肾综合征出血热疫苗候选毒株A16株的分子特性

为研究肾综合征出血热疫苗候选毒株A16株的分子基础,应用RT－PCR方法扩增并测定了A16株的M和S片段的序列,结果A16株M片段的全基因序列共3615个核苷酸,编码1133个氨基酸。S片段的全基因序列共1770个核苷酸,编码429个氨基酸。A16株M片段核苷酸全基因序列及其的氨基酸与HTN型毒株同源率为75.5%-90.3%和85.9%-97.1%,即A16与HTN同型毒株间的差异高达9.7%-24.3%和2.9%-14.1%,而与SEO型的同源率比较,核苷酸和氨基酸分别仅为70.2%-70.8%和73.4%-76.7%.S片段的序列同源率与M片段的结果相类似,即核苷酸和氨基酸的同源率与HTN型毒株分别为76.0%－905和92.1%-97.7%,与SEO型为67.0%-67.7%和81.7%-82.6%。结果显示,A16株虽然属于HTN型,但该毒株为HTN型病毒的新亚型病毒株。     

汉坦病毒K24株M和S片段基因的结构特征

为研究汉坦病毒K24株M和S片段基因的结构特征,从病毒感染细胞提取细胞总RNA,经35个循环的一次性PCR得到了与理论值相符合产物,将RT-PCR产物直接克隆T载体,经K24毒株M片段的全基因序列共3 653个核苷酸,四种核苷酸的酶切和PCR鉴定正确的克隆通过柱纯化后进行序列测定,结果比例分别为A 30.44%,T30.14%,G20.72%,C 18.70%,GC含量为39.42%,AT含量为60.58%,编码1 133个氨基酸.S片段的全基因序列共1 772个核苷酸,四种核苷酸的比例分别为A31.30%,G26.05%,T22.79%,C19.77%,GC含量为45.81%,AT含量为54.19%,共编码429个氨基酸.K24株M片段核苷酸全基因和氨基酸与HTN型毒株同源率分别为70.7%～71.2%和75.9%～76.9%,与SEO型为95.2%～99.2%和95.9%～99.4%.S片段与HTN 76～118和SEO SR-11病毒的比较,核苷酸同源率分别为74.0%和95.6%,氨基酸同源率为83.3%和97.9%,与M片段的结果相类似.M片段氨基酸与SEO型10个毒株存在4个氨基酸替代.应用DNASTAR软件的系统发生分析方法分别绘出了基于M片段核苷酸及推导氨基酸的系统发生树.     

汉坦病毒K24株M和S片段基因的结构特征

为研究汉坦病毒K24株M和S片段基因的结构特征,从病毒感染细胞提取细胞总RNA,经35个循环的一次性PCR得到了与理论值相符合产物,将RT－PCR产物直接克隆T载体,要K24毒株M片段的全基因序列共3653个核苷酸,四种核苷酸的酶切和PCR鉴定正确的克隆通过柱纯化后进行序列测定,结果比较分别为A30.44%,T30.14%,G20.72%,C18.70%,GC含量为39.42%,AT含量为60.58%,编码1133个氨基酸。S片段的全基因序列共1772个核苷酸,四种核苷酸的比例分别为A31.30%,G26.05%,T22.79%,C19.77%,GC含量为45.81%,AT含量为54.19%,共编码429个氨基酸。K24株M片段核苷酸全基因和 在酸与HTN型毒株同源率分别为70.7%－71.2%和75.9%-76.9%,与SEO型为95.2%-99.2%和95.9%-99.4%。S片段与HTN76－118和SEO SR－11病毒的比较,核苷酸同源率分别为74.0%和95.6%,氨基酸同源率为83.3%和97.9%,与M片段的结果相类似。M片段氨基酸与SEO型10个毒株存在4个氨基酸替代。应用DNASTAR软件的系统发生分析方法分别绘出了基于M片段核苷酸及氨基酸的系统发生树。     

汉坦病毒汉滩型特殊新亚型的发现

应用RT－PCR扩增了皖南山区分离株AH09的M和S片段全基因,克隆于T载体,纯化后测定序列。结果AH09株M片段的全基因序列共3625个核苷酸,编码1135个氨基酸;S片段的全基因序列共1724个核苷酸,编码430个氨基酸。M和S片段全基因核苷酸和氨基酸与汉坦病毒各型株的代表株和HTN型毒株的同源性比较表明,AH09株分枝与HTN型接近,与其它各型病毒则相距较远,故确定为HTN型毒株,但AH09株与HTN型毒株的M和S片段全基因序列有差异,其差异分别高达23．6％和20．4％,经种系发生分析,AH09株是迄今为止所发现的HTN型病毒中差异最大的新基因亚型病毒株,AH09株病毒M片段的氨基酸与HTN型相差13．5％至14．8％,而S片段仅相差7％－8．1％,说明AH09毒株的变异主要发生在M片段。而ORF和3‘端的NCR区核苷酸序列分析比较说明,病毒的变异更主要集中在该片段的3‘端的NCR区。     

我国肾综合征出血热疫苗生产株LR1株的全基因组序列分析

为测定我国肾综合征出血热疫苗生产株LR1株的全基因组序列 ,了解该株分子基础 ,从提取的细胞总RNA逆转录PCR扩增 ,产物纯化后克隆T载体纯化后测序 ,结果证明 ,LR1株全基因组序列由L6 5 33、M36 16、S片段的16 92个核苷酸组成 ,依各自读码框架分别编码 2 15 1、1135、42 9个氨基酸。序列同源比较分析表明 ,LR1毒株与国外HTN型毒株高度同源 ,属同一亚型 ,尤其与HTN代表株 76 - 1183个片段同源率高达 99 3%～ 99 8% ,而与国内的HTN型病毒差异较大 ,同源率仅为 79 4%～ 84 6 %。氨基酸比较也显示了同样的结果。     

汉滩病毒西安分离株84FLi的L片段核苷酸序列测定以及分析

采用逆转录 聚合酶链式反应 (RT PCR) ,分节段扩增汉滩病毒 84FLi株的L基因片段cDNA ,纯化的PCR产物片段直接用于序列测定或克隆入 pND18 T载体中进行测序。结果表明 ,84FLi株的L片段cDNA由 6 5 33个核苷酸组成 ,四种核苷酸的比例分别为A33.39% ,C16 .4 3% ,G2 0 .74 % ,T2 9.4 4 %。GC含量为 37.17% ,AT含量为6 2 .83%。推导出的最大开放读码框架为从 38到 6 4 93,共编码 2 15 1个氨基酸。序列同源性分析表明 ,84FLi株核苷酸与HTN型国际标准毒株 76 118的同源性为 83.7% ,差异性为 18.8% ;而与中国株A9的同源性高达 97.6 % ,差异性仅为 2 .4 %。与SEO型代表Seoul80 39的同源性为 75 .2 % ,6 6 .1%～ 6 6 .5 %。L片段的氨基酸比较分析表明 ,L片段与HTN型间的同源性为 97.5 %～ 98.0 % ,而与SEO型的同源性为 83.5 %～ 85 .5 %。与PUC、TUL、SN和AND等其它型汉滩病毒的同源性仅为 6 8.6 %～ 6 9.6 %。结果表明 84FLi株属于HTN型 ,并与分离自国内的HTN型病毒高度同源     

汉滩病毒西安分离株84FLi的L片段核苷酸序列测定以及分析

采用逆转录-聚合酶链式反应(RT-PCR),分节段扩增汉滩病毒 84FLi株的L基因片段cDNA,纯化的PCR产物片段直接用于序列测定或克隆入pND18-T载体中 进行测序.结果表明,84FLi株的L片段cDNA由6533个核苷酸组成,四种核苷酸的比例分别为A33.39%,C16.43%,G20. 74%,T29.44%.GC含量为37.17%,AT含量为62.83%.推导出的最大开放读码框架为从38到649 3 ,共编码2151个氨基酸.序列同源性分析表明,84FLi株核苷酸与HTN型国际标准毒株76-11 8的同源性为83.7%,差异性为18.8%;而与中国株A9的同源性高达97.6%,差异性仅为2.4%. 与SEO型代表Seoul80-39的同源性为75.2%,66.1%～66.5%.L片段的氨基酸比较分析表明, L 片段与HTN型间的同源性为97.5%～98.0%,而与SEO型的同源性为83.5%～85.5%.与PUC、TUL 、SN和AND等其它型汉滩病毒的同源性仅为68.6%～69.6%.结果表明84FLi株属于HTN型,并与分离自国内的HTN型病毒高度同源.     

我国肾综合出血热疫苗生产株LR1株的基因组序列分析

为测定我国肾综合征出血热疫苗生产株LR1株的全基因组序列,了解该株分子基础,从提纯的细胞总RNA逆转录PCR扩增,产物纯化后克隆T载体纯化后测序,结果证明,LR1株全基因组序列由L6533、M3616、S片段的1692个核苷酸组成,依各自读码框架分别编码21511、1135、429个氨基酸。序列同源比较分析表明,LR1毒株与国外HTN型毒株高度同源,属同一亚型,尤其与HTN代表株75－1183个片段同源率高达99．3％－99．8％,而与国内的HTN型病毒差异较大,同源率仅为79．4％－84．6％。氨基酸比较也显示了同样的结果。     

汉坦病毒中国疫苗株Z37M片段的克隆及序列分析

汉坦病毒Z37株是从褐家鼠体内分离到的,用于生产双价肾综合征出血热疫苗的病毒毒株之一,血清分型为SEO型。利用RT-PCR方法扩增Z37株M基因片段cDNA,克隆入质粒载体,进行核苷酸序列测定及分析。Z37株M基因片段由3651个核苷酸组成,只有一个开放读码框架,共编码1133个氨基酸。与HTN型病毒(76-118、A9、HV-114)的核苷酸和氨基酸同源性分别为71.8%～72.1%、76.2%～76.7%,与SEO型(R22、L99、80-39)的核苷酸和氨基酸同源性分别为95.3%～96.1%、95.3%～98.9%。这一结果的获得进一步从分子水平确定了Z37株的型别,并为研制M基因片段重组疫苗打下基础。     

汉坦病毒中国疫苗株Z37M片段的克隆及序列分析

汉坦病毒Z37株是从褐家鼠体内分离到的,用于生产双价肾综合征出血热疫苗的病毒毒株之一,血清分型为SEO型,利用RT－PCR方法扩增Z37株M基因片段cDNA,克隆入质粒载体,进行核苷酸序列测定及分析。Z37株M基因片段由3651个核苷酸组成,只有一个开放读码框架,共编码1133个氨基酸。与HTN型病毒（76－118、A9、HV－114）的核苷酸和氨基酸同源性分别为71.8%-72.1%、76.2%-76.7% ,与SEO型（R22、L99、80－39）的核苷酸和氨基酸同源性分别为95.3%-96.1%、95.3%-98.9%。这一结果的获得进一步从分子水平确定了Z37株的型别,并为研制M基因片段重组疫苗打下基础。     

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.     

Determination of stoichiometry of radioiodination of proteins

A sensitive method for the detection of small quantities of hydrophobic antioxidant free radical scavengers such as butylatedhydroxytoluene (BHT) and butylatedhydroxyanisole (BHA) in aqueous samples is described. The procedure involves extraction of the hydrophobic free radical scavenger into an organic solvent phase, followed by the subsequent reaction of an aliquot of this extract with the stable cation radical tris(p-bromophenyl)amminium hexachloroantimonate (TBACA). In experiments with BHT and BHA, the loss of TBACA absorbance at 730 nm was found to be linearly proportional to the amount of antioxidant added, with quantities of BHT as small as 200 pmol being easily detectable. In aqueous suspensions of dimyristoylphosphatidylcholine vesicles, assays of the aqueous BHT concentration showed that BHT partitioned strongly into the membrane phase, achieving very high BHT/phospholipid ratios. For a given concentration of BHT, partitioning into the membrane phase was greater in large, multilamellar liposomes than in either small, single-walled vesicles or in purified rat brain synaptic vesicle membranes. Direct assay of BHT and BHA in phospholipid membranes, however, was complicated by a nonspecific interaction between TBACA and the phospholipid.