Recombinant proteins, vaccines, and drugs for preventing Gram-positive pathogens

Abstract

This invention relates to the field of biomedicine, specifically to recombinant proteins, vaccines, and drugs for the prevention of Gram-positive pathogens, and more specifically to a recombinant subunit vaccine for inhibiting streptococci and / or preventing streptococcal infection. The vaccine provided by this invention has an active ingredient composed of the Sda1 active mutant mu-Sda1 and the adjuvant CpG; it effectively inhibits or prevents Gram-positive pathogens such as streptococci, Staphylococcus aureus, and pneumococci. The vaccine provided by this invention has the advantages of high efficiency, broad spectrum, and low cost. The vaccine provides a mucosal immunization route, characterized by no tissue damage, no local side effects, and ease of use, making it easy to promote and use.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to recombinant proteins, vaccines, and drugs for the prevention of Gram-positive pathogens. Background Technology

[0002] Group A Streptococcus (hereinafter referred to as A Streptococcus) is an important Gram-positive opportunistic pathogen. A Streptococcus causes a variety of diseases through infection of the upper respiratory tract mucosa and skin. Mild cases include pharyngitis, scarlet fever, impetigo, erysipelas, and cellulitis, while severe cases can lead to systemic spread and invasive infections that endanger life, such as pneumonia, bacteremia, toxic shock, and acute necrotizing fasciitis. A Streptococcus infection can also induce autoimmune diseases such as rheumatic fever and rheumatic heart disease. According to the World Health Organization, there are approximately 1.78 million new cases of A Streptococcus disease worldwide, with about 500,000 deaths annually. Furthermore, A Streptococcus infection leading to rheumatic fever and rheumatic heart disease is the leading cause of cardiovascular disease-related deaths, and therefore has always been a focus of attention for the World Health Organization and scientists.

[0003] The widespread transmission of Streptococcus aureus (A) and the high mortality rates resulting from its induced autoimmune diseases and invasive infections place a tremendous burden on global public health and medical care, urgently requiring the development of a safe, effective, and broad-spectrum A vaccine. However, the numerous serotypes of A streptococcus mean that immunity against one serotype offers no protection against another. Furthermore, the close association between A streptococcal infection and autoimmune diseases severely hinders the development of an A vaccine.

[0004] Streptococcus aureus (ALA) expresses multiple virulence factors during infection, including the ability to evade immune clearance. Neutrophils play a crucial role in ALA infection by releasing neutrophil extracellular tracts (NETs) containing their own DNA and a large number of antibacterial proteolytic enzymes (such as elastase), which capture and kill bacteria. ALA secretes various deoxyribonucleases to degrade the DNA of NETs, ​​evading the capture and clearance of neutrophil NETs. Sda1, a secreted virulence factor with DNase activity, is widely present in clinical isolates of ALA (especially the M1T1 strain, which has spread globally in recent years). It evades immune clearance by degrading the DNA in neutrophil NETs. ALA strains with the Sda1 gene knocked out or lacking the Sda1 gene show significantly reduced pathogenicity. Protein sequence and phylogenetic analysis showed that homologous proteins of Sda1 are found in important human pathogens such as Group C Streptococcus (S. equisimilis), Streptococcus pneumoniae (S. pneumoniae), and Staphylococcus aureus (S. aureus), indicating that Sda1 plays an important role in the pathogenesis of pathogens. Summary of the Invention

[0005] In view of this, the present invention provides recombinant proteins, vaccines, and drugs for the prevention of Gram-positive pathogens.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides the application of Sda1 as a target in the preparation of vaccines for preventing pathogenic bacterial infections, reagents for detecting pathogenic bacteria, and medicaments for preventing and / or treating diseases caused by pathogenic bacterial infections; the pathogenic bacteria include: pathogenic bacteria that inhibit the expression of Sda1 and / or its homologous DNases; the infection includes: human mucosal system infections. In some specific embodiments of this invention, the pathogenic bacteria include Gram-positive pathogenic bacteria; the Gram-positive pathogenic bacteria include one or more of streptococci, Staphylococcus aureus, and Streptococcus pneumoniae; the streptococci include one or more of group A streptococci, group B streptococci, group C streptococci, group G streptococci, Streptococcus pneumoniae, Streptococcus suis, or Streptococcus equi; the human mucosal system includes one or more of the respiratory system, digestive system, urinary system, genitourinary system, or skin.

[0008] This invention also provides a recombinant protein mu-Sda1, which has:

[0009] (I) Amino acid residues from position 3 to 354 of the N-terminus as shown in SEQ ID No. 6; or

[0010] (II) An amino acid sequence obtained by substituting, deleting, or adding one or two amino acid residues to the amino acid sequence described in (I), and which has the same or similar function to the amino acid sequence shown in (I); or

[0011] (III) An amino acid sequence that has at least 90% sequence identity with the sequence described in (I) or (II) and has the same or similar function as the amino acid sequence shown in (I).

[0012] In some specific embodiments of the present invention, the recombinant protein mu-Sda1 has:

[0013] (I) An amino acid sequence as shown in SEQ ID No. 6; or

[0014] (II) An amino acid sequence obtained by substituting, deleting, or adding one or two amino acid residues to the amino acid sequence described in (I), and which has the same or similar function to the amino acid sequence shown in (I); or

[0015] (III) An amino acid sequence that has at least 90% sequence identity with the sequence described in (I) or (II) and has the same or similar function as the amino acid sequence shown in (I).

[0016] Furthermore, the present invention also provides a nucleic acid molecule encoding the recombinant protein mu-Sda1, having

[0017] (I) Nucleotide sequences from 7bp to 1062bp as shown in SEQ ID No. 5 or

[0018] (II) A complementary nucleotide sequence of 7 bp to 1062 bp as shown in SEQ ID No. 5; or

[0019] (III) A nucleotide sequence that encodes the same protein as (I) or (II), but differs from the nucleotide sequence of (I) or (II) due to the degeneracy of the genetic code; or

[0020] (IV) A nucleotide sequence obtained by substituting, deleting, or adding one or two nucleotide sequences to the nucleotide sequences shown in (I), (II), or (III), and which has the same or similar function to the nucleotide sequences shown in (I), (II), or (III); or

[0021] (V) A nucleotide sequence that has at least 90% sequence identity with the nucleotide sequences described in (I), (II), (III) or (IV).

[0022] The present invention also provides a recombinant vector containing the nucleic acid molecule.

[0023] The present invention also provides a host comprising the recombinant vector.

[0024] In some specific embodiments of the present invention, the host includes one or more of Escherichia coli, yeast, or mammalian cells.

[0025] Furthermore, the present invention also provides the use of the recombinant protein mu-Sda1 in the preparation of vaccines for preventing pathogenic bacterial infections, reagents for detecting pathogenic bacteria, and medicaments for preventing and / or treating diseases caused by pathogenic bacterial infections; wherein the pathogenic bacteria include: pathogenic bacteria that inhibit the expression of Sda1 and / or DNases homologous to it; and wherein the infection includes: human mucosal system infection.

[0026] In some specific embodiments of the present invention, the pathogenic bacteria include Gram-positive pathogenic bacteria; the Gram-positive pathogenic bacteria include one or more of streptococci, Staphylococcus aureus, and Streptococcus pneumoniae; the streptococci include one or more of group A streptococci, group B streptococci, group C streptococci, group G streptococci, Streptococcus pneumoniae, Streptococcus suis, or Streptococcus equi; the human mucosal system includes one or more of the respiratory system, digestive system, urinary system, urogenital system, or skin.

[0027] The present invention also provides a vaccine comprising the recombinant protein mu-Sda1 and an acceptable adjuvant. In some embodiments of the invention, the adjuvant comprises CpG. In some embodiments of the invention, the mass ratio of the recombinant protein mu-Sda1 to the CpG is 1:1. In some embodiments of the invention, the vaccine is administered via nasal inhalation, oral administration, subcutaneous injection, intradermal injection, urogenital injection, or rectal injection.

[0028] The present invention also provides an antibody, which is obtained by immunizing animals with the vaccine.

[0029] The present invention also provides a medicament comprising the antibody and pharmaceutically acceptable excipients.

[0030] The present invention also provides a detection reagent or detection kit, comprising the antibody and a detection-acceptable adjuvant.

[0031] This invention utilizes the universality and homology of Sda1 in various pathogenic bacteria and the property of mucosal adjuvants to enhance antigenic immunogenicity to design a rational vaccine formulation. This formulation significantly induces neutralizing antibodies with broad-spectrum neutralizing activity, achieving the effects of preventing pathogen colonization and rapidly eliminating pathogens. Furthermore, it provides protection against different serotypes of Streptococcus A, exhibiting advantages of high efficiency, broad spectrum, and low cost. The vaccine provided by this invention uses mucosal immunization, which is characterized by no tissue damage, no local side effects, and ease of use, making it easy to promote and use. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0033] Figure 1A , Figure 1B Agarose gel electrophoresis images of the PCR amplification of the Sda1 and mu-Sda1 genes are shown respectively.

[0034] Figure 2 This shows an SDS-PAGE electrophoresis image of the prepared mu-Sda1 fusion protein;

[0035] Figure 3 The results of Example 2 are shown (the induction of antigen-specific antibody response in mice by intranasal immunization with vaccine solution); wherein, Figure 3 A shows the induction of antigen-specific serum IgG response in mice immunized with vaccine solution via intranasal droplets. Figure 3 B shows the antigen-specific properties of the secretory IgA response in oral wash solution induced by intranasal immunization of mice with the vaccine solution.

[0036] Figure 4 The results of Example 3 are shown (the vaccine-induced serum IgG has neutralizing activity against DNase);

[0037] Figure 5 The results of Example 4 are shown (the nasal immunization vaccine provides cross-immune protection against respiratory infections caused by different serotypes of Streptococcus aureus);

[0038] Figure 6 The results of Example 5 are shown (the nasal immunization vaccine solution provides cross-immune protection against strains that do not contain the Sda1 gene A chain). Detailed Implementation

[0039] This invention discloses recombinant proteins, vaccines, and drugs for the prevention of Gram-positive pathogens. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve these results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0040] One of the objectives of this invention is to provide a broad-spectrum single subunit vaccine for the prevention of Streptococcus aureus infection.

[0041] The second objective of this invention is to provide a recombinant gene of an active mutant of Sda1 and an expression system containing the recombinant gene.

[0042] The functions of the vaccine include (I), (II), or (III).

[0043] (I) Pathogenic bacteria that inhibit the expression of Sda1 and its homologous DNase;

[0044] (II) Prevention of infection by pathogenic bacteria that express Sda1 and its homologous DNase;

[0045] (III) Reduce or prevent the colonization and infection of human mucosal systems by pathogens expressing Sda1 and its homologous DNases.

[0046] The mu-Sda1 may specifically be either (a) or (b) as follows: (a) a protein consisting of amino acid residues from the N-terminus of sequence 3 in the sequence listing from position 22 to position 189; or (b) a protein derived from (a) by substitution and / or deletion and / or addition of one or more amino acid residues and having the same activity.

[0047] The mass ratio of the fusion protein mu-Sda1 to the adjuvant CpG is 1:1.

[0048] The pathogenic bacteria expressing Sda1 and its homologous DNase include, but are not limited to, streptococci, Staphylococcus aureus, and Streptococcus pneumoniae. The streptococci may be group A streptococci, group B streptococci, group C streptococci, group G streptococci, Streptococcus pneumoniae, Streptococcus suis, or Streptococcus equi.

[0049] The human mucosal system may be the respiratory system, digestive system, urinary and reproductive system, or skin.

[0050] The vaccine can be administered via nasal inhalation, oral administration, subcutaneous injection, intradermal injection, urogenital injection, or rectal injection.

[0051] The vaccine provided by this invention can be used for the prevention and treatment of mucosal system (respiratory, digestive, urinary, reproductive, or skin) infections caused by various pathogenic bacteria that express Sda1 and its homologous DNase.

[0052] This invention utilizes the universality and homology of Sda1 in various pathogenic bacteria and the property of mucosal adjuvants to enhance antigenic immunogenicity to design a rational vaccine formulation. This formulation significantly induces neutralizing antibodies with broad-spectrum neutralizing activity, achieving the effects of preventing pathogen colonization and rapidly eliminating pathogens. Furthermore, it provides protection against different serotypes of Streptococcus A, exhibiting advantages of high efficiency, broad spectrum, and low cost. The vaccine provided by this invention uses mucosal immunization, which is characterized by no tissue damage, no local side effects, and ease of use, making it easy to promote and use.

[0053] in:

[0054] Group A Streptococcus M1 and M3.11: from the Department of Microbiology, Minnesota State University. Reference: Wang B, Dileepan T, Briscoe S, Hyland KA, Kang J, Khoruts A, Cleary PP. Induction Of TGF-betal and TGF-betal-dependent predominant Th17 differentiation by group Astreptococcal infection. Proc Natl Acad Sci US A. 2010; 107(13): 5937-42.

[0055] Type A Streptococcus M12: Reference: Haanes EJ, Cleary PP. Identification of adivergent M protein gene and an M protein-related gene family in Streptococcus pyogenes serotype 49. J. Bacteriol. 1989 Dec; 171(12): 6397-6408.

[0056] Vector pET28a(+): Novagen, Cat. No. 69846-3. Escherichia coli BL21gold(DE3)plysS: Purchased from Beijing Kangwei Reagent Biotechnology Co., Ltd., Product No.: CW0810A. C57BL / 6JNifdc mice: Purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., STRAIN CODE: 219.

[0057] The following examples are provided to better understand the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0058] The present invention will be further illustrated below with reference to the embodiments:

[0059] Example 1: Construction of recombinant Sda1 and mu-Sda1 and preparation of their recombinant proteins

[0060] 1. Using Streptococcus aureus (M1 type, 90226WT strain) genomic DNA as a template, the Sda1 gene was amplified by PCR using primers consisting of Primer 1 and Primer 2, yielding Sdal gene PCR amplification product 1 (approximately 1059 bp). The agarose gel electrophoresis image of the PCR amplification product is shown below. Figure 1A .

[0061] Primer 1:5'-CATG CCATGG GCTCGGGCACTATTTCAAATAATT-3' (as shown in SEQ ID No. 7);

[0062] Primer 2:5'-CCG CTCGAG TTCTATATTTTCTTGAGTTGAAT-3' (as shown in SEQ ID No. 8).

[0063] 2. Point mutation was performed using PCR to mutate amino acid His 188 in sequence 2 (SEQ ID No. 1) to Gly, obtaining the recombinant protein mu-Sda1 (approximately 1059 bp) that loses its DNase activity. The following primers were used for point mutation:

[0064] Primer 3: 5'-GTTTGATAGAAGTGGTTTAATAGCCGATAG-3' (as shown in SEQ ID No. 9)

[0065] Primer 4∶5'-CTATCGGCTATTAAACCACTTCTATCAAAC-3' (as shown in SEQ ID No. 10)

[0066] Agarose gel electrophoresis image of PCR amplification products is shown below. Figure 1B .

[0067] 3. Digest the PCR amplification product from step 2 with restriction endonucleases NcoI and Xho1, and recover the digested product.

[0068] 4. The vector pET28a was digested with restriction endonucleases NcoI and Xho1, and the vector backbone of about 5400 bp was recovered.

[0069] 5. The PCR digestion product from step 3 and the vector backbone from step 4 were ligated to obtain the recombinant plasmid pET28a-mu-Sda1 (the nucleotide sequence of this recombinant plasmid is shown in SEQ ID No. 5). Based on the sequencing results, the recombinant plasmid pET28a-mu-Sda1 is described as follows: A double-stranded DNA molecule, as shown in sequence 5 (SEQ ID No. 5) from nucleotides 7-1062 of the 5' end, was inserted between the NcoI and XhoI restriction sites of the vector pET28a. The inserted double-stranded DNA molecule and a portion of the DNA on the vector backbone form the fusion gene shown in sequence 5 (SEQ ID No. 5), expressing the recombinant protein shown in sequence 6 (SEQ ID No. 6).

[0070] 5. The recombinant plasmid pET28a-mu-Sda1 was transformed into Escherichia coli BL21gold(DE3)plysS to obtain the recombinant strain pET28a-mu-Sda1-BL21 gold(DE3)plysS.

[0071] 6. The recombinant bacteria pET28a-mu-Sda1-BL21gold(DE3)plysS obtained in step 5 was inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured at 37°C with shaking at 200 rpm until OD200. 560nm When the concentration of the sample was 0.6, IPTG was added to induce the incubation at a concentration of 1 mM, and the sample was cultured at 30°C and 160 rpm for 16 hours with shaking.

[0072] 7. Take the culture system from step 6, centrifuge at 8000 rpm for 10 minutes at 4℃ to collect the bacterial pellet, suspend the bacterial pellet in PBS buffer at pH 7.4 and sonicate it (power 200W, 4 seconds of operation followed by 8 seconds of rest, 99 cycles), then centrifuge at 12000 rpm for 20 minutes and collect the supernatant.

[0073] 8. Load the supernatant obtained in step 7 onto a GE Ni Sepharose 6 Fast Flow elution system. First, elute with solution I (pH 7.4, PBS containing 20 mM imidazole) for 10 column volumes to remove impurities. Then, elute with solution II (pH 7.4, PBS containing 200 mM imidazole) for 5 column volumes to obtain the target protein. Collect the post-column solution after elution with solution II and dialyze overnight at 4°C to remove imidazole (dialysis molecular weight cutoff 1000, buffer solution is PBS solution with pH 7.4).

[0074] Each liter of the culture system in step 6 yields 1.2 mg of protein with a purity of over 90%.

[0075] SDS-PAGE gel electrophoresis images of the mu-Sda1 recombinant protein expression and purification process are shown below. Figure 3 A. Figure 2 In the diagram, lane 1 is the protein molecular weight standard (Mw), lane 2 is the contaminating protein that failed to bind to the i Sepharose 6 Fast Flow column in step 7, lane 3 is the elution solution using solution I, and lane 4 is the elution solution using solution I. The recombinant protein mu-Sda1 has a molecular weight of approximately 41 kDa.

[0076] Example 2: Inducing antigen-specific antibody response in mice by intranasal immunization with vaccine solution.

[0077] Female C57BL / 6JNifdc mice aged 6-8 weeks were randomly divided into two groups, and the treatment was as follows:

[0078] Adjuvant control group: On days 1, 7 and 14 of the experiment, adjuvant CpG dissolved in PBS (10 μg / mouse) was instilled into the nasal cavity.

[0079] Mu-Sda1 immunization group: On day 1, day 7 and day 14 of the experiment, the vaccine solution was administered via nasal drip (each mouse was given a vaccine solution containing 10 μg of mu-Sdal recombinant protein prepared in Example 1 and 10 μg of CpG dissolved in PBS each time);

[0080] On day 24 of the experiment, blood and oral lavage fluid were collected from mice. Blood was left at room temperature for 2 hours and then centrifuged to obtain serum. Serum and oral lavage fluid samples were analyzed using ELISA to detect antigen-specific antibody reactions.

[0081] See results Figure 3 In cases A and 3B, compared to the adjuvant control group, serum IgG and secretory IgA levels in oral wash were significantly increased after vaccine immunization, indicating that vaccine immunization induces antigen-specific antibody responses.

[0082] Example 3: The serum IgG induced by the vaccine solution has neutralizing activity against DNase.

[0083] GAS culture supernatant (sup.) or recombinant Sda1 was respectively mixed with... After co-incubating for 2 hours with serum from mice, mice immunized with vaccine solution, and mice infected with Streptococcus aureus M1, the serum was added to a solution containing 5 μg of plasmid pET28a DNA. The mixture was reacted at 37°C for 45 minutes, and then 200 mM of EDTA-containing stop solution was added. The degradation status of the plasmid DNA was observed by 1% agarose gel electrophoresis.

[0084] See results Figure 4The results showed that both the culture supernatant of Streptococcus A. M1 strain (sup.) and recombinant Sda1 could degrade plasmid DNA. Serum from mice immunized with the vaccine significantly inhibited the degradation of plasmid DNA by the culture supernatant of Streptococcus A. M1 strain (sup.) or recombinant Sda1, while serum from... Serum from mice infected with Streptococcus aureus M1 strain and mice did not inhibit the degradation of plasmid DNA by GAS culture supernatant (sup.) or recombinant Sda1. This indicates that vaccine-induced serum IgG has neutralizing activity and can effectively inhibit the activity of DNases in Sda1 and Streptococcus aureus M1 strain culture supernatant.

[0085] Example 4: The nasal drop immunization vaccine solution provides cross-immune protection against respiratory infections caused by different serotypes of Streptococcus aureus.

[0086] Female C57BL / 6JNifdc mice aged 6-8 weeks were randomly divided into two groups, and the treatment was as follows:

[0087] Adjuvant control group: On days 1, 7 and 14 of the experiment, adjuvant CpG dissolved in PBS (10 μg / mouse) was instilled into the nasal cavity.

[0088] Mu-Sda1 immunization group: On day 1, day 7 and day 14 of the experiment, the vaccine solution was administered via nasal drip (each mouse was given a vaccine solution containing 10 μg of the recombinant mu-Sda1 protein prepared in Example 1 and 10 μg of CpG dissolved in PBS each time);

[0089] On day 24 of the experiment, mice were challenged with live bacteria (Streptococcus aureus M1 or M12) via nasal instillation (bacterial concentration of 2 × 10⁻⁶). 8 CFU / 10μl (10μl infused per mouse). Mice were sacrificed 24 hours after challenge, and nasal associated lymphoid tissue (NALT) was isolated, prepared into a single-cell suspension, and the number of viable group A streptococci in NALT was detected by blood agar culture.

[0090] See results Figure 5 Each black dot represents one mouse (the vertical axis refers to the number of CFU of Streptococcus A in the entire NALT of each mouse). After challenge with Streptococcus A M1, the number of viable bacteria in the NALT of mice in the Mu-Sda1 immunized group prepared in Example 1 was significantly lower than that in the adjuvant control group. After challenge with Streptococcus A M12, the number of viable bacteria in the NALT of mice in the Mu-Sda1 immunized group prepared in Example 1 was significantly lower than that in the adjuvant control group. This indicates that immunization with the vaccine solution via the respiratory mucosa can effectively promote the clearance of different serotypes of Streptococcus A from the site of infection.

[0091] Example 5: The nasal drop immunization vaccine provides cross-immune protection against A-strand strains that do not contain the Sda1 gene.

[0092] Streptococcus aureus secretes multiple virulence factors with DNase activity. Protein sequence analysis revealed that these secreted DNases are highly homologous. The results of Example 3 suggest that the vaccine-induced serum IgG has broad-spectrum neutralizing activity and can effectively inhibit the activity of multiple DNases in the culture supernatant of Streptococcus aureus M1 strain. Therefore, we challenged mice with Streptococcus aureus strain that does not contain the Sda1 gene to test the broad-spectrum cross-protective effect of the vaccine.

[0093] Female C57BL / 6JNifdc mice aged 6-8 weeks were randomly divided into two groups, and the treatment was as follows:

[0094] Adjuvant control group: On days 1, 7 and 14 of the experiment, adjuvant CpG dissolved in PBS (10 μg / mouse) was instilled into the nasal cavity.

[0095] Mu-Sda1 immunization group: On day 1, day 7 and day 14 of the experiment, the vaccine solution was administered via nasal drip (each mouse was given a vaccine solution containing 10 μg of the recombinant mu-Sda1 protein prepared in Example 1 and 10 μg of CpG dissolved in PBS each time);

[0096] On day 24 of the experiment, mice were challenged with Streptococcus aureus M1 or M3.11 (strains without the Sda1 gene) via nasal instillation (bacterial concentration of 2 × 10⁻⁶). 8 CFU / 10μl (10μl infused per mouse). Mice were sacrificed 24 hours after challenge, and nasal associated lymphoid tissue (NALT) was isolated, prepared into a single-cell suspension, and the number of viable group A streptococci in NALT was detected by blood agar culture.

[0097] See results Figure 6 Each black dot represents one mouse (the vertical axis refers to the number of CFU of Streptococcus A in the entire NALT of each mouse). After challenge with Streptococcus A M1, the number of viable bacteria in the NALT of mice in the Mu-Sda1 immunized group prepared in Example 1 was significantly lower than that in the adjuvant control group. After challenge with Streptococcus A M3.11, the number of viable bacteria in the NALT of mice in the Mu-Sda1 immunized group prepared in Example 1 was significantly lower than that in the adjuvant control group. This indicates that immunization with the vaccine solution via the respiratory mucosa can effectively promote the clearance of Streptococcus A strains that do not contain the Sda1 gene from the site of infection.

[0098] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention. sequence list <110> Institute of Microbiology, Chinese Academy of Sciences <120> Recombinant proteins, vaccines, and drugs for preventing Gram-positive pathogens <130> IM2021049I <160> 10 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1173 <212> DNA <213> Sda1 <400> 1 atgtctaaac attggagaca tctaattatt cactctgctc tgaccattct agctacgtta 60 tttttaggta tactaccaat tactgaaaat actagtagta ccatttacgc tgaatcgggc 120 actatttcaa ataattggag tatcgagcaa caccccaatt attaccatgt tgaaggtaaa 180 gcgcaactgg atattaaaaa ttttcccgaa ctttatcgta caaccgaaag ggtctataag 240 aaaagtgggc aaagtactaa acctgttaca gtttccaata tccattactc tgtacttgat 300 ggctacggcc gttctggaga agcttatggt attatcacaa aagatatgat tgacatgtct 360 gctggctatc gtgaaaaatg ggaaagcaaa ccagagccaa gtgggtggta ttcttatttc 420 ttcaaaaata ctaaccagag agccactgaa tccgactaca agcatagccc caaaaatgtg 480 agtaagattt cgaacaatat caaagctagt attctcttaa gtaacggaaa tgttcgtaac 540 ggctacctgt ttgatagaag tcatttaata gccgatagct taggaggaag accttttaga 600 aataatttga ttacgggtac ccgcacccaa aacgtaggta ataatgatcg taaaggtggg 660 atgcagtata ttgaaaataa agttttagat cacattaaaa gaaatcctaa agtccatgtt 720 tactataaag caactcctgt atatcaagga tccgaattgc tacctagagc agttttagtg 780 tctgctttat catctgatgg atttattgac gagacagttc gtgtgtttaa taatgtagca 840 ggttttaata ttgattacca aaacggtgga ctcttatctt ctactgctga cgtagatatt 900 aataacgttg aagaaaatga aatcgaaact actgatgacg agattgaaga gggaatcgaa 960 aacgagcctg acacggatgc actaaaaaaa gataacaaag atacttcttt acaagacact 1020 gtatatgtgg caagtaatgg gcaatctgat gtatactggt acaacaaaga cagtatgcct 1080 aaaactgtaa acttagagaa agttgtagaa atgagtgaac aagtagcttt gactagaggt 1140 aaacatcatt caactcaaga aaatatagaa taa 1173 <210> 2 <211> 390 <212> PRT <213> Sda1 <400> 2 Met Ser Lys His Trp Arg His Leu Ile Ile His Ser Ala Leu Thr Ile 1 5 10 15 Leu Ala Thr Leu Phe Leu Gly Ile Leu Pro Ile Thr Glu Asn Thr Ser 20 25 30 Ser Thr Ile Tyr Ala Glu Ser Gly Thr Ile Ser Asn Asn Trp Ser Ile 35 40 45 Glu Gln His Pro Asn Tyr Tyr His Val Glu Gly Lys Ala Gln Leu Asp 50 55 60 Ile Lys Asn Phe Pro Glu Leu Tyr Arg Thr Thr Glu Arg Val Tyr Lys 65 70 75 80 Lys Ser Gly Gln Ser Thr Lys Pro Val Thr Val Ser Asn Ile His Tyr 85 90 95 Ser Val Leu Asp Gly Tyr Gly Arg Ser Gly Glu Ala Tyr Gly Ile Ile 100 105 110 Thr Lys Asp Met Ile Asp Met Ser Ala Gly Tyr Arg Glu Lys Trp Glu 115 120 125 Ser Lys Pro Glu Pro Ser Gly Trp Tyr Ser Tyr Phe Phe Lys Asn Thr 130 135 140 Asn Gln Arg Ala Thr Glu Ser Asp Tyr Lys His Ser Pro Lys Asn Val 145 150 155 160 Ser Lys Ile Ser Asn Asn Ile Lys Ala Ser Ile Leu Leu Ser Asn Gly 165 170 175 Asn Val Arg Asn Gly Tyr Leu Phe Asp Arg Ser His Leu Ile Ala Asp 180 185 190 Ser Leu Gly Gly Arg Pro Phe Arg Asn Asn Leu Ile Thr Gly Thr Arg 195 200 205 Thr Gln Asn Val Gly Asn Asn Asp Arg Lys Gly Gly Met Gln Tyr Ile 210 215 220 Glu Asn Lys Val Leu Asp His Ile Lys Arg Asn Pro Lys Val His Val 225 230 235 240 Tyr Tyr Lys Ala Thr Pro Val Tyr Gln Gly Ser Glu Leu Leu Pro Arg 245 250 255 Ala Val Leu Val Ser Ala Leu Ser Ser Asp Gly Phe Ile Asp Glu Thr 260 265 270 Val Arg Val Phe Asn Asn Val Ala Gly Phe Asn Ile Asp Tyr Gln Asn 275 280 285 Gly Gly Leu Leu Ser Ser Thr Ala Asp Val Asp Ile Asn Asn Val Glu 290 295 300 Glu Asn Glu Ile Glu Thr Thr Asp Asp Glu Ile Glu Glu Gly Ile Glu 305 310 315 320 Asn Glu Pro Asp Thr Asp Ala Leu Lys Lys Asp Asn Lys Asp Thr Ser 325 330 335 Leu Gln Asp Thr Val Tyr Val Ala Ser Asn Gly Gln Ser Asp Val Tyr 340 345 350 Trp Tyr Asn Lys Asp Ser Met Pro Lys Thr Val Asn Leu Glu Lys Val 355 360 365 Val Glu Met Ser Glu Gln Val Ala Leu Thr Arg Gly Lys His His Ser 370 375 380 Thr Gln Glu Asn Ile Glu 385 390 <210> 3 <211> 1089 <212> DNA <213> Artificial Sequence <400> 3 atgggctcgg gcactatttc aaataattgg agtatcgagc aacaccccaa ttattaccat 60 gttgaaggta aagcgcaact ggatattaaa aattttcccg aactttatcg tacaaccgaa 120 agggtctata agaaaagtgg gcaaagtact aaacctgtta cagtttccaa tatccattac 180 tctgtacttg atggctacgg ccgttctgga gaagcttatg gtattatcac aaaagatatg 240 attgacatgt ctgctggcta tcgtgaaaaa tgggaaagca aaccagagcc aagtgggtgg 300 tattcttatt tcttcaaaaa tactaaccag agagccactg aatccgacta caagcatagc 360 cccaaaaatg tgagtaagat ttcgaacaat atcaaagcta gtattctctt aagtaacgga 420 aatgttcgta acggctacct gtttgataga agtcatttaa tagccgatag cttaggagga 480 agacctttta gaaataattt gattacgggt acccgcacc aaaacgtagg taataatgat 540 cgtaaaggtg ggatgcagta tattgaaaat aaagttttag atcacatta aagaaatcct 600 aaagtccatg tttactataa agcaactcct gtatatcaag gatccgaatt gctacctaga 660 gcagttttag tgtctgcttt atcatctgat ggatttattg acgagacagt tcgtgtgttt 720 aataatgtag caggttttaa tattgattac caaaacggtg gactcttatc ttctactgct 780 gacgtagata ttaataacgt tgaagaaaat gaaatcgaaa ctactgatga cgagattgaa 840 gagggaatcg aaaacgagcc tgacacggat gcactaaaaa aagataacaa agatacttct 900 ttacaagaca ctgtatatgt ggcaagtaat gggcaatctg atgtatactg gtacaacaaa 960 gacagtatgc ctaaaactgt aaacttagag aaagttgtag aaatgagtga acaagtagct 1020 ttgactagag gtaaacatca ttcaactcaa gaaaatatag aactcgagca ccaccaccac 1080 caccactga 1089 <210> 4 <211> 362 <212> PRT <213> Artificial Sequence <400> 4 Met Gly Ser Gly Thr Ile Ser Asn Asn Trp Ser Ile Glu Gln His Pro 1 5 10 15 Asn Tyr Tyr His Val Glu Gly Lys Ala Gln Leu Asp Ile Lys Asn Phe 20 25 30 Pro Glu Leu Tyr Arg Thr Thr Glu Arg Val Tyr Lys Lys Ser Gly Gln 35 40 45 Ser Thr Lys Pro Val Thr Val Ser Asn Ile His Tyr Ser Val Leu Asp 50 55 60 Gly Tyr Gly Arg Ser Gly Glu Ala Tyr Gly Ile Ile Thr Lys Asp Met 65 70 75 80 Ile Asp Met Ser Ala Gly Tyr Arg Glu Lys Trp Glu Ser Lys Pro Glu 85 90 95 Pro Ser Gly Trp Tyr Ser Tyr Phe Phe Lys Asn Thr Asn Gln Arg Ala 100 105 110 Thr Glu Ser Asp Tyr Lys His Ser Pro Lys Asn Val Ser Lys Ile Ser 115 120 125 Asn Asn Ile Lys Ala Ser Ile Leu Leu Ser Asn Gly Asn Val Arg Asn 130 135 140 Gly Tyr Leu Phe Asp Arg Ser His Leu Ile Ala Asp Ser Leu Gly Gly 145 150 155 160 Arg Pro Phe Arg Asn Asn Leu Ile Thr Gly Thr Arg Thr Gln Asn Val 165 170 175 Gly Asn Asn Asp Arg Lys Gly Gly Met Gln Tyr Ile Glu Asn Lys Val 180 185 190 Leu Asp His Ile Lys Arg Asn Pro Lys Val His Val Tyr Tyr Lys Ala 195 200 205 Thr Pro Val Tyr Gln Gly Ser Glu Leu Leu Pro Arg Ala Val Leu Val 210 215 220 Ser Ala Leu Ser Ser Asp Gly Phe Ile Asp Glu Thr Val Arg Val Phe 225 230 235 240 Asn Asn Val Ala Gly Phe Asn Ile Asp Tyr Gln Asn Gly Gly Leu Leu 245 250 255 Ser Ser Thr Ala Asp Val Asp Ile Asn Asn Val Glu Glu Asn Glu Ile 260 265 270 Glu Thr Thr Asp Asp Glu Ile Glu Glu Gly Ile Glu Asn Glu Pro Asp 275 280 285 Thr Asp Ala Leu Lys Lys Asp Asn Lys Asp Thr Ser Leu Gln Asp Thr 290 295 300 Val Tyr Val Ala Ser Asn Gly Gln Ser Asp Val Tyr Trp Tyr Asn Lys 305 310 315 320 Asp Ser Met Pro Lys Thr Val Asn Leu Glu Lys Val Val Glu Met Ser 325 330 335 Glu Gln Val Ala Leu Thr Arg Gly Lys His His Ser Thr Gln Glu Asn 340 345 350 Ile Glu Leu Glu His His His His His His 355 360 <210> 5 <211> 1089 <212> DNA <213> Artificial Sequence <400> 5 atgggctcgg gcactatttc aaataattgg agtatcgagc aacaccccaa ttattaccat 60 gttgaaggta aagcgcaact ggatattaaa aattttcccg aactttatcg tacaaccgaa 120 agggtctata agaaaagtgg gcaaagtact aaacctgtta cagtttccaa tatccattac 180 tctgtacttg atggctacgg ccgttctgga gaagcttatg gtattatcac aaaagatatg 240 attgacatgt ctgctggcta tcgtgaaaaa tgggaaagca aaccagagcc aagtgggtgg 300 tattcttatt tcttcaaaaa tactaaccag agagccactg aatccgacta caagcatagc 360 cccaaaaatg tgagtaagat ttcgaacaat atcaaagcta gtattctctt aagtaacgga 420 aatgttcgta acggctacct gtttgataga agtggtttaa tagccgatag cttaggagga 480 agacctttta gaaataattt gattacgggt acccgcacc aaaacgtagg taataatgat 540 cgtaaaggtg ggatgcagta tattgaaaat aaagttttag atcacatta aagaaatcct 600 aaagtccatg tttactataa agcaactcct gtatatcaag gatccgaatt gctacctaga 660 gcagttttag tgtctgcttt atcatctgat ggatttattg acgagacagt tcgtgtgttt 720 aataatgtag caggttttaa tattgattac caaaacggtg gactcttatc ttctactgct 780 gacgtagata ttaataacgt tgaagaaaat gaaatcgaaa ctactgatga cgagattgaa 840 gagggaatcg aaaacgagcc tgacacggat gcactaaaaa aagataacaa agatacttct 900 ttacaagaca ctgtatatgt ggcaagtaat gggcaatctg atgtatactg gtacaacaaa 960 gacagtatgc ctaaaactgt aaacttagag aaagttgtag aaatgagtga acaagtagct 1020 ttgactagag gtaaacatca ttcaactcaa gaaaatatag aactcgagca ccaccaccac 1080 caccactga 1089 <210> 6 <211> 362 <212> PRT <213> Artificial Sequence <400> 6 Met Gly Ser Gly Thr Ile Ser Asn Asn Trp Ser Ile Glu Gln His Pro 1 5 10 15 Asn Tyr Tyr His Val Glu Gly Lys Ala Gln Leu Asp Ile Lys Asn Phe 20 25 30 Pro Glu Leu Tyr Arg Thr Thr Glu Arg Val Tyr Lys Lys Ser Gly Gln 35 40 45 Ser Thr Lys Pro Val Thr Val Ser Asn Ile His Tyr Ser Val Leu Asp 50 55 60 Gly Tyr Gly Arg Ser Gly Glu Ala Tyr Gly Ile Ile Thr Lys Asp Met 65 70 75 80 Ile Asp Met Ser Ala Gly Tyr Arg Glu Lys Trp Glu Ser Lys Pro Glu 85 90 95 Pro Ser Gly Trp Tyr Ser Tyr Phe Phe Lys Asn Thr Asn Gln Arg Ala 100 105 110 Thr Glu Ser Asp Tyr Lys His Ser Pro Lys Asn Val Ser Lys Ile Ser 115 120 125 Asn Asn Ile Lys Ala Ser Ile Leu Leu Ser Asn Gly Asn Val Arg Asn 130 135 140 Gly Tyr Leu Phe Asp Arg Ser Gly Leu Ile Ala Asp Ser Leu Gly Gly 145 150 155 160 Arg Pro Phe Arg Asn Asn Leu Ile Thr Gly Thr Arg Thr Gln Asn Val 165 170 175 Gly Asn Asn Asp Arg Lys Gly Gly Met Gln Tyr Ile Glu Asn Lys Val 180 185 190 Leu Asp His Ile Lys Arg Asn Pro Lys Val His Val Tyr Tyr Lys Ala 195 200 205 Thr Pro Val Tyr Gln Gly Ser Glu Leu Leu Pro Arg Ala Val Leu Val 210 215 220 Ser Ala Leu Ser Ser Asp Gly Phe Ile Asp Glu Thr Val Arg Val Phe 225 230 235 240 Asn Asn Val Ala Gly Phe Asn Ile Asp Tyr Gln Asn Gly Gly Leu Leu 245 250 255 Ser Ser Thr Ala Asp Val Asp Ile Asn Asn Val Glu Glu Asn Glu Ile 260 265 270 Glu Thr Thr Asp Asp Glu Ile Glu Glu Gly Ile Glu Asn Glu Pro Asp 275 280 285 Thr Asp Ala Leu Lys Lys Asp Asn Lys Asp Thr Ser Leu Gln Asp Thr 290 295 300 Val Tyr Val Ala Ser Asn Gly Gln Ser Asp Val Tyr Trp Tyr Asn Lys 305 310 315 320 Asp Ser Met Pro Lys Thr Val Asn Leu Glu Lys Val Val Glu Met Ser 325 330 335 Glu Gln Val Ala Leu Thr Arg Gly Lys His His Ser Thr Gln Glu Asn 340 345 350 Ile Glu Leu Glu His His His His His His 355 360 <210> 7 <211> 34 <212> DNA <213> Artificial Sequence <400> 7 catgccatgg gctcgggcac tatttcaaat aatt 34 <210> 8 <211> 32 <212> DNA <213> Artificial Sequence <400> 8 ccgctcgagt tctatatttt cttgagttga at 32 <210> 9 <211> 30 <212> DNA <213> Artificial Sequence <400> 9 gtttgataga agtggtttaa tagccgatag 30 <210> 10 <211> 30 <212> DNA <213> Artificial Sequence <400> 10 ctatcggcta ttaaaccact tctatcaaac 30

Claims

1. Recombinant protein mu-Sda1, characterized in that, Its amino acid sequence is shown in SEQ ID No.

6.

2. A nucleic acid molecule encoding the recombinant protein mu-Sda1 as described in claim 1, characterized in that, The nucleotide sequence is shown in SEQ ID No.

5.

3. A recombinant vector comprising the nucleic acid molecule as described in claim 2.

4. A host comprising the recombinant vector as described in claim 3.

5. The host as described in claim 4, characterized in that, The host includes one or more of Escherichia coli, yeast, or mammalian cells.

6. The use of the recombinant protein mu-Sda1 as described in claim 3 in the preparation of vaccines for preventing pathogenic bacterial infections, reagents for detecting pathogenic bacteria, and medicaments for preventing and / or treating diseases caused by pathogenic bacterial infections; The pathogenic bacteria include: Streptococcus A M1 or M12; The infections include respiratory infections.

7. A vaccine, characterized in that, Includes the recombinant protein mu-Sda1 as described in claim 1 and an acceptable adjuvant.

8. The vaccine as described in claim 7, characterized in that, The adjuvant includes CpG.

9. The vaccine as described in claim 8, characterized in that, The mass ratio of the recombinant protein mu-Sda1 to the CpG is 1:

1.

10. The vaccine as described in claims 7-9, characterized in that, The vaccine can be administered via nasal inhalation, oral administration, subcutaneous injection, intradermal injection, urogenital injection, or rectal injection.

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