CN109453366B - 一种抗肿瘤蛋白质的制备方法及用途 - Google Patents

一种抗肿瘤蛋白质的制备方法及用途 Download PDF

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CN109453366B
CN109453366B CN201811356093.6A CN201811356093A CN109453366B CN 109453366 B CN109453366 B CN 109453366B CN 201811356093 A CN201811356093 A CN 201811356093A CN 109453366 B CN109453366 B CN 109453366B
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程洋
付海田
石晓丹
玄英花
张馨心
楚瑞林
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Abstract

本发明公开了一种抗肿瘤蛋白质的制备方法及用途，属于生物医药技术领域。本发明首次筛选并验证了SWQ ID NO.1所示蛋白在抗肿瘤方面的应用，CCK8增值实验结果显示PvEXP100蛋白可显著抑制肝癌细胞HepG2细胞的增殖，抑制率高达80.1％，几乎完全抑制HepG2细胞的迁移性能。本发明的PvEXP100蛋白可作为活性成分或原料制备抗肿瘤药物，具有广阔的应用前景。

Description

一种抗肿瘤蛋白质的制备方法及用途
技术领域
本发明涉及一种抗肿瘤蛋白质的制备方法及用途，属于生物医药技术领域。
背景技术
疟疾是伴随人类出现最早的疾病，至今仍是全球范围内最重要的感染性疾病之一。目前关于疟疾的研究大多数集中在不同种属疟原虫入侵机制和疟疾疫苗的研发，而对疟原虫的药用价值或潜在的临床应用价值研究较少。有临床数据表明肿瘤患者感染疟原虫后，患者体内的肿瘤生长受到抑制，揭示疟原虫在临床肿瘤治疗的应用可能性。疟原虫主要通过虫体表面或内部携带的蛋白与肝细胞或红细胞表面受体结合后侵入到机体内，但由于从疟原虫裂殖子提取蛋白困难巨大，难以高效获得。
发明内容
一种PvEXP100蛋白质在制备抗肿瘤药物方面的应用；所述PvEXP100蛋白质含有SEQ ID NO.1所示的氨基酸序列。
在本发明的一种实施方式中，所述应用包括但不限于制备抗肺癌、肝癌的药物。
在本发明的一种实施方式中，所述药物包括但不限于疫苗、抑制剂、免疫调节剂。
本发明的第二个目的是提供一种制备所述PvEXP100蛋白质的方法，包括以下步骤：
1)扩增编码所述PvEXP100蛋白质的基因，将扩增的目的基因连接到表达载体上，得到重组质粒；
2)将成功表达目的基因的重组质粒转化到表达细胞中，诱导目的蛋白的表达。
在本发明的一种实施方式中，所述方法具体包括如下步骤：
1)设计引物SEQ ID NO.3和SEQ ID NO.4所示引物，扩增目的基因，将扩增的目的基因连接到表达载体上；
2)将目的基因测序正确的重组质粒转化到表达细胞中，通过IPTG诱导目的蛋白的表达，并进一步制备纯化的目的蛋白样品。
在本发明的一种实施方式中，所述表达载体可以为原核细胞表达载体、真核细胞表达载体或昆虫细胞表达载体。
在本发明的一种实施方式中，所述的表达细胞可以为原核表达细胞、真核表达细胞或昆虫细胞。
在本发明的一种实施方式中，所述表达载体为pET28a(+)，所述表达细胞为E.coliBL21(DE3)。
本发明的第三个目的是提供一种抗肿瘤的药物，其含有PvEXP100蛋白质或其活性物质，及药学上可接受的载体。
本发明还要求保护所述PvEXP100蛋白质在制备非医药用途的产品方面的应用。
有益效果：申请人对间日疟原虫蛋白进行分析筛选，发现PvEXP100是一个输出蛋白，其位于裂殖子膜表面，本发明首次筛选并验证了PvEXP100蛋白在抗肿瘤方面的应用，CCK8增值实验结果显示PvEXP100蛋白可显著抑制肝癌细胞HepG2细胞的增殖，抑制率高达80.1％，几乎完全抑制HepG2细胞的迁移性能。本发明的另外一个目的是提供本发明所制备的蛋白PvEXP100在制备抗肿瘤药物中的应用，即以本发明的PvEXP100蛋白为活性成分或原料的抗肿瘤药物，特别是肺癌、肝癌、前列腺癌。
附图说明
图1为PvEXP100蛋白考马斯亮蓝染色示意图(A)和Western blotting检测图(B)，
图2为CCK8法检测PvEXP100对HepG2细胞的增殖抑制效果；
图3为Transwell小室迁移法检测检测PvEXP100对HepG2细胞的迁移抑制效果。
具体实施方式
为使本发明更加容易理解，下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围，下列实施例中未提及的具体实验方法，通常按照常规实验方法进行。
实施例1PvEXP100重组质粒的构建
原核表达质粒Pet28a(+)、宿主菌BL21(DE3)及诱导用的IPTG均购自北京全式金生物科技有限公司；限制性内切酶、T4DNA连接酶、pfu DNA聚合酶、dNTP均购自TakaRa公司。引物的合成和核苷酸序列测序均由苏州金唯智生物科技有限公司完成。琼脂糖亲和介质镍柱(Ni)购自QIAGEN公司；His-Taq标签抗体购自Cell Signaling Technology公司。
设计引物通过PCR获取间日疟原虫PvEXP100蛋白的基因序列，引物如下：SEQ IDNO.3：GGATCCATGTTCTGGAAAGTAAAGGGG；SEQ ID NO.4：CTCGAGCAAAAGAAGGGCAACCATCAG，其中GGATCC为SEQ ID NO.3的酶切位点BamHⅠ；CTCGAG为SEQ ID NO.4的酶切位点XhoⅠ。
以卵形疟原虫基因组为模板，通过PCR扩增获得PvEXP100基因序列，扩增程序：94℃预变性3min，94℃变性10s、50℃退火30s、72℃延伸90s，循环35次，最后72℃延伸10min。
PCR产物进行琼脂糖凝胶电泳检测目的基因扩增条带，然后经过胶回收后，用BamHⅠ及XhoⅠ限制性内切酶对PCR产物及pET28a(+)37℃酶切2h，通过T4连接酶将目的基因过夜连接到原核表达载体pET28a(+)。
将连接的重组质粒转化E.coli DH5α感受态细胞：从-80℃取出E.coli DH5α细胞。立即放置于冰上,取连接的产物4μL加入到50μl感受态细胞中，混匀冰浴30min，42℃热激90后取出置于冰浴中2min。在管中加入无抗性的LB培养基1ml，放入37℃摇床中250rpm振荡培养1h。离心后取100μl培养基将菌体重悬，涂匀在含卡那霉素(50μg/ml)的LB平板上置于37℃孵箱中倒置培养过夜后观察菌落生长情况。挑取单克隆，提取质粒后进行测序。测序结果显示，目的基因序列正确。
实施例2重组蛋白PvEXP100的表达
将测序正确的阳性单克隆接种5ml含卡那霉素的LB培养基，37℃培养过夜，将菌液接种于新鲜500ml含卡那霉素的LB培养基中，当OD600为0.6-0.8时，加入1mmol/L IPTG，诱导8h。取诱导的PvEXP100进行超声破碎裂解，经10％SDS-PAGE电泳分析表明PvEXP100蛋白主要定位于包涵体中，分子量大小与预期相符。
通过8M尿素溶解包涵体释放PvEXP100蛋白，该蛋白在碳末端带有His-tag标签，因此采用GE公司的His-tag镍柱，按试剂盒说明书进行Ni2+亲和色谱纯化。用不同浓度的咪唑将蛋白提纯，咪唑的浓度分别为20mM、50mM、100mM、150mM和250mM，150mM咪唑洗下的蛋白经12％SDS-PAGE电泳，考马斯亮蓝染色验证分析。并进一步用Western Blot分析目的蛋白纯度。
考马斯亮蓝染色结果显示所得纯化产物为目的蛋白，分子量大小与预期相符(图1A)。Western Blot结果显示所表达蛋白的纯度较高、特异性强(图1B)。
实施例3PvEXP100蛋白抗肿瘤活性鉴定
(1)CCK8法检测PvEXP100对肿瘤细胞增殖性能的抑制作用
取对数期HepG2用含有10％FBS的DMEM培养基调整细胞浓度为5×104/mL，以每孔100μL接种于96孔板，置5％CO2，37℃培养箱中培养6h，待细胞贴壁后弃上清，PBS缓冲液清洗除去未贴壁细胞。样品组分别加入100μL不同浓度(10，50，100μg/mL)的药物，空白组加入等体积培养基。继续培养24h后，每孔加入10μLCCK8溶液，继续置培养箱中培养2h后，450nm处测定吸光值。以剂量为25μg/mL抗肿瘤药物5-Fu为对照。细胞增殖率(％)＝实验组A450/对照组A450×100。
结果如图2所示，随着蛋白浓度的升高，PvEXP100蛋白对HepG2细胞增殖的抑制作用逐渐上升。当浓度为100μg/mL，该蛋白对该肿瘤细胞抑制率达到约80.1％，高于剂量为25μg/mL抗肿瘤药物5-Fu对肿瘤细胞的抑制效果(67.2％)。
(2)Transwell迁移法检测PvEXP100对肿瘤细胞迁移性能的抑制作用
PvEXP100对HEPG2迁移的影响用transwell迁移法进行测定，步骤如下：24孔板各孔中加入0.6mL含有不同PvEXP100样品的DMEM培养基(含有10％FBS)；HEPG2用1％FBS的DMEM培养基调整细胞浓度为5×105/mL，取0.1mL接种于transwell小室中，然后将小室移至含有样品的培养基的孔上方，放置过程及培养过程中防止接触面气泡的产生。孵育24h后，取出transwell小室，弃除上清，用棉签擦拭小室上室内壁细胞，并用PBS漂洗去除细胞碎片后，将小室置于用PBS配置的90％乙醇溶液中固定10min。风干后的小室置于用0.1％结晶紫溶液中染色30min，其中结晶紫母液为甲醇配制的0.5％溶液，临用前用生理盐水或PBS稀释。染色结束后，用PBS漂洗数次去除多余染料，显微镜下拍照记录细胞迁移情况。
结果如图3所示，实验组迁移至下室的肿瘤细胞数量逐渐减少，通过对各组细胞个数的统计发现，PvEXP100蛋白对HepG2细胞迁移性能的抑制率基本达到100％，与剂量为25μg/mL的抗肿瘤药物5-Fu作用相当。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
SEQUENCE LISTING
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attagcggcg gaattagcgg cgggttgagc agcggtttgg ccagcggaat aagcggcgga 180
cttagcaact ccgccgccgc ttcgaccgcc ctgctgcacg cggccgtggg gaagggaccc 240
aacatgcaca cgtgccagtc ggccgggtgc gcctcataca agagcatcac gcccagcgat 300
gcaggagact gcctaaacgg gttcatctgc aaggagtgta aaagaaccca cgcgaagaac 360
ccaaacatct gcttctactc ctccctccaa ggatttgaaa gtttatatga agcacatcta 420
gaggatttta cacaaccaac gccctacgac cgtttcaacg tcccattggt taagtccagc 480
aaaggggaga acaacagggg ggacgcctca agtgacagtg gtagggaggt ctcccctaat 540
gacgaatcgg gggatcaccg cagaggcagt ctctcacaag gaggagacga cgatggagag 600
aagggggacc tccaacgcag tggcagggat ggcaaagcag gaggtagtcg cttcccacgg 660
gcgttagaag aagaggaaga agaagaggag gaggaggaag acgacgacga cgactctgcg 720
aaagaaaaac gtggtggaca taaagggggc aactcccctc agggggggaa caacggaggg 780
aacaatttcg acgcaggtta cgaaacggag tcatttctgc agaaatcgcc tgaccatgtt 840
catagaaaag gtgatctaca caagttggcg aaagaggggg ggcgggagca gcacacaaac 900
gctcacatgc atatacacac gcacatgcac actcacacgc ataacgaatt gatgtccgga 960
aaagagggac tccttagctc ggtggagacg cacgtgcggc ttggcatcag tgaggggggc 1020
tacaacaggg gagcgtccga atctccgggg aggcacagcg gcgtgagcag cggtgcctct 1080
gtcagcatgg gcaccgctgc tcatggaggc actgctgctg agagtggcta ctccttcgcc 1140
gagtccgagc ggggcaaaga aaaaatcgtc tacaagcgac tcaaaataag cctgaacaat 1200
cacgaggagt atttcaaaag caaaatgaat aagtgtcacg tgggggggga cggagtggcc 1260
actctgtatg tgaaggtgct gctgcagatt gttaaggata agaacgacgt ctatgtggac 1320
gtgagtagga ggagcggctc gtctcacatg agcccagggt cgggtgggca gaaggggaag 1380
gaagggaaga aggcaaacgg gaggagcgaa tcggggaagg aagaggagga agaagaggac 1440
gcggaagaga gtgacgacga tgagtacatg cggaagtctc aaagcgccat ggggggggga 1500
gggtacggtt accgctcagg cgcggagacg gacagcgata gcgacagcga cagcgacagc 1560
gatggggggc gcgcggcggt gaataggtac gcctacgtgg agctgcacgg gggggcgcaa 1620
aacaaagcgg cgaacgaagc ggcgaacgat gcggctacct ggggggcggc gaaggaaccg 1680
ctctccctcc tccaggtgag ggaggacctg gacggagact ccatggggaa ctactacaag 1740
tcgcgcaacg ggttcttcaa atcgattttc aaacgggttt ttaaaaaaaa aggagactcg 1800
gatgaagacg cagggggggg ggacgacgag gacagtgatg aggagcccca gggggggaag 1860
aagaagcgga ggtggagatt cccctggaag agacgaaggg ggaagggcag ccaactgcag 1920
ggcggcgacg acaatgacga cgagggcgag agcgaggatg agtcacgcag cacaagacgt 1980
aggaggggag gcaggcgggg gctattcggt cggtccaatc gaaagggcag aggcaaaggg 2040
agagacgaaa gtgatgatgg cgatgacggc gaagaaggcg aagactcaga cgatgaagag 2100
gccagtgggg gcagtggaca aaggggggca aaaaaaggaa aacgaaacgg taagggaagc 2160
ggaaccaagg ggggcagatt cgaggagacc aaatcaaaaa tgggtagcct ctttgccaaa 2220
gtgaagagga agattctccc cgtaaaacaa aaactacaca tagaggcgtt cttcaatagc 2280
atcatcgtaa agtcatgtag gaatgccctc aagtgggagg gcaacatgtt tcggaagcag 2340
tcgctcgtcg agatgactct caaggtcccg gttaaactga aatacataaa gggggagcct 2400
ctgcatttct tccgttcggg ctacgaagtc attttaactt gccgcaactg cgatgaggtt 2460
ctcttcaact cgtgtgtgca ggtctactgc gccaagcgag cgagcaaaga ggaacgggcg 2520
gtggcgcacc acggcgggca cgcggacgga ggagccctgg ggagcggcgc aaccgcagct 2580
gccattactg ccgcttccac tgctgccgcc actgccgctg cttcccccgc atcgccgtac 2640
ctttactcgg cgagcccgtc cgacgtctcc ccgctggcaa tgatccatta caacgggcag 2700
tacttccccg gcagtgtaac ccccgaggca gactacttca gcagcgatgg gcacatgtgc 2760
gttagctact cggtcgttct cgtcacgctg atggttgccc ttcttttgta g 2811
<210> 3
<211> 27
<212> DNA
<213> 人工序列
<400> 3
ggatccatgt tctggaaagt aaagggg 27
<210> 4
<211> 27
<212> DNA
<213> 人工序列
<400> 4
ctcgagcaaa agaagggcaa ccatcag 27

Claims (1)

1.一种蛋白质在制备抗肝癌药物方面的应用，其特征在于，所述蛋白质的氨基酸序列如SEQ ID NO.1所示。
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