A组轮状病毒多个结构蛋白抗原表位的串联表达及其免疫原性的检测

从北京腹泻婴儿粪便提取的轮状病毒(rotavirus,RV)(T114株)的RNA中,克隆到轮状病毒结构蛋白基因vp4,vp6和vp7的全长cDNA,对它们编码的蛋白质序列和可能的抗原表位肽进行了预测,选择了RV主要抗原蛋白VP7、VP6和VP4的4个抗原表位肽,通过人工合成DNA的方式将这些抗原表位肽基因串联融合成一个阅读框RME(rotavirus multipleepitopes,RME)并构建原核表达载体.大肠杆菌表达的RME在ELISA反应中可被RV多克隆抗体识别,纯化的RME蛋白注射免疫小鼠可诱导特异性免疫应答,产生高滴度的同源氨基酸序列特异抗体和人RV抗体,其中针对RME的IgG抗体滴度达到l∶40 000,针对单个抗原表位EV7、EV6和EV4的IgG抗体滴度达l∶10 000～l∶20 000,针对RV Wa株的IgG抗体滴度较低为l∶2 500,但能特异地中和该病毒对MAC145细胞的侵染.上述结果为新型RV基因工程疫苗的研发提供了论据和基础.     

安徽省流行性出血热病毒杂交瘤单克隆抗体的研制与应用

用流行性出血热病毒(EHFV)李株和陈株血凝素(HAN)分别免疫Balb/C小鼠,取其脾细胞和骨髓瘤细胞SP2/O融合,杂交瘤生长孔率为97.5％和60％,阳性率各为2.5％和22％,克隆化培养阳性率达到100％。通过筛选得2株杂交瘤细胞、一株为2B_7、另一株为1H_8。其腹水单克隆抗体(McAb)滴度均达1∶16×10~4—32×10~4。1H_8的血凝抑制滴度达1∶5120。经染色体核型分析,杂交细胞染色体2B_7为80—97条,1H_8为85—105条,每个细胞均含一条中着丝点标记染色体。McAb的Ig类别和IgG亚类检测结果2B_7为IgM,1H_8为IgG_(2b)。两者对19株不同来源的EHFV具有不同的反应,1H_8McAb与19株都具有不同程度的免疫荧光反应,滴度都较高,且对5株不同来源的EHFV-HAN具有血凝抑制活性,滴度在1∶1280—5120,故1H_8为血凝抑制抗体;2B_7只对12株EHFV起反应,对7株不起反应,且没有血凝抑制活性。这说明1H_8与2B_7具有不同的特性。     

草鱼生长激素单克隆抗体杂交瘤细胞系的建立及鉴定

用经草鱼生长激素(gcGH)免疫的BALB/c小鼠脾细胞与小鼠骨髓瘤细胞(Sp2/O-Ag14)融合,经3次克隆化培养和ELISA筛选,得到7株阳性杂交瘤细胞株,其中2株为强阳性。交叉试验表明,这些杂交瘤细胞株所分泌的单克隆抗体(McAb)同大鳞大马哈鱼生长激素(sGH)、大鳞大马哈鱼促性腺激素(sGTH)及牛生长激素(bGH)均不起反应。小鼠腹水McAbs滴度高达1:1280000。受体-gcGH-McAb夹心试验表明,McAb确与有活性的gcGH有特异反应。用间接ELISA方法可检测到浓度为0.125#g/ml的gcGH。     

抗黄曲霉尿酸氧化酶单克隆抗体的制备和初步鉴定

目的:制备抗黄曲霉尿酸氧化酶单克隆抗体。方法:采用杂交瘤技术,获得2株针对重组黄曲霉尿酸氧化酶(rUOX)的杂交瘤细胞McAb17和McAb3;采用盐析和A蛋白亲合层析柱纯化该抗体。结果:McAb17和McAb3的腹水效价达到1∶512000,纯化后获得纯度大于95%的单抗,抗体亚类(型)分别为IgG1/k型和IgG2a/k型;ELISA结果显示制备的单抗与rUOX和Rasburicase(商品化rUOX)可发生特异反应。结论:制备了抗黄曲霉尿酸氧化酶单克隆抗体McAb17和McAb3,为检测rUOX在动物体内的代谢变化提供了良好的实验基础。     

猪和牛轮状病毒的分离和鉴定

我国对动物轮状病毒的研究报道尚少。用细胞培养法分离轮状病毒又比较困难。本文报道用非洲罗猴胎肾细胞MA-104,结合胰酶处理及旋转培养技术,从北京地区腹泻仔猪和犊牛的粪便中成功地分离到6株猪轮状病毒(暂名为PRV1,PRV2,PRV8,PRY11,PRV15和PRV17)和4株牛轮状病毒(暂名为BRV6555,BRV6551,BRV6571和BRV6576)及其初步鉴定结果。     

抗汉坦病毒等三种单克隆抗体细胞株的建立及分析应用

以纯化的病毒抗原加完全福氏佐剂,免疫BALB/c小鼠,再利用杂交瘤技术建立针对汉坦病毒、狂犬病毒及乙型脑炎病毒的单克隆抗体(McAb)细胞株,制备并提纯标记McAb,应用于各种实验和检测工作。结果获得了6株分泌抗汉坦病毒McAb杂交瘤细胞株、6株分泌抗狂犬病毒McAb杂交瘤细胞株、2株分泌抗乙型脑炎病毒McAb杂交瘤细胞株,共14株,并对它们的特性进行了分析。各McAb效价不尽相同,有的高达10-7,有的则不足10-3。各McAb亚型以IgG型为主,少数为IgM型;轻链以κ型为主,个别为λ型或阴性。McAb与纯化的病毒抗原有明显的特异性吸附(P<0.01)。有的McAb有相同的作用位点,有的McAb则没有,但与其它McAb有交叉反应。通过对McAb进行提纯和标记,建立了双抗体夹心ELISA法,用于病毒抗原的检测。实验表明获得的McAb有较好的生物学特性,可与相应的病毒抗原特异性结合,用于免疫学检测及其它多种用途。     

建兰花叶病毒单克隆抗体的制备及检测应用

用建兰花叶病毒(Cymbidium mosaic virus,CymMV)免疫的BALB/C鼠脾细胞与SP2/0鼠骨髓瘤细胞融合,经筛选克隆,获得3株能稳定传代并分泌抗CymMV单克隆抗体(McAb)的杂交瘤细胞(2C6、5B7和12G9),分别制备它们的单抗腹水。其中5B7和12G92株单克隆抗体腹水间接ELISA效价达10-6,3株单抗的抗体类型及亚类均为IgG1,轻链均为κ链。利用单克隆抗体建立了抗原包被间接ELISA(ACP-ELISA)检测CymMV的方法。蝴蝶兰病叶作1∶10240倍稀释、提纯CymMV病毒浓度为4.87ng/mL(每孔的病毒绝对量为0.487ng)时,该方法仍能检测到病毒。利用ACP-ELISA方法检测了田间样品,发现CymMV在兰花上发病很普遍。     

被动免疫轮状病毒NSP4&VP7双抗原重组腺病毒对感染乳鼠的排毒保护作用

为建立小鼠轮状病毒(Rotavirus,RV)感染动物模型,研究可同时表达轮状病毒NSP4 (Nonstructural protein 4)和VP7(Viral protein 7)的重组腺病毒疫苗免疫孕鼠后对新生乳鼠感染RV的被动保护作用.新生乳鼠口服异源株轮状病毒Wa、ZTR-68或SA11株后(分2次给予,每次含5×104 CCID50的RV),观察乳鼠是否有腹泻症状、肠道病理变化,检测乳鼠粪便排毒百分率;另以重组腺病毒rAd-NSP4-VP7免疫孕鼠后,检测母鼠血清抗体产生情况,并对比乳鼠粪便中RV抗原检出率初步评价疫苗的被动免疫保护作用.发现口服异源株RV的乳鼠未出现类似人类婴幼儿感染后的明显腹泻症状,但在粪便中可检测到RV抗原的存在(Wa、ZTR-68攻毒组均超过80％).经rAd-NSP4-VP7被动免疫的乳鼠接受Wa和ZTR-68攻毒后其粪便中的RV检出率比未受到被动免疫保护的对照组降低(P＜0.05).rAd-NSP4-VP7重组腺病毒免疫母鼠可显示出对孕鼠感染RV的被动免疫保护作用.     

抗HSV-1淋巴细胞杂交瘤的建立及应用单克隆抗体对HSV分型

应用HSV-1 SM44株感染的BHK-21细胞及提取的感染细胞膜蛋白抗原免疫Balb/c小鼠。以免疫小鼠脾细胞与小鼠骨髓瘤细胞系Sp_2/0融合,经ELISA筛选仅与HSV-1反应的阳性克隆及克隆化,建立了一株产生抗HSV-1型特异性McAb的杂交瘤细胞系(Mad-2)。经ELISA,IF及抗原吸收试验证明,该细胞系的培养上清及制备的小鼠腹水均与HSV-1呈特异性反应,但不与HSV-2反应。ELISA测定McAb Mad-2的腹水效价为10~(-7)Ig亚类鉴定为IgG-1。应用Mad-2和抗HSV型共同性McAb 1A12及抗HSV-2型特异性McAb CH-A9,对43株临床HSV分离株做了ELISA及IF分型。结果表明,28株口唇、眼部感染分离株和1株宫颈炎分离株均与1A12和Mad-2反应,不与CH-A9反应,为HSV-1。余14株富颈炎分离株均与1A12和CH-A9反应,而不与Mad-2反应,为HSV-2。ELISA和IF分型的结果完全一致。本研究提出作者应用的一套抗HSVMcAb有可能作为HSV型别鉴定的标准试剂。     

人心肌肌钙蛋白Ⅰ单克隆抗体及多克隆抗体的制备

目的:以重组人心肌肌钙蛋白Ⅰ(cTnⅠ)为抗原制备鼠源单克隆抗体(McAb)及兔源多克隆抗体,并鉴定抗体的特性。方法:以纯化的重组人cTnⅠ为抗原免疫BALB/c小鼠,取鼠脾细胞同Sp2/0骨髓瘤细胞融合,利用选择培养基筛选融合的杂交瘤细胞,用有限稀释法分离获得能够稳定分泌抗cTnⅠ的McAb阳性克隆,并利用体内诱生法大规模制备McAb,用辛酸-硫酸铵沉淀法纯化抗体;兔多抗制备以cTnⅠ为抗原常规免疫后取其血清;用间接ELISA和Western印迹鉴定抗体的性质。结果:经ELISA鉴定,筛选出5株能分泌cTnⅠMcAb的杂交瘤细胞株,即C5B2、C5B3、C5B4、C5B1、B1A6,效价最高的B1A6株分泌的McAb为IgG3型,纯化后效价为1∶10000,亲和常数为1.08×10-9mol/L,Western印迹鉴定表明cTnⅠMcAb有良好的特异性;兔多抗纯化后的效价为1∶8000。结论:制备了具有良好特性的cTnⅠMcAb和多克隆抗体。     

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.     

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     

Selection with two alleles and complete dominance

For a plant selection model with frequency-independent viabilities, fertilities and selfing rates, it is shown that apart from global fixation, for certain parameter combinations a protected polymorphism and facultative fixation (either allele may become fixed according to initial frequencies) may both occur. Facultative fixation requires different selling rates for the dominant and recessive type. Protection of the polymorphism requires resource allocation for male and female function. In this connection the problem of purely genetically caused population extinction is discussed.
For general frequency dependence and regular segregation, the chances for establishment of a completely recessive gene are compared to those of a completely dominant gene. It is proven that the process of establishment of the recessive gene, despite a fitness advantage, may be considerably endangered by drift effects if random mating prevails. The recessive gene may reach the same effectivity in establishment as a dominant gene, only if the recessive homozygote mates exclusively with its own type during the period of establishment.