甜菜坏死黄脉病毒内蒙分离物RNA3序列分析及编码25kD蛋白基因在大肠杆菌中的表达

以甜菜坏死黄脉病毒（ＢＮＹＶＶ）内蒙分离物的总ＲＮＡ为模板,通过反转录－ＰＣＲ扩增获得ＢＮＹＶＶＲＮＡ３全长ｃＤＮＡ。将其克隆到ｐＧＥＭ－７Ｚｆ（＋）上,得到重组质粒ｐＧＢＹ５６。序列分析结果表明,内蒙分离物ＲＮＡ３基因组全长为１７７５ｎｔ,其中包含３个开放阅读框架,分别编码２５ｋＤ蛋白、４．６ｋＤ蛋白和一种由５９个氨基酸组成的Ｎ蛋白。与法国Ｆ２分离物、德国Ｇ１分离物和日本Ｓ分离物相比,其核苷酸序列的同源性分别为９６．４％、９６．８％和９７．３％。将２５ｋＤ蛋白编码基因克隆到ｐＪＷ２上,构建了该基因的原核表达载体。ＳＤＳ－ＰＡＧＥ和Ｗｅｓｔｅｒｎｂｌｏｔｉｎｇ分析结果表明,２５ｋＤ蛋白基因在Ｅ．ｃｏｌｉＢＬ２１（ＤＥ３）中经温度（４２℃）诱导后,可特异地表达２５ｋＤ蛋白     

马铃薯Y病毒普通系外壳蛋白基因在大肠杆菌中的表达

将马铃薯Ｙ病毒普通系（ＰＶＹ０）的外壳蛋白基因克隆到表达质粒ｐＭＡＬｃ２中,构建这一基因在大肠杆菌中的表达载体ｐＭＡＬｃ２ＰＶＹ０ＣＰ。ＳＤＳＰＡＧＥ及Ｗｅｓｔｅｒｎｂｌｏｔｉｎｇ检测结果表明,这一表达栽体在Ｅ．ｃｏｌｉＤＨ５α中经ＩＰＴＧ诱导可表达分子量为７１．８ｋＤａ的特异性融合蛋白。以ａｍｙｌｏｓｅｒｅｓｉｎ亲合柱层析纯化这一融合蛋白为抗原,免疫家兔制备了效价为１∶１０２４的特异性抗血清。用该抗血清可通过对流免疫电泳、免疫双扩散及Ｗｅｓｔｅｒｎｂｌｏｔｉｎｇ对ＰＶＹ进行检测     

葡萄扇叶病毒外壳蛋白基因的克隆,序列分析及其在大肠杆菌中…

通过ＲＴ－ＰＣＲ方法把葡萄扇叶病毒（ＧＦＬＶ）外壳蛋白基因（ＣＰ ｇｅｎｅ）分成两部分扩增,扩增产物克隆入ｐｇＥＭ－５Ｚｆ（＋）载体,并通过ＢｇｌⅡ位点连接成一完整的外壳蛋白基因,通过序列分析测得全长外壳蛋白基因为１５１２ｂｐ,编码５０４个ＡＡ＇ｓ与国外株系ＧＦＬＶ－Ｆ１３相比,核苷酸同源性为８８．４％,氨基酸同源性为９５．８％。并且这一外壳蛋白基因在大肠杆菌Ｅ． ｃｏｌｉＤＨ－５α中得到了表达。     

抗甜菜坏死黄脉病毒单链抗体表达载体的构建及其表达

用ＰＣＲ方法以分泌抗甜菜坏死黄脉病毒（ＢＮＹＶＶ）单克隆抗体的杂交瘤细胞的基因组为模板,扩增了编码ＢＮＹＶＶ单抗的重链可变区（ＶＨ）基因。测序表明,该ＶＨ序列属于小鼠ＩＩ（Ａ）亚类,全长为３６０ｂｐ,编码１２０个氨基酸。将其和先前克隆的轻链基因分别插入到一个含有连接ＶＨ和ＶＬ基因的连接序列的质粒之中,构建成单链抗体（ｓｃＦｖ）基因的表达载体ｐＴＣｓｃＦｖ。将质粒在大肠杆菌中表达,ＥＬＩＳＡ法检测出     

甜菜坏死黄脉病毒外壳蛋白基因在甜菜转基因植株中的表达

甜菜坏死黄脉病毒(Beet Necrotic Yellow Vein Virus,BNYVV)是一种由甜菜多粘菌(Polymyxo be tae)传播的多分体植物病毒．基因组由4～5条单链正意RNA构成^[1]。60年代末,由Tamada首次报道^[2],这种病毒可对甜菜造成严重危害,侵染甜菜后产生丛根症状(Rhizomania),并导致甜菜产量和含糖 量的大幅度下降。除欧洲、北美及日本的严重发生以外,我国自70年代以来在东北、内蒙古及西北许多省区也有大量甜菜丛根病的发生报道^[3]。由于尚无有效药剂及措施用于甜菜丛根病或病毒传播介体的防治．在我国也无法采用大面积轮作作为防治手段,所以目前在世界各地及我国上述地区甜菜丛根病的发病面积逐年扩展,对甜菜生产和制糖业造成直接威胁。针对这一情况．本文报道了含有甜菜坏死黄脉病毒外壳蛋白基因的甜菜植株的转化再生工作,以期在甜菜亲本育种中获得新的抗性材料．为抗病毒品种的培育打下基础。     

庚型肝炎病毒包膜糖蛋白E2基因在昆虫细胞中的表达

用ＰＣＲ扩增出ＨＧＶＥ２全基因,克隆进杆状病毒表达载体ｐＦＡＳＴＢＡＣＨＴａ中,构建成重组转座载体ｐＦＡＳＴＢＡＣＥ２,转化ＤＨ１０ＢＡＣ大肠杆菌感受态细胞,筛选阳性菌落,抽提大分子质粒ＤＮＡ,获得含ＨＧＶＥ２基因的重组杆状病毒穿梭载体,转染昆虫草地夜蛾Ｓｆ９细胞,出现细胞病变后,收集含有重组病毒颗粒的培养上清,重新感染草地夜蛾Ｓｆ９单层细胞及甜菜夜蛾幼虫,分别收集Ｓｆ９细胞和甜菜夜蛾幼虫体内的血淋巴细胞,进行１２％ＳＤＳ聚丙烯酰胺凝胶电泳,可见表达的融合蛋白带,经亲和层析进行蛋白纯化,用ＥＬＩＳＡ方法检测各类血清标本,初步研究ＨＧＶＥ２糖蛋白的抗原性     

人α降钙素基因相关肽与肿瘤坏死因子的融合表达

用半酶和半化学合成的方法合成和克隆了人ａ降钙素基因相关肽基因,将该基因融合在肿瘤坏死因子之后插入表达载体ｐＳＢ－９２中,使融合基因的５＇端直接置于大肠杆菌ＰＬ启动下游,采用３０℃培养,４２℃诱导,使ＴＮＦ与ＣＧＲＰ融合蛋白在大肠杆菌中获得了表达,表达的融合蛋白既有ＣＧＲＰ的结合活性（酶标检测为阳性）又有ＴＮＦ的生理功能（对Ｌ－９２９细胞的细胞毒活性）。表达菌裂解液走ＳＤＳ－聚丙烯酰胺凝胶电泳,显示     

短芽孢杆菌α—乙酰乳酸脱羧酶基因在大肠杆菌中的克隆和表达

用ＰＣＲ方法扩增短芽孢杆菌α－乙酰乳酸脱羧酶基因,引入原核表达载体ｐＢＶ２２０中,得到重组质ｐＡＬＤ１．ｐＡＬＤ１的ＡＬＤＣ基因在大肠杆菌中高效表达,每毫升发酵液产酶８０单位以上,双原始菌株提高２００余倍。ＳＤＳ－ＰＡＧＥ蛋白质分析表明,大肠杆菌ＤＨ５α表达的ＡＬＤＣ占细胞总蛋白量的４０％以上。研究了重组质粒稳定性,大肠杆菌ＤＨ５α和ＨＢ１０１分别大无选择压力下３０℃连续培养５０代以上,４１℃诱导     

甜菜坏死黄脉病毒外壳蛋白基因的克隆和序列分析

以甜菜坏死黄脉病毒(BNYVV)内蒙分离物的RNA为模板,通过PcR扩增获得外壳蛋白(CP)基因的目的片段。将其重组到pGEM一7zf(+)并转化JMl01得到了含有完整CP基因的重组子。采用双脱氧终止法进行序列分析,结果表明CP基因为567nt,与文献(1]报道相比,氨基酸和核苷酸的同源性分别为98．4％和96．7％。     

中国甜菜坏死黄脉病毒RNA5的检测及其核苷酸序列分析

利用反转录 Ｐ Ｃ Ｒ 方法,对甜菜坏死黄脉病毒（ Ｂ Ｎ Ｙ Ｖ Ｖ）５ 个中国分离物的 Ｒ Ｎ Ａ５进行了检测,结果表明新疆分离物、黑龙江分离物和内蒙古乌拉特前旗分离物不含有 Ｒ Ｎ Ａ５ ,只有包头分离物和呼和浩特分离物有 Ｒ Ｎ Ａ５ 。序列分析结果,包头分离物和呼和浩特分离物 Ｒ Ｎ Ａ５ 基因组分别为１３３８ ｎｔ 和１３５８ ｎｔ,均只包含１ 个开放阅读框架,编码产生２６ ｋ Ｄ 蛋白。与法国 Ｆ７２ 分离物和日本 Ｄ５ 分离物相比, 各分离物间核苷酸序列同源性为９３７ ％ ～９８５ ％ ,由此推导的氨基酸序列同源性为９１８ ％ ～９８２ ％ 。     

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).