Construction And Preliminary Application Of PRRSV Major Structural Protein Cell Line

Porcine reproductive and respiratory syndrome（PRRS）is a highly contagious infectious disease caused by porcine reproductive and respiratory syndrome virus（PRRSV）,which would cause porcine reproductive and respiratory disease.PRRSV belongs to genus Arterivirus,family Arteriviridae.In 1996,the disease was first reported in our country.Clinically,vaccination is one of the primary methods for the prevention and control of PRRSV.However,traditional attenuated live vaccines have safety problems.Antibodies produced by attenuated live vaccines are indistinguishable from antibodies produced by wild virus.It is urgent for the development of novel vaccines to control this virus.Single-round infectious PRRSV replicon vaccine can produce virus particle which only had single-round infectivity with no cell to cell spread.It can stimulate immunity in vivo and it is safe.This novel vaccine can distinguish vaccined from infected animals.As a novel vaccine,single-round infectious PRRSV replicon vaccine is research direction for development of PPRSV vaccine.GP5,M,and N proteins are the major structural proteins of PRRSV,all of which have strong immunogenicity.Firstly,we constructed MARC-145 cells lines stably expressing the PRRSV GP5,M,and N,respectively.Three pairs of specific primers targeted PRRS GP5,M and N proteins coding region were designed according to PRRSV full-length infectious clone（pAJX25HB）.The target genes of ORF5,ORF6 and ORF7 were amplified by PCR using pAJX25HB plasmid as template.The amplified products were cloned into the plasmid pLenti-CMV-GFP-Hypro.The recombinant plasmids were named as pLenti-CMV-GP5-Hypro,pLenti-CMV-M-Hypro and pLenti-CMV-N-Hypro.The recombinant plasmid pLenti-CMV-GP5/M/N-Hypro,packaging plasmid ps PAX2 and outer membrane plasmid VSVG were co-transfected into 293T cells and three recombinant lentiviruses were obtained.The recombinant lentiviruses were used to infect Marc-145 cells.the infected cells were stably screened with hygromycin B to obtain three cell lines stably expressing GP5,M and N proteins,named Marc-145-GP5,Marc-145-M and Marc-145-N cell line.The genes of GP5,M and N The Marc-145 cell line were detected by RT-PCR or indirect immunofluorescence assay（IFA）.PRRSV recombinant plasmids in which PRRSV ORF5,ORF6,ORF7 start codon were mutated were constructed based on PRRSV infectious clone pAJX25HB.The resulting plasmids were named as pAJX25HB-5tb,pAJX25HB-6tb and pAJX25HB-7tb,respectively.The recombinant plasmids pAJX25HB-5tb,pAJX25HB-6tb and pAJX25HB-7tb were transfected into corresponding cell line and the MARC-145 cells.No infectious virus was detected in MARC-145expressing GP5,M or normal MARC-145 cells transfected with pAJX25HB-5tb or pAJX25HB-6tb.It was suggested that the function of GP5 and M could not be complemented by a trans-complementary expression in a cell line.Infectious virus was detected in MARC-145expressing N but not in normal MARC-145 cells transfected with pAJX25HB-7tb.However,the sequencing results showed that the mutated nt of the rescued virus was mutated to the original sequence.pAJX25HB-7tb could be rescued in MARC-145 lines expressing N protein.But reverse mutation at mutated position of the rescued mutant virus was found after several passages.Therefore,recombinant clones in which N protein coding region,N-terminal RNA-binding（NRB）region,C-terminal dimerization domain（CDD）region,6 amino acids at the N-terminus and C-terminus were deleted were constructed.The resulting plasmids pAJX25HB-63BS,pAJX25HB-63BS-CDD,pAJX25HB-63BS-NRB,pAJX25HB-63BS-N-6QS^N,pAJX25HB-63BS-N-6QS^C,pAJX25HB-63BS-N-6QS^NCwere transfected into MARC-145-N cells and in normal MARC-145 cells.No infectious virus was detected in MARC-145-N and normal MARC-145 cells transfected with these plasmids.We assumed that the sequences coding N protein are important for virus replication.In a word,we have constructed the Marc-145 cell lines expressing PRRSV the major structural protein GP5,M and N.The Marc-145 cells lines were used for recovery of recombinant plasmids with carrying mutation at start codon of GP5,M and N.The results of our study lay the foundation for exploration and development of novel vaccine.