哈氏弧菌dam基因的克隆与分析

利用兼并PCR的方法克隆得到哈氏弧菌T4的DNA腺嘌呤甲基化酶(dam)基因,序列分析表明该基因编码279个氨基酸,与其它已知弧菌的Dam具有较高的同源性,其中与副溶血弧菌Dam的相同性达95%。功能检验表明所克隆的dam基因在大肠杆菌中具有DNA腺嘌呤甲基化酶活性,能够甲基化大肠杆菌染色体DNA GATC序列中的腺嘌呤。运用染色体步移法获得dam基因上游的3251 bp DNA,发现该区域含有3个基因,其与dam在染色体上的相对排列顺序为:莽草酸激酶-脱氢奎尼酸合成酶-damX-dam。对dam上游DNA序列研究发现位于翻译起点ATG上游的78bp、112bp和477bpDNA片段皆具有启动子活性,但前者的活性明显高于后二者。     

生物信息学研究新基因LRP15

为了利用生物信息学探索网上克隆基因与预测基因功能的新方法,首先用一段1.8kbDNA片段的人类EST数据库中进行电子杂交并对相互重叠的EST片段组装,通过引物设计进行cDNA末端快速扩增,以高通量基因组序列（HTGS）数据库及SAGE库为基础,进行染色体定位及组织表达分析。通过DAS及RPS－BLAST程序预测其蛋白质结构及功能。结果显示,LRP15 cDNA全长1718bp,含一个780bp的开放读码框架,编码259个氨工酸,该基因定位于染色体3p24,在多种组织中表达。其编码蛋白含有N端富含亮氨酸重复序列及潜在的穿膜序列。研究表明,生物信息学是克隆与预测基因功能的有效方法;LRP15基因可能是一个参与细胞发育调控的新基因。     

人甲状腺受体相互作用蛋白15基因全长cDNA的克隆及其特性

通过生物信息学分析及RT PCR技术 ,从人垂体cDNA文库中克隆到甲状腺素受体相互作用蛋白 15(hTRIP15)的全长cDNA ,长度 1963bp ,编码 4 4 3氨基酸 ,同时克隆该基因不同剪接方式所形成的新的异构体 ,长度 1984bp ,编码 4 50氨基酸 .与基因组序列比较显示该基因具有 12个外显子 ,5号外显子 3′端具有 2个剪切的接点 (-ag) .搜寻UniGene数据库作染色体定位于D15S146 D15S117,该基因在生物进化上具有较高的保守性 ,从单细胞藻类到人类均有该基因同源物表达 ,亚细胞定位为核内 .Northern杂交显示 ,该基因具有 3种不同大小的转录本 ,分别约为 2 0、3 5及 4 0kb ,且在人体各组织中均有一定表达 ,其中骨骼肌、心脏及肾脏组织为高表达 .半定量RT PCR显示在一些内分泌组织均有表达 ,以肾上腺较高 .     

番茄RGA4蛋白与马铃薯Rpi-blb1蛋白的同源比较分析

为获得番茄抗晚疫病广谱性基因信息,采用同源电子克隆法,基于番茄蛋白序列数据库,以马铃薯晚疫病广谱抗性蛋白Rpi-blb1为种子序列,获得番茄疾病抗性蛋白RGA4,并进行基因电子定位和基因结构、模体、二级结构、基因进化及基因电子表达等分析,以证明两者的进化关系。结果表明:番茄RGA4蛋白(XP_004245923.1)序列具有NB-ARC和LRR8两个保守结构域,定位于第8条染色体SL2.40区间,相应基因位于8号染色体序列的57228847-57232935 bp区间,长度为4 089 bp,由2个外显子和1个内含子组成,编码988个氨基酸序列,该蛋白为不稳定分泌球形蛋白;番茄RGA4蛋白和马铃薯Rpi-blb1蛋白在二级结构分布、基因序列组成、基因定位等方面相似性较高,可确定两者为垂直同源关系,但在基因进化和模体组成方面存在差异,可能导致两者功能上的不同。研究结果可为番茄晚疫病广谱抗性基因克隆及其利用提供理论依据。     

人类SR蛋白超家族新成员——SFRS12(SRrp508)的基因克隆和特性分析

采用生物信息学方法克隆出全长3811bp的人类RC508cDNA片段,经核酸和蛋白质分析为人类新基因（Gen-Bank登记号：AF459094）,利用RT－PCR方法从人类胰脏组织中扩增出包含码508个氨基酸残基最大开放读码框架（ORF）的1680bp cDNA片段,经核酸测序证明与电子克隆结果完全一致。该基因具有启动子和TATA-box,ORF前同一相位有多个终子码,后有加尾信号,显示为客观存在基因。该基因含有12个外显子（96－2093bp)和11个内含子（140－5153bp),定位于人类5号染色体5q11.2-q12.1,无任何连锁基因存在。该基因ORF342－1868（1527）横跨10个外显子,所编码508氨基酸蛋白的全长序列与大鼠丝氨酸－精氨酸二肽富含性（SR）剪切调控蛋白86（1527）横跨10外显子,所编码508氨基酸蛋白的全长序列与全长序列与大鼠丝氨酸－精氨酸二肽富含性（SR）剪切调控蛋白86（SRrp86)高度同源,在核酸和蛋白水平的同源性分别为84％和86％,与其他已知蛋白无论在核酸水平是在氨基酸水平几乎均无整体的同源性。结果表明,所克隆的508氨基酸蛋白才是大鼠SRrp86的人类同源物,从而修正了Barnard(2000)所指出的人类同源物为人类精氨酸富含性核蛋白54（p54)这一论断,并提示它是日益增长的SR蛋白超家族的又一个新成员。该基因组织表达谱广泛,有可能具有转录因子活性,暂命名为SR相关剪切调控蛋白508（SRrp508)。国际人类基因命名委员会已将其命名为丝氨酸－精氨酸二肽含性剪切因子12（splicing factor,arginine/serine-rich),缩写为SFRZS12,化名为DKFZp564B176,SRrp86。     

猪瘟病毒石门株NS4A基因的克隆,测序及分析

采用RT-PCR技术,利用一对引物,从感染猪血中成功扩增了猪瘟病毒强毒石门株NS4A基因,片段大小为757bp,与预期大小一致。克隆后经酶切鉴定,自动化测序。序列比较表明,该基因为最保守的基因之一,其中石门株序列与ALD,GPE,HCLV及Brescia株均有很高的同源性,由其推导的氨基酸的同源性高达100％。仅与Alfort株的同源性消低。对石门株序列与同属的BVDVSD－1株和NADL株序列进行了比较。     

小鼠鼻咽部组织相对特异基因调控区的克隆与分析

通过RT－PCR证实PLUNC基因在小鼠鼻咽部具有相对特异性表达。进一步用染色体步移的方法克隆该基因5′端旁侧序列847bp,网上分析结果提示此序列具有调控活性,体外报道基因的研究亦证实这一结果。进一步的研究表明,其核心调控区位于转录起始位点上游200bp之内。这一工作将为构建鼻咽癌转基因动物提供良好的前提。     

八肋游仆虫第二类释放因子基因的克隆与序列分析

分离八肋游仆虫 (Euplotesoctocarinatus)大核eRF3基因 ,为进一步研究第二类释放因子结构与功能 ,探讨低等真核生物新生肽链释放机理提供实验素材 .以八肋游仆虫基因组DNA为材料 ,根据已知的第二类释放因子eRF3保守氨基酸序列设计引物 ,扩增克隆了该游仆虫的第二类释放因子基因片段 ,并对其核苷酸序列进行了分析 .根据测得的序列设计特异性引物 ,并利用游仆虫的端粒序列 (C4 A4 C4 A4 C4 A4 C4 )为引物 ,扩增得到该基因的全序列 .序列分析表明 ,该基因位于 2 782bp长的大核染色体上 ,编码区由 2 4 0 0bp组成 ,编码 80 0个氨基酸 ,不含内含子     

哈氏弧菌dam基因的克隆与分析

利用兼并PCR的方法克隆得到哈氏弧菌T4的DNA腺嘌呤甲基化酶(dam)基因,序列分析表明该基因编码279个氨基酸,与其它已知弧菌的Dam具有较高的同源性,其中与副溶血弧菌Dam的相同性达95%。功能检验表明所克隆的dam基因在大肠杆菌中具有DNA腺嘌呤甲基化酶活性,能够甲基化大肠杆菌染色体DNA GATC序列中的腺嘌呤。运用染色体步移法获得dam基因上游的3251 bp DNA,发现该区域含有3个基因,其与dam在染色体上的相对排列顺序为:莽草酸激酶-脱氢奎尼酸合成酶-damX-dam。对dam上游DNA序列研究发现位于翻译起点ATG上游的78bp、112bp和477bpDNA片段皆具有启动子活性,但前者的活性明显高于后二者。     

人脑内-含有ACP样结构域新基因的发现

为寻找脑内新基因,以正常成人全脑cDNA为模板,采用锚定PCR方法进行扩增,将经琼脂糖DNA电泳 鉴定获得的一约1200bp大小的特异性条带回收,并克隆入Teasy载体.用310 Genetic Analyzer进行自动测序. 所得序列进行生物信息学分析BLAST相似性分析结果证明所得序列为新序列,读框分析表明,该序列中存在 一完整编码区,编码含357个氨基酸的蛋白质.ProDom软件分析发现其含有酰基携带蛋白(ACP)样结构域. 随后,经3'RACE法克隆到该基因的全长cDNA,其全长为2024bp,染色体定位在14q11.2,含有16个外显子, 15个内含子,该基因已登录到GenBank.经设计编码区引物,从Teasy载体扩增出编码区后再克隆入pGEX-4T1 表达载体,经异丙基硫代-D-乳糖苷(IPTG)化学诱导表达.其编码区克隆人pGEX-4T1表达载体后,转入 JM109宿主菌,经IPTG诱导已得到表达.点杂交及RNA印迹表明,该基因在正常成人脑内广泛高表达.     

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.