CN101410525B - 生产多肽的细胞 - Google Patents
生产多肽的细胞 Download PDFInfo
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- CN101410525B CN101410525B CN2007800108283A CN200780010828A CN101410525B CN 101410525 B CN101410525 B CN 101410525B CN 2007800108283 A CN2007800108283 A CN 2007800108283A CN 200780010828 A CN200780010828 A CN 200780010828A CN 101410525 B CN101410525 B CN 101410525B
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Abstract
本发明描述了核酸,该核酸以5’到3’的方向包含a)编码异源多肽的第一核酸,其没有符合读码框的终止密码子,b)起始于5’剪接供体位点和终止于3’剪接受体位点的第二核酸,其包含符合读码框的翻译终止密码子和多聚腺苷酸化信号,和c)核酸,其编码i)至少跨膜结构域的片段,或ii)GPI-锚定物的信号肽。
Description
技术领域
本发明属于多肽生产的领域。其描述了含有选择性剪接核酸的核酸、包含此核酸的细胞、分离表达异源多肽细胞的方法、以及生产异源多肽的方法,在所述分离表达异源多肽细胞的方法中所利用的核酸包含编码异源多肽的第一核酸、包含选择性剪接核酸的第二核酸、以及编码至少跨膜结构域的片段或GPI-锚定物的信号肽的第三核酸。
背景技术
生产重组多肽的表达系统是本领域技术中众所周知的并且由下述文章描述,例如,Marino,M.H.,Biopharm.2(1989)18-33;Goeddel,D.V.等人,Methods Enzymol.185(1990)3-7;Wurm,F.和Bernard,A.,Curr.Opin.Biotechnol.10(1999)156-160。制药应用所用多肽和蛋白的生产优选使用哺乳动物宿主细胞,如CHO细胞、BHK细胞、NS0细胞、Sp2/0细胞、COS细胞、HEK细胞、细胞等。编码多肽的核酸优选包含在核酸,例如表达载体中引入宿主细胞。表达载体的基本元件是原核质粒增殖单位,例如包括复制起点和选择标记、真核选择标记、和一个或多个表达感兴趣的结构基因的表达盒的大肠杆菌,所述表达盒各自包含启动子、结构基因、和包含多聚腺苷酸化信号的转录终止子。为了在哺乳动物细胞中瞬时表达,可以包含哺乳动物复制起点,如SV40Ori或来自EBV的OriP。启动子可以选择组成型或诱导型的启动子。为了使转录最优化可以在5’非翻译区包含一段Kozak序列。为了mRNA加工,特别是转录终止和前体mRNA的剪接,依赖于结构基因的构造(外显子-内含子结构形式),可以包括mRNA剪接信号以及多聚腺苷酸化信号。
基因的表达表现为瞬时或永久的表达。感兴趣的多肽可以是分泌型多肽或者胞质多肽,所述分泌型多肽包含N末端延伸(也称为信号序列),此延伸对多肽通过细胞和进入胞外媒介的运输/分泌是必需的。
为了多肽的大规模生产需要建立高生产型细胞株。转染宿主细胞株如CHO细胞、NS0细胞、Sp2/0细胞、BHK细胞、COS细胞、PER.细胞或HEK细胞之后,一般而言大多数具有不同特征的克隆的获得是由于例如,从瞬时转染或稳定整合质粒表达的多肽的广泛不同。为了选择的目的,引入细胞的核酸有额外的选择性标记,例如带有对另一致死性物质抗性的基因。
转染后通过在适当的选择性培养液中生长,可以分离高生产型克隆。这个过程是费时的因而需要大笔开支。发展出几个方法来处理这个问题。
这些办法其一是基因扩增。其中用载体/质粒转染二氢叶酸还原酶缺陷(DHFR)的细胞,所述载体/质粒包含表达DHFR蛋白的第一表达盒和表达感兴趣的异源多肽的第二表达盒。通过利用去除甘氨酸、次黄嘌呤和胸腺嘧啶的培养液建立了选择性生长条件。加入甲氨喋呤(MTX)以扩增DHFR的抑制物。(Kaufman,R.J.等人,J Mol.Biol.159(1982)601-621;US4,656,134)
或者可以将报告分子融合入异源多肽,如氯霉素-乙酰氨基转移酶、荧光素酶、绿色荧光蛋白、或β-半乳糖苷酶,因为所述报告分子是高生产型细胞株所需要的而且还作为间接的选择标记。选择发生在添加的外源物质或辅因子存在的情况下。
鉴定高生产型克隆的另一个方法是,经由内部核糖体进入位点(IRES)的选择性标记基因和编码异源多肽的结构基因的连锁转录。通过这一设计异源多肽的表达可以与选择性标记的表达关联起来。
人源免疫球蛋白由特化的淋巴细胞B细胞产生。这些细胞不只分泌免疫球蛋白(sIg)它们还使免疫球蛋白作为质膜-结合型免疫球蛋白(mIg)呈现在它们的细胞外膜。这些mIg在免疫应答起始时起重要作用。呈现的质膜-结合型免疫球蛋白具有它们相应抗原的细胞受体的功能。
从1980年开始,有关分泌型和质膜-结合型免疫球蛋白的起源的文章发表面世。Early等人(Early,P.等人,Cell20(1980)313-319)报道,在小鼠中有两种编码免疫球蛋白重链的mRNA,所述mRNA源自单一免疫球蛋白μ-基因的相同原始转录本。分泌形式(sIg)和质膜-结合形式(mIg)的形成由重链前体mRNA的选择剪接所产生。对于sIg亚型,所有编码免疫球蛋白结构域的外显子和编码C末端结构域外显子之后的内含子都保留在mRNA中,而位于内含子中终止密码子下游的多聚腺苷酸化信号用来剪切和多聚腺苷酸化原始转录本。对于mIg亚型,在编码分泌型形式C末端结构域(即分别为CH3或CH4)之后的选择剪接5’供体位点连接恒定区与编码跨膜结构域的下游外显子M1和M2。在这种情况下,分泌型形式的编码末端氨基酸的序列和终止密码子、及其邻近内含子区的sIg形式的多聚腺苷酸化信号随同内含子的剪接一起被移除。
例如,编码分泌型免疫球蛋白重链形式的mRNA与编码质膜-结合型免疫球蛋白重链形式的mRNA的比例为10:1到100:1之间。这个比例主要是在前体mRNA剪接过程中确定的。翻译和翻译后控制机制只起次要的作用(参见,例如Xiang,S.D.等人,Immun.Cell Biol.79(2001)472-481)。
结合到细胞质膜上的免疫球蛋白同它的分泌型类似物具有相同的氨基酸序列和二级结构。不同的是,sIg重链C末端的延伸包含跨膜结构域。这个跨膜结构域的长度一般在约40和约75个氨基酸残基之间。对于鼠和人的免疫球蛋白,所述跨膜结构域能被再分为三个截然不同的结构区:13-67个氨基酸残基的N末端胞外区、25个氨基酸残基的中心保守的跨膜伸展区、以及3-28个氨基酸残基的C末端胞质区(Major,J.G.等人,Mol.Immunol.33(1996)179-187)。
表达载体包含可扩增的选择性基因、荧光蛋白基因和编码所需产物的基因,它们以WO01/04306中已报道方式连接使转录和翻译最优化。在WO01/38557中报道了用于筛选多重转化/转染的细胞以鉴定表达至少两个感兴趣的肽或蛋白质的细胞的方法。这两个肽/蛋白质通过IRES(内部核糖体进入位点)连接于荧光标记基因。
美国专利申请2003/0033616报道了包含成像标记转基因的转基因动物和细胞。美国专利申请2005/0032127报道了在温和条件下从细胞混合物或细胞培养物中非侵入的选择单个活细胞的方法,所述方法与通过荧光显微镜检测法检测的特异生产性能有关。鉴定和分离生产分泌型蛋白质的细胞的方法在美国专利申请2002/0168702有所报道。
在美国专利申请2004/0148647中报道了表达载体,所述载体由功能性连接于仓鼠启动子的编码感兴趣蛋白质的基因、编码荧光蛋白的基因、和优选可扩增的选择基因组成。
发明概述
本发明包含核酸,所述核酸从5’到3’的方向包含
a)编码异源多肽不具有符合读码框的翻译终止密码子的第一核酸,
b)起始于5’剪接供体位点并且终止于3’剪接受体位点的第二核酸,其包含符合读码框的翻译终止密码子和多聚腺苷酸化信号,和
c)第三核酸其编码
i)至少跨膜结构域的片段,或
ii)GPI-锚定物信号肽。
本发明另外的方面是适合于真核细胞的包含本发明的核酸的载体,以及包含根据本发明的至少一个核酸的真核细胞。
本发明还包括选择表达异源多肽的真核细胞的方法,藉此该方法包括
a)用核酸转染真核细胞,所述核酸从5’到3’的方向包含
i)编码异源多肽不具有符合读码框的翻译终止密码子的第一核酸,
ii)起始于剪接供体位点并且终止于剪接受体位点的第二核酸,其含符合读码框的翻译终止密码子和多聚腺苷酸化信号,
iii)第三核酸其编码
iiia)至少跨膜结构域的片段,或
iiib)GPI-锚定物信号肽,
b)在一定条件下培养所述转染后的细胞,所述条件包括:适于从所述核酸生产前体mRNA,适于所述前体mRNA加工,以及适于所述加工后mRNA翻译成多肽,其中所述转染细胞通过选择剪接所述前体mRNA,产生可溶性的异源多肽和质膜-结合型异源多肽,和
c)选择具有质膜-结合型异源多肽的细胞作为所述表达异源多肽的细胞。
在一个实施方案中发明的方法是选择表达免疫球蛋白真核细胞的方法,藉此该方法包括
a)用核酸转染真核细胞,所述核酸从5’到3’的方向包含
i)编码免疫球蛋白重链不具有符合读码框的翻译终止密码子的第一核酸,
ii)起始于剪接供体位点并且终止于剪接受体位点的第二核酸,其包含符合读码框的翻译终止密码子和多聚腺苷酸化信号,
iii)第三核酸其编码
iiia)至少跨膜结构域的片段,或
iiib)GPI-锚定物信号肽,
b)在一定条件下培养所述转染后的细胞,所述条件包括:适于从所述核酸生产前体mRNA、适于所述前体mRNA加工、以及适于所述加工后mRNA翻译成免疫球蛋白重链,其中所述转染细胞通过选择剪接所述前体mRNA,产生可溶性的免疫球蛋白重链和质膜-结合型免疫球蛋白重链,和
c)选择具有质膜-结合型免疫球蛋白重链的细胞作为所述表达免疫球蛋白的细胞。
另外本发明还包含生产由根据本发明的核酸编码的多肽的方法,通过
a)提供真核细胞,
b)用根据本发明的核酸转染所述真核细胞,
c)在适合所述细胞表达的条件下培养所述转染后细胞,
d)从所述细胞的培养液或胞质中回收所述多肽。
另外,本发明还包含核酸,所述核酸从5’到3’的方向包含
a)第一多克隆位点,
b)起始于5’剪接供体位点并且终止于3’剪接受体位点的核酸,其中
i)所述核酸包含翻译终止密码子和多聚腺苷酸化信号,和
ii)所述核酸在前体mRNA加工过程中不会被组成性移除,
c)第二多克隆位点。
该发明此外包含未被组成性移除的根据本发明的核酸的载体。
发明详述
本发明包含选择表达异源多肽或蛋白质的真核细胞的方法,藉此该方法包含
a)用核酸转染真核细胞,所述核酸从5’到3’的方向包含
i)编码异源多肽或蛋白质不具有翻译终止密码子的第一核酸,
ii)起始于5’剪接供体位点和终止于3’剪接受体位点的第二核酸,其包含符合读码框的翻译终止密码子和多聚腺苷酸化信号,
iii)第三核酸其编码
iiia)至少跨膜结构域的片段,或
iiib)GPI-锚定物信号肽,
b)在一定条件下培养所述转染后的细胞,所述条件包括:适于从所述核酸生产前体mRNA、适于所述前体mRNA加工、以及适于所述加工后mRNA翻译成多肽或蛋白质,其中所述转染后细胞通过选择剪接所述前体mRNA,产生可溶性的异源多肽或蛋白质以及质膜-结合型异源多肽或蛋白质,和
c)选择具有质膜-结合型异源多肽或蛋白质的细胞作为所述表达异源多肽或蛋白质的细胞。
对实施本发明有用的方法和技术在下列文献中做了描述,例如,Ausubel,F.M.(编辑),Current Protocols in Molecular Biology,卷I到III(1997);Glover,N.D.,和Hames,B.D.编辑,DNA Cloning:A PracticalApproach,卷I和II(1985),牛津大学出版社;Freshney,R.I.(编辑),Animal Cell Culture-a practical approach,IRL Press Limited(1986);Watson,J.D.等人,Recombinant DNA,第二版,CHSL Press(1992);Winnacker,E.L.,From Genes to Clones;N.Y.,VCH Publishers(1987);Celis,J.编辑,Cell Biology,第二版,Academic Press(1998);Freshney,R.I.,Culture of Animal Cells:A Manual of Basic Technique,第二版,Alan R.Liss,Inc.,N.Y.(1987)。
重组DNA技术的应用使能够生产多肽的多种衍生物。举例来说,能够在其单个或几个氨基酸位置通过置换、改造、交换、删除或插入来修饰这样的衍生物。例如,可以通过定点诱变来进行修饰或衍生。本领域技术人员可以不费力的做到这种修饰(参见例如,Sambrook,J.等人,分子克隆实验指南(1999)冷泉港实验室出版社,纽约,美国;Hames,B.D.,和Higgins,S.G.,Nucleic acid hybridization-a practical approach(1985)IRL出版社,牛津,英国)。
在本发明的范围内,使用的一些术语定义如下:
此处所用的“核酸”是指多聚核苷酸分子,例如DNA和/RNA之类。这个多聚核苷酸分子可以是天然存在的多聚核苷酸分子或合成的多聚核苷酸分子,或是一个或多个天然存在的多聚核苷酸分子或其片段与一个或多个合成的多聚核苷酸分子的组合。此定义还包含,天然存在的多聚核苷酸分子中的一个或多个核苷酸被改变(例如通过突变删除或添加)的情况。可以将核酸分离,或者将其整合入另一个核酸,如在表达载体或真核宿主细胞的染色体中。核酸的特征也在于其核酸序列由单个核苷酸组成。从对应的编码核酸推断出氨基酸的序列以及同样的从编码出的氨基酸序列得到对应的核酸是本领域公知的。因此氨基酸序列同样由其核酸表征。同样由对应的氨基酸序列给出核酸。
本申请中可互换使用的表达“质粒”或“载体”包含,例如穿梭和表达质粒还有转染质粒。通常,质粒也将包含复制起点(例如ColE1和oriP复制起点)和选择标记(例如氨苄青霉素或四环素抗性基因)以分别用于在细菌中复制和选择质粒。
“表达盒”是指这样的构建体,其包含的必需调控元件在细胞中至少表达所含有结构基因。可选的,也包含能使表达的多肽或蛋白质分泌的附加元件。
“基因”是指如在染色体或质粒上的片段,其为多肽或蛋白质表达所必需的。除编码区外,基因还包含其它功能元件,所述功能元件包含启动子、一个或多个内含子和/或外显子,以及一个或多个终止子。
本申请中所用的术语“结构基因”是指基因的编码区,即外显子,没有信号序列但是有插入的内含子。
“选择标记”是指在存在或没有对应的选择物质时,该基因使得带有这个基因的细胞能被特异的选择留下或者相反的基因。有用的正选择标记是抗生素抗性基因。这种选择标记允许被该基因转化的宿主细胞在对应抗生素存在时被正选择留下来;未转化的宿主细胞在选择培养条件下(即选择物质存在时,在选择培养液中)将不能生长或存活。选择性标记可以是正向的、负向的或双功能的。正选择标记使得带有标记的细胞选择留下,而负选择标记使得带有标记的细胞被选择消除。通常,选择标记会带有对药物的抗性或对宿主细胞代谢或分解代谢缺陷的补偿性。真核细胞有用的选择标记包含,例如氨基糖甙磷酸转移酶(APH)基因,如潮霉素磷酸转移酶(hyg)、新霉素和G418APH、二氢叶酸还原酶(DHFR)、胸苷激酶(tk)、谷氨酰胺合成酶(GS)、天冬酰胺合成酶、色氨酸合成酶(选择物质为吲哚)、组氨醇脱氢酶(选择物质为组氨醇D)以及编码嘌呤霉素、博来霉素、腐草霉素、氯霉素、Zeocin和霉酚酸抗性的基因。在WO92/08796和WO94/28143中描述了更多的标记基因。
此处所用的“调控元件”是指以顺式和/或反式存在的核苷酸序列,其为包含感兴趣的结构基因的基因进行转录和/或翻译所必需的。转录调控元件通常包含位于要表达的基因序列上游的启动子、转录起始和终止的位点、以及多聚腺苷酸化信号序列。术语“转录起始位点”指在基因中的核苷酸,对应于将被整合入原始转录本(即前体mRNA)的第一核酸;转录起始位点可能与启动子序列重叠。术语“转录终止位点”是指通常存在于将要转录的感兴趣的基因3′端的核苷酸序列,它引起RNA聚合酶终止转录。多聚腺苷酸化信号序列,或多聚腺苷酸(poly-A)附加信号为在真核mRNA3′端的特异位点的剪切提供了信号,并且转录后在细胞核内为剪切过的3’端加上约100-200个腺苷酸核苷酸(多聚腺苷酸尾)序列。多聚腺苷酸化信号序列可能在剪切位点上游约10-30个核苷酸的位置含有共有序列AATAAA。
翻译调控元件包含翻译起始(AUG)和终止密码子(TAA、TAG或TGA)。在某些构建体中还可以包含内核糖体进入位点(IRES)。
“启动子”是指控制基因或核苷酸序列转录的多聚核苷酸序列,其可操纵的连接在所述基因或核苷酸序列上。启动子含有RNA聚合酶结合和转录起始的信号。考虑在其中选择/可操纵的连接序列的表达,所用的启动子在宿主细胞的细胞类型中是有功能的。包括各种不同来源的组成型、诱导型和抑制型的大量启动子是本领域众所周知的(并在数据库如基因库中确定的),也是可作为或在克隆的多聚核苷酸中得到的(从例如,ATCC等储藏库以及其他商业或个人来源)。“启动子”包含指导结构基因转录的核苷酸序列。通常情况下,启动子位于基因的5′端非编码或非翻译区,接近结构基因的转录起始站点。启动子中在启动转录起作用的序列元件,其常常表征为共有核苷酸序列。这些启动子元件包含RNA聚合酶结合位点、TATA序列、CAAT序列、分化-特异性元件(DSE;McGehee,R.E.Jr.等人,Mol.Endocrinol.7(1993)551-60)、cAMP响应元件(CRE)、血清相应元件(SRE;Treisman,R.,Seminars in Cancer Biol.1(1990)47-58)、糖皮质激素响应元件(GRE)、以及其他转录因子的结合位点如CRE/ATF(O′Reilly,M.A.等人,J.Biol.Chem.267(1992)19938-43)、AP2(Ye,J.等人,J.Biol.Chem.269(1994)25728-34)、SP1、cAMP响应元件结合蛋白(CREB;Loeken,M.R.,Gene Expr.3(1993)253-64)和八聚体因子(总体参见,Watson等人编辑,Molecular Biology of the Gene,第4版,The Benjamin/CummingsPublishing Company,Inc.(1987),和Lemaigre,F.P.和Rousseau,G.G.,Biochem.J.303(1994)1-14)。如果启动子是诱导型启动子,则转录比率将响应诱导物质而升高。相反的,如果启动子是组成型启动子,其转录比率不受诱导物质调控。抑制型启动子也广为人知。例如,c-fos启动子只在生长激素结合到其细胞表面受体时被特异激活。四环素(Tet)调控的表达可以通过人工杂合启动子实现,所述人工杂合启动子由例如,在CMV启动子后面接着两个Tet-操纵基因位点所组成。Tet-阻遏蛋白结合到两个Tet-操纵基因位点上并阻止转录。通过加入诱导物四环素,Tet-阻遏蛋白从Tet-操纵基因位点释放而转录得以进行(Gossen,M.和Bujard,H.PNAS89(1992)5547-5551)。对于其它的诱导性启动子包含金属硫蛋白和热休克启动子,参见例如,Sambrook等人(见上文)和Gossen等人,Curr.Opin.Biotech.5(1994)516-520。在真核启动子中,已被证明具有高表达水平的强启动子是SV40早期启动子、腺病毒主要晚期启动子、小鼠金属硫蛋白-I启动子、肉瘤病毒长末端重复序列、中国仓鼠延伸因子1α(CHEF-1,参见如美国5,888,809)、人EF-1α、泛素、以及人巨细胞病毒早期启动子(CMVIE)。
“启动子”可以是组成型的或诱导型的。可能需要增强子(即作用在启动子上以增强转录的顺式作用DNA元件)与启动子结合起作用,以提高启动子单独作用获得的表达水平,而且其可能被包括作为转录调控元件。包含启动子的多聚核苷酸段通常也会包含增强子序列(如CMV或SV40)。
“可操纵的连接”是指两个或多个组分位置接近,其中如此描述的组分间有一种联系,所述联系允许它们以之预期的方式起作用。举例来说,如果启动子和/或增强子顺式控制或调节其所连接的编码序列转录,它就是可操纵的连接到编码序列上的。通常但非必需的,“可操纵的连接”的DNA序列是连续的,其中需要连续的在读码框中向两个蛋白编码区加入诸如分泌前导肽和多肽、或多肽和跨膜结构域、或多肽和GPI-锚定物信号肽、或多肽和翻译终止密码子。然而,尽管可操纵连接的启动子一般位于编码序列上游,它也不是必须与之相连续。增强子不需要与之相连。如果增强子能提高编码序列的转录,它就是可操纵的连接到编码序列上的。可操纵连接的增强子可以在编码序列的上游、中间或者下游,并且与启动子之间有相当大的距离。如果多聚腺苷酸化位点位于编码序列末端的下游,因而转录得以通过编码序列进入多聚腺苷酸化序列进行,那它就是可操纵的连接到编码序列的。通过本领域周知的重组方法来完成连接,例如使用PCR方法和/或在方便的限制性位点用连接法。如果没有方便的限制性位点,那就根据常规的操作利用合成的寡核苷酸衔接子或接头。
此处所用的术语“前体mRNA的产生”是指DNA转录成为其互补前体mRNA的过程。真核DNA由被称为外显子(编码)和内含子(非编码)的编码和非编码区组成。在DNA转录成为其互补前体mRNA的过程中,外显子和内含子的基因组结构维持不变。
此处所用的术语“前体mRNA的加工”是指转录后修饰的过程。在此步骤中,前体mRNA的内含子部分被剪接掉,即从前体mRNA上移除,加工后的mRNA其5’端加帽而其3’端进行了多聚腺苷酸化。在此步骤中,最后得到了核mRNA,即成熟的mRNA。
本申请所用的术语“跨膜结构域”是指多肽或蛋白质,所述多肽或蛋白质在DNA水平上由至少一个包含胞外区、跨膜区和胞内区的外显子编码。跨膜结构域一般包含三个不同的结构区:N末端胞外区、中间保守的跨膜伸展区、以及C末端胞质区。在一个实施方案中,跨膜结构域从N末端到C末端的方向包含胞外区和跨膜区。跨膜结构域可能还包含胞内区或胞质区。
本申请所用的术语“跨膜结构域的片段”是指跨膜结构域横跨细胞膜的部分,即位于细胞膜中的部分,即跨膜伸展区。
术语“选择性剪接核酸”是指起始于5’剪接供体位点并且终止于3’剪接受体位点的核酸。此核酸含有翻译终止密码子和多聚腺苷酸化信号。这个选择性剪接核酸包含不会从对应前体mRNA被组成性剪接掉的非编码区,诸如,举例来说,在编码免疫球蛋白重链CH3或CH4结构域的外显子之后的内含子。发生在选择性剪接核酸的5’剪接供体位点的“选择性剪接事件”是决定性事件,决定选择性剪接核酸是否从前体mRNA剪接掉或者是否至少留下部分并包含在成熟的(加工后的)mRNA中。
此处所用的术语“选择剪接”及其语法等同成分是指在真核细胞中,从单一的前体mRNA开始,由于对一个或多个内含子进行不同的加工,可以得到不同的成熟mRNA,从而表达出不同亚型的多肽。在本发明的一个实施方案中,产生的前体mRNA的单个的(即只有一个)内含子可被选择性剪接。在另一个实施方案里,第二核酸可以被选择性剪接。在此外另一个实施方案里,第二核酸作为选择性剪接内含子包含其内。不同的加工过程就是“是或不是”的决定,即在选择性剪接过程中要处理的内含子(即“选择性剪接核酸”)将会或者至少部分保留或者被剪接掉。这不需要被理解为导致不同的外显子跟随其后的分支点机制。它实际上是其中选择性剪接核酸或者被剪接掉或者至少部分存留在成熟的mRNA中的机制。通过这种机制,选择性剪接核酸以及其中包含的符合读码框的翻译终止密码子都会或者被保留或者被移除。
选择性剪接是真核细胞的重要调控机制。通过选择性剪接,可以从相同前体mRNA得到成熟mRNA中外显子的不同组合,产生由相同DNA编码的多种不同蛋白质。
此处所用的术语“表达”是指在细胞中发生进行的转录和/或翻译。所需产物在细胞中的转录水平可以基于细胞内存在的相应mRNA数量来确定。举例来说,所选序列转录来的mRNA可以由PCR或由RNA杂交定量(参见Sambrook等人,分子克隆实验指南,冷泉港实验室出版社(1989))。可以通过多种方式定量多肽,如通过ELISA、通过多肽生物活性的测定、或者使用独立于这类活性之外的测定如免疫印迹、SDS聚丙烯酰胺凝胶电泳法、核磁共振(NMR)或放射免疫法,例如使用识别并结合多肽的抗体(见Sambrook等人,1989年,见上文)。
“宿主细胞”指编码多肽或蛋白质的异源核酸引入的细胞。宿主细胞包括用于质粒增殖的原核细胞,和用于表达异源核酸的真核细胞。真核细胞优选是哺乳动物细胞。哺乳动物细胞优选是CHO细胞、BHK细胞、NS0细胞、Sp2/0细胞、COS细胞、HEK细胞、PER.细胞。
“多肽”是由肽键相连的氨基酸组成的聚合物,无论是天然产生的或人工合成的。少于约20个氨基酸残基的多肽可称为“肽”,而由多于100个氨基酸残基组成的多肽或由两个或更多多肽链组成的多肽可称为“蛋白质”。
“蛋白质”是大分子,所述大分子包含至少一个长度为100个氨基酸或以上的多肽链,或包含两个或更多的多肽链。
多肽和蛋白质可能还包含非肽的组分,如糖类基团、金属离子、脂质、羧酸酯或其组合。非肽取代物可能由其中引入该多肽或蛋白质的细胞加入,并可能随细胞类型变化。多肽和蛋白质在此处是根据其氨基酸的骨架结构而定义的;附加物诸如糖类一般不会详细说明,但仍然可能存在。
“异源DNA”或“异源核酸”指并非天然存在于特定宿主细胞的DNA分子或核酸,或一组DNA分子或一组核酸。特定宿主细胞的异源DNA分子可能含有来自宿主细胞种类的DNA(即内源性DNA),只要该宿主DNA与非宿主DNA(即异源DNA)组合。例如,DNA分子含有编码多肽的非宿主DNA部分,其可操纵的连接到包含启动子的宿主DNA部分,则所述DNA分子被认为是异源DNA分子。反过来说,异源DNA可能包含内源性结构基因可操纵的连接到异源启动子上。
由非宿主(即异源)核酸编码的肽或多肽是“异源”肽或多肽。
此处所用的术语“生物活性多肽”是指有机分子,如诸如多肽、蛋白质、糖蛋白、核蛋白、粘蛋白、脂蛋白、合成多肽或蛋白质的生物大分子,所述分子在处于或进入人造生物系统控制时会导致生物学效应,例如使用细胞株和病毒、或在动物体内的(包含但不限于鸟类和哺乳动物,也包含人在内)的生物测定。该生物学效应可以是但不局限于,酶抑制或活化、结合到受体或配体的结合位点或周边、信号触发或信号调节。在一个实施方案里,所述生物活性多肽是从包含免疫球蛋白、免疫球蛋白片段、免疫球蛋白缀合物和抗融合肽(anti fusogenic peptide)的一组多肽中选定的。
生物活性的分子不限于,例如激素、细胞因子、白细胞介素、免疫球蛋白、抗融合肽、生长因子、受体的配体、其激动剂或拮抗剂、细胞毒性试剂、抗病毒试剂、显象剂、酶抑制剂、酶的激活剂或酶活性调节剂,如变构物,以及这些的缀合物。
本申请所用的术语“氨基酸”是指羧基α-氨基酸,它可以直接或作为前体被核酸编码,包含丙氨酸(三字母密码:Ala,单字母密码:A),精氨酸(Arg,R)、天冬酰胺(Asn,N)、天门冬氨酸(Asp,D)、半胱氨酸(Cys,C)、谷氨酰胺(Gln,Q)、谷氨酸(Glu,E)、甘氨酸(Gly,G)、组氨酸(His,H)、异亮氨酸(Ile,I)、亮氨酸(Leu,L)、赖氨酸(Lys,K)、蛋氨酸(Met,M)、苯丙氨酸(Phe,F)、脯氨酸(Pro,P)、丝氨酸(Ser,S)、苏氨酸(Thr,T)、色氨酸(Trp,W)、酪氨酸(Tyr,Y)和缬氨酸(Val,V)。
“克隆载体”是诸如质粒、粘粒、噬菌粒或细菌人工染色体(BAC)的核酸,其在宿主细胞中有自主复制的能力。克隆载体通常包含一个或少量限制性内切酶识别位点,所述限制性内切酶识别位点允许载体在不丧失基本生物学功能的前提下,用可确定的方式插入核酸,以及编码选择标记的核苷酸序列,所述选择标记适用于鉴别和选择用克隆载体转化过的细胞。选择标记通常包括提供四环素、新霉素、G418或氨苄青霉素抗性的基因。
“表达载体”是编码要在宿主细胞中表达的异源多肽或蛋白质的核酸。通常情况下,表达载体包含原核质粒的增殖单位(如对于大肠杆菌,包含原核复制起点和原核选择标记)、真核筛选标记、和一个或多个表达感兴趣的核酸的表达盒,每个表达盒包含启动子、核酸、和转录终止子(包括多聚腺苷酸化信号)。基因表达通常位于启动子控制下,而这样的结构基因就是所述的“可操纵的连接”到启动子。同样,如果调控元件调节核心启动子的活性。调控元件和核心启动子就是可操纵连接的。
“多顺反子转录单元”是其中一个以上的结构基因位于相同启动子的控制下的转录单元。
“分离的多肽”或“分离的蛋白”是基本上没有细胞成分污染的多肽或蛋白质,诸如没有共价连接的糖类、脂类、或其他蛋白质杂质,以及天然状态下与多肽或蛋白一起的非蛋白质杂质。通常,分离的多肽/蛋白质的制备含有高度纯化形式的多肽/蛋白质,即至少约80%的纯度、至少约90%的纯度、至少约95%的纯度、大于95%纯度、或大于99%的纯度。显示某个特定的制备含有分离的多肽或蛋白质的方法之一是,通过制备物的十二烷基硫酸钠(SDS)-聚丙烯酰胺凝胶电泳及凝胶的考马斯亮蓝染色之后的单一条带的外观来显示。但是,术语“分离的”并不排除同样的多肽或蛋白质以另外的物理形式,如二聚体或选择性糖基化或衍生形式存在。
此处所用的术语“免疫球蛋白”是指实质上由免疫球蛋白基因编码的一个或多个多肽组成的蛋白质。这个定义包含变种,所述变种如突变形式(即替换、删除和插入一个或多个氨基酸的形式)、截短的形式、以及融合形式。公认的免疫球蛋白基因,包含不同的恒定区基因,以及无数的免疫球蛋白可变区基因。免疫球蛋白可能以多种形式存在,包含例如Fv、Fab、和F(ab)2以及单链(scFv)(如Huston,J.S.等人,PNAS USA85(1988)5879-5883;Bird,R.E.等人,Science242(1988)423-426;以及总体来说,Hood等人,Immunology,Benjamin N.Y.,第二版(1984)和Hunkapiller,T.和Hood,L.,Nature323(1986)15-16)。
每个免疫球蛋白重型和轻型多肽链,如果存在,可能包含恒定区(一般在羧基末端部分)。重链的恒定区介导了抗体的结合:i)结合到有Fc受体的细胞,如吞噬细胞,或ii)结合到有初生Fc受体(FcRn)也称为Brambell受体的细胞。它也介导了一些因子的结合,包含经典的补体系统因子如C1q组分。此外跨膜结构域可以在免疫球蛋白重链C末端恒定域的后面,即CH3或CH4域。这个跨膜结构域允许形成质膜-结合型免疫球蛋白或免疫球蛋白片段或免疫球蛋白-融合多肽。
每个免疫球蛋白重型和轻型多肽链,如果出现,可能包含可变结构域(一般在羧基末端部分)。免疫球蛋白的轻或重链的可变结构域包含不同部分,即四框架区域(FR)和三超变量区域(CDR)。
术语“至少片段”是指完整的核酸或完整的多肽的一小部分,即至少20%、至少40%、至少60%、或至少80%完整的核酸、多肽、或域。举例来说,“核酸编码免疫球蛋白CH3或CH4结构域的至少片段”是指编码完整的免疫球蛋白CH3或CH4结构域的核酸的一小部分,即至少20%、至少40%、至少60%、或至少80%的编码完整的免疫球蛋白CH3或CH4域的核酸。在一个实施方案里,免疫球蛋白重链的片段是免疫球蛋白重链的C末端片段。
术语“符合读码框的翻译终止密码子”是指接在阅读框无移码的核酸编码区之后的翻译终止密码子(TAA、TAG或TGA),所述阅读框是对前面的核酸编码区而言的,该密码子即是终止编码区翻译过程的。符合读码框的翻译终止密码子是可操纵的连接到前面的核酸编码区的。
术语“没有符合读码框的翻译终止密码子”是指在指定的核酸里缺少翻译终止密码子(TAA、TAG或TGA)和/或可以在核酸内部或其编码区末端找到翻译终止密码子的存在,但是由于一个或两个碱基对的移动,在加工后的mRNA翻译过程中不能被识别(即不符合读码框,不是可操纵的连接),因此在翻译过程中不能终止编码区。
本申请中指的“转录终止子”是50-750个碱基对长度的DNA序列,它赋予RNA聚合酶终止mRNA合成的信号。非常有效的(强)终止子在表达盒的3′端能可行的阻止RNA聚合酶从此读过,尤其是当使用强启动子的时候。无效的转录终止子,可导致形成操纵子样的mRNA,其可能是不希望的(如质粒编码)的基因表达的原因。
本申请中的“在前体mRNA加工过程中没有组成性的移除”和“没有组成性的从(对应)前体mRNA剪接掉”是指在前体mRNA加工过程中没有普遍发生的剪接加工,即核酸的特异内含子只是有时在前体mRNA加工过程中被移除。结果是,得到两个不同的成熟mRNA,其一有这个内含子的至少部份而另一个没有这个内含子。
该申请中所用的术语“GPI-锚定物”是指连接到多肽或蛋白质C末端的翻译后修饰。“GPI-锚定物”有核心结构,其包含至少一个磷酸乙醇胺残基、三甘露糖苷(trimannoside)、氨基葡萄糖残基以及肌醇磷脂。尽管这样核心结构的GPI-锚定物通常拥有某微观不均一性(microheterogeniety),因而具有GPI-锚定物的蛋白质通常是带有相同核心结构不同侧链修饰的同源GPI-锚定物的蛋白质混合物。
术语“GPI-锚定物的信号肽”是指多肽或蛋白质的C末端氨基酸序列,所述多肽或蛋白质由GPI-锚定物连上的氨基酸、可选的间隔肽和疏水肽组成。几乎所有的这种信号肽(即可选的间隔肽和疏水肽)都是由GPI-转氨酶在翻译后移除的,而且在GPI-锚定物的核心磷酸乙醇胺的氨基基团与GPI-锚定物连接的氨基酸之间形成键。
用异源核酸转染后,表达异源核酸编码的异源多肽的细胞被选出。为了选择使用标记。该标记显示了总体细胞中已成功转化的细胞,并有利于选择和分离这些细胞。可以使用不同的标记,诸如,例如可选择标记,或如绿色荧光蛋白(GFP)的可检测标记。
细胞的选择可以单步执行或通过多个步骤进行。在单一/多重步骤的程序中,第一选择可以基于如选择标记,例如可检测标记的阈值水平进行。例如,对于通过流式细胞仪的选择(如由流式细胞仪-荧光激活细胞分选),设定荧光阈值水平,而细胞荧光超过这一阈值水平都被选中。或者,可以收集样品总体细胞中荧光强度前1-15%的细胞(即具有最强检测标记的15%的细胞)、或前1-10%、或前1-5%、或前5-10%、或5-6%的细胞。另一种选择细胞的方法是免疫结合,例如结合到以蛋白A或特定的免疫球蛋白包被的磁珠。所选栏的细胞可作为基本的总体进行进一步的选择步骤,例如通过单细胞接种、培养和ELISA分析(酶联免疫吸附试验),或通过有限稀释克隆,或通过在选择培养液的选择性培养条件下培养数天以扩大数量,并进一步用流式细胞仪选择,或通过另外的具有更高阈值水平的流式细胞仪选择,这可以例如,基于前述的流式细胞仪选择中检测的荧光强度,或通过免疫沉淀法(又见例如,WO2005/020924)。根据本发明选择细胞,可以通过从以下方法集合中选取一种方法来进行,包括流式细胞仪、ELISA法、免疫沉淀、免疫亲和柱色谱法、磁珠免疫亲和分选、基于显微镜的分离方法或免疫结合。在一个实施方案里,根据本发明选择细胞,可以通过从以下方法集合中选取一种方法来进行,包括流式细胞仪、ELISA法、免疫沉淀、免疫亲和柱色谱法、磁珠免疫亲和分选、基于显微镜的分离方法或免疫结合,继之以从以下集合中选取的方法,包括单细胞接种和培养、有限稀释、或通过培养扩大数量,继之以从以下集合中选取的方法,包括流式细胞仪、免疫沉淀、免疫亲和柱色谱法、磁珠免疫亲和分选、基于显微镜的分离方法或ELISA。
本领域公知的转染方法和载体的效率非常高,因此将得到很多转染后的细胞,标记是优选的,其也允许将转染后细胞的表达产量与检测的标记“强度”相关联。因此,它具有连接感兴趣异源多肽的表达和标记的表达的功能。
本发明使用剪接(即选择性剪接)的方法从相同核酸(即从相同表达盒)表达异源多肽和标记,藉此例如没有使用IRES。本发明的标记是表达的异源多肽的质膜结合的形式。本发明的可选择标记包含作为异源多肽的N末端部分和作为至少跨膜结构域的片段或GPI-锚定物的C末端部分。因此,生产出的异源多肽和可选择标记的胞外部分(即检测到的那部分选择标记)是同样的。
本发明包含选择表达异源多肽的真核细胞的方法,藉此该方法包含
a)用核酸转染真核细胞,所述核酸从5’到3’的方向包含
i)编码异源多肽的第一核酸,其没有符合读码框的翻译终止密码子,
ii)起始于剪接供体位点并且终止于剪接受体位点的第二核酸,其包含符合读码框的翻译终止密码子和多聚腺苷酸化信号,
iii)第三核酸,其编码
iiia)至少跨膜结构域的片段,或
iiib)GPI-锚定物信号肽,
b)在一定条件下培养所述转染后的细胞,所述条件包含:适于从所述核酸生产前体mRNA、适于所述前体mRNA加工、以及适于所述加工后mRNA翻译成异源多肽,其中所述转染细胞通过选择剪接所述前体mRNA产生可溶的异源多肽和质膜-结合型异源多肽,和
c)选择具有质膜-结合型异源多肽的细胞使之成为所述表达异源多肽的细胞。
在DNA转录过程中,得到了DNA的拷贝,称之为前信使RNA(前体mRNA)。这个前体mRNA具有与模板DNA相同的构造,即它有基因组的内含子-外显子结构。只有外显子包含编码多肽的氨基酸序列信息。因此,内含子在翻译之前需要从前体mRNA移除。此过程称为RNA-剪接。
“可剪接的核酸”其特征在于至少有5′剪接供体位点、3′剪接受体的位点、以及通常位于受体上游20-50个碱基所谓的分支点。这种结构影响了RNA剪接过程中,从前体mRNA上的5′剪接供体位点到3′剪接受体位点的识别和切除核酸。在剪接步骤中,产生成熟的mRNA,多肽或蛋白质是从其翻译的。在本发明的一个实施方案里,至少有一个核酸,优选第二核酸,是含有附加调控元件的可剪接核酸,所述附加调控元件例如符合读码框的终止密码子和多聚腺苷酸化信号。
但是剪接的过程不是唯一的。这是例如,可能内含子在由前体mRNA开始的前体mRNA加工过程中未被移除,并且因此至少部分嵌入了成熟mRNA。如果符合读码框的终止密码子在这个“可选的”包括在内的内含子中存在,翻译将停止在这个终止密码子,而且产生编码多肽的变体。
识别和切除内含子往往由附加在前体mRNA的顺式作用元件调控。由于他们的功能和位置,这些元件分别被称为外显子剪接增强子(ESE)、外显子剪接沉默子(ESS)、内含子剪接增强子(ISE)或内含子剪接沉默子(Black,D.L.,Annu Rev Biochem72(2003)291-336)。
大多数真核基因的基因组DNA有内含子-外显子结构。举例来说,编码分泌形式的免疫球蛋白重链C末端结构域的外显子内部(即分别为CH3或CH4)是5′剪接供体位点。
如果这个剪接供体位点在该重链前体mRNA加工过程中无效,在此外显子之后的、其中包含终止密码子和多聚腺苷酸化信号的内含子,将至少有部分保留在成熟的mRNA里。然后mRNA翻译为以CH3或CH4结构域为末端的免疫球蛋白重链,表现为可溶免疫球蛋白。这是免疫球蛋白重链的基因在免疫球蛋白分泌细胞的主要加工途径。
如果这个剪接供体位点在该重链前体mRNA加工过程中有效,因此相连的内含子和终止密码子被移除。因而翻译不会在免疫球蛋白重链C末端结构域之后停止。此外,翻译继续进行随后的对编码跨膜结构域外显子的剪接。这种对免疫球蛋白重链基因次要的加工途径,产生在免疫球蛋白生产细胞表面出现质膜-结合的免疫球蛋白形式。
这一过程被称为“选择性剪接”,而在此过程中可选地移除掉的核酸(即内含子)被称为“选择性剪接核酸”。
如果编码异源多肽或蛋白质的核酸由/通过可选择性剪接的核酸连接于编码至少跨膜结构域的片段的核酸,或连接于编码GPI-锚定物信号肽的核酸,即可选择性剪接核酸位于这两个核酸之间,并且藉此这三个核酸被可操纵连接,则表达异源多肽或蛋白质的两个变体:可溶性变体,即只包含多肽或蛋白质的变体;质膜-结合型变体,即包含多肽或蛋白质以及跨膜结构域或GPI-锚定物的变体。
在一个实施方案中,转染的核酸包含在表达盒内。在一个实施方案里,第一核酸在其3’末端没有符合读码框的终止密码子。在另一实施方案中,第一、第二和第三核酸是可操纵连接的。在一个实施方案中,第三核酸编码至少跨膜结构域的片段。在另一实施方案中,第三核酸编码GPI-锚定物信号肽。在本发明的一个实施方案里,第一核酸编码的多肽选自一组蛋白质,其包含免疫球蛋白重链、免疫球蛋白轻链、生物活性多肽、它们的片段以及它们的融合多肽。在一个实施方案中,第三核酸编码至少免疫球蛋白跨膜结构域的片段。
更具体而言,本发明包含选择表达免疫球蛋白重链的真核细胞的方法,藉此该方法包含
a)用核酸转染真核细胞,所述核酸从5’到3’的方向包含
i)编码免疫球蛋白重链的第一核酸,其没有符合读码框的翻译终止密码子,
ii)起始于剪接供体位点并且终止于剪接受体位点的第二核酸,其包含符合读码框的翻译终止密码子和多聚腺苷酸化信号,
iii)第三核酸,其编码
iiia)至少跨膜结构域的片段,或
iiib)GPI-锚定物信号肽,
b)在一定条件下培养所述转染后的细胞,所述条件包含:适于从所述核酸生产前体mRNA、适于所述前体mRNA加工、以及适于所述加工后mRNA翻译成免疫球蛋白重链,其中所述转染细胞通过选择剪接所述前体mRNA,产生可溶性免疫球蛋白重链和质膜-结合型免疫球蛋白重链,和
c)选择具有质膜-结合型免疫球蛋白重链的细胞作为所述表达免疫球蛋白的细胞。
在一个实施方案中,本发明包含选择表达免疫球蛋白真核细胞的方法,藉此该方法包括
a)用核酸转染真核细胞,该核酸包含免疫球蛋白轻链的第一表达盒和第二表达盒,所述第二表达盒从5’到3’的方向包含
i)编码免疫球蛋白重链的第一核酸,其没有符合读码框的翻译终止密码子,
ii)起始于剪接供体位点并且终止于剪接受体位点的第二核酸,其包含符合读码框的翻译终止密码子和多聚腺苷酸化信号,
iii)第三核酸其编码
iiia)至少跨膜结构域的片段,或
iiib)GPI-锚定物信号肽,
b)在一定条件下培养所述转染后的细胞,所述条件包括:适于从所述核酸生产前体mRNA、适于所述前体mRNA加工、以及适于所述加工后mRNA翻译成免疫球蛋白重链,其中所述转染细胞通过选择剪接所述前体mRNA,产生可溶性免疫球蛋白和质膜-结合型免疫球蛋白,和
c)选择具有质膜-结合型免疫球蛋白的细胞使之成为所述表达免疫球蛋白的细胞。
在一个实施方案中,包含本发明的选择表达免疫球蛋白真核细胞的方法,藉此该方法包含
a)用两个核酸同时或相继地转染真核细胞,藉此一个核酸包含免疫球蛋白轻链的表达盒,而另一个核酸含有的表达盒从5’到3’的方向包含
i)编码免疫球蛋白重链的第一核酸,其没有符合读码框的翻译终止密码子,
ii)起始于剪接供体位点并且终止于剪接受体位点的第二核酸,其包含符合读码框的翻译终止密码子和多聚腺苷酸化信号,
iii)第三核酸其编码
iiia)至少跨膜结构域的片段,或
iiib)GPI-锚定物信号肽,
b)在一定条件下培养所述转染后的细胞,所述条件包含:适于从所述核酸生产前体mRNA、适于所述前体mRNA加工、以及适于所述加工后mRNA翻译成免疫球蛋白重链,其中所述转染细胞通过选择剪接所述前体mRNA,产生可溶性免疫球蛋白和质膜-结合型免疫球蛋白,和
c)选择具有质膜-结合型免疫球蛋白的细胞使之成为所述表达免疫球蛋白的细胞。
在一个实施方案中,第三核酸编码的跨膜结构域是免疫球蛋白跨膜结构域。在本发明的一个实施方案里,第二核酸包含仅一个5′剪接供体位点和仅一个3′剪接受体位点。在本发明的另一实施方案里,第二核酸是天然存在的免疫球蛋白重链内含子,继之以编码免疫球蛋白重链CH3或CH4域的外显子,其中在所述内含子中至少有50个连续的核苷酸被缺失。
例如,对于在真核细胞中免疫球蛋白重链的重组表达,运用了有基因组的内含子-外显子结构的核酸或仅含有编码区(即cDNA)的核酸。在两种情况下,编码免疫球蛋白重链C末端结构域的外显子后面的核酸末端都有终止密码子。此后,在基因组结构中包含可选择性剪接核酸和跨膜结构域的随后的内含子和外显子都被省略。因此,这样的核酸只能得到可溶性免疫球蛋白重链。
如果为了重组表达免疫球蛋白或其片段,至少部分保留免疫球蛋白重链基因的基因组结构,即如果保留编码C末端结构域的外显子之后的内含子(即可选择的可剪接核酸)和随后的编码跨膜结构域的外显子,可能进行选择性剪接。在选择性剪接事件中,3′端密码子和编码CH3或CH4结构域外显子的终止密码子,将分别作为或随同内含子序列被移除,而替代产生不同的成熟mRNA,其中编码区(即阅读框)由附加保留的外显子在其3′端延伸。这种mRNA被翻译成C末端延长的免疫球蛋白重链,其中包含由额外的3’外显子编码的额外跨膜结构域或其片段。这种延长的免疫球蛋白重链在免疫球蛋白组装时掺入其中,产生了质膜-结合型免疫球蛋白。令人惊讶的发现是,用根据本发明的这种核酸,可以选择转染后产生异源多肽的细胞。这种方法是普遍适用的,而且并不限于免疫球蛋白。为了实践这种方法,将重组表达异源蛋白质的核酸可操纵的连接到可选择性剪接核酸,并且这种连接符合读码框,所述核酸没有符合读码框的终止密码子,所述可选择性剪接核酸衍生自包含符合读码框的终止密码子和多聚腺苷酸化位点的免疫球蛋白。随后的第三核酸也是可变的,并且可以从编码跨膜结构域或其片段的任何核酸以及编码GPI-锚定物信号肽的任何核酸中选择。这些元件(即编码多肽的核酸、可选择性剪接核酸、核酸编码跨膜结构域或GPI-锚定物信号肽的核酸)可以从不同的基因以及不同的生物体来选择和组合。唯一的前提是,这三个核酸以这样的方式相结合,使可选择性剪接核酸的翻译终止密码子与编码多肽的核酸的阅读框符合读码框,即它可以被核糖体识别并且终止翻译。
一般而言,通过选择性剪接,可选的,异源多肽可溶性形式C末端的小部分将或可能会被作为内含子的部分由前体mRNA上移除。这个部分可选的包含了3′端密码子、3′非编码区、终止密码子和分泌形式的多聚腺苷酸化信号。因此,起始于5′剪接供体位点和终止于3′剪接受体位点(其任选的被移除)的核酸与非选择性加工变体的C末端重叠或可能重叠。
因此,使用免疫球蛋白重链基因的基因组结构至少部分保留的根据本发明的核酸,可以得到异源多肽的两个变体,短的可溶性变体和长的质膜-结合型变体。
在一个实施方案里,其中第一核酸编码免疫球蛋白重链,包含第一核酸的所有外显子、以及除一个之外全部的基因组结构的免疫球蛋白重链基因的内含子。在一个实施方案中,第三核酸编码跨膜结构域的片段或GPI-锚定物信号肽,藉此跨膜结构域的片段是由单一的外显子编码。在另一个实施方案里,该跨膜结构域是免疫球蛋白的跨膜结构域,由M1-M2-外显子融合编码,即由没有基因组的间隔内含子的单一外显子编码。在一个实施方案中,免疫球蛋白跨膜结构域由cDNA所编码。
通过往宿主细胞中引入免疫球蛋白重链基因的整体基因组结构至少部分保留的核酸,得到一方面表达可溶性异源多肽,另一方面表达质膜-结合型异源多肽的细胞。例如,为了取得两种免疫球蛋白的变体,即实现选择性剪接,不需要保留免疫球蛋白重链基因的整个基因组结构,即所有外显子和内含子。这只需要保持选择剪接位点以功能性形式存在。“功能性剪接位点”是包含5′剪接供体位点和3′剪接受体位点的核酸序列,从而使居中的核酸序列由前体mRNA上切除。内含子的识别和切除往往是由附加的顺式作用元件在前体mRNA进行调控的。由于功能和位置,这些元件分别被称为外显子剪接增强子(ESE)、外显子剪接沉默子(ESS)、内含子剪接增强子(ISE)或内含子剪接沉默子(Black,D.L.,Annu Rev Biochem72(2003)291-336,并入以供参考)。
为了选择转染后表达异源多肽的细胞可以使用不同的方法,例如但不限于,通过检测生物学活性的光谱方法如,荧光、ELISA法和其衍生的方法,或通过使用独立于这些活性的测定如,免疫印迹、SDS聚丙烯酰胺凝胶电泳、或放射免疫法(使用识别并结合到异源多肽的抗体)。由于质膜-结合型异源多肽除其C末端外,具有与可溶性异源多肽相同的氨基酸序列和二级结构,因此它可以用例如,与可溶性变体相同的抗体来测定。
多肽的质膜-结合型变体牢固的连接到表达它的细胞上。因此,质膜-结合型变体可被作为标记来分离已成功转染表达异源多肽或蛋白质(如免疫球蛋白)的核酸的细胞。在一个实施方案中,多肽是免疫球蛋白。在一个实施方案中,免疫球蛋白选自IgG、IgE和IgA组成的组。
异源多肽的可溶性变体和异源多肽的质膜-结合型变体的分子比例是从高于50:50至低于100:0,优选从高于75:25至低于100:0。例如,如果真核细胞由根据本发明编码免疫球蛋白的核酸转染,成功转染的细胞可以通过外表出现质膜-结合型免疫球蛋白来选择。
根据该发明的核酸是从5’到3’的方向包含异源多肽编码区、选择性剪接核酸和跨膜结构域或其片段的编码区或GPI-锚定物信号肽编码区的核酸。更具体而言,本发明包含的核酸包含
a)编码异源多肽的第一核酸,其没有符合读码框的翻译终止密码子,
b)起始于5’剪接供体位点并且终止于3’剪接受体位点的第二核酸,其包含符合读码框的翻译终止密码子和多聚腺苷酸化信号,和
c)第三核酸,其编码
i)至少跨膜结构域的片段,或
ii)GPI-锚定物信号肽。
在另外的实施方案中,i)选择性剪接内含子的5′剪接位点的是位于编码异源多肽的核酸正常终止密码子的5’端,ii)第二核酸是选择性剪接核酸,和iii)5′剪接位点并不是组成型的而只是在有时候使用,产生的正常加工后的异源多肽的分子比例,即可溶性多肽相对于选择性加工的异源多肽(即质膜-结合型多肽)的比例,从高于50:50至低于100:0。在一个实施方案里,第一核酸和第二核酸是可操纵的连接的,即第二核酸的翻译终止密码子与编码多肽或其片段的第一核酸的阅读框是在相同读码框的。
可以通过选择性剪接移除掉的核酸,是在编码多肽至少一个片段的核酸之后,在编码跨膜结构域至少一个片段或编码GPI-锚定物信号肽的核酸之前。在一个实施方案中,异源多肽是融合多肽,其包含感兴趣多肽的N末端和免疫球蛋白重链的CH3或CH4域或其变种的至少一个片段的C-末端。在一个实施方案里,核酸包含位于第一核酸和第二核酸和/或第二核酸和第三核酸之间的第四核酸。就是说,第四核酸是位于例如第二核酸之后(即3’剪接受体位点后)和第三核酸5’端之前。
具有位于编码异源多肽的核酸和编码跨膜结构域或编码GPI-锚定物信号肽的核酸之间的选择性剪接核酸,能够表达异源多肽的两种变体:没有跨膜结构域或GPI-锚定物的异源多肽和有跨膜结构域或GPI-锚定物的异源多肽。此异源多肽可以选自而不限于,例如激素、细胞因子、生长因子、受体配体、受体的配体、其激动剂或拮抗剂、细胞毒性试剂、抗病毒试剂、显象剂、酶抑制剂、酶的激活剂或酶活性调节剂,如变构物、免疫球蛋白、或其融合物或片段。在一个实施方案中,多肽是免疫球蛋白、免疫球蛋白重链多肽、或免疫球蛋白融合物。
本发明可实践于任何多肽、任何跨膜结构域和任何GPI-锚定物信号肽,只要藉此嵌入了可选择性剪接的核酸。更具体而言,起始于5′剪接供体位点和终止于3′剪接受体位点的核酸片段需要被适当选择。前面的多肽和随后的跨膜结构域或GPI-锚定物则可以自由选择。
本发明还包含核酸,该核酸包含
a)第一多克隆位点,
b)起始于5’剪接供体位点并且终止于3’剪接受体位点的可剪接核酸,其中
i)所述可剪接核酸包含翻译终止密码子和多聚腺苷酸化信号,和
ii)所述可剪接核酸在前体mRNA加工过程中不会被组成性的移除,
c)第二多克隆位点。
能够实践本发明,例如,但不限于,使用的第二核酸(即选择性剪接核酸),来自编码以下基因的核酸:C3b/C4b受体(1型补体受体)(Hourcade,D.等人,J.Exp.Med.168(1988)1255-1270)、人、鸡、大鼠表皮生长因子受体(EGFR)(Callaghan,T.等人,Oncogene8(1993)2939-2948;Reiter,J.L.和Maihle,N.J.,Nuc.Acids Res.24(1996)4050-4056;Petch,L.等人,Mol.CellBiol.10(1990)2973-2982)、免疫球蛋白(Ig)α、ε、γ、μ重链(Zhang,K.等人,J.Exp.Med.176(1992)233-243;Rogers,J.E.等人,Cell20(1980)303-312;Milcarek,C.和Hall,B.,Mol.Cell Biol.5(1985)2514-2520;Kobrin,B.J.等人,Mol.Cell Biol.6(1986)1687-1697;Cushley,W.等人,Nature298(1982)77;Alt,F.W.等人,Cell20(1980)293-301;Peterson,M.L.,Gene Exp.2(1992)319-327)、人PLA2受体(Ancian,P.等人,J.Biol.Chem.270(1995)8963-8970)、鸡Cek5(Connor,R.J.和Pasquale,E.B.,Oncogene11(1995)2429-2438)、人FGFR(Johnson,D.E.等人,Mol.Cell Biol.11(1991)4627-4634)。
在一个实施方案中,第二核酸是免疫球蛋白的内含子,其位于编码CH3/CH4结构域的外显子和编码至少跨膜结构域片段的外显子之间。在一个实施方案中,第二核酸是来自编码以下基因的核酸:人(hu)免疫球蛋白(Ig)α(alpha)重链、人免疫球蛋白δ(delta)重链、人免疫球蛋白ε(epsilon)重链、人免疫球蛋白γ1、γ2、γ3和γ4(gamma)重链、人免疫球蛋白μ(miu)重链、小鼠免疫球蛋白重链α型、小鼠免疫球蛋白重链δ型、小鼠免疫球蛋白重链ε型、小鼠免疫球蛋白重链γ1型、小鼠免疫球蛋白重链γ2A型、小鼠免疫球蛋白重链γ2B型、小鼠免疫球蛋白重链γ3型、和小鼠免疫球蛋白重链μ型。
在一个实施方案中,第二核酸选自编码以下基因的核酸组成的组:人免疫球蛋白γ1重链、人免疫球蛋白γ2重链、人免疫球蛋白γ3重链、人免疫球蛋白γ4重链、人免疫球蛋白ε重链(1)和人免疫球蛋白ε重链(2)。在一个实施方案中,第二核酸来自/选自编码以下基因的核酸:人免疫球蛋白δ重链、人免疫球蛋白γ1重链、人免疫球蛋白γ2重链、人免疫球蛋白μ重链、小鼠重链α型、小鼠重链γ1型、小鼠重链γ2B型、小鼠重链γ3型以及小鼠重链μ型。
可以实践本发明,例如但不限于,在编码至少跨膜结构域片段的情形下,使用的第三核酸来自编码以下基因的核酸:C3b/C4b受体(1型补体受体)(Hourcade,D.等人,J.Exp.Med.168(1988)1255-1270)、人、鸡、大鼠EGFR(Callaghan,T.等人,Oncogene8(1993)2939-2948;Reiter,J.L.和Maihle,N.J.,Nuc.Acids Res.24(1996)4050-4056;Petch,L.等人,Mol.CellBiol.10(1990)2973-2982)、免疫球蛋白(Ig)α、ε、γ、μ重链(Zhang,K.等人,J.Exp.Med.176(1992)233-243;Rogers,J.E.等人,Cell20(1980)303-312;Milcarek,C.和Hall,B.,Mol.Cell Biol.5(1985)2514-2520;Kobrin,B.J.等人,Mol.Cell Biol.6(1986)1687-1697;Cushley,W.等人,Nature298(1982)77;Alt,F.W.等人,Cell20(1980)293-301;Peterson,M.L.,Gene Exp.2(1992)319-327)、人PLA2受体(Ancian,P.等人,J.Biol.Chem.270(1995)8963-8970)、鸡Cek5(Connor,R.J.和Pasquale,E.B.,Oncogene11(1995)2429-2438)、人FGFR(Johnson,D.E.等人,Mol.Cell Biol.11(1991)4627-4634)。在一个实施方案中,第三核酸选自编码以下基因的核酸:人(hu)免疫球蛋白(Ig)α(alpha)重链、人免疫球蛋白δ(delta)重链、人免疫球蛋白ε(epsilon)重链、人免疫球蛋白γ1、γ2、γ3和γ4(gamma)重链、人免疫球蛋白μ(miu)重链、小鼠免疫球蛋白重链α型、小鼠免疫球蛋白重链δ型、小鼠免疫球蛋白重链ε型、小鼠免疫球蛋白重链γ1型、小鼠免疫球蛋白重链γ2A型、小鼠免疫球蛋白重链γ2B型、小鼠免疫球蛋白重链γ3型、和小鼠免疫球蛋白重链μ型。在一个实施方案中,第三核酸选自编码以下基因的核酸:人免疫球蛋白γ1重链、人免疫球蛋白γ2重链、人免疫球蛋白γ3重链、人免疫球蛋白γ4重链、人免疫球蛋白ε重链(1)和人免疫球蛋白ε重链(2)。在一个实施方案中,第三核酸选自编码以下基因的核酸:人免疫球蛋白δ重链、人免疫球蛋白γ1重链、人免疫球蛋白γ2重链、人免疫球蛋白μ重链、小鼠重链α型、小鼠重链γ1型、小鼠重链γ2B型、小鼠重链γ3型以及小鼠重链μ型。
第三核酸除了可以选自编码至少跨膜结构域片段的核酸组,还可以选自编码GPI-锚定物信号肽的核酸组。编码GPI-锚定物信号肽的核酸组包含来自以下基因的编码GPI-锚定物信号肽的核酸组:人碱性二酯酶、乙酰胆碱酯酶、碱性磷酸酶(肠道、肝及胎盘的)、CAMPATH-1抗原、癌胚抗原、CD55、CD59、CD90、接触蛋白-1、E48抗原、叶酸受体A和B、GPI-锚定的蛋白p137、淋巴细胞功能相关抗原-3、mDIA相互作用蛋白、5′-核苷酸酶、尿激酶纤维蛋白溶酶原激活因子;鼠的LY-6C抗原、LY-6抗原、5′-核苷酸酶、OX45抗原、干细胞抗原-2、血管细胞粘附分子-1、Qa淋巴细胞抗原2(Qa2);兔的海藻糖酶;大鼠来源的短蛋白聚糖蛋白、CD90、磷脂酰肌醇蛋白聚糖蛋白、硫酸肝素蛋白多糖、MRC OX-45抗原、5′-核苷酸酶、胰腺分泌颗粒膜主要糖蛋白(pancreatic secretory granulemembrane major glycoprotein)、T细胞表面蛋白RT6.2;酵母的DNA修复蛋白PHR1、糖磷脂锚定表面蛋白1;猪的淀粉样前体蛋白、二肽酶;布氏锥虫(Trypanosoma brucei)不同的变体的表面蛋白质、多环酸性重复蛋白(polycyclic acidic repetitive protein);刚果锥虫(Trypanosoma congolense)变体来源的表面蛋白YNat1.1;鸡来源的黑素转铁蛋白(melanotransferrin)、中性细胞粘附分子;来自石纹电鳐(Torpedo marmorata)的乙酰胆碱酯酶;仓鼠来源的朊蛋白;牛来源的5′-核苷酸酶;粘液菌来源的膜蛋白GP64、前孢子特异性抗原;和鱿鱼来源的Sgp1、Sgp2。
在表1中给出了根据本发明第二核酸的核酸序列的实例。在表2中给出的实例是,根据本发明第三核酸的核酸序列和根据本发明第三核酸核酸序列相应的氨基酸序列的实例。在表3中列出的实例是,可选的第四核酸的核酸序列和根据本发明的第四可选核酸对应的氨基酸序列。
表1列出了5′剪接供体位点、第二(可选择性剪接)核酸和3′剪接受体位点。由于第二核酸的序列通常超过1kb,列在表1的序列被缩短,显示第二核酸的约前100个和后100个核苷酸,其间由表示完整第二核酸总体大小的数字来分开。第二核酸的完全序列包含于序列列表给出的SEQ IDNO。终止密码子有下划线。
以包含前面的剪接供体位点的共有序列和由垂直线分开的第二核酸的最前6个核苷酸的格式给出剪接供体位点(又见例如,Zhang,M.Q.,HumanMol.Gen.7(1998)919-932)。同样地,通过列出第二核酸的最后6个核苷酸和随后的由垂直线分开的剪接受体位点共有序列给出剪接受体位点。紧随垂直线之后(5’剪接供体位点)和正好在垂直线之前(3’剪接受体位点)的核苷酸是第二(可剪接)核酸的第一个和最后一个核苷酸。第二核酸的终止密码子在表1中有下划线。
第二核酸可以是直接连接到编码异源多肽的核酸,即第一核酸,或是有可选的小(9到21个碱基)间隔核酸序列。在一实施方案中,可选的间隔(第五)核酸是来自第二核酸在基因组上靠前的核酸,由此得到所述第二核酸。
在表2中列出了编码跨膜结构域片段的核酸序列和GPI-锚定物信号肽的氨基酸序列的实例。这个序列可以直接在3′剪接受体位点之后或在其间有可选的间隔序列,即第四核酸。在表3中列出了第四核酸序列和第四核酸相应的氨基酸序列的实例。
在一个实施方案中,根据本发明的核酸包含在所述第二核酸和所述第三核酸之间的第四核酸。在一个实施方案中,根据本发明的核酸包含在所述第一核酸和所述第二核酸之间的第五核酸。在本发明的一个实施方案中,所述第三核酸得自与第二核酸i)相同的,或ii)不同的基因或生物体,即所述第三核酸不必要与所述第二核酸编组在同一基因组之中。
序列是来自公开可获得的基因组或数据库(如人类基因组计划,http://www.gdb.org/;小鼠基因组数据库,http://www,informatics.jax.org/;SwissProt(http://www.expasy,org/sprot/)。其中没有可获得的注解,序列已由软件ALOM预测或完成(见例如,Klein,P.等人,Biochim.Biophys.Acta787(1984)221-226)。在完整的跨膜结构域的情况下,除了SwissProt给出的序列,括号内的序列是通过ALOM预测的。
在一个实施方案里,第二核酸是选自包含SEQ ID NO:001、002、003、004、005、006、007、008、009、151、152、153、154、155、156、157、158、159、169、170、171、172、173的核酸组。在一个实施方案中,根据本发明的核酸的第二核酸是选自从SEQ ID NO:001到SEQ ID NO:009的核酸序列组。在一个实施方案中,第二核酸选自包含SEQ ID NO:002、003、156、157、170、171的核酸组。在一个实施方案中,根据本发明的核酸的第三核酸或者该核酸的片段选自SEQ ID NO:010到SEQ ID NO:018的核酸序列组,和其编码氨基酸序列是SEQ ID NO:019到SEQ IDNO:069的核酸序列的核酸序列组。在一个实施方案中,根据本发明的核酸的第三核酸或者其核酸片段选自SEQ ID NO:010、011、012、013、014、015、016、017、018、160、161、163、164、165、166、167、168、174、175、176的核酸序列组,和其编码氨基酸序列是SEQ ID NO:019到SEQID NO:069和162的核酸序列的核酸序列组。在一个实施方案中,根据本发明的核酸的第三核酸选自,或者其核酸片段选自SEQ ID NO:011、012、165、166、175的核酸序列组,和其编码氨基酸序列是SEQ ID NO:019到SEQ ID NO:036的核酸序列的核酸序列组。在另一实施方案中,根据本发明的核酸的第三核酸选自,或者其核酸片段选自SEQ ID NO:011、012、165、166、175的核酸序列组。在一个实施方案中,根据本发明的核酸的第四核酸选自SEQ ID NO:070到SEQ ID NO:078的核酸序列组和其编码氨基酸序列是SEQ ID NO:079到129的核酸序列的核酸序列组,或所述第四核酸包含选自所述核酸序列组的核酸。
表1:第二核酸的实例
表1:接上
表1:接上
表1:接上
表2:第三核酸和对应第三核酸的氨基酸的实例
来源 | 核苷酸或氨基酸序列 | SEQIDNO: |
人免疫球蛋白δ重链/跨膜伸展区 | AGCCTGTGGACCACCCTGTCCACGTTTGTGGCCCTCTTCATCCTCACCCTCCTCTACAGCGGCATTGTCACTTTCATCAAGGTGAAG | 010 |
人免疫球蛋白γ1重链/跨膜伸展区 | GGGCTGTGGACGACCATCACCATCTTCATCACACTCTTCCTGTTAAGCGTGTGCTACAGTGCCACCGTCACCTTCTTC | 011 |
人免疫球蛋白γ2重链/跨膜伸展区 | GGGCTGTGGACCACCATCACCATCTTCATCACACTCTTCCTGCTAAGCGTGTGCTACAGTGCCACCATCACCTTCTTC | 012 |
人免疫球蛋白μ重链/跨膜伸展区 | AACCTGTGGGCCACCGCCTCCACCTTCATCGTCCTCTTCCTCCTGAGCCTCTTCTACAGTACCACCGTCACCTTGTTCAAG | 013 |
鼠免疫球蛋白重链α型/跨膜伸展区 | ACTGTGACCTTCCTCACCCTCTTCCTACTGAGCTTGTTCTACAGCACAGCACTCACTGTTACA | 014 |
鼠免疫球蛋白重链γ1型/跨膜伸展区 | GGGCTCTGGACGACCATCACCATCTTCATCAGCCTCTTCCTGCTCAGTGTGTGC(TACAGCGCTGCTGTCACACTCTTCAAGGTA | 015 |
鼠免疫球蛋白重链γ2B型/跨膜伸展区 | GGGCTCTGGACGACCATCACCATCTTCATCAGCCTCTTCCTGCTCAGCGTGTGC(TACAGCGCCTCTGTCACACTCTTC) | 016 |
鼠免疫球蛋白重链γ3型/跨膜伸展区 | GGGCTCTGGACGACCATCACCATCTTCATCAGCCTCTTCCTGCTCAGCGTGTGCTACAGCGCCTCTGTCACCCTCTTC | 017 |
鼠免疫球蛋白重链μ型/跨膜伸展区 | (AACCTGTGGACCACTGCCTCCACC)TTCATCGTCCTCTTCCTCCTGAGCCTCTTCTACAGCACCACCGTCACCCTGTTC(AAGGTG) | 018 |
人乙酰胆碱酯酶/GPI-锚定物信号肽,无可选的连接肽 | GEAARRPGLPLPLLLLHQLLLLFLSHLRRL | 019 |
人碱性磷酸酶(小肠)/GPI-锚定物信号肽,无可选的连接肽 | DAAHPVAASLPLLAGTLLLLGASAAP | 020 |
人碱性磷酸酶(肝脏)/GPI-锚定物信号肽,无可选的连接肽 | SSAGSLAAGPLLVALALYPLSVLF | 021 |
人碱性磷酸酶(胎盘)/GPI-锚定物信号肽,无可选的连接肽 | DAAHPGRSVVPALLPLLAGTLLLLETATAP | 022 |
人CAMPATH-1抗原/GPI-锚定物信号肽,无可选的连接肽 | SASSNISGGIFLFFVANAIIHLFCFS | 023 |
人癌胚抗原/GPI-锚定物信号肽,无可选的连接肽 | ASGTSPGLSAGATVGIMIGVLVGVALI | 024 |
人CD55(促衰变因子)/GPI-锚定物信号肽,无可选的连接肽 | SGTTRLLSGHTCFTLTGLLGTLVTMGLLT | 025 |
人CD59/GPI-锚定物信号肽,无可选的连接肽 | NGGTSLSEKTVLLLVTPFLAAAWSLHP | 026 |
人CD90(Thy-1)抗原/GPI-锚定物信号肽,无可选的连接肽 | CEGISLLAQNTSWLLLLLLSLSLLQATDFMSL | 027 |
人接触蛋白-1(CNTN1)/GPI-锚定物信号肽,无可选的连接肽 | SGAPTLSPSLLGLLLPAFGILV | 028 |
人E48抗原/GPI-锚定物信号肽,无可选的连接肽 | DLCNEKLHNAAPTRTALAHSALSLGLALSLLAVILAPSL | 029 |
人叶酸受体A/GPI-锚定物信号肽,无可选的连接肽 | SGAGPWAAWPFLLSLALMLLWLLS | 030 |
人叶酸受体B/GPI-锚定物信号肽,无可选的连接肽 | NAGEMLHGTGGLLLSLALMLQLWLLG | 031 |
人GPI-锚定蛋白p137/GPI-锚定物信号肽,无可选的连接肽 | ARGLMNGYRGPAMDSEEDMMVTALHSLTLQTVVIHSLSSVLPGITLAIN | 032 |
来源 | 核苷酸或氨基酸序列 | SEQIDNO: |
淋巴细胞功能相关抗原-3/GPI-锚定物信号肽,无可选的连接肽,无可选的连接肽 | SSGHSRHRYALIPIPLAVITTCIVLYMNVL | 033 |
人mDIA作用蛋白(DIP)/GPI-锚定物信号肽,无可选的连接肽 | SGASSLHRHFGLLLASLAPLVLCLSLL | 034 |
人5’-核苷酸酶/GPI-锚定物信号肽,无可选的连接肽 | STGSHCHGSFSLIFLSLWAVIFVLYQ | 035 |
人尿激酶纤维蛋白溶酶原激活因子/GPI-锚定物信号肽,无可选的连接肽 | GAAPQPGPAHLSLTITLLMTARLWGGTLLWT | 036 |
鼠细胞表面蛋白LY-6C抗原/GPI-锚定物信号肽,无可选的连接肽 | NAAVPTGASTWTMAGVLLFSLSSVILQTLL | 037 |
鼠Ly-6抗原ThB/GPI-锚定物信号肽,无可选的连接肽 | NERLVSAAPGHALLSSVTLGLATSLSLLTVMALCL | 038 |
鼠5’-核苷酸酶/GPI-锚定物信号肽,无可选的连接肽 | SAASHYQGSFPLVILSLSAVIFVLYQ | 039 |
鼠OX45抗原(BCM-1或Blast-1)/GPI-锚定物信号肽,无可选的连接肽 | SSGVCWTATWLVVTTLIIHRILLT | 040 |
鼠干细胞抗原2/GPI-锚定物信号肽,无可选的连接肽 | NFSAAGLGLRASIPLLGLGLLLSLLALLQLSP | 041 |
鼠血管细胞粘附分子1/GPI-锚定物信号肽,无可选的连接肽 | AKSFYFICYLCLYLAL | 042 |
大鼠短蛋白聚糖(brevican)蛋白/GPI-锚定物信号肽,无可选的连接肽 | GNSAEGSMPAFLLFLLLQLWDT | 043 |
大鼠CD90(Thy-1)抗原/GPI-锚定物信号肽,无可选的连接肽 | CGGISLLVQNTSWLLLLLLSLSFLQATDFISL | 044 |
大鼠磷脂酰肌醇蛋白聚糖蛋白/GPI-锚定物信号肽,无可选的连接肽 | SAATRPEPHYFFLLFLFTLVLAAARPRWR | 045 |
大鼠MRC OX-45抗原/GPI-锚定物信号肽,无可选的连接肽 | SSGVHWIAAWLVVTLSIIPSILLA | 046 |
大鼠5’-核苷酸酶/GPI-锚定物信号肽,无可选的连接肽 | SAASHYQGSFPLIILSFWAVILVLYQ | 047 |
大鼠胰腺分泌颗粒膜的主要糖蛋白GP2/GPI-锚定物信号肽,无可选的连接肽 | RNTGFLLAWPTFFLPVFLAWLF | 048 |
大鼠T细胞表面蛋白RT6.2/GPI-锚定物信号肽,无可选的连接肽 | SSAGARESCVSLFLVVLPSLLVQLLCLAEP | 049 |
酵母DNA损伤修复蛋白PHR1/GPI-锚定物信号肽,无可选的连接肽 | SSGVKATQQMSMVKLVSIITIVTAFVGGMSVVF | 050 |
酵母糖磷脂锚定表面蛋白I/GPI-锚定物信号肽,无可选的连接肽 | NAATNVKANLAQVVFTSIISLSIAAGVGFALV | 051 |
猪淀粉样前体蛋白/GPI-锚定物信号肽,无可选的连接肽 | ARAAPTTSLGSLMTVSALAILGWSV | 052 |
猪二肽酶/GPI-锚定物信号肽,无可选的连接肽 | SAAPSLHLPPGSLLASLVPLLLLSLP | 053 |
布氏锥虫变体表面蛋白IL Tat1.1/GPI-锚定物信号肽,无可选的连接肽 | SNSFVIHKAPLFLAFLLF | 054 |
布氏锥虫变体表面蛋白MIT117a/GPI-锚定物信号肽,无可选的连接肽 | DSSILVTKKFALTVVSAAFVALLF | 055 |
布氏锥虫变体表面蛋白MIT118a/GPI-锚定物信号肽,无可选的连接肽 | NGSFLTSKQFAFSVVSAAFVALLF | 056 |
布氏锥虫变体表面蛋白MIT221/GPI-锚定物信号肽,无可选的连接肽 | SNSFVISKTPLWLAVLLF | 057 |
布氏锥虫变体表面蛋白MITat1.1000BC/GPI-锚定物信号肽,无可选的连接肽 | DGSFLVNKKFALMVYDFVSLLAF | 058 |
布氏锥虫变体表面蛋白MITat1.5b/GPI-锚定物信号肽,无可选的连接肽 | NGSFLTSKQFALMVSAAFVTLLF | 059 |
布氏锥虫多环酸性重复蛋白/GPI-锚定物信号肽,无可选的连接肽 | GAATLKSVALPFAIAAAALVAAF | 060 |
来源 | 核苷酸或氨基酸序列 | SEQIDNO: |
布氏锥虫变体表面蛋白TxTat1/GPI-锚定物信号肽,无可选的连接肽 | SNSFVINKAPLLLGFLLF | 061 |
刚果锥虫变体表面蛋白YNat1.1/GPI-锚定物信号肽,无可选的连接肽 | SGSSHGTKAIRSILHVALLM | 062 |
鸡黑素转铁蛋白/GPI-锚定物信号肽,无可选的连接肽 | AGNKLIQQHLLVITFVPFIILGQLQGLG | 063 |
鸡神经细胞粘附分子/GPI-锚定物信号肽,无可选的连接肽 | ATLGSPSTSSSFVSLLLSAVTLLLLC | 064 |
石纹电鳐来源的乙酰胆碱酯酶/GPI-锚定物信号肽,无可选的连接肽,无可选的连接肽 | SSSGTSSSKGIIFYVLFSILYLIFY | 065 |
仓鼠朊蛋白/GPI-锚定物信号肽,无可选的连接肽 | SSAVLFSSPPVILLISFLIFLMVG | 066 |
牛5’-核苷酸酶/GPI-锚定物信号肽,无可选的连接肽 | SAGSHCCGSFSLIFLSVLAVIIILYQ | 067 |
粘液菌膜蛋白Gp64/GPI-锚定物信号肽,无可选的连接肽 | SSATTIAFNAFVVFAIVLSVLLF | 068 |
粘液菌前孢子特异性蛋白/GPI-锚定物信号肽,无可选的连接肽 | GSASTVVASLSLIIFSMILSLC | 069 |
人成纤维细胞生长因子受体1 | TCGCCCCTGTACCTGGAGATCATCATCTATTGCACAGGGGCCTTCCTCATCTCCTGCATGGTGGGGTCGGTCATCGTCTAC | 160 |
鼠表皮生长因子受体 | AAGATACCATCTATTGCCACTGGGATTGTGGGTGGCCTCCTCTTCATAGTGGTGGTGGCCCTTGGGATTGGCCTATTCATGCGAAGACGT | 161 |
人补体受体1(C3b/C4b) | THDALIVGTLSGTIFFILLIIFLSWIILK | 162 |
人白细胞介素受体4 | TTCGAGCAGCACCTCCTGCTGGGCGTCAGCGTTTCCTGCATTGTCATCCTGGCCGTCTGCCTGTTGTGCTATGTCAGCATCACCAA-GATTAAG | 174 |
鼠白细胞介素4受体 | CGCCTTCCACTGGGGGTCACCATCTCCTGCCTCTGCATCCCGTTGTTTTGCCTGTTCTGTTACTTCAGCATTACCAAGATT | 163 |
人免疫球蛋白α重链/跨膜伸展区 | ACCATCACCTTCCTCACCCTCTTCCTGCTGAGCCTGTTCTATAGCACAGCACTGACCGTGACC | 164 |
人免疫球蛋白ε重链/跨膜伸展区 | TGGACGTGGACCGGCCTCTGCATCTTCGCCGCACTCTTCCTGCTCAGCGTGAGCTACAGCGCCGCCATCACGCTCCTCATGGTGCAG | 165 |
人免疫球蛋白γ3重链/跨膜伸展区 | GGGCTGTGGACGACCATCACCATCTTCATCACACTCTTCCTGTTAAGCGTGTGCTACAGTGCCACCGTCACCTTCTTC | 166 |
人免疫球蛋白γ4重链/跨膜伸展区 | GGGCTGTGGACGACCATCACCATCTTCATCACACTCTTCCTGCTAAGCGTGTGCTACAGTGCCACCGTCACCTTCTTC | 175 |
鼠免疫球蛋白重链ε型/跨膜伸展区 | GAGCTGTGGACCAGTATTTGTGTCTTCATCACCCTGTTCCTGCTCAGTGTGAGCTATGGGGCCACTGTCACCGTCCTC | 167 |
鼠免疫球蛋白重链δ型/跨膜伸展区 | GGCCTGTGGCCCACAATGTGCACCTTCGTGGCCCTCTTCCTGCTCACACTGCTCTACAGTGGCTTCGTCACCTTCATCAAGGTA | 176 |
鼠免疫球蛋白重链γ2A型/跨膜伸展区 | GGGCTCTGGACAACCATCACCATCTTCATCAGCCTCTTCCTGCTCAGCGTGTGTTACAGCGCCTCTGTCACACTCTTC | 168 |
表3:第四核酸和对应第四核酸的氨基酸的实例
来源 | 核酸或氨基酸序列 | SEQ ID NO: |
人免疫球蛋白δ重链 | ACCTGGCCATGACCCCCCTGATCCC | 070 |
人免疫球蛋白γ1重链 | AGCTGCAACTGGAGGAGAGCTGTGCGGAG | 071 |
人免疫球蛋白γ2重链 | AGCTGCAACTGGAGGAGAGCTGTGCGGAGGCGC | 072 |
人免疫球蛋白μ重链 | AGGGGGAGGTGAGCGCCGACGAGGA | 073 |
鼠免疫球蛋白重链α型 | AACGTCAAGAGCCACTTTCCTATGTGCTACT | 074 |
鼠免疫球蛋白重链γ1型 | GGCTGCAACTGGACGAGACCTGTGCTGAGGCCCAGGACGGGGAGCTGG | 075 |
鼠免疫球蛋白重链γ2B型 | GGCTAGACCTGGATGATATCTGTGCTG | 076 |
鼠免疫球蛋白重链γ3型 | AGCTGGAACTGAATGAGACCTGT | 077 |
鼠免疫球蛋白重链μ型 | AGGGGGAGGTGAATGCTGAGGAGGAAGGCTTTG | 078 |
人乙酰胆碱酯酶 | GFTH | 079 |
人碱性磷酸酶(小肠) | ACTT | 080 |
人碱性磷酸酶(肝脏) | CAPA | 081 |
人碱性磷酸酶(胎盘) | AGTT | 082 |
人CAMPATH-1抗原 | TSSP | 083 |
人癌胚抗原 | ITVS | 084 |
人CD55(促衰变因子) | SGTT | 085 |
人CD59 | EQLE | 086 |
人CD90(Thy-1)抗原 | KLVK | 087 |
人接触蛋白-1(CNTN1) | QVKI | 088 |
人E48抗原 | CCQE | 089 |
人叶酸受体A | AAAM | 090 |
人叶酸受体B | AMHV | 091 |
人GPI锚定蛋白p137 | GSRG | 092 |
人淋巴细胞功能相关抗原-3 | TCIP | 093 |
人mDIA作用蛋白(DIP) | YGYS | 094 |
人5’-核苷酸酶 | RIKF | 095 |
人尿激酶纤维蛋白溶酶原激活因子 | QYRS | 096 |
鼠细胞表面蛋白LY-6C抗原 | EDLC | 097 |
鼠Ly-6抗原ThB | TDLC | 098 |
鼠5’-核苷酸酶 | RIKF | 099 |
鼠OX45抗原(BCM-1或Blast-1) | DLAR | 100 |
鼠干细胞抗原2 | SSFC | 101 |
鼠血管细胞粘附分子1 | HLMF | 102 |
大鼠短蛋白聚糖蛋白/GPI-锚定物 | APSS | 103 |
大鼠CD90(Thy-1)抗原 | KLVK | 104 |
大鼠磷脂酰肌醇蛋白聚糖蛋白 | GQKT | 105 |
大鼠MRCOX-45抗原 | TLAR | 106 |
大鼠5’-核苷酸酶 | RIKF | 107 |
大鼠胰腺分泌颗粒膜的主要糖蛋白GP2 | NGTP | 108 |
大鼠T细胞表面蛋白RT6.2 | NCLY | 109 |
酵母DNA损伤修复蛋白PHR1 | GSSS | 110 |
酵母糖磷脂锚定表面蛋白1/GPI-锚定物 | SSKK | 111 |
猪淀粉样前体蛋白 | EPLS | 112 |
猪二肽酶 | NYGY | 113 |
来源 | 核酸或氨基酸序列 | SEQ ID NO: |
布氏锥虫变体表面蛋白ILTat1.1 | NTTG | 114 |
布氏锥虫变体表面蛋白MIT117a | NACK | 115 |
布氏锥虫变体表面蛋白MIT118a | EKCR | 116 |
布氏锥虫变体表面蛋白MIT221 | TTGS | 117 |
布氏锥虫变体表面蛋白MITat1.1000BC | EKCC | 118 |
布氏锥虫变体表面蛋白MITat1.5b | EDCR | 119 |
布氏锥虫多环酸性重复蛋白 | EPEP | 120 |
布氏锥虫变体表面蛋白TxTat1 | NTTA | 121 |
刚果锥虫变体表面蛋白YNat1.1 | SHLP | 122 |
鸡黑素转铁蛋白 | QCSG | 123 |
鸡神经细胞粘附分子 | TVIP | 124 |
石纹电鳐来源乙酰胆碱酯酶 | DGEL | 125 |
仓鼠朊蛋白 | DGRR | 126 |
牛5’-核苷酸酶 | RIQF | 127 |
粘液菌膜蛋白Gp64 | NNVC | 128 |
粘液菌前孢子特异性蛋白 | STTT | 129 |
在一个实施方案中,多肽是免疫球蛋白重链而跨膜结构域是免疫球蛋白重链的跨膜结构域。为了表达免疫球蛋白,本发明的核酸与编码免疫球蛋白轻链的核酸一起被引入真核宿主细胞。这些核酸可以位于同一核酸或不同的核酸。
本发明涵盖包含本发明的核酸的载体以及包含本发明载体的真核细胞。
根据本发明的核酸所编码的异源多肽的可溶性变体,可以由以下步骤来生产,包含:用本发明的核酸转染真核细胞、在适合表达所述核酸所编码多肽的条件下培养细胞、以及从细胞的细胞质或培养液中回收多肽。
提供试剂盒用于根据本发明的真核细胞的制备。该试剂盒包含含有至少两个多克隆位点和起始于5′剪接供体位点并终止于3′剪接受体位点的可剪接核酸的载体,其中i)所述核酸包含翻译终止密码子和多聚腺苷酸化信号,和ii)所述核酸在前体mRNA加工过程中不会被组成性移除。
在一个实施方案中,本发明的真核细胞是哺乳动物细胞。在一个实施方案中,哺乳动物细胞是选自下面组成的组的细胞,包含:CHO细胞、NS0细胞、Sp2/0细胞、COS细胞、K652细胞、BHK细胞、PER.C细胞和HEK细胞。
接下来提供实例、序列表和图以帮助对本发明的理解,其真正的范围在所附权利要求书中阐明。应当理解的是,可以对提出的过程进行某些修改而不背离本发明的精神。
附图说明
图1:sIgG/mIgG表达载体(A)、sIgG/mIgG表达载体pmIgGΔ-A(B)、和sIgG表达载体pIgG-A(C)的质粒图谱。所描绘载体元件在正文中有详细说明。
图2:γ链表达盒的外显子内含子结构的示意图。
A:在表达载体pmIgG-A中γ1链恒定区的结构。约7.2kb的DNA片段显示为一条黑线。用空方框和上文指明的名称代表外显子。用于IgG分泌形式[p(A)S]和膜结合形式[p(A)M]的的多聚腺苷酸化位点的位置用黑色条形显示。内含子的编号方式和3′UTR在下图显示。显示了内含子6中的Sph I限制性位点和3’UTR内突变的Sph I位点(在括号内)的相对位置。
B:如A所描述的表达载体pmIgGΔ-A中约4.8kb杂合γ1-γ3-γ4链恒定区的结构。此外,图表还显示来自三个免疫球蛋白重链恒定γ基因位点(IGHG1、IGHG3和IGHG4)的区域。
图3:在表达mIgG的克隆中,IgG的分泌和细胞表面信号的相关性。转染了质粒pmIgG-A(左)、pmIgGΔ-A(中)或作为对照的pIgG-A(右)的六个独立的Sp2/0-Ag14细胞克隆,在限定条件下培养48小时。流式细胞仪荧光强度的中值作为结合到质膜的IgG的数量的测量,通过灰色的条形表示。在相应的细胞培养上清中的分泌型IgG的浓度用黑色菱形代表。
图4:典型的FL1/FSC散点图用于在FACS优势流式细胞仪上分选细胞(BD)。通道R1包含描述的在之前已通过FL1/FSC散点图门控的活细胞群体的5.7%。在分选过程中收集落入R1的细胞。
图5:比生产率的确定。克隆5B11、9B3和J5-H3在转瓶里92个小时培养。
A:细胞生长。在开始转瓶培养后的指定时间点,用CASY细胞计数器测定每个克隆的细胞计数。
B:IgG的生产。在指定时间点(如A中的),通过蛋白A亲和层析法在细胞培养上清中测定IgG的浓度。
C:比生产率。条形图表显示了每个克隆平均的比生产率,这是从前六十九小时内的细胞计数和IgG测定计算出来的(参见表2和参见正文以了解更多详情)。标准偏差用误差棒指示。
图6:免疫荧光显微镜。表达分泌型sIgG和膜结合型mIgG的克隆5B11(图像1、4)、仅表达分泌型sIgG的克隆J5-H3(图像2、5)或未转染的Sp2/0-Ag14细胞(图像3、6)用细胞表面的IgG(图像1-3)或胞内IgG(图像4-6)标记,并且图像记录在Axiophot荧光显微镜。
图7:RNA印迹分析mRNA。
A:用[α-32p]标记的探针A(泳道1-3)或探针B(泳道4-6)杂交的RNA印迹的放射自显影。在每个泳道里有分离自克隆5B11(泳道1、4)、克隆J5-H3(泳道2、5)或未转染的Sp2/0-Ag14细胞(泳道3、6)的10μg总RNA,所述总RNA通过变性琼脂糖凝胶电泳分离开并且转移至尼龙膜。
B:编码膜结合型mIgG或分泌型sIgG的重链的两个mRNA亚型的示意图。外显子用方框表示。与探针A和B互补的区域用黑色条块标记。
图8:IgG的重链亚型的免疫印迹分析。克隆5B11(泳道1、4)、克隆J5-H3(泳道2、5)或未转染的Sp2/0-Ag14细胞(泳道3、6)是根据碱性碳酸盐提取法分别提取的(见正文)。免疫球蛋白来自包含膜整合蛋白的膜组分(泳道1-3)、或来自主要包含细胞可溶性内容物的SN级份(泳道4-6),所述免疫球蛋白通过蛋白A的下拉测定(pull down assay)被纯化,并且通过变性SDS聚丙烯酰胺凝胶电泳被分离。在印迹(blotting)和用辣根过氧化物酶二抗标记之后,重链可以由化学发光反应显现出来。从而显示出分泌型sIgG重链(sIgG HC)和膜结合型mIgG重链(mIgG HC)。较低的栏显示了上栏的印迹部分短曝光的情形。
图9:RNA印迹法印迹法分析mRNA。如图7B所示的,[α-32p]标记的探针A(左栏)或探针B(右栏)杂交的RNA印迹法印迹的放射自显影。在每个泳道里有分离自克隆1A1、1B6、1C5或1D1(泳道1-4)和克隆1D6、2D6、KO2或KO6(泳道6-9)的10μg总RNA,所述总RNA通过变性琼脂糖凝胶电泳分离开来并且转移至尼龙膜。克隆“27”只表达sIgG从而加入作为对照(泳道5、10)。可显示mIgG和sIgG亚型的重链mRNA。
图10:sIgG/IgG-GPI表达载体pIgG-GPI-B的质粒图谱。
表格说明
表1第二核酸的实例。
表2第三核酸和对应于第三核酸的氨基酸的实例。
表3第四核酸和对应于第四核酸的氨基酸的实例。
表4在sIgG/mIgG表达载体‘pmIgG-A’的构建体中,用于克隆和诱变的寡核苷酸引物
表5基于在指数增长阶段的几个时间点计算的四个不同的SPRs所确定的每个选定克隆的平均SPR。
表6表4显示,单细胞培养于96孔板3周后,不同IgG浓度水平的克隆的分布,和通过ELISA对515mIgG-分选克隆和550对照克隆的细胞培养上清的分析。
表7表5显示,在第二轮筛选中,不同IgG浓度水平的克隆的分布。
表8用TT细胞计数器( Systems)确定每个克隆在摇瓶振荡培养0、1、2、3、4和7天的细胞数。
表9通过ELISA进行的摇瓶振荡培养10天的生产力测定。
序列说明
SEQ ID NO:001第二核酸来自人免疫球蛋白δ重链
SEQ ID NO:002第二核酸来自人免疫球蛋白γ1重链
SEQ ID NO:003第二核酸来自人免疫球蛋白γ2重链
SEQ ID NO:004第二核酸来自人免疫球蛋白μ重链
SEQ ID NO:005第二核酸来自小鼠免疫球蛋白重链α型
SEQ ID NO:006第二核酸来自小鼠免疫球蛋白重链γ1型
SEQ ID NO:007第二核酸来自小鼠免疫球蛋白重链γ2B型
SEQ ID NO:008第二核酸来自小鼠免疫球蛋白重链γ3型
SEQ ID NO:009第二核酸来自小鼠免疫球蛋白重链μ型
SEQ ID NO:010第三核酸来自人免疫球蛋白δ重链
SEQ ID NO:011第三核酸来自人免疫球蛋白γ1重链
SEQ ID NO:012第三核酸来自人免疫球蛋白γ2重链
SEQ ID NO:013第三核酸来自人免疫球蛋白μ重链
SEQ ID NO:014第三核酸来自小鼠免疫球蛋白重链α型
SEQ ID NO:015第三核酸来自小鼠免疫球蛋白重链γ1型
SEQ ID NO:016第三核酸来自小鼠免疫球蛋白重链γ2B型
SEQ ID NO:017第三核酸来自小鼠免疫球蛋白重链γ3型
SEQ ID NO:018第三核酸来自小鼠免疫球蛋白重链μ型
SEQ ID NO:019第三核酸来自人类的乙酰胆碱酯酶(AChE)
SEQ ID NO:020第三核酸来自人体肠道碱性磷酸酶
SEQ ID NO:021第三核酸来自人类肝脏碱性磷酸酶
SEQ ID NO:022第三核酸来源于人胎盘碱性磷酸酶
SEQ ID NO:023第三核酸来自人CAMPATH-1抗原
SEQ ID NO:024第三核酸来自人癌胚抗原
SEQ ID NO:025第三核酸来自人CD55
SEQ ID NO:026第三核酸来自人CD59
SEQ ID NO:027第三核酸来自人CD90
SEQ ID NO:028第三核酸来自人接触蛋白-1
SEQ ID NO:029第三核酸来自人E48抗原
SEQ ID NO:030第三核酸来自人叶酸受体A
SEQ ID NO:031第三核酸来自人叶酸受体B
SEQ ID NO:032第三核酸来自人GPI锚定蛋白p137
SEQ ID NO:033第三核酸来自人淋巴细胞功能相关抗原-3
SEQ ID NO:034第三核酸来自人MDIA作用蛋白
SEQ ID NO:035第三核酸来自人5′-核苷酸酶
SEQ ID NO:036第三核酸来自人尿激酶纤维蛋白溶酶原激活因子
SEQ ID NO:037第三核酸来自小鼠细胞表面蛋白LY-6C抗原
SEQ ID NO:038第三核酸来自小鼠LY-6抗原ThB
SEQ ID NO:039第三核酸来自小鼠5′-核苷酸酶
SEQ ID NO:040第三核酸来自小鼠OX45抗原
SEQ ID NO:041第三核酸来自小鼠干细胞抗原2
SEQ ID NO:042第三核酸来自小鼠血管细胞粘附分子1
SEQ ID NO:043第三核酸来自大鼠短蛋白聚糖
SEQ ID NO:044第三核酸来自大鼠CD90抗原
SEQ ID NO:045第三核酸来自大鼠磷脂酰肌醇蛋白聚糖
SEQ ID NO:046第三核酸来自大鼠MRC OX45抗原
SEQ ID NO:047第三核酸来自大鼠5′-核苷酸酶
SEQ ID NO:048第三核酸来自大鼠胰腺分泌颗粒膜的主要糖蛋白GP2
SEQ ID NO:049第三核酸来自大鼠T细胞表面蛋白RT6.2
SEQ ID NO:050第三核酸来自酵母DNA损伤修复蛋白PHR1
SEQ ID NO:051第三核酸来自酵母糖磷脂锚定表面蛋白-1
SEQ ID NO:052第三核酸来自猪淀粉样前体蛋白
SEQ ID NO:053第三核酸来自猪二肽酶
SEQ ID NO:054第三核酸来自布氏锥虫变体表面蛋白IL Tat1.1
SEQ ID NO:055第三核酸来自布氏锥虫变体表面蛋白MIT117a
SEQ ID NO:056第三核酸来自布氏锥虫变体表面蛋白MIT118a
SEQ ID NO:057第三核酸来自布氏锥虫变体表面蛋白MIT221
SEQ ID NO:058第三核酸来自布氏锥虫变体表面蛋白MITat1.1000BC
SEQ ID NO:059第三核酸来自布氏锥虫变体表面蛋白MITat1.5b
SEQ ID NO:060第三核酸来自布氏锥虫布氏多环酸性重复蛋白
SEQ ID NO:061第三核酸来自布氏锥虫变体表面蛋白TxTat1
SEQ ID NO:062第三核酸来自刚果锥虫变体表面蛋白YNat1.1
SEQ ID NO:063第三核酸来自鸡黑素转铁蛋白
SEQ ID NO:064第三核酸来自鸡神经细胞粘附分子
SEQ ID NO:065第三核酸石纹电鳐乙酰胆碱酯酶
SEQ ID NO:066第三核酸来自仓鼠朊蛋白
SEQ ID NO:067第三核酸来自牛5′-核苷酸酶
SEQ ID NO:068第三核酸来自粘液菌膜蛋白Gp64
SEQ ID NO:069第三核酸来自粘液菌前孢子特异性抗原
SEQ ID NO:070第四核酸来自人免疫球蛋白δ重链
SEQ ID NO:071第四核酸来自人免疫球蛋白γ1重链
SEQ ID NO:072第四核酸来自人免疫球蛋白γ2重链
SEQ ID NO:073第四核酸来自人免疫球蛋白μ重链
SEQ ID NO:074第四核酸来自小鼠免疫球蛋白重链α型
SEQ ID NO:075第四核酸来自小鼠免疫球蛋白重链γ1型
SEQ ID NO:076第四核酸来自小鼠免疫球蛋白重链γ2B型
SEQ ID NO:077第四核酸来自小鼠免疫球蛋白重链γ3型
SEQ ID NO:078第四核酸来自小鼠免疫球蛋白重链μ型
SEQ ID NO:079第四核酸来自人AChE
SEQ ID NO:080第四核酸来自人肠道碱性磷酸酶
SEQ ID NO:081第四核酸来自人肝脏碱性磷酸酶
SEQ ID NO:082第四核酸来自人胎盘碱性磷酸酶
SEQ ID NO:083第四核酸来自人CAMPATH-1抗原
SEQ ID NO:084第四核酸来自人癌胚抗原
SEQ ID NO:085第四核酸来自人CD55
SEQ ID NO:086第四核酸来自人CD59
SEQ ID NO:087第四核酸来自人CD90
SEQ ID NO:088第四核酸来自人接触蛋白-1
SEQ ID NO:089第四核酸来自人E48抗原
SEQ ID NO:090第四核酸来自人叶酸受体A
SEQ ID NO:091第四核酸来自人叶酸受体B
SEQ ID NO:092第四核酸来自人GPI锚定蛋白p137
SEQ ID NO:093第四核酸来自人淋巴细胞功能相关抗原-3
SEQ ID NO:094第四核酸来自人MDIA作用蛋白
SEQ ID NO:095第四核酸来自人5′-核苷酸酶
SEQ ID NO:096第四核酸来自人尿激酶纤维蛋白溶酶原激活因子
SEQ ID NO:097第四核酸来自小鼠细胞表面蛋白LY-6C抗原
SEQ ID NO:098第四核酸来自小鼠Ly-6抗原ThB
SEQ ID NO:099第四核酸来自小鼠5′-核苷酸酶
SEQ ID NO:100第四核酸来自小鼠OX45抗原
SEQ ID NO:101第四核酸来自小鼠干细胞抗原2
SEQ ID NO:102第四核酸来自小鼠血管细胞粘附分子1
SEQ ID NO:103第四核酸来自大鼠短蛋白聚糖
SEQ ID NO:104第四核酸来自大鼠CD90抗原
SEQ ID NO:105第四核酸来自大鼠磷脂酰肌醇蛋白聚糖
SEQ ID NO:106第四核酸来自大鼠MRC OX45抗原
SEQ ID NO:107第四核酸来自大鼠5′-核苷酸酶
SEQ ID NO:108第四核酸来自大鼠胰腺分泌颗粒膜的主要糖蛋白的GP2
SEQ ID NO:109第四核酸来自大鼠T细胞表面蛋白RT6.2
SEQ ID NO:110第四核酸来自酵母DNA损伤修复蛋白PHR1
SEQ ID NO:111第四核酸来自酵母糖磷脂锚定表面蛋白-1
SEQ ID NO:112第四核酸来自猪淀粉样前体蛋白
SEQ ID NO:113第四核酸来自猪二肽酶
SEQ ID NO:114第四核酸来自布氏锥虫变体表面蛋白IL Tat1.1
SEQ ID NO:115第四核酸来自布氏锥虫变体表面蛋白MIT117a
SEQ ID NO:116第四核酸来自布氏锥虫变体表面蛋白MIT118a
SEQ ID NO:117第四核酸来自布氏锥虫变体表面蛋白MIT221
SEQ ID NO:118第四核酸来自布氏锥虫变体表面蛋白MITat1.1000BC
SEQ ID NO:119第四核酸来自布氏锥虫变体表面蛋白MITat1.5b
SEQ ID NO:120第四核酸来自布氏锥虫多环酸性重复蛋白
SEQ ID NO:121第四核酸来自布氏锥虫变体表面蛋白TxTat1
SEQ ID NO:122第四核酸来自刚果锥虫变体表面蛋白YNat1.1
SEQ ID NO:123第四核酸来自鸡黑素转铁蛋白
SEQ ID NO:124第四核酸来自鸡神经细胞粘附分子
SEQ ID NO:125第四核酸来自石纹电鳐乙酰胆碱酯酶
SEQ ID NO:126第四核酸来自仓鼠朊蛋白
SEQ ID NO:127第四核酸来自牛5′-核苷酸酶
SEQ ID NO:128第四核酸来自粘液菌膜蛋白Gp64
SEQ ID NO:129第四核酸来自煤粘液菌前孢子特异性抗原
SEQ ID NO:130人免疫球蛋白重链恒定γ1(IGHG1)基因,包含从CH1到CH3的外显子,连同所有间隔的内含子和邻近的3′非编码区。
SEQ ID NO:131由质粒pIgG-A编码的恒定区γ-链的氨基酸序列。
SEQ ID NO:132寡核苷酸引物TM-fw1
SEQ ID NO:133寡核苷酸引物TM-rv1
SEQ ID NO:134寡核苷酸引物M1-rV
SEQ ID NO:135寡核苷酸引物M2-fw
SEQ ID NO:136寡核苷酸引物TM-fw2
SEQ ID NO:137寡核苷酸引物TM-rv2
SEQ ID NO:138寡核苷酸引物mutSphI-1
SEQ ID NO:139寡核苷酸引物SphI-2
SEQ ID NO:140人免疫球蛋白重链恒定γ1(IGHG1)基因,包括带有所有插入的内含子的从CH1到M2的外显子、在内含子6中用于IgG1分泌形式的多聚腺苷酸化位点、和含有用于IgG1质膜结合形式的多聚腺苷酸化位点的3′UTR。
SEQ ID NO:141质粒pmIgG-A编码的短(sIgG)亚型的氨基酸序列。
SEQ ID NO:142质粒pmIgG-A编码的γ-链恒定区的长(mIgG)亚型的氨基酸序列
SEQ ID NO:143人IGHG1基因包括带有所有插入的内含子的从CH1到CH3的外显子、内含子6邻近5′端的部分,所述部分包括用于免疫球蛋白分泌性形式的多聚腺苷酸化位点。
SEQ ID NO:144质粒pmIgGΔ-A编码的γ-链恒定区的短(sIgG)亚型的氨基酸序列。
SEQ ID NO:145质粒pmIgGΔ-A编码的γ-链恒定区的长(mIgG)亚型的氨基酸序列。
SEQ ID NO:146从质粒pmIgGΔ-B中移除的编码序列。
SEQ ID NO:147质粒pIgG-GPI-B生成时插入的编码序列(人胎盘碱性磷酸酶的羧基端信号肽的氨基酸序列(NCBI登录号:M13077;核苷酸:1542-1643))。
SEQ ID NO:148γ-链表达盒恒定区的DNA序列,表达质粒pIgG-GPI-B从CH1到3′UTR。
SEQ ID NO:149质粒pIgG-GPI-B编码的γ-链恒定区sIgG亚型的氨基酸序列。
SEQ ID NO:150γ-链恒定区的长亚型的氨基酸序列,所述恒定区包含由质粒pIgG-GPI-B编码的人胎盘碱性磷酸酶的羧基端信号肽。
SEQ ID NO:151第二核酸来自人成纤维细胞生长因子受体1
SEQ ID NO:152第二核酸来自小鼠表皮生长因子受体
SEQ ID NO:153第二核酸来自人补体受体1(C3b/C4b)
SEQ ID NO:154第二核酸来自小鼠白细胞介素4受体
SEQ ID NO:155第二核酸来自人免疫球蛋白α重链
SEQ ID NO:156第二核酸来自人免疫球蛋白ε重链(1)
SEQ ID NO:157第二核酸来自人免疫球蛋白γ3重链
SEQ ID NO:158第二核酸来自小鼠免疫球蛋白重链ε型
SEQ ID NO:159第二核酸来自小鼠免疫球蛋白重链γ2A型
SEQ ID NO:160第三核酸来自人成纤维细胞生长因子受体1
SEQ ID NO:161第三核酸来自小鼠表皮生长因子受体
SEQ ID NO:162第三核酸来自人补体受体1(C3b/C4b)
SEQ ID NO:163第三核酸来自小鼠白细胞介素4受体
SEQ ID NO:164第三核酸来自人免疫球蛋白α重链
SEQ ID NO:165第三核酸来自人免疫球蛋白ε重链
SEQ ID NO:166第三核酸来自人免疫球蛋白γ3重链
SEQ ID NO:167第三核酸来自小鼠免疫球蛋白重链ε型
SEQ ID NO:168第三核酸来自小鼠免疫球蛋白重链γ2A型
SEQ ID NO:169第二核酸来自人白细胞介素4受体
SEQ ID NO:170第二核酸来自人免疫球蛋白ε重链(2)
SEQ ID NO:171第二核酸来自人免疫球蛋白γ4重链
SEQ ID NO:172第二核酸来自鼠免疫球蛋白δ重链
SEQ ID NO:173第二核酸来自人免疫球蛋白ε重链(2)
SEQ ID NO:174第三核酸来自人白细胞介素4受体
SEQ ID NO:175第三核酸来自人免疫球蛋白γ4重链
SEQ ID NO:176第三核酸来自小鼠免疫球蛋白重链δ型
实施例
实施例1
基因片段的克隆和真核sIgG/mIgG表达载体的构建
a)sIgG表达载体‘pIgG-A’的构建
为表达对蛋白“A”有特异性的免疫球蛋白,构建‘pIgG-A’载体。其编码免疫球蛋白的分泌形式并包含下列元件(见图1C):
1.人γ1重链的转录单元,包含
-来自人巨细胞病毒(HCMV)的即刻早期增强子和启动子,
-合成的5′端非翻译区,包含Kozak共有序列(Kozak,M.,NucleicAcids Res.15(1987)8125-48),
-包含信号序列内含子的鼠免疫球蛋白重链信号序列,
-对蛋白“A”有特异性的抗体的可变的重链的c DNA,
-小鼠/人重链杂交内含子2,包括小鼠Igμ增强子(JH3-JH4切换区)与人免疫球蛋白重链恒定γ1(IGHG1)基因相连,所述IGHG1基因包含从CH1到CH3带有所有插入内含子的外显子、和含有IgG1分泌形式重链的多聚腺苷酸化位点的毗邻的3′非编码区。
重链恒定区的核苷酸序列报道在SEQ ID NO:130里。由质粒pIgG-A编码的γ-链恒定区的氨基酸序列报道在SEQ ID NO:131。
2.人κ轻链的转录单元,包含
-来自人巨细胞病毒(HCMV)的即刻早期增强子和启动子,
-合成的5′端非翻译区,包含Kozak共有序列,
-鼠免疫球蛋白轻链信号序列包含信号序列内含子,
-对蛋白“A”有特异性的抗体的可变κ链的c DNA,
-与人免疫球蛋白κ恒定(IGKC))基因相连的小鼠内含子2Ig κ增强子,和
-人IGKC基因3′非编码区,包含多聚腺苷酸化位点。
3.作为哺乳动物细胞选择标记的新霉素磷酸转移酶的转录单元。
4.来自载体pUC18的复制起点,用于在大肠杆菌中质粒的复制。
5.在大肠杆菌赋予氨苄青霉素抗性的β-内酰胺酶基因。
b)sIgG/mIgG表达载体‘pmIgG-A’的构建
人免疫球蛋白重链恒定γ1(IGHG1)位点的5.2kb基因组片段包含外显子CH3下游内含子(内含子6)的主要部分、外显子M1、外显子M1下游的内含子(内含子7)、外显子M2、以及邻近的包含膜结合形式的γ1链的多聚腺苷酸化信号的3′非编码区(3′UTR),所述片段可以使用PCR从人基因组DNA(罗氏诊断有限公司,Mannheim,德国)扩增,所述PCR使用扩展长模板PCR系统(Expand Long Template PCR System)(罗氏诊断有限公司,德国)和表4中指定的寡核苷酸引物。
表4:用于克隆和诱变的寡核苷酸引物。
*Sph I位点的诱变所改变的核苷酸用小写字体标出。
该扩增片段对应“人14号染色体的基因组重叠群(contig)”(NCBI登录号:NT_026437,反向互补)的87203513-87208691核苷酸。亚克隆PCR产物的测序与相应的14号染色体序列比较,显示了98%的同一性,所有的不同之处都在内含子或3′非编码区(3′UTR)。通过基于位点定向诱变的PCR、使用表2中指定的寡核苷酸引物,毁坏外显子M2终止密码子下游219bp的Sph I限制性位点。此片段接着通过经由Sph I限制性位点克隆入免疫球蛋白γ1链表达载体pIgG-A,与内含子6的5′侧面部分相连接,从而得到完整基因组结构的γ1链转录单元。通过亚克隆构建真核表达载体‘pmIgG-A’,所述载体编码对蛋白“A”有特异性的抗体的分泌形式(sIgG)和膜结合形式(mIgG)。该质粒包含以下组分(又见图1A):
1.前述提到的人γ1重链的转录单元,组成为
-来自人巨细胞病毒(HCMV)的即刻早期增强子和启动子,
-合成的5′端非翻译区,包含Kozak共有序列,
-包含信号序列内含子的鼠免疫球蛋白重链信号序列,
-对蛋白“A”有特异性的抗体的可变重链的cDNA,
-小鼠/人重链杂交内含子2,其包含来自JH3-JH4切换区的小鼠Igμ增强子(Banerji,J.等人,Cell33(1983)729-740;Gillies,S.D.等人,Cell,33(1983)717-728),与人免疫球蛋白重链恒定γ1(IGHG1)基因相连,所述IGHG1基因包含从CH1到M2的带有所有插入的内含子的外显子、在内含子6中用于IgG1分泌形式的多聚腺苷酸化位点、和包含用于IgG1膜结合形式的多聚腺苷酸化位点的3′UTR。
重链恒定区的基因组结构在图2A中有描绘,核苷酸序列报道在SEQID NO:140中。由质粒pmIgG-A编码的γ-链恒定区的短(sIgG)亚型或长(mIgG)亚型的氨基酸序列分别报道在SEQ ID NO:141或SEQ ID NO:142。
2.人κ轻链的转录单元,包含
-来自人巨细胞病毒(HCMV)的即刻早期增强子和启动子,
-合成的5′端非翻译区,包含Kozak共有序列,
-包含信号序列内含子的鼠免疫球蛋白轻链信号序列,
-对蛋白“A”有特异性的抗体的可变的κ链的cDNA,
-与人免疫球蛋白包含信号序列内含子恒定(IGKC))基因相连的小鼠内含子2Igκ增强子(Emorine,L.等人,Nature304(1983)447-449;Picard,D.和Schaffner,W.,Nature(1984)307,80-82),和
-人IGKC基因3′非编码区,包含多聚腺苷酸化位点。
3.新霉素磷酸转移酶的转录单元作为哺乳动物细胞可选择标记。
4.来自载体pUC18的复制起点,用于在大肠杆菌中质粒的复制。
5.在大肠杆菌赋予氨苄青霉素抗性的β-内酰胺酶基因。
c)sIgG/mIgG表达载体‘pmIgGΔ-A’和‘pmIgGΔ-B’的构建
人免疫球蛋白重链恒定γ3(IGHG3)位点的1.1kb基因组片段包含外显子CH3下游内含子(内含子6)的主要部分和外显子M1,用类似实施例1b)描述的方式,通过PCR扩增所述片段(寡核苷酸见表2)。该扩增片段对应“人14号染色体的基因组重叠群”(NCBI登录号:NT_026437,反向互补)的87235195-87236300核苷酸,序列同一性达到98%。除了外显子M1里的一个沉默的单核苷酸变换,所有的不同之处都在内含子中,因此并不改变编码氨基酸序列。同样,人免疫球蛋白重链恒定γ4(IGHG4)位点的1.6kb基因组片段可以由PCR扩增出来(寡核苷酸见表2),其包含外显子M2和含有膜结合形式γ4链的多聚腺苷酸化信号的毗邻的3’非翻译区。该片段对应着“人14号染色体的基因组重叠群”(NCBI登录号:NT_026437,反向互补)的87087786-87089377核苷酸,序列同一性达到98%,并且所有的不同之处都在3’UTR。通过基于位点定向诱变的PCR,损坏外显子M2终止密码子下游212bp的Sph I限制性位点。这两个片段都通过PCR扩增(寡核苷酸参见表2)加入在M1和M2之间,并且接着通过在内含子6中的Sph I位点克隆进入免疫球蛋白γ1链表达载体,因而得到具有缺少在M1和M2之间的内含子(内含子7)的基因组结构的杂合γ1-γ3-γ4链表达盒。通过亚克隆构建真核表达载体‘pmIgGΔ-A’,所述载体编码对蛋白“A”有特异性的抗体的分泌型sIgG和膜结合型mIgG。质粒包含以下组分(又见图1B):
1.前述的杂合γ1-γ3-γ4重链转录单元,包含
-来自人巨细胞病毒(HCMV)的即刻早期增强子和启动子,
-合成的5′端非翻译区,包含Kozak共有序列,
-包含信号序列内含子的鼠免疫球蛋白重链信号序列,
-对蛋白质“A”有特异性的抗体的可变的重链的cDNA,
-小鼠/人重链杂交内含子2,包含与人IGHG1基因相连的小鼠Igμ增强子(JH3-JH4切换区),所述IGHG1包含从CH1到CH3的带有全部插入的内含子的外显子、用于IgG1分泌形式的多聚腺苷酸化位点、以及包含免疫球蛋白分泌形式的多聚腺苷酸化位点的毗邻的内含子6的5’部分,
-来自人IGHG3基因的内含子6的3’部分和外显子M1,和
-外显子M2和含有来自人IGHG4基因的免疫球蛋白膜结合形式的多聚腺苷酸化位点的3’非翻译区。
重链恒定区的基因组结构在图2B中有描绘,在SEQ ID NO:143中报道核苷酸序列。由质粒pmIgGΔ-A编码的γ-链恒定区的短(sIgG)亚型或长(mIgG)亚型的氨基酸序列分别报道在SEQ ID NO:144或SEQ ID NO:145。
2.人κ轻链的转录单元,包含
-来自人巨细胞病毒(HCMV)的即刻早期增强子和启动子,
-合成的5′端非翻译区,包含Kozak共有序列,
-包含信号序列内含子的小鼠免疫球蛋白轻链信号序列,
-对蛋白“A”有特异性的抗体的可变的κK链的cDNA,
-与人免疫球蛋白κ恒定(IGKC))基因相连的小鼠内含子2Igκ增强子,和
-人IGKC基因3′非编码区,包含多聚腺苷酸化位点。
3.新霉素磷酸转移酶的转录单元,作为哺乳动物细胞选择标记。
4.来自载体pUC18的复制起点,用于在大肠杆菌中质粒的复制。
5.在大肠杆菌赋予氨苄青霉素抗性的β-内酰胺酶基因。
通过交换编码γ和K链的可变区的cDNA序列,构建出sIgG/mIgG表达载体‘pmIgGΔ-B’。它具有与上面对pmIgGΔ-B所描述的相同构造,但是加以替代的编码对蛋白“B”有特异性的抗体。
实施例2
Sp2/0-Ag14细胞的转染和分析
a)Sp2/0-Ag14细胞的培养和电穿孔
Sp2/0-Ag14杂交瘤细胞(ATCC No.CRL-1581,Shulman,M.等人,Nature276(1978)269-270)培养在RPMI培养液中(英杰公司(InvitrogenCorp.),美国),其中补充10%超低IgG胎牛血清(FCS)(英杰公司,美国)和2mM谷氨酰胺(英杰公司,美国),在37℃、5%CO2和95%的湿度下培养。在20mM的HEPES缓冲液(N-(2-羟乙基)-哌嗪-N′-2-乙烷磺酸),pH7.0,包含137mM NaCl、5mM KCl、0.7mM Na2HPO4和6mM葡萄糖,4℃的条件下,每106个细胞用10μg质粒DNA电穿孔转染细胞。电穿孔在220V和960μF条件下,用Genepulser电穿孔设备(Bio-RadLaboratories公司,美国)进行。转染后,将细胞以每孔100μl培养液中5000个细胞的密度被分到96孔板中。为选择转染的细胞,在电穿孔一天后往培养液里添加500μg/ml的G418(罗氏诊断有限公司,德国)。每两至三天更换一次培养液以维持选择压力。转染后约两个星期,从96孔板分出转染瘤(transfectoma)克隆,并且进一步在适当的培养容器里增殖。在含10%二甲基亚砜(Sigma-Aldrich公司,德国)的低IgG胎牛血清中(英杰公司,美国)中对一个克隆约冻结106个细胞,并储存在液氮中。
每一组106个细胞,用质粒pmIgG-A、pmIgGΔ-A和pIgG-A转染分别得到15、19和12个独立的克隆。从每个转染中随机挑选了6个克隆作进一步的分析。
b)通过流式细胞仪进行IgG定量和分析
实施例2a)的每个克隆接种105个细胞于六孔板的2ml含有G418(如上文所述)的培养液中,并培育48小时。此后,收集上清并且通过按照厂商说明书使用′人Fc检测试剂盒′(CIS国际生物),通过均相时间分辨荧光(HTRF)免疫测定对分泌型IgG定量。为了用流式细胞仪分析质膜-结合型抗体,每个克隆取105个细胞,在4℃用PBS(磷酸盐缓冲液)洗2次,然后重悬在50μl FITC-连接的鼠抗人IgG(Dianova,德国)的F(ab’)2片段,按1:50稀释在含有1%牛血清白蛋白(罗氏诊断有限公司,德国)的PBS中,并在4℃黑暗条件下孵育一小时。在PBS洗2次后,细胞重悬在PBS中并且用BD FACSCalibur流式细胞仪(BD生物科学,美国)进行分析。对于每个克隆测定荧光强度的中值,作为对结合到细胞表面IgG数量的测量。
图3中,将每个克隆在流式细胞仪中荧光强度的中值,与其在细胞培养上清中的IgG浓度一起显示。观察到,对转染pmIgG-A或pmIgGΔ-A的克隆(即编码sIgG和mIgG两种形式的质粒)分泌型IgG数量的增加与升高的细胞表面信号相对应。这种相关性在转染pIgG-A(即只编码sIgG、抗体分泌形式的质粒)的克隆中观察不到。
实施例3
用流式细胞仪分选稳定转染的细胞
为了产生稳定表达IgG的克隆,将3×106个Sp2/0-Ag14细胞用质粒pmIgG-B由电穿孔转染。转染后一天,细胞被转移到500μg/ml的G418选择培养液并且作为库培养约两个星期。选择后,结合到转染后细胞表面的IgG被标记并且通过流式细胞仪分选信号最强的细胞。因此,将来自库的4×106个细胞在4℃用培养液洗2遍,然后与FITC-连接的鼠抗人IgG(Dianova,德国)的F(ab’)2片段共同孵育,在培养液中1:50稀释,冰上放置20分钟。经过两步培养液的洗涤后,将细胞重悬在培养液中并转移至BD FACSVantage流式细胞仪(BD生物科学,美国)。设定包含着样本细胞群中荧光强度在前5-6%的细胞的门通道(图4),并且收集通过这个通道分选的细胞。在选择培养液中培养所收集的细胞数天以扩增。用这些细胞进行如上所述的第二和继而第三分选循环。替代将分选细胞作为库来收集的是,使用第四和最后一个分选步骤使单个细胞沉积在96孔板内。该过程得到了141个独立的克隆,从其中选出了21株克隆用于如实施例2b)所述通过HTRF免疫测定进行的IgG抗体表达分析。从这些中选出的两个克隆5B11和9B3显示出最高的IgG分泌率和相应最强的细胞表面信号,使该克隆适应于转瓶无血清生长,以对其生产力进行进一步评估。
实施例4
克隆的比生产率的测定
为确定其比生产率,在确定的条件下培养克隆5B11和9B3,所述条件为:在ProCHO6k培养液(Cambrex公司,美国)中,补充1×胆固醇/脂类浓缩物(英杰公司,美国)、4mM谷氨酰胺(英杰公司,美国)和250μg/ml的G418(罗氏诊断有限公司,德国)、在100ml转瓶(Belco,美国)培养。细胞在37℃、5%CO2和95%的湿度下生长,直到静止的生长期。在指数增长阶段的数个时间点,用CASY细胞计数器测定每个克隆的细胞数,并且用蛋白A亲和层析法测定上清中IgG的浓度(见下文)。作为参考,在同等条件下培养和分析克隆J5-H3。这个克隆也来自Sp2/0-Ag14转染细胞,和稳定表达与克隆5B11和9B3相同的抗体,虽然只有其分泌形式。克隆J5-H3是通过常规的数次限制稀释及亚克隆步骤所产生的,并从1000多个克隆筛选出来,以满足工业制造过程的生产率的要求。
细胞培养上清中的IgG浓度是由使用 TMexplorer色谱单位(GE健康保健,former Amersham生物科学,瑞典)的分析亲和层析法来测定。将限定体积的细胞培养上清加入蛋白A凝胶柱,以促进IgG结合到亲和基质上。所述柱用100mM,pH5.0的柠檬酸清洗两遍,然后100mM,pH3.0的柠檬酸洗脱结合的抗体。用连续记录的洗脱液在280nm的紫外吸收光谱监控洗脱过程。在用含有确定抗体浓度的标准样品系统校准后,从整合的紫外吸光值计算样本的抗体浓度。
通过下列公式从细胞计数和细胞培养上清的IgG浓度计算每一克隆的比生产率(SPR):
其中
-“Δc(IgG)”是指时间间隔开始和结束之间,在上清中IgG浓度的差异,以[pg/ml]表示,
-“Δt”是指时间间隔的长度[d],以[天]表示。
每个克隆的平均SPR是基于在指数生长阶段的几个时间点计算的四个不同的SPR来确定的(图5和表5)。
表5:比生产率的确定(SPR)。
由膜结合型mIgG分选的5B11和9B3这两个克隆,显示出平均SPR在15和20pg·细胞-1·天-1之间,这是在适合工业规模制造克隆的范围内。这些克隆的SPR可以与能够从通过实验室常规策略在相同细胞系获得的和表达相同抗体的克隆(克隆J5-H3)的SPR相比。
实施例5
用免疫荧光显微镜检测sIgG和mIgG
通过免疫荧光显微镜检测细胞内和细胞表面结合的抗体。为了进行细胞内染色,将附着于多聚赖氨酸涂层显微镜玻片的细胞用溶于PBS的2%(V/V)的甲醛固定10分钟,用溶于PBS的2%(V/V)的Triton X-100通透10分钟,并且用PBS洗两次。非特异性结合位点用溶于PBS的3%(w/V)的BSA(牛血清白蛋白)封闭30分钟。用洗涤缓冲液(WB:0.2%(w/v)BSA、0.1%(v/v)Tween20溶于PBS)洗细胞两次,然后与R-藻红蛋白缀合的山羊抗人IgG(Dianova,德国)的F(ab’)2片段孵育一小时,所述IgG是1:50稀释在WB中。用WB洗涤四次和用双重去离子水洗涤两遍之后,把细胞封固在盖玻片下,存放在4℃黑暗条件直到显微镜观察。为了对细胞表面结合抗体染色,将105细胞用溶于PBS的冷的1%(w/v)BSA洗一遍,然后非特异性结合位点用溶于PBS的1%(w/V)的BSA冰上封闭30分钟。为了标记IgG,细胞与R-藻红蛋白连接的羊抗人IgG(Dianova,德国)的F(ab’)2片段冰上孵育一小时,所述IgG是1:50稀释在PBS中。在用冰PBS洗涤两次之后,细胞附着于多聚-L-赖氨酸涂层的显微镜玻片,并且用溶于PBS的2%(V/V)的甲醛室温固定10分钟。用PBS清洗两次后,将细胞封固在盖玻片下,并且直到显微镜观察之前都存放在4℃黑暗条件。应用Plan-APOCHROMAT40×/1.0物镜,在Axiophot荧光显微镜上分析(CarlZeiss,德国)细胞内和表面标记的细胞。
在克隆5B11中,可以在细胞表面(图6,图像1)以及细胞内(图4)检测到主要是mIgG形式的抗体,这都是表达/分泌途径的IgG形式。相比之下,在克隆J5-H3中,只可以检测到细胞内抗体(图6,图像2和5),因为这株克隆不表达mIgG。在没有转染的Sp2/0-Ag14细胞系(图6,图像3和6),用这两种方法都观察不到染色,因为这些细胞根本不表达IgG。
实施例6
通过RNA印迹检测γ-链mRNA亚型
为了界定通过原始转录本的选择性剪接/多聚腺苷酸化形成的二个重链亚型之间的比例,用RNA印迹来分析稳定转染克隆的RNA。因此,使用RNeasy技术(Qiagen,德国)从细胞提取总RNA。在每个个案中,接着通过变性琼脂糖凝胶电泳将10μg的总RNA分离,并使用RNA印迹法Max系统(Ambion公司,美国)转移至尼龙膜。为了印迹膜的杂交,使用DECAprime II试剂盒(Ambion公司,美国)通过随机引物法用[α-32P]标记两种不同的DNA探针。如同图7B所述,探针′A′与外显子CH1和CH3之间的重链mRNA互补,因此同时能杂交至两个亚型。相反,探针的‘B’仅结合到长mIgG重链mRNA的3’UTR。膜经过杂交和严格的清洗之后,通过放射自显影对其印迹进行分析。
图7A代表用来自克隆5B11、克隆J5-H3和Sp2/0-Ag14细胞系的总RNA的RNA印迹的放射自显影。用′A′探针的杂交显示了在两个抗体表达克隆(泳道1和2)中主要的1.8kb信号,其对应于编码sIgG重链的短mRNA亚型的预期大小。在所描述的长时间曝光里,在2和4kb之间也检测到一些弱信号。3.3kb条带仅在克隆5B11(泳道1)可见,代表了编码mIgG重链的长mRNA亚型,因为当用探针‘B’(泳道4)杂交印迹时,会检测到作为单一的信号的具有相同迁移模式的条带。在泳道1中1.8kb和3.3kb条带的相对信号强度的比较,说明转基因抗体重链的原始转录本主要加工为产生抗体sIgG形式的小mRNA亚型。只有较小量(少于5%)完全被剪接成长mRNA亚型,从而得到质膜结合型mIgG。
实施例7
用碱性碳酸盐提取和免疫印迹纯化和检测IgG重链亚型
为了检测mIgG重链,通过使用Fujiki本发明的碱性碳酸盐提取方法从稳定转染细胞中提取膜蛋白(Fujiki,Y.等人,J.Cell Biol.93(1982)97-102)。在每个个案中,用PBS将107细胞洗两次,然后在冰上用0.1MNaCl洗一次。在悬浮于1ml冷的0.1M pH11.5的Na2CO3之后,通过超声破碎20秒破坏细胞,在冰上孵育30分钟,然后在4℃以200000×g离心60分钟。通过这一步,将仍含有整合膜蛋白的细胞膜与可溶性内容物分离开。收集此上清并加入Triton X-100至终浓度为1%(w/v)、N-辛基-β-D-葡萄糖苷至终浓度60mM、pH7.4的TRIS-HCl至终浓度50mM和NaCI至终浓度为300mM(“SN组分”)。从而,将膜颗粒溶解在1.1ml0.1M pH11.5的Na2CO3、1%(w/v)的Triton X-100、60mM的N-辛基-β-D-葡萄糖苷、50mM pH7.4的TRIS-HCl和300mM的NaCl。在那一步骤之后,通过在15000×g离心10分钟去除不溶性成分,收集其上清作为“膜组分”。进行蛋白A的下拉实验以从膜或SN组分中纯化抗体亚型。因此,每个组分与30μl床体积的蛋白A凝胶柱(GE健康保健,先前是Amersham生物科学,瑞典)在4℃孵育60分钟,然后用含有0.02%(v/v)Igepal CA360的PBS洗4次。使结合到蛋白A基质上的蛋白质在70℃下通过还原蛋白质凝胶样品缓冲液被洗脱,以及通过SDS聚丙烯酰胺凝胶电泳(NuPAGE系统,英杰公司,美国)被分离。根据半干免疫印迹法(Ausubel,F.A.等人(编辑)Current Protocols in Molecular Biology,第2卷,John Wiley和Sons,Inc.,New York(1995))将分离的蛋白质转移到PVDF(聚偏氟乙烯)膜上。用连以辣根过氧化物酶的多克隆兔抗人IgG抗体(DakoCytomation,DAKOA/S,丹麦)标记固定在膜的IgG重链,并且通过在LUMI-成像仪F1(罗氏诊断有限公司)上的化学发光反应(LUMI-增强发光免疫印迹试剂盒,罗氏诊断有限公司)检测。
在表达抗体的两个克隆的膜和SN组分里都可以检测出sIgG重链的强信号(克隆5B11和J5-H3,参见图8,泳道1、2、4和5)。在克隆5B11的个案中,检测到迁移在sIgG重链上方的额外蛋白质(图8,泳道1)。主要在这一克隆的膜组分中发现此条带,所述组分中整合的膜蛋白被富集。其代表了较大的mIgG重链亚型,计算的分子量比sIgG重链高8kDa。在克隆J5-H3中(图8泳道2)始终没有相应的信号,因为此克隆纯粹表达抗体的分泌型形式。
实施例8
CHO-K1细胞的培养和转染
将已预先适应在悬浮培养基中无血清生长的CHO-K1细胞(ATCC号CCL-61;Puck,T.T.等人,J.Exp.Med.108(1958),945-956)培养在ProCHO4-CDM培养液中(Cambrex公司,美国),补充8mM L-丙氨酰-L-谷氨酰胺(英杰公司,美国)和1×羟色胺(HT)补充物(英杰公司,美国),在37℃、5%CO2和95%湿度条件下在悬浮瓶培养。每3-4天将细胞以2×105细胞/ml的密度分到新鲜培养液中。为了转染,每7.5×106细胞用20μg质粒DNA在总体积200μl的PBS中室温下电穿孔细胞。此电穿孔用基因脉冲发生器XCell电穿孔装置(Bio-Rad实验室,美国)在2mm间隙杯中进行,应用了单个160V脉冲15ms的方波操作流程。为了产生稳定表达IgG的克隆,6×107个细胞作为库在以上指定的带有700μg/ml G418培养液中培养两个星期左右(罗氏诊断有限公司,德国),所述G418在转染一天后加入用来选择稳定转染的细胞。
实施例9
用流式细胞仪分选稳定转染的CHO-K1细胞
选择之后,标记结合到转染细胞表面的IgG并且通过流式细胞仪分选带有最强信号细胞的细胞。因此,来自库的4×107个细胞首先通过40μm的尼龙网去除大细胞团,然后与Accumax(PAA)在37℃孵育15分钟,以分开留下的较小的细胞团。细胞在溶于培养液的1%(w/v)的BSA(见实施例8)中冰上孵育20分钟,然后用10ng/ml蛋白A Alexa Fluor488(分子探针公司,英杰公司,美国),在总体积8ml1%(w/v)BSA中在冰上于黑暗中染色细胞30分钟。通过带有1%(w/v)BSA的培养液两步洗涤之后,将细胞重悬于培养液中并且转移到BD FACSAria细胞分选仪(BD生物科学,美国)。通过前散射/侧散射(FSC/SSC)散点图来门控活的单细胞群,亚群定义为包含门控的活细胞中荧光强度在前5%的细胞,从这个亚群收集了约45,000个细胞(mIgG-分选细胞库)。不论它们的荧光,随机从FSC/SSC-选通活细胞群中收集约40,000个细胞作为对照(对照细胞库)。通过在选择培养液(参见实施例8)培养两个星期扩增收集的细胞库。接着进行第二分选循环。如上文所述,对来自mIgG-分选细胞库的细胞的细胞表面IgG染色。使用BD FACSAria细胞分选仪(BD生物科学)从活的单细胞群中分选荧光强度前4%的细胞(由FSC/SSC散点图门控)。将分选的细胞作为单个细胞收集到96孔板内。随机的从FSC/SSC门控的、未染色细胞群中收集来自对照细胞库的单个细胞。在选择培养液(参见实施例8)培养三周扩增单细胞克隆。
实施例10
通过ELISA测定IgG浓度
通过夹心ELISA法测定细胞培养上清中的免疫球蛋白浓度,其中使用连有生物素的抗人IgG的F(ab’)2片段作为捕获试剂和使用缀合过氧化物酶的抗人IgG的F(ab’)2抗体片段检测。
链霉亲和素涂层的96孔板(皮尔森Reacti-BindTM链霉亲和素涂层聚苯乙烯平板,货号15121)用0.5μg/ml连有生物素的多克隆山羊抗人IgG的F(ab’)2抗体片段(F(ab’)2<h-Fcγ>Bi;Dianova,德国,货号109-066-098)捕获抗体(0.1ml/孔)在稀释缓冲液(稀释缓冲液:含0.5%重量体积比(w/v)的牛血清白蛋白的PBS缓冲液)中在室温(RT)下摇动孵育一个小时。此后,用多于0.3ml的洗涤缓冲液(洗涤缓冲液:含1%(w/v)的吐温20的PBS)洗板3次。将含有IgG的细胞培养上清(样本)系列稀释(两倍)达到在稀释缓冲液中0.5-20ng/ml的浓度,加入孔中在室温(RT)下摇动孵育一小时。稀释缓冲液中纯化的标准抗体(0.5-20ng/ml)用于IgG蛋白质标准曲线的生成。在用0.3ml/孔的洗涤缓冲液洗板3次后,用多克隆山羊抗人F(ab’)2-特异IgG(F(ab’)2<h-Fcγ>POD;Dianova,德国,货号109-036-098)的过氧化物酶连接的F(ab’)2片段可检测结合到抗人Fcγ片段的结合复合物。之后用0.3ml/孔的洗涤缓冲液清洗板子3次,平板通过(2,2’-连氮基-双(3-乙基苯并噻唑-6-磺酸)过氧化物酶底物溶液(罗氏诊断有限公司,德国,货号1684302)显影。10-30分钟后,以空白试剂(孵育缓冲液+ABTS溶液)为对照,在Tecan Spectrafluorplus板阅读器(Tecan Deutschland GmbH,德国)测量405nm和490nm处的吸光度。根据下列公式,用在405nm处的吸光度减去490nm处的吸光度进行背景校正:
从标准曲线计算该样本的IgG成份。
实施例11
表达mIgG的CHO克隆的筛选和选择
单细胞沉积在96孔板三周后,通过ELISA分析515个mIgG-分选的克隆和550个对照克隆的细胞培养上清(参见实施例10)。表6显示了不同IgG浓度水平的克隆的分布。
表6:通过ELISA筛选96孔平板沉积的克隆
观察到,在对照方法中,所有克隆的百分之九十显示为1μg/ml和低于1μg/ml的IgG的浓度。相反,没有mIgG-分选克隆落入这个组。此外在这个方法中,有6个克隆达到高于22μg/ml的IgG浓度,然而在对照方法里,测出的最高IgG浓度为12.3μg/ml。接着将所有IgG浓度大于10μg/ml的mIgG-分选克隆和所有IgG浓度大于3μg/ml的对照克隆转移至24孔平板做第二轮筛选。通过在选择培养液(参见实施例8)培养20天使克隆过度生长,然后用测定其细胞培养上清的IgG浓度。表7显示了不同IgG浓度水平的克隆的分布。
表7:通过ELISA筛选放入24孔板的克隆
对照克隆显示出平均6.1μg/ml的IgG浓度和高达16.8μg/ml的表达水平。对比之下,mIgG-分选克隆平均表达为31.4μg/ml,在这组中观察到的克隆具有最高IgG浓度为58.9μg/ml。接着将六个IgG浓度大于45μg/ml的mIgG-分选克隆和两个IgG浓度大于10μg/ml的对照克隆转移到125ml摇瓶中作进一步评估。
实施例12
选定的CHO克隆的生产力评估
为了适应摇动,将实施例11中选定的克隆转移到125ml摇瓶,在25ml选择培养液中(参见实施例8),在标准条件下,于150rpm转动摇床上培养一周时间(参见实施例8)。此后,对于每个克隆,以1×106细胞/ml接种于25ml ProCHO5-CDM培养液(Cambrex公司,美国),补充8mM L-丙氨酰-L-谷氨酰胺(英杰公司,美国)、1×HT的补充物(英杰公司,美国)和400μg/ml的G418(罗氏诊断有限公司,德国),并在125ml摇瓶中进行如前所述的培养。在第0、1、2、3、4、7天,由TT细胞计数器Systems,德国)测定每个克隆的细胞计数,表明所有测试克隆的可比增长(参见表8)。
表8:摇瓶培养的细胞计数
在第十天收集细胞培养上清和通过ELISA测定IgG浓度。
表9:通过ELISA对第10天摇瓶培养的生产力测定
观察到,六个mIgG-分选克隆里的三个(即克隆1A1、1B6和1D6)显示在相当于两个对照克隆的范围内的表达水平。其他三个mIgG-分选克隆(即克隆1C5、1D1和2D6)显示出明显上升的IgG表达,其达到比对照克隆中观察到的高3.3倍。
实施例13
选择的CHO克隆的RNA杂交分析
适应于在摇瓶里生长(参见实施例12)的六个mIgG分选克隆和两个对照克隆,通过RNA杂交分析以确定在原始转录本的选择性剪接形成的两个重链亚型之间的比例。为了对比,对克隆′27′进行平行分析。此克隆已由仅有同一抗体的分泌形式的基因组结构的表达载体稳定转染CHO-K1细胞而获得。RNA印迹法如实施例6中详细描述的进行,并显示在图9中。
实施例14
sIgG/IgG-GPI表达载体‘pIgG-GPI-B’的构建
为了将免疫球蛋白锚定到质膜,原有的由外显子M1的3′部分编码的跨膜结构域以及由外显子M2编码的胞内区,被介导糖基磷脂酰肌醇(GPI)锚定的人胎盘碱性磷酸酶的羧基端信号肽替代。因此,在质粒pmIgGΔ-B(参见实施例1c)中的DNA序列编码的下列53个氨基酸LWTTITIFITLFLLSVCYSATVTFFKVKWIFSSVVDLKQTIVPDYRNMIRQGA(SEQ ID NO:146)
被由编码下列人胎盘碱性磷酸酶(NCBI登录号:M13077;核苷酸:1542-1643)羧基端信号肽的氨基酸序列的DNA序列所替代AGTTDAAHPGRSVVPALLPLLAGTLLLLETATAP(SEQ ID NO:147)。
在SEQ ID NO:148中给出γ-链表达盒恒定区的核酸序列,来自表达质粒pIgG-GPI-B的CH1到3′UTR。
在SEQ ID NO:149中给出质粒pIgG-GPI-B编码的γ-链恒定区的sIgG亚型的相应氨基酸序列。
在SEQ ID NO:150中给出γ-链恒定区的长亚型的相应氨基酸序列,包括由质粒pIgG-GPI-B编码的人胎盘碱性磷酸酶羧基端信号肽。
这个质粒允许表达由重链前体mRNA选择性剪接的抗体的分泌形式(sIgG)和GPI锚定形式(参见图10)。
序列表
<110>弗·哈夫曼-拉罗切有限公司
<120>生产多肽的细胞
<130>23748WO
<150>EP 06010146.6
<151>2006-05-17
<160>176
<170>专利版本3.2
<210>1
<211>2767
<212>DNA
<213>智人(Homo sapiens)
<400>1
gtgagtcacc cccaggccca gggttgggac ggggactctg aggggggcca taaggagctg 60
gaatccatac taggcagggg tgggcactgg gcaggggcgg ggctaggctg tcctgggcac 120
acaggcccct tctcggtgtc cggcaggagc acagacttcc cagtactcct gggccatgga 180
tgtcccagcg tccatccttg ctgtccacac cacgtgctgg cccaggctgg ctggcacagt 240
gtaagaggtg gatacaaccc ctcgccgtgc cctgaggagt ggcggtttcc tcccaagaca 300
ttccccacgg ctgggtgctg ggcacaggcc ttccctggtg tgaccgtgaa tgtggtcacc 360
ctgaacagct gccctctctg gggacatctg actgtccaag accacagtca gcacctctgg 420
gagccagagg ggtctccaga gacccccaga tgtcaggctt gggctcagtg cccagcgaaa 480
ggtcagcccc acacatgccc ataatgggcg cccacccaga gtgacagccc ccagcctcct 540
gccaggccca cccttttccg cccccttgag gcatggcaca cagaccagtg cgcccactgc 600
ccgagcatgg ccccagtggg atgtggtggc cacgaggggc tgtacacaca gcaggaggct 660
gtccgccctg ctcagggcct gctgcctatg ccccagctgt ccaaccaagg gaggcatgga 720
agggcccctg gtgtaagctg gagccaggca cccaggcccc cggccaccct gcagagccaa 780
ggaaaggaag acacccaagt caacaagggg cagggctgag ggctgtccca ggctcttttg 840
gcccgagggg ctgccagcag ccctgacccg gcatgggcct tccccaaaag cgaccctgtg 900
aggtggcctc acagagaacc ccctctgagg acagtgtctg accctgcctg cctcacacag 960
atgggcccca cagcagtggg caacctgggg ggcagcagcc caacctgacc ctgcagggac 1020
tgccccctgc agcagcagct gcttctcagt cccccaacct ccctgtcccc gccagagggt 1080
cttccccgaa gctgcagccc caacccatgg ctgcccacct ggaaccggga ctccctgtcc 1140
actgccccct ccccttcggg gccccatctg tgctggggcc caggttcggc ctacagattc 1200
ccatcattgc catggcctcc tgaccttgcc tatccacccc caaccaccgg ctccatgctg 1260
accctccccc aggctcccac gcccagctgg ccggccatcc ccaggcacag acagtctggg 1320
atctcacagg ttagcctgga ccatccacct ggccagacct gggagaggct ggaagctgcc 1380
ctgccaccat gctccagggc cccaggttgc agtactatgg ggtgagggtg tgtgtgcaca 1440
cctgtgtgta cctaggatat ccgagtgtac ccttgtgccc ccaagcacaa gtctccctcc 1500
caggcagtga ggcccagatg gtgcagtggt tagagctgag gcttatccca cagagaaccc 1560
tggcgccttg gtcaaggaag cccctatgcc tttcttgcct cgatttcccc tcttgtctgc 1620
tgagccagca ggggccacgt cctgggctgc tgtgaggagg aagcaagttg gtgctaggag 1680
gggctcctgt gtgtgcatgg gcgggagggg tgcaggtatc tgagcacccc ggtctccact 1740
tgagagagca gggcaggagc tccctgaccc acccagacta cacacgctgt gtccacgtgt 1800
ctcccattat ctgtggcaga ggatccggct tctttctcaa tttccagttc ttcacaaagc 1860
aatgcctttg taaaatgcaa taagaaatac tagaaaaatg atatgaacag aaagacacgc 1920
cgattttttg ttattagatg taacagacca tggccccatg aaatgatccc ggaccagatc 1980
cgtccacacc cgccactcag cagctctggc cgagctcaca gtacaaccac aataaactct 2040
tgttgaatga actctaggaa gtctgtgacg tggctggttc ttgtcaatgc ttcctgcctg 2100
cccacaggct cttcctcgtg gatggggctg tgcttgccac ggaagcgcgt ttttcccggc 2160
ctaggcttgc cttgggcccc actgccgtct ccagctggag atgaccttct atacacacat 2220
ttgctcatga cagacccttg cttagccccc ttccatggct ccctcctgct gctgggataa 2280
aatcaccttg cctggatatc ccctcctggg cccctttcca ccctccttag tcagcacccc 2340
cagttcaggg cacctgcttt ccccgctgcg gagaagccac tctctccttg ctgcccggct 2400
gtgtcttgcc ttccacacct tgtcacagtg gccacttcct aaggaaggcc tccctgtgtg 2460
caggtgtgca gaagtgcccc agcctcccgt cacctttgtc acgggagccc aatccatgag 2520
agtctatggt tctgtctgtc tgccccactc agggcagcga caagtccagg cggggaggac 2580
acagtaggca gagatttgtc gaggggacat atgagcaaga gggtgaggct gggagctccc 2640
tggagataac cacgcctcct gggaagactc gccgtcattt cagctccacg ctgtgcgggg 2700
gtgggtggag gggtagcctg gccctcatga ccagggagct tctcactcag cccctgttcc 2760
tccccag 2767
<210>2
<211>1287
<212>DNA
<213>智人
<400>2
gtaaatgagt gccacggccg gcaagccccc gctccccagg ctctcggggt cgcgcgagga 60
tgcttggcac gtaccccgtg tacatacttc ccaggcaccc agcatggaaa taaagcaccc 120
agcgcttccc tgggcccctg cgagactgtg atggttcttt ccacgggtca ggccgagtct 180
gaggcctgag tggcatgagg gaggcagagt gggtcccact gtccccacac tggcccaggc 240
tgtgcaggtg tgcctgggcc gcctagggtg gggctcagcc aggggctgcc ctcggcaggg 300
tgggggattt gccagcgtgg ccctccctcc agcagcagct gccctgggct gggccacgag 360
aagccctagg agcccctggg gacagacaca cagcccctgc ctctgtagga gactgtcctg 420
ttctgtgagc gccctgtcct ccgacccgca tgcccactcg ggggcatgcc tagtccatgt 480
gcgtagggac aggccctccc tcacccatct acccccacgg cactaacccc tggcagccct 540
gcccagcctc gcacccgcat ggggacacaa ccgactccgg ggacatgcac tctcgggccc 600
tgtggagaga ctggtccaga tgcccacaca cacactcagc ccagacccgt tcaacaaacc 660
ccgcactgag gttggccggc cacacggcca ccacacacac acgtgcacgc ctcacacacg 720
gagcctcacc cgggcgaacc gcacagcacc cagaccagag caaggtcctc gcacacgtga 780
acactcctcg gacacaggcc cccacgagcc ccacgcggca cctcaaggcc cacgagccgc 840
tcggcagctt ctccacatgc tgacctgctc agacaaaccc agccctcctc tcacaaggtg 900
cccctgcagc cgccacacac acacagggga tcacacacca cgtcacgtcc ctggccctgg 960
cccacttccc agtgccgccc ttccctgcag ctggggtcac atgaggtgtg ggcttcacca 1020
tcctcctgcc ctctgggcct cagggaggga cacgggagac ggggagcggg tcctgctgag 1080
ggccaggtcg ctatctaggg ccgggtgtct ggctgagccc cggggccaaa gctggtgccc 1140
agggcgggca gctgtgggga gctgacctca ggacattgtt ggcccatccc ggccgggccc 1200
tacatcctgg gtcctgccac agagggaatc acccccagag gcccaagccc agggggacac 1260
agcactgacc acccccttcc tgtccag 1287
<210>3
<211>1301
<212>DNA
<213>智人
<400>3
gtaaatgagt gccacggccg gcaagccccc gctccccagg ctctcggggt cgcgcgagga 60
tgcttggcac gtaccccgtc tacatacttc ccgggcaccc agcatggaaa taaagcaccc 120
agcgctgccc tgggcccctg cgagactgtg atggttcttt ccgtgggtca ggccgagtct 180
gaggcctgag tggcatgagg gaggcagagc gggttccact gtccccacac tggcccaggc 240
tgtgcaggtg tgcctgggcc gcctagggtg gggctcagcc aggggctgcc ctcggcaggg 300
tgggggattt gccagcgtgg ccctccctcc agcagcagct gccctgggct gggccacggg 360
aagccctagg agcccctggg gacagacaca cagcccctgc ctctgtagga gactgtcctg 420
tcctgtgagc gccctgtcct ccgacctcca tgcccactcg ggggcatgcc tagtccatgt 480
gcgtagggac aggccctccc tcacccatct acccccacgg cactaacccc tggctgccct 540
gcccagcctc gcacccgcat ggggacacaa ccgactccgg ggacatgcac tctcgggccc 600
tgtggaggga ctggtccaga tgcccacaca cacactcagc ccagacccgt tcaacaaacc 660
ccgcgctgag gttggccggc cacacggcca ccacacacac acgtgcacgc ctcacacacg 720
gagcctcacc cgggcgaacc gcacagcacc cagaccagag caaggtcctc gcacacgtga 780
acactcctca gacacaggcc cccacgagcc ccacgcggca cctcaaggcc cacgagccgc 840
tcggcagctt ctccacatgc tgacctgctc agacaaaccc agccctcctc tcacaaggtg 900
cccctgcagc cgccacacac acacaggccc ccacacacag gggaacacac gccacgtcgc 960
gtccctggca ctggcccact tcccaatgcc gcccttccct gcagctgagg tcacatgagg 1020
tgtgggcttc accatcctcc tgccctctgg gcctcaggga gggacacagg agatggggag 1080
cgggtcctgc tgagggccag gtcgctatct agggctgggt gtctggctga gtcccggggc 1140
caaagctggt gcccagggca ggcagctgtg gggagctgac ctcaggacac tgttggccca 1200
tcccggccgg gccctacatc ctgggtcctg ccacagaggg aatcaccccc agaggcccga 1260
gcccagcagg acacagtatt gaccacccac ttcctgtcca g 1301
<210>4
<211>1914
<212>DNA
<213>智人
<400>4
gtaaacccac cctgtacaac gtgtccctgg tcatgtccga cacagctggc acctgctact 60
gaccctgctg gcctgcccac aggctcgggg cggctggccg ctctgtgtgt gcatgcaaac 120
taaccgtgtc aacggggtga gatgttgcat cttataaaat tagaaataaa aagatccatt 180
caaaagatac tggtcctgag tgcacgatgc tctggcctac tggggtggcg gctgtgctgc 240
acccaccctg cgcctcccct gcagaacacc ttcctccaca gcccccaccc ctgcctcacc 300
cacctgcgtg cctcagtggc ttctagaaac ccctgaattc cctgcagctg ctcacagcag 360
gctgacctca gacttgccat tcctcctact gcttccagaa agaaagctga aagcaaggcc 420
acacgtatac aggcagcaca caggcatgtg tggatacaca tggacagaca cggacacaca 480
caaacacatg gacacacaga gacgtgctaa cccatgggca cacacataca cagacatgga 540
cccacacaca aacatatgtg gacacacatg tacaaacatg cacaggcaca caaagagaac 600
actgactaca ggcacacaca cacacgggca cactacccac aggcacacaa cagatggaca 660
cgcgtacaca gacatgcaca cacccacagg cacaacacgt gcgcatgccg gccggccccc 720
gcccacattc tcccagggcc ctgccggata ctctgtccct gcagcagttt gctccctgcg 780
ctgtgctggc cccggggctt tgggcccagg ctctgcttgt ccttctgtct ctgcttggag 840
gtgctgccat ggcacccagc ttgggctctg cctggggagc ggaggcccca gggatagcat 900
gtgacccctg ctgaggccag gctcctgatg aaggcagcag atagccccca cacccaccgg 960
tgagcagaac cagagcctgt gccatgtgct gagagcaggc agtgactaag catatgggcc 1020
cagagggcag agtggctgcc ctgggcagct gctcctctta gcaggaggcc tcaggagatg 1080
agctagagca agtctgcccc tgcaaatacc acctgctccc caacccacag cagggagcag 1140
gcgaggtcag acagcagcag cccgggaagg accgagcccc agcagggaag gcagggcccg 1200
agtgaggtct ccacacccaa cgcacagtgc tgtctctaac tggggccacc tccgagtccc 1260
cgccacactc ttggcccttt ggagtcctgg gctccaggtg tctcccaagg gcccatctgt 1320
gcaggggatg caaccccccg aatgtcctca tcccactgtg gagctcaggt ctctgtctgc 1380
tccctgggtc ctggcagggt aggacaagtc cgccaggatg tccccatgca gactctgctc 1440
caagagggag ctggagagtc agggccttgg tgagggagtc aggatcgggt tccccccagc 1500
tcagtcctcc cacctgccag cccccacagc acagggcagg gccacacccc ctgcttcccc 1560
ctccaggaga gtcaggacat gctggccgct gctccgctgg ggccccgccc tccagccccc 1620
accttggtct gtgtgctgca tcccccacgc tctctctgcc accccaggac tctgaggaaa 1680
agacctcaga gtcccagccc tgcccagcct cggcctgtgc ccccgctgca tcaggctttc 1740
aggggcccag cccatgccct gggcagtgcc cgagcccccc tgcacttgct ctccccaccc 1800
ctgggtgcag cacagcctag gggccaaggg tgggcctaga ggatgggccc cgggggggct 1860
ttgctgggtg ccaccccagc ctgaccctat tcccccgtgc tgtgtctcct gcag 1914
<210>5
<211>2432
<212>DNA
<213>小家鼠(Mus musculus)
<400>5
gtaaacccac caatgtcagc gtgtctgtga tcatgtcaga gggagatggc atctgctact 60
gagccaccct gcctgtccct actcctgaaa taaactctgt gctcatccaa agtatccctg 120
cacttccatc cagtgcctgt ccatcatcct tagggtctac agaacacagg gaggggtcag 180
ggcccaggga gggagaaaca ccaccaccta agcttgtaag gcctcggaga ctttgaagac 240
ccttcatgcc tagagtatat gtgtatgcat gtctatgtat tggtgagtgc ttatggatgg 300
gtccatgtgc cttcatgtgt gtacctgtgt gcagcaagag aggagtgagt ttgtgtgcac 360
ctatggttta taggtatgta tatgggtata agtacatatg tggacacact atatctatgt 420
ccctgtgagc aagtgtgtgc ctgtgcatac ctatgagcat gcatatatct atgtgtgttc 480
atgttggtgg aaacatgaga gtgtgtgcac ctgccctgtg aatgtgcaat gagtgaatgg 540
gaagtaagaa taaactgggg atgaaatcca aggcgagcag actcagccaa gaaggcaaag 600
ctcatgggag agtccccagg gcagtgggtt gctgcacaag tactctgagt ggtgggtggt 660
gtggctgttt attagaccgc agcttctaac ccaaacaaga cagggataga tggagcaaaa 720
gggtagcacc atcaaggctg gtgccgtgtg ggggtcacaa tagagatgat ggtatgagaa 780
tgaagggatg aacacagggg cactgggaag aagatgccac gcaccctcag taggccatcc 840
ctgaatggaa acatgcattc agggtgacca ctgactcatg agaaagacat atagcatggc 900
acagacaacc ctcagcagca ccctgctctt cacacatgca catgggcata caccctgttc 960
tgcatacatg cacatgggca tacatgcaca gacactcaca tttgcacact cacacagacc 1020
tgaaccctgg aggaagatga agttcacagc ccacacccag cttctggaaa gcaaggggag 1080
tcccctccaa gcccccggga gaatttccag gcccgacact gacaactaca cacaatggac 1140
aagggcatag gcccctccag gtgccccctt tctaacctaa gtctcccttc ccatcctgag 1200
cccaagtatc acaaaccaga agcaggataa gaggcaaatc tgggaagaag caggagcagg 1260
ccatggagac cactcccaca caggcctcgg cctcctccat gggcacccct agaacacaaa 1320
gttctacaac caggtatact tggagcccag gtgtccacaa gagggcagtg ggggccctgc 1380
tcctgtctca caggccttca gaacttcggg gggggggggg aggagttgct ggtttctagc 1440
aacttccctc tgctctcaga gcaagagtgg agtaaaggcc catgcatgcc ctggatactc 1500
tccagcttga aagtccagtt ccctgagaat agcaagagca acagaccagc ttgggaaacc 1560
tgactcagaa aagtaccata gccacccatg gaatccaagg cctggcccat gacagagaat 1620
cctagtaagg aggagtcaat aggtgaaaga ctgaatcccc acagggagag ggggcagaga 1680
agctggagaa tcacgatagt cgggaaagat gatgagtgag cggctggagg gtaatggatg 1740
gctggctaca aggatgaacg atggggatga tggatggatt gatggatggc tgcagtgaca 1800
gaaatacgag tggttgacta tcagtacaca tttcctactg ccacttaaaa tgaaactctg 1860
tgcgcagaga acctgaggac agcagggttg gctctactgg gggtctttac tgttgcttaa 1920
cccccagtca cagctcttaa gaatgcaaca gagtcctctg accacatgtc caggccttgg 1980
tagtctaact ttggcccaga aatcacattg gcctgtatct gtttgaaggt gtctgatctt 2040
acacaaccct agtacccaag ccaagattag cctaagaact ggggtttagg gaggtattgc 2100
taaatatcta gaaaggactg agacctaagg agccttgggt tcaaactctt ctcagagttt 2160
gacagaaaca actctcttac catgacaggt ctctaattaa ggtttcccta agagagagag 2220
atgggtcttg gggccatctc aagaactgct gggattccag tcccttacct gggaccctgc 2280
ctcctcatta tccctgaaca agtcccttcc cgggaataag ggacccctac caccactgtc 2340
cccaaagccc aaggaggcat agaagcagaa accttaagtc tgagccccca tccccatggg 2400
tttggatcta gaaggtcatc tatgtcttac ag 2432
<210>6
<211>1411
<212>DNA
<213>小家鼠
<400>6
gtaaatgatc ccagtgtcct tggagccctc tggtcctaca ggactctgac acctacctcc 60
acccctccct gtgtaaataa agcacccagc actgccttgg gaccctgcaa taatgtcctg 120
gtgatttctg agatgtagag tctagctagg tcatggaatg aggggtctcc atggtttgag 180
gcctgagttg tgactaagga aaaacccata ggcctacact gccacaccca gcacttttga 240
atttgcctga catgaaaaga atttacctct ccctggaaag tggagcctta tccctaggca 300
gttcccttac cagaccttcc tctagcttgc actttgttct gggcacagaa tgtgtctaac 360
cccccaaagc aaggaagaca caacctctac ctccctcact ctgtccttac cccttttcct 420
ggctaagcat ctcactgagt gcgctgaata gatgcatgtg gccacagtct tgcagacaga 480
cccttgccat ctctccactc agctttccag aggctaagtc tagcccgtat ggtgataatg 540
cagggagctc tatgctatct cagtgctatc agactcccaa gtggaggatg aacatggacc 600
cattaaaacc aacctgcgca gcaacaccct gccaataagg cccgtatgtg aaaatgtgca 660
cacatctaca catgcacagg cacacacaca cacacatgca tgggcacaca cacatacaga 720
gagagagaat cacagaaact cccatgagca tcctatacag tactcaaaga taaaaaggta 780
ccaggtctac ccacatgatc atcctcggca tttacaagtg ggccaactga tacagataaa 840
acttttctat gccaaggacg ccaacatata cacaagtccg ctcatgacaa atctgtccct 900
gaacctcaga ctggcgcccg tgactcatac agtggacact cctccaaagc tgtatagctt 960
cctttacttc cctgtgtgta ctttctctga agtacactca tcacacagaa gaggccctgt 1020
gattactctg gccctctgtt cttggtcatc agagaataga cagaagatca ggcaaactac 1080
acagacactt cccacaatca tcacaggccc tgactctgct ctccagtctc aaaactgaag 1140
gctggagcac acagaataag ctcctgcaca ggccaggcca gtatcgggtc cagtgtgtct 1200
gactgagccc agggacaaaa tggcagcact ttggggaact gaggtttctg gtccaagaag 1260
gagagatgga ggcccaggga gggtctgctg acccagccca gcccagccca gctgcagctt 1320
tctcctgggc ctccatacag cctcctgcca cacagggaat ggccctagcc ccaccttatt 1380
gggacaaaca ctgaccgccc tctctgtcca g 1411
<210>7
<211>1357
<212>DNA
<213>小家鼠
<400>7
gtaaatgagc tcagcaccca caaagctctc aggtcctaag agacactggc acccatatcc 60
atgcatccct tgtataaata aagcatccag caaagcctgg taccatgtaa aactgtcctg 120
gttctttcca aggtatagag catagctcac gggctgatag gtctggccag ggttggagga 180
cagccttgtc tataggaaga gaatgaggtt tctgcactgc atgactcaga ggtcatgagt 240
cattctgcct tggactgttg gggcttggct ttaggcagtg ccttttcctt gccttcccat 300
gaaccagcag ctgccagaca tagagataat cctaggaagc ctcaaatgga taagcacaca 360
aacccacctc ccttaggctg ttcctgtccc cctgccccat ttctctactt aggggttttt 420
ctgagtctat tgtggagtga cacatggcca ggggcattcc atagaccttt gtcatctata 480
ctctcaactc aggcagcttt tcacagacga ggtctgcaca cccatacaaa tggctcactc 540
ctgcctgtgc cacctggggc tgaggcacat ggctcttgat gccccaaggg agggactatt 600
agatagccac actcatgcta aatcctgacc ctgctgaaca ccatccagtc catatagcac 660
atgtatcctc atgctcgtga gcacaaaacg catttaacac actgggacaa ctcctctgtg 720
cccagcacag cacctacatc cagcaatgta tcaccataca cacggccaaa aattcaatgc 780
ccacgtttct gccatcacaa tcagacacac ctttcctctt ctgaggacac tccattctcc 840
gctcaaaaca agacctctga agccagattc atctctggta cctcggggtc atgcttcaac 900
cccacatgaa taattcaaac catagccata atggtctgaa tcacttcaca ctgggatgtt 960
cccaagttca ggcaagaaaa gccacaggct ctgctgatga ctgaagaata gcaaagggtc 1020
agtccagctg tatagccact gttgacctgg gtcacaggcc ctgctgaccc tccaccttct 1080
cctgtactga aggaatgaaa gatgagacaa gcatagaggg cacttgaata atccaggtca 1140
ctctgaggtc catccaaggc attattggac tcaggtggag gaagctgaga ctgatgtccc 1200
agagggaaag gaaggaaagc aggccccggg gagggtctgc tgtcccagtc aggctggaga 1260
tctctcctct gacttcatgc agacatgtct gcctcacagg gaatctctcc cagcatcaac 1320
catgttggga caaacactga ctgtcctctc tgttcag 1357
<210>8
<211>1472
<212>DNA
<213>小家鼠
<400>8
gtaaatgaga acagcaccta gccattcctc gggtcttaca agacactgat accagcccta 60
accggtgaac cctataaata aagcacccag agatgggacc ttgtgagatt atcttggttc 120
tttacatggc acatagttca tgatacacct cagccacagg ctgtggggtc tggccagggt 180
tcaaggtgta agttaacatc caagaaagaa caaggtctta tactgccaga cccagggcat 240
gcaagtggac ctgcccttgc cagagatcat ccctctctgc atagcaagtt tgacccaagg 300
ggccctcttc atactcttcc ccacaaccag caactgttct gtgatgagtc tggagataga 360
aatatcgccc tagaaaatat ccaaagaaag gaacacagaa agtgtatccc acctcaacac 420
gaaccccacc ttcatttgtc tgtccttccc tctaccgtga ctcccctacc ttgacctagg 480
aggtctctgt atgtattaca tagacaggca ggaagactca tacatgaagc ccgttcatct 540
ccaccacacc cctagcgata gggataggga tgagtactcc taagagacta cctcccatat 600
gacattcact cacacctaag ttccaagaca ctgagagact tatgacccta gctgccccca 660
aggaagtaac taatcaaata cctacataga tgctgagacc agaactatta gaaccagcct 720
gctaagcatc actcaaccca catggtacct ccaagcctac atacatccac tgatacctca 780
cctaggcagc atgaatagca accaaaccca tcttagtgtc gggatatccc tagacactac 840
ctaaatgtac acatttggac cagtgaagat gaaatagctc tcatgggtac ctaactgttt 900
tcccctaaca cacacaagta ccttcagacc cattctgtcc ctctatgcct aagcccctat 960
tttcatccct tacagttaac actctcccat agctgtataa cctgactcac ttcccaatgt 1020
ggaccttcta ggaagcccag gtatcatcaa acgcaggccc tactctcact ctgtccctct 1080
gtactcagtc accagggaac aagcagtagg tcaggcaagc tgcagagcta ttacctacag 1140
ggcttccaag ccttgaatca gccaagccag ccccagtact gagggatagg gtagacaaaa 1200
agggttcctg catagcccag gccagcatca ggtccagtgt atctggctct acccagggac 1260
aaaacggcag cacattgggg agctgaggtt tctggttcac agagaacata aaggagagat 1320
ggccccaggg agggtcagat ggccccaggg agggtctgct gacccagtca ggctgcagct 1380
ttctcctggg cctccatgca gcctcctgcc acacagggaa tggccctagc tctaccttgt 1440
tgggacaaac actgactttc ctctctgttc ag 1472
<210>9
<211>1860
<212>DNA
<213>小家鼠
<400>9
gtaaacccac actgtacaat gtctccctga tcatgtctga cacaggcggc acctgctatt 60
gaccatgcta gcgctcaacc aggcaggccc tgggtgtcca gttgctctgt gtatgcaaac 120
taaccatgtc agagtgagat gttgcatttt ataaaaatta gaaataaaaa aaatccattc 180
aaacgtcact ggttttgatt atacaatgct catgcctgct gagacagttg tgttttgctt 240
gctctgcaca caccctgcat acttgcctcc accctggccc ttcctctacc ttgccagttt 300
cctccttgtg tgtgaactca gtcaggctta caacagacag agtatgaaca tgcgattcct 360
ccagctactt ctagatatat ggctgaaagc ttgcctaacc tggtgcaggc agcattcagg 420
cacatatata gacacacatg catttataca tagatatata ggtacacatg tgtagacaca 480
tacatgaatg tgtattcatg gacacacaga caaaggtaca catatataca catgagttca 540
tgcgcacaca catgcatgga cacttacaaa cgccttcaga gacaaatagg catagacaca 600
caaccactca cagaaacaga taccaatatg catggtcctg tgtacacaga aacagactat 660
aggcaaatat acacaaataa actatataga tacaaagata tgcatataca cacatgtaca 720
gaaacatctt cacatgtgta cactaacatg tgaacaggta tagcacacag atacacctgg 780
actctgacca gggctgtaat ctccaaggct cacggctcag agagcctaca ctaggctggg 840
tcactgatac tcctcaggag cccactctat gattgggaga gataacccca ggtacaaagt 900
atgcctatct gtctcaacac catggggcag aagatactcc actaaccacc catgacagaa 960
agttagcctt ggctgtgtct ccattaatag aacacctcag aagaccaatg tgaaattgcc 1020
taacccactc acacccaccc tgatctccag ttcaaaatgc agaaaacata atgcagttgt 1080
ccaaaagatg ccccaaccac acacacacac acacacacac acacacacac acacacacac 1140
acacacacac acacacacca tcaaggagcc tctgtaagga gtcaccaccc aataacactg 1200
cctctttggg ctcatatcct ggacattctt catattcata tccatttggg gcctaggctt 1260
tagatatccc caagggctca tctttacagg gatcagagat cccaataaat gccctggtcc 1320
cacagcctcc ctcaggtatc tgtctgttta tctcttggta ccaagaccca acattgctgg 1380
caggggtagg acaagcaacg cacgggaact ctgatcaaag aaagtcatga gatgcctgag 1440
tccttcagga agtaaggagg gacaacctct ggtatccctg ttcttattgc taaagcccaa 1500
gagacaggga gacctgctct aaattctcag tctaaacagc accgatggca ccacctgctc 1560
agggaaagtc cagagcacac caatatcatt ttgccacagt tcctgagtct gcctttaccc 1620
aggtccatac attgcatctg tcttgcttgc tctgctgccc cagggctcct ggaacaaagg 1680
ctccaaatta gtgtgtccta cagcttggcc tgttctgtgc ctccgtctag cttgagctat 1740
taggggacca gtcaatactc gctaagattc tccagaacca tcagggcacc ccaaccctta 1800
tgcaaatgct cagtcacccc aagacttggc ttgaccctcc ctctctgtgt cccttcatag 1860
<210>10
<211>87
<212>DNA
<213>智人
<400>10
agcctgtgga ccaccctgtc cacgtttgtg gccctcttca tcctcaccct cctctacagc 60
ggcattgtca ctttcatcaa ggtgaag 87
<210>11
<211>78
<212>DNA
<213>智人
<400>11
gggctgtgga cgaccatcac catcttcatc acactcttcc tgttaagcgt gtgctacagt 60
gccaccgtca ccttcttc 78
<210>12
<211>78
<212>DNA
<213>智人
<400>12
gggctgtgga ccaccatcac catcttcatc acactcttcc tgctaagcgt gtgctacagt 60
gccaccatca ccttcttc 78
<210>13
<211>81
<212>DNA
<213>智人
<400>13
aacctgtggg ccaccgcctc caccttcatc gtcctcttcc tcctgagcct cttctacagt 60
accaccgtca ccttgttcaa g 81
<210>14
<211>63
<212>DNA
<213>小家鼠
<400>14
actgtgacct tcctcaccct cttcctactg agcttgttct acagcacagc actcactgtt 60
aca 63
<210>15
<211>84
<212>DNA
<213>小家鼠
<400>15
gggctctgga cgaccatcac catcttcatc agcctcttcc tgctcagtgt gtgctacagc 60
gctgctgtca cactcttcaa ggta 84
<210>16
<211>78
<212>DNA
<213>小家鼠
<400>16
gggctctgga cgaccatcac catcttcatc agcctcttcc tgctcagcgt gtgctacagc 60
gcctctgtca cactcttc 78
<210>17
<211>78
<212>DNA
<213>小家鼠
<400>17
gggctctgga cgaccatcac catcttcatc agcctcttcc tgctcagcgt gtgctacagc 60
gcctctgtca ccctcttc 78
<210>18
<211>84
<212>DNA
<213>小家鼠
<400>18
aacctgtgga ccactgcctc caccttcatc gtcctcttcc tcctgagcct cttctacagc 60
accaccgtca ccctgttcaa ggtg 84
<210>19
<211>30
<212>PRT
<213>智人
<400>19
Gly Glu Ala Ala Arg Arg Pro Gly Leu Pro Leu Pro Leu Leu Leu Leu
1 5 10 15
His Gln Leu Leu Leu Leu Phe Leu Ser His Leu Arg Arg Leu
20 25 30
<210>20
<211>26
<212>PRT
<213>智人
<400>20
Asp Ala Ala His Pro Val Ala Ala Ser Leu Pro Leu Leu Ala Gly Thr
1 5 10 15
Leu Leu Leu Leu Gly Ala Ser Ala Ala Pro
20 25
<210>21
<211>24
<212>PRT
<213>智人
<400>21
Ser Ser Ala Gly Ser Leu Ala Ala Gly Pro Leu Leu Val Ala Leu Ala
1 5 10 15
Leu Tyr Pro Leu Ser Val Leu Phe
20
<210>22
<211>30
<212>PRT
<213>智人
<400>22
Asp Ala Ala His Pro Gly Arg Ser Val Val Pro Ala Leu Leu Pro Leu
1 5 10 15
Leu Ala Gly Thr Leu Leu Leu Leu Glu Thr Ala Thr Ala Pro
20 25 30
<210>23
<211>26
<212>PRT
<213>智人
<400>23
Ser Ala Ser Ser Asn Ile Ser Gly Gly Ile Phe Leu Phe Phe Val Ala
1 5 10 15
Asn Ala Ile Ile His Leu Phe Cys Phe Ser
20 25
<210>24
<211>27
<212>PRT
<213>智人
<400>24
Ala Ser Gly Thr Ser Pro Gly Leu Ser Ala Gly Ala Thr Val Gly Ile
1 5 10 15
Met Ile Gly Val Leu Val Gly Val Ala Leu Ile
20 25
<210>25
<211>29
<212>PRT
<213>智人
<400>25
Ser Gly Thr Thr Arg Leu Leu Ser Gly His Thr Cys Phe Thr Leu Thr
1 5 10 15
Gly Leu Leu Gly Thr Leu Val Thr Met Gly Leu Leu Thr
20 25
<210>26
<211>27
<212>PRT
<213>智人
<400>26
Asn Gly Gly Thr Ser Leu Ser Glu Lys Thr Val Leu Leu Leu Val Thr
1 5 10 15
Pro Phe Leu Ala Ala Ala Trp Ser Leu His Pro
20 25
<210>27
<211>32
<212>PRT
<213>智人
<400>27
Cys Glu Gly Ile Ser Leu Leu Ala Gln Asn Thr Ser Trp Leu Leu Leu
1 5 10 15
Leu Leu Leu Ser Leu Ser Leu Leu Gln Ala Thr Asp Phe Met Ser Leu
20 25 30
<210>28
<211>22
<212>PRT
<213>智人
<400>28
Ser Gly Ala Pro Thr Leu Ser Pro Ser Leu Leu Gly Leu Leu Leu Pro
1 5 10 15
Ala Phe Gly Ile Leu Val
20
<210>29
<211>39
<212>PRT
<213>智人
<400>29
Asp Leu Cys Asn Glu Lys Leu His Asn Ala Ala Pro Thr Arg Thr Ala
1 5 10 15
Leu Ala His Ser Ala Leu Ser Leu Gly Leu Ala Leu Ser Leu Leu Ala
20 25 30
Val Ile Leu Ala Pro Ser Leu
35
<210>30
<211>24
<212>PRT
<213>智人
<400>30
Ser Gly Ala Gly Pro Trp Ala Ala Trp Pro Phe Leu Leu Ser Leu Ala
1 5 10 15
Leu Met Leu Leu Trp Leu Leu Ser
20
<210>31
<211>26
<212>PRT
<213>智人
<400>31
Asn Ala Gly Glu Met Leu His Gly Thr Gly Gly Leu Leu Leu Ser Leu
1 5 10 15
Ala Leu Met Leu Gln Leu Trp Leu Leu Gly
20 25
<210>32
<211>49
<212>PRT
<213>智人
<400>32
Ala Arg Gly Leu Met Asn Gly Tyr Arg Gly Pro Ala Met Asp Ser Glu
1 5 10 15
Glu Asp Met Met Val Thr Ala Leu His Ser Leu Thr Leu Gln Thr Val
20 25 30
Val Ile His Ser Leu Ser Ser Val Leu Pro Gly Ile Thr Leu Ala Ile
35 40 45
Asn
<210>33
<211>30
<212>PRT
<213>智人
<400>33
Ser Ser Gly His Ser Arg His Arg Tyr Ala Leu Ile Pro Ile Pro Leu
1 5 10 15
Ala Val Ile Thr Thr Cys Ile Val Leu Tyr Met Asn Val Leu
20 25 30
<210>34
<211>27
<212>PRT
<213>智人
<400>34
Ser Gly Ala Ser Ser Leu His Arg His Phe Gly Leu Leu Leu Ala Ser
1 5 10 15
Leu Ala Pro Leu Val Leu Cys Leu Ser Leu Leu
20 25
<210>35
<211>26
<212>PRT
<213>智人
<400>35
Ser Thr Gly Ser His Cys His Gly Ser Phe Ser Leu Ile Phe Leu Ser
1 5 10 15
Leu Trp Ala Val Ile Phe Val Leu Tyr Gln
20 25
<210>36
<211>31
<212>PRT
<213>智人
<400>36
Gly Ala Ala Pro Gln Pro Gly Pro Ala His Leu Ser Leu Thr Ile Thr
1 5 10 15
Leu Leu Met Thr Ala Arg Leu Trp Gly Gly Thr Leu Leu Trp Thr
20 25 30
<210>37
<211>30
<212>PRT
<213>小家鼠
<400>37
Asn Ala Ala Val Pro Thr Gly Ala Ser Thr Trp Thr Met Ala Gly Val
1 5 10 15
Leu Leu Phe Ser Leu Ser Ser Val Ile Leu Gln Thr Leu Leu
20 25 30
<210>38
<211>35
<212>PRT
<213>小家鼠
<400>38
Asn Glu Arg Leu Val Ser Ala Ala Pro Gly His Ala Leu Leu Ser Ser
1 5 10 15
Val Thr Leu Gly Leu Ala Thr Ser Leu Ser Leu Leu Thr Val Met Ala
20 25 30
Leu Cys Leu
35
<210>39
<211>26
<212>PRT
<213>小家鼠
<400>39
Ser Ala Ala Ser His Tyr Gln Gly Ser Phe Pro Leu Val Ile Leu Ser
1 5 10 15
Leu Ser Ala Val Ile Phe Val Leu Tyr Gln
20 25
<210>40
<211>24
<212>PRT
<213>小家鼠
<400>40
Ser Ser Gly Val Cys Trp Thr Ala Thr Trp Leu Val Val Thr Thr Leu
1 5 10 15
Ile Ile His Arg Ile Leu Leu Thr
20
<210>41
<211>32
<212>PRT
<213>小家鼠
<400>41
Asn Phe Ser Ala Ala Gly Leu Gly Leu Arg Ala Ser Ile Pro Leu Leu
1 5 10 15
Gly Leu Gly Leu Leu Leu Ser Leu Leu Ala Leu Leu Gln Leu Ser Pro
20 25 30
<210>42
<211>16
<212>PRT
<213>小家鼠
<400>42
Ala Lys Ser Phe Tyr Phe Ile Cys Tyr Leu Cys Leu Tyr Leu Ala Leu
1 5 10 15
<210>43
<211>22
<212>PRT
<213>褐家鼠(Rattus norvegicus)
<400>43
Gly Asn Ser Ala Glu Gly Ser Met Pro Ala Phe Leu Leu Phe Leu Leu
1 5 10 15
Leu Gln Leu Trp Asp Thr
20
<210>44
<211>32
<212>PRT
<213>褐家鼠
<400>44
Cys Gly Gly Ile Ser Leu Leu Val Gln Asn Thr Ser Trp Leu Leu Leu
1 5 10 15
Leu Leu Leu Ser Leu Ser Phe Leu Gln Ala Thr Asp Phe Ile Ser Leu
20 25 30
<210>45
<211>29
<212>PRT
<213>褐家鼠
<400>45
Ser Ala Ala Thr Arg Pro Glu Pro His Tyr Phe Phe Leu Leu Phe Leu
1 5 10 15
Phe Thr Leu Val Leu Ala Ala Ala Arg Pro Arg Trp Arg
20 25
<210>46
<211>24
<212>PRT
<213>褐家鼠
<400>46
Ser Ser Gly Val His Trp Ile Ala Ala Trp Leu Val Val Thr Leu Ser
1 5 10 15
Ile Ile Pro Ser Ile Leu Leu Ala
20
<210>47
<211>26
<212>PRT
<213>褐家鼠
<400>47
Ser Ala Ala Ser His Tyr Gln Gly Ser Phe Pro Leu Ile Ile Leu Ser
1 5 10 15
Phe Trp Ala Val Ile Leu Val Leu Tyr Gln
20 25
<210>48
<211>22
<212>PRT
<213>褐家鼠
<400>48
Arg Asn Thr Gly Phe Leu Leu Ala Trp Pro Thr Phe Phe Leu Pro Val
1 5 10 15
Phe Leu Ala Trp Leu Phe
20
<210>49
<211>30
<212>PRT
<213>褐家鼠
<400>49
Ser Ser Ala Gly Ala Arg Glu Ser Cys Val Ser Leu Phe Leu Val Val
1 5 10 15
Leu Pro Ser Leu Leu Val Gln Leu Leu Cys Leu Ala Glu Pro
20 25 30
<210>50
<211>33
<212>PRT
<213>白色念珠菌(Candida albicans)
<400>50
Ser Ser Gly Val Lys Ala Thr Gln Gln Met Ser Met Val Lys Leu Val
1 5 10 15
Ser Ile Ile Thr Ile Val Thr Ala Phe Val Gly Gly Met Ser Val Val
20 25 30
Phe
<210>51
<211>32
<212>PRT
<213>某种酵母
<400>51
Asn Ala Ala Thr Asn Val Lys Ala Asn Leu Ala Gln Val Val Phe Thr
1 5 10 15
Ser Ile Ile Ser Leu Ser Ile Ala Ala Gly Val Gly Phe Ala Leu Val
20 25 30
<210>52
<211>25
<212>PRT
<213>猪
<400>52
Ala Arg Ala Ala Pro Thr Thr Ser Leu Gly Ser Leu Met Thr Val ser
1 5 10 15
Ala Leu Ala Ile Leu Gly Trp Ser Val
20 25
<210>53
<211>26
<212>PRT
<213>猪
<400>53
Ser Ala Ala Pro Ser Leu His Leu Pro Pro Gly Ser Leu Leu Ala Ser
1 5 10 15
Leu Val Pro Leu Leu Leu Leu Ser Leu Pro
20 25
<210>54
<211>18
<212>PRT
<213>布氏锥虫(Trypanosoma brucei)
<400>54
Ser Asn Ser Phe Val Ile His Lys Ala Pro Leu Phe Leu Ala Phe Leu
1 5 10 15
Leu Phe
<210>55
<211>24
<212>PRT
<213>布氏锥虫
<400>55
Asp Ser Ser Ile Leu Val Thr Lys Lys Phe Ala Leu Thr Val Val Ser
1 5 10 15
Ala Ala Phe Val Ala Leu Leu Phe
20
<210>56
<211>24
<212>PRT
<213>布氏锥虫
<400>56
Asn Gly Ser Phe Leu Thr Ser Lys Gln Phe Ala Phe Ser Val Val Ser
1 5 10 15
Ala Ala Phe Val Ala Leu Leu Phe
20
<210>57
<211>18
<212>PRT
<213>布氏锥虫
<400>57
Ser Asn Ser Phe Val Ile Ser Lys Thr Pro Leu Trp Leu Ala Val Leu
1 5 10 15
Leu Phe
<210>58
<211>23
<212>PRT
<213>布氏锥虫
<400>58
Asp Gly Ser Phe Leu Val Asn Lys Lys Phe Ala Leu Met Val Tyr Asp
1 5 10 15
Phe Val Ser Leu Leu Ala Phe
20
<210>59
<211>23
<212>PRT
<213>布氏锥虫
<400>59
Asn Gly Ser Phe Leu Thr Ser Lys Gln Phe Ala Leu Met Val Ser Ala
1 5 10 15
Ala Phe Val Thr Leu Leu Phe
20
<210>60
<211>23
<212>PRT
<213>布氏锥虫
<400>60
Gly Ala Ala Thr Leu Lys Ser Val Ala Leu Pro Phe Ala Ile Ala Ala
1 5 10 15
Ala Ala Leu Val Ala Al Phe
20
<210>61
<211>18
<212>PRT
<213>布氏锥虫
<400>61
Ser Asn Ser Phe Val Ile Asn Lys Ala Pro Leu Leu Leu Gly Phe Leu
1 5 10 15
Leu Phe
<210>62
<211>20
<212>PRT
<213>刚果锥虫(Trypanosoma congolense)
<400>62
Ser Gly Ser Ser His Gly Thr Lys Ala Ile Arg Ser Ile Leu His Val
1 5 10 15
Ala Leu Leu Met
20
<210>63
<211>28
<212>PRT
<213>鸡(Gallus sp.)
<400>63
Ala Gly Asn Lys Leu Ile Gln Gln His Leu Leu Val Ile Thr Phe Val
1 5 10 15
Pro Phe Ile Ile Leu Gly Gln Leu Gln Gly Leu Gly
20 25
<210>64
<211>26
<212>PRT
<213>鸡
<400>64
Ala Thr Leu Gly Ser Pro Ser Thr Ser Ser Ser Phe Val Ser Leu Leu
1 5 10 15
Leu Ser Ala Val Thr Leu Leu Leu Leu Cys
20 25
<210>65
<211>25
<212>PRT
<213>石纹电鳐(Torpedo marmorata)
<400>65
Ser Ser Ser Gly Thr Ser Ser Ser Lys Gly Ile Ile Phe Tyr Val Leu
1 5 10 15
Phe Ser Ile Leu Tyr Leu Ile Phe Tyr
20 25
<210>66
<211>24
<212>PRT
<213>某种仓鼠(Hamster sp.)
<400>66
Ser Ser Ala Val Leu Phe Ser Ser Pro Pro Val Ile Leu Leu Ile Ser
1 5 10 15
Phe Leu Ile Phe Leu Met Val Gly
20
<210>67
<211>26
<212>PRT
<213>欧洲牛(Bos taurus)
<400>67
Ser Ala Gly Ser His Cys Cys Gly Ser Phe Ser Leu Ile Phe Leu Ser
1 5 10 15
Val Leu Ala Val Ile Ile Ile Leu Tyr Gln
20 25
<210>68
<211>23
<212>PRT
<213>粘菌(slime mold)
<400>68
Ser Ser Ala Thr Thr Ile Ala Phe Asn Ala Phe Val Val Phe Ala Ile
1 5 10 15
Val Leu Ser Val Leu Leu Phe
20
<210>69
<211>22
<212>PRT
<213>粘菌
<400>69
Gly Ser Ala Ser Thr Val Val Ala Ser Leu Ser Leu Ile Ile Phe Ser
1 5 10 15
Met Ile Leu Ser Leu Cys
20
<210>70
<211>25
<212>DNA
<213>智人
<400>70
acctggccat gacccccctg atccc 25
<210>71
<211>29
<212>DNA
<213>智人
<400>71
agctgcaact ggaggagagc tgtgcggag 29
<210>72
<211>33
<212>DNA
<213>智人
<400>72
agctgcaact ggaggagagc tgtgcggagg cgc 33
<210>73
<211>25
<212>DNA
<213>智人
<400>73
agggggaggt gagcgccgac gagga 25
<210>74
<211>31
<212>DNA
<213>小家鼠
<400>74
aacgtcaaga gccactttcc tatgtgctac t 31
<210>75
<211>48
<212>DNA
<213>小家鼠
<400>75
ggctgcaact ggacgagacc tgtgctgagg cccaggacgg ggagctgg 48
<210>76
<211>27
<212>DNA
<213>小家鼠
<400>76
ggctagacct ggatgatatc tgtgctg 27
<210>77
<211>23
<212>DNA
<213>小家鼠
<400>77
agctggaact gaatgagacc tgt 23
<210>78
<211>33
<212>DNA
<213>小家鼠
<400>78
agggggaggt gaatgctgag gaggaaggct ttg 33
<210>79
<211>4
<212>PRT
<213>智人
<400>79
Gly Phe Thr His
1
<210>80
<211>4
<212>PRT
<213>智人
<400>80
Ala Cys Thr Thr
1
<210>81
<211>4
<212>PRT
<213>智人
<400>81
Cys Ala Pro Ala
1
<210>82
<211>4
<212>PRT
<213>智人
<400>82
Ala Gly Thr Thr
1
<210>83
<211>4
<212>PRT
<213>智人
<400>83
Thr Ser Ser Pro
1
<210>84
<211>4
<212>PRT
<213>智人
<400>84
Ile Thr Val Ser
1
<210>85
<211>4
<212>PRT
<213>智人
<400>85
Ser Gly Thr Thr
1
<210>86
<211>4
<212>PRT
<213>智人
<400>86
Glu Gln Leu Glu
1
<210>87
<211>4
<212>PRT
<213>智人
<400>87
Lys Leu Val Lys
1
<210>88
<211>4
<212>PRT
<213>智人
<400>88
Gln Val Lys Ile
1
<210>89
<211>4
<212>PRT
<213>智人
<400>89
Cys Cys Gln Glu
1
<210>90
<211>4
<212>PRT
<213>智人
<400>90
Ala Ala Ala Met
1
<210>91
<211>4
<212>PRT
<213>智人
<400>91
Ala Met His Val
1
<210>92
<211>4
<212>PRT
<213>智人
<400>92
Gly Ser Arg Gly
1
<210>93
<211>4
<212>PRT
<213>智人
<400>93
Thr Cys Ile Pro
1
<210>94
<211>4
<212>PRT
<213>智人
<400>94
Tyr Gly Tyr Ser
1
<210>95
<211>4
<212>PRT
<213>智人
<400>95
Arg Ile Lys Phe
1
<210>96
<211>4
<212>PRT
<213>智人
<400>96
Gln Tyr Arg Ser
1
<210>97
<211>4
<212>PRT
<213>小家鼠
<400>97
Glu Asp Leu Cys
1
<210>98
<211>4
<212>PRT
<213>小家鼠
<400>98
Thr Asp Leu Cys
1
<210>99
<211>4
<212>PRT
<213>小家鼠
<400>99
Arg Ile Lys Phe
1
<210>100
<211>4
<212>PRT
<213>小家鼠
<400>100
Asp Leu Ala Arg
1
<210>101
<211>4
<212>PRT
<213>小家鼠
<400>101
Ser Ser Phe Cys
1
<210>102
<211>4
<212>PRT
<213>小家鼠
<400>102
His Leu Met Phe
1
<210>103
<211>4
<212>PRT
<213>褐家鼠
<400>103
Ala Pro Ser Ser
1
<210>104
<211>4
<212>PRT
<213>褐家鼠
<400>104
Lys Leu Val Lys
1
<210>105
<211>4
<212>PRT
<213>褐家鼠
<400>105
Gly Gln Lys Thr
1
<210>106
<211>4
<212>PRT
<213>褐家鼠
<400>106
Thr Leu Ala Arg
1
<210>107
<211>4
<212>PRT
<213>褐家鼠
<400>107
Arg Ile Lys Phe
1
<210>108
<211>4
<212>PRT
<213>褐家鼠
<400>108
Asn Gly Thr Pro
1
<210>109
<211>4
<212>PRT
<213>褐家鼠
<400>109
Asn Cys Leu Tyr
1
<210>110
<211>4
<212>PRT
<213>白色念珠菌
<400>110
Gly Ser Ser Ser
1
<210>111
<211>4
<212>PRT
<213>某种酵母
<400>111
Ser Ser Lys Lys
1
<210>112
<211>4
<212>PRT
<213>猪
<400>112
Glu Pro Leu Ser
1
<210>113
<211>4
<212>PRT
<213>猪
<400>113
Asn Tyr Gly Tyr
1
<210>114
<211>4
<212>PRT
<213>布氏锥虫
<400>114
Asn Thr Thr Gly
1
<210>115
<211>4
<212>PRT
<213>布氏锥虫
<400>115
Asn Ala Cys Lys
1
<210>116
<211>4
<212>PRT
<213>布氏锥虫
<400>116
Glu Lys Cys Arg
1
<210>117
<211>4
<212>PRT
<213>布氏锥虫
<400>117
Thr Thr Gly Ser
1
<210>118
<211>4
<212>PRT
<213>布氏锥虫
<400>118
Glu Lys Cys Cys
1
<210>119
<211>4
<212>PRT
<213>布氏锥虫
<400>119
Glu Asp Cys Arg
1
<210>120
<211>4
<212>PRT
<213>布氏锥虫
<400>120
Glu Pro Glu Pro
1
<210>121
<211>4
<212>PRT
<213>布氏锥虫
<400>121
Asn Thr Thr Ala
1
<210>122
<211>4
<212>PRT
<213>刚果锥虫
<400>122
Ser His Leu Pro
1
<210>123
<211>4
<212>PRT
<213>鸡
<400>123
Gln Cys Ser Gly
1
<210>124
<211>4
<212>PRT
<213>鸡
<400>124
Thr Val Ile Pro
1
<210>125
<211>4
<212>PRT
<213>石纹电鳐
<400>125
Asp Gly Glu Leu
1
<210>126
<211>4
<212>PRT
<213>某种仓鼠
<400>126
Asp Gly Arg Arg
1
<210>127
<211>4
<212>PRT
<213>欧洲牛
<400>127
Arg Ile Gln Phe
1
<210>128
<211>4
<212>PRT
<213>粘菌
<400>128
Asn Asn Val Cys
1
<210>129
<211>4
<212>PRT
<213>粘菌
<400>129
Ser Thr Thr Thr
1
<210>130
<211>2074
<212>DNA
<213>智人
<400>130
cctccaccaa gggcccatcg gtcttccccc tggcaccctc ctccaagagc acctctgggg 60
gcacagcggc cctgggctgc ctggtcaagg actacttccc cgaaccggtg acggtgtcgt 120
ggaactcagg cgccctgacc agcggcgtgc acaccttccc ggctgtccta cagtcctcag 180
gactctactc cctcagcagc gtggtgaccg tgccctccag cagcttgggc acccagacct 240
acatctgcaa cgtgaatcac aagcccagca acaccaaggt ggacaagaaa gttggtgaga 300
ggccagcaca gggagggagg gtgtctgctg gaagccaggc tcagcgctcc tgcctggacg 360
catcccggct atgcagcccc agtccagggc agcaaggcag gccccgtctg cctcttcacc 420
cggaggcctc tgcccgcccc actcatgctc agggagaggg tcttctggct ttttccccgg 480
ctctgggcag gcacaggcta ggtgccccta acccaggccc tgcacacaaa ggggcaggtg 540
ctgggctcag acctgccaag agccatatcc gggaggaccc tgcccctgac ctaagcccac 600
cccaaaggcc aaactctcca ctccctcagc tcggacacct tctctcctcc cagattccag 660
taactcccaa tcttctctct gcagagccca aatcttgtga caaaactcac acatgcccac 720
cgtgcccagg taagccagcc caggcctcgc cctccagctc aaggcgggac aggtgcccta 780
gagtagcctg catccaggga caggccccag ccgggtgctg acacgtccac ctccatctct 840
tcctcagcac ctgaactcct ggggggaccg tcagtcttcc tcttcccccc aaaacccaag 900
gacaccctca tgatctcccg gacccctgag gtcacatgcg tggtggtgga cgtgagccac 960
gaagaccctg aggtcaagtt caactggtac gtggacggcg tggaggtgca taatgccaag 1020
acaaagccgc gggaggagca gtacaacagc acgtaccgtg tggtcagcgt cctcaccgtc 1080
ctgcaccagg actggctgaa tggcaaggag tacaagtgca aggtctccaa caaagccctc 1140
ccagccccca tcgagaaaac catctccaaa gccaaaggtg ggacccgtgg ggtgcgaggg 1200
ccacatggac agaggccggc tcggcccacc ctctgccctg agagtgaccg ctgtaccaac 1260
ctctgtccct acagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga 1320
tgagctgacc aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga 1380
catcgccgtg gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc 1440
cgtgctggac tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag 1500
gtggcagcag gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta 1560
cacgcagaag agcctctccc tgtctccggg taaatgagtg cgacggccgg caagcccccg 1620
ctccccgggc tctcgcggtc gcacgaggat gcttggcacg taccccctgt acatacttcc 1680
cgggcgccca gcatggaaat aaagcaccca gcgctgccct gggcccctgc gagactgtga 1740
tggttctttc cacgggtcag gccgagtctg aggcctgagt ggcatgaggg aggcagagcg 1800
ggtcccactg tccccacact ggcccaggct gtgcaggtgt gcctgggccg cctagggtgg 1860
ggctcagcca ggggctgccc tcggcagggt gggggatttg ccagcgtggc cctccctcca 1920
gcagcacctg ccctgggctg ggccacggga agccctagga gcccctgggg acagacacac 1980
agcccctgcc tctgtaggag actgtcctgt tctgtgagcg ccctgtcctc cgacctccat 2040
gcccactcgg gggcatgcga cagattgagg atcc 2074
<210>131
<211>330
<212>PRT
<213>人工
<220>
<223>由质粒pIgG-A编码的γ链恒定区的氨基酸序列A
<400>131
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
225 230 235 240
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330
<210>132
<211>40
<212>DNA
<213>人工
<220>
<223>TM-fw1
<400>132
ggccgagtct gaggcctgag tggcatgagg gaggcagagt 40
<210>133
<211>48
<212>DNA
<213>人工
<220>
<223>TM-rv1
<400>133
aactggatcc atgtagaaaa gaggagaagc cccgggggtc catgtagt 48
<210>134
<211>47
<212>DNA
<213>人工
<220>
<223>M1-rv
<400>134
gatccacttc accttgaaga aggtgacggt ggcactgtag cacacgc 47
<210>135
<211>49
<212>DNA
<213>人工
<220>
<223>M2-fw
<400>135
caccttcttc aaggtgaagt ggatcttctc ctcggtggtg gacctgaag 49
<210>136
<211>33
<212>DNA
<213>人工
<220>
<223>TM-fw2
<400>136
ctccagcagc agctgccctg ggctgggcca cga 33
<210>137
<211>50
<212>DNA
<213>人工
<220>
<223>TM-rv2
<400>137
cagggatccc ccgaggtgca gctggaccag cctcctcctg accgtgtttt 50
<210>138
<211>68
<212>DNA
<213>人工
<220>
<223>mutSphI-1
<400>138
ctaccccaga cctccgctgc ttggtgcctg cagggcactg ggggccaggt gtcccctcag 60
caggacgt 68
<210>139
<211>62
<212>DNA
<213>人工
<220>
<223>mutSphI-2
<400>139
cctgctgagg ggacacctgg cccccagtgc cctgcaggca ccaagcagcg gaggtctggg 60
gt 62
<210>140
<211>6949
<212>DNA
<213>智人
<400>140
cctccaccaa gggcccatcg gtcttccccc tggcaccctc ctccaagagc acctctgggg 60
gcacagcggc cctgggctgc ctggtcaagg actacttccc cgaaccggtg acggtgtcgt 120
ggaactcagg cgccctgacc agcggcgtgc acaccttccc ggctgtccta cagtcctcag 180
gactctactc cctcagcagc gtggtgaccg tgccctccag cagcttgggc acccagacct 240
acatctgcaa cgtgaatcac aagcccagca acaccaaggt ggacaagaaa gttggtgaga 300
ggccagcaca gggagggagg gtgtctgctg gaagccaggc tcagcgctcc tgcctggacg 360
catcccggct atgcagcccc agtccagggc agcaaggcag gccccgtctg cctcttcacc 420
cggaggcctc tgcccgcccc actcatgctc agggagaggg tcttctggct ttttccccgg 480
ctctgggcag gcacaggcta ggtgccccta acccaggccc tgcacacaaa ggggcaggtg 540
ctgggctcag acctgccaag agccatatcc gggaggaccc tgcccctgac ctaagcccac 600
cccaaaggcc aaactctcca ctccctcagc tcggacacct tctctcctcc cagattccag 660
taactcccaa tcttctctct gcagagccca aatcttgtga caaaactcac acatgcccac 720
cgtgcccagg taagccagcc caggcctcgc cctccagctc aaggcgggac aggtgcccta 780
gagtagcctg catccaggga caggccccag ccgggtgctg acacgtccac ctccatctct 840
tcctcagcac ctgaactcct ggggggaccg tcagtcttcc tcttcccccc aaaacccaag 900
gacaccctca tgatctcccg gacccctgag gtcacatgcg tggtggtgga cgtgagccac 960
gaagaccctg aggtcaagtt caactggtac gtggacggcg tggaggtgca taatgccaag 1020
acaaagccgc gggaggagca gtacaacagc acgtaccgtg tggtcagcgt cctcaccgtc 1080
ctgcaccagg actggctgaa tggcaaggag tacaagtgca aggtctccaa caaagccctc 1140
ccagccccca tcgagaaaac catctccaaa gccaaaggtg ggacccgtgg ggtgcgaggg 1200
ccacatggac agaggccggc tcggcccacc ctctgccctg agagtgaccg ctgtaccaac 1260
ctctgtccct acagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga 1320
tgagctgacc aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga 1380
catcgccgtg gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc 1440
cgtgctggac tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag 1500
gtggcagcag gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta 1560
cacgcagaag agcctctccc tgtctccggg taaatgagtg cgacggccgg caagcccccg 1620
ctccccgggc tctcgcggtc gcacgaggat gcttggcacg taccccctgt acatacttcc 1680
cgggcgccca gcatggaaat aaagcaccca gcgctgccct gggcccctgc gagactgtga 1740
tggttctttc cacgggtcag gccgagtctg aggcctgagt ggcatgaggg aggcagagcg 1800
ggtcccactg tccccacact ggcccaggct gtgcaggtgt gcctgggccg cctagggtgg 1860
ggctcagcca ggggctgccc tcggcagggt gggggatttg ccagcgtggc cctccctcca 1920
gcagcacctg ccctgggctg ggccacggga agccctagga gcccctgggg acagacacac 1980
agcccctgcc tctgtaggag actgtcctgt tctgtgagcg ccctgtcctc cgacctccat 2040
gcccactcgg gggcatgcct agtccatgcg cgtagggaca ggccctccct cacccatcta 2100
cccccacggc actaacccct ggcagccctg cccagcctcg cacccgcatg gggacacaac 2160
cgactccggg gacatgcact ctcgggccct gtggagggac tggtgcagat gcccacacac 2220
acactcagcc cagacccgtt caacaaaccc cgcactgagg ttggccggcc acacggccac 2280
cacacacaca cgtgcacgcc tcacacacgg agcctcaccc gggcgaaccg cacagcaccc 2340
agaccagagc aaggtcctcg cacacgtgaa cactcctcgg acacaggccc ccacgagccc 2400
cacgcggcac ctcaaggccc acgagccgct cggcagcttc tccacatgct gacctgctca 2460
gacaaaccca gccctcctct cacaaggtgc ccctgcagcc gccacacaca cacaggcccc 2520
cacacgcagg ggaacacacg ccacgtcgcg tccctggcac tggcccactt cccaatacag 2580
cccttccctg cagctggggt cacatgaggt gtgggcttca ccatcctcct gccctctggg 2640
cctcagggag ggacacggga gacggggagc gggtcctgct gagggccagg tcgctatcta 2700
gggccgggtg tctggctgag tcccggggcc aaagctggtg cccagggcgg gcagctgtgg 2760
ggagctgacc tcaggacatt gttggcccat cccggccggg ccctacatcc tgggccccgc 2820
cacagaggga atcaccccca gaggcccaag cccaggggga cacagcactg accaccccct 2880
tcctgtccag agctgcaact ggaggagagc tgtgcggagg cgcaggacgg ggagctggac 2940
gggctgtgga cgaccatcac catcttcatc acactcttcc tgttaagcgt gtgctacagt 3000
gccaccgtca ccttcttcaa ggtcggccgc acgttgtccc cagctgtcct tgacattgtc 3060
ccccatgctg tcacaaactg tctctgacac tgtcccacag gctgtcccca cctgtccctg 3120
acgctgtccc ccatgctctc acaaactgtc cctgacattg tccccaatgc tgcccccacc 3180
tgtccaacag tgtcccccag gctctcccca catgtccccg acactgtccc ccatgctgtc 3240
cccatctgtc cccaacactg tcccccaccc tgtccccctt tgtccccaac actgtccccc 3300
acagtttcca cctgtccctg acactgtccc ccatgctttc cccacctgtc cctgacacca 3360
tcccccactc tgtcccctat agttcctggc cctgtccccc acgctgtccc ctacagtacc 3420
tggcactgtc ccccatgctg tcccctcctg tatgaaaccc tgtcccacat gctgtcccca 3480
cctgtccgtg acaatatccc ccacactgtc cccacctgtc cccgacactc tcctccacgt 3540
tgttcttacc taaacccgac actttcctcc atgctgtccc cacccatctc cgacactgta 3600
ccccacgttg tccccacctg tcctcaacac tgtcccccat gctgtcccca cctgtcccca 3660
acactctcct ccatgctgtc cccacctgtc cctgatattg tcccccatgc agtctccacc 3720
tgtccccaat gctgtccccc aggctgtacc taccagtaca acactgtccc ccatgctgtc 3780
cccacctgtc cctgacactg tcccccacgc tgtcccctcc tgtccccgac actgtccccc 3840
acactgtccc cacctgtccc caacactatc ctccatgctg tcccctcctg tccccacctg 3900
tcccctacac tgtcccccat gctgtcccca ccagtcccca aaactttcct ccacactgtc 3960
cccacctgtc cccaacactg tcccccacgc tatcccccct gtccccgaca atgtccccac 4020
tgtttcctcc tgttccctcc tatccctgac actgtccgcc atgctgtccc cacctgtccc 4080
tgacactgtc tcccactctg tcccctataa tccctgacac tgtcccccac gccgtcccct 4140
cccgtatgca ccactgtccc ccaagctgtc cccacctgtc ctcaacacag tcccccatgc 4200
tgtccccacc tgtccccaac actctcctcc atgtccccac ctgtccctga tattgtcccc 4260
catgcagtcc ccacctgtcc ccgatgctgt cccccgggct gtacctacca gtccaacact 4320
gtcccccaca ctctccccac ctgtccctga tactgtcccc catgctgtcc ccacctgtcc 4380
cggacactgt tctccacgat ctcccctcct gtccctgaca ctgtccccca cactgtcccc 4440
acctgtcccc aacactatcc tccatcctgt cccaacctgt ctcctacact gtcccccatg 4500
ctgtccccac cagtccccaa cactgtcctc catgctgtcc cccatgtccc caacactgtc 4560
ccccatgcta cctcccctgc ccctgacaat gtccccactg tttcctgtcc cctcctatcc 4620
ctgacactgt cccccatgct gtccccacct gtcccccaca tggtctccac cggtccctga 4680
cactgtctcc cactctgtcc cctataatcc ctgacactgt cccccacacc gtcccctcct 4740
gtatgcacca cggtccccca tgctgtcccc acctgtccct gatgctgtcc tccacacagt 4800
ccccacctct ccctgacact gtccccatct ctccccaaca ctctcctcca tgctgtcctc 4860
aactgtcccc aacactcttc cacactctgt ctccacctgt ccctgacact gtcccccaca 4920
ctgtcctcac ctgtgtctga cactgtcccc cacgctgtcc ccacctgtcc ctgacgctgt 4980
cttctgtgct gtccacatgc tgttggtgcc ctggctctgc tctctatcac caagcctcag 5040
agcaggcagt ggtgaggcca tggcacctgg gtggcatgag gggccggatg ggcctcaggg 5100
gcagggctgt ggcctgcgtg gactgacagg tgggtgggcc ttgggggcag agaggtggcc 5160
tcagtgccct gaggggtggg tggggctcgg gggcagggct gtggcctcgc tcacccctgt 5220
gctgtgcctt gcctacaggt gaagtggatc ttctcctcgg tggtggacct gaagcagacc 5280
atcatccccg actacaggaa catgatcgga cagggggcct agggccaccc tctgcggggt 5340
gtccagggcc gcccagaccc cacacaccag ccatgggcca tgctcagcca ccacccaggc 5400
cacacctgcc cccgacctca ccgccctcaa ccccatggct ctctggcctc gcagttgccc 5460
tctgaccctg acacacctga cacgcccccc ttccagaccc tgtgcatagc aggtctaccc 5520
cagacctccg ctgcttggtg cctgcagggc actgggggcc aggtgtcccc tcagcaggac 5580
gtccttgccc tccggaccac aaggtgctca cacaaaagga ggcagtgacc ggtatcccag 5640
gcccccaccc aggcaggagc tggccctgga gccaaccccg tccacgccag cctcctgaac 5700
acaggcgtgg tttccagatg gtgagtggga gcatcagccg ccaaggtagg gaagccacag 5760
caccatcagg ccctgttggg gaggcttccg agagctgcga aggctcactc agacggcctt 5820
cctcccagcc cgcagccagc cagcctccat tccgggcact cccgtgaact cctgacatga 5880
ggaatgacgt tgttctgatt tcaagcaaag aacgctgctc tctggctcct gggaacagtc 5940
tcggtgccag caccacccct tggctgcctg cccacactgc tggattctcg ggtggaactc 6000
gacccgcagg gacagccagc cccagagtcc gcactgggga gagaaggggc caggcccagg 6060
acactgccac ctcccaccca ctccagtcca ccgagatcac tcggagaaga gtctgggcca 6120
tgtggccgct gcaggagccc cacagtgcaa gggtgaggat agcccaagga agggctgggc 6180
atctgcccag acaggcctcc cacagaaggc tggtgaccag gtcccaggcg ggcaagactc 6240
agccttggtg gggcctgagg acagaggagg cccaggagca tcggggagag aggtggaggg 6300
acaccgggag agccaggggc gtggacacag ccagaactca tcacagaggc tggcgtccag 6360
tcccgggtca tgtgcagcag gaacaagcag ccactctggg ggcaccaggt ggagaggcaa 6420
gacgacaaag agggtgcccg tgttcttgcg aaagcggggc tgctggccac gagtgctgga 6480
cagaggcccc cacgctctgc tgcccccatc acgccgttcc gtgactgtca cgcagaatct 6540
gcagacagga agggagactc gagcgggagt gcggccagag cctgcctcgg ccgtcaggga 6600
ggactcccgg gctcactcga aggaggtgcc accatttcag ctttggtagc ttttcttctt 6660
cttttaaatt ttctaaagct cattaattgt ctttgatgtt tcttttgtga tgacaataaa 6720
atatcctttt taagtcttgt acttcgtgat gggagccgcc ttcctgtgtc cacgcgcctc 6780
ctgcccccgg tgggaagcac ggtcaggagg aggctggtcc agctgcacct cgggggctcc 6840
ctgcactcgc cccccgcctc ctgcagccac acgcattgcc cgagcgaccc tccctggccc 6900
ctgtcactac atggaccccc ggggcttctc ctcttttcta catggatcc 6949
<210>141
<211>330
<212>PRT
<213>智人
<400>141
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
225 230 235 240
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330
<210>142
<211>399
<212>PRT
<213>智人
<400>142
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
225 230 235 240
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Glu Leu Gln Leu Glu Glu Ser Cys
325 330 335
Ala Glu Ala Gln Asp Gly Glu Leu Asp Gly Leu Trp Thr Thr Ile Thr
340 345 350
Ile Phe Ile Thr Leu Phe Leu Leu Ser Val Cys Tyr Ser Ala Thr Val
355 360 365
Thr Phe Phe Lys Val Lys Trp Ile Pne Ser Ser Val Val Asp Leu Lys
370 375 380
Gln Thr Ile Ile Pro Asp Tyr Arg Asn Met Ile Gly Gln Gly Ala
385 390 395
<210>143
<211>4608
<212>DNA
<213>智人
<400>143
cctccaccaa gggcccatcg gtcttccccc tggcaccctc ctccaagagc acctctgggg 60
gcacagcggc cctgggctgc ctggtcaagg actacttccc cgaaccggtg acggtgtcgt 120
ggaactcagg cgccctgacc agcggcgtgc acaccttccc ggctgtccta cagtcctcag 180
gactctactc cctcagcagc gtggtgaccg tgccctccag cagcttgggc acccagacct 240
acatctgcaa cgtgaatcac aagcccagca acaccaaggt ggacaagaaa gttggtgaga 300
ggccagcaca gggagggagg gtgtctgctg gaagccaggc tcagcgctcc tgcctggacg 360
catcccggct atgcagcccc agtccagggc agcaaggcag gccccgtctg cctcttcacc 420
cggaggcctc tgcccgcccc actcatgctc agggagaggg tcttctggct ttttccccgg 480
ctctgggcag gcacaggcta ggtgccccta acccaggccc tgcacacaaa ggggcaggtg 540
ctgggctcag acctgccaag agccatatcc gggaggaccc tgGccctgac ctaagcccac 600
cccaaaggcc aaactctcca ctccctcagc tcggacacct tctctcctcc cagattccag 660
taactcccaa tcttctctct gcagagccca aatcttgtga caaaactcac acatgcccac 720
cgtgcccagg taagccagcc caggcctcgc cctccagctc aaggcgggac aggtgcccta 780
gagtagcctg catccaggga caggccccag ccgggtgctg acacgtccac ctccatctct 840
tcctcagcac ctgaactcct ggggggaccg tcagtcttcc tcttcccccc aaaacccaag 900
gacaccctca tgatctcccg gacccctgag gtcacatgcg tggtggtgga cgtgagccac 960
gaagaccctg aggtcaagtt caactggtac gtggacggcg tggaggtgca taatgccaag 1020
acaaagccgc gggaggagca gtacaacagc acgtaccgtg tggtcagcgt cctcaccgtc 1080
ctgcaccagg actggctgaa tggcaaggag tacaagtgca aggtctccaa caaagccctc 1140
ccagccccca tcgagaaaac catctccaaa gccaaaggtg ggacccgtgg ggtgcgaggg 1200
ccacatggac agaggccggc tcggcccacc ctctgccctg agagtgaccg ctgtaccaac 1260
ctctgtccct acagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga 1320
tgagctgacc aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga 1380
catcgccgtg gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc 1440
cgtgctggac tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag 1500
gtggcagcag gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta 1560
cacgcagaag agcctctccc tgtctccggg taaatgagtg cgacggccgg caagcccccg 1620
ctccccgggc tctcgcggtc gcacgaggat gcttggcacg taccccctgt acatacttcc 1680
cgggcgccca gcatggaaat aaagcaccca gcgctgccct gggcccctgc gagactgtga 1740
tggttctttc cacgggtcag gccgagtctg aggcctgagt ggcatgaggg aggcagagcg 1800
ggtcccactg tccccacact ggcccaggct gtgcaggtgt gcctgggccg cctagggtgg 1860
ggctcagcca ggggctgccc tcggcagggt gggggatttg ccagcgtggc cctccctcca 1920
gcagcacctg ccctgggctg ggccacggga agccctagga gcccctgggg acagacacac 1980
agcccctgcc tctgtaggag actgtcctgt tctgtgagcg ccctgtcctc cgacctccat 2040
gcccactcgg gggcatgcct agtccatgcg cgtagggaca ggccctccct cacccatcta 2100
cccccacggc actaacccct ggcagccctg cccagcctcg cacccacatg gggacacaac 2160
cgactcctgg ggacatgcac tctcgggccc tgtggaggga ctggtccaga tgcccacaca 2220
cacactcagc ccagacccgt tcaacaaacc ccgcactgag gttggccggc cacacggcca 2280
ccacacacac acgtgcacgc ctcacacacg gagcttcacc cgggcgaacc gcacagcacc 2340
cagaccagag caaggtcctc gcacacgtga acactcctcg gacacaggcc cccacgagcc 2400
ccacgcggca cctcaaggcc cacgagccgc tcggcagctt ctccacttgc tgaccagctc 2460
agacaaaccc agccctcctc tcacaaagtg cccctgcagc cgccacacac acacaggccc 2520
ccacacacag gggaacacac gccacgtcac gtccctggca ctggcccact tcccaataca 2580
gcccttccct gcagctgagg tcacatgagg tgtgggcttc accatcctcc tgccctctgg 2640
gcctcaggga gggacacggg agacggggag tgggtcctgc tgagggccag gcctctatct 2700
agggccgggt gtctggctga gtcccggggc caaagctggt gcccagggcg ggcagctgtg 2760
gggagctgac ctcaggacac tgttggccca tcccggccgg gccctacatc ctgggccccg 2820
ccacagaggg aatcaccccc agaggcccaa gcccaggggg acacagcact gaccaccccc 2880
ttcctgtcca gagctgcaac tggaggagag ctgtgcggag gcgcaggacg gggagctgga 2940
cgggctgtgg acgaccatca ccatcttcat cacactcttc ctgctaagcg tgtgctacag 3000
tgccaccgtc accttcttca aggtgaagtg gatcttctcc tcggtggtgg acctgaagca 3060
gaccatcgtc cccgactaca ggaacatgat aaggcagggg gcctagggcc accctctgcg 3120
gggtgtccag ggccgcccag accccacaca cgagccgtgg gccatgctca gccaccaccc 3180
aggccacacc tgccccctga cctcaccgcc ctcaacccca tggctctctg gcttcgcagt 3240
cgccctctga gccctgaaac gccccccttc cagaccctgt gcatagcagg tctaccccag 3300
acctccgctg cttggtgcca gggcactggg ggccaggtgt cccctcagca ggacgtccct 3360
gccctctgga ccaccaggtg ctcacacaaa aggaggtaac cggcatccca ggcccccact 3420
caggcaggac ctcgccctgg agccaacccc gtccacgcca gcctcctgaa cacaggcatg 3480
gtttccagat ggtgagtggg agcatcagtc gccaaggtag ggaagccaca gcaccatcag 3540
gccctgttgg ggaggcttcc gagagctgcg aaggctcact cagacggcct tcctcccagc 3600
ccgcagccag ccagcctcca ttccgggcac tcccgtgaac tcctgacatg aggaatgagg 3660
ttgttctgat ttcaagcaaa gaacgctgct ctctggctcc tgagaacagt ctcggtgcca 3720
gcaccacccc ttggctgcct gcccacactg ctggattctc gggtggaact cgacccgcag 3780
ggacagccag ccccagagtc cgcactgggg agagaagggg ccaggcccag gacactgcca 3840
cctcccaccc actccagtcc accgagatca ctcagagaag agcctgggcc atgtggccgc 3900
tgcaggagcc ccacagtgca agggtgagga tagcccaagg aagggctggg catctgccca 3960
gacaggcctc ccacagaagg ctggtgacca ggtcccaggc gggcaagact cagccttggt 4020
ggggcctgag gacagaggag gcccaggagc atcggggaga gaggtggagg gacaccggga 4080
gagccaggag cgtggacaca gccagaactc accacagagg ctggcgtcca gtcccgggtc 4140
acgtgcagca ggaacaagca gccactctgg gggcaccagg tggagaggca agacgacaaa 4200
gagggtgccc gtgttcttgc gaaagcgggg ctgctggcca cgagtgctgg acagaggccc 4260
ccacgctctg ctgcccccat cacgccgttc cgtgactgtc acgcagaatc cacagacagg 4320
aagggaggct cgagcgggac tgcggccagc gcctgcctcg gccgtcaggg aggactcctg 4380
ggctcactcg aaggaggtgt caccatttca gctttggctt ttcttcttct tttaaatttt 4440
ctaaagctca ttaattgtct ttgatgtttc ttttttgatg acaataaaat atccttttta 4500
agtcttgtac ttcgtgatgg gagccacctt cctgtgtcca cgcgcctcct gcccccggtg 4560
gaaaacacgg tcaggaggag gctggtccag ctgcacctcg ggggatcc 4608
<210>144
<211>330
<212>PRT
<213>人工
<220>
<223>由质粒pmIgGδ-A编码的γ链恒定区的短(sIgG)亚型的氨基酸序列
<400>144
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
225 230 235 240
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330
<210>145
<211>399
<212>PRT
<213>人工
<220>
<223>由质粒pmIgGδ-A编码的γ链恒定区的长(mIgG)亚型的氨基酸序列
<400>145
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
225 230 235 240
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Glu Leu Gln Leu Glu Glu Ser Cys
325 330 335
Ala Glu Ala Gln Asp Gly Glu Leu Asp Gly Leu Trp Thr Thr Ile Thr
340 345 350
Ile Phe Ile Thr Leu Phe Leu Leu Ser Val Cys Tyr Ser Ala Thr Val
355 360 365
Thr Phe Phe Lys Val Lys Trp Ile Phe Ser Ser Val Val Asp Leu Lys
370 375 380
Gln Thr Ile Val Pro Asp Tyr Arg Asn Met Ile Arg Gln Gly Ala
385 390 395
<210>146
<211>53
<212>PRT
<213>智人
<400>146
Leu Trp Thr Thr Ile Thr Ile Phe Ile Thr Leu Phe Leu Leu Ser Val
1 5 10 15
Cys Tyr Ser Ala Thr Val Thr Phe Phe Lys Val Lys Trp Ile Phe Ser
20 25 30
Ser Val Val Asp Leu Lys Gln Thr Ile Val Pro Asp Tyr Arg Asn Met
35 40 45
Ile Arg Gln Gly Ala
50
<210>147
<211>34
<212>PRT
<213>智人
<400>147
Ala Gly Thr Thr Asp Ala Ala His Pro Gly Arg Ser Val Val Pro Ala
1 5 10 15
Leu Leu Pro Leu Leu Ala Gly Thr Leu Leu Leu Leu Glu Thr Ala Thr
20 25 30
Ala Pro
<210>148
<211>4551
<212>DNA
<213>人工
<220>
<223>γ链表达盒恒定区的DNA序列,来自表达质粒pIgG-GPI-B的从CH1到3′UTR
<400>148
cctccaccaa gggcccatcg gtcttccccc tggcaccctc ctccaagagc acctctgggg 60
gcacagcggc cctgggctgc ctggtcaagg actacttccc cgaaccggtg acggtgtcgt 120
ggaactcagg cgccctgacc agcggcgtgc acaccttccc ggctgtccta cagtcctcag 180
gactctactc cctcagcagc gtggtgaccg tgccctccag cagcttgggc acccagacct 240
acatctgcaa cgtgaatcac aagcccagca acaccaaggt ggacaagaaa gttggtgaga 300
ggccagcaca gggagggagg gtgtctgctg gaagccaggc tcagcgctcc tgcctggacg 360
catcccggct atgcagcccc agtccagggc agcaaggcag gccccgtctg cctcttcacc 420
cggaggcctc tgcccgcccc actcatgctc agggagaggg tcttctggct ttttccccgg 480
ctctgggcag gcacaggcta ggtgccccta acccaggccc tgcacacaaa ggggcaggtg 540
ctgggctcag acctgccaag agccatatcc gggaggaccc tgcccctgac ctaagcccac 600
cccaaaggcc aaactctcca ctccctcagc tcggacacct tctctcctcc cagattccag 660
taactcccaa tcttctctct gcagagccca aatcttgtga caaaactcac acatgcccac 720
cgtgcccagg taagccagcc caggcctcgc cctccagctc aaggcgggac aggtgcccta 780
gagtagcctg catccaggga caggccccag ccgggtgctg acacgtccac ctccatctct 840
tcctcagcac ctgaactcct ggggggaccg tcagtcttcc tcttcccccc aaaacccaag 900
gacaccctca tgatctcccg gacccctgag gtcacatgcg tggtggtgga cgtgagccac 960
gaagaccctg aggtcaagtt caactggtac gtggacggcg tggaggtgca taatgccaag 1020
acaaagccgc gggaggagca gtacaacagc acgtaccgtg tggtcagcgt cctcaccgtc 1080
ctgcaccagg actggctgaa tggcaaggag tacaagtgca aggtctccaa caaagccctc 1140
ccagccccca tcgagaaaac catctccaaa gccaaaggtg ggacccgtgg ggtgcgaggg 1200
ccacatggac agaggccggc tcggcccacc ctctgccctg agagtgaccg ctgtaccaac 1260
ctctgtccct acagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga 1320
tgagctgacc aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga 1380
catcgccgtg gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc 1440
cgtgctggac tccgacggct ccttcttcct tacagcaag ctcaccgtgg acaagagcag 1500
gtggcagcag gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta 1560
cacgcagaag agcctctccc tgtctccggg taaatgagtg cgacggccgg caagcccccg 1620
ctccccgggc tctcgcggtc gcacgaggat gcttggcacg taccccctgt acatacttcc 1680
cgggcgccca gcatggaaat aaagcaccca gcgctgccct gggcccctgc gagactgtga 1740
tggttctttc cacgggtcag gccgagtctg aggcctgagt ggcatgaggg aggcagagcg 1800
ggtcccactg tccccacact ggcccaggct gtgcaggtgt gcctgggccg cctagggtgg 1860
ggctcagcca ggggctgccc tcggcagggt gggggatttg ccagcgtggc cctccctcca 1920
gcagcacctg ccctgggctg ggccacggga agccctagga gcccctgggg acagacacac 1980
agcccctgcc tctgtaggag actgtcctgt tctgtgagcg ccctgtcctc cgacctccat 2040
gcccactcgg gggcatgcct agtccatgcg cgtagggaca ggccctccct cacccatcta 2100
cccccacggc actaacccct ggcagccctg cccagcctcg cacccacatg gggacacaac 2160
cgactcctgg ggacatgcac tctcgggccc tgtggaggga ctggtccaga tgcccacaca 2220
cacactcagc ccagacccgt tcaacaaacc ccgcactgag gttggccggc cacacggcca 2280
ccacacacac acgtgcacgc ctcacacacg gagcttcacc cgggcgaacc gcacagcacc 2340
cagaccagag caaggtcctc gcacacgtga acactcctcg gacacaggcc cccacgagcc 2400
ccacgcggca cctcaaggcc cacgagccgc tcggcagctt ctccacttgc tgaccagctc 2460
agacaaaccc agccctcctc tcacaaagtg cccctgcagc cgccacacac acacaggccc 2520
ccacacacag gggaacacac gccacgtcac gtccctggca ctggcccact tcccaataca 2580
gcccttccct gcagctgagg tcacatgagg tgtgggcttc accatcctcc tgccctctgg 2640
gcctcaggga gggacacggg agacggggag tgggtcctgc tgagggccag gcctctatct 2700
agggccgggt gtctggctga gtcccggggc caaagctggt gcccagggcg ggcagctgtg 2760
gggagctgac ctcaggacac tgttggccca tcccggccgg gccctacatc ctgggccccg 2820
ccacagaggg aatcaccccc agaggcccaa gcccaggggg acacagcact gaccaccccc 2880
ttcctgtcca gagctgcaac tggaggagag ctgtgcggag gcgcaggacg gggagctgga 2940
cggggccggc accaccgacg ccgcgcaccc ggggcggtcc gtggtccccg cgttgcttcc 3000
tctgctggcc gggaccctgc tgctgctgga gacggccact gctccctagg gccaccctct 3060
gcggggtgtc cagggccgcc cagaccccac acacgagccg tgggccatgc tcagccacca 3120
cccaggccac acctgccccc tgacctcacc gccctcaacc ccatggctct ctggcttcgc 3180
agtcgccctc tgagccctga aacgcccccc ttccagaccc tgtgcatagc aggtctaccc 3240
cagacctccg ctgcttggtg ccagggcact gggggccagg tgtcccctca gcaggacgtc 3300
cctgccctct ggaccaccag gtgctcacac aaaaggaggt aaccggcatc ccaggccccc 3360
actcaggcag gacctcgccc tggagccaac cccgtccacg ccagcctcct gaacacaggc 3420
atggtttcca gatggtgagt gggagcatca gtcgccaagg tagggaagcc acagcaccat 3480
caggccctgt tggggaggct tccgagagct gcgaaggctc actcagacgg ccttcctccc 3540
agcccgcagc cagccagcct ccattccggg cactcccgtg aactcctgac atgaggaatg 3600
aggttgttct gatttcaagc aaagaacgct gctctctggc tcctgagaac agtctcggtg 3660
ccagcaccac cccttggctg cctgcccaca ctgctggatt ctcgggtgga actcgacccg 3720
cagggacagc cagccccaga gtccgcactg gggagagaag gggccaggcc caggacactg 3780
ccacctccca cccactccag tccaccgaga tcactcagag aagagcctgg gccatgtggc 3840
cgctgcagga gccccacagt gcaagggtga ggatagccca aggaagggct gggcatctgc 3900
ccagacaggc ctcccacaga aggctggtga ccaggtccca ggcgggcaag actcagcctt 3960
ggtggggcct gaggacagag gaggcccagg agcatcgggg agagaggtgg agggacaccg 4020
ggagagccag gagcgtggac acagccagaa ctcaccacag aggctggcgt ccagtcccgg 4080
gtcacgtgca gcaggaacaa gcagccactc tgggggcacc aggtggagag gcaagacgac 4140
aaagagggtg cccgtgttct tgcgaaagcg gggctgctgg ccacgagtgc tggacagagg 4200
cccccacgct ctgctgcccc catcacgccg ttccgtgact gtcacgcaga atccacagac 4260
aggaagggag gctcgagcgg gactgcggcc agcgcctgcc tcggccgtca gggaggactc 4320
ctgggctcac tcgaaggagg tgtcaccatt tcagctttgg cttttcttct tcttttaaat 4380
tttctaaagc tcattaattg tctttgatgt ttcttttttg atgacaataa aatatccttt 4440
ttaagtcttg tacttcgtga tgggagccac cttcctgtgt ccacgcgcct cctgcccccg 4500
gtggaaaaca cggtcaggag gaggctggtc cagctgcacc tcgggggatc c 4551
<210>149
<211>330
<212>PRT
<213>人工
<220>
<223>由质粒pIgG-GPI-B编码的γ链恒定区的sIgG亚型的氨基酸序列
<400>149
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Pro
100 105 110
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
115 120 125
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
130 135 140
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
145 150 155 160
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
165 170 175
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
180 185 190
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
195 200 205
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Ala Cys Pro Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
225 230 235 240
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330
<210>150
<211>380
<212>PRT
<213>人工
<220>
<223>γ链恒定区的长亚型的氨基酸序列,包括由质粒pIgG-GPI-B编码的人胎盘碱性磷酸酶的羧基末端信
号肽
<400>150
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
225 230 235 240
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Glu Leu Gln Leu Glu Glu Ser Cys
325 330 335
Ala Glu Ala Gln Asp Gly Glu Leu Asp Gly Ala Gly Thr Thr Asp Ala
340 345 350
Ala His Pro Gly Arg Ser Val Val Pro Ala Leu Leu Pro Leu Leu Ala
355 360 365
Gly Thr Leu Leu Leu Leu Glu Thr Ala Thr Ala Pro
370 375 380
<210>151
<211>2061
<212>DNA
<213>智人
<400>151
gtacacactg taacttctcc tctcgatgtc ctgccctcgc cacgggcacg gggggagcat 60
gcatttccag gcttggggag acacagaggc aggagagctg gaagaatggg ctcctgcctg 120
cctggtgcca catcctgccc cagctttgag ggctgaatcc tctcagctca gagaagagct 180
tccctagttt tgcaggcgca cggaggcctc ccacccgtac acctgcctct gctctccccc 240
tggccattgc aatgcatgcc cgggatctgg gtttaccctg cagcccgact cctccagggc 300
aggctccttc cgtccttggg tccagcagcc tcccccctcc gctgggtact gagcatctga 360
aaagtctcag atagggccct acacctcttc tcccttctca agtgctcagc ccaggggtgg 420
ggcttgctgc ttgcaaagag cagcccaccc tcctgtactg ccatcctatt gcctttgaag 480
aeggaggtca gcctggacag gtgaactgag ttgccttcag aaaggcctgt ctcaggcagc 540
cgcagcaggc ttcacgatgt ccatgtatcc tgtttgcctg ctgccgtctc tcctctccca 600
agaggggagg tctgcgtgtt gagccaggga ggaaggagcc tgcagcctgc tcagggtggt 660
ggctggtgac tgcggggcca gggcttgctg ctgtctctcg gaatgtcctg catgtgggtc 720
tggctgttcc tgctggcact gccctcttct ctctcccctc ctttcccact ctgccctgct 780
tgcccgtcag ggtctccgga tgcccctgct ggaggcagag aagcgttctg tttttatcat 840
cgtccctgtc ttggtcctgt ccgtgctccc catgtgctcc caatcagttg tcttcgttcc 900
tcaacatcag ggaaagaaaa aacagcaaca cttctccctg tcatcagctc tgctgtctcc 960
ttgtcaggcc ttgggacgtt ctctgggctc agtctagggt cgtgcctcat gcctcatgcc 1020
tctctgggaa ggttcccagg ggcgccgcct ccatggttgg ctggtgggtt ttaaagggga 1080
aggttgggag gaggcctgtg gaaggaggag ggccgtccac acccgtgggg caaagcgtgt 1140
tccatgaaac cagtgctgtg cgtgctggga tgaaagcctc ccagcctctc gtgctgaagg 1200
gaaaggtgga ccgaactggc ttagctgctg tggcaatggg gtggggggtg agtcctaatc 1260
agaaggtggg agaagagaat aacaaaatgg aactccgccc cctgccgcca catttcctta 1320
agttttagga gttggggcct atgagtagaa tttgaaagaa ctaccaaaaa agcttatttt 1380
tggaaattaa gtcagctttg cctgtggttt ggctaggaat aaatatttac ctgtgtgccc 1440
ctacagagat gacacagacc caaagtggga cattgagcaa ggcacaggct ggttcccctc 1500
ctgtcctctg tcctcattct gtgagtgcca ccatcaccag gtactgtggc ctgcagaaat 1560
aaacgaggca ccaaccctgc cctcagggac cccgtgtctt cttagggaca gaggactcag 1620
aaacatctgt gtccccgcat gttaagtgca tgcacacagt gctgaggctt tgcagagaag 1680
ggccatggct ccctgaggag accaagaaag gcacatagaa gtgacttctc agctggccct 1740
tgcaggatcc ctgggtgcct gtacctctcc agtccagctg agcaaggagg ctcaggaatt 1800
aatgaagatg gggcttgtgt agcacgatgg ttctaggcca tcccttttcc atggctcaag 1860
tccattgaag gcagcctcca aaatcagggg gttgcctgat cctcctagtg ccacaatccc 1920
agtcctagct agaacttgcc atggttcttc tccctaaaag ttcccagggg acaggagaca 1980
ggtgttgctt ttctaatgga gcgggacctc gcatggacgg ggccctccaa gcctgctaac 2040
accctgttcg cactgactca g 2061
<210>152
<211>2726
<212>DNA
<213>小家鼠
<400>152
tacgttcaat ggcagtggat cttaaagacc ttttggatct aagaccagaa gccatctctg 60
actcccctct caccttccag tttcttccaa atcctctggg ccagccagag gtctcagatt 120
ctgccctctt gccctgtgcc caccttgttg accactggac agcatatgtg atggctactg 180
ctagtgccag cttcacaaga ggttaacact acggactagc cattcttcct atgtatctgt 240
ttctgcaaat acagccgctt tacttaagtc tcagcacttc ttagtctcct cttttcctct 300
cagtagccca aggggtcatg tcacaaacat ggtgtgaagg gctactttgt caaatgaaaa 360
ggtctatctt ggggggcatt tttttctttt ctttttttct tgaaacacat tgcccagcaa 420
agccaataaa tttctctcat cattttgttt ctgataaatt cttactattg attgaaatgc 480
ttccctgagg aacattaaat tatgtatgaa tttccattca aagtcatcat gcagcagtgg 540
tatcaagcag gcagcatcta gtgcagaact gagattgtct acagaatgcc tgcttgatgc 600
tcagccttat atcccagtta cggacatgtc tacaaatagc ccttctctta ccatctctcc 660
cttaacttca tgatgtggta gctgattcct tctgccatca actgaaatgg caaaataaag 720
gaaagcctga atcctgtctc ccctcttagt ttccaaggac acttgcccag aaaaatgcgc 780
cccccccccc ccgtatcccc aggcagcatc caggactttc ttaaatggtt ctctgtacaa 840
tcccaccttt aaaacagtca gtgaccattc tttgtaatca ttactttatt aatggttttc 900
ttttctaaac cacctgcctt tgctgttgta gcttctcaat acatgctctg agacaaatca 960
ttccttatat attccaatat tgctgaaaac tcattaattt ttactaatag ggggttgttt 1020
tctaaagctc aggaaatgac ttgtactccc taagtaggaa atgttgcaaa ccaccatttc 1080
atgatgtaca gttgattcaa ttaagtcagg tctgtgagga ctttggttcc ttatctatat 1140
gtaaataatg ctgataccaa atctttgcta ttcattaaca tgtaatcttg caaataataa 1200
taaatttaac agaaactcaa attgaggtag attcttaagg ataatagctt tcagcctagc 1260
cataaaagaa atgaaaaaat agtctttaaa gatcccaaat tcagaattag taatattaat 1320
tcagatagtc cttcgtaaga ttgaaaaaca tgacttctga tttctcatct ttccaaagtt 1380
agacttgacc ttggtgagat ggcaaagtta aaaaattcct ggggtttaag gatatattgg 1440
ggcaacagtc acagcaacgg tgatgctttt tatatctaaa tcctgtcagg ttttgtttcc 1500
actgagctca ttcattcacc tagggatctc attaaaagta ggggaccggg ctggtgagat 1560
ggctcagcag gtaagagcac ccgactgctc ttccgaaggg cctgagttca aatcccagaa 1620
accacatggt ggctcacaac catccgtaac aagatctgac tccctcttct ggtgtgtctg 1680
aagacagcta cagtgtactt acatataata aataaataaa tcttaaaaaa aaaaaaaaag 1740
taggggaccc tatcagaggt ctcaatgtgc ctggaaggtc tcccaacatg gctgtgggaa 1800
aatgcctgac aatttttttt caattgcatg tgtgttctcc gagaaaggat aagatgcaag 1860
tttatataag aagcaggatt tacttatact aaaatgcctt acacgtaagg ttcaggtagc 1920
atcgtggcag agagggtaga aagactgtaa gagccagagg accaggatgt ccgctcttag 1980
tgtcttctat atatgaaaga aagctgtacc cgtgaattct caaacagttg tctacatatc 2040
acctgcacaa tggccgttct ggaaatctaa aaaaaacaaa aaacaaacaa acaaataaat 2100
aaataaataa atatcaaaaa atcaaaaacc acccctagat gaaaagctac aagtaattaa 2160
aagctgctaa aagggggaga atcaattttc tccagagaca agtccctgat aggtcatcca 2220
atttcgggtg atcagcccta agcacataca catataagca atactaaacg gacacagctg 2280
gtctatatac ttacaacttg gggcttgaag gaagttggtg gggaaggggg gagagtagta 2340
atgatgtatg taaatacagt actcatgtac gaaactcccc aaaaataaag aattcttaaa 2400
attattaaaa actggtattc ttgtcttgga ctattttctg aattttatat taatattttc 2460
atcatcaaaa gactataaaa tgattataat aatgagagaa gttcagatga aacatcaact 2520
gtcttaaatg tagaaaagga aactgacttc aggcagagaa gctaaaaatg ttggcaacct 2580
tgccttggga cttagccacc agtcatttcc tctatataag tcacccatat cccaatttct 2640
gagataattt taaatgttat cactgtgtca aaggtgcagg acatgtatgg aatgcccata 2700
ttaccttctc ttgtctctca ttccag 2726
<210>153
<211>5686
<212>DNA
<213>智人
<400>153
gggtgagttg gcagcaacat ctcttggttt aagagttcca gcacagcgat agtactttct 60
agccacatct cagcaaggaa actaggctat tgccacctgc tcttaagagg cttgaacaca 120
ggtgttaact cctgattgaa atgaacaaag ataggagaag attaggggga aaatctgtat 180
ccttgctgga aaccagggca gtgcacatat aaagagtatg ctgttcactg gatgggaagg 240
aaaaaaactt agaagtgtag tagtcaaagc acacaaacaa ccctaaccca gagtagacat 300
tgctggaaga aagggaagac catgtagcag ctgtgtgaga gaatgaatct taatgataac 360
agcatgatcc cttgctaggg ctgccatcaa aaagtacagg ccttcctcgt tttattgtac 420
ttcgcagatg ttatgctttt tacaaattga acgcttgtgg gaacgctgtg taagcatgtt 480
cgtcggcatc atttatccaa cagcgtgtgt tgacttcgtg tctctgtgta gcattttgat 540
tattctcaca gtatcccaga tgttttcatt attatcatgt ctgtgatagt gatctgtcat 600
cagtgatctt tgatgttact attgtcattg tttggggtcc ctacgaactg cacccatata 660
agacagaaaa cttaatcaat aaatgtgcgt gctttgactg ctccatggac tagacattcc 720
ccttctgtct ccctctcttc aggactccct aatccctgag acacaataat actaaaatga 780
ctccaattaa taaccctaca atagccttta agtgttgaca tgaagggaag agtcatgcat 840
ctcttacttt aaatcaaaag ctagagatga ttaagcttac tgaggaagat acgttgaaaa 900
ccaagatagg ccaaaagcca ttcctcatgt gccaaacagc tagaaagttg taaaggcaaa 960
gtaaaaatac ttgaaggaaa tttaaaatgc tcttccagtg aacacatgta tgataagaag 1020
gtaaaacagc cttattgctg atatggaaga agttttagtg gtctagacat aagatcaaac 1080
tagaaacatt tcctgaagcc aaagcgtaat cctgagcaag gccgtaactc tcttcaattc 1140
tgtgaaggct gacagaagct ggaagctagc agaatttggt tcctgaggtt taaggaaaga 1200
agccctctcc ataacataaa ggtccaaggt gaagcagcca gtgctcatat agaagttgca 1260
gcaagttatc tagaaaatct agctaagatc actgacgaag gtgttacact aaataacaga 1320
ttttccatgt agacaaaaca gccatctatt ggactttcat agctagagag gagaagccaa 1380
tacctgggtt caaagcttca aagaacaggc tgacttttta ggagctaatg ccgctggtga 1440
ctttgagttg aagccaatgt tcaccgacca ttctaaaaat cctagggccc ttaagcatta 1500
agttaaatat accttgcctg tgtctataaa tggaagaaca aagcctgatg acagaggtat 1560
gtttgcggca tagtttactg aatattttaa gcccactgtt gagatctagt gctcagaaaa 1620
acagattcct ttcaaaatat tactgctcat tgacattgca cctaagcagc caagagctct 1680
aatggagaag gacatggaga tgaatggagt tttcctgcct gctaacaaca gcagccattc 1740
tgcagcccat ggctgaagga gtaattttga ctttcaagtt tcactattta agaaatacat 1800
tttgtaaggt gagtccccac agacagtaat tcctctgatg gatctgaaca aaataagttg 1860
aaaactgctg gaaagaattc atcattctac ataccattaa gaacattcgt gattcatggg 1920
aagagcttaa aatatcaaca ttaataggag tttggtagaa gttaattcca accctcatgg 1980
ctgactttga gaggttcaag acttcagcgg aggaaataac tgaagatgtg gtgaaaatcg 2040
caagagaact acaattagaa gtggagcctg aagacgtgac tgaattgcga caaattcatg 2100
atcaaacttg aacagatgag gagttgcttc tcatgaatca gcagagaaag tggttttttt 2160
gaggtggagt gtcttcctgg tgatgatgac atgagcattg tgaaaatgac cacaaaggat 2220
acagaatatt acaaaaactt aggtgataaa gcagcagcag tatcgggagg attaactcca 2280
attttgaaag cagttcagag ggtaaaatgt tatcaaacag cattgcatgc tagaaaaatc 2340
ttttgtgaaa ggaagagtcc atcaatacag caattttatt gctgtctcag cttaagaaat 2400
tgccacagcc accccagcct tcagcaacca ccaccctgat cagtcagcag ccatcaacct 2460
tgaggcaaga ccctccacca acaaaaagat tacaaattat tgaaggtcag ataatcttta 2520
gtattttctt atcaataaac ctttttttaa agtatgactt tgtttagaca taatgctatt 2580
tcacacttaa tagactaccg tatagtataa acacaacttt tagactcact gagaaatcaa 2640
aaaattcatc tgactcattt gttgtgatat gtgttttatt gtggtggtct ggaactgaac 2700
ctgcaatata tttgaggaat gtctgtaaca cgaagtgggc ggcttaatac aacagaagtt 2760
tttggagttc cattctagag actggaaatc caaaatcaag gttttgggag ggccacgctc 2820
cctctgaatc ctgtagggaa ggatccttca ttgcatcttc cagcttctgg agccacaggc 2880
cttccttggc atgtggcagc agaactccaa tttctgtctc tgtcctcata tggccatctt 2940
ccctctgggt ctgtgtcctc atatgctgtt ctctctgtgc atgtctgttt ccaaagtttt 3000
cccttttaat aaggacacca gtcacactgg attagggcct accttcatga cctcacctta 3060
acttgattgc gtctgcaaag atcgtatttc caaataaggt cacatccaca gggactggag 3120
gttaagactt cagcatactc aggggagaca caattcaacc tataacagag cccctttgaa 3180
aatgtcaggc atcaggtgct cacagcacac ggtccagctg ttggtgctca cccttgctca 3240
gcacacggtg gaggctctgc ctcaatctca ttcttcatgg gaacagctaa gccaggtagc 3300
gaatacgagt tacgtttgtg tcaccacagc agacactgaa aggaagtttg tggtgtccag 3360
cacatttgcc agcataggcc tcacagacca caacacctag aatctgacaa aaattagaat 3420
aaatccgatg tctccttttc tctctttttt taagcatttt taaatacaat tggtaaattt 3480
cctgactgta ccctactcat cttgcattgc tgaataagtg aggaacctct ctatggcatg 3540
aagcagaaaa ccattgagct tatgagaaga aacaggaaat acatgtaaaa gagaccagtc 3600
tgaaaaatca ggagaaattt tacaaaagag atttactgcc aagaaaccag ggaaactttc 3660
agaacactct ccttttcata tttgtaacaa gattcaattg tacaaatact tctgtgaaac 3720
tagccatgaa gtgtgtacat gaatgaatat atgtattcag tgtgtttata tatttcattt 3780
catacatgtg atctatatca tatatatata tatatatata tatatatatc acaatgaata 3840
ttgcagaaaa ttaaagcaaa gagtaagaag agttaaccaa atgttccctg aatgcagtgg 3900
gaaaagatga aaaggagaag acaggcaagc acagtggctc acacatgtaa tcccaacata 3960
ttgagaggcc ggggcaagag gatcacttga gcccaggagc tcaagaccag cctggacaac 4020
atggcaaaaa cccatctcta caaaaaagta taaaaattag ctgggcatgg cagcatgtga 4080
ctgtagtcac agctaggcag gagaatgaag tgggaggatc acctgagccc aggaggctga 4140
ggcttcagtg agccttgatg gcaaccctgc actccagcct ggatgacaga atgagaccct 4200
gtctcaaaaa aggaaaaaga gaagacatat gtaacacagt gtcaggattc aaaatacatg 4260
tagcaaaaat tccagaaggg taagcaaata agcccatacc tagccatcac tagggaaact 4320
tctgtgtttc agggatagag aaaaaaactc tgattccaga aataaaatgt gaaaatataa 4380
tacccgcaaa agaaataaat ttattgacac cacattgatt ctatattgta taagatatat 4440
tgtataacac taaattgtag aagatagcac ttctcttgag ccaagctaat atgcacacct 4500
agaggctcca ggtacacatt aaaggataag caaagacaca aaaattgtac catttaagta 4560
tgtttcccag gcaattcata agcaaatttt taaaaaggag aatgatgaat tccaaaggaa 4620
atctcaatga gataggatgt aggtaaaaag aaaaaacaac aataaaaaaa acatacacac 4680
aaaatcttgg caatgtaata ataatttatt aataatctct ctaaactcat agaatatatc 4740
aataaaacta gaaaatgtga gacagcaaga aaagataggc tagacttaca ttacagaaaa 4800
gatgagtaga ctgactatat agagtttgac tctaaaattg agaaaagagg tttaaatatg 4860
tgtcacgtgt atatgtataa tatacatgca cacatataaa atgaaattcc tcactggaaa 4920
aaatattaag tttaatttcc aaattattag aatgactaaa acagttgaat ctatctgttc 4980
catatagtaa ttgaggggga aggaaaagga atagcaagga atccattttt ctcttccacc 5040
agaaataagc tccctcatgt ttgggtcagg tcactttatt cattgctttc tccacatcct 5100
catcattagg catcatgaaa tcaatgaaaa ggcagtgaaa gaatgatgat gaaagctaat 5160
caaatgtatc atttacctca atacgtttgg tttaatcttc ttgttgaaag acaaattctc 5220
ctagacagga tcaaaagcaa catatcttgt atttataaaa aacatgtctc aagtaaaatg 5280
acatagtatt atacacacac acacacatac acacacacac acacattaag ggaaaatgca 5340
gacaaaaaat ggcagaaata atatttagag aaagatagaa tttaagaaaa aaaattgtca 5400
aacaggtctg caaaataatt gttttatttt ggtgccagtt acagtccgca atgaagcgaa 5460
gatattaaga agcaaagtgg gccaggagaa attagagcag aaatatctct ttcaatagtg 5520
agagaatagg aaaaagaagt agaaaagctg ggaacaatag gtaaagttta ggctaggcct 5580
tagacttctc ctgcattgta atccctctgg tttgccacat atgcatgctg tcaggaagtt 5640
gatgaggtat gtacagcaca atttattttc cattttttgc ctttag 5686
<210>154
<211>1260
<212>DNA
<213>小家鼠
<400>154
gtgagtatca gggtcgtagg ctgtgaggat ctctacagcc gtgtatattc tctgttcaga 60
aattccctct ggctgaccgt ggcgttttcc gttctaggat gcctctaact atacatccct 120
gtggctgtgg tgtggtgatc cgagggctgc actaacacac agatttctgt ggttgtcact 180
gttcagggcc acagaatgac tgagtcttct ttctatacag tatgcagtag ctggatagtc 240
aggactgggg aaggttgtgt gaggttgact cggtggtacc gctgaggagg aaaagggctt 300
gtccagagca ggcgtgatgt acagcgcaca ttgttttttt cagcaagtaa tgaaaatctg 360
tgactgagtg accttggggg ctgcggtggt gaggagagct cacgggaatc ctggagcagt 420
gtagctggcg tgtcaaaagc agaaacgcag gagatgggtc agtgcgccga ggcccggagt 480
ttaaatcccc agagcccacg taaaagcctt gcgcagtagc acgcatgcgt aatcctgatg 540
gagcaattag gagaagccgg tggccggcta gcctgtgcag actgtgaaaa cagagcatct 600
gaagctgtgt gaaaggctag ctcgcctgtg ctcctcagat cctgacatac gccatctcga 660
gcaaaagatt aacaatcgat taaaacccac acatgaaaac aaagaaacca gttagctcca 720
gataaaagca aatgccagga aggaaaggag gcagagggca ggggtggagg agcaggtggg 780
agaggatgac gtcagagaaa tctcagtggc cgtgaacaag gaagatattc cagggcttgt 840
tctgaatgtg tgatctttta aaccatgtgg ccctcttagc ctgtgagaaa agtccatttc 900
tagccctgct gaacagagaa gcccagggga gcaaccaact ggccttcggt cactcagcaa 960
ggacctagta gaggctggct ttgaacttga gccctccagg gcacttgacc tctgagcttt 1020
gggaaaacaa gcgaggcagc ttcacgctgt tgtgatctgg acttggatca gggccagtga 1080
ggacagggac agggatgggt gtgataagga cttggtgtgt aagagggaga gttggggaga 1140
caggtgggag ggctatctgt agccaggtaa ccaagggtcc caggggaacc cccagtgtgg 1200
acgcggactg cacatgacac agggcggcct ccccattcat gactgttttt ctccttgcag 1260
<210>155
<211>2627
<212>DNA
<213>智人
<400>155
gtaaacccac ccatgtcaat gtgtctgttg tcatggcgga ggtggacggc acctgctact 60
gagccgcccg cctgtcccca cccctgaata aactccatgc tcccccaagc agccccacgc 120
ttccatccgg cgcctgtctg tccatcctca gggtctcagc acttgggaaa gggccagggc 180
atggacaggg aagaataccc cctgccctca gcctcggggg gcccctggca cccccctgag 240
cctttccacc ctggtgtgag tgtgagttgt gagtgtgaga gtgtgtggtg caggaggcct 300
cgctggtgtg agatcttagg tctgccaagg caggcacagc ccaggatggg ttctgagaga 360
tgcacatgcc ccggacagtt ctgagtgagc agtggcatgg ccgtttgtcc ctgagagagc 420
cgcctctggc tgtagctggg agggaatagg gagggtaaaa ggagcaggct agccaagaaa 480
ggcgcaggta gtggcaggag cggcgaggga gtgaggggct ggactccagg gccccactgg 540
gaggacaagc tccaggaggg ccccaccacc ctagtgggtg ggcctcagga cgtcccactg 600
acgcatgcag gaaggggcac ctccccttaa ccacactgct ctgtacgggg cacgtgggca 660
caggtgcaca ctcacactca catatacgcc tgagccctgc aggagcggaa cgttcacagc 720
ccagacccag ttccagaaaa gccaggggag tcccctccca agcccccaag ctcagcctgc 780
tcccctaggc ccctctggct tccctgtgtt tccactgtgc acagatcagg caccaactcc 840
acagacccct cccaggcagc ccctgctccc tgcctggcca agtctcccat cccttcctaa 900
gcccaactag gacccaaagc atagacaggg aggggccacg tggggtggca tcagaagcag 960
gccagtgaga cagggcctgc ccagggccct ctgcatgcct ctggcttctg cctggggctc 1020
ccaggagtgt aagaacagtc ccacaaccac tgtggggaca cctggcaccc agactcccac 1080
aagggggcag tggggcccct gctcgtgcct tagacatctt ccgggcctcc ccagggcccc 1140
ccgccttctg gctgcctccc tctgctctca gggccaaggt gaggtggagg ccactgtcac 1200
ccctgagggt ccagtcacca gagggtaatt gagagcaaca ggtcactcgg ggaagccctg 1260
ccacagagaa gccctccagc ccatgggacc caggacctgg cccaggggag gggcttttaa 1320
agagaggggg aaagagggag aatcaacaga tgaggggctg aaccagcaga cagagatcag 1380
gcagacacat gggtagatcc taggacatat aatgaatgga tgggtggatg gaggattggt 1440
agacggagga tggatgggtg ggtaaatggc tggatggagg atggacagat ggatatatga 1500
tggatggatg aaggacgggt ggatggagga tggatgagtg gatgaatgaa ggatggaaga 1560
tggatggatg gatgggtgga tggactgatg gatgatggat ggatggatgg atggatgatg 1620
gatggatggg taggcggatg gaggatggaa gggtggatgg aggatggaag atagatggag 1680
gggtgaatgg aggatgggtg gactgacgga ggattgagga tactggggtg ggtgggtggg 1740
tagatctatg gaggataggt gtatggaaga taattggatg gagaattgct ttatgaatgg 1800
atgaatgaag agatggaaaa tagctttata gatggatggg tgaatggatg gatggataga 1860
tggaagaagg atgaatggat ggaaaatagc tttatagata tatgggtgga tatttaagtg 1920
atagccttac attaatagat gaatggagga tgaatggttg ggtgagtggg taggagtgtt 1980
actgatggag gggtggatat acggataata gctttataga tggatggatg gatagatgga 2040
tggaaggata gaaagacagg tgaatgactg gatgtatcag catatgacaa gcaggtacag 2100
ctgtacatgg gaggtctatg ccctgagacc ctgaggaaaa tgaggatgcc cgtgctggtg 2160
gccctcacct ggccctcgct tgtaacccct cagccacatt ccctgggaag gcaacagagg 2220
cctctggtct tgcccattca acctttggca cactgagtgt cagacccagg tctctgtctt 2280
ggacccagat ctccttgagg gtgggtgtgt ctggtcctct ctggccccgg gacccagtca 2340
ctgaatacgt ggctgggact gagacggggt ggggtgggag gggcgggagg gtacctcggg 2400
ctcaagcttc ccttggagaa gcagatggtg tccactttct gccctgccaa gtctctccct 2460
gaagtgccct aagaatgtca aagacagaag gtcccagccc ctcacctggg actctgcctc 2520
ctcatcctcc ctgggggagt ctcaggcctt agatggggac ccagacccca ctgtccccag 2580
accccaagga agcatagccg ctgttcacac gagtctgggc ctggcag 2627
<210>156
<211>1937
<212>DNA
<213>智人
<400>156
gtaaatgacg tactcctgcc tccctccctc ccagggctcc atccagctgt gcagtgggga 60
ggactggcca gaccttctgt ccactgttgc aatgacccca ggaagctacc cccaataaac 120
tgtgcctgct cagagcccca ggtacaccca ttcttgggag cgggcagggc tgtgggcagg 180
tgcatcttgg cacagaggaa tgggcccccc aggaggggca gtgggaggag gtgggcaggg 240
ctgagtcccc ccaggagagg cggtgggagg aggtgggcag ggctgaggtg ccactcatcc 300
atctgccttc gtgtcagggt tatttgtcaa acagcatatc tgcagggact catcacagct 360
accccgggcc ctctctgccc ccactctggg tctaccccct ccaaggagtc caaagaccca 420
ggggaggtcc tcagggaagg ggcaagggag cccccacagc cctctctctt gggggcttgg 480
cttctacccc cctggacagg agcccctgca cccccaggta tagatgggca cacaggcccc 540
tccaggtgga aaaacagccc taagtgaaac ccccacacag acacacacga cccgacagcc 600
ctcgcccaag tctgtgccac tggcgttcgc ctctctgccc tgtcccgcct tgccgagtcc 660
tggccccagc accggggccg gtggagccga gcccactcac accccgcagc ctccgccacc 720
ctgccctgtg ggcacaccag gcccaggtca gcagccaggc cccctctcct actgcccccc 780
accgcccctt ggtccatcct gaatcggccc ccaggggatc gccagcctca cacacccagt 840
ctcgcccact cacgcctcac tcaaggcaca gctgtgcaca cactaggccc catagcaact 900
ccacagcacc ctgtaccacc accagggcgc catagacacc ccacacgtgg tcacacgtgg 960
cccacactcc gcctctcacg ctgcctccag cgaggctact gccaagccct tcctctgagc 1020
catacctggg ccgctggatc ccagagagaa atggagaggc cctcacgtgg tgtcctccag 1080
tccaaccctc cctgtcaccc tgtcagcagc agcaccccac agccaaacac aggatggatg 1140
cgtgggctcc atcccccact cacccacacc ggaaccccag agcaggctac gtgcccctca 1200
cagacctcaa acccacatgt gcatctgaca ccccagatcc aaacgctccc cccggtcatg 1260
cacaccaagg gcacagcacc caccaaatcc acacggaaac acgggcaccg ggcaccccat 1320
gagcacaaag cccctccatg tctgaagaca gtccctgcac accgtcacag ccatacattc 1380
agcttcactc tcacgtccca gcccacctgc acccagctct gggcctggag cagcagaaag 1440
aggtgtgagg gcccgaggcg ggacctgcac ctgctgatga cccgggacca gcaggcagct 1500
cacggtgttg gggaagggag tggagggcac ccagggcagg agccagaggg accaggctgg 1560
tgggcggggc cgggccgggg tagggccagg aggcagctct ggacacccac aggcctgggc 1620
tcatagtcca caccaggaca gcccctcaga gcacccatgc agtgagtccc aggtcttggg 1680
agccaggccg cagagctcac gcatccttcc gagggccctg agtgaggcgg ccactgctgt 1740
gccgaggggt tgggtccttc tctggggagg gcgtggggtc tagagaggcg gagtggaggt 1800
aaccagaggt caggagagaa gccgtaagga acagagggaa aatggggcca gagtcggggc 1860
gcagggacga gaggtcagga gtggtcggcc tggctctggg ccgttgactg actcgggacc 1920
tgggtgccca ccctcag 1937
<210>157
<211>1301
<212>DNA
<213>智人
<400>157
gtaaatgagt gcgacggccg gcaagccccc gctccccggg ctctcggggt cgcgcgagga 60
tgcttggcac gtaccccgtg tacatacttc ccgggcaccc agcatggaaa taaagcaccc 120
agcgctgccc tgggcccctg cgagactgtg atggttcttt ccacgggtca ggccgagtct 180
gaggcctgag tggcatgagg gaggcagagc gggtcccact gtccccacac tggcccaggc 240
tgtgcaggtg tgcctgggcc gcctagggtg gggctcagcc aggggctgcc ctcggcaggg 300
tgggggattt gccagcgtgg ccctccctcc agcagcagct gccctgggct gggccacggg 360
aagccctagg agcccctggg gacagacaca cagcccctgc ctctgtagga gactgtcctg 420
tcctgtgagc gccctgtcct ccgacccgca tgcccactcg ggggcatgcc tagtccatgt 480
gcgtagggac aggccctccc tcacccatct acccccacgg cactaacccc tggcagccct 540
gcccagcctc gcacccgcat ggggacacaa ccgactccgg ggacatgcac tctcgggccc 600
tgtggagaga ctggtccaga tgcccacaca cacactcagc ccagacccgt tcaacaaacc 660
ccgcactgag gttggccggc cacacggcca ccacacacac acgtgcacgc ctcacacacg 720
gagcctcacc cgggcgaacc gcacagcacc cagaccagag caaggtcctc gcacacgtga 780
acactcctcg gacacaggcc cccacgagcc ccacgcggca cctcaaggcc cacgagccgc 840
tcggcagctt ctccacatgc tgaccagctc agacaaaccc agccctcctc tcacaaggtg 900
cccctgcagc cgccacacac acacaggccc ccacacacag gggaacacac gccacgtcgc 960
gtccctggca ctggcccact tcccaataca gcccttccct gcagctgggg tcacatgagg 1020
tgtgggcttc accatcctcc tgccctctgg gcctcaggga gggacacggg agacggggag 1080
tgggtcctgc tgagggccag gtcgctatct agggccgggt gtgtggctga gtcccggggc 1140
caaagctggt gcccagggcg ggcagctgtg gggagctgac ctcaggacac tgttggccca 1200
tcccggccgg gccctacatc ctgggtcctg ccacagaggg aatcaccccc agaggcccga 1260
gcccagcagg acacagcact gaccaccctc ttcctgtcca g 1301
<210>158
<211>1553
<212>DNA
<213>小家鼠
<400>158
gtaacacctc cctccatccc tcctaggcct ccatgtagct gtggtgggga aggtggatga 60
cagacatccg ctcactgttg taacaccagg aagctacccc aataaacact cagtgcctga 120
ttagagccct gggtgcctgt tcttggggaa ggcaggttat gggcagaaat atcttggcct 180
gaaagaaggg acaccccaag agaaggacag gagtgaagca tggctcaccc atctgtctat 240
gtgttggata tttgacaaat aggacatcac aggacttcag cataggtacc cctggtcctt 300
cctgctcttc actggatatc atgcacctga tctctagaga tgcagctaaa atgagccagt 360
ctgggaaacc tcagcaccca cctctcagtc ttgcaagctc ctgctcccag acattcctgg 420
atactaaacc ccttcaggta gagaaacagc caaagtcaac atctaggacg caggactcaa 480
catggtcctg ctccttccct ctctactcaa cagccattga ggctgagccc accgccccaa 540
ccgcctgcct tgccaaatga tcacaccagg cctggtgctc ctcgtcttac tacctaaact 600
cactccaacc caaattcatc ccaaggacca gaatgggctg acagcctcat acagtcaggt 660
tcccccatct atgacatgtt ttcacatgca tgcacacaca cacacacaca cacacacaca 720
cacacacaca cacacacaca cacacaggct tcattgagct ctctggttta gcaatagccc 780
aaagcaagcc atacatccat cccagttcca gaaggataag aaaaccagaa ccaagacaca 840
cccacaccta ttccataccc aaccaacagc acatatggct tacacacctg agattagtgg 900
ctcccatcat gtacacacac atgcacacaa aggagcccat acatacccat catttccaga 960
ggtaagtatc taacctttgg atctgagata tctctgagga acaccaatgg cagagtcaac 1020
cagcacctca gcctccagac taaatcctta cattttggcc caccccaagc catgagagat 1080
ggaggagggt ggaggcctga gctgcgggaa agcagagaca ggaagatgga ctgtttggtg 1140
agagtagtaa accagacaat ggggagacta aggcaggagt agagccccta caaggcccag 1200
agtctgcttt agagtccatg tgtcctgacc tgcccctcag atgccacaac caagatttct 1260
ggttccagag catgcatgca ggccctagaa atggacctat gagctcagag ccttcctaga 1320
gagccctggg tactctctga acaaaaggca attctgtgta gaggcatcct gtggccaaag 1380
accctaagac agtcatacac acacacacac acacacacac acacacacac aacacacaca 1440
acacacacaa cacaggtagg ttttatcatg ctctttggtt tagcaatagc cctgttgatg 1500
gtgggggata ctgggtcact gtgggcactg gagtagaaag agggaatgaa cag 1553
<210>159
<211>1354
<212>DNA
<213>小家鼠
<400>159
gtaaatgagc tcagcacaca caatgctcct gggtcctaat ggacactggc acccatatcc 60
atgcatccct tgtataaata aagcacccag caaagcctgg gaccatgtaa aactgtcctg 120
gttctttcca aggtatagag catagctcac gggctgatag ttctagccag ggttggagga 180
cagccttgtc tttaggtttc tgcactgcag gactcagagc tcatgagtca tcttggcctg 240
aagagtggaa cttagcttta ggcagtatct ttcctttact tcccacaaac cagcagctgc 300
cagacataga gataatccta ggaagcctca aatggagaag cacacaaacc cacctccctc 360
aggctgttcc tctccccagg ccccacttct ttacctaagg gtttctctga gtctattgta 420
gaggcacaca tggccaggga tattccggag acccttagca tccatacact caactcaggc 480
agcttttcac aggtgaggtc tgcacatcca tacagacggc tcactcctgc ctgtgccacg 540
tggggctgag gcacatggct cttgctgccc caagggaggg actattagat agccacagtc 600
atgctgaatc ctgacccatt caaattagcc tgctgaacac catccagtcc atatagcaca 660
tgtatccaca tgcacgtgtg cacacacaca tttaacacac tgggacaact cctctgtgcc 720
ctgcacagca cctacatcca gcaatgtatc accatacaca cggccaaaaa ttcaatgccc 780
acgtttctgc catcacaaac agacacacct ttcctctctg tggccactgc attatatgct 840
caacacaaga cctctgaagc cagatccatc tctggtacct cagggtcatg cttcaacccc 900
acatgaatta tgcaaaccat agccataatg gtctgaatca cttcacattg gaatggtccc 960
aagatcaggc aagaaaagcc acaggctctg ttgatgactg aaggacagca aagggtcagt 1020
ccagctgtat aaccactgtt gacctgggtc acagaccctg ctgaccctcc atattctctt 1080
gtactgaatg aatgaaaaat gagacaagca tagagggccc ttgcacaatc taggtcagta 1140
tgaggtctac ccgaggcatc attggagtca gctggaggaa gctgagactg atgtcccaga 1200
gggaaaggaa aagaaagaag gctccaggaa gggtctgctg taccagtcag gctggagctc 1260
tctcctctac ttcatgcaaa catgtctgca tcacagggaa tctctcccag caccaaccat 1320
gttgggacaa acactgactg tcctctctgt tcag 1354
<210>160
<211>81
<212>DNA
<213>智人
<400>160
tcgcccctgt acctggagat catcatctat tgcacagggg ccttcctcat ctcctgcatg 60
gtggggtcgg tcatcgtcta c 81
<210>161
<211>90
<212>DNA
<213>小家鼠
<400>161
aagataccat ctattgccac tgggattgtg ggtggcctcc tcttcatagt ggtggtggcc 60
cttgggattg gcctattcat gcgaagacgt 90
<210>162
<211>29
<212>PRT
<213>智人
<400>162
Thr His Asp Ala Leu Ile Val Gly Thr Leu Ser Gly Thr Ile Phe Phe
1 5 10 15
Ile Leu Leu Ile Ile Phe Leu Ser Trp Ile Ile Leu Lys
20 25
<210>163
<211>81
<212>DNA
<213>小家鼠
<400>163
cgccttccac tgggggtcac catctcctgc ctctgcatcc cgttgttttg cctgttctgt 60
tacttcagca ttaccaagat t 81
<210>164
<211>63
<212>DNA
<213>智人
<400>164
accatcacct tcctcaccct cttcctgctg agcctgttct atagcacagc actgaccgtg 60
acc 63
<210>165
<211>87
<212>DNA
<213>智人
<400>165
tggacgtgga ccggcctctg catcttcgcc gcactcttcc tgctcagcgt gagctacagc 60
gccgccatca cgctcctcat ggtgcag 87
<210>166
<211>78
<212>DNA
<213>智人
<400>166
gggctgtgga cgaccatcac catcttcatc acactcttcc tgttaagcgt gtgctacagt 60
gccaccgtca ccttcttc 78
<210>167
<211>78
<212>DNA
<213>小家鼠
<400>167
gagctgtgga ccagtatttg tgtcttcatc accctgttcc tgctcagtgt gagctatggg 60
gccactgtca ccgtcctc 78
<210>168
<211>78
<212>DNA
<213>小家鼠
<400>168
gggctctgga caaccatcac catcttcatc agcctcttcc tgctcagcgt gtgttacagc 60
gcctctgtca cactcttc 78
<210>169
<211>3111
<212>DNA
<213>智人
<400>169
gtgagtatca agaggcctaa gcaatggtaa tctccactct ccattcttcc cctgtggcca 60
gacacttccc ctggctgagt ctctgggctt ttatatcata ggatgcctct aatggcaatc 120
ctgccattag atacacctgc cgtggtgtat ctgccaggta ggcaggctag gctgcagtaa 180
caaacaagcc cacaatttcc atggcttaac actatgggaa tatatttctt gctcacgtaa 240
caagctaacg tgaatgttgc tggtttgtag gtggtttccc tccctgtaga aatctgggga 300
gtgaggttct ttccatcttg tggtgccgtc attctccagg acaaagattc ttacctactt 360
ttgtgtcctg gtttcctttg gcagcctggt gaagcctatg gacctcattt cagaatattt 420
ttaaatacat aaaatcccag cctgggcaat atagtgaaac ccccatctgt acaaaaatta 480
gccaggcatg gtggcatgca cctgtagtcc caggtactgg gaaggctgag gtgggaggat 540
cacttgagcc caggagtttg aggctgcagt gagccgtgat cgtaccactt tactcccacc 600
tgggtgacag agcaagagcc catctctaaa aataaataaa tacaatgaaa taaaataaaa 660
taaatagaac tacagaggaa actaattgta ttgaaatgca gttataaaac atttaaacac 720
atttttaatc tagagatata tgtgcttctt tattaagatc tataaataat aagttctagg 780
ggtagctcgc ataaatactg taatttcaaa gtagataagc ataaataata ctttatgata 840
ctgaaattgt gatgtgatat gagaatagct gtgagttttg ttttgctggg gacaggatca 900
gtgatgctgt cattactggg gtctcttccc tccattcttt ttttaaaatt gtattttatt 960
ttatttttaa aattttaaaa taaatagaga cagggtatca ctatgttgcc caggctgctt 1020
ttgacctcct gggctccagt gatcttccca tcttggcttc ccaaagtgct gggattacaa 1080
gtgggagcca gtgttcctgg ccccttcctc cattcttaat ggaaggagat gctaggtgtg 1140
agaggttagg gaaagtaaag atgtaatttc tttcccatcc aagttctcag acccctgaat 1200
tctacctgca gccatgttgg tccatcaacc ccaagtgaag aatccctgct ctagggcccc 1260
accattgtct gtatccagcc agcagaagag gcgtgattat ggagatcaca tctgcttctt 1320
gaaagcagac agcccggaag tgggccgcat cacttcctct caaattctat tggtgaaaat 1380
ggtcacatga ctacacatag ccacaaagga ggctgggaac tttctcactt ggaacctaca 1440
tcccagaaac aactcttttc agtgaggtat cccacaggtc tttcgcagta gaaatattga 1500
ttatctcaca taaaatgaag tcttacaaat ggacctactg ggttttgtac agcagccaag 1560
tgatatctct tcccttctgc tgtcttccct tctgccatcc ttcacatggt ggcattgtat 1620
ccttagactt gccacccatg ccctcaggtt ggccgttgca cactgtctta cataaagcag 1680
gaaggaaagg aaaggctgct acgagagagt gtaccttgtg catctctttt ttaatcagga 1740
agcaaacatc tttctagaag cttccctagc aaaattcccc ttacatctca ttggccaaga 1800
ctgttacatg ttacatggtt actgttatta cttgctcatt gcaaggaaga ctgggaactc 1860
aaatgcctgg aaaaaggaac aggataatcg tgattggctc aagccttagg gtgggcatgg 1920
ctccctgaca agggagagag gaaaaagctg ttgagtgaag aagactgctt cagtttcccc 1980
atctgtataa tgggaggagt aagggctgtc gtaaaaactc aatgaaagaa gattcttcaa 2040
cgtggtaggt gcagtggcag ctggcagtac cctgaccctg ccaccgcaca gccctctcag 2100
cattgctcat cctgcactgt ggatatcagt tgagccacgt gtctcctgcc ctgggctgtg 2160
agctccatag gcagggtctc catggctgta tctccagaac ccagcacaga accaggtgct 2220
tgggaaagtt ttgaattgat tctcatctgc cattggcatg gggaagggaa ctagcttgta 2280
tgaaacagat aacaatgtat gggaccctca ttcattattt cagcaaatat ttgctgagtt 2340
cctcctacat ggctagccct gtgctagaca ctggggaatc ggcgatgaac aaagcagata 2400
gaaatcccca ctcttgtgga gctgacattc tggagggaga gacaaaaagc aaacatataa 2460
agaaagaaag aaatcacatg gatctggatg acagtgagtg ctgggaagaa aataaaagca 2520
gaggaagggg atggagcgat gggcaggggg caacggtagg gagggtgtcg gggaaaactt 2580
tttggagaat gtgacgatga aagtgaacaa ggagaagtca accgtgttga gatgatggca 2640
gctaatgatg tggacaggcc actctgttct gagtgcatta tctattgatt catcatgtca 2700
tcctcgcaac agccctgcac gatcaattct gtcattaacc ccatagtaca gatgaggatg 2760
cggaggcaca gagaagataa gggacttgtc ctgtgtcaca cagcaaggag ccatccggct 2820
cctaagttgg tgcatttgac ttctgtgctt ccggaaagaa agagcagcaa gtttaagatc 2880
tggaggtggc actgagcttt ggaggagcag ggggcaatga ggtggccggt gtgacgagga 2940
ctcaatgtgc aagagggaga gtggtgggga gatgaggtgg aggggtggtc ggcggtcaga 3000
tcgtggaggg tctcggacga gggtcctgac cctgggtctc cagtcctggg aagtggagcc 3060
caggctgtac catggctgac ctcagctcat ggcttcccct cccacttcca g 3111
<210>170
<211>2093
<212>DNA
<213>智人
<400>170
gtaaatgacg tactcctgcc tccctccctc ccagggctcc atccagctgt gcagtgggga 60
ggactggcca gaccttctgt ccactgttgc aatgacccca ggaagctacc cccaataaac 120
tgtgcctgct cagagcccca ggtacaccca ttcttgggag cgggcagggc tgtgggcagg 180
tgcatcttgg cacagaggaa tgggcccccc aggaggggca gtgggaggag gtgggcaggg 240
ctgagtcccc ccaggagagg cggtgggagg aggtgggcag ggctgaggtg ccactcatcc 300
atctgccttc gtgtcagggt tatttgtcaa acagcatatc tgcagggact catcacagct 360
accccgggcc ctctctgccc ccactctggg tctaccccct ccaaggagtc caaagaccca 420
ggggaggtcc tcagggaagg ggcaagggag cccccacagc cctctctctt gggggcttgg 480
cttctacccc cctggacagg agcccctgca cccccaggta tagatgggca cacaggcccc 540
tccaggtgga aaaacagccc taagtgaaac ccccacacag acacacacga cccgacagcc 600
ctcgcccaag tctgtgccac tggcgttcgc ctctctgccc tgtcccgcct tgccgagtcc 660
tggccccagc accggggccg gtggagccga gcccactcac accccgcagc ctccgccacc 720
ctgccctgtg ggcacaccag gcccaggtca gcagccaggc cccctctcct actgcccccc 780
accgcccctt ggtccatcct gaatcggccc ccaggggatc gccagcctca cacacccagt 840
ctcgcccact cacgcctcac tcaaggcaca gctgtgcaca cactaggccc catagcaact 900
ccacagcacc ctgtaccacc accagggcgc catagacacc ccacacgtgg tcacacgtgg 960
cccacactcc gcctctcacg ctgcctccag cgaggctact gccaagccct tcctctgagc 1020
catacctggg ccgctggatc ccagagagaa atggagaggc cctcacgtgg tgtcctccag 1080
tccaaccctc cctgtcaccc tgtcagcagc agcaccccac agccaaacac aggatggatg 1140
cgtgggctcc atcccccact cacccacacc ggaaccccag agcaggctac gtgcccctca 1200
cagacctcaa acccacatgt gcatctgaca ccccagatcc aaacgctccc cccggtcatg 1260
cacaccaagg gcacagcacc caccaaatcc acacggaaac acgggcaccg ggcaccccat 1320
gagcacaaag cccctccatg tctgaagaca gtccctgcac accgtcacag ccatacattc 1380
agcttcactc tcacgtccca gcccacctgc acccagctct gggcctggag cagcagaaag 1440
aggtgtgagg gcccgaggcg ggacctgcac ctgctgatga cccgggacca gcaggcagct 1500
cacggtgttg gggaagggag tggagggcac ccagggcagg agccagaggg accaggctgg 1560
tgggcggggc cgggccgggg tagggccagg aggcagctct ggacacccac aggcctgggc 1620
tcatagtcca caccaggaca gcccctcaga gcacccatgc agtgagtccc aggtcttggg 1680
agccaggccg cagagctcac gcatccttcc gagggccctg agtgaggcgg ccactgctgt 1740
gccgaggggt tgggtccttc tctggggagg gcgtggggtc tagagaggcg gagtggaggt 1800
aaccagaggt caggagagaa gccgtaagga acagagggaa aatggggcca gagtcggggc 1860
gcagggacga gaggtcagga gtggtcggcc tggctctggg ccgttgactg actcgggacc 1920
tgggtgccca ccctcagggc tggctggcgg ctccgcgcag tcccagaggg ccccggatag 1980
ggtgctctgc cactccggac agcagcaggg actgccgaga gcagcaggag gctctgtccc 2040
ccacccccgc tgccactgtg gagccgggag ggctgactgg ccaggtcccc cag 2093
<210>171
<211>1285
<212>DNA
<213>智人
<400>171
gtaaatgagt gccagggccg gcaagccccc gctccccggg ctctcggggt cgcgcgagga 60
tgcttggcac gtaccccgtg tacatacttc ccgggcgccc agcatggaaa taaagcaccc 120
agcgctgccc tgggcccctg cgagactgtg atggttcttt ccacgggtca ggccgagtct 180
gaggcctgag tggcatgagg gaggcagagc gggtcccact gtccccacac tggcccaggc 240
tgtgcaggtg tgcctgggcc gcctagggtg gggctcagcc aggggctgcc ctcggcaggg 300
tgggggattt gccagcgtgg ccctccctcc agcagcacct gccctgggct gggccacgag 360
aagccctagg agcccctggg gacagacaca cagcccctgc ctctgtagga gactgtcctg 420
ttctgtgagc gccctgtcct ccgacccgca tgcccactcg ggggcatgcc tagtccatgt 480
gcgtagggac aggccctccc tcacccatct acccccacgg cactaacccc tggcagccct 540
gcccagcctc gaacccacat ggggacacaa ccgactccgg ggacatgcac tctcgggccc 600
tgtggaggga ctggtccaga tgcccacaca cacactcagc ccagacccgt tcaacaaacc 660
ccgcactgag gttggccggc cacacggcca ccacacacac acgtgcacgc ctcacacacg 720
gagcctcacc cgggcgaacc gcacagcacc cagaccagag caaggtcctc gcacacgtga 780
acactcctca gacacaggcc cccacgagcc ccacgcggca cctcaaggcc cacgagccgc 840
tcggcagctt ctccacatgc tgaccagctc agacaaaccc agccctcctc tcacaaggtg 900
cccctgcagc cgccacacac acaggggaac acacgccacg tcgcgtccct ggcactggcc 960
cacgtcccaa tacagccctt ccctgcagct ggggtcacat gaggggtggg tttcaccatc 1020
ctcctgccct ctggggctca gggagggaca cgggagacgg ggagtgggtc ctgctgaggg 1080
ccaggtcgct atctagggcc gggtgtctgg ctgagcccca gggccaaagc tggtgcccag 1140
ggtggacagc ttccgggagc tgacctcagg acattgttgg cccatcccgg ccgggcccta 1200
catcctgggc cccgccacag agggaatcac ccccagaggc ccaagcccag ggggacacag 1260
cactgaccac ccccttcctg tccag 1285
<210>172
<211>6350
<212>DNA
<213>小家鼠
<400>172
gtaagtcaca actgggtaag agtgtcaagc aaggacagca cttggtacct atgatagaca 60
aatactcctg tttgggaaga ggaggtctgc attgtctaga taagaggaac actgtgctta 120
tctgtttcag tttaaaagac agaactacat aacacttcac cctttctaca acacttagat 180
ttttcagccc tctcctctat ggtgtttcct gaaccctgaa gaaagtgata tagatgttta 240
tttattttct agggccactg ttccaaagaa acaatcacgt attattagca gcttgaccag 300
gtgtgaattt cccccagtat ccacttctgc tccctgtggg atgaagtccc tccataagta 360
aagttgaagg ctgtgggcat aaatgcaaat gcctgtaatc caattttaca agtccatgac 420
atacatttaa caaacaatag cagttacttc tctactggag cctatgaact ccccagctat 480
aggtttttcc cttctgcaga gcagagctta aatccaatag ggaagaagtt ggttgtaccc 540
ataactgcca taccactatt acaccaataa gcacatctta catagcaaga caattagtgt 600
agcactcaaa gtccatgttc aagagagaat gttggcaaca gcctatataa catctcccag 660
cacagaaaag aaaaggaaag gaaaggaaag gaaaggaaag gaaaggaaag gaaaggaaag 720
gaaaggaaag gaaaggaaag gaaaggaaag gaaagaaaag aaaagaaaag aaaagaaaag 780
aaaagaaaag aaaagaaaag aaaagaaaag aaaagaaaag aaaagaaaag aaaagaaagg 840
aaaagaaagg aaaagaaagg aagacagcca gaaggcagct tccacttcag tcctagccca 900
tgttttctat gtcttacagc caaggttagt gaagtctcta gcagtaggaa tttatcatct 960
acttctggca tgcaagtaaa atcaatggtg ataacttttg tggtttgtgg ctccttggga 1020
gcctccctga acaataattt atgcagaggt agcccagctt tgccactggg gcttccatta 1080
aacaaacttt ggattgaggg agtgtctttt tctacctgca cacagtgatt cctatgtttt 1140
aactcttaat gagtcactgt tgtttgttgt tgttgttatt gttgttgtta ttgttgttgt 1200
tgttgttgtt tctatcaacc cagacactat agggctataa ggcaggtgga agcatcactg 1260
gctccagctg ttctcagtca tgctcttcct atcaatgttt gtgtaatgac tgcccctttt 1320
actgtcggtt ttctatgggt gcctatggat aacgctgggt tgggcgaaac ccttgatggc 1380
agcttttccc actgcttgct tatccatgta ggtgagagca cattcactct cacttgagag 1440
gcttaggcct ttatgaagtc tgcctgacat atgggatagg taggcttata tgcttaccca 1500
tcatgcaccc atgtgctggt accataagca agatcagacc tcacctgttc atgtagcatg 1560
cagaggatca gaatatttca caagaatgcc agaagctctc attactctct ggccttctct 1620
taaagatcac atcctctaac aaagcttacc aaagccacta acatctgagt tagtcctact 1680
caggtacaca tttcttaatg ctccagcaaa agcatctcta cccacttcat tgggcactaa 1740
acaccacttc tcacatgttt agagtttttt gttaagactt taaattatct ttcagtcatg 1800
tctgatgttc atgggcacct cagccagtaa taactgagtg tttctggtgt gtgcaggttt 1860
ttgtctgttt tgtgttgttt atatgccagg aaaaagctgt ttagtacaga tctaattggc 1920
attccccttc cttcatggtg tggatatttc ttttgagtta aaacatcccc tcttgtggca 1980
gggggagggc tagaggtcaa tggaatgatt tcccgagttt ccttggataa atgaaacctt 2040
tgtctcaggg aaggaagaat ggttctactt ctgtttctcc acttcatgat gctgactcta 2100
gcctcttcca tgccccgtgt ctccaggcta tgaaggaatc cagcatgctt ttctctcttc 2160
cttccttatc ttcttcatgg gttccaagtg ggttcctgtg gtttccacag gaaaggcttt 2220
ttagcacaaa tctgatctgt actgtctttc ctttatcccc tgccagctct gagacagaaa 2280
gccagcatct ctatcagaat ctctcttttg ccttcagaat cagagaccac tgtgaaaggc 2340
agcatccgtg catctttttt tccattgtac tccttcatta cgtgagcaag atttttctaa 2400
gcagatagct tagccttcat cgcacatctt agcacacact gcaaatgtca cacagacagc 2460
ttgctatgcc atcccatgta tactattttc aaggattgca ataccttctc tgatctgaaa 2520
tccattcaag taaaacaaca tgttgcaggt ccaattcaag aggcatgcca cctcatacca 2580
tataaaagaa gatggtcagc aaaaattcct gctctctcag acaactcaga cttgggagca 2640
gaaattccta tcacctctag cagggagtaa acaactacta ctacagctgg aagcatacta 2700
tctcctacca taaaagatgc tagcttctct gacaccttag acctgaggca aggaatcctc 2760
tgcctcattt ctttcgtctg ctggaaagcc tacaggacta agggctgctg taagcttgac 2820
acaagcttga ggttgaaagg tgtcagacag gcttcacctg aggggcccta tatatgagtg 2880
tagtgaggat ggaacaggct acgtggggca tcctccggca tcttgtcctc agagtggcct 2940
tcagagccca ggctcaagtc agccatactt ccccacttag actctggaaa tgccacagta 3000
tgtccacaac ctccacaccc agctacacaa aattgagaga gggacctagt gggactgcag 3060
caatcacatg aggccatacc tggaccagcc aagctgccag tataacagtc gtggctctga 3120
gattttgaaa gcttggtctg ttgtctatgc ctcgggatct ttgggatgcc ttaggatcca 3180
tcgagaaagt tctagagatc atgcctgttt ctggctgcac tgtactgact gagaacaggg 3240
gctacagcac cactgtttct catggcctac ttcctgccag gtggtgcctc attagtactg 3300
aaaggagcct gacctacact tgccccaaat tggccttttc ttttcccctg tggcattggt 3360
tcctcaaagg acactaaggt tttctcttac ttgactccct caggatctct ctacagattt 3420
tcacaggtca ctcctcagag ctagagtcat accagcatga ctatcctctg cccacattca 3480
cacagcatgc ttaggaagtc tgggcatgct ctgtagtcat cttcattagg ttgttcccac 3540
ctgcctctct atctgcccct gccccacttc cccttgttct gcctgttggt ctctccctac 3600
tctgtgcttc acatgtcttg aggtagatac atctgcctga gtcccacata acacttcttc 3660
atcacaccaa cccctgagaa caataatgtt cttagttctg tcgactagca cctcctgact 3720
tgtctctatg atgccattct tcccctccta cccatccatc accctgttgc caaggcactc 3780
tctgagacat cttgtttcta catagataca gaagaacatt tgattaggca tgtaggccat 3840
ggtgaaagaa aaacaccaag taaactttta acatcacaat tttatttaaa actactagag 3900
aggactcagc aatgctcaaa gagactaaat tcccaggtga aaggagatgg gatgttgtat 3960
tctaggcaca gacagacaga gcagtgggac atatgtaatc accaagaaat cagccaaatt 4020
tttaataaac accttagctg ccgagaggga tgagaacaca gaagaatccc aaattctgag 4080
ccaatcacca ctcacttaac ctcccaccca ggacactgct aagcaagaaa caccttcgag 4140
cacagtgtgg gcagaatatc catcttgtac cagctccaag gcctgctgaa tataagggtt 4200
atcccaaagc acctagagcc agaagtcaac tatgataccc cagctccagc acagctcagt 4260
tcccagttcc aaaaaacagg tcttccatgc ctggtggata agatgatatg cacttgtatc 4320
tcacccccag actctacaat ccatgtatat acaatgccta gcatgcaatc agtaattaca 4380
agatacaaat gattgataac caagacagag acagtacatg ggattagacc tgaggtacgg 4440
gaaaggttgg gaaagcattc atagtgtccc ttgctacaag aagtcccaga gctttgcagg 4500
gtgtgagcag attctaacct cccccattgg caagtcttct caaagctgct tctgctacta 4560
gagaaccctc tttcccagcc acctgccact atttatcact tatctgagtt ttccccatga 4620
ttctaggatg ctaccacctc ctgcctgagt cagacggtcc ttccaggaga cctgatggtc 4680
ctgcccttgc ctgagacctt tctaggctga atggtcatca tgtccactgt ctggttagct 4740
tggttgcctc tgtgttaagt tgcttcatac tacatgtagc aaggaaagtt acctttccac 4800
ttctcaggac actgtaaaga agcacttctg taaattatag tgagaaacga tcaataataa 4860
atgaaatgaa caaacataaa cctctcttat ctctatggga cctgccctaa ggcctcttgt 4920
ggatgcctga aaccttgagt gttcttaagc catgtgtcac ttcagtcatt aacctgtggc 4980
tgaggtggat cctgactgac aaacagacag gcatgtagac agcttgaagc ctggacaaag 5040
agaaggttga cgttccaggc aaagcaagag cctatcacat tgttccaaag tgtgtgattt 5100
agaatcagaa gatttcttgt ttaaaatata ctcaaaaagt tataaaatat cagccaggac 5160
tagtcataaa gcttaaatct gagagagaga gagagagaga gagagagaga gagaggtgtt 5220
catgtatata catatgtgtg tgtgtgtata tatatgtatg tatatgtatg tatatgtgta 5280
tattggtgtt tgggtgtcag attccctgga attaaagtta taaacagtta tgagctgcaa 5340
agtaggcttt ccaatgtctt gttctccaac cataggtaat tgaaactgga aactacaggt 5400
aaaggaggct gccaagtgtt tcttattatg ctaagattca gtgttctaca atattcacat 5460
gatttcactt tgatatggtg aaatgccttt aggccaacgc atccacagac ttcacagtga 5520
ccccagctga gcttacatat aatgtcaaac agcacttgtt gaatagatac ccaggcagcc 5580
ctgtgaccca tggtgttttc cttaattctt cccatctgcc ttggactttc acaccttgag 5640
ttgagatgtg ctttctgtag aaatgtattt ccctaggtct gtacatcagc caccaccctt 5700
tctgatcttg gaccatactt cagtctccag ctgggatatt catatgcatg cttagatatg 5760
gcataaccat cgttaacgtc ttctgttacc tttaggatta agttaccgta gctaggatgc 5820
tttccaatct ctccctccat ttgcaaaggc tcagggttcc ttctgactct ggagtagtac 5880
tctttcccaa tgagtgcacc atgaccacta ttcttcctta gaatgttact cccctttctc 5940
tgcctttttc atggcacatc tttctgagaa aacctacctg gtctagctgc tctttctggg 6000
tcttcttaag tagtaaccta actcctgcac catccgtgtg gcagtgacat agatccacat 6060
caggtttgga ttcagatatt ccaatcccat gcagaccaat ctatgtgtga gattccagga 6120
tccagcaggc tggggcatgg gttgaggggt aggattcttt ggggcaaagt ggttagtagg 6180
cacttgggac tggaagtgat aagtattagc gggaggaggg catatgatca aaacattgct 6240
gctggatgtg aaagctgtac cttcaggttc agtgttcagg agaccaggtt aataagaaat 6300
gatctttctg tagggtggag accttctcat gagcactagt tcttccctag 6350
<210>173
<211>1739
<212>DNA
<213>小家鼠
<400>173
gtaacacctc cctccatccc tcctaggcct ccatgtagct gtggtgggga aggtggatga 60
cagacatccg ctcactgttg taacaccagg aagctacccc aataaacact cagtgcctga 120
ttagagccct gggtgcctgt tcttggggaa ggcaggttat gggcagaaat atcttggcct 180
gaaagaaggg acaccccaag agaaggacag gagtgaagca tggctcaccc atctgtctat 240
gtgttggata tttgacaaat aggacatcac aggacttcag cataggtacc cctggtcctt 300
cctgctcttc actggatatc atgcacctga tctctagaga tgcagctaaa atgagccagt 360
ctgggaaacc tcagcaccca cctctcagtc ttgcaagctc ctgctcccag acattcctgg 420
atactaaacc ccttcaggta gagaaacagc caaagtcaac atctaggacg caggactcaa 480
catggtcctg ctccttccct ctctactcaa cagccattga ggctgagccc accgccccaa 540
ccgcctgcct tgccaaatga tcacaccagg cctggtgctc ctcgtcttac tacctaaact 600
cactccaacc caaattcatc ccaaggacca gaatgggctg acagcctcat acagtcaggt 660
tcccccatct atgacatgtt ttcacatgca tgcacacaca cacacacaca cacacacaca 720
cacacacaca cacacacaca cacacaggct tcattgagct ctctggttta gcaatagccc 780
aaagcaagcc atacatccat cccagttcca gaaggataag aaaaccagaa ccaagacaca 840
cccacaccta ttccataccc aaccaacagc acatatggct tacacacctg agattagtgg 900
ctcccatcat gtacacacac atgcacacaa aggagcccat acatacccat catttccaga 960
ggtaagtatc taacctttgg atctgagata tctctgagga acaccaatgg cagagtcaac 1020
cagcacctca gcctccagac taaatcctta cattttggcc caccccaagc catgagagat 1080
ggaggagggt ggaggcctga gctgcgggaa agcagagaca ggaagatgga ctgtttggtg 1140
agagtagtaa accagacaat ggggagacta aggcaggagt agagccccta caaggcccag 1200
agtctgcttt agagtccatg tgtcctgacc tgcccctcag atgccacaac caagatttct 1260
ggttccagag catgcatgca ggccctagaa atggacctat gagctcagag ccttcctaga 1320
gagccctggg tactctctga acaaaaggca attctgtgta gaggcatcct gtggccaaag 1380
accctaagac agtcatacac acacacacac acacacacac acacacacac aacacacaca 1440
acacacacaa cacaggtagg ttttatcatg ctctttggtt tagcaatagc cctgttgatg 1500
gtgggggata ctgggtcact gtgggcactg gagtagaaag agggaatgaa cagtcagtgg 1560
ggaaaggaca tctgcctcta gggctgaaca gagactggag cagtctcaga gcaggtggga 1620
tggggacctc tgccactcta gcttcatcag aactgcatga gacaagtatg gggtctaccc 1680
cctccccact gtcacctgga gtctggggaa gctaactggc tggtcccacc ccatcccag 1739
<210>174
<211>93
<212>DNA
<213>智人
<400>174
ttcgagcagc acctcctgct gggcgtcagc gtttcctgca ttgtcatcct ggccgtctgc 60
ctgttgtgct atgtcagcat caccaagatt aag 93
<210>175
<211>78
<212>DNA
<213>智人
<400>175
gggctgtgga cgaccatcac catcttcatc acactcttcc tgctaagcgt gtgctacagt 60
gccaccgtca ccttcttc 78
<210>176
<211>84
<212>DNA
<213>小家鼠
<400>176
ggcctgtggc ccacaatgtg caccttcgtg gccctcttcc tgctcacact gctctacagt 60
ggcttcgtca ccttcatcaa ggta 84
Claims (6)
1.核酸,其序列为SEQ ID NO:140或SEQ ID NO:143所示的序列。
2.包含根据权利要求1的核酸的真核细胞。
4.选择表达异源多肽的真核细胞的方法,由此该方法包括
a)用根据权利要求1的核酸转染真核细胞,
b)在一定条件下培养所述转染的细胞,所述条件包括:适于从所述核酸产生前体mRNA、适于所述前体mRNA的加工、以及适于所述加工后mRNA翻译成多肽,其中所述转染的细胞通过选择性剪接所述前体mRNA产生可溶的异源多肽和质膜-结合的异源多肽,和
c)选择具有质膜-结合的异源多肽的细胞作为所述表达异源多肽的细胞,
其中所述异源多肽包含人免疫球蛋白重链恒定γ1。
5.根据权利要求4的方法,其特征在于通过选自流式细胞术、ELISA、免疫沉淀、免疫亲和柱色谱法或磁珠免疫亲和分选的方法实施所述选择细胞。
6.生产由根据权利要求1的核酸编码的异源多肽或蛋白质的方法,包括
a)提供真核细胞,
b)用根据权利要求1的核酸转染所述真核细胞,
c)在适合表达所述核酸的条件下培养所述转染的细胞,
d)从培养液或细胞的胞质中回收所述多肽或蛋白质。
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AU2007251766B2 (en) | 2013-06-20 |
ES2573027T3 (es) | 2016-06-03 |
JP4870208B2 (ja) | 2012-02-08 |
US8541204B2 (en) | 2013-09-24 |
CA2651478A1 (en) | 2007-11-22 |
JP2009537124A (ja) | 2009-10-29 |
CA2651478C (en) | 2015-02-03 |
US20130109058A1 (en) | 2013-05-02 |
BRPI0712073A2 (pt) | 2012-03-06 |
US8354516B2 (en) | 2013-01-15 |
EP2027273B1 (en) | 2016-04-13 |
IL193721A0 (en) | 2011-08-01 |
KR101202498B1 (ko) | 2012-11-16 |
IL193721A (en) | 2014-09-30 |
KR20090003327A (ko) | 2009-01-09 |
BRPI0712073B1 (pt) | 2019-11-12 |
EP2208792A3 (en) | 2010-10-27 |
BRPI0712073B8 (pt) | 2021-05-25 |
ES2543629T3 (es) | 2015-08-20 |
CN101410525A (zh) | 2009-04-15 |
US20090311725A1 (en) | 2009-12-17 |
WO2007131774A1 (en) | 2007-11-22 |
EP2208792A2 (en) | 2010-07-21 |
EP2027273A1 (en) | 2009-02-25 |
MX2008013514A (es) | 2008-10-28 |
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