实施例
实施例1:制备SEQ ID NO:2氨基酸序列的缺失型p43蛋白(p43-1)
目的片断的PCR扩增
以中国专利申请CN101225371A中公开的p43蛋白的表达质粒为模版,以表1所示的引物组(引物由上海捷瑞生物工程有限公司合成),采用KOD-plus(TOYOBO)反应体系分别PCR扩增SEQ ID NO:7的核苷酸序列(表2),1%琼脂糖凝胶电泳结果表明,扩增得到了大小与预期相符合的目的条带(图1)。
表1
表2
表达载体的构建
PCR产物经琼脂糖凝胶电泳分离、割胶回收、纯化;利用EcoR I和Xho I(TaKaRa)分别双酶切各个PCR产物及pTIG-Trx质粒载体。以T4DNA(TaKaRa)连接酶4℃连接过夜,连接产物转化E.coli TOP10感受态细胞(天根生化科技(北京)有限公司)。
筛选具有外源DNA插入的阳性克隆。经ABI 3700型测序仪、采用T7promoter和T7 terminator引物双向测序,结果证明得到了SEQ ID NO:7核苷酸序列的目的基因。
分别转化pTIG-Trx-p43系列缺失体至E.coli BL21(DE)感受态细胞(天根生化科技(北京)有限公司),经小量培养、质粒抽提及EcoR I和Xho I双酶切鉴定具有正确目的基因插入的单菌落,扩大培养,建立带有SEQ ID NO:7核苷酸序列表达载体的工程菌。
将构建好的工程菌接种于液体LB中,200rpm、37℃培养至O.D.600为0.4-0.6,加入终浓度为1mM的IPTG,200rpm、37℃继续培养4h。离心收集菌体,再以20mM Tris(pH8.0)重悬菌体,超声裂菌,SDS-PAGE分析表达形式(图2)。
扩大培养后收集得到的菌体,加入1mM PMSF冰浴条件下超声破碎菌体,4℃9000rpm离心30min,收集上清。以HisTrap FF亲和层析柱对目的蛋白进行纯化,收集洗脱液,冷冻干燥浓缩,得到SEQ ID NO:2氨基酸序列的缺失型p43蛋白(图3),具体纯化流程如图4。
实施例2:制备SEQ ID NO:3氨基酸序列的缺失型p43蛋白(p43-2)
如实施例1相同的方法制备SEQ ID NO:3氨基酸序列的缺失型p43蛋白,不同的是用表2所示的相应引物合成SEQ ID NO:8核苷酸序列的目的基因。
实施例3:制备SEQ ID NO:4氨基酸序列的缺失型p43蛋白(p43-3)
如实施例1相同的方法制备SEQ ID NO:4氨基酸序列的缺失型p43蛋白,不同的是用表2所示的相应引物合成SEQ ID NO:9核苷酸序列的目的基因。
实施例4:制备SEQ ID NO:5氨基酸序列的缺失型p43蛋白(p43-4)
如实施例1相同的方法制备SEQ ID NO:5氨基酸序列的缺失型p43蛋白,不同的是用表2所示的相应引物合成SEQ ID NO:10核苷酸序列的目的基因。
实施例5:制备SEQ ID NO:6氨基酸序列的缺失型p43蛋白(p43-5)
如实施例1相同的方法制备SEQ ID NO:6氨基酸序列的缺失型p43蛋白,不同的是用表2所示的相应引物合成SEQ ID NO:11核苷酸序列的目的基因。
实施例6:p43系列缺失体蛋白抑制肿瘤新生血管生成实验
材料和方法:M199,FBS及胰酶购自Gibico;Transwell购自corning;Matrigel购自BD Biosciences;纤维粘蛋白购自Sigma;荧光染料Calcein AM购自Biotium。
细胞培养:人脐静脉内皮细胞株(Human Umbilical Vein EndothelialCells,HUVEC)细胞由中国药科大学药物筛选中心袁胜涛副教授惠赠。细胞培养于含10%FBS的M199培养基中,37℃、5%CO2孵箱中培养。待细胞处于指数生长期时进行实验。
内皮细胞管腔形成抑制实验
Matrigel胶在冰上融化后,加入预冷的96孔板中(60μl/孔),37℃放置30分钟使其凝固。每孔加入用含10%FBS的M199培养液稀释的HUVEC(3.0×104个细胞/100μl/孔)。同时分别加入一系列终浓度为50μmol/ml的p43全长及缺失体样品,37℃孵育4h,加荧光染料Calcein AM染色,荧光显微镜下观察,随机选三个视野拍照(×100)。
实验结果表明:p43-1、p43-2、p43-3、p43-4和p43-5缺失型蛋白抑制血管内皮细胞管腔形成的活性明显高于p43蛋白。p43蛋白作用的管腔还没有完全断裂、有比较完整的管腔结构,而p43-1、p43-2、p43-3、p43-4和p43-5缺失型蛋白作用的管腔几乎都已断裂(图5)。
内皮细胞迁移抑制实验
以细胞迁移实验作为p43缺失体活性的定量检测方法,迁移抑制率作为活性高低的评价标准。把缺失体蛋白的迁移抑制率与全长人p43蛋白进行比较。HUVEC经血清饥饿4-5h。在Transwell的下室面涂上纤维粘蛋白,置超净台内风干。胰酶消化细胞,用PBS润洗,以含0.2%FBS的M199培养基重悬、计数。取细胞悬液180μl加入内室(105个细胞)。外室加入10%FBS的M199培养基,540μl/孔。将p43全长及缺失体于内室中加入20μl,外室中加入60μl(终浓度为50μmol/ml),混匀,并以PBS作为阴性对照。将培养板置于37℃、5%CO2培养箱内培养16-18h。弃去孔中培养液,用90%乙醇固定细胞10min。用棉签轻轻擦去内室上层未迁移的细胞,0.1%结晶紫室温染色10min,用PBS漂净多余染料,以10%乙酸10μl/孔抽提10min。以酶标仪在595nm波长测定光吸收。根据以下公式计算迁移抑制率:迁移抑制率=(A对照-A处理)/A对照×100%。
在相同条件下,p43-1、p43-2、p43-3、p43-4和p43-5缺失型蛋白的血管内皮细胞的迁移抑制活性明显高于全长的p43蛋白(表3)。
表3、p43缺失体对HMEC-1迁移抑制结果
序列表
<110>信谊药厂
<120>N端缺失型p43蛋白及其在肿瘤治疗药物中的应用
<130>090786
<160>11
<170>PatentIn version 3.3
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gatgctgttc tgaagagact ggagcagaag ggtgcagagg cagatcaaat cattgaatat 60
cttaagcagc aagtttctct acttaaggag aaagcaattt tgcaggcaac tttgagggaa 120
gagaagaaac ttcgagttga aaatgctaaa ctgaagaaag aaattgaaga actgaaacaa 180
gagctaattc aggcagaaat tcaaaatgga gtgaagcaaa tagcatttcc atctggtact 240
ccactgcacg ctaattctat ggtttctgaa aatgtgatac agtctacagc agtaacaacc 300
gtatcttctg gtaccaaaga acagataaaa ggaggaacag gagacgaaaa gaaagcgaaa 360
gagaaaattg aaaagaaagg agagaagaag gagaaaaaac agcaatcaat agctggaagt 420
gccgactcta agccaataga tgtttcccgt ctggatcttc gaattggttg catcataact 480
gctagaaaac accctgatgc agattctttg tatgtggaag aagtagatgt cggagaaata 540
gccccaagga cagttgtcag tggcctggtg aatcatgttc ctcttgaaca gatgcaaaat 600
cggatggtga ttttactttg taacctgaaa cctgcaaaga tgaggggagt attatctcaa 660
gcaatggtca tgtgtgctag ttcaccagag aaaattgaaa tcttggctcc tccaaatggg 720
tctgttcctg gagacagaat tacttttgat gctttcccag gagagcctga caaggagctg 780
aatcctaaga agaagatttg ggagcagatc cagcctgatc ttcacactaa tgatgagtgt 840
gtggctacat acaaaggagt tccctttgag gtgaaaggga agggagtatg tagggctcaa 900
accatgagca acagtggaat caaa 924
<210>8
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<213>p43-2核苷酸序列
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aagggtgcag aggcagatca aatcattgaa tatcttaagc agcaagtttc tctacttaag 60
gagaaagcaa ttttgcaggc aactttgagg gaagagaaga aacttcgagt tgaaaatgct 120
aaactgaaga aagaaattga agaactgaaa caagagctaa ttcaggcaga aattcaaaat 180
ggagtgaagc aaatagcatt tccatctggt actccactgc acgctaattc tatggtttct 240
gaaaatgtga tacagtctac agcagtaaca accgtatctt ctggtaccaa agaacagata 300
aaaggaggaa caggagacga aaagaaagcg aaagagaaaa ttgaaaagaa aggagagaag 360
aaggagaaaa aacagcaatc aatagctgga agtgccgact ctaagccaat agatgtttcc 420
cgtctggatc ttcgaattgg ttgcatcata actgctagaa aacaccctga tgcagattct 480
ttgtatgtgg aagaagtaga tgtcggagaa atagccccaa ggacagttgt cagtggcctg 540
gtgaatcatg ttcctcttga acagatgcaa aatcggatgg tgattttact ttgtaacctg 600
aaacctgcaa agatgagggg agtattatct caagcaatgg tcatgtgtgc tagttcacca 660
gagaaaattg aaatcttggc tcctccaaat gggtctgttc ctggagacag aattactttt 720
gatgctttcc caggagagcc tgacaaggag ctgaatccta agaagaagat ttgggagcag 780
atccagcctg atcttcacac taatgatgag tgtgtggcta catacaaagg agttcccttt 840
gaggtgaaag ggaagggagt atgtagggct caaaccatga gcaacagtgg aatcaaa 897
<210>9
<211>888
<212>DNA
<213>p43-3核苷酸序列
<400>9
gaggcagatc aaatcattga atatcttaag cagcaagttt ctctacttaa ggagaaagca 60
attttgcagg caactttgag ggaagagaag aaacttcgag ttgaaaatgc taaactgaag 120
aaagaaattg aagaactgaa acaagagcta attcaggcag aaattcaaaa tggagtgaag 180
caaatagcat ttccatctgg tactccactg cacgctaatt ctatggtttc tgaaaatgtg 240
atacagtcta cagcagtaac aaccgtatct tctggtacca aagaacagat aaaaggagga 300
acaggagacg aaaagaaagc gaaagagaaa attgaaaaga aaggagagaa gaaggagaaa 360
aaacagcaat caatagctgg aagtgccgac tctaagccaa tagatgtttc ccgtctggat 420
cttcgaattg gttgcatcat aactgctaga aaacaccctg atgcagattc tttgtatgtg 480
gaagaagtag atgtcggaga aatagcccca aggacagttg tcagtggcct ggtgaatcat 540
gttcctcttg aacagatgca aaatcggatg gtgattttac tttgtaagct gaaacctgca 600
aagatgaggg gagtattatc tcaagcaatg gtcatgtgtg ctagttcacc agagaaaatt 660
gaaatcttgg ctcctccaaa tgggtctgtt cctggagaca gaattacttt tgatgctttc 720
ccaggagagc ctgacaagga gctgaatcct aagaagaaga tttgggagca gatccagcct 780
gatcttcaca ctaatgatga gtgtgtggct acatacaaag gagttccctt tgaggtgaaa 840
gggaagggag tatgtagggc tcaaaccatg agcaacagtg gaatcaaa 888
<210>10
<211>723
<212>DNA
<213>p43-4核苷酸序列
<400>10
caaaatggag tgaagcaaat agcatttcca tctggtactc cactgcacgc taattctatg 60
gtttctgaaa atgtgataca gtctacagca gtaacaaccg tatcttctgg taccaaagaa 120
cagataaaag gaggaacagg agacgaaaag aaagcgaaag agaaaattga aaagaaagga 180
gagaagaagg agaaaaaaca gcaatcaata gctggaagtg ccgactctaa gccaatagat 240
gtttcccgtc tggatcttcg aattggttgc atcataactg ctagaaaaca ccctgatgca 300
gattctttgt atgtggaaga agtagatgtc ggagaaatag ccccaaggac agttgtcagt 360
ggcctggtga atcatgttcc tcttgaacag atgcaaaatc ggatggtgat tttactttgt 420
aacctgaaac ctgcaaagat gaggggagta ttatctcaag caatggtcat gtgtgctagt 480
tcaccagaga aaattgaaat cttggctcct ccaaatgggt ctgttcctgg agacagaatt 540
acttttgatg ctttcccagg agagcctgac aaggagctga atcctaagaa gaagatttgg 600
gagcagatcc agcctgatct tcacactaat gatgagtgtg tggctacata caaaggagtt 660
ccctttgagg tgaaagggaa gggagtatgt agggctcaaa ccatgagcaa cagtggaatc 720
aaa 723
<210>11
<211>699
<212>DNA
<213>p43-5核苷酸序列
<400>11
tttccatctg gtactccact gcacgctaat tctatggttt ctgaaaatgt gatacagtct 60
acagcagtaa caaccgtatc ttctggtacc aaagaacaga taaaaggagg aacaggagac 120
gaaaagaaag cgaaagagaa aattgaaaag aaaggagaga agaaggagaa aaaacagcaa 180
tcaatagctg gaagtgccga ctctaagcca atagatgttt cccgtctgga tcttcgaatt 240
ggttgcatca taactgctag aaaacaccct gatgcagatt ctttgtatgt ggaagaagta 300
gatgtcggag aaatagcccc aaggacagtt gtcagtggcc tggtgaatca tgttcctctt 360
gaacagatgc aaaatcggat ggtgatttta ctttgtaacc tgaaacctgc aaagatgagg 420
ggagtattat ctcaagcaat ggtcatgtgt gctagttcac cagagaaaat tgaaatcttg 480
gctcctccaa atgggtctgt tcctggagac agaattactt ttgatgcttt cccaggagag 540
cctgacaagg agctgaatcc taagaagaag atttgggagc agatccagcc tgatcttcac 600
actaatgatg agtgtgtggc tacatacaaa ggagttccct ttgaggtgaa agggaaggga 660
gtatgtaggg ctcaaaccat gagcaacagt ggaatcaaa 699