Novel coronavirus multivalent antigen as well as preparation method and application thereof
A coronavirus, a new type of technology, applied in the field of biomedicine, can solve problems such as reducing the protective effect of vaccines
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Embodiment 1
[0051] Example 1: Design of novel coronavirus broad-spectrum protective vaccine RBD-tr2-WTV2
[0052] Analysis of new coronavirus mutant strains: In January and February 2020, the D614G mutation of the new coronavirus S protein appeared. In June 2020, the D614G mutant virus strain became the main epidemic strain in the world. Cell and animal experiments showed that the D614G mutation enhanced Compared with the original strain, the infection of D614G new coronavirus will not enhance the pathogenicity. In December 2020, a new mutant strain 501Y.V1 (also called VOC-202012 / 01, belonging to B.1.1.7lineage) appeared, which has 23 mutations compared with the original strain, which may have a greater impact on the S protein The three mutations are N501Y, 69-70del (deletion) and P681H, where the N501Y mutation enhances the binding ability of the S protein to the receptor ACE2 protein, and 501Y.V1 has a stronger transmission ability, but does not enhance the pathogenicity. Also in Dece...
Embodiment 2
[0055] Example 2: Detection of protein expression by Western blot
[0056] The amino acid sequence of the designed RBD-tr2-WTV2 antigen is SEQ ID NO: 4 (signal peptide and histidine sequence are not given in the sequence), and the nucleotide sequence for expressing the antigen is SEQ ID NO: 5, nucleotide The sequence elements from N-terminal to C-terminal are (1) Kozak sequence gccacc; (2) sequence encoding MERS-S protein signal peptide: MIHSVFLLMFLLTPTES (SEQID NO: 6); (3) New coronavirus (GenBank on NCBI: QHR63250 ) S protein RBD (R319-K537); (4) New coronavirus (GenBank on NCBI: QHR63250) S protein RBD (R319-K537), which contains three point mutations are K417N, E484K, N501Y; (5) 6 histidine; (6) stop codon. The gene sequence was synthesized in Suzhou Jinweizhi Co., Ltd., and cloned into pCAGGS plasmid through EcoRI and XhoI restriction sites to obtain pCAGGS-RBD-tr2-WTV2 plasmid.
[0057] The pCAGGS plasmid expressing RBD-tr2-WTV2 was transfected into HEK293T cells, and ...
Embodiment 3
[0058] Example 3: Expression and purification of RBD-tr2-WTV2
[0059] Use HEK293F cells suitable for mass expression of proteins to express RBD-tr2-WTV2 antigen: transfect HEK293F cells with the plasmid pCAGGS-RBD-tr2-WTV2, collect the supernatant after 5 days, centrifuge to remove the precipitate and filter through a 0.22 μm filter membrane, further Remove impurities. The cell supernatant was adsorbed by a nickel affinity column (Histrap, GE Healthcare) at 4°C, and washed with buffer A (20 mM Tris, 150 mM NaCl, pH 8.0) to remove non-specific binding proteins. Then use buffer B (20mM Tris, 150mM NaCl, pH 8.0, 1000mM imidazole) to gradiently elute the target protein from Histrap, and set the ratio of buffer B to 2%, 5%, 10%, 20% in turn , 30%, 50%, 100%, see the results figure 2 . Concentrate the eluted protein solution corresponding to 10% imidazole with a 10kD concentrator tube, and change the liquid more than 30 times until the final volume of buffer A is less than 1ml....
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