Zika virus E protein conjugate vaccine and preparation method thereof
A protein-binding vaccine, Zika virus technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, viruses, etc., can solve problems such as long-term toxicity, non-degradability, and limited clinical application of nanoparticle vaccines
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Embodiment 1
[0074] Embodiment 1: Expression and purification of E protein
[0075] The gene sequence of the E protein is derived from the envelope protein of the Zika virus BeH818995 strain (Genebank number: KU365777); 6 histidine residues are fused to the C-terminus of the E protein gene sequence source (1-409 amino acid residues). After the synthetic gene sequence was cloned into pET21a expression vector, it was transformed into Escherichia coli BL21(DE3) for high-level expression. Synthetic DNA sequences were determined by restriction enzyme digestion and DNA sequence analysis. Activate Escherichia coli cells expressing E protein, inoculate 200ml LB medium at a ratio of 1%, and culture with shaking until OD 600 Equal to 0.6-0.8, add IPTG with a final concentration of 0.5mmol / L, and induce at 37°C for 3-4 hours. Centrifuge at 4° C. and 10000 rpm for 10 minutes, collect the bacterial cells, and resuspend in 50 mM Tris-HCl buffer (pH 8.0). Ultrasonication was performed for 1 hour in an...
Embodiment 2
[0078] Embodiment 2: Preparation and purification of vaccine
[0079] (1) Preparation of E-PS-1
[0080] E protein has a cysteine residue located on the surface of the E protein molecule; the sulfhydryl group of cysteine can be used to covalently bind β-glucan. Such as figure 2 Sodium periodate was dissolved in a volume of 5 mL of 50 mM acetate buffer (pH 5.6) to a final concentration of 40 mM. β-glucan (PS-OH) was dissolved in a volume of 5 ml of 50 mM acetate buffer (pH 5.6) to make a final concentration of 10 mg / mL. The two were mixed and placed at room temperature in the dark for 20 minutes. Subsequently, unreacted sodium periodate was removed by dialysis against PBS buffer (pH 7.4). Oxidized β-glucan (5 mL) with a concentration of 2 mg / mL and aminoethyl-maleimide ester (5 mL) with a concentration of 0.2 mg / mL were reacted overnight in PBS buffer (pH 7.4), and the reaction temperature is 4°C. Unreacted aminoethyl-maleimide ester was removed by dialysis against P...
Embodiment 3
[0085] Embodiment 3: the purity of gel filtration analysis vaccine
[0086] E-PS-1 and E-PS-2 were identified by analytical Superdex 200 gel filtration column (1.0cm×30cm), the eluent was PBS buffer (pH 7.4), and the flow rate was 0.5mL / min. Such as Figure 5 As shown, E protein, E-PS-1 and E-PS-2 all present a single elution peak. Compared with the elution peak of E protein (15.4mL), the elution time of E-PS-1 and E-PS-2 was obviously earlier. This indicates that covalent attachment to β-glucan significantly enhances the hydration volume of E protein. The peak elution time of E-PS-1 and E-PS-2 was 7.7mL, indicating that the hydrazone bond and disulfide bond as connecting bridges did not change the hydration volume of the conjugate.
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