Htsa-slodm fusion protein and its use in preparing group a streptococcus vaccine

Abstract

The application discloses a HtsA-SLOdm fusion protein and application thereof in preparation of a group A streptococcus vaccine. The HtsA-SLOdm fusion protein is obtained by fusing and recombining a heme transport lipoprotein (HtsA) and a streptolysin protein containing a double-mutation site (SLOdm), can induce production of HtsA protein and SLOdm protein specific IgG antibodies in serum, has strong immunogenicity, can induce high-level IgG1 and IgG2a antibody titers, and the humoral immune response in inducing production of SLOdm protein specific IgG antibodies in serum is stronger than that of a single protein. In addition, the HtsA-SLOdm fusion protein can promote skin wound healing of mice, reduce bacterial content and inflammatory response at a skin infection site of the mice, accelerate wound healing, has almost no hemolytic activity, has high safety, and can be used for preparing a group A streptococcus vaccine.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and in particular to a HtsA-SLOdm fusion protein and application thereof in preparation of a group A streptococcus vaccine. BACKGROUND

[0002] Group A Streptococcus (GAS, also known as Streptococcus pyogenes) is a common pathogenic bacterium in clinic, which can cause various infections and complications, including pharyngitis, impetigo, necrotizing fasciitis, streptococcal toxic shock syndrome, rheumatic heart disease and acute streptococcal glomerulonephritis, etc. At present, the main way for the treatment after group A streptococcus infection is to take relevant antibiotics, however, the abuse of antibiotics can easily lead to the generation of drug-resistant strains, and the way of prevention through vaccine to resist infectious diseases caused by group A streptococcus is becoming more and more important.

[0003] In related technologies, after nearly a century of research and development of group A streptococcus vaccine by scientists, many group A streptococcus proteins have been studied at the animal level or in clinical trials, and the feasibility of the proteins as candidate antigens of group A streptococcus vaccine has been evaluated. However, due to the complexity of group A streptococcus infection, it is difficult to control the infection of group A streptococcus by blocking the function of a single virulence protein. Therefore, scientists have proposed a new research strategy for group A streptococcus protein vaccine, that is, combined immunization of multiple virulence proteins of group A streptococcus to control group A streptococcus infection. The strategy is to prepare different proteins separately and then mix them for combined immunization, or to design several proteins into a fusion protein and then immunize in the form of the fusion protein. And because the fusion protein can produce and purify several proteins as one protein without the need to prepare each component of the fusion protein separately, the fusion protein not only has good safety and non-serotype-dependent characteristics, but also simplifies the production process and helps to reduce production costs. However, the immune effect of the current group A streptococcus protein vaccine based on combined immunization is still not ideal, and there is a certain hemolytic toxicity, which is difficult to achieve the expected protection purpose.

[0004] Therefore, it is urgent to seek a group A streptococcus protein vaccine with excellent immune protection and good safety. SUMMARY

[0005] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, the present application provides a HtsA-SLOdm fusion protein, which can induce the production of HtsA protein and SLOdm protein specific IgG antibodies in serum, has strong immunogenicity; can induce high level IgG1 and IgG2a antibody titers, and the humoral immune response when inducing the production of SLOdm protein specific IgG antibodies in serum is stronger than that of a single protein; can promote the healing of mouse skin wounds, reduce the bacterial content at the infected site of mouse skin, reduce the inflammation at the infected wound site of mouse skin, and accelerate wound healing.

[0006] The present application also provides a biological material related to the above-mentioned fusion protein.

[0007] The present application also provides the use of the above-mentioned fusion protein or the above-mentioned biological material in the preparation of a group A streptococcus vaccine product.

[0008] The present application also provides a recombinant vaccine.

[0009] The present application also provides the use of the above-mentioned fusion protein or biological material or recombinant vaccine in the preparation of a drug for preventing diseases caused by group A streptococcus.

[0010] In a first aspect of the present application, a fusion protein is provided, comprising the following elements:

[0011] (1) a heme transport lipoprotein;

[0012] (2) a double-site mutated streptolysin protein, the amino acid sequence of which is shown in SEQ ID NO: 4, 277-817;

[0013] The heme transport lipoprotein is connected to the double-site mutated streptolysin protein through a linker.

[0014] The fusion protein according to the embodiments of the present application has at least the following beneficial effects:

[0015] The fusion protein of the present application is obtained by fusing recombinantly a hematin transport lipoprotein (HtsA) and a streptolysin O protein containing double mutation sites (SLOdm), and can induce the production of HtsA protein and SLOdm protein specific IgG antibodies in serum, has strong immunogenicity; can induce high level IgG1 and IgG2a antibody titers, and the humoral immune response in inducing the production of SLOdm protein specific IgG antibodies in serum is stronger than that of single protein; can promote the healing of mouse skin wounds, reduce the bacterial content at the infected site of mouse skin, reduce the inflammation at the infected wound site of mouse skin, and accelerate wound healing. In addition, the HtsA-SLOdm fusion protein has high safety and no hemolytic activity, and can be used for preparing group A streptococcus vaccine.

[0016] In some embodiments of the present application, the amino acid sequence represented by SEQ ID NO: 4 at positions 277-817 is as follows:

[0017] ESNKQNTASTETTTTNEQPKPESSELTTEKAGQKTDDMLNSNDMIKLAPKEMPLESAEKEEKKSEDKKKSEEDHTEEINDKIYSLNYNELEVLAKNGETIENFVPKEGVKKADKFIVIERKKKNINTTPVDISIIDSVTDRTYPAALQLANKGFTENKPDAVVTKRNPQKIHIDLPGMGDKATVEVNDPTYANVSTAIDNLVNQWHDNYSGGNTLPARTQYTESMVYSKSQIEAALNVNSKILDGTLGIDFKSISKGEKKVMIAAYKQIFYTVSANLPNNPADVFDKSVTFKELQRKGVSNEAPPLFVSNVAYGRTVFVKLETSSKSNDVEAAFSAALKGTDVKTNGKYSDILENSSFTAVVLGGDAAEHNKVVTKDFDVIRNVIKDNATFSRKNLAYPISYTSVFLKNNKIAGVNNRTEYVETTSTEYTSGKINLSHQGAYVAQYEILWDEINYDDKGKEVITKRRWDNNWYSKTSPFSTVIPLGANSRNIRIMARECTGLAFEWWRKVIDERDVKLSKEINVNISGSTLSPYGSITYK*.

[0018] wherein “*” is a stop codon.

[0019] In some embodiments of the present application, the hematin transport lipoprotein is a streptococcal hematin transport lipoprotein.

[0020] In some embodiments of the present application, the heme transport lipocalin comprises an amino acid sequence as set forth in SEQ ID NO: 4 from 1 to 266.

[0021] In some embodiments of the present application, the heme transport lipocalin comprises an amino acid sequence as set forth in SEQ ID NO: 4 from 1 to 266.

[0022] KETEQQRIVATSVAVVDICDRLNLDLVGVCDSKLYTLPKRYDAVKRVGLPMNPDIELIASLKPTWILSPNSLQEDLEPKYQKLDTEYGFLNLRSVEGMYQSIDDLGNLFQRQQEAKELRQQYQDYYRAFQAKRKGKKKPKVLILMGLPGSYLVATNQSYVGNLLDLAGGENVYQSDEKEFLSANPEDMLAKEPDLILRTAHAIPDKVKVMFDKEFAENDIWKHFTAVKEGKVYDLDNTLFGMSAKLNYPEALDTLTQLFDHVGDHP.

[0023] In some embodiments of the present application, the amino acid sequence of the linker is (GGGGS) n or (GGGS) m , and n and m are independently selected from positive integers from 1 to 5.

[0024] In some embodiments of the present application, the linker can be (GGGGS)5, (GGGGS)4, (GGGGS)3, (GGGGS)2, (GGGS)5, (GGGS)4, or (GGGS)3, etc.

[0025] In some embodiments of the present application, the amino acid sequence of the linker is as set forth in SEQ ID NO: 4 from 267 to 276.

[0026] In some embodiments of the present application, the amino acid sequence of the fusion protein is as set forth in SEQ ID NO: 4.

[0027] In some embodiments of the present application, the fusion protein further comprises a tag.

[0028] In some embodiments of the present application, the tag is connected to the middle of the fusion protein and / or the N terminus or / and the C terminus.

[0029] According to some embodiments of the present application, the tag sequence comprises at least one of the tag sequences facilitating solubilization, purification and detection of the fusion protein.

[0030] It can be understood that the fusion protein of the present application can be linked to one or more tag sequences; the plurality of tag sequences can comprise a combination of a plurality of identical tag sequences, or a combination of a plurality of different tag sequences.

[0031] For example, the tag facilitating solubilization of the fusion protein includes but is not limited to a nus tag sequence or a maltose binding protein tag sequence; the tag facilitating purification of the fusion protein includes but is not limited to a strep tag sequence, a His tag sequence, a GST tag sequence, a pelB signal tag sequence or an ompA signal tag sequence; the tag facilitating detection of the fusion protein includes but is not limited to a horseradish peroxidase (HRP) tag sequence, a β-galactosidase tag sequence, a luciferase tag sequence, a green fluorescent protein (GFP) tag sequence, a HcRed tag sequence, a DsRed tag sequence or a cyan fluorescent protein (CFP) tag sequence.

[0032] In some embodiments of the present application, the tag can be a His tag sequence.

[0033] In a second aspect of the present application, a biological material related to the fusion protein of any one of the first aspect is provided, and the biological material is any one of A1) to A4):

[0034] A1), a nucleic acid molecule encoding the fusion protein of any one of the first aspect;

[0035] A2), an expression cassette containing the nucleic acid molecule of A1);

[0036] A3), a recombinant vector containing the nucleic acid molecule of A1) or the expression cassette of A2);

[0037] A4), a recombinant biological cell containing the nucleic acid molecule of A1), the expression cassette of A2) or the recombinant vector of A3).

[0038] In some embodiments of the present application, the nucleic acid molecule of A1) can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA, etc.

[0039] In some embodiments of the present application, the nucleic acid molecule of A1) comprises a nucleotide sequence as shown in SEQ ID NO: 1 from 1 to 798, and a nucleotide sequence as shown in SEQ ID NO: 1 from 829 to 2451.

[0040] In some embodiments of the present application, the nucleotide sequence of the nucleic acid molecule encoding the fusion protein in A1) comprises:

[0041] A11), the nucleotide sequence as shown in SEQ ID NO: 1; or

[0042] A12), the nucleotide sequence as shown in A11) with one or several substitutions and / or deletions and / or additions of nucleotides, and the nucleotide sequence having the same function as the nucleic acid molecule as shown in A11); or

[0043] A13), the nucleotide sequence having at least 80% homology with the nucleotide sequence as shown in A11), and the nucleotide sequence having the same function as the nucleic acid molecule as shown in A11).

[0044] In some embodiments of the present application, the expression cassette in A2) refers to the DNA capable of expressing the fusion protein in the host cell, which can include not only the promoter initiating the transcription of the DNA molecule encoding the fusion protein, but also the terminator terminating the transcription of the DNA molecule encoding the fusion protein. Further, the expression cassette can also include the replication initiation site, the transcription initiation sequence, the enhancer sequence, the selection element or the reporter gene.

[0045] In some embodiments of the present application, the recombinant vector in A3) can be a plasmid, a cosmid, a bacteriophage or a viral vector. The recombinant vector can be a cloning vector or an expression vector.

[0046] In some embodiments of the present application, the backbone vector of the recombinant vector is pBAD-HisA vector.

[0047] In some embodiments of the present application, the recombinant vector in A3) can be specifically the recombinant plasmid obtained by inserting the nucleic acid molecule into pBAD-HisA vector. The pBAD-HisA vector can stably and efficiently express the fusion protein with good specificity and high sensitivity.

[0048] In some embodiments of the present application, the recombinant biological cell in A4) can be bacteria (such as Escherichia coli or Bacillus subtilis, etc.), algae, fungi (such as yeast or Aspergillus, etc.), insect cells (such as S2 fruit fly cells or Sf9 cells, etc.) or animal cells (such as CHO cells, COS cells, NSO cells, HeLa cells, BHK cells or HEK 293T cells, etc.). The recombinant biological cell does not include reproductive material.

[0049] In a third aspect of the present application, the fusion protein of any one of the first aspect or the biological material of any one of the second aspect is provided for use in the preparation of a group A streptococcus vaccine product.

[0050] In a fourth aspect of the present application, a recombinant vaccine is provided, comprising the fusion protein of any one of the first aspect or the biomaterial of any one of the second aspect.

[0051] In some embodiments of the present application, the recombinant vaccine further comprises a pharmaceutically acceptable excipient.

[0052] In some embodiments of the present application, the pharmaceutically acceptable excipient comprises at least one of a pharmaceutically acceptable carrier, excipient, diluent or adjuvant.

[0053] In some embodiments of the present application, the carrier comprises a viral vector (adenovirus or lentivirus) or a nanomaterial.

[0054] In some embodiments of the present application, the diluent can comprise at least one of physiological saline, PBS, glucose, water, sterile isotonic aqueous buffer.

[0055] In some embodiments of the present application, the adjuvant comprises at least one of alum, other compounds of aluminum, Freund's complete adjuvant (FCA), poly-ICLC, STING agonist, imiquimod.

[0056] In some embodiments of the present application, the recombinant vaccine is a group A streptococcus vaccine.

[0057] In some embodiments of the present application, the administration mode of the recombinant vaccine comprises at least one of subcutaneous, intramuscular, intradermal, intravenous administration routes.

[0058] In a fifth aspect of the present application, the fusion protein of any one of the first aspect or the biomaterial of any one of the second aspect or the recombinant vaccine of any one of the fourth aspect is used for preparing a medicament for preventing diseases caused by group A streptococcus.

[0059] In some embodiments of the present application, the diseases caused by group A streptococcus comprise skin inflammation, pharyngitis, impetigo, necrotizing fasciitis, streptococcal toxic shock syndrome, sepsis, rheumatic heart disease and acute streptococcal glomerulonephritis.

[0060] Other features and advantages of the present application will be set forth in the following description. BRIEF DESCRIPTION OF DRAWINGS

[0061] The present application will be further described with reference to the following drawings and examples, wherein:

[0062] Figure 1 A schematic diagram for constructing the HtsA-SLOdm fusion protein of the present application;

[0063] Figure 2Purification and identification results of the expression of the HtsA-SLOdm fusion protein of the application, wherein M is Marker, 1 is a bacterial whole protein lysate, 2 is a eluted impure protein, 3-6 are eluted and concentrated with 10 mM, 25 mM, 50 mM and 100 mM imidazole Ni column equilibration buffer, respectively;

[0064] Figure 3 Hemolytic activity detection results of the HtsA-SLOdm fusion protein of the application, wherein A is a hemolysis experiment chart; B is a hemolysis rate line chart;

[0065] Figure 4 Antigen-specific antibody detection results in the serum of the mouse immunized with the HtsA-SLOdm fusion protein vaccine of the application, wherein A is the HtsA protein-specific IgG antibody level in the serum of the mouse immunized for 7 days, 21 days and 35 days; B is the SLOdm protein-specific IgG antibody level in the serum of the mouse immunized for 7 days, 21 days and 35 days; C is the anti-HtsA antibody IgG1 titer in the serum of the mouse immunized with the HtsA protein and the HtsA-SLOdm fusion protein; D is the anti-SLOdm antibody IgG1 titer in the serum of the mouse immunized with the SLOdm protein and the HtsA-SLOdm fusion protein; E is the anti-HtsA IgG2a titer in the serum of the mouse immunized with the HtsA protein and the HtsA-SLOdm fusion protein; F is the anti-SLOdm antibody IgG2a titer in the serum of the mouse immunized with the SLOdm protein and the HtsA-SLOdm fusion protein; G is the ratio of anti-HtsA antibody IgG1 / IgG2a; H is the ratio of anti-SLOdm antibody IgG1 / IgG2a (n=7, *p<0.05, **p<0.01, ***p<0.001, #p<0.05 indicates compared with the HtsA group, the SLOdm group and the HtsA+SLOdm group);

[0066] Figure 5 Wound area, body weight change, survival rate of the mouse after immunization with the HtsA-SLOdm fusion protein vaccine of the application, wherein A is a representative mouse wound chart; B is the wound area of the mouse; C is the body weight change of the mouse; D is the survival rate of the mouse;

[0067] Figure 6 Bacterial content of the skin wound of the mouse after immunization with the HtsA-SLOdm fusion protein vaccine of the application, wherein A is the bacterial content detection result of the skin wound of the mouse, B is a statistical result;

[0068] Figure 7 Pathological conditions of the skin infection wound of the mouse after immunization with the HtsA-SLOdm fusion protein vaccine of the application, the scale is 50 μm. DETAILED DESCRIPTION

[0069] The concept and the technical effects of the present application will be described clearly and completely in combination with the embodiments below, so as to fully understand the objects, features and effects of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present application without creative efforts are within the protection scope of the present application.

[0070] The terms "preferably", "more preferably" and the like in the description of the present application refer to embodiments of the present application which can provide certain benefits under certain circumstances. However, other embodiments can also be preferred under the same or other circumstances. In addition, the description of one or more preferred embodiments does not imply that other embodiments are not useful, nor does it imply that these other embodiments are excluded from the scope of the present application.

[0071] When a numerical range is disclosed herein, the range is to be construed as continuous, and includes the minimum and maximum values of the range, as well as every value between the minimum and maximum values. Further, when ranges are provided, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein are to be understood as encompassing any and all sub-ranges subsumed therein. Still further, when a range, a list, or a group of items is provided, the range, list, or group of items can be combined. In other words, unless otherwise indicated, all ranges disclosed herein are to be understood as encompassing any and all sub-ranges subsumed therein.

[0072] In the description of the present application, the term "and / or" includes all and any combination of one or more of the associated listed items.

[0073] In the description of the present application, the description of the terms "one embodiment", "some embodiments", "certain embodiments", "example", "specific example", or "some examples" and the like means that a particular feature, structure, material or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present application. The illustrative appearances of the above terms in various places in the specification are not necessarily referring to the same embodiment or example of the present application. Furthermore, the particular features, structures, materials, or characteristics can be combined in any suitable manner in one or more embodiments or examples.

[0074] In the description of the present application, the term "fusion protein" refers to a protein obtained by artificially connecting two or more genes encoding functional proteins together, and then expressing the proteins. The protein product obtained by connecting the coding regions of two or more genes end to end under artificial conditions, and then expressing the genes under the control of a regulatory sequence is a fusion protein.

[0075] In the description of the present application, the term "linker" refers to a short peptide used to connect two functional proteins, and the length thereof can be adjusted according to actual needs. The linker can provide a certain flexibility for each functional protein in the fusion protein, so that each functional protein can exert its respective function.

[0076] In the description of the present application, the term "amino acid" refers to the basic unit constituting a protein, which endows the protein with a specific molecular structure form, so that the molecule has biochemical activity. For example, the "amino acid" used in the present application includes the following 20 natural amino acids: alanine (Ala or A), glycine (Gly or G), isoleucine (Ile or I), asparagine (Asn or N), arginine (Arg or R), lysine (Lys or K), cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), glutamine (Gln or Q), histidine (His or H), leucine (Leu or L), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), valine (Val or V), and tyrosine (Tyr or Y).

[0077] Unless otherwise specified in the examples, the conventional conditions or the conditions recommended by the manufacturer are used. Unless otherwise specified, the reagents or instruments used are conventional products that can be obtained by commercial purchase.

[0078] Inventive Concept:

[0079] The present application is based on the finding that the hemoglobin transport lipoprotein HtsA and the streptolysin double-site mutant SLOdm protein are promising two Group A streptococcus vaccine candidate antigens. These two proteins are highly conserved in almost all types of Group A streptococcus, and theoretically, a vaccine against HtsA protein and SLOdm protein will cover almost all molecular types of Group A streptococcus. Immunization with HtsA protein or streptolysin double-site mutant SLOdm protein alone can produce high IgG antibody titers, and can improve the survival rate of mice in a mouse bacteremia model, and produce a better protective effect on mice. However, the immunoprotection effect caused by immunization with HtsA protein or SLOdm protein alone is limited, specifically, the resistance to Group A streptococcus is limited. In order to achieve better protection, the present application constructs a fusion expression vector pBAD-htsA-slodm of HtsA and SLOdm protein, expresses and purifies the HtsA-SLOdm fusion protein, detects the immunogenicity of the HtsA-SLOdm fusion protein, and evaluates the protective effect thereof as a vaccine using a mouse skin infection model.

[0080] The experimental process and results of the present application are described in detail below in connection with specific examples.

[0081] Example 1: Expression and purification of HtsA-SLOdm fusion protein

[0082] 1. Obtain the HtsA-SLOdm fusion protein expression vector:

[0083] First, find the htsA and slo gene sequences in the MGAS5005 strain of group A streptococcus from the GenBank database, use a point mutation kit to mutate the proline at position 427 to leucine and the tryptophan at position 535 to phenylalanine in the SLO protein in the recombinant expression plasmid pGEX-slo, and construct a double-site mutant recombinant expression plasmid pGEX-slodm; connect the htsA gene sequence (798 bp) without the signal peptide (1-28 AA) and the slodm gene sequence (1623 bp) without the signal peptide (1-31 AA) together through a connecting chain Gly4SerGly4Ser (30 bp) to form a fusion gene htsA-slodm (2451 bp, synthesized by Shanghai Shengong Bioengineering Co., Ltd.), wherein the construction schematic of the HtsA-SLOdm fusion protein is shown in Figure 1 The nucleotide sequence of the fusion gene htsA-slodm is shown below:

[0084] AAAGAGACTGAACAGCAGAGAATTGTAGCCACTTCGGTTGCTGTGGTTGATATCTGTGACCGTTTAAATTTAGACCTCGTTGGGGTTTGTGATAGTAAATTATATACCCTTCCTAAACGCTATGATGCTGTTAAGCGTGTGGGTTTACCCATGAATCCTGATATAGAGTTGATTGCTTCTTTGAAACCAACTTGGATTTTGAGTCCCAATTCTTTACAAGAAGATTTGGAACCCAAGTATCAAAAATTGGATACTGAGTATGGTTTTTTGAACTTACGAAGTGTTGAGGGCATGTACCAGTCCATTGATGATTTAGGGAACCTTTTCCAACGTCAACAAGAAGCAAAAGAATTGCGCCAGCAATACCAGGACTATTATCGTGCTTTCCAAGCTAAACGTAAGGGGAAGAAAAAGCCTAAAGTGCTTATTCTTATGGGCTTGCCAGGTAGTTATTTGGTGGCGACGAACCAATCTTATGTAGGGAATCTTTTGGACTTGGCAGGTGGTGAGAATGTTTATCAGTCAGATGAGAAAGAATTTCTATCAGCTAATCCTGAAGACATGCTGGCTAAGGAGCCTGACTTGATTTTACGAACAGCTCATGCCATTCCAGACAAGGTAAAAGTGATGTTTGACAAAGAATTTGCTGAAAATGATATTTGGAAACATTTTACGGCAGTCAAGGAAGGGAAAGTCTATGATTTGGACAATACCCTGTTTGGCATGAGTGCTAAATTGAACTACCCAGAAGCCTTGGACACCTTAACACAGCTTTTTGACCACGTGGGAGATCATCCG GGCGGCGGCGGCAGCGGCGGCGGCGGCAGC

[0085] In the above nucleotide sequence, the underlined sequence is the linker sequence (i.e., the base sequence shown in 799-828), and the bold sequence is the mutation site.

[0086] Then, the fusion gene htsA-slodm synthesized above is used as a template, htsA-slodm-F (5'-GGCGGC GAGCT C AAAGAGACTGAACAGCAGAG-3' (SEQ ID NO: 2), and the underlined part is the Sac I enzyme cutting site) and htsA-slodm-R (5'-CCG GAATTC TTAGTTTTCACTTGATAAGATTG-3' (SEQ ID NO: 3), and the underlined part is the EcoR I enzyme cutting site) are primers, and the htsA-slodm fusion gene is amplified by PCR, and the htsA-slodm fusion gene is connected to the pBAD-HisA vector to construct the prokaryotic expression vector pBAD-htsA-slodm.

[0087] 2. Expression and purification of HtsA-SLOdm fusion protein

[0088] The prokaryotic expression vector pBAD-HtsA-SLOdm constructed above is transfected into E. coli, and high-purity His-HtsA-SLOdm fusion protein is obtained by using L-arabinose induction and Ni-NTA affinity column chromatography. The specific method is as follows:

[0089] 10 mL of activated bacterial solution is added to 300 mL of LB medium containing Amp, and cultured at 37°C and 200 rpm until OD600 is about 0.6, then the remaining bacterial solution is added with L-(+) arabinose to a final concentration of 0.04%; 37°C, 200 rpm continues to induce 6h; then the bacterial solution induced for expression is centrifuged at 4000 rpm and 4°C for 10 min, the bacterial body is collected, washed twice with 25 mL of 1×PBS, and finally the bacterial body is resuspended with 25 mL of Ni column equilibration buffer and stored in a -80°C refrigerator; the bacterial body stored at -80°C is repeatedly frozen and thawed for 3 times, then ultrasonicated on ice for 25 min, and the ultrasonic conditions are set to 35% power, 5s on and 5s off, and the ultrasonic is performed until the bacterial solution becomes clear and transparent; the bacterial solution after ultrasonication is centrifuged at 10000 rpm and 4°C for 30 min to remove the bacterial body precipitate, and the supernatant is collected for use, and 30 μL of the supernatant is taken for SDS-PAGE.

[0090] The recombinant protein is purified by using the Ni-NTA affinity chromatography purification column of Beijing ZhiShiJin Company. The specific steps are as follows:

[0091] (1) After mixing the Ni-NTA resin, 1.5 mL of the resin was taken and left at room temperature for 30 min;

[0092] (2) The column was washed with 10 times the column volume of dd H2O;

[0093] (3) The column was equilibrated with 10 times the column volume of the equilibration buffer;

[0094] (4) The collected supernatant sample was slowly loaded into the chromatography column;

[0095] (5) The resin was washed with 10 times the column volume of the equilibration buffer and the sample effluent was collected;

[0096] (6) The nickel column was eluted with 10 mL of buffer containing 10 mM imidazole, 25 mM imidazole, 50 mM imidazole, 100 mM imidazole and 500 mM imidazole, respectively, and the eluate was collected. The content and purity of His-HtsA-SLOdm were detected by 12% SDS-PAGE;

[0097] (7) The column was washed with 10 times the column volume of the equilibration buffer and 10 times the column volume of dd H2O, and 5 mL of 20% ethanol was added to the column, which was stored at 4°C.

[0098] The expression, purification and identification results of the HtsA-SLOdm fusion protein are shown in Figure 2 , where M is Marker, 1 is the bacterial whole protein lysate, 2 is the impure protein eluted, and 3-6 are the eluted concentrated containing 10 mM, 25 mM, 50 mM and 100 mM imidazole Ni column equilibration buffer, respectively.

[0099] The amino acid sequence of the HtsA-SLOdm fusion protein obtained above was analyzed and identified by sequencing, and the results showed that the amino acid sequence was as follows:

[0100] KETEQQRIVATSVAVVDICDRLNLDLVGVCDSKLYTLPKRYDAVKRVGLPMNPDIELIASLKPTWILSPNSLQEDLEPKYQKLDTEYGFLNLRSVEGMYQSIDDLGNLFQRQQEAKELRQQYQDYYRAFQAKRKGKKKPKVLILMGLPGSYLVATNQSYVGNLLDLAGGENVYQSDEKEFLSANPEDMLAKEPDLILRTAHAIPDKVKVMFDKEFAENDIWKHFTAVKEGKVYDLDNTLFGMSAKLNYPEALDTLTQLFDHVGDHP GGGGSGGGGSESNKQNTASTETTTTNEQPKPESSELTTEKAGQKTDDMLNSNDMIKLAPKEMPLESAEKEEKKSEDKKKSEEDHTEEINDKI YSLNYNELEVLAKNGETIENFVPKEGVKKADKFIVIERKKKNINTTPVDISIIDSVTDRTYPAALQLANKGFTENKPDA VVTKRNPQKIHIDLPGMGDKATVEVNDPTYANVSTAIDNLVNQWHDNYSGGNTLPARTQYTESMVYSKSQIEAALN VNSKILDGTLGIDFKSISKGEKKVMIAAYKQIFYTVSANLPNNPADVFDKSVTFKELQRKGVSNEAPPLFVSNVAYGRT VFVKLETSSKSNDVEAAFSAALKGTDVKTNGKYSDILENSSFTAVVLGGDAAEHNKVVTKDFDVIRNVIKDNATFSRKNL AYPISYTSVFLKNNKIAGVNNRTEYVETTSTEYTSGKINLSHQGAYVAQYEILWDEINYDDKGKEVITKRRWDNNWY SKTSPFSTVIPLGANSRNIRIMARECTGLAFEWWRKVIDERDVKLSKEINVNISGSTLSPYGSITYK* (SEQ ID NO: 4).

[0101] wherein "*" is a stop codon, the linker is underlined, and the mutated amino acid is bolded. The sequencing result is consistent with the expectation, and the HtsA-SLOdm fusion protein is successfully obtained.

[0102] Example 2: Hemolytic activity detection

[0103] The hemolytic activity of the HtsA-SLOdm fusion protein, SLOdm protein and HtsA protein described above was detected. The inventors found in previous studies that the wild-type SLO protein has hemolytic effect on human red blood cells, and is not suitable for direct use as an antigen. The hemolytic effect of the double-site mutated SLOdm protein on red blood cells is significantly reduced. In order to detect whether the HtsA-SLOdm fusion protein also has low cell hemolysis, the inventors detected the hemolytic activity of the HtsA-SLOdm fusion protein, and the SLOdm protein and HtsA protein, and the specific detection method is as follows:

[0104] The ACK group (positive control group), PBS (negative control group) and different concentrations of HtsA protein group, SLOdm protein group and HtsA-SLOdm fusion protein group (experimental group) were set respectively, and the concentration gradient was set as 20 μg / mL, 100 μg / mL, 200 μg / mL and 500 μg / mL. Then the collected human red blood cells were diluted into 2% human red blood cell suspension with PBS, and different concentrations of SLOdm protein and HtsA-SLOdm fusion protein were added to the cell suspension respectively, with the addition of PBS as the negative control and the addition of red blood cell lysate as the positive control, and incubated at 37°C for 30 minutes; centrifuged at 1000g for 1 minute, and carefully aspirated 100 μL of supernatant into a new 96-well plate, and used the enzyme label instrument to detect the absorbance value at 540nm, and analyzed the data to evaluate whether it had hemolytic activity.

[0105] The detection results are shown in Figure 3 As shown in the table, only the red blood cell lysate group (ACK, positive control group) has a higher absorbance value, and the OD540 values of the PBS (negative control group) and different concentrations of HtsA protein, SLOdm protein and HtsA-SLOdm fusion protein experimental groups are all lower, indicating that only the positive control group ACK (red blood cell lysate) has hemolysis phenomenon. Further, when the protein concentration of the experimental group is 500 μg / mL, the hemolysis rate of HtsA protein is 1.57%, the hemolysis rate of SLOdm protein is 0.41%, and the hemolysis rate of HtsA-SLOdm fusion protein is 0.63%, indicating that the fusion of HtsA protein and SLOdm protein at high concentration helps to further reduce its hemolysis rate (almost no hemolytic activity).

[0106] The above results show that the HtsA-SLOdm fusion protein has almost no hemolytic activity and can be used for subsequent mouse subcutaneous immunization experiments.

[0107] Example 3: Immunogenicity detection of HtsA-SLOdm fusion protein vaccine

[0108] The level of specific antibodies in serum is an important indicator for evaluating the immunogenicity of the vaccine, among which the total IgG antibody can reflect the intensity of the overall humoral immune response induced by the vaccine, and the IgG1 and IgG2a subclass antibodies further reveal the type tendency of the immune response (humoral immunity or cellular immunity).

[0109] To evaluate the immunogenicity of the HtsA-SLOdm fusion protein vaccine, this embodiment constructs a mouse back subcutaneous multi-point immunization model, detects the titers of HtsA and SLOdm specific IgG antibodies and their subtypes IgG1 and IgG2a in the serum of immunized mice by ELISA technology, and analyzes their trends. Among them:

[0110] 1. Construction of multiple point immunization model on the back of mice:

[0111] Take 200 μL PBS or 200 μL PBS (containing 10 μL aluminum adjuvant and 25 μg protein) and inject through the skin on the back of the mice, inject in 4 points, about 50 μL sample per point.

[0112] 2. Immunization treatment of fusion protein vaccine:

[0113] Mix the recombinant protein with aluminum adjuvant and then immunize BALB / c mice through multiple points on the back of the mice, the experiment is divided into 5 groups:

[0114] ① Mock (PBS + aluminum adjuvant) group;

[0115] ② HtsA protein + aluminum adjuvant immunization group;

[0116] ③ SLOdm protein + aluminum adjuvant immunization group;

[0117] ④ HtsA protein + SLOdm protein + aluminum adjuvant immunization group;

[0118] ⑤ HtsA-SLOdm fusion protein + aluminum adjuvant immunization group;

[0119] Each group has 15 mice, 10 μL aluminum adjuvant per mouse, 25 μg protein per mouse for single protein immunization group, 25 μg HtsA and SLOdm protein per mouse for combined immunization group, 25 μg protein per mouse for HtsA-SLOdm fusion protein immunization group, a total of 3 immunizations, with an interval of 14 days between each immunization. Collect the mouse serum before immunization (0 days) and 7 days, 21 days and 35 days after immunization, and use ELISA method to detect the IgG antibody titer in the serum to determine whether the HtsA-SLOdm fusion protein has immunogenicity.

[0120] The detection results are shown in Figure 4 , the HtsA-SLOdm fusion protein can induce the production of HtsA protein and SLOdm protein specific IgG antibodies in the serum, and is significantly higher than the PBS group, after three immunizations, the antibody titer against HtsA and SLOdm protein in the mouse serum is as high as 1 / 1562500~1 / 312500 Figure 4 A and B), indicating that the HtsA-SLOdm fusion protein has strong immunogenicity.

[0121] It is worth noting that compared with the HtsA + SLOdm protein combined immunization group, the HtsA-SLOdm fusion protein can produce higher IgG antibody titer against HtsA or SLOdm after 7 days of the first immunization Figure 4indicated that the immunogenicity of the HtsA-SLOdm fusion protein was superior to that of the simple mixture of the HtsA protein and the SLOdm protein.

[0122] In addition, the Th2 type (involved in humoral immunity) and Th1 type (involved in cellular immunity) IgG subclasses IgG1 and IgG2a antibodies were detected to evaluate the type of immune response induced by the vaccine. The results are shown in Figs. 6G and 6H. Figure 4 As shown in Figs. 6C to 6F, the HtsA protein, the SLOdm protein and the HtsA-SLOdm fusion protein can induce high levels of IgG1 and IgG2a antibody titers. The titers of the HtsA- and SLOdm-specific IgG antibodies in the serum and the IgG1 / IgG2a ratio are both greater than 2, indicating that the HtsA-SLOdm fusion protein mainly induces humoral immunity. Figure 4 As shown in Figs. 6G and 6H, the HtsA-SLOdm fusion protein induces a stronger humoral immune response than the SLOdm single protein in inducing the production of SLOdm protein-specific IgG antibodies in the serum.

[0123] The above results show that the HtsA-SLOdm fusion protein of the present application has strong immunogenicity and is superior to the simple mixture of the HtsA protein and the SLOdm protein. In addition, the detection results of IgG1 / IgG2a antibodies show that the HtsA-SLOdm fusion protein mainly induces humoral immunity, and the humoral immune response in inducing the production of SLOdm protein-specific IgG antibodies in the serum is stronger than that of the SLOdm single protein.

[0124] Example 4: Evaluation of the protective effect of the HtsA-SLOdm fusion protein vaccine

[0125] In this embodiment, a challenge experiment model (mouse skin infection model) was constructed to evaluate the immune protection effect of the HtsA-SLOdm fusion protein vaccine on mice.

[0126] 1. Construction of a mouse skin infection model and immunization

[0127] The immunization was performed according to the method of the above-mentioned Example 3, and the challenge experiment of group A streptococcus was performed one week after the third immunization. Specifically, the group A streptococcus MGAS5005 strain was inoculated in 0.5% THYE medium one day in advance, and cultured at 37°C, 5% CO2 overnight. After recovery, the bacteria were inoculated in 0.5% THYE medium, and cultured at 37°C, 5% CO2 for 2-3 h. When OD600=0.5, the bacteria were collected by centrifugation, and 1×10 8CFU / mL bacterial solution for later use. Ten mice from each group were selected and after the mice were anesthetized, the right abdominal skin of the mice was punched and 10 μL of PBS diluted bacterial solution containing 1 x 10 7 7 The skin tissues of the mice infected with bacteria for 48 h were collected, fixed at 4°C overnight, and subjected to HE staining and Gram staining. The changes in the bacterial content in the skin abscesses of the mice and the pathological conditions were compared between the different groups. The size of the abscesses and the body weight of the mice were observed and measured for 14 consecutive days.

[0128] 2. Survival rate, wound area, and body weight of the mice

[0129] Seven days after the third immunization, 10 mice from each of the control group, the HtsA group, the SLOdm group, the HtsA+SLOdm group, and the HtsA-SLOdm fusion protein group were randomly selected and the right abdominal skin of the mice was punched and 10 μL of PBS diluted bacterial solution containing 1 x 10 7 CFUA group of streptococcal bacteria. The wound of the mice was observed every day until the first mouse recovered from the skin wound. The body weight, survival rate, and wound area of the mice were measured every day.

[0130] The results showed that the wound area of the HtsA-SLOdm fusion protein immunization group was smaller than that of the PBS control group. The skin wound of the mice in the HtsA-SLOdm fusion protein immunization group basically recovered on the 9th day after infection (as shown in A and B of Figure 5 ). The body weight of the mice was higher than that of the control group (as shown in C of Figure 5 ). On the 4th day after the mice were infected with bacteria, the control group of mice began to die, while the HtsA-SLOdm fusion protein immunization group did not die until the 10th day after the challenge, with a survival rate of 100%. In comparison, the survival rate of the control group was only 30% on the 10th day (as shown in D of Figure 5 ).

[0131] The above results show that immunization with the HtsA-SLOdm fusion protein can promote the healing of skin wounds in mice and has a certain immunoprotective effect on mice.

[0132] 3. Bacterial content in the skin wound of the mice

[0133] After the skin wound of the mice was infected with group A streptococcal bacteria for 48 h, the bacterial content in the skin wound was measured. The mice were anesthetized and sacrificed, and the skin tissue at the infection site was collected. Half of the tissue was ground in sterile 1 x PBS, and 10 μL of the ground solution was diluted 10-fold and plated.

[0134] The results are shown in Table 6, which shows that the bacterial content at the wound of the mice immunized with the HtsA-SLOdm fusion protein is significantly lower than that of the PBS control group, indicating that immunization with the HtsA-SLOdm fusion protein reduces the bacterial content at the site of skin infection in mice.

[0135] 4. Detection of pathological conditions of mouse skin infection wound:

[0136] The expression of F4 / 80 and CD31 at the wound of mouse skin infection was detected by immunohistochemical staining, and the wound of mouse skin infection was also subjected to Masson staining, Gram staining and HE staining. The specific method is as follows:

[0137] First, 48 hours after the construction of the mouse group A streptococcus skin infection model, 5 mice were selected from each group and sacrificed by cervical dislocation, and the skin tissue at the infection site of the mice was collected. The skin tissue was fixed with 4% paraformaldehyde at room temperature for 24 hours, and then subjected to HE staining, Masson's staining and IHC staining of F4 / 80 and CD31.

[0138] (1) HE staining:

[0139] The paraffin section was deparaffinized, and then the section was subjected to hematoxylin staining, eosin staining and dehydration and mounting. Finally, microscopic examination was performed, and the images were collected and analyzed.

[0140] (2) Masson staining:

[0141] The section was prepared, immersed in Masson A solution, mixed and dyed with equal volumes of Masson B solution and Masson C solution, differentiated, immersed in Masson D solution, immersed in Masson E solution, dyed with Masson F solution, mounted, and finally subjected to microscopic examination. The images were collected and analyzed. The specific operation steps were performed according to the instructions of the kit (Wuhan Sivier Biological Technology Co., Ltd., Catalog No.: G1006-20ML).

[0142] (3) IHC staining of F4 / 80 and CD31:

[0143] The paraffin section was deparaffinized, the tissue section was subjected to antigen repair, washing, blocking, incubation with primary antibody, incubation with secondary antibody, and then the color developing solution was added. The nucleus was then restained and dehydrated and mounted, and finally subjected to microscopic examination. The images were collected and analyzed. The specific operation steps were performed according to the instructions of the kit.

[0144] (4) Gram staining:

[0145] The paraffin section was deparaffinized, primary dyed, mordant dyed, decolorized, restained, dehydrated and mounted, and finally examined under a microscope.

[0146] The detection results are shown in Table 6. Figure 7The results show that the F4 / 80 of the PBS group is obviously more than that of the HtsA-SLOdm fusion protein immunization group and the other protein immunization group, indicating that the PBS group has more macrophages and higher inflammation degree at the skin infection wound than the HtsA-SLOdm fusion protein immunization group and the other protein immunization group; and the CD31 protein expression at the skin infection wound of the HtsA-SLOdm fusion protein immunization group and the other protein immunization group is higher than that of the PBS group, indicating that the HtsA-SLOdm fusion protein immunization group and the other protein immunization group have stronger endothelial neogenesis than the PBS group.

[0147] In addition, the Masson staining results show that the collagen fibers at the skin infection wound of the HtsA-SLOdm fusion protein immunization group and the other protein immunization group are obviously more than that of the PBS group. The gram staining experiment can dye the gram-positive bacteria into purple, and the results show that the bacterial content of the skin of the HtsA-SLOdm fusion protein group is obviously lower than that of the PBS group.

[0148] The above results show that the immunization of the HtsA-SLOdm fusion protein can protect the mice, reduce the inflammation at the skin infection wound of the mice, and accelerate the wound healing.

[0149] In summary, the present application provides a HtsA-SLOdm fusion protein and its application in preparing a group A streptococcus vaccine. The HtsA-SLOdm fusion protein is obtained by fusing and recombining a heme transport lipoprotein HtsA and a streptococcal hemolysin double-site mutant SLOdm protein. The HtsA-SLOdm fusion protein can induce the production of HtsA protein and SLOdm protein specific IgG antibodies in serum, has strong immunogenicity, can induce high level IgG1 and IgG2a antibody titers, and the humoral immune response in inducing the production of SLOdm protein specific IgG antibodies in serum is stronger than that of a single protein. The HtsA-SLOdm fusion protein can promote the skin wound healing of mice, reduce the bacterial content at the skin infection of mice, reduce the inflammation at the skin infection wound of mice, and accelerate the wound healing. In addition, the HtsA-SLOdm fusion protein has high safety, has no hemolytic activity, and can be used for preparing a group A streptococcus vaccine.

[0150] The above embodiments of the present application are described in detail, but the present application is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the purpose of the present application. In addition, the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

Claims

1. A fusion protein, characterized in that, obtained by sequentially connecting the following elements; (1) a hemopexin-like protein; the amino acid sequence of the hemopexin-like protein is shown in SEQ ID NO: 4 at positions 1-266; (2) a two-site mutated streptolysin protein; the amino acid sequence of the two-site mutated streptolysin protein is shown in SEQ ID NO: 4 at positions 277-817; the hemopexin-like protein and the two-site mutated streptolysin protein are connected by a linker; the linker is selected from any one of (GGGGS)5, (GGGGS)4, (GGGGS)3, (GGGGS)2, (GGGS)5, (GGGS)4, (GGGS)3.

2. The fusion protein of claim 1, wherein, the amino acid sequence of the linker is shown in SEQ ID NO: 4 at positions 267-276; the amino acid sequence of the fusion protein is shown in SEQ ID NO:

4.

3. Biomaterials associated with the fusion protein according to claim 1 or 2, characterized in that, the biomaterial is any one of A1) to A4): A1), a nucleic acid molecule encoding the fusion protein of claim 1 or 2; A2), an expression cassette containing the nucleic acid molecule of A1); A3), a recombinant vector containing the nucleic acid molecule of A1) or the expression cassette of A2); A4), a recombinant biological cell containing the nucleic acid molecule of A1), the expression cassette of A2), or the recombinant vector of A3).

4. The biomaterial of claim 3, wherein, in the nucleic acid molecule of A1), the nucleotide sequence of the nucleic acid molecule encoding the fusion protein comprises: A11), a nucleotide sequence shown in SEQ ID NO: 1; or A12), a nucleotide sequence obtained by substitution and / or deletion and / or addition of one or more nucleotides of the nucleotide sequence shown in A11), and encoding the same amino acid as the nucleic acid molecule shown in A11).

5. Use of the fusion protein of any one of claims 1 to 2 or the biomaterial of any one of claims 3 to 4 in the preparation of a group A streptococcus vaccine product.

6. A recombinant vaccine, characterized in that, comprising the fusion protein of any one of claims 1 to 2 or the biomaterial of any one of claims 3 to 4.

7. The recombinant vaccine according to claim 6, characterized in that, further comprising a pharmaceutically acceptable excipient.

8. Use of the fusion protein of any one of claims 1 to 2 or the biomaterial of any one of claims 3 to 4 or the recombinant vaccine of any one of claims 6 to 7 in the preparation of a medicament for preventing a disease caused by group A streptococcus.

9. Use according to claim 8, characterized in that, the disease caused by group A streptococcus includes skin inflammation, pharyngitis, impetigo, necrotizing fasciitis, streptococcal toxic shock syndrome, septicemia, rheumatic heart disease, and acute streptococcal glomerulonephritis.

Images