rmlB基因在单核细胞增生李斯特菌耐药性、生物被膜形成和毒力方面的作用

【目的】探究单核细胞增生李斯特菌(Listeria monocytogenes,Lm)rmlB基因在细菌耐药、生物被膜形成和毒力方面的作用。【方法】通过同源重组的方法敲除Lm染色体上的rmlB基因,比较野生株与rmlB缺失株在耐药性方面的差异;利用微孔板法观测rmlB缺失菌株生物被膜形成能力的变化;利用RT-PCR检测缺失菌株中主要毒力基因转录表达,并观察rmlB缺失对细菌溶血活性的影响。【结果】同野生菌株相比,rmlB缺失菌株对头孢菌素和杆菌肽等作用位点在细菌细胞壁和细胞膜的敏感性显著增加(P≤0.01),生物被膜形成能力显著降低(P≤0.01),细菌主要毒力基因hly的转录表达及溶血活性也发生显著降低(P≤0.01)。【结论】rmlB基因在Lm生物被膜形成和耐受作用位点位于细胞壁和细胞膜的抗生素及细菌毒力方面具有重要作用。     

亮氨酸脱氢酶与葡萄糖脱氢酶高效共表达制备L-叔亮氨酸北大核心CSCD

【目的】通过不同双基因共表达策略对亮氨酸脱氢酶和葡萄糖脱氢酶基因在大肠杆菌中表达影响的研究,获得具有高辅酶再生效率的双酶共表达重组生物催化剂,实现L-叔亮氨酸"一锅法"高效不对称合成。【方法】以来自于蜡状芽孢杆菌(Bacillus cereus)的亮氨酸脱氢酶(LDH)和来自芽孢菌属(Bacillus sp.)的葡萄糖脱氢酶(GDH)为模板,考察单质粒共表达,双质粒共表达和融合表达等3种共表达策略对重组细胞中亮氨酸脱氢酶和葡萄糖脱氢酶活的影响,比较不同酶活比例和不同催化剂形式对三甲基丙酮酸不对称还原制备L-叔亮氨酸效率的影响。【结果】研究发现不同共表达策略对亮氨酸脱氢酶和葡萄糖脱氢酶的影响存在明显差异。亮氨酸脱氢酶在不同策略下均能够正常表达,而葡萄糖脱氢酶在融合表达时没有活力,当C端含有组氨酸标签时,表达蛋白活性低。通过表达优化,获得3株亮氨酸脱氢酶和葡萄糖脱氢酶高效表达且具有不同酶活比例的重组菌。比较粗酶液和全细胞形式下的催化效率,发现酶活比例及催化剂形式对不对称还原反应效率具有重要影响。确定单质粒串联表达C端不含His标签重组菌E.coli BL21/p ET28a-L-SD-AS-G为最佳催化剂,以粗酶液进行转化时,完全转化0.5 mol/L底物所需菌体量为15 g/L,辅酶量为0.1 mmol/L。【结论】采用单质粒共表达策略,成功构建出1株具有较高亮氨酸脱氢酶和葡萄糖脱氢酶活性的重组菌,实现高效催化TMP合成L-Tle。     

高致病性2型猪链球菌截短型类枯草杆菌丝氨酸蛋白酶基因的鉴定和功能研究

【目的】克隆表达高致病性2型猪链球菌05ZYH33株的SspA截短型基因,验证其是否具有酶学活性,并构建该基因的缺失突变株细菌,探讨其在2型猪链球菌致病过程中所起的作用【。方法】构建SS2的SspA截短型基因05SSU0811原核表达质粒,表达并纯化05SSU0811蛋白,运用丝氨酸蛋白酶底物Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide(pNa),通过显色反应检测表达产物的酶学活性;运用同源重组技术敲除05SSU0811基因,多重交叉PCR筛选敲除株并测序鉴定,动物试验分析05SSU0811基因缺失对细菌毒力的影响。【结果】成功表达并纯化05SSU0811蛋白,浓度约为3.5 g/L。丝氨酸蛋白酶活性测定试验证实其具有酶学活性;获得05SSU0811基因缺失突变株,小鼠攻毒试验表明,野生株攻毒的20只小鼠全部死亡,基因缺失突变株攻毒组死亡9只,死亡率45%,两组间死亡率有显著性差异。表明05SSU0811基因缺失的菌株毒力较野生株明显下降。【结论】05SSU0811基因编码的截短型丝氨酸蛋白酶仍然具有酶学活性,SS2的截短型基因SspA在高致病性2型猪链球菌的致病性方面具有一定作用。     

大肠杆菌BL21(DE3)lpxM突变株的构建

目的:lpxM基因失活可以产生极低内毒素活性的脂多糖。构建大肠杆菌BL21(DE3)的lpxM突变株,并考察该突变株的生长状态和表达重组蛋白的能力。方法:构建同源臂长500bp左右的打靶载体,借助Red同源重组系统,使E.coliBL21(DE3)的lpxM基因发生插入失活,再导入编码FLP位点特异性重组酶的质粒pCP20去除抗性基因。PCR鉴定发生插入突变的菌株,应用SDS-PAGE分析突变前后的脂多糖,考查对重组蛋白表达的影响。结果:PCR鉴定结果说明lpxM基因发生了插入突变。与出发株比较,突变株的脂多糖电泳图谱发生了明显变化,但其生长状态与表达重组蛋白的能力与出发株基本一致。结论:大肠杆菌BL21(DE3)的lpxM突变株可以用于重组蛋白的表达。     

斜卧青霉L-06内切葡聚糖酶Ⅰ基因的克隆与表达

【目的】克隆斜卧青霉L-06的内切葡聚糖酶Ⅰ基因(egI),并实现其在大肠杆菌内的高效表达。【方法】利用RT-PCR技术克隆了斜卧青霉L-06的内切葡聚糖酶Ⅰ基因(egI),并将egI基因克隆到原核表达载体中,构建了重组质粒pET32a-egI。【结果】转化至大肠埃希菌Rosetta(DE3),经IPTG诱导重组蛋白表达,SDS-PAGE检测结果表明:重组表达产物的相对分子质量约为80 kD,与预期相符。重组表达的菌悬液,经破碎离心,取其上清液,进行纤维素酶活性染色,获得了活性条带。DNS法测得内切酶活力为2.56 IU/mL。【结论】构建了斜卧青霉L-06内切葡聚糖酶Ⅰ的原核表达系统。     

大肠杆菌O138 O-抗原基因簇的破译及Gne的生物信息学鉴定

利用鸟枪法对大肠杆菌E .coliO138O 抗原基因簇进行测序 ,序列全长 14 139bp ,用生物信息学的方法进行序列分析 ,共发现 11个基因 ,分别为鼠李糖合成酶基因 (rmlB ,rmlD ,rmlA ,rmlC)、UDP GalNAcA合成酶基因 (gne ,gna)、糖基转移酶基因 (3个 )、O 抗原转运酶基因 (wzx)和O 抗原聚合酶基因 (wzy)。发现一种稀有单糖UDP Gal NAcA的合成途径 ,对合成该糖的第一种酶Gne进行了生物信息学鉴定 ,另外用PCR方法筛选出了针对大肠杆菌O138的特异基因     

过表达pps基因和aroG~(fbr)基因对北京棒杆菌L-色氨酸合成的影响

【目的】通过增加北京棒杆菌(Corynebacterium pekinense)PD-67芳香族氨基酸合成的前体物质磷酸烯醇式丙酮酸(PEP)的供应,解除终产物对芳香族氨基酸合成途径中第一个酶同时也是关键酶3-脱氧-D-阿拉伯庚酮糖-7-磷酸合酶(DS)的反馈抑制并提高抗反馈抑制的DS的活力,使碳流更多地流向芳香族氨基酸合成途径,从而积累更多L-色氨酸。【方法】运用PCR技术扩增北京棒杆菌PD-67磷酸烯醇式丙酮酸合酶基因pps,与表达载体连接构建重组质粒pXPS;运用重叠PCR技术定点突变大肠杆菌(Escherichia coli)受苯丙氨酸调控的DS基因aroG,使相应的编码氨基酸序列发生突变:Leu175Asp,新的基因命名为aroGfbr,与表达载体连接构建重组质粒pXA;构建pps和aroGfbr的共表达重组质粒pXAPS。将3个重组质粒分别转入菌株PD-67,构建工程菌株PD-67/pXPS、PD-67/pXA和PD-67/pXAPS。通过摇瓶发酵研究工程菌株的发酵特性。【结果】酶活分析结果表明,pps基因和aroGfbr基因在北京棒杆菌PD-67中均实现了表达。工程菌株PD-67/pXA粗酶液DS抗反馈抑制分析表明,AroGfbr已解除酪氨酸和苯丙氨酸的反馈抑制。过表达pps基因和aroGfbr基因分别使工程菌L-色氨酸产量提高12.1%和26.8%,双基因共表达可使工程菌的产酸量提高35.9%。【结论】北京棒杆菌PD-67pps基因的过表达以及大肠杆菌来源的解除反馈抑制的aroGfbr的过表达均有助于增加PD-67 L-色氨酸的合成,而双基因的共表达可以进一步提高L-色氨酸的积累量。     

钝齿棒杆菌GlnK在氮代谢调控及L-精氨酸合成中的功能

【目的】钝齿棒杆菌是重要的氨基酸生产菌株,本研究针对氮代谢PⅡ信号转导蛋白GlnK展开相关功能研究,分析其在钝齿棒杆菌氮代谢调控及L-精氨酸合成中的作用。【方法】以GlnK蛋白为研究对象,通过基因敲除等遗传方法获得过表达、敲除及敲弱glnK的重组钝齿棒杆菌,研究GlnK对NH_4~+吸收的影响,通过RT-qPCR和酶活测定,从转录水平和蛋白水平上揭示GlnK对氮代谢和L-精氨酸合成相关基因表达水平及酶活的影响,通过5-L发酵罐发酵产L-精氨酸研究GlnK对L-精氨酸合成的影响。【结果】过表达glnK能明显促进NH_4~+的吸收,而敲除glnK后则会抑制NH_4~+的摄取;RT-qPCR和酶活测定发现,相比于野生型菌株Cc5-5,glnK过表达菌株Cc-glnK中与铵吸收相关的基因,表达量平均上调约4.58倍,L-精氨酸合成基因簇中基因的表达水平平均上调1.50倍。Cc-glnK中氮代谢相关蛋白的酶活平均提高46.97%;L-精氨酸合成途径上7个关键酶的酶活平均提高30.00%;5-L发酵罐发酵各重组菌株结果表明,Cc-glnK菌株的产量可达49.53 g/L,产率为0.516 g/(L·h),相比于出发菌株Cc5-5,其L-精氨酸产量提高了28.65%。【结论】过表达GlnK能促进NH_4~+的吸收及利用,并通过影响L-精氨酸合成途径上关键基因的表达水平,提高关键酶的酶活,最终提高L-精氨酸的产量。本研究为后续探索钝齿棒杆菌氮代谢调控机制及代谢工程改造钝齿棒杆菌生产L-精氨酸提供了一种新的策略。     

大肠杆菌O150 O-抗原基因簇的破译和dTDP-鼠李糖合成酶基因的鉴定

利用鸟枪法对大肠杆菌O150 O-抗原基因簇进行测序,序列全长13551bp,用生物信息学的方法进行序列分析,共发现11个基因,分别为鼠李糖合成酶基因(rmlB、rmlD、rmlA、rmlC)糖基转移酶基因(3个)、O-抗原转运酶基因(wzx)和O-抗原聚合酶基因(wzy),另外还有两个基因功能未知。用PCR的方法筛选出了针对大肠杆菌O150的特异基因,可以用于基因芯片或PCR方法对大肠杆菌O150的快速检测。另外,通过进化分析发现大肠杆菌O150的O-抗原基因簇中携带有典型的大肠杆菌鼠李糖合成酶基因,并且这些基因参与了O-抗原基因簇间的重组以形成新的基因簇的过程。     

改造大肠杆菌卟啉代谢途径对重组过氧化物酶活性的影响

【目的】原核表达某些需辅因子的外源蛋白时往往酶活偏低,为提高酶活和减少外加辅因子的成本,我们尝试在大肠杆菌中表达外源过氧化氢-过氧化物酶的同时提高大肠杆菌中与该酶辅因子相关的合成代谢。【方法】本研究克隆了中度嗜盐菌Halomonas elongata DSM2581的过氧化氢-过氧化物酶CAT-POD(catalase-peroxidase)编码基因kat G的ORF,构建原核表达载体p ET28a-kat G,实现了CAT-POD在大肠杆菌中的重组表达。由于CAT-POD活性依赖其活性中心血红素,而血卟啉是血红素的骨架,通过构建原核表达载体p UC19-tac-hem A,将编码5-氨基乙酰丙酸合成酶的hem A基因在大肠杆菌中过量表达,提高卟啉的含量,从而提高重组蛋白CAT-POD的酶活。【结果】最终的CAT酶活达到了377 U/m L,为对照组的7.5倍。【结论】本研究为工业生产高活性CAT-POD提供了有效的方案,也为体外重组表达含辅因子的蛋白提供可借鉴的思路。     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

A Unique Protease Cleavage Site Predicted in the Spike Protein of the Novel Pneumonia Coronavirus (2019-nCoV) Potentially Related to Viral Transmissibility

正Dear Editor,In December 2019, a novel human coronavirus caused an epidemic of severe pneumonia(Coronavirus Disease 2019,COVID-19) in Wuhan, Hubei, China(Wu et al. 2020; Zhu et al. 2020). So far, this virus has spread to all areas of China and even to other countries. The epidemic has caused 67,102 confirmed infections with 1526 fatal cases     

Curcuminoids as inhibitors of thioredoxin reductase: A receptor based pharmacophore study with distance mapping of the active site

Curcumin is the yellow pigment of turmeric that interacts irreversibly forming an adduct with thioredoxin reductase (TrxR), an enzyme responsible for redox control of cell and defence against oxidative stress. Docking at both the active sites of TrxR was performed to compare the potency of three naturally occurring curcuminoids, namely curcumin, demethoxy curcumin and bis-demethoxy curcumin. Results show that active sites of TrxR occur at the junction of E and F chains. Volume and area of both cavities is predicted. It has been concluded by distance mapping of the most active conformations that Se atom of catalytic residue SeCYS498, is at a distance of 3.56 from C13 of demethoxy curcumin at the E chain active site, whereas C13 carbon atom forms adduct with Se atom of SeCys 498. We report that at least one methoxy group in curcuminoids is necessary for interation with catalytic residues of thioredoxin. Pharmacophore of both active sites of the TrxR receptor for curcumin and demethoxy curcumin molecules has been drawn and proposed for design and synthesis of most probable potent antiproliferative synthetic drugs.     

Synthesis and antimicrobial studies of hydrazone derivatives of 4-[3-(2,4-difluorophenyl)-4-formyl-1H-pyrazol-1-yl]benzoic acid and 4-[3-(3,4-difluorophenyl)-4-formyl-1H-pyrazol-1-yl]benzoic acid

Microbial resistance to antibiotics is an unresolved global concern, which needs urgent and coordinated action. One of the guidelines of the Centers for Disease Control and Preventions (CDC) to combat antibiotic resistance is the development of new antibiotics to treat drug-resistant bacteria. In our effort to find new antibiotics, we report the synthesis and antimicrobial studies of 30 new pyrazole derivatives. These novel molecules have been synthesized by using readily available starting materials and benign reaction conditions. Some of these molecules have shown activity with MIC values as low as 0.78?µg/mL against four bacterial strains; Staphylococcus aureus, methicillin-resistant S. aureus, Bacillus subtilis, and Acinetobacter baumannii. Furthermore, active molecules are non-toxic to mammalian cell line.

     

Comparative morphology of the carpel in the Liliaceae: Hewardieae, Petrosavieae, and Tricyrteae

The young pistils in the melanthioid tribes, Hewardieae, Petrosavieae and Tricyrteae, are uniformly tricarpellate and syncarpous. They lack raphide idioblasts. All are multiovulate, with bitegmic ovules. The Petrosavieae are marked by the presence of septal glands and incomplete syncarpy. Tepals and stamens adhere to the ovary in the Hewardieae and the Petrosavieae but not in the Tricyrteae. Two vascular bundles occur in the stamens of the Hewartlieae and Tricyrtis latifolia. Ventral bundles in the upper part of the ovary of the Hewardieae are continuous with compound septal bundles and placental bundles in the lower part. Putative ventral bundles occur in the alternate position in the Tricyrteae and putative placental bundles in the opposite. position in the Petrosavieae. The dichtomously branched stigma in each carpel of the Tricyrteae is supplied by a bifurcated dorsal bundle.     

Novel compounds with potent CDK9 inhibitory activity for the treatment of myeloma

Cyclin-dependent kinases (CDKs) and Polo-like kinases (PLKs) play key role in the regulation of the cell cycle. The aim of our study was originally the further development of our recently discovered polo-like kinase 1 (PLK1) inhibitors. A series of new 2,4-disubstituted pyrimidine derivatives were synthesized around the original hit, but their PLK1 inhibitory activity was very poor. However the novel compounds showed nanomolar CDK9 inhibitory activity and very good antiproliferative effect on multiple myeloma cell lines (RPMI-8226).