CN113637079B - 抗setd3的单克隆抗体及其用途 - Google Patents
抗setd3的单克隆抗体及其用途 Download PDFInfo
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- CN113637079B CN113637079B CN202110829627.8A CN202110829627A CN113637079B CN 113637079 B CN113637079 B CN 113637079B CN 202110829627 A CN202110829627 A CN 202110829627A CN 113637079 B CN113637079 B CN 113637079B
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Abstract
本发明提供了一种抗SETD3的单克隆抗体及其用途，所述单克隆抗体能识别SETD3蛋白，所述单克隆抗体包括重链可变区和轻链可变区：所述重链可变区具有如SEQIDNO：1‑SEQIDNO：3所示的氨基酸序列的三个互补决定区；所述轻链可变区具有如SEQIDNO：4‑SEQIDNO：6所示的氨基酸序列的三个互补决定区。本发明提供的抗SETD3的单克隆抗体对体外纯化蛋白、体外培养哺乳动物细胞内外源SETD3蛋白、小鼠组织SETD3蛋白均具有良好的识别特异性；与市售抗体相比，该单克隆抗体识别特异性更强。
Description
技术领域
本发明属于生物技术领域，涉及一种抗SETD3的单克隆抗体及其用途。
背景技术
SETD3是含有SET结构域的甲基转移酶家族的一员，在亲缘关系上与SETD4和SETD6蛋白相近，结构也比较类似。SETD3在二级结构上主要包含催化结构域(SET domain)和底物结合结构域(Rubis-subs-bind domain)。SETD3在不同物种中相对比较保守，在酵母中没有发现其同源蛋白。在人体中，SETD3在多种组织中均有表达，在睾丸和骨骼肌组织中表达量最高。在肌原细胞分化的过程中，SETD3能够促进骨骼肌分化；SETD3和FoxM1共结合在血管内皮生长因子(vascular endothelial-derived growth factor，VEGF)信号通路的启动子区域并负调控VEGF表达；SETD3在不同的癌症中具有调控功能等。
近年来，Drozak和Gozani实验室同时证明了SETD3可以催化修饰β-Actin蛋白的第73位组氨酸，该位点的修饰对β-Actin蛋白的稳定性及其组装细胞骨架的能力非常重要，这些研究首次揭示了组氨酸甲基化修饰的重要作用。其中，Gozani课题组的研究通过SETD3与SETD6结构对比发现SETD3蛋白的酶活位点为313位酪氨酸(SETD3 Y313)。作者发现β-Actin蛋白H73甲基化修饰调节成肌纤维的聚合和解聚，同时也影响细胞的迁移能力。作者还发现Setd3基因敲除雌鼠的难产率超过80％，且其生崽数减少。除此之外，SETD3被证明在多种癌症中具有调控功能。
总之，SETD3蛋白作为首个在多细胞生物中被发现的组氨酸甲基转移酶，它的深入研究对于开拓了解组氨酸甲基化修饰的生物学功能具有重要的意义。因此，亟需开发一种效价好，特异性高的SETD3蛋白的单克隆抗体。
发明内容
为了解决所述技术问题，本发明提供了一种抗SETD3的单克隆抗体及其用途，可以特异性识别SETD3蛋白，抗体特异性良好且效价高，与市售抗体相比，该单克隆抗体识别特异性更强。
在本发明的第一方面，提供了一种抗SETD3的单克隆抗体，所述单克隆抗体能识别SETD3蛋白，所述单克隆抗体包括重链可变区和轻链可变区：
所述重链可变区具有如SEQ ID NO：1-SEQ ID NO：3所示的氨基酸序列的三个互补决定区；
所述轻链可变区具有如SEQ ID NO：4-SEQ ID NO：6所示的氨基酸序列的三个互补决定区。
进一步地，所述重链可变区的氨基酸序列如SEQ ID NO：7所示；所述轻链可变区的氨基酸序列如SEQ ID NO：8所示。
进一步地，所述单克隆抗体还包括：
所述单克隆抗体的氨基酸序列经取代、缺失和/或增加一个或多个氨基酸得到的具有相同功能的抗体；
或者包括具有与所述重链可变区具有至少80％的同源性的氨基酸序列的重链可变区；和与所述轻链可变区具有至少80％的同源性的氨基酸序列的轻链可变区；
或者所述单克隆抗体的N端和/或C端连接标签得到的抗体。
在另外一些实施方案中，VH和/或VL氨基酸序列可以与上述序列85％、90％、95％、96％、97％、98％或99％同源。具有与上述序列的VH和VL区高度(即80％或更高)同源的VH和VL区的抗体可以如下获得：诱变(例如定点诱变或PCR介导的诱变)编码SEQIDNO：1-6的核酸分子，然后用此处所述的功能试验检测编码的被改变抗体的保留的功能。
所述单克隆抗体包括：人抗体、人源化或嵌合抗体。
在其他实施方式中，可采用将可变区基因转化为scFv基因，一旦获得编码VH和VL片段的DNA片段，即可通过标准重组DNA技术进一步操作这些DNA片段，例如将可变区基因转化为全长抗体链基因、Fab片段基因或scFv基因。
在这些操作中，编码VL或VH的DNA片段与编码另外一种蛋白质如抗体恒定区或柔性连接体的另一个DNA片段有效连接。如本文使用的术语“有效连接”意思是两个DNA片段连接在一起，使得这两个DNA片段编码的氨基酸序列保持符合阅读框。
在本发明的第二方面，本发明提供了一种编码所述单克隆抗体的核酸分子，所述核酸分子包括编码所述重链可变区的核酸分子和编码所述轻链可变区的核酸分子。
在本发明的第三方面，提供了一种包含所述核酸的表达载体，所述表达载体能够在原核或者真核宿主细胞中表达所述核酸。
所述载体可以是常规的载体；具体可以为质粒载体、噬菌体载体、病毒载体；
在本发明的第四方面，提供了一种包含所述的表达载体的工程菌或真核宿主细胞。
在本发明的第五方面，提供了所述的SETD3蛋白的单克隆抗体在制备SETD3蛋白试剂或试剂盒中的用途。
在本发明的第六方面，提供了所述的SETD3蛋白的单克隆抗体在制备SETD3蛋白胶体金检测试剂盒的质控抗体中的用途。
在本发明的第七方面，提供了一种SETD3蛋白的胶体金快速检测试纸条，包括：
底板，
粘合在所述底板上且依次搭接的样品吸收垫、结合垫、层析基质和吸水垫；其中，
所述结合垫上涂有所述的SETD3蛋白的单克隆抗体包被的胶体金复合物；所述层析基质上靠近所述结合垫的一侧设有质控线C，所述层析基质上靠近所述吸水垫的一侧设有检测线T；所述质控线C上涂有包被有抗鼠IgG二抗；所述检测线T上包被有所述的SETD3蛋白的单克隆抗体。
本发明实施例中的一个或多个技术方案，至少具有如下技术效果或优点：
本发明提供的抗SETD3的单克隆抗体对体外纯化蛋白、体外培养哺乳动物细胞内外源SETD3蛋白、小鼠组织SETD3蛋白均具有良好的识别特异性；与市售抗体相比，该单克隆抗体识别特异性更强。
附图说明
为了更清楚地说明本发明实施例中的技术方案，下面将对实施例描述中所需要使用的附图作一简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其它的附图。
图1为实施例1中用于免疫小鼠的SETD3蛋白纯化图；
图2为SDS-PAGE检测SETD3抗体纯化结果；抗体上样量从左到右增加；
图3为ELISA实验检测SETD3抗体效价；横坐标为抗体稀释比例，纵坐标为A450吸光度；
图4为Western Blot实验鉴定纯化后抗体与SETD3蛋白之间的特异性识别作用。泳道1为纯化的GST标签的SETD3融合蛋白；泳道2为GST标签蛋白；左图为考马斯亮蓝染色表征两种蛋白的上样量相当，右图为SETD3抗体对两个样品识别情况；
图5为在哺乳动物细胞PC9细胞系中构建SETD3敲低或过表达的细胞系，用1-H9抗体检测的结果；
图6为在哺乳动物细胞HeLa-S3细胞系中敲除SETD3基因，用WB方法检测SETD3的蛋白水平结果；
图7为利用WB实验检测SETD3蛋白水平的结果；
图8为SETD3截短突变的小鼠进行检测的结果；
图9为纯化抗体与SETD3市售抗体特异性比较。
图10为IgG重链的可变区序列对比图；
图11为IgG轻链的可变区序列对比图。
具体实施方式
下文将结合具体实施方式和实施例，具体阐述本发明，本发明的优点和各种效果将由此更加清楚地呈现。本领域技术人员应理解，这些具体实施方式和实施例是用于说明本发明，而非限制本发明。
在整个说明书中，除非另有特别说明，本文使用的术语应理解为如本领域中通常所使用的含义。因此，除非另有定义，本文使用的所有技术和科学术语具有与本发明所属领域技术人员的一般理解相同的含义。若存在矛盾，本说明书优先。
除非另有特别说明，本发明中用到的各种原材料、试剂、仪器和设备等，均可通过市场购买得到或者可通过现有方法制备得到。
下面将结合实施例及实验数据对本申请的单克隆抗体及其制备方法与应用效果进行详细说明。下列实施例中未注明的具体实验条件和方法，通常按照常规条件如：J.萨姆布鲁克等主编，科学出版社，1992，分子克隆实验指南(第三版)；D.L.斯佩克特等，科学出版社，2001，细胞实验指南等书中所述的条件，或按照制造厂商所建议的条件。
实施例1单克隆抗体及其制备方法
1、克隆构建及蛋白原核表达
(1)将SETD3 CDS序列克隆在pGEX-6p-1载体上，获得重组载体pGEX-6p-1-SETD3。
所用引物对序列为：正向引物(5’-ATGGGTAAGAAGAGTCGAG-3’；SEQ ID NO：9)；反向引物(5’-TTACTCCTTAACTCCAGCAGTG-3’；SEQ ID NO：10)，克隆到pGEX-6p-1载体的EcoRⅠ-NotⅠ酶切位点。
(2)蛋白表达及纯化：
①质粒转化：
将构建好的SETD3原核表达质粒转化至BL21感受态细胞，涂布在含氨苄抗性的LB平板上，待长出单克隆之后，挑单克隆摇菌。
②摇菌&表达：
摇过夜菌，第二天早上转接大量，1：100～1：50稀释。37℃摇菌至OD600＝0.4-0.8，用IPTG(0.1-0.3mM)诱导,16-25℃诱导表达6-8小时。
③收菌&裂菌：
将诱导好的菌液用4℃离心机收菌，用Milli Q水洗一遍，加入适量Lysis buffer,以及蛋白酶体抑制剂，溶菌酶，PMSF(1：100)，冰上放置30℃，超声裂菌。裂完之后用4℃离心机高速离心2次，每次30min,留上清。
④结合GSTbeads:
将GST-beads用lysis buffer洗三次，每次用2500rpm 3min离心，洗完之后加入到上清液中，4℃结合4-6小时。
⑤洗beads&洗脱蛋白：
离心去上清，将GST-beads用lysis buffer洗30分钟，再用Wash buffer洗两次，每次30分钟，洗完之后将液体吸干净，用Elutionbuffer洗脱蛋白，4-6小时。
⑥浓缩&透析蛋白
若蛋白浓度较低，用浓缩管浓缩，边浓缩边置换PBS buffer；若蛋白浓度较高，可用PBS直接透析。
⑦蛋白储存
透析好的蛋白利用考马斯亮蓝染色定量之后加10-20％甘油，液氮速冻，储存于-80℃，
(3)蛋白纯化相关试剂配方
Lysisi buffer：Tris-HCL:50mM，NaCl:150mM，NP-40:0.05％，pH＝7.5；
Wash buffer：50mM Tris-HCl，pH＝8.0；
Elutionbuffer：Tris-Hcl:50mM，pH＝8.0，Glutathion:20mM；
蛋白纯化图如图1所示。
2、动物免疫
将纯化的人源SETD3蛋白作为抗原免疫小鼠，选用5-8周龄Balb/C小鼠2只；第一次主注射使用弗氏完全佐剂，以后加强注射使用弗氏不完全佐剂，均与等体积抗原充分混匀后注射。免疫方法采用背部多点注射。免疫量为主注射100μg抗原/只实验小鼠，加强注射50μg抗原/只实验小鼠。
免疫周期如表1所示；
表1
3、抗血清检测
(1)从小鼠尾静脉取少量血，制备抗血清。
(2)ELISA方法检测抗血清效价。
4、细胞融合及亚克隆
(1)骨髓瘤细胞制备
融合前一周，复苏SP2/0细胞，正常培养至对数期。
(2)脾细胞制备
选定要融合的小鼠，融合当天用颈椎脱臼法处死，取脾，标准流程收集脾细胞并计数。
(3)细胞融合
按1:3-1：10的比例混合骨髓瘤细胞和脾细胞，标准流程进行细胞融合操作，随后用HATDMEM完全培养基培养，融合后3天即可以看到杂交瘤细胞，第7天换1/2HAT完全培养基，第8天换1/2HT培养基。融合后10天左右开始进行筛选检测。
细胞融合结果：融合后用HAT选择性培养基培养，显微镜下观察，看到多个生长的杂交瘤细胞，证明融合操作成功。
(4)融合筛选
吸取细胞上清100μL/孔进行间接ELISA检测。根据ELISA结果，判断阳性孔。用单道移液器挑检整板检测出的阳性孔，进行第二次复检，进一步确认阳性孔。
(5)亚克隆
对复筛的阳性孔细胞做两轮亚克隆。因为第一次亚克隆得到的阳性孔细胞株尚不稳定，有可能包含多个杂交瘤细胞，普遍认为第二次亚克隆后杂交瘤细胞为单个细胞株，并确定为阳性。
第一次亚克隆有限稀释阳性孔中细胞，至多个孔中，加HTDMEM培养基培养，7天左右在显微镜下观察，间接ELISA检测有克隆生长的孔，取OD值高的孔为阳性孔；挑取阳性孔的细胞进行第二次亚克隆，检测出稳定阳性的杂交瘤细胞株，作为最终制备单抗的细胞，并扩大培养，获得杂交瘤细胞株。
(6)单克隆抗体亚型鉴定
用美国Southern Biotech的单抗亚型鉴定试剂盒分别测各个上清的亚型。准备好包被有免疫原蛋白的板条，50ng/孔，每个克隆收集600μL上清，分别滴加到6个对应蛋白的酶标孔中，100μL/孔。37℃孵育1h，PBST洗三遍，将稀释好的分型二抗抗IgM，IgA，IgG1，IgG2a，IgG2b，IgG3的抗体加入6个孔中，37℃孵育1h，PBST洗三遍，TMB显色。有信号反应的孔对应的鉴定二抗亚型即为该抗体的亚型。
(7)确定的阳性细胞株号及亚型：
经过两轮亚克隆及复检，确定了阳性细胞株及亚型，交由客户鉴定，最终确定的阳性细胞株号及亚型为1H9:IgG1。
5、腹水制备及抗体纯化
(1)腹水制备
将上述阳性细胞扩大培养并注射至Balb/C小鼠(经弗氏不完全佐剂致敏)的腹腔，一般7-10日可见小鼠腹部隆起即代表有腹水产生。当小鼠有明显腹水产生时及时抽取腹水。
(2)腹水纯化
将上述细胞的腹水，进行纯化，纯化后抗体纯度大于90％。纯化方法如下：
(3)辛酸硫酸铵+DEAE离子柱法纯化(IgG1,IgG2a,IgG2b,IgG3亚型抗体):
(4)腹水离心，吸出淡黄色液体计算体积，用4倍体积的60mM醋酸缓冲液(pH 4.0)1:3稀释，逐滴加入辛酸(终浓度为25μL/mL稀释腹水)，室温搅拌30min，然后4℃静置2h以上，使其充分沉淀。
(5)10000r/min，4℃，20min，收集上清，加入1/10体积的10×PBS(0.1M
,pH7.4)。每毫升上述混合液加0.277g固体硫酸铵(0℃条件下，45％饱和硫酸铵为0.291g/mL)，继续静置至少60min以上。
(6)10000r/min，4℃，20min，弃上清，将沉淀溶解于少量PBS中。对PBS透析，4℃透析过夜。
(7)检测抗体浓度和纯度。测得抗体浓度为3mg/mL。利用考马斯亮蓝染色试验检测纯化抗体纯度，图2结果显示出重链和轻链条带，没有其它杂带，表明抗体纯度较高。
6、抗体轻链和重链可变区测序
(1)培养杂交瘤细胞
复苏杂交瘤细胞株后培养，待细胞数扩增至约1×107时，1000rpm×5min，离心收集细胞。
(2)提取细胞RNA
超净工作台环境下，将1mLTrizol试剂加入离心细胞，静置5min，加入氯仿2mL，剧烈摇晃15sec，室温静置3min，12000rpm×15min，吸取上层水样层至新的EP管，加入0.5mL异丙醇，室温静置10min。12000rpm×10min。弃上清，加入1mL 75％乙醇，7500rpm×5min，干燥沉淀，加入50μL双蒸水。琼脂糖电泳鉴定纯度并定量，保存于-70℃备用。
(3)反转录制备cDNA
细胞总RNA 1μL，RNase Free ddH2O 6μL，oligo dT Primer 0.5μL，PRIME ScriptRT Enzyme Mix I 0.5μL，5×Prime Script Buffer 2μL，混匀，37℃15min，85℃5s。
(4)扩增cDNA
用小鼠IgG VHVL引物库，分别扩增上述cDNA。5×Prime Star Buffer 10μL，dNTP 4μL，cDNA 1μL，上游引物1μL，下游引物1μL，PrimeSTAR 0.5μL，补水至50μL。按以下反应条件进行PCR反应:94℃温育5min，94℃变性45s，63℃退火45s，72℃延伸1min，30个循环后72℃延伸10min。
(5)琼脂糖凝胶电泳及胶回收
将上述PCR产物进行琼脂糖凝胶电泳，观察电泳结果，将分子量在250-350bp的扩增产物送交测序。
(6)抗体重链测序结果如SEQ ID NO：7所示，IgG重链的可变区序列对比图如图10所示。
(7)抗体轻链测序结果如SEQ ID NO：8所示，IgG轻链的可变区序列对比图如图11所示。
表2
实施例2、抗体效价检测
利用ELISA实验确定抗体的效价，在板子上每孔包被抗原(原核纯化人源SETD3蛋白)50ng，将纯化的单克隆抗体按表2比例稀释，测量每孔的A450吸光度，结果如表3和图3所示；
表3
| 效价 | 1K | 2K | 4K | 8K | 16K | 32K | 64K | 128K | 256K |
| 吸光度 | 0.580 | 0.579 | 0.480 | 0.441 | 0.340 | 0.230 | 0.163 | 0.109 | 0.077 |
由表3和图3可知，抗体效价可达到1：128000。
实施例3、抗体特异性检测
1、利用体外纯化的SETD3蛋白检测抗体特异性。
在体外原核纯化GST标签的SETD3蛋白或纯GST标签蛋白，利用Western Blot实验检测纯化抗体的识别特异性，如图4所示，左图为考马斯亮蓝染色证明两种蛋白上样量情况，右图为Western Blot实验检测抗体特异性，结果表明，SETD31-H9单克隆抗体对GST-SETD3具有很好的识别特异性，对等量的GST蛋白没有识别。
2、在细胞水平检测抗体的特异性
(1)在哺乳动物细胞PC9细胞系中构建SETD3敲低或过表达的细胞系，用1-H9抗体检测，如图5所示。本发明实施例中的SETD3敲低或过表达的细胞系的构建的方法为：SETD3shRNA序列为：sh1:5’-GCTTTGGTTTGAGAGCAACAA-3’(SEQ ID NO：11)；sh2:5’-GAAGAAGATGAAGTTCGGTAT-3’(SEQ ID NO：12)。将SETD3 shRNA序列克隆到plko.1载体上(酶切位点为EcoR I和AgeI)，敲低质粒在helper质粒psPAX2和pMD2.G的帮助下包装成敲低慢病毒，用慢病毒感染细胞，经过puromycin筛选得到稳定敲低SETD3的细胞系。
由图5可知，敲低SETD3的细胞中，用SETD3单克隆抗体检测到SETD3蛋白表达量降低，而过表达SETD3的细胞中也可以检测到外源SETD3条带。
(2)在哺乳动物细胞HeLa-S3细胞系中敲除SETD3基因，用WB方法检测SETD3的蛋白水平，结果如图6所示，本发明实施例中的敲除SETD3基因的方法为：利用Crispr-cas9的方法进行基因敲除。设计SETD3 sgRNA序列为：5’-GTCAGGTTCAAGATTTCCTT-3’(SEQ ID NO：13)。利用BbsI内切酶将此序列克隆到PX459载体中，将载体转染进入细胞，经过puromycin筛选之后，分单克隆，单克隆细胞长起来之后鉴定基因型和敲除效果，即得到敲除SETD3的细胞系。
由图6可知，敲除SETD3的细胞中蛋白水平消失。这些结果表明，SETD31-H9单克隆抗体在哺乳动物细胞中具有很高的识别特异性。
3、在小鼠组织水平检测抗体的特异性
(1)为了检测SETD31-H9抗体是否可用于小鼠水平上的研究，我们提取了小鼠肝脏组织，利用WB实验检测SETD3蛋白水平，HeLa S3敲低细胞系作为对照，如图7所示，结果表明，SETD3在小鼠组织中可检测到单一条带，其大小与在HeLa S3细胞中检测到的一致。
(2)为了证明SETD31-H9抗体在小鼠组织中检测到的条带确实是SETD3蛋白，我们利用SETD3截短突变的小鼠进行检测，如图8所示，本发明实施例中，SETD3截短突变的小鼠的方法为：利用Crispr-cas9的方法进行基因敲除。设计小鼠Setd3 sgRNA序列为：5’-AGGTCACTCTCCTCGTATCTGGG-3’(SEQ ID NO：14)。利用BsaI内切酶将sgRNA克隆到pUC57-Kan-T7-sgRNA载体上，体外转录得到sgRNA，将其显微注射到小鼠受精卵中，待小鼠成年进行基因鉴定。得到的小鼠虽然产生了移码，但是随后出现了终止密码子造成翻译提前终止，得到的小鼠表达截短的SETD3蛋白，含502个氨基酸(野生型SETD3具有594个氨基酸)。
由图8可知，截短了SETD3的小鼠组织中，1-H9抗体检测到较短的单一条带，证明该抗体可特异性识别小鼠组织SETD3，可用于小鼠水平上的研究。
4、比较SETD3单克隆抗体与市售SETD3抗体特异性。
我们在Abclonal抗体公司购入SETD3多克隆抗体(货号A8071)，在SETD3敲低的细胞样中比较两种抗体的性能，结果如图9所示，与此市售抗体相比，虽然两者都可以识别SETD3蛋白，但是市售抗体出现两条杂带，我们纯化的抗体条带更单一，表示出其更好的特异性。
最后，还需要说明的是，术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素，而且还包括没有明确列出的其他要素，或者是还包括为这种过程、方法、物品或者设备所固有的要素。
尽管已描述了本发明的优选实施例，但本领域内的技术人员一旦得知了基本创造性概念，则可对这些实施例作出另外的变更和修改。所以，所附权利要求意欲解释为包括优选实施例以及落入本发明范围的所有变更和修改。
显然，本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样，倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内，则本发明也意图包含这些改动和变型在内。
序列表
<110> 武汉大学
<120> 抗SETD3的单克隆抗体及其用途
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Claims (8)
1.一种抗SETD3的单克隆抗体，其特征在于，所述单克隆抗体能识别SETD3蛋白，所述单克隆抗体包括重链可变区和轻链可变区：
所述重链可变区具有如SEQ ID NO：1-SEQ ID NO：3所示的氨基酸序列的三个互补决定区；
所述轻链可变区具有如SEQ ID NO：4-SEQ ID NO：6所示的氨基酸序列的三个互补决定区。
2.根据权利要求1所述的一种抗SETD3的单克隆抗体，其特征在于，所述重链可变区的氨基酸序列如SEQ ID NO：7所示；所述轻链可变区的氨基酸序列如SEQ ID NO：8所示。
3.一种编码权利要求1-2任一所述单克隆抗体的核酸分子，其特征在于，所述核酸分子包括编码所述重链可变区的核酸分子和编码所述轻链可变区的核酸分子。
4.一种包含权利要求3所述核酸的表达载体，其特征在于，所述表达载体能够在原核或者真核宿主细胞中表达所述核酸。
5.一种包含权利要求4所述的表达载体的工程菌或真核宿主细胞。
6.权利要求1-2任一所述的SETD3蛋白的单克隆抗体在制备SETD3蛋白检测试剂或试剂盒中的用途。
7.权利要求1-2任一所述的SETD3蛋白的单克隆抗体在制备SETD3蛋白胶体金检测试剂盒的质控抗体中的用途。
8.一种SETD3蛋白的胶体金快速检测试纸条，其特征在于，包括：
底板，
粘合在所述底板上且依次搭接的样品吸收垫、结合垫、层析基质和吸水垫；其中，
所述结合垫上涂有权利要求1-2任一所述的SETD3蛋白的单克隆抗体包被的胶体金复合物；所述层析基质上靠近所述结合垫的一侧设有质控线C，所述层析基质上靠近所述吸水垫的一侧设有检测线T；所述质控线C上涂有包被有抗鼠IgG二抗；所述检测线T上包被有权利要求1-2任一所述的SETD3蛋白的单克隆抗体。
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| SETD3 acts as a prognostic marker in breast cancer patients and modulates the viability and invasion of breast cancer cells;Nourhan Hassan;《Sci Rep》;20200210;全文 * |
| SETD3 protein is the actin-specific histidine N-methyltransferase.;Sebastian Kwiatkowski;《eLife》;20181211;全文 * |
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