盘基网柄菌DJ-1蛋白的原核表达及抗体制备

盘基网柄菌(Dictyostelium discoideum)是研究神经退行性病变的模式生物,可用以研究帕金森病相关基因DJ-1的作用和致病机理。本研究设计特异性引物扩增了人类DJ-1的盘基网柄菌同源基因片段DAM,并利用酶切位点Bam HⅠ和HindⅢ将DAM插入质粒载体p QE-30产生重组载体pPROF696,序列测定后对DJ-1进行了生物信息学分析。同时,通过电穿孔法将pPROF696转入大肠杆菌M15,并利用IPTG诱导DJ-1蛋白表达。经SDS-PAGE分析、Western-blot印迹和His标签树脂对DJ-1粗蛋白纯化后进行抗体制备,并在大肠杆菌M15转化株和盘基网柄菌转化株与野生型内对抗体进行检验。结果表明:本研究成功构建了盘基网柄菌DJ-1的原核表达载体pPROF696,所制备的盘基网柄菌DJ-1多克隆抗体能成功检测大肠杆菌M15和盘基网柄菌野生型与转化株中的DJ-1蛋白。这为今后检测盘基网柄菌细胞内DJ-1的表达水平,建立其与盘基网柄菌表现型相关性,标记DJ-1的亚细胞定位和进一步研究DJ-1在帕金森病中的作用机理奠定了基础。     

盘基网柄菌DdLIS1蛋白生物信息学分析

LIS1蛋白是一种与人类无脑回疾病以及细胞癌变相关的重要蛋白。对盘基网柄菌DdLIS1进行生物信息学分析,探究盘基网柄菌能否作为研究人类无脑回疾病及细胞癌变机制的模型。现从NCBI中的Genank找到盘基网柄菌DdLIS1的氨基酸序列,随后进行blastp找到模式生物中相似序列,利用理化性分析网站ProtScale、ProtParam分析DdLIS1的理化性质,通过NCBI中的保守结构域库(CDD)分析DdLIS1的保守结构域,使用MEGA6.0并选用邻位连接法构建系统进化树,分别使用PredictProtein、SWISS-MODEL网站预测Dd LIS1蛋白的二级结构、三维结构。结果得出DdLIS1蛋白全长为419,属于亲水性蛋白,有7个保守结构域,属于WD40家族,与人类和小鼠的氨基酸序列相似性为72%。二级结构中β折叠所占比例最高,为49.40%,α螺旋、随机卷曲分别占该蛋白7.16%、43.44%,与三级结构一致。以上结果说明DdLIS1与LIS1高度相似,有助于盘基网柄菌能够作为研究人类无脑回疾病以及细胞癌变机制的模型。     

将盘基网柄菌用于本科细胞生物学综合性实验教学初探——电穿孔转化绿色荧光蛋白

盘基网柄菌(Dictyostelium discoideum)是一个应用广泛的模式生物,非常适合用来研究胞质分裂、细胞运动、吞噬作用、趋化性、趋电性、信号转导以及个体发育过程中的细胞分化。该实验主要介绍电穿孔技术转化绿色荧光蛋白标记肌动蛋白基因质粒(Lifeact-GFP)进入盘基网柄菌活细胞中,抗性筛选(潮霉素B)获得阳性克隆子,最后借助荧光显微镜观察绿色荧光蛋白标记的微丝在盘基网柄菌的分布情况。综合性实验训练可提高学生的学习兴趣和综合运用理论知识的能力,进而可培养学生的科学研究思维。     

盘基网柄菌早衰蛋白结构与功能生物信息学分析

早衰蛋白(presenilin, PS)是γ分泌酶的组成成分,其突变可造成阿尔兹海默病(Alzheimer disease,AD)的形成。因PS所造成AD的发病机理较复杂,因而我们想探究一下模式生物盘基网柄菌(Dictyostelium discoideum)是否能够作为研究早衰蛋白的模型。从NCBI网站中得到盘基网柄菌早衰蛋白DdPsenA和DdPsenB氨基酸序列,随后利用ProtScale、ProtParam理化性分析工具、保守结构域、TMHMM2跨膜区分析网站、进化树软件MEGA6.0、二级结构PredictProtein以及三级结构SWISS-Model服务器对上述蛋白进行生物信息学分析。DdPsenA的氨基酸长度为622,是亲水性蛋白,其相对分子质量为69 502.79,等电点为4.53。DdPsenB的氨基酸长度为473,属于疏水性蛋白,其相对分子质量为52 558.74,等电点为4.49,与DdPsenA同属于Peptidase_A22B超家族。跨膜分析出DdPsenA和DdPsenB均有8个跨膜区。DdPsenA与人类presenilin-2序列相似性为67%,Dd PsenB与人类presenilin-1序列相似性分别为60%。盘基网柄菌可以作为研究早衰蛋白功能和作用机制的模型。     

盘基网柄菌发育中的细胞粘附分子及其信号转导

在盘基网柄菌发育早期,DdCAD-1和csA调节了变形虫细胞间的粘着,调控该过程的机制类似于胚胎发育中上皮细胞层的闭合。完成网柄菌发育的一个必需分子是gpl50异嗜性粘附分子。盘基网柄菌β-连环蛋白同源物Aardvark(Aar)的缺乏使细胞间失去粘着连接,Aar也有信号转导功能,调控了前孢子细胞基因的表达。因此,细胞间的粘着是盘基网柄菌发育的一个重要组成部分,并与调控形态发生过程的信号转导有密切相互作用关系。     

基于保守序列挖掘的乙型肝炎病毒进化研究

乙型肝炎是一种十分严重的全球性传染疾病,乙型肝炎病毒(Hepatitis B virus,HBV)是导致乙型肝炎的直接原因。而HBV突变是乙肝病毒进化过程中的一个重要部分,近几年,国内外针对HBV突变进行了广泛研究。但是,对乙肝病毒序列中保守序列的研究为仍处于起步阶段。本文首先采用MEME(Multiple EM for motif elicitation)算法挖掘HBV基序(生物序列中的保守序列片段,即Motif),并提出了一种新的度量标准保守指数(Conserved index,CI),然后对HBV序列进行系统发育分析,最后对构建的系统发育树进行可靠性评价。结果表明,新的度量标准CI可以有效地利用MEME方法挖掘出多个保守序列,进行HBV序列的系统发育树构建,进而分析HBV序列之间的进化关系,并可以找出样本可能的祖先序列。本文的实验方法对HBV大数据集分析方法的研究有积极地启示作用。     

盘基网柄菌DJ-1蛋白的亚细胞定位研究

DJ-1基因的突变或缺失导致帕金森病相关症状,但其在帕金森病中的作用以及其亚细胞位置尚存在争议。盘基网柄菌是研究神经退化性疾病的模式生物,通过绿色荧光蛋白(GFP)标记DJ-1蛋白,利用荧光蛋白技术及DJ-1与GFP共定位技术在正常和氧化情况下研究其在盘基网柄菌的亚细胞定位,可以为探索DJ-1蛋白亚细胞位置与致病机制之间的联系奠定基础。研究结果表明,正常情况下盘基网柄菌DJ-1蛋白位于细胞质内,一旦受到氧化应激, DJ-1蛋白则转移至线粒体,这个亚细胞位置转移与DJ-1蛋白C117位点的氧化相关。该研究为探索DJ-1蛋白如何在氧化应激条件下完成对细胞的保护提供了实验依据。     

利用简并引物克隆黑莓、树莓肌动蛋白基因片段及表达分析

采用同源克隆法,利用设计的一对肌动蛋白(actin)基因简并引物,从黑莓、树莓和悬钩子杂种3个悬钩子植物品种均得到一条743 bp的actin cDNA片段,推测编码247个氨基酸.序列比对发现,克隆的actin基因推导的氨基酸序列和其他植物的肌动蛋白氨基酸序列具有高度保守性,经数据库搜索后将来自黑莓和树莓品种的actin cDNA片段在GenBank中登录,登录号分别为HQ439558和HQ439559.不同来源的植物actin蛋白系统进化树的聚类结果表明,树莓和悬钩子杂种亲缘关系较为密切.actin基因在3个悬钩子植物品种不同组织中的RT-PCR分析结果表明,其在检测的不同组织中均有一定程度的表达量,可能都属于组成型表达的肌动蛋白基因.研究结果为利用actin基因作为内参进一步研究黑莓和树莓其他基因的丰度奠定基础.     

小麦WOX转录因子基因的全基因组鉴定与分析

小麦是全球主要粮食作物之一。本研究运用生物信息学技术手段对普通小麦(Triticum aestivum L.)的WOX基因进行了全基因组鉴定、系统发育树构建、序列分析。结果显示:小麦基因组一共包含了26个WOX基因,其中在Phytozome数据库中收录有序列信息的有20个,可分为3个亚族,各亚族内的WOX基因具有相似的基因结构,内含子相位为0或1;该家族基因的蛋白质基序Motif 1、Motif 2、Motif 5和Motif 9较为保守,其中Motif 1和Motif 2推测为核心基序;该家族基因启动子包含ABRE、CAT-box、CGTCA-motif、RY-element等元件,说明小麦WOX基因可能参与调控胚乳或种子的发育,或参与分生组织的形成;Ta WOX6、Ta WOX8、Ta WOX10可能参与小麦穗和茎秆的发育过程;Ta WOX15、Ta WOX18、Ta WOX20在小麦胚乳中的表达量非常高,而在别的部位的表达量几乎为零,说明这3个基因与小麦胚乳的形成发育密切相关。该研究结果为进一步探讨小麦WOX基因的功能与分子作用机理奠定了基础。     

盘基网柄菌(Dictyostelium discoideum)吞噬作用中肌动蛋白多聚化及其调节

肌动蛋白是盘基网柄菌(Dictyostelium discoideum)细胞吞噬过程中的关键组分,通过其细胞内的定位和多聚化形式在确定的时间和地点连接特定的分子,使吞噬过程得以完成。profilin是肌动蛋白多聚化的重要调节分子,在磷脂酰肌醇信号转导与细胞骨架相交处起关键作用。许多小分子G蛋白参与细胞骨架调节,CAP蛋白是两者间重要连接分子。所以,吞噬作用是细胞内诸分子协同作用的结果。     

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.     

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.     

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.     

Detection of EBV and HHV6 in the Brain Tissue of Patients with Rasmussen’s Encephalitis

Rasmussen’s encephalitis (RE) is a rare pediatric neurological disorder, and the exact etiology is not clear. Viral infection may be involved in the pathogenesis of RE, but conflicting results have reported. In this study, we evaluated the expression of both Epstein-Barr virus (EBV) and human herpes virus (HHV) 6 antigens in brain sections from 30 patients with RE and 16 control individuals by immunohistochemistry. In the RE group, EBV and HHV6 antigens were detected in 56.7% (17/30) and 50% (15/30) of individuals, respectively. In contrast, no detectable EBV and HHV6 antigen expression was found in brain tissues of the control group. The co-expression of EBV and HHV6 was detected in 20.0% (6/30) of individuals. In particular, a 4-year-old boy had a typical clinical course, including a medical history of viral encephalitis, intractable epilepsy, and hemispheric atrophy. The co-expression of EBV and HHV6 was detected in neurons and astrocytes in the brain tissue, accompanied by a high frequency of CD8⁺ T cells. Our results suggest that EBV and HHV6 infection and the activation of CD8⁺ T cells are involved in the pathogenesis of RE.     

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).     

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.