A ferritin-based multi-antigen universal influenza vaccine and its preparation method and application
A technology of influenza vaccine and ferritin, which is applied to the multi-antigen universal influenza vaccine based on ferritin and the fields of preparation and application thereof, and achieves the effects of high operability and technical value, good broad-spectrum immune effect and simple operation.
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experiment example 1
[0108] A kind of FRT nanoparticle derived from horse spleen (purchased from Sigma-Aldrich Co.LLC., the FRT nanoparticle carrier described in the following Experimental Examples 2-14 is the same as Experimental Example 1) as a carrier, the surface of which is covalently bound to the M2e antigen. Antigen influenza vaccine M2e.FRT, its preparation method is as follows:
[0109] 1) Artificially synthesized M2e polypeptide antigen (SEQ ID NO.1) derived from A / Puerto Rico / 8 / 1934 H1N1 influenza virus.
[0110] 2) Prepare a FRT carrier solution with a final concentration of 1 mg / mL in 0.1M PBS (pH 7.4), and add 10 times the molar amount of NHS-PEG to it n -Mal cross-linking agent, 4 ° C shaker in the dark for 1 h, so that the amino terminal of FRT is modified with maleimide group, and then take out and remove the unreacted residual cross-linking agent. 0.5mg / mL PEG-FRT solution modified by PEG cross-linking agent, mixed with M2e polypeptide antigen 5 times the molar weight of FRT sub...
experiment example 2
[0113] A single-antigen influenza vaccine NP.FRT with FRT nanoparticles as the carrier and NP antigen distributed on the surface.
[0114] Compared with Experimental Example 1, except that the artificially synthesized NP polypeptide antigen (SEQ ID NO.2) derived from A / Puerto Rico / 8 / 1934H1N1 influenza virus was used to replace the M2e antigen and FRT for covalent cross-linking, its preparation method Same as experimental example 1. Finally, the NP.FRT single-antigen influenza vaccine with NP antigen covalently bound to the surface was prepared, and the covalent binding rate of NP antigen on the surface was about 15 NP antigen polypeptide molecules coupled to each NP.FRT molecule. (This example is used as a comparative example)
[0115] SEQ ID NO.2: QIASNENMETMESSTL-C
experiment example 3
[0117] A single-antigen influenza vaccine HA.FRT with FRT as a carrier surface covalently bound with HA full-length protein antigen, its preparation method is as follows:
[0118] 1) A / Puerto Rico / 8 / 1934H1N1 influenza virus HA full-length protein (SEQ ID NO.3) was recombinantly expressed and purified, with a final purity of over 90%.
[0119] 2) Referring to the method described in Experimental Example 1, a FRT carrier solution with a final concentration of 1 mg / mL was prepared with 0.1M PBS (pH 7.4), and 10 times the molar amount of NHS-PEG was added to it. n -Mal cross-linking agent, 4 ° C shaker in the dark for 1 h, so that the amino terminal of FRT is modified with maleimide group, and then take out and remove the unreacted residual cross-linking agent. 0.5 mg / mL of PEG-FRT solution modified by PEG cross-linking agent, and the recombinant HA antigen after reduction treatment with tris(2-carboxyethyl)phosphine-hydrochloride (TCEP·HCl) which is 2 times the molar amount of FR...
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