Study On The Tandem Expression And Immune Efficacy Of Relevant Protective Protein About Porcine Virus Diarrhea

Porcine epidemic diarrhea virus,porcine transmissible gastroenteritis virus and porcine rotavirus can cause acute diarrhea,vomiting and mortality in neonatal piglets,resulting in significant economic losses to the pig industry.Three viruses often cause mixed infection on piglets.Although the vaccine has been developed in recent years,the effect is not ideal.Porcine diarrhea virus infection is still a problem that cannot be ignored in China's pig industry.In order to control the diseases,safely,efficient and convenient vaccine researches is one of the methods.1.Cloning and bioinformatics analysis of PEDV COE,TGEV SA and PoRV VP7 gene The primers were separately designed based on the complete genes of PEDV CV777 strain(Accession No.AF353511),TGEV Purdue strain(Accession No.DQ811789.2)and PoRV OSU strain(Accession No.KJ450849.1)from Genbank.The target fragments were separately amplified from PEDV-HN13 strain,TGEV-TZ-10-2016 strain and PoRV-GD-01-2015 strain.The target genes were connected to pGEM-T Easy.With enzyme digestion identification,pGEM-T-COE,pGEM-T-SA and pGEM-T-VP7 recombinant plasmids were obtained.Construction of eukaryotic expression vector with SnapGene.The fusion protein COE-SA-VP7 was analyzed with kinds of biology software.The results indicated that the expected fusion protein COE-SA-VP7 was 56.77kDa and had good immunogenicity.2.Construction of eukaryotic expression vector pFastBacHT-COE-SA-VP7 pGEM-T-VP7 and pFastBacHTA plasmids were digested by XhoI and KpnI.The target fragments were connected to pFastBacHTA.pFastBacHT-VP7 recombinant plasmids were obtained.pGEM-T-SA and pFastBacHT-VP7 plasmids were digested by EcoRI and XhoI.The target fragments were connected to pFastBacHT-VP7.pFastBacHT-SA-VP7 recombinant plasmids were obtained.pGEM-T-COE and pFastBacHT-SA-VP7 plasmids were digested by BamHI and EcoRI,The target fragments were connected to pFastBacHT-SA-VP7.pFastBacHT-COE-SA-VP7 recombinant plasmids were obtained.pFastBacHT-COE-SA-VP7 recombinant plasmids was transformed into DH10Bac competent cells to construct the recombinant baculovirus shuttle plasmid.Then,the rBac-COE-SA-VP7 recombinant baculovirus was obtained by transfecting into Sf9 insect cells under LipofectamineTM 3000.Total RNA was extracted from Sf9 cells transfected with rBac-COE-SA-VP7.The fragment of COE,SA,VP7 and COE-SA-VP7 were amplified with RT-PCR,which showed that pFastBacHT-COE-SA-VP7 had successfully transcribed in Sf9 cells.The results of the indirect immune fluorescence test showed that the fusion protein expressed by the rBac-COE-SA-VP7 recombinant baculovirus,it can be recognized by the anti-PEDV,anti-TGEV and anti-PoRV antibody.The results of Western-blot showed that the 56.77kDa fusion protein expressed by recombinant baculovirus could be specifically recognized by antibody against His-tagged.3.Optimization and identification of COE-SA-VP7 fusion protein eukaryotic expression vectorsIn this study,The COE-SA-VP7-TAT and L-COE-SA-VP7-L target fragments were connected to pFastBacHTA.With PCR and enzyme digestion identification,pFastBacHT-COE-SA-VP7-TAT and pFastBacHT-L-COE-SA-VP7-L recombinant plasmids were obtained.pFastBacHT-COE-SA-VP7-TAT and pFastBacHT-L-COE-SA-VP7-L recombinant plasmids was transformed into DH10Bac competent cells to construct the recombinant baculovirus shuttle plasmid.Then,the rBac-COE-SA-VP7-TAT and rBac L-COE-SA-VP7-L recombinant baculovirus was obtained by transfecting into Sf9 insect cells under LipofectamineTM 3000.Total RNA was extracted from Sf9 cells transfected with rBac-COE-SA-VP7-TAT and rBac L-COE-SA-VP7-L.The target fragment was amplified with RT-PCR,which showed that pFastBacHT-COE-SA-VP7-TAT and pFastBacHT-L-COE-SA-VP7-L had successfully transcribed in Sf9 cells.The result of the indirect immune fluorescence test showed that the fusion protein expressed by the rBac-COE-SA-VP7-TAT and rBac L-COE-SA-VP7-L recombinant baculovirus,it can be recognized by the anti-PEDV,anti-TGEV and anti-PoRV antibody.