CN111763260A - 一种PTEN-Long多克隆抗体及其制备方法和用途 - Google Patents
一种PTEN-Long多克隆抗体及其制备方法和用途 Download PDFInfo
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- CN111763260A CN111763260A CN202010530721.9A CN202010530721A CN111763260A CN 111763260 A CN111763260 A CN 111763260A CN 202010530721 A CN202010530721 A CN 202010530721A CN 111763260 A CN111763260 A CN 111763260A
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- pten
- long
- protein
- antibody
- polyclonal antibody
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Abstract
本发明提供了一种新的PTEN‑Long兔多克隆抗体及其制备方法。该抗体由如SEQ ID NO.1所示的氨基酸序列的抗原免疫日本大耳白兔后,分离纯化血清获得,其与PTEN‑Long蛋白结合能力强。本发明的PTEN‑Long兔多克隆抗体的亲和力高,对病毒、肿瘤、代谢和神经方面疾病的诊断和治疗具有指导意义,具备良好的应用前景。
Description
技术领域
本发明属于生物技术领域,尤其涉及一种PTEN-Long兔多克隆抗体的制备方法和用途。
背景技术
PTEN基因编码的蛋白具有蛋白磷酸酶和脂质磷酸酶活性,是第一个具有磷酸酶活性的抑癌基因,也是继p53和Rb基因之后,与肿瘤发生密切相关的一种抑癌基因,其主要机制为PTEN是PI3K/Akt通路的主要负调控因子。
PTEN的功能缺陷在人类多种肿瘤中广泛存在。同源性磷酸酶-张力蛋白L亚型即PTEN-Long(NP_001291646.4)是一个新鉴定的PTEN亚型,n端加入了173个氨基酸。PTEN-Long是一个新的通过其蛋白磷酸酶活性对抗磷酸化泛素的噬菌体负调节剂,其定位于线粒体外膜,PTEN-Long缺失促进各种线粒体损伤,诱导的吞噬作用,进而引起许多重要的人类疾病,包括神经退行性疾病,如帕金森氏症的关键过程。
进一步的研究表明,PTEN-Long基因在正常的组织,细胞,体液(血清,尿液,前列腺液)等样本中的表达水平与病变组织中存在明显差异,通过检测PTEN-Long的表达水平可以辅助肿瘤,代谢病,神经疾病,心血管病等的诊断。抗原抗体检测是检测基因表达非常通用的方法,但是其检测的准确度、灵敏度等依赖于优良的抗体,目前未见PTEN-Long抗体的任何报道。
发明内容
为了解决前述问题,本发明提供一种新的高亲和力的PTEN-Long抗体的其制备方法及其应用。
本发明PTEN-Long抗体,该抗体是由氨基酸序列如SEQ ID NO.1所示所述的蛋白作为免疫原制备得到的抗体。
优选地,所述抗体为多克隆抗体。
优选地,所述多克隆抗体为兔多克隆抗体。
本发明还提供了一种蛋白,其氨基酸序列如SEQ ID NO.1所示。
本发明还提供了制备前述的PTEN-Long抗体的方法,所述方法包括如下步骤:
1)制备氨基酸序列如SEQ ID NO.1所示的蛋白抗原;
2)接种免疫;
3)采血;
4)分离纯化,即可。
优选地,步骤2)中,接种免疫的对象为兔,优选为日本大耳白兔;和/或每只兔子接种的免疫原的剂量400~600μg/次,优选500μg/次接种4~5次。
优选地,接种免疫原时,加入佐剂;优选地,首次免疫时,将免疫原与等体积的完全弗氏佐剂制成乳化剂,背部皮下多点注射,之后每次免疫所用佐剂为不完全弗氏佐剂,其用量与免疫原等体积。
优选地,步骤4)中,所述分离纯化的方法包括:Protein A亲和纯化、HABP亲和纯化及抗原亲和纯化;
其中,Protein A亲和纯化为:
取Protein A亲和柱,上样,用平衡缓冲液进行淋洗,再用洗脱液洗脱,收集洗脱峰,其中,平衡缓冲液为包含浓度为50mM的Tris以及浓度为100mM的NaCl的溶液,洗脱液为包含浓度为100mM的Glycin以及浓度为10mM的NaCl的溶液;
HABP亲和纯化为:取HABP亲和柱,上样,用平衡缓冲液淋洗,收集流穿液,其中,平衡缓冲液为包含浓度为50mM的Tris以及浓度为100mM的NaCl的溶液;
抗原亲和纯化为:取抗原亲和层析,上样,用平衡缓冲液淋洗再用洗脱液洗脱,收集洗脱峰,其中,平衡缓冲液为包含浓度为50mM的Tris以及浓度为100mM的NaCl的溶液,洗脱液为包含浓度为100mM的Glycin以及浓度为10mM的NaCl的溶液。
本发明还提供了一种检测用试剂,它是以前述的PTEN-Long抗体为活性成分,加上其他可接受的辅助性成分和/或载体制备而成的检测试剂。
本发明还提供了前述的PTEN-Long抗体在制备肿瘤、代谢病、神经疾病、心血管病的诊断试剂和/或治疗药物(特别是肝癌)中的用途。
本发明的PTEN-Long兔多克隆抗体能够充分结合PTEN-Long蛋白,且PTEN-Long兔多克隆抗体对PTEN-Long蛋白的亲和力特别高(如表1所示)。
本发明的PTEN-Long兔多克隆抗体可以用于分析组织,细胞,体液(血清,尿液,前列腺液)等样本中的PTEN-Long表达水平,辅助肿瘤,代谢病,神经疾病,心血管病等的诊断,由于本发明方法制备得到的PTEN-Long兔多克隆抗体的亲和力高,其用于检测的灵敏度高,可以达到准确、高效检测的目的。
显然,根据本发明的上述内容,按照本领域的普通技术知识和惯用手段,在不脱离本发明上述基本技术思想前提下,还可以做出其它多种形式的修改、替换或变更。
以下通过具体实施方式对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1表达载体测序结果图。
图2抗原氨基酸序列融合蛋白表达工艺选择图,M=marker。
图3抗原氨基酸序列融合蛋白镍离子亲和层析纯化图,M=marker。
图4PTEN-Long兔多克隆抗体纯化后的SAS-PAGE图。
图5PTEN-Long兔多克隆抗体的Western Blotting检测图。
图6PTEN-Long兔多克隆抗体的免疫荧光检测
具体实施方式
本发明实验中使用的试剂、原料均为市售品。
实施例1本发明PTEN-Long兔多克隆抗体抗原的制备
1、文库构建,获得免疫抗体蛋白
选取PTEN-Long蛋白的一部分氨基酸序列,在一端增加其他氨基酸,设计氨基酸序列如SEQ ID NO.1所示的抗原蛋白。
SEQ ID NO.1:
MCHHHHHHHHHHNLINKAKTVEGIMELQAQVVESAKKARISEATDGLSDFLKSQTSAEDTLKSIKLSEAKEMAIRELDAQGVSDFYKNKINNAKTVEGVVALKDLILNSLAKPEVEPEAKPEVKPEVKPEAKPGSLNLPSAAAAPPVARAPEAAGGGSRSEDYSSSPHSAAAAARPLAAEEKQAQSLQPSSSRRSSHYPAAVQSQAAAERGASATAKSRAISILQKKPRHQQLLP。
通过PCR扩增目的基因或使用全合成法合成目的基因序列后将目的基因插入对应表达载体(pET24),其中,目的基因序列(SEQ ID NO.2)为:
ATGTGTCACCATCACCACCATCACCACCATCACCACAATCTTATTAATAAAGCTAAAACTGTTGAAGGAATTATGGAGCTTCAGGCACAAGTTGTTGAGTCAGCCAAAAAAGCACGTATTTCAGAGGCAACAGATGGTTTATCTGATTTCTTGAAGTCTCAAACTTCAGCAGAAGACACCCTTAAATCAATTAAACTTTCAGAAGCTAAGGAGATGGCTATTCGTGAGTTAGATGCTCAGGGTGTTAGTGACTTTTATAAGAACAAAATTAACAATGCTAAGACTGTAGAAGGAGTTGTTGCGCTTAAAGACCTTATTCTTAACTCCTTAGCTAAACCAGAGGTTGAACCAGAAGCTAAACCAGAGGTTAAGCCAGAAGTTAAACCAGAAGCTAAGCCAGGATCCCTGAATCTGCCGAGTGCAGCAGCCGCCCCGCCGGTGGCAAGAGCACCTGAAGCAGCAGGCGGTGGTAGTCGCAGCGAAGATTATAGCAGCAGCCCGCATAGTGCAGCAGCAGCCGCACGTCCGCTGGCAGCAGAAGAAAAACAGGCCCAGAGCCTGCAGCCGAGTAGTAGCCGCCGTAGCAGTCATTATCCGGCCGCCGTGCAGAGCCAGGCAGCAGCAGAACGCGGCGCAAGCGCCACCGCAAAAAGTCGTGCCATTAGCATTCTGCAGAAAAAACCGCGTCATCAGCAGCTGCTGCCGTAA。
测序验证质粒(图1)含有正确的插入基因序列后进行质粒放大生产并传递下游表达。
2、表达可行性实验
对转入表达载体的大肠杆菌BL21(DE3)菌株使用如下条件进行蛋白表达可行性小试,各条件的培养体积均为200mL:
a:自诱导(工艺一):ZY培养基(加氨苄),37℃培养16小时和25℃培养16小时;
b:IPTG诱导(工艺二):TB培养基(加氨苄),OD600=0.8时加入0.4mM IPTG,18℃培养16小时。
将上述两种表达得到的菌体通过高压均质机破碎方法破碎菌体后,通过SDS-PAGE检测,得到如图2的结果,由此可得,自诱导发酵可以获得大量的目的蛋白。
3、放大培养和纯化
3.1培养发酵
根据可行性小试表达结果选定自诱导进行抗原氨基酸序列表达发酵。
3.2抗原氨基酸序列纯化
a:菌体破碎:使用高压均质机破碎方法破碎菌体后离心去除沉淀,收集上清;
b:层析柱纯化:用平衡液(配方:20mM Tris,500mM NaCl pH8.0)平衡镍离子亲和层析柱后,用上清上样,目标蛋白与层析柱相结合,待未与层析柱结合的蛋白流穿后,使用咪唑(配方:20mM Tris,500mM NaCl,0.5M Imi pH8.0)梯度洗脱目标蛋白。
c:换液和检测:将洗脱得到的含目标蛋白的组分使用Sephendex G25换液至保存缓冲液中,随后使用BCA法定量蛋白浓度。目标蛋白的纯度通过考马斯亮蓝染色SDS-PAGE法鉴定(如图3)。
如图3所示,证明本发明得到了纯的目标蛋白(PTEN-Long兔多克隆抗体抗原)。
3.3质量控制
扩大表达获取的蛋白样本采取SDS-PAGE和UV OD280定量方法进行QC检测。
4抗原氨基酸测序
取纯化得到的蛋白,测序验证获得的蛋白的氨基酸序列的确如SEQ ID NO.1所示。
实验结果证明,本发明制备得到了氨基酸序列如SEQ ID NO.1所示的PTEN-Long兔多克隆抗体抗原。
实施例2本发明PTEN-Long兔多克隆抗体的制备
取实施例1制备得到的目标蛋白:PTEN-Long兔多克隆抗体抗原,采用如下方法制备PTEN-Long兔多克隆抗体。
1、动物免疫
免疫日本大耳白兔,每只兔子免疫抗原剂量500μg,首次免疫将免疫原与等体积(357μL)的完全弗氏佐剂制成乳化剂,背部皮下多点注射,间隔2~3周取相同剂量免疫原等体积不完全弗氏佐剂制成乳化剂,背部皮下多点注射,免疫四次后测定血清效价,血清效价合格后杀兔取血。血清效价不合格的间隔2~3周取相同剂量免疫原等体积不完全弗氏佐剂制成乳化剂进行第五次免疫,血清效价合格后杀兔取血。
2、采血及效价检测
于末次免疫后一周,经兔耳缘静脉取血3~4mL,4℃静置过夜后,1500g离心15min分离上层血清备检;
取免疫原蛋白,用包被缓冲液稀释成0.1μg/mL、1μg/mL、5μg/mL,然后用单道移液器在96孔板每孔中加入100μL,轻拍板子使样品混匀,用保鲜膜封严,4℃下包被过夜;用洗涤液按200μL/孔洗板1次,扣干酶标板;再用封闭液按300μL/孔封闭酶标板,室温下封闭1小时;用洗涤液按300μL/孔洗板2次后加样(将经过梯度稀释的样品及样品稀释剂以100μL/孔加样),同时加入检测抗体,以100μL/孔加至96孔板内,室温下作用2小时;用洗涤液按200μL/孔洗板3次,以200μL/孔加入显色液室温放置12min;以50μL/孔加入终止液终止反应;用酶标仪进行检测:测定波长为450nm。
3、PTEN-Long兔多克隆抗体纯化方法
3.1 Protein A亲和纯化
用超纯水冲洗Protein A亲和柱,用平衡缓冲液(50mM Tris,100mM NaCl,pH 8.0)平衡,将过滤好的兔血清稀释上样,用平衡缓冲液进行淋洗,用洗脱液(100mM Glycin,10mMNaCl,pH 3.0)洗脱,收集洗脱峰后用2MTris缓冲液中和。
3.2 HABP亲和纯化
用超纯水冲洗HABP亲和柱,用平衡缓冲液(50mM Tris,100mM NaCl,pH 8.0)平衡,取前一步得到的洗脱样品上样,用平衡缓冲液进行淋洗,收集流穿液,用洗脱液(100mMGlycin,10mM NaCl,pH 3.0)洗脱,收集洗脱峰后用2MTris缓冲液中和。
3.3抗原亲和纯化
抗原亲和层析柱(负载了本发明PTEN-Long蛋白)用超纯水冲洗,然后用平衡缓冲液(50mM Tris,100mM NaCl,pH 8.0)平衡。将前一步过完HABP柱得到的流穿样品上样抗原亲和层析柱,上样完毕后,用平衡缓冲液淋洗。洗脱缓冲液(100mM Glycin,10mM NaCl,pH3.0)洗脱,收集洗脱峰,用Tris缓冲液中和。
4、PTEN-Long兔多克隆抗体的纯度检测
纯化后的PTEN-Long兔多克隆抗体经考马斯亮蓝染SDS-PAGE凝胶检测结果如图4,Western Blotting检测如图5,免疫荧光检测如图6,证明本发明制备得到了目标抗体。
5、PTEN-Long兔多克隆抗体对PTEN-Long蛋白(SC7-2)的亲和力检测
通过血清滴度试验来检测PTEN-Long兔多克隆抗体对PTEN-Long蛋白(SC7-2)的亲和力,发现:在PTEN-Long蛋白(SC7-2)浓度为0.1μg/mL时,稀释倍数达128000时,PTEN-Long兔多克隆抗体的亲和力也非常高。具体实验数据见表1。
表1:血清滴定测试数据表
实验结果说明,本发明的PTEN-Long兔多克隆抗体可以很好地识别PTEN-Long蛋白,并有效结合PTEN-Long蛋白,且亲和力非常高,说明本发明通过选择特定的抗原片段,以本发明方法制备得到了亲和力高的PTEN-Long兔多克隆抗体,其可以应用于多个领域。
综上,本发明PTEN-Long兔多克隆抗体亲和力高,可以很好地识别PTEN-Long蛋白,并有效结合PTEN-Long蛋白,可以用于分析组织,细胞,体液(血清,尿液,前列腺液)等样本中的PTEN-Long蛋白含量,辅助肿瘤,代谢病,神经疾病,心血管病等的诊断。
SEQUENCE LISTING
<110> 四川大学华西医院
<120> 一种PTEN-Long多克隆抗体及其制备方法和用途
<130> GYKH1648-2020P0110116CC
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 235
<212> PRT
<213> Artificial Sequence
<220>
<223> 抗原的氨基酸序列
<400> 1
Met Cys His His His His His His His His His His Asn Leu Ile Asn
1 5 10 15
Lys Ala Lys Thr Val Glu Gly Ile Met Glu Leu Gln Ala Gln Val Val
20 25 30
Glu Ser Ala Lys Lys Ala Arg Ile Ser Glu Ala Thr Asp Gly Leu Ser
35 40 45
Asp Phe Leu Lys Ser Gln Thr Ser Ala Glu Asp Thr Leu Lys Ser Ile
50 55 60
Lys Leu Ser Glu Ala Lys Glu Met Ala Ile Arg Glu Leu Asp Ala Gln
65 70 75 80
Gly Val Ser Asp Phe Tyr Lys Asn Lys Ile Asn Asn Ala Lys Thr Val
85 90 95
Glu Gly Val Val Ala Leu Lys Asp Leu Ile Leu Asn Ser Leu Ala Lys
100 105 110
Pro Glu Val Glu Pro Glu Ala Lys Pro Glu Val Lys Pro Glu Val Lys
115 120 125
Pro Glu Ala Lys Pro Gly Ser Leu Asn Leu Pro Ser Ala Ala Ala Ala
130 135 140
Pro Pro Val Ala Arg Ala Pro Glu Ala Ala Gly Gly Gly Ser Arg Ser
145 150 155 160
Glu Asp Tyr Ser Ser Ser Pro His Ser Ala Ala Ala Ala Ala Arg Pro
165 170 175
Leu Ala Ala Glu Glu Lys Gln Ala Gln Ser Leu Gln Pro Ser Ser Ser
180 185 190
Arg Arg Ser Ser His Tyr Pro Ala Ala Val Gln Ser Gln Ala Ala Ala
195 200 205
Glu Arg Gly Ala Ser Ala Thr Ala Lys Ser Arg Ala Ile Ser Ile Leu
210 215 220
Gln Lys Lys Pro Arg His Gln Gln Leu Leu Pro
225 230 235
<210> 2
<211> 708
<212> DNA
<213> Artificial Sequence
<220>
<223> 表达抗原的目标基因的核苷酸序列
<400> 2
atgtgtcacc atcaccacca tcaccaccat caccacaatc ttattaataa agctaaaact 60
gttgaaggaa ttatggagct tcaggcacaa gttgttgagt cagccaaaaa agcacgtatt 120
tcagaggcaa cagatggttt atctgatttc ttgaagtctc aaacttcagc agaagacacc 180
cttaaatcaa ttaaactttc agaagctaag gagatggcta ttcgtgagtt agatgctcag 240
ggtgttagtg acttttataa gaacaaaatt aacaatgcta agactgtaga aggagttgtt 300
gcgcttaaag accttattct taactcctta gctaaaccag aggttgaacc agaagctaaa 360
ccagaggtta agccagaagt taaaccagaa gctaagccag gatccctgaa tctgccgagt 420
gcagcagccg ccccgccggt ggcaagagca cctgaagcag caggcggtgg tagtcgcagc 480
gaagattata gcagcagccc gcatagtgca gcagcagccg cacgtccgct ggcagcagaa 540
gaaaaacagg cccagagcct gcagccgagt agtagccgcc gtagcagtca ttatccggcc 600
gccgtgcaga gccaggcagc agcagaacgc ggcgcaagcg ccaccgcaaa aagtcgtgcc 660
attagcattc tgcagaaaaa accgcgtcat cagcagctgc tgccgtaa 708
Claims (10)
1.一种PTEN-Long抗体,其特征在于:该抗体是由氨基酸序列如SEQ ID NO.1所示所述的蛋白作为免疫原制备得到的抗体。
2.如权利要求1所述的PTEN-Long抗体,其特征在于:所述抗体为多克隆抗体。
3.如权利要求1所述的PTEN-Long抗体,其特征在于:所述多克隆抗体为兔多克隆抗体。
4.所述一种蛋白,其特征在于:其氨基酸序列如SEQ ID NO.1所示。
5.一种制备如权利要求1~3任意一项所述的PTEN-Long抗体的方法,其特征在于:所述方法包括如下步骤:
1)制备氨基酸序列如SEQ ID NO.1所示的蛋白抗原;
2)接种免疫;
3)采血;
4)分离纯化,即可。
6.根据权利要求5所述的方法,其特征在于:步骤2)中,接种免疫的对象为兔,优选为日本大耳白兔;和/或每只兔子接种的免疫原的剂量400~600μg/次,优选500μg/次接种4~5次。
7.根据权利要求5所述的方法,其特征在于:接种免疫原时,加入佐剂;优选地,首次免疫时,使用完全弗氏佐剂,之后每次免疫所用佐剂为不完全弗氏佐剂。
8.根据权利要求5所述的方法,其特征在于:步骤4)中,所述分离纯化的方法包括:Protein A亲和纯化、HABP亲和纯化及抗原亲和纯化;
其中,Protein A亲和纯化为:
取Protein A亲和柱,上样,用平衡缓冲液进行淋洗,再用洗脱液洗脱,收集洗脱峰,其中,平衡缓冲液为包含浓度为50mM的Tris以及浓度为100mM的NaCl的溶液,洗脱液为包含浓度为100mM的Glycin以及浓度为10mM的NaCl的溶液;
HABP亲和纯化为:取HABP亲和柱,上样,用平衡缓冲液淋洗,收集流穿液,其中,平衡缓冲液为包含浓度为50mM的Tris以及浓度为100mM的NaCl的溶液;
抗原亲和纯化为:取抗原亲和层析,上样,用平衡缓冲液淋洗再用洗脱液洗脱,收集洗脱峰,其中,平衡缓冲液为包含浓度为50mM的Tris以及浓度为100mM的NaCl的溶液,洗脱液为包含浓度为100mM的Glycin以及浓度为10mM的NaCl的溶液。
9.一种检测用试剂,其特征在于,它是以权利要求1~3任意一项所述的PTEN-Long抗体为活性成分,加上其他可接受的辅助性成分和/或载体制备而成的检测试剂或药物制剂。
10.权利要求1~3任意一项所述的PTEN-Long抗体在制备肿瘤、代谢病、神经疾病、心血管病的诊断试剂和/或治疗药物中的用途。
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