The Expression Strategy And Immune Activity Of Virus Vaccine Capsid Protein Antigen In E.coli

Capsid protein is one kind of constitutive protein of virus with strong antigenicity and it has become a target especially in the development of virus subunit vaccine.At present,there are some genetic engineering vaccines targeting at capsid protein,such as vaccines of hepatitis B virus(HBV),human papilloma virus(HPV)and hepatitis E virus(HEV).HBV,HPV and HEV belong to DNA virus.Compared with RNA virus,DNA virus has low frequency of mutation in genome and the capsid protein coded by DNA is less likely to change.From the point of the structure,the viral capsid proteins above are consist of one or more monomers and can form virus-like particle structure(VLPs).Porcine circovirus(PCV)is one kind of animal DNA virus and has similar features with human viruses above.The capsid protein of PCV is consist of one monomer and when it is expressed in eukaryotic expression system,it can form VLPs directly.When it is expressed in prokaryotic expression system,it can also form VLPs by assembling in vitro.Based on the features,the way to study in PCV capsid protein vaccine may be applied in the vaccine research of human virus or other animal virus targeting at capsid protein.PCV is a new epidemic strain of porcine circovirus,much information of PCV3 remains to be studied like the location of neutralizing epitope and decoy epitope in capsid protein and the structure of capsid protein.As for all reasons above,we regarded the PCV3 capsid protein as the research object and put forward some strategies to express PCV3 capsid protein in E.coli based on the bioinformatics analysis and expressed soluble recombinant capsid protein of PCV3.The results are described as bellow.1.Reconstruction and solubility research of PCV3 recombinant capsid proteinIn our study,we found that full-length PCV3 capsid protein was expressed with low expression in E.coli.In order to acquire soluble PCV3 capsid protein with high expression,we put forward two kinds of construction strategies: 1)Construction of PCV3 capsid protein with N-terminal truncated.2)Construction of PCV3 capsid protein with N-terminal replaced.Another point,for the reason of avoiding immune resources waste resulted by decoy epitope exposure,we designed another kind of recombinant protein of PCV3 capsid protein in which the decoy epitope was replaces by neutralizing epitope.(1)Expression of full-length PCV3 capsid proteinIn order to express full-length PCV3 capsid protein,we obtained the whole coding gene sequence of PCV3 capsid protein by splicing overlap extension PCR,constructed the expression plasmid L-pET28 a and expressed full-length PCV3 capsid protein in E.coli(named as L protein).The result indicated that the expressed L protein is soluble but with a low expression level less than 5%.(2)Expression of the N-terminal truncated PCV3 capsid proteinIn order to find reasons that resulted low expression of L protein,we constructed the N-terminal truncated PCV3 capsid protein.We acquired partial coding sequence of PCV3 capsid protein from L-pET28 a by PCR deleting the gene sequence which codes N-terminal Arg-rich peptide of PCV3 capsid protein.After constructing the NORpET28 a plasmid,we expressed the N-terminal truncated PCV3 capsid protein in E.coli(named as NOR protein).We found that although the expression level of NOR protein was higher reaching about 40%,NOR protein was expressed as inclusion body.(3)Expression of the N-terminal replaced PCV3 capsid proteinIn order to make the expressed PCV3 capsid protein soluble and abundant,we used the N-terminal Arg-rich acid sequence of PCV2 b capsid protein to replace the Nterminal Arg-rich acid sequence of PCV3 capsid protein and constructed DL-pET28 a plasmid.The N-terminal replaced PCV3 capsid protein was expressed in E.coli(named as DL protein)and the DL protein was soluble with high expression level about 30%.(4)Expression of the decoy epitope replaced PCV3 capsid proteinIn order to express the decoy epitope replaced PCV3 capsid protein,we used the method of bioinformatics to predict the neutralizing epitope and decoy epitope location of PCV3 capsid protein and constructed DLCS-pET28 a plasmid in which the coding sequence of decoy epitope was replaced by coding sequence of neutralizing epitope based on DL-pet28 a.And the protein was successfully expressed in E.coli with high expression and good solubility(named as DLCS protein).The results above indicate that the N-terminal Arg-rich peptide is the reason that cause the low expression of PCV3 capsid protein;Deleting the N-terminal Arg-rich peptide can improve the expression level of PCV3 capsid protein in E.coli but the solubility is too bad;However,N-terminal Arg-rich peptide of PCV2 b capsid protein replacing the N-terminal Arg-rich peptide of PCV3 capsid protein can make the expressed PCV3 capsid protein soluble and abundant;The expressed decoy epitope replaced PCV3 capsid protein(DLCS)which is constructed on the base of DL protein is also soluble with high expression.2.The preliminary research about immune activity of DL and DLCS proteinThe DL and DLCS protein was purified by ammonium sulfate precipitation.Then the DL or DLCS protein combined with oil-in-water adjuvant was injected individually into ICR mice.We had detected that there was special antibody in the serum of mice by indirect ELISA.And the recall experiment in vivo proved that DL or DLCS protein can stimulate memory immune cells.That is to say,DL and DLCS may have good immune activity.Above all,we successfully expressed reconstructed PCV3 capsid protein in E.coli with good solubility and high expression level by different expression strategies.We hope that the study of PCV3 capsid protein expression strategies can provide ideas for other virus capsid protein expression in E.coli.