大蒜渣ACEI活性肽的制备及其稳定性研究

目的以蒜渣为原料,对制备ACEI(血管紧张素转化酶抑制剂)活性肽的工艺及酶解产物的稳定性进行研究。方法以水解液的抑制活性为试验指标,从9种蛋白酶中筛选出4种较佳蛋白酶单独或联合水解蒜渣。结果蛋白酶1单独水解效果较好。通过单因素和正交试验得出最佳水解条件:加酶量(E/S)6%、底物浓度8%、pH 7、50℃、3 h,在上述条件下酶解产物对ACE的抑制率为88.81%。考察了大蒜渣ACEI活性肽的热稳定性、耐酸碱性和抗肠道酶降解能力,结果表明,该活性肽具有良好的耐高温性能;在酸性条件下活性下降较快,当pH≥8.3时活性稳定;在模拟的胃肠道环境中具有良好的抗胃蛋白酶和胰蛋白酶裂解的能力。结论大蒜渣ACEI活性肽经口服或者静脉注射后很可能具有良好的降血压效果。     

半夏凝集素基因的克隆与氨基酸序列初步分析

目的 克隆半夏凝集素基因并对其氨基酸序列生物信息与已报道相关基因进行对比分析,为抗虫基因利用和转基因育种研究奠定基础.方法 根据已报道的植物凝集素基因序列设计特异引物,以半夏叶片DNA为模板进行PCR扩增,获得特异性片段,将其连接到测序载体上,进行序列测定,用分析软件分析序列信息.结果 克隆1 069 bp的半夏凝集素基因(pta),开放阅读框全长804bp,编码268个氨基酸残基;预测相对分子质量和等电点分别为2.91×104和7.77,功能区完整,具有1条信号肽和3个甘露糖结合区;已在GenBank中登记(登录号AY725425).结论 克隆的半夏凝集素基因序列信息完整,具有典型的甘露糖结合位点,可以作为抗虫基因用于抗虫育种.     

Hassallidin A, a glycosylated lipopeptide with antifungal activity from the cyanobacterium Hassallia sp

Hassallidin A (1), a new antifungal glycosylated lipopeptide, was isolated from an epilithic cyanobacterium collected in Bellano, Italy, identified as Tolypothrix (basionym Hassallia) species. Chemical, mass spectrometric, and spectroscopic analyses, including one- and two-dimensional NMR, were performed to determine an esterified eight-residue cyclic peptide linked with a carbohydrate and a fatty acid residue. Chiral GC-MS analysis revealed the occurrence of the nonproteinogenic amino acids D-allo-Thr, D-Thr, D-Tyr, D-Gln, and dehydroaminobutyric acid (Dhb) within the peptide moiety. The additional components of hassallidin A could be identified as alpha,beta-dihydroxytetradecanoic acid (Dht) and mannose. This is the first report on a cyclic peptide of cyanobacterial origin that contains both a fatty acid and a carbohydrate moiety. Compound 1 exhibits antifungal activity against Aspergillus fumigatus and Candida albicans with MIC values of 4.8 microg/mL for both test organisms.     

Influences of AMY1 gene copy number and protein expression on salivary alpha-amylase activity before and after citric acid stimulation in splenic asthenia children

OBjECTIVE:To compare the correlations between salivary alpha-amylase(sAA) activity and amylase,alpha 1(salivary) gene(AMY1) copy number or its gene expression between splenic asthenia and healthy children,and investigate the reasons of attenuated sAA activity ratio before and after citric acid stimulation in splenic asthenia children.METHODS:Saliva samples from 20 splenic asthenia children and 29 healthy children were collected before and after citric acid stimulation.AMY1 copy number,sAA activity,and total sAA and glycosylated sAA contents were determined,and their correlations were analyzed.RESULTS:Although splenic asthenia and healthy children had no differences in AMY1 copy number,splenic asthenia children had positive correlations between AMY1 copy number and sAA activity before or after citric acid stimulation.Splenic asthenia children had a higher sAA glycosylated proportion ratio and glycosylated sAA content ratio,while their total sAA content ratio and sAA activity ratio were lower compared with healthy children.The glycosylated sAA content ratio was higher than the total sAA content ratio in both groups.Splenic asthenia and healthy children had positive correlations between total sAA or glycosylated sAA content and sAA activity.However,the role played by glycosylated sAA content in sAA activity in healthy children increased after citric acid stimulation,while it decreased in splenic asthenia children.CONCLUSION:Genetic factors like AMY1 copy number variations,and more importantly,sAA glycosylation abnormalities leading to attenuated sAA activity after citric acid stimulation,which were the main reasons of the attenuated sAA activity ratio in splenic asthenia children compared with healthy children.     

The isolation and structure elucidation of Tasiamide B,a 4-amino-3-hydroxy-5-phenylpentanoic acid containing peptide from the marine Cyanobacterium Symploca sp

A new cytotoxic peptide, which displayed an IC(50) value of 0.8 microM against KB cells, has been isolated from the marine cyanobacterium Symploca sp. The planar structure of tasiamide B (1), deduced by 2D NMR experiments, contains the unusual amino acid-derived residue 4-amino-3-hydroxy-5-phenylpentanoic acid (Ahppa). The configuration of 1 was deduced by HPLC analysis of degradation products.     

人GPx1基因的克隆及其序列分析

 目的克隆中国人谷胱甘肽过氧化物酶(GPx1)的cDNA。方法用逆转录 聚合酶链反应(RT-PCR),以中国人胎盘组织总RNA为模板,扩增中国人谷胱甘肽过氧化物酶(GPx1)的cDNA,进行序列分析。计算机分析其二级结构并比较已发现的人GPx家族的5种GPx氨基酸序列。结果从中国人胎盘组织中克隆的GPx1基因,与国外文献报道相比,只有1个氨基酸残基发生变异,即leu²²变为Val²²,该变化不影响GPx1活性中心的结构;人GPx的硒结合区及活性中心区域的氨基酸序列是高度保守的。结论首次克隆了中国人体特异的胞质内GPx1基因,为其生物学活性及结构与功能的关系的研究创造了条件。     

342bp人骨形态发生蛋白2成熟肽基因的克隆表达

目的探讨克隆人骨形态发生蛋白2(hBMP2)成熟肽的最精简的基因序列并原核表达方法。方法剔除所有信号肽和中间前肽的非必需基因序列,以RT-PCR方法克隆扩增出hBMP2的342 bp的成熟肽基因片段,将其与原核表达载体PBV220酶切后相连接并测序,构建PBV220-hBMP2的真核基因原核表达系统,在DH5α大肠杆菌下扩增并温度诱导表达hBMP2蛋白质,SDS-PAGE检测表达蛋白。结果RT-PCR扩增出hBMP2的成熟肽基因342 bp片段与Genbank公布的序列完全一致,且经DNA电泳和蛋白电泳鉴定,构建PBV220-hBMP2原核表达系统可导入DH5α大肠杆菌,并在温度诱导下表达出约16 kD的蛋白条带,诱导培养4 h后的蛋白表达量达到最高峰。结论成功扩增342 bp的成熟肽hBMP2基因片段能在原核大肠杆菌上有效表达生产hBMP2蛋白质。     

石菖蒲配伍冰片对高脂血症大鼠内皮素和降钙素基因相关肽的影响

目的：探讨石菖蒲配伍冰片对高脂血症大鼠内皮素、降钙素基因相关肽、神经肽Y的影响。方法：大鼠高脂饲料喂养17d，造成高脂血症模型，灌胃给药7d，采用放射免疫法测定脑组织内皮素、降钙素基因相关肽、神经肽Y含量。结果：石菖蒲配伍冰片组脑组织内皮素含量明显下降，降钙素基因相关肽含量明显升高。结论：石菖蒲配伍冰片能降低高脂血症大鼠脑组织中内皮素含量，并升高降钙素基因相关肽含量，有舒张脑血管、改善脑供血的作用。     

Microspinosamide, a new HIV-inhibitory cyclic depsipeptide from the marine sponge Sidonops microspinosa

Microspinosamide (1), a new cyclic depsipeptide incorporating 13 amino acid residues, was isolated from extracts of an Indonesian collection of the marine sponge Sidonops microspinosa. Its structure was elucidated by extensive NMR and mass spectral analyses, and by chemical degradation and derivatization studies. The tridecapeptide 1 incorporates numerous uncommon amino acids, and it is the first naturally occurring peptide to contain a beta-hydroxy-p-bromophenylalanine residue. Microspinosamide (1) inhibited the cytopathic effect of HIV-1 infection in an XTT-based in vitro assay with an EC(50) value of approximately 0.2 microg/mL.     

Apratoxin E, a cytotoxic peptolide from a guamanian collection of the marine cyanobacterium Lyngbya bouillonii

A collection of the marine cyanobacterium Lyngbya bouillonii from Guam afforded apratoxin E (1), a new peptide-polyketide hybrid of the apratoxin class of cytotoxins. The planar structure of 1 was elucidated by NMR spectroscopic analysis and mass spectrometry. Configurational assignments of stereocenters in the peptide portion were made by chiral HPLC analysis of the acid hydrolysate. The relative configuration in the polyketide moiety was assigned by comparison of NMR data including proton-proton coupling constants with those of the known analogues. Apratoxin E (1) displayed strong cytotoxicity against several cancer cell lines derived from colon, cervix, and bone, ranging from 21 to 72 nM, suggesting that the alpha,beta-unsaturation of the modified cysteine residue is not essential for apratoxin activity. The 5- to 15-fold reduced activity compared with apratoxin A (2) is attributed to the dehydration in the long-chain polyketide unit, which could affect the conformation of the molecule.     

铁皮石斛赤霉素3-氧化酶基因的克隆及表达分析

目的克隆珍稀濒危药用植物铁皮石斛Dendrobium officinale赤霉素3-氧化酶(gibberellin 3-oxidases,GA3ox)基因(Do GA3ox),并进行生物信息学和表达分析。方法采用RACE和RT-PCR技术获得Do GA3ox基因c DNA全长;利用生物信息学软件预测其编码蛋白的理化性质、结构域及亚细胞定位等分子特性;用DNASTAR 7.0和MEGA 6.0软件分别对其进行氨基酸多序列比对和进化关系分析;借助实时荧光定量PCR(q RT-PCR)检测基因表达模式。结果克隆到Do GA3ox基因(Gen Bank注册号KT597694),c DNA全长1 318 bp,编码一条由353个氨基酸组成的多肽,相对分子质量为39 052.5,等电点6.21;Do GA3ox蛋白不含跨膜域和信号肽,具有GA3ox的保守结构域DIOX_N(40~130)和2OG-Fe II_Oxy(197~299);Do GA3ox与大葱、油棕、海枣、野茶树及蓝猪耳GA3ox蛋白一致性分别为55%、56%、54%、51%、50%,与单子叶植物大葱、油棕和海枣的亲缘关系较近。q RT-PCR实验结果显示Do GA3ox基因在铁皮石斛种子共生(非共生)萌发1~3级时,其相对表达量呈现出先升高后降低再升高的趋势,分别为未萌发种子的13.44(5.21)、7.28(2.32)和9.40(6.21)倍,由此可知,该基因在共生萌发种子中的表达量高于非共生萌发种子。结论首次克隆得到1个Do GA3ox基因的全长c DNA,其转录本在共生和非共生不同萌发级别种子中的表达特性说明该基因在铁皮石斛种子萌发中起着重要调控作用。     

人干细胞因子基因大肠杆菌表达产物结构分析及活性研究

 目的确证人干细胞因子基因在大肠杆菌表达产物的正确性。方法通过蛋白质杂交，氨基酸末端序列分析，质谱分析及动物体内生物活性的研究，对人干细胞因子基因大肠杆菌表达产物——重组人干细胞因子(rhSCF)分子进行了鉴定。结果大肠杆菌表达产物rhSCF N端15个氨基酸序列及C端2个氨基酸序列与理论推测一致，产物的相对分子质量为18 583.75，质量肽谱与理论序列的重叠率为98.2%，动物试验亦表明当与粒细胞集落刺激因子(G CSF)联用具有显著的外周血干(祖)细胞动员作用。结论人干细胞因子基因大肠杆菌表达产物与理论推测完全一致，为其中试及临床试验奠定了基础。     

陆英HMGS基因cDNA克隆、不同器官中的差异表达及生物信息学分析

目的克隆陆英Sambucus chinensis 3-羟基-3-甲基戊二酰辅酶A合成酶(HMGS)基因并分析其差异表达。方法采用实时荧光定量PCR(RT-PCR)方法获得HMGS基因c DNA序列并对HMGS蛋白进行理化性质、蛋白二级结构及三维结构预测分析,并预测了该蛋白功能;利用RT-PCR方法检测了HMGS基因在陆英的根状茎、地上茎、叶、花中的表达情况。结果克隆获得的HMGS基因c DNA全长为1 401 bp,编码466个氨基酸。生物信息学预测HMGS蛋白无跨膜区,不含信号肽。HMGS基因主要在陆英的花和根状茎中表达较高,在叶中表达相对较低。结论首次从陆英中克隆了HMGS基因,为进一步阐明该基因在陆英萜类化合物代谢途径中的重要作用奠定基础。     

独行菜磷酸甲羟戊酸激酶LaPMK基因克隆、生物信息学分析及原核表达

目的从独行菜Lepidium apetalum中克隆萜类合成途径关键酶磷酸甲羟戊酸激酶(phosphomevalonate kinase,PMK)基因,进行序列分析和原核表达。方法根据独行菜转录组数据中La PMK基因序列设计特异性引物,克隆得到La PMK基因的开放阅读框(open reading frame,ORF),构建p ET32a-La PMK原核表达载体,在大肠杆菌BL21(DE3)中用IPTG诱导表达La PMK融合蛋白。结果 La PMK基因ORF全长为1 518 bp(Genbank注册号KT004541),编码505个氨基酸。生物信息学分析结果显示La PMK蛋白没有跨膜区,不含信号肽,位于细胞质中,含有GHMP激酶家族特异的N末端和C末端的保守结构域。系统进化树结果显示La PMK蛋白与芜菁的PMK蛋白具有92%的序列相似性,亲缘关系较近。构建p ET32a-La PMK原核表达载体,在大肠杆菌中成功表达La PMK融合蛋白。结论克隆了La PMK基因,建立其稳定的原核表达体系,为La PMK蛋白纯化、抗体的制备,进一步研究La PMK基因在独行菜萜类合成途径中的作用奠定了基础。     

眼针对血管性痴呆大鼠模型学习记忆障碍及血清CGRP ET的影响

目的:探讨眼针对血管性痴呆(VD)大鼠学习记忆障碍及血清中降钙素基因相关肽(CGRP)及内皮素(ET)的影响。方法:4-血管阻断法制成血管性痴呆大鼠模型。取眼针肝区、心区、肾区。共治疗30天,并与西药尼莫通相对照。实验前后分别进行Y-型迷宫测试及实验结束后腹腔静脉取血测量血清中CGRP和ET。结果:与对照组比较,模型组大鼠学习记忆明显障碍,眼针组、尼莫通组学习记忆能力明显改善,与模型组比较(P<0.01)。模型组与对照组相比,眼针组、尼莫通组与模型组相比CGRP和ET变化有显著差异(P<0.01)。结论:眼针能改善VD大鼠学习记忆障碍及调节血清中降钙素基因相关肽(CGRP)及内皮素(ET)两者的平衡,从而减轻缺血再灌注损伤。     

Cloning and biochemical characterization of the hectochlorin biosynthetic gene cluster from the marine cyanobacterium Lyngbya majuscula

Cyanobacteria, or blue-green algae, are a rich source of novel bioactive secondary metabolites that have potential applications as antimicrobial or anticancer agents or useful probes in cell biology studies. A Jamaican collection of the cyanobacterium Lyngbya majuscula has yielded several unique compounds including hectochlorin ( 1) and the jamaicamides A-C ( 5- 7). Hectochlorin has remarkable antifungal and cytotoxic properties. In this study, we have isolated the hectochlorin biosynthetic gene cluster ( hct) from L. majuscula to obtain details regarding its biosynthesis at the molecular genetic level. The genetic architecture and domain organization appear to be colinear with respect to its biosynthesis and consists of eight open reading frames (ORFs) spanning 38 kb. An unusual feature of the cluster is the presence of ketoreductase (KR) domains in two peptide synthetase modules, which are predicted to be involved in the formation of the two 2,3-dihydroxyisovaleric acid (DHIV) units. This biosynthetic motif has only recently been described in cereulide, valinomycin, and cryptophycin biosynthesis, and hence, this is only the second such report of an embedded ketoreductase in a cyanobacterial secondary metabolite gene cluster. Also present at the downstream end of the cluster are two cytochrome P450 monooxygenases, which are likely involved in the formation of the DHIV units. A putative halogenase, at the beginning of the gene cluster, is predicted to form 5,5-dichlorohexanoic acid.     

湿热因素对大鼠胃黏膜水通道蛋白3、4基因表达的影响

目的探讨湿热因素对大鼠胃黏膜水通道蛋白（AQP）3、4基因表达的影响。方法30只大鼠随机分成3组，造模20d。对照组正常喂饲；脾虚组隔日喂饲，非喂饲日灌喂番泻叶水煎液；模型组20％蜂蜜水自由饮用，隔日灌服油脂，造模开始后第16～20日每日20时-次日8时将大鼠置入人工气候箱中，造模结束后，采用FQ-PCR方法测定各组胃黏膜AQP3、AQP4基因表达。结果模型大鼠在造模过程中出现湿热证的特征性表现；模型组AQP3、AQP4基因表达水平高于对照组、脾虚组（P〈0．05）。结论通过对大鼠施加内、外湿因素的造模方法可复制出湿热证的证候特征；AQP3的异常表达可能是湿热证的发生机制之一。     

降钙素基因相关肽的生物活性及其临床意义

降钙素基因相关肽是一种有降钙素基因表现生物活性多肽 ,广泛分布于神经、心血管系统 ,具有巨大的扩张血管、降低血压及正性肌力作用 ,并参与体内一些激素的调节。在某些心血管疾病的发生和防治中具有十分重要的意义。     

源于鲨鱼肝的一种新基因的克隆表达及其对肝肿瘤细胞的抑制作用

目的：从鲨鱼肝脏中克隆表达一种新基因psll2，纯化该基因的表达产物，研究其对肝肿瘤细胞的抑制作用。方法与结果：我们前面已报道发现了鲨肝细胞再生刺激因子（sHRSF），根据sHRSF的N端氨基酸序列设计了引物，并利用RT-PCR方法从鲨鱼再生肝组织的，6-RNA中扩增到大小约为350bp的cDNA片段，序列分析表明350bp的片段含有一个开放阅读框（ORF），含有333个碱基，编码111个氨基酸残基，其编码的N端氨基酸序列与天然sHRSF的前7个氨基酸一致。该肽被命名为PSL12（来源于鲨肝的分子量约为12kD的肽）。该cDNA被连接到质粒pGEM—T-Easy，获得重组质粒pGEM—T-psll2。根据cDNA序列分析和质粒pET-32a的多克隆位点（BamHI和SalⅠ）的序列，设计并合成了psll2基因的特异性引物，质粒pGEM—T-psll2作为模板，进行了PCR扩增。将此PCR产物插入pET-32a得到表达质粒pET-32a—psll2，并将它转化入E．coli的BL21菌株。经IPTG诱导，带有组氨酸标签的融合蛋白的表达量约为40％。用金属螯合层析纯化融合蛋白后，再用FXa切割融合蛋白，经ResourceQ和MonoQ柱层析得到PSL12纯品，它可抑制肝肿瘤细胞株SMMC．7721和HepG2的增殖。结论：从鲨鱼肝脏中获得了一种新基因psll2。该基因的表达产物PSL12能显著抑制体外培养的肝肿瘤细胞的增殖。