Recombinant S protein, recombinant plasmid and recombinant bacterium of novel coronavirus and application of recombinant S protein, recombinant plasmid and recombinant bacterium in preparation of exosome drug or exosome vaccine
A coronavirus and exosome technology, applied in the field of vaccines, can solve the problems of poor immunogenicity, enhanced respiratory diseases, and enhanced dependent infection, and achieves a wide range of cell adhesion molecules, simple preparation methods, and enhanced therapeutic effects Effect
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[0041] The present invention also provides a preparation method for the above-mentioned exosome drug or exosome vaccine, comprising the following steps: after mixing the above-mentioned recombinant S protein or the recombinant S protein obtained by using the above-mentioned recombinant bacteria with exosomes, performing electrotransduction, The precipitate is collected by centrifugation to obtain the exosome drug or exosome vaccine.
[0042] The electrotransduction described in the present invention preferably includes: taking 10 μg of exosomes and adding them to 200 μl of electrical buffer, putting them in EP tubes, and labeling tube A; adding 10 nmol of the recombinant S protein and 200 μl of electrical buffers into 1.5 ml of EP tubes tube, label tube B; mix the liquids in tubes A and B, transfer to a pre-chilled shock cuvette, and place on ice; then proceed to electrotransduction.
[0043] The parameter of electric transduction described in the present invention preferably ...
Embodiment 1
[0049] 1.1 Find the amino acids of S1 and S2 of the new coronavirus in the Uniprot protein database, use the 5' sequence of the S1 protein and the 3' of the S2 protein to use the flexible (linker) 3 connected.
[0050] 1.2 Digest the 5' and 3' ends of the recombinant S protein coding gene with NheI and EcorV, connect with the pET-28a plasmid using T4 ligase, transform into competent cells, obtain the recombinant plasmid, and obtain stable expression by monoclonal screening Positive colonies of the recombinant S protein.
[0051] 1.3 The above cell lines were expanded and cultured, secreted, expressed and purified to obtain the purified recombinant S protein of the new coronavirus.
[0052] 1.31 Inoculate the cell line of the recombinant S protein in 50 μg / ml ampicillin lysing broth (LB), shake the bacteria at 37°C at 160 rpm overnight, and inoculate the colony of the genetically engineered bacteria in LB / kam according to the inoculation amount of 3%. In the culture medium, s...
Embodiment 2
[0058] 2.1 The exosome-carrying new coronavirus spray vaccine prepared in Example 1 was used for subcutaneous immunization of mice
[0059] Female BALB / c mice aged 6-8 weeks were divided into 2 groups (vaccine particle group and negative control group), with 8 mice in each group, which were inhaled through the nose respectively. Dissolve the vaccine in a volume of 200 µL of PBS. Blood was collected from the tail of the mice every two weeks, and the serum was separated for antibody detection. Serum samples were stored at -20°C until use.
[0060] The level and titer of Delta-specific IgG antibody in serum of immunized mice were detected by indirect ELISA. Coat the ELISA plate with 20 μg / ml S1 or S2 protein, overnight at 4°C. Wash three times with 0.05% PBST, block with 1% BSA-PBST blocking solution, and incubate at room temperature for 1 h. Wash 5 times with 0.05% PBST, add 200μl diluted serum (1:1000 dilution), incubate at 37°C for 2h; wash 5 times with PBST, add HRP-goat ...
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