CN1310758A - 基因克隆的新方法 - Google Patents
基因克隆的新方法 Download PDFInfo
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Abstract
本发明涉及用于分离膜结合蛋白的编码基因的方法,其以将一种功能性蛋白与一种融合蛋白本身相融合为特征,而且与现有的在融合蛋白中载有被抗体识别的表位的TMT方法不同。该方法使选择性分离膜结合蛋白的编码基因成为可能。
Description
技术领域
本发明涉及一种选择性而且有效地分离膜结合蛋白的编码基因的新型基因克隆方法。
背景技术
细胞中合成的蛋白质根据其各自特征可以被分成定位于细胞内细胞器如细胞核、线粒体、细胞质等中的那些蛋白;通过结合至细胞膜而发挥功能的那些蛋白,例如受体和通道分子;以及通过分泌至细胞外而发挥功能的那些蛋白,例如生长因子和细胞因子等。特别是,结合至细胞膜的蛋白质分子负责重要的生物学功能,例如对生长因子和分化因子的细胞反应、炎症反应、细胞-细胞相互作用、激素反应等等,因此这些分子可以成为各类疾病的诊断和治疗药物的靶分子。
近年来,如以基因组计划为代表,实施了利用随机方法的大规模基因克隆方法,而且堆积了大量的基因序列信息例如大量的EST(表达序列标签)(Matsubara,K.人造器官(Artificial Organs)(1996)20,823-827)。然而,通过这些EST鉴定具有预期功能的蛋白质决非易事,而且为了从基因序列信息预测和分析所编码蛋白的功能,需要大量的时间和努力。因此,长期以来人们期待着一种在随机cDNA克隆阶段筛选,或者至少在某种程度上筛选预计具有预期功能的蛋白的编码基因的方法。
利用蛋白质定位进行的克隆方法被发展成为对此类问题的一个解决方法。例如,分泌至细胞外的蛋白质具有对分泌至关重要的包括15到30个左右氨基酸残基的氨基酸序列,其通常被称为分泌信号序列或引导序列。
Tashiro,K.等关注于该分泌性蛋白合成的特征,而且发展了一种特异性筛选分泌性蛋白的编码基因的克隆方法(Tashiro,K.等,科学(Science)(1993)261,300-603)。当将正常分泌至细胞外的蛋白质如白细胞介素-2(IL-2)受体的信号序列删除时,它们不能表达于细胞膜上。如果融合有编码分泌信号序列的cDNA,则该IL-2受体能够作为一种融合蛋白而被重新表达于细胞膜上。由于用识别IL-2受体的抗体可以筛选表达IL-2受体融合蛋白的细胞,因此可以分离对引入细胞的信号序列已发挥功能的蛋白进行编码的cDNA。该方法因其选择性地克隆信号序列的编码基因而通常被称为SST(信号序列捕获)方法。通过基本上相同的原理还发展了一种针对酵母的克隆方法(美国专利号5,536,637)。
然而,即使通过该方法获得了编码一种包括信号序列的蛋白的基因片段,人们还是无法知道其是否为一种分泌性蛋白,或者其是否为一种膜结合蛋白。同样,该方法需要利用包括5’末端的cDNA文库,而用于有效构建一个选择性地包括5’末端的cDNA文库的技术不一定是容易而通用的技术。
最近,Ishihara等和Nakauchi等报道了TMT(跨膜捕获)方法,其更选择性地克隆一个编码膜结合蛋白的基因(Yoshikazu Ichihara和YoshikazuKurozawa,日本分子生物学会年会摘要(1998),No.3-509-P-533,Nakauchi等,WO98/03645)。Ichihara等的方法基于同上述SST方法相反的原理。换句话说,将IL-2受体的胞外区和含有由cDNA所编码的细胞膜结合区的一种蛋白融合,IL-2受体表达于细胞膜表面,而利用抗IL-2受体的抗体对细胞进行筛选。该方法的模式实验利用抗IL-2受体的抗体证实了Ⅰ型或Ⅱ型膜结合蛋白或者糖基磷脂酰肌醇(GPI)锚定型膜结合蛋白与IL-2受体的融合分子在细胞膜上的表达。
然而,当引入cDNA文库时,在所选的cDNA中还包括不含跨膜区和膜结合区的蛋白。换句话说,对该TMT方法获得的膜结合蛋白编码基因的克隆选择性不一定高。这表明,例如虽然理论上所有不具有跨膜区和GPI锚的融合蛋白可以被分泌,但根据融合蛋白的结构和氨基酸组成,并非由跨膜区和GPI锚所致的非特异性凝集也可以发生于细胞膜上。
此外,在该TMT方法的情况下,被抗体识别的表位表达于融合蛋白中。因此,即使以上述方式表达的融合蛋白被非特异性地吸收于细胞膜上,只要该表位暴露,该抗体将识别并结合该表位。同样,细胞膜表面那些将被分泌至细胞外的分子也可以被该抗体识别。因此,希望进一步提高对该TMT方法所获表达膜结合蛋白之细胞的选择性。
发明内容
本发明解决了TMT方法的问题并提供了一种具有更高选择性的基因克隆方法。
本发明的一个特征为通过将功能性蛋白连接至融合蛋白本身来分离编码膜结合蛋白的基因,其与携带识别抗体的表位的传统TMT方法不同。因此,本方法能够选择性分离编码膜结合蛋白的基因。
换言之,本发明提供:
(1)用于分离膜结合蛋白的编码基因的一种方法,该方法包括以下步骤:
(ⅰ)将包括对一种抗原具有结合亲和力的可分泌性功能性蛋白的编码DNA以及连接于该功能性蛋白编码DNA 3’侧下游的cDNA的载体引入细胞,
(ⅱ)对抗原具有结合亲和力的可分泌性功能性蛋白与所述cDNA所编码蛋白的融合蛋白在细胞内表达,
(ⅲ)通过将在细胞膜上表达该融合蛋白的细胞与抗原相接触,筛选结合该抗原的细胞,并且
(ⅳ)从所筛选的细胞中分离插入到所述载体中的cDNA,
(2)如(1)所述的方法,其中通过在载体上功能性蛋白编码DNA 3’侧下游的限制性酶切位点处引入cDNA来获得在步骤(ⅰ)中引入细胞的载体,
(3)如(1)所述的方法,其中通过将包括编码一功能性蛋白的DNA和连接于该功能性蛋白编码DNA 3’侧下游的cDNA的DNA引入载体来获得在步骤(ⅰ)中引入细胞的载体,
(4)如(1)至(3)中任何一个所述的方法,其中通过编码肽连接序列的DNA使编码所述功能性蛋白的DNA和其3’侧下游的cDNA连接,
(5)如(1)至(4)中任何一个所述的方法,其中所述cDNA源于哺乳动物细胞的cDNA文库。
(6)如(1)至(5)中任何一个所述的方法,其中在步骤(ⅰ)中引入细胞的载体在编码功能性蛋白的DNA 5’侧的上游包括编码分泌信号序列的DNA,
(7)如(1)至(6)中任何一个所述的方法,其中该功能性蛋白为一种抗体,
(8)如(1)至(7)中任何一个所述的方法,其中对抗原具有结合亲和力的功能性蛋白为一种单链抗体,其优选为单价或二价,
(9)如(1)至(8)中任何一个所述的方法,其中该载体包括一种DNA,在该DNA中编码所述抗体恒定区的DNA连接于编码一种单链抗体的DNA3’侧的下游,
(10)如(1)至(9)中任何一个所述的方法,其中将抗原结合于一种支持物,
(11)如(10)所述的方法,其中支持物用于细胞培养,
(12)如(1)至(11)中任何一个所述的方法,其包括确定从细胞获得的基因是否包含新的序列,
(13)如(12)所述的方法,其包括筛选cDNA文库以获得由细胞所获基因的全长基因,该基因包括一新的序列,
(14)如(13)所述的方法,其包括分离由细胞所获基因的全长基因,该基因包括一新的序列,
(15)用于分离膜结合蛋白的编码基因的试剂盒,该试剂盒包括具有用于在编码对一种抗原具有结合亲和力的可分泌性功能蛋白的DNA 3’侧下游插入cDNA的限制性酶识别位点的载体,以及
(16)如(15)所述的试剂盒进一步还包括结合抗原的支持物和/或待引入载体的细胞。
通过本发明的方法可分离的膜结合蛋白,可如Ⅰ型或Ⅱ型膜结合蛋白以及GPI锚定型膜结合蛋白等。Ⅰ型或Ⅱ型膜结合蛋白为包括跨膜区的蛋白,而且在由所表达多肽的N末端侧或C末端侧分泌至细胞外后结合于细胞膜。跨膜区为穿透细胞膜内侧和外侧的区域,而且因为该跨膜区保持在细胞膜内,所以蛋白质以固定于细胞膜上的形式存在。跨膜区通常由蛋白质的氨基酸序列内富含疏水性氨基酸残基的区域组成。例如,一种市售的计算机程序GCG序列分析软件包(Genetic Computer Group,Oxford MolecularGroup,Inc.)可以很容易地预测蛋白质是否具有跨膜区。GPI锚定型膜结合蛋白为经过GPI的修饰并通过GPI锚定于细胞膜脂质层的蛋白质(GPI锚定型膜结合蛋白)。
在本发明分离方法的第一步((ⅰ))中,将包括编码对一种抗原具有结合亲和力的可分泌性功能蛋白的DNA以及连接于功能性蛋白编码DNA 3’侧下游的cDNA的载体引入细胞。
“对一种抗原具有结合亲和力的功能性蛋白”意指功能上能够结合某种抗原的蛋白。作为功能性蛋白,优选与抗原的结合常数为107M或更高。更优选108M或更高,还更优选109M或更高。功能性蛋白特别为抗体、抗体片段、单链抗体等。抗体包括两条重链(H链)和两条轻链(L链),这些H链和L链通过二硫键结合形成单个抗体分子。H链和L链均由一个可变区(V区,Fv)和一个恒定区(C区,Fc)组成。抗体片段为抗体中对抗原具有结合亲和力的部分蛋白,例如可以为Fab、F(ab’)2、Fv等。单链抗体(此后称为单链Fv(scFv))为对抗原具有结合亲和力的蛋白,在该蛋白中通过一个连接序列将H链Fv和L链Fv相连,如单价单链抗体和二价单链抗体。单价单链抗体具有包括一个H链Fv和L链Fv的抗原结合位点,而二价单链抗体具有通过一个连接序列将两个单价单链抗体分子相连的结构,因而具有两个抗原结合位点。
为了各种目的,例如改善结合常数,可以将抗体、抗体片段或单链抗体删除、插入和/或用其它氨基酸残基置换一个或多个氨基酸残基,或者将其与其它肽或多肽融合,而这些都包括在本发明的功能性蛋白之中。同样,修饰的抗体可以用作抗体、抗体片段或单链抗体。修饰抗体的实例为嵌合抗体和人源化抗体。嵌合抗体包括源自不同动物抗体的V区和C区。人源化抗体包括源自动物而并非人的抗体的互补决定区(CDR)以及源自人的抗体的框架区(FR)和C区。
对本发明的功能性蛋白具有结合亲和力的抗原可以是任何物质,只要其具有抗原性。实例为蛋白质、肽和糖等,优选为蛋白质。用作抗原的蛋白质如细胞或微生物表达的蛋白、血清蛋白、细胞因子、细胞内蛋白、膜蛋白等。
通过众所周知的方法可以获得编码抗体的DNA。换言之,它们可以分离自产生抗体的细胞,例如杂交瘤、抗原致敏的永生化淋巴细胞以及在引入一种抗体基因后产生重组抗体的细胞。此外,也可以利用已经分离并插入到载体中的DNA。只要能够用于本发明,编码抗体的DNA的来源和类型是无须讨论的。
通过下面通常采用的方法可以由编码抗体的DNA构建编码抗体片段或单链抗体的DNA。通过连接编码抗体H链V区(H链Fv)、编码连接序列以及编码L链V区(L链Fv)的DNA获得编码单价单链抗体的DNA。连接序列不限,只要其能够在空间上重建H链Fv和L链Fv以使它们具有抗原亲和力。其优选为肽连接序列,例如包括12至19个氨基酸残基(Huston,J.S.等,美国国家科学院院报(1988)85,5879-5883)。特别为具有下述氨基酸序列的肽连接序列:GlyGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer((Gly4Ser)3)(SEQ ID NO:1)。通过利用编码一种肽连接序列的DNA连接两个编码单价单链抗体的DNA分子的5’末端和3’末端构建编码二价单链抗体的DNA。连接两个单链抗体的肽连接序列包括,例如GlyGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer((Gly4Ser)3)(SEQ ID NO:1)的氨基酸序列。
在本发明中为了提高克隆效率,例如当利用单链Fv作为功能蛋白时,优选C末端含有少量的疏水性氨基酸,特别是可以利用如在下面实施例中所述的转角(elbow)区已被删除的单链Fv。同样,在本发明中还优选通过在单链Fv的C末端进一步连接一个分泌性蛋白来源的结构域,例如编码下面实施例中所述抗体恒定区氨基酸的DNA,以增加稳定性和表达效率。
分泌功能性蛋白,可以利用分泌信号序列。换言之,在编码对一种抗原具有结合亲和力的功能性蛋白的DNA 5’侧上游连接一个编码分泌信号序列的DNA便足够了。作为分泌信号序列,采用对用于cDAN文库表达和蛋白分泌的细胞适宜的分泌信号序列。分泌信号序列可以为任何分泌性蛋白的信号序列,只要其能够分泌该功能性蛋白。优选的动物来源的分泌信号序列为那些来自哺乳动物的分泌信号序列,例如人免疫球蛋白(Kabat,E.等,免疫学重要蛋白的序列,US Department of Health and HumanServices(1991))、细胞因子以及细胞因子受体的信号序列。
连接至编码功能性蛋白的DNA 3’侧下游的cDNA优选来自cDNA文库。cDNA文库可使用经众所周知的方法获得的文库或者市售的文库。通过从目的样品中分离mRNA并由所分离的mRNA合成cDNA可以制备cDNA文库。
可使用能从中分离mRNA的来源如哺乳动物、除哺乳动物外的动物、植物、酵母、细菌或蓝绿藻,优选哺乳动物。哺乳动物的实例包括人、猴、兔、大鼠、小鼠等,特别优选人。除哺乳动物外的动物为例如果蝇(Drosophila)等昆虫。
可以从其中分离mRNA的样品来源可以是任何来源,例如取自活体的细胞、建立的细胞系、胚胎、组织、血液或者器官。代表性的实例为成骨细胞、造血干细胞、平滑肌细胞、神经元、基质细胞、ES细胞、肝脏、肠、肺、肾脏、淋巴结等。
通过常用的方法在常用缓冲液存在下悬浮用于分离的样品可以进行mRNA的分离。为了制备完整的mRNA作为mRNA分离的第一步,可以采用例如鸟苷超速离心方法(Chirgwin,J.M.等,生物化学(Biochemistry)(1979)18,5294-5299)或者AGPC方法(Chomczynski,P.和Sacchi,N.,分析生物化学(Anal.Biochem.)(1987)162,156-159)等。然后,为了从完整的mRNA中纯化mRNA,可以利用例如mRNA纯化试剂盒(Pharmacia)等。例如,QuickPrep mRNA纯化试剂盒(Pharmacia)也可以作为用寡聚dT通过亲和纯化浓缩mRNA的市售试剂盒。
利用反转录酶由所获的mRNA合成cDNA。可以使用市售的反转录酶。通过利用与mRNA的多聚A互补的寡聚dT或者利用随机序列寡核苷酸作为引物可以合成与mRNA互补的单链cDNA。例如,AMV反转录酶第一链cDNA合成试剂盒(Seikagaku公司)等可以用来合成cDNA。通过DNA聚合酶由所获的单链cDNA制备双链cDNA。
此外,为了特定的目的还可以利用常用的方法对cDNA文库进行选择性地富集。为了特定的目的,例如为了获得表达量不同的基因的cDAN,可以利用差异克隆的方法(Lau,L.F.和Nathans,D.,欧洲分子生物学组织杂志(EMBO J.)(1985)4,3145-3151)、差异显示方法(Liang,P.和Pardee,A.B.,科学(1992)257,967-971)、扣除克隆的方法(核酸研究(Nucleic AcidsResearch)(1988)16,10937)或者基因表达的系列分析方法(SAGE方法)(Velculescu,V.E.等,科学(1995)270,484-487)。SST方法(Tashiro,K.等,科学(1993)261,300-603)和在美国专利号5,536,637中所述的方法也可以用来富集编码分泌性蛋白的cDNA。
载体可以为任何载体,只要其可以转化细胞并表达其中含有的DNA。表达载体优选在待转化细胞中可以操作的载体。表达载体的实例为质粒载体和病毒来源的载体。
将所获的cDNA连接至一个载体。在这种情况下,可以通过将cDNA引入已经包含在载体中的功能性蛋白编码DNA的3’侧下游而将cDNA引入载体。为了这个目的,在编码功能性蛋白的DNA的3’侧下游设计适当的限制性酶位点,例如多克隆位点,并将cDNA引入该位点。同样,可以将cDNA首先连接到编码功能性蛋白的DNA的下游,然后可以将所获的DNA引入载体。可以将DNA构建体引入载体DNA中包含的适当的限制性酶位点。在制备载体时,可以将编码功能性蛋白的DNA和位于3’侧下游的cDNA直接连接,或者可以通过编码肽连接序列的DNA连接以便能够使功能性蛋白易于结合抗原。
表达载体优选包括目的DNA在细胞中表达所需的表达调节区。启动子/增强子可以作为表达调节区,具体可以利用人EF1α启动子、HCMV启动子或者SV40启动子等。利用常用的方法可以将以这种方式制备的表达载体引入细胞。此类方法的实例为电穿孔法(欧洲分子生物学组织杂志(1982)1,841-845)、磷酸钙法(病毒学(Virology)(1973)52,456-467)、脂质体法、DEAE葡聚糖法等。
用于转化的细胞可以为任何细胞,只要载体中包含的分泌信号序列和表达调节区在细胞内优选为动物细胞,例如COS、CHO或者BAF3等中发挥功能。
在本发明方法的第二步((ⅱ))中,将对抗原具有结合亲和力的可分泌性功能蛋白与由一种cDNA编码的蛋白的融合蛋白在细胞内进行表达。具体为,利用含有编码上述融合蛋白的DNA的载体转化细胞,并在适于细胞生长的条件下进行培养。按照常用的方法进行培养。例如,DMEM、MEM、RPMI1640以及IMDM可以用作培养基,而且可以与添加血清的溶液例如胎牛血清(FCS)一起使用。
为了在细胞内表达DNA,可以使用诱导DNA表达的系统。例如,如果使用利用四环素的表达调节系统或针对如细胞因子、脂多糖(LPS)、类固醇激素等的刺激而表达的启动子/增强子,那么通过刺激细胞有可能诱导细胞内DNA的表达。在DNA表达时产生含有功能性蛋白和cDNA的基因产物的融合蛋白。在cDNA编码一种膜结合蛋白时,融合蛋白在粗面内质网(ER)上的合成过程中分泌信号序列被去除,而融合蛋白表达于细胞膜上。在将编码肽连接序列的DNA连接在编码功能性蛋白的DNA和cDNA之间时,表达的融合蛋白在功能性蛋白和cDNA之间含有肽连接序列。
本发明方法的第三步((ⅲ))涉及通过将细胞膜上表达融合蛋白的细胞与抗原接触来筛选结合抗原的细胞。抗原优选结合于一种支持物。支持物的实例为用于细胞培养的那些支持物,优选培养板,例如塑料板、多孔板、培养板或珠。磁珠可以用作珠。利用常用的方法可以将抗原结合于支持物。例如,通过在适当的缓冲液存在下将抗原加入培养板,放置过夜,然后洗涤,可以将抗原结合于支持物。通过特异性结合抗原的抗体可以将抗原结合于支持物。例如,在加入特异性结合抗原的抗体并固定于培养板上后,可以加入抗原以使其结合于支持物。或者,首先可以结合不与支持物和细胞结合的抗原,然后利用特异性结合支持物上的固定抗原的抗体可以将细胞结合于支持物。在与未结合于支持物和细胞的抗原结合后,通过交联剂例如DMS(dimethylsulberimidate)、BS3[双(磺基琥珀酰亚氨)辛二酸酯(bis(sulfosuccinimididyl)suberate)]、以及DSS(二琥珀酰亚氨基辛二酸酯,disuccinimidyl suberate)可以将抗原和细胞交联。
将未结合抗原的细胞移去,通过在细胞可以结合培养板上的抗原的条件下温育并在细胞结合抗原后于适当的条件下洗涤培养板,可以筛选结合于抗原的细胞。流式细胞术(FACS)也可以用来筛选结合于抗原的细胞。收集通过此类方法筛选的细胞。通过重复这些方法二至数次,可以更选择性地获得目的细胞。
本发明方法的第四步((ⅳ))涉及从筛选的细胞中分离插入到载体中的cDNA。首先从已含有载体并结合于培养板上的细胞中提取载体,分离在载体中所包含的cDNA。当利用质粒载体时,提取质粒载体,引入大肠杆菌,在其中扩增,并制备来分离cDNA。随后,确定所分离基因的核苷酸序列。或者,基于载体上的核苷酸序列设计PCR引物,利用其对cDNA进行扩增,并确定核苷酸序列。当利用逆转录病毒载体时,以类似的方式通过PCR扩增cDNA,并确定核苷酸序列。
本发明的方法可能包括用于确定上述分离的基因是否包含新序列的分析步骤。利用DNA数据库,例如GENBANK、EMBL等,进行序列的同源性(氨基酸残基的等同性)检索可以分析所分离DNA序列的新颖性。可以遵照在“Wilbur,W.J.和Lipman,D.J.,美国国家科学院院报(1983)80,726-730”中所述的算法来确定蛋白质的同源性。
本发明的方法还可以包括筛选cDNA文库来获得上述分离基因的全长基因的步骤。按照常用的方法,如下可以对cDNA文库进行筛选。首先,对所分离基因的一个片段进行标记,用作探针,并同cDNA文库进行杂交。然后利用该标记物检测与分离基因的片段结合的cDNA克隆。
本发明的方法还可以包括对上述分离基因的全长基因进行分离的步骤。这可以通过如上所述筛选cDNA文库,分离通过通常已知的方法检测的cDNA克隆,并确定其核苷酸序列来进行。
此外,本发明包括一种用于分离编码上述膜结合蛋白的基因的试剂盒。本发明的试剂盒包括一种载体,其具有用于在编码对一种抗原具有结合亲和力的可分泌性功能蛋白的DNA3’侧下游插入cDNA的限制性酶识别位点。本发明的试剂盒进一步优选包括结合有一种抗原的支持物和/或其中待引入载体的细胞。另外,还可以包括用于淘洗的溶液、用于细胞与抗原桥联的交联剂、cDNA文库、用于通过溶解筛选的细胞收集DNA的溶液等。
附图简述
图1用示意图显示表达克隆载体pTMT-SR345的结构。图中的“SR345”表示人IL-6受体胞外区,“NEOr”新霉素抗性基因,“EF1α”肽链延伸因子1α的启动子/增强子区,“SV40E”SV40早期启动子/增强子,而“Ampr”为氨苄青霉素抗性基因。
图2用示意图显示表达克隆载体pTMT-scFv的结构。图中“scFv”表示单链抗体,而“Ig’s”为抗体分泌信号肽。其它符号同图1。
图3显示利用已经引入各种类型质粒DNA的COS-7细胞通过分选回收的菌落数。
图4用示意图显示表达克隆载体pTMT-BvGS3的结构。图中“hPM1-BvGS3”表示二价单链抗体。其它符号同图1和图2。
图5显示在利用抗人源化PM-1抗体的兔多克隆抗体通过流式细胞仪对已经引入各种类型质粒DNA的COS-7细胞进行分析时获得的直方图。
图6显示在利用抗小鼠抗IL-6受体抗体MT-18通过流式细胞仪对已经引入各种类型质粒DNA的COS-7细胞进行分析时获得的直方图。
图7用示意图显示表达克隆载体pTMT-shPM1F-κ的结构。图中“shPM1-κ”表示一种单链抗体。其它符号同图1和图2。
实施本发明的最佳模式
本发明的克隆方法可以按照下面所描述的特异地实施,但是本发明决非仅限于此。
实施例1:表达克隆载体pTMT-SR345的构建
构建了表达克隆载体pTMT-SR345。SR345,由表达克隆载体pTMT-SR345中所包含的DNA编码,是人IL-6受体的胞外区部分,包括从N端起的345个氨基酸残基。在表达克隆载体pTMT-SR345中,由插入在编码SR345的DNA下游的cDNA编码的蛋白与SR345以融合蛋白的形式表达。SR345的核苷酸序列和氨基酸序列一起示于SEQ IN NO:2中。
首先,为了从IL-6受体的cDNA扩增大约1.1kb的含有编码SR345的cDNA的片段(Yamasaki,K.等,科学(1988)241,825-828),设计了PCR引物IL6R1(SEQ ID NO:3)和IL6R2(SEQ ID NO:4)。将含有10mMTris-HCl(pH8.3)、50mM KCl、0.1mM dNTPs、1.5mM MgCl2、各100pmol的上述引物、100ng模板DNA(编码IL-6受体的cDNA)以及5单位AmpliTaq Gold酶的PCR反应混合物(100ml)在94℃进行变性,按照94℃ 1分钟、55℃ 1分钟以及72℃ 1分钟温育30个循环,最后于72℃温育10分钟。收集并通过1%的低熔点琼脂糖凝胶电泳纯化扩增的DNA片段,EcoRⅠ消化,并插入到表达载体pCOS1的EcoRⅠ位点中。将其转染到大肠杆菌中,并制备质粒来获得DNA片段以正确的方向插入的那些质粒。质粒HEF-PMh-gγ1(见WO92/19759)通过EcoRⅠ和SmaⅠ消化删除所含的基因并用EcoRⅠ-NotⅠ-BamHⅠ接头(TaKaRa)连接构建成表达载体pCOS1。
随后,用以下方法去除在SR345上游侧的EcoRⅠ位点。首先,由EcoRⅠ对质粒进行部分消化,收集通过在一个位点的切割所获得的线性分子。将其通过DNA聚合酶I(Klenow片段)使末端钝性化,自身连接,并转染到大肠杆菌中以获得表达克隆载体pTMT-SR345。表达克隆载体pTMT-SR345的结构示于图1中。
实施例2:表达载体pTMT-scFv的构建
构建了表达载体pTMT-scFv。利用识别人IL-6受体的人源化单克隆抗体PM-1的可变区以及一个连接序列区设计由在表达载体pTMT-scFv中包含的DNA编码的单链抗体(scFv)。在表达载体pTMT-scFv中,由插入在编码scFv的DNA下游的cDNA编码的蛋白与scFv以融合蛋白的形式表达。scFv基因的核苷酸序列和氨基酸序列一起示于SEQ IN NO:5中。
1)编码抗体V区的DNA片段的扩增
通过PCR扩增人源化PM1抗体H链和L链V区的基因(Sato,K等,癌症研究(Cancer Res.)(1993)53,851-856)。设计H链V区的反向引物TMT1(SEQ ID NO:6)使其可以与编码H链V区N末端的DNA杂交而且包括一个SalⅠ限制性酶识别位点。设计H链V区的正向引物LINK1(SEQ IDNO:7)使其可以与编码H链V区C末端的DNA杂交而且包括连接序列区的5’末端序列。同样,设计L链V区的反向引物LINK3(SEQ ID NO:8)使其可以与编码L链V区N末端的DNA杂交而且包括连接序列区的3’末端序列。设计L链V区的正向引物SCP-C(SEQ ID NO:9)使其可以与编码形成L链恒定区转角位点的氨基酸序列的核苷酸序列杂交而且还包括HindⅢ限制性酶识别位点、编码FLAG肽(SEQ ID NO:10)的核苷酸序列以及两个重复翻译终止密码子。
将含有10mM Tris-HCl(pH8.3)、50mM KCl、0.1mM dNTPs、1.5mMMgCl2、各100pmol的上述引物、100ng模板DNA以及5单位AmpliTaq Gold酶的PCR反应混合物(100ml)在94℃变性9分钟,按照94℃ 30秒以及60℃ 1分钟温育30个循环,最后于60℃温育5分钟。利用1.5%的低熔点琼脂糖凝胶对PCR产物进行纯化。
2)编码连接序列区的DNA片段的扩增
通过PCR方法利用人源化的单链抗体表达载体pSCFVT7-hM21(见WO95/14041)对编码包括氨基酸序列(Gly4Ser)3的连接序列区的DNA片段进行扩增。设计反向引物LINK2(SEQ ID NO:11)使其可以与该连接序列区的5’末端杂交而且还包括H链V区3’末端DNA序列。设计正向引物LINK4(SEQ ID NO:12)使其可以与连接序列区的3’末端杂交而且还包括L链V区5’末端DNA序列。利用100ng模板DNA(pSCFVT7-hM21)在上述条件下进行PCR,并利用1.5%的低熔点琼脂糖凝胶对PCR产物进行纯化。
3)人源化PM1抗体单链Fv的构建
上面制备的编码H链和L链V区的DNA片段,以及编码连接序列区的DNA片段用PCR方法组装,加入反向引物TMT1和正向引物TMT2(SEQID NO:13)来扩增编码人源化PM1的scFv的全长DNA片段。设计正向引物TMT2使其可以与编码HindⅢ限制性酶识别位点和FLAG肽的DNA序列杂交,而且还包括两个重复的翻译终止密码子以及EcoRⅠ限制性酶识别位点。如下进行初级PCR:将含有10mM Tris-HCl(pH8.3)、50mM KCl、0.1mMdNTPs、1.5mM MgCl2、大约各100ng的上述PCR产物以及5单位AmpliTaqGold酶的PCR反应混合物98ml首先在94℃变性,然后按照94℃ 2分钟、55℃ 2分钟以及72℃ 2分钟进行2个循环来连接各DNA片段。以下列方式进行次级PCR:将100pmol的每种引物加入到上面的PCR反应溶液中,按照94℃ 30秒和60℃ 1分钟进行30个循环,最后将混合物在60℃温育5分钟。
PCR产物用1.5%的低熔点琼脂糖凝胶纯化后,由SalⅠ和NotⅠ对其消化,并插入到包括人EF1α启动子和抗体的前导序列(SEQ ID NO:14)的表达载体pSFLAG中。进行DNA测序后,获得了质粒pTMT-scFv,其含有包括正确DNA序列的DNA片段。表达载体pTMT-scFv的结构以示意图示于图2中。如下所述进行pSFLAG的构建。设计并合成两个分别为正义和反义方向的重叠的寡核苷酸S-FLAGl(SEQ ID NO:15)和S-FLAG2(SEQ IDNO:16)以便编码EcoRⅠ限制性酶识别位点、抗体的前导序列(SEQ ID NO:14)、FLAG肽(SEQ ID NO:10)以及KpnⅠ、NotⅠ和BamHⅠ限制性酶识别位点。将含有合成的寡核苷酸各100pmol的反应混合物在96℃温育5分钟,在20分钟内将温度降至65℃,并在65℃温育5分钟。然后,在20分钟内将温度降至42℃,将混合物进一步温育5分钟,通过在20分钟内将温度降至室温使两种寡核苷酸退火。将该DNA片段插入到由EcoRⅠ和BamHⅠ消化的pCOS1中。
实施例3:SR345-gp130和scFv-gp130融合蛋白表达系统的构建
(A)SR345-gp130
细胞因子信号转导分子gp130是一种Ⅰ型膜结合蛋白(Taga,T.等,细胞(Cell)(1989)58,573-581、Saito,M.等,免疫学杂志(J.Immunol.)(1992)148,4066-4071)。将小鼠gp130 cDNA的一部分连接至表达载体pTMT-SR345上编码可溶型IL-6受体(SR345)的cDNA的下游,以便在COS细胞中表达一种包括SR345和小鼠gp130的一部分序列的融合蛋白。根据两种类型的融合蛋白在gp130部分区域中的差别构建了它们。它们中一种为膜结合融合蛋白(SR345-mgpTMIC),其中连接了gp130的跨膜区和随后的胞内区,而另一种为分泌性融合蛋白(SR345-mgpIC),其中只连接了gp130的胞内区。SEQ ID NO:17显示全长小鼠gp130的氨基酸序列和核苷酸序列。
1)膜结合融合蛋白SR345-mgpTMIC表达载体的建立
将全长小鼠gp130 cDNA用EcoRⅠ进行消化获得大约1.1kb的EcoRⅠ片段。该EcoRⅠ片段编码小鼠gp130(C末端)第603位至第917位的氨基酸,而且含有小鼠gp130胞外区的一部分(15个氨基酸)以及随后跨膜区和胞内区的全部。将该EcoRⅠ片段插入到pTMT-SR345表达载体的EcoRⅠ位点中以建立膜结合融合蛋白SR345-mgpTMIC表达载体。
2)分泌性融合蛋白SR345-mgpIC表达载体的建立
为了获得编码小鼠gp130胞内区的cDNA片段,合成已加入HindⅢ和EcoRⅠ位点的PCR引物mgp2(SEQ ID NO:20;包括编码SEQ ID NO:17氨基酸序列的第646位氨基酸至第651位氨基酸的DNA)和mgp3(SEQ IDNO:19;包括编码SEQ ID NO:17氨基酸序列(C末端)第912位氨基酸至第917位氨基酸的DNA),并利用这些引物获得了大约1kb的小鼠gp130的cDNA片段。该大约1kb的cDNA片段编码SEQ ID NO:17的氨基酸序列中第646位氨基酸至第917位氨基酸,而且其对应于缺少从N末端起6个氨基酸的胞内区。将由此获得的cDNA片段用EcoRⅠ进行消化,并插入到pTMT-SR345表达载体的EcoRⅠ位点中以制备分泌性融合蛋白SR345-mgpIC表达载体。
(B)scFv-gp130
在表达载体pTMT-scFv中,将小鼠gp130 cDNA的一部分连接于scFvcDNA的下游以便在COS细胞中表达一种包括scFv和小鼠gp130的部分区域的融合蛋白。根据两种类型的融合蛋白在gp130部分区域中的差别构建了它们。它们中一种为膜结合融合蛋白(scFv-mgpTMIC),其中连接了gp130的跨膜区和随后的胞内区,而另一种为分泌性融合蛋白(scFv-mgpIC),其中只连接了gp130的胞内区。
1)膜结合融合蛋白scFv-mgpTMIC表达载体的建立
为了获得编码小鼠gp130整个胞内区和跨膜区的cDNA片段,合成已加入HindⅢ位点的PCR引物mgp1(SEQID NO:18;包括编码SEQID NO:17氨基酸序列的第603位氨基酸至第608位氨基酸的DNA)和已加入NotⅠ位点的mgp3(SEQ ID NO:19;包括编码SEQ ID NO:17氨基酸序列(C末端)第912位氨基酸至第917位氨基酸的DNA),并利用这些引物获得了大约1.1kb的小鼠gp130的cDNA片段。该大约1.1kb的cDNA片段编码SEQID NO:17的氨基酸序列中(C末端)第603位氨基酸至第917位氨基酸,而且该片段包括小鼠gp130胞外区的一部分(15个氨基酸)和整个随后的跨膜区和胞内区。将由此获得的cDNA片段用HindⅢ和NotⅠ进行消化,并插入到pTMT-scFv表达载体的HindⅢ-NotⅠ位点中以制备膜结合融合蛋白scFv-mgpTMIC表达载体。
2)分泌性融合蛋白scFv-mgpIC表达载体的建立
为了获得编码小鼠gp130胞内区的cDNA片段,合成已加入HindⅢ-EcoRⅠ位点的PCR引物mgp2(SEQ ID NO:20;包括编码SEQ ID NO:17氨基酸序列的第646位氨基酸至第651位氨基酸的DNA)和已加入NotⅠ位点的mgp3(SEQ IDNO:19;包括编码SEQ IDNO:17氨基酸序列(C末端)第912位氨基酸至第917位氨基酸的DNA),并利用这些引物通过PCR获得了大约1kb的小鼠gp130的cDNA片段。
该大约1kb的cDNA片段编码SEQ ID NO:17的氨基酸序列中(C末端)第646位氨基酸至第917位氨基酸,而且其对应于缺少从N末端起6个氨基酸的胞内区。将由此获得的cDNA片段用HindⅢ和NotⅠ进行消化,并插入到pTMT-scFv表达载体的HindⅢ-NotⅠ位点中以制备分泌性融合蛋白scFv-mgpIC表达载体。
实施例4:由COS细胞的表达
将上述每种类型表达载体转染到COS细胞中,瞬时表达融合蛋白,并且证实了在细胞膜上表达融合蛋白的细胞通过淘洗选择性凝集。以不含基因的表达载体转染的COS细胞被用作阴性对照。阳性对照为用表达载体P3.19转染并用相应的抗体淘洗的COS细胞,载体P3.19是通过向载体pCOS1中引入编码HM1.24抗原蛋白(WO 98/14580)的DNA制备的。
1)转染到COS细胞中
利用Lipofect AMINE PLUSTM试剂(GIBCO-BRL)将质粒DNA转染到COS-7细胞中。即,在转染前一天将COS-7细胞以1×105个细胞/孔(6孔板)接种,培养过夜并用无血清DMEM培养基(GIBCO-BRL)洗涤,然后向其中加入0.8ml相同的培养基。在将1μg的质粒DNA和6μl的PLUS试剂分别加至0.1ml无血清DMEM培养基中后,在室温下将混合物温育15分钟,与0.1ml的LipofectAMINE溶液(4μl的LipofectAMINE/0.1ml的无血清DMEM培养基)混合,并在室温下继续温育15分钟。随后,将该混合物加至上述COS-7细胞并在37℃温育3小时。向其中加入含有20%胎牛血清(GIBCO-BRL)的DMEM培养基(1ml)(终浓度10%的血清)。培养过夜后,将培养基更换为3ml含有10%胎牛血清的DMEM培养基,并在37℃和5%CO2条件下温育3天。
2)淘洗皿的制备
在利用表达载体pTMT-SR345时,按照Seed,B.等,美国国家科学院院报(1987)84,3365-3369的方法制备用小鼠抗人IL-6受体抗体MT18(参见已公开未审查的日本专利申请第Hei 2-288898号)包被的培养皿。即,将小鼠抗IL-6受体抗体加到50mM Tris-HCl(pH9.5)中至10μg/ml。室温下将由此制备的抗体溶液(3ml)在60mm直径细胞培养皿中温育2小时。用0.15MNaCl溶液洗涤培养皿3次后,加入含有5%胎牛血清、1mM EDTA和0.02%NaN3的PBS,然后在封闭后按照下面所述进行淘洗。
在利用pTMT-scFv时,制备两种类型的淘洗皿。一种用可溶型IL-6受体(SR344)(Yasukawa,K.等,生物化学杂志(1990)108,673-676)包被,而另一种用上述小鼠抗IL-6受体抗体包被。用50mM Tris-HCl(pH9.5)将SR344的浓度调节至2μg/ml,并如上所述制备淘洗皿。在利用阴性对照pCOS-1时,采用由上述小鼠抗IL-6受体抗体包被的培养皿。在利用阳性对照HM1.24抗原蛋白表达载体P3.19时,采用由抗HM1.24抗原的抗体包被的培养皿。
3)淘洗
将pCOS-1或pTMT-SR345转染的COS-7细胞用含有1mM EDTA的PBS使其从培养板上脱离,用含有5%胎牛血清的PBS洗涤一次,悬于2ml含有5%胎牛血清和0.02%NaN3的PBS中,并加入到由小鼠抗IL-6受体抗体包被的淘洗板中。
通过三种不同的方法对pTMT-scFv转染的COS-7细胞进行淘洗。在一种方法中,将COS-7细胞如上所述使其脱壁并用含有5%胎牛血清的PBS洗涤一次后,悬于500μl含有2μg/ml SR344、5%胎牛血清和0.02%NaN3的PBS中,并在冰上温育1小时。将细胞用冰冷的PBS洗涤三次后,重悬于含有0.2mM交联剂双(磺基琥珀酰亚氨)辛二酸酯(BS3;PIERCE)和50mMHepes(pH8.0)的PBS中,并在冰上进一步温育30分钟。然后,加入1MTris-HCl(pH8.0)至50mM,并在冰上继续温育10分钟以去除过量的交联剂。在用PBS洗涤细胞后,将其加至由小鼠抗IL-6受体抗体包被的淘洗板中。在第二种方法中,将与SR344预温育的COS-7细胞不经交联剂处理加至由小鼠抗IL-6受体抗体包被的淘洗板中。在第三种方法中,将COS-7细胞加至由SR344直接包被的培养板中。冰上温育、Tris-HCl处理以及洗涤的时间在三种方法中都相似。
将由HM1.24抗原蛋白表达载体P3.19转染的COS-7细胞加至由抗HM1.24抗原的抗体(WO 98/14580)包被的淘洗板中。室温下将上述各种类型的COS-7细胞在各种淘洗板中温育2小时后,将淘洗板用含有5%胎牛血清和0.02%NaN3的PBS轻轻洗涤三次,并利用含有0.6%SDS和10mMEDTA的溶液从结合至淘洗皿上的细胞中收集质粒DNA。通过利用电穿孔将所回收质粒DNA的1/5转染入大肠杆菌DH5a中,并通过出现的氨苄青霉素抗性菌落的数量对淘洗的聚集作用进行评价。结果示于图3中。
在利用表达载体pTMT-SR345时,SR345-mgpIC产生比SR345-mgpTMIC多的菌落,因此未观察到针对膜结合蛋白的特异性。在另一方面,在利用表达载体pTMT-scFv时,在所有的淘洗方法中,scFv-mgpTMIC产生比scFv-mgpIC多的菌落,因此表达膜结合蛋白的细胞被特异性地富集。特别是在使用交联剂时,选择性更加明显。
因此,上述结果显示通过将功能蛋白(单链抗体)表达为细胞表面融合蛋白比通过仅仅将一种被该抗体识别的表位表达为融合蛋白更有选择性地而且更有效地获得编码膜结合蛋白的cDNA。
通常,重复淘洗数次提高克隆选择性,但是如本实施例所示,在本发明中(其中功能蛋白表达在细胞膜上)在第一次淘洗中便观察到显著的选择性。因此,通过进一步淘洗数次可以非常有效而且选择性地完成编码一种膜结合蛋白的基因的克隆。
实施例5:利用人源化二价单链Fv构建融合蛋白表达系统
1.人源化PM1抗体二价单链Fv表达载体的构建
基于人源化PM1抗体Fv构建了一种二价单链Fv表达载体。设计具有二价可变区的人源化PM1抗体单链Fv(hPM1-BvGS3)以便通过一个包括(Gly4Ser)3的肽连接序列(SEQ ID NO:1)连接在实施例2中所述的人源化PM1抗体单链Fv2的两个分子。hPM1-BvGS3的氨基酸序列和核苷酸序列示于SEQ ID NO:21中。
表达载体pTMT-BvGS3的构建按照下述方法进行。通过PCR方法对编码人源化PM1抗体单链Fv并在其C末端具有一个包括(Gly4Ser)3的连接序列的基因进行扩增。TMT-1(SEQ ID NO:6)被用作反向引物。同样,设计正向引物BvGS3(SEQ ID NO:22)使其可以与编码L链V区C末端的DNA杂交而且包括编码连接序列和SalⅠ限制性酶识别位点的核苷酸序列。在与上述相同的条件下利用100ng pTMT-scFv作为模板DNA进行PCR,而且利用1.5%的低熔点琼脂糖凝胶对PCR产物进行纯化。
将纯化的PCR产物用限制性酶SalⅠ进行消化,并插入到克隆载体pBluescriptⅡ(Stratagene)中。DNA测序后,用限制性酶SalⅠ对含有包括正确DNA序列的DNA片段的质粒进行消化,以获得编码人源化PM1抗体单链Fv并在其C末端具有一个包括(Gly4Ser)3的连接序列的基因。随后,通过将上述获得的DNA片段插入到pTMT-scFv中获得hPM1-BvGS3表达载体TMT-BvGS3。hPM1-BvGS3表达载体TMT-BvGS3的结构以示意图示于图4中。
2.融合蛋白hPM1-BvGS3-gp130表达载体的构建
在表达载体TMT-BvGS3中,将小鼠gp130 cDNA的一部分连接于编码hPM1-BvGS3的cDNA的下游以构建一种包括hPM1-BvGS3和小鼠gp130的一部分区域的融合蛋白表达系统。根据所连接的gp130部分区域的差别构建两种类型的融合蛋白。其中一种为连接了gp130的跨膜区及随后的胞内区的膜结合融合蛋白(BvGS3-mgpTMIC),另一种为只连接了gp130的胞内区的分泌性融合蛋白(BvGS3-mgpIC)。BvGS3-mgpTMIC和BvGS3-mgpIC分别在实施例3(B)的1)和2)中制备,而且通过将这些插入到pTMT-BvGS3的HindⅢ-NotⅠ位点中,构建了膜结合融合蛋白表达载体pTMT-BvGS3-mgpTMIC和分泌性融合蛋白表达载体pTMT-BvGS3-mgpIC。
实施例6:通过流式细胞仪对表达的分析
将上面构建的每种类型的表达载体,pTMT-BvGS3、pTMT-BvGS3-mgpIC和pTMT-BvGS3-mgpTMIC转染到COS-7细胞中,瞬时表达该融合蛋白,并利用流式细胞仪(FACScan,Beckton Dickinson)分析在细胞膜上的表达。通过两种类型的方法进行表达分析。一种涉及通过兔抗人源化PM-1抗体的多克隆抗体进行检测,而另一种涉及在可溶性IL-6受体抗体的存在下通过小鼠抗IL-6受体抗体MT-18进行检测。结果证实膜结合融合蛋白BvGS3-mgpTMIC以可以识别可溶性IL-6受体的形式大量表达于细胞膜上。将仅用表达载体pCOS-1转染的细胞用作阴性对照。
1)向COS-7细胞中的转染
利用转染试剂盒FuGENETM6(Boehringer-Mannheim)将质粒DNA转染到COS-7细胞中。
即,在转染前一天将COS-7细胞以5×104个细胞/孔(6孔板)接种,于37℃和5%CO2的条件下在2ml含有10%胎牛血清的DMEM培养基(GIBCO-BRL)中培养过夜。在转染当天,将6μl FuGENETM6加入到0.1ml无血清DMEM培养基中,在室温下温育5分钟后,将其与2μg的质粒DNA混合,并在室温下继续温育15分钟。然后,将该混合物加入到上述COS-7细胞中并在37℃和5%CO2的条件下温育3天。
2)COS-7细胞的染色
将上述COS-7细胞用含有1mM EDTA的PBS使其脱壁,用含有5%胎牛血清的PBS洗涤,悬于50μl FACS缓中液(含有2%胎牛血清和0.05%NaN3的PBS)中,并通过下面两种类型的方法进行染色。
A)用抗人源化PM-1抗体的兔多克隆抗体染色将一种抗人源化PM-1抗体的兔多克隆抗体(2μg/反应)加入到上述COS-7细胞中,在冰上温育30分钟,用1ml FACS缓冲液洗涤两次,并悬于50μl FACS缓冲液中。随后加入FITC(异硫氰酸荧光素)标记的山羊抗兔IgG(AMERICAN QUAREX)2μl/反应和用于单独染色死细胞的2.5μg/反应的PI(碘化丙锭),并在冰上避光温育30分钟。温育后,将细胞用1ml的FACS缓冲液洗涤两次并重悬于0.5ml的FACS缓冲液中,用流式细胞仪进行分析。
B)用小鼠抗IL-6受体抗体MT-18染色
将可溶型IL-6受体(3μg/反应)加入上述COS-7细胞中并于冰上温育4小时,用1ml FACS缓冲液洗两次,重悬于50μl FACS缓冲液中。然后,加入2μg/反应的小鼠抗IL-6受体抗体MT-18,并于冰上温育30分钟。此后用1ml FACS缓冲液将细胞洗两次,重悬于50μl FACS缓冲液中。随后加入FITC标记的山羊抗小鼠IgG2b(Dainippon Seiyaku)2μl/反应和用于单独染色死细胞的PI(碘化丙锭)2.5μg/反应,并在冰上避光温育30分钟。温育后,将细胞用1ml的FACS缓冲液洗涤两次并重悬于0.5ml的FACS缓冲液中,用流式细胞仪进行分析。
3)分析利用流式细胞仪进行的表达
通过PI和FSC(前向散射)的分析表明存在由PI染色的细胞群(死细胞)。死细胞因其被FITC非特异性地染色而干扰分析。因此,圈定不被PI染色的细胞群(活细胞),并仅对该群进行分析。
用抗人源化PM-1抗体的兔多克隆抗体染色,在细胞膜上未观察到分泌性蛋白hPM1-BvGS3的表达,然而,膜结合融合蛋白BvGS3-mgpTMIC的表达显示为最强。根据该事实推测具有一个跨膜区的BvGS3-mgpTMIC并未被分泌而是插入细胞膜中。然而,另一方面,虽然BvGS3-mgpIC为一种分泌性融合蛋白,但是在细胞膜上仍检测到BvGS3-mgpIC的表达。这被认为是由于BvGS3-mgpIC在分子大小和结构、连接于其下游的mgpIC中所含的疏水区等特征上与BvGS3的特征不同。换言之,推测由于在特征上的差别,BvGS3-mgpIC不会向BvGS3那样迅速通过细胞膜,而需要更长的时间来通过。结果,定位于细胞膜上的数量增加,通过抗人源化PM-1的兔多克隆抗体检测到所有表位向细胞外突出的分子。
另一方面,在用小鼠抗IL-6受体的抗体MT-18染色的情况下,与由抗人源化PM-1抗体的兔多克隆抗体染色的结果相似,虽然未检测到分泌性蛋白hPM1-BvGS3的表达,但膜结合融合蛋白BvGS3-mgpTMIC的表达最强。然而,对于分泌性融合蛋白BvGS3-mgpIC,结果与通过抗人源化PM-1的兔多克隆抗体所得的结果不同,在细胞膜上几乎未检测到任何表达。这提示,虽然膜结合融合蛋白BvGS3-mgpTMIC表达于细胞膜上形成一种可以识别可溶型IL-6受体的功能性构象,但是大多数分泌性融合蛋白BvGS3-mgpIC尽管定位于细胞膜上,仍不具有可以识别可溶型IL-6受体的功能性构象。结果示于图6中。
因此,通过流式细胞仪获得的结果表明当将单纯一个由抗体识别的表位表达为融合蛋白时,即使分泌性融合蛋白如果其定位于细胞膜上也将被筛选为假阳性。另一方面,当将本发明的功能性蛋白(例如,单链抗体)以融合蛋白的形式表达于细胞表面时,揭示了更有选择性且更有效地克隆编码膜结合蛋白的cDNA的可能性。
实施例7
1.人源化PM1抗体单链Fv的设计
为了提高克隆效率,设计了三种其它类型的单链Fv及其二价单链Fv。由于在构建上述人源化PM1抗体单链Fv时加入的转角区(SEQ ID NO:5,从第242位到第256位的氨基酸序列)含有具有高疏水性的氨基酸残基,所以设计下面三种类型的人源化PM1抗体单链Fv以便更稳定的细胞外表达。换言之,为了去除C末端的疏水区,设计了转角区缺失的单链Fv并指定为shPM1(ΔEL)(SEQ ID NO:23)。同样,由于发现通过在单链Fv的C末端增加某种分泌性蛋白衍生的结构域可以增加稳定性和表达效率,将由人x链恒定区或人膜型μ链恒定区的第4外显子(Dorai,H和Gillies,S.D.,核酸研究,17,6412,1989)编码的氨基酸序列加到单链Fv的C末端(SEQ IDNO:23)。虽然人x链恒定区的第107位氨基酸原本为半胱氨酸,但此次由丝氨酸残基取代(SEQ ID NO:24)。同样,将已删除跨膜区和胞内区的序列(SEQ ID NO:25)用作由人膜型μ链恒定区的第4外显子编码的氨基酸序列。将在单链Fv C末端增加了上述各序列的单链Fv称为shPM1-Kappa(SEQID NO:26)和shPM1-MCH4(SEQ ID NO:27)。
2.shPM1(ΔEL)表达载体的构建
通过PCR方法扩增编码shPM1(ΔEL)的基因。利用了反向引物EF-1(SEQ ID NO:28)和正向引物SCP-C2(SEQ ID NO:29)。在上述条件下利用100ng的pTMT-scFv作为模板DNA进行PCR,并利用1.5%低熔点琼脂糖凝胶对PCR产物进行纯化。正向引物SCP-C2与编码L链V区C末端的DNA杂交,并向其中加入HindⅢ-NotⅠ识别位点和FLAG肽(SEQ ID NO:10)。
在用EcoRⅠ和NotⅠ对纯化的PCR产物进行消化后,将消化的产物插入到pSFLAG载体中以获得shPM1(ΔEL)表达载体pTMT-shPM1F。此外,通过与实施例5中的相似的方法获得了具有一个二价可变区并且缺失转角区的shPM1(ΔEL)-BvGS3(SEQ ID NO:30)的表达载体pTMT-shPM1F-BvGS3。
3.shPM1-κ表达载体的构建
通过PCR组装构建了编码人源化PM1抗体单链Fv(SEQ ID NO:23)与人κ链恒定区(SEQ ID NO:24)的融合蛋白的基因。换言之,在通过PCR分别扩增编码人源化PM1抗体单链Fv和人κ链恒定区的基因后,通过它们的互补性进行组装并通过外部引物对全长基因进行扩增。
首先,通过PCR扩增编码人κ链恒定区的基因。设计反向引物κ1(SEQID NO:31)使其可以与编码人κ链恒定区的转角区和下面氨基酸序列(SEQID NO:24)的第12位Pro至第21位Gly的核苷酸序列杂交。设计正向引物κ2(SEQ IDNO:32)使其可以与编码人κ链恒定区C末端(SEQ ID NO:24)的第101位Ser至第11l位Ser的核苷酸序列杂交并包括编码限制性酶HindⅢ和NotⅠ识别位点和FLAG肽(SEQ ID NO:10)的核苷酸序列以及两个终止密码子。通过利用这些引物,将原本为一个半胱氨酸残基的SEQ ID NO:24的第107位氨基酸被一个丝氨酸残基取代。在与上述方式相似的条件下,以人源化PM1抗体L链表达载体RV1-PM1a(见WO92/19759)作为模板利用上述两类引物进行PCR。利用1.5%低熔点琼脂糖凝胶对PCR产物进行纯化。
然后,以相同的方式扩增编码人源化PM1抗体单链Fv的基因。在与上述相同的条件下,以pTMT-scFv为模板DNA利用EF1(SEQ ID NO:28)作为反向引物,SCP-K(SEQ ID NO:33)作为正向引物进行PCR。设计正向引物SCP-K使其可以与在SEQ ID NO:5中所示的编码单链Fv C末端的核苷酸序列杂交并包括与PCR扩增的κ链基因5’末端互补的核苷酸序列。以同样的方式对PCR产物进行纯化。
利用在实施例2-3)中所示的方法对编码shPM1-κ的全长cDNA片段进行扩增。即,通过初次PCR对各100ng的上述DNA片段进行组装,然后加入反向引物EF-1(SEQ ID NO:28)和正向引物k2(SEQ ID NO:32)各100ng来扩增全长cDNA片段。
将PCR产物用1.5%低熔点琼脂糖凝胶纯化后,利用限制性酶EcoRⅠ和NotⅠ进行消化,并将其插入到pSFLAG载体中以获得shPM1-κ表达载体pTMT-shPM1F-K(图7)。此外,利用与在实施例5中所述相似的方法,获得了具有一个二价可变区的单链Fv和shPM1-Kappa-BvGS3(SEQ ID NO:34)表达载体pTMT-shPM1FK-BVGS3。
4.shPM1-MCH4表达载体的构建
通过PCR组装构建编码人源化PM1抗体单链Fv(SEQ ID NO:23)和人μ链恒定区部分序列(SEQ ID NO:25)的融合蛋白的基因。即,通过PCR方法分别扩增编码人源化PM1抗体单链Fv和人μ链恒定区部分序列的基因,并通过它们的互补性进行组装。然后通过外部引物扩增全长基因。
首先,通过PCR扩增编码人μ链恒定区的基因。设计反向引物MCH4-1(SEQ ID NO:35)使其可以与编码人μ链恒定区第4外显子的5’末端核苷酸序列杂交而且包括人源化PM1抗体单链Fv(SEQ ID NO:23)3’末端的核苷酸序列。设计正向引物MCH4-2.1(SEQ ID NO:36)使其可以与编码人膜型μ链恒定区的胞外区的核苷酸序列杂交,而且包括限制性酶HindⅢ识别位点。在与上述方式相似的条件下,以通过常用方法从人骨髓瘤细胞系CL-4细胞中获得的cDNA作为模板利用上述两类引物进行PCR。利用1.5%低熔点琼脂糖凝胶对PCR产物进行纯化。
然后,以相同的方式扩增编码人源化PM1抗体单链Fv的基因。在与上述相同的条件下,以pTMT-scFv为模板DNA利用EF1(SEQ ID NO:28)作为反向引物,SCP-Mu(SEQ ID NO:37)作为正向引物进行PCR。设计正向引物SCP-Mu使其可以与在SEQ ID NO:A中所示的编码单链Fv C末端的核苷酸序列杂交,而且包括与通过PCR扩增的μ链部分序列基因5’末端互补的核苷酸序列。以同样的方式对PCR产物进行纯化。
利用在实施例2-3)中所示的方法对编码shPM1-MCH4的全长cDNA片段进行扩增。即,通过初次PCR对各100ng的上述DNA片段进行组装,然后加入反向引物EF-1(SEQ ID NO:28)和正向引物MCH4-2.2(SEQ IDNO:38)各100pmol来扩增全长cDNA片段。设计正向引物MCH4-2.2使其可以与上面扩增的编码人膜型μ链部分序列的核苷酸序列的3’末端杂交,而且包括编码FLAG肽的核苷酸序列、两个终止密码子以及限制性酶NotⅠ识别位点。
将PCR产物用1.5%低熔点琼脂糖凝胶纯化后,利用限制性酶EcoRⅠ和NotⅠ进行消化,并将其插入到pSFLAG载体中以获得shPM1-MCH4表达载体pTMT-shPM1F-MCH4(图7)。此外,利用与在实施例5中所述相似的方法,获得了具有一个二价可变区的单链Fv和shPM1-MCH4-BvGS3(SEQID NO:39)表达载体pTMT-shPM1FM-BvGS3。
实施例8:通过TMT方法利用shPM1-κ表达载体(图7)对STX561 cDNA文库的筛选
1.STX561 cDNA文库的制备
利用常用的方法制备来自小鼠造血基质细胞系STX561的mRNA,并将由其合成的cDNA插入到TMT表达载体shPM1-κ中来制备STX561 cDNA文库。利用cDNA合成试剂盒(STRATAGENE,cDNA合成试剂盒)制备cDNA文库。基本上按照STRATAGENE cDNA合成试剂盒的操作,并进行了下述的调整。换言之,将GIBCO-BRL的SuperscriptⅡ用作反转录酶,将NotⅠ-dT引物(Pharmacia Biotech,第一链cDNA合成试剂盒所附引物)作为第一链合成的引物,将HindⅢ-SmaⅠ位点接头用作加至cDNA 5’末端的接头,而且将Pharmacia Biotech的Size sep400离心层析柱用作大小分级的柱子。
具体如下所述制备cDNA文库:启始材料为5μg的mRNA,首先利用NotⅠ-dT引物(Pharmacia Biotech,第一链cDNA合成试剂盒所附引物)由反转录酶(SuperscriptⅡ,GIBCO-BRL)从3’多聚A尾合成第一链。然后,在用DNA聚合酶合成第二链后,将cDNA的两个末端钝性化,并加入HindⅢ-SmaⅠ位点接头(Takara)。用HindⅢ和NotⅠ对两个末端消化后,进行大小分级分离(Pharmacia Biotech,Size sep 400离心层析柱)以去除大小为0.5kb或更小的cDNA片段。将收集的cDNA插入到TMT表达载体shPM1-κ的HindⅢ-NotⅠ位点中,并通过电穿孔方法将载体引入大肠杆菌DH10B(electroMAX DH10B,GIBCO-BRL)中来制备STX561 cDNA文库。
通过分成1000个克隆/库将STX561 cDNA文库分库,其中两个库(库号:#kappa-1,#kappa-6)一共2000克隆用于通过TMT方法进行筛选。
2.通过淘洗对STX cDNA文库的筛选
1)向COS-7细胞中的转染
利用FuGENETM6(Boehringer-Mannheim)将从#kappa-1和#kappa-6制备的质粒DNA各2μg转染到COS-7细胞中。
即,在转染前一天将COS-7细胞以1×105个细胞/孔(6孔板)接种,于37℃和5%CO2的条件下在2ml含有10%胎牛血清的DMEM培养基(GIBCO-BRL)中培养过夜。在转染当天,将6μl FuGENETM6加入到0.1ml无血清DMEM培养基中,在室温下温育5分钟后,将其与2μg的质粒DNA混合,并在室温下继续温育15分钟。然后,将该混合物加入到上述COS-7细胞上并在37℃和5%CO2的条件下温育3天。
2)淘洗板的制备
按照Seed,B.等,美国国家科学院院报(1987)84,3365-3369的方法制备由山羊抗小鼠IgG抗体(Dainippon Seiyaku,山羊抗小鼠IgG(H+L链))包被的淘洗板。即,将山羊抗小鼠IgG抗体加到50mM Tris-HCl(pH9.5)中至1Oμg/ml。将由此制备的抗体溶液(3ml)加至60mm直径细胞培养皿中,并在室温下温育3小时。用O.15M NaCl溶液洗涤3次后,加入含有5%胎牛血清、1mM EDTA和0.02%NaN3的PBS,然后在封闭后按照下面所述进行淘洗。
3)淘洗
将如上所述转染的COS-7细胞用含有1mM EDTA的PBS使其从培养板上脱离,用含有5%胎牛血清的PBS洗涤一次,悬于50μl FACS缓冲液(含有2%胎牛血清和0.05%NaN3的PBS)中。
将可溶型IL-6R(2μg)加入到细胞悬液中,并在冰上放置90分钟。随后,在用FACS缓冲液洗涤两次后,将细胞悬于50μl FACS缓冲液中。然后,向细胞悬液中加入1.5μg小鼠抗IL-6受体抗体MT-18,并将细胞悬液在冰上放置30分钟。将细胞用FACS缓冲液洗涤两次,悬于2ml含有5%胎牛血清和0.02%NaN3的PBS中,并加入到用山羊抗小鼠IgG抗体包被的淘洗板中。
室温下将上述各种COS-7细胞在各种淘洗板中温育大约2小时后,将淘洗板用含有5%胎牛血清和0.02%NaN3的PBS轻轻洗涤三次,并利用Hirts溶液(含有0.6%SDS和10mM EDTA的溶液)从结合至淘洗板上的细胞中收集质粒DNA。通过电穿孔方法将所回收质粒DNA的一半转染到40μl大肠杆菌DH10B(electroMAX DH10B,GIBCO-BRL)中,在1ml SOC培养基中温育1小时后,取50μl样品用于滴度检测,并接种于LB-氨苄青霉素(100μg/ml)培养板上。另一方面,将其余的培养物转移至LB-氨苄青霉素(100μg/ml)液体培养基500ml中并培养。培养过夜后,利用质粒DNA纯化试剂盒(Plasmid-Maxi,QIAGEN)制备质粒DNA,并将其在-20℃冻存。
利用3μl FuGENTM6(Boehringer-Mannheim)将每个库获得的质粒DNA各1μg转染到COS-7细胞中,并如上所述进行第二次淘洗以及质粒DNA的回收和制备。
3.所获cDNA克隆的核苷酸序列以及推导的氨基酸序列的分析
在第一次和第二次淘洗后,从用于滴度检测的培养板中随机收集菌落,分别于2ml LB-氨苄青霉素(100μg/ml)液体培养基中培养后,制备质粒DNA。然后,通过限制性酶分析利用SmaⅠ和NotⅠ筛选另一个cDNA插入片段,并从5’侧进行测序,对它们的核苷酸序列和推导的氨基酸序列分析的结果显示,通过利用TMT方法可以选择性地筛选膜结合蛋白的基因。结果示于表1中。
表1
第一次淘洗
库名称(1000个克隆/库) | 分析的克隆数 | 具有跨膜区的克隆数 | 详细资料(插入片段大小、氨基酸残基数、跨膜区数) |
kappa-1 | 11 | 1 | 细胞色素氧化酶(0.75kb,44aa,1TM) |
kappa-6 | 7 | 3 | NADH脱氢酶(1.7kb,88aa,2TM)NADH脱氢酶(1.7kb,88aa,2TM)ATP合成酶(0.85kb,42aa,1TM) |
第二次淘洗
库名称(1000个克隆/库) | 分析的克隆数 | 具有跨膜区的克隆数 | 详细资料(插入片段大小、氨基酸残基数、跨膜区数) |
kappa-1 | 11 | 4 | ATP合成酶(0.85kb,42aa,1TM)ATP合成酶(0.85kb,42aa,1TM)ATP合成酶(0.85kb,42aa,1TM)细胞色素氧化酶(0.75kb,44aa,1TM) |
kappa-6 | 11 | 3 | NADH脱氢酶(1.2kb,81aa,2TM)NADH脱氢酶(3.5kb,58aa,2TM)多聚T(0.9kb,35aa,1TM) |
由第一次淘洗,获得了已知的膜结合蛋白,细胞色素氧化酶(1个克隆)、NADH脱氢酶(2个克隆)以及ATP合成酶(1个克隆)。在另一方面,由第二次淘洗获得了已知的膜结合蛋白,细胞色素氧化酶(1个克隆)、NADH脱氢酶(2个克隆)以及ATP合成酶(3个克隆)。上述所有均为定位于线粒体内膜上的膜结合蛋白。例如,已知ATP合成酶为一个跨膜区型蛋白,细胞色素氧化酶为两个跨膜区型蛋白,而NADH脱氢酶为15个跨膜区型蛋白。这些结果揭示TMT方法不仅能够分离Ⅰ型膜结合型蛋白,而且可以分离具有多个跨膜区的蛋白。
在第二次淘洗中获得的多聚T序列的克隆可能是由于包括多聚A的cDNA以相反的方向插入。多聚T被翻译成疏水性苯丙氨酸串联排列的氨基酸序列,而且由于其极其富于疏水性而被认为已经分离。
此外,在所收集克隆中含有的膜结合蛋白的百分率在第二次淘洗中比在第一次淘洗中高。这表明通过反复淘洗选择性地富集了膜结合蛋白。
因此,在一个真正的cDNA文库筛选系统中,TMT方法被表明是用于选择性地克隆Ⅰ型膜结合蛋白和包括多个跨膜区的膜结合蛋白的一种有效方法。
工业实用性
由于结构问题,人们认为抗体分子在分泌过程中累积于细胞膜上时以及由于融合蛋白的非天然结构和由于氨基酸的组成而凝集时不容易发挥抗原结合活性。因此,如在本发明中,利用由识别一种抗原的抗体制备的淘洗板可以选择性地筛选在细胞表面功能性地表达抗体融合蛋白的细胞。即,本发明提供了一种通过有效去除在细胞表面具有低或无抗原结合活性的融合蛋白的细胞而非常具有选择性地克隆编码细胞膜结合蛋白的基因的方法。
序列表<110>CHUGAI SEIYAKU KABUSHIKI KAISHA<120>基因克隆的新方法<130>C1-001DP1PCT<150>JP 1998-138652<151>1998-05-20<150>JP 1998-279876<151>1998-10-01<160>39<210>1<211>15<212>PRT<213>人工序列<220><223>对人工序列的描述:肽连接序列<400>1Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser1 5 10 15<210>2<211>1035<212>DNA<213>人<220><221>CDS<222>(1)..(1035)<400>2atg ctg gcc gtc ggc tgc gcg ctg ctg gct gcc ctg ctg gcc gcg ccg 48Met Leu Ala Val Gly Cys Ala Leu Leu Ala Ala Leu Leu Ala Ala Pro1 5 10 15gga gcg gcg ctg gcc cca agg cgc tgc cct gcg cag gag gtg gca aga 96Gly Ala Ala Leu Ala Pro Arg Arg Cys Pro Ala Gln Glu Val Ala Arg
20 25 30ggc gtg ctg acc agt ctg cca gga gac agc gtg act ctg acc tgc ccg 144Gly Val Leu Thr Ser Leu Pro Gly Asp Ser Val Thr Leu Thr Cys Pro
35 40 45ggg gta gag ccg gaa gac aat gcc act gtt cac tgg gtg ctc agg aag 192Gly Val Glu Pro Glu Asp Asn Ala Thr Val His Trp Val Leu Arg Lys
50 55 60ccg gct gca ggc tcc cac ccc agc aga tgg gct ggc atg gga agg agg 240Pro Ala Ala Gly Ser His Pro Ser Arg Trp Ala Gly Met Gly Arg Arg65 70 75 80ctg ctg ctg agg tcg gtg cag ctc cac gac tct gga aac tat tca tgc 288Leu Leu Leu Arg Ser Val Gln Leu His Asp Ser Gly Asn Tyr Ser Cys
85 90 95tac cgg gcc ggc cgc cca gct ggg act gtg cac ttg ctg gtg gat gtt 336Tyr Arg Ala Gly Arg Pro Ala Gly Thr Val His Leu Leu Val Asp Val
100 105 110ccc ccc gag gag ccc cag ctc tcc tgc ttc cgg aag agc ccc ctc agc 384Pro Pro Glu Glu Pro Gln Leu Ser Cys Phe Arg Lys Ser Pro Leu Ser
115 120 125aat gtt gtt tgt gag tgg ggt cct cgg agc acc cca tcc ctg acg aca 432Asn Val Val Cys Glu Trp Gly Pro Arg Ser Thr Pro Ser Leu Thr Thr
130 135 140aag gct gtg ctc ttg gtg agg aag ttt cag aac agt ccg gcc gaa gac 480Lys Ala Val Leu Leu Val Arg Lys Phe Gln Asn Ser Pro Ala Glu Asp145 150 155 160ttc cag gag ccg tgc cag tat tcc cag gag tcc cag aag ttc tcc tgc 528Phe Gln Glu Pro Cys Gln Tyr Ser Gln Glu Ser Gln Lys Phe Ser Cys
165 170 175cag tta gca gtc ccg gag gga gac agc tct ttc tac ata gtg tcc atg 576Gln Leu Ala Val Pro Glu Gly Asp Ser Ser Phe Tyr Ile Val Ser Met
180 185 190tgc gtc gcc agt agt gtc ggg agc aag ttc agc aaa act caa acc ttt 624Cys Val Ala Ser Ser Val Gly Ser Lys Phe Ser Lys Thr Gln Thr Phe
195 200 205cag ggt tgt gga atc ttg cag cct gat ccg cct gcc aac atc aca gtc 672Gln Gly Cys Gly Ile Leu Gln Pro Asp Pro Pro Ala Asn Ile Thr Val
210 215 220act gcc gtg gcc aga aac ccc cgc tgg ctc agt gtc acc tgg caa gac 720Thr Ala Val Ala Arg Asn Pro Arg Trp Leu Ser Val Thr Trp Gln Asp225 230 235 240ccc cac tcc tgg aac tca tct ttc tac aga cta cgg ttt gag ctc aga 768Pro His Ser Trp Asn Ser Ser Phe Tyr Arg Leu Arg Phe Glu Leu Arg
245 250 255tat cgg gct gaa cgg tca aag aca ttc aca aca tgg atg gtc aag gac 816Tyr Arg Ala Glu Arg Ser Lys Thr Phe Thr Thr Trp Met Val Lys Asp
260 265 270ctc cag cat cac tgt gtc atc cac gac gcc tgg agc ggc ctg agg cac 864Leu Gln His His Cys Val Ile His Asp Ala Trp Ser Gly Leu Arg His
275 280 285gtg gtg cag ctt cgt gcc cag gag gag ttc ggg caa ggc gag tgg agc 912Val Val Gln Leu Arg Ala Gln Glu Glu Phe Gly Gln Gly Glu Trp Ser
290 295 300gag tgg agc ccg gag gcc atg ggc acg cct tgg aca gaa tcc agg agt 960Glu Trp Ser Pro Glu Ala Met Gly Thr Pro Trp Thr Glu Ser Arg Ser305 310 315 320cct cca gct gag aac gag gtg tcc acc ccc atg cag gca ctt act act 1008Pro Pro Ala Glu Asn Glu Val Ser Thr Pro Met Gln Ala Leu Thr Thr
325 330 335aat aaa gac gat gat aat att ctc ttc 1035Asn Lys Asp Asp Asp Asn Ile Leu Phe
340 345<2l0>3<211>40<212>DNA<213>人工序列<220><223>对人工序列的描述:“IL6R1”,一种人工合成的引物序列<400>3ttcgaattcc caccatgctg gccggtcggct gcgcgctgct 40<210>4<211>36<212>DNA<213>人工序列<220><223>对人工序列的描述:“IL6R2”一种人工合成的引物序列<400>4ttcgaattcg aagagaatat tatcatcgtc tttatt 36<210>5<211>768<212>DNA<213>人工序列<220><221>CDS<222>(1)..(768)<220><223>对人工序列的描述:一种特别设计的单链Fv基因序列<400>5cag gtc caa ctg cag gag agc ggt cca ggt ctt gtg aga cct agc cag 48Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln1 5 10 15acc ctg agc ctg acc tgc acc gtg tct ggc tac tca att acc agc gat 96Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp
20 25 30cat gcc tgg agc tgg gtt cgc cag cca cct gga cga ggt ctt gag tgg 144His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp
35 40 45att gga tac att agt tat agt gga atc aca acc tat aat cca tct ctc 192Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu
50 55 60aaa tcc aga gtg aca atg ctg aga gac acc agc aag aac cag ttc agc 240Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser65 70 75 80ctg aga ctc agc agc gtg aca gcc gcc gac acc gcg gtt tat tat tgt 288Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
85 90 95gca aga tcc cta gct cgg act acg gct atg gac tac tgg ggt caa ggc 336Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110agc ctc gtc aca gtc tcc tca ggt ggt ggt ggt tcg ggt ggt ggt ggt 384Ser Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
115 120 125tcg ggt ggt ggc gga tcg gac atc cag atg acc cag agc cca agc agc 432Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
130 135 140ctg agc gcc agc gtg ggt gac aga gtg acc atc acc tgt aga gcc agc 480Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser145 150 155 160cag gac atc agc agt tac ctg aat tgg tac cag cag aag cca gga aag 528Gln Asp Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys
165 170 175gct cca aag ctg ctg atc tac tac acc tcc aga ctg cac tct ggt gtg 576Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser Gly Val
180 185 190cca agc aga ttc agc ggt agc ggt agc ggt acc gac ttc acc ttc acc 624Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr
195 200 205atc agc agc ctc cag cca gag gac atc gct acc tac tac tgc caa cag 672Ile Ser Ser Leu Glu Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln
210 215 220ggt aac acg ctt cca tac acg ttc ggc caa ggg acc aag gtg gaa atc 720Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile225 230 235 240aaa cga act gtg gct gca cca tct gtc ttc atc ttc ccg cca tct gat 768Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
245 250 255<210>6<211>32<212>DNA<213>人工序列<220><223>对人工序列的描述:“TMT1”一种人工合成的引物序列<400>6ggtgtcgact cccaggtcca actgcaggag ag 32<210>7<211>32<212>DNA<213>人工序列<220><223>对人工序列的描述:“LINK1”一种人工合成的引物序列<400>7ctcgtcacag tctcctcagg tggtggtggt tc 32<210>8<211>38<212>DNA<213>人工序列<220><223>对人工序列的描述:”LINK3“一种人工合成的引物序列<400>8gacatccaga tgacccagag cccaagcagc ctgagcgc 38<210>9<211>63<212>DNA<213>人工序列<220><223>对人工序列的描述:”SCP-C“一种人工合成的引物序列<400>9gctgaattct tattatttat cgtcatcgtc tttgtagtca agcttatcag atggcgggaa 60gat 63<210>10<211>9<212>PRT<400>10Met Asp Tyr Lys Asp Asp Asp Asp Lys1 5<210>11<211>34<212>DNA<213>人工序列<220><223>对人工序列的描述:“LINK2”,一种人工合成的引物序列<400>11aaccaccacc acctgaggag actgtgacga ggct 34<210>12<211>35<212>DNA<213>人工序列<220><223>对人工序列的描述:“LINK4”,一种人工合成的引物序列<400>12aggctgcttg ggctctgggt catctggatg tccga 35<210>13<211>36<212>DNA<213>人工序列<220><223>对人工序列的描述:“TMT2”,一种人工合成的引物序列<400>13atccgcggcc gcttattatt tatcgtcatc gtcttt 36<210>14<211>19<212>PRT<400>14Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val Asp Ser<210>15<211>106<212>DNA<213>人工序列<220><223>对人工序列的描述:“S-FLAG1”,一种人工合成的寡核苷酸序列<400>15aattcccacc atgggatgga gctgtatcat cctcttcttg gtagcaacag ctacaggtgt 60cgactccgac tacaaagacg atgacgataa aggtaccgcg gccgcg 106<210>16<211>106<212>DNA<213>人工序列<220><223>对人工序列的描述:“S-FLAG2”,一种人工合成的寡核苷酸序列<400>16gatccgcggc cgcggtacct ttatcgtcat cgtctttgta gtcggagtcg acacctgtag 60ctgttgctac caagaagagg atgatacagc tccatcccat ggtggg 106<210>17<211>2995<212>DNA<213>Mus musculus<220><221>CDS<222>(29)..(2839)<400>17gaattccgga catctagagg cagcgaactt gtttccgatt catgctttat catttcttaa 60tttcgtatgt tgggaacatc cctgcaag atg tca gca cca agg att tgg cta 112
Met Ser Ala Pro Arg Ile Trp Leu
1 5gcg caa gct ttg ctt ttt ttc ctc acc act gaa tct ata ggc caa ctt 160Ala Gln Ala Leu Leu Phe Phe Leu Thr Thr Glu Ser Ile Gly Gln Leu
10 15 20ttg gaa ccg tgt ggt tac atc tac cct gaa ttt cca gtt gtc cag cgc 208Leu Glu Pro Cys Gly Tyr Ile Tyr Pro Glu Phe Pro Val Val Gln Arg25 30 35 40ggc tcg aac ttc act gcc att tgt gtg ctg aag gag gcg tgt ctg cag 256Gly Ser Asn Phe Thr Ala Ile Cys Val Leu Lys Glu Ala Cys Leu Gln
45 50 55cat tac tac gtg aat gcc agc tac atc gtg tgg aag acc aac cat gct 304His Tyr Tyr Val Asn Ala Ser Tyr Ile Val Trp Lys Thr Asn His Ala
60 65 70gct gtt ccc agg gag cag gtc act gtc atc aac aga acc acg tcc agt 352Ala Val Pro Arg Glu Gln Val Thr Val Ile Asn Arg Thr Thr Ser Ser
75 80 85gtc acg ttc aca gac gtg gtc ctc ccg agc gtg cag ctc acc tgc aac 400Val Thr Phe Thr Asp Val Val Leu Pro Ser Val Gln Leu Thr Cys Asn
90 95 100atc ctg tcc ttt ggg cag atc gag cag aat gtg tat gga gtc acc atg 448Ile Leu Ser Phe Gly Gln Ile Glu Gln Asn Val Tyr Gly Val Thr Met105 110 115 120ctt tca ggc ttt cct cca gat aaa cct aca aat ttg act tgc att gtg 496Leu Ser Gly Phe Pro Pro Asp Lys Pro Thr Asn Leu Thr Cys Ile Val
125 130 135aat gag ggg aag aat atg ctg tgc cag tgg gac ccc gga agg gag act 544Asn Glu Gly Lys Asn Met Leu Cys Gln Trp Asp Pro Gly Arg Glu Thr
140 145 150tac ctt gaa aca aac tac act ttg aaa tca gag tgg gca aca gag aag 592Tyr Leu Glu Thr Asn Tyr Thr Leu Lys Ser Glu Trp Ala Thr Glu Lys
155 160 165ttt cct gat tgc cag tca aag cat ggc act tca tgt atg gtc agc tac 640Phe Pro Asp Cys Gln Ser Lys His Gly Thr Ser Cys Met Val Ser Tyr
170 175 180atg ccc acc tat tat gtc aac att gaa gtc tgg gtg gaa gca gag aat 688Met Pro Thr Tyr Tyr Val Asn Ile Glu Val Trp Val Glu Ala Glu Asn185 190 195 200gcc ctt ggg aag gtc tcc tca gag tct atc aat ttt gac ccc gtg gat 736Ala Leu Gly Lys Val Ser Ser Glu Ser Ile Asn Phe Asp Pro Val Asp
205 210 215aaa gtg aaa ccc acc cca cca tat aat tta tca gtg acc aac tca gaa 784Lys Val Lys Pro Thr Pro Pro Tyr Asn Leu Ser Val Thr Asn Ser Glu
220 225 230gaa tta tcc agt ata tta aag cta tca tgg gtc agt tca ggg ctg ggc 832Glu Leu Ser Ser Ile Leu Lys Leu Ser Trp Val Ser Ser Gly Leu Gly
235 240 245ggt ctt tta gat cta aag tct gac atc caa tat agg acc aaa gat gcc 880Gly Leu Leu Asp Leu Lys Ser Asp Ile Gln Tyr Arg Thr Lys Asp Ala
250 255 260tca act tgg atc cag gtc cct ctt gaa gat aca atg tot cct cga act 928Ser Thr Trp Ile Gln Val Pro Leu Gle Asp Thr Met Ser Pro Arg Thr265 270 275 280tcc ttc act gtg cag gac ctc aag cct ttt aca gaa tat gtg ttt agg 976Ser Phe Thr Val Gln Asp Leu Lys Pro Phe Thr Glu Tyr Val Phe Arg
285 290 295atc cgg tcc att aag gac agt ggg aag ggc tac tgg agt gac tgg agt 1024Ile Arg Ser Ile Lys Asp Ser Gly Lys Gly Tyr Trp Ser Asp Trp Ser
300 305 310gag gag gct agt ggg acc aca tac gaa gac aga cca tcc aga cca cca 1072Glu Glu Ala Ser Gly Thr Thr Tyr Glu Asp Arg Pro Ser Arg Pro Pro
315 320 325agt ttc tgg tat aag aca aat cca tcc cat ggg cag gaa tat aga tct 1120Ser Phe Trp Tyr Lys Thr Asn Pro Ser His Gly Gln Glu Tyr Arg Ser
330 335 340gta cgg ctc ata tgg aag gca ctg cct ctt tct gaa gcc eat ggg aaa 1168Val Arg Leu Ile Trp Lys Ala Leu Pro Leu Ser Glu Ale Asa Gly Lys345 350 355 360atc ttg gat tat gaa gtg att ctt acg cag tca aag tcc gtc tca caa 1216Ile Leu Asp Tyr Glu Val Ile Leu Thr Gln Ser Lys Ser Val Ser Gln
365 370 375acg tac aca gtc act ggc aca gag ctg acc gtg aat ctc acc aat gac 1264Thr Tyr Thr Val Thr Gly Thr Glu Leu Thr Val Asn Leu Thr Asn Asp
380 385 390cgc tat gtc gcg tct cta gca gca aga aac aag gtg ggc aaa tca gct 1312Arg Tyr Val Ala Ser Leu Ala Ala Arg Asn Lys Val Gly Lys Ser Ala
395 400 405gca gct gtc ctc acc atc ccc agc ccc cac gtc aca gct gct tat tct 1360Ala Ala Val Leu Thr Ile Pro Ser Pro His Val Thr Ala Ala Tyr Ser
410 415 420gta gtg aat ctt aaa gca ttt cca aaa gat aac ctg ctc tgg gtg gaa 1408Val Val Asn Leu Lys Ala Phe Pro Lys Asp Asn Leu Leu Trp Val Glu425 430 435 440tgg aca cct cca cct aaa ccc gtg agc aag tac atc tta gag tgg tgt 1456Trp Thr Pro Pro Pro Lys Pro Val Ser Lys Tyr Ile Leu Glu Trp Cys
445 450 455gtg ttg tca gag aac gca ccc tgt gtt gaa gac tgg cag cag gaa gac 1504Val Leu Ser Glu Asn Ala Pro Cys Val Glu Asp Trp Gln Gln Glu Asp
460 465 470gct acc gtg aat cgg acc cac ttg aga gga cgc ctc ctg gag agc aag 1552Ala Thr Val Asn Arg Thr His Leu Arg Gly Arg Leu Leu Glu Ser Lys
475 480 485tgc tat caa atc aca gta act ccc gta ttc gcc acg ggg ccc gga ggc 1600Cys Tyr Gln Ile Thr Val Thr Pro Val Phe Ala Thr Gly Pro Gly Gly
490 495 500tct gag tcc ttg aag gcg tac ctc aaa caa gcc gct cct gcc aga gga 1648Ser Glu Ser Leu Lys Ala Tyr Leu Lys Gln Ala Ala Pro Ala Arg Gly505 510 515 520ccg act gtt cgg aca aag aaa gtg ggg aaa aat gaa gct gtc tta gcg 1696Pro Thr Val Arg Thr Lys Lys Val Gly Lys Asn Glu Ala Val Leu Ala
525 530 535tgg gac cag att cct gtg gac gac csg aat ggc ttc att aga aac tac 1744Trp Asp Gln Ile Pro Val Asp Asp Gln Asn Gly Phe Ile Arg Asn Tyr
540 545 550tcc ata tct tac aga acc agc gtg gga aag gag atg gtt gtg cat gtg 1792Ser Ile Ser Tyr Arg Thr Ser Val Gly Lys Glu Met Val Val His Val
555 560 565gat tct tct cac acg gag tac acg ctg tcc tct ctg agt agt gat acg 1840Asp Ser Ser His Thr Glu Tyr Thr Leu Ser Ser Leu Ser Ser Asp Thr
570 575 580ttg tac atg gtc cga atg gcc gcg tac aca gat gaa ggt ggg aaa gat 1888Leu Tyr Met Val Arg Met Ala Ala Tyr Thr Asp Glu Gly Gly Lys Asp585 590 595 600ggg ccg gaa ttc act ttt aca aca cca aag ttc gct caa gga gaa ata 1936Gly Pro Glu Phe Thr Phe Thr Thr Pro Lys Phe Ala Gln Gly Glu Ile
605 610 615gaa gcc ata gtc gtg cct gtg tgc tta gcc ttc ctc ctg aca acc ctg 1984Glu Ala Ile Val Val Pro Val Cys Leu Ala Phe Leu Leu Thr Thr Leu
620 625 630ctg ggc gtc ttg ttc tgc ttt aac aaa cga gac cta att aaa aaa cac 2032Leu Gly Val Leu Phe Cys Phe Asn Lys Arg Asp Leu Ile Lys Lys His
635 640 645atc tgg cct aat gtt cct gat cct tcc aag agt caL att gcc cag tgg 2080Ile Trp Pro Asn Val Pro Asp Pro Ser Lys Ser His Ile Ala Gln Trp
650 655 660tca cct cac acc ccc cca agg cac aat ttt aac tcc aaa gat caa atg 2128Ser Pro His Thr Pro Pro Arg His Asn Phe Asn Ser Lys Asp Gln Met665 670 675 680tac tcg gac ggc aat ttc act gat gta agc gtt gtg gaa ata gaa gca 2176Tyr Ser Asp Gly Asn Phe Thr Asp Val Ser Val Val Glu Ile Glu Ala
685 690 695aac aac aag aag cct tgt cca gat gac ctg aag tcc gtg gac ctg ttc 2224Asn Asn Lys Lys Pro Cys Pro Asp Asp Leu Lys Ser Val Asp Leu Phe
700 705 710aag aag gag aaa gtg agt aca gaa ggg cac agc agt ggc atc ggg ggc 2272Lys Lys Glu Lys Val Ser Thr Glu Gly His Ser Ser Gly Ile Gly Gly
715 720 725tct tca tgc atg tcc tcc tcc agg ccc agc atc tcc agc aac gag gag 2320Ser Ser Cys Met Ser Ser Ser Arg Pro Ser Ile Ser Ser Asn Glu Glu
730 735 740aat gag tct gct cag agc acc gcc agc acg gtc gag tac tcc act gtg 2368Asn Glu Ser Ala Gln Ser Thr Ala Ser Thr Val Glu Tyr Ser Thr Val745 750 755 760gtg cac agc ggc tac agg cac cag gtc ccg tcc gtg caa gtg ttc tca 2416Val His Ser Gly Tyr Arg His Gln Val Pro Ser Val Gln Val Phe Ser
765 770 775agg tcc gag tcc acc cag ccc ctg cta gac tcg gag gag cgg cca gaa 2464Arg Ser Glu Ser Thr Gln Pro Leu Leu Asp Ser Glu Glu Arg Pro Glu
780 785 790gac ctg cag ctg gtg gat agt gta gac ggt ggg gat gag atc ttg ccc 2512Asp Leu Gln Leu Val Asp Ser Val Asp Gly Gly Asp Glu Ile Leu Pro
795 800 805agg caa ccg tat ttc aag cag aac tgc agt cag cct gaa gcc tgt cca 2560Arg Gln Pro Tyr Phe Lys Gln Asn Cys Ser Gln Pro Glu Ala Cys Pro
810 815 820gag att tca cat ttt gaa agg tca aac cag gtt ttg tcc ggc aat gag 2608Glu Ile Ser His Phe Glu Arg Ser Asn Gln Val Leu Ser Gly Asn Glu825 830 835 840gag gat ttt gtc aga ctg aag cag cag cag gtt tca gat cac att tct 2658Glu Asp Phe Val Arg Leu Lys Gln Gln Gln Val Ser Asp His Ile Ser
845 850 855cag ccc tat gga tcc gag caa cgg agg ctg ttt cag gaa ggc tct aca 2704Gln Pro Tyr Gly Ser Glu Gln Arg Arg Leu Phe Gln Glu Gly Ser Thr
860 865 870gcg gat gct ctt ggc acg ggg gct gat gga cag atg gag aga ttt gaa 2752Ala Asp Ala Leu Gly Thr Gly Ala Asp Gly Gln Met Glu Arg Phe Glu
875 880 885tct gtt gga atg gag acc aca att gat gaa gaa att ccc aaa agt tac 2800Ser Val Gly Met Glu Thr Thr Ile Asp Glu Glu Ile Pro Lys Ser Tyr
890 895 900ttg cca cag act gta aga caa ggt ggc tac atg ccg cag tgaaggactg 2849Leu Pro Gln Thr Val Arg Gln Gly Gly Tyr Met Pro Gln905 910 915gctcctgaac ttcagcagga actgcaaaat aaagctaaag acgagtggct tcagatgaga 2909aacagtcctc actccctgaa gataggcatt gcctctaagg acaaagtcac acctgggccg 2969tctccattcc agagtagctg gaattc 2995<210>18<2l1>27<212>DNA<213>人工序列<220><223>对人工序列的描述:“mgp1”,一种人工合成的引物序列<400>18cccaagcttg aattcacttt tacaaca 27<210>19<211>29<212>DNA<213>人工序列<220><223>对人工序列的描述:“mgp3”,一种人工合成的引物序列<400>19tttgcggccg cgaattccag ctactctgg 29<210>20<211>33<212>DNA<213>人工序列<220><223>对人工序列的描述:“mgp2”,一种人工合成的引物序列<400>20cccaagcttg aattcaaaaa acacatctgg ctt 33<210>21<211>1662<212>DNA<213>人工序列<220><221>CDS<222>(11)..(1648)<220><223>对人工序列的描述:“gpM1-BvGS3”,一种特别设计的单链Fv基因序列<400>21gaattccacc atg gga tgg agc tgt atc atc ctc ttc ttg gta gca aca 49
Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr
1 5 10gct aca ggt gtc gac tcc cag gtc caa ctg cag gag agc ggt cca ggt 97Ala Thr Gly Val Asp Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly
15 20 25ctt gtg aga cct agc cag acc ctg agc ctg acc tgc acc gtg tct ggc 145Leu Val Arg Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly30 35 40 45tac tca att acc agc gat cat gcc tgg agc tgg gtt cgc cag cca cct 193Tyr Ser Ile Thr Ser Asp His Ala Trp Ser Trp Val Arg Gln Pro Pro
50 55 60gga cga ggt ctt gag tgg att gga tac att agt tat agt gga atc aca 241Gly Arg Gly Leu Glu Trp Ile Gly Tyr Ile Set Tyr Ser Gly Ile Thr
65 70 75acc tat aat cca tct ctc aaa tcc aga gtg aca atg ctg aga gac acc 289Thr Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Met Leu Arg Asp Thr
80 85 90agc aag aac cag ttc agc ctg ags ctc agc agc gtg aca gcc gcc gac 337Ser Lys Asn Gln Phe Ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp
95 100 105acc gcg gtt tat tat tgt gca aga tcc cta gct cgg act acg gct atg 385Thr Ala Val Tyr Tyr Cys Ala Arg Ser Leu Ala Arg Thr Thr Ala Met110 115 120 125gac tac tgg ggt caa ggc agc ctc gtc aca gtc tcc tca ggt ggt ggt 433Asp Tyr Trp Gly Gln Gly Ser Leu Val Thr Val Ser Ser Gly Gly Gly
130 135 140ggt tcg ggt ggt ggt ggt tcg ggt ggt ggc gga tcg gac atc cag atg 481Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met
145 150 155acc cag agc cca agc agc ctg agc gcc agc gtg ggt gac aga gtg acc 529Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr
160 165 170atc acc tgt aga gcc agc cag gac atc agc agt tac ctg aat tgg tac 577Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Tyr Leu Ash Trp Tyr
175 180 185cag cag aag cca gga aag gct cca aag ctg ctg atc tac tac acc tcc 625Gln Gln Lys Pro Gly Lys Ala Pro Lye Leu Leu Ile Tyr Tyr Thr Ser190 195 200 205aga ctg cac tct ggt gtg cca agc aga ttc agc ggt agc ggt agc ggt 673Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly
210 215 220acc gac ttc acc ttc acc atc agc agc ctc cag cca gag gac atc gct 721Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala
225 230 235acc tac tac tgc caa cag ggt aac acg ctt cca tac ecg ttc ggc caa 769Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln
240 245 250ggg acc aag gtg gaa atc aaa tct aga ggt ggt ggt ggt tcg ggt ggt 817Gly Thr Lys Val Glu Ile Lys Ser Arg Gly Gly Gly Gly Ser Gly Gly
255 260 265ggt ggt tcg ggt ggt ggc gga tcg gtc gac tcc cag gtc caa ctg cag 865Gly Gly Ser Gly Gly Gly Gly Ser Val Asp Ser Gln Val Gln Leu Gln270 275 280 285gag agc ggt cca ggt ctt gtg aga cct agc cag acc ctg agc ctg acc 913Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln Thr Leu Ser Leu Thr
290 295 300tgc acc gtg tct ggc tac tca att acc agc gat cat gcc tgg agc tgg 961Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp His Ala Trp Ser Trp
305 310 315gtt cgc cag cca cct gga cga ggt ctt gag tgg att gga tac att agt 1009Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp Ile Gly Tyr Ile Ser
320 325 330tat agt gga atc aca acc tat aat cca tct ctc aaa tcc aga gtg aca 1057Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr
335 340 345atg ctg aga gac acc agc aag aac cag ttc agc ctg aga ctc agc agc 1105Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu Arg Leu Ser Ser350 355 360 365gtg aca gcc gcc gac acc gcg gtt tat tat tgt gca aga tcc cta gct 1153Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Leu Ala
370 375 380cgg act acg gct atg gac tac tgg ggt caa ggc agc ctc gtc aca gtc 1201Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly Ser Leu Val Thr Val
385 390 395tcc tca ggt ggt ggt ggt tcg ggt ggt ggt ggt tcg ggt ggt ggc gga 1249Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
400 405 410tcg gac atc cag atg acc cag agc cca agc agc ctg agc gcc agc gtg 1297Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val
415 420 425ggt gac aga gtg acc atc acc tgt aga gcc agc cag gac atc agc agt 1345Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser430 435 440 445tac ctg aat tgg tac cag cag aag cca gga aag gct cca aag ctg ctg 1393Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lya Leu Leu
450 455 460atc tac tac acc tcc aga ctg cac tct ggt gtg cca agc aga ttc agc 1441Ile Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser
465 470 475ggt agc ggt agc ggt acc gac ttc acc ttc acc atc agc agc ctc cag 1489Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln
480 485 490cca gag gac atc gct acc tac tac tgc caa cag ggt aac acg ctt cca 1537Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro
495 500 505tac acg ttc ggc caa ggg acc aag atg gaa atc aaa cga act gtg gct 1585Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala510 515 520 525gca cca tct gtc ttc atc ttc ccg cca tct gat aag ctt gac tac aaa 1633Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Lys Leu Asp Tyr Lys
530 535 540gac gat gac gat aaa taataagcgg ccgc 1662Asp Asp Asp Asp Lys
545<210>22<211>72<212>DNA<213>人工序列<220><223>对人工序列的描述:“BvGS3”,一种人工合成的引物序列<400>22ggagtcgacc gatccgccac cacccgaacc accaccaccc gaaccaccac cacctttat 60ttccaccttg gt 72<210>23<211>780<212>DNA<213>人工序列<220><221>CDS<222>(1)..(780)<220><223>对人工序列的描述:“shPM1(ΔEL)”,一种特别设计的单链Fv基因序列<400>23atg gga tgg agc tgt atc atc ctc ttc ttg gta gca aca gct aca ggt 48Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15gtc gac tcc cag gtc caa ctg cag gag agc ggt cca ggt ctt gtg aga 96Val Asp Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg
20 25 30cct agc cag acc ctg agc ctg acc tgc acc gtg tct ggc tac tca att 144Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile
35 40 45acc agc gat cat gcc tgg agc tgg gtt cgc cag cca cct gga cga ggt 192Thr Ser Asp His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly
50 55 60ctt gag tgg att gga tac att agt tat agt gga atc aca acc tat aat 240Leu Glu Trp Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn65 70 75 80cca tct ctc aaa tcc aga gtg aca atg ctg aga gac acc agc aag aac 288Pro Ser Leu Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn
85 90 95cag ttc agc ctg aga ctc agc agc gtg aca gcc gcc gac acc gcg gtt 336Gln Phe Ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val
100 105 110tat tat tgt gca aga tcc cta gct cgg act acg gct atg gac tac tgg 384Tyr Tyr Cys Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp
115 120 125ggt caa ggc agc ctc gtc aca gtc tcc tca ggt ggt ggt ggt tcg ggt 432Gly Gln Gly Ser Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly
130 135 140ggt ggt ggt tcg ggt ggt ggc gga tcg gac atc cag atg acc cag agc 480Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser145 150 155 160cca agc agc ctg agc gcc agc gtg ggt gac aga gtg acc atc acc tgt 528Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys
165 170 175aga gcc agc cag gac atc agc agt tac ctg aat tgg tac cag cag aag 576Arg Ala Ser Gln Asp Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys
180 185 190cca gga aag gct cca aag ctg ctg atc tac tac acc tcc aga ctg cac 624Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His
195 200 205tct ggt gtg cca agc aga ttc agc ggt agc ggt agc ggt acc gac ttc 672Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
210 215 220acc ttc acc atc agc agc ctc cag cca gag gac atc gct acc tac tac 720Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr225 230 235 240tgc caa cag gga aat act tta cca tac acg ttc ggc caa ggg acc aag 768Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys
245 250 255gtg gaa atc aaa 780Val Glu Ile Lys
260<210>24<211>321<212>DNA<213>人<220><221>CDS<222>(1)..(321)<400>24cga act gtg gct gca cca tct gtc ttc atc ttc ccg cca tct gat gag 48Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu1 5 10 15cag ttg aaa tct gga act gcc tct gtt gtg tgc ctg ctg aat aac ttc 96Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
20 25 30tat ccc aga gag gcc aaa gta cag tgg aag gtg gat aac gcc ctc caa 144Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
35 40 45tcg ggt aac tcc cag gag agt gtc aca gag cag gac agc aag gac agc 192Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
50 55 60acc tac agc ctc agc agc acc ctg acg ctg agc aaa gca gac tac gag 240Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu65 70 75 80aaa cac aaa gtc tac gcc tgc gaa gtc acc cat cag ggc ctg agc tcg 288Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln G1y Leu Ser Ser
85 90 95ccc gtc aca aag agc ttc aac agg gga gag tct 321Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Ser
100 105<210>25<211>363<212>DNA<213>人<220><221>CDS<222>(1)..(363)<400>25gtg gcc ctg cac agg ccc gat gtc tac ttg ctg cca cca gcc cgg gag 48Val Ala Leu His Arg Pro Asp Val Tyr Leu Leu Pro Pro Ala Arg Glu1 5 10 15cag ctg aac ctg cgg gag tcg gcc acc atc acg tgc ctg gtg acg ggc 96Gln Leu Asn Leu Arg Glu Ser Ala Thr Ile Thr Cya Leu Val Thr Gly
20 25 30ttc tct ccc gcg gac gtc ttc gtg cag tgg atg cag agg ggg cag ccc 144Phe Ser Pro Ala Asp Val Phe Val Gln Trp Met Gln Arg Gly Gln Pro
35 40 45ttg tcc ccg gag aag tat gtg acc agc gcc cca atg cct gag ccc cag 192Leu Ser Pro Glu Lys Tyr Val Thr Ser Ala Pro Met Pro Glu Pro Gln
50 55 60gcc cca ggc cgg tac ttc gcc cac agc atc ctg acc gtg tcc gaa gag 240Ala Pro Gly Arg Tyr Phe Ala His Ser Ile Leu Thr Val Ser Glu Glu65 70 75 80gaa tgg aac acg ggg gag acc tac acc tgc atg gcc cat gag gcc ctg 288Glu Trp Asn Thr Gly Glu Tnr Tyr Thr Cys Val Ala His Glu Ala Leu
85 90 95ccc aac agg gtc acc gag agg acc gtg gac aag tcc acc gag ggg gag 336Pro Asn Arg Val Thr Glu Arg Thr Val Asp Lys Ser Thr Glu Gly Glu
100 105 110gtg agc gcc gac gag gag ggc ttt gag 363Val Ser Ala Asp Glu Glu Gly Phe Glu
115 120<210>26<211>1101<212>DNA<213>人工序列<220><221>CDS<222>(1)..(1101)<220><223>对人工序列的描述:“shPM1-Kappa”,一种特别设计的单链Fv基因序列<400>26atg gga tgg agc tgt atc atc ctc ttc ttg gta gca aca gct aca ggt 48Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15gtc gac tcc cag gtc caa ctg cag gag agc ggt cca ggt ctt gtg aga 96Val Asp Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg
20 25 30cct agc cag acc ctg agc ctg acc tgc acc gtg tct ggc tac tca att 144Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile
35 40 45acc agc gat cat gcc tgg agc tgg gtt cgc cag cca cct gga cga ggt 192Thr Ser Asp His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly
50 55 60ctt gag tgg att gga tac att agt tat agt gga atc aca acc tat aat 240Leu Glu Trp Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn 65 70 75 80cca tct ctc aaa tcc aga gtg aca atg ctg aga gac acc agc aag aac 288Pro Ser Leu Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn
85 90 95cag ttc agg ctg aga ctc agc agc gtg aca gcc gcc gac acc gcg gtt 336Gln Phe Ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val
100 105 110tat tat tgt gca aga tcc cta gct cgg act acg gct atg gac tac tgg 384Tyr Tyr Cys Ala Arg Ser Leu Ala Ars Thr Thr Ala Met Asp Tyr Trp
115 120 125ggt caa ggc agc ctc atc aca gtc tcc tca ggt ggt ggt ggt tcg ggt 432Gly Gln Gly Ser Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly
l30 135 140ggt ggt ggt tcg ggt ggt ggc gga tcg gac atc cag atg acc cag agc 480Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser145 150 155 160cca agc agc ctg agc gcc agc gtg ggt gac asa gtg acc atc acc tgt 528Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys
165 170 175aga gcc agc cag gac atc agc agt tac ctg aat tgg tac cag cag aag 576Arg Ala Ser Gln Asp Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys
180 185 190cca gga aag gct cca aag ctg ctg atc tac tac acc tcc aga ctg cac 624Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His
195 200 205tct ggt gtg cca agc aga ttc agc sgt agc ggt agc ggt acc gac ttc 672Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
210 215 220acc ttc acc atc agc agc ctc cag cca gag gac atc gct acc tac tac 720Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr225 230 235 240tgc caa cag gga aat act tta cca tac acg ttc ggc caa ggg acc aag 768Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys
245 250 255gtg gaa atc aaa cga act gtg gct gca cca tct gtc ttc atc ttc ccg 816Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro
260 265 270cca tct gat gag cag ttg aaa tct gga act gcc tct gtt gtg tgc ctg 864Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu
275 280 285ctg aat aac ttc tat ccc aga gag gcc aaa gta cag tgg aag gtg gat 912Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp
290 295 300aac gcc ctc caa tcg ggt aac tcc cag gag agt gtc aca gag cag gac 960Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp305 310 315 320agc aag gac agc acc tac agc ctc agc agc acc ctg acg ctg agc aaa 1008Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys
325 330 335gca gac tac gag aaa cac aaa gtc tac gcc tgc gaa gtc acc cat cag 1056Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln
340 345 350ggc ctg agc tcg ccc gtc aca aag agc ttc aac agg gga gag tct 1101Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Ser
355 360 365<210>27<211>1143<212>DNA<213>人工序列<220><221>CDS<222>(1)..(1143)<220><223>对人工序列的描述:“shPM1-MCH4”,一种特别设计的单链Fv基因序列<400>27atg gga tgg agc tgt atc atc ctc ttc ttg gta gca aca gct aca ggt 48Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15gtc gac tcc cag tgc caa ctg cag gag agc ggt cca ggt ctt gtg aga 96Vel Asp Ser Gln Vel Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg
20 25 30cct agc cag acc ctg agc ctg acc tgc acc gtg tct ggc tac tca att 144Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile
35 40 45acc agc gat cat gcc tgg agc tgg gtt cgc cag cca cct gga cga ggt 192Thr Ser Asp His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly
50 55 60ctt gag tgg att gga tac att atg tat agt gga atc aca acc tat aat 240Leu Glu Trp Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn65 70 75 80cca tct ctc aaa tcc aga gtg aca atg ctg aga gac acc agc aag aac 288Pro Ser Leu Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn
85 90 95cag ttc agc ctg aga ctc acc agc gtg aca gcc gcc gac acc gcg gtt 336Gln Phe Ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val
100 105 110tat tat tgt gca aga tcc cta gct cgg act acg gct atg gac tac tgg 384Tyr Tyr Cys Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp
115 120 125ggt caa ggc agc ctc gtc aca gtc tcc tca ggt ggt ggt ggt tcg ggt 432Gly Gln Gly Ser Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly
130 135 140ggt ggt ggt tcg ggt ggt ggc gga tcg gac atc cag atg acc cag agc 480Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp lle Gln Met Thr Gln Ser145 150 155 160cca agc agc ctg agc gcc agc gtg ggt gac aga gtg acc atc acc tgt 528Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys
165 170 175aga gcc agc cag gac atc agc agt tac ctg aat tgg tac cag cag aag 576Arg Ala Set Gln Asp Ile Set Ser Tyr Leu Asn Trp Tyr Gln Gln Lys
180 185 190cca gga aag gct cca aag ctg ctg atc tac tac acc tcc aga ctg cac 624Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His
195 200 205tct ggt gtg cca agc aga ttc agc ggt agc ggt agc ggt acc gac ttc 672Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
210 215 220acc ttc acc atc agc agc ctc cag cca gag gac atc gct acc tac tac 720Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr225 230 235 240tgc caa cag gga aat act tta cca tac acg ttc ggc caa ggg acc aag 768Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys
245 250 255gtg gaa atc aaa gtg gcc ctg cac agg ccc gat gtc tac ttg ctg cca 816Val Glu Ile Lys Val Ala Leu His Arg Pro Asp Val Tyr Leu Leu Pro
260 265 270cca gcc cgg gag cag ctg aac ctg cgg gag tcg gcc acc atc acg tgc 864Pro Ala Arg Glu Gln Leu Aaa Leu Arg Glu Ser Ala Thr Ile Tnr Cys
275 280 285ctg gtg acg ggc ttc tct ccc gcg gac gtc ttc gtg cag tgg atg cag 912Leu Val Thr Gly Phe Ser Pro Ala Asp Val Phe Val Gln Trp Met Gln
290 295 300agg ggg cag ccc ttg tcc ccg gag aag tat gtg acc agc gcc cca atg 960Arg Gly Gln Pro Leu Ser Pro Glu Lys Tyr Val Thr Ser Ala Pro Met305 310 315 320cct gag ccc cag gcc cca ggc acg tac ttc gcc cac agc atc ctg acc 1008Pro Glu Pro Gln Ala Pro Gly Arg Tyr Phe Ala His Ser Ile Leu Thr
325 330 335gtg tcc gaa gag gaa tgg aac acg ggg gag acc tac acc tgc gtg gcc 1056Val Ser Glu Glu Glu Trp Asn Thr Gly Glu Thr Tyr Thr Cys Val Ala
340 345 350cat gag gcc ctg ccc aac agg gtc acc gag agg acc gtg gac aag tcc 1104His Glu Ala Leu Pro Asn Arg Val Thr Glu Arg Thr Val Asp Lys Ser
355 360 365acc gag ggg gag gtg agc gcc gac gag gag ggc ttt gag 1143Thr Glu Gly Glu Val Ser Ala Asp Glu Glu Gly Phe Glu
370 375 380<210>28<211>18<212>DNA<213>人工序列<220><223>对人工序列的描述:“EF-1”,一种人工合成的引物序列<400>28cagacagtgg ttcaaagt 18<210>29<211>107<212>DNA<213>人工序列<220><223>对人工序列的描述:“SCP-C2”,一种人工合成的引物序列<400>29aaagcggccg cttattattt atcgtcatcg tctttgtagt ctgaagcttt gatttccacc 60ttggtccctt ggccgaacgt gtatggtaaa gtatttccct gttggca 107<210>30<211>1557<212>DNA<213>人工序列<220><221>CDS<222>(1)..(1557)<220><223>对人工序列的描述:“shPM1(ΔEL)-BvGS3”,一种特别设计的单链Fv基因序列<400>30atg gga tgg agc tgt atc atc ctc ttc ttg gta gca aca gct aca ggt 48Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15gtc gac tcc cag gtc caa ctg cag gag agc ggt cca ggt ctt gtg aga 96Val Asp Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg
20 25 30cct agc cag acc ctg agc ctg acc tgc acc gtg tct ggc tac tca att 144Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile
35 40 45acc agc gat cat gcc tgg agc tgg gtt cgc cag cca cct gga cga ggt 192Thr Ser Asp His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly
50 55 60ctt gag tgg att gga tac att agt tat agt gga atc aca acc tat aat 240Leu Glu Trp Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn65 70 75 80cca tct ctc aaa tcc aga gtg aca atg ctg aga gac acc agc aag aac 288Pro Ser Leu Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn
85 90 95cag ttc agc ctg aga ctc agc agc gtg aca gcc gcc gac acc gcg gtt 336Gln Phe Ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val
100 105 110tat tat tgt gca aga tcc cta gct cgg act acg gct atg gac tac tgg 384Tyr Tyr Cys Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp
115 120 125ggt caa ggc agc ctc gtc aca gtc tcc tca ggt ggt ggt ggt tcg ggt 432Gly Gln Gly Ser Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly
130 135 140ggt ggt ggt tcg ggt ggt ggc gga tcg gac atc cag atg acc cag agc 480Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser145 150 155 160cca agc agc ctg agc gcc agc gtg ggt gac aga gtg acc atc acc tgt 528Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys
165 170 175aga gcc agc cag gac atc agc agt tac ctg aat tgg tac cag cag aag 576Arg Ala Ser Gln Asp Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys
180 185 190cca gga aag gct cca aag ctg ctg atc tac tac acc tcc aga ctg cac 624Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His
195 200 205tct ggt gtg cca agc aga ttc agc ggt agc ggt agc ggt acc gac ttc 672Sar Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
210 215 220acc ttc acc atc agc agc ctc cag cca gag gac atc gct acc tac tac 720Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr225 230 235 240tgc caa cag ggt aac acg ctt cca tac acg ttc ggc caa ggg acc aag 768Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys
245 250 255gtg gaa atc aaa ggt ggt ggt ggt tcg ggt ggt ggt ggt tcg ggt ggt 816Val Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
260 265 270ggc gga tcg gtc gac tcc cag gtc caa ctg cag gag agc ggt cca ggt 864Gly Gly Ser Val Asp Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly
275 280 285ctt gtg aga cct agc cag acc ctg agc ctg acc tgc acc gtg tct ggc 912Leu Val Arg Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly
290 295 300tac tca att acc agc gat cat gcc tgg agc tgg gtt cgc cag cca cct 960Tyr Ser Ile Thr Ser Asp His Ala Trp Ser Trp Val Arg Gln Pro Pro305 310 315 320gga cga ggt ctt gag tgg att gga tac att agt tat agt gga atc aca 1008Gly Arg Gly Leu Glu Trp Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr
325 330 335acc tat aat cca tct ctc aaa tcc aga gtg aca atg ctg aga gac acc 1056Thr Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Met Leu Arg Asp Thr
340 345 350agc aag aac cag ttc agc ctg aga ctc agc agc gtg aca gcc gcc gac 1104Ser Lys Asn Gln Phe Ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp
355 360 365acc gcg gtt tat tat tgt gca aga tcc cta gct cgg act acg gct atg 1152Thr Ala Val Tyr Tyr Cys Ala Arg Ser Leu Ala Arg Thr Thr Ala Met
370 375 Arggac tac tgg ggt caa ggc agc ctc gtc aca gtc tcc tca ggt ggt ggt 1200Asp Tyr Trp Gly Gln Gly Ser Leu Val Thr Val Ser Ser Gly Gly Gly385 390 395 400ggt tcg ggt ggt ggt ggt tcg ggt ggt ggc gga tcg gac atc cag atg 1248Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met
405 410 415acc cag agc cca agc agc ctg agc gcc agc gtg ggt gac aga tgt acc 1296Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr
420 425 430atc acc tgt aga gcc agc cag gac atc agc agt tac ctg aat tgg tac 1344Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Tyr Leu Asn Trp Tyr
435 440 445cag cag aag cca gga aag gct cca aag ctg ctg atc tac tac acc tcc 1392Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser
450 455 460aga ctg cac tct ggt gtg cca agc aga ttc agc ggt agc ggt agc ggt 1440Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly465 470 475 480acc gac ttc acc ttc acc atc agc agc ctc cag cca gag gac atc gct 1488Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala
485 490 495acc tac tac tgc caa cag gga aat act tta cca tac acg ttc ggc caa 1536Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln
500 505 510ggg acc aag gtg gaa atc aaa 1557Gly Thr Lys Val Glu Ile Lys
5l5<210>31<211>29<212>DNA<213>人工序列<220><223>对人工序列的描述:“Kappa1”,一种人工合成的引物序列<400>31ccgccatctg atgagcagtt gaaatctgg 29<210>32<211>54<212>DNA<213>人工序列<220><223>对人工序列的描述:“Kappa2”,一种人工合成的引物序列<400>32ttatttatcg tcatcgtctt tgtagtcaag cttagactct cccctgttga agct 54<210>33<211>29<212>DNA<213>人工序列<220><223>对人工序列的描述:“SCP-K”,一种人工合成的引物序列<400>33ttcaactgct catcagatgg cgggaagat 29<210>34<211>1878<212>DNA<213>人工序列<220><221>CDS<222>(1)..(1878)<220><223>对人工序列的描述:“shPM1-Kappa-BvGS3”,一种特别设计的单链Fv基因序列<400>34atg gga tgg agc tgt atc atc ctc ttc ttg gta gca aca gct aca ggt 48Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15gtc gac tcc cag gtc caa ctg cag gag agc ggt cca ggt ctt gtg aga 96Val Asp Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg
20 25 30cct agc cag acc ctg agc ctg acc tgc acc gtg tct ggc tac tca att 144Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile
35 40 45acc agc gat cat gcc tgg agc tgg gtt cgc cag cca cct gga cga ggt 192Thr Ser Asp His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly
50 55 60ctt gag tgg att gga tac att agt tat agt gga atc aca acc tat aat 240Leu Glu Trp Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn65 70 75 80cca tct ctc aaa tcc aga gtg aca atg ctg aga gac acc agc aag aac 288Pro Ser Leu Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn
85 90 95cag ttc agc ctg aga ctc agc agc gtg aca gcc gcc gac acc gcg gtt 336Gln Phe Ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val
100 105 110tat tat tgt gca aga tcc cta gct cgg act acg gct atg gac tac tgg 384Tyr Tyr Cys Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp
115 120 125ggt caa ggc agc ctc gtc aca gtc tcc tca ggt ggt ggt ggt tcg ggt 432Gly Gln Gly Ser Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly
130 135 140ggt ggt ggt tcg ggt ggt ggc gga tcg gac atc cag atg acc cag agc 480Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser145 150 155 160cca agc agc ctg agc gcc agc gtg ggt gac aga gtg acc atc acc tgt 528Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys
165 170 175aga gcc agc cag gac atc agc agt tac ctg aat tgg tac cag cag aag 576Arg Ala Ser Gln Asp Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys
180 185 190cca gga aag gct cca agg ctg ctg atc tac tac acc tcc aga ctg cac 624Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His
195 200 205tct ggt gtg cca agc aga ttc agc ggt agc ggt agc ggt acc gac ttc 672Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
210 215 220acc ttc acc atc agc agc ctc cag cca gag gac atc gct acc tac tac 720Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr225 230 235 240tgc caa cag ggt aac acg ctt cca tac acg ttc ggc caa ggg acc aag 768Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys
245 250 255gtg gaa atc aaa ggt ggt ggt ggt tcg ggt ggt ggt ggt tcg ggt ggt 816Val Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
260 265 270ggc gga tcg gtc gac tcc cag gtc caa ctg cag gag agc ggt cca ggt 864Gly Gly Ser Val Asp Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly
275 280 285ctt gtg aga cct agc cag acc ctg agc ctg acc tgc acc gtg tct ggc 912Leu Val Arg Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly
290 295 300tac tca att acc agc gat cat gcc tgg agc tgg gtt cgc cag cca cct 960Tyr Ser Ile Thr Ser Asp His Ala Trp Ser Trp Val Arg Gln Pro Pro305 310 315 320gga cga ggt ctt gag tgg att gga tac att agt tat agt gga atc aca 1008Gly Arg Gly Leu Glu Trp Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr
325 330 335acc tat aat cca tct ctc aaa tcc aga gtg aca atg ctg aga gac acc 1056Thr Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Met Leu Arg Asp Thr
340 345 350agc aag aac cag ttc agc ctg aga ctc agc agc gtg aca gcc gcc gac 1104Ser Lys Asn Gln Phe Ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp
355 360 365acc gcg gtt tat tat tgt gca aga tcc cta gct cgg act acg gct atg 1152Thr Ala Val Tyr Tyr Cys Ala Arg Ser Leu Ala Arg Thr Thr Ala Met
370 375 380gac tac tgg ggt caa ggc agc ctc gtc aca gtc tcc tca ggt ggt ggt 1200Asp Tyr Trp Gly Gln Gly Ser Leu Val Thr Val Ser Ser Gly Gly Gly385 390 395 400ggt tcg ggt ggt ggt ggt tcg ggt ggt ggc gga tcg gac atc cag atg 1248Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met
405 410 415acc cag agc cca agc agc ctg agc gcc agc gtg ggt gac aga gtg acc 1296Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr
420 425 430atc acc tgt aga gcc agc cag gac atc agc agt tac ctg aat tgg tac 1344Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Tyr Leu Asn Trp Tyr
435 440 445cag cag aag cca gga aag gct cca aag ctg ctg atc tac tac acc tcc 1392Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser
450 455 460aga ctg cac tct ggt gtg cca agc aga ttc agc ggg agc ggt agc ggt 1440Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly465 470 475 480acc gac ttc acc ttc acc atc agc agc ctc cag cca gag gac atc gct 1488Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala
485 490 495acc tac tac tgc caa cag gga aat act tta cca tac acg ttc ggc caa 1536Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln
500 505 5l0ggg acc aag gtg gaa atc aaa cga act gtg gct gca cca tct gtc ttc 1584Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe
515 520 525atc ttc ccg cca tct gat gag cag ttg aaa tct gga act gcc tct gtt 1632Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val
530 535 540gtg tgc ctg ctg aat aac ttc tat ccc aga gag gcc aaa gta cag tgg 1680Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp545 550 555 560aag gtg gat aac gcc ctc caa tcg ggt aac tcc cag gag agt gtc aca 1728Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr
565 570 575gag cag gac agc aag gac agc acc tac agc ctc agc agc acc ctg acg 1776Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr
580 585 590ctg agc aaa gca gac tac gag aaa cac aaa gtc tac gcc tgc gaa gtc 1824Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val
595 600 605acc cat cag ggc ctg agc tcg ccc gtc aca aag agc ttc aac agg gga 1872Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly
610 615 620gag tct 1878Glu Ser625<210>35<211>29<212>DNA<213>人工序列<220><223>对人工序列的描述:“MCH4-1”,一种人工合成的引物序列<400>35gtggaaatca aagtggccct gcacaggcc 29<210>36<211>68<212>DNA<213>人工序列<220><223>对人工序列的描述:“MCH4-2.1”,一种人工合成的引物序列<400>336tagtcaagct tctcaaatcc ctcttcgtcg gcgctaacct ctccttcggt ggacttgtcc 60acggtcct 68<210>37<211>29<212>DNA<213>人工序列<220><223>对人工序列的描述:“SCP-Mu”,一种人工合成的引物序列<400>37tgcagggcca ctttgatttc caccttggt 29<210>38<211>53<212>DNA<213>人工序列<220><223>对人工序列的描述:“MCH4-2.2”,一种人工合成的引物序列<400>38aaagcggccg cttattattt atcgtcatcg tctttgtagt caagcttctc aaa 53<210>39<211>1920<212>DNA<213>人工序列<220><221>CDS<222>(1)..(1920)<220><223>对人工序列的描述:“shPM1-MCH4-BvGS3”,一种特别设计的单链Fv基因序列<400>39atg gga tgg agc tgt atc atc ctc ttc ttg gta gca aca gct aca ggt 48Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15gtc gac tcc cag gtc caa ctg cag gag agc ggt cca ggt ctt gtg aga 96Val Asp Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg
20 25 30cct agc cag acc ctg agc ctg acc tgc acc gtg tct ggc tac tca att 144Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile
35 40 45acc agc gat cat gcc tgg agc tgg gtt cgc cag cca cct gga cga ggt 192Thr Ser Asp His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly
50 55 60ctt gag tgg att gga tac att agt tat agt gga atc aca acc tat aat 240Leu Glu Trp Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn65 70 75 80cca tct ctc aaa tcc aga gtg aca atg ctg aga gac acc agc aag aac 288Pro Ser Leu Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn
85 90 95cag ttc agc ctg aga ctc agc agc gtg aca gcc gcc gac acc gcg gtt 336Gln Phe Ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val
100 105 110tat tat tgt gca aga tcc cta gct cgg act acg gct atg gac tac tgg 384Tyr Tyr Cys Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp
115 120 125ggt caa ggc agc ctc gtc aca gtc tcc tca ggt ggt ggt ggt tcg ggt 432Gly Gln Gly Ser Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly
130 135 140ggt ggt ggt tcg ggt ggt ggc gga tcg gac atc cag atg acc cag agc 480Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser145 150 155 160cca agc agc ctg agc gcc agc gtg ggt gac aga gtg acc atc acc tgt 528Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys
165 170 175aga gcc agc cag gac atc agc agt tac ctg aat tgg tac cag cag aag 576Arg Ala Ser Gln Asp Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys
180 185 190cca gga aag gct cca aag ctg ctg atc tac tac acc tcc aga ctg cac 624Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His
195 200 205tct ggt gtg cca agc aga ttc agc ggt agc ggt agc ggt acc gac ttc 672Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
210 215 220acc ttc acc atc agc agc ctc cag cca gag gac atc gct acc tac tac 720Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr225 230 235 240tgc caa cag ggt aac acg ctt cca tac acg ttc ggc caa ggg acc aag 768Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys
245 250 255gtg gaa atc aaa ggt ggt ggt ggt tcg ggt ggt ggt ggt tcg ggt ggt 816Val Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
260 265 270ggc gga tcg gtc gac tcc cag tgc caa ctg cag gag agc ggt cca ggt 864Gly Gly Ser Val Asp Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly
275 280 285ctt gtg aga cct agc cag acc ctg agc ctg acc tgc acc gtg tct ggc 912Leu Val Arg Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly
290 295 300tac tca att acc agc gat cat gcc tgg agc tgg gtt cgc cag cca cct 960Tyr Ser Ile Thr Ser Asp His Ala Trp Ser Trp Val Arg Gln Pro Pro305 310 315 320gga cga ggt ctt gag tgg att gga tac att agt tat agt gga atc aca 1008Gly Arg Gly Leu Glu Trp Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr
325 330 335acc tat aat cca tct ctc aaa tcc aga gtg aca atg ctg aga gac acc 1056Thr Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Met Leu Arg Asp Thr
340 345 350agc aag aac cag ttc agc ctg aga ctc agc agc gtg aca gcc gcc gac 1104Ser Lys Asn Gln Phe Ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp
355 360 365acc gcg gtt tat tat tgt gca aga tcc cta gct cgg act acg gct atg 1152Thr Ala Val Tyr Tyr Cys Ala Arg Ser Leu Ala Arg Thr Thr Ala Met
370 375 380gac tac tgg ggt caa ggc agc ctc gtc aca gtc tcc tca ggt ggt ggt 1200Asp Tyr Trp Gly Gln Gly Ser Leu Val Thr Val Ser Ser Gly Gly Gly385 390 395 400ggt tcg ggt ggt ggt ggt tcg ggt ggt ggc gga tcg gac atc cag atg 1248Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met
405 410 415acc cag agc cca agc agc ctg agc gcc agc gtg ggt gac aga gtg acc 1296Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr
420 425 430atc acc tgt aga gcc agc cag gac atc agc agt tac ctg aat tgg tac 1344Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Tyr Leu Ash Trp Tyr
435 440 445cag cag aag cca gga aag gct cca aag ctg ctg atc tac tac acc tcc 1392Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser
450 455 460aga ctg cac tct ggt gtg cca agc aga ttc agc ggt agc ggt agc ggt 1440Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly465 470 475 480acc gac ttc acc ttc acc atc agc agc ctc cag cca gag gac atc gct 1488Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala
485 490 495acc tac tac tgc caa cag gga aat act tta cca tac acg ttc ggc caa 1536Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln
500 505 510ggg acc aag gtg gaa atc aaa gtg gcc ctg cac agg ccc gat gtc tac 1584Gly Thr Lys Val Glu Ile Lys Val Ala Leu His Arg Pro Asp Val Tyr
515 520 525ttg ctg cca cca gcc cgg gag cag ctg aac ctg cgc gag tcg gcc acc 1632Leu Leu Pro Pro Ala Arg Glu Gln Leu Asn Leu Arg Glu Ser Ala Thr
530 535 540atc acg tgc ctg gtg acg ggc ttc tct ccc gcg gac gtc ttc gtg cag 1680Ile Thr Cys Leu Val Thr Gly Phe Ser Pro Ala Asp Val Phe Val Gln545 550 555 560tgg atg cag agg ggg cag ccc ttg tcc ccg gag aag tat gtg acc agc 1728Trp Met Gln Arg Gly Gln Pro Leu Ser Pro Glu Lys Tyr Val Thr Ser
565 570 575gcc cca atg cct gag ccc cag gcc cca ggc cgg tac ttc gcc cac agc 1776Ala Pro Met Pro Glu Pro Gln Ala Pro Gly Arg Tyr Phe Ala His Ser
580 585 590atc ctg acc gtg tcc gaa gag gaa tgg aac acg ggg gag acc tac acc 1824Ile Leu Thr Val Ser Glu Glu Glu Trp Ash Thr Gly Glu Thr Tyr Thr
595 600 605tgc gtg gcc cat gag gcc ctg ccc aac agg gtc acc gag agg acc gtg 1872Cys Val Ala His Glu Ala Leu Pro Asn Arg Val Thr Glu Arg Thr Val
610 615 620gac aag tcc acc gag ggg gag gtg agc gcc gac gag gag ggc ttt gag 1920Asp Lys Ser Thr Glu Gly Glu Val Ser Ala Asp Glu Glu Gly Phe Glu625 630 635 640
Claims (16)
1.用于分离膜结合蛋白的编码基因的方法,所述的方法包括如下步骤:
(ⅰ)将包括编码对一种抗原具有结合亲和力的可分泌性功能蛋白的DNA以及连接于该功能蛋白编码DNA 3’侧下游的cDNA的载体引入细胞,
(ⅱ)在细胞内表达对抗原具有结合亲和力的可分泌性功能蛋白与由该cDNA所编码蛋白的融合蛋白,
(ⅲ)通过将在细胞膜上表达融合蛋白的细胞与抗原相接触,筛选结合抗原的细胞,并且
(ⅳ)从所筛选的细胞中分离插入到载体中的cDNA。
2.权利要求1所述的方法,其中通过在功能蛋白编码DNA 3’侧下游的限制性酶切位点处将cDNA引入载体来获得在步骤(ⅰ)中引入细胞的载体。
3.权利要求1所述的方法,其中通过将包括编码功能蛋白的DNA和连接于该功能蛋白编码DNA 3’侧下游的cDNA的DNA引入载体来获得在步骤(ⅰ)中引入细胞的载体,
4.权利要求1至3中任何一个所述的方法,其中通过编码肽连接序列的DNA连接编码功能蛋白的DNA和其3’侧下游的cDNA。
5.权利要求1至4中任何一个所述的方法,其中该cDNA源于哺乳动物细胞的cDNA文库。
6.权利要求1至5中任何一个所述的方法,其中在步骤(ⅰ)中引入细胞的载体在编码功能蛋白的DNA 5’侧的上游包括编码分泌信号序列的DNA。
7.权利要求1至6中任何一个所述的方法,其中该功能蛋白为抗体。
8.权利要求1至7中任何一个所述的方法,其中对抗原具有结合亲和力的功能蛋白为一种单链抗体。
9.权利要求1至8中任何一个所述的方法,其中该载体包括一种DNA,在该DNA中编码抗体恒定区的DNA连接于编码一种单链抗体的DNA 3’侧的下游。
10.权利要求1至9中任何一个所述的方法,其中将该抗原结合于一种支持物。
11.权利要求10所述的方法,其中该支持物用于细胞培养。
12.权利要求1至11中任何一个所述的方法,其包括确定从细胞获得的基因是否包含新的序列。
13.权利要求12所述的方法,其包括筛选cDNA文库以获得由细胞所获基因的全长基因,该基因包括新的序列。
14.权利要求13所述的方法,其包括分离由细胞所获基因的全长基因,该基因包括新的序列。
15.用于分离膜结合蛋白的编码基因的试剂盒,该试剂盒包括具有用于在编码对一种抗原具有结合亲和力的可分泌性功能蛋白的DNA 3’侧下游插入cDNA的限制性酶识别位点的载体。
16.权利要求15所述的试剂盒进一步还包括结合抗原的支持物和/或待引入载体的细胞。
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JP138652/1998 | 1998-05-20 | ||
JP13865298 | 1998-05-20 | ||
JP27987698 | 1998-10-01 | ||
JP279876/1998 | 1998-10-01 |
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CN99808906A Pending CN1310758A (zh) | 1998-05-20 | 1999-04-30 | 基因克隆的新方法 |
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US (3) | US6610485B1 (zh) |
EP (1) | EP1081224B1 (zh) |
JP (1) | JP3471006B2 (zh) |
KR (1) | KR100518958B1 (zh) |
CN (1) | CN1310758A (zh) |
AT (1) | ATE329020T1 (zh) |
AU (1) | AU753310B2 (zh) |
CA (1) | CA2331917A1 (zh) |
DE (1) | DE69931776T2 (zh) |
IL (1) | IL139705A0 (zh) |
NO (1) | NO20005810L (zh) |
TW (1) | TW593679B (zh) |
WO (1) | WO1999060113A1 (zh) |
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EP1081224B1 (en) * | 1998-05-20 | 2006-06-07 | Chugai Seiyaku Kabushiki Kaisha | Novel method for cloning a gene |
JP5021143B2 (ja) | 2000-09-14 | 2012-09-05 | 株式会社リバース・プロテオミクス研究所 | 医療および他の用途に用いる化合物の発見および創製のための方法 |
US20090137416A1 (en) | 2001-01-16 | 2009-05-28 | Regeneron Pharmaceuticals, Inc. | Isolating Cells Expressing Secreted Proteins |
IL161296A0 (en) * | 2001-10-13 | 2004-09-27 | Asterion Ltd | Glycosylphosphatidylinositol containing polypeptides |
US20080020376A1 (en) * | 2004-03-10 | 2008-01-24 | Akopian Armen N | Enriched Cdna Expression Libraries and Methods of Making and Using Same |
JP6393260B2 (ja) * | 2012-07-06 | 2018-09-19 | イノベーティブ テクノロジーズ イン バイオロジカル システムズ エセ.エレ. | 蛍光融合ポリペプチド、該ポリペプチドを含むバイオセンサー及びそれらの使用 |
TWI675044B (zh) | 2012-11-14 | 2019-10-21 | 美商再生元醫藥公司 | 重組細胞表面捕捉蛋白質 |
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US5474899A (en) * | 1987-05-13 | 1995-12-12 | Cistron Biotechnology, Inc. | Selective immunoassay for IL-1 β |
US5223409A (en) | 1988-09-02 | 1993-06-29 | Protein Engineering Corp. | Directed evolution of novel binding proteins |
US5283173A (en) * | 1990-01-24 | 1994-02-01 | The Research Foundation Of State University Of New York | System to detect protein-protein interactions |
IE914075A1 (en) * | 1990-11-23 | 1992-06-03 | Gen Hospital Corp | Inhibition of cell adhesion protein-carbohydrate¹interactions |
US6099842A (en) | 1990-12-03 | 2000-08-08 | The United States Of America As Represented By The Department Of Health And Human Services | Recombinant immunotoxin composed of a single chain antibody reacting with the human transferrin receptor and diptheria toxin |
US5571894A (en) * | 1991-02-05 | 1996-11-05 | Ciba-Geigy Corporation | Recombinant antibodies specific for a growth factor receptor |
WO1993007286A1 (en) | 1991-09-30 | 1993-04-15 | The United States Of America, Represented By The Secretary, Department Of Health And Human Services | Recombinant immunotoxins |
JP2879303B2 (ja) | 1993-01-14 | 1999-04-05 | 佑 本庶 | cDNAライブラリーの作製方法、および新規なポリペプチドとそれをコードするDNA |
US5817308A (en) * | 1994-02-14 | 1998-10-06 | University Of Rochester | Tolerogenic fusion proteins of immunoglobulins and methods for inducing and maintaining tolerance |
US5858752A (en) * | 1995-06-07 | 1999-01-12 | The General Hospital Corporation | Fucosyltransferase genes and uses thereof |
CA2223155C (en) | 1995-06-07 | 2003-08-12 | Zymogenetics, Inc. | Secretion leader trap cloning method |
JP2002238557A (ja) * | 1996-07-23 | 2002-08-27 | Fujisawa Pharmaceut Co Ltd | トランスメンブレントラップ法 |
JPH1132779A (ja) * | 1997-07-24 | 1999-02-09 | Fujita Gakuen | 蛋白質の遺伝子クローニング法 |
US6030834A (en) * | 1997-12-30 | 2000-02-29 | Chiron Corporation | Human IKK-beta DNA constructs and cells |
CA2329986A1 (en) | 1998-04-30 | 1999-11-11 | Dolores Cahill | New method for the selection of clones of an expression library involving rearraying |
EP1081224B1 (en) | 1998-05-20 | 2006-06-07 | Chugai Seiyaku Kabushiki Kaisha | Novel method for cloning a gene |
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- 1999-04-30 WO PCT/JP1999/002341 patent/WO1999060113A1/ja active IP Right Grant
- 1999-04-30 KR KR10-2000-7012901A patent/KR100518958B1/ko not_active IP Right Cessation
- 1999-04-30 US US09/700,820 patent/US6610485B1/en not_active Expired - Fee Related
- 1999-04-30 AT AT99918302T patent/ATE329020T1/de not_active IP Right Cessation
- 1999-04-30 IL IL13970599A patent/IL139705A0/xx unknown
- 1999-04-30 AU AU36283/99A patent/AU753310B2/en not_active Ceased
- 1999-04-30 DE DE69931776T patent/DE69931776T2/de not_active Expired - Lifetime
- 1999-04-30 CN CN99808906A patent/CN1310758A/zh active Pending
- 1999-04-30 CA CA002331917A patent/CA2331917A1/en not_active Abandoned
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KR100518958B1 (ko) | 2005-10-06 |
NO20005810L (no) | 2001-01-22 |
NO20005810D0 (no) | 2000-11-17 |
DE69931776D1 (de) | 2006-07-20 |
EP1081224A4 (en) | 2003-07-02 |
ATE329020T1 (de) | 2006-06-15 |
AU3628399A (en) | 1999-12-06 |
US7338797B2 (en) | 2008-03-04 |
JP3471006B2 (ja) | 2003-11-25 |
IL139705A0 (en) | 2002-02-10 |
KR20010043688A (ko) | 2001-05-25 |
TW593679B (en) | 2004-06-21 |
CA2331917A1 (en) | 1999-11-25 |
AU753310B2 (en) | 2002-10-17 |
DE69931776T2 (de) | 2007-05-16 |
US20050019780A1 (en) | 2005-01-27 |
WO1999060113A1 (fr) | 1999-11-25 |
EP1081224B1 (en) | 2006-06-07 |
US20030175794A1 (en) | 2003-09-18 |
US6610485B1 (en) | 2003-08-26 |
EP1081224A1 (en) | 2001-03-07 |
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