穿膜肽-LacI HPM载体的构建及其DNA结合活性测定

目的：借助穿膜肽TAT高效跨膜的特性和LacI前头肽突变体（LacI HPM）高亲和力结合DNA的特性,构建新型基因转导载体。方法：PCR扩增LacI、Lacl基因前头肽序列、前头肽序列突变体、TAT序列的编码基因,构建前头肽序列突变体和TAT的原核表达载体,可溶性表达TAT-LacI HPM融合蛋白并纯化,在缇冲液中氧化获得TAT-LacI HPM二聚体并浓缩,PCR检测二聚体融合蛋白与质粒的体外结合能力。结果：获得了pET-28a（-4-）-LacI HPM及pET-28a（＋）-TAT-LacI HPM表达质粒,表达纯化并获得二聚化融合蛋白,体外结合实验确定TAT-Lac／HPM二聚体融合蛋白与检测质粒DNA具有特异的高亲和力结合活性。结论：构建了穿膜呔TAT-LacI HPM,为进一步研究其作为新型DNA转运载体的可行性奠定了基础。     

细胞穿膜肽的穿膜活性与序列特征的关系

细胞穿膜肽(cell penetrating peptides,CPPs)是一种小分子多肽,能够容易地穿过细胞膜.这类分子,尤其是具有靶向功能的CPPs为高效率投送药物到靶细胞带来希望.因此,对其展开研究对于生物医学有着一定的意义.本工作主要从序列水平对具有不同穿膜活性的CPPs进行研究,试图找出影响CPPs穿膜活性的因素,以及不同活性CPPs与非穿膜肽(Non CPPs)序列上的差异,并引入一种分析生物序列的方法.我们基于CPPsite数据库和不同的文献获取CPPs和Non CPPs序列,并进一步从CPPs序列中提取具有高、中、低穿膜活性的穿膜肽(HCPPs、MCPPs、LCPPs)用于构建数据集.基于这些数据集,开展了以下研究:首先,利用方差分析的方法,对不同活性的CPPs以及Non CPPs的氨基酸及二级结构组成进行分析,发现氨基酸的静电与疏水相互作用对CPPs的穿膜活性起到了重要影响,同时螺旋结构和无规卷曲也会影响CPPs的穿膜活性;其次,使用理化性质与长度将不同活性的CPPs展示在二维平面上,发现在某些特殊的性质下不同活性的CPPs与Non CPPs可以产生聚簇现象,HCPPs、MCPPs以及LCPPs和Non CPPs被分成了三簇,这种现象显示了它们之间的差异;最后,本文引入了生物序列理化质心的概念,将组成序列的残基看作质点,进而把序列抽象成质点系进行研究,并将此方法应用到CPPs的分析中,通过PCA方法将不同活性的CPPs投射到三维平面上,结果发现绝大部分CPPs聚在一起,部分LCPPs与Non CPPs聚在一起.此工作对于CPPs的设计,以及理解不同活性CPPs序列上的差异具有一定的意义.另外,本文引入的生物序列理化质心的分析方法也可以用于其他生物问题的分析,同时它们可以作为某些生物分类问题的输入参数,在模式识别中起到一定的作用.     

穿膜肽的结构特点和穿膜机制

穿膜肽（penetratin）是果蝇的触角足同源异形域的DNA结合结构域第三个片段的商品名,由16个氨基酸残基组成,它可以介导多种疏水大分子进入活体细胞质内而不破坏细胞膜的完整性;其最大的特点是可以介导多种大分子进入细胞内,并且无需外源能量,分子作为整体插入细胞内,穿膜过程不需解折叠。该发现开辟了药物进入细胞的新的介导途径。该文介绍穿膜肽的结构特点、穿膜机制、应用及局限性。     

穿膜肽的内化机制及其应用

穿膜肽是一类具有特殊穿膜功能的多肽分子,能携带其它分子甚至超分子颗粒穿膜进入细胞内部．早期研究认为,其进胞是一种无需受体、也不存在饱和状态的非经典胞吞行为．近年研究表明,其穿膜机制可能与其含有的氨基酸种类有很大关系．现在,穿膜肽的穿膜过程称为巨型胞饮行为,它与传统的胞吞形式很相似．当然,还可能存在着其它的进胞方式而没有被证明或发现．关于穿膜肽的应用也是人们最感兴趣的,在很多领域的研究都在进行并不断取得进展．不论是生物界还是医学界,穿膜肽都被认为将是一类非常有发展潜力的多肽分子．     

膜穿透肽的应用与穿膜机制研究

膜穿透肽(membrane penetrating peptide,MPP)能引导大分子物质穿透细胞膜.应用MPP为载体,引导神经营养分子通透血脑屏障进入神经元,能有效治疗中枢神经系统疾病;在基因治疗方面, MPP引导干扰小RNA进行基因治疗,避免了使用病毒载体等一些传统基因治疗方法的毒副作用.穿膜机制研究证实 MPP通透细胞膜的过程分为三个阶段:与细胞表面结合;细胞巨胞饮摄取 MPP;MPP从胞饮体中逃逸入胞质,其中最后阶段是限速步骤.随着对多肽片段的深入研究和穿膜机制的逐渐明晰,MPP的应用将会更为深入和广泛.     

穿膜肽的研究现状及应用

以细胞内物质为靶标的药物(大分子、蛋白质、多肽及核酸)只有穿透细胞膜才能进一步发挥其药效。细胞穿透多肽(穿膜肽)是由少于30个氨基酸残基组成的小肽,它们能够通过与细胞膜相互作用而穿透细胞膜这一天然屏障。穿膜肽大致分为宿主防御肽、基于信号序列的穿膜肽和富含精氨酸的穿膜肽;穿膜肽进入细胞的机制尚未完全阐明,存在倒置微团模型、地毯式模型及打孔模型等假说。穿膜肽能够携带各种物质进入细胞的特性受到人们的关注。我们就穿膜肽的种类、穿膜机制,及其在生物影像学和生物递送系统中的应用做一综述。     

细胞穿膜肽的研究进展

细胞穿膜肽是一类能携带大分子物质进入细胞的短肽,其穿膜能力不依赖经典的胞吞作用。经过对天然存在的细胞穿膜肽的生物化学性质研究,已经逐渐掌握了细胞穿膜肽的一些共有特性,这类物质均为带有正电荷的长短不等的多肽片段,其中富含精氨酸、赖氨酸等碱性氨基酸残基,二级结构皆具有α-螺旋的空间构象。利用这些特性,目前已人工合成了穿透力更强、效率更高的穿膜肽PEP-1、MPG,并且成功地携带大分子物质进入细胞发挥生物学活性。     

穿膜肽介导的新型基因转运载体的功能研究

目的：借助穿膜肽TAT高效跨膜的特性和LacI前头肽突变体（LacI HPM）高亲和力结合DNA的特性,建立-种安全高效、无基因插入片段大小限制的基因转导系统。方法：在TAT-LacI HPM片段C端和N端分别添加GST标签,构建pET-28a（＋）-TAT-LacI HPM-GST和pGEX-GST-TAT-LacI HPM重组表达载体,可溶性表达TAT-LacI HPM-GST及GST-TAT-LacI HPM融合蛋白并纯化,获得TAT-LacI HPM二聚体,免疫荧光检测TAT-LacI HPM融合蛋白穿过HeLa细胞膜的情况,观察EGFP的表达,用免疫印迹检测TAT-LacI HPM融合蛋白介导质粒DNA进入细胞的能力。结果：表达、纯化并获得二聚体融合蛋白,体内实验表明其具有跨膜能力,能介导带有LacI结合序列的DNA质粒进入细胞,并在转染细胞里检测到了目的蛋白。结论：初步证实TAT-LacI HPM融合蛋白作为-种新型通用性非病毒DNA转运载体的可行性,为评价这种新型DNA疫苗载体在提高免疫效果方面的可行性奠定了前期实验基础。     

穿膜肽TAT和R9的生物学效应的体内体外研究

本文比较了两种常用穿膜肽TAT和R9的体外穿膜效率、对细胞影响以及小鼠活体内不同器官的穿膜效率。非特异性穿膜肽TAT和R9分别为11个氨基酸和9个氨基酸的短肽,能够有效地穿过细胞膜进入细胞。本研究纯化制备出融合蛋白TAT-EGFP及R9-EGFP多肽,用相同浓度的活性TAT-EGFP和R9-EGFP处理肺癌细胞,观察比较二者的穿膜活性及效率;设置不同浓度梯度检测两种穿膜肽是否对细胞产生影响;通过小鼠腹腔注射TAT-EGFP和R9-EGFP活性肽段,研究二者在活体能够穿膜到达的靶位置,并比较分析二者穿膜的效果器官分布。结果显示制备获得的两种融合蛋白TAT-EGFP和R9-EGFP均能有效穿过实现穿膜功能,两种穿膜肽对乳腺癌细胞影响较小。活体试验证明两种穿膜肽在心、肝、脾、肺、肾器官均有分布,R9主要富集在小鼠的肾和肝脏中,TAT在6种被研究的器官都有富集,两种细胞穿膜肽都能透过血脑屏障到达脑部。相对来说,TAT在小鼠活体中的穿膜效果更好,荧光富集多,R9整体穿膜效果较弱。本研究分析比较了两种穿膜肽的细胞安全性浓度和活体的穿膜能力,为TAT和R9两种穿膜肽在后续试验中的合理有效的选择和使用,提供了基础数据和指导性依据。     

细胞穿膜肽在肿瘤靶向治疗及疾病诊断中的应用

近年来细胞穿膜肽(cell-penetrating peptides,CPP)在生物医药领域被广泛应用,它为生物分子的胞内递送提供了有效的策略。关注CPP在肿瘤治疗及疾病诊断中的作用,并重点介绍其在肿瘤靶向治疗和医学影像诊断中的应用及优势。同时,根据CPP在药物传递系统中的特点,改进CPP存在的不足,扩大其联合用药的可能性,这也成为CPP研究的热点。对CPP及其在肿瘤等疾病的诊断及治疗中的应用作一综述,并简述其优化及改进策略,以期促进CPP在临床中的应用。     

抗菌肽P7抑制大肠杆菌的非膜作用机制

【目的】研究抗菌肽P7抑制大肠杆菌的非膜作用机制。【方法】P7与溴化乙锭竞争结合大肠杆菌基因组DNA的荧光光谱,分析P7与DNA的结合方式;流式细胞术分析P7与大肠杆菌基因组DNA结合对细菌细胞周期的影响;采用磁珠富集和PCR扩增相结合的方法分析P7特异结合的DNA序列;通过实时荧光定量PCR分析P7对大肠杆菌DNA复制和SOS损伤修复基因表达的影响;核酸染料的荧光分析研究P7对大肠杆菌DNA和RNA合成的影响。【结果】P7以嵌插的方式作用于大肠杆菌基因组DNA碱基对并形成肽-DNA复合物,使溴化乙锭-DNA复合体系的荧光强度减弱。P7可以显著增加大肠杆菌细胞周期中S期细胞数目,抑制大肠杆菌DNA复制。P7特异性结合rnh A使该基因表达水平显著下调2.24倍。同时,在肽的影响下参与大肠杆菌DNA复制相关的ssb、dna G、lig B和rnh A基因的表达水平显著下调(P<0.05),DNA损伤修复的rec A和rec N基因显著上调(P<0.05)。P7可降低大肠杆菌DNA和RNA的合成。【结论】P7特异性地结合rnh A序列引起大肠杆菌DNA的损伤并抑制大肠杆菌的DNA复制。在P7的影响下,参与大肠杆菌DNA复制相关的基因的表达水平下调,DNA损伤修复基因显著上调,同时抑制大肠杆菌DNA和RNA的合成。     

Cellular uptake of Aib-containing amphipathic helix peptide

Cell-penetrating peptides (CPPs) are useful tools for the delivery of hydrophilic bioactive molecules, such as peptides, proteins, and oligonucleotides, across the cell membrane. To realize the delivery of therapeutic macromolecules by CPPs, the CPPs are required to show resistance to protease and no cytotoxicity. In order to produce potent non-toxic and protease-resistant CPPs with high cellular uptake, we designed an amphipathic helix peptide using α-aminoisobutyric acid (Aib, U) and named it MAP(Aib). In the MAP(Aib) molecule, five Aib residues are aligned on the hydrophobic face of the helix and five lysine (K) residues are aligned on the hydrophilic face. MAP(Aib) showed potent resistance to trypsin and pronase compared with MAP, an amphipathic helix peptide formed by usual amino acids. Fluorescein-labeled MAP(Aib) efficiently traversed the A549 cell membrane, diffusing into the cytoplasm and slightly into the nucleus without exerting any cytotoxicity. In contrast, MAP was poorly taken up by the cell. These results indicate that the incorporation of Aib residues into CPPs markedly improves cellular uptake and MAP(Aib) may be a useful tool for the delivery of hydrophilic macromolecules.     

Design of a Tumor Homing Cell-Penetrating Peptide for Drug Delivery

The major drawbacks with conventional cancer chemotherapy are the lack of satisfactory specificity towards tumor cells and poor antitumor activity. In order to improve these characteristics, chemotherapeutic drugs can be conjugated to targeting moieties e.g. to peptides with the ability to recognize cancer cells. We have previously reported that combining a tumor homing peptide with a cell-penetrating peptide yields a chimeric peptide with tumor cell specificity that can carry cargo molecules inside the cells. In the present study, we have used a linear breast tumor homing peptide, CREKA, in conjunction with a cell-penetrating peptide, pVEC. This new chimeric peptide, CREKA–pVEC, is more convenient to synthesize and moreover it is better in translocating cargo molecules inside cancer cells as compared to previously published PEGA–pVEC peptide. This study demonstrates that CREKA–pVEC is a suitable vehicle for targeted intracellular delivery of a DNA alkylating agent, chlorambucil, as the chlorambucil–peptide conjugate was substantially better at killing cancer cells in vitro than the anticancer drug alone.     

Antisense effect of pyrrolidine-based oxy-peptide nucleic acids in Escherichia coli

To investigate the antisense effect of a pyrrolidine-based oxy-peptide nucleic acid (POPNA), we carried out the LacZ reporter assay using a 12-mer trans-l-POPNA conjugated with a cell-penetrating peptide (antisense reagent). The antisense effect of the conjugated POPNA (inhibition of LacZ activity) was comparable to that shown by a Nielsen-type peptide nucleic acid. Furthermore, the conjugated POPNA could switch the LacZ activity over a wide range of ambient temperatures.     

Simulation study of interaction mechanism between peptide and asymmetric membrane

Cell-penetrating peptides (CPPs) are widely used as drug carriers, owing to their superior ability to cross cell membrane both alone and with cargos, such as genes and other particles. Understanding the translocation mechanism of CPP is significant for many therapeutic purposes, including targeting drug and gene delivery. In this study, we performed a coarse-grained molecular dynamics simulation to investigate the interaction mechanism between polyarginine peptides and asymmetric membranes. Results showed that peptides can penetrate through the lipid bilayer by inducing a hydrophilic hole formation in the asymmetric membrane. Furthermore, the lengthy peptide chain length (R4–R16 peptides) and high membrane asymmetry positively affect peptide penetration. Our study provides insights into the molecular-level interactions between peptides and asymmetric membranes, as well as suggestions for targeted gene and drug delivery.     

Mechanistic aspects of CPP-mediated intracellular drug delivery: Relevance of CPP self-assembly

In recent years, cell-penetrating peptides have proven to be an efficient intracellular delivery system. The mechanism for CPP internalisation, which first involves interaction with the extracellular matrix, is followed in most cases by endocytosis and finally, depending on the type of endocytosis, an intracellular fate is reached. Delivery of cargo attached to a CPP requires endosomal release, for which different methods have recently been proposed. Positively charged amino acids, hydrophobicity and/or amphipathicity are common to CPPs. Moreover, some CPPs can self-assemble. Herein is discussed the role of self assembly in the cellular uptake of CPPs. Sweet Arrow Peptide (SAP) CPP has been shown to aggregate by CD and TEM (freeze-fixation/freeze-drying), although the internalised species have yet to be identified as either the monomer or an aggregate.     

On the mechanism of non-endosomial peptide-mediated cellular delivery of nucleic acids

Recently, we described a new strategy for the delivery of nucleic acids into mammalian cells, based on an amphipathic peptide of 27 residues called MPG, which was designed on the basis of a hydrophobic domain derived from a fusion sequence associated with a nuclear localization sequence and separated by a linker. This peptide carrier constitutes a powerful tool for the delivery of nucleic acids in cultured cells, without requiring any covalent coupling. We have examined the conformational states of MPG in its free form and complexed with a cargo, as well as its ability to interact with phospholipids, and have investigated the structural consequences of these interactions. In spite of its similarity to the similarly designed cell-penetrating peptide Pep-1, MPG behaves significantly differently from the conformational point of view. Circular dichroism (CD) analysis reveals a transition from a nonstructured to a β-sheet conformation upon interaction with phospholipids. We propose that the membrane crossing process involves formation of a transient transmembrane pore-like structure. Partial conformational change of MPG is associated with formation of a complex with its cargo, and an increase in sheet content occurs upon association with the cell membrane.     

Significance of C-terminal sequence elements for Petunia flavanone 3beta-hydroxylase activity

The time-specific requirement of Rho proteins for the S phase progression of vascular endothelial cells was determined by reversibly introducing inhibitor proteins with a cell-penetrating peptide. We found evidence of the reversibility of protein transduction. The removal of extracellular protein caused the transduced protein to decay in a manner sensitive to low temperatures. The time required for a 50% decay correlated with the protein size. The time-specific transduction of the inhibitor proteins uncovered a critical period requiring Rho proteins in the G₁–S transition phase. Reversible protein transduction may thus be a powerful tool to investigate the time-specific role of signaling proteins.     

Mechanism of action and specificity of antimicrobial peptides designed based on buforin IIb

Buforin IIb-a synthetic analog of buforin II that contains a proline hinge between the two α-helices and a model α-helical sequence at the C-terminus (3× RLLR)-is a potent cell-penetrating antimicrobial peptide. To develop novel antimicrobial peptides with enhanced activities and specificity/therapeutic index, we designed several analogs (Buf III analogs) by substitutions of amino acids in the proline hinge region and two α-helices of buforin IIb, and examined their antimicrobial activity and mechanism of action. The substitution of hydrophobic residues ([F(6)] and [V(8)]) in the proline hinge region with other hydrophobic residues ([W(6)] and [I(8)]) did not affect antimicrobial activity, while the substitution of the first four amino acids RAGL with a model α-helical sequence increased the antimicrobial activity up to 2-fold. Like buforin IIb, Buf III analogs penetrated the bacterial cell membranes without significantly permeabilizing them and were accumulated inside Escherichia coli. Buf III analogs were shown to bind DNA in vitro and the DNA binding affinity of the peptides correlated linearly with their antimicrobial potency. Among the Buf III analogs, the therapeutic index of Buf IIIb and IIIc (RVVRQWPIG[RVVR](3) and KLLKQWPIG[KLLK](3), respectively) were improved 7-fold compared to that of buforin IIb. These results indicate that Buf III analogs appear to be promising candidates for future development as novel antimicrobial agents.