苏云金芽孢杆菌Cyt蛋白研究进展

苏云金芽孢杆菌 (Ｂａｃｉｌｌｕｓｔｈｕｒｉｎｇｉｅｎｓｉｓ,简称Ｂｔ)是目前世界上应用最广泛、最成功的微生物杀虫剂 ,也是公认的无公害生物农药。苏云金芽孢杆菌最主要的杀虫活性物质是杀虫晶体蛋白 (ＩｎｓｅｃｔｉｃｉｄａｌＣｒｙｓ ｔａｌＰｒｏｔｅｉｎｓ,ＩＣＰｓ) ,包括晶体蛋白 (Ｃｒｙ)和Ｃｙｔ蛋白 (Ｃｙｔ)两大类。这两类蛋白在氨基酸序列及其基因同源性上相距甚远 ,但它们均能在靶标害虫中肠内被激活成毒素 ,并在昆虫中肠细胞膜上形成孔道 ,进而引起中肠细胞胶样渗透裂解 (ｃｏｌｌｏｉｄ ｏｓｍｏｔｉｃｌｙｓｉｓ) ,最…     

苏云金芽孢杆菌cry基因研究进展

从cry基因的分类、cry基因与转座因子的关系、cry基因的表达调控以及cry基因的作用机理等 4个方面综述了cry基因的研究进展 ,并简要展望了其研究和应用前景。     

苏云金芽孢杆菌质粒的检测及功能的初步研究

用改进的酶碱综合法检测了苏云金芽孢杆菌4个亚种11个野生菌株的内生质粒,获得良好的制备结果和重视性,对野生菌株YBT－1463及其无质粒突变株BMB171的形态和生理生化特性的比较研究结果表明,YBT－1463的内生质粒携带杀虫晶体蛋白基因,但不携带抗生素的抗性基因,且与该菌株对19种C源和12种N源的利用无关。     

苏云金芽孢杆菌cry2Aa操纵元蛋白对杀虫蛋白晶体形成作用的研究

利用重叠PCR的方法,通过两次PCR扩增,分别获得cry2A10操纵元的orf1、orf1 orf2与cry2Ab5基因的融合片段。融合片段经BamHⅠ和EcoRⅠ双酶切与pHT315连接,分别构建了基因融合片段的原核表达载体pFU(orf1 2Ab)和pFU(orf1 orf2 2Ab),电转化Bt无晶体突变株4Q7后,扫描电镜下可观察到典型的方形晶体,通过SDS-PAGE可检测到60kD大小的蛋白表达带。结果表明,cry2Ab5可在cry2a0的启动子帮助下有效转录和表达,并在orf2产物帮助下形成蛋白晶体。     

苏云金芽孢杆菌菌株YBT1535转cry1C基因菌的构建

利用电脉冲将ｃｒｙ１Ｃ基因转子苏云金孢杆菌野生菌株ＹＢＴ１５３５,筛选得到３个转化子。质粒电泳、ＰＣＲ扩增及Ｓｏｕｔｈｅｒｎ杂交结果均证明,基因ｃｒｙ１Ｃ已转入菌株ＹＢＴ１５３５。生物测定结果表明,３个转化子对甜菜夜蛾的毒力比出发菌ＹＢＴ１５３５均有显著的提高,转化子ＹＢＴ１５３５－１和ＹＢＴ１５３５－３对小菜蛾和棉铃虫的生物活性与出发菌ＹＢＴ１５３５相近,而转子化子ＹＢＴ１５３５－２则有一定幅度     

苏云金芽孢杆菌几丁质酶的研究进展

苏云金芽孢杆菌制剂作为无公害农药已经得到社会的认可,如果再开发其几丁质酶抑制真菌和杀虫增效功能,不仅可充分利用这一农业微生物菌种资源,也将给予传统生物农药以新的生命力。综述了苏云金芽孢杆菌几丁质酶方面研究的国内外最新进展。     

中国苏云金芽孢杆菌的分布与cry基因的多样性

采集全中国27个省、自治区及4个直辖市昆虫孳生地粉尘、土壤等样品1080份,在其中的406份中分离到苏云金芽孢杆菌965株.镜检可观察到大菱形、小菱形、方形、长方形、圆形、椭圆形、镶嵌形和不规则形等8种主要形态的伴孢晶体;采用cryⅠ、cryⅡ、cryⅢ、cryⅣ和cryⅤ基因的通用引物对221株Bt分离株进行的PCR检测结果表明各类基因的含量依次为cryⅠ＞cryⅡ＞cryⅤ＞cryⅢ基因,分别占被检菌株的75.6%、67.9%、58.4%和14.5%,没有检测到cryⅣ基因,共得到10种基因组合类型.对其中含有cryⅠ基因的菌株分别以cryIAc、cryIC和cryIE基因的特异性引物进行PCR检测,得到20株同时含有cryIAc、cryIC、cryⅡ和cryⅤ优良基因组合的Bt分离株,其中菌株Bt-15A3对棉铃虫、甜菜夜蛾及小菜蛾均表现出高毒力,具有生产开发潜力.     

球形芽孢杆菌Mtx1蛋白和苏云金杆菌Cyt1Aa晶体蛋白的协同作用

采用常规的生物测定方法确定了纯化的球形芽孢杆菌(Bacillus sphaericus)的缺失信号肽的97kDa营养期杀蚊毒素(Mosquitocidal toxin 1,Mtx1)蛋白和苏云金芽孢杆菌(Bacillus thuringiensis)27.3kDa的Cyt1Aa晶体蛋白对致倦库蚊(Culex quinquefasciatus)幼虫的杀虫活性。结果表明Mtx1和Cyt1Aa不同比例的混合物对致倦库蚊的毒力比单独毒素蛋白高,经统计分析表明两毒素蛋白对目标蚊幼虫具有明显的协同作用。在LC98处理浓度下,Mtx1和Cyt1Aa按3∶1混合的混合物LT50值比单独Mtx1的提前了6.36h。表明Cyt1Aa和Mtx1对致倦库蚊具有协同毒杀作用,提高对目标蚊虫的毒力、缩短半致死时间。该结果为深入研究Mtx1和Cyt1Aa的杀蚊作用方式奠定了基础,同时为其在蚊虫防治中的应用提供了新的思路和方法。     

苏云金芽孢杆菌cry1Ab13基因的克隆及表达研究

BtC0 0 5是我国自行分离的对多种害虫具有毒杀作用的苏云金芽孢杆菌 ,经PCR RFLP系统鉴定 ,它含有cry1Ab基因。Southernblot结果显示 :PstI酶切C0 0 5质粒所得的 8 5kb长的DNA片段为cry1Ab基因的阳性杂交带。以pUCP1 9为载体 ,克隆了该片段并证明其含有cry1Ab基因。对其进行亚克隆和测序 ,结果表明该基因编码区为 3 4 6 8bp ,其编码的蛋白含1 1 5 5个氨基酸 ,分子量为 1 3 0 6kD ,等电点为pH4 845。该基因已在GenBank基因库中注册 ,Accessionnumber为AF2 5 4 6 4 0 ,并为国际Bt杀虫晶体蛋白基因命名委员会正式命名为cry1Ab1 3。将cry1Ab1 3基因在Bt无晶体突变株cryB- 中表达 ,蛋白质电泳结果表明在 1 3 0kD处有表达带 ,并证明CryAb对小菜蛾有较高的杀虫活性。     

苏云金芽孢杆菌新型营养期杀虫蛋白Vip3A研究

营养期杀虫蛋白（Vegetative Insecticidal Protein,Vip）Vip3A是苏云金芽孢杆菌（Bacillus thuringiensis,Bt）在营养生长对数中期分泌产生的一类新型杀虫蛋白。Vip3A广泛存在于Bt中,与晶体杀虫蛋白（Insecticidal Crystal Proteins,ICPs）没有任何同源性,不具有多样性,在遗传上比较保守。其N-端存在一趋化信号传导因子相似序列,C-端存在着纤维素结合结构域。Vip3A蛋白通过诱发细胞凋亡,最终导致细胞死亡,这与Bt δ-内毒素的作用机理完全不同。本文主要对Vip3A杀虫蛋白的发现、特性及作用机理作一简要综述。     

利用表面等离子体共振仪检测黄瓜花叶病毒

目的：研究一种便捷、高效地检测黄瓜花叶病毒（CMV）的方法。方法：利用表面等离子体共振（SPR）技术检测CMV。首先用11-MUA修饰SPR金片,再用EDC／NHS活化,之后通过NHS酯基与CMV抗体结合,用BSA封闭未结合的NHS酯基。将SPR金片装入SPR仪,通入待检样品,通过折射率变化实时监测实验过程。结果：该方法检测CMV的灵敏度能够达到10ng／mL,具有良好的特异性,与同属的花生矮化病毒、番茄不孕病毒无交叉反应。结论：建立的SPR方法操作简单、灵敏度高、特异性好,是一种新的高效检测CMV的方法。     

Functional Demonstration of Connexin—Protein Binding Using Surface Plasmon Resonance

Surface plasmon resonance (SPR) allows examination of protein-protein interactions in real time, from which both binding affinities and kinetics can be directly determined. We have used the SPR technique to search for proteins in heart tissue that would be candidate binding partners for the cardiac gap junction protein, connexin43 (Cx43). Heart lysate showed a strong, pH-dependent binding to the carboxyl terminus (CT) of Cx43 (amino acids 254-382) covalently linked to an SPR cuvette. Binding was inhibited by the presence of v-src transfected 3T3 cell lysate, suggesting that binding partners in these two lysates may compete for overlapping epitopes on Cx43CT. The combined application of proteomic and functional studies is expected to identify which proteins within heart tissue interact with Cx43 and what roles they may play in gap junction function.     

表面等离子体激元共振与生物分子相互作用分析

对表面等离子体激元共振（surface plasmon resonance, SPR）的原理和在生物学研究方面的应用进行了综述.这种技术可以直接原位、实时地跟踪生物学实验研究系统,而不需要附加参数如进行标记等手段,具有高敏感性,也可以连续监测吸附或解吸附过程,目前有关的应用涉及到生物学结合分析、动力学及亲和力测定、免疫识别研究、结构与活性研究和核酸研究等多个领域.     

偏振干涉角度调制SPR传感技术研究及应用进展

针对一种新兴生物检测方法——表面等离子体波共振(SPR)技术,文中SPR传感系统采用偏振干涉和角度调制方案,使SPR传感灵敏度与光复反射系数的模和相位都相关,从而实现较大线形范围内的高灵敏测量。同时开展了该SPR传感系统在环保领域的应用研究,SPR共振信号可实时随甲烷含量线性改变,气体检测灵敏度达到1 070ppm,实验结果验证了这种SPR传感技术的检测性能并显示了其在环保监测领域的应用潜力。     

Sensitive and Label-Free Detection of DNA by Surface Plasmon Resonance

While an array of technologies based on radioactive labels or luminescent tags are dominant in modern biomedical research on DNA, surface plasmon resonance (SPR) and SPR imaging measurements are sensitive, rapid, and label-free. This review summarizes recent advances in the development of SPR and coupled techniques and their applications in DNA research, including the gene analysis at trace levels and studies of DNA–protein and DNA–drug interactions.     

Measuring the Binding Stoichiometry of HIV-1 Gag to Very-Low-Density Oligonucleotide Surfaces Using Surface Plasmon Resonance Spectroscopy

The interaction of the HIV Gag polyprotein with nucleic acid is a critical step in the assembly of viral particles. The Gag polyprotein is composed of the matrix (MA), capsid (CA), and nucleocapsid (NC) domains. The NC domain is required for nucleic acid interactions, and the CA domain is required for Gag-Gag interactions. Previously, we have investigated the binding of the NC protein to d(TG)(n) oligonucleotides using surface plasmon resonance (SPR) spectroscopy. We found a single NC protein is able to bind to more than one immobilized oligonucleotide, provided that the oligonucleotides are close enough together. As NC is believed to be the nucleic acid binding domain of Gag, we might expect Gag to show the same complex behavior. We wished to analyze the stoichiometry of Gag binding to oligonucleotides without this complication due to tertiary complex formation. We have therefore analyzed Gag binding to extremely low oligonucleotide density on SPR chips. Such low densities of oligonucleotides are difficult to accurately quantitate. We have determined by Fourier transform ion cyclotron (FTICR) mass spectrometry that four molecules of NC bind to d(TG)(10) (a 20-base oligonucleotide). We developed a method of calibrating low-density surfaces using NC calibration injections. Knowing the maximal response and the stoichiometry of binding, we can precisely determine the amount of oligonucleotide immobilized at these very-low-density surfaces (<1 Response Unit). Using this approach, we have measured the binding of Gag to d(TG)(10). Gag binds to a 20-mer with a stoichiometry of greater than 4. This suggests that once Gag is bound to the immobilized oligonucleotide, additional Gag molecules can bind to this complex.     

Enantioselective Surface Plasmon Resonance Sensor Based on C60 Fullerene‐Glutathione Self‐Assembled Monolayer (SAM)

A novel enantioselective surface plasmon resonance (SPR) sensor based on a self‐assembled monolayer of C₆₀ fullerene as the chiral selector is proposed. A binding assay, apparent affinity constant, and apparent dissociation binding constant were used to analyze and study the enantioselectivity of C₆₀ fullerene‐glutathione film for L‐histidine, which was chosen as the model analyte. The apparent affinity constant for the complex formed by L‐histidine with C₆₀ fullerene‐glutathione film was 5.2 x 10⁹ M^‐1. The proposed SPR sensor can be used for the assay of L‐histidine in the 10^‐10 – 10^‐7 mol/L concentration range. Chirality 26:129–131, 2014. © 2014 Wiley Periodicals, Inc.     

At-line monitoring of bioreactor protein production by Surface Plasmon Resonance

An innovative and automated method for the at-line monitoring of secreted protein was developed by harnessing a Surface Plasmon Resonance-based biosensor to a bioreactor. The proof of concept was performed by following at-line the relative concentration of a secreted protein produced by transient transfection of mammalian cells in a bioreactor. Our results suggest that our approach can be readily applied to the at-line determination of both protein concentration and bioactivity. Our experimental setup and strategy can thus satisfy the needs related to the development of novel bioprocess control protocols in the context of the new process analytical technology that arises in the biopharmaceutical industry.     

基于表面等离子体共振和电化学联用的DNA传感器研究进展

表面等离子共振(surface plasmon resonance,SPR)技术旨在检测物体表面附近折射率的变化,其特点是无标记、实时、灵敏和快速,该技术多用于研究分子的相互作用,包括动力学、效率常数和大分子构象变化等。电化学(electrochemical,EC)技术是一项用于定性定量研究电子转移、物质氧化还原、界面吸附等过程的成熟技术,具有简单、低成本和设备小型化的优点。现有的DNA杂交技术,例如光学、电化学或压电转导技术,主要关注于提高DNA杂交检测系统的选择性和灵敏度。传统的SPR在DNA分析方面,由于无法测量折射率的极小变化而在超灵敏检测中的应用受到限制。因此,随着纳米材料的研发和联用技术的飞速发展,SPR与EC联用的生物传感器研究越来越成为人们关注的热点。近年来,关于SPR和EC联用在DNA检测方面的综述鲜有报道。对SPR和EC检测DNA的技术原理、联用方法、应用进展等方面作出了简要的介绍,以期为表面等离子共振和电化学联用的DNA传感器相关研究提供参考。     

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance (SPR)

Protein-protein interactions are pivotal to most, if not all, physiological processes, and understanding the nature of such interactions is a central step in biological research. Surface Plasmon Resonance (SPR) is a sensitive detection technique for label-free study of bio-molecular interactions in real time. In a typical SPR experiment, one component (usually a protein, termed ''ligand'') is immobilized onto a sensor chip surface, while the other (the ''analyte'') is free in solution and is injected over the surface. Association and dissociation of the analyte from the ligand are measured and plotted in real time on a graph called a sensogram, from which pre-equilibrium and equilibrium data is derived. Being label-free, consuming low amounts of material, and providing pre-equilibrium kinetic data, often makes SPR the method of choice when studying dynamics of protein interactions. However, one has to keep in mind that due to the method''s high sensitivity, the data obtained needs to be carefully analyzed, and supported by other biochemical methods. SPR is particularly suitable for studying membrane proteins since it consumes small amounts of purified material, and is compatible with lipids and detergents. This protocol describes an SPR experiment characterizing the kinetic properties of the interaction between a membrane protein (an ABC transporter) and a soluble protein (the transporter''s cognate substrate binding protein).