CN115433723A

CN115433723A - Recombinant protein, vaccine and medicament for preventing gram-positive pathogenic bacteria

Info

Publication number: CN115433723A
Application number: CN202110623941.0A
Authority: CN
Inventors: 王北难; 毕帅
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2021-06-04
Filing date: 2021-06-04
Publication date: 2022-12-06

Abstract

The invention relates to the field of biological medicines, relates to recombinant protein, vaccine and medicine for preventing gram-positive pathogenic bacteria, and particularly relates to a recombinant subunit vaccine for inhibiting streptococcus and/or preventing streptococcus infection. The active ingredients of the vaccine provided by the invention consist of Sda1 active mutant mu-Sda1 and adjuvant CpG; has the effect of effectively inhibiting or preventing gram-positive pathogenic bacteria such as streptococcus, staphylococcus aureus, pneumococcus and the like. The vaccine provided by the invention has the advantages of high efficiency, broad spectrum and low price. The vaccine provided by the invention adopts a mucosal immunity way, has the characteristics of no tissue damage, no local side effect and simple and convenient use, and is easy to popularize and use.

Description

Recombinant protein, vaccine and medicament for preventing gram-positive pathogenic bacteria

Technical Field

The invention relates to the field of biological medicine, in particular to recombinant protein, vaccine and medicine for preventing gram-positive pathogenic bacteria.

Background

Group A streptococci (Group A streptococci, hereinafter referred to as A streptococci) are important gram-positive conditional pathogenic bacteria. A chain bacteria causes various diseases through infection of upper respiratory mucosa and skin parts of a human body, wherein the mild people comprise pharyngitis, scarlet fever, impetigo, erysipelas and cellulitis, and the severe people can cause systemic spread and invasive infection to endanger life, such as pneumonia, bacteremia, toxic shock, acute necrotizing myofascitis and the like; infection with A streptococci also induces autoimmune diseases such as rheumatic fever and rheumatic heart disease. According to the world health organization, about a patient with the A-chain bacteria disease is counted around the world, more than 178 ten thousand new cases and about 50 ten thousand dead cases are counted each year. Moreover, rheumatic fever and rheumatic heart disease caused by the infection of the A-streptococci are the leading causes of death cases of cardiovascular diseases, so that the A-streptococci are always valued by the world health organization and scientists.

The wide spread of A-streptococci, high mortality caused by infection-induced autoimmune diseases and invasive infection cause huge burden on world public health and medical treatment, and the development of safe, effective and broad-spectrum A-streptococci vaccines is urgently needed. Due to the multitude of serotypes of A, immunity is developed against one serotype while there is no protective effect against another serotype. In addition, A-chain infection is closely related to autoimmune diseases, which seriously hampers the development process of A-chain vaccine.

The A-chain bacterium expresses multiple virulence factors during the infection process, including escape immune clearance. The middle-hundred million granulocytes play an important role in resisting infection of A chain bacteria, and capture and kill bacteria by releasing DNA containing itself to form a fiber Network Structure (NETs) containing a large amount of proteolytic enzymes (such as elastase and the like) with antibacterial activity. The A chain bacteria secrete various deoxyribonucleases to degrade the DNA of the NETs, and escape the capturing and removing effects of the neutrophil NETs. Sda1 is a secretory virulence factor with Dnase activity, widely exists in A chain bacterium clinical isolates (especially M1T1 strains widely spread in the world in recent years), and breaks NETs escape immune clearance by degrading DNA in neutrophil NETs. The pathogenicity of the A chain strain with the Sda1 gene knocked out or the Sda1 gene deleted is obviously reduced. Protein sequence and phylogenetic analysis showed: the important pathogenic bacteria of human, such as streptococcus group C (S.equisimilis), pneumococcus (S.pneumoconiae), staphylococcus aureus (S.aureus) and the like, all contain Sda1 homologous protein, which indicates that Sda1 plays an important role in pathogenic bacteria pathogenic process.

Disclosure of Invention

In view of the above, the present invention provides a recombinant protein, a vaccine and a medicament for preventing gram-positive pathogenic bacteria.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides application of Sda1 as a target in preparation of vaccines for preventing pathogenic bacteria infection, reagents for detecting pathogenic bacteria and medicines for preventing and/or treating diseases caused by pathogenic bacteria infection; the pathogenic bacteria comprise: inhibiting a pathogenic bacterium that expresses Sda1 and/or a dnase homologous thereto; the infection comprises: infection of human mucosal system. In some embodiments of the invention, the pathogenic bacteria comprise gram-positive pathogenic bacteria; the gram-positive pathogenic bacteria comprise one or more of streptococcus, staphylococcus aureus and pneumococcus; the streptococcus comprises one or more of group A streptococcus, group B streptococcus, group C streptococcus, group G streptococcus, streptococcus pneumoniae, streptococcus suis or streptococcus equi; the human mucosal system comprises one or more of the respiratory system, the digestive system, the urinary system, the urogenital system, or the skin.

The present invention also provides a recombinant protein mu-Sda1 having:

(I) The amino acid residue from the 3 rd to the 354 th positions of the N tail end of the amino acid sequence shown as SEQ ID No. 6; or

(II) an amino acid sequence obtained by substituting, deleting or adding one or two amino acid residues in the amino acid sequence shown in (I), and the amino acid sequence has the same or similar functions with the amino acid sequence shown in (I); or

(III) an amino acid sequence which has at least 90% sequence identity with the sequence described in (I) or (II) and which is functionally identical or similar to the amino acid sequence shown in (I).

In some embodiments of the invention, the recombinant protein mu-Sda1 has:

(I) And an amino acid sequence shown as SEQ ID No. 6; or

(II) an amino acid sequence obtained by substituting, deleting or adding one or two amino acid residues in the amino acid sequence shown in the (I), and the amino acid sequence has the same or similar functions with the amino acid sequence shown in the (I); or

In addition, the invention also provides a nucleic acid molecule for encoding the recombinant protein mu-Sda1, which has

(I) 7 bp-1062 bp of the nucleotide sequence shown as SEQ ID No.5 or

(II) a complementary nucleotide sequence of 7 bp-1062 bp of the nucleotide sequence shown as SEQ ID No. 5; or

(III) a nucleotide sequence which encodes the same protein as the nucleotide sequence of (I) or (II) but which differs from the nucleotide sequence of (I) or (II) due to the degeneracy of the genetic code; or

(IV) a nucleotide sequence obtained by substituting, deleting or adding one or two nucleotide sequences with the nucleotide sequence shown in (I), (II) or (III), and the nucleotide sequence has the same or similar functions with the nucleotide sequence shown in (I), (II) or (III); or

(V) a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence of (I), (II), (III) or (IV).

The invention also provides a recombinant vector comprising the nucleic acid molecule.

The invention also provides a host containing the recombinant vector.

In some embodiments of the invention, the host comprises one or more of escherichia coli, yeast, or mammalian cells.

In addition, the invention also provides the application of the recombinant protein mu-Sda1 in preparing vaccines for preventing pathogenic bacteria infection, reagents for detecting pathogenic bacteria and medicines for preventing and/or treating diseases caused by pathogenic bacteria infection; the pathogenic bacteria comprise: inhibiting a pathogenic bacterium that expresses Sda1 and/or a dnase homologous thereto; the infection comprises: infection of the human mucosal system.

In some embodiments of the invention, the pathogenic bacteria comprise gram-positive pathogenic bacteria; the gram-positive pathogenic bacteria comprise one or more of streptococcus, staphylococcus aureus and pneumococcus; the streptococcus comprises one or more of group A streptococcus, group B streptococcus, group C streptococcus, group G streptococcus, streptococcus pneumoniae, streptococcus suis or streptococcus equi; the human mucosal system comprises one or more of the respiratory system, the digestive system, the urinary system, the urogenital system, or the skin.

The 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 by nasal inhalation, orally, subcutaneously, intradermally, urogenital, or intraanal.

The invention also provides an antibody, and the antibody is obtained by immunizing an animal with the vaccine.

The invention also provides a medicament comprising the antibody and pharmaceutically acceptable auxiliary materials.

The invention also provides a detection reagent or a detection kit, which comprises the antibody and a detection auxiliary agent acceptable in detection science.

The invention designs a reasonable vaccine formula by utilizing the universality and homology of Sda1 in various pathogenic bacteria and the characteristic of enhancing antigen immunogenicity by a mucosal immune adjuvant, obviously induces a neutralizing antibody with broad-spectrum neutralizing activity, achieves the effects of preventing pathogenic bacteria from colonizing and rapidly removing the pathogenic bacteria, has a protection effect on different serotype A chain bacteria, and has the advantages of high efficiency, broad spectrum and low price. The vaccine provided by the invention adopts a mucosal immunity way, has the characteristics of no tissue damage, no local side effect and simple and convenient use, and is easy to popularize and use.

Drawings

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

FIGS. 1A and 1B show agarose gel electrophoresis patterns during PCR amplification of Sda1 and mu-Sda1 genes, respectively;

FIG. 2 shows an SDS-PAGE electrophoresis of the preparation of mu-Sda1 fusion proteins;

FIG. 3 shows the results of example 2 (nasal immunization of mice with vaccine drops induced an antigen-specific antibody response); wherein, FIG. 3A shows that vaccine drops of nasal immunised mice induce an antigen specific serum IgG response; FIG. 3B shows that vaccine drops immunize nose mice to induce secretory IgA response in antigen-specific oral wash;

FIG. 4 shows the results of example 3 (serum IgG induced by the vaccine solution has neutralizing activity against DNase);

FIG. 5 shows the results of example 4 (nasal drops of the immunizing vaccine solution provided cross-immune protection against respiratory infections with different serotype A streptococci);

figure 6 shows the results of example 5 (nasal drop immunization vaccine solution provides cross-immune protection against a-chain strains that do not contain the Sda1 gene).

Detailed Description

The invention discloses a recombinant protein, vaccine and medicament for preventing gram-positive pathogenic bacteria, and a person skilled in the art can realize the prevention by properly improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

One of the objectives of the present invention is to provide a broad spectrum single subunit vaccine for the prevention of A chain bacteria infection.

The second object of the present invention is to provide a recombinant gene of a mutant having Sda1 activity and an expression system comprising the recombinant gene.

The function of the vaccine comprises (I) or (II) or (III)

(I) Inhibiting pathogenic bacteria that express Sda1 and DNA enzymes homologous thereto;

(II) preventing infection of pathogenic bacteria expressing Sda1 and dnase homologous thereto;

(III) reduce or prevent colonization and infection of the human mucosal system by pathogenic bacteria expressing Sda1 and dnase homologous thereto.

The mu-Sda1 may specifically be (a) or (b) as follows: (a) Protein consisting of amino acid residues from the 22 nd to 189 th positions of the N tail end of a sequence 3 in a sequence table; (b) And (b) a protein derived from the protein (a) by substitution and/or deletion and/or addition of one or more amino acid residues and having the same activity.

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

The pathogenic bacteria expressing Sda1 and DNase homologous thereto include but are not limited to Streptococcus, staphylococcus aureus, and pneumococcus. The streptococcus can be group A streptococcus, group B streptococcus, group C streptococcus, group G streptococcus, streptococcus pneumoniae, streptococcus suis or streptococcus equi.

The human mucosal system may be the respiratory system, the digestive system, the urogenital system or the skin.

The vaccine is administered by nasal inhalation, oral administration, subcutaneous injection, intradermal injection, urogenital injection or anal injection.

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 pathogenic bacteria expressing Sda1 and DNA enzyme homologous with the Sda 1.

The invention designs a reasonable vaccine formula by utilizing the universality and homology of Sda1 in various pathogenic bacteria and the characteristic of enhancing antigen immunogenicity by a mucosal immune adjuvant, obviously induces a neutralizing antibody with broad-spectrum neutralizing activity, achieves the effects of preventing pathogenic bacteria from colonizing and quickly removing the pathogenic bacteria, has a protection effect on different serotype A chain bacteria, and has the advantages of high efficiency, broad spectrum and low price. The vaccine provided by the invention adopts a mucosal immunity way, has the characteristics of no tissue damage, no local side effect and simple and convenient use, and is easy to popularize and use.

Wherein:

type a streptococcus, type M1 and type M3.11: from the microbial line at minnesota state university, reference: wang B, dieptant, briscoe S, hyland KA, kang J, khoruts a, clean pp.induction Of TGF-beta and TGF-beta-dependent predominant Th17differentiation by group a transcriptional introduction. Proc natl acadsi U S2010; 107 (13): 5937-42..

Group a streptococcus, type M12: reference: haanes EJ, clean PP, identification of a divergent M protein gene and an M protein-related gene family in Streptococcus genus serotype 49.J. Bacteriol.1989Dec;171 (12): 6397-6408.

Vector pET28a (+): novagen, cat.No.69846-3. Coli BL21gold (DE 3) plysS: purchased from Beijing medicine, biotech, inc., product number: CW0810A. C57BL/6JNIFdc mice: purchased from Beijing Wintolite laboratory animal technology, inc., STRAIN CODE:219.

the following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are all conventional ones unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

The invention is further illustrated by the following examples:

example 1 construction of recombinant Sda1 and mu-Sda1 and preparation of recombinant proteins thereof

1. The Sda1 gene was PCR-amplified using a Primer composed of Primer 1 and Primer 2 using genomic DNA of A-chain bacterium (M1 type, 90226WT strain) as a template, to obtain Sdal gene PCR amplification product 1 (about 1059 bp). The agarose gel electrophoresis pattern of the PCR amplification product is shown in FIG. 1A.

Primer 1：5’-CATGCCATGGGCTCGGGCACTATTTCAAATAATT-3' (shown as SEQ ID No. 7);

Primer 2：5’-CCGCTCGAGTTCTATATTTTCTTGAGTTGAAT-3' (as shown in SEQ ID No. 8).

2. Point mutation was performed by PCR to mutate the amino acid His 188 at position 188 in sequence 2 (SEQ ID No. 1) to Gly to obtain recombinant protein mu-Sda1 (about 1059 bp) with lost dnase activity, using the following primers for point mutation, respectively:

primer 3:5' GTTTGATAGAAGTGGTTTAATAGCCGATAG-

Primer 4: 5 'CTATCGGCTATTAAACCATTTCTATCAAAC-3' (shown as SEQ ID No. 10)

The agarose gel electrophoresis pattern of the PCR amplification product is shown in FIG. 1B.

3. The PCR amplification product of step 2 was double digested with restriction enzymes NcoI and Xho1, and the digested product was recovered.

4. The vector pET28a was double-digested with restriction enzymes NcoI and Xho1, and the vector backbone of about 5400bp was recovered.

5. And (3) connecting the PCR enzyme digestion product in the step (3) with the vector framework in the step (4) to obtain a recombinant plasmid pET28a-mu-Sda1 (the nucleotide sequence of the recombinant plasmid is shown as SEQ ID No. 5). Based on the sequencing results, the results for recombinant plasmid pET28a-mu-Sda1 are described below: a double-stranded DNA molecule represented by nucleotides 7 to 1062 from the 5' -end in sequence 5 of the sequence listing (SEQ ID No. 5) was inserted between the NcoI and XhoI cleavage sites of vector pET28 a. The inserted double-stranded DNA molecule and partial DNA on the carrier skeleton form a fusion gene shown in a sequence 5 (SEQ ID No. 5) of a sequence table, and express a recombinant protein shown in a sequence 6 (SEQ ID No. 6) of the sequence table.

5. The recombinant plasmid pET28a-mu-Sda1 is transformed into Escherichia coli BL21gold (DE 3) plysS to obtain the recombinant bacterium pET28a-mu-Sda1-BL21gold (DE 3) plysS.

6. The recombinant bacterium pET28a-mu-Sda1-BL21gold (DE 3) plysS obtained in step 5 was inoculated into LB liquid medium containing 50. Mu.g/mL kanamycin, and cultured at 37 ℃ with shaking at 200rpm until OD _560nm When the concentration was 1mM, IPTG was added at 0.6, and the mixture was cultured at 30 ℃ for 16 hours with shaking at 160 rpm.

7. The culture system of step 6 was centrifuged at 8000rpm and 4 ℃ for 10 minutes to collect the pellet, the pellet was suspended in PBS buffer (pH7.4) and sonicated (power 200W, 4 seconds of operation with 8 seconds intervals, 99 cycles), and then centrifuged at 12000rpm for 20 minutes to collect the supernatant.

8. The supernatant obtained in step 7 was applied to Ni Sepharose6Fast Flow of GE, and eluted with 10 column volumes of solution I (pH 7.4, PBS containing 20mM imidazole) to remove foreign proteins, followed by elution with 5 column volumes of solution II (pH 7.4, PBS containing 200mM imidazole) to obtain the target protein, and the solution after passing through the column when eluted with solution II was collected and dialyzed overnight at 4 ℃ to remove imidazole (dialysis cut-off of 1000, buffer solution is PBS solution at pH 7.4).

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

The SDS-PAGE gel of the expression of the purified mu-Sda1 recombinant protein is shown in FIG. 3A. In FIG. 2, lane 1 is a protein molecular weight standard (Mw), lane 2 is a hetero-protein that failed to bind to the I Sepharose6Fast Flow column in step 7, lane 3 is an elution solution using solution I, lane 4 is an elution solution using solution I, and the recombinant protein mu-Sda1 has a molecular weight of about 41kDa.

EXAMPLE 2 nasal drop immunization of vaccine mice induces antigen-specific antibody responses

Female C57BL/6JNIFdc mice of 6-8 weeks of age were randomly divided into two groups, which were grouped as follows:

adjuvant control group: the CpG adjuvant (10 mug/mouse) dissolved in PBS is respectively dripped into the nasal cavity on the 1 st day, the 7 th day and the 14 th day of the experiment;

Mu-Sda1 immunization group: the vaccine solutions were instilled intranasally on days 1, 7 and 14 of the experiment (each mouse was administered with 10. Mu.g of the mu-Sdal recombinant protein prepared in example 1 and 10. Mu.g of CpG in PBS);

on day 24 of the experiment, blood and oral lavage of mice were taken. The blood was left at room temperature for 2 hours and centrifuged to obtain serum. Serum and oral lavage samples were tested for antigen-specific antibody responses by ELISA.

The results are shown in fig. 3A and 3B, and compared with the adjuvant control group, the levels of serum IgG and secretory IgA in oral wash after immunization with the vaccine solution are significantly increased, indicating that immunization with the vaccine solution induces antigen-specific antibody response.

Example 3 serum IgG induced by the vaccine solution has neutralizing activity against dnase.

GAS culture supernatant (sup.) or recombinant Sda1 with a protein derived from

After incubating mice, mice immunized with the vaccine solution, and mice infected with A chain bacterium M1 for 2 hours, the cells were added to pET28aDNA containing 5. Mu.g of plasmid, reacted at 37 ℃ for 45 minutes, added with 200mM reaction terminator containing EDTA, and electrophoresed in 1% agarose to observe the degradation of plasmid DNA.

The results are shown in FIG. 4, and show that plasmid DNA can be degraded by both the culture supernatant (sup.) of the M1 strain of A chain bacteria or the recombinant Sda1, and the degradation of the plasmid DNA by the culture supernatant (sup.) of the M1 strain of A chain bacteria or the recombinant Sda1 is remarkably inhibited by the serum from the mice immunized by the vaccine solution, and the degradation of the plasmid DNA by the serum from the M1 strain of A chain bacteria or the recombinant Sda1 strain is obviously inhibited

Sera from mice and mice infected with strain M1 of A.cannot inhibit the degradation of plasmid DNA by GAS culture supernatant (sup.) or recombinant Sda 1. The serum IgG induced by the vaccine liquid has neutralization activity and can effectively inhibit the activity of DNase in culture supernatants of Sda1 and A chain bacterium M1 strains.

Example 4 nasal drops of the immunizing vaccine provide cross-immune protection against respiratory tract infections caused by different serotype a streptococci.

Mu-Sda1 immunization group: the vaccine solutions were instilled intranasally on days 1, 7 and 14 of the experiment (each mouse was administered with 10. Mu.g of the mu-Sda1 recombinant protein prepared in example 1 and 10. Mu.g of CpG in PBS);

on day 24 of the experiment, mice were challenged with live bacteria (A. Streptococci type M1 or M12) by nasal instillation (the concentration of the bacteria solution was 2X 10) ⁸ CFU/10. Mu.l, 10. Mu.l instilled per mouse). Mice were sacrificed 24 hours after challenge, nasal Associated Lymphoid Tissue (Nasal Associated Lymphoid Tissue or NALT for short) was isolated to prepare a single cell suspension, and viable count of type a streptococcus in NALT was detected by blood plate culture.

The results are shown in FIG. 5, each black dot represents 1 mouse (the ordinate refers to the number of CFU of A.chain bacteria in the entire NALT of each mouse). After M1 type challenge of A-type streptococcus, the number of live bacteria in the NALT of the Mu-Sda1 immune group mouse prepared in example 1 is obviously lower than that of an adjuvant control group, and after M12 type challenge of A-type streptococcus, the number of live bacteria in the NALT of the Mu-Sda1 immune group mouse prepared in example 1 is obviously lower than that of the adjuvant control group, so that the vaccine liquid can effectively promote the elimination of different serotype A-type streptococci at an infected part through respiratory mucosa immunity.

Example 5 nasal drops of the immunization vaccine solution provide cross-immune protection against a-chain strains that do not contain the Sda1 gene.

A chain bacterium secretes a plurality of virulence factors with DNase activity, protein sequence analysis finds that the secretory DNase has high homology, and the result of example 3 indicates that serum IgG induced by the vaccine liquid has broad-spectrum neutralizing activity and can effectively inhibit the activity of a plurality of DNase in culture supernatant of an A chain bacterium M1 strain. Therefore, the broad-spectrum cross protection effect of the vaccine liquid is detected by using the A chain strain without Sda1 gene to challenge mice.

on day 24 of the experiment, mice were challenged by nasal instillation with A.chain M1 or M3.11 (strains not containing the Sda1 gene) (the concentration of the bacterial liquid was 2X 10) ⁸ CFU/10. Mu.l, 10. Mu.l instilled per mouse). Mice were sacrificed 24 hours after challenge, nasal-associated lymphoid tissue (Nasal associated lymphoid tissue or NALT for short) was isolated to prepare single cell suspensions, and the number of viable streptococcus a in NALT was detected by blood plate culture.

The results are shown in FIG. 6, each black dot represents 1 mouse (the ordinate indicates the CFU number of A.chain in the whole NALT of each mouse). After the M1 type streptococcus A is attacked, the number of live bacteria in the NALT of the Mu-Sda1 immune group mouse prepared in the example 1 is obviously lower than that of an adjuvant control group, and after the M3.11 type streptococcus A is attacked, the number of live bacteria in the NALT of the Mu-Sda1 immune group mouse prepared in the example 1 is obviously lower than that of the adjuvant control group, so that the vaccine liquid can effectively promote and eliminate the A chain strain without the Sda1 gene at an infected part through respiratory mucosa immunization.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

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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 (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 (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 acccgcaccc aaaacgtagg taataatgat 540

cgtaaaggtg ggatgcagta tattgaaaat aaagttttag atcacattaa 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 (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 (Artificial Sequence)

<400> 7

catgccatgg gctcgggcac tatttcaaat aatt 34

<210> 8

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

ccgctcgagt tctatatttt cttgagttga at 32

<210> 9

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gtttgataga agtggtttaa tagccgatag 30

<210> 10

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

ctatcggcta ttaaaccact tctatcaaac 30

Claims

Application of Sdal as a target in preparing vaccines for preventing pathogenic bacteria infection, reagents for detecting pathogenic bacteria and medicines for preventing and/or treating diseases caused by pathogenic bacteria infection;

the pathogenic bacteria comprise: inhibiting a pathogenic bacterium that expresses Sdal and/or a DNase homologous thereto;

the infection comprises: infection of the human mucosal system.
2. The use of claim 1, wherein the pathogenic bacteria comprise gram-positive pathogenic bacteria;

the gram-positive pathogenic bacteria comprise one or more of streptococcus, staphylococcus aureus and pneumococcus;

the streptococcus comprises one or more of group A streptococcus, group B streptococcus, group C streptococcus, group G streptococcus, streptococcus pneumoniae, streptococcus suis or streptococcus equi;

the human mucosal system comprises one or more of the respiratory system, the digestive system, the urinary system, the urogenital system, or the skin.
3. Recombinant protein mu-Sdal, characterized in that it has:

(I) The amino acid residue from the 3 rd to the 354 th positions of the N tail end of the amino acid sequence shown as SEQ ID No. 6; or

(II) an amino acid sequence obtained by substituting, deleting or adding one or two amino acid residues in the amino acid sequence shown in (I), and the amino acid sequence has the same or similar functions with the amino acid sequence shown in (I); or

(III) an amino acid sequence which has at least 90% sequence identity with the sequence described in (I) or (II) and which is functionally identical or similar to the amino acid sequence shown in (I).
4. The recombinant protein mu-Sdal according to claim 3, characterized in that it has:

(I) And an amino acid sequence shown as SEQ ID No. 6; or

(II) an amino acid sequence obtained by substituting, deleting or adding one or two amino acid residues in the amino acid sequence shown in (I), and the amino acid sequence has the same or similar functions with the amino acid sequence shown in (I); or

(III) an amino acid sequence which has at least 90% sequence identity with the sequence described in (I) or (II) and which is functionally identical or similar to the amino acid sequence shown in (I).
5. Nucleic acid molecule encoding the recombinant protein mu-Sdal according to claim 3 or 4, having

(I) 7 bp-1062 bp of the nucleotide sequence shown as SEQ ID No.5 or

(II) a complementary nucleotide sequence of 7 bp-1062 bp of the nucleotide sequence shown as SEQ ID No. 5; or

(III) a nucleotide sequence which encodes the same protein as the nucleotide sequence of (I) or (II) but which differs from the nucleotide sequence of (I) or (II) due to the degeneracy of the genetic code; or

(IV) a nucleotide sequence obtained by substituting, deleting or adding one or two nucleotide sequences with the nucleotide sequence shown in (I), (II) or (III), and the nucleotide sequence has the same or similar functions with the nucleotide sequence shown in (I), (II) or (III); or

(V) a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence of (I), (II), (III) or (IV).
6. A recombinant vector comprising the nucleic acid molecule of claim 5.
7. A host comprising the recombinant vector of claim 6.
8. The host of claim 7, wherein the host comprises one or more of E.coli, yeast, or mammalian cells.
9. Use of the recombinant protein mu-Sdal according to claim 3 or 4 for the preparation of a vaccine for the prevention of pathogenic bacterial infection, a reagent for the detection of pathogenic bacteria, a medicament for the prevention and/or treatment of diseases caused by pathogenic bacterial infection;

the pathogenic bacteria comprise: inhibiting a pathogenic bacterium that expresses Sdal and/or a DNase homologous thereto;

the infection comprises: infection of the human mucosal system.
10. The use of claim 9, wherein the pathogenic bacteria comprise gram-positive pathogenic bacteria;

the gram-positive pathogenic bacteria comprise one or more of streptococcus, staphylococcus aureus and pneumococcus;

the streptococcus comprises one or more of group A streptococcus, group B streptococcus, group C streptococcus, group G streptococcus, streptococcus pneumoniae, streptococcus suis or streptococcus equi;

the human mucosal system comprises one or more of the respiratory system, the digestive system, the urinary system, the urogenital system, or the skin.
11. Vaccine comprising the recombinant protein mu-Sdal according to claim 3 or 4 and an acceptable adjuvant.
12. The vaccine of claim 11, wherein said adjuvant comprises CpG.
13. The vaccine of claim 12, wherein the mass ratio of the recombinant protein mu-Sdal to CpG is 1:1.
14. the vaccine of claims 11-13, wherein the vaccine is administered by nasal inhalation, orally, subcutaneously, intradermally, urogenital, or intraanal injection.
15. An antibody obtained by immunizing an animal with the vaccine of claims 11-14.
16. A medicament comprising the antibody of claim 15 and a pharmaceutically acceptable excipient.
17. A detection reagent or a detection kit comprising the antibody of claim 15 and a detectably acceptable auxiliary.