CN106519005A - 磷酸化nfat3突变体及其应用 - Google Patents
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Abstract
本发明公开了一种磷酸化NFAT3突变体及其应用，所述NFAT3的第259位由Ser突变为Ala。NFAT3的Ser259位点的磷酸化影响皮肤癌细胞的增殖，具有抗癌应用价值，可用于靶向NFAT3的药物研发以及皮肤癌检测。
Description
技术领域
本发明涉及分子生物学、基因工程技术以及生物医药领域，具体涉及皮肤癌检测领域。
背景技术
皮肤癌是临床上最常见的恶性肿瘤之一，可大致分为恶性黑色素瘤和非黑色素瘤性皮肤癌，后者主要包括鳞状细胞癌和基底细胞癌。皮肤癌严重者可危及生命，其发病率逐年上升，已经成为了一个全球性的公共健康问题。多种危险因素可导致皮肤癌的发生，包括内源性因素(遗传因素、基因突变等)和外源性因素(如紫外线暴露，化学致癌物质和其他环境压力)。早期诊断和治疗，可以减少因该病所造成的死亡和畸型。然而，皮肤癌发生的确切分子机制仍不清楚，这严重的影响了对于该病的治疗和预防。
活化T细胞核因子(NFAT)在活化的T细胞的核提取物中被认为是与白介素-2(IL-2)启动子结合的诱导型核酸结合因子。NFAT可分布于细胞核和细胞质。细胞核中NFAT作为活化蛋白1(AP-1)的转录因子。纯化的NFAT是一个高度磷酸化的蛋白，能够被钙调磷酸酶(Calcineurin)去磷酸化。NFAT转录因子在很多脊椎动物的组织、器官的发育过程中起关键性的作用。此外，NFAT信号的失调与细胞恶性转化及肿瘤发展也密切相关。NFAT3属于NFAT家族。已报道NFAT3作为Ras-JNK1/2-AP-1诱导的NIH-3T3细胞转化的负调控因子。NFAT3在骨关节炎、乳腺癌细胞、非小细胞肺癌、嗜铬细胞瘤细胞、胶质瘤细胞系中的作用均有报道。NFAT3可以被p38MAPK磷酸化Ser168和Ser170以及被JNK1和JNK2磷酸化Ser213和Ser217位点。丙氨酸替换Ser168和Ser170可促进NFAT3的核转运并增加NFAT3介导的转录活性，而JNK1和JNK2的磷酸化位点的突变抑制NFAT3转录活性。
发明内容
本发明提供一种新的磷酸化NFAT3突变体及其应用。
本发明提供了一种磷酸化NFAT3突变体，NFAT3的第259位由Ser突变为Ala。
一种所述的突变体在制备用于皮肤癌检测的试剂上的应用。
一种皮肤癌检测试剂盒，包括所述的突变体。
一种所述的突变体在制备用于抑制皮肤癌细胞增殖的试剂上的应用。
所述皮肤癌细胞是人皮肤鳞状癌细胞A431。
本发明具有以下有益效果：NFAT3的Ser259位点的磷酸化影响皮肤癌细胞的增殖，具有抗癌应用价值，可用于靶向NFAT3的药物研发以及皮肤癌检测。
附图说明
图1是点突变后对人永生化表皮细胞HaCaT增殖影响图，其中vector为空载体；
图2为点突变对人永生化表皮细胞HaCaT软琼脂克隆形成影响图；
图3为点突变后对人皮肤鳞状癌细胞A431增殖影响图；
图4为点突变对人皮肤鳞状癌细胞A431平板克隆形成影响图；
图5为Ser259位磷酸化NFAT3在人正常皮肤和皮肤癌组织中表达情况的免疫荧光染色图；
图6为Ser259位磷酸化NFAT3在人正常皮肤和皮肤癌组织中表达情况的统计结果图；
图7为Ser259位磷酸化NFAT3在人正常皮肤和三种不同类型的皮肤癌组织中表达情况的统计结果图。
具体实施方式
下面通过具体实施例结合附图对本发明做进一步的说明。
下述实施方式中所用的方法如无特别说明均为常规方法，具体步骤可参见《Molecular Cloning:A Laboratory Manual》(Sambrook,J.,Russell,David W.,3rdedition,2001,NY,Cold Spring Harbor)。所用DNA引物均由Invitrogen合成；NFAT3表达质粒pEGFP-C1-NFAT3购自Addgene；各种限制性内切酶购自TaKaRa公司；逆转录酶购自LifeTechnologies公司；点突变试剂盒购自Agilent Technologies公司；DMEM培养基、胎牛血清均购自HyClone公司；转染试剂Lipofectamine购自Invitrogen公司；黑色素瘤细胞培养基为含10％胎牛血清的DMEM培养基。人皮肤组织芯片购自Biomax。抗体购自Santa Cruz。如无特别说明，本实施方式采用的其它试剂均为市售商品。
1.构建表达野生型NFAT3和NFAT3-S259A突变体的慢病毒质粒
将pEGFP-C1-NFAT3质粒中NFAT3的编码序列亚克隆至慢病毒表达载体pCDH-puro，获得表达野生型NFAT3(NFAT3-wt)慢病毒质粒，命名为pCDH-NFAT3。pEGFP-C1-NFAT3质粒可以购自Addgene，质粒编号如#10961。pCDH-puro的序列如SEQ ID NO：3所示。
根据试剂盒Quick Change Lightning Site-directed Mutagenesis Kit(Agilent Life Sciences,Santa Clara,CA,USA)说明书设计上游引物NFAT-S259-F:gcccaccccagccGCCccgcggcctgc(SEQ ID NO:1)和下游引物NFAT-S259-R:gcaggccgcggGGCggctggggtgggc(SEQ ID NO:2)，以pCDH-NFAT3为模板，使用点突变试剂盒将NFAT3的259位Ser突变成Ala，获得表达NFAT3-S259A突变体的慢病毒质粒，命名为pCDH-NFAT3-S259A。
2.S259A点突变抑制NFAT3对人永生化表皮细胞HaCaT增殖的促进作用
100mm培养皿培养293T细胞至50％覆盖率。将质粒pCDH/Δ8.2/VSV-G按4：3：2的比例转染293T细胞，转染后6小时更换新鲜培养基。转染48小时后，收集培养上清，用0.45μm滤膜过滤，即可获得空载体对照慢病毒或表达野生型NFAT3或NFAT3-S259A突变体的慢病毒。
在60mm培养皿中培养人永生化表皮细胞HaCaT至90％-100％覆盖率，细胞1：6传至6孔板，按细胞悬液和慢病毒液1：1加入慢病毒感染细胞。细胞感染24h后更换含有嘌呤霉素的培养基筛选，用未感染病毒细胞作为筛选阴性对照。待嘌呤霉素筛选至未感染病毒细胞全部死亡，存活的感染病毒的细胞为稳定株。命名为HaCaT-vector(此为用pCDH-puro参与构建的慢病毒感染并用嘌呤霉素筛选过的HaCaT细胞)，HaCaT-NFAT3-wt(此为用过表达NFAT-wt的慢病毒，即pCDH-NFAT3参与构建的慢病毒，感染后用嘌呤霉素筛选后的HaCaT细胞株)和HaCaT-NFAT3-S259A(此为用过表达NFAT-S259A的慢病毒，即pCDH-NFAT3-S259A参与构建的慢病毒，感染后用嘌呤霉素筛选后的HaCaT细胞株)。
在60mm培养皿中培养稳定株细胞HaCaT-vector，HaCaT-NFAT3-wt和HaCaT-NFAT3-S259A至90％-100％覆盖率，消化收集细胞，按每孔2000个细胞的密度重新接种到96孔板中，继续培养3d。分别在接种后0hr、24hrs、48hrs、72hrs时加入MTS，4hrs后检测。细胞增殖结果如图1所示，S259A点突变能够抑制NFAT3对人永生化表皮细胞HaCaT增殖的促进作用。
3.S259A点突变抑制NFAT3对人永生化表皮细胞HaCaT软琼脂克隆形成的促进作用
将3％低熔点Agarose与两倍体积的37℃完全培养基混合得到含有1％的Agarose。在六孔板每孔加入2ml 1％Agarose混合物，室温静置30分钟至下层胶完全凝固。在60mm培养皿中培养稳定株细胞HaCaT-vector，HaCaT-NFAT3-wt和HaCaT-NFAT3-S259A至90％-100％覆盖率，消化收集细胞，将消化好的细胞调整密度至5000个/ml。取1ml细胞液与1ml含0.8％Agarose的混合物混合后将2ml混合物加入到下层胶已凝固的6孔板孔中。4℃静置30分钟后放入培养箱。14天后，用0.005％的结晶紫溶液对细胞进行染色，2小时候后用清水漂洗至能看到明显的细胞克隆，晾干拍照。如图2所示，S259A点突变能够抑制NFAT3对人永生化表皮细胞HaCaT软琼脂克隆形成的促进作用。
4.S259A点突变抑制NFAT3对人皮肤鳞状癌细胞A431平板克隆形成的促进作用
如上述实施方式相同的方法，用空载体对照慢病毒或表达野生型NFAT3或NFAT3-S259A突变体的慢病毒感染皮肤癌细胞A431，嘌呤霉素筛选获得稳定株，命名为A431-vector，A431-NFAT3-wt和A431-NFAT3-S259A，分别和空载体慢病毒、表达野生型NFAT3的慢病毒和表达NFAT3-S259A突变体的慢病毒对应，命名方式同上述HaCaT。
在60mm培养皿中培养稳定株细胞A431-vector，A431-NFAT3-wt和A431-NFAT3-S259A至90％-100％覆盖率，消化收集细胞，按每孔250个细胞的密度重新接种到6孔板中，继续培养7d后，用2％的结晶紫溶液对细胞进行染色，15min后用清水漂洗，晾干。如图3所示，S259A点突变可以抑制NFAT3对人皮肤鳞状癌细胞A431平板克隆形成的促进作用。
5.S259A点突变显著回复NFAT3对人皮肤鳞状癌细胞A431在裸鼠皮下成瘤的能力的促进作用
在100mm培养皿中培养稳定株细胞A431-vector，A431-NFAT3-wt和A431-NFAT3-S259A至90％-100％覆盖率，消化收集细胞。将5×106稳定株细胞注射到4周龄BALB/c裸鼠后腿靠近背部皮下，观察肿瘤生长情况，并用游标卡尺测量肿瘤大小，肿瘤体积(mm3)的计算方法为长×宽×宽/2。如图4所示，S259A点突变显著回复NFAT3对人皮肤鳞状癌细胞A431在裸鼠皮下成瘤的能力的促进作用。
6.人皮肤组织标本中Ser259位点磷酸化NFAT3的表达检测和统计分析
人皮肤组织芯片SK801b经脱蜡、梯度水化、抗原修复后，用5％羊血清室温封闭1小时。NFAT3-S259磷酸化抗体4℃孵育过夜，二抗室温孵育2小时。用荧光显微镜观察结果并拍照(代表性免疫荧光照片如图5所示)，将照片转变成8-bit灰度后，用ImageJ计算其平均灰度值。如图6所示，NFAT3-S259磷酸化水平在皮肤癌(Cancer)组织中的表达显著高于正常组织(Normal)对照。进一步根据组织类型将皮肤癌组织分为鳞状细胞癌(squamous cellcarcinoma),基底细胞癌(basal cell carcinoma)和黑色素瘤(malignant melanoma)，如图7所示，在三种类型的肿瘤中，NFAT3-S259磷酸化表达水平均显著高于正常组织，且在黑色素瘤中表达最高。
以上内容是结合具体的实施方式对本发明所作的进一步详细说明，不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干简单推演或替换。
SEQUENCE LISTING
<110> 深圳大学
<120> 磷酸化NFAT3突变体及其应用
<130> 16I23534
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 27
<212> DNA
<213> NFAT-S259上游引物序列
<400> 1
gcccacccca gccgccccgc ggcctgc 27
<210> 2
<211> 27
<212> DNA
<213> NFAT-S259下游引物序列
<400> 2
gcaggccgcg gggcggctgg ggtgggc 27
<210> 3
<211> 7377
<212> DNA
<213> pCDH-puro序列
<400> 3
acgcgtgtag tcttatgcaa tactcttgta gtcttgcaac atggtaacga tgagttagca 60
acatgcctta caaggagaga aaaagcaccg tgcatgccga ttggtggaag taaggtggta 120
cgatcgtgcc ttattaggaa ggcaacagac gggtctgaca tggattggac gaaccactga 180
attgccgcat tgcagagata ttgtatttaa gtgcctagct cgatacaata aacgggtctc 240
tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta 300
agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact 360
ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg 420
cccgaacagg gacctgaaag cgaaagggaa accagagctc tctcgacgca ggactcggct 480
tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt 540
gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta agcgggggag 600
aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa aatataaatt 660
aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc ctggcctgtt 720
agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc ttcagacagg 780
atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg tgcatcaaag 840
gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc aaaacaaaag 900
taagaccacc gcacagcaag cggccactga tcttcagacc tggaggagga gatatgaggg 960
acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca ttaggagtag 1020
cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg ggaataggag 1080
ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcc tcaatgacgc 1140
tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac aatttgctga 1200
gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc aagcagctcc 1260
aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg gggatttggg 1320
gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt tggagtaata 1380
aatctctgga acagattgga atcacacgac ctggatggag tgggacagag aaattaacaa 1440
ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga 1500
acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa 1560
ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat 1620
agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt 1680
tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg 1740
tggagagaga gacagagaca gatccattcg attagtgaac ggatctcgac ggttaacttt 1800
taaaagaaaa ggggggattg gggggtacag tgcaggggaa agaatagtag acataatagc 1860
aacagacata caaactaaag aattacaaaa acaaattaca aaaattcaaa attttatcga 1920
tactagtatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac 1980
gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga 2040
tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg 2100
ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg 2160
caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctcg tttagtgaac 2220
cgtcagatcg cctggagacg ccatccacgc tgttttgacc tccatagaag attctagagc 2280
tagcgaattc gaatttaaat ggatccgcgg ccgcaaggat ctgcgatcgc tccggtgccc 2340
gtcagtgggc agagcgcaca tcgcccacag tccccgagaa gttgggggga ggggtcggca 2400
attgaacggg tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact 2460
ggctccgcct ttttcccgag ggtgggggag aaccgtatat aagtgcagta gtcgccgtga 2520
acgttctttt tcgcaacggg tttgccgcca gaacacagct gaagcttcga ggggctcgca 2580
tctctccttc acgcgcccgc cgccctacct gaggccgcca tccacgccgg ttgagtcgcg 2640
ttctgccgcc tcccgcctgt ggtgcctcct gaactgcgtc cgccgtctag gtaagtttaa 2700
agctcaggtc gagaccgggc ctttgtccgg cgctcccttg gagcctacct agactcagcc 2760
ggctctccac gctttgcctg accctgcttg ctcaactcta cgtctttgtt tcgttttctg 2820
ttctgcgccg ttacagatcc aagctgtgac cggcgcctac gctagatgac cgagtacaag 2880
cccacggtgc gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc 2940
gcgttcgccg actaccccgc cacgcgccac accgtcgatc cggaccgcca catcgagcgg 3000
gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg 3060
gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg 3120
gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg 3180
cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc gtggttcctg 3240
gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc 3300
cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc 3360
cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc 3420
gaaggaccgc gcacctggtg catgacccgc aagcccggtg cctgagtcga caatcaacct 3480
ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg 3540
ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc 3600
attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt 3660
gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc 3720
attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg 3780
gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact 3840
gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt 3900
gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg 3960
gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc 4020
cctcagacga gtcggatctc cctttgggcc gcctccccgc ctggtacctt taagaccaat 4080
gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg gactggaagg 4140
gctaattcac tcccaacgaa aataagatct gctttttgct tgtactgggt ctctctggtt 4200
agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca 4260
ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa 4320
ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagta gtagttcatg 4380
tcatcttatt attcagtatt tataacttgc aaagaaatga atatcagaga gtgagaggaa 4440
cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa 4500
taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta 4560
tcatgtctgg ctctagctat cccgccccta actccgccca gttccgccca ttctccgccc 4620
catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta 4680
ttccagaagt agtgaggagg cttttttgga ggcctagact tttgcagaga cggcccaaat 4740
tcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac 4800
aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc 4860
acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg 4920
cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct 4980
tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac 5040
tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga 5100
gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat 5160
aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac 5220
ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct 5280
gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg 5340
ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg 5400
ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt 5460
cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg 5520
attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac 5580
ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga 5640
aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt 5700
gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt 5760
tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga 5820
ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc 5880
taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct 5940
atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata 6000
actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca 6060
cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga 6120
agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga 6180
gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg 6240
gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga 6300
gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt 6360
gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct 6420
cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca 6480
ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat 6540
accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga 6600
aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc 6660
aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg 6720
caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc 6780
ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt 6840
gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca 6900
cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg 6960
aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc 7020
ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc 7080
gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact atgcggcatc agagcagatt 7140
gtactgagag tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac 7200
cgcatcaggc gccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg 7260
gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg 7320
gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtgc caagctg 7377
Claims (5)
1.一种磷酸化NFAT3突变体，其特征在于，NFAT3的第259位由Ser突变为Ala。
2.一种如权利要求1所述的突变体在制备用于皮肤癌检测的试剂上的应用。
3.一种皮肤癌检测试剂盒，其特征在于，包括权利要求1所述的突变体。
4.一种如权利要求1所述的突变体在制备用于抑制皮肤癌细胞增殖的试剂上的应用。
5.如权利要求4所述的应用，其特征在于，所述皮肤癌细胞是人皮肤鳞状癌细胞A431。
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