Ni2+高效结合肽的筛选与作用研究

利用噬菌体随机十二肽库和金属亲和层析对重金属Ni2 进行结合肽筛选。经4轮生物淘洗、噬菌体扩增和DNA测序,获得一组多肽序列。GenBank Blast分析未发现同源序列,Clustal W多重序列比对也未找到Ni2 金属结合肽结合基序,但可能含有多聚组氨酸(His)2-5。噬菌体单克隆金属离子螯合树脂的亲和力测定和反筛、抑菌解毒试验表明:展示有金属结合肽的噬菌体不仅对Ni2 具有高亲和力,而且对其它金属离子也有作用,Cu2 、Ni2 、Co2 、Zn2 等金属离子对金属结合肽的亲和力显著高于Cd2 和Cr2 ,展示金属结合肽的噬菌体对重金属Ni2 具有一定的耐受和解毒作用。显微形态学观察也显示金属结合肽与金属螯合树脂的作用。对于了解重金属与多肽的相互作用机理以及环境重金属修复等均具有重要意义和价值。     

Ni2+高效结合肽的筛选与作用研究

利用噬菌体随机十二肽库和金属亲和层析对重金属Ni2+进行结合肽筛选。经4轮生物淘洗、噬菌体扩增和DNA测序,获得一组多肽序列。GenBank Blast分析未发现同源序列,Clustal W多重序列比对也未找到Ni2+金属结合肽结合基序,但可能含有多聚组氨酸（His）2－5。噬菌体单克隆金属离子螯合树脂的亲和力测定和反筛、抑菌解毒试验表明:展示有金属结合肽的噬菌体不仅对Ni2+具有高亲和力,而且对其它金属离子也有作用,Cu2+、Ni2+、Co2+、Zn2+等金属离子对金属结合肽的亲和力显著高于Cd2＋和Cr2＋,展示金属结合肽的噬菌体对重金属Ni2+具有一定的耐受和解毒作用。显微形态学观察也显示金属结合肽与金属螯合树脂的作用。对于了解重金属与多肽的相互作用机理以及环境重金属修复等均具有重要意义和价值。     

脓毒症单核巨噬细胞特异性结合肽的筛选

利用噬菌体随机肽库展示技术,筛选出与脓毒症单核/巨噬细胞特异性结合的短肽,探索脓毒症治疗的新方法.分别以经过脂多糖(lipopolysaccharide, LPS)处理的人外周血单核细胞株(THP-1)细胞作为筛选的靶细胞,以未经LPS处理的THP-1细胞作为非特异性噬菌体吸附细胞,对噬菌体随机环七肽库进行4轮“差减"筛选,经过细胞ELISA验证阳性噬菌体克隆,对获得的阳性克隆进行DNA测序及生物信息学分析,并进一步利用免疫荧光实验,鉴定噬菌体克隆与LPS处理THP-1细胞的结合特异性.4轮筛选后,随机挑取的噬菌体克隆,测序后得到可与LPS处理的THP-1细胞特异性结合肽.对去冗余后的七肽进行Clustal W多序列比对分析和BlastP蛋白同源相似性分析,细胞免疫荧光检测确定获得的噬菌体展示七肽可与LPS处理的THP-1细胞特异性结合.噬菌体随机肽库技术为脓毒症单核/巨噬细胞表面靶位的筛选提供了高效、快捷的筛选体系,实验获得的多肽基序具有高度保守性和细胞特异性,这些多肽的生物活性将是下一步的研究内容.     

利用噬菌体随机肽库筛选抗柯萨奇病毒B3多肽的研究

本实验将柯萨奇病毒B3型(CVB3)大量扩增,应用蔗糖密度梯度离心法纯化病毒。利用噬菌体随机9肽库进行筛选,3轮淘洗后,测定噬菌体克隆抗病毒复制能力。提取阳性克隆DNA并进行测序,推导外源多肽的氨基酸序列。结果表明:3个具有明显抗病毒复制能力的噬菌体阳性克隆被筛选出来,使TCID50由10-7.5SFU/mL分别降至10-5.25、10-6、10-5.5SFU/mL,由此证明可以应用噬菌体肽库来筛选具有抗病毒作用的多肽,本研究为抗病毒多肽制剂的研究奠定了基础。     

利用噬菌体随机肽库筛选抗柯萨奇病毒B3多肽的研究

本实验将柯萨奇病毒B3型(CVB3)大量扩增,应用蔗糖密度梯度离心法纯化病毒.利用噬菌体随机9肽库进行筛选,3轮淘洗后,测定噬菌体克隆抗病毒复制能力.提取阳性克隆DNA并进行测序,推导外源多肽的氨基酸序列.结果表明3个具有明显抗病毒复制能力的噬菌体阳性克隆被筛选出来,使TCID50由10-7.5SFU/mL分别降至10-5.25、10-6、10-5.5SFU/mL,由此证明可以应用噬菌体肽库来筛选具有抗病毒作用的多肽,本研究为抗病毒多肽制剂的研究奠定了基础.     

双靶向MMP-14和金属离子的结合肽筛选与分子模拟

以基质金属蛋白酶-14(MMP-14)催化结构域为靶标,通过噬菌体随机十二肽库 筛选和分子模拟、细胞免疫荧光、金属离子亲和层析以及体外细胞作用测定等技 术,进行了双靶向MMP-14和金属离子小分子结合多肽的筛选与研究.经4轮筛选, 噬菌体得到有效富集并获得13条不同的多肽序列.序列分析显示,可能的一致序列 有：AHQLH、HHXH、EI/LPLL/I.分子模拟与对接进一步确认一致序列AHQLH、HHTH 、LPLL与MMP-14催化结构域的氨基酸120~125区域良好分子对接并具有一定的专 一性,多条MMP-14结合肽不仅靶向MMP-14,同时结合金属离子.细胞生物学研究确 认,所测定的结合肽噬菌体对MMP-14诱导表达的MG63细胞具有良好的结合作用,揭 示结合肽对MMP-14的靶向结合特性,并且合成的AHQLH、LPLL一致序列多肽对MG63 细胞活力具有一定的抑制能力.这些新的和具有一定MMP-14专一性的一致序列可望 用于靶向MMP-14抗肿瘤药物的研发和利用.     

抗人白细胞介素15中和抗体识别抗原表位的噬菌体肽库筛选与鉴定

目的:用噬菌体呈现随机七肽库筛选能与抗人白细胞介素15(h IL-15)中和抗体特异性结合的模拟抗原表位肽,并初步鉴定其免疫原性。方法:以抗h IL-15中和抗体为靶分子,用生物淘洗法从噬菌体呈现线性七肽库中筛选与之结合的噬菌体克隆,用噬菌体ELISA和竞争抑制ELISA鉴定阳性噬菌体克隆;化学合成筛选得到的多肽,并与匙孔血蓝蛋白(KLH)偶联免疫BALB/c小鼠,检测其免疫原性。结果:经过3轮体外筛选后随机挑取50个阳性噬菌体克隆,ELISA检测结果显示其中15个克隆与抗h IL-15抗体有较强的结合能力,DNA测序结果得到的结构相似群为MTPFWQK、MSPFNQK、MIPYWQK和MIPFHQK;竞争性ELISA结果显示4个序列均能与IL-15竞争性地结合抗IL-15单抗;小鼠免疫实验结果显示4组多肽均能诱导IL-15特异性免疫反应。结论:筛选得到能与抗h IL-15中和抗体特异性结合,且具有免疫原性的模拟抗原7肽序列,为进一步开发h IL-15相关的多肽疫苗提供了依据。     

噬菌体展示技术筛选bFGF模拟短肽

目的：通过噬菌体展示技术筛选得到与FGFR结合的bFGF模拟短肽,为bFGF肽类抑制剂的研发提供实验基础。方法：以Balb/c 3T3细胞为靶标,以COS-7细胞作消减,对噬菌体随机七肽库进行4轮生物淘洗,再采用ELISA检测单克隆噬菌体对Balb/c 3T3亲和性和特异性,选取阳性克隆进行DNA测序分析。结果：从富集的噬菌体中获得12个阳性克隆,获得一组疏水性七肽及共同基序PR。结论：利用肽类新药开发的重要工具--噬菌体展示技术,得到2段bFGF的受体结合模拟肽,可望作为bFGF抑制剂的先导肽。     

利用噬菌体随机肽库筛选可结合猪繁殖与呼吸综合症病毒的多肽序列

【目的】采用完整的猪繁殖与呼吸综合症病毒(Porcine Reproductive and Respiratory Syndrome Virus,PRRSV)颗粒筛选噬菌体肽库,以获得能高亲和力结合并能抑制该病毒复制的特异性多肽。【方法】用纯化的病毒粒子包被ELISA板,再用M13噬菌体随机12肽库进行筛选。经过3轮淘筛,ELISA鉴定噬菌体单克隆与PRRSV的亲和力,选取与PRRSV具有高亲和力的噬菌体单克隆进行DNA测序,据此推导多肽的氨基酸序列。通过TCID50检测其抗病毒复制能力,同时人工合成FITC标记的展示肽用于PRRSV的检测。【结果】经筛选和鉴定得到17个阳性噬菌体克隆能与PRRSV呈高亲和力结合,DNA测序发现各克隆之间有部分共有基序,其中2个克隆体外能明显抑制PRRSV的复制,使TCID50由10-7.3/0.1mL分别降至10-3.2、10-3.6/0.1mL,而FITC标记该展示肽能够在5mg/L工作浓度检测PRRSV。【结论】通过噬菌体肽库能够筛选到具有抗病毒作用的阳性噬菌体克隆,为进一步开发高效PRRSV的诊断和治疗试剂奠定基础。     

从噬菌体随机肽库中筛选流感病毒神经氨酸酶的抑制多肽

目的:从噬菌体呈现12肽库中筛选与流感病毒神经氨酸酶特异性结合的肽。方法:以甲三型流感病毒裂解疫苗原液为靶分子,经过3轮生物淘选,从噬菌体随机肽库中筛选与之结合的噬菌体。用ELISA方法鉴定噬菌体克隆与靶分子的结合力,用荧光方法测定噬菌体克隆对流感病毒A/Sydney/5/97(H3N2)神经氨酸酶的抑制活性。对筛选到的阳性克隆进行DNA序列测定并推导出相应的氨基酸序列。结果:经过3轮筛选后,42个噬菌体克隆与靶分子有高度亲和力,23个噬菌体克隆对流感病毒A/Sydney/5/97(H3N2)神经氨酸酶有抑制活性。对27个噬菌体克隆的测序结果表明,分别有10个和2个克隆的序列是一致的,其氨基酸序列分别为KSLSRHDHIHHH和WPRHHHSASVQT。结论:通过噬菌体肽库筛选到抑制流感病毒神经氨酸酶的12肽,为进一步研究对流感病毒神经氨酸酶有抑制活性的分子药物奠定了基础。     

Antimicrobial activity and mechanism of action of a novel cationic α-helical octadecapeptide derived from heat shock protein 70 of rice

Hsp70(241–258), an octadecapeptide derived from the heat shock protein 70 (Hsp70) of rice (Oryza sativa L. japonica), is a novel cationic α-helical antimicrobial peptide (AMP) that contains four lysine, two arginine, and two histidine residues. The antimicrobial activity of Hsp70(241–258) against Porphyromonas gingivalis, a periodontal pathogen, and Candida albicans, an opportunistic fungal pathogen, was quantitatively evaluated using a chemiluminescence method that measures ATP derived from viable cells. The 50% growth-inhibitory concentrations of Hsp70(241–258) against P. gingivalis and C. albicans cells were 63 μM and 70 μM, respectively. Hsp70(241–258) had little or no hemolytic activity even at 1 mM, and showed negligible cytotoxicity up to 300 μM. The degrees of calcein leakage from large unilamellar vesicles, which mimic the membranes of Gram-negative bacteria, and 3,3′-dipropylthiadicarbocyanine iodide release from P. gingivalis cells induced by the addition of Hsp70(241–258) increased in a concentration-dependent manner. When Hsp70(241–258) was added to calcein-acetoxymethyl ester-loaded C. albicans cells, calcein release from the cells increased in a concentration-dependent manner. Flow cytometric analysis also showed that the percentages of C. albicans cells stained with propidium iodide, a DNA-intercalating dye, increased as the concentration of Hsp70(241–258) added was increased. Therefore, Hsp70(241–258) appears to exhibit antimicrobial activity against P. gingivalis and C. albicans through membrane disruption. These results suggest that Hsp70(241–258) could be useful as a safe and potent AMP against P. gingivalis and C. albicans in many fields of health care, especially in the control of oral infections.     

Disulfide bond polymerization of a cyclic peptide derived from the surface loop 4 of class 1 OMP of Neisseria meningitidis

Two peptides derived from the surface loop 4 of class1 Outer Membrane Protein (OMP) of Neisseriameningitidis were synthesized on solid phase usingthe Boc/Bzl strategy: one containing the entire loop4 cyclized and the other representing the polymerizedcyclic loop 4. To test a more efficient cyclic peptidepresentation, in the present study astrategy was developed to obtain polymers of cyclic peptides. Inorder to obtain the polymeric cyclic peptide, twoprotecting groups for cysteine were used – Acm andMob. The Cys(Acm)-protected cyclic peptide wasobtained after removing the Mob group. Thepolymerization reaction was carried out bysimultaneous deprotection/oxidation of S-Acmwith iodine. Analysis of the polymeric cyclic peptidein Tris-tricine-SDS-PAGE showed different bandswith molecular weights higher than expected for thecorresponding monomeric cyclic peptide. Both peptideswere used in immunization of four different mouse strains.The antisera raised against the peptides wereevaluated by ELISA and Western blotting vs. OMPpreparation of N. meningitidis. The titersraised against the polymerized cyclic peptide werehigher than the ones raised against the cyclicpeptide. The antisera elicited did not showbactericidal activity. Nevertheless, the antiseraelicited against the polymeric cyclic peptide in theCBA/J mouse strain showed opsonic activity. Theantibodies raised against the polymeric cyclic peptidewere successfully used as probes in Western blottingexperiments to verify the display of loop 4 peptide onthe surface of filamentous phage M13.     

Entry of a cationic lytic-type peptide into the cytoplasm via endocytosis-dependent and -independent pathways in human glioma U251 cells

Cationic lytic-type peptides have been studied for clinical application in various infections and cancers. This study aimed to determine the functions of our specially designed lytic peptide. To investigate the functional mechanism at the cell membrane level, we used giant unilayer vesicles (GUVs) mimicking cell membranes. In GUVs treated with FITC-labeled lytic peptide (lytic-FITC), fluorescence increased in a time-dependent manner. However, no inner fluorescence was detected in GUVs treated with lytic peptide and calcein. Next, distribution of lytic-FITC peptide on the cell membrane and in the cytoplasm was examined in a living human glioma U251 cell line. In the immunocytochemical study, some lytic peptide stains colocalized with early endosome antigen protein 1 (EEA-1). In cells treated with lytic peptide, the immunofluorescence intensity of lytic peptide increased in a concentration and treatment time-dependent manner. Cytotoxic activity of lytic peptide decreased after pretreatment with the endocytosis inhibitors cytochalasin D, chlorpromazine and amiloride. These findings suggest that lytic peptide exerts cytotoxic activity after cellular uptake via an endocytosis pathway. In conclusion, the influx mechanism of lytic peptide was shown to include not only disintegration and pore formation at the cell membrane, but also cell entry via endocytosis dependent and independent pathways.     

Disulfide bond polymerization of a cyclic peptide derived from the surface loop 4 of class 1 OMP of Neisseria meningitidis

Summary Two peptides derived from the surface loop 4 of class 1 Outer Membrane Protein (OMP) ofNeisseria meningitidis were synthesized on solid phase using the Boc/Bzl strategy: one containing the entire loop 4 cyclized and the other representing the polymerized cyclic loop 4. To test a more efficient cyclic peptide presentation, in the present study a strategy was developed to obtain polymers of cyclic peptides. In order to obtain the polymeric cyclic peptide, two protecting groups for cysteine were used — Acm and Mob. The Cys(Acm)-protected cyclic peptide was obtained after removing the Mob group. The polymerization reaction was carried out by simultaneous deprotection/oxidation ofS-Acm with iodine. Analysis of the polymeric cyclic peptide in Tris-tricine-SDS-PAGE showed different bands with molecular weights higher than expected for the corresponding monomeric cyclic peptide. Both peptides were used in immunization of four different mouse strains. The antisera raised against the peptides were evaluated by ELISA and Western blotting vs. OMP preparation ofN. meningitidis. The titers raised against the polymerized cyclic peptide were higher than the ones raised against the cyclic peptide. The antisera elicited did not show bactericidal activity. Nevertheless, the antisera elicited against the polymeric cyclic peptide in the CBA/J mouse strain showed opsonic activity. The antibodies raised against the polymeric cyclic peptide were successfully used as probes in Western blotting experiments to verify the display of loop 4 peptide on the surface of filamentous phage M13.     

Polypeptide synthesis using an expressed peptide as a building block for condensation with a peptide thioester: application to the synthesis of phosphorylated p21Max protein(1-101).

An expressed peptide proved to be useful as a building block for the synthesis of a polypeptide via the thioester method. A partially protected peptide segment, for use as a C-terminal building block, could be prepared from a recombinant protein; its N-terminal amino acid residue was transaminated to an alpha-oxoacyl group, the side-chain amino groups were then protected with t-butoxycarbonyl (Boc) groups, and. finally, the alpha-oxoacyl group was removed. On the other hand, an O-phosphoserine-containing peptide thioester was synthesized via a solid-phase method using Boc chemistry. These building blocks were then condensed in the presence of silver ions and an active ester component. During the condensation, epimerization at the condensation site could be suppressed by the use of N,N-dimthylformamide (DMF) as a solvent. Using this strategy, a phosphorylated partial peptide of the p21Max protein, [Ser(PO3H2)2.11]-p21Max(1-101), was successfully synthesized.     

人胰岛素原C肽的合成及专一性抗体制备

利用固相多肽合成中的Boc途径合成了人胰岛素原C肽 ,经TSK柱层析一步纯化 ,产物达HPLC及毛细管电泳均一 ,蛋白质序列分析和质谱分析符合理论值 ,总回收率高达 41 %。为了提高寡肽抗体的专一性 ,我们将丙烯酰化的C肽经催化聚合形成了以聚丙酰为骨架 ,C肽为侧链的聚合体 ,分子量约为 2 5kD。免疫新西兰大白兔 1月 ,获得专一性抗体。用酶联免疫吸附法测得抗血清滴度达 2 .5× 1 0 4 ,与BSA无交叉反应。聚丙酰C肽抗原是制备C肽抗体的一种新的尝试 ,而且也可能成为新的多肽工程疫苗之一 ,为其他传染性疾病多肽疫苗的研究提供新的途径     

噬菌体肽库技术的应用

噬菌体肽库是由大量带有不同肽段的单个噬菌体组成的重组噬菌体库,通过分析筛选到的多肽的结构和序列,可以了解蛋白质分子之间的相互作用。随着生物技术的发展,噬菌体肽库技术在基因治疗、抗原表位定位、确定核酸结合蛋白、基因疫苗研究和药物筛选等方面得到广泛应用并取得了很大进展。     

Predicted calmodulin-binding sequence in the gamma subunit of phosphorylase b kinase

A basic, amphiphilic alpha helix is a structural feature common to a variety of inhibitors of calmodulin and to the calmodulin-binding domains of myosin light chain kinases. To aid in recognizing this structural feature in sequences of peptides and proteins we have developed a computer algorithm which searches for sequences of appropriate length, hydrophobicity, helical hydrophobic moment, and charge to be considered as potential calmodulin-binding sequences. Such sequences occurred infrequently in proteins of known crystal structure. This algorithm was used to find the most likely site in the catalytic (gamma) subunit of phosphorylase b kinase for interaction with calmodulin (the delta subunit). A peptide corresponding to this site (residues 341-361 of the gamma subunit) was synthesized and found to bind calmodulin with approximately an 11 nM dissociation constant. A variant of this peptide in which an aspartic acid at position 7 in its sequence (347 of the gamma subunit) was replaced with an asparagine was found to bind calmodulin with approximately a 3 nM dissociation constant.     

血管钠肽对低氧大鼠心脏钠尿肽C受体表达的影响

目的 :探讨低氧对大鼠心脏钠尿肽C受体 (NPR C)表达的调节作用 ,以及血管钠肽 (VNP)对这一过程的影响。方法 :将大鼠随机分为 3组 :对照组、低氧组 (3～ 2 8d)和VNP(2 5～ 75 μg/kgbw) 低氧组 ,采用放射免疫的方法测定大鼠血浆心房钠尿肽 (ANP)的浓度 ,并采用定量PCR的方法分析NPR C的mRNA水平。结果 :低氧 2 8d大鼠血浆ANP浓度显著高于正常大鼠 (P <0 .0 5 ) ,而且每天注射 75 μg/kgbw的VNP使ANP浓度进一步升高 (P <0 .0 1)。低氧 3d对大鼠心脏NPR C的mRNA的量没有显著影响 ;低氧 7d使大鼠心脏NPR C的mRNA的拷贝数显著升高 (P <0 .0 5 ) ;低氧 14d、2 8d使大鼠心脏NPR C的mRNA的拷贝数进一步升高 (P <0 .0 1)。每日注射 2 5μg/kgbw的VNP对低氧诱导的大鼠心脏NPR C表达没有显著影响 ;5 0 μg/kgbw的VNP显著降低低氧大鼠心脏NPR C的表达 (P <0 .0 5 ) ;75 μg/kgbw的VNP进一步降低低氧大鼠心脏NPR C的表达 (P <0 .0 1)。 结论 :VNP可以升高低氧大鼠的血浆ANP水平 ;低氧可以使大鼠心脏NPR C表达增加 ,而且具有时间依赖性 ,而VNP对这一过程有抑制作用 ,并且呈剂量依赖性     

Facile synthesis of peptide nucleic acids and peptide nucleic acid‐peptide conjugates on an automated peptide synthesizer

Peptide nucleic acids (PNAs) are DNA mimics with a neutral peptide backbone instead of the negatively charged sugar phosphates. PNAs exhibit several attractive features such as high chemical and thermal stability, resistance to enzymatic degradation, and stable binding to their RNA or DNA targets in a sequence‐specific manner. Therefore, they are widely used in molecular diagnosis of antisense‐targeted therapeutic drugs or probes and in pharmaceutical applications. However, the main hindrance to the effective use of PNAs is their poor uptake by cells as well as the difficult and laborious chemical synthesis. In order to achieve an efficient delivery of PNAs into cells, there are already many published reports of peptides being used for transport across the cell membrane. In this protocol, we describe the automated as well as cost‐effective semi‐automated synthesis of PNAs and PNA‐peptide constructs on an automated peptide synthesizer. The facile synthesis of PNAs will be helpful in generating PNA libraries usable, e.g. for high‐throughput screening in biomolecular studies. Efficient synthetic schemes, the automated procedure, the reduced consumption of costly reagents, and the high purity of the products are attractive features of the reported procedure. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.