猪圆环病毒2型感染对伪狂犬疫苗免疫应答的影响

为明确PCV2感染对伪狂犬(PR)疫苗免疫应答的影响,本研究采用阻断ELISA方法对单独接种猪PR疫苗组(A组)及PCV2人工感染3周后接种猪PR疫苗组(PA组)不同时相血清中的猪PR病毒gB抗体进行检测;同时对不同时相前腔静脉血进行CD4+/CD8+流式细胞术及血常规分析。结果表明,在PCV2感染后2周至5周间,A组白细胞含量均高于PA组,随后PA组白细胞恢复至与A组略高的正常水平;在整个实验中,除接种猪PR疫苗后1周(WPI)和9周(WPI)外的所有时相PA组的淋巴细胞含量均略高于A组;PCV2感染后可使记忆/激活Th细胞数量略有升高,幼稚型Th细胞含量的下降;PCV2感染后2周～7周PA组Tc细胞均高于A组,在9WPIPA组Tc细胞数量显著下降(p0.05);除9WPI外,A组的S/N值均低于PA组。结果表明,PCV2感染看降低机体产生针对PRVgB特异性抗体水平,而且在一定程度上降低了幼稚型Th细胞及Tc细胞含量。     

重组猪细小病毒VP2基因的猪伪狂犬病毒rPRV-VP2株的构建及纯化

参考GenBank中登录的猪细小病毒(PPV)(M38367.1)基因序列,设计1对可以扩增出约1.6kb PPV VP2基因片段的特异性引物,上、下游引物的5′端均引入BamHⅠ酶切位点,PCR扩增得到VP2目的片段后,连接到pMD18-T载体中得到重组质粒pMD-VP2。取本实验室构建的含绿色荧光蛋白标记基因的伪狂犬病毒(PRV)通用转移质粒pG和构建好的pMD-VP2质粒,用BamHⅠ对质粒pG和pMD-VP2进行酶切,然后用CIAP对酶切产物进行去磷酸化处理,纯化回收后将VP2基因插入pG质粒中获得重组伪狂犬病毒转移质粒pGVP2。用脂质体转染法将PRV HB-98株全基因组与质粒pGVP2共转染ST细胞,得到携带有PPV VP2外源抗原基因和绿色荧光蛋白标记基因的重组伪狂犬病毒rPRV-VP2株。结合VP2基因PCR扩增和绿色荧光观察,经5轮病毒空斑纯化得到了纯化的重组病毒rPRV-VP2株。     

Effects of a porcine reproductive and respiratory syndrome virus infection on the development of the immune response against pseudorabies virus

The aim of this study was to investigate the effects of a porcine reproductive and respiratory syndrome virus (PRRSV) infection on the development of the immune response after pseudorabies virus (PRV) vaccination in pigs. Pigs were intranasally inoculated with the European PRRSV strain, Lelystad virus ter Huurne, and were vaccinated intramuscularly with PRV 2 weeks later (LV-PRV group). Control pigs were vaccinated with PRV only (PRV group). Eight weeks after PRV vaccination, pigs from both groups were challenged intranasally with wild-type PRV. We measured the lymphoproliferative, and the cytolytic responses to PRV of peripheral blood mononuclear cells (PBMC), isolated from blood samples. In addition, serum samples were examined for antibodies against PRV and LV. One week after PRV vaccination, PBMC proliferated abundantly to PRV in both groups. However, in the LV-PRV group the lymphoproliferative response declined after 1 week, whereas, in the PRV group, the lymphoproliferative response was high for 3 weeks and declined thereafter (P<0.05). After challenge, the lymphoproliferative response was 1 week earlier and was consistently and significantly higher in the PRV group than in the LV-PRV group. The PRV-specific killing was higher at 3 weeks after PRV vaccination and 5 weeks after PRV challenge 19+/-3 and 24+/-6%, respectively, in the PRV group, compared to 7+/-4 and 6+/-9%, respectively, in the LV-PRV group (P<0.05). However, later after vaccination and challenge the cytolytic response was identical in both groups. The antibody titre against PRV developed equally in both groups. After challenge, no PRV virus was isolated from both groups. From these results we conclude that, although PRRSV infection did cause changes in the time course of the T-lymphocyte response after PRV vaccination, PRRSV infection did not inhibit the development of vaccine-induced protection after PRV.     

表达猪细小病毒VP2蛋白和猪IL-18重组伪狂犬病毒的构建

为了获得表达猪细小病毒(PPV)VP2蛋白和细胞因子猪白细胞介素-18(IL-18)的重组猪伪狂犬病毒(PRV)。将PPV VP2基因和猪IL-18基因分别插入到PRV转移质粒PG中,得到重组质粒PG18-VP2。利用脂质体转染法将重组转移质粒PG18-VP2与猪PRV弱毒株DNA共转染猪睾丸(ST)细胞,以EGFP荧光标记,通过5轮蚀斑筛选纯化,成功获得表达PPV VP2蛋白和猪IL-18且带EGFP标记的重组伪狂犬病毒IL18-VP2-rPRV。用重组病毒感染ST细胞,12h后在荧光显微镜下可见明亮的绿色荧光;通过RT-PCR证实感染细胞中含有PPV VP2和猪IL-18mRNA;Western-blot试验结果显示,重组病毒能表达具有生物活性的PPV VP2蛋白和猪IL-18。重组病毒经连续20次传代后感染细胞仍能发出绿色荧光,PCR检测表明VP2及IL-18基因在重组病毒中能稳定遗传。为进一步研究表达PPV VP2蛋白和猪IL-18的重组猪伪狂犬病毒的免疫效力奠定了基础。     

表达猪细小病毒VP2基因的重组伪狂犬病病毒构建及其免疫原性

猪细小病毒(porcine parvovirus,PPV)和伪狂犬病病毒(pseudorabies virus,PRV)均可引起母猪的繁殖障碍疾病。本试验以PRV基因工程弱毒株rPRVSMX为载体,构建了表达PPV流行毒株VP2基因的重组PRV(rPRVSMX-VP2)。Western blot和IFA试验均证明了重组病毒在细胞中成功表达了VP2蛋白。动物试验结果证明重组病毒可以诱导猪体产生特异性的PPV抗体,首免后40 d血凝抑制抗体(HI)为1∶192±73.9,虽然低于PPV商品化灭活疫苗免疫后所产生的HI抗体,但此时免疫系统被认为是激活的水平;重组病毒诱导产生的PRV抗体与载体毒株rPRVSMX诱导产生的抗体相当。由此可见该重组毒株经过优化后,可以作为二联疫苗的候选毒株防控猪伪狂犬病和细小病毒病。     

猪伪狂犬病病毒高免血清的制备及应用

用伪狂犬病病毒及抗体阴性羊,以不同免疫剂量,间隔一定时间,多次免疫不同类型的猪伪狂犬病病毒(Bartha-K61株),制备猪伪狂犬病病毒高免血清,特异性强,效价可达1:2 048以上。本研究为伪狂犬病活疫苗或毒种的鉴定及外源病毒检验提供了具有良好特异性和高效价的中和用血清。     

Electroporation improves the immune response induced by a DNA vaccine against pseudorabies virus glycoprotein B in pigs

This study was performed to determine whether electroporation can be used to enhance the efficacy of a DNA vaccine against pseudorabies virus (PrV) in pigs. Immune responses to PrV were measured in pigs following a single intramuscular injection of plasmids encoding PrV glycoprotein B, with or without electroporation. Plasmid injection coupled with electroporation increased production of specific antibodies against PrV and peripheral blood mononuclear cells proliferated in response to stimulation with PrV glycoproteins. These results show that electroporation can improve the performance of a DNA vaccine against PrV in pigs. However, additional work is required to maximise the effectiveness of the vaccination protocol.     

Generation and immunogenicity of a recombinant pseudorabies virus expressing cap protein of porcine circovirus type 2

Porcine circovirus type 2 (PCV2) is associated with post-weaning multisystemic wasting syndrome (PMWS), which is an important economical disease affecting the pig industry worldwide. In order to develop an effective vaccine for PMWS, a recombinant pseudorabies virus (PRV) was generated and tested in piglets in this study. The PCV2 open reading frame 2 (ORF2) gene was inserted into pIECMV plasmid and co-transfected with PRV Tk-/gE-/LacZ+ genome into IBRS-2 cells to generate a recombinant Tk-/gE-/ORF2(+) virus. The expression of PCV2 ORF2 gene in the recombinant virus was confirmed by Western blotting and indirect immunofluorescence assay (IFA). Four-week-old piglets were immunized by the recombinant virus, and the immunogenicity of PRV Tk-/gE-/ORF2(+) was tested by PRV-enzyme-linked immunosorbent assay (ELISA), PRV neutralizing assay, ORF2-ELISA and ORF2 specific lymphocyte proliferation response. PRV Tk-/gE-/ORF2(+) elicited significant humoral immune responses to both PRV and PCV2, and the PCV2-specific lymphocyte proliferation response could be detected on day 49 of this experiment. These findings suggest that the recombinant PRV Tk-/gE-/ORF2(+) may be a potential vaccine against both PCV2 and PRV.     

一例猪伪狂犬病病毒的诊断

2015年11月,当地某养殖户15日龄仔猪出现角弓反张、精神沉郁等疑似猪伪狂犬病病毒感染的典型神经症状,但查看免疫程序,已免疫猪伪狂犬病疫苗（Bartha-k61株）。经gE基因血清学调查和针对gE基因的PCR诊断,证实该病例为猪伪狂犬病病毒感染所致。     

共表达猪细小病毒VP2蛋白和猪圆环病毒2型Cap蛋白的重组伪狂犬病毒的构建及其鉴定

为研制猪伪狂犬病毒(PRV)、猪细小病毒(PPV)和猪圆环病毒2型(PCV2)3联苗,本研究将PPV VP2基因和PCV2 Cap基因通过口蹄疫病毒2A基因串联得到VP2-2A-Cap(V2C)基因,将其插入pEP-CMV-in转移载体构建pEP-V2C-in重组表达质粒,再通过Red/ET两步重组法在大肠杆菌中构建了pPRV-V2C-ΔgE和pPRV-ΔgE突变体,该突变体用磷酸钙法转染BHK-21细胞获得重组病毒rPRV-V2C-ΔgE和rPRV-ΔgE。Western blot分析表明rPRV-V2C-ΔgE能够在BHK-21细胞内表达融合蛋白VP2-2A-Cap,而且目的蛋白能够被2A蛋白裂解为64 ku和28 ku两个片段。重组病毒生长曲线和噬斑大小测定结果显示rPRV-V2C-ΔgE在BHK-21细胞中的增殖滴度与亲本株rPRV无显著差异,表明外源基因的插入不影响rPRV的增殖。本研究为PRV、PPV和PCV2 3联重组活载体疫苗的研制奠定了基础。     

伐昔洛韦对猪伪狂犬病病毒复制的抑制作用

本研究确定伐昔洛韦的体外和体内抗猪伪狂犬病病毒作用,确定伐昔洛韦对Vero细胞的最大无毒质量浓度为4g/L,抑制PRV野毒在Vero细胞中复制的最低有效质量浓度为3g/L。用1个LD50的PRV对小鼠进行攻毒,在攻毒后48h给服不同剂量的伐昔洛韦,确定了伐昔洛韦能够为小鼠提供完全保护的最低剂量为3 mg/只。PRV攻毒后,未给服伐昔洛韦组小鼠表现典型的神经症状并全部死亡,而给药组小鼠临床健康并全部存活。与给药组小鼠相比,未给药组小鼠脑、肺组织中的PRV病毒载量明显高于给药组小鼠,且随时间延长一直增加。脑、肺组织呈现PRV感染的特征性病理变化。伐昔洛韦给药组小鼠相应组织中的病毒载量和脾脏T淋巴细胞分泌PRV特异性Th1型细胞因子水平在攻毒后72h逐渐降低。试验结果显示,伐昔洛韦无论在体内、体外均能够有效抑制PRV的复制。     

Immunogenicity of a recombinant pseudorabies virus expressing ORF1-ORF2 fusion protein of porcine circovirus type 2

Porcine circovirus type 2 (PCV2) is associated with post-weaning multisystemic wasting syndrome (PMWS). Pseudorabies (PR) is also an important infectious disease in swine and sometimes co-infect with PCV2. An attenuated pseudorabies virus (PRV) has been successfully used as a vector for live viral vaccines. In this study, a recombinant PRV expressing ORF1-ORF2 fusion protein of PCV2 was constructed and its immunogenicity was tested in mice and pigs. The ORF1 and partial ORF2 gene of PCV2 Yu-A strain were amplified by PCR and inserted into a transfer vector. The recombinant transfer plasmid was co-transfected with the EcoRI digested genome of vector virus (PRV TK-/gE-/LacZ+) into IBRS-2 cells. The recombinant pseudorabies virus PRV-PCV2 was purified by plaque purification and identified by PCR and Southern blotting. Expression of the ORF1-ORF2 fusion protein by the recombinant PRV-PCV2 virus was demonstrated by Western blotting analysis. The growth properties of the recombinant virus in cells were similar to that of the parent vector virus. In animal experiments, PRV-PCV2 elicited strong anti-PRV and anti-PCV2 antibodies in Balb/c mice as indicated by PRV-neutralizing assay, anti-PCV2 ELISA and PCV2 specific lymphocyte proliferation assay, respectively. And PRV-PCV2 immunization protected mice against a lethal challenge of a virulent PRV Ea strain. In pigs, PRV-PCV2 elicited significant immune response towards PRV and PCV2 as indicated by PRV-ELISA, PRV neutralizing assay and PCV2 specific lymphocyte proliferation assay, respectively. This is a first step toward the development of a potential candidate divalent vaccine against PRV and PCV2 infections.     

表达PCV2 CAP蛋白和猪IL-18重组PRV PGO18株的生物学特性

为了探讨表达猪圆环病毒2型(PCV2)CAP蛋白和猪IL-18的重组猪伪狂犬病病毒PGO18作为疫苗候选株的潜在价值,对其在ST细胞上的培养特性,ST、VERO、IBRS-2、PK-15和IPEC细胞上增殖情况,生长特性,遗传稳定性,对小鼠安全性及其理化特性等生物学特性进行了研究。结果表明,重组病毒PGO18株与亲本株猪伪狂犬病病毒HB98具有相似的培养特性和生长特性,在ST、VERO和IBRS-2细胞上的增殖滴度TCID50与亲本株(107.75/0.1mL)相当,遗传稳定性良好,安全性试验表明对小鼠是安全的;理化特性试验说明该病毒具有猪伪狂犬病病毒的通性。     

PCV2 ORF2蛋白重组PRV病毒PGO株的生物学特性

PCV2ORF2蛋白重组PRV病毒生物学试验研究表明:重组病毒PGO株在PK15细胞、ST细胞、VERO细胞、IBRS-2细胞上病毒增殖滴度分别为104.125/0.1、106.125/0.1、104.625/0.1、106.25/0.1mL。RT-PCR、倒置荧光显微镜观察、IPMA试验结果表明插入基因在重组病毒中进行表达。该重组病毒具有PRV病毒的通性;安全性试验结果表明该重组病毒对小鼠是安全的;遗传稳定性研究表明包含有PCV2ORF2基因的重组伪狂犬病毒具有稳定的遗传性状;这些研究为研发PCV2病毒重组伪狂犬病毒疫苗奠定了基础。     

The porcine humoral response to detergent extracted Aujeszky's disease (pseudorabies) virus antigens

Twenty Aujeszky's disease (AD) virus antigens were demonstrated by crossed immunoelectrophoresis in a Triton-X-100 detergent extract of virus-infected PK-1a cells. Eight of these antigens were shown to be glycosylated based on their ability to be specifically bound by the lectin Ricinus communis agglutinin II. Pigs nasally infected with AD virus showed a significant serum antibody titer to seven of the known glycosylated antigens and to four additional antigens. The antibody titer to these antigens persisted for at least 116 days. Pigs which were vaccinated parenterally with the whole detergent extract survived a nasal challenge of 10(8 . 5) PFU of virulent AD virus. The antibody response of these vaccinated pigs on the day of challenge was essentially identical to the recovery response previously observed in non-vaccinated nasally infected pigs. These results indicate that the optimum components of future AD virus subunit vaccines and their complementary diagnostic reagents should be selected from these 11 antigens.     

CpG序列对猪伪狂犬疫苗免疫效果的影响

将CpG DNA同猪伪狂犬疫苗联合免疫接种初生仔猪,检测仔猪的体液及细胞免疫反应,试验结果表明,CpG DNA与猪伪狂犬疫苗共同免疫的仔猪,其特异性抗体滴度、淋巴细胞白介素-2诱生活性以及淋巴细胞增殖反应均显著高于伪狂犬疫苗免疫组和对照组,证明CpG DNA能显著增强仔猪对常规疫苗的细胞和体液免疫反应.     

猪细小病毒VP2蛋白病毒样颗粒在伪狂犬病病毒表达系统中的表达

利用PCR技术从猪细小病毒（PPV）SC1株基因组中扩增VP2全基因,并将其插入真核表达载体pPI-2.EG-FP中,构建转移载体pPI-2.EGFP.VP2。采用脂质体介导法将猪伪狂犬病病毒（PRV）SA215株DNA与pPI-2.EGFP.VP2DNA共转染Vero细胞,待出现细胞病变后收集病毒液。经空斑纯化,并同时采用检测PPV VP2基因的PCR方法筛选获得重组病毒PRV SA215/VP2株。以兔抗VP2的多克隆抗体建立的免疫荧光技术可检测到Vero细胞发出的特异性荧光,表明PPV VP2成功插入到PRV SA215基因组中,并获得表达。进一步电镜观察表明,感染PRV SA215/VP2的Vero细胞中可同时观察到PRV与PPV 2种类病毒样颗粒。结果表明,成功实现了利用PRV载体表达PPV VP2蛋白病毒样颗粒,为进一步研制PPV病毒样颗粒疫苗奠定了基础。     

The use of recombinant DNA technology for the development of a bluetongue virus subunit vaccine

The double-stranded RNA gene coding for the surface antigen responsible for inducing neutralising antibodies has been isolated, converted to DNA, and cloned in the plasmid pBR322. So far, only plasmids containing inserts smaller than the gene have been obtained. Possible strategies for the development of a bluetongue virus subunit vaccine are discussed.     

人参茎叶总皂苷联合亚硒酸钠对猪伪狂犬病灭活疫苗免疫的增强作用

为了探讨人参茎叶总皂苷(ginseng stem-leaf saponins,GSLS)联合亚硒酸钠(Na2SeO3,简称Se)对伪狂犬病病毒(PRV)灭活疫苗免疫的增强作用,本试验给小鼠口服GSLS后,接种添加了Se的PRV灭活疫苗,并检测免疫后小鼠血清中PRV gB抗体及其亚类(IgG1和IgG2a)水平、淋巴细胞...