猪多杀性巴氏杆菌检测技术研究进展

猪多杀性巴氏杆菌(Pasteurella multocida,Pm)是导致猪呼吸系统疾病的一类重要病原菌,给世界养猪业带来了巨大的经济损失。准确、敏感、快速的Pm检测方法有助于在临床上了解Pm的流行情况,从而采取相应的预防、治疗和综合防控措施。对Pm的病原学、分子生物学、免疫学及分子分型方法的研究现状、原理和优缺点等进行综述,为进一步建立Pm标准检测方法提供参考。     

河北省小麦品种和种质资源抗白粉病鉴定与抗病基因分子标记检测

白粉病是河北省小麦生产的重要常发病害,明确小麦审定品种和高代品系中所携带的抗病基因对合理利用和布局已知抗源、实现对小麦白粉病的有效防控具有重要意义。本研究结合人工接种白粉病菌株E09和E20与抗病基因连锁(或共分离)标记对1956-2018年间河北省371份小麦材料(含审定品种256份、高代品系115份)进行苗期抗白粉病鉴定和抗病基因检测。结果表明:供试材料中,抗E09的材料占6.2%,抗E20的占11.9%,兼抗两个菌株的材料占4.9%;部分材料携带Pm1c、Pm2、Pm4b、Pm21、Pm24和Pm35基因,未检测到Pm12基因。Pm8基因在供试材料中所占比例较高,接近50%。供试材料中抗病审定品种比例远大于高代品系,说明小麦抗白粉病种质创新仍为当务之急,需要引起重视。在用连锁或共分离标记进行抗病基因检测时,通过计算某基因对两个菌株抗病反应型与标记检测结果一致的材料比例,发现Pm12、Pm21和Pm35等基因的标记检测效率较高,同时这些基因的标记也方便使用,可优先考虑用这些标记检测目的基因。     

浸麻类芽孢杆菌葡甘露聚糖降解酶的异源表达及功能研究

【目的】克隆表达浸麻类芽孢杆菌(Paenibacillus macerans)的葡甘露聚糖降解酶,研究其性质和功能,丰富葡甘露聚糖降解酶资源,了解浸麻类芽孢杆菌降解葡甘露聚糖机制。【方法】检索浸麻类芽孢杆菌的葡甘露聚糖降解酶基因,构建重组菌株,表达纯化重组酶,系统研究其功能及在降解葡甘露聚糖中的作用。【结果】克隆表达了5个葡甘露聚糖降解酶组分。结果显示Pm Man1和Pm Man2为内切β-甘露聚糖酶,Pm Glc1、Pm Glc2和Pm Glc3为外切β-葡萄糖苷酶。其中Pm Glc1只能水解p NPβGlc,Pm Glc2能水解二糖和人参皂苷的β-1,6-葡萄糖苷键,而Pm Glc3对β-葡萄糖苷键的选择性较为广泛。Pm Man1、Pm Man2、Pm Glc2和Pm Glc3能够降解葡甘露寡糖,Pm Man1和Pm Man2可以降解葡甘露聚糖。共同降解葡甘露聚糖时,Pm Glc2和Pm Glc3与Pm Man2具有协同效应,且Pm Glc3与Pm Man2的协同作用更为显著。【结论】从浸麻类芽孢杆菌中获得了4种葡甘露聚糖降解酶,阐明了该菌葡甘露聚糖降解酶系成员的作用,丰富了酶资源...     

54份CIMMYT小麦材料抗白粉病分析

选用来自我国不同地区的20个白粉病菌毒性菌株,对54个CIMMYT小麦品种(系)进行抗病性分析.结果表明:(1)34个品种(系)含有抗病基因,以Pm8基因出现频率最高,有15个品种(系)携带该基因;(2)参试主效基因中,Pm1、Pm3e、Pm5、Pm6和Pm7基因已丧失对我国白粉菌的抗性,Pm16和Pm20基因的抗性最强;(3)50个1B/1R易位系品种(系)中31个含有抗病基因,48%的抗病1B/1R易位系可检测到Pm8基因.根据田间成株期病程曲线下面积(AUDPC)聚类分析结果,可将54份材料分为高抗、中抗、中感和高感4类,7个品种(系)不含任何主效抗病基因而田间表现中到高的抗性,是典型慢病性品种.     

猪链球菌和猪多杀性巴氏杆菌多重实时荧光PCR方法的建立

【背景】猪链球菌(Streptococcus suis,SS)和猪多杀性巴氏杆菌(Pasteurella multocida,Pm)都是能引起宿主致病的人畜共患病原菌,常出现混合感染,临床诊断上易与猪瘟、猪丹毒等混淆。【目的】快速、有效鉴别猪链球菌病和猪多杀性巴氏杆菌病,建立一种能同时检测2种病原的多重实时荧光定量PCR检测方法。【方法】基于猪链球菌的gdh基因和猪多杀性巴氏杆菌的plpE基因,设计2对特异引物及TaqMan探针,以细菌16S rRNA基因设计通用引物及探针,通过对反应条件优化,建立了一种能同时检测猪链球菌和猪多杀性巴氏杆菌的多重实时荧光定量PCR检测方法。【结果】该方法能够特异性地检测猪链球菌和猪多杀性巴氏杆菌,与细菌分离后的测序结果验证完全一致。此方法对重组质粒标准品的最低检出浓度分别为4.53×10²copies/μL和3.97×10²copies/μL。重复性试验结果显示,该方法的组内和组间变异系数均小于3%。【结论】本实验所建立的方法准确、简便、可靠,能够用于2种病原菌的同时检测,为猪链球菌病和猪多杀性巴氏杆菌病的防治提供了有效的检测工具,具有重要的流行病学意义和临床应用价值。     

马氏珠母贝(Pinctada fucata martensii)PmHEX基因的功能研究

几丁质是软体动物贝壳有机框架的重要成分,其代谢在贝壳矿化中发挥重要作用。β-N-乙酰-己糖胺酶(HEX, EC3.2.1.52)是几丁质代谢的关键水解酶。为了探究马氏珠母贝β-N-乙酰-己糖胺酶(Pm HEX)(登录号:MF555152)在贝壳形成中的作用,本研究利用原位杂交(ISH)技术检测Pm HEX基因在外套膜的定位,结果显示Pm HEX的mRNA主要分布于外侧褶的外上皮细胞、中褶的内侧上皮细胞和内褶上皮细胞。利用RNAi技术抑制Pm HEX表达后,Pm HEX在边缘区和套膜区的表达量均显著下调;SEM观察发现实验组的棱柱层和珍珠层的微观结构都出现不同程度的紊乱。综上所述,Pm HEX可能通过影响几丁质代谢,参与马氏珠母贝贝壳棱柱层和珍珠层的矿化过程。     

小麦白粉病抗性基因的聚合及其分子标记辅助选择

采用了在早代进行抗性鉴定、淘汰感病株、保留抗病株继续种植、较晚世代（F4代）进行抗性鉴定结合分子标记辅助选择的策略,提高了选到聚合抗性植株的效率。利用与Pm2、Pm4α、Pm8、Pm21紧密连锁或共分离的RFLP标记和PCR标记（SCAR标记）,对含有这些基因的优良品系间配制的杂交组合的F4代进行了分子标记辅助育种选择,并结合抗性鉴定,筛选到14株Pm4α Pm2I的植株,16株Pm2 Pm4α的植株,6株Pm8 Pm21的植株。应该引起注意的是,Pm2 Pm4α对混合白粉病菌的抗性达到高抗至免疫水平,而Pm2和Pm4α单独存在时抗性较差,表明聚合抗病基因植株的抗性提高了,为培育具有持久性抗性的品系或品种提供了新思路,它在实践和理论研究上都将具有重要意义。     

马氏珠母贝磺基转移酶PmCHST9基因的分子特征与表达分析

糖胺聚糖在抗病毒、消炎和抗氧化等免疫反应发挥重要作用。磺基转移酶(sulfotransferase)是糖胺聚糖生物合成的关键酶,Carbohydrate sulfotransferase 9(CHST9)可以将磺酸基从3'-磷酸腺苷酰-5'磷酸硫酸盐(PAPS)转移到半乳糖残基非还原末端的4位碳上。为研究CHST9在马氏珠母贝免疫调节中的作用,本研究克隆得到马氏珠母贝CHST9(Pinctada martensii CHST9,Pm CHST9)c DNA全长序列并检测其在不同组织中的表达模式。利用RACE技术,得出Pm CHST9序列全长1 388 bp,其中5'UTR为122 bp,3'UTR为192 bp,开放阅读框(ORF)为1 074 bp,编码357个氨基酸;Pm CHST9氨基酸序列分子量为42 889.2 Da,等电点为9.28,脂溶性系数为81.32,总平均亲水性为-0.493;氨基酸序列分析得出Pm CHST9具有典型的磺基转移酶-2结构域和一个跨膜结构域;多序列比对结果显示Pm CHST9与其它物种的CHST9同源性较低;荧光定量PCR检测结果表明Pm CHST9在鳃中表达量最高,其次是足和肝胰腺。综上所述,Pm CHST9可能参与马氏珠母贝的免疫调节作用,为进一步阐述Pm CHST9在马氏珠母贝中的免疫机制提供基础资料。     

实验用小型猪呼吸道支原体检测及方法比较

目的通过常用的三种不同方法对支原体的检测,了解实验用小型猪支原体感染情况,为今后实验用小型猪支原体检测方法国家标准的制定提供参考。方法采用培养法、PCR和ELISA方法分别对20头小型猪的气管、肺和血清进行检测。结果三种检测方法中,PCR方法支原体阳性检出率为15%,ELISA方法为20%,而培养法结果均为阴性。结论目前在普通级小型猪中存在支原体的感染。检测方法中PCR和ELISA方法较培养法更省时,敏感性更高。     

马氏珠母贝(Pinctada fucata martensii)PmHEXL基因的分子特征与表达分析

几丁质作为有机框架主要成分参与贝壳的形成。β-N-乙酰-己糖胺酶(Beta-hexosaminidase/Beta-N-acetylhexosaminidase, HEX)属于糖苷水解酶家族20,在几丁质水解过程发挥重要作用。为了探究Pm HEXL在马氏珠母贝贝壳形成中的作用,本研究利用RACE技术克隆获得Pm HEXL基因c DNA全长序列并检测其在不同组织的表达模式。研究显示,Pm HEXL基因序列全长2 760 bp,其中5'UTR为167 bp,3'UTR为268 bp,开放阅读框(ORF)为2 325 bp,编码774个氨基酸;预测其相对分子量为89.59 kD,理论等电点为5.93;SMART软件分析PmHEXL蛋白质序列,发现它具有典型的GH20、GH20b结构域和CHB-HEX结构域;多序列比对结果显示Pm HEXL与其它物种的HEX具有较高的保守性;qPCR表达分析显示Pm HEXL在外套膜套膜区表达量最高,边缘区次之。综上所述,Pm HEXL可能参与马氏珠母贝贝壳的形成过程。     

Structure-based design,synthesis and biological evaluation of a newer series of pyrazolo[1,5-a]pyrimidine analogues as potential anti-tubercular agents

In-depth study of structure-based drug designing can provide vital leads for the development of novel, clinically active molecules. In this present study, twenty six novel pyrazolo[1,5-a]pyrimidine analogues (6a-6z) were designed using molecular docking studies. The designed molecules were synthesized in good yields. Structural elucidation of the synthesized molecules was carried out using IR, MS, ¹H NMR and ¹³C NMR spectroscopy. All the synthesized compounds were evaluated for their in-vitro anti-tubercular activity against H37Rv strain by Alamar Blue assay method. Most of the synthesized compounds displayed potent anti-tubercular activities. Amongst all the tested compounds 6p, 6g, 6n and 6h exhibited promising anti-tubercular activity. Further, these potent compounds were gauged for MDR-TB, XDR-TB and cytotoxic study. None of these compounds exhibited potent cytotoxicity. Stability of protein ligand complex was further evaluated by molecular dynamics simulation for 10 ns. All these results indicate that the synthesized compounds could be potential leads for further development of new potent anti-tubercular agents.     

分子印迹技术用于生物大分子的识别

分子印迹技术是一种人工合成具有分子识别功能的介质的一种新技术,近年来在许多领域都得到很大的发展。本文介绍了分子印迹技术的发展现状,尤其对生物大分子的分子印迹技术进行了详细论述,对生物大分子印迹采用的功能单体、印迹分子的种类、印迹的方法、印迹的机理、存在的问题和应用的前景等分别进行了讨论。     

From complexation to computation: Recent progress in molecular logic

The new thrusts in molecular logic are gathered together in this short review, while paying attention to the seeds from which these developments have arisen. The original demonstration of a few basic logic operations has now been extended to cover many of the one- and two-input varieties and even some of the three-input types. Many kinds of inputs and outputs have emerged, including various chemical species and some physical properties. The latter can include heat, light and, arguably, polarity. Reconfigurable logic has grown up to include a range of examples. Even superposable logic has proved possible with molecular systems. Numerical processors have flowered in recent years with several diverse approaches being revealed in recent years. Photochemical concepts such as photoinduced electron transfer (PET), internal charge transfer (ICT) and electronic energy transfer (EET) can be discerned among the designs in the field.     

分子标记及其在鸟类分子生态学研究中的应用

分子生态学是一门相当新的分支学科,从前人的研究工作来看,分子标记在这个学科中应用非常广泛,本文描述了RFLP、RAPD、MinisatelliteDNA,MicrosatelliteDNA和AFLP这5个分子标记的优缺点及其在鸟类分子生态学中的应用和研究概况。     

放线菌的分子分类

在放线菌分类学研究中,最初是根据形态特征、生理生化特征等表观分类学特征进行研究。随着分子生物学的飞速发展,放线菌的分类鉴定亦从传统的表型分类进入到各种基因型分类水平。分子分类在放线菌分类研究中起到越来越重要的作用,目前放线菌的分子分类研究主要包括:G Cmol%测定、DNA杂交、核酸结构分析以及DNA指纹图谱分析等方面。     

Ab initio Molecular Electrostatic Potential Grid Maps for Quantitative Similarity Calculations of Organic Compounds

The program MolSim designed to calculate the similarity of different molecules quantitatively in a fast and easy way is described. The molecular similarity is estimated for the molecular shape as well as for the electrostatic potentials of the molecules derived from ab initio calculations. A grid-based method is used to determine the steric and electrostatic similarities between a lead compound and the corresponding test set by calculating the Spearman correlation coefficient. The superpositioning of the molecules was accomplished with the SEAL algorithm incorporating a Monte Carlo simulated annealing approach while preserving the conformational flexibility of the calculated structures.The ability of the program was tested on a set of Sandalwood odour compounds, a class of substances that is difficult to analyse with respect to its structure-activity relationship because of the structural diversity of Sandalwood odour compounds, in contrast to their high selectivity and pronounced structural specificity. The application of the program on a small test set of these compounds showed that the program is able to explain the Sandalwood odour activity correctly.Electronic Supplementary Material available.     

The expanding small heat-shock protein family,and structure predictions of the conserved “α-crystallin domain”

The ever-increasing number of proteins identified as belonging to the family of small heat-shock proteins (shsps) and -crystallins enables us to reassess the phylogeny of this ubiquitous protein family. While the prokaryotic and fungal representatives are not properly resolved, most of the plant and animal shsps and related proteins are clearly grouped in distinct clades, reflecting a history of repeated gene duplications. The members of the shsp family are characterized by the presence of a conserved homologous -crystallin domain, which sometimes is present in duplicate. Predictions are made of secondary structure and solvent accessibility of this domain, which together with hydropathy profiles and intron positions support the presence of two similar hydrophobic -sheet-rich motifs, connected by a hydrophilic -helical region. Together with an overview of the newly characterized members of the shsp family, these data help to define this family as being involved as stable structural proteins and as molecular chaperones during normal development and induced under pathological and stressful conditions.Correspondence to: W.W. de Jong     

分子探针在细胞凋亡检测中的研究进展

分子探针应用于细胞凋亡检测具有直观、无创伤和实时动态观察凋亡分子信息的特点,在疾病早期诊断、监测疾病发展进程、评估疾病治疗效果、发展新的治疗方案等方面具有较好的应用前景.综述了近年来靶向细胞凋亡探针的设计、作用机制、应用等相关的研究进展,并根据亲和组件的作用机制对分子探针进行分类介绍.     

分子印迹技术研究新进展

分子印迹技术是在近十几年来才发展起来的一门边缘科学技术。它结合了高分子化学、生物化学等学科 ,是模拟抗体 -抗原相互作用的一种新技术 ,具有选择性识别位点的性质。现已应用于色谱分离、抗体和受体模拟物、固相萃取、生物传感器等领域。