CN108486125A - 一种核酸分子及其在制备人源单域抗体中的应用 - Google Patents
一种核酸分子及其在制备人源单域抗体中的应用 Download PDFInfo
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Abstract
本发明提供了一种核酸分子及其在制备单域抗体中的应用,所述核酸分子包括了宿主动物的免疫球蛋白基因或其片段,其特征在于:包括了宿主动物的IgH 5’‑端增强子、IgM的转换元件(Sμ)和IgM序列,所述IgM序列全部为宿主动物IgM序列,其中IgM缺失CH1序列(IgM‑dCH1),IgG的表达控制元件来源于宿主动物,包括Sγ、TM1、TM2和PolyA等序列,IgG的Cγ序列(Hinge、CH2和CH3)来源于人的序列(Igγ‑dCH1)。本发明保证了转基因动物B‑细胞的正常发育,并且转基因动物表达的是人的单域抗体,减少后期的人工改造和提高了抗体的成药性。
Description
技术领域
本发明属于生物技术领域,主要是一种核酸分子及其应用。
背景技术
单域抗体(Heavy-chain-only)又称纳米抗体(Nanobody),是指只有重链的免疫球蛋白抗体。与完整抗体的的质量(150-160kDa)相比,单域抗体要小得多,只有大约50kDa。
单域抗体在自然界(骆驼科动物和鲨鱼)也存在。第一个单域抗体是从骆驼中分离出来的,它利用特殊的VHH序列。另一种制作单域抗体的方法是利用转基因动物的方法来完成:2003年Nguyen等,2005年Zou等利用Igγ2a的CH1突变-构建转基因载体,转基因小鼠高表达了单域抗体;2007年Zou等证明在没有小鼠轻链(Kappa和Lambda链)的情况小,小鼠也能自然产生单域抗体。
单域抗体的特点是分子量小,能够结合一些隐蔽的抗原表位,特别适用于比较难得到抗体的靶点,如一些癌症的治疗和图影诊断;可以口服治疗大肠杆菌引起的腹泻以及消化道疾病如炎症性肠病和大肠癌等;单域抗体可以通过血脑屏障,并且和完整的抗体相比更容易渗透到大型肿瘤当中,因此有可能开发出治疗脑肿瘤的药物和双特异性抗体。容易进行抗体的改造,减少制药成本。
发明内容
本发明的目的在于提供一种核酸分子,可以用于制备全人单域抗体,减少后期的人工改造和提高了抗体的成药性。
本发明的目的是通过以下措施实现的:
一种核酸分子,包括了免疫球蛋白基因或其片段,其特征在于:包括IgG基因(Igy)和IgG转换元件(switch regionγ,Sγ)和/或IgM基因(IgHCμ)和IgM转换元件(Sμ);其中IgM基因/IgG基因缺失CH1功能。
进一步的,所述Sμ、Sγ、IgHCμ为宿主动物序列。
上述IgHCμ包括宿主动物IgM的CH2、CH3、CH4外显子及之间的序列,以及TM1、TM2、polyA信号等。具体的,所述IgHCμ结构如图3-1所示。
上述核酸分子还包括宿主动物的IgH 5’-端的增强子(5’-En)。所述增强子位于转换元件(Sμ或Sγ)的5′-端。具体的,其结构如图1和3所示。IgH 5’-端增强子与IgH的VDJ基因片段的重组和转录有关,并能增强抗体的表达量和抗体亚型的转变。IgM的CH1序列可以是完全缺失或者CH1序列中发生突变。在IgM的表达过程中只用CH2、CH3和CH4以及TM1和TM2等序列。
重链的IgM的CH2、CH3、CH4、TM1和TM2等(其中CH1缺失,IgM-dCH1)序列及其5’-增强子和表达调控等序列都来源于宿主动物序列(如图3绿色所示)。IgH 5’-端的增强子(5’-En),IgM的转换元件(switch region,Sμ)序列,IgM的CH2、CH3、CH4(去除外显子CH1和之后的内含子序列,IgM-dCH1)以及TM1、TM2和PolyA以及之间的所有序列都来源于宿主动物,保证IgM的在宿主动物中高表达和B-细胞发育。当宿主动物为小鼠时,用小鼠IgH 5’-端的增强子(5’-En),小鼠IgM的转换元件(switch region,Sμ)序列,小鼠IgM的CH2、CH3、CH4(去除外显子CH1和之后的内含子序列,IgM-dCH1)以及TM1、TM2和PolyA以及之间的所有序列,保证小鼠的B-细胞发育和抗体成熟。
更具体地,所述IgH 5’-端增强子、IgH Cμ的转换元件(Sμ)和IgM-dCH1序列为SEQID No.2所示。
上述Igγ包括人的Hinge、CH2、CH3外显子及之间的序列,缺失CH1序列或CH1序列中有突变的IgG命名为Igγ-dCH1。所述Igy-dCH1表达调控序列都来源于宿主动物序列,包括宿主动物的转换元件(switch region,Sγ)序列和宿主动物的终止信号(PolyA)、TM1和TM2等序列。如来源于小鼠的Igγ3、Igγ1、Igγ2a和Igy2b的表达调控元件等。具体地,所述Igy元件结构如图1-1所示:人的Hinge、CH2和CH3外显子序列在小鼠的Igγ表达调控元件(Sγ、TM1、TM2和PloyA等)的控制下得以表达。进一步提高了IgG的表达量和特异性。更具体的,所述小鼠5’-En,Sγ1和Igγ1-dCH1序列为SEQ ID NO.1。人的Igγ1的Hinge、CH2和CH3替代小鼠的Igγ1的CH1、Hinge、CH2和CH3的序列为SEQ ID NO.3。
上述Igγ序列,可以包括人Igγ的各种亚型,即Igγ3、Igγ1、Igγ2和/或Igγ4等;所述Igγ3,Igy1,Igy2,和(或)Igy4包括Hinge、CH2和CH3外显子以及之间的序列(去除外显子CH1和之后的内含子序列,Igy-dCH1)。外显子序列与宿主动物的表达调控元件的序列匹配以保证Igγ的表达框架。
上述Igγ-dCH1的表达调控元件可以来源于(以小鼠为例):小鼠的Igγ3、Igγ1、Igγ2a和/或Igγ2b等。
上述核酸分子还包括IgH 3’-位置表达调控序列(Local Control Region)。所述LCR是宿主动物IgH 3’-位置表达调控(LCR)序列。如图2、图5、图6和图7所示。
上述核酸分子包括了人IgH重链V-区序列或片段,人IgH D-区序列或片段,和人IgH J-区序列或片段。重链的V-区、D-区和J-区都来源于人如图6和图7。
例如:具体地,上述核酸分子含有多个(或全部)人的免疫球蛋白基因的(IgH)V-区,D-区和J-区序列,链接小鼠免疫球蛋白(IgH)的5’-增强子(5’-En),再链接小鼠的Igy的转换元件(switch region,Sγ)序列,其次链接人Igγ的Hinge,CH2和CH3序列,以及小鼠的PolyA,TM1和TM2序列,最后链接小鼠重链IgH的3’-位置表达调控(LCR)序列(如图6)。转基因小鼠表达人的IgG等单域抗体。
或者具体地,上述核酸分子含有多个(或全部)人的免疫球蛋白基因的(IgH)V-区,D-区和J-区序列后,链接小鼠免疫球蛋白(IgH)的5’-增强子(5’-En),转换元件(switchregion,Sμ)序列和IgM的CH2,CH3,CH4以及PolyA,TM1和TM2等序列,再链接小鼠的Igy的转换元件(switch region,Sγ)序列,其次链接人Igγ的Hinge,CH2和CH3序列,以及小鼠的PolyA,TM1和TM2等序列,最后链接小鼠重链IgH的3’-位置表达调控(LCR)序列(如图7)。转基因小鼠表达鼠的IgM和人的IgG等单域抗体。
上述人免疫球蛋白基因包括部分或全部人的重链(IgH)的V-区,D-区,J-区,还包括人Cμ-区序列和Cγ-区序列的CH1序列有突变或缺失。上述小鼠免疫球蛋白基因包括小鼠的重链的5’-端增强子(5’-Enhancer)序列,转换元件(switch region,Sμ和Sγ)序列,终止信号(PolyA),TM1和TM2以及小鼠重链IgH的3’-位置表达调控(LCR)等序列。
一种载体,包含上述的核酸分子。
一种含有上述核酸分子或载体的原核生物。一种含有上述核酸分子或载体的细胞,包括任何一种来源于这些核酸分子的转基因动物的细胞,还包括但不限于来源于转基因动物的淋巴细胞、杂交瘤、抗体表达细胞等。
一种人源抗体,由上述任一核酸分子重排、编码制得;包括任何一种来源于上述核酸分子或核酸分子转基因动物的人源抗体,包括但不限于抗体蛋白质、抗体DNA、cDNA序列和经过进一步修饰,改造的抗体等。
一种含有上述核酸分子或载体、细胞或抗体的转基因动物。动物可以是猪、牛、马、鼠、大鼠、兔、鸡和羊等哺乳动物。
上述核酸分子、载体、细胞或动物在编码DNA、cDNA、mRNA,表达氨基酸序列、蛋白质、载体,培养杂交瘤、细胞株、转基因动物中的应用。
尤其是,将上述转基因载体转入动物基因组内获得的转基因动物,或者与宿主动物自身免疫球蛋白基因缺失的动物进行交配的后代,宿主动物本身的重链和轻链被失活,只表达人抗体蛋白的基因工程动物。利用抗原免疫转入经过上述改造后的人免疫球蛋白基因的动物,获得的全人单域抗体。
例如,通过以上的改造,含有上述核酸、载体、细胞或转基因动物能表达宿主动物的单域IgM和人的单域IgG抗体。
采用上述核酸分子或载体制备转基因动物的方法,包括以下一些步骤:
(1)所述核酸分子的获得;
(2)将所述核酸分子构建载体;
(3)向宿主动物细胞(包括干细胞,诱导干细胞和体细胞)或胚胎导入上述载体;
(4)将含有上述载体的细胞植入宿主动物的胚胎内(嵌合体的制备)或体细胞克隆;
(5)繁殖杂合,纯合的转基因动物(包括与宿主动物自身免疫球蛋白基因缺失的动物交配)。
上述宿主动物是指上述核酸分子可以应用的转基因动物,比如猪、牛、马、鼠、大鼠、兔、鸡和羊等哺乳动物;上述载体包括酵母人工染色体(YAC),细菌人工染色体(BAC),质粒和DNA片段等,上述载体导入方法包括:电穿孔、病毒感染、脂质体介导和显微注射等。
SEQ ID.NO1、SEQ ID.NO2、SEQ ID.NO3作为某(几)次具体的实施方式,上述核酸分子的序列如所示,但这并不作为对本发明范围的限制,本领域技术人员可以根据其对核苷酸序列作出一些非本质的改进和调整,如对核苷酸的删除、增加、替换等。
本发明所述IgM基因缺失CH1功能,本领域技术人员采用常规方法使CH1功能缺失,例如缺失CH1序列或CH1序列突变等,可以简称为IgM-dCH1。
有益效果
1.本发明将免疫球蛋白基因载体的C-区的CH1序列具有突变或缺失,制备单域抗体转基因动物,生产单域抗体。
2.本发明实现了采用人的免疫球蛋白基因或片段生产人单域抗体,在不同抗原的刺激条件下,可以获得高亲和力的单域抗体。
3.本发明直接获得全人的单域抗体,减少后期的人工改造和提高了抗体的成药性。
4.本发明优点在于:转基因动物的IgH的转基因元件中,采用转基因宿主动物本身的IgH5’-端增强子,转换元件Sμ和IgM-dCH1序列,保证了转基因动物的B-细胞的正常发育;采用转基因宿主动物本身的Igγ的转换元件(switch region,Sγ)序列和转基因宿主动物本身Igγ的终止信号(PolyA)和TM1,TM2序列来控制人的Igγ-dCH1(只有Igγ的Hinge、CH2和CH3等序列)的表达,这样有利于DNA的重组、突变和BCR(B-细胞受体)的信号传递,使得人的Igγ-dCH1在抗原刺激下成熟。转基因动物的IgH转基因元件中是利用人的V-区、D-区、J-区和γ序列,所以,转基因动物表达的是人的单域抗体(IgG),减少后期的人工改造和提高了抗体的成药性。
5.本发明主要用途有:制备人的治疗性单域抗体,具体的:(1)治疗相关疾病,特别是脑部疾病和实体肿瘤;(2)制备双特异性抗体;(3)制备CAT-T。
附图说明:
图1:Igy结构及构建示意图。具体的,构建鼠的IgH 5’-增强子,鼠/人Igγ1嵌合表达元件(鼠Sγ1、TM1、TM2和PolyA等序列和人的Hinge、CH2和CH3序列)示意图。通过同源重组(Recombineering)技术和反筛重组技术(Counter-selection Recombineering)将载体转入人的IgG C-区5’-增强子、IgM和IgD的BAC中。Puromycin(Puro)筛选基因可以在细菌和细胞中分别进行筛选。Lox是一个34bp的DNA序列。
图2:构建一个核酸分子的IgG C-区示意图:将图1的DNA片段,与鼠IgH的3’-LCR通过同源重组技术(同源臂序列)相链接。Puromycin(Puro)和Zeocin(Zeo)筛选基因可以在细菌和细胞中分别进行筛选。Frt和Lox是一个34bp的DNA序列,可以通Flpo或CRE蛋白质或表达质粒去除筛选基因,在DNA序列中只留下一个Frt和Lox的序列。
图3:构建鼠的IgH 5’-增强子,鼠IgM-dCH1表达元件:鼠IgH 5’-增强子、Sμ、CH2、CH3、CH4以及TM1、TM2和PolyA等序列。通过同源重组技术(同源臂序列)将载体转入人的IgGC-区5’-增强子、IgM和IgD的BAC中。Puromycin(Puro)筛选基因可以在细菌和细胞中分别进行筛选。Lox是一个34bp的DNA序列。
图4:鼠和人Igy1-dCH1嵌合表达调控元件结构示意图:通过反筛重组技术(同源臂序列)将人Igy的Hinge、CH2和CH3序列取代鼠Igy的CH1、Hinge、CH2和CH3序列(绿色为小鼠序列,红色为人序列)。
图5:构建一个核酸分子的IgG-dCH1 C-区示意图:将图3和4的DNA片段,与鼠IgH的3’-LCR通过同源重组技术(同源臂序列)相链接。Puromycin(Puro)和Zeocin(Zeo)筛选基因可以在细菌和细胞中分别进行筛选。Frt和Lox是一个34bp的DNA序列,可以通Flpo或CRE蛋白质或表达质粒去除筛选基因,在DNA序列中只留下一个Frt和Lox的序列。
图6:其中一个IgH转基因核酸分子(转基因小鼠Ha)的组成元件:将人的IgH V-区、D-区、J-区与新的IgH C-区(Igγ1-dCH1)和3’-LCR链接,形成一个人单域抗体表达核酸分子(红色是人的DNA序列,绿色是小鼠DNA序列)。
图7:其中一个IgH转基因核酸分子(转基因小鼠Hb)的组成元件:将人的IgH V-区、D-区、J-区与新的IgH C-区(IgM-dCH1和Igy1-dCH1)和3’-LCR链接在一起,形成一个人单域抗体表达核酸分子(红色是人的DNA序列,绿色是小鼠DNA序列)。
图1-7是转基因的关键基因结构图。
图8:小鼠免疫球蛋白重链基因IgH的J-区基因打靶:小鼠IgH的J-区由J1、J2、J3和J4组成,用基因打靶技术,将整个J-区的序列切除。没有J-区序列的同源小鼠就不能产生鼠源的Ig(包括IgM和IgG等)。
图9:单域小鼠(Ha)人的IgHV2-26PCR结果,433bp PCR产物。
图10:单域抗体转基因小鼠(Ha)血清中人IgG Elisa结果。
图11:单域小鼠(Hb)的人IgHV2-26PCR结果,433bp PCR产物。
图12:单域抗体转基因小鼠(Hb)血清中人IgG Elisa结果。
图13:小鼠IgHJ-区序列的基因打靶(基因打靶后J-区PCR产物大小为732bp,正常J-区PCR产物的大小为2422bp)。
图14:OVA和HEL免疫小鼠后获得的特异性和亲和力高的单域抗体统计表。
具体实施方法:
下面是具体实施例证对本发明进行具体描述,在此指出以下实施例证只用于对本发明进行进一步说明,不能理解为对本发明保护范围的限制,本领域的技术是由熟练人员可以根据上述发明内容对本发明作出一些非本质的改进和调整。
实施例
将改造的人免疫球蛋白基因重链核酸分子载体转入小鼠体内,再免疫含有这个核酸分子的转基因小鼠获得全人单域抗体,具体步骤如下:
1.免疫球蛋白基因载体的构建
1)免疫球蛋白重链基因的构建(如图6)
首先改造人的Igy1的CH1序列:通过反筛重组和基因合成技术,将人Igγ的CH1序列去除,保证Hinge、CH2和CH3及其所有序列的表达框架;再获得小鼠IgH 5’-端增强子构建鼠5’-增强子和鼠/人Igy1的嵌合表达元件,再将这个表达元件同源重组到人IgH C-区的BAC中如图1。获得鼠3’-位置表达调控序列(LCR)并建立筛选基因和同源臂。同时将两个基因片段同源重组形成一个核酸大分子如图2。最后将人的IgH V-区、与图2的核酸大分子形成一个免疫球蛋白重链基因的基因簇如图6所示(绿色为小鼠序列,红色为人的序列)。包括了依次为免疫球蛋白重链基因人的V区、D区、J区、小鼠的IgH 5’-En、Sγ、人的Igy1-dCH1和小鼠的LCR。采用小鼠IgH 5’-端增强子,Igγ的转换元件(Sγ)序列,人的Hinge、CH2、CH3、和小鼠的TM1和TM2序列以及其之间的序列和PolyA信号序列等形成鼠/人Igγ-dCH1嵌合表达元件。LCR是小鼠IgH 3’-位置表达调控序列。
2)免疫球蛋白重链基因的改造(如图7)
首先改造小鼠IgM的CH1序列:通过反筛重组技术或DNA合成技术,将IgM的CH1序列去除,保证CH2、CH3和CH4及其所有序列的表达框架(如图3);同时通过反筛重组直接将人的Igy1的Hinge、CH2和CH3等序列取代鼠Igy1的CH1、Hinge、CH2和CH3等序列(如图4)。再获得小鼠IgH 3’-位置表达调控序列(LCR),同时建立DNA片段两端的同源背,通过同源重组获得一种单域核酸分子的C-区序列和3’-LCR(如图5)。再通过同源重组技术将上面改造好的DNA片段转入含人免疫球蛋白重链基因(Ig)的YAC或BAC载体上,构建免疫球蛋白重链基因的基因簇如图7所示(绿色为小鼠序列,红色为人的序列)。包括了依次为免疫球蛋白重链基因人的V区、D区、J区、小鼠的5’-En、Sμ、mIgM-dCH1和Sγ、人的Igy-dCH1和小鼠的LCR(如图7),其中,mIgM-dCH1经基因改造(如图3),鼠源IgH 5’-增强子,转换元件(Sμ)序列,IgM的CH2、CH3、CH4、TM1和TM2序列以及其之间的序列和相关PolyA信号序列等等都是小鼠的序列;人的Igγ-dCH1经基因改造(如图4),是采用小鼠Igγ的转换元件(Sγ)序列,人Igγ的Hinge、CH2、CH3、和小鼠Igy的TM1和TM2序列以及其之间的序列和相关PolyA信号序列等形成鼠/人Igγ-dCH1嵌合表达元件(如图5)。LCR是小鼠IgH 3’-位置表达调控序列。
2、人单域抗体转基因小鼠的培育
1)转人免疫球蛋白重链基因小鼠的培育
A.转人免疫球蛋白重链基因小鼠(单域小鼠Ha)的培育
利用已有的常规转基因技术将步骤1的1)中构建的人免疫球蛋白重链基因载体(图6)转入小鼠体内。通过PCR检测和ELISA检测双标准筛选获得的转人免疫球蛋白重链转基因小鼠为单域抗体小鼠(Ha)。
使用的PCR鉴定引物为:
人的IgHV2-26PCR:
For:GACACACTTTGCTACACACTCCTG
Rev:GCACAGTAATATGTGGCTGTGTCC
PCR产物大小为:433bp
PCR检测结果如图9所示。说明:人IgHV2-26转基因小鼠DNA PCR结果,阳性小鼠有433bp的PCR带(1%凝胶电泳)
人的IgV3-11PCR:
For:TGTTTGCAGGTGTCCAGTGTCAGG
Rev:GACAGGAAGCCAGCCCTCAGC
PCR产物大小为:686bp
Elisa检测转基因小鼠:使用的ELISA鉴定的抗体为:一抗(包埋)Abcam(ab97221)和二抗Millipore(AP113P),以健康成年人和健康小鼠血清作为对照。
单域抗体转基因小鼠血清中人IgG Elisa结果如图10所示。说明:单域抗体转基因小鼠血清中人IgG Elisa检测,用正常小鼠为阴性对照,人血清为阳性对照。转基因小鼠血清中有明显的IgG表达。
B.转人免疫球蛋白重链基因小鼠(单域小鼠Hb)的培育
利用已有的常规转基因技术将步骤1的2)中构建的人免疫球蛋白重链基因载体(图7)转入小鼠体内。通过PCR检测和ELISA检测双标准筛选获得的转人免疫球蛋白重链转基因小鼠为单域抗体小鼠(Hb)。
使用的PCR鉴定引物为:
人的IgHV2-26PCR:
For:GACACACTTTGCTACACACTCCTG
Rev:GCACAGTAATATGTGGCTGTGTCC
PCR产物大小为:433bp
PCR检测结果如图11所示。说明:人IgHV2-26转基因小鼠DNA PCR结果,阳性小鼠有433bp的PCR带(1%凝胶电泳)
人的IgV3-11PCR:
For:TGTTTGCAGGTGTCCAGTGTCAGG
Rev:GACAGGAAGCCAGCCCTCAGC
PCR产物大小为:686bp
Elisa检测转基因小鼠:使用的ELISA鉴定的抗体为:一抗(包埋)Abcam(ab97221)和二抗Millipore(AP113P),以健康成年人和健康小鼠血清作为对照。
单域抗体转基因小鼠血清中人IgG Elisa结果如图12所示。说明:单域抗体转基因小鼠血清中人IgG Elisa检测,用正常小鼠为阴性对照,人血清为阳性对照。转基因小鼠血清中有明显的IgG表达。
2)免疫球蛋白重链基因敲除小鼠的培育(图8)
利用基因打靶技术,构建免疫球蛋白重链基因敲除小鼠。选择小鼠免疫球蛋白重链基因的IgH J-区作为基因敲除位点(敲除位点及基因敲除效果见图8),获得免疫球蛋白重链基因敲除小鼠。通过PCR检测和ELISA检测双标准筛选获得的免疫球蛋白重链基因敲除小鼠。
使用的PCR鉴定引物为:
For:GGGAAAGAATGAGCAAATGCAAGC
Rev:TTCTGTGTTCCTTTGAAAGCTGGAC
PCR结果如图13所示。PCR产物:J-区基因打靶后PCR产物大小为732bp,正常J-区PCR产物的大小为2422bp。
使用的ELISA鉴定的抗体为:sigma(M8644)和sigma(A8786),以健康成年人和健康小鼠血清作为对照。
3)小鼠免疫球蛋白kappa轻链基因敲除小鼠的培育(参考CN105441455A)
4)杂交组合获得人源化抗体转基因小鼠
将第二步的Ha)或Hb)与2)、3)步获得的小鼠进行杂交繁育,经过PCR和ELISA检测,最终获得高表达人单域IgG抗体小鼠,而不表达(或低表达)鼠免疫球蛋白IgG。
转基因小鼠血清中人IgG含量的Elisa检测:
结果:正常小鼠血清的IgG含量:1-3mg/ml
正常人血清的IgG含量:3.5-15mg/ml
转基因小鼠(Ha)血清中的人IgG含:0.001-0.3mg/ml
转基因小鼠(Hb)血清中的人IgG含:0.01-0.5mg/ml
(说明:转基因小鼠中的人IgG基因是经过改造后的IgG,其中CH1及其相关的内含子缺失(IgG-dCH1),因此,IgG表示量要比正常人的IgG含量少。并且,转基因小鼠饲养在无菌和清洁环境下,其血清中的IgG也要少一些)
3.人单域抗体的获得
对人单域抗体转基因小鼠进行免疫,获得B-细胞,再结合噬菌体展示技术,获得治疗性人单域抗体或者制备双特异性抗体。
A.OVA免疫获得的人源化抗体转基因小鼠
选择8周龄的小鼠进行免疫。
初免:
①用PBS稀释OVA(Sigma A7641)抗原,终浓度为2mg/ml,加入20ug CpG(ODN1826,tlrl-1826,Invivogen),加入适量氢氧化铝(vac-alu-50,Invivogen),使氢氧化铝浓度为1%。
②将①中准备好的抗原0.75mL与CFA佐剂(Sigma F5881)按1∶1混合,用MIXPACTM注射器使之乳化,每只小鼠,注射剂量为200ul(0.2mg),进行皮下注射免疫。
二免:
①免疫后第21天进行二免,用PBS稀释抗原,终浓度为1.0mg/ml,加入10ug CpG,加入适量氢氧化铝,使氢氧化铝浓度为1%。
②将①中准备好的抗原0.75mL与IFA佐剂按1∶1混合,用MIXPACTM注射器使之乳化,每只小鼠注射剂量为200ul(0.1mg),进行腹腔注射免疫。
三免:
①免疫后第21天后进行三免,用PBS稀释抗原,终浓度为1.0mg/ml,加入10ug CpG,加入适量氢氧化铝,使氢氧化铝浓度为1%。
②直接注射抗原蛋白,按①中方法配制,每只小鼠注射剂量为200ul(0.1mg)进行腹腔注射免疫。
四免:
①免疫后第21天后进行四免,用PBS稀释抗原,终浓度为1.0mg/ml,加入10ug CpG,加入适量氢氧化铝,使氢氧化铝浓度为1%。
②直接注射抗原蛋白,按①中方法配制,每只小鼠注射剂量为200ul(0.1mg)进行腹腔
注射免疫。
加强免疫:
在第四免疫后21天,对Elisa结果符合要求的小鼠,开展加强免疫,然后获得脾胀的B-细胞进行。杂交瘤培养和筛选
1)小鼠免疫检测
第4次免疫后10天,分别取小鼠血进行ELISA检测,以免疫前小鼠作为对照,分别检测免疫后小鼠血清中鼠IgG的含量和人IgG的含量:
小鼠血清中人IgG的检测:用抗原包埋96孔板,用hlgG-HRP(Millipore,AP113P))检测经过稀释后免疫血清中的特异抗体含量。
将免疫后的人源化特异抗体在小鼠中表达量较高(血清稀释at 1∶8000),其ElisaOD450>大于1.0的免疫小鼠,用于制备单域抗体。
2)单域抗体的获得
收集小鼠的脾脏B-细胞,进行高表达特异抗体B-细胞的分离(hIgG+CD138+Ag+,FACS分离等),制备mRNA,cDNA,噬菌体展示,获得特异性和亲和力高的单域抗体(KD在0.03-5.6nM之间,如表14)。
SEQUE0CELlSTlNG
<110>金迈博生物科技有限公司
<120>一种核酸分子及其在人源化抗体中的应用
<160>3
<210>1
<211>15999
<212>DNA
<213>小鼠5’-En、Sγ1、人lgγ1-dCH1和小鼠TM1、TM2、PolyA序列(人、鼠和合成DNA)
<220>
<221>
<222>(1)..(8)
<223>Not1酶切位点(合成序列)
<222>(9)..(370)
<223>同源臂(人的序列)
<222>(433)..(1444)
<223>鼠IgH 5’-增强子(鼠的序列)
<222>(3278)..(8527)
<223>鼠Igγ1转换元件(Sγ1)(鼠的序列)
<222>(8648)..(8692)
<223>人Igγ1 Hinge(人的序列)
<222>(8811)..(9141)
<223>人Igγ1 CH2(人的序列)
<222>(9238)..(9560)
<223>人Igγ1 CH3(人的序列)
<222>(10862)..(10992)
<223>鼠TM1(鼠的序列)
<222>(11810)..(11890)
<223>鼠TM2(鼠的序列)
<222>(12611)..(13477)
<223>鼠PolyA信号序列(鼠的序列)
<222>(13837)..(13870)
<223>Lox(合成序列)
<222>(13871)..(15192)
<223>pGK-Puromycin筛选基因(合成序列)
<222>(15193)..(15226)
<223>Lox(合成序列)
<222>(15227)..(15508)
<223>同源臂(同源重组与鼠3’-LCR链接)(鼠的序列)
<222>(15509)..(15516)
<223>AsiSI(合成序列)
<222>(15517)..(15991)
<223>同源臂(同源重组到人lg C-区序列)(人的序列)
<222>(15992)..(15999)
<223>Not1(合成序列)
GCGGCCGCCG AGATGCCTGA ACAAACCAGG GGTCTTAGTG ATGGCTGAGG AATGTGTCTC60AGGAGCGGTG TCTGTAGGAC TGCAAGATCG CTGCACAGCA GCGAATCGTG AAATATTTTC120TTTAGAATTA TGAGGTGCGC TGTGTGTCAA CCTGCATCTT AAATTCTTTA TTGGCTGGAA180AGAGAACTGT CGGAGTGGGT GAATCCAGCC AGGAGGGACG CGTAGCCCCG GTCTTGATGA240GAGCAGGGTT GGGGGCAGGG GTAGCCCAGA AACGGTGGCT GCCGTCCTGA CAGGGGCTTA300GGGAGGCTCC AGGACCTCAG TGCCTTGAAG CTGGTTTCCA AGAGAAAAGG ATTGTTTATC360TTAGGAGGCA TCTTAGTGAT TGAGTCAAGG GAGAAAGGCA TCTAGCCTCG GTCTCAAAAG420GGTAGTTGCT GTCTAGAGAG GTCTGGTGGA GCCTGCAAAA GTCCAGCTTT CAAAGGAACA480CAGAAGTATG TGTATGGAAT ATTAGAAGAT GTTGCTTTTA CTCTTAAGTT GGTTCCTAGG540AAAAATAGTT AAATACTGTG ACTTTAAAAT GTGAGAGGGT TTTCAAGTAC TCATTTTTTT600AAATGTCCAA AATTCTTGTC AATCAGTTTG AGGTCTTGTT TGTGTAGAAC TGATATTACT660TAAAGTTTAA CCGAGGAATG GGAGTGAGGC TCTCTCATAA CCTATTCAGA ACTGACTTTT720AACAATAATA AATTAAGTTT CAAATATTTT TAAATGAATT GAGCAATGTT GAGTTGGAGT780CAAGATGGCC GATCAGAACC AGAACACCTG CAGCAGCTGG CAGGAAGCAG GTCATGTGGC840AAGGCTATTT GGGGAAGGGA AAATAAAACC ACTAGGTAAA CTTGTAGCTG TGGTTTGAAG900AAGTGGTTTT GAAACACTCT GTCCAGCCCC ACCAAACCGA AAGTCCAGGC TGAGCAAAAC960ACCACCTGGG TAATTTGCAT TTCTAAAATA AGTTGAGGAT TCAGCCGAAA CTGGAGAGGT1020CCTCTTTTAA CTTATTGAGT TCAACCTTTT AATTTTAGCT TGAGTAGTTC TAGTTTCCCC1080AAACTTAAGT TTATCGACTT CTAAAATGTA TTTAGAATTC ATTTTCAAAA TTAGGTTATG1140TAAGAAATTG AAGGACTTTA GTGTCTTTAA TTTCTAATAT ATTTAGAAAA CTTCTTAAAA1200TTACTCTATT ATTCTTCCCT CTGATTATTG GTCTCCATTC AATTCTTTTC CAATACCCGA1260AGCATTTACA GTGACTTTGT TCATGATCTT TTTTAGTTGT TTGTTTTGCC TTACTATTAA1320GACTTTGACA TTCTGGTCAA AACGGCTTCA CAAATCTTTT TCAAGACCAC TTTCTGAGTA1380TTCATTTTAG GAGAAAGACT TTTTTTTTAA ATGAATGCAA TTATCTAGAC TTATTTCAGT1440TGAACATGCT GGTTGGTGGT TGAGAGGACA CTCAGTCAGT CAGTGACGTG AAGGGCTTCT1500AAGCCAGTCC ACATGCTCTG TGTGAACTCC CTCTGGCCCT GCTTATTGTT GAATGGGCCA1560AAGGTCTGAG ACCAGGCTGC TGCTGGGTAG GCCTGGACTT TGGGTCTCCC ACCCAGACCT1620GGGAATGTAT GGTTGTGGCT TCTGCCACCC ATCCACCTGG CTGCTCATGG ACCAGCCAGC1680CTCGGTGGCT TTGAAGGAAC AATTCCACAC AAAGACTCTG GACCTCTCCG AAACCAGGCA1740CCGCAAATGG TAAGCCAGAG GCAGCCACAG CTGTGGCTGC TGCTCTTAAA GCTTGTAAAC1800TGTTTCTGCT TAAGAGGGAC TGAGTCTTCA GTCATTGCTT TAGGGGGAGA AAGAGACATT1860TGTGTGTCTT TTGAGTACCG TTGTCTGGGT CACTCACATT TAACTTTCCT TGAAAAACTA1920GTAAAAGAAA AATGTTGCCT GTTAACCAAT AATCATAGAG CTCATGGTAC TTTGAGGAAA1980TCTTAGAAAG CGTGTATAGA AAGTAGGGAC TCTGGGGTCC CAGGTTCAAT CCCAGCATTG2040TTATTACAGT AACTAAGGGA ACAGAAGCAA CTCTGGATTG AAGGAGGGAA AGACAAGTAG2100AGGGAAGCCC AAGCTGCTCT GAGGGACTCA GGGTATGTGG GGGATCCAGA GAGCCAGGGG2160AACTCGAGAG CTCAGGACAG AAGACTGAGC ACCTGCAAAA GAGCTGAGGC TGGTAAGAGT2220AACAAGGTAA CCTGGGTAAC AGGTTCAGCA GCTCCAGGGG AGCCAGGAGA GATGGAAGTG2280TGGGGAGCCA GGCAGAGCAG CTCCATGACA GCCAGGACAG GTGGAAGTGT GGTACCCAGG2340CAGAGCAGCT CCATGACAGC CAGGACAGGT GGAATTGTGG TGACCCAGGC AGAGCAGCTC2400CAGGGCAGCC AGGACAGGTG GAAGTGTGGT GACCCAGGCA GAGCATCTAT AGGGGAGCCA2460GGACAGGTGG GAGTGTGGTG ACCCAGGCAG AGCAGCTCCA GGGCATCCAG GACAGGTGGA2520ATTGTGGTGA CCCAGGCAGA GCAGCTACAG GGGAGCCAGG AAAGGTGGGA GTGTGGTGAC2580CCAGGCAGAG CAGCTACAGG GGAGCCAGGA CAGGTGGGAG TGTGGTGACC CAGGCAGAGC2640AGCTATAGGG CAGCCAGGAC AGGTGGAAGT GTGGTGACCC AGGCAGAGCA GCTACAGGGG2700AGCCAGGAAA GGTGGGAGTG TGGTGACCCA GGCAGAGCAG CTACAGGGGA GCCAGGAAAG2760GTGGGAGTGT GGTGACCCAG GCAGAGCAGC TACAGGGGAG CCAGGAAAGG TGGGAGTGTG2820GTGACCCAGG CAGAGCAGCT ACAGGGCAGC CAGGACAGGT GGAAGTGTGG TGACCCAGGC2880AGAGCAGCTC CATGGCACCC AGGACAGGTG GAAGTGTGGT GACCCAGGCA GAGTAGCTAC2940AGGGCAGCCA GGACAGGTGG AAGTGTGGTG ACCCAGGCAG AGCAGCTTCA GGGCAGCCAG3000GACAGGTGGA AGTGTGGTGA CCCAGGCAGA GCAGCTTCAG GGCAGCCAGG ACAGGTGGAA3060GTGTGGTGAC CCAGGCAGAG CAGCTTCAGG GCAGCCAGGA CAGGTGGGAG TGTGGTGACC3120CAGGCAGAGC AGCTCCAGGG GAGCCAGGAC AGGTGGGAGT GTGGTGACCC AGGCAGAGCA3180GCTCCAGGGC AGCCAGGACA GGTGGAAGTG TTGTGACCCA GGCAGAGCAT CTATAGGGTA3240GCCAGGACAG GTGGAATTGT GGTGACCCAG GCAGAGCAGC TCCAGGGCAG CCAGGACAGG3300TGGGAGTGTG GTGACCCAGG CAGAGCATCT ATAGGGGAGC CAGGACAGGT GGAAGTGTGG3360TGACCCAGGC AGAGTAGCTA CAGGGAGCCA GGACAGGTGG AAGTGTGGTG ACCCAGGCAG3420AGTAGCTCTT GGGGAGCCTG GACAGGTGGG AGTGTGGTGA CCCAGGCAGA GCAGCTACAG3480GGGAGCTAGG ACAGGTGGAA GTGTGGTGAC CCAGGCAGAG TAGCTCTTGG GGAGCCAGGA3540CAGGTGGAAG TGTGGTGACC CAGGCAGAGC ATCTATAGGG GAGCCAGGAC AGGTGGGAGT3600GTGGGGATCC AGGTAAGGCA GGACTGGGGA GCCAGGACAG GTGGAAGTGT GGTGACCCAG3660GCAGAGCAGC TCCAGGGCAG CCAGGACAGG TGGAAGTGTG GTGACCCAGG CAGAGCAGCT3720CCATGGCAGC CAGGACAGGT GGAAATGTGG TGACCCAGGC AGAGCAGCTC CAGGGCAGCC3780AGGACAGGTG GAAGTGTGGT GACCCAGGCA GAGCATCTAT AGGGGAGCCA GGACAGGTGG3840GAGTGTGGTG ACCCAGGCAG AGCAGCTCCA TGGCAGCCAG GACAGGTGGG AGTGTGGTGA3900CCCAGGCAGA GCAGCTCATG GCAGCCAGGA CAGGTGGGAG TGTGGTGACC CAGTCAGAGC3960AGCTGCAGGG CAGCCAGGAC AGGTGGGAGT GTGGTGACCC AGGCAGAGCA TCTATAGGGG4020AACCAGGACA GGTGGAAGTG TGGTGACCCA GGCAGAGCAG CTGCAGGGCA GCCAGGACAG4080GTGGGAGTGT GGTGACCCAG GCAGAGCATC TATAGGGGAA CCAGGACAGG TGGAAGTGTG4140GTGACCCAGG CAGAGCAGCT ATAGGGAGCC AGGACAGGTG GAAGTGTGTT GATCCAGACA4200AAACACCTAC AGGGGAGACA AATCAGCTAG ATTGGGCGGA TCCTGGCAGA TCAGCTTCAG4260GGGACCTAGG CAAGTGGAAC TGTGGGGACC CGTGTAGGGC AGCTGTAGGG AAATCAGGAC4320AGGTACAAGT GTGTGGATCC ATGCAGTGTA GTGCCTTGGG AGCCTGAACA GATAGAAGGG4380TGGGGATACA GGCAGGGTAG CTATAGGGAA TCCAGTTGAG GTGGAAGAAT GGGGATCCAG4440GCAGAGTAGC TATAGGGCAG CCAGGAGAAA TGGAAGAATG CAGATCCAAA CAGAAGAGCT4500ACAGAGGAGC CAAGACAACT AGAAGTGTGT GAATCCAGGC AGAGCAGTGC CTTAGGAGCA4560AGGACAGGGA AGCTATAGGG AAACCAGGAC AGGTGGAAGA ATGGGGATCC AGGTGCTGCA4620GCTACAGGTA AGCAGGGACA GGTGGAAGTG TGGAGACCCA GGCAGAGCAG CTATAGGGCA4680GCCAGGACAG GTGGGAGTGT GGGGATCCAG GTAAGGCAGG ACTGGGGAGT CAAGGTAGGT4740GGAATGTGAA GTTCCAGGCA GAACAGGTCC AGGGTGCCAG GACAGGTACA AGTTTAGTAG4800TTATAGAGGA ACAGGGGCAG GTTAGAATGA AGGATGGGCA TCCCGGGTGA GCAAATACAA4860GGGAACTGAT GGCAAATGGA AGGGCAGGGA CCCAGACTAA ATGGCTACAG AGAAGCTGAG4920GCAGGTAAGA GTGTGGGAAC CCAGTCAAAA ACCACAGAAG AGCAGGAGCT AATTGGCACG4980GGCTGGGGTG CATGCTGGCT ACTCATAGGG AAGCTGGGAT AAGTAGTAGT TGGGGATTCT5040AAGCAGTCAC AGAGAAGCTG ATCCCGGTGA GAGTACGGGG TACACAGCTG AGCAAATACT5100ACATAGCTGG AGCTGATGGC TGTATAAGGT ACCAGGCTGA GCAGCTGAAG GTAACCTGGA5160CCTAGTGGGG GTGTGGGAGA CCTGGCTGAG CAGCTACCAA GGATCAGGGA TAGACATGTA5220AGCAGTCAAG CTCAGCTACT ACATGAGAGC TGGAGCTAGT ATGAAGGTGG AGGTCCAGTT5280GAGTGTCTTT AGAGAAACTG AGGCAAGTGG GAGTGCAGAG ATCCAGGCTG AGCAGCTCCA5340GCTTAGCTGG TATAGGTGAC AGGATGGGGG ATAACAAGGC TAAGAACACA CAGAGAGCAG5400GATCTCCTGG GTAGGTTACA GGTCAAGACT GAGTAGAATC AGGGGAGCTG AGGTTGGGAG5460TAATGCAGAA TTCCAGACTT AGCAGTCCAG GCAAACTAAA CCAAGTGGGA GTGTGGGAGT5520CCTAACTGAA CAAATACCAG GCATATGAAG CTGATAAGTG TGTATAAAGT ACCAAGCTGA5580GCAGCTACAG GAGAGCTGGG ATAGCTATGT GGGGAGACCA GGTTAAGCAA ACAGTGGAGA5640GCAAGATAAA GTCTTAATGT ATGCATCCAG GCTGAATAGA CACAGGGGAG CTGAGGAAGG5700CAGTACTAGA GGATTCTAGG CTTAGAAGTC ACAGGGAAAC TGAGGCCTGG GTGAGGGTGG5760GCATCCTAGC TGGAAAAATC ACCAGGGAGC TGGAGCTGAT GGGTATAAAA AGGTACCAGG5820TTGAGCAGCT ACAGGAGAGC TAGGACATGT GGGGATGTTT TGTTCCAGGC TGAACAACTG5880TAGAGCATCA GGGGGAGGTG GAACTTTAAG AAGTCAGGCT GAGCAGCTAC AGGAGAGCTG5940CAGCTATTCG GTATGTGGAG GTCCAGCCAG AGCAGTTACA GGGTAGCTGG GATAAATGGG6000GCTGGAGAAC CAGGCTAAGA AGACACAGGG GAGCAGGTTC TAGTCTGCAT AGGAGTGGGG6060ATCCAGGTCG AGTACACACA GAAGAGAAAA GGGTAGGTAG TAATGGAGGA TTCTAGACTC6120AGCTATAACT GAGTGGTAGT GTGAGTGTTC TAGCTGGAAA AAAAAATACC ATGAAGCTGG6180AGCTGATGGG TATAAAAGGA ACGAGCCTGA GAAACTGTAG GGTATCTGGG GTGGATGGGG6240ATGTGGGGAG TCATGCTGAG CTGCTACAAG GGCAGTGTGG CCTCTAGGAG TGTAGGGGAC6300CAAACTGAGC ATCTACAGGG AAACTGAGGC AAACTGGAGT GTAGGGCTCC AGAATGAGCA6360ACTGCAGGTC AGCTGAGGCC GGTAAGAGTG TGGGGAACCA GACTGGACAG TTAGTTGCAA6420GTTAGCCAGG GTAGGTGAGA GTTCTATTAG AGAAGCCTCA GCAGAATGGG AAGTGGGAAC6480TAGGAAACCA GGATGAGCAC CTAACAACAG AAGTGAGGCA GGTTAGGGTG TAGGGGATGA6540GCTGTGCAGC TACTGTGGAG CACGCAGACT GGAATAGAAG AGGTTCTGGC TGAACACTTG6600AAGGGAACCA GGTAGGCAGG AGGCAGTGTG TACAGACAGC TGAATGAGAC ATCATGCAGG6660GCCAGTTCCC TGCCCTGAGC TACATTAGCT GGGACCAGGG CCAGCGGTTG AGGAACCAGG6720CAGAGGTAAA ATGGTGGTGT GATAGGAAGC CAATGGCAGA GGGGAGGGAG AAGTTATGCT6780TATGTCATGC TGGAATGTAG GAAGGGGAAA GAGCCAGGAT GTCTAGGCTG GAGCTGATCC6840GGCTGTCTGC TCTGATGGCA GCAACAGGCC TGAGCTTCTC TGGACTCAAG AAGCCAGGGC6900AACAAAATAA AGGGGGCCTA GCAGAGCAAA GACACTGCTA GCACTGGGAT CAGGAAAACA6960GGACAAGACT CCCGATCCAG GAGGTCATGG GAGGGAAGGA GAAGACTACA GGGGACTGTC7020CTTGGGAAAG AGTAAGGGCC CACTGGAGGG AGTGCTCAGG AAGCAAGCCC ATTGACAGGG7080GAGAACAAGG CTGGTGGACG TCTGGATGGG CAGTAGGCAG CCCCAAGTCC CAGGAGGGAG7140AGAAGAGGCA GATAGGAAAA CAGGTCAGGT CTAGCAGAGG CCTACTGAAG TACTCTCCTC7200AGGACAGAAC CCTGAATACT GGAAAATGCG GAACTGCTGC AGGCACAAAG AATAGCTGAG7260GTCTAAGAGT AAAACAGACT AGGGGATGAG AGGACCTTAG GAAGAGCCTT TGGCTGAGCA7320GGAACAAGAA CAGGGGAAAT CCTAGGGCTG ACATTGCCAG TGGAAACATA CAGGCTGGAG7380CTCTTTAGTC AGGAGCTCCA GCTGTGATCT AGACATCAGG CAGGAAGATC AAATCTGTCC7440CAACAATACA GGGGACAGAG GCTCAACCTA GAGTGTGAGC ATCAGGGGCT GTGCAGGAGA7500TTTCAGAGCT CAGGTGCAGC AGAGACTAGC ATGGCCCTGG GGATAAAGGG AAGGATCCAA7560GGGACAAGGG GATAATCCTG GGGAGGTAAG GGCCAGCTTC GTGACAGAAG GTGGTGGTGT7620CCAACTTCAA GAGCCCTGTG CTACAATTTA AAAAAAAAAA AAAAGGAAAG GGACTTCTCT7680GTGTTTGGCA ACACAAGTGC GATGCACAGG CAGGAAGATC AAATCTGTCC CAACAATACA7740GGGGACAGAG GGTCAACCTA CAAAAGGAAA GAACCTGGGG CAGTGTGAAG ACAACACTGT7800AGAAGCCAAG GCTGAGTTCA CTGAGCTCTC GTTAGTGAGA CTACACAGCA AGGAGGTGGC7860GGGCACTGAG CAGTGAGGCC CCGGGAAGTG GGGGTGATGG TGGTGACGGT GGTAACTGTT7920AAGAACTGGG GGAAAGAATT GTGGAGAACC AAGCTAAATA GTTATGTCAA ACCACATGTT7980TAGGAGCCTG GGTTGACTTC ATAGGGAGTA GGCATGGAGG CTAATCTAGA GGTTTGTGTA8040TAGGCAAGAA GTGAATCCTG ACCCAAGAAT AGAGAGTGCT AAACGGACTT AGTTCAAAGA8100CAACTGAAAA AGACAATGCC TGCAAAACAA AGCTAAGGCC AGAGCTCTTG GACTATGAAG8160AGTTCAGGGA ACCTAAGAAC AGGGACCATC TGTGTACAGG CCAAGGCCGG TAGAAGCAGC8220CTAGGAAGTG TCAAGAGCCA ACGTGGCTGG GTGGGCAAAG ACAGGAAGGG ACTGTTAGGC8280TGCAGGGATG TGCCGACTTC AATGTGCTTC AGTATTGTCC AGATTGTGTG CAGCCATATG8340GCCCAGGTAT AAGAGGTTTA ACAGTGGAAC ACAGATGCCC ACATCAGACA GCTGGGGGGC8400GGGGGTGAAC ACAGATACCC ATACTGGAAA GCAGGTGGGG CATTTTCCTA GGAACGGGAC8460TGGGCTCAAT GGCCTCAGGT CTCATCTGGT CTGGTGATCC TGACATTGAT AGGCCCAAAT8520GTTGGATATC ACCTACTCCA TGTAGAGAGT CGGGGACATG GGAAGGGTGC AAAAGAGCGG8580CCTTCTAGAA GGTTTGGTCC TGTCCTGTCC TGTCTGACAG TGTAATCACA TATACTTTTT8640CTTGTAGAGC CCAAATCTTG TGACAAAACT CACACATGCC CACCGTGCCC AGGTAAGCCA8700GCCCAGGCCT CGCCCTCCAG CTCAAGGCGG GACAGGTGCC CTAGAGTAGC CTGCATCCAG8760GGACAGGCCC CAGCCGGGTG CTGACACGTC CACCTCCATC TCTTCCTCAG CACCTGAACT8820CCTGGGGGGA CCGTCAGTCT TCCTCTTCCC CCCAAAACCC AAGGACACCC TCATGATCTC8880CCGGACCCCT GAGGTCACAT GCGTGGTGGT GGACGTGAGC CACGAAGACC CTGAGGTCAA8940GTTCAACTGG TACGTGGACG GCGTGGAGGT GCATAATGCC AAGACAAAGC CGCGGGAGGA9000GCAGTACAAC AGCACGTACC GTGTGGTCAG CGTCCTCACC GTCCTGCACC AGGACTGGCT9060GAATGGCAAG GAGTACAAGT GCAAGGTCTC CAACAAAGCC CTCCCAGCCC CCATCGAGAA9120AACCATCTCC AAAGCCAAAG GTGGGACCCG TGGGGTGCGA GGGCCACATG GACAGAGGCC9180GGCTCGGCCC ACCCTCTGCC CTGAGAGTGA CCGCTGTACC AACCTCTGTC CCTACAGGGC9240AGCCCCGAGA ACCACAGGTG TACACCCTGC CCCCATCCCG GGATGAGCTG ACCAAGAACC9300AGGTCAGCCT GACCTGCCTG GTCAAAGGCT TCTATCCCAG CGACATCGCC GTGGAGTGGG9360AGAGCAATGG GCAGCCGGAG AACAACTACA AGACCACGCC TCCCGTGCTG GACTCCGACG9420GCTCCTTCTT CCTCTACAGC AAGCTCACCG TGGACAAGAG CAGGTGGCAG CAGGGGAACG9480TCTTCTCATG CTCCGTGATG CATGAGGCTC TGCACAACCA CTACACACAG AAGAGCCTCT9540CCCTGTCTCC GGGTAAATGA TAATGTCCTG GTGATTTCTG AGATGTAGAG TCTAGCTAGG9600TCATGGAATG AGGGGTCTCC ATGGTTTGAG GCCTGAGTTG TGACTAAGGA AAAACCCATA9660GGCCTACACT GCCACACCCA GCACTTTTGA ATTTGCCTGA CATGAAAAGA ATTTACCTCT9720CCCTGGAAAG TGGAGCCTTA TCCCTAGGCA GTTCCCTTAC CAGACCTTCC TCTAGCTTGC9780ACTTTGTTCT GGGCACAGAA TGTGTCTAAC CCCCCAAAGC AAGGAAGACA CAACCTCTAC9840CTCCCTCACT CTGTCCTTAC CCCTTTTCCT GGCTAAGCAT CTCACTGAGT GCGCTGAATA9900GATGCATGTG GCCACAGTCT TGCAGACAGA CCCTTGCCAT CTCTCCACTC AGCTTTCCAG9960AGGCTAAGTC TAGCCCGTAT GGTGATAATG CAGGGAGCTC TATGCTATCT CAGTGCTATC10020AGACTCCCAA GTGGAGGATG AACATGGACC CATTAAAACC AACCTGCGCA GCAACACCCT10080GCCAATAAGG CCCGTATGTG AAAATGTGCA CACATCTACA CATGCACAGG CACACACACA10140CACACATGCA TGGGCACACA CACATACAGA GAGAGAGAAT CACAGAAACT CCCATGAGCA10200TCCTATACAG TACTCAAAGA TAAAAAGGTA CCAGGTCTAC CCACATGATC ATCCTCGGCA10260TTTACAAGTG GGCCAACTGA TACAGATAAA ACTTTTCTAT GCCAAGGACG CCAACATATA10320CACAAGTCCG CTCATGACAA ATCTGTCCCT GAACCTCAGA CTGGCGCCCG TGACTCATAC10380AGTGGACACT CCTCCAAAGC TGTATAGCTT CCTTTACTTC CCTGTGTGTA CTTTCTCTGA10440AGTACACTCA TCACACAGAA GAGGCCCTGT GATTACTCTG GCCCTCTGTT CTTGGTCATC10500AGAGAATAGA CAGAAGATCA GGCAAACTAC ACAGACACTT CCCACAATCA TCACAGGCCC10560TGACTCTGCT CTCCAGTCTC AAAACTGAAG GCTGGAGCAC ACAGAATAAG CTCCTGCACA10620GGCCAGGCCA GTATCGGGTC CAGTGTGTCT GACTGAGCCC AGGGACAAAA TGGCAGCACT10680TTGGGGAACT GAGGTTTCTG GTCCAAGAAG GAGAGATGGA GGCCCAGGGA GGGTCTGCTG10740ACCCAGCCCA GCCCAGCCCA GCTGCAGCTT TCTCCTGGGC CTCCATACAG CCTCCTGCCA10800CACAGGGAAT GGCCCTAGCC CCACCTTATT GGGACAAACA CTGACCGCCC TCTCTGTCCA10860GGGCTGCAAC TGGACGAGAC CTGTGCTGAG GCCCAGGACG GGGAGCTGGA CGGGCTCTGG10920ACGACCATCA CCATCTTCAT CAGCCTCTTC CTGCTCAGTG TGTGCTACAG CGCTGCTGTC10980ACACTCTTCA AGGTCAGCCA TACTGTCCCC ACAGTGTCTA CAATGTCCTC ATACTCTTCC11040CCATACTGTC CCTGTGGTGA CCTATACCCC ACACTGTCCC ATGCTAATGA CCACAGTCTT11100ACATGCTATG TAATGCTGTC TACCCTTCTG TATGCACAGT CTCACAATGT CCCATGCAGT11160CTCCACGATG CTCCATACTG TCCCCATTCC AACCCATGCT GCCCCTTGTT CCCCGCTATG11220CTGTCCCATG CTATTGTCTG TATTTTCATG CTCTTTTCAC ACTGTCCCTA GTGTCACATT11280CTGCCCATGT TGTCCACCAC ATTGTCCCCA CTCTGTACAC AGCCTCACAC TGTACCCTGC11340TACCCGATAA TGTTCCCTGT TGTCCCCAAC TCTCTCCCTG CACCATTTGT CAACTGTCCC11400CTGAATTCCC ATGTTGTTCC CACACTGTTA GTGTGTAATG TGCTCTGTCC CAGGTGTACC11460TTGTTCCGTG CTGTCTCACT TCATCGCCCA TTCTGTCCTT GTACTAACCC CACTCTATCA11520CCACACTGTC CCTATGCACT GCCCACATTG TCCTCATACT GTCCCATTTT GTATCTTCAT11580CCTGTCCCCA TAGTGTCCAA TGATCTACCC CACACTATTC CCACTTCATG CCCCTACAAT11640TTCCCTATTC CATTCCTCTC TGGTCACCAT GCCATCCTTC CCACTCCTGC ACAGCTGGAG11700AGGGACTCCC GGGATGAGTC CTTGCCCAGA TGAGCTACCT ATCTAGAGGA GTCTTCAGGT11760GGGAAGGGAA TGCAGTCTTG ATCTTGGTCT TATTCACCCT GTCTCACAGG TAAAGTGGAT11820CTTCTCCTCG GTGGTGGAGC TGAAGCAGAC ACTGGTTCCT GAATACAAGA ACATGATTGG11880GCAAGCACCC TAGGCCACCT CCTGTAATGG CATTTCCCAG GCCCCGAAGG ACCCTGTCCA11940ATATGCCAAG CAGCACAACT GAGATCACAC TGTCTGCTCA TCTCGCTTTC CTCCGACCCC12000GAGACTCAGC TACTCTCAAA TTTTCCCTCT CTGAAGGACC ATGTGGACAT TACATTGCTC12060CAGGCCACAG CCACCAGGAC CTAAAACACC ATCACAGCAG CACCAAAGAC ACTGGATAGA12120CCCACAAGGG CAATAGTTTC CTCAACAGTA TATCCAAACT GTTGGGACAA ACGAGCAATC12180ACTGAAGAAG TGACAAGTTC CCACAATGTC AGTGTCCAGC TGAGAAGGGA CAAAAAGTGG12240TACCAGCCCT GTCCACACCA CCTTCTAATT CACAGGAATA CGTGATAGAA GAGGCAGGTT12300GTAGATCCGA AAGATGAGAC AGATTTTATC AACTCCAGAA AGAGCTGGGT CCAACTGAAT12360TATTCTAGCG ACCTTGGCAT TGTCATGACC TGCCATGACC TTCCTCCTTA ACACTTCGAT12420AAACCCTGGG ATATGGAAAA TGCCTGTGTT TCTCAGGGTT TGGGAAAGAA CCATCCATGT12480TGGGATTCTT GTGTAGATCC TCCTTCTGGT CACAGATGCA ATACACTGGA TTTTCAGGCA12540AAGGAGCAAA TTCACAGACA ACTCTGGCCC TACAGCCCTA AGACCTAGAC ACCACCATCT12600CCTTGGAATT ATCAAATTTA ACACCCGGCA CACAACAAAG AAGGACTGGG ACTTTGAGGC12660CTTTGTGTAG CCCTAGAGGG GGCAGAGGCC ACTGAGCAGG GATTGGGTGA TCACAAGGAC12720CTCCTGGAGA GGGACCTGAG GAGCAGGTTC CAATTGGGCC AAAGAAAGAA GAACAACAAT12780AGAGATGAAG GATGCTGGAA AGAGCCATGG TACAGCAGTC TTGTCCTTCA GACATGACTC12840TTACAGCCCA GGACTCTTAC AGTAGCTAGC TGGAGCAGAA GTCCAAGGGA TTACCATGCC12900CTAGGGCCAC AGGCTACTGG AGGGTGGAGT GAGTCTACTA CACAGGTCCA ATGCCTGTTT12960CTCCATTGCT TCTCAGCCAA TGAGAAATCA GAGTCTCCAC CTCCAAGAAA AAGGAAGGTG13020GAAATGAAAG GTGAGCACCT GCCTTCCCGT GACTGGCAGA AAGATCTCCA CGGACTCAAG13080GCTTTGACTT CAAACTTCCC GTAGATCCCT GATGTCTTTA AGGACTCTGT CTGCTTTGTT13140GGTTTTGTTT GTTTGTTTTG TACTTTGTCA ATAAAACATT TTTCAAATAT TCTACAATTG13200CTGTGCTCTT CCTATGCAAA CTGGCCCTGG CACTTCAAAA CATGGCACAG TTAAGTTGAC13260CAGTGGGCCA TGCAGAGCAT ACTACCCCTC CTGGTCCTGT GTCCTACCAG ATATCCTCTA13320AGTGTCCCTT CACTGCTGGG CTCCAGTTCT GCTGCCCTGA ACCACACAGG TCCTCAGTCT13380GTCCTCCCTA TGGGCAGTTT CATCCCAGCC AGAAGCTGCC CTGTGGCCCC TAGGCTGCCC13440AGGCATGGTC TCCCACACCA ACCACACAAA CTAAGAAGCC TGTCCCATAC ATTGACCTCA13500GGCTCAGTGA TAACATTTCT ATAGAAAAAG GAAAGGAATA AGAAAAAAAA AACTCCATAT13560TACATGGGGG CTGCCTACCT TGCTGATGCT TATTGTCTCA AGCAAGTTTG GAGAAGTTCC13620AAATCAGGCA CTGACAGGGT GGGTTCTCAG CCATGCTCTT CTTACCTGGT AGGTGCCTTC13680TCCCCCATGG CACCTCACAG GCTCTCCATC TGTGTGTGTC TGGGTCCTGA TCTCTTCTCA13740TAAGTACAAA GTCAGGCTGG AAGAGGTACA CCCTAGCCCT CATTATAACT TACCAGTTTA13800TGATCCTGTC TGCAAACATC TGAGGTCCCT GTGGCTATAA CTTCGTATAA TGTATGCTAT13860ACGAAGTTAT CTACCGGGTA GGGGAGGCGC TTTTCCCAAG GCAGTCTGGA GCATGCGCTT13920TAGCAGCCCC GCTGGGCACT TGGCGCTACA CAAGTGGCCT CTGGCTCGCA CACATTCCAC13980ATCCACCGGT AGGCGCCAAC CGGCTCCGTT CTTTGGTGGC CCCTTCGCGC CACCTTCTAC14040TCCTCCCCTA GTCAGGAAGT TCCCCCCCGC CCCGCAGCTC GCGTCGTGCA GGACGTGACA14100AATGGAAGTA GCACGTCTCA CTAGTCTCGT GCAGATGGAC AGCACCGCTG AGCAATGGAA14160GCGGGTAGGC CTTTGGGGCA GCGGCCAATA GCAGCTTTGC TCCTTCGCTT TCTGGGCTCA14220GAGGCTGGGA AGGGGTGGGT CCGGGGGCGG GCTCAGGGGC GGGCTCAGGG GCGGGGCGGG14280CGCCCGAAGG TCCTCCGGAG GCCCGGCATT CTGCACGCTT CAAAAGCGCA CGTCTGCCGC14340GCTGTTCTCC TCTTCCTCAT CTCCGGGCCT TTCGACCTGC AGCAGCACGT GTTGACAATT14400AATCATCGGC ATAGTATATC GGCATAGTAT AATACGACAA GGTGAGGAAC TAAACCATGA14460CCGAGTACAA GCCCACGGTG CGCCTCGCCA CCCGCGACGA CGTCCCCCGG GCCGTACGCA14520CCCTCGCCGC CGCGTTCGCC GACTACCCCG CCACGCGCCA CACCGTCGAC CCGGACCGCC14580ACATCGAGCG GGTCACCGAG CTGCAAGAAC TCTTCCTCAC GCGCGTCGGG CTCGACATCG14640GCAAGGTGTG GGTCGCGGAC GACGGCGCCG CGGTGGCGGT CTGGACCACG CCGGAGAGCG14700TCGAAGCGGG GGCGGTGTTC GCCGAGATCG GCCCGCGCAT GGCCGAGTTG AGCGGTTCCC14760GGCTGGCCGC GCAGCAACAG ATGGAAGGCC TCCTGGCGCC GCACCGGCCC AAGGAGCCCG14820CGTGGTTCCT GGCCACCGTC GGCGTCTCGC CCGACCACCA GGGCAAGGGT CTGGGCAGCG14880CCGTCGTGCT CCCCGGAGTG GAGGCGGCCG AGCGCGCCGG GGTGCCCGCC TTCCTGGAGA14940CCTCCGCGCC CCGCAACCTC CCCTTCTACG AGCGGCTCGG CTTCACCGTC ACCGCCGACG15000TCGAGGTGCC CGAAGGACCG CGCACCTGGT GCATGACCCG CAAGCCCGGT GCCTGAGCGG15060GACTCTGGGG TTCGAATAAA GACCGACCAA GCGACGTCTG AGAGCTCCCT GGCGAATTCG15120GTACCAATAA AAGAGCTTTA TTTTCATGAT CTGTGTGTTG GTTTTTGTGT GCGGCGCGCC15180GTTTAAACGC GGATAACTTC GTATAATGTA TGCTATACGA AGTTATAGAC CGACGTGACA15240ACTTCCCAGC CCACTCACAG ACTTTCCTGA GCAATAAATT GATGCAAAAC CACAAATTCC15300TACTCTCAAA AACAAACATT AACAAAGGAT TGGGGGAGGG GGTCAGGGGT AGTATGGTGG15360CGTTGGGCAG GGTAAACTCA GGGTACCCAT TGTCTAATGT CTGAGACATA ACTTGAACAT15420ATGTGTAGCT GCAGCCAAAG ATGAACAAGT GATGGTATTT GTGTCCTCTT CAGACCCGAT15480ACCAGGTCAT TAAGCTTGAG ATCTGGACGC GATCGCCAGC CTCCAGAATG GTGGGAAATA15540AGTTTCTGTT GTTTCTCAGC CACCACGTCT GTAGTATGTG GAAGTCATCA GAATCAAAAT15600TGAGTCACCT GTGGTTTTTT TTTTTTCTAA ATCCCTGACA AATAGAGCCT AGGAAGGCCA15660AGAAGAGAAG AGGGTTCTCA TCCATAAACA CTTGATAACA AAAACTATCA CCAAGGACTC15720TACAAAAACT GCAACTGGCA CAAAGACCAT CACAACCTTA CACAGAAAGT ACTTCTGTGA15780GGACATCTTC CCAGCAACGG GCTGTCCAAC CTCAGACTGG CATTGCCTTT GTTATTGGTC15840CTTGTAGAGA GGGTAATTAT CTCAAAGCAA TCATGTAATC CTCCTCATTT TTCCTTTGAA15900AGCCTTGGTC TCCCTTTGCC TCCCTGAATA CGCACATAGC TGATCATGGC AGGTGTATCC15960CACTGCAGTG CTCTACCTCC AAATAGATAT CGCGGCCGC 15999
<210>2
<211>11862
<212>DNA
<213>小鼠5’-En、Sμ和IgM-dCH1序列(人、鼠序列和合成的序列)
<220>
<221>
<222>(1)..(8)
<223>Not1酶切位点(合成序列)
<222>(9)..(370)
<223>同源臂(人的序列)
<222>(433)..(1444)
<223>鼠IgH 5’-增强子(鼠的序列)
<222>(2550)..(4451)
<223>鼠IgM转换元件(Sμ)(鼠的序列)
<222>(5674)..(6012)
<223>鼠IgM CH2(鼠的序列)
<222>(6292)..(6609)
<223>鼠IgM CH3(鼠的序列)
<222>(6717)..(7111)
<223>鼠IgM CH4(鼠的序列)
<222>(7112)..(7341)
<223>鼠PolyA信号序列(鼠的序列)
<222>(8910)..(9025)
<223>鼠TM1(鼠的序列)
<222>(9144)..(9149)
<223>鼠TM2(鼠的序列)
<222>(9265)..(9556)
<223>鼠PolyA信号序列(鼠的序列)
<222>(9700)..(9733)
<223>Lox(合成序列)
<222>(9734)..(11055)
<223>pGK-Puromycin筛选基因(合成序列)
<222>(11056)(110g9)
<223>Lox(合成序列)
<222>(11090)..(11371)
<223>同源臂(同源重组与鼠Igγ1的Sγ1链接)(鼠的序列)
<222>(11372)..(11379)
<223>AsiSI(合成序列)
<222>(11380)..(11854)
<223>同源臂(同源重组到人Ig C-区序列)(人的序列)
<222>(11855)..(11862)
<223>Not1(合成序列)
GCGGCCGCCG AGATGCCTGA ACAAACCAGG GGTCTTAGTG ATGGCTGAGG AATGTGTCTC60AGGAGCGGTG TCTGTAGGAC TGCAAGATCG CTGCACAGCA GCGAATCGTG AAATATTTTC120TTTAGAATTA TGAGGTGCGC TGTGTGTCAA CCTGCATCTT AAATTCTTTA TTGGCTGGAA180AGAGAACTGT CGGAGTGGGT GAATCCAGCC AGGAGGGACG CGTAGCCCCG GTCTTGATGA240GAGCAGGGTT GGGGGCAGGG GTAGCCCAGA AACGGTGGCT GCCGTCCTGA CAGGGGCTTA300GGGAGGCTCC AGGACCTCAG TGCCTTGAAG CTGGTTTCCA AGAGAAAAGG ATTGTTTATC360TTAGGAGGCA TCTTAGTGAT TGAGTCAAGG GAGAAAGGCA TCTAGCCTCG GTCTCAAAAG420GGTAGTTGCT GTCTAGAGAG GTCTGGTGGA GCCTGCAAAA GTCCAGCTTT CAAAGGAACA480CAGAAGTATG TGTATGGAAT ATTAGAAGAT GTTGCTTTTA CTCTTAAGTT GGTTCCTAGG540AAAAATAGTT AAATACTGTG ACTTTAAAAT GTGAGAGGGT TTTCAAGTAC TCATTTTTTT600AAATGTCCAA AATTCTTGTC AATCAGTTTG AGGTCTTGTT TGTGTAGAAC TGATATTACT660TAAAGTTTAA CCGAGGAATG GGAGTGAGGC TCTCTCATAA CCTATTCAGA ACTGACTTTT720AACAATAATA AATTAAGTTT CAAATATTTT TAAATGAATT GAGCAATGTT GAGTTGGAGT780CAAGATGGCC GATCAGAACC AGAACACCTG CAGCAGCTGG CAGGAAGCAG GTCATGTGGC840AAGGCTATTT GGGGAAGGGA AAATAAAACC ACTAGGTAAA CTTGTAGCTG TGGTTTGAAG900AAGTGGTTTT GAAACACTCT GTCCAGCCCC ACCAAACCGA AAGTCCAGGC TGAGCAAAAC960ACCACCTGGG TAATTTGCAT TTCTAAAATA AGTTGAGGAT TCAGCCGAAA CTGGAGAGGT1020CCTCTTTTAA CTTATTGAGT TCAACCTTTT AATTTTAGCT TGAGTAGTTC TAGTTTCCCC1080AAACTTAAGT TTATCGACTT CTAAAATGTA TTTAGAATTC ATTTTCAAAA TTAGGTTATG1140TAAGAAATTG AAGGACTTTA GTGTCTTTAA TTTCTAATAT ATTTAGAAAA CTTCTTAAAA1200TTACTCTATT ATTCTTCCCT CTGATTATTG GTCTCCATTC AATTCTTTTC CAATACCCGA1260AGCATTTACA GTGACTTTGT TCATGATCTT TTTTAGTTGT TTGTTTTGCC TTACTATTAA1320GACTTTGACA TTCTGGTCAA AACGGCTTCA CAAATCTTTT TCAAGACCAC TTTCTGAGTA1380TTCATTTTAG GAGAAAGACT TTTTTTTTAA ATGAATGCAA TTATCTAGAC TTATTTCAGT1440TGAACATGCT GGTTGGTGGT TGAGAGGACA CTCAGTCAGT CAGTGACGTG AAGGGCTTCT1500AAGCCAGTCC ACATGCTCTG TGTGAACTCC CTCTGGCCCT GCTTATTGTT GAATGGGCCA1560AAGGTCTGAG ACCAGGCTGC TGCTGGGTAG GCCTGGACTT TGGGTCTCCC ACCCAGACCT1620GGGAATGTAT GGTTGTGGCT TCTGCCACCC ATCCACCTGG CTGCTCATGG ACCAGCCAGC1680CTCGGTGGCT TTGAAGGAAC AATTCCACAC AAAGACTCTG GACCTCTCCG AAACCAGGCA1740CCGCAAATGG TAAGCCAGAG GCAGCCACAG CTGTGGCTGC TGCTCTTAAA GCTTGTAAAC1800TGTTTCTGCT TAAGAGGGAC TGAGTCTTCA GTCATTGCTT TAGGGGGAGA AAGAGACATT1860TGTGTGTCTT TTGAGTACCG TTGTCTGGGT CACTCACATT TAACTTTCCT TGAAAAACTA1920GTAAAAGAAA AATGTTGCCT GTTAACCAAT AATCATAGAG CTCATGGTAC TTTGAGGAAA1980TCTTAGAAAG CGTGTATACA ATTGTCTGGA ATTATTTCAG TTAAGTGTAT TAGTTGAGGT2040ACTGATGCTG TCTCTACTTC AGTTATACAT GTGGGTTTGA ATTTTGAATC TATTCTGGCT2100CTTCTTAAGC AGAAAATTTA GATAAAATGG ATACCTCAGT GGTTTTTAAT GGTGGGTTTA2160ATATAGAAGG AATTTAAATT GGAAGCTAAT TTAGAATCAG TAAGGAGGGA CCCAGGCTAA2220GAAGGCAATC CTGGGATTCT GGAAGAAAAG ATGTTTTTAG TTTTTATAGA AAACACTACT2280ACATTCTTGA TCTACAACTC AATGTGGTTT AATGAATTTG AAGTTGCCAG TAAATGTACT2340TCCTGGTTGT TAAAGAATGG TATCAAAGGA CAGTGCTTAG ATCCGAGGTG AGTGTGAGAG2400GACAGGGGCT GGGGTATGGA TACGCAGAAG GAAGGCCACA GCTGTACAGA ATTGAGAAAG2460AATAGAGACC TGCAGTTGAG GCCAGCAGGT CGGCTGGACT AACTCTCCAG CCACAGTAAT2520GACCCAGACA GAGAAAGCCA GACTCATAAA GCTTGCTGAG CAAAATTAAG GGAACAAGGT2580TGAGAGCCCT AGTAAGCGAG GCTCTAAAAA GCACAGCTGA GCTGAGATGG GTGGGCTTCT2640CTGAGTGCTT CTAAAATGCG CTAAACTGAG GTGATTACTC TGAGGTAAGC AAAGCTGGGC2700TTGAGCCAAA ATGAAGTAGA CTGTAATGAA CTGGAATGAG CTGGGCCGCT AAGCTAAACT2760AGGCTGGCTT AACCGAGATG AGCCAAACTG GAATGAACTT CATTAATCTA GGTTGAATAG2820AGCTAAACTC TACTGCCTAC ACTGGACTGT TCTGAGCTGA GATGAGCTGG GGTGAGCTCA2880GCTATGCTAC GCTGTGTTGG GGTGAGCTGA TCTGAAATGA GATACTCTGG AGTAGCTGAG2940ATGGGGTGAG ATGGGGTGAG CTGAGCTGGG CTGAGCTAGA CTGAGCTGAG CTAGGGTGAG3000CTGAGCTGGG TGAGCTGAGC TAAGCTGGGG TGAGCTGAGC TGAGCTTGGC TGAGCTAGGG3060TGAGCTGGGC TGAGCTGGGG TGAGCTGAGC TGAGCTGGGG TAAGCTGGGA TGAGCTGGGG3120TGAGCTGAGC TGAGCTGGAG TGAGCTGAGC TGGGCTGAGC TGGGGTGAGC TGGGCTGAGC3180TGGGCTGAGC TGGGCTGAGC TGGGGTGAGC TGAGCTGGGG TGAGCTGAGC TGAGCTGGGG3240TGAGCTGAGC TGAGCTGGGG TGAGCTGGGG TGAGCTGAGC TGGGGTGAGC TGAGCTGAGC3300TGGGGTGAGC TGAGCTGGGG TGAGCTGAGC TGAGCTGGGG TGAGCTGAGC TGAGCTGAGC3360TGAGCTGAGC TGGGGTGAGC TGAGCTGAGC TGAGCTGGGG TGAGCTGGGG TGAGCTGAGC3420TGAGCTGGAG TGAGCTGAGC TGGGCTGAGC TGGGGTGAGC TGGGCTGAGC TGGGGTGAGC3480TGAGCTGAGC TGAGCTGAGC TGGGGTGAGC TGAGCTGAGC TGGGGTGAGC TGAGCTGGGG3540TGAGCTGGGC TGAGCTGAGC TGAGCTGAGC TGAGCTGAGC TGAGCTGAGC TGAGCTGAGC3600TGAGCTGAGC TGAGCTGAGC TGAGCTGAGC TGAGCTGGGG TGAGCTGAGC TGAGCTGGGC3660TGAGCTGGGG TGAGCTGGGC TGAGCTGGGC TGAGCTGGGC TGAGCTGGGG TGAGCTGAGC3720TGGGGTGAGC TGAGCTGAGC TGGGCTGAGC TGAGCTGAGC TGGGGTGAGC TGAGCTGAGC3780TGGGGTGAGC TGAGCTGAGC TGAGCTGGGG TGAGCTGAGC TGGGCTGAGC AGGGCTGAGC3840TGGGGTGAGC TGAGCTGAGC TGGGGTGAGC TGGGCTGAGC TGGGCTGAGC TGAGCTGAGC3900TGGGCTGAGC TGGGCTGAGC TGGGCTGAGC TGGGCTGAGC TGGGCTGAGC TGGGGTGAGC3960TGAGCTGAGC TGGGGTGAGC TGGGGTGAGC TGAGCTGGGG TGAGCTGAGC TGGGGTGAGC4020TGAGCTGAGC TGGGGTGAGC TGAGCTGGGG TGAGCTGAGC TGAGCTGGGG TGAGCTGAGC4080TGAGCTGGGG TGAGCTGAGC TAGGGTGAAC TGGGCTGGGT GAGCTGGAGT GAGCTGAGCT4140GAGGTGAACT GGGGTGAGCC GGGATGTTTT GAGTTGAGCT GGGGTAAGAT GAGCTGAACT4200GGGGTAAACT GGGATGAGCT GTGGTGAGCG GAGCTGGATT GAACTGAGCT GTGTGAGCTG4260AGCTGGGGTC AGCTGAGCAA GAGTGAGTAG AGCTGGCTGG CCAGAACCAG AATCAATTAG4320GCTAAGTGAG CCAGATTGTG CTGGGATCAG CTGTACTCAG ATGAGCTGGG ATGAGGTAGG4380CTGGGATGAG CTGGGCTAGC TGACATGGAT TATGTGAGGC TGAGCTAGCA TGGGCTGGCC4440TAGCTGATGA GCTAAGCTTG AATGAGCGGG GCTGAGCTGG ACTCAGATGT GCTAGACTGA4500GCTGTACTGG ATGATCTGGT GTAGGGTGAT CTGGACTCAA CTGGGCTGGC TGATGGGATG4560CGCCAGGTTG AACTAGGCTC AGATAAGTTA GGCTGAGTAG GGCCTGGTTG AGATGGTTCG4620GGATGAGCTG GGAAAAGATG GACTCGGACC ATGAACTGGG CTGAGCTGGG TTGGGAGACC4680ATGAATTGAG CTGAACTGAG TGCAGCTGGG ATAAACTGGG TTGAGCTAAG AATAGACTAC4740CTGAATTGTG CCAAACTCGG CTGGGATCAA TTGGAAATTA TCAGGATTTA GATGAGCCGG4800ACTAAACTAT GCTGAGCTGG ACTGGTTGGA TGTGTTGAAC TGGCCTGCTG CTGGGCTGGC4860ATAGCTGAGT TGAACTTAAA TGAGGAAGGC TGAGCAAGGC TAGCCTGCTT GCATAGAGCT4920GAACTTTAGC CTAGCCTGAG CTGGACCAGC CTGAGCTGAG TAGGTCTAAA CTGAGTTAAA4980AATCAACAGG GATAATTTAA CAGCTAATTT AACAAGCCTG AGGTCTGAGA TTGAATGAGC5040AGAGCTGGGA TGAACTGAAT GAGTTTCACC AGGCCTGGAC CAGTTAGGCT AGGACCTCGT5100TCTATAGAGG CAGACTGTGT GCTACAGTGG AGTTTCAAGA TGATTCCATG AGTCCTCCCC5160GCCCCCAACA TAACCCACCT TCCTCCTACC CTACACGCCT GTCTGGTGTG TAAATCCCAG5220CTTTGTGTGC TGATACAGAA GCCTGAGCCC CTCCCCCACC TCCACCTACC TATTACTTTG5280GGATGAGAAT AGTTCTCCCA GCCAGTGTCT CAGAGGGAAG CCAAGCAGGA CAGGCCCAAG5340GCTACTTGAG AAGCCAGGAT CTAGGCCTCT CCCTGAGAAC GGGTGTTCAT GCCCCTAGAG5400TTGGCTGAAG GGCCAGATCC ACCTACTCTA GAGGCATCTC TCCCTGTCTG TGAAGGCTTC5460CAAAGTCACG TTCCTGTGGC TAGAAGGCAG CTCCATAGCC CTGCTGCAGT TTCGTCCTGT5520ATACCAGGTT CACCTACTAC CATATCTAGC CCTGCCTGCC TTAAGAGTAG CAACAAGGAA5580ATAGCAGGGT GTAGAGGGAT CTCCTGTCTG ACAGGAGGCA AGAAGACAGA TTCTTACCCC5640TCCATTTCTC TTTTATCCCT CTCTGGTCCT CAGCTGTCGC AGAGATGAAC CCCAATGTAA5700ATGTGTTCGT CCCACCACGG GATGGCTTCT CTGGCCCTGC ACCACGCAAG TCTAAACTCA5760TCTGCGAGGC CACGAACTTC ACTCCAAAAC CGATCACAGT ATCCTGGCTA AAGGATGGGA5820AGCTCGTGGA ATCTGGCTTC ACCACAGATC CGGTGACCAT CGAGAACAAA GGATCCACAC5880CCCAAACCTA CAAGGTCATA AGCACACTTA CCATCTCTGA AATCGACTGG CTGAACCTGA5940ATGTGTACAC CTGCCGTGTG GATCACAGGG GTCTCACCTT CTTGAAGAAC GTGTCCTCCA6000CATGTGCTGC CAGTGAGTGG CCTGGGCTAA GCCCAATGCC TAGCCCTCCC AGATTAGGGA6060AGTCCTCCTA CAATTATGGC CAATGCCACC CAGACATGGT CATTTGCTCC TTGAACTTTG6120GCTCCCCAGA GTGGCCAAGG ACAAGAATGA GCAATAGGCA GTAGAGGGGT GAGAATCAGC6180TGGAAGGACC AGCATCTTCC CTTAAGTAGG TTTGGGGGAT GGAGACTAAG CTTTTTTCCA6240ACTTCACAAC TAGATATGTC ATAACCTGAC ACAGTGTTCT CTTGACTGCA GGTCCCTCCA6300CAGACATCCT AACCTTCACC ATCCCCCCCT CCTTTGCCGA CATCTTCCTC AGCAAGTCCG6360CTAACCTGAC CTGTCTGGTC TCAAACCTGG CAACCTATGA AACCCTGAAT ATCTCCTGGG6420CTTCTCAAAG TGGTGAACCA CTGGAAACCA AAATTAAAAT CATGGAAAGC CATCCCAATG6480GCACCTTCAG TGCTAAGGGT GTGGCTAGTG TTTGTGTGGA AGACTGGAAT AACAGGAAGG6540AATTTGTGTG TACTGTGACT CACAGGGATC TGCCTTCACC ACAGAAGAAA TTCATCTCAA6600AACCCAATGG TAGGTATCCC CCCTTCCCTT CCCCTCCAAT TGCAGGACCC TTCCTGTACC6660TCATAGGGAG GGCAGGTCCT CTTCCACCCT ATCCTCACTA CTGTCTTCAT TTACAGAGGT6720GCACAAACAT CCACCTGCTG TGTACCTGCT GCCACCAGCT CGTGAGCAAC TGAACCTGAG6780GGAGTCAGCC ACAGTCACCT GCCTGGTGAA GGGCTTCTCT CCTGCAGACA TCAGTGTGCA6840GTGGCTTCAG AGAGGGCAAC TCTTGCCCCA AGAGAAGTAT GTGACCAGTG CCCCGATGCC6900AGAGCCTGGG GCCCCAGGCT TCTACTTTAC CCACAGCATC CTGACTGTGA CAGAGGAGGA6960ATGGAACTCC GGAGAGACCT ATACCTGTGT TGTAGGCCAC GAGGCCCTGC CACACCTGGT7020GACCGAGAGG ACCGTGGACA AGTCCACTGG TAAACCCACA CTGTACAATG TCTCCCTGAT7080CATGTCTGAC ACAGGCGGCA CCTGCTATTG ACCATGCTAG CGCTCAACCA GGCAGGCCCT7140GGGTGTCCAG TTGCTCTGTG TATGCAAACT AACCATGTCA GAGTGAGATG TTGCATTTTA7200TAAAAATTAG AAATAAAAAA AATCCATTCA AACGTCACTG GTTTTGATTA TACAATGCTC7260ATGCCTGCTG AGACAGTTGT GTTTTGCTTG CTCTGCACAC ACCCTGCATA CTTGCCTCCA7320CCCTGGCCCT TCCTCTACCT TGCCAGTTTC CTCCTTGTGT GTGAACTCAG TCAGGCTTAC7380AACAGACAGA GTATGAACAT GCGATTCCTC CAGCTACTTC TAGATATATG GCTGAAAGCT7440TGCCTAACCT GGTGCAGGCA GCATTCAGGC ACATATATAG ACACACATGC ATTTATACAT7500AGATATATAG GTACACATGT GTAGACACAT ACATGAATGT GTATTCATGG ACACACAGAC7560AAAGGTACAC ATATATACAC ATGAGTTCAT GCGCACACAC ATGCATGGAC ACTTACAAAC7620GCCTTCAGAG ACAAATAGGC ATAGACACAC AACCACTCAC AGAAACAGAT ACCAATATGC7680ATGGTCCTGT GTACACAGAA ACAGACTATA GGCAAATATA CACAAATAAA CTATATAGAT7740ACAAAGATAT GCATATACAC ACATGTACAG AAACATCTTC ACATGTGTAC ACTAACATGT7800GAACAGGTAT AGCACACAGA TACACCTGGA CTCTGACCAG GGCTGTAATC TCCAAGGCTC7860ACGGCTCAGA GAGCCTACAC TAGGCTGGGT CACTGATACT CCTCAGGAGC CCACTCTATG7920ATTGGGAGAG ATAACCCCAG GTACAAAGTA TGCCTATCTG TCTCAACACC ATGGGGCAGA7980AGATACTCCA CTAACCACCC ATGACAGAAA GTTAGCCTTG GCTGTGTCTC CATTAATAGA8040ACACCTCAGA AGACCAATGT GAAATTGCCT AACCCACTCA CACCCACCCT GATCTCCAGT8100TCAAAATGCA GAAAACATAA TGCAGTTGTC CAAAAGATGC CCCAACCACA CACACACACA8160CACACACACA CACACACACA CACACACACA CACACACACA CACACACCAT CAAGGAGCCT8220CTGTAAGGAG TCACCACCCA ATAACACTGC CTCTTTGGGC TCATATCCTG GACATTCTTC8280ATATTCATAT CCATTTGGGG CCTAGGCTTT AGATATCCCC AAGGGCTCAT CTTTACAGGG8340ATCAGAGATC CCAATAAATG CCCTGGTCCC ACAGCCTCCC TCAGGTATCT GTCTGTTTAT8400CTCTTGGTAC CAAGACCCAA CATTGCTGGC AGGGGTAGGA CAAGCAACGC ACGGGAACTC8460TGATCAAAGA AAGTCATGAG ATGCCTGAGT CCTTCAGGAA GTAAGGAGGG ACAACCTCTG8520GTATCCCTGT TCTTATTGCT AAAGCCCAAG AGACAGGGAG ACCTGCTCTA AATTCTCAGT8580CTAAACAGCA CCGATGGCAC CACCTGCTCA GGGAAAGTCC AGAGCACACC AATATCATTT8640TGCCACAGTT CCTGAGTCTG CCTTTACCCA GGTCCATACA TTGCATCTGT CTTGCTTGCT8700CTGCTGCCCC AGGGCTCCTG GAACAAAGGC TCCAAATTAG TGTGTCCTAC AGCTTGGCCT8760GTTCTGTGCC TCCGTCTAGC TTGAGCTATT AGGGGACCAG TCAATACTCG CTAAGATTCT8820CCAGAACCAT CAGGGCACCC CAACCCTTAT GCAAATGCTC AGTCACCCCA AGACTTGGCT8880TGACCCTCCC TCTCTGTGTC CCTTCATAGA GGGGGAGGTG AATGCTGAGG AGGAAGGCTT8940TGAGAACCTG TGGACCACTG CCTCCACCTT CATCGTCCTC TTCCTCCTGA GCCTCTTCTA9000CAGCACCACC GTCACCCTGT TCAAGGTAGT GTGGTTGTGG GGCTGAGGAC ACAGGGCTGG9060GACAGGGAGT CACCAGTCCT CACTGCCTCT ACCTCTACTC CCTACAAGTG GACAGCAATT9120CACACTGTCT CTGTCACCTG CAGGTGAAAT GACTCTCAGC ATGGAAGGAC AGCAGAGACC9180AAGAGATCCT CCCACAGGGA CACTACCTCT GGGCCTGGGA TACCTGACTG TATGACTAGT9240AAACTTATTC TTACGTCTTT CCTGTGTTGC CCTCCAGCTT TTATCTCTGA GATGGTCTTC9300TTTCTAGACT GACCAAAGAC TTTTTGTCAA CTTGTACAAT CTGAAGCAAT GTCTGGCCCA9360CAGACAGCTG AGCTGTAAAC AAATGTCACA TGGAAATAAA TACTTTATCT TGTGAACTCA9420CTTTATTGTG AAGGAATTTG TTTTGTTTTT CAAACCTTTC CTGCGGTGTT GACAGCCCAA9480GGATTATCTG AATAGAGCTT AGGAACTGGA AATGGAACAG TGCAGTCTGA TGGTACTTAA9540GGGAGAAAGA GGGAAAGGAG GTGTGGAAGA AGAAAAAAGA GAAGCAGAGG GGGAGGGGAG9600AAGGGAGAGG GAGAGGGAGA GGGAGAGGGA GAGGGAGAGG GAGAGGGAGA GAGAGAGAGA9660GAGAGAGAGA GAGAGAGAGA GAGAGAGAGA GCATGCACTA TAACTTCGTA TAATGTATGC9720TATACGAAGT TATCTACCGG GTAGGGGAGG CGCTTTTCCC AAGGCAGTCT GGAGCATGCG9780CTTTAGCAGC CCCGCTGGGC ACTTGGCGCT ACACAAGTGG CCTCTGGCTC GCACACATTC9840CACATCCACC GGTAGGCGCC AACCGGCTCC GTTCTTTGGT GGCCCCTTCG CGCCACCTTC9900TACTCCTCCC CTAGTCAGGA AGTTCCCCCC CGCCCCGCAG CTCGCGTCGT GCAGGACGTG9960ACAAATGGAA GTAGCACGTC TCACTAGTCT CGTGCAGATG GACAGCACCG CTGAGCAATG10020GAAGCGGGTA GGCCTTTGGG GCAGCGGCCA ATAGCAGCTT TGCTCCTTCG CTTTCTGGGC10080TCAGAGGCTG GGAAGGGGTG GGTCCGGGGG CGGGCTCAGG GGCGGGCTCA GGGGCGGGGC10140GGGCGCCCGA AGGTCCTCCG GAGGCCCGGC ATTCTGCACG CTTCAAAAGC GCACGTCTGC10200CGCGCTGTTC TCCTCTTCCT CATCTCCGGG CCTTTCGACC TGCAGCAGCA CGTGTTGACA10260ATTAATCATC GGCATAGTAT ATCGGCATAG TATAATACGA CAAGGTGAGG AACTAAACCA10320TGACCGAGTA CAAGCCCACG GTGCGCCTCG CCACCCGCGA CGACGTCCCC CGGGCCGTAC10380GCACCCTCGC CGCCGCGTTC GCCGACTACC CCGCCACGCG CCACACCGTC GACCCGGACC10440GCCACATCGA GCGGGTCACC GAGCTGCAAG AACTCTTCCT CACGCGCGTC GGGCTCGACA10500TCGGCAAGGT GTGGGTCGCG GACGACGGCG CCGCGGTGGC GGTCTGGACC ACGCCGGAGA10560GCGTCGAAGC GGGGGCGGTG TTCGCCGAGA TCGGCCCGCG CATGGCCGAG TTGAGCGGTT10620CCCGGCTGGC CGCGCAGCAA CAGATGGAAG GCCTCCTGGC GCCGCACCGG CCCAAGGAGC10680CCGCGTGGTT CCTGGCCACC GTCGGCGTCT CGCCCGACCA CCAGGGCAAG GGTCTGGGCA10740GCGCCGTCGT GCTCCCCGGA GTGGAGGCGG CCGAGCGCGC CGGGGTGCCC GCCTTCCTGG10800AGACCTCCGC GCCCCGCAAC CTCCCCTTCT ACGAGCGGCT CGGCTTCACC GTCACCGCCG10860ACGTCGAGGT GCCCGAAGGA CCGCGCACCT GGTGCATGAC CCGCAAGCCC GGTGCCTGAG10920CGGGACTCTG GGGTTCGAAT AAAGACCGAC CAAGCGACGT CTGAGAGCTC CCTGGCGAAT10980TCGGTACCAA TAAAAGAGCT TTATTTTCAT GATCTGTGTG TTGGTTTTTG TGTGCGGCGC11040GCCGTTTAAA CGCGGATAAC TTCGTATAAT GTATGCTATA CGAAGTTATA TGACATCTTG11100TTGTGGAATA GCAAACAGAG AAGACTGTTT GGACATCGAT TTAAGGCAAT AGAAAGTCTT11160TACTAGCCAA CCTGTGACTA CACTGGGTGT TCAGGATCTC AGTGTAGCCC TGAACCTTTC11220TCAGGGTGAG ATTTTAGGTA AAAATTATGT TCTGGGTTGA CATAGTTCAG TTAACAAGAA11280CACTTAGCCA GAAACAGAAC TACAAGACCC AAAAAGCAAA GTTAAAACAT TTACAAACTT11340TCCTAGAACT GTATGAACTT TAATGGATTA GGCGATCGCC AGCCTCCAGA ATGGTGGGAA11400ATAAGTTTCT GTTGTTTCTC AGCCACCACG TCTGTAGTAT GTGGAAGTCA TCAGAATCAA11460AATTGAGTCA CCTGTGGTTT TTTTTTTTTC TAAATCCCTG ACAAATAGAG CCTAGGAAGG11520CCAAGAAGAG AAGAGGGTTC TCATCCATAA ACACTTGATA ACAAAAACTA TCACCAAGGA11580CTCTACAAAA ACTGCAACTG GCACAAAGAC CATCACAACC TTACACAGAA AGTACTTCTG11640TGAGGACATC TTCCCAGCAA CGGGCTGTCC AACCTCAGAC TGGCATTGCC TTTGTTATTG11700GTCCTTGTAG AGAGGGTAAT TATCTCAAAG CAATCATGTA ATCCTCCTCA TTTTTCCTTT11760GAAAGCCTTG GTCTCCCTTT GCCTCCCTGA ATACGCACAT AGCTGATCAT GGCAGGTGTA11820TCCCACTGCA GTGCTCTACC TCCAAATAGA TATCGCGGCC GC 11862
<210>3
<211>1280
<212>DNA
<213>人Igγ1的Hinge、CH2和CH3序列以及小鼠Igγ1的同源臂【人和鼠序列(Swa1酶切片段)】
<220>
<221>
<222>(1)..(131)
<223>同源臂(同源重组与鼠Igγ1的5’-端)(鼠的序列)
<222>(132)..(176)
<223>人Igγ1Hinge(人的序列)
<222>(295)..(625)
<223>人Igγ1CH2(人的序列)
<222>(722)..(1044)
<223>人1gγ1CH3(人的序列)
<222>(1045)..(1280)
<223>同源臂(同源重组与鼠Igγ1的3’-端)(鼠的序列)
AAATGTTGGA TATCACCTAC TCCATGTAGA GAGTCGGGGA CATGGGAAGG GTGCAAAAGA60GCGGCCTTCT AGAAGGTTTG GTCCTGTCCT GTCCTGTCTG ACAGTGTAAT CACATATACT120TTTTCTTGTA GAGCCCAAAT CTTGTGACAA AACTCACACA TGCCCACCGT GCCCAGGTAA180GCCAGCCCAG GCCTCGCCCT CCAGCTCAAG GCGGGACAGG TGCCCTAGAG TAGCCTGCAT240CCAGGGACAG GCCCCAGCCG GGTGCTGACA CGTCCACCTC CATCTCTTCC TCAGCACCTG300AACTCCTGGG GGGACCGTCA GTCTTCCTCT TCCCCCCAAA ACCCAAGGAC ACCCTCATGA360TCTCCCGGAC CCCTGAGGTC ACATGCGTGG TGGTGGACGT GAGCCACGAA GACCCTGAGG420TCAAGTTCAA CTGGTACGTG GACGGCGTGG AGGTGCATAA TGCCAAGACA AAGCCGCGGG480AGGAGCAGTA CAACAGCACG TACCGTGTGG TCAGCGTCCT CACCGTCCTG CACCAGGACT540GGCTGAATGG CAAGGAGTAC AAGTGCAAGG TCTCCAACAA AGCCCTCCCA GCCCCCATCG600AGAAAACCAT CTCCAAAGCC AAAGGTGGGA CCCGTGGGGT GCGAGGGCCA CATGGACAGA660GGCCGGCTCG GCCCACCCTC TGCCCTGAGA GTGACCGCTG TACCAACCTC TGTCCCTACA720GGGCAGCCCC GAGAACCACA GGTGTACACC CTGCCCCCAT CCCGGGATGA GCTGACCAAG780AACCAGGTCA GCCTGACCTG CCTGGTCAAA GGCTTCTATC CCAGCGACAT CGCCGTGGAG840TGGGAGAGCA ATGGGCAGCC GGAGAACAAC TACAAGACCA CGCCTCCCGT GCTGGACTCC900GACGGCTCCT TCTTCCTCTA CAGCAAGCTC ACCGTGGACA AGAGCAGGTG GCAGCAGGGG960AACGTCTTCT CATGCTCCGT GATGCATGAG GCTCTGCACA ACCACTACAC ACAGAAGAGC1020CTCTCCCTGT CTCCGGGTAA ATGATCCCAG TGTCCTTGGA GCCCTCTGGT CCTACAGGAC1080TCTGACACCT ACCTCCACCC CTCCCTGTGT AAATAAAGCA CCCAGCACTG CCTTGGGACC1140CTGCAATAAT GTCCTGGTGA TTTCTGAGAT GTAGAGTCTA GCTAGGTCAT GGAATGAGGG1200GTCTCCATGG TTTGAGGCCT GAGTTGTGAC TAAGGAAAAA CCCATAGGCC TACACTGCCA1260CACCCAGCAC TTTTGAATTT 1280
Claims (22)
1.一种核酸分子,包括了免疫球蛋白基因或其片段,其特征在于:包括IgG基因(Igγ)和IgG转换元件(Sγ)和/或IgM基因(IgHCμ)和IgM转换元件(Sμ);其中IgM基因/IgG基因缺失CH1功能。
2.如权利要求1所述的核酸分子,所述Sμ、Sγ、IgHCμ为宿主动物序列。
3.如权利要求1或2所述的核酸分子,所述IgHCμ包括宿主动物IgM的CH2、CH3、CH4外显子及之间的序列,以及TM1、TM2、polyA信号。
4.如权利要求1-3任一所述的核酸分子,所述IgHCμ结构如图3-1所示。
5. 如权利要求1-4任一所述的核酸分子, 所述Igγ包括Hinge、CH2、CH3外显子及之间的序列,以及TM1、TM2、polyA信号。
6.如权利要求1-5任一所述的核酸分子,所述TM1、TM2、polyA信号为宿主动物序列。
7.如权利要求1-6任一所述的核酸分子,所述Igγ结构如图1-1所示。
8.如权利要求1-7任一所述的核酸分子,包括宿主动物IgH重链的5'-端的增强子(5’-Enhancer)。
9.如权利要求1-8任一所述的核酸分子,所述Sγ为Sγ1、Sγ3、Sγ2a和/或Sγ2b。
10. 如权利要求1-9任一所述的核酸分子,所述Sμ的核苷酸序列如SEQ ID NO.2(2550)..(4451)所示。
11. 如权利要求1-10任一所述的核酸分子,所述增强子的核苷酸序列如SEQ ID NO.2的(433)..(1444)所示。
12.如权利要求1-11任一所述的核酸分子,IgG基因为宿主动物/人IgG嵌合元件或全人IgG序列。
13.如权利要求12所述的核酸分子,所述Igγ序列包括人Igγ的单种或多种亚型,和宿主动物的格种Igγ亚型的位置转换元件(Sγ)。
14.如权利要求13所述的核酸分子,人Igγ亚型包括Igγ3、Igγ1、Igγ2和/或Igγ4。
15. 如权利要求1-14任一所述的核酸分子,包括人或宿主动物IgH重链的3’-位置表达调控序列(Local Control Region)。
16. 如权利要求1-14任一所述的核酸分子,包括了人IgH重链 V-区序列或片段,人IgHD-区序列或片段,和人IgH J-区序列或片段。
17.如权利要求1-16所述的核酸分子,其结构组成如图6或图7所示。
18.一种载体,包含如权利要求1-17所述的核酸分子。
19.一种细胞,包含如权利要求1-17任一所述核酸分子或权利要求18所述载体。
20.一种人源抗体,来源于权利要求1-17任一核酸分子或权利要求18所述载体或或权利要求19所述细胞。
21.权利要求1-17任一所述核酸分子或权利要求18所述载体或权利要求19所述细胞在编码DNA、cDNA、mRNA,表达氨基酸序列、蛋白质、载体,培养杂交瘤、细胞株、转基因动物和/或制备人源单域抗体中的应用。
22.采用权利要求1-17任一所述核酸分子或权利要求17所述载体或权利要求18所述细胞制备转基因动物的方法,包括以下步骤:
(1)所述核酸分子的获得;
(2)将所述核酸分子构建入载体;
(3)向宿主动物细胞或胚胎导入所述载体;
(4)将含有上述载体的细胞植入宿主动物的胚胎内或体细胞克隆;
(5)繁殖杂合、纯合的转基因动物。
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CN201810261005.8A CN108486125B (zh) | 2018-03-27 | 2018-03-27 | 一种核酸分子及其在制备人源单域抗体中的应用 |
PCT/CN2018/083162 WO2019184014A1 (zh) | 2018-03-27 | 2018-04-16 | 一种核酸分子及其在制备人源单域抗体中的应用 |
JP2020564296A JP2021512650A (ja) | 2018-03-27 | 2018-04-16 | 核酸分子およびヒト化型シングルドメイン抗体調製における核酸分子の応用 |
US16/982,602 US20210017253A1 (en) | 2018-03-27 | 2018-04-16 | Nucleic acid molecules and applications thereof in preparing human single-domain antibody |
AU2018416756A AU2018416756A1 (en) | 2018-03-27 | 2018-04-16 | Nucleic acid molecule and use thereof in preparing humanized single-domain antibody |
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108486126A (zh) * | 2018-03-27 | 2018-09-04 | 重庆金迈博生物科技有限公司 | 一种核酸分子及其在人源化抗体中的应用 |
WO2022048604A1 (zh) * | 2020-09-04 | 2022-03-10 | 北京仁源欣生生物科技有限公司 | 一种非人哺乳动物或其子代的制备方法及其应用 |
CN117587070A (zh) * | 2024-01-19 | 2024-02-23 | 北京仁源欣生生物科技有限公司 | 一种经遗传修饰的非人哺乳动物的制备方法及应用 |
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IL303529A (en) | 2020-12-09 | 2023-08-01 | Trianni Inc | Heavy chain antibodies only |
Citations (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5633425A (en) * | 1990-08-29 | 1997-05-27 | Genpharm International, Inc. | Transgenic non-human animals capable of producing heterologous antibodies |
EP2003960A2 (en) * | 2006-03-31 | 2008-12-24 | Medarex, Inc. | Transgenic animals expressing chimeric antibodies for use in preparing human antibodies |
WO2009143472A2 (en) * | 2008-05-23 | 2009-11-26 | Aliva Biopharmaceuticals, Inc. | Method of generating single vl domain antibodies in transgenic animals |
US20120204278A1 (en) * | 2009-07-08 | 2012-08-09 | Kymab Limited | Animal models and therapeutic molecules |
US20130167256A1 (en) * | 2010-03-31 | 2013-06-27 | Ablexis Llc | Genetic engineering of non-human animals for the production of chimeric antibodies |
EP2820947A1 (en) * | 2013-07-05 | 2015-01-07 | B Cell Design | Transgenic non-human mammal for producing chimeric human immunoglobulin E antibodies |
CN104755493A (zh) * | 2012-08-03 | 2015-07-01 | Sab有限责任公司 | 用于在转基因动物中生产人类抗体的复杂的染色体工程 |
CN105274116A (zh) * | 2015-10-21 | 2016-01-27 | 重庆市畜牧科学院 | 一种制备人源化抗体的核酸分子及其应用 |
CN105441455A (zh) * | 2015-10-21 | 2016-03-30 | 重庆市畜牧科学院 | 一种嵌合核酸分子及其在人源化抗体制备中的应用 |
CN105695415A (zh) * | 2010-06-17 | 2016-06-22 | 科马布有限公司 | 动物模型及治疗分子 |
CN105777894A (zh) * | 2016-03-12 | 2016-07-20 | 长春力太生物技术有限公司 | 通过转基因啮齿类动物制备人源化驼类单域抗体的方法 |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU2007219159B8 (en) * | 2006-01-25 | 2012-06-28 | Roger Kingdon Craig | Generation of heavy-chain only antibodies in transgenic animals |
GB0618345D0 (en) * | 2006-09-18 | 2006-10-25 | Univ Erasmus | Binding molecules |
WO2014141192A1 (en) * | 2013-03-15 | 2014-09-18 | Erasmus University Medical Center | Generation of heavy chain-only antibodies |
US11547099B2 (en) * | 2014-10-22 | 2023-01-10 | Crescendo Biologies Limited | Transgenic mice |
GB201500464D0 (en) * | 2015-01-12 | 2015-02-25 | Crescendo Biolog Ltd | Method of producing optimised therapeutic molecules |
US11578118B2 (en) * | 2017-10-20 | 2023-02-14 | Fred Hutchinson Cancer Center | Systems and methods to produce B cells genetically modified to express selected antibodies |
-
2018
- 2018-03-27 CN CN201810261005.8A patent/CN108486125B/zh active Active
- 2018-04-16 JP JP2020564296A patent/JP2021512650A/ja active Pending
- 2018-04-16 US US16/982,602 patent/US20210017253A1/en active Pending
- 2018-04-16 EP EP18911822.7A patent/EP3770261B1/en active Active
- 2018-04-16 AU AU2018416756A patent/AU2018416756A1/en not_active Abandoned
- 2018-04-16 WO PCT/CN2018/083162 patent/WO2019184014A1/zh unknown
Patent Citations (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5633425A (en) * | 1990-08-29 | 1997-05-27 | Genpharm International, Inc. | Transgenic non-human animals capable of producing heterologous antibodies |
EP2003960A2 (en) * | 2006-03-31 | 2008-12-24 | Medarex, Inc. | Transgenic animals expressing chimeric antibodies for use in preparing human antibodies |
WO2009143472A2 (en) * | 2008-05-23 | 2009-11-26 | Aliva Biopharmaceuticals, Inc. | Method of generating single vl domain antibodies in transgenic animals |
US20120204278A1 (en) * | 2009-07-08 | 2012-08-09 | Kymab Limited | Animal models and therapeutic molecules |
US20130167256A1 (en) * | 2010-03-31 | 2013-06-27 | Ablexis Llc | Genetic engineering of non-human animals for the production of chimeric antibodies |
CN105695415A (zh) * | 2010-06-17 | 2016-06-22 | 科马布有限公司 | 动物模型及治疗分子 |
CN104755493A (zh) * | 2012-08-03 | 2015-07-01 | Sab有限责任公司 | 用于在转基因动物中生产人类抗体的复杂的染色体工程 |
EP2820947A1 (en) * | 2013-07-05 | 2015-01-07 | B Cell Design | Transgenic non-human mammal for producing chimeric human immunoglobulin E antibodies |
CN105274116A (zh) * | 2015-10-21 | 2016-01-27 | 重庆市畜牧科学院 | 一种制备人源化抗体的核酸分子及其应用 |
CN105441455A (zh) * | 2015-10-21 | 2016-03-30 | 重庆市畜牧科学院 | 一种嵌合核酸分子及其在人源化抗体制备中的应用 |
CN105777894A (zh) * | 2016-03-12 | 2016-07-20 | 长春力太生物技术有限公司 | 通过转基因啮齿类动物制备人源化驼类单域抗体的方法 |
Non-Patent Citations (6)
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108486126A (zh) * | 2018-03-27 | 2018-09-04 | 重庆金迈博生物科技有限公司 | 一种核酸分子及其在人源化抗体中的应用 |
WO2022048604A1 (zh) * | 2020-09-04 | 2022-03-10 | 北京仁源欣生生物科技有限公司 | 一种非人哺乳动物或其子代的制备方法及其应用 |
CN117587070A (zh) * | 2024-01-19 | 2024-02-23 | 北京仁源欣生生物科技有限公司 | 一种经遗传修饰的非人哺乳动物的制备方法及应用 |
CN117587070B (zh) * | 2024-01-19 | 2024-04-30 | 北京仁源欣生生物科技有限公司 | 一种经遗传修饰的非人哺乳动物的制备方法及应用 |
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AU2018416756A1 (en) | 2020-10-15 |
JP2021512650A (ja) | 2021-05-20 |
EP3770261A1 (en) | 2021-01-27 |
EP3770261A4 (en) | 2021-04-14 |
US20210017253A1 (en) | 2021-01-21 |
WO2019184014A1 (zh) | 2019-10-03 |
EP3770261B1 (en) | 2024-03-27 |
CN108486125B (zh) | 2024-01-05 |
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