A surface protein of fusicatenibacter saccharivorans and screening method and application thereof

Abstract

The application discloses a kind of fowl secretory bacterium surface proteins and its screening method and application, belong to biotechnology field.The application can be soluble high-efficiency expression in escherichia coli by screening protective antigen from fowl secretory bacterium heparin binding protein, combined with antigen prediction, soluble analysis and structure-oriented protein design, successfully obtained protein A0A3Q9GGY1, A0A3S9QKJ0 and six-site mutant PLO that can be soluble high-efficiency expression in escherichia coli, has good immunoprotective efficiency, and has accumulated experience for the screening and design of high-yield antigen, and has laid a foundation for the research and development of fowl secretory bacterium vaccine.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a surface protein of Cryptococcus pyogenes, its screening method, and its application. Background Technology

[0002] Cryptococcus pyogenes ( Trueperella pyogenes Cryptococcus pyogenes is a normal component of the skin and mucous membrane biomes of the upper respiratory tract, digestive tract, and urogenital tract in animals such as pigs, cattle, and sheep. It is also an important opportunistic pathogen. Cryptococcus pyogenes infection typically occurs after physical or microbial trauma to the skin or mucous membranes of animals. The bacteria, carried by the animal itself, invade the body through the wound, leading to infection. Cryptococcus pyogenes infection can cause localized purulent necrosis, chronic inflammation, and even systemic sepsis, affecting animal growth, meat quality, milk production, and reproductive performance. In severe cases, it can lead to animal death, causing significant economic losses to the livestock industry. Currently, the control of this bacterium mainly relies on antibiotics, but problems such as drug resistance and biofilm formation are becoming increasingly prominent, making vaccination a viable option for infection control.

[0003] Vaccine development for Cryptococcus pyogenes began in the 1940s. Early research focused on inactivated whole-cell vaccines or toxoid-based vaccines. While these vaccines could induce high levels of antibodies in animals and alleviate clinical symptoms, they were unable to completely block bacterial colonization and the formation of purulent foci. Furthermore, these vaccines were complex in composition, contained diverse antigens, and their inactivation process was difficult to control precisely, easily leading to unstable immune responses, inflammatory reactions, and even immunization failure. Screening for Cryptococcus pyogenes protein antigens that could be mass-produced and possessed good protective efficacy is crucial for developing novel vaccines against this bacterium.

[0004] In contrast, subunit vaccines with well-defined components and high purity are safer, and their effectiveness has been validated in multi-component protein vaccines such as those against Group B meningococcus (MenB). Currently, the antigens available for Cryptococcus pyogenes vaccine development are limited, and pyolysin (PLO) is an important protective antigen for this bacterium. PLO belongs to the cholesterol-dependent cytolysin (CDC) family, and its strong cytolytic activity limits its direct use as a vaccine component; it must be inactivated before it can be used in vaccines. The surface of the PLO molecule contains multiple hydrophobic amino acid residues. The exposed hydrophobic amino acid residues on the cholesterol recognition motif (CRM) and undecapeptide (UDP) regions of its D4 domain mediate recognition and binding to the eukaryotic cell membrane. The binding of the D4 domain to the membrane triggers rotation of the β5α2 ring of the PLO's D2 domain around the glycyl-glycine motif (GGM), thereby disrupting the hydrogen bond interaction between β5 and β4 in the D2 domain. The exposed β4 binds to β1 in adjacent PLO monomers, inducing the release of β5 from the β4 of neighboring monomers. This process transmits the oligomerization forces of the monomers, ultimately leading to further structural rearrangement on the cell membrane surface, forming transmembrane β-barrel pores capable of lysing cells. The exposed hydrophobic amino acid residues on the surface of the D4 domain's CRM and UDP, along with GGM, are the key molecular basis for PLO's cytotoxic activity. The exposed hydrophobic amino acid residues on the PLO surface are also a key factor leading to its easy aggregation and low solubility in E. coli expression systems. Protein solubility directly affects its soluble expression, correct folding, product stability, and ultimately, its immunoprotective efficacy in E. coli. Therefore, screening soluble expression antigens and conducting structure-guided rational design to improve the soluble expression level of antigens are crucial for advancing vaccine development. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a surface protein of Cryptococcus pyogenes and its screening method and application, so as to solve the technical problem that the vaccine composition against Cryptococcus pyogenes is complex and easily leads to unstable immune response or even immune failure.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A surface protein of *Cryptospirae* is provided, wherein the surface protein of *Cryptospirae* is a six-site mutant PLO, and the amino acid sequence of the six-site mutant PLO is shown in SEQ ID NO: 9; a screening method for the surface protein of *Cryptospirae* includes the following steps:

[0007] S1. Preliminary extraction of surface proteins from Cryptococcus pyogenes, and screening of target proteins based on surface exposure, conservation, and strain distribution.

[0008] S2. Recombinant proteins were prepared using the GST fusion expression system;

[0009] S3. Determine the immunoprotective level of the recombinant protein and screen for surface proteins of Cryptococcus pyogenes.

[0010] Based on the above technical solution, the present invention can be further improved as follows:

[0011] Furthermore, S1 specifically involves: extracting heparin-binding proteins from Cryptorchidica pyogenes, analyzing the homology and distribution of the proteins in strains with published genomes, and screening for target proteins based on surface exposure, conservation, and strain distribution rate.

[0012] Furthermore, S2 specifically refers to:

[0013] S201. Perform structure-guided protein design on the target protein, and replace six residues in PLO to form a six-site mutant PLO, so as to improve the solubility and conformational stability of the protein in aqueous solution.

[0014] S202. Insert the coding gene into the vector to obtain a recombinant plasmid; transform the recombinant plasmid into Escherichia coli to obtain a recombinant strain; culture the recombinant strain, induce protein expression and purify it to obtain the recombinant protein.

[0015] The present invention also discloses an antigen composition for preventing infection with Cryptococcus pyogenes, comprising the aforementioned Cryptococcus pyogenes surface protein.

[0016] The present invention also discloses the application of the above-mentioned Cryptococcus pyogenes surface protein in the preparation of a drug for preventing Cryptococcus pyogenes infection.

[0017] The beneficial effects of this invention are as follows:

[0018] 1. This invention extracted 116 proteins using heparin agarose gel electrophoresis, from which 59 target proteins were screened. Through solubility prediction and rational design, soluble expression of 8 proteins (A0A3Q9GGY1, A0A3Q9GI33, A0A3Q9GH58, A0A3Q9GHP5, A0A3Q9GMF6, A0A3S9QKG1, A0A3S9QPC2, A0A3S9QKJ0) and 1 six-site mutant PLO (L116S / V167T / G357S / L405Q / W497N / L524D) was successfully achieved. More than 10 mg of recombinant protein could be prepared per liter of induction culture. The yield of the six-site mutant PLO was 9 times higher than that of PLO. A0A3Q9GGY1 showed the highest expression, with 30 mg of recombinant protein prepared per liter of induction culture. The challenge experiment showed that the protection rates of A0A3S9QKJ0, PLO and its six-site mutant PLO were all higher than 80%, which was better than other proteins.

[0019] 2. This invention constructs a system encompassing "surface protein screening - assessment of physiological or virulence effects at key pathogenic stages - soluble expression assessment - immunoprotection rate determination," enabling high-throughput screening of protective antigens from the heparin-binding proteome of *Cryptobacillus pyogenes*. Simultaneously, by utilizing 3D structures to conduct structure-guided surface hydrophilication and detoxification design of PLO, a six-site mutant PLO was obtained, exhibiting significantly increased soluble expression levels, significantly reduced cytolytic activity, and good immunoprotective efficacy. This invention provides a reference technical route for high-throughput screening of soluble proteins and addressing the common problem of soluble expression of exogenous proteins in *E. coli* expression systems, while also providing optional antigens for developing highly effective vaccines. Attached Figure Description

[0020] Figure 1 A pie chart showing the gene coverage statistics for the samples;

[0021] Figure 2 This is a banding diagram of surface proteins of Cryptococcus pyogenes;

[0022] Figure 3 A 3D structural diagram of A0A3S9QPC2;

[0023] Figure 4 The distribution of hydrophobic amino acid residues exposed on the PLO surface;

[0024] Figure 5 Ramachandran plot for PLO (A0A380MBA8);

[0025] Figure 6 Ramachandran plot of the six-site mutant PLO;

[0026] Figure 7 Electrophoresis images of recombinant proteins A0A3Q9GGY1, A0A3Q9GI33, six-site mutant PLO, A0A3S9QKJ0, and A0A3S9QPC2;

[0027] Figure 8 Electrophoresis images of recombinant proteins A0A3Q9GH58, A0A3S9QKG1, A0A3Q9GHP5, and A0A3Q9GMF6. Detailed Implementation

[0028] The specific embodiments of the present invention are described below to facilitate understanding of the invention by those skilled in the art. Unless otherwise specified, specific conditions are applied according to conventional conditions or the manufacturer's recommendations. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various modifications are obvious as long as they fall within the spirit and scope of the invention as defined and determined by the appended claims. All inventions utilizing the concept of this invention are protected.

[0029] The following examples use Cryptococcus pyogenes (Cryptobacter pyoderma) Trueperella pyogenes The strain is ZSH-2020, which was preserved and provided by the Institute of Veterinary Medicine and Animal Drugs, Chongqing Academy of Animal Sciences. ZSH-2020 strain was deposited at the China General Microbiological Culture Collection Center on November 14, 2023, at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.28995.

[0030] Cryptococcus pyogenes ( Trueperella pyogenes The TP2 strain has been published in the literature "A Virulent Trueperella pyogenes Isolate, Which Causes Severe Bronchoconstriction in Porcine Precision-Cut Lung Slices", and its genome data comes from the NCBI GenBank database (GenBank: CP033903).

[0031] TSB and TSA culture media were purchased from BD Biosciences, Inc. (USA); fetal bovine serum was purchased from Hyclone Biosciences, Inc. (USA); bacterial membrane protein extraction kit was purchased from Shanghai Bebo Biotechnology Co., Ltd.; reduced glutathione and heparin agarose gels were purchased from Solarbio; GST affinity columns were purchased from Zhongke Senhui Microsphere Technology (Suzhou) Co., Ltd.; protein purification instrument was purchased from Suzhou Saipu Instrument Co., Ltd.; Quick Start Bradford was purchased from Bio-Rad Biomedical Products (Shanghai) Co., Ltd.; and defibrinated sheep blood was purchased from Hunan Bickman Holdings Co., Ltd.

[0032] Four-week-old female SPF Kunming mice were purchased from Spiford (Beijing) Biotechnology Co., Ltd.

[0033] Example 1: Extraction of surface proteins from Cryptococcus pyogenes

[0034] 1. Bacterial culture

[0035] Freeze-dried Cryptococcus pyogenes (ZSH-2020 strain) was inoculated into TSB medium containing 8% fetal bovine serum and cultured at 37°C with shaking at 150 rpm for 48 h. The culture was then inoculated at a 1% inoculum onto TSB medium containing 8% fetal bovine serum and subcultured three times. The subcultured cultures were then inoculated into TSA medium containing 8% fetal bovine serum and cultured at 37°C for 24 h. Single colonies were picked and inoculated into TSB medium containing 8% fetal bovine serum and cultured at 37°C with shaking at 150 rpm for 15 h. Finally, a 1% inoculum was inoculated onto TSB medium containing 8% fetal bovine serum and cultured at 37°C with shaking at 150 rpm for 15 h.

[0036] 2. Gene transcription analysis

[0037] Freshly cultured bacteria were injected intraperitoneally into mice, with 0.2 mL injected into each mouse. Mice mortality was observed. Mice were then euthanized on day 3 post-infection, and lung tissue was harvested. Using *Cryptobacter pyogenes* TP2 strain (GenBank: CP033903) as a reference strain, gene transcription of *C. pyogenes* in the lungs of challenged mice was detected and analyzed. The gene transcription detection and analysis were performed by Sangon Biotech (Shanghai) Co., Ltd.

[0038] Using the genes annotated in the genome of *Cryptospira pyogenes* strain TP2 (GenBank: CP033903) as a reference, transcripts of 2072 *Cryptospira pyogenes* genes were detected in the lung tissue of a mouse infection model, such as... Figure 1 As shown, different colors represent different percentage ranges, the percentage value represents the percentage of the detected gene region to the total length of the corresponding gene region, the number of genes that can be detected in the interval is in parentheses, and the pie chart shows the percentage of genes that can be detected to the total number of genes in different coverage intervals.

[0039] Multiple analyses of gene transcripts revealed high abundance of 312 gene transcripts, with high-level transcription of genes encoding A0A380MBA8(PLO), A0A3Q9GGY1, A0A3Q9GI33, and A0A3S9QPC2 detected (Table 1), indicating high expression levels in lung tissue of the infection model. Among the detected genes, those related to metabolism, particularly amino acid metabolism, carbohydrate metabolism, and cofactor and vitamin metabolism, were the most numerous.

[0040] Table 1. Statistics of some high-transcriptional-level genes of Cryptococcus pyogenes in lung tissue of mouse infection model

[0041]

[0042] Wherein, “1t” is the group number, indicating that Table 1 shows the statistical data of some high-transcriptional-level genes of Cryptococcus pyogenes in the lung tissue of the first group of mouse infection models; “Count” indicates the number of genes; “TPM” is Transcripts Per Million, representing the number of transcripts per million; and “FPKM” is Fragments Per Kilobase of transcript per Million mapped fragments, representing the number of mapped fragments per million of transcripts per kilobase length.

[0043] 3. Extraction of heparin-binding protein

[0044] Fresh cultures of *Cryptobacter pyogenes* were centrifuged at 6000×g for 10 min. The bacterial pellet was washed three times with Tris-HCl (0.02 mol / L, pH=8.0) and then thoroughly resuspended. Bacterial surface proteins were extracted using a bacterial membrane protein extraction kit. 5 mL of heparin-agarose gel suspension was washed three times with pre-chilled PBS solution. The extracted proteins were mixed with the heparin-agarose gel and incubated at 4°C with slow vertical rotation for 1 h. The gel was then washed three times with pre-chilled PBST solution (containing 0.5 M NaCl and 0.5% Tween-20) and eluted with 8 mol / L urea solution. The extracted proteins were detected by 12% SDS-PAGE and Coomassie Brilliant Blue staining.

[0045] The results are as follows Figure 2As shown in the figure, M represents the relative molecular mass standard of the protein; the number "1" indicates the protein extracted from the sample using the heparin agarose gel extraction kit; and the number "2" indicates the protein extracted using the same kit. The figure shows that after separation by SDS-PAGE and Coomassie brilliant blue staining, the protein bands of *Cryptobacillus pyogenes* underwent significant changes after adsorption onto the heparin agarose gel, indicating that some proteins were enriched.

[0046] 4. Identification of heparin-binding proteins

[0047] Protein samples were analyzed by LC-MS / MS. LC-MS / MS analysis and database search were performed by Sangon Biotech (Shanghai) Co., Ltd. After LC-MS / MS analysis and database search, and washing away impurities with 0.5 mol / L NaCl solution, 116 types of Cryptococcus pyogenes proteins were extracted from the samples using a heparin agarose gel extraction kit.

[0048] For protein extraction, surface proteins of *C. pyogenes* were first extracted using a bacterial membrane protein extraction kit, and then proteins were extracted from these surface proteins using heparin agarose gel electrophoresis. The bacterial membrane protein extraction kit can extract approximately 700 proteins from *C. pyogenes*, and as with other extraction methods, the extracted proteins may contain cytoplasmic proteins. The extracted proteins were then adsorbed onto the heparin agarose gel, and impurities were washed away with a 0.5 mol / L NaCl solution. Proteins with high affinity for heparin were concentrated and extracted, reducing the protein content and increasing the proportion of surface proteins.

[0049] Example 2: Screening for target proteins

[0050] 1. Screening of target proteins

[0051] PsortB 3.0 was used to predict protein subcellular localization, Deep TMHMM to predict transmembrane domains, Phobius to predict transmembrane domains and signal peptides, and SignalP 5.0 to predict signal peptides. BLAST analysis was used to analyze protein homology and distribution in strains with published genomes. VaxiJen 2.0 was used to predict protein immunogenicity, with the model set to a "bacterial" model and a threshold of 0.5. Target proteins were screened based on criteria including surface exposure, high conservation (homology greater than 90%), and widespread distribution in strains (distribution rate greater than 90%).

[0052] The results are shown in Table 2. The identified proteins can be divided into cell wall proteins, secretory proteins, lipoproteins, and membrane proteins, with 10, 58, 26, and 22 types, respectively. A total of 31 substrate-binding proteins (SBPs) of ATP binding cassette (ABC) transporters were identified, of which 6 proteins (Table 2) were predicted to be located in the cell wall and were counted as cell wall proteins; the other SBPs were still counted as lipoproteins.

[0053] Based on VaxiJen predictions, 59 potential candidate antigens were screened according to the criteria of surface exposure, high conservation, and wide distribution in the strains. Among them, 27 ABC transporters SBP were selected as potential candidate antigens.

[0054] Table 2. Results of target protein screening for Cryptococcus pyogenes

[0055]

[0056] The superscript "S" indicates the ABC transporter component SBP, and the superscript "A" indicates the target protein.

[0057] For target protein screening, multiple bioinformatics tools were integrated to construct a surface protein system screening process, combining the physiological and virulence functions of proteins for screening. Proteins related to bacterial physiology and virulence, such as ABC transporter substrate-binding proteins, PLO, and enzymes, were identified from the surface proteins. Among them, ABC transporter substrate-binding proteins were the most abundant functional category. These lipoproteins play a central role in bacterial nutrient uptake, virulence exertion, and cell membrane homeostasis maintenance, making them important vaccine targets. Subsequently, VaxiJen was used for initial immunogenicity screening, followed by refined screening based on surface exposure, high conservation, and high cross-strain distribution. To ensure effective contact of membrane proteins with the immune system, their extracellular domains were limited to greater than 250 amino acids. To mitigate experimental errors, BLAST analysis was used to analyze their conservation and distribution among 56 genetically diverse strains. These 56 *Cryptospira pyogenes* strains, whose genome sequences are published in GenBank, were isolated from pigs, cattle, sheep, and other hosts from multiple regions worldwide, reflecting the genetic diversity of this bacterium. Using the above screening principles and analytical methods, 59 target proteins were screened out, including previously reported antigens such as PLO, verifying the reliability of the screening strategy.

[0058] 2. Selection and design of target proteins

[0059] Target protein priority selection:

[0060] (1) Cell wall proteins, secretory proteins, lipoproteins, or membrane proteins with large extracellular domains;

[0061] (2) The abundance of gene transcripts was high in the lung tissue of the infection model;

[0062] (3) Known or predicted based on conserved structural domains to play an important physiological or toxicological role in key pathogenic processes;

[0063] (4) High solubility, without key factors affecting soluble expression such as surface hydrophobic patches and potential aggregation sites.

[0064] Measures to assess the likelihood of achieving soluble and efficient protein expression in E. coli:

[0065] (1) Download the 3D structure of the protein from AlphaFold DB and RCSB PDB;

[0066] (2) First, SOLpro was used to predict the solubility of the protein when it was expressed efficiently in E. coli. Then, ChimeraX was used to analyze the hydrophobic spots exposed on the surface of the protein in the 3D structure. Aggrescan3D identified the hydrophobic residues on the protein surface that are prone to protein aggregation.

[0067] (3) The reliability of the prediction results of ChimeraX and Aggrescan 3D was analyzed by 3D structure analysis. After comprehensive analysis, the target proteins were selected for priority testing from the target proteins.

[0068] Structure-guided protein design is employed to target important antigens, aiming to improve protein solubility and conformational stability in aqueous solutions. Specifically, structure-guided protein design involves:

[0069] (1) ChimeraX was used to analyze the hydrophobic spots exposed on the surface of the protein 3D structure, and Aggrescan3D was used to identify hydrophobic residues on the protein surface that are prone to protein aggregation.

[0070] (2) Based on the results of 3D structural analysis, hydrophilic amino acid residues were used to replace the exposed hydrophobic residues on the protein surface to improve the water solubility of the protein, and large side chain residues were introduced into the flexible region to restrict the conformational changes of the protein.

[0071] (3) Use the homology modeling platform swiss-model to evaluate the quality of the protein structure obtained by the engineering design.

[0072] The likelihood of soluble expression of target proteins in *E. coli* was analyzed using software such as SOLpro, Aggrescan3D, and ChimeraX. The results assessed that most ABC transporter SBPs are likely to be solublely expressed in *E. coli*. Considering factors such as sufficient surface exposure, high abundance of gene transcripts in lung tissue of infection models, known or predicted important physiological or virulence roles in key pathogenic processes based on conserved domains, and high solubility, proteins such as A0A3Q9GGY1 were selected as priority targets for validation (Table 3). A0A3Q9GGY1, A0A3Q9GI33, A0A3Q9GH58, A0A3Q9GHP5, A0A3Q9GMF6, and A0A3S9QKG1 are ABC transporter SBPs, which may play roles in nutrient acquisition, promoting virulence, establishing and maintaining cell membrane homeostasis, and adhering to host cells. A0A3S9QPC2 is a membrane protein of the UPF0182 family, with an extramembrane domain consisting of approximately 700 residues (…). Figure 3 It may be related to transport processes or biofilm formation, and is abundant in the biofilm matrix of *Micrococcus faecalis*. A0A3S9QKJ0 is a cell wall localization protein, an S8 family serine peptidase, with a C5a peptidase domain at its N-terminus. C5a peptidase is involved in streptococcal adhesion and invasion of host cells.

[0073] Table 3 Target protein information

[0074]

[0075] Three-dimensional structural analysis of PLO revealed surface hydrophobic patches containing multiple exposed hydrophobic residues, primarily concentrated on the cholesterol recognition motif (CRM) at the top of domain 4 and on the undecapeptide (UDP). Figure 4 ). Figure 4 The yellow area represents hydrophobic amino acid residues on the surface of protein molecules, the white area represents amino acid residues with near-neutral hydrophobicity / hydrophilicity, and the blue area represents the least hydrophobic amino acid residues.

[0076] Furthermore, the glycyl-glycine motif (GGM) is located in the flexible region of PLO and is involved in the monomer oligomerization process. Therefore, a six-site residue substitution was performed on PLO to form a six-site mutant PLO (L116S / V167T / G357S / L405Q / W497N / L524D). L116S / V167T / L405Q / W497N / L524D replaces surface hydrophobic residues, with W497N and L524D located in the UDP and CRM, respectively. The G357S mutation restricts the flexibility of the GGM, inhibits oligomerization, and stabilizes the monomer conformation. According to SOLpro's predictions, the solubility of the six-site mutant PLO is significantly improved compared to the two-site mutant PLO (W497N / L524D). According to the Swiss-model structural assessment (Table 4), mutations targeting G357 alter the residue distribution at that site. Replacing G357 with S residues has the least impact on the overall structure of PLO compared to other residues. The L116S / V167T / L405Q / W497N / L524D mutation has no significant impact on the overall structure of PLO. The MolProbit score and conflict score of the six-site mutant PLO are higher than the PLO score, indicating a very slight decrease in the overall stereochemical quality of the structure. This may be due to a few local atoms being too close together while the distribution pattern of other residues remains unchanged. The mutation does not alter the β-sheet region or the α-helix distribution. Figure 5 and Figure 6 This reflects that the six-site mutant PLO still maintains very high structural integrity, without global structural damage or misfolding.

[0077] Table 4. MolProbability Results of PLO and its Six-Site Mutant PLO

[0078]

[0079] For important virulence factors such as PLO, structure-guided protein design was employed. Hydrophilic amino acid residues were used to replace exposed hydrophobic residues on the protein surface to improve water solubility, and large side-chain residues were introduced into flexible regions to restrict conformational changes. The structure quality of the engineered proteins was evaluated using the homology modeling platform Swiss-model.

[0080] By using sequence and 3D structure analysis to assess key factors affecting soluble expression of target proteins, such as solubility, surface hydrophobic plaques, and potential aggregation sites, we screened for novel antigens that can be efficiently expressed in soluble form in Escherichia coli and have high immunoprotective efficacy.

[0081] Six proteins, A0A3Q9GGY1, A0A3Q9GI33, A0A3Q9GH58, A0A3Q9GHP5, A0A3Q9GMF6, and A0A3S9QKG1, all have signal peptides at their N-termini. Expression was performed on the portion following the signal peptide that possesses a complete 3D structure. A0A3S9QPC2 is a membrane protein; expression was performed on the portion outside the cell membrane that forms a compact 3D structure. A0A3S9QKJ0 was expressed using its C5a peptidase domain. PLO is a secreted protein with a signal peptide at its N-terminus; expression was performed on the portion following the signal peptide that possesses a complete 3D structure. PLO and the six-site mutant PLO are identical in sequence except for the site-directed mutation. To improve soluble expression levels, the expression sequence initiation sites described in Table 3 were determined based on the SOLpro soluble expression score.

[0082] The amino acid sequence of A0A3Q9GGY1 is as follows:

[0083] GKSNTPATTAAGESTTSAKSSAPSGQLNLGVAYETTNYDPSTTSSALAMGTNWHVVEGLYEFDMSNYKLFPALAAGEPKKISDTEYEITLRDGAKFSDGKDVTTKDFLESYKRTTADTSIYKQFFT FIDSVAAKDDKTISVKLKYPFAALAERLVDVKVIPASSNQKDMTAKPVGTGPFKYETISNTVVEAVPNEFYNGPKPAKVAKMHWDVLKDDTARLAAALGGTIDVMETVPSATKDQLKAAGWTLDEV PGYNNPFLMFNTTKAPFDKKDVRKAFHYAIDREKLVKDAMGGDATVASSFLPKSNPMYKEAAEQFTMDTAKAKAAFEAAGLKEITLITTDHPWIANLAPQIRKDLEAAGLTVNVQSMASGDLYANF ADVDNPTFDVALAPGDPSVFGIDPGIIINWWYGDNVWTQKRGAWGKTNPEGFKKLSEIVSAAEQATGDAAKAKWGEAQDLIADEAPIFPLFHRTMITGINGAKVVGGHGIGTTGLNFVGASVK (SEQ ID NO: 1).

[0084] The amino acid sequence of A0A3Q9GI33 is as follows:

[0085] STDAGAAGASLDASNSVSIGYILEPVGLDPTRVSGAALDQLVIDNVYEGLTTTDQNGEVTAKLAQSWEISEDSLTYTFHLRDGVTFHDGSAFDANDVVATLQASAAQDSANPDHKLMERFASATAIDPLTVEVKLSAPDSRFLNTMSTDAAMMVPSDNSVDLNTASNGTGPYKIGPWQTGSSITLERNENYWGKPAANKKAIFRYYKDQAAANNALESGELDILNVFNNDTVARFSNRDDFVMNEGHQTSWMTLAFNHRHPDLQDERVRRAIRKAIDKDGLIAALGGQFHRIGSMVGPGEAWFDESLTAIDAYDPQGARELLRQAGKENLTFTLRVSNSYDPMISEYMKSQLAKVGITLNIEQIEFATWLDQVYHGANYEITMVLHTDPWTLMYYANPKYYWNYDNPKIQAMVNEVIESSAIAERDEKLKAVARAVSEDAAADWLFTPKALTFARKGTSGYPTSRTGSRFPAYNITTP (SEQ ID NO: 2).

[0086] The amino acid sequence of A0A3Q9GH58 is:

[0087] GDDAAQTSTTATEGFRAADLSAIEKNDELAAMVPAQIAADGKLVLGTNIFFAPAEFYAPDGVTPQGYDIDLGKALGKILGLETEFQHAEFAAIIPGIGSRYEAGIANFTINPERQEVVDMIQYLEAGSAWAVPAGNPKGFDQTAPCGTVVGVQTGTYQEEVLTDMNAKACKDNPIQIQSFSEQSAVTLRVASGQIDAMYTDSPVADYAISQNAGKIERIGEVEDSAGYGVVAAKDNPELTKVLQAALQKLMDDGHLKAIFTTWGITEGVATQASLNPAS (SEQ ID NO: 3).

[0088] The amino acid sequence of A0A3Q9GHP5 is:

[0089] SNTGDFPRTVTHSAGETTIKARPESIVVLDMAALDTIDALGAGDRVVGTATSAVPTWLKDKEGIDYSALTSVGSLKEPDMEAIAKLKPDLVVIGNRSAKYYEEFSKNFTTIDATHSWKVSDYSATVPKNVEMVAKAIGADAKGTSAAEAIRTKLAGYTGAAKDKGNALVVMTSAGELSLHDRGSRWAPIWDVFGFGEAYKKATPDEGHKGDKVSFETIKEINPDWMFVVDRDAAIGKVNPGQTAAQVLDNELVKATNAAKNNRIVYLTPERWYIVMTGATNFPAMLDEIADAIQ (SEQ ID NO: 4).

[0090] The amino acid sequence of A0A3Q9GMF6 is:

[0091] GNAQSAKVTTPNTESSTTASMDAAQYPQTVKACDTSFDIKKAPERVMFINSTGTSALLDLGLIDRVVARIGVVETSTYSQEDAKKIKDIEVIESAVKGGGHYNVSTEAVIEKHPDLIIASNPADLDVQKLYDAGIAIYVPEEFCVRDNTEKVTFDNVYREIRTFAKVFGVTDRAEKVIEKLKGEVKASGSTKVSGTAAAVFVVPGDSKFYAYGNSSMVTPQFEALGLKNVYGNEKKRVFTVSTESLLEHNPDRIIILHQGDEQGAIETFKQAHGASDLRAVREGNLIGMPFPLTDPPSSLSVEGLKYLQNKLK (SEQ ID NO: 5).

[0092] The amino acid sequence of A0A3S9QKG1 is:

[0093] GASSADATVKLVVGASPVPHAPILQFVADNLAAGAGLDLEVKEYTDYVQPNVGLDAGELDANFFQHLPYLEAQIKDRGYDLEHGVGIHIEPFGIYSKKVTSLADVAEGGVVLVTNDPSNQARGLKLLAAEGLITLADVANPTIYDVKDNPKNLQIRESEAPAIPVQLPDVDLAIINGNFALEAGLVPAKDALALESGENNPYANILAWKKGTKKIAAVKKLDELLHSPEVAQFIKDTYPNGEVTAAF (SEQ ID NO: 6).

[0094] The amino acid sequence of A0A3S9QPC2 is:

[0095] QRNIDATRHAYDMAHVQTRQYDARTDVEPGQLRKDSETAAQIRLLDPNIVDPSFNQLQQNRQYYQFRDPLTVDRYQIGNENRDTVIAVRELNLDGLDAERRSWVNDHTVYTHGFGVAAAYGNTITSRGDPAFWEAGIPSSGQLGEYEPRVYFGQQSPSYSIVGAPEGSDPWELDYPDDTAPNGQVMNTYTGNGGPSVGSVFERLMFSIKMRSTELFFSDRVTANSQILFDRNPHERVRKVAPYLTLDTKAVPAVVDMDNDPSTRKDLVWIIDGYTTSNNYPYSARETLTEATADSTTTGLGQPRPQEINYIRNSIKAVVNAYDGSVTLYQWDNEDPIINAWKSIFPGQITELSEMPGDLIAHVRYPEDLFKIQRSLLARYHVTDAKSFYSGGDFWNVPLEPTAAAGPTTPKQPPYYLTLKMPGQDETAFSLTTSYIPGGRTNRNIMTGFLAASGDAGNQTGVKGENYGKLRLLELPRDLTVPGPGQAENTMLTNPKVSTALNLLQQGGTEIMKGNLLSLPVGDGLLYVQPIYVQASSGTQYPTLQYVLTLFGDNVGFAPTLDESLDQ (SEQ ID NO: 7).

[0096] The amino acid sequence of A0A3S9QKJ0 is:

[0097] GAPDAPPAPPAPPAAATERPEKNVPVIVTLERQPKGSTDKAMVEKIANDLAAKYNMSIRRQFSYLVNAFSAYVPVSAIEDLALEKGVAAVDRMRVYYPSMESAVKLTQVVQASQKHDVDGQGLVVSIVDTGIDLNHQDMRLDDGVAVRVKPEAGFTDKVPYGYNFADETTQVKDKTASQHGMHVAGIVAANAPDGADVIKNGRIDGVAPNAQLLAMKVFSNDPKKPGAAADDVMAAVEESVKHGADVINLSLGHPNGHEGQALGEQRVIANARAAGVEVIVAAGNEGQNGSAKGVTDDQLGLLDDGTVGGPSTGTDAWSVASVENSTIVNSAGTAKKDKEEYTFSYQLQVGSSDGSPVEIVDAGWGTVQETLGKNFSGKFVLIQRGAKEGEEPITFGDKFRNAIIAKAAGVIVYNHKQGGDEFLGMGGLEGITIPGAFIGHKDGVKLAEMIKAGKTTIALTNMRVVVANPDSMRPSSFTSWGAGPELGFKPEIAGIGGNVYSTVNDHKYDTKSGTSMAAPHVAGVAALMIQKAEADNPQRPRSEIVLRNRVALSNTAKILEKDGVPFAPRQIGAGLVQVQDALETKVLATVDDSPVVALKEVAATKSFTVTLANESDQPRTFSAGATCVVNEEEKPDSKTTTYCSKTDTITASTDEVTVPAGGKTEVTYTLNVSGADHWTQGWVTFEAKDEGQPNLSVPYLGFAGDWNA (SEQ ID NO: 8).

[0098] The amino acid sequence of the six-site mutant PLO (L116S / V167T / G357S / L405Q / W497N / L524D) is as follows:

[0099] AGLGNSSGLTDGLSAPRASISPMDKVDLKSAQETNETSVDKYIRGLKYDPSGVLAVKGESIENVPVTKDQLKDGTYTVFKHERKSFNNSRSDISAFDANNAHVYPGALVLANKDLAKGSPTSIGIARAPQTVSVDLPGLTDGKNKVVINNPTKSSVTQGMNGLLDGWIQRNSKYPDHAAKISYDETMVTSKRQLEAKLGLGFEKVSAKLNVDFDAIHKRERQVAIASFKQIYYTASVDTPTSPHSVFGPNVTAQDLKDRGVNNKNPLGYISSVSYGRQIFVKLETTSTSNDVQAAFSGLFKAKFGNLSTEFKTKYADILNKTRATVYVVSGSARGGVEVATGNIDALKKIIKEESTFSTKVPAVPVSYAVNFLKDNQQAAVRSSGDYIETTATTYKSGEITFRHGGGYVAKFRLKWDEISYDPQGKEIRTPKTWSGNWVGRTAGFRETIQLPANARNIHVEAGEATGLANDPWWTVINKKNLPLVPHREIVLKGTTDNPWVEENVKP (SEQ ID NO: 9).

[0100] Example 3 Preparation of Recombinant Protein

[0101] Based on the codon preference of *E. coli*, the protein-coding gene was optimized by introducing a BamHI recognition site at the 5′ end and an XhoI recognition site at the 3′ end. The gene sequence was synthesized chemically by Sangon Biotech (Shanghai) Co., Ltd. The gene was cloned into pGEX-4T-1 via the restriction endonucleases BamHI and XhoI recognition sites, and the recombinant plasmid was transformed into *E. coli* BL21(DE3). The recombinant strain was cultured at 37°C with shaking until the cell concentration reached approximately OD600 for 1 hour. IPTG was added to a final concentration of 0.1 mmol / L, and expression was induced at 28°C for 6 hours. The culture was centrifuged at 5000 × g for 10 minutes at 4°C. 50 mL of TBS (50 mM Tris-HCl + 0.15 M NaCl, pH=8.0) was added to every 500 mL of culture medium to suspend the bacterial pellet. The bacterial suspension was then subjected to ultrasonic lysis in an ice-water bath at 300 W, with a 2-second upswing followed by a 3-second downswing, for a total lysis time of 10 minutes. The lysate was centrifuged at 12000×g for 15 min, and the supernatant was filtered through a 0.22 μm filter. Recombinant protein was affinity purified from the supernatant using a protein purifier and a GST affinity column at a flow rate of 1 mL / min. After loading, the protein was washed with TBS to baseline, followed by elution with elution buffer (50 mM Tris-HCl + 10 mM reduced glutathione, pH=8.0), and the eluent was collected. After dialysis, the purification effect was analyzed by 10% SDS-PAGE and Coomassie Brilliant Blue staining, and the protein concentration was determined using Quick Start Bradford assay.

[0102] The recombinant plasmid pGEX-4T-1 containing the gene encoding the target protein was transformed into BL21(DE3) competent cells. Expression was induced by IPTG at 28℃ for 6 h, resulting in soluble expression of the target protein. The recombinant protein was obtained by GST affinity purification. Figure 7 and Figure 8 ). Figure 7 In the diagram, M represents the relative molecular mass standard of the protein; the number "1" represents A0A3Q9GGY1; the number "2" represents A0A3Q9GI33; the number "3" represents PLO; the number "4" represents the six-site mutant PLO (L116S / V167T / G357S / L405Q / W497N / L524D); the number "5" represents A0A3S9QKJ0; and the number "6" represents A0A3S9QPC2. Figure 8 In the diagram, M represents the relative molecular mass standard of the protein; the number "1" represents A0A3Q9GH58; the number "2" represents A0A3S9QKG1; the number "3" represents A0A3Q9GHP5; and the number "4" represents A0A3Q9GMF6.

[0103] Concentration determinations showed that, except for PLO, other proteins could yield no less than 10 mg of recombinant protein per liter of bacterial culture; A0A3Q9GGY1 had the highest yield, yielding 30 mg of recombinant protein per liter of bacterial culture; the six-site mutant PLO (L116S / V167T / G357S / L405Q / W497N / L524D) had a 9-fold higher yield than PLO, yielding 10 mg of recombinant protein per liter of bacterial culture.

[0104] Example 4: Determination of Immunoprotective Level

[0105] 1. Hemolytic activity and stability assay

[0106] The hemolytic activity of PLO and its six-site mutant PLO was examined. The hemolytic activity of PLO and its six-site mutant PLO was measured on the day of purification and after storage at -20℃ for 2 months. Changes in hemolytic activity within 2 months were compared to determine their stability. 2 mL of defibrinated sheep blood was centrifuged at 1000×g for 10 min, the supernatant plasma and leukocytes were discarded, and the lower red blood cells were mixed thoroughly with physiological saline, centrifuged at 1000×g for 10 min, and the supernatant was discarded. This process was repeated 3 times. A 2% (v / v) red blood cell suspension was prepared with physiological saline and stored at 4℃ for later use. PLO and its six-site mutant PLO were serially diluted with physiological saline. 50 μL of each dilution was added to each well of a microagglutination plate, followed by 50 μL of the red blood cell suspension. The plates were mixed, incubated at 37℃ for 30 min, and the red blood cell lysis was observed in each well. The hemolytic titer unit (HTU) of PLO and its six-site mutant PLO is expressed as the lowest dilution factor that can cause hemolysis in a sample. The hemolytic activity is expressed as the hemolytic titer unit per milligram of protein.

[0107] The hemolytic activity of PLO and its six-site mutant PLO was reflected by their ability to lyse sheep erythrocytes. Hemolytic activity was measured on the day of purification. The hemolytic activity of PLO was 969.69 HTU / mg, while that of the six-site mutant PLO was 3.51 HTU / mg. The hemolytic activity of the six-site mutant PLO in lysing sheep erythrocytes was 276.26 times lower than that of PLO. After storage at -80℃ for 2 months, the hemolytic activity of PLO was 2.38 HTU / mg, while that of the six-site mutant PLO was 3.26 HTU / mg, indicating that the stability of the six-site mutant PLO was better than that of PLO.

[0108] 2. Animal immunization settings

[0109] Recombinant PLO was inactivated overnight at 4°C with 0.5% formaldehyde solution; other recombinant proteins were not inactivated. The purified recombinant protein (50 μg / mL) and aluminum gel adjuvant were mixed at a volume ratio of 5:1 and thoroughly shaken. 0.2 mL was injected intramuscularly into the leg of each mouse, and immunizations were repeated twice, with a two-week interval. Mice were divided into groups according to the injected protein; the control group received PBS without recombinant protein, with 20 mice in each group.

[0110] 3. Determination of recombinant protein protection rate

[0111] Fresh cultures of *Cryptobacter pyogenes* ZSH-2020 were centrifuged at 6000×g for 10 min, washed twice, and then resuspended in physiological saline at 1 / 2 or 1 / 4 volume of the original culture. Using a pipette, 1 mL of the bacterial suspension was slowly pipetted along the tube wall into a sterile test tube containing 9 mL of physiological saline. The tube was then shaken to mix thoroughly, thus preparing a 1:10 dilution. This method was repeated to continuously dilute to 1:10. 10 From 1:10 8 1:10 9 1:10 10 For each diluted sample, pipette 1 mL of sample into a sterile culture dish, performing two replicates for each dilution. Add 1 mL of physiological saline to two sterile culture dishes as blank controls. Immediately pour 15 mL-20 mL of TBA medium cooled to 46℃-50℃ into the culture dish and rotate to mix thoroughly. Place horizontally until the agar solidifies, then invert the plate and incubate at 37℃ for 96 hours. Count the colonies on plates with a count between 30-300. Each colony counted represents one colony-forming unit (CFU). Calculate the concentration of the original bacterial suspension using the following formula:

[0112] ;

[0113] Where a is the concentration of the original bacterial suspension, CFU / mL; b is the average number of colonies in the bacterial suspensions taken at different dilutions, CFU; c is the dilution factor; and V is the volume of the bacterial suspension taken, mL.

[0114] On day 14 after the second immunization, mice were injected intraperitoneally with 0.2 mL of freshly cultured lethal dose ZSH-2020 strain. After 21 days of observation, the number of dead mice in the control group and each immunization group during the challenge observation period was counted. The mouse mortality rate M and protein immunoprotection rate N were calculated using the following formulas:

[0115] ;

[0116] ;

[0117] Where M is the mortality rate of mice, %; m0 is the initial number of mice, mice; m1 is the number of mice that died, mice; N is the protein immunoprotection rate, %; M' is the mortality rate of mice in the control group, %; M'' is the mortality rate of mice in the protein immunoprotection group, %.

[0118] Mice were immunized twice with the recombinant protein, and a challenge test was performed 14 days after the second immunization. Based on the viable bacterial count, each mouse was intraperitoneally injected with 8.8 × 10⁸ g of the protein. 8 CFU (Cryptospirosis Cryptococcus). During the 21-day observation period, 2 mice survived in the control group, while 18, 17, 18, and 16 mice survived in the PLO, six-site mutant PLO, A0A3S9QKJ0, and A0A3Q9GGY1 recombinant protein immunization groups, respectively. The immunoprotection rates were 88.89%, 83.33%, 88.89%, and 77.78%, respectively, which were higher than the immunoprotection rates of other proteins (Table 5).

[0119] Table 5 Protein Immunoprotection Rate

[0120]

Claims

1. A surface protein of Cryptococcus pyogenes, characterized in that, The surface protein of *Cryptobacillus pyogenes* is a six-site mutant PLO, and the amino acid sequence of the six-site mutant PLO is shown in SEQ ID NO:

9.

2. An antigen composition for preventing infection with Cryptococcus pyogenes, characterized in that, The antigen composition comprises the surface protein of Cryptococcus pyogenes as described in claim 1.

3. The use of the Cryptococcus pyogenes surface protein of claim 1 in the preparation of a medicament for preventing Cryptococcus pyogenes infection.

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