| Porcine reproductive and respiratory syndrome(PRRS), commonly known as blue ear disease,is made up of Porcine reproductive and respiratory syndrome virus(PRRSV), caused a sexually transmitted diseases. It mainly causes reproductive failure in female pigs and piglets respiratory disorder. High morbidity and mortality in pigs cause great economic losses. The measures of prevention and control of PRRS in addition to strict biosecurity measures, however, the most effective way is vaccination. Clinic mainly uses the attenuated vaccine and inactivated vaccine to prevent disease, but its existence defects such as poor immune effect and returning to poison, make people’s eyes have to turn to other new vaccines to control the occurrence and prevalence of the PRRS. This research through the separation of PRRS chongqing strain identification, select the GP5 and M gene, construct PRRS virus-like particles(VLPs), made into a vaccine, in assessing the immune activity of mice, lay a foundation as a new development of PRRS vaccine.1 Identification and genetic diversity of porcine reproductive and respiratory syndrome virus isolated from Chong Qing The visceral organs were collected from swines at a Chong Qing farm. Once the paghogen was idetified as the porcine reproductive and respiratory syndrome virus via RT-PCR identification, we used reverse transcription making c DNA. Both NSP2 and GP5 gene were amplified by PCR, then they were sequenced. The NSP2 sequence analysis indicated that homology between CQ and classical VR-2332 strain was 66.7%, while it was as high as 98.8% compared with HP-PRRSV variation SD and JXA1 in nucleotide. And the GP5 sequence analysis indicated that there were variations in individual bases. It was suggested that the CQ strain was a HP-PRRSV variation. At the same time, the patho-material was inoculated in Marc-145 cells, then the CPE appeared. The viral titer is 10-5.67TCID50/0.1m L by Reed-Muench.2 Construction of Porcine Reproductive and Respiratory Syndrome Virus-like Particles In order to construct the VLPs of highly PRRSV in this research, as templet of full-length GP5 and M c DNA of the highly pathogenic PRRSV Chongqing strain identified before, the GP5 and M proteins were expressed by the recombinant baculovirus system. The constructed VLPs was identified by SDS-PAGE and Western blotting as well as transmission electron microscopy after purified by sucrose gradient centrifugation. The result showed that GP5 and M proteins of PRRSV were expressed by the recombinant r Bacmid-GP5-M in baculovirus-insect expressing system to automatically assemble into VLPs, PRRSV-VLPs were observed in transmission electron microscopy. The expressed proteins have similar immunogenicity with natural proteins of PRRSV by SDS-PAGE and Western blotting. The diameter of the VLPs is about 50 nm in the electron microscopy.3 Immune responses in mice vaccinated with virus-like particles of porcine reproductive and respiratory syndrome virus BALB/c mice immunized intramuscularly three times with different doses(5.0, 10.0, 20.0, 50.0and 100.0 μg) of the PRRSV-VLPs vaccine, commercial vaccines consisting of inactivated PRRSV vaccine and normal saline(NS) were used as positive and negative controls, respectively. Ig G titers to GP5 were significantly higher in all groups of mice vaccinated with the VLPs than in control mice.Neutralizing antibodies were only detected in mice vaccinated with 50.0 and 100.0 μg of the VLPs.Cytokine levels were determined in all experimental groups and positive controls. Mice immunized with 20.0 μg of the VLPs produced a significantly higher amount of interleukin(IL-4) than mice immunized with the commercial inactivated PRRSV vaccine and NS. In addition, immunization with the commercial vaccine induced higher production of IFN- γ in mice than mice vaccinated with VLPs. The overall rule is not obvious. These data together demonstrate that PRRSV-VLPs can produce higer levels of cellular immunity and humoral immunity in mice and PRRSV-VLPs vaccine is a kind of potencial vaccine to prevent PRRS. |
