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。     

人粒细胞巨噬细胞集落刺激因子在重组痘苗病毒中的 …

目的 观察人ＧＭ－ＣＳＦ（粒细胞－巨噬细胞集落刺激因子）对重组痘苗病毒免疫效果的影响。方法 大鼠体内观察。结果 从核酸和蛋白水平均证实重组痘苗病毒ＲＶＪＳＢ１１７５Ｄ／ＧＭ－ＣＳＦ可同时表达ＧＭ－ＣＳＦ及ＨＳＶ－１ｇＤ（单纯疱疹病毒ｇＤ糖蛋白），但ＲＶＪＳＢ１１７５Ｄ／ＧＭ－ＣＳＦ免疫大鼠产生的抗－ＨＳＶ１ｇＤ特异性抗体水平与ＲＶＪＳＢ１１７５Ｄ组相比无显著性差异。结论 人ＧＭ－ＣＳＦ在大鼠体内对     

核酶对鸭乙型肝炎病毒感染体内抗病毒作用效果的观察

目的观察核酶在体内抗病毒作用效果。方法选择鸭乙型肝炎（ＤＨＢＶＬＪ－７６）及其鸭动物模型作为检测系统，将针对ＤＨＢＶＰｒｅ－Ｓ７３６位点的核酶（ＲｚＤＳ）插入ｐＪ１２０质粒构建成ｐＪ－ＲｚＤＳ。与痘苗病毒天坛７６１株（ＶＶ）同源重组后获得含ＲｚＤＳ的重组痘苗病毒（Ｖ－ＲｚＤＳ）。用ＤＨＢＶ感染１日龄北京鸭，分别在感染的同时、感染后２４小时和７２小时注射Ｖ－ＲｚＤＳ和ＶＶ，１０天后检测血清中ＤＨＢＶＤＮＡ和ＤＨＢｓＡｇ的含量。结果北京鸭在感染ＤＨＢＶ的同时注射Ｖ－ＲｚＤＳ，其ＤＨＢＶＤＮＡ和ＤＨＢｓＡｇ的含量均低于ＶＶ对照组，两组之间有显著性差异。结论提示ＲｚＤＳ对ＤＨＢＶ基因组复制和表达均有抑制作用，其抑制率分别为４５％和６３％。     

丙型肝炎病毒结构蛋白在痘苗病毒中的表达

为研究中国丙型肝炎病毒（ＨＣＶ）的抗原性及在细胞内的加工，将丙型肝炎病毒（ＨＣＶ）５’非编码区（ＮＴＲ）和结构基因（Ｃｏｒｅ＋Ｅ１＋Ｅ２／ＮＳ１）插入痘苗病毒表达载体ｐＪＳＡ１１７５中，转染ＴＫ－１４３细胞，经纯化得到丙型肝炎（ＨＣＶ）重组痘苗病毒ｖＪＳＡ１１７５ＣＥ株。Ｓｏｕｔｈｅｒｎｂｌｏｔ杂交表明，ＨＣＶ结构基因存在于痘苗病毒之中。Ｗｅｓｔｅｒｎｂｌｏｔ分析发现，ｖＪＳＡ１１７５ＣＥ表达蛋白带位于９０ｋＤａ，为一多聚蛋白；此蛋白为分泌型，分泌量与细胞裂解物内量大致相同     

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2型单纯疱疹病毒糖蛋白D的重组痘苗病毒对动物的免疫效果

用表达２型单纯疱疹病毒（ＨＳＶ－２）糖蛋白Ｄ的重组痘苗病毒（Ｒ－ｇＤ－Ｖ）免疫ＣＢＡ小鼠，通过间接免疫荧光（ＩＦ）（观察小鼠抗体滴度变化）和四甲基偶氮唑蓝法（ＭＴＴ）动态观察细胞毒性Ｔ细胞活性（ＣＴＬ），均见增高并检测了其产生的抗体对动物的保护作用。同时对Ｒ－ｇＤ－Ｖ和２型单纯疱疹病毒（ＨＳＶ－２）产生的二次ＣＴＬ活性进行了比较，结果表明，Ｒ－ｇＤ－Ｖ产生抗体３周达高峰（１：６４０），７周下降为１：３２０，抗体对病毒攻击动物有保护作用，其中和指数（Ｎ）为６３。Ｒ－ｇＤ－Ｖ在鼠体内产生初次ＣＴＬ８ｄ达高峰，１２ｄ后消失；二次ＣＴＬ持续８周之久，且ＣＴＬ活性明显高于初次。Ｒ－ｇＤ－Ｖ和ＨＳＶ－２相比较产生ＣＴＬ在统计学上有差异（Ｐ＜０．０５）。说明Ｒ－ｇＤ－Ｖ能引起体液免疫和细胞免疫，对动物有保护作用。     

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。     

CD34基因克隆和表达

ＣＤ３４分子是高度糖基化的Ⅰ型跨膜蛋白，主要表达于多功能造血干细胞、祖细胞表面，在成熟血细胞表面无ＣＤ３４抗原表达，提示ＣＤ３４分子在早期造血调控方面起着重要的作用。本文采用ＲＴ－ＰＣＲ方法，从高表达人ＣＤ３４抗原的ＫＧ－１ａ细胞系中成功地克隆出人ＣＤ３４抗原全长ｃＤＮＡ，并将此基因插入克隆“载体ｐＵＣ１８ＨｉｎｃＩＩ酶切位点，构建了重组质粒ｐＵＣ１８－３４。用双酶切ｐＵＣ１８－３４质粒，将分离得到的基因片段插入痘苗病毒载体的ＳｍａⅠ位点，构建了重组质粒ＰＪＳＡ１１７５－３４。采用Ｌｉｐｏｆｅｃｔｉｎ方法，ＰＪＳＡ１１７５－３４转染已被野生痘苗病毒感染的ＴＫ￣－１４３细胞，用ＢｕｄＲ和ＬａｃＺ双筛选，获得带有人ＣＤ３４抗原全长ｃＤＮＡ的重组痘苗病毒。经活细胞荧光染色和ＡＰＡＡＰ检测，表明重组痘苗病毒能特异地表达人ＣＤ３４抗原。人ＣＤ３４抗原ｃＤＮＡ克隆和表达，为进一步研究其结构和功能的关系以及研究造血调控机理奠定了基础。     

表达2型单纯疱疹病毒糖蛋白D的重组痘苗病毒对动物…

用表达２型单纯疱疹病毒糖蛋白Ｄ的重组痘苗病毒免疫ＣＢＡ小鼠，通过间接免疫荧光和甲甲基偶氮唑蓝法动态观察细胞毒性Ｔ细胞活性，均见增高并检测了其产生的抗体和动物的保护作用。同时对Ｒ－ｇＤ－Ｖ和２型单纯疱疹病毒产生的二次ＣＴＬ活性进行了比较，结果表明，Ｒ－ｇＤ－Ｖ产生抗体３周达高峰，７周下降为１：３２０，抗体对病毒击动物有保护作用，其中和指数为６３ 。     

HSV—1 SM44株糖蛋白D基因真核表达载体的构建及其免？…

目的 构建ＨＳＶ－１型ＳＭ４４株糖蛋白Ｄ（ｇＤ）基因的真核表达载体,并用此重组质粒直接免疫小鼠,探讨ＨＳＶ－１ ｇＤ基因作为基因疫苗的可能性。方法 从ＨＳＶ－１基因组中扩增ｇＤ的全编码基因,克隆入载体ｐＵＣ１９中,测序鉴定后转入真核表达载体ｐｃＤＮＡ３．１（＋）。所得重组质粒ｐｃＤ－ＮＡ－ｇＤ以电穿孔法转染ＣＨＯ细胞,并以荧光染色法鉴定表达效果。用ｐｃＤＮＡ－ｇＤ免疫小鼠,ＥＬＩＳＡ法检测基因免疫     

Transactivation by herpes simplex virus proteins ICP4 and ICP0 in vaccinia virus infected cells.

Vaccinia virus recombinants containing the sequences from herpes simplex virus type 1 (HSV-1) encoding the immediate early (IE)(alpha) proteins ICP4 and ICP0, under the control of a mutated vaccinia virus 11K late promoter, were constructed. A cDNA copy of the gene encoding ICPO and an ICP4-encoding genomic segment were each inserted into the vaccinia virus genome at the thymidine kinase (TK) locus by homologous recombination. Steady-state analyses revealed that RNAs homologous to the IE-0 and IE-4 sequences accumulated in cells infected by recombinants with the kinetics of a typical vaccinia late mRNA. Western blot analyses demonstrated that the expression level of both ICPO and ICP4, produced by the recombinant viruses, was comparable to that in HSV-1-infected cells at late times postinfection. Both proteins synthesized in cells infected by the recombinants were located in the nucleus as revealed by immunofluorescence. Although in vitro studies reveal that extracts from vaccinia-virus-infected cells lose the ability to transcribe genes that contain RNA polymerase II promoters (Puckett and Moss (1983), Cell 35, 441-448) both ICPO and ICP4 expressed by the recombinant viruses can transactivate plasmids containing a reporter gene driven by the promoters for the HSV-1 TK and glycoprotein C genes. Nuclear extracts prepared from cells infected with the vaccinia virus vector expressing ICP4 exhibited sequence-specific DNA-binding activity.     

Vaccinia virus DNA ligase is nonessential for virus replication: Recovery of plasmids from virus-infected cells

The essentiality of the vaccinia virus DNA ligase gene, SalF 15R, for virus growth was tested by insertional mutagenesis. A plasmid containing E. coli gpt inserted within a large deletion in the DNA ligase gene was transfected into vaccinia virus-infected cells and recombinant viruses selected by three cycles of plaque purification in the presence of mycophenolic acid (MPA). Surprisingly, in some isolates, which replicated in a manner indistinguishable from wild type (WT) virus, the WT gene was replaced by the gpt allele, demonstrating that the DNA ligase gene is nonessential for growth in cultured cells. In other isolates the entire plasmid was integrated into the virus genome by a single crossover event and a functional copy of the DNA ligase was retained. Southern blot analyses of the latter, drug-resistant viruses indicated extra DNA fragments, of sizes inconsistent with predicted viral structures, which represent the plasmid products of homologous recombination. Hirt extracts from cells infected with such multiply plaque purified virus isolates yielded plasmids that produced ampicillin-resistant colonies after transformation of E. coli. These plasmids were of two structures, representing either the original plasmid used for transfection, or a plasmid containing the WT ligase gene rescued by recombination with the virus genome. Similarly, insertional mutagenesis of the vaccinia virus thymidine kinase (TK) gene with gpt yielded plasmids containing mutant or wild type TK alleles when recombinant viruses were selected in MPA. Such plasmids were not isolated when TK minus viruses were selected in 5-bromodeoxyuridine (BUdR).     

Construction of recombinant swinepox viruses and expression of the classical swine fever virus E2 protein

To explore the swinepox virus (SPV) as a potential live vector for immunization, a vector was developed for the construction of a recombinant SPV carrying foreign genes. In this system, a foreign gene placed under the strong vaccinia virus promoter P(11) can be inserted into the viral thymidine kinase (TK) gene, and the recombinant virus can be isolated in a non-selective medium by the co-expression of E. coli lacZ gene. Compared with the wild type virus, the TK(-)recombinant SPV showed a modest level of attenuation in porcine cells while more attenuation was observed in monkey or human cells. Using this system, a recombinant virus expressing the E2 glycoprotein of classical swine fever virus (CSFV) was produced. Engineered with the gX signal sequence of the pseudorabies virus, and transmembrane domain of E2, the E2 protein was expressed as a dimeric form in the cytoplasm of the infected cells.     

Simultaneous expression of the Lassa virus N and GPC genes from a single recombinant vaccinia virus

A new transfer vector was constructed that directs the insertion of two heterologous genes into the vaccinia virus thymidine kinase (TK) gene during a single recombination event. This vector, pDAVAC2, contains bidirectional vaccinia P7.5 early/late promoter elements and two unique cloning sites. cDNA clones containing the complete coding sequences for the Lassa virus (Josiah strain) nucleoprotein (N) and glycoprotein (GPC) genes were inserted into the vaccinia TK gene using this transfer vector. The recombinant virus, V-LSGN-II, expressed proteins in cell culture that appeared to be authentic with respect to electrophoretic mobility, glycosylation, and post-translational cleavage. Indirect immunofluorescence (IFA) of recombinant virus-infected cells demonstrated both the bright granular and diffuse patterns of staining characteristic of the Lassa nucleoprotein and glycoprotein, respectively. Electron-dense inclusion bodies typical of arenavirus-infected cells were observed by electron microscopy in V-LSN and V-LSGN-II-infected cells, but not in V-LSGPC-infected cells. Mice inoculated with V-LSGN-II by intraperitoneal injection developed serum antibodies that reacted with authentic Lassa proteins in immunofluorescence and radioimmune precipitation assays. This recombinant virus represents an additional candidate for a Lassa fever vaccine and demonstrates the feasibility of expressing any two genes of interest in a single recombinant vaccinia virus through the use of the transfer vector pDAVAC2.     

Construction of a pigeonpox virus recombinant: expression of the Newcastle disease virus (NDV) fusion glycoprotein and protection of chickens against NDV challenge

Summary A pigeonpox transfer plasmid was constructed by cloning a 2.5 kb DNA fragment containing the viral thymidine kinase (TK) gene in the psp65 plasmid. The vaccinia virus P11K promoter followed by the NDV fusion (F) gene was inserted in the TK gene. The F gene was transferred to the viral genome by homologous recombination in pigeonpox virus infected CEF cells, transfected with the recombinant plasmid. Recombinant viruses were selected with BUdR and screened for their ability to induce fusion between adjacent cells. Because of the unexpected growth advantage of the TK+ WT over the TK– recombinants, viral purification was needed to obtain stable recombinants expressing a glycosylated and cleaved F protein. Vaccination of chickens by the follicular method induced high anti-F antibody titers and good protection against challenge with the virulent Italian NDV strain. Half of the oculonasal vaccinated chickens showed anti F antibodies and also half of them were protected. Although protection seems to be correlated with antibody titers, no neutralizing antibodies were found.     

Construction of recombinant fowlpox viruses as vectors for poultry vaccines

Plasmid vectors have been constructed which allow the construction of infectious fowlpox virus (FPV) recombinants expressing foreign genes. The foreign genes were inserted within the thymidine kinase (TK) gene of FPV contained in these vectors. To facilitate the selection of recombinants the Escherichia coli xanthine guanine phosphoribosyl transferase (Ecogpt) gene was developed as a dominant selectable marker. This marker operates in a wide variety of cell types and obviates the need for TK⁻ cell lines for selection of TK⁻ recombinants when foreign genes have been inserted within the TK gene of FPV. The general approach adopted was to construct plasmid vectors in which the FPV TK was interrupted by the Ecogpt gene under the control of a poxvirus promoter in tandem with a gene of interest under the control of another poxvirus promoter. Selection of viruses expressing the Ecogpt gene simultaneously selects for recombinants carrying both the Ecogpt gene and the gene of interest. Using this approach a series of plasmid vectors was constructed in which the FPV TK gene was interrupted by the Ecogpt gene under the control of the P7.5 vaccinia virus promoter in tandem with the A/PR/8/34 haemagglutinin gene under the control of the PL11 vaccinia virus promoter. A recombinant FPV constructed using these plasmids had the expected genome arrangement, expressed influenza haemagglutinin, and induced haemagglutination-inhibiting antibodies when inoculated into chickens. These techniques should allow the construction of a variety of recombinant FPVs expressing poultry vaccine antigens. Such recombinants should be a very cost-effective means of delivering vaccines to poultry.     

DNA immunization with a herpes simplex virus 2 bacterial artificial chromosome

Construction of a herpes simplex virus 2 (HSV-2) bacterial artificial chromosome (BAC) is described. BAC vector sequences were inserted into the thymidine kinase gene of HSV-2 by homologous recombination. DNA from cells infected with the resulting recombinant virus was transformed into E. coli, and colonies containing the HSV-2 BAC (HSV2-BAC) were isolated and analyzed for the expected genotype. HSV2-BAC DNA was infectious when transfected back into mammalian cells and the resulting virus was thymidine kinase negative. When used to immunize mice, the HSV2-BAC DNA elicited a strong HSV-2 specific antibody response that was equal to or greater than live virus immunization. Further, HSV2-BAC immunization was protective when animals were challenged with a lethal dose of virus. The utility of the HSV2-BAC for construction of recombinant virus genomes was demonstrated by elimination of the HSV-2 glycoprotein D (gD) gene. A recombinant HSV-2 BAC with the gD gene deleted was isolated and shown to be incapable of producing infectious virus following transfection unless an HSV gD gene was expressed in a complementing cell line. Immunization of mice with the HSV2 gD-BAC also elicited an HSV-2 specific antibody response and was protective. The results demonstrate the feasibility of DNA immunization with HSV-2 bacterial artificial chromosomes for replicating and nonreplicating candidate HSV-2 vaccines, as well as the utility of BAC technology for construction and maintenance of novel HSV-2 vaccines. The results further suggest that such technology will be a powerful tool for dissecting the immune response to HSV-2.     

Analysis of canine herpesvirus gB, gC and gD expressed by a recombinant vaccinia virus

Summary.  The genes encoding the canine herpesvirus (CHV) glycoprotein B (gB), gC and gD homologues have been reported already. However, products of these genes have not been identified yet. Previously, we have identified three CHV glycoproteins, gp145/112, gp80 and gp47 using a panel of monoclonal antibodies (MAbs). To determine which CHV glycoprotein corresponds to gB, gC or gD, the putative genes of gB, gC, and gD of CHV were inserted into the thymidine kinase gene of vaccinia virus LC16mO strain under the control of the early-late promoter for the vaccinia virus 7.5-kilodalton polypeptide. We demonstrated here that gp145/112, gp80 and gp47 were the translation products of the CHV gB, gC and gD genes, respectively. The antigenic authenticity of recombinant gB, gC and gD were confirmed by a panel of MAbs specific for each glycoprotein produced in CHV-infected cells. Immunization of mice with these recombinants produced high titers of neutralizing antibodies against CHV. These results suggest that recombinant vaccinia viruses expressing CHV gB, gC and gD may be useful to develop a vaccine to control CHV infection. Accepted November 20, 1996 Received October 10, 1996