Cloning and efficient expression of <Emphasis Type="Italic">Bacillus sp.</Emphasis> BH072 <Emphasis Type="Italic">tasA</Emphasis> gene in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The Bacillus strain BH072 isolated from a honey sample showed strong antifungal activity against phytopathogen. Gene cloning test demonstrated that the strain had a tasA gene encoding an antifungal TasA protein. Although the wild strain simultaneously produced various antifungal substances, only the physicochemical property and antifungal activity of TasA protein were unclear due to the difficulty in extraction. In this study, tasA gene encoding the protein from Bacillus sp. BH072 was amplified by using the polymerase chain reaction (PCR) method and cloned into pET 28a (+) vector, and then expressed in host cells Escherichia coli BL21 (DE3). The expressed proteins were collected by centrifugation and ultrasonic treatment, and then purified by using nickel-nitrilotriacetic acid (Ni-NTA) metal affinity column and dialysis methods. The result of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) test showed that an expected protein band appeared with a size of 31 kDa. The expressed products possessed antifungal activity against the phytopathogenic indicator strain Botrytis cinerea. A genetically engineered strain tasA of E. coli was established in this study which can efficiently express Tas A protein.     

Antibacterial and antifungal activities of different parts of Tribulus terrestris L. growing in Iraq

Antimicrobial activity of organic and aqueous extracts from fruits, leaves and roots of Tribulus terrestris L., an Iraqi medicinal plant used as urinary anti-infective in folk medicine, was examined against 11 species of pathogenic and non-pathogenic microorganisms: Staphylococcus aureus, Bacillus subtilis, Bacillus cereus, Corynebacterium diphtheriae, Escherichia coli,Proteus vulgaris, Serratia marcescens, Salmonella typhimurium, Klebsiella pneumoniae, Pseudomonas aeruginosa and Candida albicans using microdilution method in 96 multiwell microtiter plates. All the extracts from the different parts of the plant showed antimicrobial activity against most tested microorganisms. The most active extract against both Gram-negative and Gram-positive bacteria was ethanol extract from the fruits with a minimal inhibitory concentration (MIC) value of 0.15 mg/ml against B. subtilis,B. cereus, P. vulgaris and C. diphtheriae. In addition, the same extract from the same plant part demonstrated the strongest antifungal activity against C. albicans with an MIC value of 0.15 mg/ml.     

Construction of recombinant industrial Saccharomyces cerevisiae strain with bglS gene insertion into PEP4 locus by homologous recombination

The bglS gene encoding endo-1,3-1,4-β-glucanase from Bacillus subtilis was cloned and sequenced in this study. The bglS expression cassette, including PGK1 promoter, bglS gene fused to the signal sequence of the yeast mating pheromone α-factor (MFals), and ADH1 terminator with G418-resistance as the selected marker, was constructed. Then one of the PEP4 allele of Saccharomyces cerevisiae WZ65 strain was replaced by bglS expression cassette using chromosomal integration of polymerase chain reaction (PCR)-mediated homologous recombination, and the bglS gene was expressed simultaneously. The recombinant strain S. cerevisiae (SC-βG) was preliminarily screened by the clearing hydrolysis zone formed after the barley β-glucan was hydrolyzed in the plate and no proteinase A (PrA) activity was measured in fermenting liquor. The results of PCR analysis ofgenome DNA showed that one of the PEP4 allele had been replaced and bglS gene had been inserted into the locus of PEP4 gene in recombinant strains. Different endo-1,3-1,4-β-glucanase assay methods showed that the recombinant strain SC-βG had high endo-1,3-1,4-β-glucanase expression level with the maximum of 69.3 U/(h-ml) after 60 h of incubation. Meanwhile, the Congo Red method was suitable for the determination of endo-1,3-1,4-β-glucanase activity during the actual brewing process. The current research implies that the constructed yeast strain could be utilized to improve the industrial brewing property of beer.     

假单胞茵产弹性蛋白酶基因的克隆与表达

从假单胞菌（Pseudomonassp．）XZG36中克隆弹性蛋白酶基因,构建原核表达载体,实现其在大肠杆菌（Escherichiacoli）中的高效表达,并对表达产物进行酶学性质分析,为微生物发酵生产弹性蛋白酶奠定基础．以假单胞菌基因组DNA为模板,PCR扩增弹性蛋白酶基因,并将其开放阅读框（0RF）克隆至融合表达载体pET30a（＋）进一步IPTG诱导表达;表达产物经His·Bind亲和层析纯化后对弹性蛋白酶进行酶学性质分析．实验成功克隆了弹性蛋白酶基因,DNA基因片段为1672bp、编码497个氨基酸残基的多肽,与预计长度相符合;实现了其在E．coli中的高效表达,表达量约占菌体总蛋白的20％;经SDS-PAGE分析,相对分子质量为48000,与预期的一致;提纯后的表达蛋白SDS-PAGE分析可见单一条带,纯度可达92％以上．表达蛋白具有良好活性．     

Purification and characterization of a novel antifungal protein from Bacillus subtilis strain B29"

An antifungal protein was isolated from a culture of Bacillus subtilis strain B29. The isolation procedure comprised ion exchange chromatography on diethylaminoethyl (DEAE)-52 cellulose and gel filtration chromatography on Bio-Gel P-100.The protein was absorbed on DEAE-cellulose and Bio-Gel P-100. The purified antifungal fraction was designated as B29I, with a molecular mass of 42.3 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), pl value 5.69 by isoelectric focusing (IEF)-PAGE, and 97.81% purity by high performance liquid chromatography (HPLC). B29I exhibited in-hibitory activity on mycelial growth in Fusarium oxysporum, Rhizoctonia solani, Fusarium moniliforme, and Sclerotinia scle-rotiorum. The 50% inhibitory concentrations (IC50) of its antifungal activity toward Fusarium oxysporum and Rhizoctonia solani were 45 and 112 μmol/L, respectively. B291 also demonstrated an inhibitory effect on conidial spore germination of Fusarium oxysporum and suppression of germ-tube elongation, and induced distortion, tumescence, and rupture of a portion of the germi-nated spores.     

酯酶产生菌的筛选、鉴定及系统发育分析

利用溴甲酚紫染色透明圈法和发酵测酶活的方法,从黄皮果、芒果自然发酵过程中筛选出5株酯酶产生菌,其中HP-4菌株活性最高,经生理生化及16SrDNA鉴定,初步断定该茵为芽孢杆菌属,丰富了微生物菌种资源库,同时为酯酶的工业化生产提供了一定参考。     

亲和色谱法从融合蛋白GST-IL-6中纯化重组人白细胞介素-6

研究用亲和融合谷胱甘肽 S 转移酶 (GST)的方法纯化重组人白细胞介素 6(IL 6)的发酵和纯化工艺 ,使用含有质粒pHZl818的E .coliJMl0 9在 2XYT培养基中进行发酵表达 ,IL 6表达为与谷胱甘肽 S 转移酶 (GST)融合的融合蛋白GST IL 6.融合蛋白存在可溶的活性蛋白和不可溶的包含体两种形式 ,此包含体无活性且无法复性 ,无法用亲和层析回收 ,通过实验优化摇床发酵的诱导温度和转速 ,以增加可溶融合蛋白的表达 .菌体超声破碎液后 ,上清液用作亲和柱层析 ,可将融合蛋白提纯至 80 00 ,每升发酵液可得 10mg融合蛋白 ,用凝血酶裂解处理 6h ,亲和标志物GST被特异性切除 ,裂解得到的IL 6用离子交换柱层析可纯化至 95 00 ,MTT法测得纯化的IL 6生物学活性为 1.0 2× 10 8IU/mg .     

Purification and characterization of a novel antifungal protein from Bacillus subtilis strain B29

An antifungal protein was isolated from a culture of Bacillus subtilis strain B29. The isolation procedure comprised ion exchange chromatography on diethylaminoethyl (DEAE)-52 cellulose and gel filtration chromatography on Bio-Gel® P-100. The protein was absorbed on DEAE-cellulose and Bio-Gel® P-100. The purified antifungal fraction was designated as B29I, with a molecular mass of 42.3 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), pI value 5.69 by isoelectric focusing (IEF)-PAGE, and 97.81% purity by high performance liquid chromatography (HPLC). B29I exhibited inhibitory activity on mycelial growth in Fusarium oxysporum, Rhizoctonia solani, Fusarium moniliforme, and Sclerotinia sclerotiorum. The 50% inhibitory concentrations (IC₅₀) of its antifungal activity toward Fusarium oxysporum and Rhizoctonia solani were 45 and 112 μmol/L, respectively. B29I also demonstrated an inhibitory effect on conidial spore germination of Fusarium oxysporum and suppression of germ-tube elongation, and induced distortion, tumescence, and rupture of a portion of the germinated spores.     

神经干细胞分化相关的一个新基因的研究

1IntroductionNeural stemcells(NSCs)are a subtype of progenitorcells in the nervous systemthat can differentiate intoneurons and glia[1-3].Due to their feature of self-re-newal,NSCs have expectations for treatment of ner-vous system diseases such as Parkin…     

SEB超抗原受体拮抗剂在大肠杆菌中的高效可溶性表达、纯化及活性初步研究

目的:在大肠杆菌(BL21)中构建可溶性表达的金黄色葡萄球菌B型肠毒素(SEB)受体拮抗剂。方法:首先确定SEB受体桔抗剂的基因序列,然后用含有SEB受体桔抗剂的基因序列重组质粒表达载体PGEX-4T-1转化大肠杆菌BL21(DE3),利用IPTG诱导表达获得蛋白,产物经GST柱纯化后,利用ELISA检测其与SEB的结合能力并进行其体内外药效学实验。结果:该质粒成功转化为可溶性表达,ELISA结果显示表达产物可与SEB特异性结合。结论:本研究成功对SEB受体拮抗剂GST可溶性表达并对其活性进行初步分析。     

柿叶挥发油抑菌活性的研究

采用水蒸气蒸馏法提取柿叶挥发油,并用滤纸片法研究柿叶挥发油对大肠杆菌、金黄色葡萄球菌及枯草芽孢杆菌的抑菌活性和最低抑菌浓度.结果表明,柿叶挥发油对金黄色葡萄球菌和枯草芽孢杆菌具有较强的抑制作用,而对大肠杆菌抑制作用较弱,对金黄色葡萄球菌、枯草芽孢杆菌和大肠杆菌的最低抑菌浓度分别为3.13μL/mL、3.13μL/mL和25.00μL/mL.柿叶挥发油较好的抗微生物活性表明它可能是一种较好的临床抗菌药物.     

ICA69基因工程融合抗原的制备

基于基因工程技术 ,用PCR法扩增出编码ICA6 9的cDNA片段 ,直接克隆到pSPORT 1质粒上 ,经DNA序列测定 ,插入到GST融合蛋白表达载体 pGEX 2T ,构成重组质粒 p2T ICA6 9,得到的表达产物GST ICA6 9融合蛋白用间接ELISA法检测其免疫原性 .测序结果表明 ,所获PCR产物已正确重组到PGEX 2T表达型质粒中 .重组质粒在原核细胞中表达的融合蛋白具有免疫原性 ,并能应用于Ⅰ型糖尿病病人血清中抗ICA6 9抗体的检测 .所获得的表达产物为重组ICA6 9融合抗原 ,有助于提高Ⅰ型糖尿病的预报率和确诊率 .     

Keggin型杂多酸的抑菌作用

合成了1种多氧酸盐K5BW12O40.15H2O,并通过紫外和红外光谱对其结构进行了表征.利用纸片法研究了K5BW12O40.15H2O、H4SiW12O40.xH2O和H3PW12O40.xH2O对大肠杆菌、枯草芽孢杆菌、酵母菌、黑曲霉和青霉的抑菌活性.结果表明,3种物质对大肠杆菌、枯草芽孢杆菌、酵母菌和黑曲霉均有不同程度的抑菌效果,但对青霉的抑菌效果较差.     

斑节对虾酚氧化酶原在大肠杆菌中的表达

将斑节对虾酚氧化酶原（prophenoloxidase,proPO）基因克隆进pET28a（＋）,在大肠杆菌BL21菌株中诱导表达,SDS-PAGE和Western-blotting分析均能检测到一条分子量为79．4kDa的特异性条带,与推导的融合蛋白理论分子量82．4kDa基本相符;包涵体变性后对经Ni—NTAagarose纯化的变性液进行复性,表现出0．3851U／mg．min的PO活性;不同条件下的诱导表达结果显示,18℃时诱导培养能检测到目的蛋白的可溶性表达,经诱导9h后可溶性表达比例达到最高,约占菌体可溶性蛋白总量的s．56％;可溶性表达的目的蛋白纯化后经胰酶消化,可检测到分子量为60kDa、42．7kDa的特异性条带,并表现出0．4249U／mg．min的PO活性．     

Fusion expression of pedA gene to obtain biologically active pediocin PA-1 in Escherichia coli

Two heterologous expression systems using thioredoxin (trxA) as a gene fusion part in Escherichia coli were developed to produce recombinant pediocin PA-1. Pediocin PA-1 structural gene pedA was isolated from Pediococcus acidilactici PA003 by the method of polymerase chain reaction (PCR), then cloned into vector pET32a(+), and expressed as thioredoxin-PedA fusion protein in the host strain E. coli BL21 (DE3). The fusion protein was in the form of inclusion body and was refolded before purification by nickel-iminodiacetic acid (Ni-IDA) agarose resin column. Biological activity of recombinant pediocin PA-1 was analyzed after cleavage of the fusion protein by enterokinase. Agar diffusion test revealed that 512-arbitrary unit (AU) recombinant pediocin PA-1 was obtained from 1 ml culture medium of E. coli (pPA003PED1) using Listeria monocytogenes as the indicator strain. Thioredoxin-PedA fusion gene was further cloned into pET20b(+). Thioredoxin-PedA fusion protein was detected in both the periplasmic and cytoplasmic spaces. The recombinant pediocin PA-1 from the soluble fraction attained 384 AU from 1 ml culture medium of E. coli (pPA003PED2). Therefore, biologically active pediocin PA-1 could be obtained by these two hybrid gene expression methods.     

Effects of testing versus review on rote and conceptual learning from prose

This study investigated the effect of reading ability level(high, low) and type of study activity (immediate rote test, immediate conceptual test, structured review, unstructured review, no review control) on rote and conceptual learning outcomes. Subjects were 110 sophomore and junior level high school students. The students studied a 2100 word passage and were then given either a posttest (rote or conceptual) or a set of directions leading to structured or unstructured review or a control group filler task. Rote and conceptual delayed retention tests were administered one week later. A 2×5 factorial design was used. Results indicated no significant effects for type of study activity. As expected, high reading ability students performed better than low reading ability students across all conditions.     

Magnaporthe oryzae MTP1 gene encodes a type III transmembrane protein involved in conidiation and conidial germination

In this study the MTP1 gene, encoding a type III integral transmembrane protein, was isolated from the rice blast fungus Magnaporthe oryzae. The Mtp1 protein is 520 amino acids long and is comparable to the Ytp1 protein of Saccharomyces cerevisiae with 46% sequence similarity. Prediction programs and MTP1-GFP (green fluorescent protein) fusion expression results indicate that Mtp1 is a protein located at several membranes in the cytoplasm. The functions of the MTP1 gene in the growth and development of the fungus were studied using an MTP1 gene knockout mutant. The MTP1 gene was primarily expressed at the hyphal and conidial stages and is necessary for conidiation and conidial germination, but is not required for pathogenicity. The Deltamtp1 mutant grew more efficiently than the wild type strain on non-fermentable carbon sources, implying that the MTP1 gene has a unique role in respiratory growth and carbon source use.     

鼠尾藻脂溶性物质抗食物腐败菌的初步研究

采用圆形纸片法,对鼠尾藻脂溶性物质及经柱层析分离得到的不同洗脱组分进行抗食物腐败菌实验.结果表明,鼠尾藻粗脂对大肠杆菌的抑菌圈为1.25mm.乙醇洗脱组分具有较强的抗4种食物腐败菌的活性,其对枯草杆菌、大肠杆菌、金黄色葡萄球菌和假单胞杆菌的抑菌圈分别达到3.0mm,4.0mm,5.0mm,2.5mm.     

苏云金芽孢杆菌及其在害虫防治上的应用(综述)

苏云金芽孢杆菌 (Bacillusthuringiensis,简称Bt)自 1 90 1年发现以来 ,一直在微生物学、昆虫学和生物化学等方面受到研究者的关注。由于Bt能形成对许多昆虫具特异性毒力的杀虫蛋白 ,因此从 1 959年开始Bt已被制成杀虫剂防治鳞翅目害虫。近 1 0年来 ,随着分子生物学和遗传工程技术的成熟和发展 ,使人们增加了有关晶体形成的分子生物学知识。Bt的杀虫活性主要是其能产生含有杀虫晶体蛋白 (ICPs)的伴孢晶体。一些杀虫蛋白基因 (又称cry基因 )已经成功用于转基因抗虫植物中。本文将从Bt微生物学、Bt制剂在害虫防治中以及Btcry基因在转基因抗虫植物中的应用等方面作一综述。