Study On The Insertion Of Foreign Genes Between ORF7 And 3’UTR Of Porcine Reproductive And Respiratory Syndrome Virus

PRRS is a highly contagious infectious disease caused by PRRSV,which would cause porcine reproductive and respiratory disease.In 1996,the disease was first reported in our country and the virus was isolated,then PRRSV spread widely in our country.The now available vaccines for controlling PRRSV do not distinguish itself from filed isolates.Furthermore,PRRSV inactivated vaccine has limited protection against PRRSV infection.Attenuated vaccine has a risky of virulence reversion and poor cross protection against heterogenous strains.Therefore,the development of marker PRRSV vaccine and the use of PRRSV as a viral vector recombinant vector expressing foreign gene has become one of the future research directions of PRRS vaccine.In this study,based on PRRSV full-length cDNA clone pAJX25HB,BstB Ⅰ、I SbfⅠ restriction sites and the transcriptional regulation sequence(TRS)were inserted between the ORF7 and 3’UTR by SOE-PCR.Then the foreign gene EGFP、PRRSV-ORF4、EAV-ORF4、EAV-ORF5a was inserted into between BstB I with Sbf I,and the recombinant plasmids was obtained:pAJX25HB-7 3BS-TRS-EGFP、pAJX25HB-73BS-TRS-PRRSV-ORF4、pAJX25HB-73BS-TRS-EAV-ORF4、pAJX25HB-73BS-TRS-EAV-ORF5a,respectviely.All the recombinant plasmids were transfected into BHK-21 cells to obtain packaged virus particles,and then packaged virus particles were transferred to MARC-145 cells to obtain recombinant virus.The biological characteristics of the rescued virus were then studied in MARC-145 cells.PRRSV typical cytopathic effect(CPE)was abserved in MARC-145 cells and results of RT-PCR and IFA showed that the replication of PRRSV genome and the specific expression of PRRSV N protein in MARC-145 cells.The results of RT-PCR amplification,enzyme digestion and sequencing showed that the passaged recombinant PRRSV virus contain insertion of exogenous gene sequence;The multi-step growth curve showed that all the recombinant viruses have a similar replicative characteristic with the parent virus.All virus titer reached the highest titer at 36 hours post infection(hpi).All the recombinant viruses was significantly higher than that of the parental virus vJX25HB at each time-point before 48hpi.rJX25HB-73BS-TRS-PRRSV-ORF4 have a lowest titer at 60hpi.The initial viral titer of rJX25HB-73BS-TRS-EGFP is the lowest,this may be due to insertion of the largest number of foreign gene into the PRRSV genome compared to other inserted genes.For the viruses with insertion of exogenous gene of EAV rJX25HB-73BS-TRS-EAV-ORF4 and rJX25HB-73BS-TRS-EAV-ORF5a,have a similar growth curves.In summary,we did a preliminary study on the insertion and expression of foreign gene between ORF7 and 3 ’UTR of PRRSV.Therefore,this study has laid an important foundation for the study of the recombinant PRRSV vaccine expressing foreign gene.