Site-specific mutagenesis carrier protein and application thereof in preparation of vaccines
A technology of site-directed mutagenesis and carrier protein, which is applied in the preparation method of peptides, vaccines, multivalent vaccines, etc., can solve the problems of complicated separation and purification process, uneven binding process, and reduced polysaccharide protein binding efficiency, and achieve the optimization of modification conditions, Simple and easy modification reaction, protein harmless effect
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Embodiment 1
[0049] Construction of the carrier protein expression plasmid of embodiment 1 site-directed mutation
[0050] 1: Selection of mutation sites
[0051] The three-dimensional structures of HID and HIN47 were predicted using the online software phyre2, respectively.
[0052] 10 key amino acids were selected on the fusion proteins HIN47-HID and HID-HIN47 respectively. After these key amino acid positions are replaced, not only can the polysaccharide be bound at a fixed point, but it is expected to further reduce the residual protease activity of HIN47, which is conducive to improving the final polysaccharide binding product. stability. The information of the specific mutation site is shown in Table 1-Table 2, wherein the amino acid position refers to the position on the sequence shown in SEQ ID NO: 2-3.
[0053] Table 1 HIN47-HID mutation site
[0054]
[0055] Table 2 HID-HIN47 mutation sites
[0056]
[0057]
[0058] 2: Acquisition of expression plasmid
[0059] Ac...
Embodiment 2
[0070] Expression and purification of embodiment 2 mutant carrier protein
[0071] Lys-azido-incorporated expression and purification of mutant protein
[0072] The expression plasmid pET9a-HIN47-HID-K522 obtained in Example 1 was cultured in LB medium at 37°C for 12 to 16 hours, and then amplified twice until the OD value of the bacterial solution reached 0.6 to 1.0, and Lys- azido to a final concentration of 1 mM, continue to amplify at 37°C for 30 minutes, add IPTG to a final concentration of 0.5mM, and arabinose to a final concentration of 0.2%, and induce expression at 24°C for 12 hours before collecting the cells.
[0073] The collected cells were balanced and resuspended with Ni-NTA-Bind-Buffer, ultrasonically disrupted, centrifuged to remove cell debris, subjected to Ni-NTA metal chelate affinity chromatography, fully washed with Ni-NTA-Wash-Buffer, and finally washed with Ni-NTA-Elute-Buffer elution, the protein sample HIN47-HID--K522 that obtains preliminary purific...
Embodiment 3
[0074] Example 3 Coupling of mutant carrier protein and polysaccharide through copper-free catalysis
[0075] The copper-free catalyzed Click reaction needs to be realized by the ring tension of cyclooctyne, and the modification is coupled with DIBO (polysaccharide with cyclooctyne structure) to perform copper-free catalyzed Click reaction with azide-containing groups.
[0076] The Click reaction system is as follows:
[0077] HIN47-HID--K522 protein 1 microgram per microliter, DIBO-pneumonia type 3 polysaccharide, the molecular weight is 200KD, 400KD, each 2Mm, 4 ℃ vertical suspension for 2 hours, the results show different molecular weight (200KD, 400KD) pneumonia 3 All types of polysaccharides can be successfully modified on the protein F3-HIN47-HID--K522-200, F3-HIN47-HID--K522-400 (see figure 1 , line1, 2).
[0078] After the above reaction conditions, about 90% of the protein can be coupled to the polysaccharide within 1 hour, and the reconstituted product after the re...
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