鸡贫血病毒vp3基因的克隆和分子特征

目的 获得来源于浙江地区鸡组织中的鸡贫血病毒(CA)vp3基因并分析其变异情况.方法 根据GenBank登录的vp3序列设计特异引物,利用PCR方法从鸡组织中获得7个CAV的vp3基因克隆,将其克隆到载体进行测序.结果 40份鸡血清中共检出CAV阳性16份,阳性率40.0％.序列测定结果表明,vp3基因长366 bp,...     

传染性非典型肺炎病毒和疫苗的研究

2002年11月16日中国广东省报道首例传染性非典型肺炎(严重急性呼吸系统综合征，SARS)。2003年7月5日，WHO宣布最后一个SARS疫区中国台湾被解除，标志着SARS得到了初步控制，但是我们应意识到对于SARS，我们还知之甚少，还有许多问题需要我们去解决。为此就SARS病毒和疫苗研究等方面的问题进行了探讨。     

重组Semliki森林病毒疫苗的研制现状

Semliki森林病毒引起的Semliki脑炎是一种自然疫原性疾病。该病毒可诱导感染的宿主产生细胞、体液和黏膜免疫应答,可通过分子生物学技术改造为理想的疫苗载体,从而表达病毒和细菌的多种蛋白。这些病原体包括流行性感冒病毒、马流感病毒、Ⅰ型人类免疫缺陷病毒、猴免疫缺陷病毒、猪瘟病毒、罗氏病病毒、乙型肝炎病毒、猪园环病毒Ⅱ型、布鲁氏菌、肉毒梭菌等,本文对Semliki森林病毒介导的病原体疫苗的研制现状加以阐述。     

丙型病毒性肝炎预防性疫苗的研究近况

丙型肝炎症毒(HCV)是肠道外非甲非乙肝炎的主要致病因子，呈世界范围性，其致病常引起持续感染，并且HCV的慢性感染和慢性肝脏疾病，肝硬化及肝癌明显相关。因此，运用疫苗保护免受HCV感染很有必要。     

慢病毒介导β-珠蛋白基因添加在β-地中海贫血基因治疗中的应用

目的:构建全长人β-珠蛋白基因慢病毒载体,稳定转染K562细胞使得β-珠蛋白高效表达,为β-珠蛋白基因添加治疗奠定基础。方法:采用PCR扩增人正常β-珠蛋白基因,加上Sph I和Not I两个酶切位点进行T载体克隆,获得重组质粒p UC57-β-globin。用限制性内切酶连接目的基因片段和慢病毒载体,筛选获得慢病毒表达载体LV-β-globin,测序。将慢病毒载体转染293T细胞,包装病毒。用LV-β-globin感染K562细胞。采用流式细胞仪检测荧光效率,运用实时PCR及Western Blotting检测K562细胞中β-珠蛋白mRNA及蛋白表达情况。结果:通过PCR获得长度为2 128 bp的目的基因。连接p UC57载体和慢病毒表达载体,慢病毒表达载体LV-β-globin构建成功,序列正确。LV-β-globin转染K562细胞72 h后,显示大量绿色荧光表达,荧光效率为94.8%。β-珠蛋白mRNA 2-ΔΔCt值为4.080±0.078,高于对照组;β-珠蛋白灰度值为1.063±0.082,高于对照组,差异均有统计学意义(P<0.05)。结论:成功构建并包装含人β-珠蛋白基因的慢病毒载体并高效稳定表达。     

乳球菌介导重组消化道病毒疫苗的研制现状

消化道病毒是消化性疾病的一类病原体,研制疫苗防治该类病毒感染是当前研究的热点之一.乳球菌是一种新型疫苗载体,此文综述了乳球菌介导的轮状病毒、HBV、HCV和肠道病毒71型等消化道病毒疫苗的构建及其作用机制等方面的研制现状.     

子宫颈癌细胞株表达kiss-1基因重组慢病毒的构建

目的 探讨体内表达kiss-1基因的方法 治疗子宫颈癌的可行性,构建可表达kiss-1基因的重组慢病毒.方法 采用RT-PCR方法 ,根据制备两端含有酶切位点的kiss-1基因的cDNA片段,通过常规分子生物学手段在确保信号肽酶的识别点的条件下,构建可以表达kiss-1基因的慢病毒包装专用质粒pLenti6/V5D-TOPO,与其他辅助质粒共同转染293细胞,获得能表达kiss-1的重组慢病毒.结果 测序证实克隆kiss-1的cDNA与Genebank提供的序列完全一致;经限制性内切酶物理图谱方法 证实已经成功地将kiss-1和cDNA重组到含NT4的信号肽和前导序列下游.经RT-PCR方法 证明重组慢病毒在人子宫颈癌Hela细胞中可以表达kiss-1基因.结论成功构建了能够表达kiss-1的重组慢病毒.     

改进方案构建人糖皮质激素受体α基因正、反义重组逆病毒载体

目的构建人糖皮质激素α(hGRα)cDNA的表达重组体，为较大DNA片段的克隆提供借鉴，并为利用反义核酸技术探讨hGRα功能及其作用机制提供实验材料。方法L—PCR扩增hGRα cDNA，克隆至T载体；随后通过改进的连接方案进行亚克隆。结果限制性酶增和序列分析表明克隆及亚克隆成功。结论成功构建于hGRα cDNA的逆转录病毒载体pLXSN正义及反义重组子。     

脊髓灰质炎疫苗重组株病毒在我国的循环及其致病性

我国自1994年10月以后已无脊髓灰质炎(脊灰)本土野病毒引起的病例.现在从急性弛缓性麻痹病例粪便标本中分离到的脊灰病毒都是3个血清型的疫苗衍生株,并以Ⅱ型为主.近几年每年Ⅱ型分离株的数量均多于同年I型株加Ⅲ型株之和.1997～1999年从贵州、云南省分离到的Ⅱ型疫苗株中,除了疫苗变异株外,还发现了不同血清型间的重组株,如VP1片段来自SabinⅡ,而3D片段分别来自SabinⅢ(S2×S3)和SabinI(S2×S1),它们的基因序列与脊灰疫苗株SabinⅡ型有差异.能从未服苗儿童的粪便标本中分离到这类毒株,表明在外环境中有这类毒株在循环.这些毒株可以致病,其致病性与该儿童是否接受了疫苗全程免疫有很大的相关性.重组株的发现提示我们,它们在自然环境中已有循环.从贵州省Ⅱ型病例发生时间和区域上看,病例有聚集分布的趋势,但目前尚处于小范围的循环中,其原因与对策有待进一步研究.     

转基因转状病毒疫苗植物表达载体的构建及GUS基因表达

目的：选用植物细胞表达轮状病毒结构蛋白，拟构建有效植物细胞表达系统。方法：利用PT－PCR扩增A组轮状病毒外壳蛋白VP4基因片段，经双酶切后与植物表达载体连接，转化感受态细菌TG1。利用地高辛标记探针进行斑点杂交检测，筛选出阳性克隆，拟用该克隆转化马铃薯细胞，研制新型轮状病毒疫苗。本研究还利用β－葡萄糖苷酸酶（GUS）基因转化马铃薯细胞。结果：A组轮状病毒SA11株VP4基因产物大小与设计相同，为960bp。在被检测的未知载体中，有四个显紫色斑，可判定其为带有VP4cDNA的转化载体。用肉眼或显微镜可到马铃薯组织细胞中的蓝色物质。结论；在转基因植物组织器官中观察到GUS的活性，获得外源基因转化的条件。拟用本研究的表达载体转化马铃薯细胞，研制新型轮状病毒疫苗。     

Construction and immunogenicity of a recombinant fowlpox vaccine coexpressing S1 glycoprotein of infectious bronchitis virus and chicken IL-18

Infectious bronchitis virus (IBV) poses a major threat to the chicken industry worldwide. In this study, we developed a recombinant fowlpox virus (rFPV) vaccine expressing the IBV S1 gene and chicken interleukin-18 gene (IL-18), rFPV-S1/IL18. Recombinant plasmid pSY-S1/IL18 was constructed by cloning chicken IL-18 into fowlpox virus transfer plasmid containing S1 gene and transfected into the chicken embryo fibroblasts cell pre-infected with S-FPV-017 to generate rFPV-S1/IL18. Expression of the recombinant proteins was confirmed by RT-PCR and IFA. We also constructed the recombinant fowlpox virus rFPV-S1 without IL-18. One-day-old chickens were vaccinated by wing-web puncture with the two rFPVs, and the induced humoral and cellular responses were evaluated. There was a significant difference in ELISA antibody levels (P<0.05) elicited by either rFPV-S1 or rFPV-S1/IL18. The ratios of CD4(+) to CD8(+) in chickens immunized with rFPV-S1/IL18 were significantly higher (P<0.05) than in those immunized with rFPV-S1. All chickens immunized with rFPV-S1/IL18 were completely protected (20/20) after challenge with the virulent IBV HN99 strain 43 days after immunization, while only 15 out of 20 of the chickens immunized with the rFPV-S1 were protected. Our results show that the protective efficacy of the rFPV-S1 vaccine could be enhanced significantly by simultaneous expression of IL-18.     

构建SARS病毒E2蛋白保护性抗原原核表达载体

目的 构建SARS病毒E2蛋白保护性抗原片段的原核表达载体，为SARS的诊断和预防奠定基础。方法 采用人工合成法合成SARS病毒E2蛋白3’端cDNA；采用常规分子克隆方法将此cDNA片段克隆入原核表达载体pBV220中，经温度诱导和SDS-PAGE电泳证明外源蛋白表达。结果 合成的E2蛋白cDNA经序列测定表明，与报道的SARS病毒相应序列一致，内切酶酶切及PCR均得到456bp的cDNA片段，与实验设计一致，证明该cDNA片段插入了pBV220载体中；SDS-PAGE电泳表明有外源蛋白表达。结论 成功合成并克隆出SARS病毒E2蛋白原核表达载体。     

Novel DNA-launched Venezuelan equine encephalitis virus vaccine with rearranged genome

Novel live-attenuated V4020 vaccine was prepared for Venezuelan equine encephalitis virus (VEEV), an alphavirus from the Togaviridae family. The genome of V4020 virus was rearranged, with the capsid gene expressed using a duplicate subgenomic promoter downstream from the glycoprotein genes. V4020 also included both attenuating mutations from the TC83 VEEV vaccine secured by mutagenesis to prevent reversion mutations. The full-length infectious RNA of V4020 vaccine virus was expressed from pMG4020 plasmid downstream from the CMV promoter and launched replication of live-attenuated V4020 in vitro or in vivo. BALB/c mice vaccinated with a single dose of V4020 virus or with pMG4020 plasmid had no adverse reactions to vaccinations and developed high titers of neutralizing antibodies. After challenge with the wild type VEEV, vaccinated mice survived with no morbidity, while all unvaccinated controls succumbed to lethal infection. Intracranial injections in mice showed attenuated replication of V4020 vaccine virus as compared to the TC83. We conclude that V4020 vaccine has safety advantage over TC83, while provides equivalent protection in a mouse VEEV challenge model.     

Effect of infectious bursal disease (IBD) vaccine on Salmonella Enteritidis infected chickens

BackgroundChickens infected with both infectious bursal disease virus (IBDV) and Salmonella had higher mortality. In this work, we investigated the effect of IBDV vaccine (modified live-virus bursal disease vaccine, Nobilis strain 228E®) on experimentally infected chickens with Salmonella Enteritidis (SE).MethodsFour experimental groups were included in this study, negative control group, 228E®group, 228E® + SE infected group, and SE infected group. Chickens were ocularly administrated 228E® at 12 days of age and orally infected with S. Enteritidis at 13 days of age. Sera, intestinal fluid, blood, cloacal swabs and tissue samples were collected at 1, 2 and 3 weeks post vaccination (PV).ResultsThe recorded mortalities were higher in the 228E® + SE infected group, compared to the SE infected group. The anti-S. Enteritidis serum antibody titer and the intestinal mucosal IgA level were higher in the SE infected group at 2 and 3 weeks PV, compared to 228E® + SE infected group. S. Enteritidis fecal shedding and organ colonization were significantly higher in the 228E® + SE infected group than the SE infected group at 2 and 3 weeks PV. The 228E® + SE group had significantly lower bursa to body weight ratios at 2 and 3 weeks PV, as well as had higher bursal lesion scores than the SE infected group. IBDV vaccine depressed the specific-SE systemic and mucosal antibody responses, but did not affect the specific-SE cellular immune responses.ConclusionChickens administrated IBDV vaccine, followed by S. Enteritidis infection, could cause a significant effect on the bursa of Fabricius, resulting in failure of systemic and mucosal antibody responses to the S. Enteritidis and reduce the elimination and the clearance of S. Enteritidis.     

Viral load and clinical disease enhancement associated with a lentivirus cytotoxic T lymphocyte vaccine regimen

Effective DNA-based vaccines against lentiviruses will likely induce CTL against conserved viral proteins. Equine infectious anemia virus (EIAV) infects horses worldwide, and serves as a useful model for lentiviral immune control. Although attenuated live EIAV vaccines have induced protective immune responses, DNA-based vaccines have not. In particular, DNA-based vaccines have had limited success in inducing CTL responses against intracellular pathogens in the horse. We hypothesized that priming with a codon-optimized plasmid encoding EIAV Gag p15/p26 with co-administration of a plasmid encoding an equine IL-2/IgG fusion protein as a molecular adjuvant, followed by boosting with a vaccinia vector expressing Gag p15/p26, would induce protective Gag-specific CTL responses. Although the regimen induced Gag-specific CTL in four of seven vaccinated horses, CTL were not detected until after the vaccinia boost, and protective effects were not observed in EIAV challenged vaccinates. Unexpectedly, vaccinates had significantly higher viral loads and more severe clinical disease, associated with the presence of vaccine-induced CTL. It was concluded that (1) further optimization of the timing and route of DNA immunization was needed for efficient CTL priming in vivo, (2) co-administration of the IL-2/IgG plasmid did not enhance CTL priming by the Gag p15/p26 plasmid, (3) vaccinia vectors are useful for lentivirus-specific CTL induction in the horse, (4) Gag-specific CTL alone are either insufficient or a more robust Gag-specific CTL response is needed to limit EIAV viremia and clinical disease, and (5) CTL-inducing vaccines lacking envelope immunogens can result in lentiviral disease enhancement. Although the mechanisms for enhancement associated with this vaccine regimen remain to be elucidated, these results have important implications for development of lentivirus T cell vaccines.     

Evaluation of the immunogenicity of a recombinant glycoprotein-based Chandipura vaccine in combination with commercially available DPT vaccine

Chandipura virus (CHPV) belongs to family Rhabdovoridae and has emerged as an encephalitis causing pathogen with high mortality among pediatric population from three Indian states. The recombinant glycoprotein (rGp) was shown to be an excellent vaccine candidate as evaluated in a murine model. As the disease is predominantly rural, to ensure maximum coverage for Chandipura vaccine, an attempt was made to evaluate combination of rGp and a commercially available DPT vaccine (CHP–DPT). When CHP–DPT was used for immunization of mice, 90% seroconversion against rGp with high antibody titers (1:1200 by ELISA and 1:320 by neutralization test) was observed and did not differ from mice immunized with rGp alone (P > 0.05). Similarly seroconversions and antibody titers against DPT were comparable in mice immunized with DPT alone or in combination with rGp. Seroconversions and antibody titers ranged from 90 to 100% and 1:1200 to 1:2400 respectively. Intracerebral challenge with homologous CHPV strain resulted in 90% survival in rGp alone and CHP–DPT groups. Lymphocyte proliferative responses were also comparable. Thus, neither components of the candidate combination vaccine inhibited immune response to the other component. Substantial decrease of CHPV RNA and absence of histopathological changes in the brains of surviving immunized mice after challenge than the unimmunized controls further confirm efficacy of the vaccine even after intracerebral challenge. In conclusion, a combination vaccine seems feasible for use in a restricted area where the disease is endemic and should be subjected to additional studies required for future use in humans.     

Venezuelan equine encephalitis vaccine with rearranged genome resists reversion and protects non-human primates from viremia after aerosol challenge

Live-attenuated V4020 vaccine for Venezuelan equine encephalitis virus (VEEV) containing attenuating rearrangement of the virus structural genes was evaluated in a non-human primate model for immunogenicity and protective efficacy against aerosol challenge with wild-type VEEV. The genomic RNA of V4020 vaccine virus was encoded in the pMG4020 plasmid under control of the CMV promoter and contained the capsid gene downstream from the glycoprotein genes. It also included attenuating mutations from the VEE TC83 vaccine, with E2-120Arg substitution genetically engineered to prevent reversion mutations. The population of V4020 vaccine virus derived from pMG4020-transfected Vero cells was characterized by next generation sequencing (NGS) and indicated no detectable genetic reversions. Cynomolgus macaques were vaccinated with V4020 vaccine virus. After one or two vaccinations including by intramuscular route, high levels of virus-neutralizing antibodies were confirmed with no viremia or apparent adverse reactions to vaccinations. The protective effect of vaccination was evaluated using an aerosol challenge with VEEV. After challenge, macaques had no detectable viremia, demonstrating a protective effect of vaccination with live V4020 VEEV vaccine.     

Development and efficacy of a novel live-attenuated QX-like nephropathogenic infectious bronchitis virus vaccine in China

In this study, we attenuated a Chinese QX-like nephropathogenic infectious bronchitis virus (IBV) strain, YX10, by passaging through fertilized chicken eggs. The 90th passage strain (YX10p90) was selected as the live-attenuated vaccine candidate strain. YX10p90 was found to be safe in 7-day-old specific pathogen free chickens without induction of morbidity or mortality. YX10p90 provided nearly complete protection against QX-like (CH I genotype) strains and partial protection against other two major Chinese genotype strains. YX10p90 also showed no reversion to virulence after five back passages in chickens. An IBV polyvalent vaccine containing YX10p90 was developed and showed that it could provide better protection against major Chinese IBV virulent strains than commercial polyvalent vaccines. In addition, the complete genome sequence of YX10p90 was sequenced. Multiple-sequence alignments identified 38 nucleotide substitutions in the whole genome which resulted in 26 amino acid substitutions and a 110-bp deletion in the 3′ untranslated region. In conclusion, the attenuated YX10p90 strain exhibited a fine balance between attenuation and immunogenicity, and should be considered as a candidate vaccine to prevent infection of Chinese QX-like nephropathogenic IBV.     

Biological and molecular characterization of ArkGA: A novel Arkansas serotype vaccine that is highly attenuated,efficacious, and protective against homologous challenge

Almost all commercial poultry are vaccinated against avian coronavirus infectious bronchitis virus (IBV) using live attenuated vaccines mass administered by spray at day of hatch. Although many different types of IBV vaccines are used successfully, the ArkDPI serotype vaccine, when applied by spray, does not infect and replicate sufficiently to provide protection against homologous challenge. In this study, we examined a different Ark vaccine strain (Ark99), which is no longer used commercially due to its reactivity in one day old chicks, to determine if it could be further attenuated by passage in embryonated eggs but still provide adequate protection. Further attenuation of the Ark99 vaccine was achieved by passage in embryonated eggs but ArkGA P1, P20, and P40 (designated ArkGA after P1) were still too reactive to be suitable vaccine candidates. However, ArkGA P60 when given by spray had little or no vaccine reaction in one day old broiler chicks, and it induced protection from clinical signs and ciliostasis following homologous challenge. In addition, vaccinated and challenged birds had significantly less challenge virus, an important measure of protection, compared to non-vaccinated and challenged controls. The full-length genomes of viruses from egg passages 1, 20, 40, and 60 were sequenced using the Illumina platform and the data showed single nucleotide polymorphisms (SNPs) had accumulated in regions of the genome associated with viral replication, pathogenicity, and cell tropism. ArkGA P60 accumulated the most SNPs in key genes associated with pathogenicity (polyprotein gene 1ab) and cell tropism (spike gene), compared to previous passages, which likely resulted in its more attenuated phenotype. These results indicate that the ArkGA P60 vaccine is safe for spray vaccination of broiler chicks and induces suitable protection against challenge with pathogenic Ark-type virus.