Expression Of African Swine Fever Virus Penton Protein And Preparation Of Its Antibody

African swine fever(ASF)is a highly deadly infectious disease caused by the African swine fever virus(ASFV),which has a serious impact on pig farming in China and many other countries.The viral genome encodes more than 150 proteins which of them is around50 structural.Penton is encoded by the H240 R gene of the virus.It participates in the assembly of virus particles and has the effect of stabilizing the capsid structure.In this study,the penton protein was expressed in prokaryotic E.coli cells,rabbit and murine antibodies were prepared,and then the Bac-to-Bac insect baculovirus system was established to express the soluble penton protein.Our results will lay a foundation for further research on the biological function of penton protein.1.Prokaryotic expression and polyclonal antibody preparation of penton proteinThe penton gene was synthesized by the biological company and connected to the vector p Cold II to construct a prokaryotic expression recombinant plasmid(named p Cold-penton)and convert E.coli Rosetta receptive cells.The recombinant penton protein was induced at15? with optimal IPTG concentration and identified by SDS-PAGE method.The results showed that penton recombinant protein mainly aggregated into inclusion bodies in E.coli.Therefore,these aggregates were denatured in 8M urea followed by refolding and then purified using a Ni-NTA affinity chromatography column.The purified protein was mixed with Freund's adjuvant,and injected into BALB/c mice via intraperitoneal injection.The inclusion body protein was purified by the SDS-PAGE gel-cutting method and immunized the New Zealand female rabbits via subcutaneous injection on the back.The polyclonal antibodies in the serum of mice and rabbits were detected by indirect ELISA and Western blot.The results showed that murine-derived polyclonal antibody titer was 1:102400,and the level of rabbit-derived polyclonal antibody could reach 1:409600.2.Recombinant penton protein was expressed in the Bac-to-Bac baculovirus expression systemIn order to obtain soluble penton protein,we ligated insect cell expression vector p Fast Bac1 and p Cold-penton plasmid after restriction digestion,transformed E.coli Rosetta competent cells,and sequenced the positive clones identified by PCR and double restriction digestion.The correct recombinant p Fast Bac1-penton plasmid was transformed into DH10 Bac competent cells.The positive clones were identified by blue-white spot screening,bacterial liquid PCR and double enzyme digestion.Furthermore,the recombinant Bacmidpenton bacmids obtained were transfected into the insect Bm N cells with non-lipid cationic polymer transfection reagent.The cells were observed under a microscope.The results showed that the infected cells had obvious cytopathic changes at 72 hours after transfection,compared with the non-infection control cells.The cells with high-titer recombinant baculovirus expressing penton was passaged four times.The supernatant of cells infected with the recombinant virus was collected and checked for the gene expression and protein expression of penton by PCR and Western blot,respectively.Our results showed that the soluble expression of penton protein was performed in insect cell line Bm N.In summary,this study first performed prokaryotic expression of penton protein in E.coli.The fusion protein inducted by IPTG was mainly expressed in inclusion bodies and was refolded in 8M urea denaturation.The penton protein was purified with affinity column or gel cutting methods.Rabbit-derived and murine-derived polyclonal antibodies were prepared for penton.Moreover,the penton gene was connected to the eukaryotic expression vector p Fast Bac1 to obtain the recombinant plasmid and transfected into insect Bm N cells.The recombinant baculovirus with penton protein was obtained and expressed in Bm N cells.The prepared rabbit-derived and murine-derived polyclonal antibodies could recognize penton protein expressed in insect cells.Our results have laid the foundation for further investigation of the biological function of penton protein.