【作者】 洪琦；

【导师】 陈焕春； 熊远著；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2005， 博士

【摘要】 口蹄疫、猪细小病毒病与伪狂犬病是危害全球养猪业的三种重要传染病,分别由口蹄疫病毒(foot-and-mouth disease virus,FMDV)、猪细小病毒(porcine parvovirus,PPV)、伪狂犬病病毒(pseudorabies virus,PRV)引起。当前猪细小病毒病和伪狂犬病等猪繁殖障碍性传染病在我国很多猪场流行,造成大量死胎、流产及猪生产能力下降,危害严重。口蹄疫则是人畜共患的急性、烈性传染病,由于其传染性极强,而传统灭活疫苗在生产使用过程中有灭活不彻底导致病毒逃逸工厂而发生口蹄疫的风险,因此口蹄疫基因工程疫苗的研究受到高度重视。 伪狂犬病病毒具有不感染人、基因组容量大、重组病毒遗传稳定等特点,适合改造成表达外源基因的活病毒表达载体,以伪狂犬病病毒基因缺失株为表达载体构建基因工程疫苗,将其它病原的保护性抗原基因插入PRV基因缺失疫苗株中,就可构建成二价或多价基因工程疫苗,从而在猪场疫病免疫中起到一针防多病的作用。 1.口蹄疫与伪狂犬病二价基因工程疫苗的研究 将口蹄疫病毒衣壳蛋白前体P1-2A基因和蛋白酶3C基因共同插入到重组载体pIECMV中,构建重组伪狂犬病病毒转移载体pIEP1-2A-3C。采用脂质体介导法将pIEP1-2A-3C与伪狂犬病病毒基因缺失标志疫苗株PRV TK~-/gE~-/LacZ~+基因组共转染,构建了表达口蹄疫病毒P1-2A和3C基因的重组伪狂犬病病毒TK/gE~-/P1-2A-3C,并用空斑筛选与PCR鉴定的方法纯化重组病毒。进行Western blot鉴定和测定重组病毒在不同细胞上的增殖滴度,结果表明重组病毒构建正确且外源基因获得有效表达,其增殖滴度与亲本株相比差异不明显。遗传稳定性和安全性检测表明重组病毒稳定性、安全性良好,重组病毒免疫BALB/c小鼠后用ELISA检测血清中FMDV抗体,并测定FMDV中和抗体效价,结果表明重组病毒可诱导小鼠产生明显的免疫反应,从而为伪狂犬病与口蹄疫二价基因工程疫苗的研制应用打下基础。 2.口蹄疫—猪细小—伪狂犬病三价基因工程疫苗的研究 将口蹄疫病毒衣壳蛋白前体P1-2A基因和猪细小病毒VP2基因共同插入到重组载体pIECMV中,构建重组伪狂犬病病毒转移载体pIEPI-2A-VP2,经PCR和酶切鉴定证实质粒构建正确且两个表达框为反向插入。采用脂质体介导法将pIEP1-2A-VP2与伪狂犬病病毒基因缺失标志疫苗株PRV TK~-/gE~-/LacZ~+基因组共转染,构建了共表达口蹄疫病毒P1-2A基因和细小病毒VP2基因的重组伪狂犬病病毒PRV TK~-/gE~-/P1-2A-VP2,并更多还原

【Abstract】 Pseudorabies (PR), foot-and-mouth disease (FMD), and porcine parvovirus disease are three important infectious diseases in swine worldwide. The gene-deleted pseudorabies virus (PRV) has been used as the vaccine for eradication program of PR and a live-viral vector to develop bivalent or multivalent genetic engineering vaccine. In this study, A recombinant plasmid pEPl-2A-3C was constructed by inserting the P1-2A structural protein precursor gene and proteinase 3C gene of FMDV into PRV expression vector pIECMV. The genomic DNA of PRV TK/gE/LacZ+ strain and pEEPl-2A-3C were co-transfected into PK-15 cell with lipofection, and purified recombinant virus PRV TK’/gE7Pl-2A-3C was screened out with PCR method by examining P1-2A-3C gene. The recombinant virus was confirmed by PCR and Western blot assay, and the test of virus titers showed that insertion of P1-2A-3C gene did not influence proliferation of recombinant virus. Its virulence was reduced considerably and equaled with that of parent strain PRV TK7gE7LacZ+, and PRV TK7gE7Pl-2A-3C is genetically stable for at least 20 passages. Compared with other controlling groups, mice injected by PRV TK7gE7Pl-2A-3C were induced higher anti-FMDV antibody titer. The recombinant virus should be useful for the further research of bivalent genetic engineering vaccine against FMD and pseudorabies.Gene fragment of capsid proteins precursor P1-2A of FMD virus and VP2 gene of porcine parvovirus were chosen as the target antigen genes. Based on homologous recombination, a recombinant PRV co-expressing protein precursor P1-2A of FMDV and VP2 protein of PPV had been constructed using PRV TK7gE7LacZ+ mutant as the vector virus. By plaque purification and PCR detection, recombinant virus PRV TK7gE7P1-2A-VP2 was acquired. To evaluate its immunogenicity, BALB/c mice were vaccinated with the recombinant PRV, and the humoral response was measured by indirect ELISA and a serum neutralization test (SNT). After booster inoculation, mice sera showed high titers of antibodies against PRV, FMDV and PPV both in ELISA and SNT. Safety of the recombinant PRV and its protection against PRV were confirmed in a mouse model. The results indicate that the recombinant PRV would be a suitable candidate vaccine strain to develop a novel trivalent vaccine against PRV, FMDV and PPV in the pig industry.更多还原