薄荷伪造桥虫NPV感染斜纹夜蛾幼虫分离的一种新NPV特性的研究

用薄荷伪造桥虫核型多角体病毒(Argroyamma agnata NPV)(以下简称Aa NPV)在室内感染斜纹夜蛾(Prodenia litura)幼虫,从死虫体内分离到一种NPV。经电镜观察,多角体蛋白分析、病毒核酸的限制性内切酶酶解分析等研究,证明此多角体直径为1.5—2.6/μm,病毒粒子为杆状,其大小为100—150×420nm,病毒粒子为多粒包埋型。提纯的多角体蛋白只有一种多肽,分子量为33,500d,提纯的病毒粒子的结构多肽至少有15种,其分子量范围为15,600     

辛德毕斯病毒在BHK—21细胞中繁殖过程的研究

辛德毕斯病毒(Sindbis virus)在BHK—21细胞中繁殖的一步生长曲线表明,病毒感染后2小时便可产生大量的子代病毒;感染后6小时,病毒滴度达到最高峰,为109TCID50/ml电镜技术显示了病毒粒子的形态结构与形态发生过程。本文还对SbV的代谢合成动态及对宿主细胞的影响进行了研究和讨论。     

酵母细胞外蛋白质和多肽的产生及分泌

白地霉(Geotrichum candidum)AS 2．198的突变株NX47和橙黄红酵母(Rhodotoru-ta aurantiacam)AS 2．280蛋白质的合成与分泌和多肽的合成均在培养前期与细胞生长同步进行。合成的蛋白质全部或大部分贮存在胞周间,并随即通过细胞壁从胞周间分泌到细胞外,培养基的组成和培养时间对蛋白质的组份无显著的影响。合成的多肽全部贮存在细胞内。突变株NX47缓慢地分泌蛋白质,快速分泌多肽。其分泌能力取决于分为三层的细胞壁结构。多肽在培养前期不分泌,直到进入对数生长后期,才通过原生质膜和细胞壁由细胞内迅速地分泌到细胞外。分泌时间比其合成滞后一天以上。菌株AS 2．280的细胞壁没有三层结构,却能快速分泌蛋白质,但不分泌多肽。     

家蚕核型多角体病毒结构多肽的双向电泳分析

用等电聚焦-聚丙烯酰胺梯度凝胶电泳的双向电泳法和银染色方法,分析了家蚕核型多角体病毒粒子和核衣壳的结构多肽。该病毒单粒包埋型病毒粒子含约96种多肽,核衣壳含约72种多肽。病毒粒子制剂和核衣壳制剂中含有较多量的分子量为31K的多肽,用蔗糖梯度离心、离心洗涤、碱处理甚至蛋白酶酶解和去污剂处理,都不能将其除去。向病毒制剂中加入纯化的多角体蛋白后作双向电泳,发现外加多角体蛋白改变原等电点面与上述31K多肽重合。对31K多肽的来源进行了讨论。     

葡萄扇叶病毒移动蛋白在寄主体内的动态检测和免疫金标记

利用葡萄扇叶病毒法国分离物F13(Grapenive fanleaf virus, GFLVF13)移动蛋白抗体对杭州分离物(GFLVH)移动蛋白进行Western blot,分析表明移动蛋白在接种GFLVH 3d后的苋色藜(Chenopodium amaranticolor)系统叶中就可检测到,随着时间推移,其积累量逐渐升高,接种16d后达到最高值。接种32d后的病叶已经枯黄,但移动蛋白积累量并没有减少。超薄切片电镜观察发现,在感染GFLVH的昆诺藜(C.quinoa)和苋色藜的叶肉组织薄壁细胞中,病毒粒子呈纵列整齐地排列在小管状结构中,在胞间连丝中也发现有管状结构。免疫金标记显示胶体金能定位在细胞质、细胞壁和胞间连丝上,在管状结构也发现有少量的金粒子。这些结果进一步说明了GFLV是通过管状结构实现细胞间移动的。     

杆状病毒转移载体的构建及绿色荧光蛋白在斜纹夜蛾幼虫中的表达

为构建斜纹夜蛾核型多角体病毒 （SpltMNPV）的重组病毒,以该病毒日本C3株基因组DNA为PCR扩增模板,根据GenBank SpltMNPV中国G2株基因序列,设计了两对引物分别扩增多角体蛋白基因的5′端侧翼序列（含启动子）和3′端侧翼序列（含终止子）,将这两个片段依次克隆于pUC18质粒载体后,再将绿色荧光蛋白(GFP)基因亚克隆到上述载体的多角体蛋白基因启动子和终止子之间,获得转移载体pSplt-gfp。将pSplt-gfp与野生型SpltMNPV 基因组DNA共转染Spli细胞,通过同源重组和有限稀释法筛选,获得了以gfp基因替代多角体蛋白基因的重组病毒SpltMNPV-gfp。SpltMNPV-gfp感染Spli细胞和斜纹夜蛾幼虫,分别在感染24h和48h后可发现绿色荧光蛋白的表达。该重组病毒的获得,为建立斜纹夜蛾核型多角体病毒表达体系奠定了基础。     

柑桔碎叶病毒研究

用汁液摩擦接种方法对柑桔碎叶病毒(Citrus tatter leaf virus,CTLV)进行了进一步的生物学鉴定.结果表明,该病毒除在豇豆上引起枯斑外,还侵染克里芙兰烟(Nicotianaclevilandii)产生系统斑驳和轻花叶,侵染昆诺藜(Chenopodium quinoa)和苋色藜(C.amaranticolor)引起系统坏死和花叶,侵染鸡冠花(Celosia cristata)引起局部环斑.这些草本寄主均可作为CTLV的指示植物.在发病的柑桔叶汁液中,测得CTLV的稀释限点为10~(-5).经汁液摩擦接种,可以将CTLV从克里芙兰烟传播到木本指示植物柑桔和枳橙上.感病植物材料的组织超微结构观察结果表明:CTLV感染柑桔、枳橙、克里芙兰烟和昆诺藜均使其叶肉薄壁细胞的叶绿体出现淀粉沉积、叶绿体片层结构解体和消失;病毒在受感染的植株叶脉韧皮部细胞中紧密聚集,形成病毒结晶体;这种结构也出现在叶肉薄壁细胞中.这是关于该病毒组织病变的首次报道.用直接负染方法从感染CTLV的柑桔(枳橙)、克里芙兰烟和昆诺藜病叶中均能检查到线状病毒粒子,用改良的Deriick’s免疫电镜方法和琼脂双扩散方法测定均显示该病毒…     

新城疫病毒F蛋白中两段七肽重复序列的克隆和表达

从新城疫病毒 (NDV)中国强毒株F4 8E9和弱毒株长春株F蛋白的cDNA中亚克隆出两段七肽重复序列(HeptadRepeatRegion ,HR1,HR2 ) ,将HR1和HR2分别插入表达载体pGEX 6p 1,在大肠杆菌BL2 1(DE3 )中表达 ,将与载体中的GST(GlutathioneS Trasferase)融合表达的可溶性融合蛋白用GST亲和层析柱纯化。纯化的融合蛋白用蛋白酶酶切后 ,先用GST亲和层析柱除去GST ,再加热进一步纯化。纯化的HR1和HR2质谱分析其分子量 ,结果表明 ,强株的HR1和HR2的分子量分别为 7 10 3kD和 6 3 0 1kD ,弱株的HR1和HR2的分子量分别为 7 10 7kD和6 3 0 9kD ,强弱株HR1和HR2的分子量都基本一致。本工作为研究HR1、HR2的结构以及它们在NDV与宿主细胞融合中的作用奠定了基础。     

菜白蝶的一种新病毒I.病毒的分离、鉴定

从菜白蝶罹病死亡的幼虫里,分离到两种昆虫病毒。一种足巳知的颗粒体病毒;另一种是无波诎膜的非包涵体病毒,病毒外壳呈典型的六角形,廿面对称体,粒子直径大小约25亳微米,衣壳山许多壳粒(Capsomer)构成,完整和空壳病毒粒于的外周有12个壳粒围绕,壳粒中空。一些病毒粒子有着明显的核心(Core),核心球形,直径大小约15毫微米。 提纯的病毒经0 01％吖啶橙(Acridine orange)染色,在荧光显徽镜下观察呈红色荧光。它能够单独感染,使菜白蝶幼虫致病,有很高的毒性。试验证明这株非包涵体病毒为菜白蝶的一种新病毒。     

烟草花叶病毒的普通株和油菜株RNA的体外翻译比较

对烟草花叶病毒普通株(TMVc)及另一毒株,油菜花叶病毒株(YMV15)的RNA,在麦胚无细胞提取液和兔网织红细胞裂解液中的体外翻译产物,利用SDS-聚丙烯酰胺凝胶电泳分离,放射自显影,荧光放射自显影以及放射免疫沉淀法进行了分析。结果证明,在YMV15-RNA的体外翻译产物中检测出有与天然的YMVIr外壳蛋白分子量(16 5K)一致的,并被TMV。抗血清和金黄葡萄球菌(Staphylococus aureus)细胞悬浮液所沉淀的多肽。V8蛋白酶酶解图谱也证明该产物与天然外壳蛋白相同。而TMVc-RNA的体外翻译产物中则完全不产生其外壳蛋白(M．W．17．5K),在TMV。抗血清沉淀反应中也未见任何沉淀带产生。属于同一分类组的同一病毒的不同毒株,其翻译策略很可能不一样,对此本文进行了讨论。     

Polypeptide Synthesis in Simian Virus 5-Infected Cells

Polypeptide synthesis in three different cell types infected with simian virus 5 has been examined using high-resolution polyacrylamide slab gel electrophoresis, and all of the known viral polypeptides have been identified above the host cell background. The polypeptides were synthesized in infected cells in unequal proportions, which are approximately the same as they are found in virions, suggesting that their relative rates of synthesis are controlled. The nucleocapsid polypeptide (NP) was the first to be detected in infected cells, and by 12 to 14 h the other virion structural polypeptides were identified, except for the polypeptides comprising the smaller glycoprotein (F). However, a glycosylated precursor (F(0)) with a molecular weight of 66,000 was found in each cell type, and pulse-chase experiments suggested that this precursor was cleaved to yield polypeptides F(1) and F(2). No other proteolytic processing was found. In addition to the structural polypeptides, the synthesis of five other polypeptides, designated I through V, has been observed in simian virus 5-infected cells. One of these (V), with a molecular weight of 24,000, was found in all cells examined and may be a nonstructural viral polypeptide. In contrast, there are polypeptides present in uninfected cells that correspond in size to polypeptides I through IV, and similar polypeptides have also been detected in increased amounts in cells infected with Sendai virus. These findings, and the fact that the synthesis of all four of these polypeptides is not increased in every cell type, suggest that they represent host polypeptides whose synthesis may be enhanced upon infection. When a high salt concentration was used to decrease host cell protein synthesis in infected cells, polypeptides IV and (to a lesser extent) I were synthesized in relatively greater amounts than other cellular polypeptides, as were the viral polypeptides. The possibility that these polypeptides may play some role in virus replication is discussed.     

Herpesvirus sylvilagus I. Polypeptides of virions and nucleocapsids.

Herpesvirus sylvilagus was propagated in juvenile cotton tail rabbit kidney cells and purified from the cytoplasmic fraction of the infected cells. The purification procedure included zonal centrifugation through a 5 to 30% dextran t-10 gradient, followed by equilibrium centrifugation in a 5 to 50% potassium tartrate gradient. H. sylvilagus formed one band after centrifugation through the tartrate gradient at a density of 1.22 g/cm3. Contamination of the purified virus preparation by cellular proteins was less than 0.2% as determined by the removal of radioactivity from an artificially mixed sample containing [35S]methionine-labeled control cells and nonlabeled infected cells. H. sylvilagus nucleocapsids were isolated from infected cell nuclei and purified by sedimentation through a 36% sucrose cushion, followed by equilibrium centrifugation in 5 to 50% tartrate gradient. Forty-four polypeptides ranging in molecular weight from 18,000 to 230,00 were resolved when [35S]methionine-labeled enveloped H. sylvilagus was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Seventeen polypeptides found within the enveloped virus were also identified with the nucleocapsid. Six additional nucleocapsid polypeptides han no counterparts within the enveloped virus. The major polypeptide within both the virus and the nucleocapsid had a molecular weight of 150,000.     

Identification of a transformation-specific protein induced by a Rous sarcoma virus.

Using antisera obtained from rats bearing Schmidt-Ruppin strain Rous sarcoma virus-induced tumors, we have idnetified a protein with an apparent molecular weight of 56,000 daltons and an isoelectric point of 6.3 in extracts of chick embryo fibroblasts transformed by a wild-type nondefective Rous sarcoma virus (Schmidt-Ruppin strain). This protein was not found in cells infected by trnasformation-defective mutants with either a partial or complete deletion of the src gene, nor in cells infected by a nontransforming avian leukosis virus. The 56,000 dalton molecular weight protein was found to be synthesized at both the permissive and nonpermissive temperatures in cells infected by either of two conditionallethal mutants that are temperature-sensitive in cell transformation. The amount of this protein, however, accumulated in cells infected by these temperature-sensitive mutants, relative to the structural polypeptides, differed significnatly from that seen with the nondefective virus. Pulsechase experiments indicate that the protein is extremely unstable, with a half-life of about 20 min, and does not serve as a precursor to any of the detectable virion polypeptides. Furthermore, incubation of the rat antiserum with purified, disrupted virus did not affect its immunoreactivity to this particular protein. We conclude that this 56,000 dalton molecular weight protein is a nonstructural protein specific to cells transformed by Rous sarcoma virus.     

Intracellular synthesis of measles virus-specified polypeptides.

The intracellular synthesis of measles-specified polypeptides was examined by means of polyacrylamide gel electrophoresis of cell extracts. Since measles virus does not efficiently shut off host-cell protein synthesis, high multiplicities of infection were used to enable viral polypeptides to be detected against the high background of cellular protein synthesis. The cytoplasm of infected cells contained viral structural polypeptides with estimated molecular weights of 200,000, 80,000, 70,000, 60,000, 41,000, and 37,000. All of these structural polypeptides, with the exception of P1, the only virion glycoprotein (molecular weight congruent to 80,000), were also found in the nuclei. In addition, two nonstructural polypeptides with estimated molecular weights of 74,000 and 72,000 were also present in the cytoplasm of infected cells. The initial synthesis of the smaller, nonstructural polypeptide began later in infection than the structural polypeptides. Pulse-chase experiments failed to detect any precursor-product relationships. The intracellular glycosylation and phosphorylation of the viral polypeptides were found to be similar to those found in purified virions.     

Synthesis, stability, and cleavage of Newcastle disease virus glycoproteins in the absence of glycosylation.

Polypeptides synthesized in Newcastle disease virus (NDV)-infected CHO cells in the absence of glycosylation were characterized. Incorporation of either [3H]mannose of [3H]glucosamine into NDV polypeptides was inhibited to greater than 99% by the antibiotic tunicamycin. Under these conditions, infected cells synthesized proteins which comigrated on polyacrylamide gels with the viral L protein, nucleocapsid protein, membrane protein, and a polypeptide with a molecular weight of 55,000 (P55). These cells did not synthesize polypeptides with the size of the hemagglutinin-neuraminidase (HN) protein or the fusion (F0) protein. They did, however, synthesize new polypeptides with molecular weights of 75,000 (P75), 67,000 (P67), and 52,000 (P52). Peptide analysis revealed that P75 was a host cell protein whose synthesis is enhanced by tunicamycin. P67 corresponded to the unglycosylated forms of the glycoproteins were found to be relatively stable in infected cells. P55, previously thought to correspond to the cleaved form of F0, was found to be a unique viral protein which is associated with intracellular nucleocapsid structures.     

Tumor antigens of human ad5 in transformed cells and in cells infected with transformation-defective host-range mutants

We have studied the polypeptides associated with the expression of the transforming region of the Ad5 genome by immunoprecipitating antigens (using the double antibody and protein A-Sepharose techniques) from cells infected with wild-type (wt) Ad5 or transformation-defective host range (hr) mutants and from cells transformed by Ad5. Three different antisera were used: P antiserum specific for early viral products (Russell et al., 1967) and two different hamster tumor antisera. Immunoprecipitation of antigens from wt-infected KB cells followed by SDS-polyacrylamide gel electrophoresis of precipitated proteins revealed that a major polypeptide having a molecular weight of approximately 58,000 was detected with all three antisera and with both the double antibody and the protein A-Sepharose techniques, while P antiserum also precipitated polypeptides of molecular weights 72,000, 67,000 and 44,000, which probably represent the DNA binding protein and related polypeptides, respectively. With the double antibody technique, in addition to the proteins mentioned above, P antiserum and the hamster tumor antisera precipitated a 10,500 dalton polypeptide which was not detected when the protein A-Sepharose procedure was used. Using either the double antibody or the protein A-Sepharose technique, we found that hr mutants from complementation group II failed to induce the synthesis of the 58,000 dalton protein, whereas mutants from complementation group I produced normal or near normal amounts. Using the double antibody technique, we found that the 10,500 dalton protein was absent or made in reduced amounts by group I mutants. A 58,000 dalton protein was detected in a number of different Ad5-transformed cell lines, including the 293 human line, the 14b hamster line and several transformed rat cell lines. This observation and the fact that transformation negative group II mutants fail to induce the synthesis of a 58,000 dalton polypeptide suggest that this protein is one of the Ad5-specific products necessary for cell transformation.     

Purification of scrapie agent from infected animal brains and raising of antibodies to the purified fraction

The fraction (P4) containing scrapie infectivity was obtained by treatment of scrapie-infected mouse brains with the detergent sarcosyl, differential centrifugation, and proteolytic enzyme digestion. Scrapie infectivity in the P4 fraction was purified 239-2,390 times with respect to protein. Similar fractions were also prepared from the brain of a sheep naturally infected with scrapie. Morphological observation of the P4 fractions revealed that the main components were unique rods of 3-5 X 60-200 nm, which resembled scrapie-associated fibrils (SAF) or prion rods. The P4 fractions formed three major broad bands of polypeptides with molecular weights (MWs) of about 24.5K, 21K, and 17K dalton (Kd) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and some low MW polypeptides were also present in the fraction. Rabbits immunized with this fraction prepared from mouse brains raised antibodies against the three major polypeptides.     

Detection of adenovirus type 2-induced early polypeptides using cycloheximide pretreatment to enhance viral protein synthesis.

(35S) methionine-labeled polypeptides synthesized by adenovirus type 2-infected cells have been analyzed by polyacrylamide gradient gel electrophoresis and autoradiography. Cycloheximide (CH) was added to infected cultures to accumulate early viral mRNA relative to host cell mRNA. This allowed viral proteins to be synthesized in increased amounts relative to host proteins after removal of CH and pulse-labeling with (35S)methionine. During the labeling period arabinosyl cytosine was added to prevent the synthesis of late viral proteins. This procedure facilitated the detection of six early viral-induced polypeptides, designated EP1 through EP6 (early protein), with apparent molecular weights of 75,000 (75K), 42K, 21K, 18K, 15K, and 11K. Supportive data were obtained by coelectrophoresis of (35S)- and (3H)methionine-labeled polypeptides from infected and uninfected cells, respectively. Three of these early polypeptides have not been previously reported. CH pretreatment enhanced the rates of synthesis of EP4 and EP6 20- to 30-fold and enhanced that of the others approximately twofold. The maximal rates of synthesis of the virus-induced proteins varied, in a different manner, with time postinfection and CH pretreatment. Since CH pretreatment appears to increase the levels of early viral proteins, it may be a useful procedure to assist their isolation and functional characterization.