Construction of recombinant PRRSV ORF5 gene by DNA shuffling technique / 生物工程学报

Recombinant PRRSV △2ORF5 gene was constructed using DNA shuffling from four genetically different strains of PRRSV to study its heterologous cross-neutralizing ability. The △2ORF5 mutant gene was cloned into the vector pET-32a and transferred into E. coli BL21. SDS-PAGE confirmed that the molecular weight of the recombinant △2ORF5 was about 42 kDa, consistent with the predicted result. Then the purified recombinant protein was injected into BALB/c mouse to obtain polyclonal antibody. Western blotting analysis with mouse-anti-△2ORF5 polyclonal serum indicated that the parental virus recombinant GP5 protein reacted with the specific antibodies. Four parental viruses could be inhibited by the anti-△2ORF5 polyclonal antibody and the inhibition rates were higher than 53%. This work has laid a foundation for further development vaccine for PRRSV.

Expression of Open Reading Frame 5 Protein of Porcine Reproductive and Respiratory Syndrome Virus Using Semliki Forest Virus Expression System

The ORF5 gene encodes a major envelope glycoprotein (GP5), which is one of the three major proteins of porcine reproductive and respiratory syndrome virus (PRRSV). The GP5 protein has been known to be a 24.5-26kDa N-glycosylated envelope protein. The GP5 is involved in inducing neutralizing antibodies. For this reason, the GP5 is primary candidate for the PRRSV subunit vaccine. To produce the native form of GP5 in mammalian cells, we have cloned the ORF5 gene from PRRSV CNV-1 into the Semliki Forest virus (SFV)-based expression vector, resulting in recombinant pSFV-ORF5. By the infection with recombinant pSFV-ORF5 to BHK-21 cells, the GP5 expression was confirmed by immunocytochemistry and immunoblotting assay. The recombinant virus particle harboring ORF5 gene was infectious to BHK-21 and MARC-145. The RNA synthesis and expression of GP5 in the infected cell was also confirmed by RT-PCR.

Study on immunological responses to eukaryotic expression vector containing PRRSV ORF5 and CpG motifs / 中国免疫学杂志

Objective:To enhance the immune efficiency of porcine reproductive and respiratory syndrome virus(PRRSV)DNA vaccine,the ORF5 genes were used as candidate genes to construct the recombinant plasmids CpG-pVAX1-ORF5 by pVAX1 as eukaryotic expression vector.The aim is to analyze the immune responses and the effects of CpG-ODN induced by GP5 recombinant plasmids of PRRSV.Methods:Piglets were immunized with recombinant DNA plasmids which expressed PRRSV GP5 for three inoculations.The PRRSV antibody in serum,the concentration of IL-2 and the lymphocyte proliferation test(MTT) in peripheral blood of vaccinated piglets were detected.The vaccinated pigs were challenged intranasally with PRRSV SD2.Results:GP5 DNA immunization with CpG resulted in the production of both PRRSV antibodies and cellular immune(a significant enhancement of a lymphoproliferative response ).The CpG-pVAX1-ORF5 was showed significantly better protection from the PRRSV challenge compared with the control plasmid.This immune response was characterized by a significantly decreased frequency of viraemia with decrease of 80% compared with the control group.There were little clinical symptoms and protection in some extent from lung damage.Conclusion:The results indicate that CpG-ODN could be used as immune adjuvant of PRRSV DNA vaccine.