Construction Of A Recombinant Pseudorabies Virus And Porcine Parvovirus VP2 Gene Expressing Green Fluorescent Protein

Porcine parvovirus (PPV) and pseudorabies virus (PRV) are two important etiological agents causing swine reproductive failures, which occurr in high preval ence and cause great morbidity in infected swine in our country, leading to great economic losses to swine industry. The conventional vaccines have been used to prevent PPV and PrV infections. However, the existence of inevitable shortcomings, in order to better control of PPV and PRV infection, A more safe and effective bivalent genetic engineering vaccine against PPV and PRV should be developed to solve the the best way to question.。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. A pair of primers of VP2 gene was designed according to PPV NADL-2 genomic sequence reported by Ana I Ranz. In the primer, downstream 5'end introduced BamHI restriction sites, Containing PPV NADL-2 strain of all the GH plasmid sequence as a template, amplification of the complete PPV VP2 gene and part of VP1 gene,The construction of the recombinant plasmid which the amplified VP2 gene was cloned into PMD18-T vector. Identified by PCR to prove the VP2 gene insert and the recombinant plasmid named as PT-GFP. Digested with BamHI amplified VP2 gene and plasmid vector PG and recovery,will be to phosphorlation of vector PG after fragment VP2 link, Construction of the recombinant plasmid PG-VP2,amplified by PCR, restriction enzyme digestion, sequencing proved that the success of the transfer vector. Proved by the enzyme digestion fragment was inserted into VP2 fragment and inserted into the right direction to prove.PRV Min-A DNA and recombinant plasmid PG-VP2 DNA were co-transfected into PK-15 cells with lipofectamine, Fluorescence microscopy in 48 hours will be observed green fluorescent, straw will be used fluorescence aspiration of the lesion cells, after three consecutive passages have been purified virus. And have been identified by PCR and restriction enzyme digestion successfully constructed recombinant viruses. The recombinant virus was developed for the next two or trivalent vaccine price, providing a convenient selection.