反向线性探针杂交技术检测慢性乙型肝炎病毒拉米夫定耐药研究

目的:建立起简单、快速、灵敏、准确的慢性乙型肝炎病毒拉米夫定耐药位点的反向线性探针(reverse line probe,RLP)检测方法.方法:根据HBV野生及耐药基因序列设计通用探针、3'、5'对称加有poly-C的特异性探针和5'标记生物素的扩增引物.将探针线性固定在硝酸纤维膜上,使HBV PCR扩增产物与探针进行杂交.通过优化杂交条件,建立RLP检测方法.利用该方法对重庆地区86个慢性乙肝病人进行检测,同时与直接测序结果比较.结果:半巢式PCR可对103拷贝/ml的血清样本进行有效特异扩增,新建的RLP检测方法可对PCR扩增产物在1 ng/ml以上,或血清样本中突变型DNA占野生型DNA比例为5％以上的均可有效检测,86例临床的检测灵敏度为100％,野生型和耐药型的检测准确性分别为98.09％(103/105)、100％ (43/43),与直接测序法比,RLP检测野生与耐药混合型准确性更好.结论:反向线性探针杂交检测方法检测HBV拉米夫定耐药位点方便、灵敏、准确,是HBV拉米夫定治疗有效的监控工具,该方法适合临床应用.     

基于连接酶检测反应的心血管相关基因多态性相关性研究

心血管疾病是世界上发病率高、危害大的一大类疾病。研究表明AGT M235T,ACE I/D,ApoE,LPL D9N,LPL N291S,FV R506Q基因多态性与心血管疾病有密切联系。本文就基于连接酶检测反应的心血管相关基因多态性相关性进行了研究。     

检测乙型肝炎病毒血清标志物四种方法学的分析

胶体金免疫层析法(GICT)、酶联免疫吸附试验(ELISA)、化学发光免疫测定法(CLIA)以及聚合酶链式反应(PCR)4种方法在HBV血清学检测中各有优势,但在实际工作中单独使用任何一种方法都有其局限性,只有联合检测乙肝标志物和HBV-DNA水平才能为临床医师的诊疗方案提供可靠依据。     

基于连接酶检测反应的并行分型系统检测AGT M235T和ACE I/D基因多态性

基因多态性蕴藏着大量遗传信息,分型技术是研究多态性的主要研究手段。本文建立了一个基于连接酶检测反应的基因多态性并行检测系统,该系统应用连接酶检测反应与基因芯片技术。对AGT M 235T 3种基因型和ACE I/D 3种基因型分型,同直接测序分型结果一致。将该系统应用于168份临床样本,经过统计学分析,用卡方检验发现高血压组和正常组AGT M 235T多态性存在显著差异(2χ=6.191,P<0.05),但ACE I/D多态性没有显著差异(χ2=5.241,P>0.05)。     

乙型肝炎病毒(HBV)DNA免疫的初步研究

用聚合酶链反应（ＰＣＲ）扩增了编码ＨＢＶｓＡｇ的基因序列,将其插入到ｐｃＤＮＡ３载体中,位于人巨细胞病毒（ＣＭＶ）早期启动子下游．重组质粒ｐｃＤＮＡ３－ｓＡｇ转染细胞后检测到ＨＢｓＡｇ表达．用纯化后的重组质粒直接注射到ＢＡＬＢ／Ｃ小鼠骨骼肌内,诱发实验小鼠产生了抗ＨＢｓＡｇ特异性抗体．ＰＣＲ扩增未检测到注射部位及肝脏细胞染色体中有外源ＨＢＶＤＮＡ整合     

用酶联免疫法检测乙型肝炎病毒表面抗原的安全模拟方法

用酶联免疫法检测乙型肝炎病毒表面抗原是医院常用的实验方法,也是医学检验专业学生必须掌握的酶免疫标记技术实验方法。做这个实验需要乙型肝炎病毒表面抗原阳性患者的血清和正常人的血清,乙型肝炎病毒表面抗原阳性患者的血清传染性很强,正常人的血清也不是十分安全,也可能有其它病毒。学生在做这个实验时,如果操作技术不熟练,自我安全保护意识差,就有被感染及污染实验室的可能。为了能够让学生安全地上好这个实验,我们采用了安全模拟方法。     

旱獭——乙型肝炎病毒及肝癌研究的良好的动物模型

<正> 绪言乙型肝炎是我国常见的一种传染病,具有传染性强、传播途径复杂、流行面广泛、发病率较高等特点,且部分乙型肝炎病人可成慢性肝炎并继发肝癌。在我国,原发性肝癌的发病率也很高,对人民挺康危害很大。所以,对乙型肝炎病毒(HBV)及肝癌的研究是摆在我     

连接酶反应介导的荧光策略用于高效检测尿嘧啶-DNA糖基化酶的活性

目的:灵敏检测尿嘧啶-DNA糖基化酶(UDG)活性有利于生物医学研究和疾病预后.这里,构建一种连接酶反应介导的荧光策略用于高效检测UDG活性.方法:在本策略中,两条短寡核苷酸链分别与发夹探针环部序列的一半杂交,形成含缺口的DNA复合物.在UDG作用下,发夹探针环部的单个U碱基被移除,产生无嘌呤/无嘧啶(AP)位点.AP位点能抑制缺口处的连接酶反应,使位于发夹探针末端的立足点和链迁移区域仍彼此邻近,从而引发杂交链式反应(HCR),产生大量G四倍体(G4)结构.最后,G4与N甲基卟啉IX(NMM)结合,产生增强的荧光信号.结果:本策略检测限低至0.000 20U/mL,并能区分UDG与其它DNA糖基化酶.结论:本策略为生物医学研究和疾病预后提供了一种有潜力的工具用于灵敏定量检测UDG活性.     

乙型肝炎病毒转基因小鼠外源基因的复制和传代稳定性观察

目的研究乙型肝炎病毒(HBV)转基因小鼠外源基因的复制、传代稳定性.方法通过nest-PCR和Southern分子杂交等检测方法,对48#、87#、90#三个系列中各15只整合阳性小鼠定期进行血清中HBV DNA存在情况以及对各系列繁育F1-F5代小鼠外源基因整合传代进行检测.结果各系列转基因鼠血清都有HBV DNA存在,并以1月龄时血清中HBV DNA阳性率最高,但随月龄改变,其DNA阳性率也变化,并存在系列、个体及月龄上的差异.结论HBV基因可在转基因小鼠体内复制、传代,且目前已稳定传至第五代(F5).     

Control of the SCF(Skp2-Cks1) ubiquitin ligase by the APC/C(Cdh1) ubiquitin ligase

Skp2 and its cofactor Cks1 are the substrate-targeting subunits of the SCF(Skp2-Cks1) (Skp1/Cul1/F-box protein) ubiquitin ligase complex that regulates entry into S phase by inducing the degradation of the cyclin-dependent kinase inhibitors p21 and p27 (ref. 1). Skp2 is an oncoprotein that often shows increased expression in human cancers; however, the mechanism that regulates its cellular abundance is not well understood. Here we show that both Skp2 and Cks1 proteins are unstable in G1 and that their degradation is mediated by the ubiquitin ligase APC/C(Cdh1) (anaphase-promoting complex/cyclosome and its activator Cdh1). Silencing of Cdh1 by RNA interference in G1 cells stabilizes Skp2 and Cks1, with a consequent increase in p21 and p27 proteolysis. Depletion of Cdh1 also increases the percentage of cells in S phase, whereas concomitant downregulation of Skp2 reverses this effect, showing that Skp2 is an essential target of APC/C(Cdh1). Expression of a stable Skp2 mutant that cannot bind APC/C(Cdh1) induces premature entry into S phase. Thus, the induction of Skp2 and Cks1 degradation in G1 represents a principal mechanism by which APC/C(Cdh1) prevents the unscheduled degradation of SCF(Skp2-Cks1) substrates and maintains the G1 state.     

液体分布环对喷淋塔中烟气流场影响的数值模拟

湿法烟气脱硫是目前广泛使用的烟气脱硫技术.喷淋塔是湿法脱硫系统的核心设备,它的优化设计对于提高脱硫效率至关重要.通过对南京工程学院湿法烟气脱硫系统喷淋塔的数值模拟,分析了液体分布环(LDR)对塔内烟气流场的影响.研究结果对喷淋塔的优化设计具有一定的指导作用和实际意义.     

Multiplex detection of KRAS and BRAF mutations using cationic conjugated polymers

The mutation detections of KRAS and BRAF genes are of significant importance to predict the responses to anti-cancer therapy and develop new drugs. In this paper, we developed a multi-step fluorescence resonance energy transfer (FRET) assay for multiplex detection of KRAS and BRAF mutations using cationic conjugated polymers (CCP). The newly established detection system could detect as low as 2% mutant DNAs in DNA admixtures. By triggering the emission intensity change of CCP and the dyes labeled in the DNA, four possible statuses (three mutations and one wildtype) can be differentiated in one extension reaction. The detection efficiency of this new method in clinical molecular diagnosis was validated by determining KRAS and BRAF mutations of 51 formalin-fixed paraffin-embedded (FFPE) ovary tissue samples. Furthermore, the result of the CCP-based multi-step FRET assay can be directly visualized under UV light so that no expensive instruments and technical expertise are needed. Thus, the assay provides a sensitive, reliable, cost-effective and simple method for the detection of disease-related gene mutations.     

cDNA cloning and functional analysis of 4-coumarate --CoA: ligase (4CL) gene in Chinese white aspen

cDNA encoding 4-coumarate: CoA ligase (4CL) was isolated from the secondary developing xylem of Chinese white aspen (Populus tomentosa) by RT-PCR for the first time, which was 1619 bp in length. The coding sequence and putative amino acid sequence showed 97.53% and 97.00% identity to that of Pt4CL1 in quaking aspen (P. tremuloids), respectively. Molecular analysis indicated that 4CL was encoded by multiple genes in P.tomentosa, its mRNA was highly accumulated in xylem and the expression of 4CL revealed a biphasic pattern in one growing season, almost in phase with the expression of other related enzymes in lignin biosynthesis. Transgenic research showed that expression of antisense 4CL cDNA led to the decreasing of lignin content in transgenic tobaccos, among which the average reduction was 10.3% and the highest could be up to 18.9%. These data suggested that 4CL gene was a potential gene used in altering lignin biosynthesis by biotechnology for producing new materials of papermaking.     

Polymerization reaction in restricted space of layered double hydroxides （LDHs）

This paper reported the preparation of styrene sulfonate intercalated layered double hydroxides (LDHs)material, SS-LDHs by coprecipitation method, followed by in-situ polymerization of the monomers in the interlayer space of LDHs. The polymerization reaction was monitored by UV and NMR. It is confirmed that when the reaction occurred at 100℃ for 24 h, part of monomers did not react. When the reaction was carried out at 150~C, the polymerization of the intercalated monomers is complete to afford the polymer intercalated product PSS-LDHs. During the polymerization process, the layered structure remains well. At the same time the gallery height increases with the lengthening of reaction time. This is preliminarily because that the PSS becomes more swelling with the amount of water it absorbs due to its hygroscopicity property.     

(S)-N-(2-氨基苯基)四氢吡咯-2-甲酰胺的合成及其在直接不对称Aldol反应中的应用

以(S)-Pro1ine为原料三步合成(S)-N-(2-氨基苯基)四氢吡咯-2甲酰胺1，总产率64．25％．并用于催化直接不对称Aldol反应，产率65％～84％，ee值26．5％～40．7％．     

吸附强化异构反应过程分析

提出以吸附强化可逆异构反应的变压循环过程,构建了平衡条件下的模型,考虑了线性和扩展Langmuir两种类型的吸附等温线。根据模型计算反应所能达到的转化率,结果表明脱附物料中反应物的转化率显著高于反应平衡转化率。考察了脱附物料转化率的变化规律,当采用线性吸附等温线时,只有选择性系数、吸附压力和脱附压力的取值对转化率有影响：脱附物料转化率随选择性系数增大而增大,极限值为1;脱附物料转化率随吸附压力增大而增大,极限值为α_B/AK_p/（1+α_B/AK_p）。当采用扩展Langmuir等温线时,饱和吸附量Q_m的取值不影响脱附物料转化率,而Langmuir常数b_B的取值对其有影响。此外还分析了气相与吸附相物料相对比例、脱附过程逆向反应对强化效果的影响。     

利用不对称交叉偶联反应合成S-(+)-萘普生

用β-氨基烷基膦的NiCl2配合物为手性催化剂,以6-甲氧基-2-溴萘和a-溴代丙酸酯的有机锌试剂为原料,通过不对称交叉偶联反应合成S-(+)-萘普生.     

胡椒基类化合物与苯醌光反应的CIDNP研究

光照的¹H化学诱导动态核极化(CIDNP)技术用于研究胡椒基类化合物与苯醌的光化反应。Photo-CIDNP谱上胡椒基的亚甲基质子(—O—CH₂—O—)在氘乙腈中产生发射极化信号,在氘苯中产生增强吸收信号,证实了在乙腈和苯中分别形成“溶剂分离离子化自由基对(SSIP)”和中性的半醌-苯并间二氧杂环戊烯自由基对(RP)。在氘苯中,产物三苯基原甲酸酯衍生物的次甲基质子的异常增强吸收,表明该产物由中性自由基对(RP)形成。