A fusion protein KP-Ag1 and its application as a Klebsiella pneumoniae vaccine antigen

Abstract

The present invention provides a fusion protein KP-Ag1 used as a Klebsiella pneumoniae vaccine antigen, which is composed of KP6 protein, KP21 protein and KP24 protein connected by a flexible amino acid Linker; wherein, the amino acid sequence of the KP6 is SEQ ID NO:4, the amino acid sequence of the KP21 is SEQ ID NO:5, and the amino acid sequence of the KP24 is SEQ ID NO:6. The present invention also provides its expression vector and recombinant engineering diagram, as well as its application as a Klebsiella pneumoniae antigen. The antigen protein prepared by the method described in the present invention can effectively stimulate the body to produce a higher humoral immune response, and can provide obvious protection against lethal-dose Klebsiella pneumoniae infection, and can be used as a candidate antigen for Klebsiella pneumoniae vaccine.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a fusion protein KP-Ag1 and its application as a Klebsiella pneumoniae vaccine antigen. Background Art

[0002] In 2017, the World Health Organization first released a list of 12 key pathogens that seriously endanger human health. Among them, Enterobacteriaceae bacteria represented by Klebsiella pneumoniae (KP) were classified as extremely important (Tacconelli E, et al. Lancet Infect Dis. 2018). KP is a Gram-negative bacterium and is one of the most common opportunistic pathogens clinically. KP can colonize in the intestine, nasopharynx, axilla and other parts, and the digestive tract is the main colonization site. The colonization rates vary greatly in different regions. The colon colonization rate in Western countries is 5% - 35%, while it is generally higher in Asian countries, and even as high as over 85% in some countries (such as Malaysia) (Zhang Xin, et al. Chinese Journal of Tuberculosis and Respiratory Diseases. 2020). KP can cause infections in various parts of the body, commonly seen in elderly patients, malnourished, patients with chronic diseases and systemic failure, and can cause systemic or local infections such as pneumonia, urinary tract infection, meningitis, sepsis, etc. (Lee CR, et al. Front Cell Infect Microbiol. 2017). According to different virulence and pathogenic characteristics, KP is currently divided into two categories. One is classical Klebsiella pneumoniae (cKP), which mainly causes hospital-acquired infections such as pneumonia, urinary tract infection, sepsis, etc., and has a high drug resistance rate, commonly seen in people with underlying diseases or low immunity. The other is hypervirulent Klebsiella pneumoniae (hvKP), which mainly causes infections in healthy people without underlying diseases in the community, and the most common is liver abscess, 66% of which is caused by KP infection (Russo TA, et al. Clin Microbiol Rev. 2019).

[0003] In recent years, the drug resistance situation of Klebsiella pneumoniae (KP) to various common antibiotics has become increasingly severe. The results of the CHINET China Bacterial Resistance Surveillance in 2021 showed that the isolation rate of KP has jumped to the second among Gram-negative bacilli, second only to Escherichia coli, reaching 19.8% in 2021. Its resistance rate to ampicillin has reached as high as 91.8%, and the resistance rate to piperacillin is close to 50%. Especially with the wide clinical application of carbapenems, the detection rate of carbapenem-resistant Klebsiella pneumoniae (CRKP) has increased year by year. The resistance rates of KP in China to imipenem and meropenem have increased from about 3% in 2005 to 23.1% and 24.4% respectively in 2021, and the situation is very serious (https: / / www.chinets.com / Document). Moreover, KP infections also have a very high mortality rate. According to statistics: 22-32% of patients with community-acquired pneumonia caused by KP need to be admitted to the intensive care unit (ICU) for treatment, and the mortality rate is as high as 45%-72%; in addition, KP accounts for 5-20% of sepsis cases caused by Gram-negative bacterial infections, and the mortality rate is as high as 27.4%-37% (Paczosa MK, et al. Microbiol Mol Biol Rev. 2016). More importantly, some studies have shown that the fatality rate of KP infections sensitive to carbapenem antibiotics is 20%-30%, while the fatality rate of CRKP infections is significantly higher, reaching 40%-70% (Iredell J, et al. BMJ. 2016). Due to its super drug resistance and pathogenicity, CRKP is known as the "king of superbugs". Considering that the drug resistance situation of KP is already very serious, especially the widespread prevalence of CRKP, it has made antibiotic treatment extremely difficult, and it is urgent to develop new effective prevention and treatment measures. Vaccine development is one of the most promising strategies.

[0004] Since the emergence of research on KP vaccines in the 1970s, various types of vaccine research have continuously emerged. Early studies mainly focused on inactivated, attenuated, and bacteriolysate vaccines. These vaccines have complex components, making quality control difficult and potentially having residual toxicity. Due to safety considerations, they failed to enter clinical research (Ahmad TA, et al. Vaccine. 2012). Subsequently, research on ribosomal vaccines began, but due to their intracellular components and limited immunoprotection, in-depth research was not carried out. Next were polysaccharide vaccines. The antigens used in polysaccharide vaccines mainly include capsular polysaccharides and LPS. However, since Klebsiella pneumoniae capsular polysaccharides (K-antigens) have more than 80 serotypes and LPS (O-antigens) have 12 serotypes, and the K-antigens and O-antigens vary greatly among different serotypes. Although studies have shown that such vaccines exhibit good immunogenicity and safety in human trials and passive immunity also has good protective effects, their cross-protective ability against other serotypes is limited, objectively restricting their application (Jenney AW, et al. J Clin Microbiol. 2006). In recent years, research has mainly focused on recombinant protein vaccines. A variety of secreted proteins and outer membrane proteins have shown certain protective effects against KP infection, such as outer membrane proteins (OmpA, OmpK36, FepA, OmpK17, OmpW), Colicin I receptor protein, adhesin MrkD protein, fimbrial protein, cell surface iron-regulated protein, and toxoid, etc. (Zhang BZ, et al. Front Immunol. 2021), which are currently the most promising vaccine types. Due to the slow progress of antigens with high specificity for Klebsiella pneumoniae, there has been no breakthrough in the research and development of related vaccines for the diagnosis and prevention and treatment of Klebsiella pneumoniae infection, and no effective drugs have been marketed. Summary of the Invention

[0005] By using the method of reverse vaccinology, the whole genome of Klebsiella pneumoniae was analyzed in the present invention to screen out potential antigenic determinants, which were cloned, expressed, and purified through high-throughput methods, and their protective evaluation was carried out in animals. As a result, more than 150 protective candidate antigens were screened out, and the protective evaluation of 42 of these candidate antigens was carried out in animals, thus screening out a series of protective candidate antigens.

[0006] Aiming at the high infection rate, high pathogenicity, and high drug resistance of Klebsiella pneumoniae, the present invention provides a Klebsiella pneumoniae vaccine protein antigen KP-Ag1, which can be used to prepare a recombinant subunit vaccine against Klebsiella pneumoniae.

[0007] The present invention first provides a fusion protein KP-Ag1, which is composed of KP6 protein, KP21 protein and KP24 protein connected by a flexible amino acid Linker; wherein, the amino acid sequence of KP6 is SEQ ID NO:4, the amino acid sequence of KP21 is SEQ ID NO:5, and the amino acid sequence of KP24 is SEQ ID NO:6.

[0008] In one embodiment according to the present invention, the fusion protein has the following structure:

[0009] KP6-L 1 -KP21-L 2 -KP24;

[0010] wherein, L 1 and L 2 are each independently a Linker selected from (GGGGS) m and (Gly) n , and m is an integer selected from 1-4, and n is an integer selected from 6-8; preferably GGGGS.

[0011] In one embodiment according to the present invention, the amino acid sequence is SEQ ID NO:2.

[0012] The present invention also provides the coding gene of the above fusion protein, and its nucleotide sequence is SEQ ID NO:1.

[0013] The present invention further provides an expression vector, which contains a backbone plasmid and the above coding gene.

[0014] In one embodiment according to the present invention, the backbone plasmid is selected from any one of pGEX series vectors, pET series vectors or pQE series vectors; preferably pET30a plasmid.

[0015] In one embodiment according to the present invention, the nucleotide sequence of the expression vector is SEQ ID NO:3.

[0016] The present invention also provides a recombinant engineering bacterium, which contains the above expression vector;

[0017] Preferably, the host bacterium is Escherichia coli XL1-blue strain, BL21 series strains or HMS174 series strains, preferably Escherichia coli BL21.

[0018] Another aspect of the present invention provides the use of the above-mentioned fusion protein in the preparation of a preparation for diagnosing, preventing or treating Klebsiella pneumoniae infection; preferably, the preparation further comprises a pharmaceutically acceptable adjuvant, and 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 bovis BCG adjuvant.

[0019] Yet another aspect of the present invention provides a subunit vaccine for preventing or treating Klebsiella pneumoniae infection, which contains the above-mentioned fusion protein; preferably, it further comprises a pharmaceutically acceptable adjuvant, and 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 bovis BCG adjuvant.

[0020] The beneficial effects of the above technical solutions of the present invention are as follows:

[0021] 1) The fusion protein KP-Ag1 provided by the present invention contains three proteins derived from Klebsiella pneumoniae and can simultaneously generate specific immune responses against the three proteins;

[0022] 2) The fusion protein KP-Ag1 provided by the present invention can be expressed in the prokaryotic expression system - Escherichia coli, with low cost and high yield;

[0023] 3) When the pET30a vector is selected, the KP-Ag1 fusion protein provided by the present invention is expressed in a soluble form;

[0024] 4) The purification conditions of the fusion protein KP-Ag1 provided by the present invention are mild, the steps are simple, no denaturing agent needs to be added, it is easy to scale up, has good repeatability, and has a good recovery rate.

[0025] 5) The fusion protein KP-Ag1 provided by the present invention can induce animals to produce specific antibodies: The subunit vaccine prepared with the recombinant KP-Ag1 protein of the present invention can be immunized by subcutaneous (intramuscular) injection, and can stimulate the body to produce high-titer IgG antibodies, confirming its good immunogenicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a double digestion identification result diagram of the recombinant plasmid pET30a-KP-Ag1; wherein,

[0027] Lane M: Nucleic acid (DNA) molecular weight standard (Marker), from top to bottom, the sizes are: 5000, 3000, 2000, 1500, 1000, 750, 500, 250, 100 bp; Lane 1: Identification result of the recombinant expression plasmid pET30a-KP-Ag1 digested with Nde1 and Xho1. The separated fragments after digestion are approximately 5400 bp and 1400 bp; Lane 2: Plasmid pET30a-KP-Ag1;

[0028] Figure 2 It is the identification result map of the induced expression of KP-Ag1 protein; among them,

[0029] Lane M: Protein molecular weight standard (Marker), from top to bottom, the sizes are: 170 kDa, 130 kDa, 100 kDa, 70 kDa, 55 kDa, 40 kDa, 35 kDa, 25 kDa, 15 kDa, 10 kDa; Lane 1: Supernatant after cell disruption; Lane 2: Precipitate after cell disruption. The size of the target protein is approximately 47 kDa and is expressed in both the supernatant and the precipitate;

[0030] Figure 3 It is the cation exchange chromatography map; among them,

[0031] It can be seen from the map that the cation exchange column has a good capture effect on the target protein;

[0032] Figure 4 It is the hydrophobic chromatography map; among them,

[0033] The target protein mainly exists in peak 1, and there are also impurity proteins in peak 2;

[0034] Figure 5 It is the SDS-PAGE electrophoresis result map of the purified KP-Ag1 protein;

[0035] Lane 1: Protein molecular weight standard (Marker), from top to bottom, the sizes are: 170 kDa, 130 kDa, 100 kDa, 70 kDa, 55 kDa, 40 kDa, 35 kDa, 25 kDa, 15 kDa, 10 kDa; Lane 2: KP-Ag1 protein after cation exchange chromatography; Lane 3: KP-Ag1 protein (peak 1) after hydrophobic chromatography; Lane 4: KP-Ag1 protein (peak 2) after hydrophobic chromatography; The target protein KP-Ag1 mainly exists in peak 1 and has good purity;

[0036] Figure 6 It is the HPLC identification result map of the purified KP-Ag1 protein. Specific implementation mode

[0037] To make the technical problems, technical solutions, and advantages to be solved by the present invention clearer, the following will be described in detail with reference to the accompanying drawings and specific embodiments.

[0038] The strains and various reagents used in the present invention are as follows:

[0039] The plasmid pET30a was purchased from GE Healthcare;

[0040] The Escherichia coli strain BL21 was purchased from Shanghai Chaoyan Biotechnology Co., Ltd.;

[0041] The Klebsiella pneumoniae international standard strain 700721 was purchased from ATCC;

[0042] The restriction endonucleases Nde I and Xho I, and protein Marker were purchased from Dalian TakaRa;

[0043] The plasmid extraction kit and gel recovery kit were purchased from Omega, USA;

[0044] The cation exchange chromatography column (HiTrap SP HP 5ml) and hydrophobic chromatography column (HiTrap Phenyl HP 5ml) were from GE Healthcare, USA.

[0045] Example 1: Gene synthesis and subcloning

[0046] Gene synthesis and subcloning

[0047] 1. The synthesis of the DNA sequence encoding KP-Ag1 (SEQ ID NO: 1) and the ligation of the sequence with pET30a were synthesized by Shanghai Sangon Biological Engineering Co., Ltd.

[0048] 2. Transformation of the recombinant plasmid: Take 1 tube of Escherichia coli BL21 competent cells (Shanghai Chaoyan Biotechnology Co., Ltd.) from the -80°C refrigerator, and add 0.5 μl of the synthesized pET30a-KP-Ag1 plasmid. Incubate on ice for 50 min, heat shock in a 42°C metal bath for 90 s, and quickly incubate on ice for 2 min. Add 600 μl of LB blank medium, mix well, and place it in a 37°C shaker and shake at 220 rpm for 1 h. Centrifuge each tube at 5000 rpm at room temperature for 3 min, discard 300 μl of the supernatant, resuspend the cells again, take 200 μl and spread it on a Kana-resistant LB plate. Invert the plate and culture it in a 37°C incubator for 24 h. Pick well-separated colonies on the transformation plate, inoculate them into Kana-resistant LB medium, and culture them overnight at 37°C with shaking.

[0049] 3. Double digestion identification

[0050] Take the bacterial solution cultured overnight in a 37°C shaker, and extract the plasmid of the positive clone according to the steps in the instruction manual using a Quick Plasmid Mini-Preparation Kit (Tiangen Biochemical Technology Co., Ltd.). Use Nde1 (Takara) and Xhol (Takara) for enzyme digestion and incubate in a 37°C water bath for half an hour. The reaction system is as follows:

[0051]

[0052] Prepare a 1.0% agarose gel containing EB (Shanghai Junsheng Biotechnology Co., Ltd.) at 0.5 μg / ml. Add 1 μl of 6× Loading buffer to each of the above enzyme digestion reaction systems, and perform electrophoresis on the gel at 80 V for 20 min. Observe the enzyme digestion results using a UV scanner. It was found that the plasmid of the positive clone was cut into two fragments. The large fragment was approximately 5400 bp, which was part of the expression vector pET30a, and the small fragment was approximately 1400 bp, which was the inserted DNA fragment encoding KP-Ag1( Figure 1 ).

[0053] Example 2: Identification of the expression form of KP-Ag1 antigen protein

[0054] 1. Induced expression of KP-Ag1

[0055] Take 100 μL of the overnight-cultured pET30a-KP-Ag1 / BL21 bacterial solution and add it to 10 mL of LB medium with Kana resistance. Culture at 37°C with 180 rpm for 3 h. When the OD600 reaches 0.6 - 0.8, add IPTG to a final concentration of 200 μM, and then place it in a shaker for induced expression at 25°C for 5 h. Take out the induced bacterial solution, centrifuge at 12000 rpm for 5 min, discard the supernatant, add 1 mL of lysis buffer (50 mM citric acid, pH 5.0), mix well, ultrasonically lyse for 3 min, and then centrifuge at 14000 rpm at 4°C for 15 min to separate the supernatant and precipitate.

[0056] 2. SDS-PAGE electrophoresis

[0057] Pour the 10% separating gel into the gel plate, add distilled water to flatten the gel, and let it solidify at room temperature for 30 min. Pour out the upper-layer distilled water, then pour in the stacking gel, immediately insert the comb, and let it solidify at room temperature for 30 min for standby. Take 10 μL of each of the treated samples for loading and perform SDS-PAGE electrophoresis. First, run the electrophoresis at 80 v for 30 min, then adjust the voltage to 180 v and run for 1 - 2 h. Take out the gel, place it in the Coomassie Brilliant Blue staining solution and shake for staining, then place it in the decolorizing solution and shake for decolorization, and observe the results under the imaging system. pET30a-KP-Ag1 / BL21 was expressed in a soluble form under the induction condition of 25°C, and there were also a large amount of proteins in the precipitateFigure 2 )。

[0058] Example 3: Preparation of KP-Ag1 Protein Antigen

[0059] 1. Amplification culture to obtain the protein

[0060] Take 400 μL of the pET30a-KP-Ag1 / BL21 bacterial solution stored in a 4°C refrigerator for standby, add it to 30 mL of LB medium containing Kana resistance for primary activation. After culturing at 37°C with 200 rpm for 5 - 6 h, take 20 mL of the primary-activated bacterial solution and add it to 2000 mL of LB medium containing Kana resistance for secondary activation. When the OD600 reaches 1.0 after culturing at 37°C for 3 - 4 h, add 200 μL of IPTG (final concentration 200 μM), place it in a shaker at 25°C for induction for 5 h, then centrifuge at 12000 rpm for 15 min to collect the bacterial cells. After resuspending the bacterial cells with another 50 mL of lysis buffer (the same as in Example 2), ultrasonically lyse the bacterial solution for 3 min (200 V), and collect the supernatant for subsequent purification.

[0061] 2. Preliminary purification of KP-Ag1 by cation exchange chromatography

[0062] Take a cation exchange chromatography column (HiTrap SP HP 5 ml), equilibrate the chromatography system and the SP HP chromatography column with lysis buffer (the same as in Example 2), then load the above supernatant, and perform linear gradient elution with wash buffer (50 mM citric acid, pH 5.0, 1 M NaCl). Set the elution flow rate at 5 ml / min, the elution gradient is from 0 to 100% of wash buffer, and the elution volume is 50 ml. Collect the eluted target protein and store it at 4°C for standby. The chromatogram is as shown in Figure 3 shown, and the electrophoresis result is as shown in Figure 5 shown.

[0063] 3. Fine purification of KP-Ag1 by hydrophobic chromatography

[0064] Take a hydrophobic chromatography column (HiTrap Phenyl HP 5ml), and equilibrate the chromatography system and the SP HP column with buffer A (20 mM PB, pH 7.5, 1.5 M ammonium sulfate). Then mix the cation exchange chromatography sample with 20 mM PB, pH 7.5, 3 M ammonium sulfate buffer at a ratio of 1:1 and load the mixture onto the column. Perform linear gradient elution using wash buffer (20 mM PB, pH 7.5), set the elution flow rate at 5 ml / min, the elution gradient with wash buffer from 0 to 100%, and the elution volume at 50 ml. Collect the target protein eluted, and replace the buffer of the peak 1 sample with 20 mM PB, pH 7.5, 150 mM NaCl through a desalting column, and store it at 4 °C for later use. The chromatogram is as shown in Figure 4 shown, and the electrophoresis result is as shown in Figure 5 shown.

[0065] 4. Detection of the purification effect of KP-Ag1 by HPLC

[0066] Use a C3 column (purchased from Agilent) to detect the purity of the purified KP-Ag1 protein. Equilibrate the column with 0.1% TFA aqueous solution, load 5 μl of the sample, and elute with 0.1% TFA acetonitrile solution. Set the column temperature at 60 °C and the flow rate at 0.5 ml / min. The elution program is: 0% - 100% B, 30 min. The mass spectrometry diagram is as shown in Figure 6 shown, and the results show that the purity of KP-Ag1 is about 96% ( Figure 6 ), meeting the purity requirements of biopharmaceuticals for protein drugs. Among them, the preparation of 0.1% TFA aqueous solution: Mix 1 ml of TFA with 1 L of grade I water and filter through a 0.22 μm filter membrane. The preparation of 0.1% TFA acetonitrile solution: Mix 1 ml of TFA with 1 L of acetonitrile.

[0067] Example 4: Immunization of animals and antibody detection

[0068] Dilute the KP-Ag1 antigen with PBS and add Al(OH) with a concentration of 1 mg / mL 3 to prepare the vaccine; Immunize BALB / C mice by bilateral thigh muscle injection with a No. 5 half-sized needle on days 0, 7, and 14. The injection volume for each mouse is 100 μL, and the antigen content is 50 μL. The blank control group is immunized with the same volume of PBS; On the 7th day after the last immunization, collect the orbital blood of BALB / C mice and detect the antigen-specific IgG response level in the mice after immunization by ELISA.

[0069] 1. Preparation of the liquid

[0070] 1) Preparation of the coating solution: Weigh Na 2 CO 3 1.6 g, NaHCO3 2.9 g, dissolved in 1 L ddH 2 O, and the pH was adjusted to 9.6 with a pH meter;

[0071] 2) Preparation of blocking solution: 1 g bovine serum albumin, dissolved in 100 mL antibody diluent (1:100);

[0072] 3) Preparation of antibody diluent: Dissolve phosphate in 1 L ddH 2 O, then add 500 μL Tween 20, and adjust the pH to 7.4 with a pH meter;

[0073] 4) Preparation of washing solution: The same as the antibody diluent

[0074] 5) Chromogenic solution (TMB), a product of Tiangen Company;

[0075] 6) Preparation of termination solution (2M H 2 SO 4 ): Pour 22.2 mL concentrated sulfuric acid into 177.8 mL ddH 2 O.

[0076] 2. ELISA to detect the antibody titer produced by immunizing mice with the KP-Ag1 fusion protein

[0077] 1) Dilute the purified KP-Ag1, antigen proteins of No. 6, No. 21, and No. 24 to 1 μg / mL with the coating solution;

[0078] 2) Coating: Add the antigen protein diluent to the enzyme-linked immunosorbent assay (ELISA) plate, 100 μL / well, incubate overnight at 4°C, wash 3 times with the washing solution, dry in air, wrap with plastic wrap, and store in a 4°C refrigerator for later use;

[0079] 3) Blocking: Add 200 μL / well of blocking solution to the ELISA plate, incubate in a 37°C incubator for 2 hours, and wash 3 times;

[0080] 4) Dilute the serum serially at 1:1000, 1:2000, 1:4000, 1:8000, etc.;

[0081] 5) Take the blocked ELISA plate, add the diluted serum sequentially, 100 μL / well, incubate in a 37°C incubator for 1 h, wash 3 times, and dry in air;

[0082] 6) Dilute the preserved solution of HRP-labeled goat anti-mouse IgG antibody 1:10000 to prepare the antibody working solution;

[0083] 7) Add the diluted antibody working solution, 100 μL / well, incubate in a 37°C incubator for 40 min, wash three times, and dry in air;

[0084] 8) Add 100 μL / well of substrate chromogenic solution (TMB), and react for 5 min at room temperature in the dark;

[0085] 9) Add stop solution (2M H 2 SO 4 ), and immediately measure the OD value at a wavelength of 450 nm using an ELISA reader;

[0086] 10) Result judgment: A 样品 / A 阴性 value ≥ 2.1 is positive (negative control is mouse serum diluted 1:1000 before immunization).

[0087] Results: The geometric mean titer of KP-Ag1 specific IgG antibody produced by immunizing mice with KP-Ag1 protein antigen was 1:512000; the geometric mean titers of specific IgG antibodies against antigen proteins No. 6, No. 21, and No. 24 were 1:128000; 1:64000; 1:256000 respectively; the antibody positive rate reached 100% on the 7th day after the last immunization, indicating that the KP-Ag1 fusion protein constructed by the present invention has good immunogenicity.

[0088] Example 5: Immunization and challenge protection experiment of animals

[0089] Dilute KP-Ag1 antigen with PBS, and add Al(OH) with a concentration of 1 mg / mL 3 to prepare the vaccine; immunize BALB / C mice by bilateral thigh muscle injection with a 5-gauge needle on days 0, 7, and 14, with an injection volume of 100 μL per mouse and an antigen content of 50 μL. The blank control group was immunized with the same volume of PBS; on the 7th day after the last immunization, challenge the mice with 3×10 7 CFU dose of Klebsiella pneumoniae standard strain 700721 by intratracheal instillation, observe the survival status of the mice, with an observation period of 10 days, record the number of dead mice every day, and calculate the survival rate of the mice after the observation period. The results of three consecutive animal experiments showed that: compared with the control group, the survival rate of mice in the KP-Ag1 immunized group was significantly improved, and the protection rate was about 80 - 100%, indicating that it can be used as a candidate antigen for vaccine research and development.

[0090] The above is the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A fusion protein KP-Ag1, characterized in that, it is composed of KP6 protein, KP21 protein and KP24 protein connected by a flexible amino acid Linker; wherein, the amino acid sequence of the KP6 is SEQ ID NO:4, the amino acid sequence of the KP21 is SEQ ID NO:5, and the amino acid sequence of the KP24 is SEQ ID NO:6; the amino acid sequence of the fusion protein KP-Ag1 is SEQ ID NO:

2.

2. The encoding gene of the fusion protein KP-Ag1 according to claim 1, and its nucleotide sequence is SEQ ID NO:

1.

3. An expression vector, characterized in that, it contains a backbone plasmid and the encoding gene according to claim 2.

4. The expression vector according to claim 3, characterized in that, the backbone plasmid is selected from any one of pGEX series vectors, pET series vectors or pQE series vectors.

5. The expression vector according to claim 4, characterized in that, the backbone plasmid is pET30a plasmid.

6. The expression vector according to claim 3 or 4, characterized in that, the nucleotide sequence of the expression vector is SEQ IDNO:

3.

7. A recombinant engineering bacterium, characterized in that, it contains the expression vector according to any one of claims 3-6.

8. The recombinant engineering bacterium according to claim 7, characterized in that, the host bacterium is Escherichia coli XL1-blue strain, BL21 series strains or HMS174 series strains.

9. The recombinant engineering bacterium according to claim 8, characterized in that, the host bacterium is Escherichia coli BL21.

10. The application of the fusion protein KP-Ag1 according to claim 1 in the preparation of a preparation for preventing or treating Klebsiella pneumoniae infection.

11. The application according to claim 10, characterized in that, the preparation further contains a pharmaceutically acceptable adjuvant.

12. The application according to claim 11, characterized in that, the adjuvant is selected from any one of adjuvants aluminum hydroxide adjuvant, aluminum phosphate adjuvant, aluminum monostearate adjuvant, MF59, complete Freund's adjuvant, incomplete Freund's adjuvant and Mycobacterium bovis BCG adjuvant.

13. The application according to claim 12, characterized in that, the adjuvant is aluminum hydroxide adjuvant.

14. A subunit vaccine for preventing or treating Klebsiella pneumoniae infection, characterized in that, it contains the fusion protein KP-Ag1 according to claim 1.

15. The vaccine according to claim 14, characterized in that, it further contains a pharmaceutically acceptable adjuvant.

16. The vaccine according to claim 15, characterized in that, the adjuvant is selected from any one of adjuvants aluminum hydroxide adjuvant, aluminum phosphate adjuvant, aluminum monostearate adjuvant, MF59, complete Freund's adjuvant, incomplete Freund's adjuvant and Mycobacterium bovis BCG adjuvant.

17. The vaccine according to claim 16, characterized in that, the adjuvant is aluminum hydroxide adjuvant.

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