Synthetic Smac Peptide Enhances Chemo-sensitivity of Bladder Cancer Cells

The effects of synthetic Smac peptide （SmacN7） on chemotherapeutic sensitivity of bladder cancer cells were investigated. SmacN7 penetratin peptide was synthesized and delivered into T24 cells. MTT assay was used to evaluate the viability of T24 cells induced by low-dosage of MMC Flow cytometry was used to analyze the proportions of apoptosis. Western blot was used to detect the expression of XIAP and Caspase-3. The activity of Caspase-3 was measured and the effect of SmacN7 combined with MMC on T24 cell lines was also determined. The results showed that SmacN7 penetratin peptide could successfully interact with endogenous XIAP, increase the proportions of apoptosis of T24 cell lines induced by low-dosage of MMC in a dose-and time-dependent manner. An obvious down-regulation of XIAP expression and up-regulation of Caspase-3 was identi-fied by Western blot. The activity of Caspase-3 in experimental group was significantly increased as compared with that in the control group. As compared with MMC group, the viability of T24 cells in SmacN7 penetratin peptide＋MMC group was markedly decreased to 2.22 and 3.61 folds at 24 h and 48 h respectively. It was concluded that SmacN7 penetratin peptide could act as a cell-permeable IAP inhibitor, inhibit the proliferation, induce apoptosis and enhance the chemo-sensitivity of bladder cancer cells to MMC. These findings indicate that SmacN7 penetratin peptide may be a very promising ageut for bladder cancer treatment when used in combination with chemotherapy.

Development of a Synthetic Peptide ELISA Assay for the Detection of Foot-and-Mouth Disease Virus Nonstructural Protein Antibodies

In order to develop an ELISA assay with synthetic peptides for the detection of antibody to the nonstructural proteins of foot-and-mouth disease virus, specific peptides were synthesized by a solid-phase method according to FMDV NSPs B-cell epitopes, and were conjugated to carrier protein BSA. An ELISA system was developed to detect FMDV NSPs antibody with the conjugated proteins as the coating antigen. The optimal coating concentration of the antigen was determined as 2.5 μg mL-1. The comparative study of this assay with UBI NSP ELISA kit and national commercial 3ABC ELISA kit in the detection of 199 serum samples showed that they were very coincident, and the identity rates were 96.48 and 97.48%, respectively. The development of ELISA using the synthetic peptides as coating antigen is specific, reproducible, stable, and easy, and can be used to differentiate FMDV infected pigs from immunized pigs.

Detection of anti-HEV by synthetic peptides

Three synthetic peptides representing distinct antigenic epitopes from three genomic regions of hepatitis E virus (HEV) were used to develop enzyme-linked immunoserbent assay ELISA for detecting antibodies to HEV. Both IgG and IgM antibodies to HEV were d

拟Smac合成肽羧甲基壳聚糖磁性纳米复合物的制备及体外抗肿瘤性能研究

目的:探讨拟Smac合成肽羧甲基壳聚糖磁性纳米复合物的制备及联合恒定外磁场体外对膀胱癌细胞的凋亡促进作用。方法:以羧甲基壳聚糖为骨架与具有超顺磁性的Fe3O4纳米粒合成纳米载体粒子,将拟Smac合成肽SmacN7与其结合,制备拟Smac合成肽羧甲基壳聚糖磁性纳米复合物,并通过透射电镜、振动样磁强计等考察其理化性质;Hoechst33258染色观察外加磁场下纳米复合物诱导膀胱癌T24细胞凋亡的形态,MTT比色分析法观察其对肿瘤细胞的生长抑制作用。结果:拟Smac合成肽羧甲基壳聚糖磁性纳米粒子的粒径约为46.2nm;磁化曲线提示具有超顺磁性;载药量和包封率分别为(31.8±3.6)%和(65.2±2.4)%并具有良好的药物控释性能;外加磁场下磁性纳米粒子能使肿瘤细胞呈现明显的凋亡形态改变并表现出显著的生长抑制活性。结论:拟Smac合成肽羧甲基壳聚糖磁性纳米复合物具有粒径小、较强的磁响应性、载药量和包封率高和良好的药物缓释性能,联合恒定外磁场具有明显的促进肿瘤细胞凋亡的作用,为膀胱肿瘤的生物治疗提供了新的切入点。

拟Smac促凋亡多肽的合成及其生物活性的初步研究

目的探讨拟Smac多肽的合成及其对膀胱癌细胞的促凋亡生物活性。方法应用固相多肽合成技术,合成具有细胞膜穿透性SmacN7融合多肽,经RP-HPLC纯化、纯度分析,用质谱仪定性鉴定;通过荧光显微镜观察细胞凋亡形态、细胞增殖抑制率测定及流式细胞仪分析,研究其对低剂量丝裂霉素C诱导的膀胱癌T24细胞的凋亡促进作用。结果可穿透性融合多肽SmacN7产物峰纯度达95%以上,分子量为3278.08,质谱鉴定结果与合成预期结果完全一致;50～500μg/LSmacN7作用12～48h,肿瘤细胞出现典型的凋亡形态学改变;随着SmacN7浓度的增加或作用时间的延长,细胞增殖抑制率出现明显增加,药物作用12h、24h、48h后增殖抑制率分别为(9.62±1.07)%～(61.48±1.15)%、(24.17±1.02)%～(72.86±1.68)%、(43.24±1.15)%～(84.91±1.74)%;肿瘤细胞凋亡率也明显增加,分别为(6.12±1.16)%～(49.81±2.11)%、(13.47±1.15)%～(64.54±2.27)%、(28.91±1.08)%～(82.36±2.19)%。结论固相合成的拟Smac融合多肽SmacN7为高纯度的目的肽,能够稳定地转入细胞内且利用率高,并有明显促进低剂量丝裂霉素C诱导的膀胱癌T24细胞凋亡的生物活性,为进一步研究膀胱肿瘤的生物治疗积累了有价值的资料。

Smac促凋亡机制及其与肺癌的关系

由于线粒体蛋白Smac具有促进细胞凋亡作用,因而成为肿瘤研究中重要的凋亡调控因子之一。本文概述了Smac的结构、细胞定位和组织分布;通过Smac与IAPs、TRAIL和其它凋亡调节因子的相互作用,阐述了其促进肿瘤细胞凋亡的作用机制;并从表达、预后和治疗等方面论述了Smac在肺癌中的研究进展。研究表明,Smac表达状况与肺癌的发生发展、预后评估及化疗效果相关。而且,Smac模拟小分子肽能通过级联放大效应增加肺癌细胞化疗敏感性,因此,Smac对肺癌的预后评价和治疗等均有重要的作用。

Smac合成肽靶向下调凋亡抑制蛋白XIAP提高胰腺癌细胞化疗敏感性

目的:探讨包含目的蛋白功能结构域的Smac合成肽能否增强胰腺癌细胞的化疗药物敏感性及其作用机制。方法:化学合成SmacN7细胞可穿透融合多肽,共沉淀实验观察SmacN7细胞穿透肽与胰腺癌Panc-1细胞内的XIAP的相互作用,应用流式细胞术检测SmacN7细胞穿透肽与顺铂、5-FU联用对Panc-1细胞凋亡的影响,MTT法检测SmacN7细胞穿透肽应用前后Panc-1细胞的化疗药物敏感性。结果:SmacN7融合多肽能与内源性XIAP结合,明显下调Panc-1细胞XIAP表达水平,显著增强顺铂或5-FU诱导的Panc-1细胞凋亡,使其对顺铂、5-FU的药物半数抑制浓度(IC50)分别降低1.98倍、2.62倍。结论:应用包含目的蛋白功能结构域的Smac合成肽能靶向下调胰腺癌Panc-1细胞XIAP表达,显著提高其化疗敏感性,为胰腺癌的生物治疗协同化疗提供了新思路。

人工合成拟Smac多肽增强膀胱癌细胞化疗敏感性的实验研究

背景与目的:Smac是目前发现的惟一能直接同时抑制多个IAPs家族成员活性的凋亡相关蛋白,其N端的4个氨基酸残基AVPI(Ala-Val-Pro-Ile)是重要的促凋亡结构域,我们通过人工合成可穿透细胞的促凋亡融合多肽SmacN7,探讨其对膀胱癌化疗药物敏感性的促进作用。方法:噻唑蓝(MTT)检测SmacN7融合多肽对低剂量丝裂霉素C(MMC)诱导的膀胱癌T24细胞的相对存活率的影响:Annexin V/PI双标流式细胞术检测T24细胞的凋亡1;Western印迹检测SmacN7融合多肽与MMC联用后T24细胞内XIAP、Caspase-3的表达;同时检测Caspase-3活性及SmacN7融合多肽与MMC联用对T24细胞的杀伤作用。结果:SmacN7融合多肽能穿透细胞并与内源性XIAP结合,增加低剂量MMC诱导的T24细胞凋亡并呈时间和浓度依赖性:并能显著降低细胞内XIAP的表达水平,增强Caspase-3的表达及活性:在24 h和48 h,SmacN7+MMC组与单用MMCtg相比,T24细胞的存活率分别降低55%和72.7%。结论:人工合成可穿透细胞的促凋亡融合多肽SmacNT能促进化疗药物诱导的膀胱癌T24细胞凋亡,抑制细胞增殖,增强膀胱癌细胞对MMC的化疗药物敏感性。

人工合成Smac拟肽促进前列腺癌细胞凋亡的实验研究

目的探讨人工合成的小分子Smac拟肽对前列腺癌细胞增殖的抑制作用和诱导细胞调亡作用。方法应用固相多肽合成技术人工合成Smac融合多肽,以激光共聚焦显微镜观察肽段的细胞定位;以不同浓度处理前列腺癌细胞,应用MTT比色法、Hoechst染色、流式细胞术检测细胞增殖和凋亡情况。结果激光共聚焦检测显示小分子肽段能进入细胞内,没有表现出某一特定细胞器的聚集性;MTT结果显示Smac拟肽对前列腺癌LNCap和PC-3细胞的增殖抑制具有浓度依赖性和时间依赖性(高浓度Smac拟肽);Hoechst染色发现LNCap和PC-3细胞经高浓度(200μg/mL)作用48 h时细胞凋亡明显;同样浓度Smac拟肽作用LNCap和PC-3细胞48 h后用流式细胞仪分析,结果同样显示细胞明显凋亡。结论小分子Smac拟肽能有效地诱导前列腺癌细胞的凋亡,为前列腺癌的生物治疗提供了新的思路。

人工合成拟Smac融合多肽促低剂量丝裂霉素C诱导膀胱癌细胞凋亡的研究

目的：探讨人工合成拟Smac融合多肽（SmacN7）对低剂量丝裂霉素C（MMC）诱导的膀胱癌T24细胞凋亡的促进作用。方法：运用固相多肽合成技术人工合成SmacN7细胞可穿透性融合多肽，将0．05mg／ml MMC诱导的膀胱癌T24细胞与50～500μg／L的SmacN7融合多肽共同孵育4～48h，采用Annexin—V荧光染色技术检测肿瘤细胞凋亡情况；应用流式细胞术和MTT比色法检测诱导后T24细胞凋亡率、增殖抑制率与SmacN7的时间和浓度的效应关系。结果：50-500μg／L SmacN7作用4～48h，肿瘤细胞出现典型的凋亡形态学改变，随着SmacN7浓度的增加或药物作用时间的延长，肿瘤细胞凋亡率也增加，12h为5．67％～56．12％，24h为14．54％～65．24％，48h为31．48％～87．23％，同时细胞增殖抑制率出现明显增加，药物作用12h、24h、48h后，增殖抑制率分别为9．58％～63．42％、28．94％～72．3％、44．7％～87．12％。结论：SmacN7能够有效地促进低剂量MMC诱导的膀胱癌T24细胞凋亡，并具有时间和浓度依赖性，为膀胱肿瘤的生物治疗提供了新思路。

Rationally designed synthetic peptide as versatile calibrant to improve the accuracy of protein sequence analysis using MALDI mass spectrometry

Matrix-assisted laser desorption/ionization(MALDI)mass spectrometry(MS)plays an indispensable role in analyzing protein covalent structures.The reliable identification of amino acid residues and modifications relies on the mass accuracy,which is highly dependent on calibration.However,the accuracy provided by the currently available calibrants still needs further improvement in terms of compatibility with multiple tandem MS modes or ion polarity modes,calibratable range,and minimizing suppression of and interference with analyte signals.Here aiming at developing a versatile calibrant to solve these problem,we designed a synthetic peptide format of calibrant R_x(GDP_n)_m(referred to as“Gly-Asp-Pro,GDP”)according to the chemical natures of amino acids and polypeptide fragmentation rules in tandem MS.With four types of amino acid residues selected and arranged through rational designs,a GDP peptide produces highly regulated fragments that give rise to evenly spaced signals in each tandem MS mode and is compatible with both positive and negative ion modes.In internal calibration,its regulated fragmentation pattern minimizes interference with analyte signals,and using a single peptide as the input minimizes suppression of the analyte signals.As demonstrated by analyses of proteins including monoclonal antibody and Aβ-42,these features allowed significant increase of the mass accuracy and precision,which improved sequence coverage and sequence resolution in sequence analyses(including de novo sequencing).This rational design strategy may also inspire further development of synthetic calibrants that benefit structural analysis of biomolecules.

Growth and differentiation of human induced pluripotent stem cell(hiPSC)-derived kidney organoids using fully synthetic peptide hydrogels

Human induced pluripotent stem cell(hiPSC)-derived kidney organoids have prospective applications ranging from basic disease modelling to personalised medicine.However,there remains a necessity to refine the bio-physical and biochemical parameters that govern kidney organoid formation.Differentiation within fully-controllable and physiologically relevant 3D growth environments will be critical to improving organoid reproducibility and maturation.Here,we matured hiPSC-derived kidney organoids within fully synthetic self-assembling peptide hydrogels(SAPHs)of variable stiffness(storage modulus,G′).The resulting organoids con-tained complex structures comparable to those differentiated within the animal-derived matrix,Matrigel.Single-cell RNA sequencing(scRNA-seq)was then used to compare organoids matured within SAPHs to those grown within Matrigel or at the air-liquid interface.A total of 13,179 cells were analysed,revealing 14 distinct clusters.Organoid compositional analysis revealed a larger proportion of nephron cell types within Transwell-derived organoids,while SAPH-derived organoids were enriched for stromal-associated cell populations.Notably,dif-ferentiation within a higher G’SAPH generated podocytes with more mature gene expression profiles.Addi-tionally,maturation within a 3D microenvironment significantly reduced the derivation of off-target cell types,which are a known limitation of current kidney organoid protocols.This work demonstrates the utility of syn-thetic peptide-based hydrogels with a defined stiffness,as a minimally complex microenvironment for the selected differentiation of kidney organoids.

Immune Responses to Six Synthetic Peptides of Capsid Protein with Sera from HIV-1 Infected Individuals

Many B cell epitopes within p24 of human immunodeficiency virus type 1 （HIV-1） were identified, while most of them were determined by using murine monoclonal antibodies reacting with overlapping peptides of p24. Therefore these epitopes may not represent the actual epitopes recognized by the HIV-1 infected individuals. In the present study, immune responses of 67 HIV-1 positive sera from Yunnan Province, China to five peptides on p24 of HIV-1 and one of HIV-2 were analyzed. All of 67 sera did not recognize peptide GA-12 on HIV-1 and peptide AG-23 on HIV-2, which indicated that GA-12 was not human B cell epitope and AG-23 did not cross-react with HIV-1 positive serum. Except 13 sera （19.4%）, all remaining sera did not recognize peptides NI-15, DR-16, DC-22 and PS-18, which indicated that these four peptides represented B cell linear epitopes of HIV-1 p24 in some HIV-1 infected individuals but not the immuno-dominant epitopes in most individuals.

Selection and application of serotypical synthetic peptides derived from hepatitis C virus NS5A region

Background Numerous studies have reported a relationship between hepatitis C virus （HCV） genotype and the response to interferon therapy. Despite high sensitivity and specificity, genotyping methods can be performed only on HCV RNA positive samples. Serotyping might be a rapid and cost effective method for determining HCV genotypes, especially in patients with previously undetectable HCV RNA. In this study, an enzyme linked immunosorbent assay （ELISA） method for HCV serotyping with the genotype specific, synthetic peptides derived from HCV nonstructural 5a （NS5A） region was developed. Methods Based on 45 sequences, representing HCV genotypes 1-6 from Genebank, we synthesised 305 overlapping 30-mer peptides within NS5A region at positions 2182-2343 of HCV. All peptides for antigenic reactivity were tested by enzyme immunoassay with 69 human sera with antiHCV positive representing genotype 1-6. Forty hepatitis C patient sera were serotyped using serotype specific, synthetic peptides and genotyped by sequencing analysis. Results The correspondence of amino acids in HCV NS5A region with amino acids in positions 2182-2343 was very low among different genotype peptides. The highly conserved sequences were residues 2182-2211 （R1）, 2272-2301 （R7） and 2302-2331 （R9）： the highly variable 2212-2241 （R3） and 2257-2286 （R6）. Using 305 peptides, antigenic regions were located in R3, R7 and R9. Eighteen peptides from highly conserved region representing genotypes 1 to 6 showed broad immunoreactivity with sera containing antibody to all HCV genotypes. Immunoreactivity of the peptides from highly variable region was stronger with similar genotype sera. Twelve unique peptides showed highly, genotype specific, reactivity with types 1 and 3 sera. Type 2 genotype specific peptides had cross reaction with type 3 serum. No type 4, 5 or 6 specific peptides were selected. The serotyping results showed high agreement with sequencing analysis. Conclusions The major antigenic regions in HCV NS5A region were at 2212-2241（R3）, 2272-2301（R7） and 2302-2331（R9）. Eighteen peptides from highly conserved region show genotype independent, immunoreactivity, useful for antiHCV antibody test. Twelve peptides from highly variable region show genotype 1 and 3 dependent immunoreactivity, useful for determining HCV serotype, especially for patients with previously undetectable HCV RNA.

The synthetic fragment of rabbit blastocyst peptide＿2 and its physiological significance relevant to

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ACTIVATION OF TCELLS BY SYNTHETIC PEPTIDES OF SUPERANTIGEN TSST-1 UNDER ASSISTANCE OF ASSITANT MOLECULES

根据以往对超抗原ＴＳＳＴ－１分子的Ｔ细胞表位预测结果，从ＴＳＳＴ－１分子中选择了一段序列，合成了两个交叠肽，Ｔ３４和Ｔ５８；观察它们在辅助分子辅助下的促Ｔ细胞增殖能力。结果发现：Ｔ３４和Ｔ５８单独虽均不能活化人的ＰＢＭＣ或小鼠脾细胞，但在ＣＤ２８的共刺激或ＰＭＡ的辅助下却可活化人的ＰＢＭＣ和小鼠的脾细胞。提示：Ｔ３４和Ｔ５８不具备ＭＨＣ结合位，但含有Ｔ细胞表位，两者的Ｔ细胞表位可能位于两肽的共同序列内，即ＴＳＳＴ－１（１２５～１５８）。

PRRSV非结构蛋白Nsp1α合成肽多克隆抗体的制备及应用

本研究尝试利用PRRSV Nsp1α合成肽进行多克隆抗体的制备,并对抗体的特性进行了分析。首先,利用生物信息学技术预测获得PRRSV非结构蛋白Nsp1α的抗原肽序列;随后,按照此序列合成获得抗原肽,并将其与KLH载体蛋白进行偶联;接下来,将偶联好的抗原肽与弗氏佐剂混合,通过免疫大白兔制备针对Nsp1α合成肽的多克隆抗体;最后,利用ELISA、Western blot及间接免疫荧光法对多克隆抗体的特性进行了分析。结果显示:该多克隆抗体ELISA的效价超过105;利用Western blot可以检测到特异的Nsp1α表达条带,包括瞬时转染的样品和不同毒株病毒感染的样品;利用间接免疫荧光法分析,该多克隆抗体可有效地区分病毒感染和未感染的样品。结果表明,PRRSV Nsp1α合成肽多克隆抗体可以有效地应用于PRRSV Nsp1α表达检测,为进一步研究PRRSV Nsp1α的功能提供了一个有效的工具。

深低温保存下高效抗冻多肽的合理设计和机理探讨

The development of effective antifreeze peptides to control ice growth has attracted a significant amount of attention yet still remains a great challenge.Here,we propose a novel design method based on in-depth investigation of repetitive motifs in various ice-binding proteins(IBPs)with evolution analysis.In this way,several peptides with notable antifreeze activity were developed.In particular,a designed antifreeze peptide named AVD exhibits ideal ice recrystallization inhibition(IRI),solubility,and biocompatibility,making it suitable for use as a cryoprotective agent(CPA).A mutation analysis and molecular dynamics(MD)simulations indicated that the Thr6 and Asn8 residues of the AVD peptide are fundamental to its ice-binding capacity,while the Ser18 residue can synergistically enhance their interaction with ice,revealing the antifreeze mechanism of AVD.Furthermore,to evaluate the cryoprotection potential of AVD,the peptide was successfully employed for the cryopreservation of various cells,which demonstrated significant post-freezing cell recovery.This work opens up a new avenue for designing antifreeze materials and provides peptide-based functional modules for synthetic biology.

毕赤酵母分泌型遗传操作工具的构建与优化

为了解决毕赤酵母遗传操作效率低且流程繁琐的问题,提高其应用价值,构建并优化了分泌型遗传操作工具。首先,以α-淀粉酶为报告蛋白,通过融合自主复制序列构建非整合型表达质粒;其次,借助通用引物和POE-PCR,快速筛选评估与目的蛋白最匹配的内源信号肽;最后,构建以亚磷酸盐为唯一磷源的营养依赖型筛选标记。结果表明:2种非整合型质粒表达α-淀粉酶的酶活分别是整合型质粒的2倍和1.6倍;以FLO10、EXG1和MSB 2为信号肽引导的α-淀粉酶酶活分别是常规信号肽α-MF的2.5倍、1.94倍和1.75倍;亚磷酸脱氢酶基因(ptxD)成功筛选出异源表达α-淀粉酶的重组菌株。研究简化了基因操作流程,改进了遗传操作工具,开发的绿色安全且低成本的营养依赖型筛选标记,有助于毕赤酵母分泌表达系统的应用和推广。

Identification and Characterization of Peptides Binding AgEG1 from a Phage Display Library

Endoglucanases are the main cellulolytic enzymes digestion as well as its good kinetic properties make it an attractive of Anoplophora glabripennis. Their high activities in cellulose target for development of cellulase inhibitors. In this study, random pepfide phage display technology was employed to identify peptides that bound the AgEG1, a member of endoglucanase isozymes. Phage clones with peptide LPPNPTK and XPP （X is residue T, L, A or H） motif frequently occurred in the selected phage population and showed a higher phage recovery than other clones. Peptide LPPNPTK was chemically synthesized and characterized tor its binding activities to AgEG1. The synthetic peptide exhibited high specificity for AgEG1. The peptide LPPNPTK has the potential to be developed into inhibitors of the endoglucanase of A. glabripennis.