Exogenous Gene Insertion Between Porcine Reproductive And Respiratory Syndrome Virus (PRRSV) ORF1b And ORF2a

Porcine Reproductive and Respiratory Syndrome(PRRS),the pathogen is Porcine Reproductive and Respiratory Syndrome Virus(PRRSV),which is susceptible to all pigs.In 1987,the disease was first discovered in the United States,and then spead the global swine industry..The disease was reported in Taiwan,China in 1991.The spread of PRRSV pose a serious threat to pig industry in China and the world.There is no effective means for the prevention and control of PRRSV.The commercial inactivated and attenuated vaccines have great limitations for the prevention of PRRSV.These vaccines do not provide sufficient immune protection agaist PRRSV infection.The attenuated vaccine is unsafe and have poor cross-protection to heterologous strains.Furthermore,all the commercial vaccines can not distinguish vaccined from infected animals.It is ugent for us to develop the bivalent or even polyvalent vaccines to control PRRSV and other porcine pathogens.PRRSV contains at least 10 open reading frames,and most of the adjacent open reading frames overlap with each other.However,there is only a nucleotide interval between ORF1 b and ORF2 a of PRRSV.The insertion of exogenous genes at this site do not destroy the original open reading frame of PRRSV and it is suitable site for insertion of foreign gene.In this study,two restriction sites,Bstb I and Sbf I,transcriptional regulatory sequence 6(TRS 6),were inserted between the ORF1 and ORF2 of PRRSV based on a infectious clone p GXAM by overlap extension PCR(SOE-PCR).And then,the recombinant clones in which green fluorescent protein(GFP)and foot-and-and-mouth disease VP1 were inserted between ORF1 b and ORF2 a of PRRSV through two restriction enzymes Bstb I and SBF I were constructed.The resuting recombinant plasmids p GXAM-TRS,pGXAM-GFP,p GXAM-FMDV-A-VP1 and p GXAM-FMDV-O-VP1 and p GXAM were transfected into suckling hamster kidney cells(BHK-21 cells).300 ?L supernatant was collected after 48 hours transfection(hpi)and inoculated into African green monkey kidney cells(Marc-145 cells).The cytopathic changes were observed and passaged continuously in Marc-145 cells.The recombinant viruses were confirmed by RT-PCR and IFA and the biological characteristics of the recombinant virus were identified.The results of RT-PCR and sequencing of targeted regio showed that the recombinant viruses were stable for at least three generations.The growth curve showed that the rescued virus had similar growth kinetics with its parent strains.In this study,we constructed recombinant PRRSV virues expressing exogenous genes(GFP,-FMDV-A-VP1 and FMDV-O-VP1).The results of this study lay a solid foundation for the development of recombinant bivalent or even multivalent vaccine for prevention of PRRSV and other swine pathogens.