CN119841912B

CN119841912B - Meningococcal outer membrane protein synthetic peptide and application thereof

Info

Publication number: CN119841912B
Application number: CN202510336441.7A
Authority: CN
Inventors: 刘晓满; 董文娜; 王齐; 付丽丽
Original assignee: Beijing Huanuotai Biomedical Technology Co ltd
Current assignee: Beijing Huanuotai Biomedical Technology Co ltd
Priority date: 2025-03-21
Filing date: 2025-03-21
Publication date: 2025-06-27
Anticipated expiration: 2045-03-21
Also published as: CN119841912A

Abstract

The present application relates to the technical field of biomedicine, and in particular to a synthetic peptide of meningococcal outer membrane protein and its application. The present application discloses a synthetic peptide of meningococcal outer membrane protein and its application, wherein the amino acid sequence of the polypeptide is at least one of (I) to (IV): (I) the amino acid sequence shown in SEQ ID NO.1; (II) the amino acid sequence shown in SEQ ID NO.2; (III) the amino acid sequence shown in SEQ ID NO.3; (IV) the amino acid sequence shown in SEQ ID NO.4; The present application discloses a synthetic peptide of meningococcal outer membrane protein and its application, and discloses the polypeptide sequence. The vaccine made of the polypeptide or its polymer can effectively deal with the diversity of meningococcal PorA and PorB antigens, is easy to synthesize on a large scale, and has good application prospects.

Description

Meningococcal outer membrane protein synthetic peptide and application thereof

Technical Field

The application relates to the technical field of biological medicines, in particular to a meningococcal outer membrane protein synthetic peptide and application thereof.

Background

Meningococcus is the main causative agent of epidemic cerebrospinal meningitis (epidemic cerebrospinal meningitis) and fulminant sepsis, and is the only pathogenic bacterium that can cause epidemic cerebrospinal meningitis. Meningococci have 13 different serogroups, of which A, B, C, Y and W135 are the major pathogenic groups.

A variety of membrane and extracellular components are currently used in meningococcal vaccine development, including class 1,2 and 3 outer membrane proteins (porin proteins), class 4 outer membrane proteins (Rmp proteins) and class 5 outer membrane proteins (Opacity proteins). PorA protein is a P1 type outer membrane protein, porB protein is a P2 or P3 type outer membrane protein, and N.meningitidis expresses both types of proteins. PorA and PorB are key antigens that have been used in vaccine formulations and are considered ideal antigens for eliciting potent bactericidal antibodies. However, meningococcus effectively evades immune responses by antigenicity and phase transition. For example, the secondary structure of PorA and PorB proteins suggests that both have 8 exposed cyclic structures (loopI to VIII), respectively. Wherein loopI and IV are the variable regions of PorA and loopI, V, VI and VII are the variable regions of PorB, the epitopes that determine the serotype and serotype of the strain being predominantly present on both variable regions. PorA and PorB, the highly immunogenic surface loops of which are the primary reasons for eliciting an immune response that is not broadly protective, make PorAN_SNh proteins derived from a particular meningococcal strain tend to preferentially provide immunity against that particular strain.

Thus, in order to effectively cope with meningococcal PorA and PorB antigen diversity, it is critical to develop novel polypeptides for the synthesis of meningococcal outer membrane protein synthetic peptide vaccines.

Disclosure of Invention

Aiming at the defects of the prior art, the application provides a meningococcal outer membrane protein synthetic peptide and application thereof, in particular to a polypeptide or a polypeptide polymer thereof for a meningococcal outer membrane protein synthetic peptide vaccine, and a vaccine containing the polypeptide or the polypeptide polymer thereof and application of the vaccine.

The application discloses a meningococcal outer membrane protein synthetic peptide and application thereof, and a vaccine prepared from the polypeptide or a polymer thereof can effectively cope with the diversity of meningococcal PorA and PorB antigens, is easy to synthesize on a large scale, and has good application prospect.

In a first aspect, the present application provides a polypeptide, which adopts the following technical scheme:

According to the application, through analyzing the protein sequence of eleven domestic group B meningococcal strains, the mutation situation of the porA and porB antigen sites is researched, and simultaneously, the analysis and prediction of the porA and porB antigen sites are carried out by combining a computer-aided method, so that a conserved antigen site polypeptide sequence is obtained, wherein the amino acid sequence of the polypeptide is at least one of (I) to (IV):

(I) An amino acid sequence as shown in SEQ ID NO. 1;

(II) an amino acid sequence shown as SEQ ID NO. 2;

(III) an amino acid sequence shown as SEQ ID NO. 3;

(IV) the amino acid sequence shown in SEQ ID NO. 4;

(V) a sequence having at least 90% homology to the sequences of (I) - (IV);

(VI) has an amino acid sequence obtained by substituting, deleting or adding one or two amino acid sequences with the amino acid sequence shown in any one of (I) to (V), and has the same or similar functions with the amino acid sequence shown in (I) to (V).

Further preferably, the amino acid sequence of the polypeptide is a sequence shown as SEQ ID NO. 1-4.

In a second aspect, the present application provides a polypeptide polymer, which adopts the following technical scheme:

A polypeptide polymer comprises a polypeptide, wherein the amino acid sequence of the polypeptide is at least one of (I) to (IV):

(I) An amino acid sequence as shown in SEQ ID NO. 1;

(II) an amino acid sequence shown as SEQ ID NO. 2;

(III) an amino acid sequence shown as SEQ ID NO. 3;

(IV) the amino acid sequence shown in SEQ ID NO. 4;

(V) a sequence having at least 90% homology to the sequences of (I) - (IV);

Preferably, a carrier protein is also included.

Preferably, the carrier protein comprises at least one of albumin, porin, diphtheria toxin, tetanus toxin, heat labile enterotoxin, ferritin.

In a third aspect, the application provides an application of a polypeptide or a polypeptide polymer in preparing a meningococcal outer membrane protein synthetic peptide vaccine, which adopts the following technical scheme:

the application of the polypeptide or the polypeptide polymer in preparing meningococcal outer membrane protein synthetic peptide vaccine.

In a fourth aspect, the application provides a meningococcal outer membrane protein synthetic peptide vaccine, which adopts the following technical scheme:

A meningococcal outer membrane protein synthetic peptide vaccine comprising a polypeptide as described above or a polypeptide polymer as described above.

Preferably, the vaccine further comprises an immunoadjuvant.

Further preferably, the immunoadjuvant comprises at least one of aluminum adjuvant, squalene, tocopherol, MPL, LPA, cpG, and QS-21.

In a fifth aspect, the present application provides a method for preparing a polypeptide, which adopts the following technical scheme:

a method for producing a polypeptide, comprising the steps of:

attaching an amino acid constituting the C-terminal of a peptide having an α -amino protecting group to a resin in the presence of an alkaline agent using the resin as a starting material;

removing the protecting group with a uncapping agent;

and (3) sequentially connecting all amino acids forming the peptide according to the sequence from the C end to the N end of the peptide to react so as to form peptide connecting resin, and then cutting the peptide and purifying the crude product to obtain the polypeptide.

In a specific embodiment, a method for producing a polypeptide comprises the steps of:

(1) Deprotection reaction, namely, in N-methyl pyrrolidone solution of hexahydropyridine with the volume percentage of 15-30%, reacting for 25-40 minutes at the temperature of 20-28 ℃, removing 9-fluorenylmethoxycarbonyl protecting groups on resin amino groups, drying with nitrogen, and washing with N-methyl pyrrolidone;

(2) The activation of amino acid, namely, each amino acid with 9-fluorenylmethoxycarbonyl protecting group for synthesis is reacted with 1-hydroxybenzotriazole to synthesize amino acid-1-hydroxybenzotriazole ester;

(3) The condensation reaction, namely automatically adding the various amino acids, the resin and the diisopropylcarbodiimide into a reactor by using a polypeptide synthesizer, reacting for 0.5-2.5 hours at 20-28 ℃, drying by nitrogen, and washing the resin by using N-methylpyrrolidone;

(4) Acetylation reaction, namely reacting an acetylimidazole N-methyl pyrrolidone solution with the weight and volume percentage of 1.5% -4% (g/mL) with the resin obtained in the step (3) for 20-40 minutes at 20-28 ℃, drying by nitrogen, and washing the resin by methanol;

(5) Continuously repeating the steps (1) - (4) from the C end to the N end according to the amino acid sequence in the synthesis process, and cleaning with N-methylpyrrolidone after the reaction is finished to obtain dry polypeptide resin;

(6) Separating polypeptide from resin, namely adding a cracking reagent into dried polypeptide resin, stirring at a constant speed for 1-4 hours, then placing the dried polypeptide resin at 0 ℃ for reaction for 10 minutes, recovering to room temperature, volatilizing trifluoroacetic acid, adding tert-butyl methyl ether and diethyl ether into a polypeptide solution, stirring, washing, and filtering to obtain a polypeptide solution;

(7) Ultrafiltration for purification of the polypeptide and aseptic treatment, wherein the polypeptide is ultrafiltered under 20-28deg.C by using membrane package, and is preserved by sterilizing with 0.22 μm on-line filter.

Preferably, the cracking reagent comprises trifluoroacetic acid, triisopropylsilane, ethanedithiol, phenol and water, wherein the volume ratio of the trifluoroacetic acid, the triisopropylsilane, the ethanedithiol, the phenol and the water is 85:8:3:3:1.

In a sixth aspect, the present application provides a method for preparing a polypeptide polymer, which adopts the following technical scheme:

A method for preparing a polypeptide polymer, comprising the steps of:

activating the carrier protein by using 3-sulfo-N-hydroxysuccinimide ester sodium salt of 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid, and then filtering and desalting to obtain the activated carrier protein;

mixing the sequences of the polypeptides in equal proportion to obtain a polypeptide mixture;

coupling the polypeptide mixture with the activated carrier protein to obtain the polypeptide polymer.

Preferably, the mass ratio of the 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt to the carrier protein is (0.5-1): 3-7.

Preferably, the volume to mass ratio of the polypeptide mixture to the activated carrier protein is (0.1-1) mL (4-8) mg.

The application provides a broader spectrum of immunoprotection against neisseria meningitidis strains by designing conserved PorA protein polypeptides such that the immune response is directed primarily against conserved epitopes and enhancing the immunogenicity of the synthetic polypeptides by coupling to carrier protein carriers, such that vaccines comprising the synthetic polypeptides are directed against neisseria meningitidis strains.

In a seventh aspect, the application provides an application of a meningococcal outer membrane protein synthetic peptide vaccine, which adopts the following technical scheme:

Use of a meningococcal outer membrane protein synthetic peptide vaccine, comprising at least one of:

(1) The application of the composition in preparing medicines for preventing and/or treating meningococcal induced diseases;

(2) The application of the composition in preparing medicines for preventing and/or treating epidemic cerebrospinal meningitis;

(3) The application of the composition in preparing medicines for preventing and/or treating fulminant septicemia.

In summary, the present application includes at least one of the following beneficial technical effects:

the application discloses a meningococcal outer membrane protein synthetic peptide and application thereof, and discloses a polypeptide sequence, and a vaccine prepared from the polypeptide or a polymer thereof can effectively cope with the diversity of meningococcal PorA and PorB antigens, is easy to synthesize on a large scale, and has good application prospect.

Drawings

FIG. 1 is a graph showing the results of ELISA detection of antigen-specific serum IgG;

FIG. 2 is a graph showing the results of ELISA detection of antigen-specific secretory IgA.

Detailed Description

The experimental methods of the present application, in which specific conditions are not specified in the following examples, are generally carried out under conventional conditions or under conditions recommended by the manufacturer, and the experimental materials and reagents are commercially available unless otherwise specified.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

The terms "comprising" and "having" and any variations thereof, are intended to cover a non-exclusive inclusion. For example, a process, method, apparatus, article, or device that comprises a list of steps is not limited to the elements or modules listed but may alternatively include additional steps not listed or inherent to such process, method, article, or device.

The following description of the embodiments of the present application will be made more apparent and fully by reference to the accompanying drawings, in which some, but not all embodiments of the application are shown. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to fall within the scope of the application.

Example 1 solid phase Synthesis of meningococcal outer membrane protein synthetic peptide vaccine polypeptide antigen

The preparation method is carried out by adopting a Merrifield solid-phase synthesis method, wherein the solid-phase carrier is RINK AMIDE MBHA resin, and the instrument is an ABI 433A full-automatic polypeptide synthesizer.

1. Preparation of synthetic raw materials:

the sequence of the meningococcal outer membrane protein synthetic peptide vaccine polypeptide is the amino acid sequence shown in SEQ ID NO.1, the amino acid sequence shown in SEQ ID NO.2, the amino acid sequence shown in SEQ ID NO.3 and the amino acid sequence shown in SEQ ID NO. 4.

Suitable 9-fluorenylmethoxycarbonyl (Fmoc) modified amino acids were prepared according to the above polypeptide antigen sequences and 1mmol synthetic scale and added to the corresponding Cartridge (amino acid-containing vials). RINK AMIDE MBHA resin was also weighed as required, placed in the reaction chamber and the reaction chamber was loaded into the synthesizer. N-methylpyrrolidone (NMP), caproyl imidazole (AIM), piperidine (PIP), methanol and the like are placed in the corresponding reagent bottles.

2. Preparation of meningococcal outer membrane protein synthetic peptides:

(6) Separating polypeptide from resin, namely adding a cracking reagent (the volume ratio of the components is trifluoroacetic acid to triisopropylsilane to ethanedithiol to phenol to water=85:8:3:3:1) into dried polypeptide resin, uniformly stirring for 1-4 hours, then placing the mixture at 0 ℃ for reaction for 10 minutes, recovering the mixture to room temperature, volatilizing the trifluoroacetic acid, adding tert-butyl methyl ether and diethyl ether into a polypeptide solution, stirring and washing, and filtering the polypeptide solution;

EXAMPLE 2 preparation of polypeptide Polymer

A preparation method of polypeptide polymer comprises the following steps:

(1) Filtering the albumin solution through a 0.45 mu m filter membrane, and measuring UV280 to obtain an albumin solution with the concentration of 1 mg/mL;

(2) Weighing 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid 3-Sulfo-N-hydroxysuccinimide ester sodium salt (Sulfo-SMCC), and dissolving in water to obtain a Sulfo-SMCC solution with the concentration of 4 mg/mL;

(3) Adding 0.2mL of Sulfo-SMCC solution into 5mL of albumin solution, standing at room temperature in a dark place for 2h, filtering with 0.2 μm, desalting, and removing superfluous Sulfo-SMCC to obtain activated albumin;

(4) Mixing an amino acid sequence shown in SEQ ID NO.1, an amino acid sequence shown in SEQ ID NO.2, an amino acid sequence shown in SEQ ID NO.3 and an amino acid sequence shown in SEQ ID NO.4 according to a mass ratio of 1:1:1:1 to obtain a polypeptide mixture;

(5) 6mg of the polypeptide mixture was dissolved in 0.6mL of 1 XPBS (pH 7.2) solution, thiol groups in the polypeptide were detected by Ellman reagent, and the UV absorbance OD value of the Nano spectrophotometer was >0.15 at λ=412 nm;

(6) Adding 0.6mL of the polypeptide mixture solution obtained in the step (5) into about 6mg of activated albumin, and standing for 4h at room temperature in a dark place;

(7) After the reaction, the sulfhydryl group in the polypeptide is detected by an Ellman reagent, and the ultraviolet absorption value OD value is measured by a Nano spectrophotometer at lambda=412 nm and is less than 0.03, which indicates that the crosslinking rate of the polypeptide and albumin is over 80 percent, and the polypeptide polymer is obtained.

EXAMPLE 3 preparation of meningococcal outer membrane protein synthetic peptide vaccine

The preparation method of the meningococcal outer membrane protein synthetic peptide vaccine comprises the following steps:

200. Mu.g of the polypeptide polymer prepared in example 2 was mixed with 1mL of CpG solution (concentration: 200. Mu.g/mL) to obtain a meningococcal outer membrane protein synthetic peptide vaccine.

EXAMPLE 4 meningococcal outer membrane protein synthetic peptide vaccine mice immunization and immunogenicity detection

1. Immunization of animals

(1) Selecting female C57BL/6 mice of 6-8 weeks of age for immunization, wherein the female C57BL/6 mice are randomly divided into two groups, and 6 groups are CpG control groups and synthetic peptide vaccine groups respectively;

(2) The animals of the CpG control group are given with CpG solution, the synthetic peptide vaccine group is given with the meningococcal outer membrane protein synthetic peptide vaccine prepared in the example 3, each mouse is given with 10 mug of polypeptide polymer antigen and 10 mug of CpG each time, the injection amount is 50 mug/animal, and the specific grouping and the number are shown in the table 1;

(3) The immunization mode is nasal drip immunization, each mouse is immunized 3 times, and nasal drops are respectively carried out on the 1 st day, the 7 th day and the 14 th day;

(4) One week after the last immunization, the tail of the anesthetized mice was bled and oral lavage fluid was obtained.

Table 1 immunized mice groups and numbering

2. ELISA detects antigen-specific serum IgG as follows:

(1) Coating antigen, namely diluting meningococcal lysate to 10 mug/mL by using coating liquid, and coating the meningococcal lysate by using a 100 mug/Kong Jiazhi 96-well ELISA plate at 4 ℃ overnight;

(2) Blocking, namely spin-drying the coated ELISA plate, washing the plate 5 times with 300 mu L of washing liquid per hole, beating, adding blocking liquid (2% BSA) into the plate at 300 mu L/hole, and standing at 37 ℃ for 2 hours;

(3) Diluting standard substance (mouse IgG) to 200ng/mL with sample diluent, and diluting with 2-fold gradient;

(4) Spin-drying the sealed ELISA plate, washing the plate with 300 mu L of washing liquid for 5 times, beating to dry, and placing samples (a gradient diluted standard product and a diluted to-be-detected product) in 100 mu L/Kong Jiazhi ELISA plate from the dilution plate for 1.5h at room temperature;

(5) Adding enzyme-labeled working solution, namely diluting goat anti-mouse IgG antibody with secondary antibody diluent, spin-drying the enzyme-labeled plate, washing the plate 5 times with 300 mu L of washing solution per hole, beating, and placing the diluted secondary antibody in 100 mu L/Kong Jiazhi enzyme-labeled plate at room temperature for 1h;

(6) Color development, namely washing the plate 5 times by using 300 mu L of washing liquid in each hole, and spin-drying. The bottom is wiped with absorbent paper. The color development liquid is moved from a refrigerator at 4 ℃ to a light-proof room temperature for preservation in advance for 30 minutes, 100 mu L/hole of the color development liquid is added, and the color development is carried out for 15 minutes at room temperature in a light-proof way;

(7) Stopping the reaction by adding a stopping solution at a concentration of 50 mu L/hole, and reading the plate within 5 minutes;

(8) And (3) reading the plate, namely placing the ELISA plate into an ELISA reader, setting a medium-speed vibration for 10 seconds, and reading by OD 450.

The results are shown in figure 1, where the sera one week after immunization with meningococcal outer membrane protein synthetic peptide vaccine contained higher levels of meningococcal specific IgG class antibodies than the control CpG group.

3. ELISA detection of antigen-specific secretory IgA the detection steps were as follows:

(3) Diluting the standard substance (mouse IgA) to 200ng/mL with sample diluent, and performing 2-time gradient dilution; diluting the sample to be tested (immunized mouse oral lavage liquid) to 1000-10000 times with PBS;

(5) Adding enzyme-labeled working solution, namely diluting goat anti-mouse IgA antibody with secondary antibody diluent, spin-drying the enzyme-labeled plate, washing the plate 5 times with 300 mu L of washing solution per hole, beating, and placing the diluted secondary antibody in 100 mu L/Kong Jiazhi enzyme-labeled plate at room temperature for 1h;

The results are shown in figure 2, where the oral lavage fluid one week after immunization of the meningococcal outer membrane protein synthetic peptide vaccine contained higher levels of meningococcal specific secretory IgA antibodies than the control CpG group.

Claims

1. A preparation method of a polypeptide polymer is characterized by comprising the following steps:

Mixing the sequences of the polypeptides shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 in equal proportion to obtain a polypeptide mixture;

2. The method of producing a polypeptide polymer according to claim 1, wherein the mass ratio of the sodium salt of 4- (N-maleimidomethyl) cyclohexane-1-carboxylate 3-sulfo-N-hydroxysuccinimide ester to the carrier protein is (0.5-1): 3-7.

3. The method of producing a polypeptide polymer according to claim 1, wherein the polypeptide mixture is prepared by dissolving 6mg in 0.6mL of 1 XPBS to prepare a solution, and the volume/mass ratio of the polypeptide mixture solution to the activated carrier protein is (0.1-1) mL (4-8) mg.

4. The method of producing a polypeptide polymer according to claim 1, wherein the carrier protein comprises at least one of albumin, porin, diphtheria toxin, tetanus toxin, heat-labile enterotoxin, and ferritin.

5. A polypeptide polymer prepared by the method of any one of claims 1 to 4.

6. Use of the polypeptide polymer of claim 5 for the preparation of antisera that bind to meningococcal lysate.