A Klebsiella pneumoniae vaccine fusion antigen mHla-EpiVac and its preparation method and application

Abstract

The present invention discloses a Klebsiella pneumoniae vaccine fusion antigen mHla-EpiVac, the amino acid sequence of the antigen protein is shown in SEQ ID NO: 1. Wherein mHla is a non-toxic mutant of Staphylococcus aureus α-hemolysin, which can form a heptamer as a carrier protein of the epitope; EpiVac contains three immunodominant epitopes of Klebsiella pneumoniae, which are connected by a linker. The present invention also discloses a preparation method and application of the antigen protein. The antigen protein prepared by the method of the present invention can effectively stimulate the body to produce an efficient humoral response and a cellular immune response, and can provide a significant protective effect against a lethal dose of Klebsiella pneumoniae infection, and can be used as a candidate antigen for the Klebsiella pneumoniae vaccine.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and pharmaceuticals, and relates to a Klebsiella pneumoniae vaccine candidate antigen and a preparation method thereof, as well as application of the antigen protein in the preparation of a Klebsiella pneumoniae recombinant protein vaccine. Background Art

[0002] In 2017, the World Health Organization (WHO) released its first list of 12 priority pathogens that pose a serious threat to human health. Among them, Enterobacteriaceae, 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 one of the most common opportunistic pathogens in clinical practice. KP can colonize in the intestines, nasopharynx, and axillae, with the digestive tract being the primary site of colonization. Colonization rates vary significantly across regions, ranging from 5% to 35% in Western countries to generally higher rates in Asian countries, reaching over 85% in some countries (such as Malaysia) (Zhang Xin, et al. Chinese Journal of Tuberculosis and Respiratory Diseases, 2020). Klebsiella pneumoniae (KP) can cause infections throughout the body and is common in the elderly, those with malnutrition, chronic illness, and systemic failure. It can cause systemic or localized infections such as pneumonia, urinary tract infection, meningitis, and sepsis (Lee CR, et al. FrontCell Infect Microbiol. 2017). KP is currently divided into two types based on virulence and pathogenicity. One type, classical K. pneumoniae (cKP), primarily causes hospital-acquired infections such as pneumonia, urinary tract infection, and sepsis. It has a high rate of drug resistance and is common in people with underlying diseases or compromised immune systems. The other type, hypervirulent K. pneumoniae (hvKP), primarily causes infections in healthy individuals without underlying diseases in the community. The most common cause of infection is liver abscess, 66% of which is caused by KP infection (Russo TA, et al. Clin Microbiol Rev. 2019).

[0003] In recent years, Klebsiella pneumoniae (KP) resistance to various common antibiotics has become increasingly severe. The 2022 CHINET China Antimicrobial Resistance Surveillance Results show that the isolation rate of KP has jumped to second place among Gram-negative bacilli, second only to Escherichia coli, reaching 13.99% in 2022. Its resistance to ampicillin has reached as high as 88.58%, and its resistance to piperacillin has exceeded 50%. In particular, with the widespread clinical use of carbapenems, the detection rate of carbapenem-resistant Klebsiella pneumoniae (CRKP) has increased annually. The resistance rate of KP in my country to imipenem and meropenem has risen from approximately 3% in 2005 to 22.6% and 24.2% in 2022, respectively, creating a serious situation (https: / / www.chinets.com / Document). Furthermore, KP infection has an extremely high mortality rate. According to statistics, 22-32% of patients with community-acquired pneumonia caused by KP require ICU admission, with a mortality rate as high as 45-72%. Furthermore, KP accounts for 5-20% of cases of sepsis caused by Gram-negative bacteria, with a mortality rate as high as 27.4-37% (Paczosa MK, et al. Microbiol Mol Biol Rev. 2016). More importantly, studies have shown that the mortality rate of KP infections sensitive to carbapenems is 20-30%, while the mortality rate of CRKP infections is significantly higher, reaching 40-70% (Iredell J, et al. BMJ. 2016). Due to its extremely high drug resistance and pathogenicity, CRKP has been dubbed the "king of superbugs." Given the already severe KP resistance situation, especially the widespread prevalence of CRKP, antibiotic treatment has become increasingly difficult. The development of new and effective prevention and treatment methods is urgently needed, and vaccine development is one of the most promising strategies.

[0004] Since the emergence of K. pneumoniae vaccine research in the 1970s, various vaccine types have been explored. Early research focused on inactivated, attenuated, and split bacterial vaccines. However, these vaccines are complex in composition, difficult to control for quality, and may contain residual toxicity, preventing them from entering clinical trials due to safety concerns (Ahmad TA, et al. Vaccine. 2012). Later, research on ribosomal vaccines began, but due to their intracellular nature, their protective potential was limited and they were not studied in depth. The next approach is polysaccharide vaccines, which primarily use capsular polysaccharides and LPS. However, since K. pneumoniae capsular polysaccharide (K-antigen) has over 80 serotypes and LPS (O-antigen) has 12 serotypes, the K-antigen and O-antigen profiles vary significantly between serotypes. Although studies have shown that these vaccines have demonstrated good immunogenicity and safety in human trials and have also provided good protection after passive immunization, their limited cross-protection against other serotypes has objectively limited their application (Jenney AW, et al. J Clin Microbiol. 2006). In recent years, research has mainly focused on recombinant protein vaccine research. A variety of secretory 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 proteins, adhesin MrkD proteins, fimbriae proteins, cell surface iron regulatory proteins and toxins (Zhang BZ, et al. Front Immunol. 2021), which are currently the most promising types of vaccines.

[0005] α-hemolysin (Hla) is a member of the perforin family secreted by Staphylococcus aureus. Hla can self-assemble into a heptamer structure on the host cell membrane, leading to cell lysis and death. A mutant Hla lacking the two β segments that form the pore structure (mHla) retains the ability to assemble into heptamers but loses its biological activity (Zou JT, et al. Front Immunol. 2021). mHla can be used as a universal carrier protein to improve the immunogenicity of protein antigens. First, oligomer formation increases the size of the antigenic protein. Second, fusion with mHla promotes the exposure of hidden epitopes on the antigenic protein (Zou JT, et al. PLoS Pathog. 2021). Summary of the Invention

[0006] In view of the high infection rate, high pathogenicity and high drug resistance of Klebsiella pneumoniae, the present invention provides a Klebsiella pneumoniae vaccine fusion protein antigen mHla-EpiVac, which can be used in the preparation of a recombinant subunit vaccine of Klebsiella pneumoniae.

[0007] The Klebsiella pneumoniae vaccine fusion antigen mHla-EpiVac provided by the present invention has the amino acid sequence shown in SEQ ID NO: 1. Preferably, the fusion antigen contains three immunodominant epitopes derived from Klebsiella pneumoniae.

[0008] The amino acid sequences of the three immunodominant epitopes are FepA396-423, as shown in SEQ ID NO: 2, OmpA148-165, as shown in SEQ ID NO: 3, and OmpW144-163, as shown in SEQ ID NO: 4.

[0009] The present invention also provides a method for preparing the Klebsiella pneumoniae vaccine fusion antigen mHla-EpiVac, which mainly comprises the steps of:

[0010] 1) Synthesis and subcloning of mHla-EpiVac;

[0011] 2) Inducible expression of mHla-EpiVac;

[0012] 3) Preparation of mHla-EpiVac protein antigen;

[0013] 4) Purification of mHla-EpiVac protein.

[0014] Specifically, step 1) comprises: connecting mHla to the FepA396-423 sequence shown in SEQ ID NO: 2 with a flexible chain, then connecting the OmpW144-163 sequence shown in SEQ ID NO: 4 with a flexible chain, and then connecting the OmpA148-165 sequence shown in SEQ ID NO: 3 with a flexible chain to form the fusion antigen mHla-EpiVac.

[0015] Step 1) further comprises using the prokaryotic expression plasmid pGEX-6p-1 having ampicillin resistance to construct a recombinant expression plasmid.

[0016] Step 3) involves scaling up the induced protein obtained in step 2).

[0017] Step 4) comprises: collecting genetically engineered bacteria expressing mHla-EpiVac; purifying mHla-EpiVac by a sequential combination of high-pressure lysis, centrifugation, cation exchange, and hydrophobic chromatography.

[0018] The Klebsiella pneumoniae vaccine fusion antigen mHla-EpiVac of the present invention can be used to prepare an immune preparation for preventing or treating Klebsiella pneumoniae.

[0019] The present invention also provides a Klebsiella pneumoniae injectable immune preparation containing the above-mentioned Klebsiella pneumoniae vaccine fusion antigen mHla-EpiVac and any one or any combination of the following adjuvants: aluminum hydroxide adjuvant, aluminum phosphate adjuvant, aluminum monostearate adjuvant, MF59, complete Freund's adjuvant, incomplete Freund's adjuvant, and Mycobacterium Calmette-Guerin adjuvant.

[0020] As described above, the present invention expresses a fusion of three epitopes with mHla, connecting mHla and FepA396-423 using a flexible linker GGGGS chain, then connecting OmpW144-163 using a flexible linker GGGGS, and finally connecting OmpA148-165 using a flexible linker GGGSG. The present invention names this fusion protein mHla-EpiVac, and its amino acid sequence is SEQ ID NO: 1.

[0021] The present invention preferably uses the prokaryotic expression plasmid pGEX-6p-1 to construct the recombinant expression plasmid. This vector has ampicillin resistance and can be used to screen positive recombinants. To facilitate subsequent industrialization, no additional tag sequences are introduced at either end of the fusion protein.

[0022] The present invention provides a method for purifying the recombinant protein antigen mHla-EpiVac from Klebsiella pneumoniae. The main technical scheme involves collecting genetically engineered bacteria expressing mHla-EpiVac; purifying mHla-EpiVac through a sequential combination of high-pressure lysis, centrifugation, cation exchange, and hydrophobic chromatography. This method is simple, produces a high-purity target protein, is easily scalable, has good reproducibility, and offers a good recovery rate.

[0023] The present invention adopts genetic engineering technology to clone and express this recombinant protein, which is convenient for separation and purification. It can be directly used in combination with adjuvants (such as aluminum hydroxide adjuvant, aluminum phosphate adjuvant, aluminum monostearate adjuvant, MF59, complete Freund's adjuvant, incomplete Freund's adjuvant, Mycobacterium Calmette-Guerin adjuvant, etc.) and is suitable for injection immunization.

[0024] The genetically engineered recombinant mHla-EpiVac protein of the present invention has the following advantages:

[0025] 1) The recombinant mHla-EpiVac antigen protein contains three immunodominant epitopes from three Klebsiella pneumoniae antigens, and can simultaneously generate specific immune responses against all three proteins;

[0026] 2) The recombinant mHla-EpiVac antigen protein can be expressed in the prokaryotic expression system, Escherichia coli, with low cost and high yield;

[0027] 3) When the pGEX-6p-1 vector is selected, the mHla-EpiVac recombinant protein is expressed in a soluble form;

[0028] 4) mHla-EpiVac purification requires mild conditions, simple steps, and does not require the addition of denaturants. It is easy to scale up, has good reproducibility, and has a good recovery rate.

[0029] 5) The recombinant mHla-EpiVac protein can induce animals to produce specific antibodies: The subunit vaccine prepared using the recombinant mHla-EpiVac 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.

[0030] 6) In addition to inducing specific humoral immunity, the recombinant mHla-EpiVac protein can also induce cellular immunity.

[0031] Klebsiella pneumoniae vaccine fusion antigen mHla-EpiVac, the amino acid sequence of the antigen protein is shown in SEQ ID NO: 1. mHla is a non-toxic mutant of Staphylococcus aureus alpha hemolysin, serving as a carrier protein for the epitope (ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTPSGSVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNFLD EpiVac contains three immunodominant epitopes of Klebsiella pneumoniae (KDNASNTQALSGGEIPGYDSTGR, NEDFNDTGKAAGLSDLSLKD, and ADSKGNYASTGVSRSEHD), connected by a linker (GGGGS, GGGGS, and GSGGSG). The present invention also discloses methods for preparing and using this antigenic protein. The antigenic protein prepared using the present method can effectively stimulate a high humoral and cellular immune response and provide significant protection against lethal doses of Klebsiella pneumoniae infection, making it a candidate antigen for a Klebsiella pneumoniae vaccine.

[0032] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The above and other features, properties and advantages of the present invention will become more apparent from the following description with reference to the accompanying drawings and embodiments.

[0034] Figure 1 :Double enzyme digestion identification results of recombinant plasmid pGEX-6p-1-mHla-EpiVac

[0035] Lane M: DNA molecular weight markers (markers), sizes from top to bottom: 5000, 3000, 2000, 1500, 1000, 750, 500, 250, and 100 bp; Lane 1: Identification of the recombinant expression plasmid pGEX-6p-1-mHla-EpiVac after double digestion with Nde1 and Xho1. The fragments separated after digestion are approximately 5900 bp and approximately 1020 bp; Lane 2: Plasmid pGEX-6p-1-mHla-EpiVac.

[0036] Figure 2 :Identification results of mHla-EpiVac protein induced expression

[0037] Lane M: Protein molecular weight markers (from top to bottom): 180 kDa, 130 kDa, 100 kDa, 70 kDa, 55 kDa, 40 kDa, 35 kDa, 25 kDa, 15 kDa, and 10 kDa; Lane 1: Supernatant from lysis; Lane 2: Pellet from lysis; Lane 3: Flow-through from binding; Lane 4: Packing material binding; Lane 5: Enzyme-digested protein; Lane 6: Enzyme-digested packing material. The target protein is approximately 37 kDa and is expressed in both the supernatant and pellet, but contains significant protein impurities and requires further purification.

[0038] Figure 3 :SDS-PAGE electrophoresis results of purified mHla-EpiVac protein

[0039] Lane M: Protein molecular weight markers, sizes from top to bottom are: 180 kDa, 130 kDa, 100 kDa, 70 kDa, 55 kDa, 40 kDa, 35 kDa, 25 kDa, 15 kDa, 10 kDa; Lane 1: mHla-EpiVac protein after cation exchange chromatography, with good purity.

[0040] Figure 4 : Specific IgG antibody titers against EpiVac after three immunizations

[0041] After three immunizations, specific IgG antibody titers against EpiVac were obtained. mHla-EpiVac immunization can effectively increase the production of antibodies, while EpiVac immunization almost does not produce corresponding antibodies.

[0042] Figure 5 in Figure 5 A to Figure 5 C: Splenocyte ELISpot after three immunizations

[0043] The number of IL-4 and IFN-γ specific cells was measured, and spleen cells from mice were taken 7 days after the third immunization for restimulation. Pictures were taken and the results were statistically analyzed 40 hours after stimulation. The experiment proved that mHla-EpiVac can induce effective cellular immunity.

[0044] Figure 6 in Figure 6 A to Figure 6 C: Protective effect of mHla-EpiVac on mice

[0045] Seven days after the third immunization, mice were treated with a lethal dose of YBQ 8×10 6 The mice were infected with CFU lungs and then monitored for ten days for survival, body weight, and clinical scores. The experiment found that mHla-EpiVac alone provided a 50% protection rate, while its combination with an aluminum adjuvant resulted in an 80% protection rate. In contrast, EpiVac was as effective as PBS. Mice immunized with mHla-EpiVac gained less weight, recovered more quickly, and showed improved clinical scores.

[0046] Figure 7 :Uptake of mHla-EpiVac by mouse BMDCs

[0047] Immature mouse BMDCs were co-incubated with mHla-EpiVac and EpiVac for 10 hours, respectively. The experiment showed that immature mouse BMDCs took up more mHla-EpiVac and less EpiVac. DETAILED DESCRIPTION

[0048] The present invention will be further described below with reference to the accompanying drawings and examples. The following examples will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that those skilled in the art may make several variations and improvements without departing from the scope of the present invention. These all fall within the scope of protection of the present invention.

[0049] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or those recommended by the manufacturer. All reagents or instruments used, if the manufacturer is not specified, are commercially available conventional products.

[0050] The strains and various reagents used in the present invention are as follows:

[0051] Plasmid pGEX-6p-1 and Escherichia coli strain BL21 were deposited by the inventor's institution; restriction enzymes Nde I and Xho I and protein markers were products of Dalian TakaRa Company; the plasmid extraction kit and gel recovery kit were products of Omega Company in the United States; the cation exchange chromatography column (HiTrapSP HP 5 ml) and hydrophobic chromatography column (HiTrap Phenyl HP5 ml) were products of GE Healthcare in the United States.

[0052] Using a reverse vaccinology approach and literature research, the inventors screened five outer membrane protein (OMPs) from KP outer membrane proteins: OmpW, FepA, OmpK17, OmpA, and OmpK36. Twenty-two loop structures and their sequences were identified. Using serum recovered from infection with the 700721 ​​standard strain, ELISA was used to screen these epitopes, identifying three highly immunogenic epitopes: FepA396-423, OmpA148-165, and OmpW144-163.

[0053] Steps for ELISA screening of highly immunogenic loops:

[0054] 1) Dilute the purified 22-loop protein to 5 μg / mL using coating solution;

[0055] 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 washing solution. After air drying, wrap the plate with plastic wrap and store in a refrigerator at 4°C until ready for use.

[0056] 3) Blocking: Add 200 μL / well of blocking solution to the ELISA plate, incubate at 37°C for 2 hours, and wash three times.

[0057] 4) Serum from the third day after infection with C. pneumoniae strain 700721 ​​was diluted 1:200;

[0058] 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.

[0059] 6) Dilute the HRP-labeled goat anti-mouse IgG antibody storage solution 1:10,000 to prepare the antibody working solution;

[0060] 7) Add diluted antibody working solution (100 μL / well), incubate at 37°C for 40 min, wash three times, and air dry.

[0061] 8) Add 100 μL / well of substrate color development solution (TMB) and react at room temperature in the dark for 5 minutes;

[0062] 9) Add stop solution (2M H2SO4) and immediately measure the OD value on a microplate reader at a wavelength of 450 nm to select loops with higher OD450 values.

[0063] The above three epitopes were fused and expressed as the fusion antigen EpiVac, and then mHla and EpiVac were fused to form the antigen protein mHla-EpiVac. This antigen was combined with an aluminum adjuvant to prepare a vaccine to immunize mice three times. The protection rate against the highly virulent strain YBQ screened in the laboratory in the early stage was 80%. After titer measurement, it was found that mHla-EpiVac could induce a highly efficient humoral immune response. ELISpot experiments proved that this protein could induce highly efficient cellular immunity and could serve as an effective vaccine candidate antigen.

[0064] Example 1: Gene synthesis and subcloning

[0065] 1. Synthesis of the amino acid sequence encoding mHla-EpiVac (SEQ ID NO: 1) and its ligation with pGEX-6p-1 were synthesized by Wuhan Jinkairui Bioengineering Co., Ltd.

[0066] 2. Transformation of the Recombinant Plasmid: Remove one tube of E. coli BL21 competent cells (Shanghai Sangon Biotechnology Co., Ltd.) from a -80°C freezer and add 4 μl of the synthesized pGEX-6p-1-mHla-EpiVac plasmid. Incubate on ice for 30 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. Spread 20 μl of the bacterial suspension onto an ampicillin-resistant LB plate. Incubate the plate upside down at 37°C in a 34°C incubator for 14 hours. Pick a well-separated colony from the transformation plate and inoculate it into ampicillin-resistant LB medium. Incubate at 37°C with shaking overnight.

[0067] 3. Double enzyme digestion identification

[0068] From the overnight culture at 37°C, shaker incubation was performed. Plasmids from positive clones were extracted using a Rapid Plasmid Extraction Kit (Tiangen Biochemical Technology Co., Ltd.) according to the manufacturer's instructions. Enzyme digestion was performed using Nde1 (Takara) and Xhol (Takara). The cells were incubated in a 37°C water bath for half an hour. The system was as follows:

[0069] Reagents Volume (ul) plasmid DNA 3 I 0.5 I 0.5 10×buffer(H) 1 <![CDATA[ddH2O]]> 5 total volume 10

[0070] A 1.0% agarose gel containing 0.5 μg / ml of EB (Shanghai Junsheng 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: a large fragment of approximately 5900 bp, representing the expression vector pGEX-6p-1, and a small fragment of approximately 1020 bp, representing the inserted DNA fragment encoding mHla-EpiVac ( Figure 1 ).

[0071] Example 2: Identification of mHla-EpiVac antigen protein expression patterns

[0072] 1. mHla-EpiVac-induced expression

[0073] Add 100 μL of an overnight pGEX-6p-1-mHla-EpiVac / BL21 culture to 10 mL of ampicillin-resistant LB medium. Incubate at 180 rpm and 37°C for 5 hours. When the OD600 reaches 0.6-0.8, add IPTG to a final concentration of 200 μM. Induce expression on a shaker at 16°C for 14 hours. Remove the induced culture and centrifuge at 10,000 rpm for 5 minutes. Discard the supernatant, add 1 mL of PBS, mix thoroughly, sonicate for 3 minutes, and centrifuge at 12,000 rpm at 4°C for 15 minutes to separate the supernatant and precipitate.

[0074] 2. SDS-PAGE Electrophoresis

[0075] Pour 10% separation gel into the gel plate, then add distilled water to flatten the gel, let it stand at room temperature for 30 minutes to solidify, drain the distilled water on the top layer, then pour in the concentrated gel, immediately insert the comb, let it stand at room temperature for 30 minutes to solidify for later 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 1-2 hours, remove the gel, place it in instant blue staining solution for shaking staining, and then place it in first-level water for shaking decolorization. Observe the results under the imaging system. pGEX-6p-1-mHla-EpiVac / BL21 is expressed in a soluble form under 16°C induction conditions, and there is also a large amount of protein in the precipitate ( Figure 2 ).

[0076] Example 3: Preparation of mHla-EpiVac protein antigen

[0077] 1. Amplification and culture to obtain protein

[0078] 100 μL of the pGEX-6p-1-mHla-EpiVac / BL21 bacterial solution stored in a 4°C refrigerator was added to 10 mL of LB medium containing Ampicillin resistance for primary activation. After incubation at 220 rpm and 37°C for 4-5 h, 10 mL of the primary activated bacterial solution was added to 2000 mL of LB medium containing Ampicillin resistance for secondary activation. The culture was cultured at 37°C for 4-5 h until the OD600 reached 1.0. 400 μL of IPTG (final concentration of 200 μM) was added and the culture was induced in a shaker at 16°C for 14 h. The cells were collected by centrifugation at 10,000 rpm for 15 min, and then 50 mL of PBS (same as in Example 2) was added to resuspend the cells. The bacterial solution was ultrasonically lysed for 3 min (200 V), and the supernatant was collected for subsequent purification.

[0079] 2. Purification of mHla-EpiVac Protein

[0080] Wash 5 mL of Glutathione Sepharose affinity medium three times with 20 mM PBS, pH 7.5. Mix the prepared centrifugation supernatant with the affinity medium and allow it to bind for 1 hour at room temperature. Remove unbound supernatant by air gravity column. Wash the affinity medium three times with PBS. Add 1 mg of PreScission protease to remove the GST tag and digest at room temperature for 2 hours. After digestion, collect the supernatant and use it for ion exchange chromatography.

[0081] A cation exchange chromatography column (HiTrapSP HP 5ml) was prepared using a PBS (same as in Example 2) equilibrated chromatography system and SP HP column. The supernatant was then loaded and eluted using a linear gradient of wash buffer (50 mM PB, pH 6.25, 1 M NaCl). The elution flow rate was set at 5 ml / min, with an elution gradient from 0 to 100% wash buffer. The elution volume was 50 ml. The eluted target protein was collected and stored at 4°C until further use. The electrophoresis results are shown in Figure 2. Figure 3 shown.

[0082] Example 4: Animal Immunization and Antibody Detection

[0083] The mHla-EpiVac antigen was diluted with PBS and Al(OH)3 at a concentration of 1 mg / mL was added to prepare the vaccine. BALB / C mice were immunized with 100 μL of 30 μL of antigen injected into the bilateral thigh muscles using a 5-gauge half-size needle on days 0, 14, and 21. The blank control group was immunized with the same volume of PBS. On the 7th day after the last immunization, tail vein blood was collected from the BALB / C mice, and the antigen-specific IgG response level of the mice after immunization was detected by ELISA.

[0084] 1. Prepare the liquid

[0085] 1) Preparation of coating solution: Weigh 1.6 g of Na2CO3 and 2.9 g of NaHCO3, dissolve in 1 L of ddH2O, and adjust the pH to 9.6 using a pH meter.

[0086] 2) Preparation of blocking solution: Dissolve 1g of bovine serum albumin in 100mL of antibody diluent (1:100);

[0087] 3) Preparation of antibody diluent: Dissolve phosphate in 1 L ddH2O, add 500 μL Tween 20, and adjust the pH to 7.4 using a pH meter.

[0088] 4) Preparation of washing solution: same as antibody dilution solution

[0089] 5) Chromogenic solution (TMB), a product of Tiangen Company;

[0090] 6) Preparation of stop solution (2M H2SO4): Pour 22.2 mL of concentrated sulfuric acid into 177.8 mL of ddH2O.

[0091] 2. ELISA detection of antibody titers produced by mice immunized with mHla-EpiVac recombinant protein

[0092] 1) Dilute the purified mHla-EpiVac, FepA396-423, OmpA148-165, and OmpW144-163 proteins to 5 μg / mL using coating solution.

[0093] 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 washing solution. After air drying, wrap the plate with plastic wrap and store in a refrigerator at 4°C until ready for use.

[0094] 3) Blocking: Add 200 μL / well of blocking solution to the ELISA plate, incubate at 37°C for 2 hours, and wash three times.

[0095] 4) Dilute the serum in serial ratios of 1:1000, 1:2000, 1:4000, 1:8000, etc.

[0096] 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.

[0097] 6) Dilute the HRP-labeled goat anti-mouse IgG antibody storage solution 1:10,000 to prepare the antibody working solution;

[0098] 7) Add diluted antibody working solution (100 μL / well), incubate at 37°C for 40 min, wash three times, and air dry.

[0099] 8) Add 100 μL / well of substrate color development solution (TMB) and react at room temperature in the dark for 5 minutes;

[0100] 9) Add stop solution (2M H2SO4) and immediately place on a microplate reader to measure the OD value at a wavelength of 450 nm;

[0101] 10) Result judgment: A 样品 ∕A 阴性 A value ≥ 2.1 was considered positive (the negative control was mouse pre-immune serum diluted 1:1000).

[0102] Results: The geometric mean titer of EpiVac-specific IgG antibodies produced by mice immunized with mHla-EpiVac protein antigen was 1:4096000; the geometric mean titer of EpiVac-specific IgG antibodies produced by the combination of mHla-EpiVac and aluminum adjuvant was 1:8192000. The geometric mean titers of IgG antibodies specific to FepA396-423, OmpA148-165, and OmpW144-16 were 1:8192000, 1:1024000, and 1:2048000, respectively. The antibody positive rate reached 100% on the 7th day after the last immunization ( Figure 4 ), indicating that the mHla-EpiVac recombinant protein constructed by the present invention has good immunogenicity.

[0103] Example 5: Animal Immunity and Virus Protection Experiment

[0104] The mHla-EpiVac antigen was diluted with PBS and Al(OH)3 at a concentration of 1 mg / mL was added to prepare the vaccine. BALB / C mice were immunized with a 5-gauge half-size needle via bilateral thigh intramuscular injection on days 0, 14, and 21. Each mouse was injected with 100 μL of PBS containing 30 μL of antigen. The blank control group was immunized with the same volume of PBS. On day 7 after the last immunization, 8×10 6 Mice were challenged with a CFU dose of the clinical strain YBQ of Klebsiella pneumoniae via tracheal instillation. The survival status of the mice was observed for 10 days, with the number of deaths recorded daily. Survival rates were calculated after the observation period. Three consecutive animal experiments showed that compared with the control group, the survival rate of mice in the mHla-EpiVac-immunized group was significantly improved, with a protection rate of approximately 50%. This rate reached 80% when combined with an aluminum adjuvant. Figure 6 in Figure 6 A to Figure 6 C), indicating that it can be used as a candidate antigen for vaccine development.

[0105] Example 6: ELISpot

[0106] The spleen of mice was collected 7 days after the third immunization and ground into homogenate under sterile conditions. The red blood cells were lysed using red blood cell lysis buffer and the cell concentration was adjusted to 1×10 7 , followed by addition to ELISpot plates (MabTech).

[0107] 1) Treatment of each group:

[0108] (1) Activation of pre-coated plates: First, add 200uL of sterile PBS to each well for washing, let it stand for two minutes, then remove it, and repeat 4 times; then add 200μL of 1640 complete culture medium to each well, let it stand at room temperature for 30 minutes, then remove it.

[0109] (2) Add cell suspension: Add the adjusted concentration of cell suspension to each experimental well, 100 μL / well.

[0110] (3) Add stimulants: 100 μL / well, as follows:

[0111] Positive control wells: Add positive stimulator working solution.

[0112] Negative control wells: add culture medium (or culture medium for resuspending cells);

[0113] Experimental wells: Add mHla-EpiVac and EpiVac (prepared with serum-free medium or RPMI 1640).

[0114] 2) Color rendering

[0115] (1) Empty the plate to remove cells and wash five times with 200 μl / well of PBS.

[0116] (2) Dilute the detection antibody (R4-6A2-biotin) to 1 μg / ml in PBS containing 0.5% fetal calf serum (PBS-0.5% FCS). Add 100 μl / well and incubate at room temperature for 2 hours.

[0117] (3) Wash five times with 200 μl / well PBS.

[0118] (4) Dilute streptavidin HRP (1:1000) in PBS-0.5% FCS and add 100 μl / well. Incubate at room temperature for 1 hour.

[0119] (5) Wash five times with 200 μl / well PBS.

[0120] (6) Add 100 μl / well of ready-to-use TMB matrix solution and develop until obvious spots appear.

[0121] (7) Stop color development by washing extensively in deionized water. Remove the plate holder from the plastic tray and rinse the underside of the membrane.

[0122] (8) Count the spots on the ELISPOT plate and record various parameters of the spots for statistical analysis

[0123] Results: Immunization with mHla-EpiVac could induce the production of specific IL-4 and IFN-γ cells. Re-stimulation with mHla-EpiVac could produce more IL-4 and IFN-γ secreting cells ( Figure 5 in Figure 5 A to Figure 5 C), indicating that mHla-EpiVac immunization can produce specific cellular immunity.

[0124] Example 7: Laser Confocal

[0125] mHla-EpiVac and EpiVac were stained with fluorescent dye AF488 according to the procedure. Fluorescent mHla-EpiVac and EpiVac were diluted with 1640 medium containing 10% FBS at a concentration of 40 μg / ml and then mixed with 1×10 5 Immature mouse BMDCs were incubated in a confocal microplate in the dark for 10 hours. The medium was discarded, and the cells were washed three times with 37°C preheated PBS and fixed with 4% paraformaldehyde for 15 minutes. The cells were washed three times with PBS and permeabilized with PBS containing 3% BSA and Triten-100 for 15 minutes, discarded, and washed three times with PBS. The cells were stained with 100 nmol / l phalloidin (absin) for 10 minutes, discarded, and washed three times with PBS. The cells were stained with DAPI (Biyuntian) for 5 minutes, discarded, and washed three times with PBS. The cells were then added with 100 μl of PBS and observed on an imaging microscope.

[0126] Results: At the same time and concentration, the uptake of mHla-EpiVac by immature BMDCs was higher than that of EpiVac. Figure 7 ), indicating that mHla-EpiVac can promote the uptake of antigens by immature BMDCs.

[0127] The above description is only a preferred embodiment of the present invention and is not intended to limit the scope of implementation of the present invention. If the present invention is modified or replaced with equivalents without departing from the spirit and scope of the present invention, it should be included in the scope of protection of the claims of the present invention.

Claims

1. A Klebsiella pneumoniae vaccine fusion antigen mHla-EpiVac, characterized in that: The amino acid sequence of the fusion antigen mHla-EpiVac is shown in SEQ ID NO:

1.

2. The fusion antigen mHla-EpiVac according to claim 1, wherein The fusion antigen contains three immunodominant epitopes derived from Klebsiella pneumoniae.

3. The fusion antigen mHla-EpiVac according to claim 2, characterized in that The amino acid sequences of the three immunodominant epitopes are shown in SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively.

4. The method for preparing the Klebsiella pneumoniae vaccine fusion antigen mHla-EpiVac according to any one of the above claims, characterized in that: Contains steps: 1) Synthesis and subcloning of mHla-EpiVac; 2) Inducible expression of mHla-EpiVac; 3) Preparation of mHla-EpiVac protein antigen; 4) Purification of mHla-EpiVac protein.

5. The method according to claim 4, wherein Step 1) specifically comprises: connecting mHla to the sequence shown in SEQ ID NO: 2 with a flexible chain, then connecting the sequence shown in SEQ ID NO: 4 with a flexible chain, and then connecting the sequence shown in SEQ ID NO: 3 with a flexible chain to form the fusion antigen mHla-EpiVac.

6. The method according to claim 4 or 5, characterized in that Step 1) involves constructing a recombinant expression plasmid using the prokaryotic expression plasmid pGEX-6p-1 having ampicillin resistance.

7. The method according to claim 4 or 5, characterized in that Step 4) comprises: collecting genetically engineered bacteria expressing mHla-EpiVac; purifying mHla-EpiVac by a sequential combination of high-pressure lysis, centrifugation, cation exchange chromatography, and hydrophobic chromatography.

8. Use of the Klebsiella pneumoniae vaccine fusion antigen mHla-EpiVac according to claim 1 in the preparation of an immune preparation for preventing or treating Klebsiella pneumoniae infection.

9. A Klebsiella pneumoniae injectable immune preparation comprising the Klebsiella pneumoniae vaccine fusion antigen mHla-EpiVac according to claim 1 and any one or any combination of the following adjuvants: aluminum hydroxide adjuvant, aluminum phosphate adjuvant, aluminum monostearate adjuvant, MF59, complete Freund's adjuvant, incomplete Freund's adjuvant, or Mycobacterium Calmette-Guerin adjuvant.

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