A Klebsiella pneumoniae KPC-Pal recombinant protein and its application

Abstract

The present invention provides a Klebsiella pneumonia KPC-Pal recombinant protein, which is composed of a KPC peptide segment extracted from the Klebsiella pneumonia KPC-2 protein and a Pal peptide segment extracted from the Klebsiella pneumonia Pal protein connected by a polypeptide linker, wherein the amino acid sequence of the Klebsiella pneumonia KPC-2 protein is SEQ ID NO: 3, and the amino acid sequence of the Klebsiella pneumonia Pal protein is SEQ ID NO: 4. The present invention also provides its expression vector and recombinant engineered bacteria, as well as its application in the preparation of a preparation for diagnosing, preventing or treating Klebsiella pneumonia infection. The Klebsiella pneumonia KPC-Pal recombinant protein provided by the present invention can effectively stimulate the body to cause a protective immune response, thereby resisting the lethal infection of Klebsiella pneumonia.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a Klebsiella pneumoniae KPC-Pal recombinant protein and an application thereof. Background Art

[0002] Klebsiella pneumoniae (KP) frequently colonizes the human intestinal tract and respiratory tract and is a common opportunistic pathogen in clinical practice (Martin RM, et al. Front Cell Infect Microbiol, 2018). It can cause infections throughout the body, particularly in the elderly, those with malnutrition, chronic alcoholism, chronic diseases, and systemic failure. It can cause systemic or localized infections such as pneumonia, urinary tract infections, meningitis, and sepsis, which can be life-threatening in severe cases (Akova M, et al. Clin Microbiol Infect, 2012). In recent years, the incidence of carbapenem-resistant K. pneumoniae (CRKP) has increased annually in clinical practice. Due to its extreme drug resistance and pathogenicity, it has been dubbed the "king of superbugs," creating significant obstacles to clinical treatment and has therefore become a hot topic of research (Munoz-Price LS, et al. Lancet Infect Dis, 2013). It poses a serious threat to human health. Given its increasingly severe drug resistance, the development of new prevention and treatment methods is urgent. Vaccination can effectively protect the body from bacterial invasion and is unaffected by antibiotic resistance mechanisms, making it a very promising prevention and treatment method.

[0003] Due to limited safety, no inactivated Klebsiella pneumoniae vaccines are currently approved for marketing. With the continuous advancement of disciplines such as immunology, molecular biology, and nanoscience, the types and composition of bacterial vaccines have undergone significant changes, with the emergence of new vaccines such as polysaccharide vaccines, polysaccharide conjugate vaccines, protein vaccines, and nanovaccines (Hackett RJ, et al. Infect Immun, 1970). Whole-bacterial and inactivated vaccines utilize complex components and have relatively low safety. The immunogenicity of the polysaccharide component alone is poor, but immune efficacy can be enhanced by combining it with other components, such as carrier proteins. Recombinant subunit vaccines are a promising vaccine type.

[0004] KPC-2 and Pal are two candidate antigens for Klebsiella pneumoniae vaccines identified by the research team through reverse vaccinology. KPC-2 is a class A serine carbapenemase located on the bacterial surface. It is the most prevalent carbapenemase globally among Enterobacteriaceae, particularly K. pneumoniae. Encoded by the plasmid-borne bla-KPC gene, it can confer resistance to most β-lactam antibiotics, including carbapenems (Wei ZQ, et al. Antimicrob Agents Chemother, 2007). KPC carbapenemases were first discovered in the United States in 1996. Subsequently, an outbreak of KPC-producing K. pneumoniae in the United States has been reported globally, including in China. As KPC-encoding genes continue to mutate, over 100 subtypes have been identified. BlaKPC-2 is the predominant subtype in my country, accounting for over 70% (Hobson CA, et al. Clin Microbiol Infect, 2021). The KPC encoding gene is located on a plasmid. The plasmid's ability to replicate and spread horizontally makes the spread of this type of drug-resistant gene more efficient and rapid. Currently, KPC carbapenemases have been detected in common clinical Gram-negative bacteria such as Acinetobacter baumannii and Pseudomonas aeruginosa, posing a significant challenge to clinical treatment (Porreca AM, et al. Curr Infect Dis Rep, 2018). Pal is a peptidoglycan-associated lipoprotein of Klebsiella pneumoniae, belonging to the OmpA family of proteins and associated with bacterial virulence. Pal from Acinetobacter baumannii has been used as an antigen in vaccine research, but no relevant reports have been published for the Klebsiella pneumoniae Pal protein. Summary of the Invention

[0005] The present invention prepares the recombinant protein KPC-Pal by fusing KPC-2 and Pal proteins through gene splicing technology. After the protein is combined with aluminum adjuvant to prepare a vaccine, it shows good immunogenicity in animal experiments and has a good protective effect against a lethal dose of Klebsiella pneumoniae in animal experiments.

[0006] The antigen KPC-Pal is spliced ​​at the genetic level by the recombinant protein antigen KPC of Klebsiella pneumoniae and the Pal peptide chain. It can be expressed at high levels and with better purity in BL21 (DE) competent cells in the pGEX-6p-1 plasmid vector system. It can induce efficient humoral immune responses in animal challenge and protection experiments, significantly improving the survival rate of mice infected with Klebsiella pneumoniae, and can be used as an effective vaccine candidate antigen.

[0007] The innovations of the present invention are: (1) a fusion antigen KPC-Pal was constructed, which is a combination of two antigens. These two antigens have not been reported for use in Klebsiella pneumoniae vaccine research; (2) a drug resistance target was used as a candidate vaccine antigen for the first time. The antibodies produced by this target can, on the one hand, inhibit or block the hydrolysis activity of KPC-2 by binding to KPC-2, thereby reducing or blocking bacterial drug resistance; on the other hand, they can also directly kill bacteria through effects such as ADCC or CDC; (3) KPC-2 can be transmitted between different bacterial species via plasmids, and the sequence is highly conserved between different KPC subtypes. Therefore, vaccines targeting KPC-2 may have good universality. Moreover, the vaccine research strategy based on drug resistance targets can also be easily extended to other drug resistance targets, providing a theoretical basis for the research of universal vaccines for multiple drug resistance targets.

[0008] The present invention provides a recombinant Klebsiella pneumoniae antigen protein, which is composed of a KPC peptide segment extracted from the Klebsiella pneumoniae KPC-2 protein and a Pal peptide segment extracted from the Klebsiella pneumoniae Pal protein connected by a polypeptide linker, wherein the amino acid sequence of the Klebsiella pneumoniae KPC-2 protein is SEQ ID NO: 3, and the amino acid sequence of the Klebsiella pneumoniae Pal protein is SEQ ID NO: 4; preferably, the KPC peptide segment comprises a polypeptide with an amino acid sequence of SEQ ID NO: 5; the Pal protein comprises a polypeptide with an amino acid sequence of SEQ ID NO: 6; the polypeptide linker consists of 4 glycines (G) and 1 serine (S); preferably, the amino acid sequence of the polypeptide linker is SEQ ID NO: 7GGGGS.

[0009] In one embodiment according to the present invention, the amino acid sequence thereof is SEQ ID NO: 2. The amino acid sequence thereof is SEQ ID NO: 2.

[0010] The present invention also provides a gene encoding the above-mentioned recombinant antigen protein, the nucleotide sequence of which is SEQ ID NO: 1.

[0011] The present invention further provides an expression vector comprising a backbone plasmid and the encoding gene.

[0012] In one embodiment of the present invention, one end of the encoding gene is connected to the coding sequence of a protein purification tag; the protein purification tag is selected from any one of His, GST, MBP, NusA or SUMO, preferably GST.

[0013] In one embodiment of the present invention, the backbone plasmid is selected from any of the pGEX series, pET series, or pQE series vectors; preferably, the pGEX-6p-1 plasmid. The prokaryotic expression plasmid pGEX-6p-1 is used to construct the recombinant expression plasmid. This vector confers ampicillin resistance and can be used for screening positive recombinants. GST tag sequences are introduced at both ends of the protein.

[0014] The present invention further provides a recombinant engineered bacterium containing the above expression vector.

[0015] In one embodiment of the present invention, the host bacteria is Escherichia coli XL1-blue, BL21 (DE) series or HMS174 series, preferably Escherichia coli BL21 (DE).

[0016] The present invention also provides a method for purifying the recombinant protein antigen KPC-Pal from Klebsiella pneumoniae. The main technical scheme involves collecting genetically engineered bacteria expressing KPC-Pal; then purifying KPC-Pal through a sequential combination of high-pressure lysis, centrifugation, affinity chromatography, and enzymatic digestion. This method is simple, produces a high-purity target protein, is easily scalable, has good reproducibility, and offers a good recovery rate.

[0017] Another aspect of the present invention provides the use of the above-mentioned recombinant antigen protein in the preparation of a preparation for diagnosing, preventing or treating Klebsiella pneumoniae infection; preferably, the preparation further comprises a pharmaceutically acceptable adjuvant, more preferably, the adjuvant is selected from any one of aluminum hydroxide adjuvant, aluminum phosphate adjuvant, aluminum monostearate adjuvant, MF59, complete Freund's adjuvant, incomplete Freund's adjuvant and Mycobacterium Calmette-Guerin adjuvant; preferably aluminum hydroxide adjuvant.

[0018] The present invention further provides a subunit vaccine for preventing or treating Klebsiella pneumoniae infection, which contains the above-mentioned recombinant antigen protein; preferably, it also contains a pharmaceutically acceptable adjuvant, more preferably, the adjuvant is selected from any one of aluminum hydroxide adjuvant, aluminum phosphate adjuvant, aluminum monostearate adjuvant, MF59, complete Freund's adjuvant, incomplete Freund's adjuvant and Mycobacterium Calmette-Guerin adjuvant

[0019] The beneficial effects of the above technical solution of the present invention are as follows:

[0020] 1) The recombinant protein antigen KPC-Pal can effectively stimulate the body to induce a protective immune response, thereby resisting the lethal infection of Klebsiella pneumoniae.

[0021] 2) The recombinant protein antigen KPC-Pal can be expressed in the prokaryotic expression system - Escherichia coli, with low cost and high yield;

[0022] 3) When the pGEX-6p-1 vector is selected, the recombinant protein antigen KPC-Pal is expressed in a soluble form;

[0023] 4) The purification conditions for KPC-Pal protein are mild, the steps are simple, no denaturing agents are required, and it is easy to scale up, has good reproducibility, and has a good recovery rate.

[0024] 5) The recombinant protein antigen KPC-Pal can induce animals to produce specific antibodies: The subunit vaccine prepared using the KPC-Pal recombinant protein of the present invention can be immunized via subcutaneous (intramuscular) injection and can stimulate the body to produce high-titer IgG antibodies, confirming its good immunogenicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The double enzyme digestion result map of the recombinant plasmid pGEX-6p-1-KPC-Pal;

[0026] Lane M is a nucleic acid (DNA) molecular weight marker, with sizes from top to bottom being: 5000 bp, 3000 bp, 2000 bp, 1500 bp, 1000 bp, 750 bp, 500 bp, 250 bp, and 100 bp; Lane 1 is the identification result of the recombinant expression plasmid pGEX-6p-1-KPC-Pal after double digestion with Xho1 and BamHI. The fragments separated after enzyme digestion are approximately 1146 bp (lane 1) and 5000 bp (lane 2);

[0027] Figure 2 This is the result of identification of the expanded induced expression of the recombinant protein KPC-Pal;

[0028] Lane M is a protein molecular weight standard (Marker), and the sizes from top to bottom are: 180kDa, 130kDa, 100kDa, 70kDa, 50kDa, 40kDa, 35kDa, and 25kDa; Lane 1 is the GST recombinant protein of KPC-Pal; Lane 2 is the soluble target protein after PP enzyme digestion: KPC-Pal; Lane 2 is the GST-Tag affinity filler precipitate after PP enzyme digestion.

[0029] Figure 3 This is a graph showing the serum specific antibody titer levels at days 7, 14, and 21 after administration of the recombinant protein KPC-Pal.

[0030] Figure 4 This is a graph showing the survival rate results of the recombinant protein KPC-Pal in the mouse challenge protection experiment. DETAILED DESCRIPTION

[0031] In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0032] Unless otherwise specified, all reagents used in this example were of analytical grade, and the progress of all chemical reactions was monitored by thin-layer chromatography.

[0033] In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0034] The strains and various reagents used in the present invention are as follows:

[0035] Plasmid pGEX-6p-1 was purchased from GE Healthcare;

[0036] Escherichia coli strain BL21 (DE) was purchased from Shanghai Shenggong Bioengineering Technology Co., Ltd.;

[0037] Restriction enzymes XhoI and BamHI and protein markers were purchased from Dalian TakaRa Co., Ltd.

[0038] Plasmid extraction kit and gel recovery kit were purchased from Omega Company, USA;

[0039] Glutathione Sepharose 4F was purchased from GE Healthcare, USA.

[0040] Example 1: Gene synthesis and subcloning

[0041] 1. The synthesis of the DNA sequence encoding the KPC-Pal recombinant protein (SEQ ID NO: 1) and the ligation of the DNA sequence with pGEX-6p-1 were commissioned to Shanghai Sangon Biotechnology Co., Ltd.

[0042] 2. Transformation of Recombinant Plasmid

[0043] Remove one tube of E. coli BL21(DE) competent cells (Shanghai Chaoyan Biotechnology Co., Ltd.) from the -80°C freezer and add 0.5 μL of the synthesized pGEX-6p-1-KPC-Pal plasmid. Incubate on ice for 50 minutes, heat shock in a 42°C metal bath for 90 seconds, and quickly incubate on ice for 2 minutes. Add 600 μL of LB blank medium, mix thoroughly, and shake at 220 rpm in a 37°C shaker for 1 hour. Centrifuge each tube at 5000 rpm for 3 minutes at room temperature. Discard 300 μL of the supernatant, resuspend the cells, and spread 200 μL onto an LB plate containing ampicillin resistance. Incubate the plate upside down at 37°C in a 34°C incubator for 24 hours. Pick well-separated colonies from the transformation plate and inoculate them into ampicillin-resistant LB medium. Incubate at 37°C with shaking overnight.

[0044] 3. Double enzyme digestion identification

[0045] Take the bacterial culture that has been cultured overnight at 37°C in a shaker and extract the plasmid of the positive clone using the Rapid Plasmid Extraction Kit (Tiangen Biochemical Technology Co., Ltd.) according to the instructions. Use XhoI (Takara) and BamHI (Takara) for enzyme digestion and incubate at 37°C in a water bath for half an hour. The system is as follows:

[0046]

[0047] A 1.0% agarose gel containing 0.01% GenRed nucleic acid dye (Beijing Jinbaite Biotechnology Co., Ltd.) was cast. 1 μl of 6× Loading buffer was added to each of the above enzyme digestion reactions. After electrophoresis at 80V for 20 minutes, the digestion results were observed using a UV scanner. The results showed that the plasmid of the positive clone was cut into two fragments. The larger fragment, approximately 5000 bp, was the expression vector pGEX-6p-1, and the smaller fragment, approximately 1146 bp, was the inserted KPC-Pal fragment encoding KPC-Pal. Figure 1 ).

[0048] 4. Preservation of original strains

[0049] Take the bacterial liquid cultured overnight at 37℃ in a shaker after identification, mix it evenly with sterile 30% glycerol in a 1:1 ratio in a bacteria-preserving tube, and store it at -80℃. The validity period is 3 years.

[0050] Example 2: Identification and preparation of KPC-Pal antigen protein expression form

[0051] 1. Obtain protein

[0052] Take 100 μL of the pGEX-6p-1-KPC-Pal / BL21 bacterial solution stored in a 4°C refrigerator and add it to 100 mL of LB medium containing ampicillin resistance for primary activation. After incubation at 200 rpm and 37°C overnight, add the activated 100 mL of bacterial solution to 2 L of LB medium containing ampicillin resistance for secondary activation. Incubate at 37°C for 3–4 h until the OD reaches 0. 600 When the pH is 1.2, add 400 μL of IPTG (final concentration 200 μM) and induce in a shaker at 25°C for 6 hours. Centrifuge at 6000 rpm for 15 minutes to collect the cells. Resuspend the cells in 80 mL of PBS and sonicate for 30 minutes. Centrifuge at 12000 rpm for 20 minutes to collect the supernatant, which is the protein solution containing KPC-Pal.

[0053] 2. Affinity chromatography and protease cleavage

[0054] The KPC-Pal protein solution was mixed with 4 mL of glutathione-Sepharose 4F and applied to an affinity chromatography column. Binding was allowed to proceed at room temperature for 2 h. The column was then washed three times with 1M NaCl-0.5% Tween-20-20 mM PBS for 40 column volumes (160 mL), followed by three washes with 300 mM NaCl-20 mM PBS for 40 column volumes (160 mL), and finally three washes with 20 mM PBS for 40 column volumes (160 mL). This yielded a large amount of GST-tagged KPC-Pal recombinant protein. 4 mL of PBS and 1 mL of PreScission protease (PPase) were then added, and the column was digested overnight at 4°C with vertical rotation. The column was then washed twice with 5 mL of PBS, and 15 mL of the digested soluble protein solution was collected.

[0055] 3. SDS-PAGE electrophoresis

[0056] Pour 10% separation gel into the gel plate, add distilled water to flatten the gel, let it stand at room temperature for 30 minutes to solidify, drain the distilled water on the upper layer, then pour in the concentrated gel, immediately insert the comb, let it stand at room temperature for 30 minutes to solidify for use. Take 10 μL of the processed samples and load them for SDS-PAGE electrophoresis. The voltage is first 80V for 30 minutes, then adjusted to 180V. After electrophoresis for 40 minutes, the gel is taken out and placed in Coomassie Brilliant Blue staining solution for shaking staining, and then placed in decolorizing solution for shaking decolorization. The results are observed under the imaging system. pGEX-6p-1-KPC-Pal / BL21 (DE) can express a GST-tagged KPC-Pal protein with a molecular weight of about 65 kDa at 25°C, and the recombinant proteins are all in the supernatant of ultrasonic lysis. Therefore, the recombinant protein is a soluble protein under the above-mentioned induction temperature conditions, and the expression level is very high at 25°C. Figure 2 Shown: Lane 2 is the target protein obtained after enzyme digestion.

[0057] Example 3: Animal immunization and antibody detection

[0058] The KPC-Pal antigen was diluted with histidine and an aluminum hydroxide adjuvant at a concentration of 10 mg / mL was added to prepare the vaccine, so that the adjuvant dose for each mouse was 0.5 mg per immunization. BALB / C mice were immunized with a 5-gauge half-type needle through bilateral thigh muscle injection on days 0, 7, and 14. The injection volume for each mouse was 200 μL, and the antigen content was 50 μg. The blank control group was immunized with the same volume of histidine. On the 7th day after the last immunization, eye blood was collected from the BALB / C mice, and the antigen-specific IgG response level of the mice after immunization was detected by ELISA.

[0059] 1. Liquid Preparation

[0060] 1) Preparation of coating solution: Weigh 1.6 g Na2CO3 and 2.9 g NaHCO3, dissolve in 1 L ddH2O, and adjust the pH to 9.6;

[0061] 2) Preparation of blocking solution: Dissolve 1g of bovine serum albumin in 100mL of antibody diluent (1:100);

[0062] 3) Preparation of antibody diluent: Dissolve phosphate in 1 L ddH2O, add 500 μL Tween-20, and adjust the pH to 7.4;

[0063] 4) Preparation of washing solution: same as antibody dilution solution;

[0064] 5) Chromogenic solution (TMB), a product of Tiangen Company;

[0065] 6) Stop solution (containing 2M H2SO4): a product of Tiangen Company.

[0066] 2. ELISA detection of antibody titers produced by mice immunized with KPC-Pal recombinant protein

[0067] 1) Dilute the purified KPC-Pal recombinant protein to 1 μg / mL using coating solution;

[0068] 2) Coating: Add the recombinant protein dilution to the ELISA plate at 100 μL / well. Incubate at 4°C overnight, then wash three times with detergent. After air drying, wrap the plate with plastic wrap and store in a refrigerator at 4°C until ready for use.

[0069] 3) Blocking: Add 200 μL / well of blocking solution to the ELISA plate, incubate at 37°C for 2 h, and wash three times.

[0070] 4) Dilute the serum in serial ratios of 1:1000, 1:2000, 1:4000, 1:8000, etc.

[0071] 5) Take the blocked ELISA plate and add diluted serum at 100 μL / well. Incubate at 37°C for 1 hour, wash three times, and air dry.

[0072] 6) Add HRP-labeled goat anti-mouse IgG antibody storage solution and dilute it at an initial dilution of 1:1000 or 1:10000 to prepare the antibody working solution;

[0073] 7) Add diluted antibody working solution (100 μL / well), incubate at 37°C for 40 min, wash three times, and air dry.

[0074] 8) Add 100 μL / well of substrate color development solution (TMB) and react at room temperature in the dark for 5 minutes;

[0075] 9) Add stop solution (2M H2SO4) and measure the OD value at 450 nm on a microplate reader within 2 h.

[0076] 10) Result judgment: A 样品 / A 阴性 A value ≥ 2.1 was considered positive (the negative control was a 1:1000 dilution of mouse pre-immune serum).

[0077] The results are as follows Figure 3 As shown, the geometric mean titer of KPC-Pal-specific IgG antibodies produced by mice immunized with KPC-Pal protein antigen was 1:128,000; the antibody positive rate reached 100% on the 7th day after the last immunization, indicating that the KPC-Pal recombinant protein constructed by the present invention has good immunogenicity.

[0078] Example 4: Determination of the protection of animals immunized with KPC-Pal recombinant protein against virus infection by immunizing mice

[0079] The purified antigen was quantitatively detected using a BCA kit and immunized BALB / C mice with aluminum hydroxide adjuvant. Each dose contained 50 μg of antigen protein and 0.5 mg of adjuvant, and the total immunization volume was 200 μL (completed with histidine buffer). The immunization and infection process was as follows: 10 mice per group were immunized intramuscularly on days 0, 7, and 14. The histidine immunization group was used as a negative control. Seven days after the third immunization, Klebsiella pneumoniae YBQ was infected through orotracheal intubation. The infection dose of each mouse was 8×10 6 CFU / 20μL, and the survival rate of mice was observed for 7 consecutive days after infection. Figure 4 shown.

[0080] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A Klebsiella pneumoniae KPC-Pal recombinant protein, characterized in that The amino acid sequence thereof is SEQ ID NO:

2.

2. The gene encoding the recombinant protein according to claim 1, whose nucleotide sequence is SEQ ID NO:

1.

3. An expression vector for expressing KPC-Pal recombinant protein, comprising a backbone plasmid and the coding gene according to claim 2.

4. The expression vector according to claim 3, wherein One end of the coding gene is connected to a coding sequence of a protein purification tag; the protein purification tag is selected from any one of His, GST, MBP, NusA or SUMO.

5. The expression vector according to claim 4, wherein The protein purification tag is a GST tag.

6. A recombinant engineered bacterium for expressing KPC-Pal recombinant protein, characterized in that: The method comprises the expression vector according to any one of claims 3 to 5.

7. The recombinant engineered bacterium according to claim 6, wherein The host bacteria are selected from Escherichia coli XL1-blue, BL21 (DE) series or HMS174 series.

8. The recombinant engineered bacterium according to claim 7, characterized in that The host bacteria is Escherichia coli BL21 (DE).

9. A subunit vaccine for preventing Klebsiella pneumoniae infection, characterized in that: Comprising the KPC-Pal recombinant protein as claimed in claim 1.

10. The subunit vaccine according to claim 9, wherein A pharmaceutically acceptable adjuvant is also included.

11. The subunit vaccine according to claim 10, wherein The adjuvant is selected from any one of aluminum hydroxide adjuvant, aluminum phosphate adjuvant, aluminum monostearate adjuvant, MF59, complete Freund's adjuvant, incomplete Freund's adjuvant and Mycobacterium Calmette-Guerin adjuvant.

12. Use of the KPC-Pal recombinant protein according to claim 1 in the preparation of a preparation for preventing Klebsiella pneumoniae infection.

13. The use according to claim 12, characterized in that The formulation also contains a pharmaceutically acceptable adjuvant.

14. The use according to claim 13, characterized in that The adjuvant is selected from any one of aluminum hydroxide adjuvant, aluminum phosphate adjuvant, aluminum monostearate adjuvant, MF59, complete Freund's adjuvant, incomplete Freund's adjuvant and Mycobacterium Calmette-Guerin adjuvant.

15. The use according to claim 14, characterized in that The adjuvant is aluminum hydroxide adjuvant.

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