Pro Gln Lys Thr Ser Asn Val Ser Cys Ala His Gly Asp Leu Gly1330 1335 1340Gln Glu Ile Phe Leu Gln Lys Thr Arg Gly Pro Val Asn Leu Asn Lys1345 1350 1355 1360Val Asn Arg Pro Gly Arg Thr Pro Ser Lys Leu Leu Gly Pro Pro Met
1365 1370 1375Pro Lys Glu Trp Glu Ser Leu Glu Lys Ser Pro Lys Ser Thr Ala Leu
1380 1385 1390Arg Thr Lys Asp Ile Ile Ser Leu Pro Leu Asp Arg His Glu Ser Asn
1395 1400 1405His Ser Ile Ala Ala Lys Asn Glu Gly Gln Ala Glu Thr Gln Arg Glu1410 1415 1420Ala Ala Trp Thr Lys Gln Gly Gly Pro Gly Arg Leu Cys Ala Pro Lys1425 1430 1435 1440Pro Pro Val Leu Arg Arg His Gln Arg Asp Ile Ser Leu Pro Thr Phe
1445 1450 1455Gln Pro Glu Glu Asp Lys Met Asp Tyr Asp Asp Ile Phe Ser Thr Glu
1460 1465 1470Thr Lys Gly Glu Asp Phe Asp Ile Tyr Gly Glu Asp Glu Asn Gln Asp
1475 1480 1485Pro Arg Ser Phe Gln Lys Arg Thr Arg His Tyr Phe Ile Ala Ala Val1490 1495 1500Glu Gln Leu Trp Asp Tyr Gly Met Ser Glu Ser Pro Arg Ala Leu Arg1505 1510 1515 1520Asn Arg Ala Gln Asn Gly Glu Val Pro Arg Phe Lys Lys Val Val Phe
1525 1530 1535Arg Glu Phe Ala Asp Gly Ser Phe Thr Gln Pro Ser Tyr Arg Gly Glu
1540 1545 1550Leu Asn Lys His Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val
1555 1560 1565Glu Asp Asn Ile Met Val Thr Phe Lys Asn Gln Ala Ser Arg Pro Tyr1570 1575 1580Ser Phe Tyr Ser Ser Leu Ile Ser Tyr Pro Asp Asp Gln Glu Gln Gly1585 1590 1595 1600Ala Glu Pro Arg His Asn Phe Val Gln Pro Asn Glu Thr Arg Thr Tyr
1605 1610 1615Phe Trp Lys Val Gln His His Met Ala Pro Thr Glu Asp Glu Phe Asp
1620 1625 1630Cys Lys Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val
1635 1640 1645His Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Arg Ala Asn Thr Leu1650 1655 1660Asn Ala Ala His Gly Arg Gln Val Thr Val Gln Glu Phe Ala Leu Phe1665 1670 1675 1680Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu Asn Val
1685 1690 1695Glu Arg Asn Cys Arg Ala Pro Cys His Leu Gln Met Glu Asp Pro Thr
1700 1705 1710Leu Lys Glu Asn Tyr Arg Phe His Ala Ile Asn Gly Tyr Val Met Asp
1715 1720 1725Thr Leu Pro Gly Leu Val Met Ala Gln Asn Gln Arg Ile Arg Trp Tyr1730 1735 1740Leu Leu Ser Met Gly Ser Asn Glu Asn Ile His Ser Ile His Phe Ser1745 1750 1755 1760Gly His Val Phe Ser Val Arg Lys Lys Glu Glu Tyr Lys Met Ala Val
1765 1770 1775Tyr Asn Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met Leu Pro Ser
1780 1785 1790Lys Val Gly Ile Trp Arg Ile Glu Cys Leu Ile Gly Glu His Leu Gln
1795 1800 1805Ala Gly Met Ser Thr Thr Phe Leu Val Tyr Ser Lys Glu Cys Gln Ala1810 1815 1820Pro Leu Gly Met Ala Ser Gly Arg Ile Arg Asp Phe Gln Ile Thr Ala1825 1830 1835 1840Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His Tyr
1845 1850 1855Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys Asp Pro His Ser Trp Ile
1860 1865 1870Lys Val Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile Met Thr Gln
1875 1880 1885Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile1890 1895 1900Met Tyr Ser Leu Asp Gly Arg Asn Trp Gln Ser Tyr Arg Gly Asn Ser1905 1910 1915 1920Thr Gly Thr Leu Met Val Phe Phe Gly Asn Val Asp Ala Ser Gly Ile
1925 1930 1935Lys His Asn Ile Phe Asn Pro Pro Ile Val Ala Arg Tyr Ile Arg Leu
1940 1945 1950His Pro Thr His Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu Leu Met
1955 1960 1965Gly Cys Asp Leu Asn Ser Cys Ser Met Pro Leu Gly Met Gln Asn Lys1970 1975 1980Ala Ile Ser Asp Ser Gln Ile Thr Ala Ser Ser His Leu Ser Asn Ile1985 1990 1995 2000Phe Ala Thr Trp Ser Pro Ser Gln Ala Arg Leu His Leu Gln Gly Arg
2005 2010 2015Thr Asn Ala Trp Arg Pro Arg Val Ser Ser Ala Glu Glu Trp Leu Gln
2020 2025 2030Val Asp Leu Gln Lys Thr Val Lys Val Thr Gly Ile Thr Thr Gln Gly
2035 2040 2045Val Lys Ser Leu Leu Ser Ser Met Tyr Val Lys Glu Phe Leu Val Ser2050 2055 2060Ser Ser Gln Asp Gly Arg Arg Trp Thr Leu Phe Leu Gln Asp Gly His2065 2070 2075 2080Thr Lys Val Phe Gln Gly Asn Gln Asp Ser Ser Thr Pro Val Val Asn
2085 2090 2095Ala Leu Asp Pro Pro Leu Phe Thr Arg Tyr Leu Arg Ile His Pro Thr
2100 2105 2110Ser Trp Ala Gln His Ile Ala Leu Arg Leu Glu Val Leu Gly Cys Glu
2115 2120 2125Ala Gln Asp Leu Tyr2130<210>3l<21l>19<212>PRT<213>人(Homo sapiens)<400>31Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe1 5 10 15Cys Phe Ser<210>32<211>24<212>PRT<213>人工序列<220><223>人工序列的描述:接头<400>32Ser Phe Ala Gln Asn Ser Arg Pro Pro Ser Ala Ser Ala Pro Lys Prol 5 10 15Pro Val Leu Arg Arg His Gln Arg
20<210>33<211>105<212>DNA<213>人工序列<220><223>人工序列的描述:接头<400>33gtcattgaac ctaggagctt tgcccagaat tcaagacccc ctagtgcgag cgctccaaag 60cctccggtcc tgcgacggca tcagagggac ataagccttc ctact 105<210>34<211>21<212>DNA<213>人工序列<220><223>人工序列的描述:寡核苷酸引物<400>34gaggaaaacc agatgatgtc a 21<210>35<211>60<212>DNA<213>人工序列<220><223>人工序列的描述:寡核苷酸引物<400>35ctttggagcg ctcgcactag ggggtcttga attctgggca aagctcctag gttcaatgac 60<210>36<211>66<212>DNA<213>人工序列<220><223>人工序列的描述:寡核苷酸引物<400>36cctagtgcga gcgctccaaa gcctccggtc ctgcgacggc atcagaggga cataagcctt 60cctact 66<210>37<211>4404<212>DNA<213>猪(Porcine)<220><221>CDS<222>(1)..(4401)<400>37atg cag cta gag ctc tcc acc tgt gtc ttt ctg tgt ctc ttg cca ctc 48Met Gln Leu Glu Leu Ser Thr Cys Val Phe Leu Cys Leu Leu Pro Leu1 5 10 15ggc ttt agt gcc atc agg aga tac tac ctg ggc gca gtg gaa ctg tcc 96Gly Phe Ser Ala Ile Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser
20 25 30tgg gac tac cgg caa agt gaa ctc ctc cgt gag ctg cac gtg gac acc 144Trp Asp Tyr Arg Gln Ser Glu Leu Leu Arg Glu Leu His Val Asp Thr
35 40 45aga ttt cct gct aca gcg cca gga gct ctt ccg ttg ggc ccg tca gtc 192Arg Phe Pro Ala Thr Ala Pro Gly Ala Leu Pro Leu Gly Pro Ser Val
50 55 60ctg tac aaa aag act gtg ttc gta gag ttc acg gat caa ctt ttc agc 240Leu Tyr Lys Lys Thr Val Phe Val Glu Phe Thr Asp Gln Leu Phe Ser65 70 75 80gtt gcc agg ccc agg cca cca tgg atg ggt ctg ctg ggt cct acc atc 288Val Ala Arg Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile
85 90 95cag gct gag gtt tac gac acg gtg gtc gtt acc ctg aag aac atg gct 336Gln Ala Glu Val Tyr Asp Thr Val Val Val Thr Leu Lys Asn Met Ala
100 105 110tct cat ccc gtt agt ctt cac gct gtc ggc gtc tcc ttc tgg aaa tct 384Ser His Pro Val Ser Leu His Ala Val Gly Val Ser Phe Trp Lys Ser
115 120 125tcc gaa ggc gct gaa tat gag gat cac acc agc caa agg gag aag gaa 432Ser Glu Gly Ala Glu Tyr Glu Asp His Thr Ser Gln Arg Glu Lys Glu
130 135 140gac gat aaa gtc ctt ccc ggt aaa agc caa acc tac gtc tgg cag gtc 480Asp Asp Lys Val Leu Pro Gly Lys Ser Gln Thr Tyr Val Trp Gln Val145 150 155 160ctg aaa gaa aat ggt cca aca gcc tct gac cca cca tgt ctt acc tac 528Leu Lys Glu Asn Gly Pro Thr Ala Ser Asp Pro Pro Cys Leu Thr Tyr
165 170 175tca tac ctg tct cac gtg gac ctg gtg aaa gac ctg aat tcg ggc ctc 576Ser Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu
180 185 190att gga gcc ctg ctg gtt tgt aga gaa ggg agt ctg acc aga gaa agg 624Ile Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Thr Arg Glu Arg
195 200 205acc cag aac ctg cac gaa ttt gta cta ctt ttt gct gtc ttt gat gaa 672Thr Gln Asn Leu His Glu Phe Val Leu Leu Phe Ala Val Phe Asp Glu
210 215 220ggg aaa agt tgg cac tca gca aga aat gac tcc tgg aca cgg gcc atg 720GIy Lys Ser Trp His Ser Ala Arg Asn Asp Ser Trp Thr Arg Ala Met225 230 235 240gat ccc gca cct gcc agg gcc cag cct gca atg cac aca gtc aat ggc 768Asp Pro Ala Pro Ala Arg Ala Gln Pro Ala Met His Thr Val Asn Gly
245 250 255tat gtc aac agg tct ctg cca ggt ctg atc gga tgt cat aag aaa tca 816Tyr Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Lys Lys Ser
260 265 270gtc tac tgg cac gtg att gga atg ggc acc agc ccg gaa gtg cac tcc 864Val Tyr Trp His Val Ile Gly Met Gly Thr Ser Pro Glu Val His Ser
275 280 285att ttt ctt gaa ggc cac acg ttt ctc gtg agg cac cat cgc cag gct 912Ile Phe Leu Glu Gly His Thr Phe Leu Val Arg His His Arg Gln Ala
290 295 300tcc ttg gag atc tcg cca cta act ttc ctc act gct cag aca ttc ctg 960Ser Leu Glu Ile Ser Pro Leu Thr Phe Leu Thr Ala Gln Thr Phe Leu305 310 315 320atg gac ctt ggc cag ttc cta ctg ttt tgt cat atc tct tcc cac cac 1008Met Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His His
325 330 335cat ggt ggc atg gag gct cac gtc aga gta gaa agc tgc gcc gag gag 1056His Gly Gly Met Glu Ala His Val Arg Val Glu Ser Cys Ala Glu Glu
340 345 350ccc cag ctg cgg agg aaa gct gat gaa gag gaa gat tat gat gac aat 1104Pro Gln Leu Arg Arg Lys Ala Asp Glu Glu Glu Asp Tyr Asp Asp Asn
355 360 365ttg tac gac tcg gac atg gac gtg gtc cgg ctc gat ggt gac gac gtg 1152Leu Tyr Asp Ser Asp Met Asp Val Val Arg Leu Asp Gly Asp Asp Val
370 375 380tct ccc ttt atc caa atc cgc tcg gtt gcc aag aag cat ccc aaa acc 1200Ser Pro Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr385 390 395 400tgg gtg cac tac atc tct gca gag gag gag gac tgg gac tac gcc ccc 1248Trp Val His Tyr Ile Ser Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro
405 410 415gcg gtc ccc agc ccc agt gac aga agt tat aaa agt ctc tac ttg aac 1296Ala Val Pro Ser Pro Ser Asp Arg Ser Tyr Lys Ser Leu Tyr Leu Asn
420 425 430agt ggt cct cag cga att ggt agg aaa tac aaa aaa gct cga ttc gtc 1344Ser Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Ala Arg Phe Val
435 440 445gct tac acg gat gta aca ttt aag act cgt aaa gct att ccg tat gaa 1392Ala Tyr Thr Asp Val Thr Phe Lys Thr Arg Lys Ala Ile Pro Tyr Glu
450 455 460tca gga atc ctg gga cct tta ctt tat gga gaa gtt gga gac aca ctt 1440Ser Gly Ile Leu GIy Pro Leu Leu Tyr Gly Glu ValGly Asp Thr Leu465 470 475 480ttg att ata ttt aag aat aaa gcg agc cga cca tat aac atc tac cct 1488Leu Ile Ile Phe Lys Asn Lys Ala Ser Arg Pro Tyr Asn Ile Tyr Pro
485 490 495cat gga atc act gat gtc agc gct ttg cac cca ggg aga ctt cta aaa 1536His Gly Ile Thr Asp Val Ser Ala Leu His Pro Gly Arg Leu Leu Lys
500 505 510ggt tgg aaa cat ttg aaa gac atg cca att ctg cca gga gag act ttc 1584Gly Trp Lys His Leu Lys Asp Met Pro Ile Leu Pro Gly Glu Thr Phe
515 520 525aag tat aaa tgg aca gtg act gtg gaa gat ggg cca acc aag tcc gat 1632Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp
530 535 540cct cgg tgc ctg acc cgc tac tac tcg agc tcc att aat cta gag aaa 1680Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Ser Ile Asn Leu Glu Lys545 550 555 560gat ctg gct tcg gga ctc att ggc cct ctc ctc atc tgc tac aaa gaa 1728Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu
565 570 575tct gta gac caa aga gga aac cag atg atg tca gac aag aga aac gtc 1776Ser Val Asp Gln Arg Gly Asn Gln Met Met Ser Asp Lys Arg Asn Val
580 585 590atc ctg ttt tct gta ttc gat gag aat caa agc tgg tac ctc gca gag 1824Ile Leu Phe Ser Val Phe Asp Glu Asn Gln Ser Trp Tyr Leu Ala Glu
595 600 605aat att cag cgc ttc ctc ccc aat ccg gat gga tta cag ccc cag gat 1872Asn Ile Gln Arg Phe Leu Pro Asn Pro Asp Gly Leu Gln Pro Gln Asp
610 615 620cca gag ttc caa gct tct aac atc atg cac agc atc aat ggc tat gtt 1920Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val625 630 635 640ttt gat agc ttg cag ctg tcg gtt tgt ttg cac gag gtg gca tac tgg 1968Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp
645 650 655tac att cta agt gtt gga gca cag acg gac ttc ctc tcc gtc ttc ttc 2016Tyr Ile Leu Ser Val Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe
660 665 670tct ggc tac acc ttc aaa cac aaa atg gtc tat gaa gac aca ctc acc 2064Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr
675 680 685ctg ttc ccc ttc tca gga gaa acg gtc ttc atg tca atg gaa aac cca 2112Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro
690 695 700ggt ctc tgg gtc ctt ggg tgc cac aac tca gac ttg cgg aac aga ggg 2160Gly Leu Trp Val Leu Gly Cys His Asn Ser Asp Leu Arg Asn Arg Gly705 710 715 720atg aca gcc tta ctg aag gtg tat agt tgt gac agg gac att ggt gat 2208Met Thr Ala Leu Leu Lys Val Tyr Ser Cys Asp Arg Asp Ile Gly Asp
725 730 735tat tat gac aac act tat gaa gat att cca ggc ttc ttg ctg agt gga 2256Tyr Tyr Asp Asn Thr Tyr Glu Asp Ile Pro Gly Phe Leu Leu Ser Gly
740 745 750aag aat gtc att gaa cct agg agc ttt gcc cag aat tca aga ccc cct 2304Lys Asn Val Ile Glu Pro Arg Ser Phe Ala Gln Asn Ser Arg Pro Pro
755 760 765agt gcg agc gct cca aag cct ccg gtc ctg cga cgg cat cag agg gac 2352Ser Ala Ser Ala Pro Lys Pro Pro Val Leu Arg Arg His Gln Arg Asp
770 775 780ata agc ctt cct act ttt cag ccg gag gaa gac aaa atg gac tat gat 2400Ile Ser Leu Pro Thr Phe Gln Pro Glu Glu Asp Lys Met Asp Tyr Asp785 790 795 800gat atc ttc tca act gaa acg aag gga gaa gat ttt gac att tac ggt 2448Asp Ile Phe Ser Thr Glu Thr Lys Gly Glu Asp Phe Asp Ile Tyr Gly
805 810 815gag gat gaa aat cag gac cct cgc agc ttt cag aag aga acc cga cac 2496Glu Asp Glu Asn Gln Asp Pro Arg Ser Phe Gln Lys Arg Thr Arg His
820 825 830tat ttc att gct gcg gtg gag cag ctc tgg gat tac ggg atg agc gaa 2544Tyr Phe Ile Ala Ala Val Glu Gln Leu Trp Asp Tyr Gly Met Ser Glu
835 840 845tcc ccc cgg gcg cta aga aac agg gct cag aac gga gag gtg cct cgg 2592Ser Pro Arg Ala Leu Arg Asn Arg Ala Gln Asn Gly Glu Val Pro Arg
850 855 860ttc aag aag gtg gtc ttc cgg gaa ttt gct gac ggc tcc ttc acg cag 2640Phe Lys Lys Val Val Phe Arg Glu Phe Ala Asp Gly Ser Phe Thr Gln865 870 875 880ccg tcg tac cgc ggg gaa ctc aac aaa cac ttg ggg ctc ttg gga ccc 2688Pro Ser Tyr Arg Gly Glu Leu Asn Lys His Leu Gly Leu Leu Gly Pro
885 890 895tac atc aga gcg gaa gtt gaa gac aac atc atg gta act ttc aaa aac 2736Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val Thr Phe Lys Asn
900 905 910cag gcg tct cgt ccc tat tcc ttc tac tcg agc ctt att tct tat ccg 2784Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser Leu Ile Ser Tyr Pro
915 920 925gat gat cag gag caa ggg gca gaa cct cga cac aac ttc gtc cag cca 2832Asp Asp Gln Glu Gln Gly Ala Glu Pro Arg His Asn Phe Val Gln Pro
930 935 940aat gaa acc aga act tac ttt tgg aaa gtg cag cat cac atg gca ccc 2880Asn Glu Thr Arg Thr Tyr Phe Trp Lys Val Gln His His Met Ala Pro945 950 955 960aca gaa gac gag ttt gac tgc aaa gcc tgg gcc tac ttt tct gat gtt 2928Thr Glu Asp Glu Phe Asp Cys Lys Ala Trp Ala Tyr Phe Ser Asp Val
965 970 975gac ctg gaa aaa gat gtg cac tca ggc ttg atc ggc ccc ctt ctg atc 2976Asp Leu Glu Lys Asp Val His Ser Gly Leu Ile Gly Pro Leu Leu Ile
980 985 990tgc cgc gcc aac acc ctg aac gct gct cac ggt aga caa gtg acc gtg 3024Cys Arg Ala Asn Thr Leu Asn Ala Ala His Gly Arg Gln Val Thr Val
995 1000 1005caa gaa ttt gct ctg ttt ttc act att ttt gat gag aca aag agc tgg 3072Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp1010 1015 1020tac ttc act gaa aat gtg gaa agg aac tgc cgg gcc ccc tgc cat ctg 3120Tyr Phe Thr Glu Asn Val Glu Arg Asn Cys Arg Ala Pro Cys His Leu1025 1030 1035 1040cag atg gag gac ccc act ctg aaa gaa aac tat cgc ttc cat gca atc 3168Gln Met Glu Asp Pro Thr Leu Lys Glu Asn Tyr Arg Phe His Ala Ile
1045 1050 1055aat ggc tat gtg atg gat aca ctc cct ggc tta gta atg gct cag aat 3216Asn Gly Tyr Val Met Asp Thr Leu Pro Gly Leu Val Met Ala Gln Asn
1060 1065 1070caa agg atc cga tgg tat ctg ctc agc atg ggc agc aat gaa aat atc 3264Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn Ile
1075 1080 1085cat tcg att cat ttt agc gga cac gtg ttc agt gta cgg aaa aag gag 3312His Ser Ile His Phe Ser Gly His Val Phe Ser Val Arg Lys Lys Glu1090 1095 1100gag tat aaa atg gcc gtg tac aat ctc tat ccg ggt gtc ttt gag aca 3360Glu Tyr Lys Met Ala Val Tyr Asn Leu Tyr Pro Gly Val Phe Glu Thr1105 1110 1115 1120gtg gaa atg cta ccg tcc aaa gtt gga att tgg cga ata gaa tgc ctg 3408Val Glu Met Leu Pro Ser Lys Val Gly Ile Trp Arg Ile Glu Cys Leu
1125 1130 1135att ggc gag cac ctg caa gct ggg atg agc acg act ttc ctg gtg tac 3456Ile Gly Glu His Leu Gln Ala Gly Met Ser Thr Thr Phe Leu Val Tyr
1140 1145 1150agc aag gag tgt cag gct cca ctg gga atg gct tct gga cgc att aga 3504Ser Lys Glu Cys Gln Ala Pro Leu Gly Met Ala Ser Gly Arg Ile Arg
1155 1160 1165gat ttt cag atc aca gct tca gga cag tat gga cag tgg gcc cca aag 3552Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys1170 1175 1180ctg gcc aga ctt cat tat tcc gga tca atc aat gcc tgg agc acc aag 3600Leu Ala Arg Leu His Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys1185 1190 1195 1200gat ccc cac tcc tgg atc aag gtg gat ctg ttg gca cca atg atc att 3648Asp Pro His Ser Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile Ile
1205 1210 1215cac ggc atc atg acc cag ggt gcc cgt cag aag ttt tcc agc ctc tac 3696His Gly Ile Met Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr
1220 1225 1230atc tcc cag ttt atc atc atg tac agt ctt gac ggg agg aac tgg cag 3744Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp Gly Arg Asn Trp Gln
1235 1240 1245agt tac cga ggg aat tcc acg ggc acc tta atg gtc ttc ttt ggc aat 3792Ser Tyr Arg Gly Asn Ser Thr Gly Thr Leu Met Val Phe Phe Gly Asn1250 1255 1260gtg gac gca tct ggg att aaa cac aat att ttt aac cct ccg att gtg 3840Val Asp Ala Ser Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile Val1265 1270 1275 1280gct cgg tac atc cgt ttg cac cca aca cat tac agc atc cgc agc act 3888Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser Thr
1285 1290 1295ctt cgc atg gag ttg atg ggc tgt gat tta aac agt tgc agc atg ccc 3936Leu Arg Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys Ser Met Pro
1300 1305 1310ctg gga atg cag aat aaa gcg ata tca gac tca cag atc acg gcc tcc 3984Leu Gly Met Gln Asn Lys Ala Ile Ser Asp Ser Gln Ile Thr Ala Ser
1315 1320 1325tcc cac cta agc aat ata ttt gcc acc tgg tct cct tca caa gcc cga 4032Ser His Leu Ser Asn Ile Phe Ala Thr Trp Ser Pro Ser Gln Ala Arg1330 1335 1340ctt cac ctc cag ggg cgg acg aat gcc tgg cga ccc cgg gtg agc agc 4080Leu His Leu Gln Gly Arg Thr Asn Ala Trp Arg Pro Arg Val Ser Ser1345 1350 1355 1360gca gag gag tgg ctg cag gtg gac ctg cag aag acg gtg aag gtc aca 4128Ala Glu Glu Trp Leu Gln Val Asp Leu Gln Lys Thr Val Lys Val Thr
1365 1370 1375ggc atc acc acc cag ggc gtg aag tcc ctg ctc agc agc atg tat gtg 4176Gly Ile Thr Thr Gln Gly Val Lys Ser Leu Leu Ser Ser Met Tyr Val
1380 1385 1390aag gag ttc ctc gtg tcc agt agt cag gac ggc cgc cgc tgg acc ctg 4224Lys Glu Phe Leu Val Ser Ser Ser Gln Asp Gly Arg Arg Trp Thr Leu
1395 1400 1405ttt ctt cag gac ggc cac acg aag gtt ttt cag ggc aat cag gac tcc 4272Phe Leu Gln Asp Gly His Thr Lys Val Phe Gln Gly Asn Gln Asp Ser1410 1415 1420tcc acc ccc gtg gtg aac gct ctg gac ccc ccg ctg ttc acg cgc tac 4320Ser Thr Pro Val Val Asn Ala Leu Asp Pro Pro Leu Phe Thr Arg Tyr1425 1430 1435 1440ctg agg atc cac ccc acg agc tgg gcg cag cac atc gcc ctg agg ctc 4368Leu Arg Ile His Pro Thr Ser Trp Ala Gln His Ile Ala Leu Arg Leu
1445 1450 1455gag gtt cta gga tgt gag gca cag gat ctc tac tga 4404Glu Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr
1460 1465<210>38<211>1467<212>PRT<213>猪(Porcine)<400>38Met Gln Leu Glu Leu Ser Thr Cys Val Phe Leu Cys Leu Leu Pro Leu1 5 10 15Gly Phe Ser Ala Ile Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser
20 25 30Trp Asp Tyr Arg Gln Ser Glu Leu Leu Arg Glu Leu His Val Asp Thr
35 40 45Arg Phe Pro Ala Thr Ala Pro Gly Ala Leu Pro Leu Gly Pro Ser Val
50 55 60Leu Tyr Lys Lys Thr Val Phe Val Glu Phe Thr Asp Gln Leu Phe Ser65 70 75 80Val Ala Arg Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile
85 90 95Gln Ala Clu Val Tyr Asp Thr Val Val Val Thr Leu Lys Asn Met Ala
100 105 110Ser His Pro Val Ser Leu His Ala Val Gly Val Ser Phe Trp Lys Ser
115 120 125Ser Glu Gly Ala Glu Tyr Glu Asp His Thr Ser Gln Arg Glu Lys Glu
130 135 140Asp Asp Lys Val Leu Pro Gly Lys Ser Gln Thr Tyr Val Trp Gln Val145 150 155 160Leu Lys Glu Asn Gly Pro Thr Ala Ser Asp Pro Pro Cys Leu Thr Tyr
165 170 175Ser Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu
180 185 190Ile Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Thr Arg Glu Arg
195 200 205Thr Gln Asn Leu His Glu Phe Val Leu Leu Phe Ala Val Phe Asp Glu
210 215 220Gly Lys Ser Trp His Ser Ala Arg Asn Asp Ser Trp Thr Arg Ala Met225 230 235 240Asp Pro Ala Pro Ala Arg Ala Gln Pro Ala Met His Thr Val Asn Gly
245 250 255Tyr Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Lys Lys Ser
260 265 270Val Tyr Trp His Val Ile Gly Met Gly Thr Ser Pro Glu Val His Ser
275 280 285Ile Phe Leu Glu Gly His Thr Phe Leu Val Arg His His Arg Gln Ala
290 295 300Ser Leu Glu Ile Ser Pro Leu Thr Phe Leu Thr Ala Gln Thr Phe Leu305 310 315 320Met Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His His
325 330 335His Gly Gly Met Glu Ala His Val Arg Val Glu Ser Cys Ala Glu Glu
340 345 350Pro Gln Leu Arg Arg Lys Ala Asp Glu Glu Glu Asp Tyr Asp Asp Asn
355 360 365Leu Tyr Asp Ser Asp Met Asp Val Val Arg Leu Asp Gly Asp Asp Val
370 375 380Ser Pro Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr385 390 395 400Trp Val His Tyr Ile Ser Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro
405 410 415Ala Val Pro Ser Pro Ser Asp Arg Ser Tyr Lys Ser Leu Tyr Leu Asn
420 425 430Ser Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Ala Arg Phe Val
435 440 445Ala Tyr Thr Asp Val Thr Phe Lys Thr Arg Lys Ala Ile Pro Tyr Glu
450 455 460Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu465 470 475 480Leu Ile Ile Phe Lys Asn Lys Ala Ser Arg Pro Tyr Asn Ile Tyr Pro
485 490 495His Gly Ile Thr Asp Val Ser Ala Leu His Pro Gly Arg Leu Leu Lys
500 505 510Gly Trp Lys His Leu Lys Asp Met Pro Ile Leu Pro Gly Glu Thr Phe
515 520 525Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp
530 535 540Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Ser Ile Asn Leu Glu Lys545 550 555 560Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu
565 570 575Ser Val Asp Gln Arg Gly Asn Gln Met Met Ser Asp Lys Arg Asn Val
580 585 590Ile Leu Phe Ser Val Phe Asp Glu Asn Gln Ser Trp Tyr Leu Ala Glu
595 600 605Asn Ile Gln Arg Phe Leu Pro Asn Pro Asp Gly Leu Gln Pro Gln Asp
610 615 620Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val625 630 635 640Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp
645 650 655Tyr Ile Leu Ser Val Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe
660 665 670Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr
675 680 685Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro
690 695 700Gly Leu Trp Val Leu Gly Cys His Asn Ser Asp Leu Arg Asn Arg Gly705 710 715 720Met Thr Ala Leu Leu Lys Val Tyr Ser Cys Asp Arg Asp Ile Gly Asp
725 730 735Tyr Tyr Asp Asn Thr Tyr Glu Asp Ile Pro Gly Phe Leu Leu Ser Gly
740 745 750Lys Asn Val Ile Glu Pro Arg Ser Phe Ala Gln Asn Ser Arg Pro Pro
755 760 765Ser Ala Ser Ala Pro Lys Pro Pro Val Leu Arg Arg His Gln Arg Asp
770 775 780Ile Ser Leu Pro Thr Phe Gln Pro Glu Glu Asp Lys Met Asp Tyr Asp785 790 795 800Asp Ile Phe Ser Thr Glu Thr Lys Gly Glu Asp Phe Asp Ile Tyr Gly
805 810 815Glu Asp Glu Asn Gln Asp Pro Arg Ser Phe Gln Lys Arg Thr Arg His
820 825 830Tyr Phe Ile Ala Ala Val Glu Gln Leu Trp Asp Tyr Gly Met Ser Glu
835 840 845Ser Pro Arg Ala Leu Arg Asn Arg Ala Gln Asn Gly Glu Val Pro Arg
850 855 860Phe Lys Lys Val Val Phe Arg Glu Phe Ala Asp Gly Ser Phe Thr Gln865 870 875 880Pro Ser Tyr Arg Gly Glu Leu Asn Lys His Leu Gly Leu Leu Gly Pro
885 890 895Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val Thr Phe Lys Asn
900 905 910Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser Leu Ile Ser Tyr Pro
915 920 925Asp Asp Gln Glu Gln Gly Ala Glu Pro Arg His Asn Phe Val Gln Pro
930 935 940Asn Glu Thr Arg Thr Tyr Phe Trp Lys Val Gln His His Met Ala Pro945 950 955 960Thr Glu Asp Glu Phe Asp Cys Lys Ala Trp Ala Tyr Phe Ser Asp Val
965 970 975Asp Leu Glu Lys Asp Val His Ser Gly Leu Ile Gly Pro Leu Leu Ile
980 985 990Cys Arg Ala Asn Thr Leu Asn Ala Ala His Gly Arg Gln Val Thr Val
995 1000 1005Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp1010 1015 1020Tyr Phe Thr Glu Asn Val Glu Arg Asn Cys Arg Ala Pro Cys His Leu1025 1030 1035 1040Gln Met Glu Asp Pro Thr Leu Lys Glu Asn Tyr Arg Phe His Ala Ile
1045 1050 1055Asn Gly Tyr Val Met Asp Thr Leu Pro Gly Leu Val Met Ala Gln Asn
1060 1065 1070Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn Ile
1075 1080 1085His Ser Ile His Phe Ser Gly His Val Phe Ser Val Arg Lys Lys Glu1090 1095 1100Glu Tyr Lys Met Ala Val Tyr Asn Leu Tyr Pro Gly Val Phe Glu Thr1105 1110 1115 1120Val Glu Met Leu Pro Ser Lys Val Gly Ile Trp Arg Ile Glu Cys Leu
1125 1130 1135Ile Gly Glu His Leu Gln Ala Gly Met Ser Thr Thr Phe Leu Val Tyr
1140 1145 1150Ser Lys Glu Cys Gln Ala Pro Leu Gly Met Ala Ser Gly Arg Ile Arg
1155 1160 1165Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys1170 1175 1180Leu Ala Arg Leu His Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys1185 1190 1195 1200Asp Pro His Ser Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile Ile
1205 1210 1215His Gly Ile Met Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr
1220 1225 1230Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp Gly Arg Asn Trp Gln
1235 1240 1245Ser Tyr Arg Gly Asn Ser Thr Gly Thr Leu Met Val Phe Phe Gly Asn1250 1255 1260Val Asp Ala Ser Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile Val1265 1270 1275 1280Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser Thr
1285 1290 1295Leu Arg Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys Ser Met Pro
1300 1305 1310Leu Gly Met Gln Asn Lys Ala Ile Ser Asp Ser Gln Ile Thr Ala Ser
1315 1320 1325Ser His Leu Ser Asn Ile Phe Ala Thr Trp Ser Pro Ser Gln Ala Arg1330 1335 1340Leu His Leu Gln Gly Arg Thr Asn Ala Trp Arg Pro Arg Val Ser Ser1345 1350 1355 1360Ala Glu Glu Trp Leu Gln Val Asp Leu Gln Lys Thr Val Lys Val Thr
1365 1370 1375Gly Ile Thr Thr Gln Gly Val Lys Ser Leu Leu Ser Ser Met Tyr Val
1380 1385 1390Lys Glu Phe Leu Val Ser Ser Ser Gln Asp Gly Arg Arg Trp Thr Leu
1395 1400 1405Phe Leu Gln Asp Gly His Thr Lys Val Phe Gln Gly Asn Gln Asp Ser1410 1415 1420Ser Thr Pro Val Val Asn Ala Leu Asp Pro Pro Leu Phe Thr Arg Tyr1425 1430 1435 1440Leu Arg Ile His Pro Thr Ser Trp Ala Gln His Ile Ala Leu Arg Leu
1445 1450 1455Glu Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr
1460 1465
Claims (12)
1.编码SEQ ID NO:39列出的POL1212氨基酸序列的DNA。
2.含有权利要求1所述的DNA的表达载体。
3.如权利要求1所述的DNA,其特征在于,该DNA具有SEQ ID NO:38的核苷酸序列。
4.含有权利要求3所述的DNA的表达载体。
5.具有SEQ ID NO:39的氨基酸序列的修饰的猪凝血因子VIII。
6.一种治疗组合物,其特征在于,该组合物含有权利要求5所述的修饰的猪凝血因子VIII和生理学上可接受的载体。
7.一种产生具有SEQ ID NO:39的氨基酸序列的修饰的猪凝血因子VIII蛋白质的方法,其特征在于,该方法包括:
在哺乳动物宿主细胞中表达编码SEQ ID NO:39所述的氨基酸序列的DNA。
8.如权利要求7所述的方法,其特征在于,编码SEQ ID NO:39的氨基酸序列的DNA还编码信号肽,通过所述信号肽使修饰的猪凝血因子VIII从哺乳动物细胞中运出。
9.如权利要求8所述的方法,其特征在于,所述信号肽具有SEQ ID NO:30的氨基酸1-19的序列。
10.一种哺乳动物细胞,其特征在于,该细胞含有并复制含有编码SEQ ID NO:39所示的POL1212氨基酸序列的DNA的表达载体。
11.如权利要求10所述的哺乳动物细胞,其特征在于,所述含有DNA的载体具有SEQ ID NO:38的核苷酸序列。
12.如权利要求11所述的细胞,其特征在于,所述宿主细胞是BHK CRL-1632。
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US09/523,656 US6458563B1 (en) | 1996-06-26 | 2000-03-10 | Modified factor VIII |
US09/523,656 | 2000-03-10 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN1416348A true CN1416348A (zh) | 2003-05-07 |
CN1191360C CN1191360C (zh) | 2005-03-02 |
Family
ID=24085870
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CNB018063179A Expired - Lifetime CN1191360C (zh) | 2000-03-10 | 2001-02-16 | 修饰的凝血因子ⅷ |
Country Status (36)
Country | Link |
---|---|
US (3) | US6458563B1 (zh) |
EP (1) | EP1280540B1 (zh) |
JP (1) | JP4044337B2 (zh) |
KR (1) | KR100485525B1 (zh) |
CN (1) | CN1191360C (zh) |
AT (1) | ATE391512T1 (zh) |
AU (2) | AU3841601A (zh) |
BE (1) | BE2016C024I2 (zh) |
BR (2) | BRPI0109131B8 (zh) |
CA (1) | CA2400295C (zh) |
CY (2) | CY1108179T1 (zh) |
CZ (1) | CZ298250B6 (zh) |
DE (1) | DE60133541T2 (zh) |
DK (1) | DK1280540T3 (zh) |
EE (1) | EE05075B1 (zh) |
ES (1) | ES2304379T3 (zh) |
FR (1) | FR16C0016I2 (zh) |
HK (1) | HK1051004A1 (zh) |
HU (2) | HU227804B1 (zh) |
IL (2) | IL151371A0 (zh) |
LT (1) | LTC1280540I2 (zh) |
LU (1) | LU93049I2 (zh) |
ME (1) | MEP8209A (zh) |
MX (1) | MXPA02008798A (zh) |
NL (1) | NL300808I2 (zh) |
NO (2) | NO331935B1 (zh) |
NZ (1) | NZ520799A (zh) |
PL (1) | PL202936B1 (zh) |
PT (1) | PT1280540E (zh) |
RS (1) | RS50364B (zh) |
RU (1) | RU2285724C2 (zh) |
SI (1) | SI1280540T1 (zh) |
SK (1) | SK286205B6 (zh) |
UA (1) | UA75064C2 (zh) |
WO (1) | WO2001068109A1 (zh) |
ZA (1) | ZA200206810B (zh) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102277379A (zh) * | 2011-08-18 | 2011-12-14 | 中国科学院遗传与发育生物学研究所 | 表达凝血因子viii的表达载体及其应用 |
CN103298483A (zh) * | 2010-11-05 | 2013-09-11 | 巴克斯特国际公司 | 具有增加的比活性的抗血友病因子viii的新型变体 |
CN111108196A (zh) * | 2017-05-09 | 2020-05-05 | 爱莫里大学 | 凝血因子变体及其用途 |
Families Citing this family (91)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7560107B2 (en) | 1996-06-26 | 2009-07-14 | Emory University | Modified factor VIII |
CA2434097A1 (en) * | 2001-01-12 | 2002-08-08 | The American National Red Cross | Methods and compositions for reducing heparan sulfate proteoglycan-mediated clearance of factor viii |
JP4634036B2 (ja) | 2001-10-05 | 2011-02-16 | エクスプレッション セラピューティクス, エルエルシー | 高レベルエキスプレッサー第viii因子ポリペプチドをコードする核酸配列およびアミノ酸配列および使用方法 |
EP1456235A4 (en) * | 2001-11-30 | 2005-08-17 | Univ Emory | VARIANTS OF DOMAIN C2 OF FACTOR VIII |
GB0207092D0 (en) * | 2002-03-26 | 2002-05-08 | Sod Conseils Rech Applic | Stable pharmaceutical composition containing factor VIII |
TWI353991B (en) | 2003-05-06 | 2011-12-11 | Syntonix Pharmaceuticals Inc | Immunoglobulin chimeric monomer-dimer hybrids |
JP4644663B2 (ja) * | 2003-06-03 | 2011-03-02 | セル ジェネシス インコーポレイテッド | ペプチド開裂部位を用いた単一ベクターからの組換えポリペプチドの高められた発現のための構成と方法 |
US7485291B2 (en) * | 2003-06-03 | 2009-02-03 | Cell Genesys, Inc. | Compositions and methods for generating multiple polypeptides from a single vector using a virus derived peptide cleavage site, and uses thereof |
ATE368687T1 (de) * | 2003-06-26 | 2007-08-15 | Merck Patent Gmbh | Thrombopoietinproteine mit verbesserten eigenschaften |
US20050059023A1 (en) * | 2003-09-16 | 2005-03-17 | Cantor Thomas L. | Methods and kits for monitoring resistance to therapeutic agents |
US7211559B2 (en) | 2003-10-31 | 2007-05-01 | University Of Maryland, Baltimore | Factor VIII compositions and methods |
CA2547569C (en) * | 2003-12-03 | 2013-04-16 | University Of Rochester | Recombinant factor viii having increased specific activity |
ES2449044T3 (es) * | 2004-05-03 | 2014-03-18 | Emory University | Procedimiento de administración de fVIII sin dominio B porcino |
WO2005123928A1 (en) * | 2004-06-08 | 2005-12-29 | Battelle Memorial Institute | Production of human coagulation factor viii from plant cells and whole plants |
LT1824988T (lt) * | 2004-11-12 | 2017-10-25 | Bayer Healthcare Llc | Nukreipta į užduotą saitą fviii modifikacija |
US20100256062A1 (en) | 2004-12-06 | 2010-10-07 | Howard Tommy E | Allelic Variants of Human Factor VIII |
US7855279B2 (en) | 2005-09-27 | 2010-12-21 | Amunix Operating, Inc. | Unstructured recombinant polymers and uses thereof |
CN101379077A (zh) | 2005-12-07 | 2009-03-04 | 夏洛特·豪泽 | 因子ⅷ和因子ⅷ-类似蛋白的小肽或者拟肽亲合性配体 |
EP1816201A1 (en) | 2006-02-06 | 2007-08-08 | CSL Behring GmbH | Modified coagulation factor VIIa with extended half-life |
AU2007236280B2 (en) * | 2006-04-11 | 2013-07-25 | Csl Behring Gmbh | Method of increasing the in vivo recovery of therapeutic polypeptides |
US7939632B2 (en) * | 2006-06-14 | 2011-05-10 | Csl Behring Gmbh | Proteolytically cleavable fusion proteins with high molar specific activity |
JP5448839B2 (ja) * | 2006-12-22 | 2014-03-19 | ツェー・エス・エル・ベーリング・ゲー・エム・ベー・ハー | インビボで長い半減期を有する修飾された凝固因子 |
EP1935430A1 (en) * | 2006-12-22 | 2008-06-25 | CSL Behring GmbH | Modified coagulation factors with prolonged in vivo half-life |
FR2913020B1 (fr) * | 2007-02-23 | 2012-11-23 | Biomethodes | Nouveaux facteurs viii pour le traitement des hemophiles de type a |
EP1988101A1 (en) * | 2007-05-04 | 2008-11-05 | Novo Nordisk A/S | Improvement of factor VIII polypeptide titers in cell cultures |
WO2009058446A1 (en) * | 2007-11-01 | 2009-05-07 | University Of Rochester | Recombinant factor viii having increased stability |
EP2149603A1 (en) | 2008-07-28 | 2010-02-03 | DRK-Blutspendedienst Baden-Württemberg-Hessen gGmbH | Factor IX variants with clotting activity in absence of their cofactor and their use for treating bleeding disorders |
US8716448B2 (en) | 2009-02-03 | 2014-05-06 | Amunix Operating Inc. | Coagulation factor VII compositions and methods of making and using same |
NZ628987A (en) | 2009-02-03 | 2015-11-27 | Amunix Operating Inc | Extended recombinant polypeptides and compositions comprising same |
PT2440239T (pt) | 2009-06-09 | 2017-10-19 | Prolong Pharmaceuticals Llc | Composições de hemoglobina |
CA2780761A1 (en) * | 2009-11-13 | 2011-05-19 | Puget Sound Blood Center | Factor viii t cell epitope variants having reduced immunogenicity |
US9050318B2 (en) | 2009-12-06 | 2015-06-09 | Biogen Idec Hemophilia Inc. | Factor VIII-Fc chimeric and hybrid polypeptides, and methods of use thereof |
US20110177107A1 (en) * | 2010-01-14 | 2011-07-21 | Haplomics, Inc. | Predicting and reducing alloimmunogenicity of protein therapeutics |
WO2012006623A1 (en) | 2010-07-09 | 2012-01-12 | Biogen Idec Hemophilia Inc. | Systems for factor viii processing and methods thereof |
AU2011274423B2 (en) | 2010-07-09 | 2016-02-11 | Bioverativ Therapeutics Inc. | Chimeric clotting factors |
KR20140036144A (ko) * | 2011-01-05 | 2014-03-25 | 익스프레션 테라퍼틱스 엘엘씨 | 고수율 현탁세포주, 시스템 및 그 제조방법 |
EP3527218A1 (en) | 2011-06-10 | 2019-08-21 | Bioverativ Therapeutics Inc. | Pro-coagulant compounds and methods of use thereof |
EA029045B1 (ru) | 2011-07-08 | 2018-02-28 | Байоджен Хемофилия Инк. | Химерные и гибридные полипептиды фактора viii и способы их применения |
EP2737311B1 (en) | 2011-07-25 | 2020-12-02 | Bioverativ Therapeutics Inc. | Assays to monitor bleeding disorders |
PL2802668T3 (pl) | 2012-01-12 | 2019-03-29 | Bioverativ Therapeutics Inc. | Sposoby zmniejszania immunogenności wobec czynnika krzepnięcia viii u pacjentów poddawanych leczeniu czynnikiem krzepnięcia viii |
NZ626945A (en) | 2012-01-12 | 2016-10-28 | Biogen Ma Inc | Chimeric factor viii polypeptides and uses thereof |
PT3564260T (pt) | 2012-02-15 | 2023-01-18 | Bioverativ Therapeutics Inc | Composições de fator viii e métodos de produção e utilização das mesmas |
KR20190094480A (ko) | 2012-02-15 | 2019-08-13 | 바이오버라티브 테라퓨틱스 인크. | 재조합 인자 viii 단백질 |
EP2666782A1 (en) * | 2012-05-22 | 2013-11-27 | Imnate Sarl | Coagulation factor VIII with reduced immunogenicity. |
EP4079316A1 (en) | 2012-06-08 | 2022-10-26 | Bioverativ Therapeutics Inc. | Procoagulant compounds |
AU2013270683A1 (en) | 2012-06-08 | 2014-12-11 | Biogen Ma Inc. | Chimeric clotting factors |
EP2870250B2 (en) | 2012-07-06 | 2022-06-29 | Bioverativ Therapeutics Inc. | Cell line expressing single chain factor viii polypeptides and uses thereof |
SG11201500045RA (en) | 2012-07-11 | 2015-02-27 | Amunix Operating Inc | Factor viii complex with xten and von willebrand factor protein, and uses thereof |
WO2014018777A2 (en) | 2012-07-25 | 2014-01-30 | Biogen Idec Ma Inc. | Blood factor monitoring assay and uses thereof |
WO2014063108A1 (en) | 2012-10-18 | 2014-04-24 | Biogen Idec Ma Inc. | Methods of using a fixed dose of a clotting factor |
EP2914293A4 (en) | 2012-10-30 | 2016-04-20 | Biogen Ma Inc | METHOD OF USE OF FVIII POLYPEPTIDE |
BR112015013311A2 (pt) | 2012-12-07 | 2017-11-14 | Haplomics Inc | indução de tolerancia e reparação de mutação do fator 8 |
DK2956477T4 (da) | 2013-02-15 | 2024-04-15 | Bioverativ Therapeutics Inc | Optimeret faktor viii-gen |
SI2968477T1 (sl) | 2013-03-15 | 2020-04-30 | Bioverativ Therapeutics Inc. | Polipeptidne formulacije faktorja VIII |
TWI745671B (zh) | 2013-03-15 | 2021-11-11 | 美商百歐維拉提夫治療公司 | 因子ix多肽調配物 |
EP3875106A1 (en) | 2013-08-08 | 2021-09-08 | Bioverativ Therapeutics Inc. | Purification of chimeric fviii molecules |
TWI667255B (zh) | 2013-08-14 | 2019-08-01 | 美商生物化學醫療公司 | 因子viii-xten融合物及其用途 |
MX2016005333A (es) | 2013-10-22 | 2017-02-15 | Dbv Tech | Método para tratar la hemofilia induciendo tolerancia a factores sanguíneos. |
EP3065769A4 (en) | 2013-11-08 | 2017-05-31 | Biogen MA Inc. | Procoagulant fusion compound |
ES2967617T3 (es) | 2013-12-06 | 2024-05-03 | Bioverativ Therapeutics Inc | Herramientas de farmacocinética poblacional y sus usos |
EP2881463A1 (en) | 2013-12-09 | 2015-06-10 | DRK-Blutspendedienst Baden-Württemberg-Hessen gGmbH | Factor IX variants with clotting activity in absence of their cofactor and/or with increased F.IX clotting activity and their use for treating bleeding disorders |
BR122023020301A2 (pt) | 2014-01-10 | 2023-12-12 | Bioverativ Therapeutics Inc. | Uso de uma proteína quimérica compreendendo uma proteína fviii |
US20160347787A1 (en) | 2014-02-04 | 2016-12-01 | Biogen Ma Inc. | Use of cation-exchange chromatography in the flow-through mode to enrich post-translational modifications |
WO2015127129A1 (en) * | 2014-02-19 | 2015-08-27 | Bloodworks | Factor viii b cell epitope variants having reduced immunogenicity |
WO2015132724A1 (en) | 2014-03-05 | 2015-09-11 | Pfizer Inc. | Improved muteins of clotting factor viii |
MA40864A (fr) | 2014-10-31 | 2017-09-05 | Biogen Ma Inc | Hypotaurine, gaba, bêta-alanine et choline pour la régulation de l'accumulation de sous-produits résiduaires dans des procédés de culture de cellules mammifères |
WO2017024060A1 (en) | 2015-08-03 | 2017-02-09 | Biogen Ma Inc. | Factor ix fusion proteins and methods of making and using same |
PE20231949A1 (es) * | 2015-10-30 | 2023-12-05 | Spark Therapeutics Inc | VARIANTES DEL FACTOR VIII REDUCIDO CON CpG, COMPOSICIONES Y METODOS Y USOS PARA EL TRATAMIENTO DE TRASTORNOS DE LA HEMOSTASIA |
SG11201804070XA (en) | 2015-11-13 | 2018-06-28 | Baxalta Inc | Viral vectors encoding recombinant fviii variants with increased expression for gene therapy of hemophilia a |
EA036944B1 (ru) | 2015-11-13 | 2021-01-19 | Баксалта Инкорпорейтед | Вирусные векторы, кодирующие рекомбинантные варианты fviii с повышенной экспрессией для генной терапии гемофилии a |
US11753461B2 (en) | 2016-02-01 | 2023-09-12 | Bioverativ Therapeutics Inc. | Optimized factor VIII genes |
US11046749B2 (en) | 2016-06-24 | 2021-06-29 | Mogam Institute For Biomedical Research | Chimera protein comprising FVIII and vWF factors, and use thereof |
MX2019006444A (es) | 2016-12-02 | 2019-10-30 | Bioverativ Therapeutics Inc | Métodos de tratamiento de artropatía hemofílica utilizando factores de coagulación quiméricos. |
CN110520149A (zh) | 2016-12-02 | 2019-11-29 | 比奥维拉迪维治疗股份有限公司 | 诱导对凝血因子的免疫耐受性的方法 |
CA3072334A1 (en) | 2017-08-09 | 2019-02-14 | Bioverativ Therapeutics Inc. | Nucleic acid molecules and uses thereof |
KR20200118089A (ko) | 2018-02-01 | 2020-10-14 | 바이오버라티브 테라퓨틱스 인크. | 인자 viii을 발현하는 렌티바이러스 벡터의 용도 |
MX2020010369A (es) | 2018-04-04 | 2020-10-22 | Sigilon Therapeutics Inc | Particulas implantables y metodos relacionados. |
BR112020022164A2 (pt) | 2018-05-18 | 2021-02-02 | Bioverativ Therapeutics Inc. | métodos de tratamento de hemofilia a |
EP3823985A1 (en) | 2018-07-16 | 2021-05-26 | Baxalta Incorporated | Gene therapy of hemophilia a using viral vectors encoding recombinant fviii variants with increased expression |
AU2019319984A1 (en) | 2018-08-09 | 2021-03-04 | Bioverativ Therapeutics Inc. | Nucleic acid molecules and uses thereof for non-viral gene therapy |
UY38389A (es) | 2018-09-27 | 2020-04-30 | Sigilon Therapeutics Inc | Dispositivos implantables para terapia celular y métodos relacionados |
TW202039546A (zh) | 2019-01-16 | 2020-11-01 | 美商巴克斯歐塔公司 | 用於a型血友病基因治療之編碼表現增加之重組fviii變異體的病毒載體 |
CA3144630A1 (en) | 2019-06-19 | 2020-12-24 | Bioverativ Therapeutics Inc. | Methods and compositions for treating hemophilia and low bone mineral density |
JP2022537555A (ja) | 2019-06-20 | 2022-08-26 | 武田薬品工業株式会社 | ウイルスベースの遺伝子療法による治療方法 |
CN114981299A (zh) | 2019-12-12 | 2022-08-30 | 武田药品工业株式会社 | 使用编码具有增加的表达的重组fviii变体的病毒载体的a型血友病的基因疗法 |
TW202246505A (zh) * | 2021-03-05 | 2022-12-01 | 俄羅斯聯邦商亞那拜恩有限公司 | 編碼凝血因子ix蛋白的密碼子優化的核酸及其用途 |
EP4314819A1 (en) * | 2021-03-31 | 2024-02-07 | Haemonetics Corporation | Hemostasis measurement device quality control formulations |
CN117858895A (zh) | 2021-06-14 | 2024-04-09 | 武田药品工业株式会社 | 使用表达增强的编码重组fviii变体的病毒载体的血友病a基因疗法 |
IL310997A (en) | 2021-08-23 | 2024-04-01 | Bioverativ Therapeutics Inc | Factor VIII gene optimization |
CA3232988A1 (en) | 2021-09-30 | 2023-04-06 | Bioverativ Therapeutics Inc. | Nucleic acids encoding factor viii polypeptides with reduced immunogenicity |
WO2024081309A1 (en) | 2022-10-11 | 2024-04-18 | Sigilon Therapeutics, Inc. | Engineered cells and implantable elements for treatment of disease |
Family Cites Families (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4757006A (en) | 1983-10-28 | 1988-07-12 | Genetics Institute, Inc. | Human factor VIII:C gene and recombinant methods for production |
EP0182448A3 (en) | 1984-08-24 | 1987-10-28 | Genetics Institute, Inc. | Production of factor viii and related products |
WO1986006101A1 (en) | 1985-04-12 | 1986-10-23 | Genetics Institute, Inc. | Novel procoagulant proteins |
JPH0387173A (ja) | 1987-09-10 | 1991-04-11 | Teijin Ltd | ヒト活性化天然型ファクター8cの製造方法及びそれに用いる形質転換体 |
DK162233C (da) | 1989-11-09 | 1992-03-16 | Novo Nordisk As | Fremgangsmaade til isolering af faktor viii fra blodplasma og pharmaceutisk praeparat indeholdende den saaledes isolerede fator viii |
US6180371B1 (en) * | 1996-06-26 | 2001-01-30 | Emory University | Modified factor VIII |
US5663060A (en) | 1992-04-07 | 1997-09-02 | Emory University | Hybrid human/animal factor VIII |
US5364771A (en) | 1992-04-07 | 1994-11-15 | Emory University | Hybrid human/porcine factor VIII |
US5563045A (en) | 1992-11-13 | 1996-10-08 | Genetics Institute, Inc. | Chimeric procoagulant proteins |
AU6291896A (en) | 1995-07-11 | 1997-02-10 | Chiron Corporation | Novel factor viii:c polypeptide analogs comprising factor v domains or subdomains |
WO1997003193A1 (en) | 1995-07-11 | 1997-01-30 | Chiron Corporation | Novel factor viii:c polypeptide analogs with altered metal-binding properties |
RU2003107100A (ru) * | 2000-09-19 | 2004-08-27 | Эмори Юниверсити (Us) | Модифицированный фактор viii |
EP1456235A4 (en) * | 2001-11-30 | 2005-08-17 | Univ Emory | VARIANTS OF DOMAIN C2 OF FACTOR VIII |
US7105745B2 (en) * | 2002-12-31 | 2006-09-12 | Thomas & Betts International, Inc. | Water resistant electrical floor box cover assembly |
-
2000
- 2000-03-10 US US09/523,656 patent/US6458563B1/en not_active Expired - Lifetime
-
2001
- 2001-02-16 UA UA2002097145A patent/UA75064C2/uk unknown
- 2001-02-16 NZ NZ520799A patent/NZ520799A/en not_active IP Right Cessation
- 2001-02-16 ME MEP-82/09A patent/MEP8209A/xx unknown
- 2001-02-16 CA CA2400295A patent/CA2400295C/en not_active Expired - Lifetime
- 2001-02-16 KR KR10-2002-7011843A patent/KR100485525B1/ko active IP Right Grant
- 2001-02-16 AU AU3841601A patent/AU3841601A/xx active Pending
- 2001-02-16 SI SI200130823T patent/SI1280540T1/sl unknown
- 2001-02-16 CN CNB018063179A patent/CN1191360C/zh not_active Expired - Lifetime
- 2001-02-16 WO PCT/US2001/005076 patent/WO2001068109A1/en active IP Right Grant
- 2001-02-16 DK DK01910853T patent/DK1280540T3/da active
- 2001-02-16 BR BRPI0109131A patent/BRPI0109131B8/pt not_active IP Right Cessation
- 2001-02-16 SK SK1439-2002A patent/SK286205B6/sk not_active IP Right Cessation
- 2001-02-16 PL PL359672A patent/PL202936B1/pl unknown
- 2001-02-16 CZ CZ20023346A patent/CZ298250B6/cs unknown
- 2001-02-16 MX MXPA02008798A patent/MXPA02008798A/es active IP Right Grant
- 2001-02-16 PT PT01910853T patent/PT1280540E/pt unknown
- 2001-02-16 RS YUP-680/02A patent/RS50364B/sr unknown
- 2001-02-16 JP JP2001566673A patent/JP4044337B2/ja not_active Expired - Lifetime
- 2001-02-16 AU AU2001238416A patent/AU2001238416B2/en active Active
- 2001-02-16 IL IL15137101A patent/IL151371A0/xx unknown
- 2001-02-16 HU HU0300586A patent/HU227804B1/hu active Protection Beyond IP Right Term
- 2001-02-16 BR BR122013026957A patent/BR122013026957A8/pt not_active Application Discontinuation
- 2001-02-16 EE EEP200200510A patent/EE05075B1/xx active Protection Beyond IP Right Term
- 2001-02-16 AT AT01910853T patent/ATE391512T1/de active
- 2001-02-16 RU RU2002124123/13A patent/RU2285724C2/ru active
- 2001-02-16 ES ES01910853T patent/ES2304379T3/es not_active Expired - Lifetime
- 2001-02-16 DE DE60133541T patent/DE60133541T2/de not_active Expired - Lifetime
- 2001-02-16 EP EP01910853A patent/EP1280540B1/en not_active Expired - Lifetime
-
2002
- 2002-06-28 US US10/187,319 patent/US7012132B2/en not_active Expired - Fee Related
- 2002-08-21 IL IL151371A patent/IL151371A/en active IP Right Grant
- 2002-08-26 ZA ZA200206810A patent/ZA200206810B/en unknown
- 2002-09-09 NO NO20024296A patent/NO331935B1/no not_active IP Right Cessation
-
2003
- 2003-05-06 HK HK03103224A patent/HK1051004A1/xx not_active IP Right Cessation
-
2004
- 2004-09-10 US US10/938,414 patent/US7122634B2/en not_active Expired - Fee Related
-
2008
- 2008-07-07 CY CY20081100709T patent/CY1108179T1/el unknown
-
2016
- 2016-04-27 LU LU93049C patent/LU93049I2/xx unknown
- 2016-04-29 FR FR16C0016C patent/FR16C0016I2/fr active Active
- 2016-05-02 BE BE2016C024C patent/BE2016C024I2/fr unknown
- 2016-05-06 LT LTPA2016014C patent/LTC1280540I2/lt unknown
- 2016-05-09 HU HUS1600020C patent/HUS1600020I1/hu unknown
- 2016-05-09 NL NL300808C patent/NL300808I2/nl unknown
- 2016-05-10 NO NO2016007C patent/NO2016007I2/no unknown
- 2016-05-10 CY CY2016011C patent/CY2016011I1/el unknown
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103298483A (zh) * | 2010-11-05 | 2013-09-11 | 巴克斯特国际公司 | 具有增加的比活性的抗血友病因子viii的新型变体 |
CN103298483B (zh) * | 2010-11-05 | 2017-04-12 | 百深有限责任公司 | 具有增加的比活性的抗血友病因子viii的新型变体 |
CN102277379A (zh) * | 2011-08-18 | 2011-12-14 | 中国科学院遗传与发育生物学研究所 | 表达凝血因子viii的表达载体及其应用 |
CN102277379B (zh) * | 2011-08-18 | 2013-07-24 | 中国科学院遗传与发育生物学研究所 | 表达凝血因子viii的表达载体及其应用 |
CN111108196A (zh) * | 2017-05-09 | 2020-05-05 | 爱莫里大学 | 凝血因子变体及其用途 |
US11633504B2 (en) | 2017-05-09 | 2023-04-25 | Emory University | Nucleic acids encoding clotting factor variants and their use |
Also Published As
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN1191360C (zh) | 修饰的凝血因子ⅷ | |
CN1205221C (zh) | 修饰的凝血因子ⅷ | |
CN1224627C (zh) | 修饰的凝血因子ⅷ | |
CN1246462C (zh) | 修饰的维生素k依赖性多肽 | |
CN1033760C (zh) | 产生具有第八因子活性的第八因子缺失突变体的方法 | |
CN1745100A (zh) | 因子ⅷ多肽 | |
CN1202128C (zh) | 修饰蛋白的免疫原性 | |
CN1138786C (zh) | 识别破骨细胞形成抑制因子结合蛋白的抗体及制备和用途 | |
CN1194014C (zh) | 促红细胞生成素与聚乙二醇的偶联物 | |
AU2007269233B2 (en) | Method of producing Factor VIII proteins by recombinant methods | |
CN1902226A (zh) | 单链胰岛素 | |
CN1076966A (zh) | 抗人白细胞介素-4的人性化单克隆抗体的克隆和表达 | |
CN1283659C (zh) | 截短的神经胶质细胞系来源的神经营养因子 | |
CN1784425A (zh) | 用结合天然脑钠尿肽前体分子氨基酸38-44的单克隆抗体检测天然脑钠尿肽前体分子的方法 | |
CN1671423A (zh) | 新型嵌合cd154 | |
CN1146664C (zh) | 包含一个IgE-结合结构域和一个HSA成分的融合多肽及其在诊断和治疗中的应用 | |
CN1419563A (zh) | 肽衍生物 | |
CN1140536C (zh) | 人血小板糖蛋白Ib/Ix的米摩托培和抗米摩托培 | |
CN1152579A (zh) | 制备环状血管活性肽的方法 | |
CN1646564A (zh) | 修饰的因子ⅷ | |
CN1241638C (zh) | Ifnar2/ifn复合物 | |
CN1039441A (zh) | 毒蛇毒液多肽及基因表达 | |
CN1171792A (zh) | 具有ii型磷脂酶a2抑制作用的新型单克隆抗体及含其一部分的蛋白质 | |
CN1254487C (zh) | 抗免疫球蛋白等模式标本的重整形单克隆抗体 | |
CN1283120A (zh) | Cd8作为细胞免疫系统的抑制剂 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
C14 | Grant of patent or utility model | ||
GR01 | Patent grant | ||
CX01 | Expiry of patent term | ||
CX01 | Expiry of patent term |
Granted publication date: 20050302 |