Studies On Dna Vaccine Against Porcine Reproductive And Respiratory Syndrome Virus And Comparison Of The Dna Vaccine Immune

Porcine reproductive and respiratory syndrome(PRRS) (commonly called blue ear disease) is characterized with either reproductive failure in pregnant sows, or respiratory tract distress particularly in sucking pigs, and is recognized as a serious swine disease. This viral disease was first discovered in China in 1996. During the summer of 2006, the large-scale devastating outbreak of PRRS in China, known locally as "high fever", resulted the remarkably high morbidity and mortality. To date, PRRS has caused enormous economic losses each year. There is an urgent and important for novel control strategies such as a "safe, efficient, and cheap" vaccines against the PRRS infection. Here we amplified six main structural protein gene, then cloned into the mammalian expression vector PCI-neo, expressed these genes in Vero cell, evaluated the humoral and cellular immune responses in BALB/c mice and swine respectively immunized with six DNA recombinant plasmids by intramuscular injection. The results of research are summarized as following:1. The gene segments of ORF2, ORF3, ORF4, ORF5, ORF6, and ORF7 was amplified by reverse transcription-polymerase chain reaction (RT-PCR) from total RNA of PRRSV SCS strain culture solution. Then, these verified correct genes were respectively cloned into the mammalian expression vector PCI-neo, and these plasmids were transfected into Vero cells. The expression of six genes were determined by RT-PCR, indirect immunofluorescence (IFA), and western blot etc. These results showed that the recombinant plasmids of pCI-ORF2, pCI-ORF3, pCI-ORF4, pCI-ORF5, pCI-ORF6, and pCI-ORF7 were normally transcribed and expressed in Vero cells.2. BALB/c mice were immunized with the DNA vaccines pCI-ORF2, pCI-ORF3, pCI-ORF4, pCI-ORF5, pCI-ORF6, and pCI-ORF7 of PRRSV (SCS strain) by intramuscular injection. The TLR3,TLR4,INF-B,BCL10 transcriptional level of thoracic gland, lung, and spleen were detected by fluorescent quantitation-PCR, using miceβ-actin gene as reference genes. These results demonstrated that DNA immunization against PRRSV activate the function of proteinum TLR3, TLR4, INF-β, and BCL10, and strengthen immunization signal transmission. The plasmid pCI-ORF5 elicited the most strongest specific cellular immune response, compared with other plasmids.3. BALB/c mice immunized DNA vaccine showed higher levels of IL-2, IL-4, and IFN-y than mice immunized with pCI-neo, then enhancing resistance to disease. Those mice receiving injection of pCI-ORF5 showed the most strongest specific cellular immune response, on the contrary, the plasmid pCI-ORF2 elicited the most weakest specific cellular immune response.4. We have previously found that vaccination of BALB/c mice with the combination plasmids pCI-ORF3, pCI-ORF4, pCI-ORF5, and pCI-ORF6 showed good immune effect. Then, swine were inoculated intramuscularly with these plasmids, the change of CD3+, CD4+, and CD8+on lymphocyte of peripheral blood were investigated. The number of antigen-specific CD3+, CD4+, and CD8+ T-lymphocytes cells were up. The swine injected with combination plasmids enhances cellular immune response.5. Swine which immunized with the plasmids of pCI-ORF3, pCI-ORF4, pCI-ORF5, and pCI-ORF6 showed the increasing production of IL-2, IL-4, and IFN-γ. and improve swine's immune response and resistance to disease.