CN112266407A - 特异结合p53蛋白的七肽、编码基因、制备方法及用途 - Google Patents
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Abstract
本发明公开了特异结合p53蛋白的七肽、编码基因、制备方法及用途。七肽的氨基酸序列为Val Cys Phe Gln Trp Asp Met，其编码基因的核苷酸序列为cgcggcagctgccaggagaag。该七肽特异性强、亲和性高、活性好。用于肺癌靶向药物递送系统的制备和肺癌的检测。
Description
技术领域
本发明涉及生物技术领域，特别涉及特异结合p53蛋白的七肽、编码基因、制备方法及用途。
背景技术
近年来，肺癌的发病率和致死率呈持续上升趋势，成为病死率位居第二位的恶性肿瘤，严重威胁人的生命健康。肺癌血供丰富，具有生长快、易转移的特点，使得肺癌的治疗更为困难。常规化疗药物对肺癌细胞没有选择性，在杀伤癌细胞的同时，也会杀伤正常组织细胞，具有比较严重的毒副作用。肿瘤靶向药物递送系统是一类能够使药物选择性地作用于肿瘤细胞的新型给药系统，对正常组织细胞没有影响，因此，能够降低药物的毒副作用。肿瘤靶向药物递送系统中的给药载体包括单克隆抗体、多肽、脂质体和纳米粒等，其中的多肽因具有高亲和性、较好的特异性和安全性的特点而受到广泛关注，目前已成为一种发展迅速且应用日益广泛的新型肿瘤靶向给药载体。
p53基因是一种抑癌基因，定位于人类染色体17p13.1，编码393个氨基酸组成的53kD的核内磷酸化蛋白，被称为p53蛋白。p53基因是细胞生长周期中的负调节因子，与细胞周期的调控、DNA修复、细胞分化、细胞凋亡等重要的生物学功能有关。p53基因分为野生型和突变型两种，其产物也有野生型和突变型。野生型p53蛋白极不稳定，半衰期仅数分钟，并具有反式激活功能和广谱的肿瘤抑制作用。突变型p53蛋白稳定性增加，半衰期延长，可被免疫组化方法检测出来。p53基因的突变(缺失)是人类肿瘤的常见事件，与肿瘤的发生、发展有关。一般认为p53过表达与肿瘤的转移、复发及不良预后相关。
目前的靶向药物递送系统中的给药载体包括单克隆抗体、多肽、脂质体和纳米粒等，其中的多肽因具有高亲和性、较好的特异性和安全性的特点而受到广泛关注，目前已成为一种发展迅速且应用日益广泛的新型靶向给药载体。靶向的多肽偶联药物递送系统，首先要发现具有靶向结合活性的多肽载体，可以通过噬菌体展示肽库筛选、化学合成活性肽的方法获取靶向多肽载体。
发明内容
发明目的：本发明目的是提供特异性强、亲和性高、活性好的特异结合p53蛋白的七肽及编码基因。该七肽以融合蛋白的形式融合表达在噬菌体外壳蛋白PVIII或PIII的N端，展示在噬菌体颗粒的表面，其保持相对独立的空间结构和生物活性，可以与p53蛋白特异结合且不影响噬菌体的包装和复制。
本发明另一目的是提供所述七肽及编码基因的制备方法。
本发明最后一目的是提供所述七肽及编码基因的用途。
技术方案：本发明提供一种特异结合p53蛋白的七肽，氨基酸序列为Val Cys PheGln Trp Asp Met，其编码基因的核苷酸序列为cgcggcagctgccaggagaag。
进一步地，所述七肽以融合蛋白的形式融合表达在噬菌体外壳蛋白PVIII或PIII的N端。
所述的特异结合p53蛋白的七肽的制备方法，包括如下步骤：
(1)将P53蛋白包被在平板上，封闭平板，将噬菌体展示随机七肽库加入包被和封闭后的平板中，进行至少4轮生物学淘选并测定每轮淘选后的噬菌体滴度；
(2)从至少第4轮计数平板上随机选择数珠噬菌体，挑选每一个噬菌体至OD0.5的E.coli ER2738菌液中，培养4-5h，后离心，取上清，重复两次，加入PEG-8000/NaCl对噬菌体进行沉淀，后离心，取上清，用TBS溶液溶解，得到每一个噬菌体单克隆的扩增产物；
(3)将噬菌体单克隆扩增产物加入到已经包被好P53蛋白的平板上，通过噬菌体ELISA测定各株噬菌体对结核分支杆菌的结合强度，并进行测序。
所述的特异结合p53蛋白的七肽在制备肺癌靶向药物递送系统中的用途。
所述的特异结合p53蛋白的七肽在肺癌检测中的用途。
有益效果：本发明可通过靶向的多肽偶联药物递送系统靶向治疗肺癌，通过筛选出与可以与p53蛋白相连的多肽，进一步抑制肺癌的发生发展，以期获得有效的抑肺癌药物。
附图说明
图1是挑选的20个噬菌体克隆P-ELISA检测的结果；横坐标为噬菌体克隆，纵坐标为OD 450值；
图2是竞争ELISA检测P53蛋白，OD值与P53蛋白浓度曲线，横坐标为P53蛋白的浓度，纵坐标为OD450值；
图3是特异性噬菌体检测小鼠模型肺部组织图，A图为对照组，B图为肺癌组。
具体实施方式
实施例主要试剂如下：
(1)LB培养基：每升含10g蛋白胨，5g酵母提取物，5g NaCl，高压灭菌，室温贮存；
(2)LB/IPTG/Xgal平板：LB培养基+15g/L琼脂粉。高压灭菌，冷却至低于70℃时，加入1mL IPTG/Xgal，混匀倒平板。平板4℃避光贮存；
(3)顶层琼脂：每升含10g蛋白胨，5g酵母提取物，5g NaCl，7g琼脂粉。高压灭菌，分成50ml等份，固体培养基室温贮存，用时微波炉融化；
(4)四环素贮液：以20mg/mL的浓度溶于70％乙醇中，-20℃避光贮存，用前摇匀；
(5)LB-Tet平板：LB培养基+15g/L琼脂粉。高压灭菌，冷却至低于70℃时，加入1mL四环素贮液，混匀倒平板，平板4℃避光贮存；
(6)PEG/NaCl：20％(w/v)PEG-8000，2.5M NaCl，高压灭菌，室温贮存；
(7)IPTG/Xgal配方：将1.25g IPTG(isopropylβ-D-thiogalactoside)和1gXgal溶于25mL二甲基甲酰胺中，-20℃避光贮存；
(8)TBS：50mM Tris-HCl(pH 7.5)，150mM NaCl。高压灭菌，室温贮存；
(9)PBST溶液：在PBS溶液中加入体积比为0.05％/0.02％的吐温20
(10)碘化物缓冲液：10mM Tris-HCl(pH8.0)，1mM EDTA，4M NaI。室温避光贮存；
(11)0.2M Glycine-HCl(pH2.2)，1M Tris-HCl(pH 9.1)，高压灭菌，室温贮存。
实施例1.噬菌体文库的扩增及纯化
接种E.coli ER2738单菌落于5-10ml LB液体培养基中，37℃，200rpm摇床孵育至对数中期(OD0.5)；加入10ul噬菌体，37℃，200rpm摇床震荡4-5h，10000g离心10min，取上清；再次10000g离心10min，取80％上清，加入1/6体积的PEG8000/NaCl，4℃静置过夜。次日取出，白色沉淀即为噬菌体。10000g离心15分钟，倒掉上清，瞬时离心后轻轻吸出残留溶液；加入1ml TBS溶液溶解白色沉淀。
实施例2.结合P53蛋白的七肽的淘选及鉴定
(1)噬菌体滴度测定
接种E.coli ER2738单菌落于5-10ml LB液体培养基中，37℃，250rpm摇床孵育至对数中期(OD600：～0.5)；微波炉加热融化顶层琼脂糖培养基，分成3mL/份分装到灭菌试管中，每个噬菌体稀释度用一管，保存于45℃备用；37℃预温LB/IPTG/Xgal琼脂平板，每个噬菌体稀释梯度取一个平板备用；用LB培养液对噬菌体进行10倍比系列稀释(建议稀释范围：扩增的噬菌体培养物上清：108-1011；未扩增的淘选洗脱物：101-104)；每个稀释度换一新鲜吸头，建议使用带滤芯吸头以避免交叉污染；当大肠杆菌菌液达对数中期时，将菌液分成200μL等份于微量离心管中，每个噬菌体稀释度用一管；每管大肠杆菌菌液中分别加入10μL不同稀释倍数的噬菌体，快速震荡混匀，室温温育1-5min；将噬菌体感染的大肠杆菌菌液加入45℃预温的顶层琼脂糖培养基管中，每次一管，快速混匀，立即倾注于37℃预温的LB/IPTG/Xgal琼脂平板上。适当倾斜平板将上层琼脂均匀铺开；待平板冷却5min后，倒置于37℃孵箱，培养过夜；检查平板，计数有～102个噬菌斑的平板上的斑数，然后，用此数目乘以稀释因子即得到每10μL噬菌体的空斑形成单位(pifu)滴度。
(2)P53蛋白特异性噬菌体的富集
以50μg/mL浓度的P53蛋白包被免疫试管并于4℃封闭过夜，TBST洗涤6次后加入噬菌体肽库，37℃振荡孵育1h，TBST洗涤10次去除未结合的噬菌体，加0.2mol/L Glycine-HCl(pH 2.2)1mL振荡10min洗脱特异结合的噬菌体，加150μL 1mol/L Tris-HCl(pH 9.1)中和，取10μL洗脱产物计数噬菌体的滴度，其余洗脱产物感染大肠杆菌ER2738扩增并纯化噬菌体，得到次级库，对次级库滴度进行测定后进入下一轮筛选程序。
(3)特异性结合噬菌体的筛选
重复步骤2的方法再进行四轮淘选，每一轮的滴度如表1所示，第1轮洗涤条件是体积比0.1％的Tween20，第2和第3轮Tween-20浓度变为0.3％(v/v)，第4轮Tween-20浓度为0.5％(v/v)。
表1特异性噬菌体富集第一轮至第四轮噬菌体投入产出表
| 轮次 | 投入滴度(pfu/ml) | 产出滴度(pfu/ml) |
| 第一轮 | 2×1011 | 3.5×109 |
| 第二轮 | 2×1011 | 2.7×1010 |
| 第三轮 | 1×1011 | 5.7×1011 |
| 第四轮 | 1×1011 |
实施例3.P53蛋白特异性噬菌体的鉴定
随机挑取第三轮筛选后滴度测定平板上的分离良好的单菌落20个，经过分别培养纯化后，用噬菌体ELISA检测各株噬菌体对P53蛋白的结合活性。具体步骤如下为：分别以P53蛋白和Blocking包被酶标板，每组三个平行，并于4℃封闭过夜，TBST洗涤6次后加入纯化后的噬菌体100μL，37℃振荡孵育1h，TBST洗涤10次去除未结合的噬菌体，加入HRP标记的鼠抗M13单克隆抗体100μL，37℃振荡孵育1h，TBST洗涤10次，加TMB底物室温避光反应5～10min，2mol/L硫酸终止反应，于450nm波长测定OD值，以P/N≥2.1为阳性。
检测结果如图1显示，有15株噬菌体表现出对P53蛋白的结合。提取这15株噬菌体ssDNA，阳性克隆的菌液DNA测序由上海生工生物技术公司完成，引物为-96gIII。实施例中涉及的核苷酸序列：cgcggcagctgccaggagaag使用DNAP53蛋白N和Swiss数据库序列进行分析。获得相应的氨基酸序列：Val Cys Phe Gln Trp Asp Met。
实施例4：序列的结合活性测定
分别以不同浓度P53蛋白和Blocking包被平板并于4℃封闭过夜，TBST洗涤6次，加入展示有ATPX3987序列的噬菌体2*1011(以相同滴度的无关噬菌体为对照)，TBST洗涤10次去除未结合的噬菌体，加入HRP标记的鼠抗M13单克隆抗体100μL，37℃振荡孵育1h，TBST洗涤10次；加TMB室温避光反应5～10min，2mol/L硫酸终止反应，于490nm波长测定OD值。结果如图2所示，展示有7肽序列的噬菌体表现出对P53蛋白的特异性结合。
实施例5.序列体内结合实验
取C57小鼠5只，采用支气管接种方法接种NCL-H460细胞，建立肺癌小鼠模型，称作肺癌组；另取C57小鼠5，采用支气管接种方法接种等量的PBS溶液，作为肺癌组对照组。分别选取不同组的C57小鼠的肺组织切片，进行特异性噬菌体免疫组化检测，如图3所示，可以发现肺癌组小鼠模型病变部位出现典型的黄色或棕色颗粒，和对照组有显著差异。提示可以应用该七肽，靶向结合p53蛋白。
Claims (5)
1.一种特异结合p53蛋白的七肽，氨基酸序列为Val Cys Phe Gln Trp Asp Met，其编码基因的核苷酸序列为cgcggcagctgccaggagaag。
2.根据权利要求1所述的七肽，其特征在于：其以融合蛋白的形式融合表达在噬菌体外壳蛋白PVIII或PIII的N端。
3.权利要求1所述的特异结合p53蛋白的七肽的制备方法，其特征在于：包括如下步骤：
(1)将P53蛋白包被在平板上，封闭平板，将噬菌体展示随机七肽库加入包被和封闭后的平板中，进行至少4轮生物学淘选并测定每轮淘选后的噬菌体滴度；
(2)从至少第4轮计数平板上随机选择数珠噬菌体，挑选每一个噬菌体至OD0.5的E.coliER2738菌液中，培养4-5h，后离心，取上清，重复两次，加入PEG-8000/NaCl对噬菌体进行沉淀，后离心，取上清，用TBS溶液溶解，得到每一个噬菌体单克隆的扩增产物；
(3)将噬菌体单克隆扩增产物加入到已经包被好P53蛋白的平板上，通过噬菌体ELISA测定各株噬菌体对结核分支杆菌的结合强度，并进行测序。
4.权利要求1所述的特异结合p53蛋白的七肽在制备肺癌靶向药物递送系统中的用途。
5.权利要求1所述的特异结合p53蛋白的七肽在肺癌检测中的用途。
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