CN119185523A - Broad-spectrum multi-subunit vaccine for preventing group B meningococcal infection and application thereof - Google Patents

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to a broad-spectrum multi-subunit vaccine for preventing serogroup B meningococcal infection and application thereof, wherein the broad-spectrum multi-subunit vaccine comprises complement regulator H binding protein fHbp; neisserial heparin binding antigen NHBA, pile protein, chimeric antigen expressed by fusion of complement regulator H binding protein fHbp with the signal peptide part removed as skeleton and the multiple variable region of an additional antigen Opa, app protein, igA protease and adjuvant CpG. The vaccine of the invention has high efficiency, broad spectrum and low price, adopts a mucous membrane immunization way, has no tissue injury, no local side effect, simple and convenient use and easy popularization.

Description

Broad-spectrum multi-subunit vaccine for preventing group B meningococcal infection and application thereof

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to a broad-spectrum multi-subunit vaccine for preventing meningococcal infection of group B and application thereof.

Background

Neisseria meningitidis (NEISSERIA MENINGITIDIS) is a gram-negative bacterium that can cause respiratory infections, also called epidemic cerebrospinal meningitis (epidemic for short), which occurs in all ages, with the highest incidence in infants under 1 year old. Humans are the only host of neisseria meningitidis, present in the upper respiratory tract of about 10% of adults, and primarily pass through the respiratory tract infections, across mucous membranes and the blood brain barrier, forming Invasive Meningitis (IMD), which can lead to long-term disability, with a severe mortality rate of up to 10% -20%.

The capsular polysaccharide of neisseria meningitidis is a major virulence factor and can be divided into 12 serogroups depending on its polysaccharide chemical composition, with most epidemic invasive meningitis being caused by a, B, C, W, Y and X6 serogroups, but the distribution of these serogroups is also affected by the geographical environment, vaccination conditions. Currently, polysaccharide vaccines with multivalent binding are aimed at A, C, W, Y and X, but the group B meningitis capsular polysaccharide has similar chemical components as polysialic acid in human glycoprotein, has poor immunogenicity and safety, and can not be used as vaccine components.

The earliest vaccine used to prevent group B meningitis was the detergent extracted Outer Membrane Vesicle (OMV) vaccine. The main antigen of OMV vaccines is porin (PorA). PorA exhibits a high degree of antigenic variability, limiting cross-immune protection against different strains, and the lack of PorA conservation is a challenge for OMVs as a broad-spectrum vaccine. The vaccine with single recombinant protein component is difficult to obtain better immune effect, and the multicomponent vaccine becomes a development trend, so that a new direction is brought for the research of the meningococcal vaccine of group B.

Bacterial strain fHbp is one of the major virulence factors of neisseria meningitidis and survives in human blood by binding factor H (fH) to the pathogenic bacteria bypassing the alternative complement pathway. Deletion of fHbp genes results in failure of fH binding, reduced viability of the strain in human serum, binding of fH by strains harboring different fHbp variants, and binding capacity correlated with fHbp expression levels (7, 8). Meanwhile, the fHbp is also the main component of two obtained batch type B meningococcal vaccines, and in general, the fHbp is used as an immune source to induce bactericidal antibodies to the same subfamily strain, and has cross immune protection.

Opa (NEISSERIAL COLONY OPACITY-associated protein adhesins), expressed in the course of pathogen infection, binds to the CEACAMs receptor, plays an important role in colonisation and infection of the respiratory epithelial cells by the pathogen, and is one of the main virulence factors of Neisseria meningitidis.

The protein neisserial heparin-binding antigen (NHBA or GNA 2132) is a surface-exposed lipoprotein characteristic of neisseria and NHBA gene sequence analysis has reported more than 400 different NHBA polypeptide variants (1). The NHBA protein is composed of about 420-490 amino acids, the N end of the amino acid sequence of the NHBA protein is about 250 residues, the sequence difference is larger, the C end of the NHBA protein is composed of about 180 amino acids, 8 beta-sheet structural domains are formed, the NHBA protein has high conservation, the recombinant NHBA immunized mouse serum antibody can be combined to the surfaces of different neisseria meningitidis strains, the complement mediated bactericidal activity is induced, and meanwhile, the bacteremia of a young mouse can be passively protected, so that the NHBA protein can provide cross immune protection to different strains, and is one of candidate antigens of a recombinant protein vaccine.

The specification of chinese invention cn20201102034337. X describes a neisseria meningitidis vaccine comprising bacterial ghosts and factor H binding protein (fHBP). The vaccine has simple components and is difficult to obtain broad-spectrum immune effect. There are currently no broad-spectrum vaccines for preventing meningococcal infection in group B, and there are many technical difficulties in developing a broad-spectrum multi-subunit vaccine suitable for use in meningococcal infection in group B. Thus, there is a need to develop a broad-spectrum multi-subunit vaccine that prevents meningococcal infection in group B.

Disclosure of Invention

The invention provides a broad-spectrum multi-subunit vaccine for preventing B group meningococcal infection and application thereof, which utilizes the conservative antigen mucous membrane of a plurality of B group meningococcus to immunize mice, effectively induces the reaction of Th17 and antibodies of the mice, effectively prevents the epidemic of B group meningococcus, provides wide protection effect for B group meningococcus, adopts a mucous membrane immunization way, has the characteristics of no tissue injury, no local side effect and simple and convenient use, and is easy to popularize and use.

The broad-spectrum multi-subunit vaccine for preventing the meningococcal infection of the group B is characterized in that the active ingredients of the vaccine consist of an ingredient A, an ingredient B, an ingredient C, an ingredient D, an ingredient E, an ingredient A and an ingredient G;

The component A is a complement regulator H binding protein fHbp, a fusion protein with the full-length or partial amino acid sequence of the fHbp, a protein in which the full-length or partial amino acid sequence of the fHbp is conjugated and connected with an adjuvant protein, a connection complex of the full-length or partial amino acid sequence of the fHbp and polysaccharide or a DNA expression vector carrying the full-length or partial encoding gene of the fHbp;

the component B is NHBA, fusion protein with the full-length or partial amino acid sequence of the NHBA, protein in which the full-length or partial amino acid sequence of the NHBA is conjugated and connected with adjuvant protein, a connection complex of the full-length or partial amino acid sequence of the NHBA and polysaccharide or a DNA expression vector carrying the full-length or partial coding gene of the NHBA;

The component C is fHbp-Opa (HV 2), a fusion protein with the full-length or partial amino acid sequence of the fHbp-Opa (HV 2), a protein in which the full-length or partial amino acid sequence of the fHbp-Opa (HV 2) is conjugated and connected with an adjuvant protein, a connection complex of the full-length or partial amino acid sequence of the fHbp-Opa (HV 2) and polysaccharide or a DNA expression vector carrying the full-length or partial encoding gene of the fHbp-Opa (HV 2);

Said component is pile, a fusion protein having said pile full-length or partial amino acid sequence, a protein in which said pile full-length or partial amino acid sequence is conjugated to an adjuvant protein, a complex of said pile full-length or partial amino acid sequence and a polysaccharide, or a DNA expression vector carrying said pile full-length or partial coding gene;

The component (A) is App, fusion protein with the whole or partial amino acid sequence of the App, protein in which the whole or partial amino acid sequence of the App is conjugated and connected with adjuvant protein, a connection complex of the whole or partial amino acid sequence of the App and polysaccharide or a DNA expression vector carrying the whole or partial coding gene of the App;

the component is IgA protease, fusion protein with the full-length or partial amino acid sequence of the IgA protease, protein conjugated and connected with adjuvant protein by the full-length or partial amino acid sequence of the IgA protease, a connecting complex of the full-length or partial amino acid sequence of the IgA protease and polysaccharide or a DNA expression vector carrying the full-length or partial coding gene of the IgA protease;

the component G is an immunological adjuvant CpG;

The vaccine has the function of providing immune protection for the group B meningococcus;

The mass ratio of the component A to the component B to the component C to the component D to the component E to the component G is 1:1:1:1:1:1.

In a preferred embodiment of the present invention, the component A is a fHbp, and the amino acid sequence after the signal peptide is removed from the fHbp is specifically shown as SEQ ID NO. 3;

The component B is NHBA, and has an amino acid sequence after removing the signal peptide and the cell wall anchoring domain of the NHBA, and is specifically shown as SEQ ID NO. 6;

The component C is fHbp-Opa (HV 2) -GST, and has the amino acid sequences of the fHbp removal signal peptide and Opa (HV 2) variable region, and GST labels, and the GST labels are specifically shown as SEQ ID NO. 14;

The component D is pile, has an amino acid sequence of pile after the signal peptide is removed, and is specifically shown as SEQ ID NO. 17;

the component is App, and the amino acid sequence of the binding domain of the App is shown as SEQ ID NO. 20;

The component is IgA Protease, and has an amino acid sequence after the IgA Protease removes a signal peptide and a cell wall anchoring domain, and the amino acid sequence is specifically shown as SEQ ID NO. 23.

The use modes of the vaccine comprise pulmonary inhalation, nasal inhalation, oral administration, subcutaneous injection, intradermal injection, genital tract injection and anal injection.

In a preferred embodiment, the vaccine is administered by nasal inhalation.

In a preferred embodiment, the vaccine comprises 10 μg/dose of component A, component B, component C, component D, component F, and component G, and the immunization times are 3 times, spaced apart by one week.

In a further aspect the invention provides the use of a vaccine as described in any of the preceding in the manufacture of a medicament for the prevention of a meningococcal infection in group B.

The vaccine has the functions of (I) efficiently inducing IgG in serum and secretory IgA in mucous membrane, (II) inducing specific T cell reaction mainly of Th17 in lung and spleen, and (III) inducing neutralizing antibody with high bactericidal activity to clinical strain. The clinical strain of the group B meningitis is 2015-16#.

The group B meningococcus comprises different group B meningococcus epidemic strains.

The prevention of the meningococcal infection of the group B is prevention of human infection caused by different meningococcal infection of the group B.

The human infection is an infection of respiratory system, digestive system, urinary system, reproductive system, skin and blood circulation system.

Th17 cells are a newly discovered T cell type in recent years, and immune memory Th17 cells generated after mucosal immunity can rapidly migrate to infected mucosal sites, and play an important role in anti-adhesion membrane bacterial infection. Different from the immunity provided by B cells, the immunity provided by T cells has the characteristic of tolerating antigen variation, can provide cross immunity protection for allogeneic bacteria, and is a theoretical basis for constructing novel vaccines. The cytokine IL-17 released after the activation of Th17 cells activates neutrophil granulocyte and macrophage phagocytosis, effectively killing pathogenic bacteria entering the body. The vaccine component selected is capable of inducing a Th17 cell-based immune response.

Antigens such as fHbp and NHBA are located on the meningococcal surface of group B, and antibodies directed against these virulence factors can specifically neutralize the pathogenic effects of virulence factors or act against meningococcal infection of group B through complement-received antibody-dependent bactericidal activity.

The inventor has found through long-term intensive research that the combined use of fHbp, NHBA, fHbp-Opa (HV 2), pipe, app and IgAprotease can induce and induce neutralizing antibodies and activate Th17 cell responses, thereby playing a more effective role in protecting group B meningococcus. Thus, the combination of fHbp, NHBA, fHbp-Opa (HV 2), pipe, app, and IgA protease can have a broad spectrum bactericidal effect against group B meningococcus. The mucosal adjuvant can promote the vaccine subunit to be taken up by antigen processing cells at a mucosal site, remarkably enhance the immunogenicity and the immune effect of the vaccine subunit, and simultaneously avoid local tissue reaction caused by the adjuvant in muscle or subcutaneous immunity.

The antigen adopted by the multi-linked recombinant protein vaccine (hereinafter referred to as Men.B-V6) is ubiquitous in Neisseria, and the homology is more than 90%. The immunogenicity of the antigen is enhanced by fully utilizing the universality and homology of various antigens in neisseria, mucosal pathway immunity and mucosal immunity adjuvant, the activation of Th17 cells and the level of antibodies are obviously improved, the effects of preventing pathogenic bacteria from colonising and rapidly eliminating pathogenic bacteria are achieved, and the neisseria meningitidis strain B has the protection effect, and has the advantages of high efficiency, broad spectrum and low price. Meanwhile, the vaccine provided by the invention adopts a mucosal immunization way, has the characteristics of no tissue injury, no local side effect and simple and convenient use, and is easy to popularize and use;

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are required to be used in the description of the embodiments will be briefly described below.

FIG. 1 is a diagram of polyacrylamide gel electrophoresis during preparation of fHbp, NHBA, fHbp-Opa (HV 2), pile, app, igA Protease proteins;

FIG. 2 is a graph showing the results of antigen-specific serum IgG responses from men.B-V6 immune-induced mice in example 2;

FIG. 3 is a graph showing the results of antigen-specific secretory IgA response by men.B-V6 immune-induced mice in example 2;

FIG. 4 is a graph showing the results of the Th17 immune cell response specific for the antigen induced by men.B-V6 immunization of mice in example 3.

Detailed Description

The invention is further illustrated by the following description of specific embodiments:

the experimental methods in the following examples, which are conventional methods unless otherwise specified, are provided for better understanding of the present invention, but are not limited thereto. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores. The quantitative tests in the following examples were all set up in triplicate and the results averaged.

CpG reference Iho S,Maeyama J,Suzuki F.CpG oligodeoxynucleotides as mucosal adjuvants.Hum Vaccin Immunother.2015;11(3):755-60.

Vector pET28a (+): novagen, cat. No.69846-3. Vector pGEX6P-1: vast, organism, cat.P0005. Vector pCold-SUMO, sea-based biotechnology Co., ltd, cat.C1801M. Coli BL21 (DE 3) was purchased from Beijing full gold Biotechnology Co., ltd, product number CD601-02.(ICR) IGS mice-purchased from CHARLES RIVER; STRAIN CODE:201.

Example 1

Preparation of complement binding proteins (fHbp)

1. And (3) taking genome DNA of the group B meningitis clinical strain 2015-16# as a template, and carrying out PCR amplification by using a primer pair consisting of F1 and R1 to obtain a PCR amplification product.

F1:5’-CATCATATGGTCGCCGCCGACATCGGCGC-3’;

R1:5’-GATCTCGAGCTACTGTTTGCCGGCGATGC-3’。

2. The PCR amplified product of step 1 was digested with restriction enzymes NdeI and XhoI, and the digested product was recovered.

3. The vector pET28a (+) was digested with the restriction enzymes NdeI and XhoI, and the vector backbone of about 5400bp was recovered.

4. And (3) connecting the enzyme digestion product in the step (2) with the vector skeleton in the step (3) to obtain the recombinant plasmid pET28a-fHbp. Based on the sequencing results, the recombinant plasmid pET28a-fHbp was obtained by inserting a double-stranded DNA molecule shown in the 28 th to 274 th nucleotides from the 5' end of sequence 1 of the sequence table between NdeI and XhoI cleavage sites of the vector pET28a (+). The inserted double-stranded DNA molecules and partial DNA on the carrier framework form a fusion gene shown in a sequence 2 of a sequence table, and the fusion protein shown in a sequence 3 of the sequence table is expressed.

5. The recombinant plasmid pET28a-fHbp is introduced into escherichia coli BL21 (DE 3) to obtain recombinant bacteria.

6. The recombinant bacteria obtained in the step 5 were inoculated into LB liquid medium containing 50. Mu.g/ml kanamycin, cultured at 37℃and 220rpm with shaking until OD560 nm=0.6, induced by adding IPTG and the concentration thereof was 40. Mu.g/ml, cultured at 37℃and 220rpm with shaking for 4 hours.

7. The culture system of step 6 was centrifuged at 3000rpm at 4℃for 20 minutes to collect the cell pellet, the cell pellet was suspended in PBS buffer at pH7.4 and sonicated (power: 200W, intermittent 6 seconds every 4 seconds of operation, 99 cycles), and then centrifuged at 12000rpm for 20 minutes to collect the supernatant.

8. The supernatant obtained in step 7 was applied to a GE company Ni Sepharose 6Fast Flow, eluting with 10 column volumes with a solution I (pH 7.4, the solvent was water, 20mM Na2HPO4 and 500mM NaCl) to remove the impurity protein, eluting with a solution II (pH 7.4, the solvent was water, 200mM imidazole, 20mM Na2HPO4 and 500mM NaCl) to obtain the target protein, and collecting the post-column-passing solution when eluting with the solution II, and then designating it as a fHbp solution.

10 Mg of protein with purity of more than 90% can be obtained per liter of the culture system of the step 6.

Preparation of NHBA

1. And (3) taking genomic DNA of the group B meningitis clinical strain 2015-16# as a template, and carrying out PCR amplification by using a primer pair consisting of F1 and R1 to obtain a PCR amplification product.

F1:5’-CCGCCATGGCGAATGGCGGTAGCAATTTT-3’;

R1:5’-CCGCTCGAGTGCGGCCGCAAGCTTGTCG-3’。

2. The PCR amplified product of step 1 was digested with restriction enzymes NcoI and XhoI, and the digested product was recovered.

3. The vector pET28a (+) was digested with restriction enzymes NcoI and XhoI, and a vector backbone of about 6000bp was recovered.

4. And (3) connecting the enzyme digestion product of the step (2) with the vector skeleton of the step (3) to obtain a recombinant plasmid pET28a-NHBA. Based on the sequencing result, the recombinant plasmid pET28a-NHBA is described as a double-stranded DNA molecule shown in 397-1284 nucleotides from the 5' end of the sequence 4 of the sequence table inserted between NcoI and XhoI cleavage sites of the vector pET28a (+). The inserted double-stranded DNA molecule and partial DNA on the carrier skeleton form a fusion gene shown in a sequence 5 of a sequence table, and the fusion protein shown in a sequence 6 of the sequence table is expressed.

5. The recombinant plasmid pET28a-NHBA is introduced into escherichia coli BL21 (DE 3) to obtain recombinant bacteria.

6. The recombinant bacteria obtained in the step 5 were inoculated into LB liquid medium containing 50. Mu.g/mL kanamycin, cultured at 37℃and 220rpm with shaking until OD560 nm=0.6, induced by adding IPTG and the concentration thereof was 40. Mu.g/mL, cultured at 37℃and 220rpm with shaking for 4 hours.

7. The culture system of step 6 was centrifuged at 4℃and 6000rpm for 10 minutes to collect the cell pellet, the cell pellet was suspended in PBS buffer at pH7.4 and sonicated (power: 200W, intermittent 8 seconds every 4 seconds of operation, 99 cycles), and then centrifuged at 12000rpm for 20 minutes to collect the supernatant.

8. The supernatant obtained in step 7 was applied to a GE company Ni Sepharose 6Fast Flow, eluting with 10 column volumes with a solution I (pH 7.4, the solvent was water, 20mM Na2HPO4 and 500mM NaCl) to remove the impurity protein, eluting with a solution II (pH 7.4, the solvent was water, 200mM imidazole, 20mM Na2HPO4 and 500mM NaCl) to obtain the target protein, and collecting the post-column-passing solution when eluting with the solution II, and then designated as NHBA solution.

24 Mg of protein with purity of more than 90% can be obtained per liter of the culture system of the step 6.

Preparation of fHbp-Opa (HV 2)

1. The Opa is taken as a membrane penetrating protein, the structure is more complicated, 16 transmembrane regions are provided, the soluble protein cannot be obtained, the virulence factor fHbp is taken as a molecular scaffold of an extramembranous soluble region of the Opa, the extramembranous soluble region VR2 loop of the Opa and the fHbp form a chimeric antigen, and the chimeric antigen can generate immune protection. Has an amino acid sequence that deletes the signal peptide for fHbp, and simultaneously deletes the 240 th aspartic acid, and inserts VR2 loop in the Opa membrane outside soluble region. The method comprises the steps of taking a group B meningococcal clinical strain 2015-16# genome DNA as a template, carrying out PCR amplification by using a primer pair consisting of F1 and R1 to obtain a PCR amplification product, and sequencing to obtain an HV2 sequence:

F1:5′-TGAAAACCGTAGCCTACGGACACGTTAGGCATCA-3′

R1:5′-CAGCATCCGCCGCTTGGGTGGCATACGCCATATCG-3′

2. the genome DNA of the meningococcal standard strain MC58 of group B is used as a template, and a primer pair consisting of F1 and R1 is used for PCR amplification to obtain a PCR amplification product, and a fHbp skeleton is obtained:

F1:5′-TGTTCCAGGGGCCCCTGGGATCCGTCGCCGCCGACATCGGT-3′

R1:5′-ATATCGGCCTTGCCGCCAAGCAACTCGAGCGGCCGCATCGTG-3′

4. the vector pGEX6P-1 was digested with the restriction enzymes BamHI and XhoI.

5. And (3) carrying out homologous recombination on the enzyme digestion products of the steps (2) and the vector skeleton of the step (4) to obtain a recombinant plasmid pGEX6P-1-fHbp-Opa (HV 2). Based on the sequencing results, the recombinant plasmid pGEX6P-1-fHbp (Opa) was described as a double-stranded DNA molecule shown in sequence 7 and sequence 8 of the sequence Listing inserted between BamHI and XhoI cleavage sites of the vector pGEX 6P-1. The inserted double-stranded DNA molecule and partial DNA on the carrier skeleton form a fusion gene shown in a sequence 9 of a sequence table, and the fusion protein shown in a sequence 10 of the sequence table is expressed.

6. Recombinant plasmid pGEX6P-1-fHbp-Opa (HV 2) was introduced into E.coli BL21 (DE 3) to obtain recombinant bacteria.

7. The recombinant bacteria obtained in the step 5 were inoculated into LB liquid medium containing 50. Mu.g/mL kanamycin, and were shake-cultured at 37℃and 220rpm until OD560 nm=0.6, and were induced by adding IPTG, and shake-cultured at 37℃and 220rpm for 4 hours.

8. The culture system of step 7 was centrifuged at 3000rpm at 4℃for 20 minutes to collect the cell pellet, the cell pellet was suspended in PBS buffer at pH7.4 and sonicated (power: 200W, intermittent 8 seconds every 4 seconds of operation, 99 cycles), and then centrifuged at 12000rpm for 20 minutes to collect the supernatant.

9. The supernatant obtained in the step 8 is loaded on GST rap HP, firstly, elution is carried out by using an equilibrium buffer solution for 10 column volumes to remove the impurity proteins, and then the target proteins are obtained by eluting with an eluent.

10. And (3) further purifying the eluent obtained in the step (9) by using an AKTA pulsifer to obtain purer target protein.

Pile preparation

1. And (3) taking the genomic DNA of the group B meningococcal clinical strain 2015-16# as a template, and carrying out PCR amplification by using a primer pair consisting of F1 and R1 to obtain a PCR amplification product.

F1:5’-CCGCCATGGCCTGCTTATCAAGACTACACAGC-3’;

R1:5’-CCGCTCGAGGCTGGCAGATGAAGCGTCGC-3’。

2. The PCR amplified product of step 1 was digested with restriction enzymes NcoI and XhoI, and the digested product was recovered.

3. The vector pCold-sumo (+) was digested with restriction enzymes NcoI and XhoI, and a vector backbone of about 6000bp was recovered.

4. And (3) connecting the enzyme digestion product of the step (2) with the vector skeleton of the step (3) to obtain a recombinant plasmid pCold-sumo-pile. Based on the sequencing result, the recombinant plasmid pCold-sumo-pile was described as a double-stranded DNA molecule comprising nucleotides 85-504 of sequence 11 of the sequence table inserted between NcoI and XhoI cleavage sites of the vector pCold-sumo (+). The inserted double-stranded DNA molecule and partial DNA on the carrier skeleton form a fusion gene shown in a sequence 12 of a sequence table, and the fusion protein shown in a sequence 13 of the sequence table is expressed.

5. The recombinant plasmid pCold-sumo-pile was introduced into E.coli BL21 (DE 3) to obtain recombinant bacteria.

6. The recombinant strain obtained in the step 5 was inoculated into LB liquid medium containing 50. Mu.g/mL of ampicillin, cultured at 37℃and 220rpm with shaking until OD560 nm=0.6, and induced by adding IPTG to a concentration of 40. Mu.g/mL, and cultured at 37℃and 220rpm with shaking for 4 hours.

7. The culture system of step 6 was centrifuged at 3000rpm at 4℃for 20 minutes to collect the cell pellet, the cell pellet was suspended in PBS buffer at pH7.4 and sonicated (power: 200W, intermittent 8 seconds every 4 seconds of operation, 99 cycles), and then centrifuged at 12000rpm for 20 minutes to collect the supernatant.

8. The supernatant obtained in step 7 was applied to a GE company Ni Sepharose 6Fast Flow, eluting with 10 column volumes with a solution I (pH 7.4, the solvent was water, 20mM Na2HPO4 and 500mM NaCl) to remove the impurity protein, eluting with a solution II (pH 7.4, the solvent was water, 200mM imidazole, 20mM Na2HPO4 and 500mM NaCl) to obtain the target protein, and collecting the post-column-passing solution when eluting with the solution II, and then designated as pile solution.

12 Mg of protein with purity of more than 90% can be obtained per liter of the culture system of the step 6.

Preparation of App

1. And (3) taking the genomic DNA of the group B meningococcal clinical strain 2015-16# as a template, and carrying out PCR amplification by using a primer pair consisting of F1 and R1 to obtain a PCR amplification product.

F1:5′-CATGCCATGGGGATCCGAAAAAGACAACG-3′

R1:5′-CCGCTCGAGCTCGAGGTCGACGCCTAATT-3′

2. The PCR amplified product of step 1 was digested with restriction enzymes NcoI and XhoI, and the digested product was recovered.

3. Vector pET28a was digested with the restriction enzymes NcoI and XhoI.

4. And (3) connecting the enzyme digestion product in the step (2) with the vector skeleton in the step (3) to obtain the recombinant plasmid pET28a-App. Based on the sequencing result, the result of the recombinant plasmid pET28a-App is described as a double-stranded DNA molecule in which nucleotides 3229-4362 from the 5' -end of sequence 14 of the sequence table are inserted between NcoI and XhoI cleavage sites of the vector pET28a-App. The inserted double-stranded DNA molecule and partial DNA on the carrier skeleton form a fusion gene shown in a sequence 15 of a sequence table, and the fusion protein shown in a sequence 16 of the sequence table is expressed.

5. The recombinant plasmid pET28a-App is introduced into escherichia coli BL21 (DE 3) to obtain recombinant bacteria.

6. Inoculating the recombinant bacteria obtained in the step 5 into LB liquid medium containing 100 mug/mL ampicillin, culturing at 37 ℃ and 220rpm in a shaking way until OD560 nm=0.6, cooling the medium to 15 ℃, adding IPTG for induction, culturing at 15 ℃ and 220rpm in a shaking way for 24 hours.

7. The culture system of step 6 was centrifuged at 3000rpm at 4℃for 20 minutes to collect the cell pellet, the cell pellet was suspended in PBS buffer at pH7.4 and sonicated (power: 200W, intermittent 8 seconds every 4 seconds of operation, 99 cycles), and then centrifuged at 12000rpm for 20 minutes to collect the supernatant.

8. Loading the supernatant obtained in the step 7 on Ni Sepharose 6Fast Flow, eluting with an equilibrium buffer solution for 10 column volumes to remove the impurity proteins, eluting with 0, 20, 50, 100 and 150mM imidazole respectively to obtain target proteins, and collecting the solution after column passing when eluting with 150mM imidazole.

9. And (3) further purifying the eluent obtained in the step (8) by using an AKTA pulsifer to obtain purer target protein.

IgA Protease preparation

1. And (3) taking the genomic DNA of the group B meningococcal clinical strain 2015-16# as a template, and carrying out PCR amplification by using a primer pair consisting of F1 and R1 to obtain a PCR amplification product.

F1:5′-CATGCCATGGGCATTGGTCAGAGACGATGTCG-3′

R1:5′-ACGCCTCGAG GTTCTCGGCATAAGGATTGTACAAT-3′

2. The PCR amplified product of step 1 was digested with restriction enzymes NcoI and XhoI, and the digested product was recovered.

3. Vector pET28a was digested with the restriction enzymes NcoI and XhoI.

4. And (3) connecting the enzyme digestion product in the step (2) with the vector skeleton in the step (3) to obtain the recombinant plasmid pET28a-IgA Protease. Based on the sequencing result, the recombinant plasmid pET28a-IgA Protease was described as a double-stranded DNA molecule of the nucleotide sequence No. 82-2904 from the 5' -end, which was inserted between the NcoI and XhoI cleavage sites of the vector pET28a, in sequence 17 of the sequence table. The inserted double-stranded DNA molecule and partial DNA on the carrier skeleton form a fusion gene shown in a sequence 18 of a sequence table, and the fusion protein shown in a sequence 19 of the sequence table is expressed.

5. The recombinant plasmid pET28a-IgA Protease is introduced into escherichia coli BL21 (DE 3) to obtain recombinant bacteria.

6. Inoculating the recombinant strain obtained in the step 5 into LB liquid medium containing 100 mug/mL kanamycin, culturing at 37 ℃ and 220rpm in a shaking way until OD560 nm=0.6, cooling the medium to 15 ℃, adding IPTG for induction, and culturing at 15 ℃ and 220rpm in a shaking way for 24 hours.

7. The culture system of step 6 was centrifuged at 4℃and 6000rpm for 10 minutes to collect the cell pellet, the cell pellet was suspended in PBS buffer at pH7.4 and sonicated (power: 200W, intermittent 8 seconds every 4 seconds of operation, 99 cycles), and then centrifuged at 12000rpm for 20 minutes to collect the supernatant.

8. Loading the supernatant obtained in the step 7 on Ni Sepharose 6Fast Flow, eluting with an equilibrium buffer solution for 10 column volumes to remove the impurity proteins, eluting with 0, 20, 50, 100 and 150mM imidazole respectively to obtain target proteins, and collecting the solution after column passing when eluting with 150mM imidazole.

9. And (3) further purifying the eluent obtained in the step (8) by using an AKTA pulsifer to obtain purer target protein.

The polyacrylamide gel electrophoresis diagram of the process of preparing fHbp, NHBA, fHbp-Opa (HV 2) and pile, app, igAprotease proteins is shown in figure 1. Wherein men.B recombinant protein fHbp(Mw≈34kd),NHBA(Mw≈30kd),fHbp-Opa(HV2)-GST(Mw≈59kd),pile(Mw≈27kd),App(Mw≈42kd),IgA Protease(Mw≈107kd).

Example 2

Men.b-V6 immunization induced antibody responses in mice, and 6-8 week old female C57BL/6 mice were randomized into two groups, the grouping being as follows:

CpG group, i.e. nasal instilling CpG solution respectively on experiment day 1, day 7 and day 14;

Group Men.B-V6 vaccine solutions (vaccine solutions were obtained by mixing 6 recombinant proteins prepared in example 1 with CpG solution, and each mouse was given 10. Mu.g and 10. Mu.g of CpG per 6 recombinant proteins) were instilled via nasal cavity on days 1, 7 and 14 of the experiment, respectively. On experiment day 21, tail blood and oral lavage fluid were taken after anesthetizing the mice, ELIAS serum IgG, oral lavage fluid IgA. The results are shown in FIG. 2 and FIG. 3. Compared to the CpG group, men.b-V6 group induced significantly antigen-specific serum IgG and secretory IgA.

Example 3

Men.b-V6 mucosal immunity induces a Th17 cell immune response, and 6-8 week old female C57BL/6 mice were randomly divided into two groups, the grouping being as follows:

CpG group, i.e. nasal instilling CpG solution respectively on experiment day 1, day 7 and day 14;

Group Men.B-V6 vaccine solutions (vaccine solutions were obtained by mixing 6 recombinant proteins prepared in example 1 with CpG solution, and each mouse was given 10. Mu.g and 10. Mu.g of CpG per 6 recombinant proteins) were instilled via nasal cavity on days 1, 7 and 14 of the experiment, respectively. On experiment day 21, spleen and lung tissue were taken to extract cells, and men.b-V6 antigen-specific IL-17+ cells were measured using ELISPOT. The results are shown in FIG. 4, and compared with CpG groups, GBSV6 groups significantly induce antigen-specific Th17 cells in spleen and lung tissues, indicating that Men.B-V6 nasal immunized mice induce Th17 cell responses.

Example 4

Complement-mediated sterilization assay to prepare mouse antisera men.b-V6 mucosal immunity induces an immune response to Th17 cells, 6-8 week old female C57BL/6 mice were randomly grouped and the grouping was treated as follows:

TABLE 1 formulation grouping of multicomponent vaccine components

On experiment 1, 7 and 14, control (CpG adjuvant) vaccine solutions (vaccine solutions were obtained by mixing 6 recombinant proteins prepared in example 1 with CpG solutions according to Table 1 group) were added through nasal cavity, mice were immunized, on experiment 21, tail-broken blood was taken and heat-inactivated at 56℃for 30 minutes before the test, serum from young rabbits was used as complement source, and the bactericidal titer of serum was defined as the serum dilution corresponding to 50% decrease of the remaining CFU before the reaction after incubation of bacteria with the reaction mixture for 60 minutes, the results are shown in Table 2:

Table 2B clinical trials for sterilizing force of meningitis BC50 (Titer)

The above examples are given for clarity of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.

While certain specific embodiments of the invention have been described in detail by way of example, it will be appreciated by those skilled in the art that the above examples are for illustration only and are not intended to limit the scope of the invention. Those skilled in the art will also appreciate that many modifications may be made to the embodiments without departing from the scope and spirit of the invention. The scope of the invention is defined by the appended claims.

Claims (10)

1. A broad-spectrum multi-subunit vaccine for preventing serogroup B meningococcal infection is characterized in that the active ingredients of the vaccine comprise an ingredient A, an ingredient B, an ingredient C, an ingredient D, an ingredient E, an ingredient A and an ingredient G;

The component A is a complement regulator H binding protein fHbp, a fusion protein with the full-length or partial amino acid sequence of the fHbp, a protein in which the full-length or partial amino acid sequence of the fHbp is conjugated and connected with an adjuvant protein, a connection complex of the full-length or partial amino acid sequence of the fHbp and polysaccharide or a DNA expression vector carrying the full-length or partial encoding gene of the fHbp;

the component B is NHBA, fusion protein with the full-length or partial amino acid sequence of the NHBA, protein in which the full-length or partial amino acid sequence of the NHBA is conjugated and connected with adjuvant protein, a connection complex of the full-length or partial amino acid sequence of the NHBA and polysaccharide or a DNA expression vector carrying the full-length or partial coding gene of the NHBA;

The component C is fHbp-Opa (HV 2), a fusion protein with the full-length or partial amino acid sequence of the fHbp-Opa (HV 2), a protein in which the full-length or partial amino acid sequence of the fHbp-Opa (HV 2) is conjugated and connected with an adjuvant protein, a connection complex of the full-length or partial amino acid sequence of the fHbp-Opa (HV 2) and polysaccharide or a DNA expression vector carrying the full-length or partial encoding gene of the fHbp-Opa (HV 2);

Said component is pile, a fusion protein having said pile full-length or partial amino acid sequence, a protein in which said pile full-length or partial amino acid sequence is conjugated to an adjuvant protein, a complex of said pile full-length or partial amino acid sequence and a polysaccharide, or a DNA expression vector carrying said pile full-length or partial coding gene;

The component (A) is App, fusion protein with the whole or partial amino acid sequence of the App, protein in which the whole or partial amino acid sequence of the App is conjugated and connected with adjuvant protein, a connection complex of the whole or partial amino acid sequence of the App and polysaccharide or a DNA expression vector carrying the whole or partial coding gene of the App;

the component is IgA protease, fusion protein with the full-length or partial amino acid sequence of the IgA protease, protein conjugated and connected with adjuvant protein by the full-length or partial amino acid sequence of the IgA protease, a connecting complex of the full-length or partial amino acid sequence of the IgA protease and polysaccharide or a DNA expression vector carrying the full-length or partial coding gene of the IgA protease;

the component G is an immunological adjuvant CpG;

The mass ratio of the component A to the component B to the component C to the component D to the component E to the component G is 1:1:1:1:1:1.

2. A broad spectrum multi-linked subunit vaccine for preventing meningococcal infection from serogroup B according to claim 1, wherein:

the component A is the fHbp, and has an amino acid sequence after the signal peptide is removed from the fHbp, and is specifically shown as SEQ ID NO. 3;

The component B is NHBA, and has an amino acid sequence after removing the signal peptide and the cell wall anchoring domain of the NHBA, and is specifically shown as SEQ ID NO. 6;

The component C is fHbp-Opa (HV 2) -GST, and has the amino acid sequences of the fHbp removal signal peptide and Opa (HV 2) variable region, and GST labels, and the GST labels are specifically shown as SEQ ID NO. 14;

The component D is pile, has an amino acid sequence of pile after the signal peptide is removed, and is specifically shown as SEQ ID NO. 17;

the component is App, and the amino acid sequence of the binding domain of the App is shown as SEQ ID NO. 20;

The component is IgA Protease, and has an amino acid sequence after the IgA Protease removes a signal peptide and a cell wall anchoring domain, and the amino acid sequence is specifically shown as SEQ ID NO. 23.

3. A broad spectrum multi-subunit vaccine for preventing meningococcal infection within group B according to claim 1 or 2, wherein said vaccine is administered by pulmonary inhalation, nasal inhalation, oral administration, subcutaneous injection, intradermal injection, genital tract injection, or anal injection.

4. A broad spectrum multi-subunit vaccine for preventing meningococcal infection within group B as claimed in claim 3, wherein said vaccine is administered by nasal inhalation.

5. The broad-spectrum multi-subunit vaccine for preventing meningococcal infection of group B of claim 4, wherein the amount of component A, component B, component C, component D, component F, component Y and component G is 10 μg/time, and the number of immunizations is 3 times at intervals of one week.

6. Use of the vaccine of any one of claims 1-5 in the manufacture of a medicament for preventing a group B meningococcal infection.

7. The method of claim 6, wherein the vaccine has the functions of (I) efficiently inducing IgG in serum and secretory IgA in mucous membrane, (II) inducing specific T cell reaction mainly including Th17 in lung and spleen, and (III) inducing neutralizing antibody with high bactericidal activity to clinical strain.

8. The method of claim 6, wherein the group B meningococcus comprises different epidemic strains of group B meningococcus.

9. The method according to claim 6, wherein the preventing of meningococcal infection in group B is preventing infection in humans by different meningococcal infection in group B.

10. The method according to claim 9, wherein the human is infected with respiratory, digestive, urinary, reproductive, skin and blood systems.