Epstein—Barr病毒膜抗原在昆虫细胞sf9中的表达,纯 …

将Ｅｐｓｔｅｉｎ－Ｂａｒｒ（ＥＢ）病毒膜抗原基因（ＥＢＶ－ＭＡ）称ＭＡ１和截去穿膜区的ＭＡ基因称ＭＡ２分别插入杆状病毒表达载体ｐＶＬ－９４１。将两种重组质粒分别与杆状病毒ＤＮＡ共转染ｓｆ９细胞后获得Ｂａｃｕｌｏ－ＭＡ１和Ｂａｃｕｌｏ－ＭＡ２两种重组病毒，表达产物分别位于重组病毒感染的细胞表面或释放到细胞培养液中。免疫荧光方法检查，在感染的细胞表面表达产物与抗ＭＡ的单克隆抗体特异性地结合，ＳＤＳ－Ｐ     

应用痘苗病毒载体表达猴轮状病毒VP4抗原基因

把编码猴轮状病毒（Ｒｈｅｓｕｓｒｏｔａｖｉｒｕｓ，ＲＲＶ）Ｖｐ４抗原的第４基因片段插入到痘苗病毒表达载体ｐＪＳＡ１１７５的Ｐ７．５启动子下游，构建成在痘苗病毒Ｐ７．５启动子调控下表达猴轮状病毒Ｖｐ４抗原基因的重组质粒ＰＪＳＡ１１７５－ＶＰ４。应用磷酸钙沉淀技术将ＰＪＳＡ１１７５－ＶＰ４ＤＮＡ转入ＴＫ－１４３细胞，在ＢＵＤＲ和Ｘ－ｇａｌ存在下筛选蓝色蚀斑。经３代以上纯化和病毒增殖，获重组病毒Ｒ－ＶＪＳＡ１１７５－Ｖｐ４。蚀斑滴定其满度达到１５×１０１１ＰＦＵ／Ｌ。经核酸杂交试验证明所获得的重组痘苗病毒带有猴轮状病毒Ｖｐ４抗原基因。用重组病毒感染ＴＫ－１４３细胞（或Ｖｅｒｏ细胞），在感染后４８ｈ，用酶免疫法（ＥＩＡ）检测受染细胞上清液和细胞裂解液中表达的猴轮状病毒Ｖｐ４抗原基因均呈阳性反应。本试验为本研究室轮状病毒基因工程疫苗的一部分，为深入了解轮状病毒基因结构及其功能在方法学上奠定了必要的基础。     

检测Epstein—Barr病毒特异性细胞毒性T淋巴细胞方？…

目的 建立一种非放射性、简便易行的可检测特异性细胞毒性Ｔ淋巴细胞的方法，并且初步应用于Ｅｐｓｔｅｉｎ－Ｂａｒｒ病毒的细胞免疫应答。方法 用重组的ＥＢＶ－ＬＭＰ１痘苗病毒、ＴＫ＾＋痘苗病毒和杆状病毒系统表达的ＥＢＶ－ＬＭＰ！蛋白分别免疫Ｂａｌｂ／Ｃ小鼠，用Ｐ８１５细胞和乳酸脱氢酶法检测ＥＢ病毒特异性细胞毒性Ｔ细胞的杀伤效应。结果 重组ＥＢＶ－ＬＭＰ１痘苗病毒免疫组原发ＣＴＬ水平和体外诱生的二次ＣＴＬ     

用细菌／杆状病毒系统在昆虫细胞中表达HIV—2外膜糖蛋白gp105和跨膜糖蛋白g

目的 用细菌／杆状病毒（Ｂａｃ ｔｏ Ｂａｃ）表达系统在昆虫细胞Ｓｆ９中表达ＨＩＶ－２外膜糖蛋白ｇｐ１０５跨膜糖蛋白ｇｐ３６,为研制艾滋病疫苗和诊断试剂奠定基础。方法 分别将ＨＩＶ－２外膜蛋白ｇｐ１０５和跨膜蛋白ｇｐ３６全基因序列克隆到杆状病毒转座载体ｐＦａｓｔ Ｂａｃ ＴＨａ和ｐＦａｓｔ Ｂａｃ ＴＨｂ中我角体启动子下游,构建成重组转座载体ｐＦａｓｔ Ｂａｃ ＨＴａ－ｐｇ１０５和ｐＦａｓｔ Ｂａｃ ＨＴｂ－ｇｐ３６,利用细菌／杆状病毒（Ｂａｃ ｔｏ Ｂａｃ）表达系统筛选重组杆状病毒,并在昆虫细胞Ｓｆ９中表达ＨＩＶ－２的ｇｐ１０５和ｇｐ３６。结果 ＳＤＳ－ＰＡＧＥ分析结果表明,ｐｇ１０５基因表达产物为－６６０００ｕ糖蛋白,ｐｇ３６基因则表达－４１０００ｕ糖蛋白,与天然产物一致。Ｗｅｓｔｅｒｎ ｂｌｏｔ结果显示：     

检测Epstein-Barr病毒特异性细胞毒性T淋巴细胞方法的建立及其初步研究

目的建立一种非放射性、简便易行的可检测特异性细胞毒性Ｔ淋巴细胞的方法，并且初步应用于Ｅｐｓｔｅｉｎ－Ｂａｒ病毒的细胞免疫应答。方法用重组的ＥＢＶ－ＬＭＰ１痘苗病毒、ＴＫ＋痘苗病毒和杆状病毒系统表达的ＥＢＶ－ＬＭＰ１蛋白分别免疫Ｂａｌｂ／Ｃ小鼠，用Ｐ８１５细胞和乳酸脱氢酶法检测ＥＢ病毒特异性细胞毒性Ｔ细胞的杀伤效应。结果重组ＥＢＶ－ＬＭＰＩ痘苗病毒免疫组原发ＣＴＬ水平和体外诱生的二次ＣＴＬ水平均高于ＴＫ＋痘苗病毒免疫组和正常组；杆状病毒系统表达的ＥＢＶ－ＬＭＰ１蛋白免疫组的ＣＴＬ水平也明显高于正常鼠。结论本法可以较好的反映ＥＢ病毒特异性细胞毒性Ｔ细胞的水平，而且再一次说明ＬＭＰ１基因能够诱发特异性的细胞免疫。     

应用痘苗病毒体表达猴轮状病毒VP4抗原基因

把编码猴轮状病毒Ｖｐ４抗原的第４基因片段插入到痘苗病毒表达载体ｐＪＳＡ１１７５的Ｐ７．５启动子下游，构建成在痘苗病毒Ｐ７．５启动子调控下表达猴轮状病毒Ｖｐ４抗原基因的重组质粒ｐＪＳＡ１１７５－Ｖｐ４。应用磷酸钙沉淀技术将ｐＪＳＡ１１７５－Ｖｐ４ＤＮＡ转入ＴＫ－１４３细胞，在ＢＵＤＲ和Ｘ－ｇａｌ 存在下筛选蓝色蚀斑。     

表达单纯疱疹病毒Ⅱ型糖蛋白D的重组痘苗病毒活疫苗株的建立

我们以前曾报道，表达单纯疱疹病毒Ⅱ型糖蛋白Ｄ（ＨＳＶ－２ｇＤ）的重组痘苗病毒（实验疫苗株）能保护被免疫小鼠抵抗致死量ＨＳＶ－２病毒的攻击。在此工作基础上，严格按人用疫苗研究要求的实验条件，成功地建立了表达ＨＳＶ－２ｇＤ的重组痘苗病毒活疫苗株。首先将经聚合酶链反应（ＰＣＲ）修饰的ＨＳＶ－２ｇＤ基因插入痘苗表达质粒ｐＪＳＢ１１７５，置于痘苗病毒Ｐ７５Ｋ早／晚期启动子控制下。将此重组质粒用Ｌｉｐｏｆｅｃｔｉｎ方法转染已受野型ＴＫ＋痘苗病毒天坛７６１株感染的人胚肺二倍体细胞。经同位素探针（３２Ｐ－ＨＳＶ－２ｇＤ）原位杂交法和３轮蚀斑纯化，筛选出基因组内整合有ＨＳＶ－２ｇＤ基因的重组痘苗病毒。斑点和Ｓｏｕｔｈｅｒｎ杂交证实，ＨＳＶ－２ｇＤ基因已插入痘苗病毒基因组内预期的ＴＫ区段，间接免疫荧光检测显示，重组病毒感染细胞后能有效地表达ＨＳＶ－２ｇＤ蛋白。     

表达单纯疱疹病毒Ⅱ型糖蛋白D的重组痘苗病毒活疫苗…

我们以前曾报道，表达单纯疱疹病毒Ⅱ型糖蛋白的重组痘苗病毒能保护被免疫小鼠抵抗致死量ＨＳＶ－２病毒的攻击。在此工作基础上，严格按人用疫苗研究要求的实验条件，成功地建立了表达ＨＳＶ－２ｇＤ的重组痘苗病毒活疫苗株，首先将经聚合酶链反应修饰的ＨＳＶ－２ｇＤ基因插入痘苗表达质粒ｐＪＳＢ１１７５，置于痘苗病毒Ｐ７．５Ｋ早／晚期启动子控制下，将同位重组质粒用Ｌｉｐｏｆｅｃｔｉｎ方法转染已受野型ＴＫ＾＋痘苗病毒天     

利用穿梭质粒快速构建含乙型肝炎病毒表面抗原（HBsAg …

目的 应用新型杆状病毒表达系统快速构建含有ＨＢｓＡｇ基因的重组杆状病毒，高效表达ＨＢｓＡｇ，为ＨＢＶ诊断试剂、疫苗及治疗研究提供依据。方法 构建含有ＨＢｓＡｇ基因的供体质粒ｐＦＢ－ＢＳ，转化Ｂａｃ－ｔｏ－Ｂａｃ杆状病毒表达试剂盒中的ＤＨ１０Ｂａｃ致敏菌，利用其含有的细菌Ｔｎ７转座繁忙将ＨＢｓＡｇ基因重组至穿梭质粒Ｂａｃｍｉｄ上，快速筛选出含有ＨＢｓＡｇ基因的重组杆状病毒。结果 此重组病毒能在昆虫细     

中国狂犬病毒疫苗株（5aG）糖蛋白基因在痘苗病毒中的表达及免疫效果观察

将克隆到的中国狂犬病毒疫苗株（５ａＧ）的糖蛋白基因重组到痘苗病毒ＴＫ区，并在痘苗病毒Ｐ１１启动子的控制下，构建了狂犬－痘苗重组病毒（ＶＶａＧ）。经间接免疫荧光和Ｗｅｓｔｅｒｎ免疫印染证明，重组病毒ＶＶａＧ能良好地表达狂犬病毒糖蛋白，其分子量约为６６００。用ＶＶａＧ免疫小鼠，７ｄ便可诱生较高的狂犬病毒中和抗体，２１ｄ达４１６９，并能１００％保护狂犬病毒本毒株和国际标准攻击毒（ＣＶＳ）的致死量攻击。     

蓝舌病毒L3基因的克隆与表达

目的　通过高效表达,研究蓝舌病毒（ＢＴＶ） ＶＰ３ 的功能,为后续ＢＴＶ病毒样颗粒的装配作准备。方法　克隆出完整的ＢＴＶ１３ Ｌ３ 基因,将其插入杆状病毒表达载体进行表达。结果　获得了含有全长Ｌ３ 基因的克隆,ＶＰ３ 在昆虫细胞中得到了高效表达,表达蛋白占细胞总蛋白的１０％ ～１５％ ,ＶＰ３ 与ＶＰ７ 共表达可装配出ＢＴＶ 核心样颗粒。结论　在昆虫细胞中表达ＢＴＶＶＰ３ 蛋白具有生物学活性,可用于ＢＴＶ病毒样颗粒的装配研究     

Antigenic profile of African horse sickness virus serotype 4 VP5 and identification of a neutralizing epitope shared with bluetongue virus and epizootic hemorrhagic disease virus.

African horse sickness virus (AHSV) causes a fatal disease in horses. The virus capsid is composed of a double protein layer, the outermost of which is formed by two proteins: VP2 and VP5. VP2 is known to determine the serotype of the virus and to contain the neutralizing epitopes. The biological function of VP5, the other component of the capsid, is unknown. In this report, AHSV VP5, expressed in insect cells alone or together with VP2, was able to induce AHSV-specific neutralizing antibodies. Moreover, two VP5-specific monoclonal antibodies (MAbs) that were able to neutralize the virus in a plaque reduction assay were generated. To dissect the antigenic structure of AHSV VP5, the protein was cloned in Escherichia coli using the pET3 system. The immunoreactivity of both MAbs, and horse and rabbit polyclonal antisera, with 17 overlapping fragments from VP5 was analyzed. The most immunodominant region was found in the N-terminal 330 residues of VP5, defining two antigenic regions, I (residues 151-200) and II (residues 83-120). The epitopes were further defined by PEPSCAN analysis with 12mer peptides, which determined eight antigenic sites in the N-terminal half of the molecule. Neutralizing epitopes were defined at positions 85-92 (PDPLSPGE) for MAb 10AE12 and at 179-185 (EEDLRTR) for MAb 10AC6. Epitope 10AE12 is highly conserved between the different orbiviruses. MAb 10AE12 was able to recognize bluetongue virus VP5 and epizootic hemorrhagic disease virus VP5 by several techniques. These data will be especially useful for vaccine development and diagnostic purposes.     

Expression of the outer capsid protein VP5 of two bluetongue viruses, and synthesis of chimeric double-shelled virus-like particles using combinations of recombinant baculoviruses.

We have previously reported the assembly of virus-like particles (VLPs), consisting of the four major structural proteins of bluetongue virus (BTV), in Spodoptera frugiperda cells coinfected with recombinant baculoviruses (French et al. (1990). J. Virol. 64, 5695-5700). In this paper we report further studies using this system to assemble heterologous VLPs containing the outer capsid proteins (VP2 and VP5) of a range of different BTV serotypes. S. frugiperda cells were coinfected with three recombinant baculoviruses; a dual recombinant expressing VP3 and VP7 (of BTV-17 and -10, respectively) in combination with a single recombinant expressing VP2 of BTV-1, -2, -10, -11, 13, or -17 and an additional single recombinant expressing VP5 of BTV-2, BTV-10, or BTV-13. The resultant VLPs were purified and analyzed by electronmicroscopy, Western immunoblotting, and hemagglutination assays to determine whether double-shelled VLPs had been assembled. In the course of these experiments the VP2 proteins of all six available serotypes were successfully incorporated into VLPs. Particles from two different combinations of chimeric VLPs (having VP2 derived from BTV-1 or that of BTV-17) were used to raise antisera in guinea pigs. Both of these sera showed high neutralizing antibody titers against live BTV, indicating that heterologous VLPs may have potential for use in anti-BTV vaccines.     

VP4 protein of porcine rotavirus strain OSU expressed by a baculovirus recombinant induces neutralizing antibodies

The complete VP4 gene of porcine rotavirus strain OSU has been inserted into a baculovirus expression vector under the control of the polyhedrin promoter. The VP4 outer capsid protein, which is a major neutralization antigen in rotavirus, was expressed in high yield in Spodoptera frugiperda cells. Reactivity with polyclonal and monoclonal antibodies suggested that neutralizing epitopes were functionally unaltered on the expressed VP4. The VP4 produced in this system also induced antibodies in guinea pigs which inhibited hemagglutination of OSU and neutralized its infectivity to high titer. The available evidence suggests that the VP4 expressed in insect cells maintained its antigenic configuration and may prove useful in elucidation of (1) the extent of VP4 polymorphism among human and animal rotaviruses and (2) the distribution of VP4 among these viruses.     

Changes in the outer capsid proteins of bluetongue virus serotype ten that abrogate neutralization by monoclonal antibodies

Six neutralizing monoclonal antibodies (Mabs) and nine neutralization resistant viral variants (escape-mutant viruses (EMVs)) were used to further characterize the neutralization determinants of bluetongue virus serotype 10 (BTV10). The EMVs were produced by sequential passage of a highly cell culture adapted United States prototype strain of BTV10 in the presence of individual neutralizing Mabs. Mabs were characterized by neutralization and immune precipitation assays, and phenotypic properties of EMVs were characterized by neutralization assay. Sequencing of the gene segments encoding outer capsid proteins VP2 and VP5 identified mutations responsible for the altered phenotypic properties exhibited by individual EMVs. Amino acid substitutions in VP2 were responsible for neutralization resistance in most EMVs, whereas an amino acid substitution in VP5, without any change in VP2, was responsible for the neutralization resistance of one EMV. The data confirm that VP2 contains the major neutralization determinants of BTV, and that VP5 also can influence neutralization of the virus. The considerable plasticity of the neutralization determinants of BTV has significant implications for future development of non-replicating vaccines.     

Electron microscopic and molecular characterization of turnip vein-clearing virus

Summary Cattle are proposed to be reservoir hosts of bluetongue virus (BTV) because infected animals typically have a prolonged cell-associated viremia. Enriched populations of bovine monocytes, erythrocytes and lymphocytes were inoculated with BTV serotype 10 (BTV 10) and the infected cells then were examined by transmission electron microscopy to characterize the interaction of BTV with bovine blood cells. Replication of BTV 10 in monocytes and stimulated (replicating) lymphocytes was morphologically similar to that which occured in Vero cells, with formation of viral inclusion bodies and virus-specific tubules. In contrast, BTV 10 infection of unstimulated (non-replicating) lymphocytes and erythrocytes did not progress beyond adsorption, after which virus particles persisted in invaginations of the cell membrane. Studies with core particles and neutralizing monoclonal antibodies established that outer capsid protein VP2 is necessary for attachment of BTV 10 to erythrocytes. These in vitro virus-cell interactions provide a cogent explanation for the pathogenesis of BTV infection of cattle, especially the prolonged cell associated viremia that occurs in BTV-infected cattle.     

Isolation of a capsid protein of bluetongue virus that induces a protective immune response in sheep

A method to purify the neutralization specific antigen of bluetongue virus P2 in large amounts has been developed. The purified protein is free from virus-specified or cellular contaminants and its immunological specificity has been preserved. The purification is based on the observation that protein P2 can be dissociated from the virion by treatment with monovalent or divalent salts. The salt concentration required to solubilize the outer capsid proteins is pH dependent and in general decreases with a decrease in pH. P2 purified by extraction from polyacrylamide gels does not induce immune-precipitating or neutralizing antibodies. The response against P5, on the other hand, is much less conformational dependent and P5 purified from gels readily induces P5-precipitating antibodies in rabbits. These antibodies do not neutralize the virus. Purified P2, immunoabsorbed with anticore serum to remove trace amounts of P7, was injected into sheep. An initial dose of 50 micrograms of P2 was sufficient to induce P2-precipitating antibodies as well as neutralizing and hemagglutination-inhibiting antibodies. These sheep were fully protected against challenge with a virulent strain of the same BTV serotype. Lower doses of P2 still provided a significant level of protection even though no neutralizing antibodies could be detected.     

Identification of a bluetongue virus serotype 1-specific ovine helper T-cell determinant in outer capsid protein VP2.

Ovine T-cell lines (including one clone [101A]), which are specific for Bluetongue virus serotype 1 (BTV1), have been established and characterized. Although these T-cell lines react with different isolates of BTV1 (including those from South Africa, Australia, Nigeria, and Cameroon), they do not react with heterologous BTV serotypes. Antigen specificity of these T-cells was studied using purified virus particles, infectious subviral particles (ISVP) and cores, or using individual BTV structural proteins that were either isolated by SDS-PAGE or expressed by recombinant strains of vaccinia virus. The results showed that each of the T-cell lines reacted with outer capsid protein VP2 (the BTV protein exhibiting most serotype-specific variation and the major neutralization antigen). However, all of the uncloned T-cell lines also reacted with either the core structural proteins or the outer capsid protein VP5. In contrast, the T-cell clone 101A only reacted with outer capsid protein VP2. Cell surface marker analysis showed that 101A has a helper T-cell phenotype (CD5+, CD4+, CD8-, T-19-). The T-cell lines and clone 101A all produced large amounts of interleukin 2 (IL-2) when stimulated with purified BTV1 virus particles, or with VP2 (up to 120 IU/ml from 2 x 10(5) T-cells). BTV serotype-specific antigenic sites, for B cells and at least one site for ovine helper T-cells, are therefore located within VP2.     

Complete nucleotide and deduced amino acid sequence of genome segment 5 encoding the outer capsid protein, VP5, of a U.S. isolate of bluetongue virus serotype 11.

The complete nucleotide sequence of the RNA genome segment coding for the outer capsid protein, VP5, of the United States prototypic strain of bluetongue virus (BTV) serotype 11 was determined from two overlapping cDNA clones. The genome segment was found to be 1638 nucleotides in length with a single open reading frame coding for a 526 amino acid protein of MW 59,278 and having a net charge of -4.0 at neutral pH. Comparisons of the predicted amino acid sequence of VP5 of BTV 11 with those of the United States serotypes 2, 10, and 13 and two isolates of BTV 1 from Australia and South Africa confirmed earlier reports that VP5 is a conserved protein with no clear regions of variability. A computer generated consensus sequence suggested VP5 of BTV 2 to be representative of the average VP5 sequences reported thus far.     

The amino acid sequence of the outer coat protein VP2 of neutralizing monoclonal antibody-resistant, virulent and attenuated bluetongue viruses.

Monoclonal antibodies which reacted with four different epitopes were used to select neutralization-resistant variants of Australian bluetongue virus serotype 1 (BTV1AUS; isolate CS156). Nucleotide sequencing of the VP2 outer coat protein gene of these variants showed that two of them contained alterations within the previously defined neutralization site at amino acids 328 to 335 (Gould et al., 1988). Comparison of VP2 sequences of several BTV serotypes, in addition to nucleotide sequence changes in a number of variants, suggested that this neutralization site was larger and contained 19 amino acids, the conformation of which could be affected by other regions of the VP2 protein. Nucleotide sequencing of neutralization-resistant variants revealed a total of four other regions of VP2 affecting the ability of monoclonal antibodies to neutralize the virus and these results support the notion that the neutralization site in VP2 was conformation dependent. The complete nucleotide sequence of the VP2 gene of virulent BTV1AUS (C5156) was determined directly from viral nucleic acid isolated from the blood of a sheep suffering clinical bluetongue disease. Comparison of the VP2 sequence of this virulent virus with that previously published for an avirulent, laboratory strain (Gould, 1988), indicated that the passage of virulent virus approximately 20 times in tissue culture over the last decade, not only led to attenuation but resulted in the appearance of ten nucleotide changes in the VP2 gene. Six of these nucleotide changes were silent, two resulted in conservative amino acid substitutions and two generated radical amino acid changes. However, in a separate experiment, a single passage of the virulent virus in tissue culture while leading to attenuation did not result in a nucleotide change in the VP2 outer coat protein gene.