从HFRS患者的PBMC建立BLCL及其意义

体外研究汉滩病毒(Hantaan virus,HTNV)S基因及其5'端、3'端在BLCL(EBV-transformed Blymphoblastoidcellline,BLCL)内稳定表达的意义,为核蛋白T细胞表位的研究奠定基础.本研究从肾综合征出血热(HFRS)患者的外周血单个核细胞(PBMC)建立与HTNV特异性CTL同源的患者自身的EB病毒转化的B淋巴母细胞系,将含有不同HTNV S基因片段的重组真核表达载体转染入BLCL,获得长期稳定表达,作为靶细胞系,为下一步进行CTL杀伤试验提供靶细胞系.设计4条引物,用PCR方法从PBV220-S22原核质粒中扩增出S基因全读码框(37-1326bp)及S基因5'端(37-501bp),S基因3'端(502-1326bp),用TA克隆将其克隆入pcDNA3.1/V5-His-TOPO载体中,成功构建pcDNA3.1-S及pcDNA3.1-S-N、pcDNA3.1-S-C真核表达载体,并通过脂质体转染至BLCL细胞中,进行了稳定表达.间接免疫荧光成功检测到pcDNA3.1-S及pcDNA3.1-S-N、pcDNA3.1-S-C在BLCL细胞中的表达.pcDNA3.1-S及pcDNA3.1-S-N、pcDNA3.1-S-C真核表达载体有较高的转染效率,目的基因能在宿主细胞中长期稳定表达,有利于研究HTNV-S基因在T细胞表位研究中的意义.建立的特异性CTL克隆对表达完整NP、NP羧基和氨基端肽段的靶细胞均有比较明显的杀伤效应,平均杀伤率分别为50.2%、39.0%和25.4%.HTNV-NP优势T细胞表位可能主要位于病毒核蛋白的羧基端.     

汉滩病毒S基因及其5'端真核表达载体的构建及在细胞中的表达

体外研究汉滩病毒(HTNV)S基因及其5'端表达的意义,为核蛋白T细胞表位的研究奠定基础.设计2套引物,用PCR方法从PBV220-S22原核质粒中扩增出S基因全读码框(37-1326bp)及S基因5'端(37-501bp),用TA克隆将其克隆入pcDNA3.1/V5-His-TOPO载体中,成功构建pcDNA3.1-S及pcDNA3.1-S-N真核表达载体,并通过脂质体转染至Vero细胞中,进行了瞬时表达.间接免疫荧光成功检测到pcDNA3.1-S及pcDNA3.1-S-N在Vero细胞中的表达.pcDNA3.1-S及pcDNA3.1-S-N真核表达载体有较高的转染效率,目的基因能在宿主细胞中表达,有利于研究HTNV-S基因在T细胞表位研究中的意义.     

人sTNFR1基因的克隆以及在NIH3T3细胞中的稳定表达

以He1a细胞的总RNA为模板,用RT—PCR方法扩增sTNFR1全编码区基因片段,构建含有目的片段的T载体克隆及真核表达载体pcDNA3．1（-）重组质粒亚克隆,将重组质粒和脂质体共同转染NIH3T3细胞系,G418筛选稳定转染细胞株．经核苷酸序列测序和酶切鉴定,成功构建了pcDNA3．1（-）-sTNFR1真核表达质粒,脂质体法建立了高效表达sTNFRI的稳定转染细胞系,并经RT—PCR和Western Blotting鉴定．人sTNFR1基因能在NIH3T3细胞系中稳定表达,为今后的研究打下了基础．     

grp78真核表达载体构建及其稳定转染HeLa细胞系建立

目的构建人grp78基因真核表达载体,并建立稳定高表达grp78的人宫颈癌HeLa细胞系。方法用RT—PCR方法从人宫颈癌HeLa细胞中扩增grp78基因编码区,将PCR产物克隆到pcDNA3．1（＋）真核表达载体,构建重组质粒pcDNA3．1（＋）／grp78并测序鉴定。用构建成功的pcDNA3．1（＋）／grp78真核表达载体转染入人宫颈癌HeLa细胞,经G418筛选获得grp78稳定高表达的HeLa细胞系,并用RT—PCR及Western印迹方法鉴定。结果成功构建pcDNA3．1（＋）／grp78真核表达载体,筛选获得稳定高表达人grp78的HeLa细胞系。结论grp78真核表达载体的成功构建和稳定高表达grpTS的HeLa细胞系的建立为进一步研究grp78的功能奠定了基础。     

Ag85A真核表达重组体的构建及稳定转染细胞系的建立

构建结核杆菌抗原85A（AgS5A）的真核表达重组体,转染L929细胞,建立稳定转染细胞系。从质粒V1 Jns．tPA—Ag85A中经PCR扩增出Ag85A基因,利用DNA重组技术将其插入到真核表达载体peDNA3．1／myc—HisA中,经酶切和测序鉴定后,脂质体转染法转染L929细胞,通过G418选择培养,建立稳定转染细胞系,Western Blot检测Ag85A的表达。成功构建pcDNA3．1／mye—HisA—Ag85A真核表达载体并稳定转染L929细胞,成功表达了目的基因。为进一步研究Ag85ADNA疫苗对结核杆菌的免疫防护作用奠定了基础。     

Cx26基因重表达抑制人鼻咽癌细胞系HNE1的恶性增殖

间隙连接蛋白 (Cx)基因在胚胎发育、细胞生长、分化以及细胞内环境的稳定过程中起重要调节作用 .肿瘤发生与Cx基因的表达及功能异常密切相关 ,肿瘤细胞常存在Cx基因表达下调或缺失 .将人Cx2 6基因编码区cDNA序列 ,亚克隆于真核表达载体pcDNA3 1(+) ,采用脂质体转染 ,将重组表达载体pcDNA3 1(+) Cx2 6转入鼻咽癌细胞系HNE1,使Cx2 6基因在HNE1中重表达 ,探讨Cx2 6基因对鼻咽癌细胞系HNE1的生物学功能的影响 .研究结果表明 :Cx2 6基因的重表达 ,抑制HNE1细胞生长 ,细胞周期阻滞于G0 G1期 ,HNE1细胞的克隆形成能力下降 ,裸鼠致瘤能力减弱 .     

pcDNA3与pIRES1.neo筛选克隆效率的比较

将绿色荧光蛋白(GFP)报告基因分别插入pIRES1.neo与pcDNA3二真核表达载体,构建pGFP-IRES1.neo和pcDNA3-GFP,转染小鼠B16细胞和人HepG2细胞,经G418筛选出抗性克隆,在倒置荧光显微镜下观察,鉴定共表达克隆数;结果在两个细胞系中,pcDNA3-GFP共表达率分别为0%和4%,而pIRES1.neo-GFP共表达率为75%和100%,差异非常显著;因此在筛选稳定表达目的基因细胞克隆的实验中,pIRES1.neo是一个较理想的载体。     

血红素加氧酶-1对肝癌细胞细胞周期调控因子的影响

目的观察血红素加氧酶-1（heme oxygenase 1,HO-1）对人肝癌细胞HepG2细胞周期调控因子的影响。方法构建含有野生型和突变型HO-1基因的重组载体pcDNA3．1（＋）-wtHO-1和pcDNA3．1（＋）-mHO-1G143H。利用脂质体介导的方法将构建好的重组载体转染肝癌细胞系HepG2,以空载体转染作为对照组。通过G418筛选建立稳定表达野生型和突变型HO-1的HepG2肝癌细胞系。经半定量RT—PCR、Western印迹检测转染细胞系中HO-1 mRNA和蛋白的表达水平。在HO-1表达改变的稳转细胞系中,利用Western印迹检测转染细胞系中P21、P27蛋白表达水平。结果成功实现了野生型和突变型HO-1在HepG2细胞中的过表达;野生型和突变型HO-1过表达均能诱导抑癌基因p21和p27的表达。结论HO．1过表达诱导抑癌基因p21和p27的表达与血红素分解产物无关。HO-1可能通过其它机制调节p21和p27的表达。     

TCRBV12—3重组载体构建及其抗肿瘤作用的初步研究

目的：构建pEGFP．C3-TCRBVl2—3表达载体并初步研究其抗肿瘤作用。方法：TCRBVl2—3基因片段从pGEM-T—TCRBVl2-3载体上酶切并克隆至pEGFP—C3载体中,通过脂质体将pEGFP—C3．TCRBVl2-3表达载体转染外周血单个核细胞（PBMCs）,48小时后荧光显微镜观察转染效率。PBMCs细胞、pEGFP-C3载体转染的PBMCs细胞、pEGFP—C3-TCRBVl2-3表达载体转染的PBMCs细胞分别与肝癌细胞BEL-7402和宫颈癌细胞HeLa共培养24h,显微镜观察肿瘤细胞的生长情况。结果：测序证实TCRBVl2-3基因片段成功亚克隆至pEGFP—C3载体中,荧光显微镜证实重组体转染PBMCs细胞48h后可有效表达绿色荧光。显微镜观察发现pEGFP—C3-TCRBVl2-3载体转染的PBMCs对肝癌细胞有杀伤作用．但对宫颈癌杀伤作用不明显。结论：成功构建pEGFP—C3一TCRBVl2—3表达载体,初步证实TCRBVl2．3对肝癌细胞有杀伤作用。     

pcDNA3．1表达载体转染对细胞生长的影响

为了探讨真核表达载体转染对细胞生长的影响,通过脂质体介导将pcDNA3.1( )表达载体DNA转染鼻咽癌细胞系HNE1,G418筛选后,Southern杂交鉴定稳定表达细胞株,以HNE1细胞为对照,观察pcDNA3.1( ）／HNE1克隆细胞的生物学特性;结果显示,在pcDNA3.1( )/HNE1阳性克隆中,一株细胞克隆培养过程中发生自溶性死亡,一株细胞生长明显受到抑制,另一株细胞生长无明显影响,揭示在宿主细胞中pcDNA3.1( )DNA与宿主基因组DNA发生了随机整合,从而表现不同的细胞生物学改变。     

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.     

The structure, reproduction and taxonomy of Vidalia and Osmundaria (Rhodophyta, Rhodomelaceae)

Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.     

New or rare chromosome counts in Artemisia L. (Asteraceae, Anthemideae) and related genera from Kazakhstan

Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.     

肝癌中HBV和HCV基因和抗原的分布及意义

采用原位分子杂交方法检测HCV RNA及HBV X基因;采用免疫组织化学方法研究HCV核心抗原,非结构区C33c抗原及HBxAg在肝细胞肝癌中的定位及分布.结果表明(1)HCV RNA、HBV X基因在肝细胞肝癌组织检出率分别为40%(55/136)和82%(112/136).HCV RNA定位于癌细胞的胞浆内,阳性细胞呈散在、灶状及弥漫分布三种形式;HBV X基因在肝癌细胞中的分布呈胞浆型、核型及核浆型,阳性细胞也呈上述三种分布形式;(2)HCV C33c抗原、核心抗原在肝细胞肝癌中的阳性率为81%(133/164)及86%(141/164).C33c抗原定位于癌细胞及肝细胞的胞浆内;核心抗原既定位于癌细胞核中,又可定位于胞浆中.C33c抗原阳性细胞以灶状分布为主;而核心抗原阳性细     

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.