CN112979757A - 特异靶向人肝癌细胞的多肽 - Google Patents
特异靶向人肝癌细胞的多肽 Download PDFInfo
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Abstract
本发明属于生物医学领域,具体涉及对人肝癌细胞有特异性结合的多肽及其用途。该多肽为以下任意(1)多肽的氨基酸序列为:IPHGSLYTFMSA(IA12,SEQ ID No.1),DYHDPSLPTLRK(DK12,SEQ ID No.2),TITHVHKISETP(TP12,SEQ ID No.3);(2)在(1)所述的多肽分子中经过缺失、插入或置换一个或几个氨基酸且与(1)所述的多肽分子具有相同生物学功能的多肽衍生物。本发明的多肽能够与肝癌细胞特异性结合,而与正常的肝上皮细胞无特异性作用,而且作用效果明显,为临床早期诊断和靶向药物的研发提供了可靠的科学依据。
Description
技术领域
本发明属于生物医学领域,具体涉及对肝癌细胞BEL-7402有特异性结合的多肽及其用途。
背景技术
肝癌是我国临床上最常见的恶性肿瘤之一,发病率在所有恶性肿瘤中位居第四,死亡率位居第三,患者的五年生存率仅为10%左右,且发病率和死亡率呈逐年上升趋势。由于肝癌早期临床症状不明显、恶性程度高、进展速度快,发现时已是晚期,导致我国每年死于肝癌的患者高达11万人。因此,早期诊断、早期治疗,是改善肝癌患者预后的重要突破口。
现有的治疗肝癌药物通常具有毒副作用较大、药物用量大、容易产生后天耐药等缺点,而且肝癌对大多现有的化疗药物不敏感,更为重要的是,大多数药物还要经过肝脏代谢,又给肝脏带来损伤和负担。靶向治疗,是在细胞分子水平上,针对已经明确的致癌抗原发挥作用的一种治疗方式。可设计相应的治疗药物,药物进入体内会特异地选择致癌位点来相结合而发挥作用,使肿瘤细胞特异性死亡,而不会波及肿瘤周围的正常组织细胞,所以研发用于肝癌的治疗的分子靶向药物日益受到重视和关注。
噬菌体展示技术是分子生物学领域一种重要的筛选靶向药物的技术(荣获2018年诺贝尔化学奖)。噬菌体展示技术的主要原理是目的基因或编码多肽和蛋白质的基因通过基因工程技术克隆到噬菌体表面蛋白基因的适当位置上,让其随着噬菌体DNA的扩增而表达在噬菌体表面,由于外源基因和噬菌体基因的兼容性,外源基因的表达产物多肽或蛋白质仍可以保持其原有的空间结构和相应的生物学活性。我们利用噬菌体展示技术,采用肝正常上皮细胞和肝癌细胞差减筛选的方式,从噬菌体多肽库中筛选出能够与靶细胞特异性结合的目的噬菌体,并对其DNA进行测序,即可得到相应多肽的编码序列。这一技术实现了蛋白质或多肽基因型和表现型之间的联系,并且具有操作简便,可以高通量检测的特点,从而成为筛选肿瘤细胞特异性结合肽的高效手段,为肿瘤的早期检测和靶向药物的研究提供了新的方向。
发明内容
本发明的目的在于提供的多肽,能特异性与肝癌细胞靶向结合,而与正常肝上皮细胞没有结合,这在肝癌的早期诊断和靶向药物的研发等方面具有重要作用。
本实验即以人正常肝上皮细胞HL-7702为对照,采用噬菌体展示多肽库对人肝癌细胞BEL-7402进行减数筛选。经过三轮淘选后,通过蓝白筛选试验挑选出能够与肝癌细胞发生特异性结合的噬菌体阳性克隆,并用ELISA实验验证噬菌体与肝癌细胞结合的特异性。然后以大肠杆菌为载体,扩增纯化噬菌体后提取其DNA进行测序,经翻译后得到能够与肝癌发生特异性结合的多肽的编码序列,并人工合成荧光标记的阳性多肽,进一步验证阳性多肽与人肝癌细胞的靶向结合作用,进而为研发肝癌的早期诊断试剂和靶向治疗药物提供实验基础。
为了实现上述目的,本发明采用如下技术方案:与人肝癌细胞特异性结合的多肽,多肽为以下任意:(1)多肽的氨基酸序列为:IPHGSLYTFMSA(IA12,SEQ ID No.1),DYHDPSLPTLRK(DK12,SEQ ID No.2),TITHVHKISETP(TP12,SEQ ID No.3);
(2)在(1)所述的多肽分子中经过缺失、插入或置换一个或几个氨基酸且与(1)所述的多肽分子具有相同生物学功能的多肽衍生物。
该多肽对肿瘤细胞有靶向结合,与肿瘤细胞特异性结合。
所述的肿瘤细胞为肝癌细胞。
多肽在制备肿瘤诊断试剂盒中的应用,该试剂盒中包含所述多肽或多肽偶联物。
与人肝癌细胞特异性结合的多肽在制备用于治疗肝癌药物中的应用,该药物包含所述的多肽与药物活性成分,或包含所述的多肽与递药载体。该药物为任何药物治疗学上可接受的剂型,该药物优选的剂型为注射制剂。
该药物为任何药物治疗学上可接受的剂量。
与现有技术相比,本发明的效果在于:本发明使用噬菌体展示技术具有操作简便、高通量的淘选、高效率,可以筛选模拟表位、展示多肽或蛋白质与其包含在噬菌体内部的基因密码的连接、重组噬菌体易于纯化等优点。我们运用噬菌体展示技术所筛选出的多肽能够与肝癌细胞特异性结合,而与正常的肝上皮细胞几乎无结合作用,而且作用效果明显,为临床研发治疗肝癌的靶向药物提供了可靠的科学依据。
附图说明
图1 噬菌体展示技术筛选与肝癌细胞特异性结合的噬菌体阳性克隆图。A为噬菌体筛选的模式图;B为经过3轮筛选后与肝癌细胞BEL-7402结合的阳性噬菌体洗脱液的滴定图;C为每轮筛选后洗脱液的滴定数;D为每轮筛选后阳性噬菌体的富集。
图 2 ELISA鉴定1-30号噬菌体阳性克隆与人肝正常上皮细胞HL-7702和肝癌细胞BEL-7402结合力的OD450nm结果图。
图 3 噬菌体阳性克隆的测序结果图。
图4 荧光标记多肽与人肝正常上皮细胞HL-7702和肝癌细胞BEL-7402靶向结合的免疫荧光结果图。A为多肽IA12分别与肝正常上皮细胞HL-7702和肝癌细胞BEL-7402结合的免疫荧光结果图;B为多肽DK12与肝正常上皮细胞HL-7702和肝癌细胞BEL-7402结合的免疫荧光结果图;C为多肽TP12与与肝正常上皮细胞HL-7702和肝癌细胞BEL-7402结合的免疫荧光结果图。
图5 荧光标记多肽与人肝正常上皮细胞HL-7702和肝癌细胞BEL-7402结合能力的流式细胞检测结果图。A为多肽IA12与肝正常上皮细胞HL-7702和肝癌细胞BEL-7402结合的流式结果图;B为多肽DK12与肝正常上皮细胞HL-7702和肝癌细胞BEL-7402结合的流式结果图;C为多肽TP12与肝正常上皮细胞HL-7702和肝癌细胞BEL-7402结合的流式结果图。
具体实施方式
以下所述仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
实施例1
1.实验材料
1.1噬菌体肽库、细胞和宿主菌。
噬菌体12肽库、大肠杆菌E.coli ER2738、人肝癌BEL-7402细胞、人正常肝上皮HL-7702细胞。
1.2 实验试剂
RPMI 1640培养基、胰蛋白酶、FITG标记兔抗鼠、胎牛血清、酵母粉、蛋白胨、琼脂粉、四环素贮存液、吐温-20(tween-20)、牛血清蛋白BSA、M13噬菌体单链DNA提取试剂盒、IPTG、X-gal、PEG-8000、TMB。
1.3 实验工作液
1×PBS、LB液体培养基、LB-Tet固体平板、顶层琼脂、IPTG/X-gal工作液、IPTG/X-gal平板、PEG-NaCl、TBS缓冲液、0.1%TBST、0.5%TBST、4%多聚甲醛固定液、TBS-NaN3液的配制、3%BSA封闭液、碘化钠缓冲液、TE缓冲液、TMB工作液、四环素贮存液。
2.实验方法
2.1大肠杆菌的培养。
(1)大肠杆菌的复苏:从-80ºC冰箱中取出大肠杆菌甘油冻存液,用接种环取少量划线于LB/Tet固体平板上,然后将此LB/Tet固体平板倒置于37ºC的电热恒温培养箱中培养过夜,使用时挑取单菌落即可。
(2)大肠杆菌的培养:在15ml离心管中加入10ml LB/Tet液体培养基,挑取大肠杆菌单菌落加入其中。然后将离心管置于恒温振荡器中培养过夜,待菌液的OD600值为0.5时即可进行相关实验。
2.2 噬菌体展示多肽库的减数筛选。
(1)准备细胞:先将6孔培养板经poly-lysine预处理,取人肝癌BEL-7402细胞和人正常肝上皮HL-7702细胞,分别用胰蛋白酶处理后铺于其中,培养至细胞成功贴壁且生长状态良好后进行筛选。
(2)制备菌液:筛选当天,将大肠杆菌ER2738接种于20ml LB/Tet液体培养基中,置于37ºC恒温振荡器中震荡培养, 待菌液的OD600值为0.5时,用于扩增筛选洗脱的噬菌体。
(3)无血清培养:吸掉细胞培养基,用PBS洗涤1次后加入无血清培养基,置于通有5%CO2的37ºC恒温细胞培养箱中1h。
(4)洗涤:吸掉封闭液,用0.1%TBST较轻地洗涤5次,每次均需旋转以使微孔的底部及边缘都被洗涤,甩干。至第2、3轮筛选时分别用0.5%TBST、1.0%TBST。
(5)封闭细胞:吸掉细胞培养基,将平板倒置于干净的纸巾上用力甩去除残存的培养基,用含1%BSA的培养基封闭人正常肝上皮细胞HL-7702和人肝癌细胞BEL-7402,置于通有5%CO2的37ºC恒温细胞培养箱中1h。
(6)吸附: 取原始多肽库10μl,加入到990μl 0.5% BSA/ PBS缓冲液中,将噬菌体稀释为1.5×1011pfu/ml,并将其加入到已封闭的人正常肝上皮细胞HL-7702中,37ºC 1h,吸附可以和人正常肝上皮HL-7702细胞结合的噬菌体,留取上清。
(7)结合:将吸附后的噬菌体上清液与人肝癌细胞BEL-7402共同孵育1h。
(8)洗涤:弃掉未结合的噬菌体,将微孔板倒置于干净的纸巾上用力拍甩,以去除残存的溶液。按上述方法用0.1%TBST洗板5次。
(9)洗脱:加入0.2M Glycine-HCl(pH2.2)1mg/mlBSA洗脱液1ml,冰上慢摇10min,然后将洗脱液吸出并转移至预先已准备好的150μl中和液(1M Tris-HCl,pH9.1)中。
(10)按照上述步骤重复操作2次。
2.3噬菌体的滴度测定。
将IPTG/X-gal平板预热于37ºC的电热恒温培养箱中;取出适量的顶层琼脂在微波炉中加热,待其完全融化后取出,在每个10ml离心管中分装3ml;将待筛选的噬菌体进行等比稀释后,取10μl与200μl的大肠杆菌菌液充分混合反应5min后,加入到3ml的顶层琼脂中,然后均匀地铺在预热的IPTG/X-gal平板上,待冷凝后置于37ºC的电热恒温培养箱过夜,观察滴定结果。
2.4噬菌体的扩增和纯化。
(1)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)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。
2.5 酶联免疫吸附试验
(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HL-7702细胞悬液,5、6、7、8行的每个小孔按照蛇形各铺100μ人肝癌BEL-7402细胞悬液,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)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。
(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。
(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。
(5)加噬菌体样品:取出封闭后的96孔板,拍干孔中液体后,加入纯化得到的阳性噬菌体,于37ºC细胞恒温培养箱中反应1h。
(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。
二抗: 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。
(7)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。
(8)终止:取出反应后的96孔板,加入2M硫酸终止反应。
(9)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。
2.6 阳性噬菌体DNA的提取及测序
(1)在上述纯化的噬菌体沉淀中加入100ul碘化物缓冲液,再加入250ul无水乙醇, 充分混匀,室温作用20min。
(2)离心:4℃,14,000rpm,10min,弃上清。
(3)清洗:用500ul 70%乙醇洗涤沉淀,短暂离心后真空干燥。
(4)30ulTE(10mM Tris-HCl,pH5.0,1mMEDTA)缓冲液重悬沉淀,制成DNA测序模板液,送与上海生工测序。
2.7 细胞免疫荧光实验
(1)准备细胞铺板:在6孔板上提前铺上载玻片,将人肝癌细胞BEL-7402和人正常上皮细胞HL-7702铺于六孔板中待用。
(2)固定:用4%多聚甲醛固定15min。
(3)封闭:弃去4%多聚甲醛,PBS洗2次,用3%BSA/PBS于37℃封闭30min。
(4)孵育荧光标记多肽:将封闭液擦拭后加入荧光标记多肽,37℃ 1h。
(5)DAPI染色:用PBS洗涤3次后加DAPI 100μl,室温,15min
(6)封片:PBS洗3次后,封片。
2.8 流式细胞技术
(1)准备细胞铺板:将人肝癌细胞BEL-7402和人正常肝上皮细胞HL-7702铺于六孔板中待用。
(2)封闭: PBS洗2次,用3%BSA/PBS于37℃,封闭30min。
(3)荧光标记多肽孵育:将封闭液弃去后加入荧光标记多肽,37℃,孵育30min。
(4)收集细胞:用PBS洗涤3次后加胰酶消化收集细胞上流式检测。
3.实验结果
如图1所示,应用噬菌体展示技术(图1A),通过3轮差减筛选筛选出能与肝癌细胞BEL-7402特异性结合的阳性噬菌体克隆(图1B)。如图1C所示,经过三轮淘选后,洗脱液的滴定数从3.8×104/1×106细胞扩增到1.2×108/1×106细胞,噬菌体的输出/输入相对值从38增加到1.2×105,提示噬菌体阳性克隆在肝癌细胞BEL-7402上富集了3000多倍(图1D)。
如图2所示,随机挑取30个携带噬菌体多肽的大肠杆菌克隆,经细胞酶联免疫分析,结果显示实验组BEL-7402 的平均吸光度值OD405与对照组HL-7702的平均吸光度值OD405之比大于2.5的克隆共有14个,分别是1、3、5、7、8、12、14、18、20、21、24、25、27、29。上述14个阳性噬菌体与人肝癌BEL-7402细胞结合作用较强,而与人源性正常肝上皮HL-7702细胞结合作用则较弱。
接下来对14个阳性噬菌体克隆进行扩增、纯化并提取其DNA测序,结果显示,1、8、12、14、18、20、21、27号阳性噬菌体的测序结果显示同样的序列,按照三联密码子的原则,翻译出多肽序列:IPHGSLYTFMSA(IA12);3、7、14、24、29号阳性噬菌体的测序结果显示同样的序列,按照三联密码子的原则,翻译出多肽序列DYHDPSLPTLRK(DK12);5、25号阳性噬菌体的测序结果显示同样的序列,按照三联密码子的原则,翻译出多肽序列:TITHVHKISETP(TP12),如图3所示。
然后人工合成绿色荧光标记的阳性多肽荧光标记多肽,分别采用免疫荧光染色实验和流式细胞技术验证该多肽与人肝癌细胞BEL-7402的靶向结合。如图4所示,该多肽序列与人源性正常肝上皮HL-7702细胞结合能力较弱,而与人肝癌细胞BEL-7402结合能力则较强。
流式细胞检测实验进一步验证了上述结果。人肝癌细胞BEL-7402分别和10 μM的上述荧光标记的5条多肽孵育后进行流式检测,其阳性细胞的百分数分别为98.3%、85.7%和80.4%(如图5)。证明该多肽能特异性与肝癌细胞靶向结合,而对正常肝上皮细胞没有影响。
序列表
<110> 辽宁中健医药科技有限公司
<120> 特异靶向人肝癌细胞的多肽
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 12
<212> PRT
<213> Artificial sequence
<400> 1
Ile Pro His Gly Ser Leu Tyr Thr Phe Met Ser Ala
1 5 10
<210> 2
<211> 12
<212> PRT
<213> Artificial sequence
<400> 2
Asp Tyr His Asp Pro Ser Leu Pro Thr Leu Arg Lys
1 5 10
<210> 3
<211> 12
<212> PRT
<213> Artificial sequence
<400> 3
Thr Ile Thr His Val His Lys Ile Ser Glu Thr Pro
1 5 10
Claims (10)
1.肿瘤靶向的新型多肽,其特征在于,该多肽为以下任意:
(1)多肽的氨基酸序列为:IPHGSLYTFMSA(IA12,SEQ ID No.1);
DYHDPSLPTLRK(DK12,SEQ ID No.2);
TITHVHKISETP(TP12,SEQ ID No.,3);
(2)在(1)所述的多肽分子中经过缺失、插入或置换一个或几个氨基酸且与(1)所述的多肽分子具有相同生物学功能的多肽衍生物。
2.根据权利要求1所述的多肽,其特征在于,该多肽对肿瘤细胞有靶向作用,与肿瘤细胞特异性结合。
3.根据权利要求1所述的多肽,其特征在于,所述的肿瘤细胞为肝癌细胞。
4.如权利要求1所述的多肽在制备肿瘤诊断试剂盒中的应用。
5.根据权利要求1所述的多肽在制备肿瘤诊断试剂盒中的应用,其特征在于,在该试剂盒中包含所述多肽或多肽偶联物。
6.如权利要求1所述的多肽在制备用于治疗肿瘤药物中的应用。
7.根据权利要求6所述的多肽在制备用于治疗肿瘤药物中的应用,其特征在于,该药物包含所述的多肽与药物活性成分,或包含所述的多肽与递药载体。
8.根据权利要求6所述的多肽在制备用于治疗肿瘤药物中的应用,其特征在于,该药物为任何药物治疗学上可接受的的剂型。
9.根据权利要求6所述的多肽在制备用于治疗肿瘤药物中的应用,其特征在于,该药物的剂型为注射制剂。
10.根据权利要求6所述的多肽在制备用于治疗肿瘤药物中的应用,其特征在于,该药物为任何药物治疗学上可接受的的剂量。
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Citations (2)
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CN108409836A (zh) * | 2018-01-08 | 2018-08-17 | 中国医科大学 | 一种多肽及其用途 |
CN108530520A (zh) * | 2018-04-19 | 2018-09-14 | 福建师范大学福清分校 | 一种锰离子结合肽及其筛选方法、亲和性能的检测方法 |
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CN108409836A (zh) * | 2018-01-08 | 2018-08-17 | 中国医科大学 | 一种多肽及其用途 |
CN108530520A (zh) * | 2018-04-19 | 2018-09-14 | 福建师范大学福清分校 | 一种锰离子结合肽及其筛选方法、亲和性能的检测方法 |
Non-Patent Citations (1)
Title |
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吴淼等: "肝癌特异性黏附肽的鉴定和分析", 现代免疫学, no. 02 * |
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