新型α-芋螺毒素基因克隆的引物筛选

目的:优化PCR条件,建立能特异扩增出α-芋螺毒素基因片段的最理想PCR条件.方法:根据α-芋螺毒素基因保守的信号肽或内含子序列和非翻译区保守核苷酸序列设计了多组特异引物,并对引物浓度和退火温度等影响因素进行优化.结果:根据α-芋螺毒素基因保守的内含子序列为引物、引物浓度为0.1 μmol/L、退火温度为50℃时,能特异的扩增出α-芋螺毒素基因片段,分子量大约分别为180bp和300bp.结论:采用优化的PCR条件,能筛选出克隆新型的α--芋螺毒素基因片段的最理想引物,为α-芋螺毒素的化学合成、活性研究和应用提供基础.     

新芋螺毒素基因的克隆及其遗传进化分析

全世界有约800种芋螺,每种芋螺产生多达2 000种的肽类毒素,这些毒素可以作用于电压门控离子通道(Na+,K+,Ca2+)、配体门控离子通道(n ACh Rs,5-HT3R,NMDAR)、G蛋白偶联受体(神经降压素和血管加压素)和神经递质转运蛋白。虽然已有大量的芋螺毒素通过毒液分离、c DNA克隆和转录组测序获得,但已发现的芋螺毒素不足其总量的0.5%。A-超家族中α-芋螺毒素基因结构包含了一个内含子和被该内含子分开的两个外显子,成熟肽具有标准的4个半胱氨酸骨架(CC-C-C)。本研究利用具有保守性的α-芋螺毒素基因内含子序列,采用多个PCR退火温度,从海南产疣缟芋螺中克隆到了1个新的具有6个半胱氨酸骨架(CC-C-C-CC)的M-超家族芋螺毒素基因和1个含有5个半胱氨酸新颖骨架(CC-C-C-C)的未知新家族芋螺毒素,并对它们的基因结构、成熟肽序列,以及与其他M-超家族芋螺毒素的遗传进化关系进行了深入分析。首次证实保守的α-芋螺毒素基因内含子序列可能存在于其他超家族中。     

特异阻断乙酰胆碱受体的海南产α*-芋螺毒素的研究

国际上被誉为"海洋药物宝库"的芋螺毒素(CTx),具有特异结合动物体内各种离子通道和受体的特殊功能。其中的α*-芋螺毒素(α*-CTxs)能特异性地作用于乙酰胆碱受体(n ACh Rs),对其亚型具有极高的选择性阻断活性。不同的亚基组合形成的各种乙酰胆碱受体亚型,在正常健康状态下和多种疾病状态下起着很重要的生理和病理学功能,这些功能至今尚不很清楚,因而发现和开发各种亚型的选择性分子探针,如α*-CTxs将有助于阐释各个亚型的精细结构和功能。深入研究特异阻断乙酰胆碱受体不同亚型的α*-CTxs的结构与功能,及其与受体相互作用的机理具有很重要的科学意义,将有助于研发出与n ACh Rs相关的多种疑难杂症的治疗药物,包括神经痛、癌症化疗、成瘾、痴呆、重症肌无力、精神分裂症、癫痫、乳腺癌、肺癌、脑脊髓炎,以及其他神经疾病等。近年来,我们实验室经过大量筛选研究,鉴定出5个海南产新颖α*-芋螺毒素,对它们的人工合成、三维结构、作用靶点、与受体相互作用机制、药理药效等进行了深入的研究。现对上述特异阻断乙酰胆碱受体不同亚型的5个海南产α*-芋螺毒素的研究进展和应用前景进行综述。     

芋螺毒素MⅦA研究进展

来自芋螺(Conus)的芋螺毒素(conotoxin)是一类具有独特神经药理活性作用的多肽,它们能够高特异选择性识别、作用其受体,这是在五千万年进化史中与其受体相互作用形成的。其中,w-芋螺毒素MⅦA来自致幻芋螺(Conusmagus),是一种高选择性的N-型电压敏感型Ca^2 通道阻断剂。研究表明它具有强大的镇痛活性,并对脑缺血所造成的神经损伤具有保护作用。本综述芋螺毒素MⅦA的相关基础、临床前及临床研究。     

芋螺毒素MrIA的串联表达、纯化及生物活性鉴定

芋螺毒素(Conotoxins,CTxs)是一类特异作用于离子通道和膜受体的小分子多肽,是研究受体结构和功能及其相关疾病的重要工具。MrIA是进入临床研究可用于镇痛治疗的一种T超家族的芋螺毒素。传统获取芋螺毒素是通过化学合成的方法,成本高、产量低。实验利用串联表达技术,构建原核表达载体,在大肠杆菌(Escherichia coli,E. coli)中成功表达了芋螺毒素MrIA(recombinant MrIA,rMrIA)。通过溴化氰切割和纯化,最终1L菌液可获得纯度高达95%的rMrIA 30mg。小鼠热板实验表明,rMrIA具有较好的镇痛活性。这为大量获得MrIA以及其他芋螺毒素小肽的表达提供了方法。     

新型α-芋螺毒素Di1.1的克隆、合成及功能研究

目的：从中国南海长距芋螺中克隆出新的芋螺毒素序列并用固相方法合成该毒素,测定其折叠后的二硫键配对方式并初步研究其药理学特性。方法：根据芋螺毒素A超家族保守的信号肽序列设计引物,通过3＇-RACE扩增,从芋螺毒腺管中克隆出新的毒素基因;采用Fmoc-固相法合成线性多肽,通过空气氧化折叠获得含二硫键的折叠产物,用两步氧化折叠法测定多肽的二硫键连接方式;用双电极电压钳技术初步研究其药理学特性。结果：发现-种新的α-芋螺毒素Dil．1的cDNA序列,其成熟肽序列为CcVIESCHSNHIDECES;该肽二硫键连接方式以C1-C4、C2-C3为主,以C1-C3、C2-C4连接为辅,对烟碱型乙酰胆碱受体各亚型活性较弱。结论：Dil．1是-种新的α4／7型芋螺毒素,其折叠方式以C1-C4、C2-C3连接为主。     

芋螺毒素的翻译后修饰

芋螺毒素是一类由芋螺毒液管分泌的生物活性小肽,能特异地作用于各种离子通道和神经递质受体.芋螺毒素的一个显著特点是具有高度的翻译后修饰,包括Glu的γ羧基化、L型氨基酸到D型氨基酸的异构化、Pro等的羟基化、Trp的溴代等.这些翻译后修饰在提高芋螺毒素分子多样性的同时,也增强了芋螺毒素的功能.现主要针对芋螺毒素中的各种翻译后修饰的发现、分布、功能和参与反应的酶系统进行综述.     

新的J超家族芋螺毒素的克隆与进化分析

目的:从中国南海芋螺中克隆新的J超家族芋螺毒素,并进行序列和进化分析。方法:以芋螺毒腺管总RNA为模板,采用3′-RACE及巢式PCR的方法扩增J超家族芋螺毒素基因,并将得到的目的基因与pMD18-T载体连接并转化大肠杆菌DH5α,经测序比对后,获得新的J超家族毒素,利用软件BioEdit、ClustalX及Mega5.05进行进化分析。结果:获得12个新的J超家族芋螺毒素前体肽序列,其氨基酸组成具有新颖性,在进化树上与已报道的J超家族毒素处于不同的进化分支。结论:12个新的J超家族毒素与已报道的毒素序列之间的同源性较低,是J超家族新成员。     

织锦芋螺ο家族芋螺毒素的序列分析

为了从织锦芋螺（Ｃｏｎｕｓｔｅｘｔｉｌｅ）中尽可能多地分离出ο家族的毒素序列和研究其应用价值,在克隆了织锦芋螺α芋螺毒素的基础上进行了织锦芋螺ο家族芋螺毒素基因的分离工作．从织锦芋螺毒管中提取ｍ ＲＮＡ,通过ＲＡＣＥ（ｒａｐｉｄ ａｍ ｐｌｉｆｉｃａｔｉｏｎ ｏｆｃＤＮＡ ｅｎｄｓ,ｃＤＮＡ 末端的快速扩增）－ＰＣＲ方法扩增获得ο家族芋螺毒素ｃＤＮＡ 片段,并进行克隆和序列分析．从织锦芋螺毒液中获得了６种新的芋螺毒素序列,且毒素序列的成熟肽部分均符合Ｃ－ Ｃ－ ＣＣ－ Ｃ－ Ｃ的保守半胱氨酸框架．这些是新的ο家族芋螺毒素序列,新序列的阐明为进一步研究其生物活性和应用打下了基础．     

芋螺毒素的药用价值研发进展

芋螺毒素是一种海洋软体动物芋螺分泌的一类用于自卫和捕食的小肽神经性毒素。芋螺毒素具有很高的药用开发价值和潜力。近年来,具有高度特异性生物活性的芋螺毒素一直广泛应用于研制特异性诊断试剂以及开发疗效特异的新药之中,并作为分子模型用于相关新药的设计。本文对近年来芋螺毒素药用开发研究的最新进展做一综述。     

The evolution of DNA polymerases with novel activities

DNA and RNA polymerases have evolved in nature to function in specific environments with specific substrates. Thus, although the commercial availability of these enzymes has revolutionized the biotechnology industry, their applications are limited. The availability of polymerases that have unnatural properties would be of even greater utility. Towards this goal, several activity-based screening and selection approaches have been developed. Using these techniques, polymerases that synthesize a variety of different polymers, including those containing 2'-O-methyl-modified nucleotides or unnatural base pairs, have been evolved. These results suggest that polymerases tailored for any specific application could soon be available.     

Chromosome specific c-DNA libraries: reduction of unspecific priming events by purification of heteronuclear RNA

Chromosome specific c-DNA libraries greatly facilitate the isolation of disease associated genes which have been previously linked to particular chromosomes. Recently, several methods have been developed and employed for the isolation of transcribed sequences from specific human chromosomes and chromosome regions. Heteronuclear (hn) RNA from somatic human/rodent cell hybrids has been used as starting material to selectively prime the synthesis of human specific c-DNAs. A drawback of this method is the high number of rodent clones found in these chromosome specific c-DNA libraries. Here, we provide direct evidence that unspecific priming events account for the majority of these rodent clones. Using an Alu consensus primer hn-RNA human specific c-DNA libraries have been established and the specificity of Alu-priming has been evaluated. Using a variety of purification schemes for isolating hn-RNA we have significantly reduced the percentage of unspecific priming events. We also included a comparison of the hn-RNA yield from different somatic hybrids prior and after purification.     

Interaction of a protooncogene product, Myb with DNAs.

The DNA-binding domain of Myb consists of three imperfect tandem repeats and the third one which is essential for sequence-specific binding was established to have a helix-turn-helix-related motif. DNA sequences recognized by Myb have been reported to contain TAACPy sequence. Here we have examined the details of Myb-binding sequence. Using DNAs with a single mutation on the various sites of two specific DNAs and some fragments of the DNA-binding domain of Myb, we have found that (i) in a specific DNA which contains only one AAC sequence, each AAC nucleotide is found to be essential for the specific binding of Myb, while any other mutations cause no serious binding loss, (ii) in a specific DNA which contains two AAC sequences separately, one AAC is not so important in the binding, and (iii) for the specific binding with DNA, at least both repeats 2 and 3 of Myb are required. These findings suggest that repeat 3 containing a helix-turn-helix-related structure recognizes the core AAC sequence and repeat 2 supports this recognition by interactions with phosphate groups of DNA.     

Translational control in cellular and developmental processes

Growing evidence indicates that translational control of specific mRNAs contributes importantly to genetic regulation across the breadth of cellular and developmental processes. Synthesis of protein from a specific mRNA can be controlled by RNA-binding proteins at the level of translational initiation and elongation, and translational control is also sometimes coupled to mRNA localization mechanisms. Recent discoveries from invertebrate and vertebrate systems have uncovered novel modes of translational regulation, have provided new insights into how specific regulators target the general translational machinery and have identified several new links between translational control and human disease.     

Simultaneous increases in specific growth rate and specific lipid content of Chlorella vulgaris through UV‐induced reactive species

A challenge in algae‐based bio‐oil production is to simultaneously enhance specific growth rates and specific lipid content. We have demonstrated simultaneous increases in both the above in Chlorella vulgaris through reactive species (RS) induced under ultraviolet (UV) A and UVB light treatments. We postulated that the changes in photosystem (PS) stoichiometry and antenna size were responsible for the increases in specific growth rate. UVB treatment excited PSII, which resulted in a twofold to sevenfold increase in PSII/PSI ratio compared to control. An excited PSII caused a 2.7‐fold increase in the specific levels of superoxide and a twofold increase in the specific levels of hydroxyl radicals. We have established that the increased specific intracellular RS (si‐RS) levels increased the PSII antenna size by a significant 10‐fold as compared to control. In addition, the 8.2‐fold increase in specific lipid content was directly related to the si‐RS levels. We have also demonstrated that the RS induced under UVA treatment led to a 3.2‐fold increase in the saturated to unsaturated fatty acid ratio. Based on the findings, we have proposed and demonstrated a UV‐based strategy, which achieved an 8.8‐fold increase in volumetric lipid productivity. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:291–299, 2014     

Involvement of cyclooxygenase-2 and prostaglandins in the molecular pathogenesis of inflammatory lung diseases

Inducible cyclooxygenase (COX-2) and its metabolites have diverse and potent biological actions that are important for both physiological and disease states of lung. The wide variety of prostaglandin (PG) products are influenced by the level of cellular activation, the exact nature of the stimulus, and the specific cell type involved in their production. In turn, the anti- and proinflammatory response of PG is mediated by a blend of specific surface and intracellular receptors that mediate diverse cellular events. The complexity of this system is being at least partially resolved by the generation of specific molecular biological research tools that include cloning and characterization of the enzymes distal to COX-2 and the corresponding receptors to the final cellular products of arachidonic metabolism. The most informative of these approaches have employed genetically modified animals and specific receptor antagonists to determine the exact role of specific COX-2-derived metabolites on specific cell types of the lung in the context of inflammatory models. These data have suggested a number of cell-specific, pathway-specific, and receptor-specific approaches that could lead to effective therapeutic interventions for most inflammatory lung diseases.     

Dependence of the specific activity of antitetanus immunoglobulin on the degree of its fragmentation

Antitetanus immunoglobulin preparations with the increasing content of Fab-fragments (15, 30, 53%) have been obtained under specific experimental conditions. Tests for specific activity have revealed an insignificant decrease (13%) in this activity in the preparation containing 15% of Fab-fragments and its sharp drop in the preparations containing 30-50% of Fab-fragments. The specific activity of antitetanus immunoglobulin has been found to be related to the degree of its fragmentation.     

Self-assembled monolayers and polymer brushes in biotechnology: current applications and future perspectives

The chemistry and topography of a surface affect biological response and are of fundamental importance, especially when living systems encounter synthetic surfaces. Most biomolecules have immense recognition power (specific binding) and simultaneously have a tendency to physically adsorb onto a solid substrate without specific receptor recognition (nonspecific adsorption). Therefore, to create useful materials for many biotechnology applications, interfaces are required that have both enhanced specific binding and reduced nonspecific binding. Thus, in applications such as sensors, the tailoring of surface chemistry and the use of micro or nanofabrication techniques becomes an important avenue for the production of surfaces with specific binding properties and minimal background interference. Both self-assembled monolayers (SAMs) and polymer brushes have attracted considerable attention as surface-active materials. In this review, we discuss both of these materials with their potential applications in biotechnology. We also summarize lithographic methods for pattern formation using combined top-down and bottom-up approaches and briefly discuss the future of these materials by describing emerging new applications.     

Enhancement of Candida rugosa lipase production by using different control fed-batch operational strategies

Simulation studies have predicted that maximum lipase activity is reached with fed-batch operation strategies. In this work, two different fed-batch operational strategies have been studied: constant substrate feeding rate and specific growth rate control. A constant substrate feeding rate strategy showed that maximum aqueous lipolytic activity (55 U/mL) was reached at low substrate feeding rates, whereas lipase tends to accumulate inside the cell at higher rates of substrate addition. In the second fed-batch strategy studied, a feedback control strategy has been developed based on the estimation of state variables (X and mu) from the measurement of indirect variables such as CER by means of mass spectrometry techniques. An on-off controller was then used to maintain the specific growth rate at the desired value by adjusting the substrate feeding rate. A constant specific growth rate strategy gave higher final levels of aqueous lipolytic activity (117 U/mL) at low specific growth rates. At higher specific growth rates the enzyme remained accumulated inside the cell, as was observed with a constant feeding fed-batch strategy. With a constant specific growth rate strategy, lipase production by Candida rugosa was enhanced 10-fold compared to a batch operation. Purification studies have demonstrated that lipolytic and esterasic specific activity ratios of Candida rugosa isoenzymes can be modified by using different operational conditions. These studies have also showed that the isoenzymes obtained in a controlled growth rate strategy are around three- to four-fold more active than those obtained in a constant feeding rate strategy.     

A Genetic Mosaic Screen of Essential Zygotic Genes in Caenorhabditis Elegans

We have devised a simple genetic mosaic screen, which circumvents the difficulties posed by phenotypic analysis of early lethal mutants, to analyze essential zygotic genes in Caenorhabditis elegans. The screen attempts to distinguish genes involved in cell type and/or lineage specific processes such as determination, differentiation or morphogenesis from genes involved in general processes such as intermediary metabolism by using the pattern of gene function to classify genes: genes required in one or a subset of early blastomeres may have specific functions, whereas genes required in all early blastomeres may have general functions. We found that 12 of 17 genes examined function in specific early blastomeres, suggesting that many zygotic genes contribute to specific early processes. We discuss the advantages and limitations of this screen, which is applicable to other regions of the C. elegans genome.