Lactococcus lactis HX21473 and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and discloses lactococcus lactis HX21473 and application thereof, the lactococcus lactis HX21473 is preserved in China General Microbiological Culture Collection Center (CGMCC) on January 20, 2025, and the preservation number is CGMCC NO.33476. The invention further discloses a preparation method of the lactococcus lactis HX21473. The invention provides a strain of lactococcus lactis HX21473 capable of efficiently expressing a foreign protein. The lactococcus lactis HX21473 has strong acid resistance, can be used as an oral vaccine live carrier, and promotes research, development and application of oral vaccines.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a type of lactococcus lactis HX21473 and its applications. Background Technology

[0002] Gastrointestinal pathogens disrupt the gastrointestinal barrier and affect health through pathogenic mechanisms such as secreting toxins or invading epithelial cells. Oral vaccines have attracted much attention because they can activate the mucosal immune system and establish a specific immune barrier at infection portals (such as the gastrointestinal mucosa). Lactococcus lactis (L. lactis), a food-grade microorganism with a long history in the biopharmaceutical and food industries, has been actively developed in recent years as an oral delivery vector and exogenous protein expression platform due to its high safety and mature genetic research, for applications in vaccine delivery, therapeutic protein expression, and functional foods. Its potential as a transiently resident or delivery microorganism in the gut has drawn attention in oral vaccines, intestinal-targeted therapies, and functional nutritional products.

[0003] However, existing Lactococcus lactis strains still face several limitations in practical applications. First, while developed engineered Lactococcus lactis host strains exhibit some tolerance to gastric acid and gastric juice, their tolerance is poor, resulting in a significantly reduced survival rate in the stomach after oral administration. This makes it difficult for them to effectively reach the ileum or colon, thus weakening their effectiveness as delivery carriers or probiotics; or requiring higher dosages, which significantly increases production costs. Second, the natural Lactococcus lactis strains to be developed generally contain endogenous plasmids, making the introduction of exogenous engineered plasmids difficult. Furthermore, these endogenous plasmids are prone to loss, rearrangement, or copy number fluctuations during continuous passage, industrial fermentation, or storage, leading to unstable expression levels. In addition, there are problems such as low expression efficiency against certain pathogenic antigens. Therefore, it is essential to conduct research on the discovery of Lactococcus lactis host expression strains. Summary of the Invention

[0004] The present invention aims to address the shortcomings of the prior art by providing a Lactococcus lactis strain HX21473, which solves the problems of insufficient tolerance of existing Lactococcus lactis strains to the acidic environment of the gastrointestinal tract and low expression efficiency of recombinant antigens.

[0005] To achieve the above objectives, the present invention provides a *Lactococcus lactis* HX21473 (i.e., *L. lactis* HX21473), which was deposited on January 20, 2025, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing), with accession number CGMCC NO. 33476.

[0006] In one feasible embodiment, the *Lactococcus lactis* HX21473 does not contain endogenous plasmids. Colony morphology characteristics: white, with regular edges, a raised center, and a smooth surface; microscopic examination of bacterial morphology: Gram-positive, oval or spherical, single, paired, or in chains.

[0007] This invention also discloses a method for culturing the aforementioned *Lactococcus lactis* HX21473. The *Lactococcus lactis* HX21473 is obtained by inoculating it into Elliker medium, GM17 medium, or a modified GM17 medium, specifically by static culturing at 30-40°C in the aforementioned medium. However, the culture medium used to culture this strain is not limited to the three media mentioned above; any culture medium suitable for culturing this strain is acceptable.

[0008] In one feasible embodiment, the modified GM17 medium comprises 2-8 g / L tryptone, 2-8 g / L caseinone, 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 glucose, 2-8 g / L lactose, and the balance being water.

[0009] The present invention also provides the application of the above-mentioned Lactococcus lactis HX21473 in the preparation of vaccines or as a platform for the expression of exogenous proteins.

[0010] In one feasible implementation, the *Lactococcus lactis* HX21473 serves as an oral delivery vector or a host for exogenous protein expression. Preferably, the *Lactococcus lactis* HX21473 serves as a live vector for oral vaccines against gastrointestinal pathogens.

[0011] In one feasible implementation, *Lactococcus lactis* HX21473 can be used as an expression host. A plasmid expression vector linked to a foreign gene fragment is electroporated into *Lactococcus lactis* HX21473 competent cells to express the target foreign protein, such as intracellularly or extracellularly. The target foreign protein includes *Helicobacter pylori* antigen proteins UreA, LpoB, and UreAB, and *Staphylococcus aureus* nuclease protein NucA, etc. Specifically, the following steps are included:

[0012] Preparation of competent cells of Lactococcus lactis HX21473;

[0013] The plasmid expression vector linked with the foreign gene was electroporated into Lactococcus lactis HX21473 competent cells to obtain recombinant Lactococcus lactis colonies;

[0014] Recombinant lactococcus colonies were cultured to express the target protein.

[0015] In one feasible manner, *Lactococcus lactis* HX21473 is cultured in a medium until OD200. 600 The solution is approximately 0.3. After washing several times with a solution containing sucrose and glycerol, Lactococcus lactis HX21473 competent cells can be obtained.

[0016] In one feasible implementation, the plasmid expression vector is pVE5523, pMG36e, pAMJ2008, pTREX1, or a derivative of any plasmid expression vector.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) This invention provides a strain of Lactococcus lactis HX21473 that can efficiently express exogenous proteins;

[0019] (2) The Lactococcus lactis HX21473 provided by the present invention has strong acid resistance. Its survival rate remains basically unchanged after incubation in simulated gastric fluid at pH 2.5 for 2 h, and the survival rate is about 22.9% after incubation for 4 h; the survival rate is still about 100% after incubation in simulated intestinal fluid at pH 6.8 for 8 h.

[0020] (3) The Lactococcus lactis HX21473 provided by the present invention can be used as a live vector for oral vaccines, promoting the research and development and application of oral vaccines. Attached Figure Description

[0021] Figure 1 The results show the identification of Lactococcus lactis HXN21; where A is the colony morphology of L. lactis HXN21 on Elliker plate, and B is the microscopic image of L. lactis HXN21.

[0022] Figure 2 The image shows the plasmid identification results of Lactococcus lactis strains; where M corresponds to the Marker, 1 corresponds to the plasmid extraction and identification results of Lactococcus lactis HXN21, and 2 corresponds to the plasmid extraction and identification results of Lactococcus lactis HX21473.

[0023] Figure 3 The images show the culture results of Lactococcus lactis HX21473; where A is a colony morphology of L. lactis HX21473 on GM17 plates; and B is a microscopic image of L. lactis HX21473.

[0024] Figure 4To evaluate the tolerance of different Lactococcus lactis strains to simulated gastrointestinal fluid; strain A corresponds to the tolerance of simulated gastric fluid; and strain B corresponds to the tolerance of simulated intestinal fluid.

[0025] Figure 5 The results of SDS-PAGE expression identification of recombinant protein UreA are shown; where M corresponds to Marker; 1 corresponds to negative control pVE5501 / HX21473; and 2 corresponds to UreA / pVE01-HX21473.

[0026] Figure 6 The results of SDS-PAGE expression identification of recombinant proteins nucA and LpoB are shown; where M corresponds to Marker; 1 corresponds to negative control pVE5501 / HX21473; 2 corresponds to pVE5523 / HX21473; and 3 corresponds to LpoB / pVE01-HX21473.

[0027] Lactococcus lactis HX21473 was deposited on January 20, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.33476. Detailed Implementation

[0028] 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.

[0029] 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.

[0030] Example 1: Lactococcus lactis HX21473

[0031] This embodiment provides a strain of Lactococcus lactis HX21473, which was deposited on January 20, 2025 at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing), with accession number CGMCC NO.33476.

[0032] (I) Acquisition and identification of Lactococcus lactis HXN21

[0033] Mix 5 times the volume of fresh milk with 1 times the volume of light cream and heat to 37-45°C. Slowly add lemon juice while stirring constantly. After the whey separates, let it stand for about half an hour. Filter through fat-free gauze to discard the whey; the resulting solid is homemade cream cheese. Ferment the homemade cream cheese sample naturally at 4-15°C for 2-3 weeks. Weigh 2g of the sample, crush it in a sterile petri dish, add 10mL of fresh M17 liquid culture medium (purchased from Qingdao Haibo), vortex to mix, and incubate overnight at 30°C. Dilute the overnight culture in 10-fold increments. -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 10 -8 Take 10 respectively -5 10 -6 10 -7 and 10 -8 100 μL of each compound was evenly spread onto Elliker plates (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), and then incubated at 30°C for approximately 40–48 h. Colony morphology on the Elliker plates was observed visually. Yellow colonies were picked and examined under a microscope using Gram staining. Colonies suspected to be *Lactococcus lactis* were repeatedly streaked on Elliker plates to isolate and purify a single colony strain, named HXN21. HXN21 forms yellow colonies on Elliker plates with neat edges, a raised center, and a smooth surface; it is Gram-positive under microscopy, oval or spherical, and can be single, paired, or in chains. Figure 1 As shown.

[0034] Genomic DNA was extracted from HXN21 according to the instructions of the Tiangen Biotech Bacterial Genomic DNA Extraction Kit. PCR amplification was performed using universal primers 27F and 1492R (27F: 5'-AGAGTTTGATCCTGGCTCAG-3' / 1492R: 5'-GGTTACCTTGTTACGACTT-3') for the bacterial 16S rDNA gene. A 50 μL reaction volume was prepared according to the Primer STAR HS DNA Polymerase (TAKARA) kit instructions. 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. The PCR products were verified by 1% agarose gel electrophoresis. Subsequently, the PCR products were sent to Shanghai Bioengineering Co., Ltd. for sequencing. The 16S rDNA gene sequence obtained from the sequencing was submitted to the NCBI GenBank database for BLAST comparison to identify the species of bacteria. The comparison results showed that the 16S rDNA sequence of this strain was more than 99% homologous to that of Lactococcus lactis. HXN21 was preliminarily identified as Lactococcus lactis.

[0035] (II) Obtaining Lactococcus lactis HX21473 without endogenous plasmids

[0036] The natural *Lactococcus lactis* HXN21 strain is difficult to introduce with exogenous plasmids and genes using conventional methods for constructing engineered lactic acid bacteria (such as electroporation), which limits its application to some extent. This embodiment employs a method of continuous passage under stress conditions, successfully eliminating the endogenous plasmids of *L. lactis* HXN21. The resulting plasmid-free progeny strain can be effectively introduced with exogenous DNA.

[0037] The specific steps for plasmid elimination are as follows:

[0038] (1) Activation of strain

[0039] Glycerol-containing L. lactis HXN21 bacteria stored at -80℃ were 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℃ for 24 h.

[0040] (2) Temperature adaptation to 39℃

[0041] 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.

[0042] (3) Elimination of endogenous plasmids in Lactococcus lactis HXN21

[0043] Inoculate the bacterial culture, which has been incubated overnight at 39°C, at a 2% inoculation rate into fresh modified GM17 liquid medium. Add neomycin stock solution to a final concentration of 4-10 μg / mL, and then incubate at 39°C for approximately 24 hours. After 4-18 passages every 24 hours, isolate single colonies by plating, culture several single colony cultures, and extract and identify the plasmid loss of the corresponding single colony strains.

[0044] After several subcultures and identifications, a plasmid-free colony strain of L. lactis HXN21 was successfully obtained, with plasmid loss as follows: Figure 2 As shown, the strain was preserved and named L. lactis HX21473.

[0045] The culture results of L. lactis HX21473 after culturing for approximately 48 hours on a commonly used Lactococcus lactis medium (GM17 medium) at 30℃ are as follows: Figure 3 As shown, the colony morphology characteristics are: white, with neat edges, a raised center, and a smooth surface; microscopic examination of the bacterial cells shows that they are Gram-positive, oval or spherical, and can be single, paired, or in chains.

[0046] (III) Identification of L. lactis HX21473

[0047] (1) Biochemical identification items

[0048] The physiological and biochemical characteristics of the strain 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. Table 1 shows that L. lactis HX21473 can utilize glucose, lactose, maltose, and D-ribose to produce acid; it cannot liquefy gelatin; it cannot oxidize inositol; it does not produce gas when utilizing glucose; and it is negative for catalase.

[0049] Table 1. Physiological and Biochemical Identification Results

[0050]

[0051] "+" indicates a positive result, and "-" indicates a negative result.

[0052] (2) Identification of 16S rDNA

[0053] Genomic DNA was extracted from strain L. lactis HX21473 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 had more than 99% homology with the 16S rDNA sequence of Lactococcus lactis.

[0054] The sequence of L. lactis HX21473 16S rDNA is shown in SEQ ID NO. 1:

[0055]

[0056] (3) Antibiotic susceptibility testing

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

[0058] Table 2 Results of Antibiotic Susceptibility Tests

[0059]

[0060] The results showed that L. lactis HX21473 exhibited strong sensitivity to seven antibiotics: erythromycin, cefazolin, penicillin, chloramphenicol, vancomycin, clindamycin, and ampicillin. Therefore, the Lactococcus lactis HX21473 selected in this example has a certain degree of safety.

[0061] Example 2: Tolerance of L. lactis HX21473 to simulated gastrointestinal fluid

[0062] Glycerol-containing *L. lactis* HX21473 culture, stored at -80℃, was inoculated into GM17 liquid medium and incubated statically at 30℃ for approximately 14 hours. The culture was then centrifuged at 6000g for 5 minutes, washed twice with PBS, and finally resuspended in PBS to adjust the bacterial concentration to approximately 10. 9 CFU / mL.

[0063] (a) Tolerance to simulated gastric juice

[0064] Take 200 μL of the above bacterial culture and add it to 4 mL of simulated gastric fluid (3.2 g / L pepsin, 2.0 g / L sodium chloride, the remainder being water; adjust the pH of the solution to 2.5 with 1 M HCl before adding pepsin). Incubate at 37℃ for different times (0 h, 2 h, 4 h). At each incubation time point, take 100 μL and perform a 10-fold serial dilution (each 10-fold dilution is performed with sterile physiological saline). 1 ~10 6 (Multiple times). Take 100 μL of each serial dilution and spread it evenly on GM17 plates. Incubate at 30℃ 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.

[0065] ×100% (1);

[0066] Note: In equation (1), t is 0, 2, or 4.

[0067] Experimental results are as follows Figure 4 As shown in Figure A, after incubation in simulated gastric fluid at pH 2.5 for 2 hours, the survival rate of HX21473 remained essentially unchanged, while the survival rate of the commonly used commercially available engineered host bacterium *L. lactis* NZ9000 decreased to 10.4%. After incubation in simulated gastric fluid at pH 2.5 for 4 hours, the survival rate of HX21473 was 22.9%, but the survival rate of NZ9000 almost dropped to 0. This demonstrates that HX21473 exhibits strong tolerance to acidic environments and is more suitable as a live vector for oral vaccine delivery.

[0068] (ii) Tolerance to simulated intestinal fluid

[0069] Add 200 μL of the above bacterial culture to 4 mL of simulated intestinal fluid (6.8 g / L potassium dihydrogen phosphate, 10 g / L trypsin, and the remainder water; adjust the pH to 6.8 with 1 M NaOH before adding trypsin). Incubate at 37℃ for different times (0 h, 4 h, 8 h), and at each incubation time point, take 100 μL for 10-fold serial dilution. Spread 100 μL of each serial dilution evenly on GM17 plates and incubate at 30℃ for 40-48 h. Select plates with colony counts between 30 and 300 for colony counting and calculate the number of viable bacteria per unit volume.

[0070] ×100% (2);

[0071] Note: In equation (2), t is 0, 4, or 8.

[0072] Experimental results are as follows Figure 4 As shown in Figure B, after incubation in simulated intestinal fluid at pH 6.8 for 4 hours, the survival rate of HX21473 (100.6%) was slightly higher than that of NZ9000 (95.8%); after incubation in simulated intestinal fluid at pH 6.8 for 8 hours, the survival rate of HX21473 (93.8%) was slightly lower than that of NZ9000 (98.1%), but the difference was not significant.

[0073] Example 3: L. lactis HX21473 as a plasmid-free engineered probiotic host for expressing exogenous proteins.

[0074] (I) Preparation of L. lactis HX21473 competent cells

[0075] Using a sterile inoculation loop, take one loopful of L. lactis HX21473 glycerol bacteria stored at -80℃ and streak it in four zones on a GM17 agar plate. Then, incubate it statically at 30℃ for approximately 48 hours. Pick a single colony and inoculate it 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). Incubate the medium at 30℃ for 24 hours. Add 2 mL of the overnight culture to 20 mL of GSGM17 medium and incubate at 30℃ for 14 hours.

[0076] 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 Approximately 0.3. Transfer the cultured bacterial solution into a clean centrifuge tube, centrifuge at 4°C and 4000g for 20 minutes, and collect the bacterial cells. Resuspend the bacterial cells in 40 mL of pre-chilled ice-cold Wash Buffer I (0.5 mol / L sucrose, 100 mL / L glycerol, balance water), centrifuge at 4000 g for 20 min at 4 °C, and collect the cells. Then resuspend the cells in 30 mL of pre-chilled ice-cold Wash Buffer II (0.5 mol / L sucrose, 100 mL / L glycerol, 50 mM EDTA, balance water), incubate on ice for 15 min, and centrifuge at 4 °C for 20 min to collect the cells. Resuspend the cells in 20 mL of pre-chilled ice-cold Wash Buffer I, centrifuge at 4 °C for 20 min, and collect the cells. Finally, suspend the cells in 800 μL of pre-chilled ice-cold Wash Buffer I, aliquot 40 μL into 1.5 mL EP tubes pre-chilled on ice, and store at -80 °C for later use.

[0077] (II) Construction of recombinant Lactococcus lactis expression system

[0078] 1. Construction of recombinant plasmid UreA / pVE01: Using plasmid pVE5523 (purchased from Fenghui Biotechnology, containing the nucA gene encoding Staphylococcus aureus nucA protein) as a template, plasmid pVE5523 was linearized using primers P3 (5'-gatatcgctagttctagattgaggc-3') and P4 (5'-gtcgaccgcatcttgtttagca-3'), while removing the nucA gene from the plasmid. Fragment UreA was amplified using primers PA-F (5'-ctaaacaagatgcggtcgacATGAAACTCACCCCAAAAGAG-3') and PA-R (5'-aatctagaactagcgatatcTTACTCCTTAATTGTTTTTAC-3'); fragment LpoB was amplified using primers P5 (5'-ctaaacaagatgcggtcgacGCGACGTACCAGAATGTTAATGA-3') and P6 (5'-aatctagaactagcgatatcTTAAAACATGCGCTTGTTGGAAGC-3'). Prepare a 50 μL reaction system according to the PrimeSTAR® HS DNA Polymerase (TAKARA) kit instructions. Amplification conditions for UreA and LpoB fragments: 98℃ for 3 min, 1 cycle; 98℃ for 10 s, 55℃ for 15 s, 72℃ for 8 min, 30 cycles; 72℃ for 1 min, 1 cycle. PCR products were validated by 1% agarose gel electrophoresis. The obtained target fragments were recovered from the gel using the thermoscientific gel recovery kit instructions. The obtained linearized vector and target fragments were ligated using the Gibson assembly method to obtain recombinant plasmids UreA / pVE01 and LpoB / pVE01. UreA / pVE01 is characterized by containing the UreA gene encoding the Helicobacter pylori UreA protein, and LpoB / pVE01 is characterized by containing the LpoB gene encoding the Helicobacter pylori LpoB protein.

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

[0080] ATGAAACTCACCCCAAAAGAGTTAGATAAGTTGATGCTCCACTACGCTGGAGAATTAGCTAGGAAACGCAAAGAAAAAGGCATTAAGCTTAACTATGTGGAAGCGGTAGCTTTGATTAGTGCCCATATTATGGAAGAAGCGAGAGCTGGTAAAAAGACTGCGGCTGAATTGATGCAAGAAGGGCGCACTCTTTTAAAACCGGATGATGTGATGGATGGTGTGGCAAGCATGATCCATGAAGTGGGTATTGAAGCGATGTTTCCTGATGGGACCAAACTCGTAACCGTGCATACCCCTATTGAGGCTAATGGTAAATTGGTTCCTGGTGAGTTGTTCTTAAAAAATGAAGACATCACTATCAACGAAGGCAAAAAAGCCGTTAGCGTGAAAGTTAAAAACGTGGGCGACAGACCGGTTCAAATCGGTTCACACTTCCATTTCTTTGAAGTGAATAGATGCCTAGACTTTGACAGAGAAAAAACTTTCGGCAAACGCTTAGACATTGCGAGCGGGACAGCGGTAAGGTTTGAGCCTGGCGAAGAAAAATCCGTAGAATTGATTGACATTGGTGGCAACAGAAGAATCTTTGGATTTAACGCATTGGTTGATAGGCAAGCAGACAACGAAAGCAAAAAAATTGCTTTACACAGAGCTAAAGAGCGTGGTTTTCATGGTGCTAAAAGCGATGACAACTATGTAAAAACAATTAAGGAGTAA

[0081] The amino acid sequence of UreA is shown in SEQ ID NO. 3:

[0082] MKLTPKELDKLMLHYAGELARKRKEKGIKLNYVEAVALISAHIMEEARAGKKTAAELMQEGRTLLKPDDVMDGVASMIHEVGIEAMFPDGTKLVTVHTPIEANGKLVPGELFLKNEDITINEGKKAVSVKVKNVGDRPVQIGSHFHFFEVNRCLDFDREKTFGKRLDIASGTAVRFEPGEEKSVELIDIGGNRRIFGFNALVDRQADNESKKIALHRAKERGFHGAKSDDNYVKTIKE-

[0083] The LpoB nucleotide sequence is shown in SEQ ID NO. 4:

[0084] GCGACGTACCAGAATGTTAATGATGCAACAAAGAACACGACAGCTTCAATTAACAGCACAGACTTACTTTTGACCGCAAACGCCATGCTGGACTCGATGTTTTCAGATCCAAATTTTGAACAGTTAAAAGGCAAACACTTGATCGAAGTCAGCGACGTAATCAATGACACTACTCAACCCAATCTGGACATGAATTTACTTACTACAGAGATCGCCCGCCAACTTCGTCTGCGTAGCAACGGTCGTTTCAATATTACGCGTGCAAGCGGAGGATCTGGAATCGAAGCCGACAGTCGCATGGTGAAGCAGCGTGAGAAGGAACGCGAATCTGAAGAATACAATCAGGATACTACTGTCGAAAAGGGAACTTTGAAAGCAGCCGATTTAAGTCTGTCCGGTAAAGTTTCTTCAATTGCAGCGTCTATCTCTTCCAGCCGCCAGCGTTTGGACTACGACTTCACATTATCTTTGACGAATCGCAAGACAGGTGAAGAAGTTTGGTCAGATGTCAAACCCATTGTCAAGAACGCTTCCAACAAGCGCATGTTTTAA

[0085] The LpoB amino acid sequence is shown in SEQ ID NO. 5:

[0086] ATYQNVNDATKNTTASINSTDLLLTANAMLDSMFSDPNFEQLKGKHLIEVSDVINDTTQPNLDMNLLTTEIARQLRLRSNGRFNITRASGGSGIEADSRMVKQREKERESEEYNQDTTVEKGTLKAADLSLSGKVSSIAASISSSRQRLDYDFTLSLTNRKTGEEVWSDVKPIVKNASNKRMF-

[0087] 2. Electroporation of L. lactis HX21473 competent cells using plasmids or recombinant plasmids: 5 μL of the recombinant plasmid or 1 μL of plasmid pVE5523 was electroporated into 40 μL of host Lactococcus lactis HX21473 competent cells. Electroporation conditions: voltage 1150V~1250V, resistance 100Ω~400Ω, pulse 25μF, time 3.1~5.0ms. 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℃ for 2 h. 100 μL of the bacterial culture was then evenly spread onto GM17 plates (containing 10 μg / mL erythromycin) and incubated at 30℃ for approximately 48 h. Transformants were validated by colony PCR using universal primers P1 / P2 (P1: 5'-ACAGTGATACTTTCTGCTGCAG-3' / P2: 5'-TAGCTAGAGCGGCGGATTTG-3') for plasmid pVE5523. Transformants matching the target band size were sent to Shanghai Bioengineering Co., Ltd. for sequencing. The recombinant lactococci with correct nucleotide sequences were named UreA / pVE01-HX21473, LpoB / pVE01-HX21473, and pVE5523 / HX21473.

[0088] The constructed and preserved empty vector plasmid pVE5501 (pVE5523-derived plasmid) was transduced into HX21473 competent cells by electroporation to construct the recombinant strain pVE5501 / HX21473 as a negative control. The plasmid pVE5501 was constructed as follows: using plasmid pVE5523 as a template, PCR amplification was performed using primers 5501-F (5'-AACCATGGGCATGCACTAGTgatatcgctagttctagattgag-3') and 5501-R (5'-ACTAGTGCATGCCCATGGTTAgtcgaccgcatcttgtttagc-3'). A 50 μL reaction mixture was prepared according to the PrimeSTAR® HS DNA Polymerase (TAKARA) kit instructions. Amplification conditions were: 98℃ for 3 min, 1 cycle; 98℃ for 10 s, 68℃ for 8 min, 30 cycles; 68℃ for 10 min, 1 cycle. The PCR products were verified by 1% agarose gel electrophoresis. The obtained target fragment was recovered from the gel using a gel recovery kit (thermo). The linearized plasmid pVE5501 was self-ligated according to the Gibson assembly method to obtain the recombinant plasmid pVE5501. The recombinant plasmid pVE5501 is obtained by replacing the nucleotide sequence between the SalI and EcoR V restriction sites on the original plasmid pVE5523 with the sequence TAACCATGGGCATGCACTAGT, thereby introducing the stop codon TAA and the multiple cloning site. This plasmid can be used as a negative control for pVE5523 and its plasmids containing exogenous fragments.

[0089] (III) Expression of recombinant proteins

[0090] Single colonies of different recombinant *Lactococcus lactis* successfully constructed in step (II) of this embodiment 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 suspension was centrifuged (4000g, 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 sonicated under the following conditions: power 180W, 3s on, 3s off, total time 5 min. The supernatant was concentrated using the trichloroacetic acid (TCA)-acetone protein concentration method. The broken whole bacterial suspension and culture supernatant were subjected to SDS-PAGE electrophoresis using a 12% PAGE separating gel to identify the expression of the target protein. The results are as follows: Figure 5 and Figure 6 As shown in the figure. It can be seen from the figure that the recombinant UreA protein ( Figure 5 Lane 2 (indicated by a red arrow), recombinant LpoB protein ( Figure 6 Lane 3 (indicated by a red arrow) and recombinant nucA protein ( Figure 6 Lane 2 (indicated by red arrows) successfully secreted and expressed the compound bacteria.

[0091] Therefore, the present invention provides a strain of Lactococcus lactis HX21473 that has strong acid resistance and can effectively express the antigen of Helicobacter pylori, a gastric pathogen. This strain can be used as an oral delivery live vector for gastrointestinal pathogen vaccines, and can also be used as an expression host for some other bacterial proteins, including but not limited to the Staphylococcus aureus nuclease protein in the above embodiments.

[0092] Those skilled in the art will recognize that the embodiments described herein are intended to help the reader understand the principles of the invention, and should be understood that the scope of protection of the invention is not limited to such specific statements and embodiments. Those skilled in the art can make various other specific modifications and combinations based on the technical teachings disclosed in this invention without departing from the spirit of the invention, and these modifications and combinations are still within the scope of protection of this invention.

Claims

1. A type of lactococcus lactis HX21473, characterized in that, The lactococcus lactis HX21473 was deposited at the China General Microbiological Culture Collection Center on January 20, 2025, with accession number CGMCC NO.33476.

2. The *Lactococcus lactis* HX21473 according to claim 1, characterized in that, The lactococcus lactis HX21473 does not contain endogenous plasmids; colony morphology characteristics: white, with neat edges, a raised center, and a smooth surface; microscopic examination of bacterial morphology: Gram-positive, oval or spherical, single, paired, or in chains.

3. The method for culturing Lactococcus lactis HX21473 according to claim 1 or 2, characterized in that, Lactococcus lactis HX21473 was obtained by inoculating it into Elliker medium, GM17 medium or modified GM17 medium.

4. The method for culturing Lactococcus lactis HX21473 according to claim 3, characterized in that, 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 glucose, 2-8 g / L lactose, and the balance being water.

5. The use of Lactococcus lactis HX21473 as described in claim 1 or 2 in the preparation of vaccines or as a platform for the expression of exogenous proteins.

6. The application according to claim 5, characterized in that, The Lactococcus lactis HX21473 is used as an oral delivery vector or a host for the expression of exogenous proteins.

7. The application according to claim 6, characterized in that, Lactococcus lactis HX21473 was used as the expression host, and the plasmid expression vector linked to the exogenous gene fragment was electroporated into Lactococcus lactis HX21473 competent cells to express the target exogenous protein.

8. The application according to claim 7, characterized in that, The target exogenous proteins include Helicobacter pylori antigen proteins UreA, LpoB, and UreAB, and Staphylococcus aureus nuclease protein NucA.

9. The application according to claim 7, characterized in that, The plasmid expression vector is pVE5523, pMG36e, pAMJ2008, pTREX1, or a derivative of any plasmid expression vector.

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