Immune Enhancement Of Recombinant Fusion Peptide TBP5 To DHV-1 Gene Engineering Subunit Vaccine

Duck viral hepatitis is an acute,lethal death of ducklings caused by duck hepatitis virus(DHV)with rapid onset,rapid spread,short duration and high mortality.Clinical expression is opisthotonos and after dissecting hepatomegaly and a large number of hemorrhagic spots can be seen.Mainly through contact spread or be infected by the respiratory tract.DHV is usually divided into 3 serotypes,DHV-1 ?DHV-2 and DHV-3.The DHV-1 is the main serotype.The disease is one of the most serious infectious diseases that endanger the duck industry,and there are different degrees of epidemic and occurrence in China,the epidemic is difficult to control.Although the duck hepatitis vaccine and inactivated vaccine have been widely promoted across the country,but in the actual production process,the immune effect is not ideal.The DHV is still outbreaks,bring serious harm to duck industry and the control is still not significantly improved.This research was amplificating the DHV-1gene,constructing the recombinant expression plasmids,transforming the recombinant plasmid into E.coli BL21(DE3)to induce the expression to obtain recombinant VP1 protein.At the same time,using the phage display technology to restracture and DHV-1 VP protein as the target protein,through screened by phage random 12 peptide library,combined with ELISA identification and competitive inhibition test,and screened to the peptide which specific binding it.The ducklings animal immunization experiment was studied by using the recombinant fusion peptide TP5-BP5(TBP5)with DHV-1VP1 Protein Gene Engineering Subunit Vaccine to explore its immune enhancement effect on DHV-1 VP1 protein subunit vaccine.The main experiment content is divided into the following three aspects:Cloning and Expression of DHV-1 VP1 GeneDHV-1 VP1 gene was amplified by RT-PCR using primers F1 and F2.The product was subjected to agar gel electrophoresis and showed a band about714 bp.Then the gene was cloned into the pET-32 a vector to screening the recombinant expression plasmid.The positive plasmid pET32a-VP1 was identified by PCR and digestion with EcoR ? and Xba ?,transformed the plasmid pET32a-VP1 into E.coil BL21(DE3)to induce expression.The expression product of positive plasmid pET-32a(+)-VP1 was analyzed by SDS-PAGE,then showed a target protein band about43.0 KD(contained TRX thioredoxin).The result indicated that DHV-1 VP1 gene can successfully expressed in E.coli BL21.Western-blot analysis showed that the specific bands were visible at 43.0 KD,consistent with the expected results.This study provides an experimental material for the preparation of the DHV-1VP1 protein gene engineering subunit vaccine.DHV-1 VP1 Screening of protein-specific binding peptides20 single phage clones were obtained using the phage display technique and DHV-1 protein as the target to affinity screen the phage random 12 peptide library for the 3 round.Combined with ELISA identification and competitive inhibition test,positive phage clones P4 and P6 that specifically bind to DHV-1 VP1 protein were obtained.Two positive phage clones(P4 and P6)were found and the sequencing results showed that they were LLSPGTHH-SPWP(P4-12)and LLADTTHHRPWT(P6-12),respectively both of which shared a conserved sequence THHXPW.MTT assay showed that DHV-1 VP1 protein binding peptide P4-12 and P6-12 had no significant effect on the proliferation of duck embryo fibroblasts(DEF).The results showed that both P4-12 and P6-12 could significantly decrease the titer of DHV-1virus and the number of copies of virus in duck embryo fibroblasts by measuring the virus titer and virus copy number which indicated that peptides P4-12 and P6-12 significantly inhibited the proliferation of DHV-1 in duck embryo fibroblasts(DEF).This study result provides novel insights on the interaction mechanism of DHV-1 and host cells.The immunoenhancement effect of the recombinant fusion peptideTBP5 on subunit vaccine of DHV-1 VP1 proteinCombining the recombinant fusion peptide TBP5 with subunit vaccine of DHV-1 VP1 protein,the immunoenhancement effect of the recombinant fusion peptide TBP5 on subunit vaccine of DHV-1 VP1 protein was evaluated through the grouping and immunity of ducklings,detecting the levels of serum IgG antibody and serum cytokines,the vaccinated protection test as well as hematoxylin-eosin staining.The results showed that in the group of the recombinant fusion peptide TBP5 coordinated subunit vaccine of DHV-1 VP1 protein,the levels of serum Ig G antibody and serum cytokines were significantly higher than those in the control group and the vaccine group.Results of animal vaccinated protection test showed that the recombinant fusionpeptide TBP5 with subunit vaccine of DHV-1 VP1 protein in immune ducklings enhanced the protective effect on liver.The above results show that the recombinant fusion peptideTBP5 has immunoenhancement effect on subunit vaccine of DHV-1VP1 protein,which lays a foundation for the further development of the new vaccine adjuvants of subunit vaccine of DHV-1 VP1 protein.