牛血清中27kDa蛋白的免疫学特性研究

为探讨牛血清中存在的HBsAg样蛋白的性质,给HBV的发病机制、治疗、预防等研究提供依据,我们采集93份牛血清,以SDS-PAGE、Westem Blot对血清中HBsAg样蛋白进行研究,发现其分子量约为27kDa：以SDS-PAGE分离牛血清中的HBsAg样蛋白,经皮下多点免疫小鼠,可产生与人HBV编码蛋白HBsAg反应的抗体。表明该27kDa蛋白可能存在与HBsAg相似的免疫学特性。     

表面抗原和表面抗体检测双阳性CD重组型乙型肝炎病毒全基因组序列分析

西藏地区藏族人群乙型肝炎病毒(Hepatitis B virus,HBV)感染率较高,而针对感染者血清中HBV表面抗原(Hepatitis B surface antigen,HBsAg)和HBV表面抗原抗体(Hepatitis B surface antibody,HBsAb)双阳性的研究一直进展缓慢,尚无明确的研究结论。为探讨西藏地区藏族人群慢性HBV感染者血清中HBsAg和HBsAb双阳性与基因组核苷酸/氨基酸突变的关系,本研究在西藏选取7个地区作为研究区域,进行多阶段抽样,选取样本进行HBV血清五项指标检测,筛选HBsAg和HBsAb均为阳性的患者血清共24份作为双阳性组,以年龄和乙型肝炎e抗原(HBeAg)等感染指标进行匹配,选取96份HBsAg阳性,HBsAb阴性患者血清作为对照组。HBV全基因组序列通过聚合酶链式反应(Polymerase chain reaction,PCR)产物直接测序获得,并进行重组分析和突变分析。852名西藏HBV感染者中,HBsAg/HBsAb双阳性率为2.82%(24/852)。双阳性组在S蛋白N端和主要亲水区(Major hydrophilic region,MHR)的突变率以及PreS缺失发生率均显著高于对照组。T1753C、C1990T和C2002T等核苷酸突变;S蛋白中V224A、PreS区D103E等氨基酸突变在两组内分布存在显著差异。HBV/CD重组型的HBsAg/HBsAb双阳性发生率与中国乙肝主要流行区域接近。HBV感染者血清HBsAg和HBsAb共存可能与S蛋白,特别是MHR内的高氨基酸突变造成的免疫逃逸有关。PreS缺失、S抗原蛋白C端V224A突变和PreS区D103E突变可能对HBsAg/HBsAb双阳性的产生具有协同作用。     

pSUPER介导的RNAi抗乙肝病毒S基因的研究

目的 研究RNAi抗乙肝病毒的作用.方法 设计并合成一段针对HBV S基因的干扰序列及一段无关的序列,克隆进pSUPER载体中,分别构建pSUPEK-HBS、pSUPEK-nonsense载体,然后将pSUPER、pSUPEK-HBS、pSUPER-nonsense转染进HepG2.2.15细胞中,用ELISA方法对细胞上清中HBsAg、HBeAg进行检测.尾静脉注射HBV转基因小鼠,取血清检测.结果 pSUPER-HBS所表达的siRNA成功的抑制了HepG 2.2.15上清及HBV转基因鼠血清中的HBsAg、HBeAg,抑制率分别达到70%、51%以及60%、42%.结论 针对HBV S区的siRNA能明显抑制HBV的复制.     

HCV E2区基因在原核系统中的表达

用提取的重组表达载体pET-E2转化BL21(DE3)感受态细胞,经IPTG诱导,再进行SDS-PAGE,可得到有一条约34 kDa的表达带,与理论推测的蛋白分子量一致,通过Western-blot鉴定,证明此带即为目的蛋白带.该产物有一个六聚组氨酸尾,主要以包涵体形式存在;计算机扫描分析考马斯亮兰染色后的蛋白胶显示目的蛋白占整个菌体蛋白的36%以上,经Ni-柱纯化的E2蛋白纯度可达95%以上;以纯化的E2蛋白为抗原,用ELISA方法检测了20份抗HCV阴阳性血清,结果表明15份抗HCV阳性血清中检出5份E2抗体阳性血清,而5份抗HCV阴性血清中没有检测到E2抗体.     

我国17株绵羊肺炎支原体分子分型及其菌体蛋白免疫印迹

【目的】分别从基因和蛋白水平研究我国部分地区绵羊肺炎支原体(Mycoplasma ovipneumoniae)分离株的分子特征,并了解其免疫原性蛋白的差异。【方法】对分离自8个地区的17株绵羊肺炎支原体进行扩增片段长度多态性(Amplified Fragment Length Polymorphism,AFLP)和十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis,SDS-PAGE)分析,采用NTsys-2.10e软件对AFLP和SDS-PAGE结果进行聚类,并用绵羊肺炎支原体模式株Y98高免血清对部分分离株进行免疫印迹分析。【结果】当相似系数分别为0.78和0.85时,绵羊肺炎支原体分离株可根据8个来源地区分成8个AFLP群和8个SDS-PAGE群;用8株分离株进行免疫印迹共出现6条蛋白条带,分子质量分别为105 kDa、83 kDa、65 kDa、42 kDa、40 kDa或26 kDa,其中83 kDa和40 kDa蛋白为8个菌株保守的免疫原性蛋白。【结论】我国部分地区绵羊肺炎支原体分离株之间存在遗传差异,不同分离株的主要免疫原性蛋白也存在一定差异,但83 kDa和40 kDa蛋白为其保守的免疫原性蛋白。本研究首次对我国部分地区绵羊肺炎支原体分离株进行了分子分型与免疫印迹分析,结果将为绵羊肺炎支原体病的新型诊断技术开发和疫苗研制奠定理论基础。     

登革2型病毒结合血管内皮细胞分子机制的初步研究

以ECV304细胞为对象分析登革病毒感染血管内皮细胞的机制.2型登革病毒(DEN2)吸附后微量蚀斑法测定ECV304细胞上清释放的病毒滴度,证实该细胞对DEN2感染有一定的敏感性.机械刮取或胰蛋白酶消化法收集ECV304细胞分离膜蛋白,SDS-PAGE见胰酶处理样品缺失一43 kDa的膜蛋白.将ECV304细胞膜蛋白与35S-Met标记的DEN2进行病毒重叠蛋白结合试验(VOPBA),有29、34和43 kDa的3种膜蛋白可与DV结合,其中29 kDa的蛋白对胰酶耐受.培养的ECV304细胞中加入重组E蛋白(rEgp)对DEN2吸附进行阻断试验,微量蚀斑法与间接免疫荧光表明rEgp抑制DEN2感染该细胞.VOPBA中rEgp可阻断病毒与细胞膜蛋白的结合.结果表明ECV304细胞表面可能存在29、34、43 kDa的3种与DEN2结合的相关蛋白,DEN2 E蛋白可直接介导DV感染血管内皮细胞.     

斑蝥素合成期的芫菁蛋白质表达差异分析

研究斑蝥素大量合成期的芫菁体内可溶性蛋白,有助于阐明斑蝥素合成相关酶蛋白的种类和合成机理.本研究采用SDS-PAGE、双向电泳技术,分析了苹斑芫菁Mylabris calida Palla在斑蝥素合成前期、大量合成早期和末期的蛋白质组成的变化.SDS-PAGE电泳分析显示,芫菁从羽化1 h到25 d过程中分子量约为80 kDa,45 kDa,30 kDa的蛋白组分存在着显著地表达差异.进而通过双向电泳分析,发现合成前期蛋白质与其它时期有明显差异:蛋白质大多分布在Pl值4～7,分子量20～80 kDa的区域,其中20～45 kDa蛋白质差异明显.芫菁羽化后1h、2d、4d的蛋白质点数目分别为471个、569个、645个;大量合成早期和合成末期为827个和999个,增长25%以上.研究结果显示:3个时期蛋白质组成的变化趋势恰好与芫菁合成斑蝥素含量显著峰值变化相吻合,表明芫菁体内斑蝥素的合成是多种蛋白参与的复杂过程,这些蛋白分子量范围在20～45 kDa.     

HBcAg和HBsAg前S1表位肽融合蛋白的表达研究

构建、表达并纯化了HBcAg与HBsAg前S1表位肽融合蛋白BTcs1,为开发新型HBV治疗和预防性疫苗提供实验依据。利用DNA重组技术,构建了HBcAg与HBsAg前S1表位肽融合蛋白原核表达质粒pBTcs1,在大肠杆菌（HB101）中进行表达,用蔗糖密度梯度超速离心对表达产物BTcs1进行纯化,用SDS-PAGE、SEC、WESTERN-BLOT和电镜进行鉴定。结果表明成功构建了HBcAg与HBsAg前S1表位肽融合蛋白原核表达质粒pBTcs1,BTcs1的表达量为20-25 mg/L,DOT-BLOT结果显示BTcs1主要分布在30%-50%蔗糖介质中,SDS-PAGE和SEC结果显示蛋白纯度>95%,WESTERN-BLOT结果显示BTcs1可以与抗HBcAg抗体和抗HBsAg前S1抗体特异杂交,显色条带在约28 kD处,电镜分析表明BTcs1可以自主组装成病毒样颗粒（VLP）,直径约为30-34 nm。本研究为进一步探讨BTcs1的功能及应用奠定了基础。     

一种联合检测乙型肝炎病毒前S1抗原与核心抗原方法的建立及其与病毒核酸检测结果的一致性

本研究以与血清中HBV DNA含量高度相关的两种HBV抗原(前S1抗原与核心抗原)为靶标,建立了联合检测这两种HBV核酸相关抗原(NRAg)的双抗体夹心法ELISA试剂.对系列稀释血清的检测表明,该试剂的平均分析灵敏度为103.2基因组拷贝/mL(95%可信限102.2-4.2基因组拷贝/mL),显著高于前S1抗原或核心抗原的单独检测.对994份HBsAg阴性血清的检测结果表明NRAg ELISA的特异性为99.7%(95%可信限:99.1%～99.9%).对271份临床慢性肝炎血清进行检测,结果NRAg ELISA与HBV DNA结果的总符合率达96.3%(95%可信限:93.3%～98.2%),NRAg ELISA的读值/临界值比(S/CO)与HBV基因组拷贝数呈正相关.利用NRAg试剂,发现了1例HBsAg"a"抗原表位突变的变异株.这些结果显示HBV NRAg ELISA与HBV DNA具有高度相关性,并能够检测出HBsAg抗原变异株,有望成为HBsAg变异株筛选的有力工具,并为广大基层医疗单位提供一种便捷的替代HBV DNA定性检测的手段.     

HBV感染中抗-(抗-HBs)独特型抗体的研究

1.75％聚乙二醇(MW6000)沉淀血清免疫复合物后,采用ELISA法检测上清中抗独特型抗体〔抗-(抗-HBs)〕。56例急性HBV感染患者血清IgM抗-(抗-HBs),第一周检出率最高(60.00％),1个月后大部分阴性,IgG抗-(抗-HBs)维持时间略长。IgM抗-(抗-HBs)较HBsAg/IgM和IgM抗-HBc消失早,将有利于急性HBV感染的诊断。27例CAH和29例CPH,IgM和IgG抗-(抗-HBs)阳性率约为38～52％,两型间无显著差异(p>0.5)。3例无症状HBsAg携带者和12例HBsAg血清疫苗接受者血清,抗-(抗-HBs)阴性。115例HBV感染者中,IgM抗-(抗-HBs)阳性者HBsAg阳性率(93.62)显著高于IgM抗-(抗-HBs)阴性者(52.31％),p<0.005;反之,HBsAg阳性血清IgM抗-(抗-HBs)阳性率(55.5S％)显著高于HBsAg阴性血清(9.68％),p<0.005;IgM抗-(抗-HBs)阳性和阴性血清的HBVDNA阳性率有显著差异(p<0.025),IgG反应到类似的结果。以上资料表明,抗-(抗-HBs)提示了一些HBV感染患者体内可能存在一种缺陷的反馈机制,导致抗-HBs产生不足而容许更活跃的HBV复制。本文证明了HBV感染患者血清中存在的抗-(抗-HBs)可能是共同决定簇“a”特异性的,还表明抗-HBs人抗-Id反应可能被纯化人血清HBsAg抑制,提示人抗-(抗-HBs)与抗-HBs结合的位点可能在HBsAg与抗-HBs结合位点内或在其附近,这和一些动物抗-(抗-HBs)的研究结果是一致的。     

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.