Lactococcus lactis hX9302 expressing exogenous protein and application thereof

Abstract

The application belongs to the technical field of biological medicine, and discloses a lactococcus lactis HX9302 for expressing exogenous proteins and application, wherein the lactococcus lactis HX9302 has been preserved in the China General Microbiological Culture Collection Center on January 20, 2025, and the preservation number is CGMCC NO.33477; the lactococcus lactis has good acid resistance and digestion enzyme tolerance, and can be used as an oral delivery live carrier of a helicobacter pylori vaccine, so as to promote the research and application of the oral vaccine.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a lactococcus lactis strain HX9302 that expresses exogenous proteins and its applications. Background Technology

[0002] Helicobacter pylori ( Helicobacter pylori , H. pylori It is a spiral-shaped, Gram-negative bacillus that can infect and colonize the gastric mucosa. H. pylori Infection is not only a significant contributing factor to various gastrointestinal diseases, mucosa-associated lymphoid tissue lymphoma (MALT lymphoma), and even serious diseases such as gastric cancer, but it is also closely related to hematological diseases, cardiovascular and cerebrovascular diseases, and extra-gastrointestinal diseases such as Alzheimer's disease. Therefore, controlling infection is crucial. H. pylori It is of great significance in terms of infection and transmission in my country and even globally.

[0003] Currently, routine clinical treatment involves triple or quadruple therapy consisting of proton pump inhibitors (PPIs), bismuth preparations, and one or two antibiotics. However, long-term use of these treatment regimens can easily lead to gastrointestinal microecological dysbiosis and induce the emergence of antibiotic-resistant strains. In contrast, vaccines offer both prevention and treatment. H. pylori Oral vaccines are an ideal choice for preventing the development of drug-resistant strains. They hold significant market potential, acting by stimulating the body's mucosal immune system to produce a specific immune response, thus providing effective immune protection and preventing pathogen invasion at the mucosal barrier level. However, the immunogenicity of oral vaccines is relatively weak, requiring higher doses to achieve effective protection. During the process from oral ingestion to the exertion of an immune effect, vaccines face multiple biological barriers, including corrosion from gastric acid, degradation by pepsin, the intestinal mucus barrier on the surface of small intestinal epithelial cells, and the difficulty in maintaining antigen activity over a long period. Therefore, the choice of delivery vector is crucial for effectively improving the immunogenicity of oral vaccines.

[0004] Lactic acid bacteria can tolerate the acidic environment of the gastrointestinal tract and the degradation by various proteases, and can temporarily colonize the intestine, which is beneficial for maintaining the homeostasis of the intestinal microecology and provides ideal conditions for the delivery and protection of exogenous proteins. Among them, Lactococcus lactis (… Lactococcus lactis , L. lactis As a food-grade microorganism, it has a long history of application in the biopharmaceutical and food industries. Due to its high safety, good gastrointestinal adaptability, and short-term colonization ability, L. lactis It has been extensively studied as a live bacteria oral delivery carrier for delivery. H. pylori Antigens. Although Lactococcus lactis is abundant in nature, its development and utilization as a vaccine vector is still relatively limited. Currently, the following prominent problems exist: some strains are sensitive to antigens. H. pyloriThe expression efficiency of the antigen is low, the growth rate is relatively slow, and the tolerance to the acidic environment of the gastrointestinal tract is insufficient. To address these issues, there is an urgent need to further screen and develop Lactococcus lactis strains with superior performance, in order to achieve efficient expression of exogenous antigens and improve acid resistance and tolerance to digestive enzymes, thereby promoting the research and application of oral vaccines. Summary of the Invention

[0005] This invention aims to address the shortcomings of existing technologies by providing a *Lactococcus lactis* strain HX9302 that expresses exogenous proteins, thereby solving the problem of existing *Lactococcus lactis* strains being unable to express exogenous proteins. H. pylori The problems include low antigen expression efficiency, relatively slow growth rate, and insufficient tolerance to the acidic environment of the gastrointestinal tract.

[0006] According to a first aspect disclosed in this invention, the present invention provides a *Lactococcus lactis* HX9302 strain expressing a foreign protein (… Lactococcus lactis HX9302, i.e. L. lactis Lactococcus lactis HX9302 was deposited at the China General Microbiological Culture Collection Center on January 20, 2025, with accession number CGMCC NO.33477.

[0007] In one feasible embodiment, the *Lactococcus lactis* HX9302 strain does not contain endogenous plasmids, allowing for the effective introduction of exogenous genes / plasmids. It grows well on the culture medium, producing white colonies with smooth surfaces, regular edges, and a central convexity. Microscopic examination reveals oval or spherical colonies, occurring singly, in pairs, or in chains, and are Gram-positive. The *Lactococcus lactis* HX9302 of this invention can efficiently express exogenous proteins and can be used to secrete and express the Helicobacter pylori antigen protein UreA, with an expression level higher than that of the commonly used engineered *Lactococcus lactis* expression host NZ9000. The culture medium is M17 medium, GM17 medium, modified M17 medium, or modified GM17 medium.

[0008] The modified M17 medium consists of 2-8 g / L tryptone, 2-8 g / L casein, 1.5-4 g / L yeast extract, 1.5-4 g / L beef extract, 1.5-4 g / L soybean peptone, 0.2-0.8 g / L ascorbic acid or sodium ascorbate, 0.1-0.4 g / L magnesium sulfate, 8-15 g / L disodium hydrogen phosphate, 2-8 g / L lactose, and the balance being water.

[0009] The modified GM17 medium consists of 2-8 g / L tryptone, 2-8 g / L casein, 1.5-4 g / L yeast extract, 1.5-4 g / L beef extract, 1.5-4 g / L soybean peptone, 0.2-0.8 g / L ascorbic acid or sodium ascorbate, 0.1-0.4 g / L magnesium sulfate, 8-15 g / L disodium hydrogen phosphate, 2-8 g / L lactose, 2-8 g / L glucose, and the balance being water.

[0010] According to a second aspect disclosed in this invention, the present invention provides the application of the above-mentioned Lactococcus lactis HX9302 expressing exogenous proteins in the preparation of vaccines.

[0011] In one feasible implementation, the *Lactococcus lactis* HX9302 is used as a live vaccine vector. Preferably, the *Lactococcus lactis* HX9302 is used as a live oral vaccine vector for *Helicobacter pylori*.

[0012] In one feasible implementation, a plasmid expression vector linked to a foreign gene is electroporated into Lactococcus lactis HX9302 competent cells for expression of the target protein; specifically, the following steps are included:

[0013] Preparation of competent cells of Lactococcus lactis HX9302;

[0014] The plasmid expression vector linked with the foreign gene was electroporated into Lactococcus lactis HX9302 competent cells to obtain recombinant Lactococcus lactis colonies;

[0015] The target protein was expressed by culturing recombinant lactococcus colonies.

[0016] Furthermore, the plasmid expression vector is pVE5523, pMG36e, pAMJ2008, pTREX1, or a derivative of any plasmid expression vector.

[0017] Furthermore, the exogenous gene is UreA, MCP, nucA, or LpoB, etc.

[0018] The preparation steps of Lactococcus lactis HX9302 competent cells include:

[0019] Stored at -80℃ L.lactis HX9302 glycerol bacteria were streaked in four zones on a GM17 plate and then incubated at 30°C for about 48 hours. A single colony was picked and inoculated into 5 mL of liquid GSGM17 medium and incubated at 30°C for 24 hours. 2 mL of the overnight culture was added to 20 mL of GSGM17 medium and incubated at 30°C for 14 hours.

[0020] Take 10 mL of the above bacterial culture and transfer it to 80 mL of GSGM17 medium. Incubate at 30°C until OD reaches 100%. 600 The concentration of the cultured bacterial culture was approximately 0.3. The cultured bacterial culture was transferred to centrifuge tubes and centrifuged at 4000g for 20 min at 4°C to collect the bacterial cells. The bacterial cells were resuspended in 40 mL of pre-chilled wash buffer I (on ice) and centrifuged at 4000g for 20 min at 4°C to collect the bacterial cells. Then, the bacterial cells were resuspended in 30 mL of pre-chilled wash buffer II (on ice) and allowed to stand on ice for 15 min. The cells were then centrifuged at 4000g for 20 min at 4°C to collect the bacterial cells. The cells were then resuspended in 20 mL of pre-chilled wash buffer I (on ice) and centrifuged at 4000g for 20 min at 4°C to collect the bacterial cells. Finally, the bacterial cells were resuspended in 800 μL of pre-chilled wash buffer I (on ice) and dispensed into 1.5 mL EP tubes (40 μL per tube) pre-ice-bathed. The tubes were stored at -80°C for later use.

[0021] Washing solution I comprises: 0.5 mol / L sucrose and 100 ml / L glycerol, with the remainder being water; washing solution II comprises 0.5 mol / L sucrose, 100 ml / L glycerol and 50 mM EDTA, with the remainder being water.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] This invention provides a strain of Lactococcus lactis HX9302 that can efficiently express exogenous proteins. This strain has good acid resistance and digestive enzyme tolerance, and can be used as an oral delivery live vector for Helicobacter pylori vaccine, promoting the research and application of oral vaccines. Attached Figure Description

[0024] Figure 1 Image showing plasmid identification of Lactococcus lactis strain: M: Marker; Lane 1: Plasmid extraction and identification results of L. lactis HX9; Lane 2: L. lactis Plasmid extraction and identification results of HX9302;

[0025] Figure 2 for L. lactis The culture results of HX9302: A is L. lactis Colony morphology of HX9302 on GM17 plate; B represents... L. lactis Microscopic image of HX9302;

[0026] Figure 3 To assess the tolerance of Lactococcus lactis strains to simulated gastric juice;

[0027] Figure 4 To assess the tolerance of Lactococcus lactis strains to simulated artificial intestinal fluid;

[0028] Figure 5SDS-PAGE images for identifying the expression of recombinant proteins UreA and MCP; where M: Marker; Lane 1: UreA / pVE5523-HX9302; Lane 2: UreA / pVE5523-NZ9000; Lane 3: MCP / pVE5523-HX9302; Lane 4: MCP / pVE5523-NZ9000;

[0029] Figure 6 The growth curves of Lactococcus lactis HX9302, NZ9000 and their derivative strains are shown; (a) corresponds to GM17 medium and (b) corresponds to modified GM17 medium.

[0030] Figure 7 SDS-PAGE expression identification of recombinant proteins pVE5523-HX9302 and LpoB / pVE5523-HX9302: M: Marker; Lane 1: Negative control pVE5501 / HX9302 lysed whole bacterial culture; Lane 2: pVE5523-HX9302 lysed whole bacterial culture; Lane 3: LpoB / pVE5523-NZ9000 lysed whole bacterial culture; Lane 4: Negative control pVE5501 / HX9302 culture supernatant; Lane 5: pVE5523-HX9302 culture supernatant; Lane 6: LpoB / pVE5523-NZ9000 culture supernatant.

[0031] Lactococcus lactis L.lactis HX9302 was deposited on January 20, 2025, at the China General Microbiological Culture Collection Center (No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), with accession number CGMCC NO.33477. Detailed Implementation

[0032] The technical solutions of various embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used are commercially available.

[0034] Example 1: Lactococcus lactis HX9302 expressing exogenous protein ( L.lactis HX9302)

[0035] The above-mentioned lactococci expressing exogenous proteins L.lactis HX9302 was prepared by the following method:

[0036] Elimination of endogenous plasmids in Lactococcus lactis strains

[0037] In the early stages of this invention, a strain with probiotic properties was isolated from naturally fermented dairy products (yogurt obtained by naturally fermenting unsterilized fresh milk at 18-25°C for 2-3 days). L. lactis HX9. The specific screening process is as follows: After shaking and mixing the naturally fermented dairy product, take 100 μL of the sample solution and dilute it with physiological saline in a 10-fold gradient (10... -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 10 -8 Take 10 respectively -5 10 -6 10 -7 10 -8 100 μL of each solution was evenly spread onto Elliker plates containing 0.5% calcium carbonate (20 g / L tryptone, 5 g / L yeast extract, 4 g / L sodium chloride, 1.5 g / L anhydrous sodium acetate, 0.5 g / L ascorbic acid, 0.4‰ bromocresol purple, 5 g / L lactose, 15 g / L agar). The plates were then incubated at 30°C for approximately 40–48 hours. Colony morphology on the Elliker plates was observed visually. Yellow colonies with neat edges, a raised center, smooth surface, and a calcified zone were selected and examined under a microscope using Gram staining. Strains that stained Gram-positive and exhibited morphological characteristics consistent with *Lactococcus lactis* (ovoid or spherical, single, paired, or in chains) were preserved as *Lactococcus lactis*. L. lactis HX9.

[0038] However, due to Lactococcus lactis L. lactis HX9 is difficult to introduce into exogenous plasmids and therefore cannot be used as an engineered host expression strain, which limits its application to some extent. This invention employs a method of using high temperature and neomycin to act on *Lactococcus lactis*, thereby... L. lactis After multiple rounds of passage, HX9 successfully eliminated the endogenous plasmids of this strain, resulting in plasmid-free progeny strains that could be efficiently introduced with exogenous DNA. The specific steps for plasmid elimination are as follows:

[0039] (I) Activation of strains

[0040] Stored at -80℃ L.lactis 10 μL of HX9 glycerol bacteria was inoculated into fresh modified GM17 liquid medium (5 g / L tryptone, 5 g / L casein, 2.5 g / L yeast extract, 2.5 g / L beef extract, 2 g / L soybean peptone, 0.4 g / L ascorbic acid, 0.25 g / L magnesium sulfate, 10 g / L disodium hydrogen phosphate, 5 g / L lactose, 5 g / L glucose, with the remainder being water), and incubated at 30°C for 24 h.

[0041] (II) Temperature adaptation to 39℃

[0042] The activated bacterial solution was inoculated into fresh modified GM17 liquid culture medium at an inoculation rate of 2%, and then incubated at a constant temperature of 39°C for 24 hours.

[0043] (III) Elimination of endogenous plasmids in Lactococcus lactis

[0044] Inoculate the bacterial culture that has been cultured overnight at 39°C into fresh modified GM17 liquid medium at an inoculation rate of 2%, add neomycin stock solution to a final concentration of 2-15 μg / mL, and then incubate at 39°C for about 24 h. After 4-24 passages every 24 h, isolate single colonies by plating, culture several single colony cultures, and extract and identify the plasmid loss of the corresponding single colony strains.

[0045] After several generations and identifications, it was successfully obtained L.lactis The plasmid loss of HX9 apoptosis colony strains is as follows: Figure 1 As shown, this strain was named L. lactis HX9302.

[0046] L.lactis The culture results of HX9302 on the commonly used lactococcus lactis medium (GM17 medium) after static incubation at 30°C for approximately 48 hours are as follows: Figure 2 As shown, the colony morphology is white, with neat edges, a raised center, and a smooth surface; microscopic examination reveals that the colony is Gram-positive, oval or spherical, and can be single, paired, or in chains.

[0047] Example 2: Lactococcus lactis L.lactis HX9302 identification

[0048] (I) Biochemical Identification Items

[0049] The physiological and biochemical characteristics of the strains were identified according to Bergey's Manual of Bacterial Identification, Second Edition, and the Manual of Systematic Identification of Common Bacteria. The results are shown in Table 1. As can be seen from Table 1, L.lactisHX9302 can produce acid using glucose, lactose, maltose, and D-ribose; it cannot liquefy gelatin; it cannot oxidize inositol; it does not produce gas when using glucose; and it is negative in the catalase test.

[0050] Table 1. Physiological and Biochemical Identification Results

[0051]

[0052] "+" indicates a positive result, and "-" indicates a negative result.

[0053] (II) Identification of 16S rDNA

[0054] Extraction strains L.lactis Using HX9302 genomic DNA as a template, PCR amplification was performed using universal primers 27F and 1492R for the bacterial 16S rDNA gene. Amplification conditions were as follows: 98℃ for 3 min, 1 cycle; 98℃ for 10 s, 55℃ for 15 s, 72℃ for 1.5 min, 30 cycles; 72℃ for 5 min, 1 cycle. PCR products were validated by 1% agarose gel electrophoresis. The PCR products were then sent to Shanghai Bioengineering Co., Ltd. for sequencing. The obtained 16S rDNA sequence was compared with the NCBI GenBank database using BLAST analysis. Based on sequence homology, the species relationship of this strain was verified. The results showed that the sequence shared over 99.5% homology with the 16S rDNA sequence of *Lactococcus lactis*.

[0055] L.lactis

[0056] (III) Antibiotic susceptibility testing

[0057] The antibiotic susceptibility of the strains was tested using the antimicrobial susceptibility disc method. Different types of antimicrobial susceptibility discs were purchased from Shifeng Biotechnology Co., Ltd. The results are shown in Table 2.

[0058] Table 2 Results of Antibiotic Susceptibility Tests

[0059]

[0060] From the above, we can see that: L.lactis HX9302 showed strong sensitivity to seven antibiotics: erythromycin, cefazolin, penicillin, chloramphenicol, vancomycin, clindamycin, and ampicillin. Therefore, the lactococcus lactis screened in this invention has a certain degree of safety.

[0061] Example 3: L.lactis HX9302's tolerance to simulated gastric fluid

[0062] Stored at -80℃ L.lactis HX9302 Glycerin Bacteria or L.lactis NZ9000 glycerol bacteria were inoculated into GM17 liquid medium and incubated statically at 30°C for approximately 14 hours. The bacterial cells were then collected by centrifugation at 6000g for 5 minutes, washed twice with sterile PBS, and then resuspended in sterile PBS to adjust the bacterial concentration to approximately 10. 9 CFU / mL; 200 μL of the resuspended bacterial solution was added to 4 mL of simulated gastric fluid (3.2 g / L pepsin, 2.0 g / L sodium chloride, the remainder being water; the pH of the solution was adjusted to 2.5 with 1 M HCl before adding pepsin; the solution was filtered through a 0.22 μm microporous membrane for sterilization). The solution was incubated at 37 °C for 2 h, and 100 μL was taken at 0 h and 2 h respectively. The bacterial solution was then diluted 10 times with sterile physiological saline in a 10-fold gradient. 1 ~10 6 Take 100 μL of each serial dilution and spread it evenly on a GM17 plate. Incubate at 30°C for 40-48 h. Select plates with colony counts between 30-300 for colony counting and calculate the number of viable bacteria per unit volume.

[0063] ×100% (1)

[0064] Note: t in equation (1) is 0 or 2.

[0065] Experimental results: After incubation for 2 hours in simulated gastric fluid at pH 2.5, the survival rate of HX9302 was approximately 73.7%, but commonly used commercially engineered host bacteria... L.lactisThe survival rate of NZ9000 decreased to 10.4% (e.g. Figure 3 As shown in the figure), HX9302 has good tolerance to acidic environments and is more suitable as a live vector for oral delivery of vaccines.

[0066] Example 4: L.lactis HX9302's tolerance to simulated intestinal fluid

[0067] Stored at -80℃ L.lactis HX9302 Glycerin Bacteria or L.lactis NZ9000 glycerol bacteria were inoculated into GM17 liquid medium and incubated statically at 30°C for approximately 14 hours. The bacterial cells were then collected by centrifugation at 6000g for 5 minutes, washed twice with sterile PBS, and resuspended in sterile PBS to adjust the bacterial concentration to approximately 10. 9 CFU / mL; 200 μL of resuspended bacterial solution was added to 4 mL of simulated artificial intestinal fluid (6.8 g / L potassium dihydrogen phosphate, 10 g / L trypsin, the remainder being water; the pH of the solution was adjusted to 6.8 with 1M NaOH before adding trypsin; the solution was filtered through a 0.22 μm microporous membrane for sterilization). The solution was incubated at 37°C for different times (0 h, 4 h, 8 h), and 100 μL was taken at 0 h, 4 h, and 8 h for 10-fold serial dilution (the operation was the same as in Example 3). 100 μL of each serial dilution was evenly spread on GM17 plates and incubated at 30°C for 40-48 h. Plates with colony counts between 30 and 300 were selected for colony counting, and the number of viable bacteria per unit volume was calculated.

[0068] ×100% (2)

[0069] Note: In equation (2), t is 0, 4 or 8.

[0070] Experimental results: After incubation for 4 hours in simulated intestinal fluid at pH 6.8, the survival rate of HX9302 (104.5%) was slightly higher than that of NZ9000 (95.8%); after incubation for 8 hours in simulated intestinal fluid at pH 6.8, the survival rate of HX9302 (109.0%) increased slightly, and was still slightly higher than that of NZ9000 (98.1%), but there was no significant difference between the two. Figure 4 As shown.

[0071] Example 5: L.lactis HX9302 was used as a plasmid-free engineered probiotic host for expressing exogenous proteins.

[0072] (I) Preparation of competent cells of Lactococcus lactis

[0073] Take one loopful using a sterile inoculation loop and store at -80℃. L.lactis HX9302 Glycerin Bacteria or L.lactis NZ9000 glycerol bacteria were streaked in four zones on GM17 plates and then incubated statically at 30°C for about 48 hours. A single colony was picked and inoculated into 5 mL of liquid GSGM17 medium (171.0 g / L sucrose, 25.0 g / L glycine, 37.3 g / L M17 medium, 5.0 g / L glucose, with the remainder being water), and then incubated statically at 30°C for 24 hours. 2 mL of the overnight culture was added to 20 mL of GSGM17 medium and incubated statically at 30°C for 14 hours.

[0074] Take 10 mL of the cultured bacterial solution and transfer it to 80 mL of GSGM17 medium. Incubate at 30°C until OD reaches 100%. 600 Approximately 0.3; Transfer the cultured bacterial solution to a clean centrifuge tube and centrifuge at 4000g for 20 min at 4°C to collect the bacterial cells; Resuspend the bacterial cells in 40 mL of pre-chilled washing buffer I (0.5 mol / L sucrose, 100 mL / L glycerol, balance water), centrifuge at 4000g for 20 min at 4°C to collect the bacterial cells; Resuspend the bacterial cells in 30 mL of pre-chilled washing buffer II (0.5 mol / L sucrose, 100 mL / L glycerol, 50 mM EDTA, balance water), incubate on ice for 15 min, then centrifuge at 4000g for 20 min at 4°C to collect the bacterial cells; Resuspend the bacterial cells in 20 mL of pre-chilled washing buffer I, centrifuge at 4000g for 20 min at 4°C to collect the bacterial cells; Finally, suspend the bacterial cells in 800 μL of pre-chilled washing buffer I and dispense into 1.5 mL pre-ice-bathed centrifuge tubes. 40 μL per EP tube, stored at -80°C for later use.

[0075] (II) Construction of recombinant plasmids UreA / pVE5523 and MCP / pVE5523

[0076] Using plasmid pVE5523 as a template, the plasmid pVE5523 was linearized using primers P3 (5'-gatatcgctagttctagattgaggc-3') and P4 (5'-gtcgaccgcatcttgtttagca-3'), while removing the plasmid's... nucAGenes. The UreA fragment was amplified using primers PA-F (5'-ctaaacaagatgcggtcgacATGAAACTCACCCCAAAAGAG-3') and PA-R (5'-aatctagaactagcgatatcTTACTCCTTAATTGTTTTTAC-3'); the MCP fragment was amplified using primers PM-F (5'-ctaaacaagatgcggtcgacAAAGACAGTGAGATCACTGAAT-3') and PM-R (5'-aatctagaactagcgatatcTTAGATGTTCTCTTTTACGTGTTTAG-3'). A 50 μL reaction volume was prepared according to the PrimeSTAR® HS DNA Polymerase (TAKARA) kit instructions. Amplification conditions were: 98℃ for 3 min per cycle; 98℃ for 10 s, 55℃ for 15 s, 72℃ for 8 min for 30 cycles; 72℃ for 10 min per cycle. PCR products were validated using 1% agarose gel electrophoresis. The obtained target fragment was recovered from the gel using the gel recovery kit (Thermo Scientific) instructions. The linearized vector and the target fragment were ligated using the Gibson assembly method to obtain recombinant plasmids UreA / pVE5523 and MCP / pVE5523. UreA / pVE5523 is characterized by containing the UreA gene encoding the Helicobacter pylori UreA protein, while MCP / pVE5523 is characterized by containing the MCP gene encoding the Helicobacter pylori MCP protein.

[0077] The nucleotide sequence of UreA is shown in SEQ ID NO. 1:

[0078] ATGAAACTCACCCCAAAAGAGTTAGATAAGTTGATGCTCCACTACGCTGGAGAATTAGCTAGGAAACGCAAAGAAAAAGGCATTAAGCTTAACTATGTGGAAGCGGTAGCTTTGATTAGTGCCCATATTATGGAAGAAGCGAGAGCTGGTAAAAAGACTGCGGCTGAATTGATGCAAGAAGGGCGCACTCTTTTAAAACCGGATGATGTGATGGATGGTGTGGCAAGCATGATCCATGAAGTGGGTATTGAAGCGATGTTTCCTGATGGGACCAAACTCGTAACCGTGCATACCCCTATTGAGGCTAATGGTAAATTGGTTCCTGGTGAGTTGTTCTTAAAAAATGAAGACATCACTATCAACGAAGGCAAAAAAGCCGTTAGCGTGAAAGTTAAAAACGTGGGCGACAGACCGGTTCAAATCGGTTCACACTTCCATTTCTTTGAAGTGAATAGATGCCTAGACTTTGACAGAGAAAAAACTTTCGGCAAACGCTTAGACATTGCGAGCGGGACAGCGGTAAGGTTTGAGCCTGGCGAAGAAAAATCCGTAGAATTGATTGACATTGGTGGCAACAGAAGAATCTTTGGATTTAACGCATTGGTTGATAGGCAAGCAGACAACGAAAGCAAAAAAATTGCTTTACACAGAGCTAAAGAGCGTGGTTTTCATGGTGCTAAAAGCGATGACAACTATGTAAAAACAATTAAGGAGTAA

[0079] The amino acid sequence of UreA is shown in SEQ ID NO. 2:

[0080] MKLTPKELDKLMLHYAGELARKRKEKGIKLNYVEAVALISAHIMEEARAGKKTAAELMQEGRTLLKPDDVMDGVASMIHEVGIEAMFPDGTKLVTVHTPIEANGKLVPGELFLKNEDITINEGKKAVSVKVKNVGDRPVQIGSHFHFFEVNRCLDFDREKTFGKRLDIASGTAVRFEPGEEKSVELIDIGGNRRIFGFNALVDRQADNESKKIALHRAKERGFHGAKSDDNYVKTIKE-

[0081] The MCP nucleotide sequence is as shown in SEQ ID NO. 3:

[0082]

[0083] The amino acid sequence of MCP is shown in SEQ ID NO. 4:

[0084] KDSEITELKKEVNLYQSLLNLCLHEGFVGIKNNKVVFKSGNLASLNNLEEQSVHFKENAESVNLQGVSYSLKSQNIDGVQYFSLAKKTGGVGEYHKNDLFKTFCTSLKEGLENAQESMQYFHQETGLLLNAAKNGEAHSTEGLGTVNKTGQDIESLYEKMQNATSLADSLNQRSNEIT QVISLIDDIAEQTNLLALNAAIEAARAGEHGRGFAVVADEVRKLAEKTQKATKEIAVVVKSMQQEANDIQTNTHDINSIVGSIKSDVEELKSTVKNNMIITSHKSCRLGKWYYEGAGKENFANTSGYRALESHHASVHAEANDLVKAVQEDHVTDSKYLEHKVHLMEDSAKHVKENI-

[0085] (III) Construction of recombinant Lactococcus lactis

[0086] 5 μL of the recombinant plasmid was transduced into 40 μL of either *Lactococcus lactis* HX9302 or NZ9000 competent cells using electroporation. Electroporation conditions: voltage 1150 V–1250 V, resistance 100 Ω–400 Ω, pulse 25 μF, time 3.2–4.8 ms. Immediately after electroporation, 1 mL of pre-cooled recovery medium (37.3 g / L M17 medium, 1.9 g / L MgCl2, 0.2 g / L CaCl2, 5.0 g / L glucose, balance water) was added, and the mixture was incubated on ice for 10 min, then incubated at 30°C for 1.5–2 h. 100 μL of the bacterial culture was then evenly spread onto GM17 plates (containing 10 μg / mL erythromycin) and incubated at 30°C for approximately 48 h. Transformants were picked from the plates and sent to Shanghai Bioengineering Co., Ltd. for sequencing. The recombinant lactococci with the correct nucleotide sequences were named UreA / pVE5523-HX9302, UreA / pVE5523-NZ9000, MCP / pVE5523-HX9302, and MCP / pVE5523-NZ9000.

[0087] (iv) Expression of recombinant proteins

[0088] Single colonies of the successfully constructed recombinant *Lactococcus lactis* were picked and inoculated into 10 mL of modified GM17 liquid medium (containing 10 μg / mL erythromycin) and incubated overnight at 30°C. The obtained bacterial suspensions were centrifuged (4000 g, 20 min), and the bacterial cells and culture supernatant were collected separately. The bacterial cells were washed twice with PBS and then resuspended. The bacterial suspension was then sonicated under the following conditions: 180 W power, 3 s on, 3 s off, total time 5 min. The supernatant was concentrated using the trichloroacetic acid (TCA)-acetone protein concentration method. SDS-PAGE electrophoresis was performed on the disrupted whole bacterial suspension and culture supernatant using a 12% PAGE separating gel to identify the expression of the target protein. The results are shown below. Figure 5 As shown, from Figure 5 As can be seen, recombinant UreA protein (lanes 1 and 2, indicated by red arrows) and recombinant MCP protein (lanes 3 and 4, indicated by red arrows) were successfully secreted and expressed in recombinant bacteria. Among them, the expression levels of recombinant UreA protein and recombinant MCP protein in Lactococcus lactis HX9302 were much higher than those in Lactococcus lactis NZ9000, indicating that Lactococcus lactis has a greater advantage as an expression host for exogenous recombinant proteins.

[0089] (v) Draw growth curves

[0090] Stored at -80℃ L.lactis HX9302, UreA / pVE5523-HX9302, MCP / pVE5523-HX9302, L.lactis Glyceryl bacteria NZ9000, UreA / pVE5523-NZ9000, and MCP / pVE5523-NZ9000 were inoculated into modified GM17 medium (containing 10 μg / mL erythromycin) and incubated statically overnight at 30°C; the OD of the bacterial culture was measured using a spectrophotometer. 600 Then, the remaining bacterial culture was centrifuged (6000g, 5min), and the cells were resuspended in sterile PBS until the bacterial culture OD was reached. 600 The value was 1.0; the resuspended bacterial culture was inoculated into fresh GM17 medium or modified GM17 medium at an inoculation rate of 2%, and incubated statically at 30°C. The OD of the bacterial culture was measured within 24 hours using a microplate reader. 600 Value changes, every 1 hour / time, and after the detection is completed, based on OD. 600 Growth curves of different strains in GM17 medium and modified GM17 medium were plotted.

[0091] The results are as follows Figure 6As shown in (a) and (b), after 10 h of culture, HX9302 and its derivative strains UreA / pVE5523-HX9302 and MCP / pVE5523-HX9302 achieved higher cell counts than NZ9000 and its derivative strains UreA / pVE5523-NZ9000 and MCP / pVE5523-NZ9000 in GM17 medium or modified GM17 medium. Furthermore, as... Figure 6 As shown in (a) and (b), after 10 hours of cultivation, the cell count of HX9302 and its derivative strains was significantly higher in the modified GM17 medium than in the GM17 medium alone. Furthermore, the cell count of NZ9000 and its derivative strains was also higher in the modified GM17 medium than in the GM17 medium alone. This indicates that the optimized culture medium formulation (modified GM17 medium) of the present invention is more suitable for the growth of *Lactococcus lactis*.

[0092] In summary, this invention provides a strain of Lactococcus lactis HX9302 that can efficiently express exogenous proteins. This strain has good acid resistance and can be used as an oral delivery live vector for Helicobacter pylori vaccine.

[0093] Example 6

[0094] Construction of plasmid LpoB / pVE5523:

[0095] Using plasmid pVE5523 as a template, plasmid pVE5523 was linearized using primers P3 (5'-gatatcgctagttctagattgaggc-3') and P4 (5'-gtcgaccgcatcttgtttagca-3'), while the nucA gene on the plasmid was removed. Fragment LpoB was amplified using primers P5 (5'-ctaaacaagatgcggtcgacGCGACGTACCAGAATGTTAATGA-3') and P6 (5'-aatctagaactagcgatatcTTAAAACATGCGCTTGTTGGAAGC-3'). Prepare a 50 μL reaction mixture according to the PrimeSTAR® HS DNA Polymerase (TAKARA) kit instructions. Amplification conditions: 98℃ for 3 min, 1 cycle; 98℃ for 10 s, 55℃ for 15 s, 72℃ for 8 min, 30 cycles; 72℃ for 10 min, 1 cycle. PCR products were validated by 1% agarose gel electrophoresis. The obtained target fragment was recovered from the gel using the gel recovery kit (Thermo Scientific) instructions. The obtained linearized vector and target fragment were ligated using the Gibson assembly method to obtain the recombinant plasmid LpoB / pVE5523. LpoB / pVE5523 is characterized by containing the LpoB gene encoding the Helicobacter pylori LpoB protein.

[0096] Plasmid electroporation of L. lactis HX9302 competent cells:

[0097] Take 1 μL of plasmid pVE5523 (purchased from Fenghui Biotechnology, containing the nucA gene encoding the Staphylococcus aureus nucA protein) or LpoB / pVE5523, and transform it into 40 μL of host Lactococcus lactis HX9302 competent cells via electroporation. Electroporation conditions: voltage 1150V, resistance 200Ω, pulse 25μF, time 4.4-4.7ms. Immediately after electroporation, add 1 mL of pre-cooled recovery medium (M17 medium (37.3g / L), MgCl2 (1.9g / L), CaCl2 (0.2g / L), glucose (5.0g / L), balance water), incubate on ice for 10 min, and then incubate statically at 30℃ for 1.5-2 h. Spread 100 μL of the bacterial culture evenly on GM17 plates (containing 10 μg / mL erythromycin) and incubate statically at 30℃ for approximately 48 h. Transformants were picked from the plate and sent to Shanghai Bioengineering Co., Ltd. for sequencing. The nucleotide sequences were correct, and the recombinant lactococcus pVE5523-HX9302 and LpoB / pVE5523-HX9302 were successfully constructed.

[0098] The procedures for culturing the recombinant strain, expressing the target protein, and identifying it were the same as those for UreA / pVE5523-HX9302, and the results were as follows. Figure 7 As shown in the figure, Staphylococcus aureus nucA protein (lane 5, indicated by the red arrow) and Helicobacter pylori LpoB protein (lane 6, indicated by the red arrow) were successfully secreted and expressed in the recombinant bacteria.

[0099] The LpoB nucleotide sequence is shown in SEQ ID NO. 5:

[0100] GCGACGTACCAGAATGTTAATGATGCAACAAAGAACACGACAGCTTCAATTAACAGCACAGACTTACTTTTGACCGCAAACGCCATGCTGGACTCGATGTTTTCAGATCCAAATTTTGAACAGTTAAAAGGCAAACAC TTGATCGAAGTCAGCGACGTAATCAATGACACTACTCAACCCAATCTGGACATGAATTTACTTACTACAGAGATCGCCCGCCAACTTCGTCTGCGTAGCAACGGTCGTTTCAATATTACCGTGCAAGCGGAGGATCT GGAATCGAAGCCGACAGTCGCATGGTGAAGCAGCGTGAGAAGGAACGCGAATCTGAAGAATACAATCAGGATACTACTGTCGAAAAGGGAACTTTGAAAGCAGCCGATTTAAGTCTGTCCGGTAAAGTTTCTTCAATT GCAGCGTCTATCTCTTCCAGCCGCCAGCGTTTGGACTACGACTTCACATTATCTTTGACGAATCGCAAGACAGGTGAAGAAGTTTGGTCAGATGTCAAACCCATTGTCAAGAACGCTTCCAACAAGCGCATGTTTTAA

[0101] The amino acid sequence of LpoB is shown in SEQ ID NO. 6:

[0102] ATYQNVNDATKNTTASINSTDLLLTANAMLDSMFSDPNFEQLKGKHLIEVSDVINDTTQPNLDMNLLTTEIARQLRLRSNGRFNITRASGGSGIEADSRMVKQREKERESEEYNQDTTVEKGTLKAADLSLSGKVSSIAASISSSRQRLDYDFTLSLTNRKTGEEVWSDVKPIVKNASNKRMF-

[0103] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A type of lactococcus expressing exogenous protein ( Lactococcus lactis HX9302, characterized in that, The Lactococcus lactis HX9302 was deposited at the China General Microbiological Culture Collection Center on January 20, 2025, with accession number CGMCC NO.33477.

2. The application of Lactococcus lactis HX9302 expressing exogenous protein as described in claim 1 in the preparation of vaccines.

3. The application according to claim 2, characterized in that, The Lactococcus lactis HX9302 was used as a live vaccine vector.

4. The application according to claim 3, characterized in that, The Lactococcus lactis HX9302 was used as a live vector for the Helicobacter pylori oral vaccine.

5. The application according to claim 2, characterized in that, The plasmid expression vector linked to the foreign gene was electroporated into Lactococcus lactis HX9302 competent cells for expression of the target protein.

6. The application according to claim 5, characterized in that, The plasmid expression vector is pVE5523, pMG36e, pAMJ2008, or pTREX1.

7. The application according to claim 5, characterized in that, The exogenous gene is UreA, MCP, nucA, or LpoB.

8. The application according to any one of claims 2 to 7, characterized in that, The lactococcus lactis HX9302 grows well on the culture medium. The colonies are white, smooth, with neat edges and a raised center. Microscopic examination of the bacterial morphology shows that they are oval or spherical, single, paired or in chains, and Gram-positive. The culture medium is M17 medium, GM17 medium, modified M17 medium or modified GM17 culture medium. The modified M17 medium comprises 2-8 g / L tryptone, 2-8 g / L casein, 1.5-4 g / L yeast extract, 1.5-4 g / L beef extract, 1.5-4 g / L soybean peptone, 0.2-0.8 g / L ascorbic acid or sodium ascorbate, 0.1-0.4 g / L magnesium sulfate, 8-15 g / L disodium hydrogen phosphate, 2-8 g / L lactose, and the balance being water. The modified GM17 medium consists of 2-8 g / L tryptone, 2-8 g / L casein, 1.5-4 g / L yeast extract, 1.5-4 g / L beef extract, 1.5-4 g / L soybean peptone, 0.2-0.8 g / L ascorbic acid or sodium ascorbate, 0.1-0.4 g / L magnesium sulfate, 8-15 g / L disodium hydrogen phosphate, 2-8 g / L lactose, 2-8 g / L glucose, and the balance being water.

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