The Design, Expression And Activity Of VP1Epitope Peptide And Its Fusion Protein

Background:For inactivated virus vaccines, if the processing is not strict, such as inadequateinactivation, this will leave a serious hidden trouble for future. Genetic engineeringvaccines can only contain the active ingredients of virus antigen and nothing else, i.e.an antigen epitope vaccine, so as to escape such a risk. Epitope-based vaccine is theuse of genetic engineering methods for the effective parts constructed recombinantprotein antigen vaccine, although only a part of the viral proteins, but has the sameimmunogenicity of the full-length viral protein. If an epitope-based vaccine protein isfused with other proteins of relative merits to express, this can further improve theepitope vaccine immunogenicity and soluble-expression chances of an epitope-basedvaccine. The lab the researcher has been with has constructed a series of eptitopepeptide vaccines based on the T cell and B cell epitopes of a FMDV VP1protein, butthese vaccines are expressed in the form of inclusion body in E. Coli, so the waydifferent epitopes arranged in a vaccine should be further explored and the effectiveaccompanied protein composing a fusion protein with an eptitope peptide vaccineshould be sreened from a number of candidates in order to significantly improve theimmunogenicity and soluble-expression chances of an epitope-based vaccine.Purpose:Optimize the serial combinations of epitopes from VP1in an epitope-basedvaccine by SWISS MODEL to construct a epitope peptide similar to the naturalspatial structure of VP1and determine the effective accompanied protein that canenhance the immunogenicity and soluble-expression chances of an epitope-based vaccine when fused with the vaccine, ultimately to develop a highly efficientepitope-based vaccine.Method:Construct the expression vector based on pET28a (+) of the VP1epitope peptidenamed JXL-1. Construct a series of fusion protein consisting of JXL-1and a proteinthat is the N-terminal first41N-terminal amino acids (N41), or the N-terminal first214N-terminal amino acids of PCV capsid protein(PCV(214aa)), or the C-teminalpart of HSP65(HLC) or the the C-teminal part of a flagellin, to form PFJ, HJC, HJCand PFJ, respectively. All fusion-protein plasmids are transformed into BL21(DE3)and induced by IPTG to express proteins. All the recombinant proteins in this researchare labeled with his-tag, so Ni-affinity chromatography will be used to purify proteins.Detect protein activity by ELISA and immunizing mice.Results:1. A series of epitope peptides have been designed, from which the optimalepitope combination is determined according to protein spatial structure predictionand named as JXL-1. The expression vector of JXL-1was successfully constructed;2. A series of fusion protein, all based on JXL-1, were respectively constructed:CJ, HJC, PFJ and NJ;3. JXL-1doesn’t express and the reason is hitherto unknown; CJ and HJCexpress in the form of inclusion body; one third of the PFJ is soluble expression, NJ iscompletely soluble expression;4. The purified PFJ has a purity is relatively low that cannot be used to immunizemice;5. The sera from mice immunized with NJ immunizing mice will can recognizeinactivated FMDV.. Conclusion:1. The epitope peptide, JXL-1, alone can not be expressed, and it can only getexpressed as a part of fusion protein;2. The fusion expression of JXL-1and N41can realize the soluble expresion in E.Coli.;3. Comparing with other accompanied proteins, N41from PCV capsid proteinhas a better ability to aid a foreign peptide to solubly express in E. Coli.;4. The activity of NJ is desirable..