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A carrier protein for site-directed mutation and its use in preparing vaccines

A carrier protein, site-directed mutagenesis technology, applied in the preparation methods of peptides, vaccines, multivalent vaccines, etc., can solve the problems of complex separation and purification process, reduced polysaccharide protein binding efficiency, and uneven binding process, and achieves optimization of modification conditions, Attenuate the protease activity, the effect of simple and easy modification reaction

Active Publication Date: 2021-06-29
CANSINO BIOLOGICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Such a binding method can reduce the steric hindrance of the combination of polysaccharide and carrier protein, retain the epitope of polysaccharide, avoid the solubility of polysaccharide itself, and reduce the side effects of polysaccharide and antiserum reaction, but there are some shortcomings:( 1) The polysaccharide-AH derivative will continue to react with the polysaccharide activated by cyanogen bromide to form a self-polymer of the polysaccharide, which reduces the binding efficiency of the polysaccharide protein; (2) EDAC mediates the binding of the ADH-derived polysaccharide to the carrier protein, It is easy to cause self-crosslinking of the carrier protein, thereby reducing the binding efficiency of the polysaccharide protein. Therefore, the immunogenicity and antibody persistence of the polysaccharide protein conjugate vaccine still need to be further improved. For example, the polysaccharide conjugate vaccine needs to be immunized three times to produce the immune effect
In the face of the problems of inhomogeneous binding process of conjugated vaccines and complicated separation and purification process in the prior art, there is an urgent need in this field for a method that can specifically modify functional groups at any site

Method used

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  • A carrier protein for site-directed mutation and its use in preparing vaccines
  • A carrier protein for site-directed mutation and its use in preparing vaccines
  • A carrier protein for site-directed mutation and its use in preparing vaccines

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Construction of carrier protein expression plasmid for site-directed mutagenesis

[0047] 1: Selection of mutation sites

[0048] The three-dimensional structures of HID and HIN47 were predicted using the online software phyre2, respectively.

[0049] 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.

[0050]

[0051]

[0052] 2: Acquisition of expression plasmid

[0053] According to the HID and HIN47 gene sequences published by NCBI Gene Bank, the full-length DNA fragmen...

Embodiment 2

[0062] Expression and purification of embodiment 2 mutant carrier protein

[0063] Lys-azido-incorporated expression and purification of mutant protein

[0064] The expression plasmid pET9a-HIN47-HID-K522 obtained in Example 1 was cultured in LB medium at 37°C for 12-16 hours, and then amplified twice until the OD value of the bacterial solution reached 0.6-1.0, 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%, induce expression at 24°C for 12 hours and collect the cells.

[0065] 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 Eluted with Ni-NTA-Elute-Buffer, the preliminary purified protein sample HIN47-HID--K522 was obtained with a pur...

Embodiment 3

[0066] Example 3 Coupling of Mutant Carrier Proteins and Polysaccharides by Copper-free Catalysis

[0067] 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.

[0068] The Click reaction system is as follows:

[0069] HIN47-HID--K522 protein 1 microgram per microliter, DIBO-pneumonia type 3 polysaccharide, molecular weight 200KD, 400KD, 2Mm each, vertical suspension at 4°C for 2 hours, the results showed 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).

[0070] 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 reactio...

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Abstract

The invention relates to a site-directed mutation carrier protein and its application in preparing vaccines, wherein the carrier protein is selected from the group consisting of diphtheria toxoid, avirulent mutant of diphtheria toxin, bacterial outer membrane protein and bacterial expression protein A fusion protein formed by one or more than two, wherein the amino acid at least one position on the carrier protein is mutated into an unnatural amino acid, and the unnatural amino acid contains an azide or alkynyl terminal group. The site-directed mutation carrier protein of the present invention reacts with the polysaccharide antigen to form a covalent bond, and at the same time, the conjugate is in a beaded state, which can effectively avoid excessive cross-linking between the carrier protein and the polysaccharide antigen, and the particle size distribution of the conjugate is remarkable. It is uniform and controllable, and provides an effective means for improving the quality of polysaccharide-protein conjugated vaccines.

Description

technical field [0001] The invention relates to the field of biopharmaceuticals, in particular, the invention relates to a site-directed mutation carrier protein and its use in preparing vaccines, especially pneumonia multivalent conjugate vaccines. Background technique [0002] Infection caused by Streptococcus pneumoniae is one of the leading causes of morbidity and mortality worldwide. Pneumonia, febrile bacteremia, and meningitis are the most common manifestations of invasive pneumococcal disease, and spread of the bacteria in the respiratory tract can lead to middle ear infection, sinusitis, or recurrent bronchitis. [0003] A polysaccharide-protein conjugate vaccine is prepared using a polysaccharide linked to a protein carrier, and the chemical bond of the polysaccharide to the protein carrier can induce an immune response against bacteria displaying the polysaccharide contained in the vaccine on its surface, thereby preventing disease. Vaccination with polysaccharid...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/34C07K14/22C07K14/285C07K14/21C07K19/00C07K1/10A61K39/09A61K39/385A61P31/04A61P11/00
CPCC07K14/34C07K14/22C07K14/285C07K14/21A61K39/092A61K39/385A61P31/04A61P11/00C07K2319/00A61K2039/70A61K2039/6031A61K2039/60A61K2039/6037A61P31/16C07K2319/55A61K2039/6043A61K2039/6068A61K2039/6087A61K2039/627
Inventor 王浩猛晏巧玲巢守柏毛慧华朱涛
Owner CANSINO BIOLOGICS INC
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