Foot-and-mouth disease virus bivalent multi-epitope recombinant virus-like particle and application thereof
A foot-and-mouth disease virus and recombinant virus technology, applied in the field of biological vaccines, can solve problems such as potential safety hazards and economic losses
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[0027] The preparation method of the carrier protein of the present invention preferably comprises connecting two hepatitis B core antigen THBcAg molecules in series using a linker to obtain the carrier protein; the amino acid sequence of the hepatitis B core antigen THBcAg molecule is shown in SEQ ID NO.2; The amino acid sequence of the linker is shown in SEQ ID NO.3.
[0028] The present invention inserts the antigenic epitope of foot-and-mouth disease virus respectively at the 78th to 82nd amino acid position of each hepatitis B core antigen THBcAg molecule of the carrier protein, and the antigenic epitope of foot-and-mouth disease virus includes the antigenic epitope of type A foot-and-mouth disease virus and the antigenic epitope of type O The antigenic epitope of foot-and-mouth disease virus, the antigenic epitope of described A type foot-and-mouth disease virus preferably comprises the recombinant B cell epitope of A type foot-and-mouth disease virus, the aminoacid seque...
Embodiment 1
[0037] 1. Design of bivalent multi-epitope recombinant protein of type A and type O foot-and-mouth disease
[0038] According to the gene sequences of A-type FMDV (AGDMM / CHA / 2013) and O-type FMDV (O / MAY98 / BY / 2010) on NCBI, two B cell expressions on the gene sequence of the structural protein VP1 of these two serotypes were respectively selected. The 134th to 161st and 200th to 213th amino acid sequences on VP1 are specially concatenated, and the linker sequence GGSSGG is introduced between different epitopes, so as to avoid the formation of new epitopes and form the recombinant B cell expression of type A FMDV Epitope gene (SEQ ID NO.4) and the recombinant B cell epitope gene (SEQ ID NO.5) of O-type FMDV.
[0039] Look up the gene sequence of the HBcAg molecule (GenBank accession number AEK93756) on NCBI, and select two molecules of HBcAg connected in series as the carrier protein of the compound epitope (i.e. dimer, amino acid sequence SEQ ID NO.1, nucleotide sequence SEQ ID ...
Embodiment 2
[0046] Preparation of recombinant virus-like particles
[0047] The three recombinant proteins obtained in Example 1 were refolded by the method of urea gradient dialysis for inclusion body protein. The purified protein was put into a dialysis bag (the molecular weight cut-off of the dialysis bag was 15KD), and then both ends of the dialysis bag were sealed and put into the refolding solution (composition: 0.5M NaCl, 50mM Tris, 0.5mM EDTA, 6M Urea, 10 % glycerol, 2mM GSH and 0.2mMGSSG, pH 7.3), and change the dialysate every 8h, and reduce the concentration of urea (6M, 4M, 2M, 0M) successively at the same time, and finally dialyze with PBS for 8h to obtain the urea after renaturation. Recombinant protein.
[0048] At the same time, negative staining electron microscopy was used to detect the recombinant protein THBc-AO. The sample was diluted to 0.8 mg / mL, tapped onto the copper grid and incubated for 5 min, negatively stained with 2% phosphotungstic acid, and observed unde...
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