CN111647569A - AT-CoaR6 fusion protein and application thereof in preparation of dual-component vaccine for resisting staphylococcus aureus infection - Google Patents

Abstract

The invention discloses a fusion protein consisting of a recombinant R structural domain protein CoaR6 of staphylococcus aureus coagulase, a single-site mutant AT of staphylococcus aureus alpha-hemolysin H35L and a recombinant R structural domain CoaR6, and application of the fusion protein in a bi-component vaccine for resisting staphylococcus aureus infection. The CoaR6 is formed by combining 6 repeated fragments derived from different clinical strains, and has high amino acid sequence consistency with Chinese epidemic strains ST239 and ST 5. The fusion protein prepared by the invention can obviously improve the immunogenicity of CoaR6, induce a mouse to generate high-level AT-resistant specific antibody and neutralizing antibody, improve the opsonophagocytosis of whole blood of the mouse to two methicillin-resistant staphylococcus aureus strains, and resist the death of the mouse caused by lethal dose of staphylococcus aureus.

Description

AT-CoaR6 fusion protein and application thereof in preparation of dual-component vaccine for resisting staphylococcus aureus infection

Technical Field

The invention discloses a fusion protein, and belongs to the technical field of polypeptide and vaccine application.

Background

Staphylococcus aureus (s. aureus) is a common gram-positive bacterium that usually inhabits the nasal cavity, skin and mucous membranes of a host and does not show symptoms of infection. Researches show that the rate of staphylococcus aureus nasal colonization in normal population can reach 30%. Meanwhile, staphylococcus aureus is a main pathogenic bacterium in various infectious diseases, including local infectious diseases such as skin and soft tissue infection, peritonitis, pneumonia and the like, and systemic infectious diseases such as bacteremia, septicemia, sepsis and the like. In recent years, the infectious diseases caused by staphylococcus aureus reach 16-21% of clinical infectious diseases. Statistics of infectious diseases treated with antibiotics in the U.S. hospital show that nearly 11% of infections and over 75,000 cases of infections are caused by staphylococcus aureus infections. In the statistics of nosocomial infectious diseases at the european centers for disease prevention and control, nearly 5,400 deaths from infection were also associated with drug-resistant staphylococcus aureus infections. Since the use of methicillin for the treatment of infectious diseases, methicillin-resistant Staphylococcus aureus has been rapidly and in ever increasing proportions transmitted in hospitals and communities. At present, the main means for treating staphylococcus aureus is still to use antibiotics, but with the continuous expansion of the infection range of drug-resistant strains, the continuous increase of drug-resistant varieties and the continuous increase of the complexity of staphylococcus aureus treatment, and the use of antibiotics can not provide the body with the capability of resisting reinfection. Therefore, the development of safe and effective staphylococcus aureus vaccines is significant for preventing and treating staphylococcus aureus infection.

Currently, three staphylococcus aureus candidate vaccines are clinically evaluated on large-scale effectiveness. The first is StaphVAX developed by Naphi Pharmaceuticals, which is a Polysaccharide conjugate vaccine consisting of two different serotypes of Staphylococcus aureus Capsular Polysaccharide (CP) CP5 and CP8, and the clinical study was performed on patients with end-stage renal disease, and two efficacy evaluations were performed. While 26% effectiveness could be observed at 54 weeks of patient vaccination in the first evaluation (n-1804), only 8% effectiveness was observed in the second, scaled-up evaluation (n-3359). Therefore, the evaluation of the clinical effectiveness of the vaccine ended up with failure. The second vaccine was V710, developed by Merck, an unadjuvanted monoclonal antigen vaccine (IsdB). The clinical study was performed on patients who had to undergo an extracardiac operation, but the vaccine did not significantly reduce the bacteremia infection in the patients who underwent the operation or the staphylococcus aureus infection at the wound site after vaccination, and even a part of patients who underwent vaccination showed a higher probability of organ failure and death after staphylococcus aureus infection. Unlike the two vaccines, SA4Ag is a multicomponent vaccine comprising four antigens, namely, coagulation factor a (cifa), Manganese ion transfer receptor C (MntC), CP5 and CP 8. SA4Ag was called off in 2018 at 12 months and no relevant findings were formally published for the phase II clinical trial of this vaccine. Researchers have generally accepted that the failure of a single-target vaccine to protect against the complex pathogenic mechanisms of staphylococcus aureus and inappropriate clinical subjects are potential causes of candidate vaccine failure.

At present, a plurality of candidate vaccines are in a clinical test stage, for example, a 4-component vaccine developed by GSK company has completed the clinical I-stage evaluation, an rLukS-PV/rAT two-component vaccine constructed by Nabi company has completed the clinical I-stage evaluation, a recombinant protein vaccine developed by the third-military medical science in China has entered the II-stage clinical test, and the like. However, no staphylococcus aureus vaccine is approved to be on the market so far, and with the continuous and deep research on the pathogenic mechanism of staphylococcus aureus, new pathogenic mechanism is continuously discovered, and the research on new targets still has important significance on the design of multi-component vaccines.

Although the staphylococcus aureus Coagulase (Coa) is not a newly discovered virulence factor, the action mechanism of the Coa Coagulase in the pathogenic process of the staphylococcus aureus is only clearly elucidated recently, and no staphylococcus aureus candidate vaccine contains Coa related components at present. Coa comprises 4 main domains, a signal peptide sequence of 26 amino acids, a D1-D2 domain at the N terminal, a central L domain and a repetitive fragment R domain at the C terminal. The D1-D2 structural domain can be combined with prothrombin in blood to form an active compound, the compound can cut fibrinogen to form insoluble fibrin monomer, the Coa R structural domain can be combined with the fibrin monomer and enriched around staphylococcus aureus thalli, and identification and killing of host cells escaping from the thalli are further protected. The D1-D2 domains are highly variable across strains, and antibodies directed against the D1-D2 domains from a single strain source are not cross-protected across strains of different Coa serotypes. The R domain is a potential vaccine target, and the monoclonal antibody 3B3 has wide protection among different strains. However, research shows that the recombinant protein has weak immunogenicity, and the immunogenicity needs to be improved so as to discuss the feasibility of the recombinant protein as a staphylococcus aureus vaccine target.

Alpha-toxin is an exotoxin secreted by staphylococcus aureus, one of the major members of the sclerostin family of proteins of staphylococcus aureus, which is capable of lysing erythrocytes to produce hemolysis. Studies have shown that mutation of amino acid 35 can render it non-toxic and that immunization of mice with the mutant can generate neutralizing antibodies against α -toxin. At present, a plurality of monoclonal antibodies aiming at alpha-toxin enter clinical tests, which indicates that the alpha-toxin is a potential vaccine target.

Therefore, the invention aims to provide a fusion protein composed of a staphylococcus aureus coagulase R structural domain recombinant protein CoaR6, a staphylococcus aureus alpha-hemolysin H35L single-site mutant AT and the recombinant R structural domain CoaR6, and application of the fusion protein in a two-component vaccine for resisting staphylococcus aureus infection.

Disclosure of Invention

Based on the above purpose, the invention firstly provides a staphylococcus aureus coagulase R structural domain recombinant protein, and the amino acid sequence of the recombinant protein is shown as SEQ ID NO. 2.

Secondly, the invention also provides a polynucleotide for coding the recombinant protein, and the sequence of the polynucleotide is shown as SEQ ID NO. 3.

Thirdly, the invention provides a fusion protein containing the recombinant protein, and the fusion protein also contains staphylococcus aureus alpha-toxin.

In a preferred technical scheme, the amino acid sequence of the staphylococcus aureus alpha-toxin is shown as SEQ ID No. 1.

More preferably, the α -toxin is located at the amino terminus of the fusion protein.

More preferably, the α -toxin is linked to the recombinant protein of the R domain of staphylococcus aureus coagulase with a G4S linker.

Fourthly, the invention provides a polynucleotide for coding the fusion protein, and the sequence of the polynucleotide is shown as SEQ ID NO. 4.

Fifth, the invention also provides an expression vector containing the polynucleotide, wherein the vector is pET21a + -AT-CoaR 6.

Sixth, the present invention provides a host cell comprising the above expression vector, wherein the host cell is Escherichia coli BL21(DE 3).

Finally, the invention provides the application of the fusion protein in preparing a vaccine for resisting staphylococcus aureus infection.

The R structure domain CoaR6 has a certain amount of B cell epitopes capable of being identified by B cells to activate downstream immune reaction, wherein partial peptide segments have higher affinity to MHC-I molecules⁵The anti-AT IgG titer was 1.43 × 10 respectively⁶anti-CoaR 6IgG titre 2.55 × 10³. The results indicate that AT-CoaR6 has good immunogenicity. In the MRSA252 intraperitoneal infection model, the survival rate of the AT-CoaR6 immune group after 7 days of challenge was 70% respectively, while the PBS group died the whole number of mice 24h after challenge (FIG. 5A). In the USA300 abdominal cavity infection model, the survival rates of the AT-CoaR6 immune group mice 7 days after challenge were 100% respectively; the PBS group mice also all died within 24h after challenge (fig. 5B). Mice immunized with AT-CoaR6 still had a 50% survival rate 10 days after MRSA252 tail vein infection, significantly higher than that of PBS control group (p)<0.05) (fig. 6A). In the USA300 tail vein infection model, although both groups of mice died within 10 days after challenge, it was still observed that the immunization of AT-CoaR6 significantly prolonged the survival time of the mice (p)<0.01). Compared with the PBS group, 90% of the mice died within 1 day after challenge; in most of the mice immunized with AT-CoaR6, the onset and death of disease occurred from day 3 (FIG. 6B). The above results indicate that the two-component vaccine AT-CoaR6 is a potential vaccine candidate for the prevention of Staphylococcus aureus infection.

Drawings

FIG. 1 is a graph of the output of the prediction scoring results for CoaR6B cell epitopes;

FIG. 2 SDS-PAGE identifies an electrophoretogram of expression conditions for pET32a + -AT-CoaR 6;

FIG. 3 electrophoretograms of different purification conditions of AT-CoaR6 recombinant protein;

FIG. 4 is a graph comparing the levels of specific antibodies in immunized mice;

FIG. 5 is a graph of survival analysis of AT-CoaR6 immunized mice after intraperitoneal infection of MRSA252 and USA 300;

FIG. 6 graph of survival analysis of AT-CoaR6 immunized mice after systemic infection with MRSA252 and USA 300.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are only illustrative and do not limit the scope of protection of the invention as defined by the claims.

Example 1 preparation of AT-CoaR6 fusion protein

1. Clinical strain R structural domain amino acid sequence analysis

(1) Clinical strain genome DNA extraction: 43 staphylococcus aureus clinical strains were isolated from pneumonia patients, 2 methicillin-resistant staphylococcus aureus strains were purchased from ATCC, and genomic DNA was extracted using Wizard whole gene extraction kit.

(2) The R domain gene was PCR amplified with the amplification primers shown in Table 1:

TABLE 1 primers for gene identification of each domain

(3) The amplification product is sent to Shanghai Biotechnology limited to purify and sequence.

(4) Analyzing a sequencing result by Geneius software, and respectively intercepting repeated fragments in each R structural domain to form a sequence file taking the repeated fragments of 27 amino acids as a unit; and (3) introducing the sequence into Excel software to count the sequence types of the repeated fragments and the occurrence frequency of each repeated fragment. The analysis results of the repeated segments are shown in Table 2, wherein the 258 repeated segments contain 22 sequence types in total, and the proportion of the sequence types with the highest occurrence frequency can reach 32.2. The number of repeats contained in the R domain of the clinical strain is shown in table 3, with 6 repeats being the most common form.

TABLE 2 frequency statistics of each repeated segment of clinical strains

TABLE 3 statistics of the number of repeat segments contained in the R domain of clinical strains

(5) The first 6 kinds of repeated fragments with the highest occurrence probability are selected to be recombined into a new R structural domain, and the amino acid sequence of the R structural domain is shown by SEQ ID NO. 2. The conversion from the amino acid sequence to the nucleotide sequence of the CoaR6 sequence is carried out by using Jcat software, and codon optimization is carried out by using E.Coli as a host, wherein the nucleotide sequence is shown by SEQ ID NO. 3.

2. Prediction of CoaR6 protein T, B epitope

The reason for the weak immunogenicity of the R domain was analyzed by predicting the T, B epitope of CoaR6 using online software. Importing the CoaR6 sequence file saved in FASTA format into the IEDB database B cell prediction website: (http:// tools.immuneepitope.org/bcell/). The Bepipred Linear Event Prediction (BLEP) method was chosen for Prediction, and the threshold was chosen to be 1.2. Fragments with a predictive value above the threshold are considered as potential B cell epitopes. The prediction of T cell epitopes using the IEDB database is divided into: MHC class II molecules (http:// tools.immuneepitope.org/mhcii/) Prediction of binding capacity of (c); MHC class I molecules (http:// tools.immuneepitope.org/mhci/) And (4) predicting the binding capacity. Higher results obtained from this prediction indicate higher affinity of the test antigen for binding to the MHC molecule.

(1) B cell epitope results as shown in figure 1, the middle horizontal line in figure 1 represents the set threshold, the presence of 9 epitopes above the threshold in this prediction method by CoaR6 and the key amino acids for each epitope are listed in table 4. This result indicates that CoaR6 has a certain number of B cell epitopes that B cells recognize to activate downstream immune responses.

TABLE 4 prediction of amino acid sequences for CoaR6B cell epitopes

(2) Prediction of the ability of CoaR6 to bind to MHC class II molecules, the top 10 peptide fragments in the prediction are listed in Table 5, and the percent Rank value of the first peptide fragment in CoaR6 is 29.27 (Balb/c). The MHC molecules have polymorphism in different inbred line mice, and the invention predicts the MHC molecule binding capacity of the CoaR6 and Balb/C and C57BL/6 inbred line mice respectively. The MHC molecules of Balb/C and C57BL/6 inbred mice are encoded by the H2-IAd and H2-IAb genes, respectively. The higher the Percentage Rank, the weaker the binding ability of the antigen peptide to MHC class II molecules. This result indicates that the peptide fragments contained in the CoaR6 belong to antigenic peptides with low affinity to MHC-class II molecules, and may result in the failure of the CoaR6 to effectively bind to MHC-class II molecules. The effective combination with MHC-II molecules is a key link for inducing the organism to generate specific immune response by the recombinant protein vaccine. Failure of the CoaR6 to bind effectively to MHC class II molecules may result in failure of the candidate vaccine to induce active immunity in the body and thus failure to exert anti-infective activity.

(3) Corresponding predictions were made for the ability of CoaR6 to bind to MHC-I molecules, and the top 10 peptides are shown in Table 6, where the top peptide percent Percentage Rank score is 0.6, indicating that CoaR6 has a high affinity for MHC-I molecules in part.

TABLE 5 prediction of CoaR6 binding Capacity to MHC class II molecules

TABLE 6 prediction of CoaR6 binding Capacity to MHC class I molecules

Selection of forms of presentation of AT and CoaR6 proteins

The fusion carrier protein is one of ways of improving immunogenicity of polypeptide without T cell epitope or weak binding ability with MHC-II molecules, and the fusion protein has the characteristics of simple preparation and single product, the invention selects CoaR6 and AT to carry out fusion expression, analyzes the corresponding relation of α -hemolysin protein structure and physiological function thereof, and has already researched and shown that α -hemolysin can form heptamer on cell membrane, wherein N end is wrapped in the interior of heptamer pore-shaped structure and is responsible for forming 'amino lock' to stabilize heptamer structure, while C end is free outside heptamer and is not involved in the exertion of α -toxin function, therefore, we select CoaR6 to be connected with C end of AT between AT and CoaR6₄S Linker, the amino acid sequence of AT is shown by SEQ ID NO.1, and the nucleotide sequence of AT-CoaR6 fusion protein is shown by SEQ ID NO. 4.

Expression and purification of AT-CoaR6 fusion protein

(1) Amplification of the AT-CoaR6 gene: AT and CoaR6 genes were amplified for overlap extension PCR using pET28a + -AT and pET32a + -CoaR6 as templates, respectively. pET28a + -AT plasmid containing AT gene was constructed and stored for the early stages of this laboratory. The overlap extension PCR primers are shown in Table 7.

TABLE 7 AT-CoaR6 overlap extension PCR primers

(2) Construction of AT-CoaR6 expression plasmid: the fragment of the AT-CoaR6 gene obtained by PCR amplification is subjected to enzyme digestion, the NheI and XhoI enzyme digestion sites are exposed, the AT-CoaR6 gene is connected with an expression vector pET21a +, and an expression plasmid pET21a + -AT-CoaR6 is constructed.

(3) Construction and expression of BL21-pET21a + -AT-CoaR6 expression engineering bacteria: pET21a + -AT-CoaR6 expression plasmid was transformed into BL21(DE3) competent cells. The AT-CoaR6 protein contains 471 amino acids in total, and the theoretical molecular weight of the AT-CoaR6 protein is 52.4 kD. The plasmid pE32a + -AT-CoaR6 with the correct construction was transformed into BL21(DE3) competent strain for a small sample expression. Expressing for 4h under inducing conditions of 37 ℃ and IPTG concentration of 0.5. mu.M under temperature condition, performing SDS-PAGE to identify the whole bacteria, supernatant and precipitate of the expression lysate to obtain the expression location and expression amount of AT-CoaR 6. As a result, as shown in FIG. 2, under the expression condition, a large amount of protein expression can be detected AT 55kD, which is consistent with the theoretical molecular weight of AT-CoaR6 and has a high expression level. Part of the recombinant protein present in the lysed supernatant was expressed as soluble intracellular expression. In FIG. 2, M, protein Marker; 1,4,7: BL21-pET32a + -AT-CoaR6 does not induce the expression of the whole bacteria, supernatant and sediment; 2,5,8: BL21-pET32a + -AT-CoaR6 induces the whole bacteria, the supernatant and the precipitate of the expression under the condition of 37 ℃; 3,6,9: BL21-pET32a + -AT-CoaR6 induces the expression of the whole bacteria, the supernatant and the precipitate AT the temperature of 28 ℃.

(4) And (3) prokaryotic expression and purification of AT-CoaR 6: BL21-pET21a + -AT-CoaR6 was expressed by IPTG induction, and intracellular soluble AT-CoaR6 was purified by first purifying it with Ni column using its His-tagged C-terminus, and the purification effect was analyzed by SDS-PAGE. As shown in A of FIG. 3, the protein band before and after the column shows that AT-CoaR6 binds well to Ni, and part of the impurity protein can be removed by gradient elution. Subsequently, since AT-CoaR6 predicts a theoretical isoelectric point of 9.20, the crude pure product of the first step is further separated in the second step using cation exchange chromatography. The second purification step, shown in panel B of FIG. 3, is better able to remove impure proteins by cation exchange chromatography. In FIG. 3, (A) purification of the AT-CoaR6 recombinant protein Ni column; 1, before column; 2, post-column; 3-8: collecting peaks; (B) purifying the AT-CoaR6 recombinant protein SPHP column; 1, before column; 2, post-column; 3-8: the peaks were collected.

Example 2 evaluation of immunogenicity of AT-CoaR6

1. Immunization of mice

6-8 week-old SPF-grade Balb/c female mice were used to assess the immunogenicity of AT-CoaR6, the mice were randomly divided into 2 groups of 8 mice each immunized with 25. mu.g of AT-CoaR6 recombinant protein and PBS, and the adjuvant used was Al (OH)₃And (4) a composite adjuvant mixed with CpG. Respectively at 0, 14 and 2Intramuscular injection was performed for 8 days.

Determination of specific antibody levels by ELISA method

Sera from immunized mice were taken AT day 42 and the anti-AT and anti-CoaR 6IgG levels in the sera from the mice were determined by ELISA. The anti-CoaR 6IgG detection ELISA method was as follows:

coating: the recombinant protein CoaR6 antigen was diluted to a final concentration of 2. mu.g/mL with ELISA coating solution, added to a 96-well plate in a volume of 100. mu.L per well, and left overnight at 4 ℃;

(a) washing: discarding the coating solution, and washing the 96-well plate by using a plate washing machine, wherein the detergent is PBST;

(b) and (3) sealing: add 200. mu.L ELISA blocking solution (2% BSA + PBST) to each well, and leave at 37 ℃ for 1 h;

(c) washing: removing the sealing liquid, and washing the 96-well plate by using a plate washing machine;

(d) adding the serum to be detected: first well 200. mu.L of ELISA diluent (0.2% BSA + PBST) was added and the remaining wells 100. mu.L of ELISA diluent. Adding 4 mu L of serum to be detected into the first hole, namely diluting the serum by 50 times, then carrying out double dilution, adding negative serum into each plate as a control, and placing the plate in a constant-temperature incubator at 37 ℃ for 1h after the serum is diluted;

(e) washing: discarding serum diluent in the 96-well plate, and washing the 96-well plate by using a plate washing machine;

(f) adding a secondary antibody: diluting the secondary antibody (Anti-Mouse IgG-HRP) in an ELISA diluent at a dilution ratio of 1:10000, adding 100 mu L of the secondary antibody into each hole, and placing the secondary antibody in a constant-temperature incubator at 37 ℃ for 1 h;

(g) washing: discarding the secondary antibody, and washing the 96-well plate by using a plate washing machine;

(h) color development: adding 100 μ L of TMB single-component color developing solution into each well, placing in dark for developing for about 3min, adding 50 μ L of LISA stop solution, and measuring double-wavelength OD with microplate reader₄₅₀-OD₆₃₀。

The anti-AT IgG detection ELISA method is used for coating AT recombinant protein with the antigen of 2 mug/mL, the first hole dilution of serum detection is 1:1000, and the other steps are the same as the above steps.

Antibody levels against AT-CoaR6 in sera of immunized mice were determined by ELISAThe detection result is shown in figure 4, and the AT-CoaR6 can stimulate the generation of anti-AT-CoaR 6IgG titer of 9.31 × 10 by immunizing mice under the action of Al + CpG adjuvant⁵The anti-AT IgG titer was 1.43 × 10 respectively⁶anti-CoaR 6IgG titre 2.55 × 10³. The results indicate that AT-CoaR6 has good immunogenicity.

Example 3 assessment of AT-CoaR6 Immunoprotective

1. Immunization of mice

6-8 week-old SPF-grade Balb/c female mice were used to evaluate the immunogenicity of AT-CoaR6, the mice were randomly divided into 3 groups of 10 mice each, immunized with 25. mu.g of AT-CoaR6 recombinant protein and PBS, and adjuvanted with Al (OH)₃And (4) a composite adjuvant mixed with CpG. Intramuscular injections were performed on days 0, 14, and 28, respectively, and the challenge was performed 38 days after the third immunization.

Active immunization against lethal dose of Staphylococcus aureus in peritoneal cavity with AT-CoaR6

(a) Recovering the frozen strains: two methicillin-resistant staphylococcus aureus USA300FPR3757 (BAA-1556)^TM) And MRSA252 (BAA-1720)^TM) Purchased from American Type culture Strain resources (ATCC) and frozen at-80 ℃. The frozen MRSA252 and the USA300 strain are streaked on a TSA plate and cultured for 18 to 24 hours at 37 ℃;

(b) MRSA252 and USA300 are picked to be monocloned in a TSB culture medium, and cultured overnight at 37 ℃ and 220rpm until saturation;

(c) transferring the overnight culture bacterial suspension 1:100 into a new TSB culture medium, and culturing the bacterial suspension at 37 ℃ and 220rpm to ensure that the OD600 is about 0.8;

(d) centrifuging at 6500rpm for 15min to collect thallus;

(e) removing the culture medium supernatant, resuspending the thallus with PBS to wash the thallus, and repeating twice;

(f) the concentration of MRSA252 was adjusted to 2 × 10¹⁰CFUs/ml, USA300 concentration adjusted to 1 × 10¹⁰Injecting CFUs/ml into abdominal cavity, wherein 5 mice are totally injected into groups with different concentrations, and 100 mu L of bacterial suspension is injected into the abdominal cavity of each mouse;

(g) the survival rate of the mice was observed.

In the MRSA252 intraperitoneal infection model, the survival rate of the AT-CoaR6 immune group after 7 days of challenge was 70% respectively, while the PBS group died the whole number of mice 24h after challenge (A in FIG. 5). In the USA300 abdominal cavity infection model, the survival rates of the AT-CoaR6 immune group mice 7 days after challenge were 100% respectively; the PBS group mice also all died within 24h after challenge (fig. 5B).

Active immunization against lethal doses of Staphylococcus aureus systemic infection with AT-CoaR6

(a) Recovering the frozen strains: the frozen MRSA252 and the USA300 strain are streaked on a TSA plate and cultured for 18 to 24 hours at 37 ℃;

(b) MRSA252 and USA300 are picked to be monocloned in a TSB culture medium, and cultured overnight at 37 ℃ and 220rpm until saturation;

(c) transferring the overnight culture bacterial suspension 1:100 into a new TSB culture medium, and culturing the bacterial suspension at 37 ℃ and 220rpm to ensure that the OD600 is about 0.8;

(d) centrifuging at 6500rpm for 15min to collect thallus;

(e) removing the culture medium supernatant, resuspending the thallus with PBS to wash the thallus, and repeating twice;

(f) the concentrations of MRSA252 and USA300 were both adjusted to 4 × 10⁹CFUs per ml, tail vein injection is carried out respectively, and 100 mu L of bacterial suspension is injected into the abdominal cavity of each mouse;

(g) the survival rate of the mice was observed.

Mice immunized with AT-CoaR6 still had 50% survival rate 10 days after MRSA252 tail vein infection, significantly higher than the PBS control group (p <0.05) (a in fig. 6). In the USA300 tail vein infection model, although both groups of mice died entirely within 10 days after challenge, it was still observed that the immunized AT-CoaR6 was able to significantly prolong the survival time of the mice (p < 0.01). Compared with the PBS group, 90% of the mice died within 1 day after challenge; in most of the mice immunized with AT-CoaR6, the onset and death of disease occurred from day 3 (FIG. 6, panel B). The above results indicate that the two-component vaccine AT-CoaR6 is a potential candidate vaccine against Staphylococcus aureus infection.

Sequence listing

<110> military medical research institute of military science institute of people's liberation force of China

<120> AT-CoaR6 fusion protein and application thereof in preparation of two-component vaccine for resisting staphylococcus aureus infection

<160>4

<170>SIPOSequenceListing 1.0

<210>1

<211>296

<212>PRT

<213> Staphylococcus aureus (Staphylococcus aureus)

<400>1

Met Ala Ser Ala Asp Ser Asp Ile Asn Ile Lys Thr Gly Thr Thr Asp

1 5 10 15

Ile Gly Ser Asn Thr Thr Val Lys Thr Gly Asp Leu Val Thr Tyr Asp

20 25 30

Lys Glu Asn Gly Met Leu Lys Lys Val Phe Tyr Ser Phe Ile Asp Asp

35 40 45

Lys Asn His Asn Lys Lys Leu Leu Val Ile Arg Thr Lys Gly Thr Ile

50 55 60

Ala Gly Gln Tyr Arg Val Tyr Ser Glu Glu Gly Ala Asn Lys Ser Gly

65 70 75 80

Leu Ala Trp Pro Ser Ala Phe Lys Val Gln Leu Gln Leu Pro Asp Asn

85 90 95

Glu Val Ala Gln Ile Ser Asp Tyr Tyr Pro Arg Asn Ser Ile Asp Thr

100 105 110

Lys Glu Tyr Met Ser Thr Leu Thr Tyr Gly Phe Asn Gly Asn Val Thr

115 120 125

Gly Asp Asp Thr Gly Lys Ile Gly Gly Leu Ile Gly Ala Asn Val Ser

130 135 140

Ile Gly His Thr Leu Lys Tyr Val Gln Pro Asp Phe Lys Thr Ile Leu

145 150 155 160

Glu Ser Pro Thr Asp Lys Lys Val Gly Trp Lys Val Ile Phe Asn Asn

165 170 175

Met Val Asn Gln Asn Trp Gly Pro Tyr Asp Arg Asp Ser Trp Asn Pro

180 185 190

Val Tyr Gly Asn Gln Leu Phe Met Lys Thr Arg Asn Gly Ser Met Lys

195 200 205

Ala Ala Asp Asn Phe Leu Asp Pro Asn Lys Ala Ser Ser Leu Leu Ser

210 215 220

Ser Gly Phe Ser Pro Asp Phe Ala Thr Val Ile Thr Met Asp Arg Lys

225 230 235 240

Ala Ser Lys Gln Gln Thr Asn Ile Asp Val Ile Tyr Glu Arg Val Arg

245 250 255

Asp Asp Tyr Gln Leu His Trp Thr Ser Thr Asn Trp Lys Gly Thr Asn

260 265 270

Thr Lys Asp Lys Trp Ile Asp Arg Ser Ser Glu Arg Tyr Lys Ile Asp

275 280 285

Trp Glu Lys Glu Glu Met Thr Asn

290 295

<210>2

<211>170

<212>PRT

<213> Staphylococcus aureus (Staphylococcus aureus)

<400>2

Ala Arg Pro Arg Phe Asn Lys Pro Ser Glu Thr Asn Ala Tyr Asn Val

1 5 10 15

Thr Thr Asn Gln Asp Gly Thr Val Ser Tyr Gly Ala Arg Pro Thr Gln

20 25 30

Asn Lys Pro Ser Lys Thr Asn Ala Tyr Asn Val Thr Thr His Ala Asn

35 40 45

Gly Gln Val Ser Tyr Gly Ala Arg Pro Thr Tyr Lys Lys Pro Ser Glu

50 55 60

Thr Asn Ala Tyr Asn Val Thr Thr Asn Gln Asp Gly Thr Val Ser Tyr

65 70 75 80

Gly Ala Arg Pro Thr Tyr Lys Lys Pro Ser Glu Thr Asn Ala Tyr Asn

85 9095

Val Thr Thr His Ala Asn Gly Gln Val Ser Tyr Gly Ala Arg Pro Thr

100 105 110

Gln Asn Lys Pro Ser Glu Thr Asn Ala Tyr Asn Val Thr Thr His Ala

115 120 125

Asn Gly Gln Val Ser Tyr Gly Ala Arg Pro Thr Gln Asn Lys Pro Ser

130 135 140

Lys Thr Asn Ala Tyr Asn Val Thr Thr His Ala Asp Gly Thr Ala Thr

145 150 155 160

Tyr Gly Leu Glu His His His His His His

165 170

<210>3

<211>553

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

ctagaataat tttgtttaac tttaagaagg agatatacat atggcacgcc cgcgctttaa 60

caagccgagc gagaccaacg cctacaatgt gaccaccaac caagacggta ccgtgagcta 120

tggtgcccgc ccgacccaaa acaaaccgag caagaccaat gcctacaacg tgaccaccca 180

tgccaatggc caagtgagct acggtgcccg cccgacctat aaaaagccga gcgagaccaa 240

tgcctacaat gtgaccacca atcaggatgg taccgtgagt tatggtgccc gcccgaccta 300

caaaaaaccg agcgaaacca atgcctacaa cgtgaccacc cacgccaatg gtcaggtgag 360

ctacggtgcc cgtccgaccc aaaataagcc gagcgaaacc aatgcctaca atgtgaccac 420

ccatgccaac ggtcaggttt cttacggtgc ccgcccgaca cagaataagc cgagcaagac 480

caatgcctac aatgtgacca cccatgcaga tggcaccgcc acctacggtc tcgagcacca 540

ccaccaccac cac 553

<210>4

<211>1413

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

atggctagcg cagattctga tattaatatt aaaaccggta ctacagatat tggaagcaat 60

actacagtaa aaacaggtga tttagtcact tatgataaag aaaatggcat gctgaaaaaa 120

gtattttata gttttatcga tgataaaaat cataataaaa aactgctagt tattagaacg 180

aaaggtacca ttgctggtca atatagagtt tatagcgaag aaggtgctaa caaaagtggt 240

ttagcctggc cttcagcctt taaggtacag ttgcaactac ctgataatga agtagctcaa 300

atatctgatt actatccaag aaattcgatt gatacaaaag agtatatgag tactttaact 360

tatggattca acggtaatgt tactggtgat gatacaggaa aaattggcgg ccttattggt 420

gcaaatgttt cgattggtca tacactgaaa tatgttcaac ctgatttcaa aacaatttta 480

gagagcccaa ctgataaaaa agtaggctgg aaagtgatat ttaacaatat ggtgaatcaa 540

aattggggac catatgatag agattcttgg aacccggtat atggcaatca acttttcatg 600

aaaactagaa atggctctat gaaagcagca gataacttcc ttgatcctaa caaagcaagt 660

tctctattat cttcagggtt ttcaccagac ttcgctacag ttattactat ggatagaaaa 720

gcatccaaac aacaaacaaa tatagatgta atatacgaac gagttcgtga tgactaccaa 780

ttgcactgga cttcaacaaa ttggaaaggt accaatacta aagataaatg gatagatcgt 840

tcttcagaaa gatataaaat cgattgggaa aaagaagaaa tgacaaatgg tggtggtggt 900

tctgcacgcc cgcgctttaa caagccgagc gagaccaacg cctacaatgt gaccaccaac 960

caagacggta ccgtgagcta tggtgcccgc ccgacccaaa acaaaccgag caagaccaat 1020

gcctacaacg tgaccaccca tgccaatggc caagtgagct acggtgcccg cccgacctat 1080

aaaaagccga gcgagaccaa tgcctacaat gtgaccacca atcaggatgg taccgtgagt 1140

tatggtgccc gcccgaccta caaaaaaccg agcgaaacca atgcctacaa cgtgaccacc 1200

cacgccaatg gtcaggtgag ctacggtgcc cgtccgaccc aaaataagcc gagcgaaacc 1260

aatgcctaca atgtgaccac ccatgccaac ggtcaggttt cttacggtgc ccgcccgaca 1320

cagaataagc cgagcaagac caatgcctac aatgtgacca cccatgcaga tggcaccgcc 1380

acctacggtc tcgagcacca ccaccaccac cac 1413

Claims (10)

1.A staphylococcus aureus coagulase R structural domain recombinant protein is characterized in that the amino acid sequence of the recombinant protein is shown in SEQ ID NO. 2.

2.A polynucleotide encoding the recombinant protein of claim 1, wherein the sequence of the polynucleotide is set forth in SEQ ID No. 3.

3.A fusion protein comprising the recombinant protein of claim 1, wherein said fusion protein further comprises s.

4. The fusion protein of claim 3, wherein the amino acid sequence of Staphylococcus aureus α -toxin is set forth in SEQ ID No. 1.

5. The fusion protein of claim 4, wherein the α -toxin is located at the amino terminus of the fusion protein.

6. The fusion protein of claim 5, wherein the α -toxin is linked to the S.aureus coagulase R domain recombinant protein with a G4S linker.

7.A polynucleotide encoding the fusion protein of claim 6, wherein the polynucleotide has the sequence shown in SEQ ID No. 4.

8. An expression vector comprising the polynucleotide of claim 7, wherein the vector is pET21a + -AT-CoaR 6.

9. A host cell comprising the expression vector of claim 8, wherein said host cell is E.coli BL21(DE 3).

10. Use of the fusion protein of any one of claims 3-6 for the preparation of a vaccine against s.

Images