CN117643623A

CN117643623A - Broad-spectrum multi-subunit vaccine for preventing B group streptococcus infection and application

Info

Publication number: CN117643623A
Application number: CN202311124028.1A
Authority: CN
Inventors: 王北难; 王杰; 王海龙
Original assignee: Beijing Huanuotai Biomedical Technology Co ltd
Current assignee: Beijing Huanuotai Biomedical Technology Co ltd
Priority date: 2023-09-01
Filing date: 2023-09-01
Publication date: 2024-03-05

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to a broad-spectrum multi-subunit vaccine for preventing group B streptococcus infection and application thereof, wherein component A is sortase A, and the sortase A has a protein sequence after signal peptide is removed; component B is CspA, and has a protein sequence after CspA removes signal peptide and cell wall anchoring domain; component C is C5a peptidase, and has a protein sequence after the signal peptide and the cell wall anchoring domain are removed by the C5a peptidase; component D is FbsB, and has a protein sequence after FbsB is removed from the signal peptide; the component penta is Srr2, and has a protein sequence of a binding domain of Srr 2; the component is hexobA, and has a protein sequence after the signal peptide and the cell wall anchoring domain are removed from the BibA; the ingredient heptanzoic 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 B group streptococcus infection and application

Technical Field

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

Background

Sortase a (SrtA) is present in highly conserved membrane surface proteins of a variety of gram-positive pathogens and is necessary for the anchoring of virulence factors for a variety of membrane surface proteins. Group B streptococcus (Group B Streptococcus, hereinafter referred to as B chain bacteria) lacking sortase A, also called streptococcus agalactiae, belongs to conditional pathogenic bacteria, is asymptomatic colonized in female vagina or gastrointestinal tract, and is a source of infection of newborns and pregnant women. Maternal colonization makes pregnant and parturients and neonates prone to various bad outcomes. Pregnant women can be infected by abortion, premature birth, stillbirth, intrauterine restriction of fetus, premature rupture of fetal membranes, urinary tract infection, upper genital tract infection, postpartum endometritis, etc. More importantly, maternal colonization increases the risk of neonatal infection, and B-chain bacteria can cause neonatal pneumonia, meningitis, septicemia and even death after being transmitted to the neonate through placenta or birth canal during delivery, so that the B-chain bacteria are always valued by world health organization and scientists. World health organization data indicate that annual GBS infections result in about 15 tens of thousands of deaths, over 50 tens of thousands of premature births and severe long-term disabilities.

The main reason for restricting vaccine development is that since there are 10 types of B-chain bacteria, each serotype lacks crossover, and it is difficult to develop a vaccine against all serotypes. Second, vaccines need to provide immune protection to maternal and fetal/neonates. Finally, since B-chain bacterial colonization of pregnant women can lead to infection of newborns from 3 months of life, world health organization and specialists suggest providing immune protection for newborns by maternal immunization, but maternal immunization may pose certain safety problems, which seriously hamper the development process of B-chain bacterial vaccines. Thus, there is an urgent need to develop a vaccine that is safe, effective, broad-spectrum, and easy to popularize and apply.

The B-chain bacteria deleted for bacterial Sortase a (Sortase a, srtA) resulted in a variety of membrane protein related functional defects, with significantly reduced adhesion to host epithelial cells. CspA (cell-surface-associated protein) is a virulence protein secreted by B-chain bacteria, and is present in B-chain bacterial strains of various serotypes, which can cleave the alpha chain of human fibrinogen and inhibit migration of neutrophils to the site of infection by degrading CXC chemokines, inhibiting host clearance of B-chain bacteria. The B-chain bacteria deleted for CspA have been shown to have significantly reduced virulence and reduced phagocytic capacity of neutrophils. The C5a peptidase is a virulence protein located on the surface of B-chain bacteria (C5 apeptidase, scpB), which is prevalent in B-chain bacteria of all serotypes. The C5a peptidase can inactivate normal complement protein C5a in a human body, can be combined with motifs generated by a plurality of adjacent fibronectin molecules in parallel, and participates in invasion of epithelial cells. Studies have demonstrated that C5a peptidase immunizes female mice via the respiratory tract, not only reduces vaginal colonization of the female mice by B-chain bacteria, but also provides immune protection to neonatal mice born by the female mice. FbsB (fiber-binding protein) is an adhesin that is ubiquitous in various serotypes of B-chain bacteria, and can bind to human Fibrinogen and attenuate invasive infections of epithelial cells by FbsB knockout strains, which are associated with the invasive ability of B-chain bacteria to enter cells. Srr2 (Serine-rich repeat 2) is a cell wall anchoring protein of the high virulent B-chain strain ST-17, an adhesin, which binds fibrinogen and plasminogen. The colonization of the mouse brain tissue by the Srr2 knockout strain is reduced, and it has been demonstrated that immunization of mice with the recombinant protein Srr2 can induce the production of their corresponding specific antibodies, providing immune protection against B-chain lethal infection. BibA (GBS immunogenic bacterial adhesin) is a multifunctional protein anchored on the surface of B chain bacteria, and the BibA deletion strain has impaired ability to adhere to human cervical and lung epithelial cells, and at the same time, bibA specifically binds complement C4 binding protein and resists the killing of neutrophils. The gene sequence of BibA is highly conserved in clinical isolates, with up to 98-100% N-terminal domain homology. Immunization of female mice with BibA has been shown to induce antibody production, and the antibodies can be transferred through the placental barrier to young mice to provide protection against infection with B chain bacteria in new-born mice.

However, there is a technical problem that the existing research results confirm that a mixture of single B-chain bacteria subunit components cannot provide comprehensive and effective protection for a plurality of serotype B-chain bacteria.

The specification of Chinese invention CN201710792641.9 describes a broad-spectrum multi-subunit vaccine for preventing group A streptococcus infection, and the active ingredients of the vaccine consist of a component A, a component B, a component C, a component D, a component E and a component A; the component A is sortase or fusion protein with sortase; the component B is C5a protease or fusion protein with the C5a protease; the component C is Spy0269 or fusion protein with the Spy 0269; the component D is SCPC or fusion protein with the SCPC; the component is SLO or fusion protein with the SLO; the component has an adjuvant CpG or other mucosal immune adjuvant. The vaccine of the invention has the advantages of high efficiency, broad spectrum and low price.

The group A streptococcus and the group B streptococcus belong to the same genus of streptococcus, the protective effect of the group A streptococcus broad-spectrum multi-subunit vaccine proves that the combination of a plurality of virulence factors as vaccine antigens has superiority, and a researcher also searches the pathogenic mechanism of the group B streptococcus and screens a series of important specific virulence factors in the pathogenic mechanism of the group B streptococcus so as to obtain the broad-spectrum and high-efficiency group B streptococcus broad-spectrum multi-subunit vaccine. However, group a streptococci and group B streptococci have different infectious groups, modes of infection, mechanisms of infectious pathology, and diseases caused by infection, and there are many technical difficulties in developing a broad-spectrum multi-linked unit vaccine suitable for infection with group B streptococci. Therefore, no vaccine of B chain bacteria exists at home and abroad at present.

Disclosure of Invention

In order to solve the technical problems, the invention provides a broad-spectrum multi-subunit vaccine for preventing B chain bacteria infection and application thereof, which utilizes the conservative antigen mucosa of a plurality of B chain bacteria to immunize mice, effectively induces the reactions of Th17 and antibodies of the mice, and provides broad-spectrum effective protection effects across serotypes for B chain bacteria with lethal doses, B chain bacteria infection with different serotypes and neonatal mice; the vaccine is high-efficiency, broad-spectrum and low-cost, 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 B-chain bacterial infection in the invention for solving the technical problems is characterized in that: the active ingredients of the vaccine consist of a first component, a second component, a third component, a fourth component, a fifth component, a sixth component and a seventh component;

the component A is sortase A, fusion protein with the whole or partial amino acid sequence of sortase A, protein with the whole or partial amino acid sequence of sortase A conjugated and connected with adjuvant protein, connection complex of the whole or partial amino acid sequence of sortase A and polysaccharide or DNA expression vector carrying the whole or partial encoding gene of sortase A;

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

the component C is C5a peptidase, fusion protein with the full length or partial amino acid sequence of the C5a peptidase, protein in which the full length or partial amino acid sequence of the C5a peptidase is conjugated and connected with adjuvant protein, a connection complex of the full length or partial amino acid sequence of the C5a peptidase and polysaccharide or a DNA expression vector carrying the full length or partial coding gene of the C5a peptidase;

the component D is FbsB, fusion protein with the full-length or partial amino acid sequence of the FbsB, protein in conjugated connection of the full-length or partial amino acid sequence of the FbsB and adjuvant protein, a connection complex of the full-length or partial amino acid sequence of the FbsB and polysaccharide or a DNA expression vector carrying the full-length or partial encoding gene of the FbsB;

the component is Srr2, fusion protein with the full-length or partial amino acid sequence of the Srr2, protein in which the full-length or partial amino acid sequence of the Srr2 is conjugated and connected with adjuvant protein, a connection complex of the full-length or partial amino acid sequence of the Srr2 and polysaccharide or a DNA expression vector carrying the full-length or partial coding gene of the Srr 2;

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

also comprises component G as immunological adjuvant CpG;

the vaccine has the functions of preventing B chain bacteria infection or maternal immunity and providing immune protection for newborn infants;

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 1:1:1:1:1:1:1.

in the optimized scheme of the invention, the component A is sortase A, and has a protein sequence after the sortase A removes signal peptide; specifically is a protein composed of an amino acid sequence shown as a sequence 3 in a sequence table;

the component B is CspA, and has a protein sequence after the CspA removes the signal peptide and the cell wall anchoring domain; specifically is a protein composed of an amino acid sequence shown as a sequence 6 in a sequence table; the component C is C5a peptidase, and has a protein sequence after the C5a peptidase removes signal peptide and cell wall anchoring domain; in particular to a protein consisting of an amino acid sequence shown as a sequence 9 in a sequence table. The component D is FbsB and has a protein sequence after the FbsB is removed from the signal peptide; specifically is a protein composed of an amino acid sequence shown as a sequence 12 in a sequence table; the component penta is Srr2, a protein sequence with a binding domain of the Srr 2; specifically is a protein composed of an amino acid sequence shown as a sequence 15 in a sequence table; the component is BibA, and a protein sequence with the BibA removed signal peptide and a cell wall anchoring domain; in particular to a protein consisting of an amino acid sequence shown as a sequence 18 in a sequence table.

The vaccine of the invention has the functions of (I) or (II) or (III) or (IV): inhibiting different serotypes of streptococci B; (ii) preventing infection of humans by different serotypes of B-chain bacteria; (iii) reducing or preventing colonization of the human mucosal system, including the genital tract of pregnant women, by different serotypes of B-chain bacteria; (IV) immunization of the mother results in immunoprotection of the newborn infant.

The different serotypes of B-chain bacteria may be the ten serotypes of B-chain bacteria currently found and reported, respectively B-chain bacteria serotype Ia, ib, II, III, IV, V, VI, VII, VIII, and IX; it may also be a subtype of the above-mentioned serotypes which may occur and a novel serotype which is not included in the above-mentioned serotypes.

In the optimized scheme, the B-chain bacteria can be specifically B-chain bacteria serotype Ib or serotype III.

The prevention of the infection of the B chain bacteria is prevention of the infection of a human mucous membrane system caused by the B chain bacteria.

In a further preferred embodiment, the human mucosal system may be the respiratory system, digestive system, urinary system, reproductive system or skin.

The vaccine is used in the modes of nasal inhalation, oral administration, subcutaneous injection, intradermal injection, genital tract injection or anal injection.

The application of the vaccine in the invention is the application of any one of the vaccines in preparing medicines for preventing B-chain bacteria infection.

The recommended use method of the vaccine provided by the invention comprises the following steps: the protein amount of the component A, the component B, the component C, the component D, the component F and the component F is 10 mug/time, the protein amount of the component G is CpG, the protein amount of the component G is 10 mug/time, the immunization times is 3 times, and the interval is one week. The immunization mode is nasal inhalation.

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. Since sortase a is located inside the bacterial cell wall, antibodies directed against sortase a do not play a role in immunoprotection. Sortase a was able to induce a Th17 cell-based immune response, B-chain bacteria in a T cell dependent manner.

In addition to SrtA, cspA, C5a peptidase, fbsB, srr2, and BibA are located on the surface of B-chain bacteria or secreted extracellularly, antibodies to these virulence factors are capable of specifically neutralizing the pathogenic effects of virulence factors or acting against B-chain bacterial infection through antibody-dependent phagocytic killing.

The inventor has found through long-term intensive research that the combined use of sortase A, cspA, C5a peptidase, fbsB, srr2 and BibA can induce the response of Th17 cells and induce protective antibodies which are not serotype dependent, thereby playing a more effective role in protecting the B chain bacteria. Thus, the combined use of sortase A, cspA, C5a peptidase, fbsB, srr2 and BibA is capable of broad spectrum protection across serotypes of B chain bacteria of different serotypes. 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 use modes of the vaccine provided by the invention comprise pulmonary inhalation, nasal inhalation, oral administration, subcutaneous injection, intradermal injection, genital tract injection, anal injection and the like.

The vaccine provided by the invention can be used for preventing and treating mucosal system (respiratory system, digestive system, urinary system, reproductive system or skin) infection caused by various gram positive bacteria.

The antigen adopted by the multi-linked recombinant protein vaccine (GBSV 6) is commonly existed in streptococcus, and the homology is more than 90%. The universality and homology of various antigens in streptococcus, mucosal pathway immunity and mucosal immunity adjuvant are fully utilized to enhance antigen immunogenicity, the level of Th17 cell activation and antibodies is obviously improved, the effects of preventing pathogenic bacteria from colonizing and rapidly eliminating pathogenic bacteria are achieved, and the vaccine has the protection effect on different serotype B chain bacteria, 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

FIG. 1 is a diagram showing polyacrylamide gel electrophoresis during the preparation of SrtA, scpB, cspA, srr, bibA and FbsB proteins according to the present invention

FIG. 2 is a graph showing the results of example 2 of the present invention (removal of streptococci at genital tract infection site B after nasal inhalation of GBSV 6)

FIG. 3 is a graph showing the results of example 3 of the present invention (immunoprotection against B-chain lethal infection after nasal inhalation of GBSV 6)

FIG. 4 is a graph showing the results of example 4 of the present invention (GBSV 6 immune-induced mice produce Th17 immune cell response specific for antigen)

FIG. 5 is a graph showing the results of example 5 in the present invention (GBSV 6 immune-induced antigen-specific serum IgG responses in mice)

FIG. 6 is a graph showing the results of example 5 in the present invention (GBSV 6 immune-induced antigen-specific secretory IgA response in mice)

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.

B streptococcal serotype Ib, B streptococcal serotype III: from the microbiological laboratory of Beijing children hospital affiliated to the university of medical science of capital in Beijing city, reference: shen AD, zhang GR, wang YH, yang yh.zhonghua Er Ke Za zhi.2005;43 (9):661-664.. CpG: reference Iho S, maeyama J, suzuki f.cpg oligodeoxynucleotides as mucosal adjuvants, hum vaccine immunother.2015;11 (3):755-60. Vector pET28a (+): novagen, cat.No.69846-3. Vector pCold-SUMO: sea-based biotechnology Co., ltd., cat.C1801M. Coli BL21 (DE 3): purchased from Beijing allThe product number of the gold Biotechnology Co., ltd., CD601-02.(ICR) IGS mice: purchased from Charles River; STRAIN CODE:201.

example 1

Preparation of sortase A (SrtA)

1. And (3) taking the genome DNA of the B chain bacteria serotype Ib 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’-CATGCCATGGGCTCTGCTCAAACGAAATCACA-3’；

R1：5’-CCGCTCGAGGAGATTAATTTGATTATATT-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 the vector backbone of about 5400bp was recovered.

4. And (3) connecting the enzyme digestion product of the step (2) with the vector skeleton of the step (3) to obtain the recombinant plasmid pET28a-SrtA. Based on the sequencing results, the recombinant plasmid pET28a-SrtA was described as follows: a double-stranded DNA molecule shown in 244 th-741 th nucleotide of the 5' tail end of the sequence 1 of the sequence table is inserted between NcoI and XhoI restriction 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-SrtA 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, and were shake-cultured at 37℃and 220rpm until OD560 nm=0.6, and were induced by adding IPTG and shaking-cultured at 37℃and 220rpm for 4 hours at a concentration of 40. Mu.g/ml.

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 naming it as a sortase A solution. 13 mg of protein with purity of more than 90% can be obtained per liter of the culture system of the step 6.

Preparation of CspA

1. And (3) taking the genome DNA of the serotype III of the chain bacteria B 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’-CATGCCATGGGCGATTCTGTCATAAATAAGCC-3’；

R1：5’-CCGCTCGAGATTGCCAATATTGATCAAATCT-3’。

2. Point mutation Using PCR method Asp172 and Ser567 of active center were mutated to Ala to obtain CspA retaining immunogenicity but losing catalytic activity, and the following primers were used for point mutation, respectively:

Asp-Ala(172)：

F1：5′-GTAGCAATTATTGCTTCAGGACTAGAT-3′

R1：5′-ATCTAGTCCTGAAGCAATAATTGCTAC-3′

Ser-Ala(567)：

F1：5′-ATGAGTGGGACAGCTATGGCTTCTCCC-3′

R1：5′-GGGAGAAGCCATAGCTGTCCCACTCAT-3′

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

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

5. 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-CspA. Based on the sequencing results, the recombinant plasmid pET28a-CspA was described as follows: a double-stranded DNA molecule shown in 106 th to 3228 th nucleotides from the 5' end of the sequence 7 of the sequence table is inserted between NcoI and XhoI restriction 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-CspA 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, and were shake-cultured at 37℃and 220rpm until OD560 nm=0.6, and were induced by adding IPTG and were shake-cultured at 37℃and 220rpm for 4 hours at a concentration of 40. Mu.g/mL.

7. The culture system of step 6 was centrifuged at 6000rpm at 4℃for 10 minutes to collect the cell pellet, the cell pellet was suspended in PBS buffer at pH7.4 and sonicated (power: 200W, 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 Ni Sepharose 6Fast Flow, 10 column volumes of the eluate were first subjected to elution with a solution I (pH 7.4, the solvent was water, 20mM Na2HPO4 and 500mM NaCl) to remove the impurity protein, and then 2 column volumes of the eluate were subjected to elution with a solution II (pH 7.4, the solvent was water, 200mM imidazole, 20mM Na2HPO4 and 500mM NaCl) to obtain the target protein, and the post-column-passing solution obtained when the eluate with the solution II was collected and designated as a CspA solution. 36 mg of protein with purity of more than 90% can be obtained per liter of the culture system of the step 6.

Preparation of C5a peptidase

1. And (3) taking genome DNA of the serotype III of the chain bacteria B 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′-ATGACCATGGGCAATACTGTGACAGAAGACACTCC-3′，R1：5′-CCGCTCGAGAGAGTGGCCCTCCAATAG。

2. Point mutation Using PCR method Asp130 and Ser512 at the active center were mutated to Ala to obtain ScpB retaining immunogenicity but losing catalytic activity, and the following primers were used for point mutation, respectively:

Asp-Ala(130)：F1：5′-GTTGCAGTGATTGCTGCTGGTTTTGAT-3′，R1：5′-ATCAAAACCAGCAGCAATCACTGCAAC-3′。

Ser-Ala(512)：F1：5′-CTTTCTGGAACTGCTATGTCTGCGCCA-3′，R1：5′-TGGCGCAGACATAGCAGTTCCAGAAAG-3′。

4. The vector pET28a (+) was digested with the restriction enzymes NcoI and XhoI.

5. And (3) connecting the enzyme digestion product of the step (3) with the vector skeleton of the step (4) to obtain the recombinant plasmid pET28a-ScpB. Based on the sequencing results, the recombinant plasmid pET28a-ScpB was described as follows: a double-stranded DNA molecule shown in 94 th to 3096 th nucleotides from the 5' end of the sequence 4 of the sequence table is inserted between NcoI and XhoI restriction 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 8 of a sequence table, and the fusion protein shown in a sequence 9 of the sequence table is expressed.

6. The recombinant plasmid pET28a-ScpB was introduced into E.coli BL21 (DE 3) to obtain a recombinant strain.

7. The recombinant bacteria obtained in the step 5 were inoculated into LB liquid medium containing 50. Mu.g/mL kanamycin, and were subjected to shaking culture at 37℃and 220rpm until OD560 nm=0.6, and were induced by adding IPTG, followed by shaking culture 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, 8 seconds every 4 seconds of operation, 99 cycles), and then centrifuged at 12000rpm for 20 minutes to collect the supernatant.

9. Loading the supernatant obtained in the step 8 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.

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

Preparation of Srr2

1. And (3) taking genome DNA of the serotype III of the chain bacteria B 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' -CATGCCATGGGCTCAGAAGCGGCAACGACCGCTAGAG-3’；R1：5’-CCGCTCGAGTTGAGCATTTACATCTGAATA-3’。

3. The vector pET28a (+) was digested with the restriction enzymes NdeI 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-Srr2. Based on the sequencing results, the recombinant plasmid pET28a-Srr2 was described as follows: a double-stranded DNA molecule shown in 571-1629 nucleotides at the 5' -end of the sequence 10 of the sequence table is inserted between NdeI and XhoI restriction 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 11 of a sequence table, and the fusion protein shown in a sequence 12 of the sequence table is expressed.

5. The recombinant plasmid pET28a-Srr2 is introduced into escherichia coli BL21 (DE 3) to obtain recombinant bacteria.

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, 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 designating it as a Srr2 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 FbsB

F1：5′-CGAGCTCGCCGGGATAACTAAAG-3′，R1：5′-ACGCGTCGACCTCTTTTATACGCGATGAG-3′。

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

3. The vector pCold-SUMO was digested with the restriction enzymes SacI and SalI.

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-FbsB. Based on the sequencing results, the recombinant plasmid pCold-SUMO-FbsB was described as follows: a double-stranded DNA molecule shown in 76-1884 nucleotides from the 5' end of sequence 13 of the sequence table is inserted between SacI and SalI 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 14 of a sequence table, and the fusion protein shown in a sequence 15 of the sequence table is expressed.

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

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

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.

Preparation of BibA

1. And (3) taking the genome DNA of the B chain bacteria serotype Ib 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' -CGAGCTCCACGCGGATACTAGTTCAGGA-3′，R1：5′-ACGCGTCGACACCTCTGGTAAGGTCTTGAA-3′。

3. The vector pCold-SUMO-BibA was digested with the restriction enzymes SacI and SalI.

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-BibA. Based on the sequencing results, the recombinant plasmid pCold-SUMO-BibA was described as follows: a double-stranded DNA molecule shown in the 102-1413 nucleotide of the sequence 16 of the sequence table from the 5' end is inserted between the SacI and SalI 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 17 of a sequence table, and the fusion protein shown in a sequence 18 of the sequence table is expressed.

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

The polyacrylamide gel electrophoresis diagram in the process of preparing SrtA, scpB, cspA, srr, bibA and FbsB proteins is shown in figure 1. Wherein the GBS recombinant protein SrtA (amino acid sequence: 82-247, mw: 19 kd), scpB (amino acid sequence: 32-1032, mw: 110 kd), cspA (amino acid sequence: 36-1076, mw: 114 kd), srr2 (amino acid sequence: 192-543, mw: 40 kd), bibA (amino acid sequence: 34-471, mw: 64 kd), fbsB (amino acid sequence: 19-628, mw: 86 kd).

Example 2

After nasal inhalation of GBSV6+ CpG, the removal of B-chain bacteria at genital tract infection sites, female ICR mice of 6-8 weeks of age were randomly divided into two groups, and the grouping treatment was as follows:

CpG group: respectively dripping PBS buffer solution into the nasal cavity on the 1 st day, the 7 th day and the 14 th day of the experiment;

GBSV6 group: on experiment 1, 7 and 14, vaccine solutions (vaccine solutions were obtained by mixing the 6 recombinant proteins prepared in example 1 with CpG solutions, and each mouse was given 10. Mu.g and 10. Mu.g of CpG each of 5 recombinant proteins) were instilled through nasal cavity.

On day 21 of the experiment, mice were challenged by genital tract instillation with live bacteria (Streptomyces B serotype Ib or III) (concentration of bacterial solution 1X 107 CFU/10. Mu.l, instilled with 10. Mu.l each). And (3) taking genital tract lavage liquid 1 day, 3 days, 6 days, 9 days, 15 days and 35 days after virus attack, and detecting the number of viable bacteria of the B chain bacteria in the genital tract lavage liquid by using blood plate culture.

The results are shown in FIG. 2, each black dot represents 1 mouse (ordinate indicates the number of CFU of B-chain bacteria in the whole genital lavage fluid of each mouse). After B chain bacteria serotype Ib or III are attacked, the number of viable bacteria in the GBSV6 group mouse genital tract lavage fluid is obviously less than that of CpG groups, which indicates that the vaccine provided by the invention can effectively clear infection of the genital tract B chain bacteria serotype Ib or III through respiratory tract mucosa immunization.

Example 3

Immunization protection against lethal infection with B-chain bacteria after nasal inhalation of GBSV6, 6-8 week old female ICR mice were randomly divided into two groups, and the grouping was handled as follows:

CpG group: nasal drops of CpG solution were applied on experiment day 1, day 7 and day 14, respectively; GBSV6 group: on experiment 1, 7 and 14, vaccine solutions (vaccine solutions were obtained by mixing the 6 recombinant proteins prepared in example 1 with CpG solutions, and each mouse was given 10. Mu.g and 10. Mu.g of CpG each of the 6 recombinant proteins) were instilled through nasal cavity, respectively.

On day 21 of the experiment, mice were challenged with live bacteria (Streptomyces B serotype Ib or III) by tail vein injection at a lethal dose (concentration of bacterial solution 2X 108 CFU/50. Mu.l, 50. Mu.l instilled per mouse). Survival of mice was observed after challenge, and the results are shown in figure 3, where GBSV6 group significantly improved survival rate (80%) of mice compared to CpG group, indicating that GBSV6 immunized mice provided immunoprotection against lethal doses of B-chain bacteria.

Example 4

GBSV6 mucosal immunity induces a Th17 cell immune response, and 6-8 week old female ICR mice are randomly divided into two groups, which are treated as follows: cpG group: nasal drops of CpG solution were applied on experiment day 1, day 7 and day 14, respectively; GBSV6 group: on experiment 1, 7 and 14, vaccine solutions (vaccine solutions were obtained by mixing the 6 recombinant proteins prepared in example 1 with CpG solutions, and each mouse was given 10. Mu.g and 10. Mu.g of CpG each of the 6 recombinant proteins) were instilled through nasal cavity, respectively. On experiment day 21, spleen and genital tract tissues were taken to extract cells, and GBSV6 antigen-specific IL-17+ cells were measured using ELISPOT. The results are shown in figure 4, where GBSV6 group significantly induced antigen-specific Th17 cells in spleen and genital tract tissues compared to CpG group, indicating that GBSV6 nasal immunized mice induced Th17 cell responses.

Example 4

GBSV6 immunization induced antibody responses in mice, 6-8 week old female ICR mice were randomly divided into three groups, the grouping being as follows: cpG group: nasal drops of CpG solution were applied on experiment day 1, day 7 and day 14, respectively; GBSV6 group: on experiment 1, 7 and 14, vaccine solutions (vaccine solutions were obtained by mixing the 6 recombinant proteins prepared in example 1 with CpG solutions, and each mouse was given 10. Mu.g and 10. Mu.g of CpG each of the 6 recombinant proteins) were instilled through nasal cavity, respectively.

On day 21 of the experiment, tail blood and genital tract lavage fluid were collected from anesthetized mice, and ELIAS measured serum IgG, genital tract lavage fluid IgA. The results are shown in FIGS. 5 and 6. Compared to the CpG group, GBSV6 group induced significantly antigen-specific serum IgG and genital IgA.

Example 5

GBSV6 nasal inhalation or intramuscular immunization of the mice, each providing cross-immunoprotection of neonatal mice against lethal infection with different serotypes of B streptococcus, randomized 6-8 week old female ICR mice into four groups, treated as follows: cpG group: the CpG solution is respectively dripped into the nasal cavity on the 1 st day, the 7 th day and the 14 th day of the experiment, the 15 th day and the male mice are matched in a cage for conception, and the newborn young mice are delivered after 4 weeks; cpg+gbsv6 group: on experiment 1, 7 and 14 days, respectively, vaccine solutions (the vaccine solutions are obtained by mixing 6 recombinant proteins prepared in example 1 with CpG solutions, each mouse is given 10 mug and 10 mug of CpG each time, and each mouse is given 6 recombinant proteins, and the mice are bred by mating with male mice in a cage, and the newborn mice are born after 4 weeks; alum group: the aluminum adjuvant solution is respectively injected into the muscle on the 1 st day, the 7 th day and the 14 th day of the experiment, the 15 th day and the male mice are matched in a cage for conception, and the newborn young mice are delivered after 4 weeks; group alum+gbsv 6: on experiment 1, 7 and 14 days, vaccine solutions were respectively injected intramuscularly (the vaccine solution is obtained by mixing the 6 recombinant proteins prepared in example 1 with Alum solution, each mouse is given 10 mug and 25 mug of Alum each time, each mouse is given 6 recombinant proteins, and on 15 days, the mice are bred in a cage with male mice, and after 4 weeks, newborn young mice are born;

2-day-old young mice, cpG group and GBSV6 group mice were challenged by intraperitoneal injection using GBSIb or type III (concentration of bacterial solution is 2×10) ⁶ CFU/20. Mu.l, 20. Mu.l each mice were injected) and the groups of neonatal mice were observedIs a survival rate of (a). The results are shown in Table 1.

TABLE 1GBSV6 nasal inhalation or intramuscular immunization of female mice, all providing cross-immunoprotection of neonatal mice against lethal infection with different serotypes of B chain bacteria

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As can be seen from the above table, the GBSV6 group not only protects the B chain bacteria serotype Ib, but also protects the B chain bacteria serotype III of different serotypes, which indicates that the GBSV6 immunized mice induce cross immune protection to the B chain bacteria of different serotypes

The above examples/experiments are only examples for clarity of illustration 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.

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Claims

1. A broad-spectrum multi-subunit vaccine for preventing group B streptococcus infection, characterized by: the active ingredients of the vaccine consist of a first component, a second component, a third component, a fourth component, a fifth component, a sixth component and a seventh component;

also comprises component G as immunological adjuvant CpG;

2. a broad spectrum multi-subunit vaccine for preventing group B streptococcus infection according to claim 1 wherein:

the component A is sortase A, and has a protein sequence after the sortase A removes signal peptide; specifically is a protein composed of an amino acid sequence shown as a sequence 3 in a sequence table;

the component B is CspA, and has a protein sequence after the CspA removes the signal peptide and the cell wall anchoring domain; specifically is a protein composed of an amino acid sequence shown as a sequence 6 in a sequence table;

the component C is C5a peptidase, and has a protein sequence after the C5a peptidase removes the signal peptide and the cell wall anchoring domain; specifically is a protein composed of an amino acid sequence shown as a sequence 9 in a sequence table;

the component D is FbsB and has a protein sequence after the FbsB is removed from the signal peptide; specifically is a protein composed of an amino acid sequence shown as a sequence 12 in a sequence table;

the component penta is Srr2, a protein sequence with a binding domain of the Srr 2; specifically is a protein composed of an amino acid sequence shown as a sequence 15 in a sequence table;

the component is hexobA, a protein sequence with the signal peptide removed by the BibA and a cell wall anchoring domain; in particular to a protein consisting of an amino acid sequence shown as a sequence 18 in a sequence table.

3. A broad spectrum multi-subunit vaccine for preventing group B streptococcus infection according to claim 1 wherein: the vaccine has the functions of (I) or (II) or (III) or (IV): inhibiting group B streptococcus of different serotypes; (ii) preventing systemic infections of the human respiratory, reproductive and systemic passages caused by group B streptococci of different serotypes; (III) providing immune protection against neonatal group B Streptococcus infection and death following maternal immunization.

4. A broad spectrum multi-subunit vaccine for preventing group B streptococcus infection according to claim 1 wherein: the group B streptococcus is specifically group B streptococcus comprising different serotypes.

5. A broad spectrum multi-subunit vaccine for preventing group B streptococcus infection according to claim 4 wherein: the different serotypes of the B chain bacteria are the serotypes of ten B chain bacteria, namely B chain bacteria serotypes Ia, ib, II, III, IV, V, VI, VII, VIII and IX; also included are subtypes of the above-mentioned serotypes which occur and novel serotypes which are not included in the above-mentioned serotypes.

6. A broad spectrum multi-subunit vaccine for preventing group B streptococcus infection according to claim 5 wherein: the group B streptococcus serotype is specifically a group B streptococcus serotype Ib or a group III streptococcus serotype.

7. A broad spectrum multi-subunit vaccine for preventing group B streptococcus infection according to claim 1 wherein: the prevention of group B streptococcus infection is the prevention of human infection caused by group B streptococcus of different serotypes.

8. A broad spectrum multi-subunit vaccine for preventing group B streptococcus infection according to claim 7 wherein: the human infection is an infection of respiratory system, digestive system, urinary system, reproductive system, skin and blood circulation system.

9. A broad spectrum multi-subunit vaccine for preventing group B streptococcus infection according to claim 1 wherein: the vaccine is used in the modes of nasal inhalation, oral administration, subcutaneous injection, intradermal injection, genital tract injection or anal injection.

10. Use of a vaccine according to any one of claims 1 to 9 in the manufacture of a medicament for the prophylaxis of group B streptococcus infection.