Structural proteins of infectious bovine rhinotracheitis virus.

Infectious bovine rhinotracheitis (IBR) virus grown in bovine embryo kidney cell cultures was concentrated and purified in Ficoll density gradients. The polypeptide composition of the virus was studied by polyacrylamide gel electrophoresis. The mature virion was found to contain 18 structural proteins with molecular weights from 250,000 to 29,000 daltons; 8 of them were glycosylated. The similarity of IBR virus protein composition to proteins of other herpetoviruses is discussed.     

Isolation and characterization of low-molecular-weight DNA-binding proteins from retroviruses

The DNA-binding proteins from Rauscher leukemia virus, baboon leukemia virus, endogenous cat virus, woolly monkey virus, bovine leukemia virus, equine infectious anemia virus, Rous sarcoma virus, Prague strain, mouse mammary tumor virus, and Mason-Pfizer monkey virus were purified to homogeneity by molecular sieving chromatography and DNA affinity chromatography. The amino acid composition shows each to be a basic protein.     

Purification of the Sendai virus nonstructural C protein expressed in E. coli,and preparation of antiserum against C protein

Summary An expression plasmid, ptac-C, was constructed by inserting the cDNA of the coding region of the Sendai virus nonstructural C protein downstream of the tac promoter ofE. coli expression plasmid ptac12-Bam. A new protein produced inE. coli after induction was purified to near homogeneity. The purified protein was found to be identical with the C protein predicted from the C gene cDNA in molecular weight, isoelectric point, amino acid composition, and the amino acid sequence at the N-terminal of the protein as well as those of several fragments obtained on V8 protease digestion. Antiserum raised against the purified protein specifically reacted with the C protein in infected cells. Using this antiserum, the localization of the C protein in infected cells was examined by immunofluorescence, which revealed that it appeared in the cytoplasm but not in nuclei.     

The molecular mass of adenovirus type 5 as determined by means of scanning transmission electron microscopy (STEM)

The mass of adenovirus type 5 was determined by means of computer-assisted scanning transmission electron microscopy (STEM). Arithmetic mean of 157 +/- 10(SD) X 10(6) daltons and mode between 160 and 170 X 10(6) daltons compare favourably with previously reported data. The advantages of the STEM-procedure over the physical and chemical techniques are: low amounts of purified virus particles are needed; visual control of the physical state of virus particles; no need to know the chemical composition or protein concentration of the virus sample.     

Composition of artificially produced and naturally occurring empty capsids of poliovirus type 1

The polypeptide composition of the empty capsids of poliovirus type 1 represented either by the 73 S component isolated from sucrose gradients or by the top component from CsCl gradients is different from that ot the whole infectious virion. One protein (VP4) is absent from the empty capsids, another (VP2) is lower in relative amount, while an additional protein (NCVP6), not present in purified virions, is found in large amounts. Artificial production of 73 S particles by borate buffer, pH 10.5, treatment of purified virions results in removal of one protein component (VP4) and the RNA of the virus. The observed differences in composition of these virus-specific particles are discussed with regard to their possible role in the architecture and the assembly of the virion.     

丙型肝炎病毒亚单位融合蛋白的表达及免疫原性测定

目的获得高纯度的丙型肝炎亚单位融合蛋白并评价其免疫原性。方法利用原核表达系统,以pET—11d作为载体,在大肠埃希菌BL21（DE3）中经IPTG诱导表达获得融合蛋白,之后采用DEAE阴离子交换和镍离子柱亲和层析进行纯化。通过Western Blot验证融合蛋白的抗原活性。同时以该蛋白免疫实验动物后测定血清中的抗体滴度。结果成功获得很高纯度的丙型肝炎亚单位融合蛋白,EIA显示融合蛋白能够诱生出高滴度的抗体。结论原核表达系统表达的丙型肝炎病毒亚单位融合蛋白具有较强的免疫原性,为丙型肝炎病毒治疗性和预防性疫苗的研制提供了一条新的途径。     

Isolation of the matrix (membrane) protein of vesicular stomatitis virus by gel filtration in guanidine hydrochloride.

The L, N and M proteins of vesicular stomatitis virus (VSV) were resolved from each other by gel filtration in the presence of 6 m-guanidine hydrochloride. Amino acid analysis for purified M protein of VSV showed that its chemical composition differed from those of influenza and SV5 M proteins.     

The paramyxovirus SV5 V protein binds two atoms of zinc and is a structural component of virions

The paramyxovirus simian virus 5 (SV5) cysteine-rich V protein has been shown to be a virus structural protein by analysis of the polypeptides of purified SV5 virions. In addition, the V protein has been identified as a component of the virus nucleocapsid core both by the analysis of the polypeptides present in radioactively labeled preparations of purified nucleocapsids and by immunoelectron microscopy. Quantitative autoradiography was used to determine that there are approximately 350 molecules of the V protein in virions. The V protein has been purified from V recombinant baculovirus-infected insect cells and by using inductively coupled argon plasma atomic emission spectroscopy it was found that each molecule of V binds two zinc atoms.     

SARS冠状病毒N蛋白基因的表达及其在SARS诊断中的应用

目的 获得重组SARS冠状病毒（SARS-CoV）N蛋白抗原，建立特异性诊断SAILS病毒感染的免疫学方法。方法 大肠埃希菌中表达SARS病毒N蛋白基因，用金属螯合层析纯化N蛋白，建立检测SARS抗体的EUSA方法。结果 大肠埃希菌中表达了SARS病毒全长N蛋白抗原，经包涵体洗涤和金属螯和纯化后得到纯度较高的重组蛋白。用重组抗原EUSA检测30名SARS患者抗体全部为阳性，30名正常人血清为阴性，30名发热非SARS患者血清为阴性。结论 SARS表达核蛋白可以在大肠埃希菌中得到高效表达，纯化的重组N蛋白具有良好抗原性，可用于检测SARS抗体。     

重组汉滩病毒核蛋白的纯化及鉴定

目的：纯化重组表达的汉滩病毒核蛋白。方法：重组菌经IPTG诱导后，表达的目的蛋白为带有谷胱甘肽转硫酶（GST）标签的融合蛋白，并以包涵体形式存在。将包涵体变性、复性后，采用Glutathione Sepharose 4B亲和色谱对核蛋白进行纯化，并用夹心ELISA和Western blot检测纯化蛋白。结果：表达产物第一次过柱亲和层析后可去除杂蛋白，获得纯化的核蛋白与谷胱甘肽转硫酶的融合蛋白，再经凝血酶酶切，第二次过柱亲和层析后获得的穿过峰为核蛋白，洗脱峰为GST。纯化的融合蛋白和纯化的核蛋白均为SDS－PAGE单点纯，并具有良好的抗原活性。结论：Glutathione Sepharose 4B亲和色谱纯化重组汉滩病毒核蛋白是一种较为有效的方法。     

鸭坦布苏病毒感染对BHK-21 细胞分泌胞外体的影响

目的:探讨鸭坦布苏病毒感染BHK-21 细胞对胞外体的影响,进一步研究鸭坦布苏病毒的致病机制。方法:利用鸭坦布苏病毒AH-F10 株感染BHK-21 细胞,并设置未感染病毒的对照组。用PEG 法提取感染病毒的BHK-21 细胞和对照组细胞上清中的胞外体,通过电镜观察、Western blot 检测对胞外体进行鉴定,并对提取的两组胞外体进行质谱鉴定分析。结果:电镜观察到纯度高的内部色浅、边缘浓染的杯状囊泡,直径30 ~ 160 nm。感染组胞外体的平均粒径大小大于对照组胞外体的平均粒径大小。Western blot 试验中,均检测出CD9、CD63 分子。应用液相质谱法从胞外体中鉴定出106 种蛋白,其中感染坦布苏病毒组共检测出84 种蛋白,对照组49 种蛋白,有27 种共同蛋白分子。结论:鸭坦布苏病毒感染BHK-21 细胞会影响细胞胞外体的分泌,影响胞外体粒径大小及其蛋白组成成分。本试验为进一步研究坦布苏病毒的感染和致病机制奠定了基础。     

Biophysical and biochemical properties of tomato aspermy virus

A virus isolated from tomato in British Columbia was identified as tomato aspermy virus (TAV). The virus was purified and compared with an isolate of TAV from chrysanthemum (CV-L) and with an isolate of cucumber mosaic virus (CMV). TAV and CV-L were serologically identical but CMV was only distantly related. The amino acid composition of TAV was similar to that of CV-L, showing only four amino acid exchanges, whereas there were 18 amino acid exchanges between TAV and CMV. TAV had a sedimentation coefficient of 98 S, a diffusion coefficient of 1.49 × 10^?7 cm²/sec and a molecular weight of 5.3 million. The molecular weight of the viral protein subunit, determined on polyacrylamide gel, was 26,300. The base percentage composition of the viral RNA was G = 23.1, A = 26.2, C = 21.6, and U = 29.0. Phosphorus analysis of the RNA indicated that the nucleic acid content of the virus was 17.7%.     

Generation of Sendai virus nucleocapsid-like particles in yeast

The gene encoding Sendai virus nucleocapsid protein was cloned into the yeast Saccharomyces cerevisiae expression vector pFGG3 under control of GAL7 promoter. The high level of recombinant Sendai virus nucleocapsid protein expression (12-14 mg/l of yeast culture) was obtained. The evaluation of recombinant proteins expression in yeast by Western blot analysis revealed specific reactivity with immune sera. Electron microscopy demonstrated the assembly of typical herring-bone structures of purified recombinant nucleocapsid protein. These structures contained host RNA, which was resistant to an RNase treatment. The nucleocapsid protein revealed stability in yeast and can be easily purified by cesium chloride gradient ultracentrifugation. The development of a simple, efficient and cost-effective system for generation of Sendai virus nucleocapsid protein might help to upgrade reagents for virus serology, and facilitate investigation of virus replication and RNA encapsidation mechanisms.     

抗SARS病毒N蛋白单克隆抗体的制备和初步应用

目的 制备抗SARS病毒N蛋白的单克隆抗体并研究其初步应用。方法 用基因重组N蛋白免疫小鼠，取免疫后的鼠脾细胞与骨髓瘤细胞融合，筛选分泌抗SAPS病毒N蛋白单克隆抗体细胞株。将阳性细胞株接种小鼠腹腔制备单克隆抗体腹水并对抗体进行纯化，分析纯化抗体的相对亲和力。选择亲和力较高的抗体制备检测SARS病毒抗原的酶联免疫诊断试剂，并对其敏感性和特异性进行分析。结果 共获得11株单克隆抗体细胞株，其中3株单抗与N蛋白具有较高的亲和力，4株纯化单抗与N蛋白反应很弱，其余4株单抗介于两者之间。用亲和力较高的单抗制备检测SARS病毒抗原的诊断试剂，其敏感性可达31PFU／ml，而且与其他呼吸道病毒无交叉反应。结论 该试剂特异性较好，可用于SARS病毒抗原的检测，其敏感性仍需用临床急性期样品进行评价。     

Purification of a baculovirus-expressed hepatitis E virus structural protein and utility in an enzyme-linked immunosorbent assay.

We report on the purification of the full-length structural protein encoded by open reading frame 2 (ORF-2) of hepatitis E virus. The ORF-2 protein, expressed in Sf9 cells by using a recombinant baculovirus vector system, was successfully purified to homogeneity. Gel electrophoresis of the purified ORF-2 protein showed a single polypeptide of 75 kDa by Coomassie blue staining and by Western blot (immunoblot) analysis. We demonstrated that the partially purified ORF-2 protein could be used successfully in a sensitive and specific enzyme-linked immunosorbent assay for the detection of antibodies to hepatitis E virus.     

Radioimmunoassay of measles virus hemagglutinin protein G

Guinea pig and rabbit antisera from animals immunized with purified measles virus hemagglutinin (G) protein were used to establish a solid-phase four-layer radioimmunoassay for quantitative measurement of the G protein. The sensitivity of the assay was 2 ng of purified G protein, and 200 μg of protein from uninfected Vero cells neither decreased the sensitivity nor reacted non-specifically in the assay. Radioimmunoassay standard dose—response curves were established and unknown values interpolated from these using the logit program of a desktop computer. Using this procedure, a measles virus growth curve in infected Vero cells was determined by measurement of G protein production. Under these same conditions, hemagglutination was not sensitive enough to detect early hemagglutinin production.Viral antigens in canine distemper virus, Newcastle disease virus, parainfluenza viruses 1–4, simian virus 5, and respiratory syncytial virus-infected cell lysates did not cross-react in the radioimmunoassay. A small degree of cross-reactivity was detected with mumps viral antigens, both with Vero cell-derived (wild-type strain) and egg-derived (Enders strain) purified virus preparations and with a cell lysate antigen prepared from wild-type mumps virus-infected Vero cells.     

人H5N1流感病毒M1基因的亚克隆及其在原核细胞的表达

目的 构建人H5N1亚型禽流感病毒A/Anhui/1/2005 M1蛋白的原核表达系统,为进一步研究M1蛋白的生物学功能和制备其诊断试剂奠定基础。方法 以该病毒基因节段七cDNA为模板,PCR扩增得到M1基因片段。将该片段亚克隆至载体pQE80-L中,构建重组质粒pQE80-L/M1,转化大肠埃希菌BL21（ DE3）。IPTG诱导重组蛋白表达。金属镍离子螯合层析纯化N末端携带多聚组氨酸标签的重组M1蛋白,免疫小鼠制备多克隆抗体。结果 获得了重组M1蛋白,能与抗H5N1亚型流感病毒血清发生特异性结合,且其免疫后能诱导机体产生特异性抗体。结论 成功获得了人H5N1亚型禽流感病毒M1蛋白在原核细胞中高效表达。     

Enzyme-linked immunosorbent assay for determination of antibodies to the envelope glycoprotein of rabies virus.

The envelope glycoprotein G of rabies virus induces neutralizing antibodies, which are important in protection against rabies. This protein was solubilized from purified virus and isolated by differential and sucrose density gradient centrifugation followed by high-performance liquid chromatography. Conditions for solubilization and purification of G were optimized by using immunoblotting and enzyme-linked immunosorbent assay techniques. The reaction with conventional antisera and monoclonal antibodies indicated that purified G protein was essentially devoid of internal viral proteins. Microdilution plates were coated with purified G protein, and sera from humans vaccinated against rabies were tested for the presence of antibodies. Results were compared with those of the rapid fluorescent focus inhibition assay, which is the standard neutralization assay for antibodies to rabies virus. The results of this comparison indicate that the enzyme-linked immunosorbent assay for G is a reliable and simple alternative to the neutralization test.     

肾综合征出血热病毒结构蛋白的纯化及免疫学特性分析

应用从肾综合征出血热（ＨＦＲＳ）患者血清中提取的ＩｇＧ与活化的Ｓｅｐｈａｒｏｓｅ４Ｂ偶联，制备亲和层析柱，用此亲和层析柱从ＨＦＲＳＶ感染的小白鼠乳鼠鼠脑中提取出２种分子量为６７０００和５５０００的病毒结构蛋白。经ＥＬＩＳＡ证明，此病毒结构蛋白能与ＨＦＲＳ患者血清ＩｇＧ及抗ＨＦＲＳ病毒的ＭｃＡｂ反应，效价为１６０。与６株抗ＨＦＲＳＶＭｃＡｈ试验表明，此病毒结构蛋白能与Ｈ７株反应。血凝试验证明，此病毒蛋白不能凝集鹅红细胞、鸽血球和人＂Ｏ＂型血红细胞。将纯化的病毒结构蛋白免疫家兔，证明其有较强的免疫原性，可刺激家兔产生特异性抗体；微量中和试验及乳鼠中和试验证明，中和效价为２５６，说明纯化的病毒结构蛋白具有中和抗原位点；免疫血清对感染乳鼠有一定保护作用。