Site-directed mutagenesis carrier protein and purpose thereof to vaccine preparation

A technology for site-directed mutagenesis and mutant protein, which is applied in the preparation methods of peptides, vaccines, and thioether preparations, etc., and can solve the problems of weak cross-protection, affecting antigenicity, affecting vaccine immunogenicity and antibody specificity, etc.

Active Publication Date: 2020-08-14
CANSINO BIOLOGICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is still no international universal meningococcal group B OMV vaccine
[0017] 2. Limitations of the protective coverage of OMV vaccines
MenB OMV vaccine has been successfully used to control the epidemic and outbreak of the disease, but its effectiveness is limited to the control of vaccine-type and subtype MenB disease outbreaks, and its effectiveness is limited for diseases caused by different types and subtypes of MenB
[0020] 3. The production process of MenB OMV affects its antigenicity
[0023] Early research put too much emphasis on protein purity, and used a more complex physical and chemical combination of repeated processes, which destroyed the natural conformation of OMV and weakened the rich antigenic sites, thus affecting the immunogenicity and antibody specificity of the vaccine , leading to poor protection and persistence of immunity
[0024] 4. "Broad-spectrum" group B meningococcal vaccines (such as Novartis 4C MenB, Bexsero) are difficult to achieve broad protection
Different countries have different types of Nm PorA in Group B, and the coverage of PorA strains of 4CMenB vaccine is affected by the PorA types of different Group B strains, which has limitations
[0028] The fHbp in the 4C MenB vaccine is Variant 1, and the effectiveness of immune serum against fHbp Variant 1 strains can reach 95

Method used

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  • Site-directed mutagenesis carrier protein and purpose thereof to vaccine preparation
  • Site-directed mutagenesis carrier protein and purpose thereof to vaccine preparation
  • Site-directed mutagenesis carrier protein and purpose thereof to vaccine preparation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0118] Example 1 Construction of the MenB protein expression plasmid for site-directed mutation

[0119] 1. Selection of mutation sites

[0120] Natural MenB undergoes lipidation modification at its N-terminus. This modification does not affect the three-dimensional structure of MenB protein and plays a role in anchoring the antigen to the cell membrane. Structural studies have shown that the first 20 amino acids at the N-terminal of the MenB protein are not folded to form a secondary structure, but are stretched, and its function is to expose the antigen part through the bacterial outer membrane to the bacterial surface. Therefore, the mutated sites are preferentially selected from the N-terminal 20 amino acids, among which the 2-10 amino acids are preferentially selected. See Tables 1-3 for information on specific mutation sites, where the amino acid positions refer to the positions on the sequences shown in SEQ ID NO: 1-3.

[0121] SEQ ID NO.1:

[0122] cgssggggsggggvtad...

Embodiment 2

[0154] Example 2 Lys-azido incorporation expression and purification of mutein

[0155] The expression plasmids pET28a-MenB-V1.55-G2, pET28a-MenB-V2.16-S3, pET28a-MenB-V3.45-S4 obtained in Example 1 were cultured in LB medium at 37°C for 12-16 hours Then, after secondary amplification until the OD value of the bacterial solution reaches 0.6~1.0, add Lys-azido to a final concentration of 1mM, 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 the cells were collected after induction of expression at 24°C for 12 hours.

[0156] 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 purified protein samples pET28a-MenB-V1.55-G2, pET28a-MenB-V2.16-S3, and p...

Embodiment 3

[0158] Example 3 Synthesis of tripalmitoyl-S-glycerocysteine ​​analogue 8

[0159] The synthetic route of tripalmitoyl-S-glycerocysteine ​​analogue 8 is as follows:

[0160] 1. Dissolve compound 1 (5g) and acetonylidene (5g) in dichloromethane (100ml). After the dissolution is complete, slowly add PTSA (0.9g) into the ice-water bath. After the addition is complete, remove the ice bath and stir at room temperature 2 hours. After the reaction, the solvent was distilled off under reduced pressure, and compound 2 was obtained by purification with silica gel chromatography.

[0161]2. Dissolve compound 2 (5g) in DMF (100ml), then add EDCI (5g), HOBT (3.5g), TEA (10g) in turn, stir for 3-5 minutes, then add compound a (6g), add After completion, it was placed in an oil bath at 80°C to react overnight. After the reaction, the solvent was distilled off under reduced pressure, and compound 3 was obtained by purification with silica gel chromatography.

[0162] 3. Compound 3 (5g) wa...

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PUM

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Abstract

The invention relates to a site-directed mutagenesis and site-directed modification protein antigen, and also relates to a site-directed mutagenesis and site-directed modification method of the protein antigen. The method comprises the following steps of introducing unnatural amino acids into specific sites of the protein antigen in a site-directed way by using a gene codon extension technology; and performing site-directed modification with the protein antigen by using the unnatural amino acids and a modifier, wherein the modifier is a receptor agonist such as tripalmitoyl-S-glyceryl cysteineand monophosphoryl lipid A. The invention further relates to application of the site-directed mutagenesis and site-directed modification protein antigen, such as a purpose as a vaccine and the like.

Description

technical field [0001] The invention relates to the field of biopharmaceuticals, in particular, the invention relates to a protein with site-directed mutation and site-directed modification and its use in preparing vaccines, especially meningococcal multivalent vaccines. Background technique [0002] Meningococcal meningitis (meningococcal meningitis) caused by Neisseria meningococcus (Nm) infection is a worldwide acute respiratory infectious disease, which still seriously endangers human health. It's children. Neisseria meningitidis can be divided into 13 serogroups according to the differences in their capsular polysaccharide structures, and all serogroups can be pathogenic. Among them, the diseases caused by Neisseria meningitidis group A, B, C, Y, and W135 accounted for more than 95% of Neisseria meningitidis-related diseases. [0003] Among them, various monovalent (A or C), bivalent (A\C) and quadrivalent (ACYW135) vaccines based on meningococcal capsular polysacchar...

Claims

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Application Information

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IPC IPC(8): C07K14/22C07K1/10A61K39/095A61K47/54A61P31/04C07C323/59C07C319/20
CPCC07K14/22A61K39/095A61K47/544A61K47/542A61P31/04C07C323/59A61K2039/70C07C2601/02A61K2039/55555A61K2039/55505A61K2039/575A61K47/646A61K39/116
Inventor 王浩猛严志红晏巧玲邵娟史建明隋秀文李军强朱涛
Owner CANSINO BIOLOGICS INC
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