猪瘟病毒野毒株持续感染细胞模型的稳定性研究

以本实验室建立的CSFV39-PK15持续感染细胞模型为实验材料,综合运用免疫荧光、RT-PCR、流式细胞仪,对其稳定性进行研究.实验结果均表明,该细胞模型有着良好的稳定性.即使在连续传至128代的CSFV39-PK15传代细胞中,CSFV仍持续存在呈免疫荧光抗体反应阳性和RT-PCR检测阳性.同时,该细胞与正常的PK-15细胞相比,细胞周期无显著差异.通过同段序列的同源性比较,发现CSFV39与CSFV石门株的同源性最高,达99.02%.     

猪瘟病毒野毒株持续感染细胞模型的建立

采用猪瘟病毒野毒珠（ＣＳＦＶ３９）感染猪肾传代细胞系ＰＫ－１５,经连续传７９代,建立了稳定的病毒持续感染细胞模型,获得ＣＳＦＶ３９－ＰＫ１５细胞株。用免疫荧光、ＲＴ－ＰＣＲ检测、透射电镜跟踪观察了ＣＳＦＶ３９－ＰＫ１５细胞株连续传代中病毒在细胞内的存在情况。结果表明第９,２９,７９代细胞仍有猪瘟病毒存在,表现出病毒持续感染的基本特征。这为深入研究猪瘟病毒持续感染机理奠定了基础。     

携带高滴度猪瘟病毒C株猪睾丸细胞株的建立及蛋白质组学分析

猪睾丸细胞(Swine testicular,ST)广泛用于猪瘟病毒(Classical swine fever virus,CSFV)的传代,为了进一步提高ST细胞增殖的CSFV滴度,我们利用有限稀释法克隆出一种稳定携带高滴度CSFVC株的单克隆细胞株(STC).间接免疫荧光实验结果显示,几乎100％STC细胞携带CSFV抗原;且STC细胞中CSFV滴度为106 TCID50/mL,与CSFV单次感染亲本ST细胞的病毒滴度104 TCID50/mL相比,具有更高的病毒产量.为了探究STC细胞持续带毒后细胞内蛋白表达水平的变化,我们通过非标记定量蛋白质组学分析发现541种蛋白表达水平发生明显变化,并选取其中差异显著且与病毒复制或细胞存活相关的两种上调蛋白(GRP94,GRP78)、两种下调蛋白(β-catenin,ISG15)进行免疫印迹验证.本试验成功获得了可以稳定携带高滴度CSFV C株的STC细胞株,并初步筛选了与细胞持续带毒有关的目标蛋白,不仅为简化猪瘟传代细胞苗生产工艺提供条件,也为研究STC细胞持续携带CSFV稳定传代的机制奠定基础.     

猪瘟病毒野毒株持续感染细胞模型的稳定性研究

以本实验室建立的CSFV39-PKl5持续感染细胞模型为实验材料，综合运用免疫荧光、RT-PCR、流式细胞仪，对其稳定性进行研究。实验结果均表明，该细胞模型有着良好的稳定性。即使在连续传至128代的CSFV39-PKl5传代细胞中，CSFV仍持续存在：呈免疫荧光抗体反应阳性和RT-PCR检测阳性。同时，该细胞与正常的PK-15细胞相比，细胞周期无显著差异。通过同段序列的同源性比较，发现CSFV39与CSFV石门株的同源性最高，达99．02％。     

猪瘟病毒的形态结构与形态发生

建立了猪瘟病毒(CSFV)弱毒疫苗Thiverval株(T株)与中 国兔化弱毒疫苗C株在MPK细胞中的感染模式。使用MPK细胞增殖CSFV,其病毒滴度明显提高,从而为应用电镜超薄切片技术研究猪瘟病毒的形态结构与形态发生提供了可能性。猪瘟病毒呈圆形颗粒,直径约为70nm。其内部是电子致密的核心,直径约为40nm,有时呈六角形;外有包膜包裹。在CSFV感染的MPK细胞质中,可观察到处于不同发育阶段的子代病毒粒子。此外,猪瘟病毒的感染能够引起某些宿主细胞超微结构上的变化。     

猪瘟病毒与宿主细胞间的相互关系

猪瘟病毒(CSFV)是一种正链RNA病毒,在培养细胞中增殖力弱,滴度低,通常不使培养细胞产生病理变化;而感染猪导致的猪瘟发病迅速,死亡率高。本文综述了近年来在CSFV增殖过程、与宿主细胞间的相互作用方面的研究进展,并对CSFV与细胞凋亡间的关系进行了探讨。     

应用免疫金银技术检测猪瘟病毒兔化弱毒株感染细胞

在应用免疫金银染色技术成功地对猪瘟病毒弱毒疫苗Thiveral株（CSFV．T株）感染的PK－15细胞进行检测的基础上,通过改进免疫金银反应的条件,进一步提高了免疫金银法的检测灵敏度和特异性。目前已将该技术应用到对猪瘟病毒兔化弱毒疫苗株（CSFVC株）感染细胞的检测,获得较为满意的实验结果。免疫金银染色技术的应用为研究CSFV在细胞中增殖的规律提供更为灵敏的检测手段,并且可望为猪瘟疫苗生产中的滴度测定提供一种简便有效的手段。     

口蹄疫病毒持续感染细胞系的快速选择及其特性研究

采用NH4Cl弱碱法处理感染口蹄疫病毒的叙利亚仓鼠肾细胞系(BHK-21), 存活细胞经过单克隆和选择,获得32株阳性克隆细胞株.随机选取一株(BHK-ROp),常规传代并采用RT-PCR,透射电子显微镜,流式细胞仪进行持续感染特性分析,结果表明,BHK-ROp第4,16,36代细胞中病毒持续存在,但不影响细胞的生长特性,表现出病毒持续感染的基本特征.可见,NH4Cl弱碱法用于建立病毒持续感染细胞系是十分有效的.     

猪瘟病毒C株共感染口蹄疫病毒影响口蹄疫病毒复制

【背景】猪瘟(Classical Swine Fever)是由猪瘟病毒(Classical Swine Fever Virus,CSFV)引起的猪高度接触性传染病,致死率极高。在临床中存在着CSFV与猪其他病原菌共感染的情况,例如CSFV与口蹄疫病毒(Foot-and-Mouth Disease Virus,FMDV)的共感染。【目的】利用CSFV与FMDV共感染猪源宿主细胞,研究CSFV与FMDV共感染对FMDV病毒复制的影响。【方法】构建体外共感染细胞模型,在正常PK-15细胞上进行CSFV共感染FMDV实验,通过观察细胞病变效应(Cytopathic Effect,CPE)、实时荧光定量PCR(RT-qPCR)、Western Blot、间接免疫荧光检测CSFV和FMDV共感染及FMDV单独感染情况下FMDV复制水平的差异。利用RT-qPCR筛选鉴定能够影响FMDV复制的CSFV蛋白。【结果】CSFVC株共感染FMDV能够抑制FMDV的复制,而且灭活的CSFV同样抑制FMDV的复制。通过筛选鉴定出CSFV的C蛋白能够抑制FMDV复制。【结论】研究发现CSFV C株共感染FMDV能够抑制FMDV复制,而其C蛋白具有抑制FMDV复制的能力。     

猪瘟病毒C株兔脾毒在SK6细胞中的增殖培养及其鉴定

猪瘟病毒(Classical swine fever virus,CSFV)是猪的最重要传染病之一,给养猪业造成巨大经济损失.传统疫苗C株在猪瘟防制中曾发挥了巨大作用,但由于猪瘟病毒逐渐产生变异,同时使用传统疫苗无法区分自然感染动物和免疫动物,从而使传统疫苗的应用受阻.因此十分必要研制新型猪瘟疫苗.用适宜的宿主细胞培养猪瘟传统弱毒疫苗C株,通过灵敏可靠的方法检测病毒在宿主细胞中的感染,是研究猪瘟病毒C株的一个重要基础环节.     

Investigation of the mechanism of temperature sensitivity of the electroreceptors of ampullae of lorenzini

In experiments on Black Sea skates (Raja clavata), the potential of the receptor epithelium of the ampullae of Lorenzini and spike activity of single nerve fibers connected to them were investigated during electrical and temperature stimulation. Usually the potential within the canal was between 0 and –2 mV, and the input resistance of the ampulla 250–400 k. Heating of the region of the receptor epithelium was accompanied by a negative wave of potential, an increase in input resistance, and inhibition of spike activity. With worsening of the animal's condition the transepithelial potential became positive (up to +10 mV) but the input resistance of the ampulla during stimulation with a positive current was nonlinear in some cases: a regenerative spike of positive polarity appeared in the channel. During heating, the spike response was sometimes reversed in sign. It is suggested that fluctuations of the transepithelial potential and spike responses to temperature stimulation reflect changes in the potential difference on the basal membrane of the receptor cells, which is described by a relationship of the Nernst's or Goldman's equation type.I. P. Pavlov Institute of Physiology, Academy of Sciences of the USSR, Leningrad. I. M. Sechenov, Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Pacific Institute of Oceanology, Far Eastern Scientific Center, Academy of Sciences of the USSR, Vladivostok. Translated from Neirofiziologiya, Vol. 12, No. 1, pp. 67–74, January–February, 1980.     

Principles of organization and evolution of systems of regulation of functions

Evolution of living organisms is closely connected with evolution of structure of the system of regulations and its mechanisms. The functional ground of regulations is chemical signalization. As early as in unicellular organisms there is a set of signal mechanisms providing their life activity and orientation in space and time. Subsequent evolution of ways of chemical signalization followed the way of development of delivery pathways of chemical signal and development of mechanisms of its regulation. The mechanism of chemical regulation of the signal interaction is discussed by the example of the specialized system of transduction of signal from neuron to neuron, of effect of hormone on the epithelial cell and modulation of this effect. These mechanisms are considered as the most important ways of the fine and precise adaptation of chemical signalization underlying functioning of physiological systems and organs of the living organism