大鲵肽在牙膏中的应用研究

采用酶法从人工饲养大鲵肉中提取得到大鲵肽,利用串联飞行时间质谱仪(MALDI-TOF/TOF)测定其分子量主要为1 422,1 521,1 746,1 828和1 879,均小于2 000,酶解产物大鲵肽为低聚肽。将提取到的大鲵肽应用到牙膏中,应用自由基清除活性评价方法研究了大鲵肽牙膏的抗氧化活性。结果表明,牙膏中添加质量分数为0.1%的大鲵肽可达到较好的抗氧化效果,其牙膏水溶液具有清除DPPH自由基、羟基自由基(·OH)、超氧阴离子自由基以及ABTS自由基的能力。     

WT1蛋白CTL表位肽基因载体的构建与鉴定

目的构建WT1(Wilms’tumor gene 1)蛋白CTL表位肽基因载体,并检测其在293T细胞中的转录。方法设计分别含WT1-126肽、WT1-235肽以及这两种肽的基因载体,并加入Th通用表位Pan-DR-Th(PADRE),应用蛋白酶体切割软件PAProC和NetChop优化各表位和间隔序列,DNA疫苗在线预测工具DyNAVacS优化真核密码子后,人工合成核苷酸序列,分别插入pUC57载体,构建pUC57-WT1质粒,测序鉴定后,酶切回收各目的片段,亚克隆至真核表达载体pcDNA3.1(+)中,构建重组真核表达质粒,转染293T细胞,RT-PCR检测各目的基因在293T细胞中的转录。采用无内毒素质粒大量提取试剂盒提取各重组质粒,采用紫外分光光度计测定质粒的纯度和浓度。结果各重组真核表达质粒经双酶切及测序鉴定证实构建正确;重组质粒携带的目的基因可在293T细胞中成功转录;各重组质粒DNA的纯度均合格,浓度在864.6～883.9μg/ml之间。结论成功构建了WT1蛋白CTL表位肽基因载体,并能在真核细胞中正常转录,为下一步在小鼠体内探讨特异性不同的CTLs群发挥抗肿瘤作用的机制奠定了基础。     

HIV自然感染与疫苗诱导产生抗体鉴别肽的原核表达及纯化

目的原核表达并纯化HIV自然感染与疫苗诱导产生抗体鉴别肽sk1、sk2、sk3及3条肽段串联序列。方法用普通PCR及重叠延伸PCR法从HIV-1 B亚型质粒扩增得到sk1、sk2、sk3基因序列及3条肽段基因片段的6种连接序列,并分别克隆入原核表达载体pET-32a(+)及pGEX4T-2中,转化大肠杆菌BL21(DE3),IPTG诱导表达,表达产物经Ni-NAT或谷胱甘肽-Sepharose-4B层析柱亲和层析纯化。SDS-PAGE分析融合肽的表达,Western blot分析融合肽的抗原性。结果分别以pET-32a(+)和pGEX4T-2为载体,共构建了18个重组表达质粒;his-sk1、his-sk123、his-sk213、his-sk231及GST-sk3、GST-sk123、GST-sk213分别在pET-32a(+)及pGEX4T-2表达载体中以可溶形式表达,且均能与HIV-1阳性血清反应,而不能与HIV DNA-天坛疫苗免疫血清反应;经大量诱导表达纯化后,GST-sk3表达量最高,可达40 mg/L,纯度超过90%。结论已成功表达并纯化了具有生物学活性的HIV自然感染与疫苗诱导产生抗体鉴别肽,为研制HIV自然感染与疫苗诱导产生抗体鉴别诊断试剂盒奠定了基础。     

牛乳铁蛋白十肽的基因改造、克隆及表达

目的克隆并表达牛乳铁蛋白十肽及其突变体M1和M2基因。方法参照大肠杆菌偏爱密码子,分别针对牛乳铁蛋白十肽及其突变体M1和M2基因,设计并合成两个具有相同黏性末端的DNA片段,同时在其基因前后分别加上天冬酰胺和甘氨酸的密码子,构成羟胺裂解位点,通过连接获得其二拷贝基因同向串联体。分别将该串联体克隆至载体pUC18上,经双酶切、PCR及测序鉴定后,构建重组表达质粒,转化大肠杆菌BL21(DE3),IPTG诱导表达。表达产物纯化后,用凝血酶切割及羟胺裂解。结果获得了牛乳铁蛋白十肽及其突变体M1和M2二拷贝基因,并在大肠杆菌BL21(DE3)中表达出相对分子质量约29000的目的蛋白条带,纯化后的蛋白经凝血酶切割后,相对分子质量约29000的目的蛋白条带消失。结论已成功克隆并表达了牛乳铁蛋白十肽及其突变体M1和M2基因,为基因工程抗真菌肽的制备奠定了基础。     

三氟乙酸及甲酸体系下重组人促红素液质肽图的比较

目的探讨流动相中添加三氟乙酸(trifluoroacetic acid,TFA)或甲酸(formic acid,FA)时,重组人促红素(erythropoietin,EPO)液质肽图中各肽段的保留时间、峰面积及离子化效率的差异。方法将重组人EPO样品进行酶切,分别在含0.1%TFA及0.1%FA的流动相体系下测定液质肽图。通过分析质谱数据对各色谱峰进行定性,记录并计算各肽段的保留时间、峰面积及质谱信号强度。结果两种添加剂体系下,各肽段的保留时间存在差异,大部分肽段的保留时间变化在1 min左右,个别肽段超过2 min;TFA体系下各肽段的峰面积相对较小,约为FA体系下的84%;FA体系下各肽段离子化效率较高,信号强度一般为TFA体系的6倍,个别肽段高达10倍以上。结论两种体系下重组人EPO的液质肽图存在较大差异,应根据实验目的选择合适的流动相添加物。     

人内毒素结合肽基因的突变及突变体蛋白表达

目的分析亲代人内毒素结合肽一级结构,选择可能影响其生物学活性的氨基酸对应碱基位点进行突变,克隆基因突变体mEBP基因并研究其蛋白表达。方法以亲代EBP基因为基础,结合DNASIS软件理化性质分析确定突变碱基,应用PCR定点诱变技术进行第5、18位谷氨酰胺→赖氨酸的定点突变。并将其克隆至原核融合表达载体pinpointXa-3,转化大肠杆菌BL21(DE3)pLysS进行表达,经Western blot鉴定。结果突变EBP基因13及52位核苷酸由C突变为A,构建的阳性重组子经酶切及测序鉴定证实与设计序列完全一致,经IPTG诱导在大肠杆菌BL21(DE3)pLysS表达生物素化的融合蛋白,Western blot鉴定显示能与抗生物素单克隆抗体结合。结论已成功获得EBP基因突变体,并在大肠杆菌以融合蛋白的形式进行表达。     

基于金属有机骨架材料富集翻译后修饰肽段的研究进展

蛋白质翻译后修饰是真核细胞用来使蛋白质功能多样化并动态协调其信号传导网络的重要机制。蛋白质磷酸化和糖基化是重要的翻译后修饰,影响着多种复杂的生物过程。然而由于其低丰度和低电离效率,用质谱法直接检测磷酸化肽段和糖基化肽段面临着许多挑战。因此,发展制备简便、成本低廉且高效富集磷酸化肽段/糖基化肽段的纳米材料吸引着众多研究者的关注。本文综述了近年来基于金属有机骨架材料（MOFs）富集磷酸化肽段/糖基化肽段的研究进展,以期为扩展MOFs在蛋白组学中的应用指明一个新的方向。     

多价EZH2肿瘤相关抗原肽的制备及其抗原性

目的人工设计制备多价EZH2肿瘤相关抗原肽,并鉴定其抗原特性。方法设计合成编码多价EZH2肿瘤相关抗原肽的DNA序列,克隆入原核表达载体p ET-30a(+)中,构建含4个EZH2肿瘤相关抗原肽段(Aa120-128、Aa165-174、Aa291-299、Aa735-742)的重组多价抗原肽原核表达质粒p ET-30a(+)-EZH2-antigen,转化E.coli BL21(DE3),经IPTG诱导表达后,用Ni-NTA树脂在变性条件下对重组多肽进行亲和层析纯化,Western blot分析重组多价EZH2肿瘤相关抗原肽的抗原特性。用重组多价EZH2肿瘤相关抗原肽免疫大耳白兔,制备多克隆抗体,间接ELISA法检测血清中抗体效价,Western bolt分析多抗的特异性。结果重组原核表达质粒p ET-30a(+)-EZH2-antigen经酶切及测序证实构建正确;表达的重组多价EZH2肿瘤相关抗原肽表观相对分子质量约4 300,主要以包涵体形式存在;纯化的多肽纯度不低于90%,浓度为0.5 mg/ml,且能被兔抗EZH2多抗和兔抗His标签多抗有效识别和结合;纯化的多价EZH2肿瘤相关抗原肽能在动物体内高效诱导抗体生成,抗血清效价为1∶64×104,且能特异性识别和结合PC3人前列腺癌细胞中表达的EZH2蛋白。结论成功制备了一种多价EZH2肿瘤相关抗原肽,该多价抗原肽相对分子质量虽小,但抗原肽密度高,可能具有更强的肿瘤相关抗原的活性,具有重要的应用前景。     

抑制新城疫病毒繁殖的九肽及其突变体基因的克隆与原核表达

目的克隆并表达抑制新城疫病毒(NDV)繁殖的九肽(Nonapeptide)及其突变体基因。方法设计并合成Non-apeptide2串联体及其突变体基因,克隆于质粒pUC18,经酶切回收目的基因,与经相同酶切的表达载体pGEX-4T-1连接,转化大肠杆菌BL21(DE3),筛选阳性菌落,抽提质粒进行鉴定。IPTG诱导表达后,进行SDS-PAGE及Western blot鉴定。结果重组表达质粒经双酶切、PCR及测序鉴定,证明构建正确。阳性重组菌的诱导表达产物经SDS-PAGE分析,在相对分子质量约29000处可见一明显条带,与理论值相符。Nonapeptide 2及其突变体蛋白的表达量分别占菌体总蛋白的41%和37%。Western blot结果显示两种蛋白均具有良好的反应原性。结论已成功克隆并表达了抗NDV繁殖的Nonapeptide及其突变体基因。     

组织激肽释放酶的研究进展

组织激肽释放酶为一类性质相似的化合物,可由肝脏、胰脏、脑神经等合成。人类多种疾病与激肽释放酶基因表达相关,该基因是高血压基因治疗研究的首选靶点。本文综述了激肽释放酶基因与疾病的相关性以及该基因重组载体的构建和表达等研究进展。     

MiR-10a in Pancreatic Juice as a Biomarker for Invasive Intraductal Papillary Mucinous Neoplasm by miRNA Sequencing

New biomarkers are needed to further stratify the risk of malignancy in intraductal papillary mucinous neoplasm (IPMN). Although microRNAs (miRNAs) are expected to be stable biomarkers, they can vary owing to a lack of definite internal controls. To identify universal biomarkers for invasive IPMN, we performed miRNA sequencing using tumor-normal paired samples. A total of 19 resected tissues and 13 pancreatic juice samples from 32 IPMN patients were analyzed for miRNA expression by next-generation sequencing with a two-step normalization of miRNA sequence data. The miRNAs involved in IPMN associated with invasive carcinoma were identified from this tissue analysis and further verified with the pancreatic juice samples. From the tumor-normal paired tissue analysis of the expression levels of 2792 miRNAs, 20 upregulated and 17 downregulated miRNAs were identified. In IPMN associated with invasive carcinoma (INV), miR-10a-5p and miR-221-3p were upregulated and miR-148a-3p was downregulated when compared with noninvasive IPMN. When these findings were further validated with pancreatic juice samples, miR-10a-5p was found to be elevated in INV (p = 0.002). Therefore, three differentially expressed miRNAs were identified in tissues with INV, and the expression of miR-10a-5p was also elevated in pancreatic juice samples with INV. MiR-10a-5p is a promising additional biomarker for invasive IPMN.     

A Case Study on the Keap1 Interaction with Peptide Sequence Epitopes Selected by the Peptidomic mRNA Display

Many protein–protein and peptide–protein interactions (PPIs) play key roles in the regulation of biological functions, and therefore, the modulation of PPIs has become an attractive target of new drug development. Although a number of PPIs have already been identified, over 100 000 unknown PPIs are predicted to exist. To uncover such unknown PPIs, it is important to devise a conceptually distinct method from that of currently available methods. Herein, an mRNA display by using a total RNA library derived from various human tissues, which serves as a unique method to physically isolate peptide epitopes that potentially bind to a target protein of interest, is reported. In this study, selection was performed against Kelch-like ECH-associated protein (Keap1) as a model target protein, leading to a peptide epitope originating from astrotactin-1 (ASTN1). It turned out that this ASTN1 peptide was able to interact with Keap1 more strongly than that with a known peptide derived from Nrf2; a well-known, naturally occurring Keap1 binder. This case study demonstrates the applicability of peptidomic mRNA display for the rapid exploration of consensus binding peptide motifs and the potential for the discovery of unknown PPIs with other proteins of interest.     

Vortex-assisted dispersive liquid–liquid microextraction trace amounts of melatonin prior to HPLC determination in fruit juice samples

In the present study, a fast, simple and sensitive vortex-assisted dispersive liquid–liquid microextraction method was developed for extraction and preconcentration of trace amounts of melatonin in fruit juice samples. The extraction conditions were optimized as extraction solvent, chloroform; volume of extracting, 200.0 μL; and vortex time, 120 s. The HPLC determinations were done using ODS Waters C₁₈ column under gradient elution. The calibration curve was linear in the range of 1.0–1000.0 ng mL^–1 and detection limit was 0.23 ng mL^–1. The validated method was applied for separation and determination of melatonin in different fruit juice samples (cherry, tomato and grape).     

Supercritical fluid extraction of lycopene from tomato juice and characterization of its antioxidation activity

A new method to extract lycopene from tomato juice using supercritical CO₂ as solvent and without the need to dry the raw material is presented. To conduct the extraction, the tomato juice was subjected to cycles of centrifugation followed by rinsing with absolute ethanol to partially remove the water present in the solid part of the juice. The influence of the temperature and pressure on the extraction efficiency and on the extract antioxidant activity was studied using a factorial experimental design. The extraction efficiency varied from 7.7% to 76.7% and only extraction temperature had a statically significant effect on the process. The reversed phase HPLC analysis showed that lycopene is the major compound of the extract. The extract that presented higher antioxidant activity was obtained at 40 °C and 350 bar with 12.7 mmol L⁻¹ Trolox/g of extract using the DPPH radical scavenging method and 61.3 mmol L⁻¹ Trolox/g of extract using the rubrene singlet oxygen quenching method.     

二维电泳-质谱分析麦拓莱霉素发酵过程中差异蛋白

引言 本课题组从土壤中筛选到一株藤黄灰链霉菌菌株,该菌株能产生一种新型的抗生素--麦拓莱霉素.麦拓莱霉素具有抗HIV蛋白酶活性和抗Coxsackie-virus B6 病毒 [1]活性,有很好的工业化前景.但目前产量还比较低,其生产过程的优化和产量的提高是亟待解决的问题.     

Hyaluronidase Alters the Lipid Profile of Cumulus Cells as Detected by MALDI-TOF MS and Multivariate Analysis

This research aimed to study the changes in lipid composition in cumulus cells using hyaluronidase according to the intracytoplasmic sperm injection protocol commonly used in human reproduction clinics. The lipid extraction was performed by the Blight‐Dyer protocol and the lipid profiles were obtained by MALDI‐TOF MS in positive and negative modes. The mass spectra data were processed with MassLynx and the statistical analysis was performed using MetaboAnalyst 2.0. Fifteen ions were selected for each mode as potential markers for differences between the groups. These ions were identified in the human metabolome database as phosphatidylserine with and without treatment, phosphatidylethanolamine in the after treatment group and phosphatidylinositol in the before treatment group, which are lipids that may be involved in cell apoptosis and signaling. We concluded that MALDI‐TOF MS coupled with multivariate analysis can be utilized as a strategy to obtain and study the lipid profiles of cumulus cells and as a tool to study the metabolic state of cumulus cells.     

Determination of Bile Acids in Piglet Bile by Solid Phase Extraction and Liquid Chromatography-Electrospray Tandem Mass Spectrometry

An LC/MS/MS‐based method was developed for the determination of individual bile acids (BA) and their conjugates in porcine bile samples. The C18‐based solid‐phase extraction (SPE) procedure was optimized so that all 19 target BA and their glycine and taurine conjugates were collected with high recoveries for standards (89.1–100.2 %). Following this, all 19 compounds were separated and quantified in a single 12 min chromatographic run. The method was validated in terms of linearity, sensitivity, accuracy, precision, and recovery. An LOD in the low ppb range with measured precisions in the range of 0.5–9.3 % was achieved. The recoveries for all of the 19 analytes in bile samples were all >80 %. The validated method was successfully applied to the profiling of BA and their conjugates in the bile from piglets treated with exogenous glucagon‐like peptide‐2 (GLP‐2) in a preclinical model of neonatal parenteral nutrition‐associated liver disease (PNALD). The method developed is rapid and could be easily implemented for routine analysis of BA and their conjugates in other biofluids or tissues.     

儿童玩具和用品中18种多环芳烃含量检测与风险分析

采用自建的快速溶剂萃取法提取18种多环芳烃,经硅胶固相柱净化,使用气相色谱三重四级杆串联质谱测定儿童玩具和用品中多环芳烃的内标定量法,选取了江苏苏州地区流通市场销售的儿童玩具和用品进行了18种多环芳烃含量的检测,对检测结果进行了风险分析。