Study On Molecular Diagnosis And Gene Vaccine Of Porcine Reproductive And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome (PRRS) is often called blue ear disease, which is characterized by respiratory disease and reproductive failure in sows and high motality in piglets. The aetiological agent, porcine reproductive and respiratory syndrome virus (PRRSV), belongs to the member of arteriviruses, a group of small, enveloped, positive-strand RNA virus. The disease appeared in the last century and caused enormous economic losses. PRRS is classified as a notifiable disease by OIE and as a list B disease by China. The diagnosis and prevention of PRRS is a very important research field. In this study, the genes of PRRSV GS strain were obtained by RT-PCR and sequencing. A molecular diagnostic method of PRRS was established by designing the specific primers. Eukaryotic expression vectors containing ORF3 and ORF5 were constructed and used to immunize mice. The results were as follows:1. The PRRSV GS strain was separated by using Marc-145 cells and its whole genome was sequenced. The structural proteins were analyzed with Accelrys and the epitopes were predicted.2. A molecular diagnostic method of PRRSV was primarily established using one-step RT-PCR. The results revealed that it is able to detect PRRSV quickly and exactly.3. The ORF3 and ORF5 sequences were inserted into pBudCE4.1 vector. The expression vector pBud-GP3 or pBud-GP5 was transfected into the Marc-145 cells by liposome mediated method. RT-PCR and immunofluorescent test showed transcription and expression of the target genes in the Marc-145 cells. The recombinant plasmids were intramuscularly injected into BALB/c mice and the anti-PRRSV antibody was tested by indirect ELISA. The result indicated that the humoral immune response was effectively induced.4. A IFN-γgene was inserted into the pBud-GP3 and pBud-GP5, respectively. The pBud-IFN-GP3 or pBud-IFN-GP5 recombinant plasmid was transfected into the Marc-145 cells. RT-PCR and immunofluorescent test showed that the target genes were transcribed and expressed in the Marc-145 cells. The recombinant plasmids were intramuscularly injected into BALB/c mice. The anti-PRRSV antibody was tested by ELISA and the T lymphocyte subset was investigated by flow-cytometry. The results showed that the pBud-IFN-GP3 and pBud-IFN-GP5 recombinant plasmids enhanced cellular immunologic response, in which the IFN-γwas acted as an immunologic adjuvant enhances immunogenicity. At the same time, to evaluate the feasibility of the CD58 as an immunologic adjuvant. The vectors pBud-CD-GP3 and pBud-CD-GP5 were constructed and transfected into the Marc-145 cells, respectively. The result showed that CD58, GP3 and GP5 were successfully expressed in vitro. These results will lay a foundation for further study of immunization of CD58 in animal models.