CN117384291A - 抗人sall4兔单克隆抗体及其制备方法和应用、多核苷酸分子、表达载体和宿主细胞 - Google Patents
抗人sall4兔单克隆抗体及其制备方法和应用、多核苷酸分子、表达载体和宿主细胞 Download PDFInfo
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Abstract
本发明涉及基因工程技术领域,公开了抗人SALL4兔单克隆抗体及其制备方法和应用、多核苷酸分子、表达载体和宿主细胞,该单克隆抗体包括重链和轻链,重链包含重链可变区,轻链包括轻链可变区;重链可变区包含3个重链互补决定区,其氨基酸序列如SEQ ID NO.8‑10所示;轻链可变区包含3个轻链互补决定区,其氨基酸序列如SEQ ID NO.3‑5所示。该单克隆抗体具有高特异性,可以特异性识别含人SALL4蛋白的细胞,适用于免疫学检测,特别是免疫组化检测。
Description
技术领域
本发明属于基因工程技术领域,特别涉及抗人SALL4兔单克隆抗体及其制备方法和应用、多核苷酸分子、表达载体和宿主细胞。
背景技术
SALL基因家族成员能编码在发育过程中高表达的假定锌指转录因子。SALL4与Oct-4和Nanog等其他全能性调节分子在发育早期就开始表达。最近的研究表明,SALL4是一种主调节分子,可在控制干细胞全能性和干细胞命运的转录调节反馈环中调节其自身表达和Oct-4表达。免疫组织化学研究表明,SALL4是原发性生殖细胞瘤和卵黄囊瘤的一个敏感且特异的诊断标记物。研究表明,SALL4在急性髓性白血病(AML)细胞中呈结构性表达,并且可能是疾病细胞中Wnt/β-catenin信号转导通路的一个效应子。此外,SALL4突变还与Duane桡骨线综合征(Okihiro综合征)和顶端肾-眼综合征等人体畸形综合征有关。
SALL4基因负责调节胚胎干细胞的自我更新,是许多肿瘤中的关键基因。SALL4是一种癌胚蛋白,研究表明,SALL4是***瘤和卵巢原始生殖细胞肿瘤敏感和特异性标记物,也是一种侵袭性表型肝细胞癌的祖细胞亚类标志物。
发明内容
本发明提供了抗人SALL4兔单克隆抗体及其制备方法和应用、多核苷酸分子、表达载体和宿主细胞。该单克隆抗体具有高特异性,可以特异性识别含人SALL4蛋白的细胞,适用于免疫学检测,特别是免疫组化检测。
为达到上述目的,本发明采用以下技术方案:
本发明提供了一种抗人SALL4兔单克隆抗体,所述单克隆抗体包括重链和轻链,所述重链包含重链可变区,所述轻链包括轻链可变区;
所述重链可变区包含3个重链互补决定区,其氨基酸序列如SEQ ID NO.8-10所示;
所述轻链可变区包含3个轻链互补决定区,其氨基酸序列如SEQ ID NO.3-5所示。
在以上技术方案中,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.2所示。
在以上技术方案中,所述重链的氨基酸序列如SEQ ID NO.6所示,所述轻链的氨基酸序列如SEQ ID NO.1所示。
本发明还提供了一种多核苷酸分子,所述多核苷酸分子编码上述单克隆抗体。
本发明还提供了一种表达载体,所述表达载体包含上述多核苷酸分子。
本发明还提供了一种宿主细胞,所述宿主细胞转化有上述表达载体。
本发明还提供了一种抗人SALL4兔单克隆抗体的制备方法,包括如下步骤:采用人SALL4蛋白C末端的第954位至第1053位氨基酸片段作为免疫原,免疫兔,从脾细胞中分离出单个抗原特异性B淋巴细胞培养,通过特定引物提取所述单克隆抗体对应的重链可变区、轻链可变区的基因扩增产物,构建到表达载体上,转染宿主细胞,获得含有所述单克隆抗体的上清,纯化,即得所述单克隆抗体,其中所述人SALL4蛋白C末端的第954位至第1053位氨基酸片段的序列如SEQ ID NO.11所示。
本发明还提供了上述单克隆抗体在制备人SALL4蛋白检测试剂和/或检测试剂盒中的应用,检测方法包括酶免疫分析法、酶联免疫吸附法、酶联免疫斑点法、免疫组织化学法、免疫荧光法、免疫印迹法和流式细胞分析法中的至少一种。
在以上技术方案中,所述检测方法优选为免疫组织化学法。
本发明的有益效果在于:本发明选取人SALL4蛋白C末端的第954位至第1053位氨基酸片段为抗原,大肠杆菌Rosetta表达的基因片段,蛋白表达在上清,最后得到纯度较高的人SALL4重组蛋白。利用人SALL4重组蛋白作为免疫原对新西兰大白兔进行免疫,基于单个B淋巴细胞筛选和培养的单克隆抗体开发技术,获得抗SALL4蛋白的兔单克隆抗体及重链、轻链序列。本发明制备得到的抗人SALL4兔单克隆抗体具有高特异性,可以特异性识别含人SALL4蛋白的细胞,适用于免疫学检测,特别是免疫组化检测。
附图说明
图1为人SALL4蛋白纯化图;
图2为构建含兔单克隆抗体重链恒定区的表达载体示意图;
图3为构建含兔单克隆抗体轻链恒定区的表达载体示意图;
图4为抗人SALL4兔单克隆抗体在人***瘤中的染色表现。
具体实施方式
为了更好地说明本发明的目的、技术方案和优点,下面将结合具体实施例对本发明做进一步描述。本发明可以许多不同的形式实施,而不应该被理解为限于在此阐述的实施例。相反,提供这些实施例,使得本公开将是彻底和完整的,并且将把本发明的构思充分传达给本领域技术人员,本发明将仅由权利要求来限定。
本发明提供一种抗人SALL4兔单克隆抗体,其氨基酸序列如下:
轻链可变区的氨基酸序列:
AQALTQTPSSVSAAVGGTVTISCQSSQNVYDANALSWYQQKPGQPPKRLIYSASTLASGVPSRFKGSGSGTQFTLTISGVQCDDAASYYCAGVYYGKIGFGGGTEVVVK(SEQ ID NO.2)
重链可变区的氨基酸序列:
QSLEESGGRLVTPGGSLTLTCTVSGFSLSSNSITWVRQAPGKGLEWIGTMNIDNSTYYMSWAKGRLTISKTSTTVTLKMTSLTTEDTATYFCARIAVVSNTDIWGPGTLVTVSF(SEQ ID NO.7)
轻链互补决定区CDR1的氨基酸序列:
QNVYDANALSW(SEQ ID NO.3)
轻链互补决定区CDR2的氨基酸序列:
LIYSASTLASGV(SEQ ID NO.4)
轻链互补决定区CDR3的氨基酸序列:
AGVYYGKIGF(SEQ ID NO.5)
重链互补决定区CDR1的氨基酸序列:
FSLSSNSIT(SEQ ID NO.8)
重链互补决定区CDR2的氨基酸序列:
WIGTMNIDNSTYYMSWAK(SEQ ID NO.9)
重链互补决定区CDR3的氨基酸序列:
YFCARIAVVSNTDI(SEQ ID NO.10)
轻链的氨基酸序列:
MDTRAPTQLLGLLLLWLPGARCAQALTQTPSSVSAAVGGTVTISCQSSQNVYDANALSWYQQKPGQPPKRLIYSASTLASGVPSRFKGSGSGTQFTLTISGVQCDDAASYYCAGVYYGKIGFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC(SEQ ID NO.1)
重链的氨基酸序列:
METGLRWLLLVAVLKGVQCQSLEESGGRLVTPGGSLTLTCTVSGFSLSSNSITWVRQAPGKGLEWIGTMNIDNSTYYMSWAKGRLTISKTSTTVTLKMTSLTTEDTATYFCARIAVVSNTDIWGPGTLVTVSFGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPMCPPPELPGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK(SEQ ID NO.6)
本发明还提供一种抗人SALL4兔单克隆抗体的制备方法,包括如下步骤:采用人SALL4蛋白C末端的第954位至第1053位氨基酸片段(SEQ ID NO.11)作为免疫原,免疫兔,从脾细胞中分离出单个抗原特异性B淋巴细胞培养,通过特定引物提取所述单克隆抗体对应的重链可变区、轻链可变区的基因扩增产物,构建到表达载体上,转染宿主细胞,获得含有所述单克隆抗体的上清,纯化,即得本发明的抗人SALL4兔单克隆抗体。
下面针对上述抗人SALL4兔单克隆抗体的制备及应用进行举例说明。
试验例1重组SALL4蛋白的表达纯化
1、蛋白表达质粒构建
采用基因合成方式得到人SALL4蛋白C末端的第954位至第1053位相应的核酸片段。
2、蛋白表达
将包含有SALL4 954-1053aa序列的pET-32a质粒,转化至大肠杆菌Rosetta菌株,并在LB琼脂平板(含有100μg/mL氨苄青霉素)上于37℃温育过夜,以获得几个单菌落转化物。
将单菌落转化物接种于10mL聚丙烯管中的2mL LB培养液(含100μg/mL氨苄青霉素)中,于37℃下,以220rpm培养3-4小时,OD600nm约为0.4-0.6,接下来,将每种菌株的2mL培养物转移到在1L烧瓶中的400mL LB表达培养基中,于37℃下,以220rpm再培养3-4小时。当OD600nm达到约0.45-0.55时,加入0.8mM IPTG,37℃诱导3-4h,将诱导完成的菌液转移至干燥的500mL离心瓶中,用电子称平衡,质量不等则加纯水,水平转子4000rpm离心10min,弃上清,离心瓶正置,菌体-20℃冰柜保存。
取30mL的破菌液(50mM Tris-300mM NaCl)在离心瓶中悬浮菌体。将悬浮完成的菌液转移至50mL圆底离心管,放入冰盒中,用冰固定。选择变幅杆,放入破菌舱内,功率350W,破菌时间3s,间隔时间3s,倒计时5min,之后置于冰水混合物中冷却5min,再重复上述步骤破菌5min。破菌完成后9000rpm离心10min得到上清液②和沉淀。
第二次破碎:量取30mL破菌液(2M尿素-PBS缓冲液)倒入沉淀中,吹匀沉淀,转移至50mL的圆底离心管中,功率350W,破菌时间3s,间隔时间3s,破碎0-5min(破碎时间按沉淀量及质地来决定,菌液置于冰块中),9000rpm离心10min得到上清液和包涵体,泳道1为上清液②、泳道8为包涵体,泳道6和泳道7为300mM和500mM咪唑洗脱下来的蛋白(如图1)。最后得到相应的蛋白片段。
其中,人SALL4蛋白第954位至第1053位氨基酸片段序列如下:
PKEILAPSVNVDPVVWNQYTSMLNGGLAVKTNEISVIQSGGVPTLPVSLG ATSVVNNATVSKMDGSQSGISADVEKPSATDGVPKHQFPHFLEENKIAVS(SEQ ID NO.11)
3、蛋白获得
使用Ni-IDA亲和纯化基质通过亲和层析从上清液②中提取纯化SALL4蛋白,纯化结果如图1所示。其中,从左至右,图1中泳道1为上清液②(简称“上2”),泳道2和泳道3分别为0.4mg/mL和0.2mg/mL的BSA,泳道4为marker,泳道5为纯化过程中的流穿液(简称“FT”),泳道6、泳道7为收集的SALL4蛋白2倍稀释液(简称“*2”)。从图1的SDS-PAGE胶图中可辨:得到了纯度为95%的重组SALL4蛋白,包含SALL4蛋白片段、His标签及Ni-IDA蛋白标签。
试验例2兔单克隆抗体的制备及纯化方法
1、动物免疫
以重组人SALL4蛋白(试验例1制备纯化获得)为免疫原,免疫3只新西兰大白兔;每只大白兔免疫300μg免疫原,首次免疫前将免疫原与等量的完全弗式佐剂(购自Sigma公司)混合制成乳化剂,在兔腹部及背部皮下多点注射;首次免疫后每间隔2周取150μg免疫原与等量的不完全弗式佐剂(购自Sigma公司)混合制成乳化剂,在兔腹部及背部皮下多点注射,加强免疫两次。三次免疫后采集兔子血清样本,用ELISA方法测定其针对人SALL4蛋白的滴度,取血清按1:243K稀释后用ELISA测滴度,取OD450nm超过0.1的兔子,用300μg免疫原就皮下多点注射加强免疫一次,三天后取脾脏。
2、分离脾细胞
在安全柜中无菌操作取出一个培养皿,加入30-40mL的基础培养基,放一个细胞筛网,将脾脏取出放于细胞筛中,将兔脾脏组织上多余的***、脂肪剪除,脾脏组织剪碎放入细胞筛网中研磨,取干净的研磨棒,用其按压处的末端对组织进行碾压研磨。膜内细胞会慢慢游离出来,经过细胞筛之后,悬浮在培养皿溶液中;用10mL基础培养基清洗细胞筛网,收集细胞筛网外基础培养基。室温下以400g离心力离心5min,去上清,留细胞,加入13mL常温的RBC红细胞裂解液(购自BioGems公司),用移液器轻柔吹散细胞团后计时1min,进行红细胞裂解,加入基础培养基37mL,混匀,终止红细胞裂解,室温下以400g离心力离心5min,去上清,留细胞,加入40mL常温放置的基础培养基,用移液器轻柔吹散细胞团,使细胞重悬,完成第一次清洗,室温下以400g离心力离心5min,去上清,留细胞,加入20mL常温放置的基础培养基,用移液器轻柔吹散细胞团,使细胞重悬;将重悬细胞经细胞筛网再次过滤,去除结团细胞,之后对细胞进行计数。
3、B淋巴细胞分选
采用中国专利CN201910125091.4(专利名称:从脾脏细胞中高效分离单个抗原特异性B淋巴细胞的方法)说明书中的方法。
4、培养后的B细胞上清用抗原包被的ELISA来鉴定阳性克隆
阳性克隆的细胞收集裂解后用Quick-RNATM MicroPrep试剂盒(购自ZYMO公司)提取RNA,并反转录成cDNA。
以cDNA为模板,采用PCR方法,将天然配对的兔单克隆抗体轻链可变区(VL)和重链可变区(VH)基因从对应阳性克隆的cDNA中被扩增出来,挑选若干个克隆进行测序,测序工作由金开瑞生物科技有限公司完成。
其中,PCR反应体系如下:4μL cDNA,1μL正向引物(10mM),1μL反向引物(10mM),12.5μL 2×Gloria HiFi(武汉爱博泰克生物科技有限公司提供),6.5μL N.F H2O。
其中,轻链可变区引物对为:
5’-tgaattcgagctcggtacccATGGACACGAGGGCCCCCAC-3’
5’-cacacacacgatggtgactgTTCCAGTTGCCACCTGATCAG-3’
重链可变区引物对为:
5’-tgaattcgagctcggtacccATGGAGACTGGGCTGCGCTG-3’
5’-gtagcctttgaccaggcagcCCAGGGTCACCGTGGAGCTG-3’
PCR扩增程序:98℃预变性30s,随后按照98℃10s,64℃30s,72℃30s的条件进行40次循环,最后在72℃保持5min,得到的反应液置于4℃保存。
5、单克隆抗体制备和纯化
(1)将步骤4中挑选出的若干个兔单克隆抗体的重链基因、轻链基因分别装载在表达载体上,所使用的哺乳动物表达载体pBR322见图2和图3。图2和图3中,pBR322 origin和f1 origin是在大肠杆菌(E.Coli)中的复制启动子,Ampcillin是质粒抗性基因,CMVimmearly promotor为在真核生物中的启动子,SV40 PA terminator是加尾信号,图2中Heavy chain constant为兔单克隆抗体重链恒定区的核苷酸序列,图3中Light chainconstant为兔单克隆抗体轻链恒定区的核苷酸序列。
(2)将含兔单克隆抗体重链恒定区(图2)和轻链恒定区(图3)的哺乳动物细胞(如CHO、HEK293等)表达载体分别用NheI和XbaI限制性内切酶常规线性化处理。
(3)将步骤4中扩增后的PCR产物进行纯化后,采取同源重组的方式,分别将重链可变区基因和轻链可变区基因构建到相应的哺乳动物表达载体中;经测序验证之后,将含有相应兔单克隆抗体轻链基因和重链基因的表达载体一起转染至293F细胞中;293F细胞活率为75%±5%的情况下,转染72-96小时获得培养上清中含有重组的识别人SALL4的兔单克隆抗体。
使用protein A亲和凝胶树脂从转染后的培养基上清中纯化出重组的识别人SALL4蛋白的兔单克隆抗体,使用SDS-PAGE凝胶电泳验证抗体纯度,验证合格后分装,于-20℃低温保存备用。挑选出的兔单克隆抗体的序列如上所述。
试验例3特异性鉴定
免疫组化组织芯片染色和鉴定
1、芯片选择:
人SALL4蛋白几乎表达于所有原始生殖细胞肿瘤,在未成熟畸胎瘤的原始神经上皮中也可有部分表达。因此选择***瘤、胚胎性癌组织作为阳性样本,肝癌、扁桃体、间变性大细胞淋巴瘤(ALCL)、霍奇金淋巴瘤肾、肝、胸腺瘤组织作为阴性对照。
2、IHC染色及分析:
样本准备,烤片:将石蜡切片按同一朝向放置在切片架上,将其放入56℃的恒温箱中烤片30min;同时将脱蜡液1缸一起放入56℃的恒温箱中;脱蜡至水:将石蜡切片连同切片架一起放入脱蜡液1缸中,再一起从恒温箱中取出置于常温,5min后,将切片取出浸入到常温脱蜡液2缸中,并按照脱蜡液2、脱蜡液3、无水乙醇1、无水乙醇2、无水乙醇3的顺序依次将石蜡切片放入缸中,脱蜡液试剂缸每缸5min,无水乙醇试剂缸每缸3min;用流水清洗切片3min。
抗原修复:0.01M Tris-EDTA修复液(pH9.0)高压热修复。
内源性过氧化物酶灭活:用PBS缓冲液浸洗3次,每次1min,去除切片上的缓冲液;将切片完全浸入到3%过氧化氢溶液中,室温,孵育10min。
封闭:用PBS缓冲液浸洗3次,每次3min,去除切片上的缓冲液;用免疫组化水笔圈定玻片上待检测组织区域,在圈定区域内滴加封闭液-PBS封闭液;将切片水平放置在底部呈有水的孵育湿盒中,于常温孵育30min,从滴加封闭液开始计时。
一抗孵育:去除封闭液,在组织切片上滴加用抗体稀释液-PBS工作液稀释的一抗,水平放置于孵育湿盒中,于常温孵育60min;去除抗体工作液,PBS缓冲液快速漂洗1次,用缓冲液PBS浸泡洗涤3次,每次3min;浸泡洗涤期间需反复上下提拉多次(一抗稀释比:1:100)。
二抗孵育:在组织切片上滴加即用型二抗工作液(剂瓶A)后水平放置于孵育湿盒中,于常温孵育25min;去除切片上的试剂,缓冲液PBS快速漂洗1次,用缓冲液PBS浸泡洗涤3次,每次3min;浸泡洗涤期间需反复上下提拉多次。
显色:在组织切片上滴加显色液工作液,显微镜下密切观察颜色变化情况,得到合适的染色强度后;将切片浸入大量蒸馏水中即可终止显色;终止显色后,将切片入流水中清洗10min。
复染:将稍沥干的切片浸入Mayer’s苏木素中复染切片1min,复染完成后,用流水清洗3min。
返蓝:将稍沥干的切片浸入碳酸锂饱和水溶液中蓝化3s,用流水清洗3min。
脱水:将清洗后的切片于无水乙醇中浸泡1次,浸泡期间需上下提拉数次,计时10s后,取出;置于恒温鼓风干燥箱中高温(54-58℃)下完全干燥。
封片:在切片中心滴加适量中性树胶,并加盖盖玻片,加胶量需适量,加封盖玻片后需完全覆盖组织,且不能有胶溢出。
切片扫描。
使用试验例2制备的抗人SALL4兔单克隆抗体在人***瘤中有特异性着色。
图4显示在人***瘤中的染色表现。结果显示,试验例2制备的抗人SALL4兔单克隆抗体染色定位准确,染色清晰且无非特异性染色,背景干净。
显然,上述实施例仅仅是为清楚地说明所作的举例,而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的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸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 (9)
1.一种抗人SALL4兔单克隆抗体,其特征在于:所述单克隆抗体包括重链和轻链,所述重链包含重链可变区,所述轻链包括轻链可变区;
所述重链可变区包含3个重链互补决定区,其氨基酸序列如SEQ ID NO.8-10所示;
所述轻链可变区包含3个轻链互补决定区,其氨基酸序列如SEQ ID NO.3-5所示。
2.根据权利要求1所述单克隆抗体,其特征在于:所述重链可变区的氨基酸序列如SEQID NO.7所示,所述轻链可变区的氨基酸序列如SEQ ID NO.2所示。
3.根据权利要求1所述单克隆抗体,其特征在于:所述重链的氨基酸序列如SEQ IDNO.6所示,所述轻链的氨基酸序列如SEQ ID NO.1所示。
4.一种多核苷酸分子,其特征在于:所述多核苷酸分子编码权利要求1-3任一项所述单克隆抗体。
5.一种表达载体,其特征在于;所述表达载体包含权利要求4所述多核苷酸分子。
6.一种宿主细胞,其特征在于:所述宿主细胞转化有权利要求5所述表达载体。
7.权利要求1所述单克隆抗体的制备方法,其特征在于:包括如下步骤:采用人SALL4蛋白C末端的第954位至第1053位氨基酸片段作为免疫原,免疫兔,从脾细胞中分离出单个抗原特异性B淋巴细胞培养,通过特定引物提取所述单克隆抗体对应的重链可变区、轻链可变区的基因扩增产物,构建到表达载体上,转染宿主细胞,获得含有所述单克隆抗体的上清,纯化,即得所述单克隆抗体,其中所述人SALL4蛋白C末端的第954位至第1053位氨基酸片段的序列如SEQ ID NO.11所示。
8.权利要求1-3任一项所述单克隆抗体在制备人SALL4蛋白检测试剂和/或检测试剂盒中的应用,其特征在于:检测方法包括酶免疫分析法、酶联免疫吸附法、酶联免疫斑点法、免疫组织化学法、免疫荧光法、免疫印迹法和流式细胞分析法中的至少一种。
9.权利要求8所述应用,其特征在于:所述检测方法为免疫组织化学法。
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