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 Staphylococcus aureus coagulase recombinant R domain protein CoaR6, a fusion protein composed of Staphylococcus aureus α-hemolysin H35L single point mutant AT and the recombinant R domain CoaR6, and the fusion protein Use in a two-component vaccine against Staphylococcus aureus infection. The CoaR6 is composed of 6 repeated fragments derived from different clinical strains, and has a high amino acid sequence identity with the popular Chinese strains ST239 and ST5. The fusion protein prepared by the invention can significantly improve the immunogenicity of CoaR6, induce mice to produce high-level anti-AT specific antibodies and neutralizing antibodies, and improve the immunity of mouse whole blood to two methicillin-resistant Staphylococcus aureus strains. Opsonophagy against lethal doses of Staphylococcus aureus-induced death in mice.

Description

AT-CoaR6 fusion protein and its application in the preparation of two-component vaccine against Staphylococcus aureus infection

technical field

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

Background technique

Staphylococcus aureus (S. aureus) is a common Gram-positive bacteria that usually parasitizes the host's nasal cavity, skin and mucous membranes, and this parasite does not show symptoms of infection. The study found that the proportion of Staphylococcus aureus colonization in the normal population can reach 30%. At the same time, Staphylococcus aureus is the main pathogen in a variety of infectious diseases, including local infectious diseases, such as skin and soft tissue infections, peritonitis, pneumonia, etc., and systemic infectious diseases, such as bacteremia, sepsis and sepsis. In recent years, infections caused by Staphylococcus aureus have reached 16-21% of clinical infections. A survey of infectious diseases treated with antibiotics in hospitals in the United States showed that nearly 11% of infections and more than 75,000 cases of infections were caused by Staphylococcus aureus. Nearly 5,400 deaths from infections were also associated with drug-resistant S. aureus infections in the European Centre for Disease Control and Prevention's tally of nosocomial infections. Since methicillin was used to treat infectious diseases, methicillin-resistant Staphylococcus aureus has spread rapidly and at an increasing rate in nosocomial and community settings. At present, the main method for the treatment of Staphylococcus aureus is still the use of antibiotics, but with the continuous expansion of the infection range of drug-resistant strains and the increasing number of drug-resistant species, the complexity of the treatment of Staphylococcus aureus continues to increase, and the use of antibiotics cannot The body provides the ability to resist reinfection. Therefore, the development of a safe and effective Staphylococcus aureus vaccine is of great significance for the prevention and treatment of Staphylococcus aureus infection.

At present, three candidate vaccines for Staphylococcus aureus have been clinically evaluated on a large scale. The first is StaphVAX developed by Naphi Pharmaceuticals, which is a polysaccharide conjugate vaccine composed of two different serotypes of Staphylococcus aureus capsular polysaccharide (Capsular Polysaccharide, CP) CP5 and CP8. The clinical research object is patients with end-stage renal disease , a total of two effectiveness evaluations were carried out. While 26% efficacy at 54 weeks of patient vaccination was observed in the first evaluation (n=1804), only 8% was observed in the second scaled-up evaluation (n=3359) effectiveness. Therefore, the clinical efficacy evaluation of this vaccine ended in failure. The second vaccine is V710 developed by Merck, which is an unadjuvanted single antigen vaccine (Iron-regulated surface determinant B, IsdB). The subjects of the clinical study were patients who needed to undergo cardiac surgery, but the vaccine did not significantly reduce bacteremia infection in surgical patients or S. Staphylococcus showed a higher probability of organ failure and death. Different from the above two vaccines, SA4Ag is a multi-component vaccine containing four antigens, namely Clumping factors A (ClfA), Manganese transporter C (MntC), CP5 and Multicomponent vaccine for CP8. The phase II clinical trial of SA4Ag was suspended in December 2018. For the phase II clinical trial of the vaccine, no relevant research results have been officially published. Researchers generally believe that the inability of single-target vaccines to combat the complex pathogenic mechanism of S. aureus and inappropriate clinical subjects are potential reasons for the failure of vaccine candidates.

At present, there are still several candidate vaccines in the clinical trial stage. For example, the 4-component vaccine developed by GSK has completed the clinical phase I evaluation, and the rLukS-PV/rAT dual-component vaccine constructed by Nabi has completed the clinical phase I evaluation. The recombinant protein vaccine developed by the former Third Military Medical University in China has entered Phase II clinical trials. But so far, no Staphylococcus aureus vaccine has been approved for marketing. With the continuous in-depth study of the pathogenic mechanism of Staphylococcus aureus, new pathogenic mechanisms are constantly being discovered, and the research on new targets is still of great significance for the design of multi-component vaccines. .

Although Coagulase (Coa) is not a newly discovered virulence factor, its mechanism of action in the pathogenic process of Staphylococcus aureus has only recently been clearly elucidated, and there is currently no candidate vaccine for Staphylococcus aureus containing Coa related components. Coa consists of 4 main domains, 26 amino acid signal peptide sequence, N-terminal D1-D2 domain, central L domain and C-terminal repeat R domain. The D1-D2 domains can combine with prothrombin in blood to form an active complex, which can cleave fibrinogen to form insoluble fibrin monomers, and the Coa R domain can bind to fibrin monomers and form an active complex. It is enriched around Staphylococcus aureus cells to protect the cells from escaping the recognition and killing of host cells. The D1-D2 domains are highly variable across strains, and antibodies directed against the D1-D2 domains from a single strain cannot produce cross-protection in strains of different Coa serotypes. The R domain is a potential vaccine target, and its mAb 3B3 has broad protection across different strains. However, studies have shown that its recombinant protein is weak in immunogenicity, and it is necessary to improve its immunogenicity to explore its feasibility as a target for Staphylococcus aureus vaccines.

α-toxin is an exotoxin secreted by Staphylococcus aureus and one of the main members of the Staphylococcus aureus porogen family of proteins, which can lyse red blood cells to produce hemolysis. Studies have shown that the mutation of amino acid 35 can make it lose its toxicity, and the mutant can produce neutralizing antibodies against α-toxin after immunizing mice. At present, a number of monoclonal antibodies against α-toxin have entered clinical trials, indicating that α-toxin is a potential vaccine target.

Therefore, the object of the present invention is to provide a Staphylococcus aureus coagulase R domain recombinant protein CoaR6, a fusion protein composed of Staphylococcus aureus α-hemolysin H35L single point mutant AT and the recombinant R domain CoaR6, and Application of the fusion protein in a two-component vaccine against Staphylococcus aureus infection.

SUMMARY OF THE INVENTION

Based on the above purpose, the present invention first provides a recombinant protein of the R domain of Staphylococcus aureus coagulase, and the amino acid sequence of the recombinant protein is shown in SEQ ID NO.2.

Secondly, the present invention also provides a polynucleotide encoding the above-mentioned recombinant protein, and the sequence of the polynucleotide is shown in SEQ ID NO.3.

Third, the present invention provides a fusion protein containing the above-mentioned recombinant protein, and the fusion protein also contains Staphylococcus aureus α-toxin.

In a preferred technical solution, the amino acid sequence of the Staphylococcus aureus α-toxin is shown in SEQ ID NO.1.

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

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

Fourth, the present invention provides a polynucleotide encoding the above fusion protein, and the sequence of the polynucleotide is shown in SEQ ID NO.4.

Fifth, the present invention also provides an expression vector containing the above-mentioned polynucleotide, and the vector is pET21a+-AT-CoaR6.

Sixth, the present invention provides a host cell containing the above-mentioned expression vector, and the host cell is Escherichia coli BL21(DE3).

Finally, the present invention provides the application of the above fusion protein in the preparation of a vaccine against Staphylococcus aureus infection.

The Staphylococcus aureus coagulase (Coa) R domain selected in the present invention is an artificial recombination R domain formed by recombining 6 repeating fragments with the highest frequency in clinical isolates of Staphylococcus aureus. The design ensures that The recombinant protein has broad antigenic representation. The R domain CoaR6 has a certain number of B cell epitopes that can be recognized by B cells to activate downstream immune responses, and some of the peptides have high affinity for MHC-I molecules. In the present invention, the single-site mutant AT of Staphylococcus aureus α-hemolysin H35L is fused with the R domain CoaR6, that is, AT-CoaR6 is used as a two-component vaccine, so that the immunogenicity of CoaR6 is improved, and the target for α-toxin is stimulated at the same time. specific and neutralizing antibodies. The anti-AT-CoaR6 IgG titer was 9.31×10 ⁵ , the anti-AT IgG titer was 1.43×10 ⁶ , and the anti-CoaR6 IgG titer was 2.55× 10 ³ . This result indicates that AT-CoaR6 has good immunogenicity. In the MRSA252 intraperitoneal infection model, the survival rate of mice in the AT-CoaR6 immunized group was 70% after 7 days of challenge, while the mice in the PBS group died 24 hours after challenge (Fig. 5A). In the USA300 intraperitoneal infection model, the survival rate of mice in the AT-CoaR6 immunized group was 100% after 7 days of challenge; the mice in the PBS group also all died within 24h after challenge (Fig. 5B). AT-CoaR6 immunized mice still had a 50% survival rate 10 days after MRSA252 tail vein infection, which was significantly higher than that of the 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 immunization with AT-CoaR6 could significantly prolong the survival time of mice (p<0.01). Compared with the PBS group, 90% of the mice died within 1 day after the challenge; most of the mice immunized with AT-CoaR6 started to develop morbidity and death from the third day (Fig. 6B). The above results indicate that the two-component vaccine AT-CoaR6 is a potential candidate vaccine against Staphylococcus aureus infection prevention.

Description of drawings

Figure 1. CoaR6B cell epitope prediction and scoring result output graph;

Figure 2. SDS-PAGE identification of pET32a+-AT-CoaR6 expression condition electrophoresis pattern;

Figure 3. Electrophoretic patterns of different purification conditions of AT-CoaR6 recombinant protein;

Figure 4. Control chart of specific antibody levels in immunized mice;

Figure 5. Analysis of the survival rate of mice immunized with AT-CoaR6 after intraperitoneal infection with MRSA252 and USA300;

Figure 6. Analysis of survival rate of AT-CoaR6 immunized mice after systemic infection with MRSA252 and USA300.

Detailed ways

The present invention will be further described below with reference to specific embodiments, and the advantages and characteristics of the present invention will become clearer with the description. However, these embodiments are only exemplary, and do not constitute any limitation to the protection scope defined by the claims of the present invention.

Example 1. Preparation of AT-CoaR6 fusion protein

1. Analysis of the amino acid sequence of the R domain of clinical strains

(1) Extraction of genomic DNA from clinical strains: 43 clinical strains of Staphylococcus aureus were isolated from patients with pneumonia, 2 strains of methicillin-resistant Staphylococcus aureus were purchased from ATCC, and genomic DNA was extracted by Wizard whole gene extraction kit.

(2) PCR amplification of R domain gene, amplification primers are shown in Table 1:

Table 1. Primers for gene identification of each domain

(3) The amplified product was purified and sequenced by Shanghai Sangon Biotechnology Co., Ltd.

(4) Geneious software analyzes the sequencing results, cuts out the repeating fragments in each R domain, and forms a sequence file with repeating fragments of 27 amino acids as a unit; import the sequence into Excel software to count the sequence types of the repeating fragments, and the number of occurrences of each repeating fragment. The analysis results of repetitive fragments are shown in Table 2. The 258 repetitive fragments contain a total of 22 sequence types, and the proportion of sequence types with the highest frequency can reach 32.2. The number of repeats contained in the R domain of clinical strains is shown in Table 3, and 6 repeats are the most common form.

Table 2. Statistics of the frequency of each repeating fragment of clinical strains

Table 3. Statistics of the number of repeats contained in the R domain of clinical strains

(5) The top 6 repeating fragments with the highest occurrence probability are selected and recombined into a new R domain whose amino acid sequence is shown in SEQ ID NO.2. The CoaR6 sequence was converted from amino acid sequence to nucleotide sequence by Jcat software, and codon-optimized with E. Coli as the host, the nucleotide sequence of which is shown in SEQ ID NO.3.

2. Prediction of CoaR6 protein T and B epitopes

The online software was used to predict the T and B epitopes of CoaR6, and the reasons for the weak immunogenicity of the R domain were analyzed. The CoaR6 sequence file saved in FASTA format was imported into the IEDB database B cell prediction website ( http://tools.immuneepitope.org/bcell/ ). The Bepipred Linear Epitope Prediction (BLEP) method was chosen for prediction, and the threshold was chosen as 1.2. Fragments with predicted values above the threshold were considered possible B-cell epitopes. The prediction of T cell epitopes using the IEDB database is divided into: the binding ability prediction of MHC-II molecules ( http://tools.immuneepitope.org/mhcii/ ); MHC-I molecules ( http:// tools. immuneepitope.org/mhci/ ) binding capacity prediction. The higher the result obtained by this prediction, the higher the binding affinity of the tested antigen to the MHC molecule.

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

Table 4. CoaR6 B cell epitope predicted amino acid sequence

(2) Prediction of the binding ability of CoaR6 to MHC-II molecules, the top 10 peptides in the prediction are listed in Table 5, and the Percentage Rank (Percentage Rank) value of the first peptide in CoaR6 /c). MHC molecules have polymorphisms in different inbred mice, and the present invention predicts the MHC molecule binding ability of CoaR6 and Balb/c and C57BL/6 two inbred mice respectively. The MHC molecules of Balb/c and C57BL/6 inbred mice are encoded by H2-IAd and H2-IAb gene groups, respectively. The higher the Percentage Rank, the weaker the binding ability of the antigenic peptide to MHC-II molecules. This result indicated that the peptides contained in CoaR6 belonged to antigenic peptides with low affinity with MHC-II molecules, which may lead to the inability of CoaR6 to effectively bind to MHC-II molecules. Effective binding to MHC-II molecules is a key link for recombinant protein vaccines to induce specific immune responses in the body. If CoaR6 cannot effectively bind to MHC-II molecules, the candidate vaccine may not be able to induce active immunity in the body and thus cannot perform anti-infection function.

(3) The ability of CoaR6 to bind to MHC-I molecules was also predicted accordingly. The top 10 peptides are listed in Table 6, and the top-ranked peptides have a Percentage Rank score of 0.6, which indicates the existence of CoaR6. Some peptides have high affinity with MHC-I molecules.

Table 5. Prediction of the binding ability of CoaR6 to MHC-II molecules

Table 6. Prediction of the binding ability of CoaR6 to MHC-I molecules

3. Selection of presentation forms of AT and CoaR6 proteins

Fusion carrier protein is one of the ways to improve the immunogenicity of polypeptides without T cell epitopes or with weak binding ability to MHC-II molecules. And the fusion protein has the characteristics of simple preparation and single product, the present invention chooses to fuse and express CoaR6 and AT. The corresponding relationship between the protein structure of α-hemolysin and its physiological function has been analyzed. It has been shown that α-hemolysin will form a heptamer on the cell membrane, in which the N-terminus is wrapped in the interior of the heptamer pore structure and is responsible for the formation of ""Aminolock" stabilizes the heptameric structure. The C-terminus is free outside the heptamer and does not participate in the function of α-toxin toxin. Therefore, we chose to attach CoaR6 to the C-terminus of AT. AT and CoaR6 are connected by G ₄ S Linker, the amino acid sequence of AT is shown in SEQ ID NO.1, and the nucleotide sequence of AT-CoaR6 fusion protein is shown in SEQ ID NO.4.

4. Expression and purification of AT-CoaR6 fusion protein

(1) Amplification of AT-CoaR6 gene: using pET28a+-AT and pET32a+-CoaR6 as templates to amplify AT and CoaR6 genes respectively for overlap extension PCR. The pET28a+-AT plasmid containing the AT gene was constructed and preserved in our 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 AT-CoaR6 gene fragment obtained by PCR amplification was digested, and the restriction sites NheI and XhoI were exposed, and the AT-CoaR6 gene was connected with the expression vector pET21a+ to construct pET21a+-AT- CoaR6 expression plasmid.

(3) Construction and expression of BL21-pET21a+-AT-CoaR6 expressing engineering bacteria: The pET21a+-AT-CoaR6 expression plasmid was transformed into BL21(DE3) competent cells. AT-CoaR6 protein contains a total of 471 amino acids, and its theoretical molecular weight is 52.4kD. The correctly constructed pE32a+-AT-CoaR6 plasmid was transformed into BL21(DE3) competent strain for small sample expression. Expression was performed for 4 h under the induction condition of temperature at 37°C and IPTG concentration of 0.5 μM, and SDS-PAGE was performed to identify the whole bacteria, supernatant and precipitation of the expressed lysate to obtain the expression location and expression level of AT-CoaR6. The results are shown in Figure 2. Under this 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 the expression level is high. Part of the recombinant protein in the lysis supernatant is intracellular soluble expression. In Figure 2, M, Protein Marker; 1,4,7: BL21-pET32a+-AT-CoaR6 uninduced expression of whole bacteria, supernatant and pellet; 2,5,8: BL21-pET32a+-AT- at 37°C Whole bacteria, supernatant and pellet of CoaR6-induced expression; 3, 6, 9: whole bacteria, supernatant and pellet of BL21-pET32a+-AT-CoaR6-induced expression at 28°C.

(4) Prokaryotic expression and purification of AT-CoaR6: BL21-pET21a+-AT-CoaR6 was induced and expressed by IPTG, and the intracellular soluble AT-CoaR6 was purified. First, using its C-terminal His tag, the Ni column purified, and its purification effect was analyzed by SDS-PAGE. The results are shown in Figure 3A. According to the protein bands before and after the column, AT-CoaR6 binds well to Ni, and some impurity proteins can be removed by gradient elution. Next, since the theoretical isoelectric point of AT-CoaR6 was predicted to be 9.20, the crude product of the first step was further separated in the second step by cation exchange chromatography. The purification result of the second step is shown in Figure B of 3, and the impurity protein can be better removed by cation exchange chromatography. In Figure 3, (A) AT-CoaR6 recombinant protein purified by Ni column; 1, before column; 2, after column; 3-8: collected peaks; (B) AT-CoaR6 recombinant protein purified by SPHP column; 1, before column; 2, post-column; 3-8: collected peaks.

Example 2. AT-CoaR6 immunogenicity evaluation

1. Immunization of Mice

The immunogenicity of AT-CoaR6 was evaluated by using SPF grade Balb/c female mice aged 6-8 weeks. The mice were randomly divided into 2 groups with 8 mice in each group, immunized with 25 μg AT-CoaR6 recombinant protein and PBS, respectively. The adjuvant was a complex adjuvant mixed with Al(OH) ₃ and CpG. Intramuscular injections were performed on days 0, 14, and 28, respectively.

2. ELISA method to measure the level of specific antibodies

The serum of the immunized mice was collected on the 42nd day, and the levels of anti-AT and anti-CoaR6 IgG in the serum of the mice were determined by ELISA. Anti-CoaR6IgG detection ELISA method is as follows:

Coating: The recombinant protein CoaR6 antigen was diluted with ELISA coating solution to a final concentration of 2 μg/mL, added to a 96-well plate in a volume of 100 μL per well, and placed at 4°C overnight;

(a) Washing: discard the coating solution, wash the 96-well plate with a plate washer, and the detergent is PBST;

(b) Blocking: add 200 μL of ELISA blocking solution (2%BSA+PBST) to each well, and place at 37°C for 1 hour;

(c) Washing: discard the blocking solution, and use a plate washer to wash the 96-well plate;

(d) Add serum to be tested: add 200 μL of ELISA diluent (0.2% BSA + PBST) to the first well, and add 100 μL of ELISA diluent to the remaining wells. Add 4 μL of the serum to be tested in the first well, that is, dilute the serum by 50 times, and then perform double dilution. Negative serum should be added to each plate as a control. After the serum dilution is completed, place it in a constant temperature incubator at 37 °C for 1 hour;

(e) Washing: discard the serum diluent in the 96-well plate, and use a plate washer to wash the 96-well plate;

(f) Adding secondary antibody: Dilute the secondary antibody (Anti-Mouse IgG-HRP) in ELISA dilution solution at a dilution of 1:10000, add 100 μL of secondary antibody to each well, and place in a constant temperature incubator at 37°C for 1 hour;

(g) Washing: discard the secondary antibody, and use a plate washer to wash the 96-well plate;

(h) Color development: add 100 μL of TMB single-component color development solution to each well, leave it in the dark for about 3 minutes, then add 50 μL of ELISA stop solution, and measure the dual-wavelength OD ₄₅₀ -OD ₆₃₀ on a microplate reader.

The anti-AT IgG detection ELISA method coats the AT recombinant protein with 2μg/mL antigen, and the dilution in the first well of serum detection is 1:1000, and the other steps are the same as above.

The level of anti-AT-CoaR6 antibody in the serum of immunized mice was detected by ELISA. The results are shown in Figure 4. AT-CoaR6 can stimulate the titer of anti-AT-CoaR6 IgG produced by immunizing mice under the action of Al+CpG adjuvant were 9.31×10 ⁵ , the anti-AT IgG titers were 1.43×10 ⁶ , and the anti-CoaR6 IgG titers were 2.55×10 ³ . This result indicates that AT-CoaR6 has good immunogenicity.

Example 3. Evaluation of AT-CoaR6 immune protection

1. Mouse Immunization

The immunogenicity of AT-CoaR6 was evaluated using 6-8-week-old SPF Balb/c female mice. The mice were randomly divided into 3 groups of 10 mice and immunized with 25 μg of AT-CoaR6 recombinant protein and PBS, respectively. The adjuvant was a complex adjuvant mixed with Al(OH) ₃ and CpG. Intramuscular injections were performed on days 0, 14, and 28, respectively, and challenge was performed 38 days after the third immunization.

2. AT-CoaR6 active immunity against lethal dose of S. aureus intraperitoneal infection

(a) Recovery cryopreserved strains: two methicillin-resistant Staphylococcus aureus strains USA300FPR3757 (BAA-1556 ^™ ) and MRSA252 (BAA-1720 ^™ ) were purchased from American Type Culture Collection (ATCC) , frozen at -80°C. The cryopreserved MRSA252 and USA300 strains were streaked on TSA plates and cultured at 37°C for 18 to 24 hours;

(b) Pick MRSA252 and USA300 monoclones in TSB medium, and cultivate overnight at 37°C and 220rpm to saturation;

(c) Transfer the overnight cultured bacterial suspension to a new TSB medium at 1:100, and cultivate the OD600 to about 0.8 at 37°C and 220 rpm;

(d) 6500rpm centrifugation for 15min to collect bacterial cells;

(e) discard the medium supernatant, resuspend the cells in PBS to wash the cells, repeat twice;

(f) The concentration of MRSA252 was adjusted to 2×10 ¹⁰ CFUs/ml, and the concentration of USA300 was adjusted to 1×10 ¹⁰ CFUs/ml for intraperitoneal injection. There were 5 mice in different concentration groups, and each mouse was intraperitoneally injected with 100 μL bacterial suspension;

(g) Observation of mouse survival rate.

In the MRSA252 intraperitoneal infection model, the survival rate of mice in the AT-CoaR6 immunized group was 70% after 7 days of challenge, while the mice in the PBS group died 24 hours after challenge (Figure 5A). In the USA300 intraperitoneal infection model, the survival rate of mice in the AT-CoaR6 immunized group was 100% after 7 days of challenge; the mice in the PBS group also all died within 24h after challenge (Fig. 5B).

3. AT-CoaR6 active immunity against lethal dose of Staphylococcus aureus systemic infection

(a) Recovery of cryopreserved strains: streak the cryopreserved MRSA252 and USA300 strains on TSA plates, and culture at 37°C for 18 to 24 hours;

(b) Pick MRSA252 and USA300 monoclones in TSB medium, and cultivate overnight at 37°C and 220rpm to saturation;

(c) Transfer the overnight cultured bacterial suspension to a new TSB medium at 1:100, and cultivate the OD600 to about 0.8 at 37°C and 220 rpm;

(d) 6500rpm centrifugation for 15min to collect bacterial cells;

(e) discard the medium supernatant, resuspend the cells in PBS to wash the cells, repeat twice;

(f) The bacterial concentrations of MRSA252 and USA300 were adjusted to 4×10 ⁹ CFUs/ml, respectively, were injected into the tail vein, and 100 μL of bacterial suspension was intraperitoneally injected into each mouse;

(g) Observation of mouse survival rate.

The mice immunized with AT-CoaR6 still had a 50% survival rate 10 days after MRSA252 tail vein infection, which was significantly higher than that of the 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 immunization with AT-CoaR6 could significantly prolong the survival time of mice (p<0.01). Compared with the PBS group, 90% of the mice died within 1 day after the challenge; most of the mice immunized with AT-CoaR6 started to develop morbidity and death on the 3rd day (Fig. 6B). The above results indicate that the two-component vaccine AT-CoaR6 is a potential candidate vaccine against Staphylococcus aureus infection.

sequence listing

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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 domain recombinant protein, characterized in that the amino acid sequence of the recombinant protein is as shown in SEQ ID NO.2. 2 . A polynucleotide encoding the recombinant protein of claim 1 , wherein the sequence of the polynucleotide is shown in SEQ ID NO.3. 3 . 3. A fusion protein comprising the recombinant protein of claim 1, wherein the fusion protein further comprises Staphylococcus aureus α-toxin. 4. The fusion protein according to claim 3, wherein the amino acid sequence of the Staphylococcus aureus α-toxin is shown 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 according to claim 5 , wherein the α-toxin is linked to the recombinant protein of the R domain of Staphylococcus aureus coagulase by a G4S linker. 7 . 7. A polynucleotide encoding the fusion protein of claim 6, wherein the sequence of the polynucleotide is shown in SEQ ID NO.4. 8. An expression vector containing the polynucleotide of claim 7, wherein the vector is pET21a+-AT-CoaR6. 9 . A host cell containing the expression vector of claim 8 , wherein the host cell is Escherichia coli BL21 (DE3). 10 . 10. The application of any one of the fusion proteins of claims 3-6 in the preparation of a vaccine against Staphylococcus aureus infection.

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