Preparation And Identification Of PRRSV Virus-like Particles With Myc And His Tags

PRRS is a highly contagious disease caused by PRRSV,leading to respiratory symptoms in fattening pigs and reproductive failure in sows.The infection rate is very high and bring a devastating impact on the pig industry worldwide.Up to now,vaccine inoculation is still the most effective measure for preventing PRRS.However,the inactivated vaccine has the disadvantages of administration of large amount of immunogen,poor immunity effect and repeated immunization,and the attenuated vaccine has the risk of transmitting virus and generating new variant by recombination with prevalent virus in pig farm.Therefore,it is necessary to develop new vaccines against PRRS.Virus-like particles?VLPs?is very similar to natural virions,having good immunogenicity and containing no infectious viral nucleic acids,so they become the promising vaccine candidate.GP5protein encoded by PRRSV ORF5 gene is the most important immunoprotective protein.ORF6 gene encodes M protein with strong immunity.Co-expression of GP5 and M protein can form heterodimers,which are the basic elements for forming infectious virus particles.ORF7 gene encodes N protein,which highly enriches in virion.Although the antibody induced by N protein does not possess the ability to neutralize the virus,N protein can induce efficient cellular immunity.In this study,ORF5,ORF6 and ORF7 genes of type 2 highly pathogenic PRRSV QH-08 strain encoded GP5,M and N protein were used to develop PRRSV VLPs labeled with Myc and His tags via Bac-toBac baculovirus expression system.In addition,our study also explored the influence of N protein on the formation of VLPs.Preparation of PRRS VLPs with tags lays foundation to develop DIVA vaccine which is capable of differentiation of infected from vaccinated animals.In this study,pFastBacdual Myc-GP5-His-M and pFastBac HTB-N recombinant plasmids are first prepared.These two recombinant plasmids were transformed into E.coli DH10Bac competent,respectively,and the corresponding bacmids integrated into the baculovirus genome were obtained by transposition mechanism.Then the two purified recombinant bacmids were transferred into SF9insect cells by liposome,respectively.After infection,the cells became larger and intracellular granules were significantly increased in comparison with the uninfected cells.The virus was collected when the cells was ready to lyse.The virus was further passaged in SF9 cells to the third generation,P3.TCID50 of P3 virus of p FastBac dual Myc-GP5-His-M was 10^-9.36/mL,and it was10^-8.94/mL for pFastBac HTB-N virus.So far,two recombinant baculoviruses containing Myc-GP5-His-N protein and His-N protein were successfully prepared and laid foundation for preparing PRRS labeled VLPs.In order to generate PRRS labeled VLPs and further study the effect of N protein on the assembly of GP5-M VLPs,highfive?HF?cells were co-infected with the third generation viruses of pFastBac dual Myc-GP5-His-M and p FastBac HTB-N prepared from sf9 at MOI 3:3 ratio.Western blotting was performed with anti-Myc and anti-His as the primary antibodies to analyze the expression of recombinant proteins.The results showed that Myc-GP5,His-M and His-N proteins were successfully expressed and the molecular weight is 26,20 and 14 kDa,respectively,consisting with the expected size.Furthermore,Transmission electron microscopy was used to observe the assembly of VLPs.The results showed that Myc-GP5 and His-M recombinant proteins could self-assemble into VLPs which were similar to PRRSV natural particles purified by sucrose density gradient centrifugation from Marc-145 cell cultivation.The VLPs particles were round or oval,and had bilayer membrane structure with the diameter of 40 to 60nm.Immunoelectron microscopic observation with rabbit serum against PRRSV M protein showed that the prepared VLPs could be labeled with gold particles just like the natural virus,showing that the expression of N protein did not affect the assembly of labeled VLPs.The VLPs were further used as coating antigens for indirect ELISA and the results showed that labeled VLPs had good reactivity with porcine serum against PRRSV.In this study,PRRSV VLPs tagged with Myc and His were successfully prepared by Bac-to-Bac baculovirus expression system for the first time.The VLPs with double tags were pretty similar to the characteristics of PRRS natural virions.Our study lays foundation for further developing new VLPs vaccine differentiating the infected and immunized pigs.