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 wa

[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. Centri

[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 protei