CN108929382B - 一种抗serinc5的抗体及其应用 - Google Patents
一种抗serinc5的抗体及其应用 Download PDFInfo
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- CN108929382B CN108929382B CN201810800332.6A CN201810800332A CN108929382B CN 108929382 B CN108929382 B CN 108929382B CN 201810800332 A CN201810800332 A CN 201810800332A CN 108929382 B CN108929382 B CN 108929382B
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- serinc5
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- monoclonal antibody
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Abstract
本发明涉及一种抗SERINC5的抗体及其应用,具体而言,本发明提供了一种抗SERINC5的抗体及功能性片段、核苷酸、载体、单克隆抗体、试剂盒及其在在检测SERINC5蛋白或SEQ ID NO:9所示的多肽序列中的应用,同时,本发明提供了一种制备单克隆抗体的方法、制备单克隆抗体杂交瘤细胞系的方法。利用本发明中的方法制备的抗体和功能性片段可有效的用于与SERINC5蛋白的反应中,如在流式细胞术检测细胞表面的SERINC5分子、结合细胞表达的SERINC5,研究病毒扩增机制等,这将为研究SERINC5的抗病毒机理提供有效的研究工具,将有助于加快本领域的研究进展,为进一步开发靶向HIV蛋白Nef的新型艾滋病药物提供新的思路。
Description
技术领域
本发明涉及分子生物学领域,具体涉及一种抗SERINC5的抗体及其应用。
背景技术
丝氨酸整合因子(SERINC)是一种膜蛋白,其家族共有5个成员,其中SERINC5是目前最新发现的抗艾滋病病毒的天然免疫分子,可阻断病毒感染新的细胞,从而显著降低人类免疫缺陷病毒(HIV)的感染能力。SERINC5蛋白对病毒传染力的影响很大,可将HIV病毒粒子的传染力降低100多倍。HIV编码了一种调节蛋白Nef来抵抗SERINC5。HIV抑制SERINC蛋白的能力对于其感染的细胞有着极大的影响,破坏抑制SERINC的这一机制可使得SERINC5蛋白能够失活病毒,是治疗HIV和表达Nef蛋白的相似病毒的一种非常强大的策略。
除了能够抑制HIV之外,近期研究发现,SERINC5蛋白的抗逆转录病毒效应似乎横跨所有的逆转录病毒,有可能是普遍存在的。SERINC5蛋白抑制了所有逆转录病毒、甚至与HIV-1亲缘关系最远的逆转录病毒的传染力。深入研究SERINC5蛋白的功能及其机制有可能会对所有有包膜病毒的治疗产生影响。
然而,SERINC家族各成员的抗病毒活性、体内表达以及抗病毒机理,科研人员还知之甚少。目前尚不清楚SERINC5蛋白阻止病毒粒子将它的基因组下载到新宿主细胞中去的确切机制。一种可能是SERINC5在物理上阻止了病毒基因组通过病毒包膜及细胞膜进入到新宿主细胞中。对于这一过程以及SERINC运作机制我们仍有许多东西尚不清楚。而SERINC5特异性单克隆抗体,特别是可用于流式细胞术检测的单克隆抗体的缺乏,阻碍了研究人员揭示SERINC5抗病毒功能的研究进度。
发明内容
本发明旨在至少在一定程度上解决相关技术中的技术问题,为此,本发明的目的在于提供一种SERINC5特异性单克隆抗体或功能性片段,该抗体或功能性片段可与SERINC5有效结合,从而可用于研究SERINC5的作用机理,这将为研究SERINC5的抗病毒机理提供有效的研究工具,将有助于加快本领域的研究进展,为进一步开发靶向HIV蛋白Nef的新型艾滋病药物提供新的思路。
为此,本发明的第一方面提供了一种抗体或功能性片段,其选自:
a)抗体或功能性片段,包含SEQ ID NO:1—SEQ ID NO:3所示氨基酸序列,和/或SEQ ID NO:4—SEQ ID NO:6所示氨基酸序列;
b)抗体或功能性片段,含有分别与SEQ ID NO:1—SEQ ID NO:3,和/或SEQ ID NO:4—SEQ ID NO:6所示的氨基酸序列同源性序列,其中至少有一条序列具有与SEQ ID NO:1—SEQ ID NO:6不低于95%、96%、97%、98%或99%的同源性;
c)a)-b)所述的抗体或功能性片段经氨基酸残基的修饰、取代、缺失或添加而形成的氨基酸衍生序列。
上面所述的抗体或功能性片段能与SERINC5结合。
其中SEQ ID NO:1—SEQ ID NO:3所示氨基酸序列分别为重链互补决定区CDR1—CDR3,SEQ ID NO:4—SEQ ID NO:6所示氨基酸序列分别为轻链互补决定区CDR1—CDR3。
在一优选例中,所述的抗体或功能性片段含有SEQ ID NO:10和/或SEQ ID NO:11所示的氨基酸序列。
在一优选例中,所述的抗体或功能性片段为鼠源抗体或功能性片段。
本发明的第二方面提供了一种核苷酸,所述核苷酸序列含有编码上述抗体或功能性片段的序列;
在一优选例中,所述的核苷酸序列包含SEQ ID NO:7和/或SEQ ID NO:8所示的序列。
本发明的第三方面提供了一种载体,所述载体含有上述的核苷酸序列。
本发明的第四方面提供了一种单克隆抗体,所述单克隆抗体由杂交瘤细胞株4D4.8,保藏编号为CCTCC NO:C2018140表达。
本发明的第五方面提供了一种制备杂交瘤细胞株4D4.8,保藏编号为CCTCC NO:C2018140的方法,所述方法包括如下步骤:
将含有SEQ ID NO:9所示的多肽序列的试剂免疫小鼠;
取免疫后小鼠的脾脏细胞与SP2/0细胞融合;
通过免疫检测反应筛选可与SERINC5蛋白反应的融合细胞。
本发明的第六方面提供了一种制备单克隆抗体的方法,即在合适的培养基中培养杂交瘤细胞株4D4.8,保藏编号为CCTCC NO:C2018140。
本发明的第七方面提供了一种试剂盒,所述试剂盒包含上述的抗体或功能性片段,或核苷酸,或载体,或单克隆抗体。
本发明的第七方面提供了抗体或功能性片段,或核苷酸,或载体,或单克隆抗体在检测SERINC5蛋白或SEQ ID NO:9所示的多肽序列中的应用。
所述检测不局限于检测SERINC5蛋白或SEQ ID NO:9所示的多肽序列是否存在,还包括筛选,使目标蛋白或多肽序列特异结合进行其它研究等,如,与SEQ ID NO:9所示的多肽或源于SERINC5ECL4的免疫原多肽特异性结合;Western blot,检测SERINC5的表达;应用于流式细胞术检测细胞表面的SERINC5分子;结合细胞表达的SERINC5,研究病毒扩增机制等中的应用等。
本发明所取得的有益效果
本发明提供了一种抗体和功能性片段,利用该抗体和功能性片段可以有效检测出SERINC5蛋白及来源SERINC5蛋白的具有免疫原性的多肽分子,此种抗体可以有效的用于与SERINC5蛋白的反应中,如在流式细胞术检测细胞表面的SERINC5分子、结合细胞表达的SERINC5,研究病毒扩增机制等,这将为研究SERINC5的抗病毒机理提供有效的研究工具,将有助于加快本领域的研究进展,为进一步开发靶向HIV蛋白Nef的新型艾滋病药物提供新的思路。
词语释义:
功能性片段:结构处于氨基酸和蛋白质之间的一类化合物,具有生物活性。
附图说明
图1为SERINC5蛋白胞外环4的氨基酸序列及其亲水性、免疫原性和表面概率强弱,及选用的用于抗体制备的多肽序列(方框标示)。
图2为ELISA筛选出阳性克隆4D4和5H12。
图3为ELISA检测4D4.6、4D4.8、4D4.11、5H4.1与多肽的结合的结果图。
图4为流式细胞术分析单克隆抗体4D4.6、4D4.8、4D4.11、5H4.1与293T-SERINC5细胞的结合的结果图,图中灰色峰为杂交瘤培养上清与293T细胞反应的结果,白色的峰为培养上清与293T-SERINC5细胞反应的结果。
图5为流式细胞术检测单克隆抗体4D4.8和5H4.1检测PBMC细胞表面SERINC5的表达的结果图。
图6为Western blot用单克隆抗体4D4.8检测SERINC5的表达的结果图。
图7为单克隆抗体4D4.8亚型分析结果。
图8为4D4.8重链可变区序列结构图。
图9为4D4.8轻链可变区序列结构图。
生物材料保藏:
本发明中杂交瘤细胞株送中国典型培养物保藏中心保藏,保藏编号为CCTCC NO:C2018140,地址位于中国武汉大学,保藏日期均为2018年6月28日,分类命名为杂交瘤细胞株4D4.8。
具体实施方式
本发明提供了能够抗SERINC5的特异性单克隆抗体及其功能性片段、编码抗体的核苷酸序列、载体及其应用。
在不实质性影响抗体活性的前提下,本领域技术人员可以对本发明的序列替换、添加和/或缺失一个或更多个(例如1、2、3、4、5、6、7、8、9或10个或更多个)氨基酸,以获得所述抗体或其功能性片段之序列的变体。它们都被视为包括在本发明保护的范围内。如在可变区将具有类似性质的氨基酸进行替换。本发明所述变体的序列可以与其来源序列有至少有95%、96%、97%、98%或99%的一致性。本发明所述的序列一致性可以使用序列分析软件测量。例如使用缺省参数的计算机程序BLAST,尤其是BLASTP或TBLASTN。
本发明的抗体可以是全长的(例如,IgM,IgG1或IgG4抗体)或可仅包含抗原结合部分(例如,Fab、F(ab’)2或scFv片段),或可以被修饰以影响功能。本发明包括具有修饰的糖基化模式的抗SERINC5抗体。在一些应用中,进行修饰以除去不期望的糖基化位点可以是有用的,或在寡糖链上不存在岩藻糖部分以例如增强抗体依赖性细胞毒性(ADCC)功能的抗体。在另一些应用中,可进行半乳糖基化修饰以改变补体依赖性细胞毒性(CDC)。
本文所使用的术语“功能性片段”指的是具有SERINC5结合活性的片段,尤其是指抗体片段如Fv、scFv(sc指单链)、Fab、F(ab’)2、Fab’、scFv-Fc片段或者双抗体(diabody)、或者通过化学修饰或通过掺入脂质体中应能够增加半寿期的任何片段,所述化学修饰例如添加聚(亚烷基)二醇如聚乙二醇(“聚乙二醇化,PEG化”)(被称为Fv-PEG、scFv-PEG、Fab-PEG、F(ab’)2-PEG或Fab’-PEG的聚乙二醇化片段)(“PEG”为聚乙二醇)。优选地,所述功能片段将由其来源抗体的重或轻可变链的部分序列构成或者包含它们,所述部分序列足以保留与其来源抗体相同的结合特异性和充分的亲和力,对于SERINC5,优选至少等于其来源抗体亲和力的1/100,在更优选方式中至少等于1/10。这种功能片段将包含最少5个氨基酸,优选其来源的抗体序列的10、15、25、50和100个连续氨基酸。
PBS:NaCl 137mmol/L,KCl 2.7mmol/L,Na2HPO4 10mmol/L,KH2PO4 2mmol/L
Western blot上样缓冲液:购自碧云天(货号:P0015)
下面参考具体实施例和附图,对本发明进行说明。需要说明的是,这些实施例仅仅是说明性的,而不能理解为对本发明的限制。实施例中未注明具体技术或条件的,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
实施例1 SERINC5的多肽免疫原的筛选
SERINC5(GENBANK ACCESSION:NM_001174072)为9次跨膜蛋白,目前尚无法制备纯化蛋白,故采用多肽免疫小鼠,制备单克隆抗体的策略。
SERINC5胞外区包含氨基端和4个胞外环,其中氨基端包含35个氨基酸;第1个胞外环包含14个氨基酸残基,且含有1个糖基化位点;第2个胞外环包含21个氨基酸残基,含有1个糖基化位点;第3个胞外环只包含7个氨基酸;第4个胞外环由53个氨基酸残基组成,且无糖基化位点。SERINC5的氨基端和第4个胞外环区包含氨基酸残基较多,且无糖基化位点,比较容易筛选出合适作为免疫原的多肽。通过DNASTAR软件分析发现氨基端C25-R35的区域免疫原性较强,但由于该区域较短只有10个氨基酸,且紧靠细胞膜,有空间位阻效应,不是理想的免疫原。处于第4个胞外环区域的G362-G375肽片段具有良好的免疫原性、亲水性和表面频率(图1),适合作为免疫原,故合成多肽并偶联钥孔血蓝蛋白(KLH),以免疫小鼠,制备单克隆抗体。
合成多肽序列如下:
GGEDTEEQQPGKEG(SEQ ID NO:9),
上述多肽的合成及与KLH(血蓝蛋白)的偶联由南京金斯瑞生物科技有限公司完成。
实施例2抗人SERINC5单克隆抗体的制备
1.免疫动物
蒸馏水溶解KLH偶联的上述多肽(SEQ ID NO:9)配制浓度为1mg/ml作为免疫原,取免疫原与弗氏完全佐剂(Sigma-Aldrich)各100μl混合震荡形成油包水乳浊液。初次免疫取6-8周龄BALB/c小鼠进行多点皮下注射200μl免疫原/佐剂混合液。在初次免疫以后,分别于第3周和第6周进行一次减半剂量的加强免疫,并与第8周采血,ELISA分析抗体滴度。
2.细胞融合
在最后一次加强免疫后2周,处死小鼠,取脾脏细胞,按照10:1的比例与SP2/0细胞(购自ATCC,货号:CRL-1581)混合,离心后按常规方法用PEG1450(Sigma-Aldrich)进行细胞融合。将融合细胞重悬于含有HAT(次黄嘌呤(h)、氨基蝶呤(a)、胸苷(t))和20%FBS(胎牛血清)的DMEM筛选培养基(Gibco,货号:10569010)中,接种96孔细胞培养板,置于5%CO2,37℃条件下培养。
3.杂交瘤细胞筛选
将2中制备的多个杂交瘤细胞培养10~12天,待杂交瘤细胞铺满孔底1/10面积时,取100μl培养上清,分别加到用实施例1中合成的多肽裸肽(SEQ ID NO:9),以10μg/ml的浓度包被酶标板中,用常规ELISA方法进行阳性克隆的筛选(HRP标记二抗购自北京义翘神州科技有限公司,货号:SSA007-200),结果发现第4块酶标板的D4孔呈强阳性,将其命名为4D4,第5块酶标板的H4呈弱阳性,将其命名为5H4(图2)。当杂交瘤细胞铺满孔底约20%面积时,用有限稀释法对阳性克隆4D4进行亚克隆,按上述的ELISA法,筛选出阳性克隆4D4.6、4D4.8、4D4.11、5H4.1(图3)。
实施例3流式细胞术检测细胞表面SERINC5
取实施例2筛选出的杂交瘤细胞4D4.6、4D4.8、4D4.11、5H4.1的培养上清各100μl,分别与1×106个293T细胞和293T-SERINC5细胞(稳定表达人SERINC5的293T细胞系)(上述2个细胞系为深圳市第三人民医院肝病研究所保存)在室温下进行孵育10分钟,加PBS离心洗涤,加0.2μl荧光二抗AF647-Anti mouse IgG(H+L)(Invitrogen,货号A-21237),室温避光孵育10分钟,PBS洗涤后,流式细胞术检测细胞表面荧光信号。结果发现,4D4.6、4D4.8、4D4.11可以识别细胞表面的SERINC5分子,而5H4.1只能识别多肽,不能识别细胞表面的SERINC5分子(图4,图中灰色峰为杂交瘤培养上清与293T细胞反应的结果,白色的峰为培养上清与293T-SERINC5细胞反应的结果)。
为检测CD4T细胞SERINC5分子表达情况,取健康人外周抗凝血100μl,分别加入100μl杂交瘤细胞4D4.8、5H4.1(阴性对照)培养上清、5μl PerCP/Cy5.5anti-human CD3(PerCP.Cy5.5CD3)(Biolegend,货号317336)、5μl FITC anti-human CD4(FITC.CD4)(Biolegend,货号357406),混匀室温孵育10分钟,加入2ml PBS洗涤后,加0.2μl荧光二抗AF647-Anti mouse IgM(Invitrogen,货号M31601),室温避光孵育10分钟,加入2ml红细胞裂解液(碧云天,货号C3702),室温孵育5分钟裂解红细胞,离心,PBS洗涤后流式细胞术检测细胞表面荧光信号。通过FSC和SSC选取淋巴细胞(图5A)进一步分析,通过CD3和CD4的表达情况,将淋巴细胞分为3群,选取双阳性的CD4T淋巴细胞(图5B)进一步分析4D4.8和5H4.1与细胞表面SERINC5的结合情况。结果类似293T-SERINC5的检测结果一样,4D4.8可以检测CD4T细胞表面的SERINC5,而5H4.1则不能。以上结果表明单克隆抗体4D4.8可以用于流式细胞术检测细胞表面SERINC5分子(图5C-D)。
实施例4 Western blot检测细胞SERINC5的表达
取4×106健康人外周血单个核细胞(PBMC)和1×106 293T细胞,按照常规方法提取全细胞蛋白,按4:1的体积比加入5×Western blot上样缓冲液,100℃处理样本10分钟,各取15μl样品,按照常规方法跑SDS-PAGE、转膜、封闭,然后用5ml杂交瘤4D4.8培养上清室温孵育1小时后,常规方法进行洗涤、HRP标记的二抗(购自北京义翘神州科技有限公司,货号:SSA007-200)孵育、ECL试剂曝光,结果在预期的位置发现SERINC5的条带,表明单克隆抗体4D4.8可以用于Western blot实验检测SERINC5的表达(图6)。
实施例5亚型分析
采用Rapid Isotyping Kits–Mouse(Thermo Scientific)小鼠单抗亚型分析试剂盒,按照试剂盒说明书分析了单克隆抗体4D4.8的亚型,结果显示4D4.8为IgM类抗体,轻链为κ链(图7)。
实施例6抗体可变区序列
将杂交瘤细胞4D4.8总数量培养到107个细胞,1000rpm离心10分钟收集细胞,并以Trizol试剂盒(Invitrogen,货号15596026)按照试剂盒说明书提取总RNA。以所述RNA为模板,合成第一链cDNA(TAKARA,货号6110A),以第一链cDNA为后续模板扩增杂交瘤细胞所对应的可变区DNA序列。扩增反应中所使用的引物序列与抗体可变区第一框架区和恒定区互补,引物序列详见表1,在50μl反应体系中,分别加入cDNA 1μl,10×PCR缓冲液5μl,上游及下游引物各1μl(25pmol),dNTP 1μl,Taq酶(Invitrogen)1μl,H2O 40μl,95℃预变性5min,进入温度循环,进行PCR扩增。反应条件为94℃变性1min,55℃退火1min,72℃延伸30秒,共30个循环,然后72℃保温10min,4℃冷却。
将扩增产物测序后,得到杂交瘤克隆4D4.8重链可变区序列结构(图8)和轻链可变区序列结构(图9)及其对应的核苷酸序列,具体序列如表2所示:
表1:引物序列
表1中K、Y、S、W和R分别为简并碱基,其中K对应G/T,Y对应C/T,S对应G/C,W对应A/T,R对应A/G。
表2:4D4.8重链和轻链序列
在本说明的描述中,参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中,对上述术语的示意性表述不必针对的是相同的实施例或示例。而且,描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外,在不相互矛盾的情况下,本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例,可以理解的是,上述实施例是示例性的,不能理解为对本发明的限制,本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。
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<110> 深圳市第三人民医院
<120> 一种抗SERINC5的抗体及其应用
<130> 20180702
<160> 28
<170> PatentIn version 3.5
<210> 1
<211> 8
<212> PRT
<213> Mus musculus
<400> 1
Gly Tyr Thr Phe Thr Ser Tyr Trp
1 5
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<211> 8
<212> PRT
<213> Mus musculus
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Ile Tyr Pro Gly Asp Gly Asp Thr
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<211> 14
<212> PRT
<213> Mus musculus
<400> 3
Ala Arg Ser Thr Tyr Gly Ser Ser Phe Ala Trp Phe Ala Tyr
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<212> PRT
<213> Mus musculus
<400> 4
Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr
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<212> PRT
<213> Mus musculus
<400> 5
Lys Val Ser
1
<210> 6
<211> 9
<212> PRT
<213> Mus musculus
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Phe Gln Gly Ser His Val Pro Trp Thr
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<212> DNA
<213> Mus musculus
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caggttcagc tccagcagtc tggggctgag ctggcaagac ctggggcttc agtgaagttg 60
tcctgcaagg cttctggcta cacctttact agctactgga tgcagtgggt aaaacagagg 120
cctggacagg gtctggaatg gattggggct atttatcctg gagatggtga tactaggtac 180
actcagaagt tcaagggcaa ggccacattg actgcagata aatcctccag cacagcctac 240
atgcaactca gcagcttggc atctgaggac tctgcggtct attactgtgc aagatcgacg 300
tacggtagta gcttcgcctg gtttgcttac tggggccaag ggactctggt cactgtctct 360
gca 363
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<212> DNA
<213> Mus musculus
<400> 8
gatgttttga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60
atctcttgca gatctagtca gagcattgta catagtaatg gaaacaccta tttagaatgg 120
tacctgcaga aaccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180
tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240
agcagagtgg aggctgagga tctgggagtt tattactgct ttcaaggttc acatgttccg 300
tggacgttcg gtggaggcac caagctggaa atcaaagctgat 342
<210> 9
<211> 14
<212> PRT
<213> 人工序列
<400> 9
Gly Gly Glu Asp Thr Glu Glu Gln Gln Pro Gly Lys Glu Gly
1 5 10
<210> 10
<211> 121
<212> PRT
<213> Mus musculus
<400> 10
Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Trp Met Gln Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Ala Ile Tyr Pro Gly Asp Gly Asp Thr Arg Tyr Thr Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Thr Tyr Gly Ser Ser Phe Ala Trp Phe Ala Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ala
115 120
<210> 11
<211> 114
<212> PRT
<213> Mus musculus
<400> 11
Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly
1 5 10 15
Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser
20 25 30
Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly
85 90 95
Ser His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
Ala Asp
<210> 12
<211> 21
<212> DNA
<213> 人工序列
<400> 12
ggatacagtt ggtgcagcat c 21
<210> 13
<211> 21
<212> DNA
<213> 人工序列
<400> 13
gatgttttga tgacccaaac t 21
<210> 14
<211> 21
<212> DNA
<213> 人工序列
<400> 14
gatattgtga tgacgcaggc t 21
<210> 15
<211> 18
<212> DNA
<213> 人工序列
<400> 15
gatattgtga taacccag 18
<210> 16
<211> 21
<212> DNA
<213> 人工序列
<400> 16
gacattgtgc tgacccaatc t 21
<210> 17
<211> 21
<212> DNA
<213> 人工序列
<400> 17
gacattgtga tgacccagtc t 21
<210> 18
<211> 21
<212> DNA
<213> 人工序列
<400> 18
gatattgtgc taactcagtc t 21
<210> 19
<211> 21
<212> DNA
<213> 人工序列
<400> 19
gatatccaga tgacacagac t 21
<210> 20
<211> 21
<212> DNA
<213> 人工序列
<400> 20
gacatccagc tgactcagtc t 21
<210> 21
<211> 21
<212> DNA
<213> 人工序列
<400> 21
caaattgttc tcacccagtc t 21
<210> 22
<211> 21
<212> DNA
<213> 人工序列
<400> 22
gacattctga tgacccagtc t 21
<210> 23
<211> 21
<212> DNA
<213> 人工序列
<400> 23
atggtgytaa ktcttytgta s 21
<210> 24
<211> 24
<212> DNA
<213> 人工序列
<400> 24
atggctgtyy tggtgctgtt cctc 24
<210> 25
<211> 22
<212> DNA
<213> 人工序列
<400> 25
atgggawgga gctgkrtctt yc 22
<210> 26
<211> 24
<212> DNA
<213> 人工序列
<400> 26
atgaartkca gctgggtcat sttc 24
<210> 27
<211> 22
<212> DNA
<213> 人工序列
<400> 27
atgaagttgt tggstgaact gg 22
<210> 28
<211> 19
<212> DNA
<213> 人工序列
<400> 28
acatttggga aggactgac 19
Claims (10)
1.一种抗体或功能性片段,其特征在于,
其重链可变区的HCDR1、HCDR2、HCDR3分别对应是SEQ ID NO:1、SEQ ID NO:2、SEQ IDNO:3所示氨基酸序列,且其轻链可变区的LCDR1、LCDR2、LCDR3分别对应是SEQ ID NO:4、SEQID NO:5、SEQ ID NO:6所示氨基酸序列;
所述的抗体或功能性片段能与SERINC5结合。
2.根据权利要求1所述抗体或功能性片段,其特征为,所述的抗体或功能性片段的重链可变区为SEQ ID NO:10所示的氨基酸序列,且轻链可变区为SEQ ID NO:11所示的氨基酸序列。
3.根据权利要求2所述抗体或功能性片段,其特征为,所述抗体或功能片段为鼠源抗体或功能片段。
4.一种多核苷酸,其特征为,所述多核苷酸序列含有编码权利要求1或2所述的抗体或功能性片段的序列。
5.根据权利要求4所述多核苷酸,其特征为,所述的多核苷酸序列包含SEQ ID NO:7和SEQ ID NO:8所示的序列。
6.一种载体,其特征为,所述载体含有权利要求4所述的多核苷酸序列。
7.一种单克隆抗体,其特征为,所述单克隆抗体由保藏编号为CCTCC NO:C2018140的杂交瘤细胞株4D4.8表达。
8.根据权利要求7所述单克隆抗体,其特征为,所述单克隆抗体为IgM抗体。
9.一种试剂盒,其特征为,所述试剂盒包含权利要求1-3中任一项所述的抗体或功能性片段,或权利要求4-5中任一项所述的多核苷酸,或权利要求6所述的载体,或权利要求7所述的单克隆抗体。
10.权利要求1-3中任一项所述的抗体或功能性片段,或权利要求4-5中任一项所述的多核苷酸,或权利要求6所述的载体,或权利要求7所述的单克隆抗体在检测SERINC5蛋白或SEQ ID NO:9所示的多肽序列中的应用。
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