Recombinant subunit vaccine of novel coronavirus South African mutant strain and application of recombinant subunit vaccine

[0018] Embodiment 1, carrier construction

[0019] By chemical synthesis, Beijing Liuhe Huada Gene Technology Co., Ltd. synthesized the fusion protein-related gene sequence of the antigenic epitope of the new coronavirus South African mutant COVID-19 South African mutant vaccine. in,

[0020] The RBD nucleotide sequence of the novel coronavirus South African mutant strain COVID-19 South African mutant strain is as follows (SEQ ID NO: 1):

[0021]

[0022] The amino acid sequence of the encoded RBD is as follows (SEQ ID NO: 2):

[0023]

[0024] The encoded SD1 amino acid sequence is as follows (SEQ ID NO: 3):

[0025]

[0026] The nucleotide sequence of SD1 is as follows (SEQ ID NO: 4):

[0027]

[0028] The encoded hFc amino acid sequence is as follows (SEQ ID NO: 5):

[0029]

[0030] The nucleotide sequence of hFc is as follows (SEQ ID NO: 6):

[0031]

[0032] RBDSD1-hFc amino acid sequence (SEQ ID NO: 7)

[0033]

[0034] The nucleotide sequence...

[0038] Example two, cell transient

[0039] ①The day before transfection, according to 2.0×10 6 cells / mL density inoculation, the cell density can reach 4.0×10 on the second day of culture 6 cells / mL or so;

[0040] ② After counting the cells on the second day of culture, the cell viability is >95%, and the viable cell density is ≥4.0×

[0041] 10 6 cells / mL, can be used directly; if the cell density is lower than 4.0×10 6 cells / mL, the cells can be collected by centrifugation (800rpm, 5min), and the cells6 cells / mL density resuspended in Transpro CD01 medium;

[0042] ③According to the optimized transient process, prepare the DNA and PEI mixture;

[0043] ④ Add the mixed solution to the culture medium for cultivation;

[0044] ⑤After 18 hours of culture, it is recommended to add a final concentration of 2mM valproic acid (VPA) and an initial culture volume of 5%+0.5%DN feed 2+DN feed B2, which can further increase the density of viable cells and protein expression. If ...

[0046] Embodiment three, protein purification

[0047] The protein extracted in Example 2 was purified by affinity chromatography and then ion exchange as follows.

[0048] 1. For the purification of RBDSD1-Fc, the following Protein A affinity chromatography method is adopted

[0049] One) the overall steps of the Protein A affinity chromatography method are as follows

[0050] Chromatography column: AT ProteinA Diamond affinity chromatography medium, column volume CV: 10ml, flow rate: 4-6ml / min, pressure limit: ≤0.3MPa.

[0051] Pretreatment: first rinse with 0.1M NaOH for at least 3 CVs, and then rinse with purified water for at least 10 CVs.

[0052] Equilibrium: Wash 2 to 3 CVs with washing buffer (0.1M HAc-NaAc, pH3.0), then fully equilibrate at least 10 CVs with binding buffer (0.02M PB, 0.5M NaCl, pH7.0) stand-by.

[0053] Sample loading: Take the filtered CHO suspension cell culture and use 5M NaCl to adjust the conductance to be consistent with the binding buffer,...