CN113527429B - 人肝癌细胞特异结合多肽及其用途 - Google Patents
人肝癌细胞特异结合多肽及其用途 Download PDFInfo
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
本发明属于生物医学领域，具体涉及一种肝癌细胞特异结合多肽及其用途。所述多肽的氨基酸序列分别为：SEQ ID NO.1：NFWSAAYPLG为TL；SEQ ID NO.2：NLVPPNTVHNST；SEQ ID NO.3：NAFFLCDRYKYR；SEQ ID NO.4：NFHLRRFVGDTC；SEQ ID NO.5：NCWDDYNTVHLS。本发明还涉及了上述多肽及其生物活性片段和衍生物作为靶向多肽与能杀伤癌细胞的制剂和显像制剂形成的药物组合物和分子影像探针及其用途。本发明所述多肽对肝癌细胞具有特异靶向结合能力，选择性强，为临床上肿瘤的早期诊断和靶向药物的研发提供了新资源和新方向。
Description
技术领域
本发明属于生物医学领域，具体涉及能与肝癌细胞特异性结合的多肽；编码该多肽的核酸；包含该多肽的组合物；及该多肽或该组合物的应用，尤其是用于肿瘤预防、治疗及诊断目的的用途。
背景技术
肝癌是严重威胁人类健康的疾病之一, 据“Global cancer statistics 2018”显示，肝癌的死亡率位居全球男性癌症病死率的第二位，其发病机制隐匿，进展快，预后差，死亡率高。50%的患者在诊断为肝癌时已经处于晚期，此时的治疗方法无论是手术还是介入等, 其效果都不是很理想。因此，肝癌的早期诊断及早期干预，对于改善肝癌的病死率有重要意义。近年来肝癌早期诊断的价值已经受到广泛重视，不过，现存的标志物普遍特异性不高。因此，寻找与肝癌细胞及组织具有较高结合特异性和敏感性的靶向分子，进而展开分子探针及靶向化疗药物载体的开发是关键。
肿瘤归巢肽(tumor homing peptides，THPs)是一类对肿瘤组织或血管具有归巢效应的多肽,它能识别和结合肿瘤组织或血管表面的特异性受体或标志物。除了原位肿瘤,THPs还能识别并结合血管源性或转移性肿瘤。因此,THPs可将抗肿瘤药物直接靶向递送至肿瘤组织、细胞中,能够减少或消除药物耐受及毒副作用。当前发现的肿瘤归巢肽通常是由5～31 个氨基酸构成的分子量很小的活性多肽。小分子多肽与抗体药物相比，克服了抗体药物因其分子量较大所致的难以渗透到肿瘤组织的缺欠，多肽易通过生理屏障，亲和力高，且易化学合成。近十年中，肿瘤归巢肽已成为一种抗癌剂的有效靶向运载工具和肿瘤区域显像剂。
噬菌体展示技术（phage display technology，PDT）是George P. Smith在1985年提出的一种快速筛选多肽、蛋白质、抗体的高通量方法，并在2018年获得了诺贝尔化学奖。该技术的原理是将外源多肽或蛋白质的DNA序列插入中噬菌体次要外壳蛋白p III基因中，外源蛋白随着衣壳蛋白的表达而展示到噬菌体表面，噬菌体展示肽库在构建、改造及筛选方面具有易操作的优点，因此形成了商业化试剂盒以供科研使用。
发明人利用噬菌体12肽库对人肝癌细胞进行差减筛选，通过DNA测序并翻译，最终得到能与人肿瘤细胞靶向结合的多肽序列。其优势在于，（1）所得到的多肽片段水溶性高且较稳定，增强了多肽的实用性；（2）建立了多肽基因型和表现型之间的联系，操作更加便利；（3）该多肽能特异亲合肿瘤细胞，具有肿瘤靶向性。基于之前利用噬菌体展示技术靶向筛选的人肝癌细胞靶向亲和多肽，发明人进一步做了验证，以证明该多肽的高效性和特异性，为进一步开展新型肿瘤靶向药物的设计和研发提供候选资源和奠定坚实基础。
发明内容
本发明的目的在于提供高亲和性、特异性结合肝癌细胞的多肽。这样的结合多肽尤其适用本发明的目的，在于提供一种靶向新型多肽及其用途，通过特异性靶向结合肝癌细胞，优选地将这种新型靶向上述多肽或其衍生产品用于制备靶向肝癌的抗肿瘤药物或显像制剂。
为实现上述目的，本发明采用以下技术方案：
本发明的首要目的在于提供能与人肝癌细胞特异靶向结合的多肽，其氨基酸序列分别为：SEQ ID NO.1 NFWSAAYPLGTL；SEQ ID NO.2：NLVPPNTVHNST；SEQ ID NO.3：NAFFLCDRYKYR；SEQ ID NO.4：NFHLRRFVGDTC；SEQ ID NO.5：NCWDDYNTVHLS。
上述肽链中单字母符号代表的氨基酸残基，见氨基酸缩略表：
表1氨基酸缩略表
本发明目的之一在于提供一种生物活性片段或衍生物，其特征在于包含权利要求1所述的任一多肽序列为核心序列，包括共价连接的化合物以及由核心序列组成的多聚体混合。
优选地，该生物活性片段或衍生物与权利要求1中所述多肽具有相同的生物学功能，即同样具有肝癌细胞特异靶向作用。
本发明目的之一在于提供一种多聚核苷酸序列，其可以编码包含权利要求1所述的SEQ ID NO.1-SEQ ID NO.5的任一项多肽序列以及权利要求2所述的多肽活性片段及其衍生物。
本发明还进一步提供了一种用于肿瘤诊断的多肽分子探针，包含氨基酸序列为SEQ ID NO.1-SEQ ID NO.5所述多肽。
本发明还进一步提供了一种显像剂，包含本发明所述多肽和显影剂或放射性核素，包含氨基酸序列为SEQ ID NO.1-SEQ ID NO.5所述多肽，用于临床肿瘤的成像和诊断。
本发明还提供一种药物组合物，包括本发明提供的的氨基酸序列为SEQ ID NO.1-SEQ ID NO.5所述的多肽作为靶向肽，作为用于肿瘤治疗的药物，也可作为靶头增加药物或载有药物的载体如纳米材料、脂质体等。
本发明提供的药物组合物还包括与所述的多肽相缀合或混合可制备靶向药物的载体。
优选地，所述的制剂为能杀伤肿瘤细胞的化学药物、生物药物、纳米药物、放射性药物、光热治疗或光动力治疗药物或包裹这些药物的载体中的任意一种。
更优选地，所述的制剂为烷化剂、抗代谢药物、抗肿瘤天然药物、抗肿瘤抗生素、激素、金属络合物或肿瘤放射靶向标瘤放射靶向标记物中的任意一种。
本发明还提供上述药物组合物在任何药物治疗学上可接受的剂量和剂型。
进一步地，所述载体为目前药剂学广泛应用的、天然的高分子材料以及人工合成的分子聚合物及它们的混合物。
本发明还提供了氨基酸序列为SEQ ID NO.1-SEQ ID NO.5所述的多肽及其生物活性片段或衍生物在制备用于预防、治疗或诊断肿瘤的药物或显像制剂中的用途。
所述肿瘤为肝癌、胰腺癌、大肠癌、肺癌、卵巢癌、乳腺癌中的任意一种，优选为肝癌。
本发明利用了噬菌体展示技术筛选肝癌特异性结合的多肽，鉴定其特异性和亲和力，为肝癌诊断试剂及靶向治疗肝癌药物的研制奠定基础。
本发明的优势是：（1）本发明的多肽片段能够特异性结合肝癌细胞，而不识别正常肝癌上皮细胞（即正常细胞）。因此，该肿瘤靶向多肽及其衍生物在肝癌肿瘤分子诊断，筛查和靶向治疗中具有重要应用价值。
（2）该多肽的肿瘤特异性结合作用，为进一步寻找新的肿瘤靶点，研究大分子间相互作用的结合位点、寻找亲和力高且具有生物活性的配体分子以及药物的筛选、疫苗和新型诊断试剂的研发等领域带来了更多希望。
（3）与传统药物相比,在生产上,应用生物工程技术，短肽类药物合成和纯化简便,容易实现规模化生产，而且兼有相对分子质量较小、免疫原性弱、活性高等优点，为选出的多肽有望成为效果好、效益高的新时代药物带来更多希望。
附图说明
图1为噬菌体展示技术对肝癌细胞特异性结合阳性噬菌体克隆的三轮减性筛选实验结果图：A为各轮噬菌体淘选后洗脱液的滴定数；B为各轮噬菌体淘选的回收率图。
图2为ELISA鉴定1-20号阳性噬菌体克隆与人肝癌细胞Huh-7亲和力的OD405结果图。
图3为20个阳性噬菌体克隆的DNA测序结果。A为阳性噬菌体克隆DNA测序统计表；B为重复率最高的SEQ.ID.NO.1的DNA测序图。
图4 为荧光标记多肽FITC-NL与肝正常上皮细胞Hl-7702和肝癌细胞Bel-7402、Huh-7靶向结合的细胞免疫荧光结果图。A为FITC-NL与肝正常上皮细胞Hl-7702、肝癌细胞Bel-7402、Huh-7结合的共聚焦结果图；B为相对荧光强度统计柱状图。
具体实施方式
以下通过具体实施例进一步说明本发明的技术方案。
实施例1利用噬菌体展示技术对肝癌细胞特异性结合阳性多肽的三轮减性筛选
1.1宿主菌E.coli ER2738的复苏、培养
制备大肠杆菌平板，取LB-TET培养平板于37度孵箱预热1小时，待E.coli ER2738菌液融化后，用接种环蘸取少量菌液均匀铺在培养平板，然后倒置于37℃的恒温培养箱中过夜培养。制备宿主菌液，从生长良好的培养板中挑取单一菌落，置于含有四环素的LB细菌培养液中，37℃、180rpm振荡过夜培养，使细菌处于对数生长期。制备好的含大肠杆菌LB-Tet平板放到4℃冰箱保存备用，宿主菌液放到-80℃冰箱保存备用。
1.2噬菌体展示十二肽库体外减性筛选
以人源性肝癌细胞Huh-7作为靶细胞，人正常肝上皮细胞Hl-7702为吸附细胞。利用噬菌体展示技术体外筛选方法，对纽英伦生物技术有限公司 (NEW ENGLAND Biolabs)的 Ph.D.-12TMPhage Display Peptide Library 进行3轮体外筛选。并尽量保证每一轮噬菌体的投入总量相同，每轮递增筛选压力，最终获得高度富集的噬菌体。具体筛选步骤如下：
1.2.1准备筛选所用细胞
挑选生长良好的人源性肝癌细胞Huh-7、人正常肝上皮细胞Hl-7702。用胰蛋白酶分别消化细胞，显微镜下观察细胞变圆变亮后，吸掉胰酶，加入细胞培养基，轻轻吹打细胞使贴壁细胞悬浮。将两种细胞悬液分别吸到预先制备好的，经过多聚赖氨酸处理的培养皿中，放到细胞孵育箱中，培养到细胞贴壁，待生长良好后用于筛选。
1.2.2准备菌液
筛选当天将过夜培养的宿主菌液加到LB培养液中。37℃、180rpm振荡约3小时。以上为筛选滴度测定及扩增使用菌液。
1.2.3封闭细胞
用吸管吸净培养皿中的液体，并在无菌滤纸上将培养皿中液体拍甩干净。用0.5%BSA封闭，37℃放置1小时。
1.2.4肽库结合
弃掉培养皿里的封闭液。加入用TBST稀释100倍的Ph.D. -12TM原始噬菌体展示十二肽库，室温下120rpm微摇作用1小时。
1.2.5洗涤
吸掉第一轮未结合的噬菌体液，把培养皿放到无菌滤纸上用力拍干。用0.1%TBST洗涤培养皿10次，每次约30秒左右，并冲洗培养皿底部及其边缘，倒掉洗涤液，在无菌滤纸上拍干（注意每次洗涤后更换一张新的干净滤纸，防治交叉污染）。
1.2.6洗脱
培养皿里加入洗脱液（0.2M Glycine-HCl，PH2.2），常温下、80rpm、微摇15min。洗脱作用结束后，轻轻吹打洗脱液后吸出，加入到含有中和缓冲液的EP管里，混匀。
1.3测定噬菌体滴度
提前摇菌，加TET到LB中，37度180rpm，3小时后，达到对数生长期（OD值达到0.6左右为佳）。测定滴度前先将LB/ IPTG/Xgal平板在37℃孵箱预热1小时以上。准备琼脂糖凝胶，微波加热至融化。取吸附后中和洗脱液（或扩增后噬菌体上清液），用LB培养基倍数稀释噬菌体。稀释范围为：吸附后中和洗脱液稀释102-105；扩增后噬菌体上清液稀释109-1012。每个EP管加入备好的菌液，并在每管里加入10μl不同稀释倍数的噬菌体液。在振荡仪上振荡5min混匀。把感染后噬菌体液加到室温融化的琼脂中，立即将混悬液加至已预热的LB/IPTG/Xgal平板里，用冷却的涂布玻璃棒均匀涂开（每个稀释倍数一个平板，并做好标记）。将涂好的平板倒置于37℃的孵箱过夜培养。第二天检查平板上的噬菌体蓝斑生长情况并计数（即数每个平板上的噬菌体斑个数）。
1.4噬菌体扩增纯化
把剩余的吸附后洗脱液进行扩增纯化，以备接下来的筛选。取无菌离心管，把备好的过夜宿主菌接种到LB培养基中，形成对数前宿主菌液。把所有的吸附后洗脱液加入到对数前宿主菌液里，于37℃、200rpm快速振荡5小时进行扩增。把扩增噬菌体液于4℃、12000rpm离心10min，把上清转到另一新的离心管里再次同等条件下离心。小心取离心管上部80%的上清液到一新的离心管里，再往离心管里加入六分之一体积的PEG/NaCl，放4℃冰箱过夜沉淀。次日，将前日沉淀50ml管于4℃、12000rpm条件下离心15min，弃上清，再次同等条件离心2min，弃掉剩余上清液。把沉淀物用1ml的TBS重悬后转移到无菌EP管里，于4℃、14000rpm条件下离心5min。上清转移到新的EP管里，再次加入六分之一体积的PEG/ NaCl，冰上沉淀1小时。于4℃、14000rpm条件下离心10min，弃掉上清液，保留沉淀。用200μl的TBS重悬沉淀物，于4℃、1000rpm离心1min，保留上清于新的EP管（此为扩增后噬菌体液），-20℃冰箱储存。
1.5第二到三轮筛选
筛选的基本步骤同第一轮。每下一轮筛选均选用前一轮扩增后的噬菌体液作为次级肽库，每轮尽量保持同第一轮基本一致的噬菌体投入量，总共进行三轮减性筛选。每轮筛选后的洗脱液均要测定噬菌体滴度，并计算噬菌体的回收率。
结果如图1所示，图1为利用噬菌体展示技术对肝癌细胞特异性结合阳性多肽的三轮减性筛选实验结果图；其中A为三轮筛选各轮洗脱液的噬菌体滴度；B为三轮筛选各轮的噬菌体的回收率，上述结果显示经过三轮淘选后能够阳性与肝癌细胞特异性结合的阳性噬菌体洗脱液的滴定数为2.6×108Pfu/ml，富集了约7000倍。
实施例2酶联免疫吸附实验检测阳性噬菌体克隆与肝癌细胞的亲和力
2.1 纯化阳性噬菌体克隆
2.1.1阳性噬菌体的扩增：在锥形瓶中加入20mlLB/Tet液体培养基，然后按1:100加入大肠杆菌菌液和待扩增的噬菌体，置于37ºC，恒温振荡器中剧烈震荡4.5h，得到噬菌体的扩增液。
2.1.2阳性噬菌体的纯化：将经上述步骤得到的噬菌体扩增液4ºC、12000r/min，离心10min，取上清后加入1/6体积PEG-NaCl沉淀过夜后，12000r/min离心15min，弃去上清液，用TBS缓冲液溶解沉淀，再次给予1/6体积PEG-NaCl，冰上孵育1h。 4ºC、14000r/min，离心15min，弃去上清，将得到的沉淀用TBS-NaN3溶解后置于4ºC冰箱保存。
2.2 酶联免疫吸附实验检测亲和力
2.2.1制备细胞96孔板，铺板规则：96孔板边缘两列16个孔分别加入100μl×PBS作为空白组；然后1、2、3、4行的每个小孔按照蛇形各铺100μ人正常肝上皮细胞Hl-7702悬液，5、6、7、8行的每个小孔按照蛇形各铺100μ人源性肝癌细胞Huh-7悬液，然后将铺好的细胞平板置于通有5%CO2的37ºC细胞恒温培养箱中过夜即可进行ELISA实验。
2.2.2固定：取出过夜铺有细胞的96孔板，拍干孔中液体后，用PBS洗涤3次，然后加入4%多聚甲醛固定20min。
2.2.3阻断：取出固定后的96孔板，拍干孔中液体后，用PBS洗涤3次，然后加入3%过氧化氢,于37ºC细胞恒温培养箱中封闭30min，用以阻断内源性过氧化物酶的活性。
2.2.4封闭：取出阻断后的96孔板，拍干孔中液体后，用PBS洗涤3次，再加入3%BSA/PBS于37ºC细胞恒温培养箱中封闭1h。
2.2.5加噬菌体样品：取出封闭后的96孔板，拍干孔中液体后，加入纯化得到的20个阳性噬菌体，于37ºC细胞恒温培养箱中反应1h。
2.2.6加一抗：取出反应后的96孔板，拍干孔中液体后，用PBS洗涤3次后加入1:4000的M13抗体，4ºC过夜。
2.2.7：取出反应后的96孔板，拍干孔中液体后，用PBS洗涤3次，加二抗,于37ºC细胞恒温培养箱中反应30min。
2.2.8加底物TMB：将PBS洗涤3次后的96孔板于避光条件下加入TMB显示剂，避光置于37ºC细胞恒温培养箱中15min。
2.2.9终止：取出反应后的96孔板，加入2M硫酸终止反应。
2.2.10结果的测定：将完成全部反应的96孔板置于酶标仪中，于405nm处测定其OD值，保存结果并进行分析。
结果如图2所示，实验组肝癌细胞Huh-7的平均吸光度值OD405与对照组正常肝上皮细胞Hl-7702的平均吸光度值OD405相比明显增强。说明上述阳性噬菌体克隆能特异的与肝癌细胞Huh-7结合而与肝正常上皮细胞Hl-7702结合作用则较弱。
实施例3测定分析阳性噬菌体克隆DNA序列
3.1.阳性单克隆噬菌体挑选
把第三轮筛选后所得的噬菌体液，进行滴度测定铺制LB平板，在生长菌斑数不足50个的平板上，随机挑取20个间隔5mm生长良好的蓝斑。把随机挑取的20个蓝斑分别加入到1ml对数前宿主菌液（同噬菌体扩增），于37℃、200rpm快速振荡4.5小时进行扩增。
3.2阳性单克隆噬菌体单链DNA提取
将扩增的单克隆噬菌体液分别于4℃、14000rpm离心30秒，取上清液转到新管中，4℃、1000rpm离心30秒，取上清的80% 转到新的无核酸酶离心管里，取300ul菌液按1:1比例加300ul甘油，-20度冰箱冻存，取此即为扩增的单克隆噬菌体液。剩下500ul菌液加200ulPEG，混匀室温静置20min，4度离心14000r，10min，弃上清，4度14000rpm离心3min，弃上清，加100ul NaI，混合均匀，加250ul无水乙醇，静置10min，4度10000rpm离心10min，弃上清，用预冷的70%乙醇轻微洗3次，晾干30min，10000rpm离心5min，弃上清，加60ul TE。
3.3 DNA纯化
取上一步60ul TE管，加40ul TE补足至100ul。向离心管里加入500ul Buffer B3，充分混匀。将混合液全部移入吸附柱，室温下，8000g，离心30秒，将滤出液再次加入吸附柱中再次过柱。倒掉收集管里的液体，将吸附柱放回收集管里。向吸附柱里加入500μlWashSolution，9000g，离心30秒。倒掉收集管里的液体，吸附柱重新放到收集管中。重复上一步骤，将空吸附柱和收集管放入离心机，9000g，离心1min。在吸附膜中央加入40μl的Elution Buffer，室温静置2min。9000g离心1min，将所得到的DNA溶液进行测序。
3.4 测定DNA序列并分析
将提取的单链DNA进行序列测定，并根据三联密码子的原则翻译出相应的氨基酸序列。用DNAstar软件将其翻译为短肽序列和结构分析，并运用NCBI Blast与已知蛋白多肽序列对比同源性；GeneBank、Swiss-prot蛋白数据库对核苷酸序列进行相似性分析。
结果如图3所示为阳性噬菌体克隆的测序结果， A为DNA测序结果中筛选出来的5个序列和重复次数，其中共测了20个阳性单克隆噬菌体，其中重复次数最高的SEQ ID NO.1为11次，其他序列重复次数分别为SEQ ID NO.2为4次，SEQ ID NO.3为3次，SEQ ID NO.4为1次，SEQ ID NO.5为1次，将5个序列与已知蛋白多肽序列对比同源性和相似性分析结果为与已知蛋白多肽序列没有同源性，与核苷酸序列没有相似性。B为重复率最高的阳性噬菌体克隆的DNA测序波形图。
实施例4 共聚焦检测FITC-阳性多肽片段FITC-NL与肝癌细胞特异靶向结合能力
根据测得的氨基酸序列，采取固相合成的方法制备荧光标记多肽FITC-NL，并采用C18反相制备柱纯化，冷冻干燥后得到白色固体即为产物。将Huh-7、Bel-7402以及Hl-7702细胞铺于带有载玻片的24孔板中，放细胞孵箱培养细胞贴壁并铺满单层。24小时后，弃去培养基用PBS洗3次，每次5min。4%多聚甲醛固定10min。PBS洗3次，每次5min。用含0.5%TritonX-100的PBS孵育10min。PBS洗3次，每次5min。加3% BSA封闭，室温静置15min。加入5μM FITC-NL，37度静置15min。PBS洗3次，每次5min。加入DAB染液，37度静置15min。将24孔板中的载玻片取出，倒扣在加有抗荧光淬灭封片剂的载玻片上，通过激光共聚焦显微镜定位FITC-NL在细胞的位置，并鉴定肝癌细胞的特异性靶向作用。
结果如图4所示，为FITC-NL与肝正常上皮细胞Hl-7702和肝癌细胞Bel-7402、Huh-7靶向结合的细胞免疫荧光结果图。其中A为共聚焦实测图，结果发现，在肝癌细胞Bel-7402、Huh-7上观察到强荧光信号，而在肝正常上皮细胞Hl-7702上则几乎检测不到荧光信号。B为相对荧光强度柱形统计图，可以发现与正常上皮细胞相比，多肽FITC-NL与肝癌细胞的结合能力显著增强，有统计学差异，***P<0.001。
序列表
<110> 辽宁中健医药科技有限公司
<120> 人肝癌细胞特异结合多肽及其用途
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Claims (2)
1.一种与人肝癌细胞特异靶向结合的多肽，其特征在于，所述氨基酸序列分别为：SEQID NO.1：NFWSAAYPLGTL。
2.权利要求1所述的多肽在制备诊断和治疗肝癌药物中的应用，这种应用是指用于肝癌精准检测的分子探针和治疗药物的药物靶头。
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| CN102532272A (zh) * | 2011-12-29 | 2012-07-04 | 陕西师范大学 | HepG2细胞表面特异性结合的多肽 |
| CN103288927A (zh) * | 2013-05-31 | 2013-09-11 | 陕西师范大学 | Sgc-7901细胞表面特异性结合的多肽 |
| CN104650190A (zh) * | 2015-01-21 | 2015-05-27 | 陕西师范大学 | 肝癌细胞表面特异性结合的多肽 |
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| CN102532272A (zh) * | 2011-12-29 | 2012-07-04 | 陕西师范大学 | HepG2细胞表面特异性结合的多肽 |
| CN103288927A (zh) * | 2013-05-31 | 2013-09-11 | 陕西师范大学 | Sgc-7901细胞表面特异性结合的多肽 |
| CN104650190A (zh) * | 2015-01-21 | 2015-05-27 | 陕西师范大学 | 肝癌细胞表面特异性结合的多肽 |
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