抗A型流感病毒聚合酶碱性蛋白1多克隆抗体的制备和鉴定

流感病毒属于正黏病毒科,为有包膜包裹的单股负链RNA病毒。它的8个基因片段编码至少16种病毒蛋白,其中3个蛋白组成流感病毒的聚合酶复合体。流感病毒聚合酶碱性蛋白1(PB1)是该复合体的组分之一,在病毒的转录、复制及重配中发挥重要的作用。为研究其功能,构建His-PB1(aa 550–755)融合蛋白原核表达质粒,IPTG诱导融合蛋白表达,通过镍柱亲和将其纯化,然后作为抗原免疫家兔。对抗血清进行间接ELISA检测,表明抗体效价可达1∶100 000。兔源PB1蛋白抗血清经亲和纯化后,用于免疫印迹检测流感病毒WSN毒株PB1蛋白以及外源转染的FLAG-PB1蛋白,结果表明该PB1抗体具有良好的特异性,同时也能特异性识别其他亚型的A型流感病毒的PB1蛋白,为进一步研究流感病毒PB1蛋白的功能奠定了基础。     

A型流感病毒RNA聚合酶及其对基因组复制和转录的调控作用

A型流感病毒是正粘病毒科成员,为单股负链分节段RNA病毒,全基因组由八个节段组成,分别编码八种结构蛋白(PB2、PB1、PA、HA、NP、NA、M1和M2)和两种非结构蛋白(NS1和NS2).核蛋白(NP)和RNA聚合酶复合体与病毒的八个RNA节段组成八个螺旋丝状的病毒核衣壳(RNP),核衣壳被双层类脂膜包裹,脂膜内为基质蛋白(M1)层,膜上镶嵌着HA、NA和M2三种膜蛋白.HA和NA为流感病毒的主要抗原.根据HA和NA抗原性的差异,A型流感病毒可分16个HA亚型和9个NA亚型[1].A型流感病毒具有广泛的宿主范围和超强的重组变异能力,对人类健康的威胁日趋严重,引起各国政府和科技工作者的广泛关注.研究RNA聚合酶的功能、揭示病毒复制和变异机理是目前抗流感病毒感染研究的热点之一.本文综述了流感病毒RNA聚合酶及其对病毒基因组复制和转录调控的研究进展.     

H3亚型鸭源流感病毒聚合酶PB1基因的序列分析

目的阐明H3亚型鸭流感病毒与其他亚型流感病毒的关系。方法对活禽市场分离的3株H3N8亚型鸭源流感病毒聚合酶PB1基因进行了序列分析。结果3株鸭源H3N8流感病毒聚合酶PB1基因核苷酸同源性为99.9%,与H9N2亚型流感病毒(DK/ST/2143/00)的同源性为96.31%~96.44%,而与H3N8亚型鸭流感病毒(Mal/Alberta/279/98)为88.65%~88.79%。系统进化树分析表明,本实验中的3株病毒属于相同的分支,且与A/duck/Hong Kong/Y439为代表的H9N2亚型禽流感病毒位于一进化分支,说明三株H3N8亚型流感病毒重排了H9N2亚型禽流感病毒的基因片段。结论不同亚型禽流感病毒在贮存宿主体内的重排以及重排病毒的新特点如鸭H3N8亚型流感病毒对禽的致病性,应当引起我们的高度重视。     

A型流感病毒RNA聚合酶及其对基因组复制和转录的调控作用

A型流感病毒是正粘病毒科成员,为单股负链分节段RNA病毒,全基因组由八个节段组成,分别编码八种结构蛋白(PB2、PB1、PA、HA、NP、NA、M1和M2)和两种非结构蛋白(NS1和NS2)。核蛋白(NP)和RNA聚合酶复合体与病毒的八个RNA节段组成八个螺旋丝状的病毒核衣壳(RNP),核衣壳被双层类脂膜包裹,脂膜内为基质蛋白(M1)层,膜上镶嵌着HA、NA和M2三种膜蛋白。HA和NA为流感病毒的主要抗原。根据HA和NA抗原性的差异,A型流感病毒可分16个HA亚型和9个NA亚型[1]。A型流感病毒具有广泛的宿主范围和超强的重组变异能力,对人类健康的威胁日趋…     

甲型流感病毒非结构蛋白的研究进展

PA、PB1和PB2以及NS1蛋白作为甲型流感病毒的非结构蛋白,虽然不直接参与病毒颗粒的组装,但是在病毒的复制周期中起到非常重要的调控作用.由PA、PB1和PB2组成的RNA聚合酶主要参与病毒mRNA的合成以及病毒基因组RNA的复制,而NS1蛋白则通过抑制宿主细胞的干扰素应激系统来拮抗宿主的抗病毒反应.通过研究甲型流感病毒非结构蛋白的结构与功能,对了解流感病毒复制及开发新型抗流感病毒药物有重要意义.     

B型肉毒毒素单克隆抗体的制备及其检测试剂盒的研制

目的 建立B型肉毒毒素(botulinum toxin B, BoNT/B)快速检测方法,研制B型肉毒毒素酶联免疫吸附测定(ELISA)检测试剂盒。方法 以B型肉毒类毒素为抗原免疫小鼠,用杂交瘤技术获得鼠源单克隆抗体(单抗),体内法扩大制备单抗。间接ELISA测定杂交瘤细胞株分泌单抗的稳定性、腹水效价以及单抗的亚型、理化性质和抗原识别表位。过碘酸钠法对抗原表位差异较大的抗体进行HRP标记,筛选最佳包被抗体和标记抗体,并确定包被抗体包被浓度和标记抗体工作浓度。将其配制成试剂盒,对试剂盒的线性、定量限、准确度和重复性等进行研究。结果 筛选、鉴定8株能稳定分泌B型肉毒毒素特异性单抗的杂交瘤细胞株。8株单抗腹水效价1∶32 000～1∶512 000,重链为IgG类、轻链为κ型,均耐碱、耐热,不耐酸,其中4C11、4D7、7F9和8D2 4株单抗抗原表位差异较大。以4D7为包被抗体、8D2为标记抗体配制试剂盒,B型肉毒毒素滴度在2～32 LD₅₀/mL范围内呈现良好的线性(r=0.996),定量限为0.96 LD₅₀/mL,试剂盒2～8℃有效期为12...     

幽门螺杆菌尿素酶B单位DNA疫苗的构建及鉴定

报告了利用淋巴细胞杂交瘤技术制备抗呼吸道合胞病毒蛋白的鼠单克隆抗体。获得 7株分泌抗呼吸道合胞病毒单克隆抗体的杂交瘤细胞株 :IF12 ,4H10 ,1D8,2B2 ,1G8,4G6 ,2D2。其中IF12 ,4H10 ,1D8,2B2 ,4G6分泌的抗体类别为IgG1,1G8为IgG2a ,2D2为IgG2b。经免疫印迹分析证实单抗 1F12 ,2B2识别病毒 4 4ku的蛋白 ,单抗1G8识别 37ku蛋白 ,2D2识别 2 1ku的病毒蛋白 ,单抗4G6识别病毒 90ku蛋白。对单抗的生物学活性进行了分析 ,单抗 4G6对呼吸道合胞病毒有明显的中和作用 ,小鼠腹水的中和滴度为 1∶32 ,其余各单抗中和滴度均小于1∶4。…     

甲型流感病毒vRNP及PB1、PB2、PA和NP蛋白的核内转运机制研究进展

与许多在细胞浆内复制的RNA病毒不同,流感病毒的复制及转录都在细胞核内进行。流感病毒感染细胞进入细胞浆后,经病毒脱壳将病毒核糖核蛋白体复合物(v RNP)释放到细胞浆。v RNP含有病毒的RNA基因和碱性聚合酶1(PB1)、碱性聚合酶2(PB2)、酸性聚合酶(PA)及核蛋白(NP)。v RNP被主动运送到细胞核内,开始病毒基因组的复制和转录。流感病毒感染细胞的晚期,在细胞浆中新合成的PB1、PB2、PA及NP蛋白也需要进入细胞核,参与新的v RNP的装配。我们简要介绍有关流感病毒v RNP和新合成的PB1、PB2、PA及NP蛋白进入细胞核的机制。     

一株针对H7N9亚型禽流感病毒血凝素蛋白茎部的单克隆抗体的表征

流感病毒血凝素蛋白(hemagglutinin,HA)的茎部区相对保守,是广谱疫苗、抗体与病毒检测制剂的重要靶点.前期研究获得了一株与H7N9亚型禽流感病毒HA蛋白茎部多肽(aa 428-452)反应的单克隆抗体(5D3-1B5).为系统评价其生物学特性,本研究测定了5D3-1B5的抗体效价(IgG、血凝抑制与病毒中和...     

马铃薯卷叶病毒单克隆抗体的制备

马铃薯卷叶病毒(Potato leafroll virus,PLRV)在植物体内数量很少,难以提纯足够量病毒进行免疫,至今未见国内有制备PLRV单克隆抗体(McAb)的报道.本文以本室以前报道的方法提纯马铃薯卷叶病毒作为抗原,直接注入Balb/c小鼠脾脏,随后从尾静脉加强注射,采用杂交瘤技术建立了4个分泌抗PILRV McAb的杂交瘤细胞株。经微量免疫双扩散法鉴定,杂交瘤细胞株A1、A3、D3分泌的McAb均属gG1亚类,C3株者属IgG2a,它们的轻链都是λ型。将0.5ml含10~8个A1,A3,C3,D3杂交瘤细胞注入     

Improvement of protein stability in protein microarrays

Protein stability in microarrays was improved using protein stabilizers. PEG 200 at 30% (w/v) was the most efficient stabilizer giving over 4-fold improvement in protein stability compared to without the stabilizer. PEG 200 above 10% (w/v) in the array solution prevented the evaporation of water in the sample and thereby improved protein stability in the microarray. When the streptavidin-biotin binding reaction was performed under optimized conditions, biotin-BSA-fluorescein isothiocyanate (FITC) was detected from 1 ng ml^–1 to 5 g ml^–1 by fluorescence analysis.     

Patterns of protein protein interactions in salt solutions and implications for protein crystallization

The second osmotic virial coefficients of seven proteins-ovalbumin, ribonuclease A, bovine serum albumin, alpha-lactalbumin, myoglobin, cytochrome c, and catalase-were measured in salt solutions. Comparison of the interaction trends in terms of the dimensionless second virial coefficient b(2) shows that, at low salt concentrations, protein-protein interactions can be either attractive or repulsive, possibly due to the anisotropy of the protein charge distribution. At high salt concentrations, the behavior depends on the salt: In sodium chloride, protein interactions generally show little salt dependence up to very high salt concentrations, whereas in ammonium sulfate, proteins show a sharp drop in b(2) with increasing salt concentration beyond a particular threshold. The experimental phase behavior of the proteins corroborates these observations in that precipitation always follows the drop in b(2). When the proteins crystallize, they do so at slightly lower salt concentrations than seen for precipitation. The b(2) measurements were extended to other salts for ovalbumin and catalase. The trends follow the Hofmeister series, and the effect of the salt can be interpreted as a water-mediated effect between the protein and salt molecules. The b(2) trends quantify protein-protein interactions and provide some understanding of the corresponding phase behavior. The results explain both why ammonium sulfate is among the best crystallization agents, as well as some of the difficulties that can be encountered in protein crystallization.     

Physical principles of protein structure and protein folding

Physical principles determining the protein structure and protein folding are reviewed: (i) the molecular theory of protein secondary structure and the method of its prediction based on this theory; (ii) the existence of a limited set of thermodynamically favourable folding patterns of α- and β-regions in a compact globule which does not depend on the details of the amino acid sequence; (iii) the moderns approaches to the prediction of the folding patterns of α- and β-regions in concrete proteins; (iv) experimental approaches to the mechanism of protein folding. The review reflects theoretical and experimental works of the author and his collaborators as well as those of other groups.     

Peptide-based covalent inhibitors of protein–protein interactions

Protein–protein interactions (PPI) are involved in all cellular processes and many represent attractive therapeutic targets. However, the frequently rather flat and large interaction areas render the identification of small molecular PPI inhibitors very challenging. As an alternative, peptide interaction motifs derived from a PPI interface can serve as starting points for the development of inhibitors. However, certain proteins remain challenging targets when applying inhibitors with a competitive mode of action. For that reason, peptide-based ligands with an irreversible binding mode have gained attention in recent years. This review summarizes examples of covalent inhibitors that employ peptidic binders and have been tested in a biological context.     

Inhibition of protein hydroperoxide formation by protein thiols

Abstract

Studies on plasma and cells exposed to hydroxyl and peroxyl radicals have indicated that there are few inhibitors of protein hydroperoxide formation. We have, however, observed a small variable lag period during bovine serum albumin (BSA) oxidation by 2-2′ azo-bis-(2-methyl-propionamidine) HCl (AAPH) generated peroxyl radicals, where no protein hydroperoxide was formed. The addition of free cysteine to BSA during AAPH oxidation also produced a lag phase suggesting protein thiols could inhibit protein hydroperoxide formation. The selective reduction of thiols on BSA by β-mercaptoethanol treatment caused the appearance of a lag period where no protein hydroperoxide was formed during the AAPH mediated oxidation. Increasing free thiol concentration on the BSA increased the lag period. Protein hydroperoxide formation began when the protein thiol concentration dropped below one thiol per BSA molecule. It is unlikely that the lag period is due to gross structural alteration of the reduced protein since blocking the free thiols with N-ethyl maleimide eliminated the lag in protein hydroperoxide formation. Protein thiols were found to be ineffective in inhibiting hydroxyl radical-mediated protein hydroperoxide formation during X-ray radiolysis. Evidence is given for protein thiol oxidation occurring via a free radical mediated chain reaction with both free cysteine and protein bound thiol. The data suggest that reduced protein thiol groups can inhibit protein hydroperoxide formation by scavenging peroxyl radicals.     

药物靶标蛋白在蛋白网络中的分布

药物靶标发现是目前生物学研究领域的热点和难点问题。从已有药物靶标中寻找规律可以为新靶标的发现总结规律,提供依据。随着功能基因组学的发展,这种组学数据的积累为这一问题的研究提供了契机。本文研究了已有靶标在蛋白网络中的分布,并分析了它们的蛋白功能域组成情况。结果显示靶标基因倾向位于网络的核心区域,并且集中在一些特定蛋白家族中。这些规律的总结将对药物研发过程中药物靶点的选择提供一定的帮助。