耻垢分枝杆菌MSMEG_6281 过表达对菌株生长和形态的影响

【目的】耻垢分枝杆菌(Mycobacterium smegmatis mc2155,mc2155)MSMEG_6281为结核分枝杆菌自溶素Rv3717的同源蛋白,通过建立过表达MSMEG_6281的耻垢分枝杆菌菌株,推测该蛋白对耻垢分枝杆菌肽聚糖代谢的影响。【方法】利用RT-PCR方法检测乙胺丁醇(Ethambutol,EMB)作用后MSMEG_6281基因的表达变化;以耻垢分枝杆菌基因组DNA为模板,采用PCR技术克隆MSMEG_6281基因,构建分枝杆菌表达质粒p VV16-MSMEG_6281,进一步建立MSMEG_6281过表达的耻垢分枝杆菌菌株;利用生长曲线检测MSMEG_6281过表达对耻垢分枝杆菌生长的影响;利用扫描电子显微镜分析MSMEG_6281过表达引起的耻垢分枝杆菌形态变化。【结果】EMB处理引起MSMEG_6281基因表达上调;构建了过表达MSMGE_6281的耻垢分枝杆菌菌株(mc2155/p VV16-MSMEG_6281);过表达MSMGE_6281的耻垢分枝杆菌生长缓慢,菌体形态由短杆状转变为长杆状。【结论】MSMGE_6281的过表达可改变耻垢分枝杆菌形态。MSMGE_6281的功能与细胞壁肽聚糖水解相关,在mc2155细胞壁形态维持方面发挥重要作用。     

耻垢分枝杆菌的蚯蚓血红蛋白样蛋白MSMEG_3312影响其大环内酯类药物的敏感性

【目的】活性氧类分子是机体有氧代谢的自然产物,可以引起氧化损伤,导致细胞DNA突变、蛋白质失活,是细菌耐药产生的原因之一。蚯蚓血红蛋白家族是能携带氧、可逆地结合氧的一类蛋白,在氧代谢过程中发挥重要作用,与活性氧类分子介导的细菌耐药相关。预测耻垢分枝杆菌(Mycobacterium smegmatis)MSMEG_3312是蚯蚓血红蛋白样蛋白,其功能可能与细菌抗药性有关。【方法】通过生物信息学预测耻垢分枝杆菌MSMEG_3312的结构特征。通过基因敲除、遗传互补和抗药性分析以及启动子表达测定等方法研究MSMEG_3312与耻垢分枝杆菌抗药的相关性。【结果】与野生菌株mc2155相比,msmeg_3312敲除菌株表现为抗大环内酯类抗生素,而且回补msmeg_3312部分丧失了这种耐药表型。此外,大环内酯类抗生素的胁迫在统计上显著下调msmeg_3312启动子的表达。另外,对作用于核糖体的其他药物,敲除菌株和野生菌株没有抗药性差异。【结论】生物信息学分析显示MSMEG_3312的氨基酸序列具有典型的蚯蚓血红蛋白保守的HHE结构域,预测其二级结构含有4个α-螺旋组成的典型蚯蚓血红蛋白结构。MSMEG_3312与耻垢分枝杆菌对大环内酯类抗生素的药物敏感性相关,其可能通过影响药物与核糖体50S亚基的作用来发挥功能。     

结核杆菌DnaA蛋白在耻垢分枝杆菌中的同源表达

目的：在耻垢分枝杆菌中表达重组结核杆菌DnaA蛋白并对表达产物进行鉴定。方法：用PCR的方法扩增结核杆菌dnaA基因并克隆至表达载体pMF406中,构建重组大肠杆菌-分枝杆菌穿梭质粒pMF-dnaA。经双酶切及测序鉴定后,用电转化的方法将重组质粒转至耻垢分枝杆菌mc2155中。用0.02%乙酰胺诱导重组耻垢分枝杆菌,对表达产物进行SDS-PAGE和Western blotting检测和鉴定。结果：重组耻垢分枝杆菌构建成功,SDS-PAGE及Western blotting结果显示该重组耻垢杆菌可以实现结核杆菌DnaA蛋白的同源高效表达。结论：结核杆菌DnaA蛋白的同源表达为结核杆菌DNA复制机制的研究奠定了基础。     

定量蛋白质组分析蚯蚓血红蛋白样蛋白MSMEG_3312介导的分枝杆菌耐红霉素机制（英文）

【目的】细菌耐药机制是个复杂的机制,系统生物学是系统性揭示耐药机制的有力研究手段。我们课题组前期研究结果显示,蚯蚓血红蛋白样蛋白msmeg_3312基因敲除后能够增加耻垢分枝杆菌对红霉素的耐药性,本文系统研究MSMEG_3312参与红霉素耐药性形成的机制。【方法】首先纯化MSMEG_3312蛋白,利用光谱及圆二色谱描述MSMEG-3312蛋白。利用定量蛋白质组学的方法比较分析敲除菌株Δmsmeg_3312与野生型菌株mc~2155蛋白表达的差异,并通过qRT-PCR进行验证。利用红霉素ELASA试剂盒测定Δmsmeg_3312与mc~2155的胞内药物浓度。【结果】光谱及圆二色谱分析确定MSMEG_3312是蚯蚓血红蛋白样蛋白。定量蛋白质组学分析发现,红霉素未处理的条件下,相比于野生型菌株mc~2155,敲除菌株Δmsmeg_3312有包括3种转运蛋白在内的8种蛋白表达水平上调,14种蛋白表达下调;而红霉素处理后,Δmsmeg_3312中有448种蛋白差异表达,其中有11种转运蛋白表达上调,26种蛋白与氨基酸合成通路相关。胞内药物浓度检测显示敲除菌株Δmsmeg_3312的胞内红霉素浓度显著低于野生型菌株。【结论】蚯蚓血红蛋白样蛋白MSMEG_3312调控改变了细菌对红霉素药物处理的反应网络,其介导的红霉素耐药是一种集合抗生素耐受机制。     

乙酰化修饰降低Ku蛋白的DNA结合活性

【目的】研究乙酰化修饰对Ku蛋白活性的影响。【方法】利用耻垢分枝杆菌为表达菌株,转入Ku蛋白表达质粒,纯化具有乙酰化修饰的Ku蛋白和无乙酰化的Ku蛋白突变体,比较两类蛋白的生化活性;分析氧化压力和酸性环境下耻垢分枝杆菌细胞内Ku蛋白乙酰化水平的变化。【结果】Ku蛋白过量表达的耻垢分枝杆菌比转入空质粒的对照菌株生长缓慢;乙酰化Ku蛋白比未发生乙酰化Ku蛋白修复断裂DNA的活性降低、DNA结合活性降低;氧化压力和酸性压力环境下,耻垢分枝杆菌细胞内Ku蛋白数量降低,乙酰化Ku蛋白数量变化不大。【结论】乙酰化修饰能够调节Ku蛋白的DNA结合活性,从而调节非同源末端连接修复系统的活性;Ku蛋白乙酰化程度升高是耻垢分枝杆菌对不良生长环境的反应。     

CRISPR-Cas9系统与mazF介导的大片段删减法在酿酒酵母染色体大片段删减中的比较

【目的】比较CRISPR-Cas9系统与maz F法这两种酿酒酵母染色体大片段删减方法。【方法】分别用上述两种方法删减了酿酒酵母长度为26.5 kb的染色体大片段YKL072W-YKL061W,并比较了两种方法的转化效率、敲除成功率。【结果】利用CRISPR-Cas9系统平均得到5个转化子,但正确率为100%;maz F法得到约100个转化子,正确率略低于前者,为93%。【结论】两种方法均能高效删减酿酒酵母染色体大片段,CRISPR-Cas9系统正确率较高,操作简便省时;maz F法相对稳定,对目的基因无PAM位点要求。     

两步PCR介导的Red重组技术快速敲除鼠疫耶尔森菌sRNA及染色体大片段

【目的】利用Red同源重组系统,通过二步PCR法建立一种适合鼠疫耶尔森菌s RNA和大片段染色体基因敲除的方法。【方法】第一步PCR先扩增出目的基因的上、下游同源臂(600–1000 bp)及卡那抗性盒,再以上、下游同源臂及卡那抗性盒等摩尔混合物为模板,通过融合PCR获得含上下游同源臂及卡那抗性盒的线性突变盒,再将此突变盒的PCR产物电转到含有pKD46质粒的鼠疫201菌株,在阿拉伯糖的诱导下,p KD46质粒表达Red重组酶,促使卡那抗性盒替换目的基因,最后对获得的重组克隆进行PCR鉴定。【结果】本研究通过两步PCR法构建600–1000 bp的同源臂,提高了同源重组效率,并将鼠疫菌sRNA RyhB1(108 bp)和RyhB2(106 bp)和染色体大片段47-2(10.4 kb)、47-3(21.6 kb)、47-3a(9.2 kb)及47-3b(6.1 kb)成功敲除。【结论】基于Red重组系统构建的二步法突变技术,是一种简单、高效的精确修饰鼠疫菌s RNA及大片段染色体的方法,适合于鼠疫菌全基因组的基因敲除,为鼠疫菌基因表达与调控、致病和毒力等研究提供有力的工具。     

分枝杆菌glpX基因的功能

【目的】构建耻垢分枝杆菌(Mycobacterium smegmatis)glpX基因敲除株,研究其在生理代谢中的功能。【方法】利用分枝杆菌噬菌体Che9c重组系统构建耻垢分枝杆菌glpX基因敲除株;比较野生株及突变株在不同碳源培养条件下的生长差异;通过荧光实时定量PCR,比较野生株在以葡萄糖或油酸为唯一碳源培养下,glpX基因的表达水平。【结果】glpX突变株在以甘油或油酸为唯一碳源的培养基中无法生长;野生株在以油酸为唯一碳源培养下,glpX基因表达上调。【结论】glpX基因编码了分枝杆菌糖异生途径必需的和非冗余的果糖1,6-二磷酸酶(fructose 1,6-bisphosphatase,FBPase)。     

分枝杆菌glpX基因的功能

摘要:【目的】构建耻垢分枝杆菌(Mycobacterium smegmatis)glpX基因敲除株,研究其在生理代谢中的功能。【方法】利用分枝杆菌噬菌体Che9c重组系统构建耻垢分枝杆菌glpX 基因敲除株;比较野生株及突变株在不同碳源培养条件下的生长差异;通过荧光实时定量PCR,比较野生株在以葡萄糖或油酸为唯一碳源培养下,glpX基因的表达水平。【结果】glpX突变株在以甘油或油酸为唯一碳源的培养基中无法生长;野生株在以油酸为唯一碳源培养下,glpX基因表达上调。【结论】glpX基因编码了分枝杆菌糖异生途径必需的和非冗余的果糖1,6-二磷酸酶(fructose 1,6-bisphosphatase,FBPase)。     

结核分枝杆菌H37有毒株与无毒株的代谢相关基因pabB和lpdA启动子活性分析

【目的】通过分析结核分枝杆菌无毒株H37Ra的全基因组序列,并与H37Rv基因组序列比较,发现pabB和lpdA预测的启动子区发生了突变。我们利用报告基因,确认启动子突变与其基因转录水平的关系,探索结核分枝杆菌H37Ra毒力丧失的内在原因。【方法】利用生物信息学方法预测这两对基因的启动子区,采用PCR技术克隆这两对基因的启动子,与分枝杆菌启动子探针载体pMC210相连,DNA测序证实连接片段正确后,用电穿孔法将重组质粒转化至耻垢分枝杆菌mc2155。利用Quantitative Real-Time RT-PCR检测报告基因lacZ转录水平的差异,进一步验证这两对基因启动子的突变对相应基因转录水平的影响。【结果】Quantitative Real Time PCR检测结果显示H37RapabB启动子活性是H37Rv pabB启动子活性的6倍(p0.05),而H37Rv lpdA启动子的活性是H37RalpdA启动子的2倍(p0.05)。【结论】pabB,lpdA的启动子在H37Ra中的突变对其启动子的活性产生了影响,其中lpdA启动子的突变可能与结核分枝杆菌H37Ra的毒力丧失有关。     

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.     

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     

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.     

鸡传染性法氏囊病病毒研究进展

传染性法氏囊病(Infection bursal disease, IBD)是由鸡传染性法氏囊病毒(Infectious bursal disease virus, IBDV)引起的鸡和火鸡的一种高度接触性传染病,给世界各国的禽养殖业带来了巨大损失.自IBDV发现至今新的变异株不断出现,分子结构的改变导致病毒致病力的改变及宿主对疫苗应答的改变,使得传统的疫苗已不能控制其流行,因此各国学者对其基因组结构和功能进行了广泛深入的研究,并积极研制新型有效的疫苗以达到防治的目的.     

Development of a Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2

In conclusion, the novel visual RT-LAMP assay is a simple, rapid, and sensitive approach for detection of SARS-CoV-2, and it is ready for application in primary care and community hospitals or health care centers, and even patients' own houses in response to the current SARS-CoV-2 epidemic because the assay does not require sophisticated equipment and skilled personnel. Furthermore, it is also ready to be used in fields for screening samples from wild animals and environments to facilitate the identification of potential intermediate hosts that mediate the cross-species transmission of SARS-CoV-2 from bats to humans.     

Perinatal Vertical Transmission of Chikungunya Virus in Ruili,a Town on the Border between China and Myanmar

正Dear Editor,Chikungunya virus (CHIKV), an arbovirus in the family of Togaviridae, genus Alphavirus, is transmitted by the A.aegyptii or A. albopictus mosquito, and causes disease in humans characterized by fever, rash, and arthralgia (Silva and Dermody 2017; Suhrbier 2019). It was first reported in 1953 in Tanzania, and caused only a few outbreaks and sporadic cases in Africa and Asia in last century. However, in the epidemic in 2004, CHIKV acquired mutations that conferred enhanced transmission by the A. albopictus mosquito(Schuffenecker et al. 2006). Since then, it has successively caused outbreaks in Africa, the Indian Ocean, South East Asia, the South America, and Europe (Zeller et al. 2016).     

Enterovirus 71 3C proteolytically processes the histone H3 N-terminal tail during infection

Highlights
1. The N-terminal tail of histone H3 is specifically cleaved during EV71 infection.
2. Viral protease 3C is identified as a protease responsible for proteolytically processing the N-terminal H3 tail.
3. Our finding reveals a new epigenetic regulatory mechanism for Enterovirus 71 in virus-host interactions.