猪肥胖基因在大肠杆菌中的高效融合表达

采用PCR方法扩增猪肥胖基因编码原成熟蛋白cDNA序列,并在5′端加上BamHⅠ位点,3′端加上EoRⅠ位点,将5′端密码子CCC转变为大肠杆菌常见密码子CCG,扩增得到459bp的片段,克隆于融合表达载体p GEX-2TBamHⅠ和EcoRⅠ位点,酶切、测序正确,经0．1mmol/LIPTG诱导表达出一条约42kD的融合蛋白,其中26kD为pGEX-2T中带有的谷胱苷肽转移酶,16kD是猪肥胖基因表达产物瘦蛋白。利用非融合表达产品制备抗血清,检测融合表达的重组蛋白,Western-blot为阳性。     

蓝氏贾第虫核纤层蛋白基因的初步研究

贾第虫一度被认为是迄今已知的最原始的真核细胞,但近来争议日盛。利用PCR和测序等技术,对蓝氏贾第虫(Giardia lamblia)的核纤层蛋白(lamin)基因进行了研究。结果表明：蓝氏贾第虫基因组中存在一个编码具有明显lamin特征的基因序列。如该基因序列的3’一端具有编码与核内膜亲和的特征性模体(motif)CaaX的序列;具有B型lamin基因所特有的高度保守的27bp片段,该片段编码高度保守的位于a螺旋杆状区的9氨基酸片段等。同时,这些序列特征又与多细胞的后生动物存在一定差异。这些事实说明在贾第虫中已经进化产生了典型真核细胞的B型lamin(基因)或至少是类似B型的lamin(基因),该生物的进化地位可能并非过去所认为的那么原始。     

云斑车蝗线粒体基因组全序列测定与分析

采用长距 PCR 扩增及保守引物步移法并结合克隆测序测定并注释了云斑车蝗 Gastrimargus marmoratus （Thunberg）的线粒体基因组全序列。结果表明：云斑车蝗线粒体基因组全序列为15 904 bp（GenBank登录号为EU527334）,A+T含量略高于非洲飞蝗Locusta migratoria,为76.04%,包括13个蛋白质编码基因,22个tRNA 基因,2个rRNA基因和一段1 057 bp的A+T富集区。蛋白质基因的起始密码子中,除COⅠ和ND5为TTG以外,均为昆虫典型的起始密码子ATN。ND5基因使用了不完全终止密码子T,其余基因均为典型的TAA或TAG。预测了22个tRNA基因的二级结构,发现tRNA^Ser（AGN）缺少DHU臂, tRNA^Ser（UGY）的反密码子环上有9个碱基。预测了云斑车蝗12S和16S rRNA二级结构,分别包括3个结构域30个茎环和6个结构域44个茎环。A+T富集区含有3个串联重复序列。     

乙肝病毒突变核心蛋白基因的克隆及序列分析

目的克隆发生长片段缺失的乙肝病毒核心蛋白基因（HBV-C）,并对其进行DNA序列和蛋白质结构分析。方法通过PCR从1株乙型肝炎病毒中扩增得到发生长片段缺失的HBV-C基因,利用TA克隆将PCR产物克隆人pUCm—T载体并进行测序、同源性比较和蛋白质结构分析。结果PCR扩增出的HBV-C基因经序列分析表明长度为454bp,其核苷酸序列缺失了220—317bp之间的98个碱基,造成从74个氨基酸起发生移码突变。结论成功克隆发生长片段缺失的HBV-C基因,为表达及功能研究奠定了基础。     

鼠疫耶尔森氏菌LcrV基因的克隆及序列分析

为了研究鼠疫耶尔森氏菌（Y.pestis）保护性抗原V蛋白,从基因库中查得Y.pestis LcrV基因DNA序列,针对序列设计合成了一对PCR扩增引物,以本所保存的Y.pestis菌种为模板进行基因扩增,结果获得长约980bp的DNA片段。将扩增产物回收纯化,克隆至pGEM-T载体,构建重组载体pGEN-T/ypV,经过PCR,酶切鉴定,并对pGEM-T/ypV中的V基因片段进行测序,分析测序结果与己知序列相同,表明获得了LcrV基因。     

利用两种不同引物扩增金龟子COⅠ序列片段的研究与对比

利用两类不同的引物,即通用引物(L1490,H2198)与特异引物(Pat,Jerry)分别对4种常见金龟子线粒体细胞色素C氧化酶I(COⅠ)基因片段序列进行扩增和测序,获得长度为689 bp与775 bp的序列。对测序结果进行遗传距离分析,并构建了4种金龟子系统进化树。结果表明,特异引物扩增序列的遗传距离在种内稳定性与种间的差异性都明显优于通用引物扩增序列,利用特异引物扩增序列所构建的系统进化树最符合实际情况,因此利用特异引物扩增序列更能够准确的对金龟子进行分类。     

人ZP3基因的RT-PCR cDNA克隆

目的：研究人ZP3基因的结构及构建人ZP3基因原核表达系统。方法：从人卵巢组织中分离出mRNA并以此作为模版,通过RT—PCR扩增出人ZP3基因cDNA片段,然后将其克隆在pUC18质粒上,并对克隆片段进行序列分析。结果：共克隆到ZP3-A（1300bp）、ZP3-B（1180bp）、ZP3-C（1200bp）和ZP3-D（1080bp）4种不同长度的人ZP3基因cDNA片段,对其中最长的ZP3-A片段的测序结果表明,它包含了人ZP3基因阅读框内的全部序列,与NCBI Sequence Viewer中公布的人ZP3 mRNA序列（NM-007155）相比较,在1275bp长的编码区内只有一个碱基不同,两者同源性达到99．92％。结论：本研究克隆到的ZP3-A cDNA片段确是人ZP3基因无疑。     

结球甘蓝抗TuMV相关基因的克隆

以结球甘蓝高抗TuMV自交不亲和系84075为材料,构建了cDNA文库。根据抗病基因保守序列（NBS－LRR）设计一对简并引物,以84075的基因组DNA和cDNA为模板,进行PCR扩增,分别扩增出一条513bp片段,扩增片段进行克隆测序。选取两个与抗病基因同源性较高的克隆片段作探针（命名Borl,Bor2）,对构建的cDNA文库进行筛选,得到一个阳性克隆（命名TuR2）,测序及序列分析表明,该基因总长为762bp,编码226个氨基酸、包含681bp的开放阅读框。与已克隆的抗病基因有不同程度的同源性。利用TuR2作探针,进行了Southern杂交、Northern杂交以及抗病性的共分离检测分析。结果表明,TuR2可能吧单拷贝形式存在,其表达是组成成型的,且无组织特异性;初步确定是一个与结球甘蓝抗TuMV相关的基因。     

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.     

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.     

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.

     

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).