Nanoparticles for resisting H1N1 subtype influenza A virus based on self-assembled ferritin as well as preparation method and application of nanoparticles

[0048] Example 1 Construction of plasmid signal peptide-SpyTag-ferritin monomer subunit (signal peptide-ST-FE) and signal peptide-SpyCatcher-anti-H1N1 subtype influenza virus hemagglutinin protein Nanobody (signal peptide-SC-HA -VHH)

[0049] First, the gene signal peptide-ST-FE and the signal peptide-SC-HA-VHH were synthesized. The gene tandem structure is as follows figure 1 , and optimized by the codons of the CHO eukaryotic expression system, synthesized by GenScript Biotechnology Co., Ltd. The amino acid sequence of the signal peptide-ST-FE (SEQ ID NO: 1) is as follows:

[0050] MGWSCIILFLVATATGVHSAHIVMVDAYKPTKGGGSGGGSGGGSRMLKALNDQLNRELYSAYLYFAMAAYFEDLGLEGFANWMKAQAEEEIGHALRFYNYIYDRNGRVELDEIPKPPKEWESPLKAFEAAYEHEKFISKSIYELAALAEEEKDYSTRAFLEWFINEQVEEEASVKKILDKLKFAKDSPQILFMLDKELSARAPKLPGLLMQGGE;

[0051] The amino acid sequence (SEQ ID NO: 2) of the signal peptide-SC-HA-VHH is as follows:

[0052] MGWSCIILFLVATATGVHSSYYHHHHHHDYDIPTTENLYFQGSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHIGGGSGGGSGGGSQVQLVESGGGLVQSGGSLRLSCAASGSMSRIITMGWYRQAPGMERELVAVIGNNDNTVYGDSVQGRFTVSRDNAKNTAYLQMNSLNAEDTAMYYCKISTLTPPHEYWGQGTQVTVSSHHHHHH;

[0053] The amino acid sequence (SEQ ID NO: 3) of the signal peptide-SpyTag is as follows:

[0054] MGWSCIILFLVATATGVHSAHIVMVDAYKPTK;

[0055] The amino acid sequence of the signal peptide-SpyCatcher (SEQ ID NO: 4) is as follows:

[0056] MGWSCIILFLVATATGVHSSYYHHHHHHDYDIPTTENLYFQGSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHI;

[0057] The amino acid sequence of the linker peptide (SEQ ID NO: 5) is as follows:

[0058] GGGSGGGSGGGS;

[0059] The amino acid sequence of the signal peptide (SEQ ID NO: 6) is as follows:

[0060] MGWSCIILFLVATATGVHS.

[0061] After the signal peptide-ST-FE and signal peptide-SC-HA-VHH genes are synthesized, the synthetic gene is used as a template to amplify the target fragment by PCR technology. The amplification primers are:

[0062] Signal Peptide-ST-FE:

[0063] Upstream primer sequence: AATCTCTAGAATGGGCTGGAGCTGCAT (SEQ ID NO: 7);

[0064] Downstream primer sequence: AATCAAGCTTTTACTCGCCGCCCTGCAT (SEQ ID NO: 8);

[0065] Signal Peptide-SC-HA-VHH

[0066] Upstream primer sequence: AATCTCTAGAATGGGCTGGAGCTGCAT;

[0067] Downstream primer sequence: AATCAAGCTTTTAGTGGTGGTGGTGGTG (SEQ ID NO: 9).

[0068] The nucleotide sequences of the signal peptide-ST-FE and signal peptide-SC-HA-VHH genes are as follows:

[0069] Signal Peptide-ST-FE:

[0070] GAATTCATGGGTTGGAGTTGCATCATCCTATTTCTAGTGGCCACCGCTACCGGCGTGCACTCTGCCCACATCGTGATGGTGGACGCCTACAAGCCCACAAAGGGCGGAGGCAGCGGCGGCGGCTCTGGCGGAGGATCTCGGATGCTGAAGGCCCTGAACGACCAGCTGAATCGGGAGCTGTACTCCGCCTACCTGTACTTTGCCATGGCCGCTTACTTCGAGGACCTGGGCCTGGAGGGCTTCGCCAACTGGATGAAAGCTCAGGCCGAGGAAGAGATCGGCCACGCCTTGAGATTCTACAACTACATCTACGACAGAAACGGCAGAGTGGAACTGGATGAGATTCCTAAGCCTCCAAAAGAGTGGGAGAGCCCCCTGAAGGCTTTCGAGGCTGCTTACGAGCATGAGAAGTTCATCTCCAAGTCCATCTACGAGCTGGCTGCTCTGGCAGAGGAAGAAAAGGATTATTCCACCAGAGCCTTCCTGGAATGGTTCATCAACGAGCAAGTCGAAGAAGAGGCCTCCGTGAAGAAGATCCTGGACAAGCTGAAGTTTGCCAAGGACTCCCCTCAGATCCTGTTCATGCTCGATAAAGAACTGTCTGCTCGGGCCCCTAAGCTGCCTGGCCTGCTGATGCAGGGCGGCGAGTGAGGATCC(SEQ ID NO:10);

[0071] Signal Peptide-SC-HA-VHH:

[0072]GAATTCATGGGTTGGAGTTGCATCATCCTATTTCTAGTGGCCACCGCTACCGGCGTGCACTCCTCCTACTACCACCACCACCACCACCACGACTACGACATTCCCACCACCGAGAACCTGTACTTCCAGGGCTCCGCCACACACATCAAGTTCTCCAAGAGAGACGAGGATGGCAAAGAGCTGGCTGGCGCTACAATGGAACTGAGAGATAGCTCTGGCAAAACAATCTCTACCTGGATCAGCGACGGCCAAGTGAAGGACTTCTACCTCTATCCTGGCAAGTACACCTTCGTGGAAACAGCTGCTCCTGATGGCTACGAGGTGGCTACCGCCATCACCTTTACCGTGAACGAGCAGGGCCAGGTCACCGTGAACGGCAAGGCCACCAAGGGCGATGCCCACATCGGCGGAGGATCTGGCGGAGGCTCCGGCGGAGGCTCTCAGGTGCAGCTGGTGGAATCTGGAGGTGGCCTGGTGCAGTCCGGCGGCAGCCTGCGGCTGTCCTGTGCCGCTTCTGGCTCCATGAGCCGGATCATCACCATGGGCTGGTACAGACAGGCCCCAGGCATGGAACGCGAGCTGGTCGCCGTGATCGGCAACAACGACAATACCGTTTACGGCGACTCCGTGCAAGGCAGATTCACCGTGTCTCGGGACAATGCCAAGAACACCGCTTATCTGCAGATGAACTCCCTGAACGCCGAGGACACCGCCATGTACTACTGCAAGATCTCCACCCTGACACCTCCTCACGAGTACTGGGGCCAGGGCACCCAGGTGACCGTGTCCTCTCACCATCACCACCATCATTGAGGATCC(SEQ IDNO:11)。

[0073] The target gene amplification system is shown in Table 1:

[0074] Table 1

[0075]

[0076] The PCR reaction program of the target gene is shown in Table 2:

[0077] Table 2

[0078]

[0079] The amplified products were electrophoresed on a 1% agarose gel, and the results were as follows figure 2 shown, is the same as the expected fragment length.

[0080] The PCR products were recovered, and the two target fragments recovered from the PCR gel were digested with the eukaryotic expression plasmid P3 plasmid using restriction enzymes Xba I and Hind III respectively; T4 DNALigase was used to connect the above two restriction fragments to construct Two target gene vector plasmids were obtained, namely plasmid signal peptide-ST-FE (p3-ST-FE) and signal peptide-SC-HA-VHH (p3-SC-HA-VHH), which were stored at -20°C.

[0081] Example 2 Linearization of Plasmid Signal Peptide-ST-FE and Signal Peptide-SC-HA-VHH

[0082] Take out the plasmid signal peptide-ST-FE and signal peptide-SC-HA-VHH stored at -20°C, and measure their concentrations by NanoDrop one to be 1200ng/μL and 890ng/μL, respectively, and use the restriction endonuclease PvuⅠ for single enzyme Cut, concrete reaction system is shown in following table 3, table 4:

[0083] Table 3 Plasmid signal peptide-ST-FE single digestion reaction system

[0084]

[0085] Table 4 Plasmid signal peptide-SC-HA-VHH single enzyme digestion reaction system

[0086]

[0087] Add each component according to the single enzyme digestion reaction system table, and place it in a 37°C water bath for 2h digestion.

[0088] During digestion, prepare a 1% agarose gel. First, prepare the gel plate, select and insert the comb according to the total enzyme digestion system, then weigh 0.5g of agarose into a 100mL beaker, weigh 50mL of 1×TAE with a graduated cylinder, and add it to it, mix well and place it in a microwave oven for heating Take it out for 30s to observe the dissolution of the agarose, repeat heating to make it fully dissolved, then add 2 μL GoldView I nucleic acid dye to it, shake it gently (at this time, force evenly to avoid bubbles), pour the gel, and let it stand at room temperature for 40min to make the gel fully solidified. Then, the single-enzyme-digested product was mixed with an appropriate amount of 6×Loading Buffer and added to the gel well. The non-single-enzyme-digested knockout plasmid and DL 10000 DNA Marker were used as controls. The electrophoresis conditions were constant voltage 110V, 600mA, 30min. After electrophoresis, the gel block was gently taken out and placed in a blue-light gel cutter for gel cutting. According to the instructions of the gel recovery kit (SanPrepColumn DNA Gel Extraction Kit), the purified single-enzyme digested vector was recovered. The purified product was recovered and the concentration and OD were determined using the NanoDrop one 260/280 , stored at -20°C. 1% agarose gel as image 3 As shown, linearized plasmids run slower than circular plasmids, as expected.

[0089] Example 3 Electrotransfection of linear signal peptide-ST-FE and signal peptide-SC-HA-VHH GS gene deletion CHO cells

[0090] (1) Preparation before electroporation of CHO suspension cells lacking GS gene. 24h before electroporation, cells were at a viable cell density of 5×10 5 The cells/mL were inoculated into a 125 mL conical flask for shaking culture, the culture volume was 30 mL, and the rotational speed of the carbon dioxide shaking incubator was set to 110 rpm.

[0091] (2) Collection of CHO suspension cells lacking GS gene. On the day of electroporation, samples were taken to calculate the viable cell density and viability (98%) of suspension culture, and 1×10 cells were collected by calculation. 7 The cell suspension volume required for each viable cell was placed in a sterile centrifuge tube, centrifuged at 1000 rpm for 5 min, and then resuspended in 400 μL of 302 medium (containing glutamine).

[0092] (3) Incubation of cells with plasmids. Add 40 μg of linearized knockout plasmid (linear plasmid p3-ST-FE or linear plasmid p3-SC-HA-VHH) to the above-treated cell suspension, mix by gently pipetting, and incubate at room temperature for 10 min.

[0093] (4) Electric shock. The parameters of the electroporator are set in advance, and the electric shock conditions are: voltage 280V, electric shock time 20ms, and electric shock once. After 10 min incubation at room temperature, the above cell plasmid mixture was quickly added to a pre-cooled 4 mm electric shock cup, and electric shock was performed once.

[0094] (5) Cell treatment after electric shock. After the electric shock, the cells after the electric shock were quickly transferred to 20 mL of pre-warmed 302 medium (containing glutamine), and the T75 cell culture flask was left to culture for 24 hours.

[0095] (6) 24h after electric shock, the cells were sampled to calculate the viability and record. The cell viability was 48% after 24h electroporation with linear plasmid p3-ST-FE, and 52% after 24h electroporation with linear plasmid p3-SC-HA-VHH.

[0096] (7) Continue culturing to a cell viability rate of more than 95% using a medium without glutamine.