CN117417455A - 一种特异性结合25-羟基维生素d3的抗原结合蛋白 - Google Patents
一种特异性结合25-羟基维生素d3的抗原结合蛋白 Download PDFInfo
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- CN117417455A CN117417455A CN202311420610.2A CN202311420610A CN117417455A CN 117417455 A CN117417455 A CN 117417455A CN 202311420610 A CN202311420610 A CN 202311420610A CN 117417455 A CN117417455 A CN 117417455A
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- antigen binding
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
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- G01N33/82—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving vitamins or their receptors
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/52—Constant or Fc region; Isotype
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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- C07K2317/565—Complementarity determining region [CDR]
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/567—Framework region [FR]
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2510/00—Genetically modified cells
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
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Abstract
本发明公开了一种特异性结合25‑羟基维生素D3的抗原结合蛋白,属于生物技术领域。本发明提供了一种与目前的抗25‑羟基维生素D3单克隆抗体差异化显著、且具有更好应用前景的抗原结合蛋白,包含两条重链和两条轻链,其中重链可变区包含如SEQ ID NO.1‑3所示的重链CDR1‑3氨基酸序列,轻链可变区包含如SEQ ID NO.4‑6所示的轻链CDR1‑3氨基酸序列。本发明的抗原结合蛋白具有良好的亲和力和特异性,可用于开发检测25‑羟基维生素D3的免疫分析方法。
Description
技术领域
本发明涉及一种特异性结合25-羟基维生素D3的抗原结合蛋白,属于生物技术领域。
背景技术
维生素D3,又称胆钙化醇,是一种脂溶性维生素。人体内的维生素D3可以通过自身合成,在紫外线的作用下,皮肤表皮层内的7-脱氢胆固醇经过一系列反应最终生成维生素D3。维生素D3具有调节人体钙磷平衡,维持骨骼健康,调节免疫功能,预防心血管疾病,调节脂肪和能量代谢等多种功能。维生素D3可以作为口服膳食补充剂,用以预防维生素D缺乏症;也可以作为药物,用以治疗佝偻病、家族性低磷血症、甲状旁腺功能减退、低钙血症和钙质疏松症等相关疾病。鱼、牛肝、鸡蛋和奶酪等食物中含有维生素D3,合理的膳食能预防人体维生素D缺乏。
在人体中,维生素D3有两种代谢形式。在维生素D3羟化酶的催化作用下,维生素D3在肝脏中被代谢为25-羟基维生素D3,即25(OH)D3,又称钙二醇;25(OH)D3可以由肾脏中的25(OH)D-1α-羟化酶进一步羟基化,形成1,25-(OH)2D3,又称钙三醇,也就是维生素D3的活性形式。1,25-(OH)2D3的半衰期短,人体血液中的浓度低;而人体血液中的25(OH)D3通常是以维生素D结合蛋白紧密结合的形式存在,稳定性好,半衰期长,其浓度与体内维生素D含量直接相关。在医学诊断中,对血浆或血清中25(OH)D3的检测可以用于确定人体的维生素D水平。
目前,针对25(OH)D3的检测方法主要有仪器分析方法和免疫分析方法。其中,仪器分析方法包括高效液相色谱法(HPLC)和液质联用法(LC-MS/MS)等,具有灵敏度高、特异性强、准确度好等优点,LC-MS/MS更是测定25(OH)D3含量的金标准。然而,仪器分析方法依赖精密的仪器、专业的操作人员和复杂的样本前处理过程,成本高,费时长,检测效率和通量低。免疫分析是基于抗原-抗体结合反应的检测方法,具有快速、灵敏、高效等优点,适用于临床诊断中即时检验(POCT)的技术需求。
抗体是免疫检测的核心试剂,传统单克隆抗体的获取需经过小鼠免疫、杂交瘤细胞融合、单克隆筛选、细胞扩培和腹水生产等制备过程。传统单克隆抗体有以下局限性:在杂交瘤细胞的冻存、复苏和传代过程中,会出现抗体基因漂变或丢失的现象,导致单克隆抗体性能下降;在腹水生产过程中,由于小鼠个体有差异,会出现溶血的现象,从而影响抗体的质量;另外,杂交瘤细胞的长期保存十分依赖液氮,需随时补充,保存成本高。基于基因工程技术的重组抗体可以规避这些局限,相比传统腹水法制备的抗体,重组抗体的优势在于:可重复性,抗体基因的信息可以永久保持,不会丢失,具有准确性和批间一致性;可重塑性,通过基因重组的方式可以改变抗体的种类或亚型,也可以增强抗体的性能;无需使用实验动物,减少对动物的伤害;纯度高,重组抗体是在无血清条件下表达的,避免血清成分污染。所以,开发抗25-羟基维生素D3重组抗体可以建立稳定性更好、一致率更高的快速免疫检测方法。
另外,现有技术中已公开针对25-羟基维生素D的多种抗体。如中国专利CN105352958A公开了一种总25-羟基维生素D检测试剂盒,其中使用的抗体能与维生素D2和D3同时反应;专利CN103857698A也公开了一种针对25-羟基维生素D2和D3的抗体;CN101273062A也公开了一种与25-羟基维生素D2和25-羟基维生素D3两种25-羟基维生素D形式均能反应的抗25-羟基维生素D抗体。但以上公开的抗体并不能区分不同种类的25-羟基维生素D(如维生素D2和D3),而且,目前针对25-羟基维生素D3的抗体由于在制备过程中引入交联基团,使得到的抗体检测效果有限。
发明内容
为解决上述问题,本发明提供了一种高灵敏、高特异的抗25-羟基维生素D3重组抗原结合蛋白。基于上述特性,该抗原结合蛋白有望用于25-羟基维生素D3的检测。
本发明的第一个目的是提供一种特异性结合25-羟基维生素D3的抗原结合蛋白,所述抗原结合蛋白含有重链可变区和轻链可变区,其中,重链可变区包含氨基酸序列如SEQID NO.1-3所示的互补决定区VH-CDR1、VH-CDR2、VH-CDR3,轻链可变区包含氨基酸序列如SEQ ID NO.4-6所示的互补决定区VL-CDR1、VL-CDR2、VL-CDR3。
进一步地,所述抗原结合蛋白可为抗体或其抗原结合片段。
进一步地,其中所述抗原结合片段包括Fab,Fab’,Fv片段,F(ab’)2,scFv,di-scFv和/或dAb。
进一步地,本发明包括那些含有CDR的抗原结合蛋白重链可变区和轻链可变区的分子,只要其CDR与上述CDR序列具有80%以上(较佳的90%以上,最佳的95%以上,如90%、91%、92%、93%、94%、95%、96%、97%、98%、99%)的同源性。
进一步地,所述抗原结合蛋白的重链可变区包含框架区VH-FR1、VH-FR2、VH-FR3、VH-FR4,相邻两个框架区之间设置互补决定区,即VH-CDR1、VH-CDR2、VH-CDR3被框架区VH-FR1、VH-FR2、VH-FR3、VH-FR4所隔开。因此抗原结合蛋白的重链可变区上依次设置有VH-FR1、VH-CDR1、VH-FR2、VH-CDR2、VH-FR3、VH-CDR3、VH-FR4。
进一步地,所述框架区VH-FR1、VH-FR2、VH-FR3、VH-FR4分别包含SEQ ID NO.7-10所示的序列或与其同源性不低于90%的序列。
进一步地,所述抗原结合蛋白的轻链可变区包含框架区VL-FR1、VL-FR2、VL-FR3、VL-FR4,相邻两个框架区之间设置互补决定区,即VL-CDR1、VL-CDR2、VL-CDR3被框架区VL-FR1、VL-FR2、VL-FR3、VL-FR4所隔开。因此抗原结合蛋白的轻链可变区上依次设置有VL-FR1、VL-CDR1、VL-FR2、VL-CDR2、VL-FR3、VL-CDR3、VL-FR4。
进一步地,所述框架区VL-FR1、VL-FR2、VL-FR3、VL-FR4分别包含SEQ ID NO.11-14所示的序列或与其同源性不低于90%的序列。
进一步地,所述抗体为鼠源抗体或人源化抗体。
进一步地,所述抗原结合蛋白还包含恒定区。
进一步地,所述恒定区的种属来源为牛、马、乳牛、猪、绵羊、山羊、大鼠、小鼠、狗、猫、兔、驴、鹿、貂、鸡、鸭、鹅或人;
进一步地,所述恒定区选自鼠源IgG1、IgG2a、IgG2b、IgG3或人源IgG1、IgG2、IgG3、IgG4、IgM中的一种。
进一步地,所述恒定区来源于小鼠;重链恒定区的氨基酸序列如SEQ ID NO.15所示;轻链恒定区的氨基酸序列如SEQ ID NO.16所示。
本发明的第二个目的是提供编码上述抗原结合蛋白的核酸分子。所述核酸分子为DNA或RNA。
本发明的第三个目的是提供含有上述核酸分子的表达载体。
进一步地,所述的表达载体可为病毒载体或非病毒载体,如DNA、RNA、病毒载体(如慢病毒、腺病毒、AAV病毒、逆转录病毒或其组合)、质粒、转座子、其他基因转移系统、脂质体纳米颗粒等。
本发明的第四个目的是提供含有上述抗原结合蛋白的宿主细胞。
进一步地,所述宿主细胞可为原核细胞或真核细胞,如植物细胞、动物细胞、微生物等。优选地,宿主细胞为中国仓鼠卵巢(CHO)细胞、人胚胎肾细胞(HEK293)、HeLa细胞、幼仓鼠肾细胞、NS0小鼠骨髓瘤细胞或其它哺乳动物细胞的一种。
本发明的第五个目的是提供含有上述抗原结合蛋白的单价抗体、二价抗体或多价抗体。
本发明的第六个目的是提供含有上述抗原结合蛋白的重组蛋白或免疫偶联物。
进一步地,所述重组蛋白含有抗原结合蛋白,以及协助表达和/或纯化的标签序列。
进一步地,所述免疫偶联物含有抗原结合蛋白,以及偶联部分,如可检测标记物。
本发明的第七个目的是提供一种用于25-羟基维生素D3的检测试剂盒,包含所述抗原结合蛋白、核酸分子、表达载体、宿主细胞、单价抗体、二价抗体、多价抗体、重组蛋白或免疫偶联物。
进一步地,所述的检测包括流式检测、细胞免疫荧光检测、酶联免疫吸附试验(ELISA)检测等。
进一步地,上述试剂盒可用于体内或体外检测25-羟基维生素D3。
本发明的有益效果:
本发明经过大量筛选,成功获得一种特异性结合25-羟基维生素D3的抗原结合蛋白,该抗原结合蛋白具有独特的可变区序列,对25-羟基维生素D3抗原具有较高的亲和力和特异性;本发明研制的重组抗原结合蛋白序列明确,可有效避免基因突变,抗原结合蛋白批间差异小、质量稳定、结构完整,更利于开发稳定的免疫分析快速检测方法。
附图说明
图1为本发明中PCR扩增获得的抗体可变区基因的琼脂糖凝胶电泳图。
图2为本发明中重组抗体的SDS-PAGE蛋白电泳图。
图3为本发明中抗25-羟基维生素D3重组抗体对25-羟基维生素D3的ic-ELISA标准曲线(浓度从低到高依次为0,1.37,4.12,12.35,37.04,111.11,333.33,1000ng/mL)。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明,以使本领域的技术人员可以更好地理解本发明并能予以实施,但所举实施例不作为对本发明的限定。
术语定义
在本发明中,术语“特异性结合”通常是指抗体通过其抗原结合域与表位结合,并且该结合需要抗原结合域和表位之间的一些互补性。根据该定义,当抗体相比于其将结合随机的,不相关的表位而言更容易通过其抗原结合域与表位结合时,抗体被称为“特异性结合”该抗原。
在本发明中,术语“分离的”或“纯化的”通常是指至少部分从在其天然状态中通常与其结合的其他分子中分离的分子(例如抗体、核酸等)。“分离的或纯化的多肽”或“分离的或纯化的核酸”基本上没有其他生物分子,如核酸、蛋白质、脂质、碳水化合物、细胞碎片和生长培养基。
在本发明中,术语“抗原结合蛋白”被以其广义使用并且意指包含与抗原或靶标结合的一部分并且任选地包含允许抗原结合部分采用促进抗原结合蛋白与抗原结合的构型的构架或框架部分的蛋白质。抗原结合蛋白的实例包括人抗体、人源化抗体;嵌合抗体;重组抗体;单链抗体;双功能抗体;三功能抗体;四功能抗体;Fab片段;F(ab’)2片段;IgD抗体;IgE抗体;IgM抗体;IgG1抗体;IgG2抗体;IgG3抗体;或IgG4抗体以及其片段。抗原结合蛋白可以包括,例如具有移植的CDR或CDR衍生物的嵌合抗原受体、替代蛋白构架或人工构架。此类构架包括,但不限于:抗体来源的构架,其包含被引入以便例如使抗原结合蛋白的三维结构稳定的突变;以及完全合成的构架,其包含例如生物相容性聚合物。
在本发明中,术语“抗体”其以最广泛意义使用,且具体涵盖,但不限于,单克隆抗体(包括包含两条轻链和两条重链的全长单克隆抗体)、多克隆抗体、多特异性抗体(例如双特异性抗体)、人源化抗体、完全人类抗体、嵌合抗体、重链抗体和骆驼化单结构域抗体(如,重链可变结构域抗体)。抗体通常具有免疫球蛋白的结构,可以包含通过二硫键互相连接的至少两条重链(HC)和两条轻链(LC)的蛋白,或其抗原结合片段。每条重链包含重链可变区(VH)和重链恒定区。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同,故其抗原性也不同。据此,可将免疫球蛋白分为五类,或称为免疫球蛋白的同种型,即IgM,IgD,IgG,IgA和IgE,其相应的重链分别为μ链,δ链,γ链,α链,ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别,又可分为不同的亚类,如IgG可分为IgG1,IgG2,IgG3,IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中第每类Ig都可以有κ链或λ链。
在本发明中,术语“可变”通常是指,抗体的可变结构域的序列的某些部分变化强烈,它形成各种特定抗体对其特定抗原的结合和特异性。然而,变异性并非均匀地分布在抗体的整个可变区中。它集中在轻链和重链可变区中的三个区段,被称为互补决定区(CDRs)或高变区(HVR)。可变域中更高度保守的部分被称为框架(FR)。天然重链和轻链的可变结构域各自包含四个FR区,大部分采用β-折叠构型,通过三个CDRs连接,形成环连接,并且在一些情况下形成β-折叠结构的一部分。每条链中的CDRs通过FR区紧密靠近在一起,并与来自另一条链的CDRs一起形成抗体的抗原结合位点,恒定区不直接参与抗体与抗原的结合。在本领域中,可以通过多种方法来定义抗体的CDR,例如基于序列可变性的Kabat定义规则、Chothia定义规则或IMGT定义规则。
本发明涉及的序列信息如下:
实施例1:抗25-羟基维生素D3单克隆抗体可变区基因分离和鉴定
复苏产抗25-羟基维生素D3单克隆抗体的杂交瘤细胞株于培养瓶中,扩培后收集5-10×106个细胞,加入1mL Trizol试剂,吹打混匀,充分混匀裂解;向裂解液中加入200μL氯仿,震荡15s后得乳浊液,在4℃静置5min后12000g离心15min;取上层450μL无色水相,加入等体积预冷异丙醇,上下颠倒混匀,在4℃静置10min后12000g离心10min;弃上清,加入1mL75%乙醇洗涤沉淀后,12000g离心10min;弃上清,沉淀用100μL RNase-free水重悬,置于-80℃保存。
以RNA为模板,用RACE 5’/3’Kit试剂盒(购自Takara公司)分别进行第一链cDNA合成和cDNA末端快速扩增,其中抗体的重链和轻链对应不同的基因特异性引物,分别命名为H-5’GSP和L-5’GSP,H-5’GSP的序列为GATTACGCCAAGCTTCTCAATTTTCTTGTCCACCTTGGTGC,L-5’GSP的序列为GATTACGCCAAGCTTCTCATTCCTGTTGAAGCTCTTGACAATGGG。如图1所示,琼脂糖凝胶电泳可得明亮目的条带,目的条带分别含VH和VL基因片段。
用胶回收试剂盒纯化目的基因至20μL,以In-fusion的方式将纯化产物克隆到线性化的pRACE质粒上,并转化Stellar感受态细胞,涂布于LB固体培养基(含氨苄青霉素)。次日,VH和VL各取6-8个单菌落,扩培后送基因测序公司进行测序,测序引物为M13-F/R通用引物。将上述测序得到的基因序列导入Kabat抗体数据库中进行比对分析,鉴定VH和VL基因以及CDR区和骨架区,其中VH基因序列长度为357bp,前面有57bp信号肽序列;VL基因序列长度为321bp,前面有60bp信号肽序列。
实施例2:重组抗体表达质粒的构建
根据测序所得抗25-羟基维生素D3单克隆抗体的重链和轻链可变区基因,设计特异性引物,以pRACE重轻链质粒为模板,PCR扩增重链和轻链可变区基因,分别同源重组至含重链恒定区和轻链恒定区的pcDNA3.4骨架质粒上,以获得含全长重链和轻链基因的重组抗体表达质粒。分别将重链和轻链表达质粒转化Top 10感受态细胞,挑取单菌落,测序验证序列准确性。取测序正确的重链和轻链表达质粒对应的单菌落,扩大培养,用去内毒素质粒提取试剂盒提取重链和轻链表达质粒。
实施例3:重组抗体的表达和纯化
从液氮罐中取一管HEK293F悬浮细胞,细胞数≥107个,在37℃水浴中快速解冻后转移至30mL预热培养基中,在125mL摇瓶中培养,终密度约为0.3×106cells/mL,培养基为义翘神州的SMM-293TII培养基。培养箱条件设置为:37℃,125rpm,5% CO2,湿度>80%,培养3-4天。细胞可长至3×106cells/mL,活率大于95%,继续传代2次,保证倍增时间为24h左右,用作种子细胞。
转染前一天按照1.5×106cells/mL密度接种HEK293F细胞至500mL摇瓶中,接种体积为120ml。次日细胞长至3×106cells/mL,细胞活率>95%,开始细胞转染。其中重链和轻链表达质粒总用量为4μg每毫升培养体积,重轻链比例为1:1.5,转染试剂为PEI,PEI的用量为质粒用量的2.5倍。质粒和PEI在新鲜培养基中混合均匀,室温静置15-20分钟,缓慢加入摇瓶中,再加入适量新鲜培养基稀释细胞最终密度至2×106cells/mL,放回摇床培养箱培养。转染20-24h后添加3.5%培养体积的补料液,并在转染后第3、5天继续添加3.5%培养体积的补料液。转染后第7天或细胞活率<60%,收获细胞培养基上清。
高速离心收集细胞培养基上清,去除细胞和细胞碎片。培养基上清经孔径为0.45μm滤膜过滤后,用Protein G亲和层析柱纯化抗体。抗体透析至0.01M PBS缓冲液中,置于-20℃保存。取少量抗体进行还原性SDS-PAGE电泳验证,如图2所示,共2个蛋白条带,其中一条为分子量为50kDa左右的重链,另一条为分子量为25kDa左右的轻链。
实施例4:重组抗体的性能测试
将上述重组抗体应用于25-羟基维生素D3的ic-ELISA检测,具体步骤如下:将包被原25-羟基维生素D3-BSA用0.05M碳酸盐缓冲液(pH 9.6)稀释至0.3μg/mL,以100μL/孔加入96孔酶标板中,37℃孵育2h;用PBST洗涤液洗涤3次,每次3min;加入200μL/孔封闭液,37℃孵育2h;加入梯度稀释的25-羟基维生素D3标准品(50μL/孔)和重组抗体(0.3μg/mL,50μL/孔),37℃孵育30min;洗涤后,加入HRP羊抗小鼠IgG(100μL/孔),37℃孵育30分钟;洗涤后,加入TMB底物溶液(100μL/孔),37℃反应15分钟;加入终止液(50μL/孔)停止反应,用酶标仪测量450nm吸光度。
所述重组抗体对25-羟基维生素D3的抑制标准曲线如图3所示,其灵敏度(IC50)为26.6ng/mL,检测限(IC10)为3.0ng/mL,说明所述重组抗体对25-羟基维生素D3具有较好的灵敏度。此外,在ic-ELISA中所述重组抗体对25-羟基维生素D2没有交叉反应,说明其具有很好的特异性,可用于25-羟基维生素D3免疫分析。
显然,上述实施例仅仅是为清楚地说明所作的举例,并非对实施方式的限定。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
Claims (10)
1.一种特异性结合25-羟基维生素D3的抗原结合蛋白,其特征在于,所述抗原结合蛋白含有重链可变区和轻链可变区,其中,重链可变区包含氨基酸序列如SEQ ID NO.1-3所示的互补决定区VH-CDR1、VH-CDR2、VH-CDR3,轻链可变区包含氨基酸序列如SEQ ID NO.4-6所示的互补决定区VL-CDR1、VL-CDR2、VL-CDR3。
2.根据权利要求1所述的抗原结合蛋白,其特征在于,所述重链可变区含有框架区VH-FR1、VH-FR2、VH-FR3、VH-FR4;框架区VH-FR1、VH-FR2、VH-FR3、VH-FR4分别包含SEQ IDNO.7-10所示的序列或与其同源性不低于90%的序列。
3.根据权利要求1所述的抗原结合蛋白,其特征在于,所述轻链可变区含有框架区VL-FR1、VL-FR2、VL-FR3、VL-FR4;框架区VL-FR1、VL-FR2、VL-FR3、VL-FR4分别包含SEQ IDNO.11-14所示的序列或与其同源性不低于90%的序列。
4.根据权利要求1所述的抗原结合蛋白,其特征在于,所述抗原结合蛋白包含恒定区。
5.根据权利要求4所述的抗原结合蛋白,其特征在于,重链恒定区的氨基酸序列如SEQID NO.15所示;轻链恒定区的氨基酸序列如SEQ ID NO.16所示。
6.编码权利要求1-5任一项所述的抗原结合蛋白的核酸分子。
7.含有权利要求6所述的核酸分子的表达载体。
8.含有权利要求1-5任一项所述的抗原结合蛋白的宿主细胞。
9.含有权利要求1-5任一项所述的抗原结合蛋白的单价抗体、二价抗体、多价抗体、重组蛋白或免疫偶联物。
10.一种用于25-羟基维生素D3的检测试剂盒,其特征在于,包含权利要求1-5任一项所述抗原结合蛋白、权利要求6所述核酸分子、权利要求7所述表达载体、权利要求8所述宿主细胞或权利要求9所述单价抗体、二价抗体、多价抗体、重组蛋白或免疫偶联物。
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