马铃薯Y病毒普通系外壳蛋白基因在大肠杆菌中的表达

将马铃薯Y病毒普通系(PVY0)的外壳蛋白基因克隆到表达质粒pMAL-c2中,构建这一基因在大肠杆菌中的表达载体pMAL-c2-PVY0CP.SDS-PAGE及Western blotting检测结果表明,这一表达栽体在E.coli DH5α中经IPTG诱导可表达分子量为71.8kDa的特异性融合蛋白.以amylose resin亲合柱层析纯化这一融合蛋白为抗原,免疫家兔制备了效价为1:1024的特异性抗血清.用该抗血清可通过对流免疫电泳、免疫双扩散及Western blotting对PVY进行检测.     

黄瓜花叶病毒CP基因原核表达及抗血清的制备

把黄瓜花叶病毒 (CMV)西番莲分离物的外壳蛋白 (CP)基因 ,通过BamHI SacI位点定向插入pET 2 2b( )载体 ,转化大肠杆菌BL2 1 (DE3)中 ,经IPTG诱导下 37℃培养 6h ,SDS PAGE电泳示表达蛋白分子质量为 31 8kDa ,表达量占菌体总蛋白的 2 8 9%,表明该蛋白得到了高效表达。用冰冷的氯化钾溶液显色 ,用表达的特异蛋白质条带制备抗原 ,免疫家兔制备出病毒特异抗血清。采用ID ELISA测定抗血清效价为 1 0 -6;抗血清和CMV几个分离物均有特异反应 ,和TMV、菌体蛋白不发生非特异性反应 ;检测病毒灵敏度达 30ng ml,能够从稀释 31 2 5倍的感病植物汁液中检测出病毒     

用IgG指示试纸同时检测多种马铃薯病毒病

用硝基纤维素膜(NCM)为载体制备的抗体IgG指示试纸,可同时检测马铃薯X病毒(PVX)、马铃薯Y病毒(PVY)、马铃薯S病毒(PVS)和马铃薯卷叶病毒(PLRV)的感染。以pH10.0的1％甘氨酸溶液为PVX、PVY和PVS的研磨缓冲液,比已报道的缓冲液效果好。在提取病毒抗原时加入4％纤维素酶,可提高病毒得率及所制备的抗血清灵敏度。     

流感疫苗单向免疫扩散试验中抗血清制备新方法的研究

流感疫苗血凝素含量检测方法为单向免疫扩散试验,其抗血清通常由WHO参比实验室提供,通过纯化病毒、蛋白酶切获得血凝素主要抗原片段,然后再免疫动物获得抗血清。该方法制备时间较长,是制约流感疫苗研发及检测的主要因素。以RT-PCR方法获取甲型H1N1流感病毒血凝素中主要抗原片段基因,构建重组质粒并在大肠杆菌中表达,表达产物经纯化、复性后,以蛋白电泳及免疫印迹方法进行了鉴定。以纯化蛋白免疫家兔,制备了相应的抗体,初步证明可用于常规的单向免疫扩散试验。以该法可以快速获得抗血清,尤其是在大流行流感疫苗的研发中,加快疫苗研发进程。     

大蒜E病毒外壳蛋白基因的原核表达及抗血清制备

设计特异性引物PCR扩增了余杭大蒜病样中的大蒜E病毒 (GarV E)的全长CP基因 ,构建原核表达载体并在大肠杆菌中过量表达 ,纯化表达产物后免疫家兔制备抗血清。Westernblot检测结果表明 ,抗血清与GarV E的CP起强的特异性反应。 7个大蒜样品中 ,内蒙古赤峰市、宁夏银川和甘肃天水等 4个样品受到GarV E的侵染。试验也证明了田间GarV E编码的CP分子量为 35kD ,不同于已报道的其他葱X病毒属成员的 2 8kD外壳蛋白     

我国水稻条纹叶枯病病原性质的研究

病样经提纯后,在电镜下获得3nm丝状和8nm分枝丝状结构,病毒样在蔗糖密度梯度离心后产生三条沉降带。经过电泳分析表明:病毒外壳蛋白为单一组分,分子量约为3.69×10~4dalton;病毒核酸有三种大小不同的组分,分子量为0.9,1.0,1.4×100dalton(混合样)。用提纯的抗原制备抗血清。提纯病毒抗原与所制备抗血清、与我国过去研究制备的抗血清及与日本制备的条纹叶枯病单克隆抗体都有血清学反应。     

侵染广西罗汉果的小西葫芦黄花叶病毒的鉴定及抗血清制备

在广西临桂罗汉果花叶畸形病株上获得了一个线状病毒分离物LGL-1,寄主范围、蚜传能力测定、病毒粒子形态和细胞病理特征研究表明,它是马铃薯Y病毒科成员.采用马铃薯Y病毒科特异性简并引物做PCR扩增,并测定了分离物LGL-1的基因组3′-末端序列,序列分析表明它是小西葫芦黄花叶病毒（ZYMV）.系统进化树分析揭示,世界范围内的ZYMV分离物主要可分为3大群体,分离物LGL-1为中国特有群体Group Ⅲ成员.原核表达制备了分离物LGL-1的外壳蛋白抗血清,明确了ZYMV是引起广西罗汉果病害的主要病毒,并且比较了原核表达法和提纯病毒法制备的抗血清,在病样的间接ELISA法检测中结果的差异.     

苹果茎沟病毒的提纯和检测

用生物学和血清学方法从苹果分离鉴定出苹果茎沟病毒G-2和TC-3分离物,采用皂土澄清、15％蔗糖垫超离心、10％～40％蔗糖梯度离心等步骤,获得提纯的病毒样品,紫外吸收比值A_(260/280)为1.15。13％SDS-PAGE法测定的病毒外壳蛋白分子量为31000。用提纯的苹果茎沟病毒G-2分离物制备的兔抗血清,PTA-ELISA测定的效价为1/64000,特异性强。用PAS-ELISA可成功地检测出苹果茎沟病毒,此外还试验了DAS-ELISA,并证明其检测苹果茎沟病毒的效果与PAS-ELISA一致。     

齿兰环斑病毒的鉴定及其抗血清的制备与应用

根据病毒生物学特性、粒体形态和血清学性质等初步确定从在厦门地区种植的表现为褪绿、褐色坏死斑症状的卡特兰(Cattleya)病株上分离的一个病毒分离物为齿兰环斑病毒(Odontoglossumringspotvirus,ORSV)。通过摩擦接种6科27种(或品种)指示植物,结果其中4科8种(或品种)表现症状,与烟草花叶病毒不同;理化性质试验表明稀释限点为5×10-5,致死温度为92℃～94℃,体外存活期超过3个月。提纯病毒为长约300nm,宽约18nm杆状粒体。SDS-PAGE电泳分析表明其外壳蛋白分子量约为17kDa。该分离物与ORSV抗血清具有强阳性反应、而与TMV抗血清反应微弱,认为该病毒为ORSV分离物。用纯化病毒免疫家兔,成功地制备出特异性抗血清,并通过Dot-ELISA法对厦门地区种植的兰花病株进行了检测研究。     

重组人骨硬化蛋白的表达、纯化及多克隆抗体制备

[目的]构建人骨硬化蛋白(Sclerostin,SOST)原核载体,表达、纯化SOST蛋白,制备与鉴定小鼠多克隆抗体。[方法]通过分子克隆获得SOST蛋白的表达菌株;利用SDS-PAGE电泳切胶纯化、尿素梯度复性获得SOST蛋白,免疫小鼠制备SOST蛋白多克隆抗体;采用ELISA法检测抗体滴度;Western blotting检测抗血清特异性;DNAMAN与MEGA软件分析SOST蛋白系统进化关系。[结果]SOST重组载体双酶切、DNA测序鉴定结果,以及蛋白表达、纯化条带大小与预测一致。ELISA法获得SOST抗血清滴度达1:6 400倍,Western blotting证实抗血清具有很好的特异性。SOST序列的系统发生分析发现动物具有显著不同的进化趋势。[结论]成功克隆、表达与纯化SOST蛋白,制备并鉴定小鼠多克隆抗体。为SOST蛋白功能研究奠定基础。     

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