CN114891069A - 特异性靶向mCNα的二元环肽配体及其获得方法 - Google Patents
特异性靶向mCNα的二元环肽配体及其获得方法 Download PDFInfo
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Abstract
特异性靶向mCNα的二元环肽配体及其获得方法,涉及生物医药领域。所述特异性靶向mCNα的二元环肽配体,由一条含有三个半胱氨酸的线性多肽链和一个小分子组成;小分子与线性多肽链的偶联使得原本的线性多肽链成为二元环肽;所述特异性靶向mCNα的二元环肽配体的氨基酸序列如序列表SEQ ID NO.1~12所示。先构建噬菌体展示二元环肽库:将二元环肽库用于mCNα蛋白配体的筛选,选取富集最多的多肽进行合成,然后与小分子2ClAc‑3.5反应得到二元环肽配体分子,表征二元环肽配体分子与mCNα的亲合力。该多肽配体对后续mCNα靶点以及钙调神经磷酸酶的研究是具有重要意义的。
Description
技术领域
本发明涉及生物医药领域,尤其是涉及基于噬菌体展示技术获得与mCNα具有高亲合力结合的特异性靶向mCNα的二元环肽配体及其获得方法。
背景技术
钙调神经磷酸酶(CN),是唯一已知的由钙和钙调蛋白(CaM)直接调节的蛋白丝氨酸/苏氨酸磷酸酶。CN将Ca2+信号与细胞反应关联,从而具有多种生物学功能,并在许多生理过程中发挥关键作用,包括免疫反应、细胞凋亡、肌肉分化、骨形成和神经元信号传导等。CN最典型的底物是活化的T细胞(NFAT)转录因子家族(Mancini M,Toker A.NFAT proteins:emerging roles in cancer progression.Nat Rev Cancer2009;9:810-820.)。CN直接与细胞质中的NFAT转录因子结合,导致它们去磷酸化并随后易位到细胞核中。由于其在T细胞活化中的作用,CN已成为开发免疫抑制剂药物的主要目标。迄今为止发现的两种最成功的抑制剂是环孢菌素A(CsA)和他克莫司(FK506),它们分别与胞内蛋白亲环蛋白A(CyPA)和FKBP12结合。生成的CyPA-CsA和FKBP12-FK506复合物抑制NFAT蛋白的去磷酸化,从而阻断它们的核输入(Liu J,Farmer JD Jr,Lane WS,Friedman J,Weissman I,Sch-reiberSL.Calcineurin is acommon target of cyclophilin-cyc-losporin A and FKBP-FK506complexes.Cell 1991;66:807-815.)。
CN是一种异二聚体磷酸酶,由催化亚基钙调磷酸酶A(CNA)和调节亚基钙调磷酸酶B(CNB)组成。除了催化结构域外,CNA还包含一个B亚基结合螺旋(BBH)、一个CaM结合结构域(CBD)和一个自身抑制结构域(AID),它们共同构成了A亚基的调节结构域(RD)。调节亚基CNB包含四个Ca2+结合环并与CAN的BBH紧密结合。mCNα是全长的钙调神经磷酸酶,包含上述的结构组成,命名为鼠源CNα(Li,S.J.;Wang,J.;Ma,L.;Lu,C.;Wang,J.;Wu,J.W.;Wang,Z.X.Cooperative autoinhibition and multi-level activation mechanisms ofcalcineurin[J].Cell Res.,2016,26(3):336-349.)。目前发现的配体与该蛋白结合的亲合力都不是很高,由于CN在许多生理过程中发挥关键作用,因此发现其新型配体是有必要的。
多肽逐渐成为研究蛋白质-蛋白质相互作用的一类重要分子(Craik,D.J.;Fairlie,D.P.;Liras,S.;Price,D.The future of peptide-based drugs[J].Chem.Biol.Drug Des.,2013,81(1):136-147.)。多肽分子量介于小分子和生物大分子之间,具有独特的性质,其能够与靶标的大表面积建立大量的非共价相互作用,产生更高的选择性和更好的亲合力。而多肽也有其固有的劣势,比如抵抗酶解稳定性差,口服给药时容易降解等。研究者们通过很多方法来提高多肽的稳定性,其中最直接最简单的方法就是将多肽环化。而通过多肽的位点特异性反应,借助含有反应性官能团的小分子将多肽环化是其中最直观最具多样性的方法之一。二元环肽无疑是环肽分子中更具治疗潜力的一种。Heinis等人利用三(溴甲基)苯分子修饰噬菌体展示多肽库筛选靶标蛋白的方法已经开发出多种用于临床的二元环肽药物(Heinis,C.;Rutherford,T.;Freund,S.;Winter,G.Phage-encoded combinatorial chemical libraries based on bicyclic peptides[J].Nat.Chem.Biol.,2009,5(7):502-507.)。
另一方面,噬菌体展示技术,即将多肽或者蛋白质展示于丝状噬菌体表面,是一种能够从大量多肽库中提取出所需要多肽的体外筛选技术,其作为一种基础的研究工具,已成为药物发现和开发中极其强大的手段。将噬菌体展示多肽库翻译后化学修饰,即通过设计的有机小分子与噬菌体展示多肽库反应,构建噬菌体展示环肽库。这种方法最直观最简单,且得到的环肽分子特别稳定。通过有机小分子的引入,丰富噬菌体展示多肽库的多样性,赋予其更多的结构与功能。筛选得到的环肽配体亲合力高,稳定性强。C.L.Wu等(Zheng,X.;Li,Z.;Gao,W.;Meng,X.;Li,X.;Luk,L.Y.P.;Zhao,Y.;Tsai,Y.H.;Wu,C.Condensationof2-((Alkylthio)(aryl)methylene)malononitrile with 1,2-Aminothiol as a NovelBioorthogonal Reaction for Site-Specific Protein Modification and PeptideCyclization[J].J.Am.Chem.Soc.,2020,142(11):5097-5103.)发展了1,2-氨基硫醇与2-((烷硫基)(芳基)亚甲基)丙二腈反应,该反应可以在生物相容的条件下,高效,特异性地迅速发生,经历硫醇-乙烯基硫醚交换,环化和二氰基甲烷离去的过程,最终形成稳定的五元噻唑环。将该反应用于噬菌体展示环肽库的构建,得到一系列靶标蛋白的高亲合力环肽配体。
本发明是基于噬菌体展示二元环肽库来筛选和生成针对mCNα的二元环肽配体。借助二元环肽的高亲合力与高稳定性,发展对钙调神经磷酸酶(CN)靶点的相关研究。
氨基酸包括所谓的标准或规范氨基酸。这20个α-氨基酸直接由通用遗传密码的密码子编码。它们是真核生物中发现的蛋白质α-氨基酸。
发明内容
本发明的第一目的在于提供合成简单、生物相容性好、构象稳定、抗酶解稳定性强,与靶标蛋白结合具有高特异性与高亲合力的特异性靶向mCNα的二元环肽配体。
本发明的第二目的在于提供特异性靶向mCNα的二元环肽配体的获得方法。
所述特异性靶向mCNα的二元环肽配体,由一条含有三个半胱氨酸的线性多肽链和一个小分子组成;所述小分子与线性多肽链的偶联使得原本的线性多肽链成为二元环肽;
所述多肽包括但不限于以下序列:
CIVLTAPNGRCELLDC(peptide 1)
CTGPHIIITDCTHHEC(peptide 2)
CIVLTAPNGRCELRDC(peptide 3)
CGVIILINGICDECHC(peptide 4)
GIVLTAPNGRCELLDC(peptide 5)
CRSNQEIPQVCVNGLC(peptide 6)
CAEDWRIPRICVTGEC(peptide 7)
CIVLTAPNGRCELVDC(peptide 8)
CIVLTAPTGRCELLDC(peptide 9)
CIVLTAPNGRCELLDC(peptide 10)
CNCLSYQDTNCYEYRC(peptide 11)
CIVLTAPNGRCELLEC(peptide 12)
所述特异性靶向mCNα的二元环肽配体的氨基酸序列如序列表SEQ ID NO.1~12所示。
所述特异性靶向mCNα的二元环肽配体的获得方法,包括以下步骤:
1)构建噬菌体展示二元环肽库:
(1)噬菌体展示线性多肽库的序列骨架(由N端到C端)如下:
C(X)9C(X)4C
其中,氨基酸均为L型氨基酸,X代表任意氨基酸,下标代表含有(X)的数量;所述噬菌体展示线性多肽库的库容量:≈3.93×109。
(2)将小分子2ClAc-3.5(李聪.稳定共价键约束型二元环肽库设计及筛选[D].厦门:厦门大学化学化工学院,2021)与上述噬菌体展示线性多肽库反应,确保反应温和、高效、无毒,反应前后噬菌体的数量与活性几乎不变,得到具有巨大序列空间的噬菌体展示二元环肽库。
2)将步骤1)中所得噬菌体展示二元环肽库用于mCNα蛋白配体的筛选,具体步骤为:将噬菌体展示二元环肽库针对mCNα靶点进行筛选,mCNα蛋白经过生物素化后固定在磁珠上,经过4轮筛选后,选取筛选后得到的噬菌体进行测序,得到多肽包含但不限于以下序列:
CIVLTAPNGRCELLDC(peptide 1)
CTGPHIIITDCTHHEC(peptide 2)
CIVLTAPNGRCELRDC(peptide 3)
CGVIILINGICDECHC(peptide 4)
GIVLTAPNGRCELLDC(peptide 5)
CRSNQEIPQVCVNGLC(peptide 6)
CAEDWRIPRICVTGEC(peptide 7)
CIVLTAPNGRCELVDC(peptide 8)
CIVLTAPTGRCELLDC(peptide 9)
CIVLTAPNGRCELLDC(peptide 10)
CNCLSYQDTNCYEYRC(peptide 11)
CIVLTAPNGRCELLEC(peptide 12)
其中,G为甘氨酸;P为脯氨酸;A为丙氨酸;V为缬氨酸;L为亮氨酸;I为异亮氨酸;M为甲硫氨酸;C为半胱氨酸;F为苯丙氨酸;Y为酪氨酸;W为色氨酸;H为组氨酸;K为赖氨酸;R为精氨酸;Q为谷氨酰胺;N为天冬酰胺;E为谷氨酸;D为天冬氨酸;S为丝氨酸;T为苏氨酸。
3)选取步骤2)中富集最多的多肽peptide 1进行合成,然后与小分子2ClAc-3.5反应得到二元环肽配体分子,表征二元环肽配体分子与mCNα的亲合力(荧光偏振实验)。
本发明借助噬菌体展示技术这一有力工具,通过构建噬菌体展示二元环肽库,从巨大的多肽序列空间中筛选得到靶标蛋白mCNα的二元环肽配体。该多肽配体对后续mCNα靶点以及钙调神经磷酸酶(CN)的研究是具有重要意义的。
与现有技术相比,本发明具有以下的效益和优势:
a.本发明所得到的二元环肽配体为小环肽,只需要常规的固相合成即可得到,合成简单。
b.本发明所涉及的都是天然氨基酸,成本低,且能很好地适合于生物体。
c.线性多肽配体存在易被酶解,构象不稳定,寿命短等问题。本发明所述二元环肽却能很好地解决这些问题,通过环化对多肽施加构象限制,提高多肽的稳定性。另外,环肽的构象熵通常低于其线性对应物,其与靶标结合时采用特定构象,熵损失较小,与线性肽相比,这会产生更高的结合亲合力。
d.钙调神经磷酸酶(CN)在许多生理过程中发挥着关键作用,目前已知的多肽配体与该类蛋白结合的亲合力都不是很高,因此发现其新型的高亲合力配体是有必要的。本发明得到的二元环肽配体,如peptide 1的环化产物,通过荧光偏振表征亲合力,KD值为35.64nM,与mCNα靶点结合的亲合力很高。
附图说明
图1为噬菌体展示二元环肽库筛选mCNα的高通量测序结果中富集数大于100条的多肽序列。
图2为peptide 1的质谱图。
图3为peptide 2的质谱图。
图4为小分子2ClAc-3.5的结构式。
图5为小分子2ClAc-3.5的质谱图。
图6为peptide 1与小分子2ClAc-3.5反应得到二元环肽配体的色谱图。
图7为peptide 1与小分子2ClAc-3.5反应得到的二元环肽配体的结构示意图。
图8为peptide 1与小分子2ClAc-3.5反应得到的二元环肽配体的质谱图。
图9为peptide 2与小分子2ClAc-3.5反应得到二元环肽配体的色谱图。
图10为peptide 2与小分子2ClAc-3.5反应得到的二元环肽配体的质谱图。
图11为荧光偏振实验中peptide 1的还原型荧光肽的结构示意图。
图12为荧光偏振实验中peptide 1的还原型荧光肽的质谱图。
图13为荧光偏振实验中peptide 1的二元环肽荧光肽的结构示意图。
图14为荧光偏振实验中peptide 1的二元环肽荧光肽的质谱图。
图15为peptide 1的二元环肽荧光肽与mCNα结合的荧光偏振饱和曲线。
具体实施方式
以下实施将结合附图对本发明作进一步的说明。
本发明包括以下步骤:
1)噬菌体展示二元环肽库用于mCNα蛋白配体的筛选。
2)选取筛选后得到的噬菌体进行测序,将测序得到的多肽合成。
3)表征得到的二元环肽配体与mCNα的亲合力。
所述特异性靶向mCNα的二元环肽配体,由一条含有三个半胱氨酸的线性多肽链和一个小分子组成;所述小分子与线性多肽链的偶联使得原本的线性多肽链成为二元环肽;所述二元环肽配体的结构示意图见图7。
所述多肽包括但不限于以下序列:
CIVLTAPNGRCELLDC(peptide 1)
CTGPHIIITDCTHHEC(peptide 2)
CIVLTAPNGRCELRDC(peptide 3)
CGVIILINGICDECHC(peptide 4)
GIVLTAPNGRCELLDC(peptide 5)
CRSNQEIPQVCVNGLC(peptide 6)
CAEDWRIPRICVTGEC(peptide 7)
CIVLTAPNGRCELVDC(peptide 8)
CIVLTAPTGRCELLDC(peptide 9)
CIVLTAPNGRCELLDC(peptide 10)
CNCLSYQDTNCYEYRC(peptide 11)
CIVLTAPNGRCELLEC(peptide 12)
所述特异性靶向mCNα的二元环肽配体的氨基酸序列如序列表SEQ ID NO.1~12所示。
所述特异性靶向mCNα的二元环肽配体的获得方法,包括以下步骤:
1)构建噬菌体展示二元环肽库:
(1)噬菌体展示线性多肽库的序列骨架(由N端到C端)如下:
C(X)9C(X)4C
其中,氨基酸均为L型氨基酸,X代表任意氨基酸,下标代表含有(X)的数量;所述噬菌体展示线性多肽库的库容量:≈3.93×109。
(2)将一个小分子,命名是2ClAc-3.5,结构式见图4,(李聪.稳定共价键约束型二元环肽库设计及筛选[D].厦门:厦门大学化学化工学院,2021.)与上述噬菌体展示线性多肽库反应,确保反应温和、高效、无毒,反应前后噬菌体的数量与活性几乎不变,得到具有巨大序列空间的噬菌体展示二元环肽库。
2)将步骤1)中所得噬菌体展示二元环肽库用于mCNα蛋白配体的筛选,具体步骤为:将噬菌体展示二元环肽库针对mCNα靶点进行筛选,mCNα蛋白经过生物素化后固定在磁珠上,经过4轮筛选后,选取筛选后得到的噬菌体进行测序,得到多肽包含但不限于以下序列:
CIVLTAPNGRCELLDC(peptide 1)
CTGPHIIITDCTHHEC(peptide 2)
CIVLTAPNGRCELRDC(peptide 3)
CGVIILINGICDECHC(peptide 4)
GIVLTAPNGRCELLDC(peptide 5)
CRSNQEIPQVCVNGLC(peptide 6)
CAEDWRIPRICVTGEC(peptide 7)
CIVLTAPNGRCELVDC(peptide 8)
CIVLTAPTGRCELLDC(peptide 9)
CIVLTAPNGRCELLDC(peptide 10)
CNCLSYQDTNCYEYRC(peptide 11)
CIVLTAPNGRCELLEC(peptide 12)
其中,G为甘氨酸;P为脯氨酸;A为丙氨酸;V为缬氨酸;L为亮氨酸;I为异亮氨酸;M为甲硫氨酸;C为半胱氨酸;F为苯丙氨酸;Y为酪氨酸;W为色氨酸;H为组氨酸;K为赖氨酸;R为精氨酸;Q为谷氨酰胺;N为天冬酰胺;E为谷氨酸;D为天冬氨酸;S为丝氨酸;T为苏氨酸。
3)选取步骤2)中富集最多的多肽peptide 1进行合成,然后与小分子2ClAc-3.5反应得到二元环肽配体分子,表征二元环肽配体分子与mCNα的亲合力(荧光偏振实验)。
以下给出具体实施例:
实施例1
利用噬菌体展示二元环肽库对mCNα蛋白进行4轮筛选富集。取50μL链酶亲和素包被磁珠于一1.5mL离心管中(筛选过程所用离心管均为低吸附离心管,第一,三轮筛选所用磁珠是链酶亲和素包被磁珠,每轮取50μL。第二,四轮筛选所用磁珠是中性亲和素包被磁珠,每次用100μL,第二,四轮筛选过程需全程避光),用1mL的1×PBS洗涤三次,弃去上清。用50μL的1×PBS重悬磁珠,均匀地平分到两个离心管中。向其中一管中加入生物素化的靶标蛋白溶液,此为实验组;另一管中加入等体积的1×PBS,此为对照组。混匀后放于摇床上室温孵育1h。将两离心管置于磁力架上,静置1min,弃去上清液。分别用1mL的1×PBS洗涤两管中的磁珠三次,弃去上清。向两管中分别加入1mL的封闭缓冲液,混匀后放于摇床上封闭2h。在磁珠与靶标蛋白孵育的时候,将用于筛选的噬菌体展示多肽库从4℃冰箱中取出,全部加入到10mL的无菌离心管中,再加入2mL的封闭缓冲液,混匀后放于摇床上同样封闭2h。将封闭好的噬菌体溶液平分到两个10mL无菌离心管中,分别记为实验组和对照组。将实验组的磁珠加入到实验组的噬菌体溶液中,对照组的磁珠加入到对照组的噬菌体溶液中。混匀后放于摇床上结合30min。将两个离心管置于磁力架上,静置1min,吸去上清。分别用1mL的洗涤缓冲液洗涤两管中的磁珠9次,弃去上清液,最后再分别用1mL的1×PBS洗涤两管中的磁珠两次,弃去上清液。注意整个洗涤过程中应至少更换3次离心管,以降低管子对磁珠的非特异性吸附。洗涤完毕后,在两管中分别加入150μL的洗脱缓冲液,混匀后放于摇床上洗脱5min。之后分别向两管中加入25μL的中和缓冲液,混匀后将两管置于磁力架上,静置1min,将上清分别转移至干净的无菌离心管中,即得到实验组和对照组洗脱下来的噬菌体溶液。重复洗脱操作一次,收集洗脱下来的噬菌体溶液。暂放于4℃冰箱,用于滴度测定和涂大板操作。经过4轮筛选后,提取筛选结果噬菌体DNA进行测序并分析多肽序列富集度。测序结果如图1。
实施例2
多肽的合成。将基因测序得到的多肽序列peptide 1,peptide 2通过多肽合成仪合成。在0.152g的Rink amide MBHA树脂上固相合成0.025mmol的多肽。脱保护分两步进行,初始脱保护时间为30s,然后在50W下脱保护3min,最高温度为80℃。将5倍过量的Fmoc保护的氨基酸溶解在N,N-二甲基甲酰胺中,通过活化剂活化来进行偶联反应。偶联反应条件为40W,5min,最高温度为80℃。偶联上一个氨基酸后,用20%的哌啶脱除Fmoc保护基,然后进行下一个氨基酸的偶联。将多肽序列中所有的氨基酸都偶联上后,用N,N-二甲基甲酰胺(3×5mL)洗涤,最后用二氯甲烷(5mL)洗涤,烘干后可得到干燥的多肽树脂。多肽树脂经切割,纯化后得到多肽固体粉末。peptide 1与peptide 2的质谱图分别见图2,图3。
实施例3
多肽与小分子2ClAc-3.5反应得到二元环肽分子。小分子2ClAc-3.5的结构式,质谱图分别见图4,图5。反应溶剂用5×PBS(pH=6.0),总反应体积500μL。首先向反应体系中加入多肽peptide 1的母液(0.2μmol,1.0equiv.),TCEP母液(0.6μmol,3equiv.)。反应30min后,向反应体系中加入2ClAc-3.5小分子母液(0.2μmol,1.0equiv.),N-乙酰-L-半胱氨酸母液(0.6μmol,3equiv.)。反应1h后,用1M的氢氧化钠调节体系的pH至7.4,继续反应过夜。反应完毕后,二元环肽产物的色谱图,结构示意图,质谱图分别见图6~8。
Peptide 2与小分子2ClAc-3.5的环化反应条件与多肽peptide 1的一致,其对应的生成二元环肽的色谱图,质谱图分别如图9和10所示。可见,本发明涉及的二元环肽分子合成过程十分简便,反应效率很高。
实施例4
荧光偏振实验饱和实验测定多肽配体与mCNα结合的亲合力。测定了peptide 1对应的二元环肽的饱和曲线。为了在多肽中引入荧光素FITC,在peptide 1的C端依次偶联A、G、K三个氨基酸(A、G两个氨基酸结构简单,没有多余的活性基团,起到间隔作用,侧链带有Mtt保护基的K是为了提供与FITC偶联的反应位点)。首先用多肽合成仪合成线性多肽链,多肽N端为Fmoc保护,C端K的侧链为Mtt保护。然后用1%的三氟乙酸脱除Mtt保护,使K的侧链氨基裸露,可进行FITC的偶联。接着进行N端脱除Fmoc保护和多肽的切割,纯化,可得到peptide 1的还原型荧光肽,其结构示意图,质谱图分别如图11和12所示。最后,采用与实施例3中相似的反应条件,将还原型荧光肽与小分子2ClAc-3.5反应得到peptide 1的二元环肽荧光肽,其结构示意图,质谱图分别如图13和14所示。
在荧光偏振饱和实验中,将二元环肽的荧光肽用10mM PBS稀释至终浓度为30nM,mCNα蛋白逐级稀释浓度范围为50-1000nM。室温下二者共孵育10min后将每个样品溶液分别加入96孔黑色酶标板中再进行数据读取。使用公式(1)对蛋白质浓度与各向异性的非线性回归分析解离常数(KD=35.64nM),图15为peptide 1的二元环肽对mCNα的荧光偏振饱和曲线:
其中,A1和A2分别是拟合曲线上的最低点和最高点对应的各向异性值,c为荧光肽的浓度,x为蛋白浓度,y为测得的荧光各向异性平均值,KD为解离常数。
综上所见,本发明是关于利用噬菌体展示二元环肽库,对mCNα进行筛选,获得了能与其高亲合力结合的二元环肽配体。本发明得到的特异性靶向mCNα的二元环肽配体,合成简单,生物相容性好。小分子构建的二元环肽结构刚性稳定,抗酶解稳定性强,极大地克服多肽类分子的固有缺陷。并且,它能与mCNα特异性结合,其亲合力(KD=35.64nM)远高于目前报道的mCNα的多肽配体,这对后续钙调神经磷酸酶(CN)参与的各种重要生理活动的研究具有重要意义。
序列表
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Claims (4)
1.特异性靶向mCNα的二元环肽配体,其特征在于其由一条含有三个半胱氨酸的线性多肽链和一个小分子组成;所述小分子与线性多肽链的偶联使得原本的线性多肽链成为二元环肽;
所述多肽包括但不限于以下序列:
CIVLTAPNGRCELLDC(peptide 1)
CTGPHIIITDCTHHEC(peptide 2)
CIVLTAPNGRCELRDC(peptide 3)
CGVIILINGICDECHC(peptide 4)
GIVLTAPNGRCELLDC(peptide 5)
CRSNQEIPQVCVNGLC(peptide 6)
CAEDWRIPRICVTGEC(peptide 7)
CIVLTAPNGRCELVDC(peptide 8)
CIVLTAPTGRCELLDC(peptide 9)
CIVLTAPNGRCELLDC(peptide 10)
CNCLSYQDTNCYEYRC(peptide 11)
CIVLTAPNGRCELLEC(peptide 12)
所述特异性靶向mCNα的二元环肽配体的氨基酸序列如序列表SEQ ID NO.1~12所示。
2.如权利要求1所述特异性靶向mCNα的二元环肽配体的获得方法,其特征在于包括以下步骤:
1)基于噬菌体展示技术构建噬菌体展示二元环肽库;
2)将步骤1)中所得噬菌体展示二元环肽库用于mCNα蛋白配体的筛选;
3)选取步骤2)中富集最多的多肽进行合成,与小分子2ClAc-3.5反应得到二元环肽配体分子,表征二元环肽配体分子与mCNα的亲合力。
3.如权利要求1所述特异性靶向mCNα的二元环肽配体的获得方法,其特征在于在步骤1)中,所述构建噬菌体展示二元环肽库的具体方法为:
(1)噬菌体展示线性多肽库的序列骨架由N端到C端如下:
C(X)9C(X)4C
其中,氨基酸均为L型氨基酸,X代表任意氨基酸,下标代表含有(X)的数量;所述噬菌体展示线性多肽库的库容量:≈3.93×109;
(2)将小分子2ClAc-3.5与上述噬菌体展示线性多肽库反应,确保反应温和、高效、无毒,反应前后噬菌体的数量与活性几乎不变,得到具有巨大序列空间的噬菌体展示二元环肽库。
4.如权利要求1所述特异性靶向mCNα的二元环肽配体的获得方法,其特征在于在步骤2)中,所述将步骤1)中所得噬菌体展示二元环肽库用于mCNα蛋白配体的筛选,具体步骤为:将噬菌体展示二元环肽库针对mCNα靶点进行筛选,mCNα蛋白经过生物素化后固定在磁珠上,经过4轮筛选后,选取筛选后得到的噬菌体进行测序,得到多肽包含但不限于以下序列:
CIVLTAPNGRCELLDC(peptide 1)
CTGPHIIITDCTHHEC(peptide 2)
CIVLTAPNGRCELRDC(peptide 3)
CGVIILINGICDECHC(peptide 4)
GIVLTAPNGRCELLDC(peptide 5)
CRSNQEIPQVCVNGLC(peptide 6)
CAEDWRIPRICVTGEC(peptide 7)
CIVLTAPNGRCELVDC(peptide 8)
CIVLTAPTGRCELLDC(peptide 9)
CIVLTAPNGRCELLDC(peptide 10)
CNCLSYQDTNCYEYRC(peptide 11)
CIVLTAPNGRCELLEC(peptide 12)
其中,G为甘氨酸;P为脯氨酸;A为丙氨酸;V为缬氨酸;L为亮氨酸;I为异亮氨酸;M为甲硫氨酸;C为半胱氨酸;F为苯丙氨酸;Y为酪氨酸;W为色氨酸;H为组氨酸;K为赖氨酸;R为精氨酸;Q为谷氨酰胺;N为天冬酰胺;E为谷氨酸;D为天冬氨酸;S为丝氨酸;T为苏氨酸。
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