检测DNA-蛋白质交联的新方法

将彗星实验进行改进以用于DNA-蛋白质交联作用的检测。利用甲醛对受试动物肝细胞的影响来判定此法是否适用于检测DNA-蛋白质交联。由于在实验中添加一定量的蛋白酶K, 可使单细胞在电泳时产生更大的迁移, 因此可以利用添加蛋白酶K前后的彗星尾距比来判断外来化合物对生物机体产生DNA损伤效应的时候是否有出现DNA-蛋白质交联作用。结果表明, 该方法快速、经济、灵敏度较高, 可以在单细胞水平对甲醛等强交联剂引发的不同组织的DNA-蛋白质交联效应进行检测, 希望该方法能成为指示DNA交联能力的有用工具。     

丙型肝炎病毒核心蛋白在大肠杆菌和毕赤酵母中表达的比较

为获得丙型肝炎病毒的核心蛋白（Core）,将克隆有Core基因的表达载体pBVIL1-Core转化大肠杆菌HB101,温度诱导表达Core蛋白。同时利用PCR方法以含有丙型肝炎病毒全基因的质粒PBR^TM／HCV为模板扩增Core基因,克隆进表达载体pPICZαA,构建表达载体pPICZαA-Core,转化毕赤酵母（Pichia pastoris）GS115,在甲醇诱导下表达Core蛋白。Western-blot显示Core蛋白在大肠杆菌中高效表达,表达蛋白量占菌体总蛋白的20％;在酵母培养上清中存在Core蛋白,证明Core蛋白在酵母系统中成功表达。     

抗病毒蛋白RC28在大肠杆菌和毕赤酵母中的表达及活性比较

RC28蛋白是从野生蕈类皱盖罗鳞伞中分离得到的新型抗病毒蛋白。前期通过3'-RACE方法,从皱盖罗鳞伞总RNA中克隆获得包含RC28编码区的c DNA,并通过TA克隆获得质粒p MD18-T-RC28。以该质粒为模板,设计特异性引物,PCR扩增RC28片段,分别插入大肠杆菌表达载体p ET28a(+)和毕赤酵母表达载体p PIC9K中,形成在N端表达组氨酸标签的重组RC28表达粒体。将这两种RC28表达质粒转化各自的宿主菌后,筛选阳性克隆,构建稳定表达体系。诱导表达后,用SDS-PAGE电泳和Western blot分析RC28的表达情况。放大表达体系后用Ni-NTA柱纯化获得RC28蛋白,并用MTT法测试重组表达蛋白质的抗病毒活性。在实验条件下,大肠杆菌和毕赤酵母中均能表达可溶性重组RC28,纯化后蛋白质得率分别是3.5mg/L发酵液及0.2mg/L发酵液。但抗病毒活性实验表明,大肠杆菌体系表达的RC28蛋白活性较差,而毕赤酵母系统表达的RC28蛋白的抗病毒活性与天然蛋白接近。     

体内甲醛交联及染色质免疫沉淀--研究DNA和蛋白质作用的新方法

甲醛交联及染色质免疫沉淀作用研究体内DNA和蛋白质相互作用的一种新方法,在染色质结构研究中获得了广泛的应用。该方法利用甲醛固定活细胞中的DNA与蛋白质,通过免疫沉淀分离复合物,从而分析蛋白质及其体内的DNA结合序列。     

丙型肝炎病毒核心蛋白在大肠杆菌和毕赤酵母中表达的比较*

为获得丙型肝炎病毒的核心蛋白(Core),将克隆有Core基因的表达载体pBVIL1-Core转化大肠杆菌HB101,温度诱导表达Core蛋白。同时利用PCR方法以含有丙型肝炎病毒全基因的质粒PBRTM/HCV为模板扩增Core基因,克隆进表达载体pPICZαA,构建表达载体pPICZαA-Core,转化毕赤酵母(Pichiapastoris)GS115,在甲醇诱导下表达Core蛋白。Western-blot显示Core蛋白在大肠杆菌中高效表达,表达蛋白量占菌体总蛋白的20%;在酵母培养上清     

毕赤酵母表达蛋白质的糖基化

毕赤酵母表达系统可对表达产物进行翻译后加工如糖基化等。通过研究其糖基化的特点、影响因素以及对重组蛋白质质产量、功能的影响,介绍如何避免过度糖基化以使表达产物更加符合人类需求。     

抗菌肽magaininⅡ的密码子优化及在大肠杆菌和毕赤酵母中的表达

Magainin Ⅱ是非洲爪蟾皮肤分泌的一种抗菌肽,在低浓度下即能够对细菌、真菌、原虫、肿瘤等都具有杀伤作用,并且与同是来源于非洲爪蟾皮肤的另一种抗菌肽PGLa在杀菌、抗肿瘤等方面具有很好的协同效应。文中分别按照大肠杆菌和毕赤酵母的密码子利用频率,对抗菌肽magainin Ⅱ以及杂合抗菌肽magainin Ⅱ-PGLa,进行了密码子优化。构建了高效表达抗菌肽magainin Ⅱ的原核表达载体,经过蛋白酶切割获得纯化的抗菌肽magainin Ⅱ。同时进一步构建了杂合抗菌肽magainin Ⅱ-PGLa的分泌型毕赤酵母表达载体,实现了杂合抗菌肽在毕赤酵母中的分泌表达。本研究为magainin Ⅱ和magainin Ⅱ-PGLa的生产与应用奠定了基础。     

毕赤酵母菌高效表达乙肝表面抗原的研究

目的:筛选高效表达HBsAg的毕赤酵母茵,制备目的蛋白.方法:从已确诊的乙肝病人血清中提取DNA,PCR扩增HBVS基因,将其分别克隆入毕赤酵母胞内表达栽体pPICZA中.构建重组质粒pPICZA-S和pPICZA-SH,经Sac I线性化后,LiCI化学法转化入酵母茵株GS115、X-33、KM71H和SMD1168.结果:诱导表达后的GS115工程茼单位体积的培养基所得的抗原含量最高,诱导培养基中加入0.1%酪蛋氨基酸后,可抑制目的蛋白的水解,有利于目的蛋白的表达,粗略估算表达量为15.3mg/L,最佳收获时间为72 h.结论:经SDS-PAGE和Westcrn-blot分析表明,所得产物为乙肝表面抗原S蛋白.     

HPV18晚期蛋白L1在毕赤酵母菌中的表达及鉴定

目的:用毕赤酵母胞内表达载体构建含人乳头瘤病毒18型(HPV18)L1基因质粒,诱导表达并进行鉴定。方法:按照毕赤酵母密码子偏爱性原则,合成全长L1基因,然后克隆到pAO819表达载体上,在体外分别构建含一个拷贝和二个拷贝的L1基因载体。线形化后转化到GS115酵母细胞,经G418抗性筛选,获高拷贝重组子并经甲醇诱导表达,表达产物采用化学发光Western blot鉴定,一抗为抗HPV18L1蛋白鼠抗血清。结果:在55kDa处有诱导蛋白免疫印迹出现,并在电镜下观察到HPV18的病毒样颗粒(VLPs),证明该表达系统能表达出HPV18 L1蛋白。结论:本实验构建的毕赤酵母表达菌株,可经甲醇诱导表达HPV18L1晚期蛋白,为进一步研制人乳头瘤病毒18型基因工程疫苗打下基础。     

极端耐热木聚糖酶基因在大肠杆菌和毕赤酵母中的高效表达

以海栖热袍菌 (Thermotoga maritima) MSB8菌株基因组DNA为模板,通过PCR扩增出木聚糖酶（XylanaseB）基因, 将此基因克隆至大肠杆菌表达载体pET_28a（+）和毕赤酵母表达载体pPIC9K,并分别转化大肠杆菌 BL21和毕赤酵母GS115。该木聚糖酶在大肠杆菌细胞中表达量高, 但不能分泌; 而在毕赤酵母细胞的表达产物可分泌至胞外。酶学性质分析表明,此酶分子量约为40kD,其最适反应温度为90℃, 最适反应pH值为6.65,且在碱性条件下稳定,具有重要的工业应用前景。     

Development and validation of a liquid chromatography-mass spectrometric method for the determination of DPC 423, an antithrombotic agent,in rat and dog plasma

A sensitive and selective LC-MS-MS method for the determination of DPC 423 (I), an antithrombotic agent, is described. This method used a solid-phase extraction from 0.1 ml plasma with an Isolute C(2) cartridge. HPLC separation was carried out on a YMC ODS-AQ C(18) column (50x2 mm) at a flow-rate of 300 microliter/min with an analysis time of 5 min. Compounds were eluted using a mobile phase of H(2)O/CH(3)CN/HCOOH: 66:34:0.1 (v/v/v), pH 4.0. A structural analogue of I was used as the internal standard to account for variations in recovery and instrument response. Mass spectrometric detection was carried out with a PE Sciex API III(+) triple quadrupole mass spectrometer equipped with a Turbo IonSpray source as the LC-MS interface. Good intra-day and inter-day assay precision (<10% CV) and accuracy (<10% difference) were observed over a concentration range of 0.005-2.5 microM in plasma. The extraction recoveries were approximately 90% and the method was found to be linear for the assay (r(2)>0.999). The method has been successfully applied to discovery and preclinical pharmacokinetic studies, including a dose range-finding study and toxicokinetic exposure studies in rat and dog.     

小麦叶片日光合高值持续时间基因型差异的初步研究

首次把“日高值光合作用持续时间”作为重要的光合特性进行了基因型比较研究,研究了不同基因型小麦开花期旗叶光合速率的日变化和日光合量的变化,提出了“日光合高值持续时间”的概念。结果表明：日光合高值持续时间和日光合量在基因型间存在显着差异．所有基因型都存在不同程度午休现象,矮秆、大穗基因型午休现象较轻,有利于日高值光合持续时间。日光合高值持续时间长,日光合量大,有利于干物质生产。     

DPC4／Smad4基因RNA干扰靶点设计和慢病毒载体制备

目的：DPC4／Smad4基因RNA干扰靶点的设计和RNA干扰靶点慢病毒载体制备。方法：针对DPC4／Smad4基因序列,并利用网站设计程序,依据RNA干扰序列设计的原则,设计多个RNA干扰靶点序列。根据设计经验和设计软件将其进行评估测定,选择最佳动力学参数靶点进入其后续的实验流程;生工生物合成含干扰序列的DNAoligo,具有严格的检测体系（PAGE纯化体系）,其两端含酶切位点粘端,直接连入酶切后的RNA干扰载体上。将连接好的产物转入制备好的细菌感受态细胞,并且对长出的克隆进行酶切鉴定。然后挑选出阳性克隆测序,进行测序比对后,鉴定阳性的克隆即为构建成功的目的基因RNA干扰慢病毒载体。将构建的慢病毒载体以及辅助包装载体质粒共转染到293T细胞。收获含有病毒的细胞培养上清,浓缩后进行滴度测定,并检测其感染性。另外应用荧光实时定量PCR检测在感染的293T细胞中敲减效果。结果：成功构建DPC4／Smad4shRNA的慢病毒载体LVshSmad4,并成功制备DPC4／Smad4shRNA慢病毒,三株病毒感染细胞后均具有有效的敲减效应,其中SHl最为显著。结论：DPC4／Smad4基因RNA干扰靶点的成功设计和RNA干扰靶点慢病毒制备,为以后探讨DPC4／Smad4基因与肿瘤的相关性治疗提供了实验基础。     

DPC4/Smad4基因RNA干扰靶点设计和慢病毒载体制备*

摘要目的：DPC4/Smad4 基因RNA干扰靶点的设计和RNA 干扰靶点慢病毒载体制备。方法：针对DPC4/Smad4基因序列,并利用网站设计程序,依据RNA干扰序列设计的原则,设计多个RNA干扰靶点序列。根据设计经验和设计软件将其进行评估测定,选择最佳动力学参数靶点进入其后续的实验流程;生工生物合成含干扰序列的DNA oligo,具有严格的检测体系（PAGE 纯化体系）,其两端含酶切位点粘端,直接连入酶切后的RNA干扰载体上。将连接好的产物转入制备好的细菌感受态细胞,并且对长出的克隆进行酶切鉴定。然后挑选出阳性克隆测序,进行测序比对后,鉴定阳性的克隆即为构建成功的目的基因RNA 干扰慢病毒载体。将构建的慢病毒载体以及辅助包装载体质粒共转染到293T 细胞。收获含有病毒的细胞培养上清,浓缩后进行滴度测定,并检测其感染性。另外应用荧光实时定量PCR 检测在感染的293T细胞中敲减效果。结果：成功构建DPC4/Smad4 shRNA的慢病毒载体LVshSmad4,并成功制备DPC4/Smad4 shRNA慢病毒,三株病毒感染细胞后均具有有效的敲减效应,其中SH1 最为显著。结论：DPC4/Smad4 基因RNA干扰靶点的成功设计和RNA 干扰靶点慢病毒制备,为以后探讨DPC4/Smad4 基因与肿瘤的相关性治疗提供了实验基础。     

Molecular interaction between kisspeptin decapeptide analogs and a lipid membrane

Kisspeptin-10 is the C-terminal decapeptide amide of kisspeptin, an endogenous ligand for GPR54, and exhibits the same binding and agonist activity as the parent molecule. Although GPR54 is a membrane-embedded protein, details of the molecular interaction between kisspeptin-10 and lipid membranes remain unclear. Here, we performed a series of structural analyses using alanine-scanning analogs of kisspeptin-10 in membrane-mimetic medium. We found that there is a close correlation between lipid membrane binding and agonist activity. For instance, the F10A and non-amidated (NH₂ → OH) analogs showed little or no GPR54-agonist activity and elicited no blue shift in tryptophan fluorescence. NMR analysis of kisspeptin-10 analog in DPC micelles revealed it to contain several tight turn structures, encompassing residues Trp3 to Phe10, but no helical conformation like that seen previously with SDS micelles. Together, our results suggest that kisspeptin-10 may activate GPR54 via a ligand transportation pathway incorporating a lipid membrane.     

抑癌基因DPC4和TGF-β1在膀胱尿路上皮癌中的表达和意义

目的探讨膀胱尿路上皮癌中TGF-β1和DPC4的表达及其意义。方法收集武汉大学人民医院病理科2000-2006年有完整临床和病理资料的膀胱尿路上皮癌存档蜡块50例和5例癌旁组织,采用免疫组织化学S-P法检测50例膀胱与癌旁组织相比,差异有显著性(P<0.05);(2)DPC4在膀胱尿路上皮癌中呈低表达,癌旁组织中呈高表达。膀胱尿路上皮尿路上皮癌和5例癌旁组织中DPC4和TGF-β1的表达水平。采用HPIAS-1000高清晰度彩色病理图文报告管理系统,对DPC4和TGF-β1的表达进行定量分析,并用SPSS13.0软件对各组免疫组织化学反应阳性颗粒的平均光密度、阳性面积率做单因素方差分析和SNK(q)检验。结果 (1)TGF-β1在膀胱尿路上皮癌中呈高表达,癌旁组织中呈低表达。膀胱尿路上皮癌癌与癌旁组织相比,差异有显著性(P<0.05)。结论 TGF-β1高表达和DPC4低表达可能促进肿瘤细胞发生免疫逃逸。在膀胱尿路上皮癌的发生和发展中发挥了重要作用。     

A fluorescence study with polarised incident light of the compression of phospholipid monolayers spread at the air/water interface: orientation processes in the glycerol region

Monolayers of phospholipids spread at the air/water interface were studied by means of fluorescence measurements. Using linearly polarised incident light and following the behaviour of a fluorescent covalently-labelled phospholipid (dansylphosphatidylethanolamine) embedded in the monolayer, it was possible to obtain information about the orientation changes at the glycerol level of the phospholipid.When using dipalmitoylphosphatidylcholine as phospholipid, the main orientation of the probe appears unchanged during the phase transition process. On the other hand, the standard deviation of the distribution function of orientations is larger in the liquid-expanded state relative to the condensed state.When using phosphatidic acid or a mixture of dipalmitoylphosphatidylcholine and phosphatidic acid as phospholipids, the same orientation of the probe is observed as with pure dipalmitoylphosphatidylcholine. This orientation stays the same whatever the ionic content of the subphase, i.e. the ionisation state of the polar head.The glycerol region of a phospholipid spread in monolayer at the air/water interface appears unaffected structurally either by the nature of the polar moiety, by its ionisation state, or by the physical state of the hydrocarbon chains.     

Effect of DPC4 gene on invasion and metastasis of colorectal carcinoma cells

To investigate the effect of DPC4 gene on invasion and metastasis of colorectal carcinomacells,the expression of DPC4 was detected in sixty-three samples of colorectal tumors and seven cases ofcolorectal mucosa.The biological behavior of tumors expressing DPC4 was evaluated (including tumorstaging,differentiation degree and metastasis).pcDNA3.1-DPC4 plasmid was constructed and transferredinto HCT116 cells not expressing DPC4.The cell models (DPC4~ -HCT116) steadily expressing DPC4 wereobtained.Compared with HCT116 and pcDNA3.1-HCT116 cells,the doubling time of DPC4~ -HCT116 cellswas lengthened obviously (P<0.01),the apoptosis rate of DPC4~ -HCT 116 cells was significantly increased(P<0.01),the cloning efficiency,cell adherency,migration and invasion ability of DPC4~ -HCT116 cells weredropped obviously (P<0.01).The number of cancer nodules was decreased significantly in abdominal cavityand liver of the nude mice inoculated with DPC4~ -HCT116 cells.The activity of MMP-9 and MMP-2 wasdetected by gelatin zymography.In comparison with HCT116 and pcDNA3.1-HCT116 cells,the activity ofMMP-9 was decreased in DPC4~ -HCT116 cells.Therefore,the down-regulation of DPC4 expression may beassociated with the carcinogenesis of colorectal carcinoma.DPC4 may inhibit the proliferation of coloncancer cell by restraining growth and inducing apoptosis,and the invasion and metastasis of colorectalcarcinoma cells.MMP-9 may be one of the downstream target genes regulated by DPC4.