【作者】 吴凤笋；

【导师】 李文刚； 卢中华；

【作者基本信息】 河南农业大学 ， 预防兽医学， 2006， 硕士

【摘要】 猪细小病毒（PPV）与伪狂犬病毒（PRV）是引起猪繁殖障碍的两个主要病原。PPV和PRV引起的疾病在我国有很高的感染率和发病率，给我国的养猪业造成了巨大的经济损失。传统的常规疫苗对防制PPV和PRV的感染有一定的效果，但是存在不可避免的缺陷，为了更好的控制PPV和PRV感染，研制更加安全有效的PPV-PRV二价基因工程疫苗是解决这一问题的最好途径。 伪狂犬病病毒基因组为一双链、线状DNA，在长约150kb的基因组上存在许多与病毒复制无关的非必需基因，这些基因可供外源基因的插入，而使之成为活载体疫苗。以含PPVNADL-2株全部序列的GH质粒为模板，扩增了完整的PPV VP2基因和部分VP1基因，并将其连接到PGEM-T Easy载体中，构建了重组质粒pVP2。为了构建猪细小病毒和伪狂犬病毒重组病毒，我们根据AnaI．Ranz等报道的PPV NADL-2株病毒基因组序列设计了一对包含其结构蛋白VP2基因的PCR引物，以复制型DNA为模板，通过PCR扩增出长约2.114bp的DNA片段，与经Stu Ⅰ和Sph Ⅰ双酶切处理的pPR128质粒DNA连接，转化大肠杆菌DH5α，构建了转移重组质粒pPR-VP2。经酶切鉴定证明插入片断是VP2片断，并证明插入方向正确。 利用脂质体介导的方法，将pPR-VP₂和PRV基因组共转染于亚单层PK-15细胞，用低熔点琼脂糖和中性红筛选重组病毒，经数次蚀斑纯化后，得到重组病毒PRV-VP2。为进一步制备抗猪细小病毒的重组伪狂犬病毒活载体疫苗奠定了基础。更多还原

【Abstract】 Porcine parvovirus （PPV） and pseudorabies virus （PRV） are two important etiological agents causing swine reproductive failures,which occurr in high prevalence and cause great morbidity in infected swine, leading to great economic losses to swine industry. The conventional vaccines have been used to prevent PPV and PRV infections. However, they possessed some disadvantages in view of safety and convenience. A bivalent genetic engineering vaccine against PPV and PRV should be developed to meet with the practical need.The genome of PRV is a linear DNA molecule of about 150kb,which includes many nonessential genes into which foreign genes can be stably inserted. This makes attenuated PRV become a promising candidate for the development of a live vaccine vector. To construct recombinant virus of PRV and PPV, a pair of primers of VP2 gene was designed according to PPV NADL-2 genomic sequence reported by Ana I Ranz. After amplification from RFDNA, the fragment which was 2.114kb long was cloned in the plasmid pPR128 which digested with StuI and SphI and transformed into strain DH5a of E.coli, yielding plasmid pPR-VP2.PRV Min-A DNA and recombinant plasmid pPR-VP₂ DNA were co-transfected into PK-15 cells with lipofectamine,and several recombinant virus strains were screened out with Low agarose and Neutral red. The recombinant virus can be further developed as a live vectored vaccine against pseudorabies and Porcine parvovirus influenza.更多还原