Vaccine fusion protein

[0175] Example 1 Construction of fusion protein based on flagellin fusion protein

[0176] Construct fusion proteins, from N-terminal to C-terminal: flagellin flagellin (FliC) N-terminal constant region (1-176 amino acids, SEQ ID NO: 6), B cell epitope hot spot region II (B2, SEQ ID NO: 6) NO: 21, SEQ ID NO: 22 and SEQ ID NO: 28 overlap), SARS-CoV-2 receptor binding domain RBD (containing B1 fragment, SEQ ID NO: 5), T cell epitope (SEQ ID NO: 24 overlaps with SEQ ID NO: 25), panT (pan-HLA-DR binding epitope/helper T cell epitope, i.e. CD4 + cellular epitope, SEQ ID NO: 4), and the flagellin FliC C-terminal constant region (amino acids 403-495, SEQ ID NO: 7). Among them, flagellin is an intramolecular adjuvant, which together with the SARS-CoV-2 antigen constitutes a recombinant vaccine, and the obtained fusion protein is called SC2V7 (the amino acid sequence is shown in SEQ ID NO: 9, and the specific structure is shown in figure 2 shown in A).

[0177] The fusion protein was constructed, from N-terminal to C-terminal: flagellin flagellin (FliC) N-terminal constant region (1-176 amino acids, SEQ ID NO: 6), panT (pan-HLA-DR binding epitope/helper T) cell epitope i.e. CD4 + Cell epitope, SEQ ID NO: 4), SARS-CoV-2 receptor binding domain RBD (containing B1 fragment, SEQ ID NO: 5), T cell epitope (SEQ ID NO: 26), and flagellin FliC C-terminal constant region (amino acids 403-495, SEQ ID NO: 7). Among them, flagellin is an intramolecular adjuvant, which together with the SARS-CoV-2 antigen constitutes a recombinant vaccine, and the obtained fusion protein is called SC2V8 (the amino acid sequence is shown in SEQ ID NO: 10, and the specific structure is shown in figure 2 shown in B).

[0178] The nucleotide sequences encoding SC2V7 and SC2V8 are shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively.

[0179] In addition, the C-terminal constant region of flagellin flagellin (FliC) was replaced by a fragment comprising the amino acid sequence shown in SEQ ID NO: 7 to a fragment comprising the amino acid sequence shown in SEQ ID NO: 8 to construct a fusion without the complete C-terminal Proteins SC2V7-1 (SEQ ID NO: 11) and SC2V8-1 (SEQ ID NO: 12), the nucleotide sequences encoding SC2V7-1 and SC2V8-1 are shown in SEQ ID NO: 15 and SEQ ID NO: 16, respectively. Show.

[0180] Example 2 Fusion protein expression verification

[0181] The nucleic acid sequences encoding SC2V7 and SC2V8 were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. and inserted between Nde I and Pst I of the pET21a expression vector (purchased from EMD Biosciences) to obtain the expression vector. The expression vector was transformed into E. coli BL21-Gold (DE3) and grown in LB medium (containing 100 µg/ml ampicillin) overnight at 37°C. Large-scale expression of the transformed E. coli in TB medium (containing 100µg/ml ampicillin), when the DO595 is 2.0-2.5, was induced by adding 1mM IPTG (purchased from Sangon Bioengineering (Shanghai) Co., Ltd.), 37°C , incubate for 4 hours. The cultured bacteria were collected, crushed under high pressure, the bacterial lysate was heated at 42°C overnight, the precipitate was collected by centrifugation, and the precipitate was resuspended in 0.2-0.25M ammonium sulfate solution at 4°C for 2 hours with slow stirring. Centrifuge, the pellet was washed with PBS-Tween buffer (10 mM Na 2 HPO 4 ,1.8 mM KH 2 PO 4 , 137 mM NaCl, 2.7 mM KCl, pH 7.4 and 0.25% Tween20) and resuspended with slow stirring overnight. After centrifugation again, the resulting pellet was resuspended in 3-3.5M urea (PBS, 20% glycerol, pH 9.0) buffer at 4°C overnight. The fusion protein was collected on a size exclusion column (HiLoad 26/60 Superdex 200 pg SECcolumn), the column was equilibrated with 3-3.5M urea (PBS, 20% glycerol, pH 9.0) buffer. Dialyzed overnight at room temperature for reconstitution with 50 mM Tris–HCl, 800 mM NaCl (pH 7.0). The reaggregated fusion protein was further concentrated and purified with 0.5M ammonium sulfate solution. After centrifugation, the pellet was resuspended with PBS-Tween buffer, and then dialyzed against PBS buffer.

[0182] The purified fusion proteins SC2V7 and SC2V8 were obtained. The bacterial solution harvested after IPTG induction and the samples collected by ultrasound were separated by 15% SDS-PAGE and then transferred to PVDF membranes. They were blocked with 1 × TBST containing 5% nonfat milk powder at room temperature for 1 h; -His-tag antibody or anti-SARS-CoV S protein RBD antibody (Sino Biological, cat#40150-T62-COV2), incubate for 1 h at room temperature; wash 3 times with 1× TBST, 5 min each; add HRP-labeled rabbit anti-mouse IgG (1:10000 dilution, purchased from Santa Cruz, USA), incubated at room temperature for 1 h; washed 3 times with 1×TBST for 5 min each, and washed twice with 1×TBS for 5 min each. ECL color development, line negative exposure analysis results. The result is as image 3 and Figure 4 shown, SC2V7 ( image 3 ) and SC2V8 ( Figure 4 ) protein expression was successful and the composition was single.

[0183] Example 3 Fusion protein RBD conformation verification

[0184] 1) The correctly folded RBD domain will be able to interact with its receptor ACE2, which is used as a principle to verify the RBD conformation in recombinant vaccines. The recombinant vaccine protein was obtained according to the method of Example 2, and the ACE2 protein (Sino biological, Cat: 10108-H08H) was coated in the ELISA microplate, and after washing, the blocking solution was added to block, and then the recombinant vaccine protein SC2V7 or SC2V8 was added to the microplate. Incubate in the well plate, add anti-RBD antibody (Sino biological, Cat: 40150-T62-COV2) to incubate after washing, add secondary antibody (Jackson ImmunoResearch, Cat: 111-035-003) to incubate after washing, and finally wash and add After the reaction substrate was incubated for an appropriate time, the reaction was terminated, and the data of each well was read to analyze the results. The results showed that both fusion proteins SC2V7 and SC2V8 could interact with ACE2 coated in the microplate ( Figure 5).

[0185] 2) The correctly folded RBD domain induces the animals to produce corresponding antibodies, which can correctly recognize the S1 protein of the new coronavirus (SARS-CoV-2), so as to verify the RBD conformation in the recombinant vaccine.

[0186] Immunized normal C57 mice: 6-8 week old C57 mice were inoculated with the fusion proteins (SC2V7 and SC2V8) prepared in Example 2. In the first week, the immunization dose was 25µg/mouse, and it was halved in the next two weeks. The immune part was injected intramuscularly in the hind leg. After three consecutive weeks of immunization, mice were sacrificed for blood collection and serum separation.

[0187] Immune serum isolation: mice were enucleated or heart blood was collected, and left standing at room temperature for 1 h until the blood coagulated. The coagulated blood was centrifuged at 3000rpm for 10min, and the supernatant was carefully removed to obtain serum. The serum was stored at -20°C after aliquoting.

[0188] Prepare 100mM carbonate coating buffer, dilute the S protein of SARS-CoV-2 virus to 20μg/ml, coat it in ELISA microplate, and incubate at room temperature for 2h. The coating supernatant was discarded, and after washing twice with PBS, the liquid in the plate was carefully removed, blocking solution was added to block, and the cells were incubated at room temperature for 2 h. After washing twice with PBS, immune serum diluted with blocking solution was added and incubated at room temperature for 2 h. After washing 4 times with PBS, appropriate concentration of secondary antibody was added and incubated at room temperature for 1 h. After washing twice with PBS, the reaction substrate was added, incubated at room temperature for 10-20 min, and an equal volume of stop solution was added to terminate the reaction. Read the absorbance data of each sample well and analyze the results.

[0189] The result is as Image 6 As shown, the immune serum after immunizing mice with recombinant vaccine protein SC2V8 (the fusion protein described in this application) can correctly recognize the new coronavirus S protein (SARS-CoV-2).