CN116375802B - 一种小窝膜联蛋白AnnA1的亲和多肽及应用 - Google Patents
一种小窝膜联蛋白AnnA1的亲和多肽及应用 Download PDFInfo
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Abstract
本发明公开了一种肿瘤靶向的亲和多肽及其应用。亲和多肽的氨基酸序列选自SEQ ID No.1~SEQ ID No.5之一,亲和多肽能够高效亲和小窝膜联蛋白Annexin A1,并衍射出生物活性片段、亲和噬菌体、进行相关编码的核苷酸序列、复合纳米材料或者噬菌体复合材料。本发明的靶向多肽,包含以上多肽的生物活性片段,展示以上多肽的噬菌体以及偶联有以上多肽的其他生物纳米材料,均可以与抗癌药物结合形成复合纳米材料,有效靶向并治疗高表达AnnA1的肿瘤,结合力强,特异性高。
Description
技术领域
本发明属于生物医学领域的一种抗癌制药的多肽及其应用,具体是一种肿瘤特异性高表达小窝膜联蛋白AnnA1的亲和多肽(亲和噬菌体)及其应用。
背景技术
靶向治疗对于癌症患者有着至关重要的作用。小窝蛋白膜联蛋白Annexin A1在多种肿瘤组织中特异性高表达,而在健康组织中不表达,因此是一个固有的肿瘤靶点。但是,搜索文献发现尚未有报道过针对该靶点的靶向多肽,而主要是通过单克隆抗体来靶向肿瘤。单克隆抗体不仅制备技术复杂、容易产生免疫反应,而且价格昂贵,在生产应用中优势较弱。
发明内容
本发明利用丝状噬菌体M13的12肽库对小窝膜联蛋白AnnA1进行亲和筛选,获得了能够与AnnA1蛋白高度亲和的多肽序列,特异性强、便捷高效。同时,本发明将提供该亲和多肽及亲和噬菌体在肿瘤靶向治疗中的应用。
本发明采用以下技术方案。
一、一种亲和多肽:所述亲和多肽的氨基酸序列选自SEQ ID No.1~SEQ ID No.5之一。
所述亲和多肽能够特异结合AnnA1蛋白。所述亲和多肽能够高效亲和小窝膜联蛋白Annexin A1(以下简称AnnA1),而Annexin A1在多种人类肿瘤中高表达,如乳腺癌、肾癌、肝癌、肺癌、脑癌和***癌。
SEQ ID No.1~SEQ ID No.5的氨基酸序列分别为:MHHHINRHHFVQ、MPMFKHRMFHTH、GHHKHGQIMPMS、MSHHKHNTNWSR、HPFLHWYNGQRT。如下表所示(表1),亲和力高、特异性强。
表1 AnnA1亲和多肽氨基酸序列表
二、一种生物活性片段:所述的生物活性片段包含权利要求1所述亲和多肽,为生物活性片段中功能区的一部分,所述的生物活性片段也具有和所述亲和多肽一致的功能,即能够与AnnA1蛋白结合。生物活性片段在制备药物中应用。
三、一种亲和噬菌体,其外壳蛋白的序列选自或包含SEQ ID No.1~SEQ ID No.6之一。所述的外壳蛋白为侧壁、末端位置。
四、一种核苷酸序列:所述核苷酸序列能编码所述亲和多肽。
五、一种核苷酸序列:所述核苷酸序列能编码所述生物活性片段。
六、一种用于肿瘤靶向的复合纳米材料:所述复合纳米材料包含所述亲和多肽或者所述生物活性片段,也包含搭载抗肿瘤药物的纳米载体。
所述的多肽或生物活性片段作为靶向功能肽段,与能够载药的纳米颗粒或纳米纤维通过静电吸附或化学修饰的方法连接,作为肿瘤靶向复合纳米材料。
所述的药物为细胞毒类药物、激素类药物、生物反应调节剂、单克隆抗体药物、及其他类药物中的任意一种。
所述的药物载体为脂质体、金属或含有金属的纳米颗粒、天然或人工合成的高分子纳米材料、无机晶体材料、碳材料、聚合物囊泡、聚合物胶束中的任意一种。
七、一种用于肿瘤靶向的噬菌体复合材料:所述复合材料包含权利要求3所述亲和噬菌体,还包含在所述亲和噬菌体上基因工程化、化学修饰或物理吸附上其他材料,而形成更进一步的噬菌体。
本发明在研发、制备与生产用于肿瘤靶向治疗的生物医学材料中的应用。
本发明的多肽及对应噬菌体特异性强,亲和能力高,且操作相对简便。本发明的多肽可以和药物载体复合构成肿瘤靶向纳米材料,也可以直接用靶向噬菌体构建肿瘤靶向生物材料。
本发明的靶向多肽,包含以上多肽的生物活性片段,展示以上多肽的噬菌体以及偶联有以上多肽的其他生物纳米材料,均可以与抗癌药物结合形成复合纳米材料,有效靶向并治疗高表达AnnA1的肿瘤(乳腺癌、肾癌、肝癌、肺癌、脑癌和***癌)。经验证,多肽和靶向噬菌体与AnnA1结合力强,特异性高,为肿瘤的靶向治疗材料研发提供了新思路。
与现有技术相比,本发明具有以下突出优势:
(1)本发明筛选出的多肽能特异结合重组人小窝膜联蛋白AnnA1,而不结合其他蛋白,具有专一性。
(2)本发明使用噬菌体展示技术筛选与AnnA1结合的小分子多肽,具有耗时较短,操作简便,成功率高的特点。
(3)本发明筛选出的AnnA1亲和肽可以利用标准化的化学合成工艺进行合成,相比抗体类试剂,其生产、纯化、保存成本大大降低。
(4)本发明筛选出的亲和肽或亲和噬菌体,能够靶向高表达AnnA1蛋白的多种肿瘤,为开发肿瘤靶向材料提供了广阔的思路,也为科研人员构建更有价值的能够治疗多种癌症的多功能靶向治疗材料提供了参考。
附图说明
附表1为实施例1每轮噬菌体文库筛选后获得噬菌体滴度统计结果。
附表2为实施例1第3、4轮噬菌体文库筛选获得亲和多肽频次统计结果。
附图1为实施例2噬菌体酶联免疫吸附实验(ELISA)结果统计图。
附图2为实施例3多肽体内靶向肿瘤验证实验成像图。
附图3为实施例4噬菌体体内靶向肿瘤验证实验统计结果图。
具体实施方式
下面通过实施例对本发明作进一步的详细说明,以下实施例是对本发明的解释而本发明并不局限于以下实施例。凡在本发明的精神和原则之内,所做的任何更改和变化,均应当包括在本发明的保护范围之内。
本发明的实施例如下:
实施例1:利用噬菌体淘选技术筛选特异结合小窝膜联蛋白AnnA1的亲和多肽。
a.目标蛋白的包被与封闭:将20μg小窝蛋白溶于50μL PBS和50μLNaHCO3的混合液,取一个新24孔板,用NaHCO3润湿其中一个孔后,加入蛋白混合液,在4℃湿盒中孵育过夜以包被目标蛋白。而后吸除剩余液,加5%BSA封闭液孵育2h,阻断非特异性结合位点;
b.噬菌体文库去背景:取10μL Ph.D.-12噬菌体文库(1*1013pfu/ml),用无菌PBS稀释至100μL。将稀释后的噬菌体文库加入24空板中,室温孵育1h,使噬菌体与孔板结合,去除背景;
c.噬菌体文库结合:吸除蛋白封闭液,用PBST洗涤6遍,加入去背景后的噬菌体,室温孵育1h,使噬菌体和蛋白充分结合;
d.洗涤:用PBST缓冲液洗涤孔板10次,以充分洗涤未结合的噬菌体;
e.洗脱:去除洗涤液,加入洗脱液(0.2M的甘氨酸-盐酸,pH 2.2,1mg/mL BSA)室温孵育10分钟,将与蛋白特异性结合的噬菌体洗脱下来;
f.中和:加入中和液(1M 的Tris-HCl,pH 9.1),使得洗脱后的噬菌体液与中和液混匀后呈中性;
g.噬菌体扩增:将洗脱中和液加入20mL ER2738菌液中(对数前期),静置20min后,37℃剧烈摇晃培养4.5h;
h.噬菌体纯化:将含有噬菌体的菌液离心,向上清中加入的PEG/NaCl沉降纯化,用100μL PBS重悬噬菌体沉淀;
上述a~h步骤为第一轮筛选,重复这些步骤4次即可得到特异性结合重组人AnnA1蛋白的亲和噬菌体。
将每一轮筛选洗脱后和扩增后的噬菌体各取5μL使用LB/IPTG/X-gal平板进行浓度滴定,次日对平板上出现的噬菌斑进行计数,根据稀释倍数得到每轮筛选所得到的噬菌体数目。计数结果如附表1所示。
附表1每轮噬菌体文库筛选后获得噬菌体滴度统计
附表1的结果显示,在每轮筛选保持投入噬菌体数目相同(1×1011pfu)的情况下,输出噬菌体数目逐轮递增,第四轮达到了最大(6.47×106pfu),这表明亲和噬菌体在四轮的筛选过程中得到了有效地富集。
测试1:基因测序获得AnnA1蛋白亲和多肽序列:对第3轮和第4轮得到的亲和噬菌体,铺平板后挑取单克隆扩增培养,对菌液进行基因测序,从而获得噬菌体表面展示的亲和多肽序列。三、四轮多肽序列与频次统计如附表2所示。
附表2第3和第4轮噬菌体文库筛选获得亲和多肽频次统计
上表中可见,有5种亲和多肽在第3轮和第4轮筛选后合计重复次数大于2,其中MPMFKHRMFHTH多肽重复次数达到47次。
测试2:噬菌体酶联免疫吸附实验(ELISA)验证并比较特异结合AnnA1蛋白的亲和多肽的结合力大小。
a.将50μL 用0.1M NaHCO3稀释的4μg/mL蛋白加入96孔酶标板中,将酶标板置于湿盒,并置于4℃过夜孵育;
b.吸出蛋白液,并将200μL的5%BSA封闭液加入孔中4度孵育1h;弃掉封闭液,用PBST溶液洗涤6遍,每次6分钟;
c.扩增并纯化亲和噬菌体,加入50μL的2×109pfu/mL的不同种亲和噬菌体(具体分别为包含有MHHHINRHHFVQ、MPMFKHRMFHTH、GHHKHGQIMPMS、MSHHKHNTNWSR、HPFLHWYNGQRT序列的噬菌体),室温孵育1h,野生噬菌体做对照;
d.吸出噬菌体溶液,并用PBST洗涤洗涤6次;
e.每孔加入100μL事先用PBST稀释好的M13噬菌体pVIII外壳蛋白抗体,37℃孵育1h;然后吸出抗体,并用PBST洗涤3次;
f.每孔加入100μL事先用PBST稀释好的HRP偶联的羊抗鼠二抗,37℃孵育1h;然后吸出抗体,并用PBST洗涤3次;
g.每孔加入100μL的TMB显示底物,孵育10-20min;
h.每孔加入100μL终止液2M H2SO4进行终止,并在通过酶标仪在450nm测吸收峰,统计所得结果见附图1。
测试3:合成亲和多肽,通过尾静脉注射的方式注射入荷瘤小鼠体内,进行荧光成像,验证各个亲和肽的体内靶向肿瘤效果。
a.订购羧基端连有生物素的亲和多肽(具体分别为包含有MHHHINRHHFVQ、MPMFKHRMFHTH、GHHKHGQIMPMS、MSHHKHNTNWSR、HPFLHWYNGQRT序列的多肽),将亲和肽溶于生理盐水中,再将多肽溶液与含有Cy5的链霉亲和素溶液孵育3h,透析过夜。
b.将上述透析完的多肽溶液向每只荷瘤小鼠体内注射200ul,在不同时间点用小动物活体成像仪进行荧光成像,结果见附图2(黑色圆圈标注部位为皮下乳腺癌肿瘤部位)。
测试4:将亲和噬菌体通过尾静脉注射的方式注射入小鼠体内,通过肿瘤titer验证各种噬菌体的体内靶向肿瘤效果。
a.扩增各种亲和噬菌体(具体分别为包含有MHHHINRHHFVQ、MPMFKHRMFHTH、GHHKHGQIMPMS、MSHHKHNTNWSR、HPFLHWYNGQRT序列的噬菌体),用PBS将噬菌体稀释到1×1012pfu/ml,每只荷瘤小鼠尾静脉注射100ul噬菌体。
b.噬菌体在体内循环1h后,用20ml PBS对荷瘤小鼠进行心脏灌流,解剖,分离肿瘤组织,称重后加入组织裂解液,用研磨棒研磨;
c.将肿瘤组织裂解液稀释后用LB/IPTG/X-gal平板进行浓度滴定,次日对平板上出现的噬菌斑进行计数,肿瘤中含有的噬菌体数目比上肿瘤质量就得到单位肿瘤质量含有的噬菌体数目,以比较噬菌体的体内靶向能力,统计结果见附图3。
本发明涉及的氨基酸序列如下:
SEQ ID No.1;
名称:亲和多肽1的氨基酸序列
来源:人工序列(Artificial Sequence)
MPMFKHRMFHTH
SEQ ID No.2;
名称:亲和多肽2的氨基酸序列
来源:人工序列(Artificial Sequence)
MHHHINRHHFVQ
SEQ ID No.3;
名称:亲和多肽3的氨基酸序列
来源:人工序列(Artificial Sequence)
GHHKHGQIMPMS
SEQ ID No.4;
名称:亲和多肽4的氨基酸序列
来源:人工序列(Artificial Sequence)
MSHHKHNTNWSR
SEQ ID No.5;
名称:亲和多肽5的氨基酸序列
来源:人工序列(Artificial Sequence)
HPFLHWYNGQRT。
Claims (5)
1.一种小窝膜联蛋白AnnA1的亲和多肽,其特征在于:所述亲和多肽的氨基酸序列为MHHHINRHHFVQ。
2.一种亲和噬菌体,其特征在于:所述亲和噬菌体的外壳蛋白的序列为MHHHINRHHFVQ。
3.一种核酸,其特征在于:所述核酸编码权利要求1所述亲和多肽。
4.权利要求1所述亲和多肽、权利要求2所述亲和噬菌体或者权利要求3所述核酸的应用,其特征在于:在制备用于乳腺癌靶向治疗的生物医学材料中的应用。
5.权利要求1所述亲和多肽、权利要求2所述亲和噬菌体或者权利要求3所述核酸的应用,其特征在于:在制备用于乳腺癌靶向治疗的药物中的应用。
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