A live attenuated vaccine strain of Klebsiella pneumoniae with relA / spoT double gene deletion, its preparation method and application

Abstract

This invention provides a live attenuated Klebsiella pneumoniae vaccine strain with double gene deletion of relA / spoT, its preparation method, and its application. By precisely regulating the metabolic stress response, the virulence risk is reduced while retaining strong immunogenicity. This vaccine has the following advantages: Safety: Double gene deletion prevents virulence recovery, making it suitable for special populations; Efficacy: Induces long-lasting humoral immunity, with a challenge protection rate of up to 90%; Process advantages: Low cost for room temperature storage and transportation, and convenient mucosal administration route.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a live attenuated vaccine strain of Klebsiella pneumoniae with relA / spoT double gene deletion, its preparation method and application. Background Technology

[0002] Traditional live attenuated vaccines have technical drawbacks: (1) Traditional live attenuated vaccines carry the risk of virulence recovery and potential adverse reactions, especially posing a higher risk to special populations (such as pregnant women and those with weakened immune systems). (2) Vaccine storage and transportation require strict infrastructure, significantly increasing distribution costs. (3) Furthermore, using mice as a research model has inherent limitations, mainly due to the significant differences between the mouse and human immune systems. Existing attenuation strategies (such as single-gene deletion) struggle to balance safety and immunogenicity, and lack stable strain construction systems suitable for clinical translation.

[0003] Therefore, it is necessary to develop a live attenuated Klebsiella pneumoniae vaccine with a stable strain construction system that can balance safety and immunogenicity and is suitable for clinical translation. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a live attenuated Klebsiella pneumoniae vaccine strain with relA / spoT double gene deletion, its preparation method, and its application, which has advantages such as good safety and strong immunogenicity.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] In a first aspect of the invention, a live attenuated vaccine strain of Klebsiella pneumoniae with double gene deletion of relA / spoT is provided, said vaccine strain being Klebsiella pneumoniae HvKp containing double gene deletion of relA and spoT.

[0007] In a second aspect of the invention, a method for preparing a live attenuated Klebsiella pneumoniae vaccine strain with relA / spoT double gene deletion is provided, the method comprising:

[0008] Using Klebsiella pneumoniae NTUH-K2044 strain DNA as a template, the upstream and downstream homologous arm fragments of relA were amplified using relA-A / B and relA-C / D primer pairs, respectively, and the upstream and downstream homologous arm fragments of spoT were amplified using spoT-A / B and spoT-C / D primer pairs.

[0009] Using relA-A / D as primer pairs, the upstream and downstream homologous arm fragments of relA were connected to obtain the homologous arm fusion fragment of relA;

[0010] Using spoT-A / D as primer pairs, the upstream and downstream homologous arm fragments of spoT were connected to obtain the homologous arm fusion fragment of spoT.

[0011] After the homologous arm fusion fragment of relA and the homologous arm fusion fragment of spoT were respectively ligated into the pKO4-Km vector, recombinant plasmids pKO4-Km-ΔrelA and pKO4-Km-ΔspoT containing homologous arms were obtained.

[0012] The recombinant plasmid pKO4-Km-ΔrelA was transformed into S17-1λpir competent cells. The correct colonies were identified as donor bacteria, and NTUH-K2044 strain was used as recipient bacteria for conjugation experiments. Single colonies with small fragments using relA-A / D primer pair and no bands using pKO4-Km-F / R primer pair were screened to obtain the ΔrelA strain.

[0013] The recombinant plasmid pKO4-Km-ΔspoT was transformed into S17-1λpir competent cells. The correctly identified colonies were used as donor bacteria, and the ΔrelA strain was used as recipient bacteria for conjugation experiments. PCR identification and screening were performed using spoT-A / D primer pair, pKO4-Km-F / R primer pair, and NTUH-K2044 strain as a positive control to obtain the Klebsiella pneumoniae attenuated live vaccine strain with relA / spoT double gene deletion, namely the ΔrelAΔspoT strain.

[0014] The nucleotide sequences of the relA-A / B primer pair are shown in SEQ ID NO.1-2; the nucleotide sequences of the relA-C / D primer pair are shown in SEQ ID NO.3-4; the nucleotide sequences of the spoT-A / B primer pair are shown in SEQ ID NO.7-8; and the nucleotide sequences of the spoT-C / D primer pair are shown in SEQ ID NO.9-10.

[0015] In a third aspect of the invention, a live attenuated vaccine for Klebsiella pneumoniae with the slyA gene deletion is provided, comprising the aforementioned live attenuated vaccine strain of Klebsiella pneumoniae with the relA / spoT double gene deletion and a pharmaceutically acceptable adjuvant.

[0016] Further, the adjuvant includes at least one of aluminum hydroxide, lecithin, Freund's adjuvant, MPLTM, IL-12, aluminum hydroxide combined with CpG ODN complex adjuvant, ISA51VG, ISA720VG, MF59, QS21, and AS03 adjuvant.

[0017] In a fourth aspect of the invention, the use of the said vaccine strain and / or the said vaccine in the preparation of a vaccine for the prevention of Klebsiella pneumoniae infection in humans and animals is provided.

[0018] The GenBank accession number for relA is KP4398, and the GenBank accession number for spot is KP5353.

[0019] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0020] This invention provides a live attenuated Klebsiella pneumoniae vaccine strain with double gene deletion of relA and spoT, its preparation method, and its application. This invention demonstrates the feasibility of constructing a live attenuated vaccine by deleting the metabolic regulatory genes relA and spoT. Immunogenicity (specific antibody levels) was tested through animal immunization experiments. Highly virulent Klebsiella pneumoniae (HvKp) was used to challenge immunized animals, and the protective effect was evaluated. Immunization experiments showed that this strain could induce high levels of specific antibody IgG. Compared with the control group, the immunized group showed a significant increase in survival rate after HvKp challenge (p<0.0001). Further studies revealed a significant decrease in HvKp colonization levels in immunized animals. The relA / spoT double gene deletion strategy provides a new approach for the development of bacterial attenuated vaccines. This method has advantages such as good safety and strong immunogenicity, and is expected to become an important technical platform for developing next-generation bacterial vaccines. This invention provides a live attenuated Klebsiella pneumoniae vaccine based on a double gene deletion strategy, which reduces virulence risk by precisely regulating metabolic stress response while retaining strong immunogenicity. This vaccine has the following advantages:

[0021] (1) Safety: Through a dual safety mechanism: relA deletion blocks the global stress response, spoT deletion inhibits the synthesis of virulence factors; multiple passage stability verifications (>50 generations without virulence recovery), dual gene deletion prevents virulence recovery, suitable for special populations.

[0022] (2) Efficacy: Induces long-term humoral immunity and enhances immunogenicity: Induces high levels of IgG antibodies (3.6 times higher than the control group); long-term protective effect (90% survival rate in challenge experiments).

[0023] (3) Process advantages: No need for complicated cold chain transportation (stable storage at 2-8℃ for 3 months), low cost of room temperature storage and transportation, suitable for mucosal immunity (nasal drop administration route), convenient mucosal administration route. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 Electrophoresis images of the upstream and downstream homologous arms (A) and fusion fragment (B) of relA and spoT. Note: Figure A: M: Trans5K DNA marker; 1: Upper homologous arm fragment of relA amplified using the WT genome as a template and relA-A / B primer pair; 3: Lower homologous arm fragment of relA amplified using the WT genome as a template and relA-C / D primer pair; 5: Upper homologous arm fragment of spoT amplified using the WT genome as a template and spoT-A / B primer pair; 7: Lower homologous arm fragment of spoT amplified using the WT genome as a template and spoT-C / D primer pair; 2, 4, 6, 8: Negative controls. Figure B: M: Trans5K DNA marker; 1: Fusion fragment amplified using relA upstream and downstream homologous arms as templates and relA-A / D primer pair as a control; 2: Large fragment amplified using the WT genome as a template and relA-A / D primer pair as a control; 4: Fusion fragment amplified using spoT upstream and downstream homologous arms as templates and spoT-A / D primer pair as a control; 5: Large fragment amplified using the WT genome as a template and spoT-A / D primer pair as a control; 3, 6: Negative controls.

[0026] Figure 2 Electrophoresis image for PCR identification of recombinant plasmid pKO4-Km-relA / spoT. Note: M: Trans5K DNA marker; 1: Fragment amplified using relA transformant as template and pKO4-Km-F / R primer pair; 2: Fragment amplified using spoT transformant as template and pKO4-Km-F / R primer pair; 3: Fragment amplified using plasmid pKO4-Km as template and pKO4-Km-F / R primer pair; 4: Negative control.

[0027] Figure 3 Electrophoresis images for PCR identification of ΔrelA (Figure A) and ΔrelAΔspoT (Figure B);

[0028] Figure 4 The levels of IgG antibodies in mice before and after immunization;

[0029] Figure 5 Survival curves for mice in the immunized group and the PBS control group after challenge;

[0030] Figure 6 The colonization level of HvKp strain in mice in the immunized group and PBS control group.

[0031] Figure 7 This refers to the results of histopathological examination. Detailed Implementation

[0032] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0033] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0034] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0035] The effects of this application will be described in detail below with reference to embodiments and experimental data. Unless otherwise specified, the molecular cloning methods, protein expression and purification methods, cell culture methods, and various detection methods mentioned in the following schemes are all traditional experimental methods, which can be obtained by consulting the literature; the relevant reagents used can be purchased from the corresponding reagent suppliers.

[0036] The highly virulent Klebsiella pneumoniae NTUH-K2044 strain of this invention was obtained by donation, and the original source can be found in the literature: Genome sequencing and comparative analysis of Klebsiella pneumoniae NTUH-K2044, a strain causing liver abscess and meningitis.

[0037] The T-carrier of this invention is pUCm-T, purchased from Shanghai Sangon Biotech, item number B522211-0001.

[0038] The vector pKO4-Km of this invention, also known as pKO3-Km-DbolA, was donated and named by Nankai University. It was obtained by modifying the pKO3-Km plasmid. The modification method is detailed in the literature: Zhang F, Yan X, Bai J, Xiang L, Ding M, Li Q, Zhang B, Liang Q, Zhou Y. Identification of the BolA Protein Reveals a Novel Virulence Factor in K. pneumoniae That Contributes to Survival in Host. Microbiol Spectr. 2022 Oct26;10(5):e0037822.doi:10.1128 / spectrum.00378-22.Epub 2022Sep 19.PMID:36121239;PMCID:PMC9603091.

[0039] Example 1: Klebsiella pneumoniae live attenuated vaccine strain with relA / spoT double gene deletion and its preparation method

[0040] 1. Primer design

[0041] Table 1

[0042]

[0043]

[0044] 2. Preparation of bacterial strains

[0045] Klebsiella pneumoniae NTUH-K2044, frozen at -80℃, and vector bacteria containing plasmid pKO4-Km were streaked onto LB agar plates and incubated at 37℃ and 30℃, respectively. The next day, single colonies were picked and inoculated onto LB broth medium and cultured with shaking at 220 rpm for 12 h.

[0046] 3. Extraction of bacterial genome and suicide plasmids

[0047] Take 1 mL of the NTUH-K2044 bacterial culture that has been cultured overnight and use a bacterial genomic DNA extraction kit to obtain the genome. Take 5 mL of the vector bacterial culture that has been cultured overnight and use a plasmid miniprep kit to obtain the plasmid pKO4-Km. After extraction, the concentrations of the genome and plasmid were detected using a micro spectrophotometer and stored at -20°C.

[0048] 4. Construction of recombinant plasmids pKO4-Km-ΔrelA and pKO4-Km-ΔspoT

[0049] (1) Amplification of upper and lower homologous arms: Using NTUH-K2044 strain DNA as a template, the upper and lower homologous arm fragments of relA were amplified using relA-A / B and relA-C / D primer pairs, respectively, and the upper and lower homologous arm fragments of spoT were amplified using spoT-A / B and spoT-C / D primer pairs, respectively. The following components were added to the amplification system:

[0050] Table 2

[0051]

[0052]

[0053] The above reaction solution was placed in a PCR instrument under the following conditions: 98℃ pre-denaturation for 5 min, 98℃ denaturation for 10 s, 60℃ annealing for 5 s, 72℃ extension at 1 kbp / min, 34 cycles, 72℃ extension for 5 min, and storage at 12℃. After the reaction, 4 μL of 6× loading buffer was added, and the results were verified by 1% agarose gel electrophoresis. Bands of the correct size were excised, recovered using a DNA gel extraction kit, and the amplified fragment concentration was detected using a micro spectrophotometer. The fragments were then stored at -20℃.

[0054] (2) Fusion of upper and lower homologous arms: Using PCR, the upper and lower homologous arm fragments were ligated using relA-A / D and spoT-A / D primer pairs, respectively. The amplification system is shown in Table 3-5. The total template volume was 1 μL to ensure consistent concentration of the upper and lower homologous arms. The fragments were verified and recovered by agarose gel electrophoresis, and the concentrations were measured and stored. The fusion fragment and plasmid pKO4-Km were digested with the restriction endonuclease XbaI. The following components were added respectively:

[0055] Table 3

[0056]

[0057] The above reaction solution was placed in a 37°C incubator for 1 hour for enzyme digestion. 2 μL of 10× loading buffer was added, and the digested product was recovered using a column PCR product purification kit, with the concentration determined.

[0058] The results of the amplification and fusion of the upper and lower homologous arms are as follows: Figure 1 As shown:

[0059] Using NTUH-K2044 strain DNA as a template, the upstream and downstream homologous arm fragments of relA were amplified using relA-A / B and relA-C / D primer pairs, with sizes of 600bp and 407bp, respectively.

[0060] The upstream and downstream homologous arm fragments of spoT were amplified using spoT-A / B and spoT-C / D primer pairs, with sizes of 578bp and 651bp, respectively.

[0061] The upstream and downstream homologous arm fragments were ligated using relA-A / D and spoT-A / D primer pairs, respectively, with sizes of 1007 bp and 1229 bp. Identification results showed that the band sizes were as expected.

[0062] (3) Screening and identification of recombinant plasmids

[0063] The fusion fragment prepared in the above steps and the pKO4-Km plasmid were ligated using T4 ligase at a ratio of 3:1. The following components were added respectively:

[0064] Table 4

[0065]

[0066] After the above reaction solution was left to stand at room temperature for 3 hours, it was placed in a 65°C water bath for 10 minutes to inactivate it.

[0067] Thaw 50 μL of LDH5α competent cells on ice, add 5 μL of ligation product, gently pipette to mix, and incubate on ice for 30 min. Place the mixture in a 42℃ water bath for 45 s heat shock, followed by an ice bath for 2 min. Then, slowly add 500 μL of preheated LB broth to the mixture and incubate at 30℃ and 130 rpm for 1 h with shaking. Spread 100 μL of the culture onto LB agar plates containing 50 μg / mL kanamycin and incubate overnight at 30℃. Select larger single colonies as templates and perform PCR identification and screening using relA-A / D and spoT-A / D primer pairs. Send the PCR products containing the target fragment to Beijing Qingke Biotechnology Co., Ltd. for sequencing. Compare the sequencing results with the genome to identify the correct pKO4-Km-ΔrelA and pKO4-Km-ΔspoT recombinant plasmids.

[0068] The fusion fragment was recovered, digested and ligated together with the knockout plasmid, and then transformed into competent cells. Single colonies containing fragments of 1469 bp and 1691 bp were screened using pKO4-Km-F / R primers. The identification results are as follows: Figure 2 As shown, the band size is as expected.

[0069] 5. Homologous recombination to construct the deletion strain ΔrelAΔspoT

[0070] (1) Obtaining strain ΔrelA

[0071] Correctly identified single colonies were cultured overnight at 30°C. Recombinant plasmids were obtained using a plasmid miniprep kit, and their concentration was measured using a micro-spectrophotometer. The plasmids were then stored at -20°C. The recombinant plasmids were transformed into S17-1λpir competent cells, and PCR screening was performed using pKO4-Km-F / R primers and pKO4-Km plasmid as a positive control. Colonies exhibiting large bands were used as donor bacteria, and NTUH-K2044 strain as recipient bacteria, and cultured at 30°C and 37°C, respectively, to the logarithmic growth phase. 1 mL of each bacterial culture was centrifuged at 10,000 rpm for 3 min, washed twice with 10 mmol / L MgSO4 solution, and resuspended in 100 μL of MgSO4. The two solutions were mixed at a 1:1 ratio, and 10 μL was added to LB agar plates and incubated overnight at 30°C.

[0072] Scrape the entire bacterial colony from the plate, resuspend it in 1 mL of PBS, dilute and spread it onto an M9 basal plate containing 50 μg / mL kanamycin. Perform PCR identification and screening using relA-A / D primers and NTUH-K2044 strain as a positive control. Select three verified colonies and mix them in 5 mL of LB broth, incubate at 30°C and 130 rpm for 4 h with shaking. Dilute and spread on LB agar plates containing 50 μg / mL kanamycin, and incubate overnight at 42°C.

[0073] PCR identification and screening were performed using relA-A / D primers. Colonies with small fragments were selected and cultured overnight at 30°C with shaking at 130 rpm. The bacterial suspension was diluted and plated onto LD plates containing 5% sucrose and cultured overnight. Larger single colonies were simultaneously inoculated onto both ordinary LB plates and LB plates containing 50 μg / mL kanamycin and incubated overnight at 30°C. Single colonies that grew normally on ordinary LB plates but not on antibiotic-resistant plates were selected. Single colonies with small fragments using relA-A / D primers and no bands using pKO4-Km-F / R primers were screened, streaked continuously, and then identified using relA-A / D primers. Finally, the PCR product containing the target fragment was sent to the company for sequencing. Successful sequencing results confirmed the identification of the ΔrelA strain.

[0074] The identified positive transformants were transformed into S17-1λpir competent cells and then conjugated into wild-type competent cells for two homologous recombination experiments. Using the wild-type strain as a control, single colonies amplifying a 1007 bp fragment were selected using the relA-A / D primer pair. Then, using the plasmid pKO4-Km as a control and the pKO4-Km-F / R primer pair, single colonies failing to amplify the fragment were selected. Colonies that amplified both fragments were identified as the deletion strain ΔrelA. The identification results are as follows: Figure 3As shown in Figure A, the band size is as expected.

[0075] (2) Obtain the ΔrelAΔspoT strain

[0076] The successfully constructed pKO4-Km-ΔspoT recombinant plasmid was transformed into S17-1λpir competent cells using the same procedure. Correctly identified colonies were used as donor bacteria, and the ΔrelA strain was used as the recipient bacteria for conjugation experiments. PCR identification and screening were performed using spoT-A / D primers, pKO4-Km-F / R primers, and NTUH-K2044 strain as a positive control. Successful sequencing verification yielded the ΔrelAΔspoT strain, which was then stored at -80°C.

[0077] The identified positive transformants were transformed into S17-1λpir competent cells and then conjugated into the deletion strain ΔrelA competent cells for two homologous recombinations. Using the wild-type strain as a control and spoT-A / D as the primer pair, single colonies capable of amplifying a 1229 bp fragment were screened. Then, using plasmid pKO4-Km as a control and pKO4-Km-F / R as the primer pair, single colonies unable to amplify the fragment were screened. Colonies that amplified both fragments correctly were identified as the deletion strain ΔrelAΔspoT. The identification results are as follows: Figure 3 As shown in B, the band size is as expected.

[0078] Example 2, Effect Experiment

[0079] I. ΔrelAΔspoT strains can stimulate humoral immune responses.

[0080] 1. Mouse immunization

[0081] High dose (2×10) 6 C57BL / 6J mice were immunized (nasally) with CFU-ΔrelAΔspoT strain twice, every 2 weeks for 4 weeks. Mice immunized with PBS served as normal controls. Serum samples were collected from mice before and after the last immunization, and IgG antibody production was assessed using enzyme-linked immunosorbent assay (ELISA).

[0082] 2. The steps of the enzyme-linked immunosorbent assay (ELISA) are as follows:

[0083] Prepare standards at concentrations of 50 ng / ml, 25 ng / ml, 12.5 ng / ml, 6.25 ng / ml, 3.12 ng / ml, and 1.56 ng / ml, respectively, and prepare HRP-labeled anti-mouse IgG antibody working solution 2 hours in advance.

[0084] Add 0.1 ml of each of the following standards (100 ng / ml, 50 ng / ml, 25 ng / ml, 12.5 ng / ml, 6.25 ng / ml, 3.12 ng / ml, and 1.56 ng / ml) sequentially to a row of 7 wells. The well containing only sample diluent is designated as the zero well. For plasma, add 100 μL of sample diluted with sample diluent to each well. Cover the plate with a sealing film and incubate at 37°C for 60 minutes. Wash three times with 1X wash buffer, soaking for approximately 1 minute each time. Add 0.1 ml of the prepared HRP-labeled anti-mouse IgG working solution to each well (except for the TMB blank wells). Incubate at 37°C for 30 minutes. Then wash five times with 1X wash buffer, soaking for approximately 1-2 minutes each time. Add 90 μL of TMB chromogenic solution to each well sequentially and incubate at 37°C in the dark for 15-20 minutes. Finally, add 0.1 ml of stop solution to each well sequentially; the blue color will immediately turn yellow. Finally, the OD value was measured at 450 nm using an ELISA reader.

[0085] Considering that ΔrelAΔspoT may be a live attenuated vaccine that can stimulate the body's immune system to respond, the concentration of IgG antibodies in the serum of C57BL / 6J mice before and after immunization was first determined by ELISA.

[0086] The results are as follows Figure 4 As shown, continuous immunization of mice for 4 weeks significantly induced the production of IgG antibodies, and the IgG concentration level was 3.6 times that of the PBS control group. In summary, this indicates that the ΔrelAΔspoT strain can induce a humoral immune response in the host and lead to an increase in serum IgG antibodies.

[0087] II. Immunization by ΔrelAΔspoT strains significantly reduces HvKp colonization levels.

[0088] The ΔrelAΔspoT strain can effectively stimulate the host's humoral immune response and produce IgG antibodies, making it a potential live attenuated vaccine. To further explore the host protective effect of ΔrelAΔspoT strain immunization, pneumonia models were constructed in mice with PBS control and immunized groups.

[0089] The results are as follows Figure 6 As shown, on day 2 of the challenge experiment, mice in the PBS control group began to die, and all died by day 4. In contrast, the survival rate of the immunized group mice was 90% at day 4. These findings indicate that immunization of mice with the ΔrelAΔspoT strain effectively resists challenge with the HvKp strain.

[0090] III. Immunological effects of ΔrelAΔspoT strains can alleviate histopathological changes.

[0091] Histopathological results such as Figure 7 As shown:

[0092] Lungs: Immunoassay group: Microscopically, the alveolar structure was intact, no abnormal exudate was observed in the alveolar cavities, no dilation or congestion of the alveolar septal capillaries was observed, and there was no inflammatory cell infiltration. The overall lung tissue structure was clear and normal. PBS group: Microscopically, the alveolar cavities were compressed and deformed, with obvious damage to the alveolar epithelial cells and alveolar epithelial cell shedding. Some alveolar septa were broken, and there was considerable hemorrhage and inflammatory cell infiltration in the alveolar cavities and alveolar septa, mainly neutrophils and eosinophils. A small amount of protein exudate was observed in some alveolar cavities, and the alveolar septa were slightly thickened in some areas. Immunoassay group: Most of the lung tissue was consolidated, with considerable hemorrhage and inflammatory cell infiltration in the consolidated areas, mainly neutrophils and eosinophils. The bronchioles showed significant proliferation, and large areas of inflammatory cell aggregation were observed in the bronchioles.

[0093] Liver: Negative control group: The liver tissue structure was clearly discernible, showing a lobular structure centered on the central vein. Hepatocytes were polygonal, with eosinophilic cytoplasm, large and round nuclei that stained lightly, and hepatocyte cords arranged radially. PBS group: Microscopically, the hepatocytes showed disordered arrangement, unclear lamina structure, slight congestion of the sinusoids, polygonal hepatocytes, some with loose, red-stained cytoplasm, mild to moderate edema and degeneration of the hepatocytes, scattered inflammatory cell infiltration within the hepatocytes, focal punctate necrosis, and a few inflammatory cell aggregates within the necrotic foci. No clear portal areas were observed. Significant congestion and a large amount of protein exudate were observed in the central vein. Immunotherapy group: Microscopically, hepatocytes were arranged radially around the central vein, the lamina structure was clear, some sinusoids were slightly congested, polygonal hepatocytes, some with loose, red-stained cytoplasm, mild edema and degeneration of the hepatocytes, scattered inflammatory cell infiltration within the hepatocytes, focal punctate necrosis, and a large number of inflammatory cell aggregates within the necrotic foci. No clear portal areas were observed. Spleen: Negative control group: Microscopically, splenic corpuscles were clearly visible, with abundant lymphoid tissue around the central artery. The red and white pulp structures were distinct, and blood cells were evenly distributed within the red pulp, with no obvious inflammation or structural abnormalities. PBS group: Microscopically, red and white pulp structures were visible. Medullary cords and sinus structures were visible within the red pulp. Significant congestion and hemorrhage were observed in the red pulp, along with a few lymphoid tissue structures and neutrophil infiltration. The white pulp consisted of irregular lymphoid follicles, with numerous histiocytes phagocytosing nuclear debris. The boundary between the white and red pulp was clear. Immunotherapy group: Congestion and hemorrhage were observed in the red pulp, along with a few lymphoid tissue structures and a few neutrophil infiltrations.

[0094] Kidneys: Negative control group: Microscopically, glomerular structures were intact, renal tubules were neatly arranged, and there was no edema, inflammatory cell infiltration, or structural abnormalities. PBS group: Microscopically, glomerular structures were visible, with no decrease in number or significant dilation and congestion. Tubular epithelial cells were arranged in a single columnar layer, with significantly loose, edematous, and vacuolated cytoplasm, loss of brush borders, and visible damage and degeneration. No clear casts were observed within the lumen. No fibrous tissue proliferation was observed in the renal interstitium, but focal congestion was visible. Immunotherapy group: Tubular epithelial cells were arranged in a single columnar layer, with slightly loose and edematous cytoplasm in a few individual tubular epithelial cells.

[0095] In summary, pathological changes were observed in the lung, liver, spleen, and kidney tissues of both the PBS and immunization groups. The lesions in the PBS group were relatively more severe, while those in the immunization group were relatively milder.

[0096] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0097] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0098] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A live attenuated Klebsiella pneumoniae vaccine strain with double gene deletion of relA / spoT, characterized in that, The method for preparing the vaccine strain includes: Using Klebsiella pneumoniae NTUH-K2044 strain DNA as a template, the upstream and downstream homologous arm fragments of relA were amplified using relA-A / B and relA-C / D primer pairs, respectively, and the upstream and downstream homologous arm fragments of spoT were amplified using spoT-A / B and spoT-C / D primer pairs. Using relA-A / D as primer pairs, the upstream and downstream homologous arm fragments of relA were ligated to obtain the homologous arm fusion fragment of relA; using spoT-A / D as primer pairs, the upstream and downstream homologous arm fragments of spoT were ligated to obtain the homologous arm fusion fragment of spoT; the homologous arm fusion fragments of relA and spoT were ligated to the pKO4-Km vector to obtain the recombinant plasmids pKO4-Km-ΔrelA and pKO4-Km-ΔspoT containing homologous arms. The recombinant plasmid pKO4-Km-ΔrelA was transformed into S17-1λpir competent cells. The correct colonies were identified as donor bacteria, and NTUH-K2044 strain was used as recipient bacteria for conjugation experiments. Single colonies with small fragments using relA-A / D primer pair and no bands using pKO4-Km-F / R primer pair were screened to obtain the ΔrelA strain. The recombinant plasmid pKO4-Km-ΔspoT was transformed into S17-1λpir competent cells. The correctly identified colonies were used as donor bacteria, and the ΔrelA strain was used as recipient bacteria for conjugation experiments. PCR identification and screening were performed using spoT-A / D primer pair and pKO4-Km-F / R primer pair to obtain the Klebsiella pneumoniae attenuated live vaccine strain with relA / spoT double gene deletion, namely the ΔrelAΔspoT strain. The nucleotide sequences of the relA-A / B primer pair are shown in SEQ ID NO. 1-2; the nucleotide sequences of the relA-C / D primer pair are shown in SEQ ID NO. 3-4; the nucleotide sequences of the spoT-A / B primer pair are shown in SEQ ID NO. 7-8; the nucleotide sequences of the spoT-C / D primer pair are shown in SEQ ID NO. 9-10; and the nucleotide sequences of the pKO4-Km-F / R primer pair are shown in SEQ ID NO. 13-14.

2. A method for preparing a live attenuated Klebsiella pneumoniae vaccine strain with double gene deletion of relA / spoT as described in claim 1, characterized in that, The method includes: Using Klebsiella pneumoniae NTUH-K2044 strain DNA as a template, the upstream and downstream homologous arm fragments of relA were amplified using relA-A / B and relA-C / D primer pairs, respectively, and the upstream and downstream homologous arm fragments of spoT were amplified using spoT-A / B and spoT-C / D primer pairs. Using relA-A / D as primer pairs, the upstream and downstream homologous arm fragments of relA were ligated to obtain the homologous arm fusion fragment of relA; using spoT-A / D as primer pairs, the upstream and downstream homologous arm fragments of spoT were ligated to obtain the homologous arm fusion fragment of spoT; the homologous arm fusion fragments of relA and spoT were ligated to the pKO4-Km vector to obtain the recombinant plasmids pKO4-Km-ΔrelA and pKO4-Km-ΔspoT containing homologous arms. The recombinant plasmid pKO4-Km-ΔrelA was transformed into S17-1λpir competent cells. The correct colonies were identified as donor bacteria, and NTUH-K2044 strain was used as recipient bacteria for conjugation experiments. Single colonies with small fragments using relA-A / D primer pair and no bands using pKO4-Km-F / R primer pair were screened to obtain the ΔrelA strain. The recombinant plasmid pKO4-Km-ΔspoT was transformed into S17-1λpir competent cells. The correctly identified colonies were used as donor bacteria, and the ΔrelA strain was used as recipient bacteria for conjugation experiments. PCR identification and screening were performed using spoT-A / D primer pair and pKO4-Km-F / R primer pair to obtain the Klebsiella pneumoniae attenuated live vaccine strain with relA / spoT double gene deletion, namely the ΔrelAΔspoT strain. The nucleotide sequences of the relA-A / B primer pair are shown in SEQ ID NO. 1-2; the nucleotide sequences of the relA-C / D primer pair are shown in SEQ ID NO. 3-4; the nucleotide sequences of the spoT-A / B primer pair are shown in SEQ ID NO. 7-8; the nucleotide sequences of the spoT-C / D primer pair are shown in SEQ ID NO. 9-10; and the nucleotide sequences of the pKO4-Km-F / R primer pair are shown in SEQ ID NO. 13-14.

3. A live attenuated Klebsiella pneumoniae vaccine with double gene deletion of relA / spoT, characterized in that, This includes the Klebsiella pneumoniae live attenuated vaccine strain with the relA / spoT double gene deletion as described in claim 1, and a pharmaceutically acceptable adjuvant.

4. The vaccine according to claim 3, characterized in that, The adjuvant includes at least one of aluminum hydroxide, Freund's adjuvant, MPLTM, IL-12, aluminum hydroxide combined with CpG ODN complex adjuvant, ISA51VG, ISA720VG, MF59, QS21, and AS03 adjuvant.

5. The use of the vaccine strain of claim 1 and / or the vaccine of any one of claims 3-4 in the preparation of a vaccine for the prevention of Klebsiella pneumoniae infection in humans and animals.

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