Cloning Of Porcine Parvovirus VP2 Gene And Construction Of Pseudonabies Virus Transfer Vector

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. Firstly, an endogenous PPV was isolated from PK-i 5 cells, identified by etiological, serological and molecular biological methods, and designated as SY-99. The genomic DNA of the virus was extracted and used to amplify its immunogenic VP2 gene by PeR. The VP2 gene of SY-99 was determined to be 1740 bp in length, coding 580 amino acids. Molecular analysis showed that VP2 gene of PPV SY-99 strain was 99.2%, 99.3%, 99.5% and 99% nucleotide identities, and 98.1%, 98.3%, 98.8% and 96.6% amino acid identities with those of NADL-2(4973), NADL-2(5075), Kresse and US-i strains, respectively. This indicated that PPV SY-99 strain is most related to Kresse strain. The VP2 gene of PPV SY-99 strain was then subcloned into a yeast expression vector (Ppicza-B). It will be expressed in pichia to develop a subunit vaccine against PPV. A fragment that contains the immediate early promoter of CMV and polyA signal of BCIH from pCR3-Uni was amplified by PeR, and cloned into AccI sites of the vector pBTK, 2 resulting a Pry transfer vector with the name pBdTK-Uni. The VP2 gene of PPV SY-99 strain and a leuciferinase reporter gene box was inserted into the PrV transfer vector, thus a transfer plasmid containing the VP2 gene of PPV SY-99 strain was generated. This transfer plasmid is the perquisite to develop a recombinant PrV expressing PPV VP2 gene as a bivalent genetic engineering vaccine against PrV and PPV. Master candidate: Changwen Li Major: Preventive Veterinary Tutors: Prof. Wenzhou Liu Prof. Guangzhi Tong...