A recombinant lactococcus lactis live vector vaccine and a preparation method and application thereof

Abstract

The application belongs to the technical field of biological pharmacy, and discloses a recombinant lactococcus lactis live carrier vaccine, which is obtained by connecting a urease subunit UreA gene with a plasmid expression vector and then electrically transforming the lactococcus lactis; the amino acid sequence of the urease subunit UreA gene is shown as SEQ ID NO. 1. The application further discloses application of the recombinant lactococcus lactis live carrier vaccine in preparation of an anti-helicobacter pylori vaccine. The recombinant lactococcus lactis live carrier vaccine provided by the application can effectively stimulate the body to produce a mucosal immune response and has good protection effect.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology and relates to an anti-Helicobacter pylori vaccine using Lactococcus lactis as a live vector and its preparation. Background Technology

[0002] Helicobacter pylori (Hp) is a spiral-shaped, Gram-negative bacterium that colonizes the surface of gastric epithelial cells. Hp infection can affect the gastrointestinal microecological environment, leading to the disruption of biological barriers and bacterial translocation. Persistent infection can cause serious gastroduodenal complications, including chronic gastritis, peptic ulcers, gastric adenocarcinoma, and gastric cancer. It has been classified as a Group 1 carcinogen by the International Agency for Research on Cancer (ARC Working Group. 1994. World Health Organization, International Agency for Research on Cancer. Schistosomes, liver flukes and Helicobacter pylori. WHO, Lyon, France).

[0003] Currently, clinical treatment typically employs triple or quadruple therapy consisting of proton pump inhibitors (PPIs), bismuth, and one or two antibiotics. However, these treatments can lead to gastrointestinal microbiota dysbiosis. Furthermore, *Helicobacter pylori* (Hp) is particularly prone to antibiotic resistance, which also hinders its treatment and eradication. Vaccines may be one of the more promising strategies for preventing and controlling Hp infection. Multiple experimental results have demonstrated that vaccination can reduce Hp colonization levels in mice, and similar results have been reported in human clinical trials (Dos Santos Viana Irineu, Cordeiro Santos Maria Luísa, Santos MarquesHanna, et al. Helicobacter pylori Vaccine development against: from ideal antigens to the current landscape[J]. Expert Rev Vaccines, 2021, 20: 989-999).

[0004] Urease is the most important protective factor against *Helicobacter pylori* (Hp), composed of UreA and UreB subunits. Its catalytic products, CO2 and NH3, can effectively neutralize gastric acid and are used as antigens in preventive and therapeutic vaccines against Hp infection (Gao Yuan, Research progress on *Helicobacter pylori*-related antigens and host immune responses [J]. Journal of Immunology, 2002(S1):102-105). The intestinal mucosal system has immune tolerance to food and symbiotic bacteria, but it has an immune response to pathogenic microorganisms. Oral administration can stimulate the intestinal mucosal immune system and simultaneously put other mucosal immune systems in a state of immune protection, blocking pathogens outside the mucosa and effectively avoiding the toxic effects of pathogens on body cells (Li Guang, Construction of recombinant lactic acid bacteria expressing novel duck-derived goose parvovirus VP2 protein [D]. Shandong Agricultural University, 2022. DOI:10.27277 / d.cnki.gsdnu.2022.000189). However, oral immunization has the problems of weak immune effect and higher required dose. There are multiple obstacles between entering the mouth and exerting the vaccine effect, including the corrosive effect of gastric acid, the degradation of pepsin, the difficulty in maintaining antigen activity in the body for a long time, and the presence of intestinal mucus barrier on the surface of small intestinal epithelial cells. In order to achieve better immune effect, the choice of delivery carrier is crucial.

[0005] Lactic acid bacteria can resist the acidic environment and digestion by various proteases in the animal's gastrointestinal tract, and colonize the intestine to participate in maintaining the balance of intestinal flora, providing conditions for the delivery and protection of exogenous proteins. Lactococcus lactis (L. lactis), as a food-grade microorganism, has high safety, and its short-term colonization makes it suitable for immunization via the mucosal route. Furthermore, the recombinant antigens expressed by L. lactis can be continuously produced in the intestine for a short period, thereby prolonging the immune stimulation cycle and enabling a more efficient immune response. Therefore, oral administration of L. lactis live vector vaccines combines the safety of inactivated vaccines with the durability of attenuated vaccines, making it a practical and feasible method.

[0006] Developing an orally administered anti-Helicobacter pylori vaccine is one of the major problems that needs to be solved in the genetic engineering of Helicobacter pylori, and it has good application prospects and social benefits. Summary of the Invention

[0007] The present invention aims to provide a recombinant live vector vaccine of Lactococcus lactis and its preparation method. The prepared vaccine secretes and expresses the UreA protein, the urease subunit of Helicobacter pylori, which can induce an immune response in the body and protect the body from Hp infection. It also has the advantages of high safety and simple preparation process.

[0008] Another object of the present invention is to provide the application of the above-mentioned recombinant lactococcus live vector vaccine in the preparation of anti-Helicobacter pylori vaccine.

[0009] The recombinant live vector vaccine of Lactococcus lactis provided by the present invention is obtained by electroporating Lactococcus lactis after linking the urease subunit UreA gene with a plasmid expression vector; the amino acid sequence of the urease subunit UreA gene is shown in SEQ ID NO.1.

[0010] SEQ ID NO.1:

[0011] MKLTPKELDKLMLHYAGELARKRKEKGIKLNYVEAVALISAHIMEEARAGKKTAAELMQEGRTLLKPDDVMDGVASMIHEVGIEAMFPDGTKLVTVHTPIEANGKLVPGELFLKNEDIT INEGKKAVSVKVKNVGDRPVQIGSHFHFFEVNRCLDFDREKTFGKRLDIASGTAVRFEPGEEKSVELIDIGGNRRIFGFNALVDRQADNESKKIALHRAKERGFHGAKSDDNYVKTIKE-

[0012] The nucleic acid sequence encoding the UreA gene, the urease subunit, is shown in SEQ ID NO.2.

[0013] SEQ ID NO.2:

[0014] ATGAAACTCACCCCAAAAGAGTTAGATAAGTTGATGCTCCACTACGCTGGAGAATTAGCTAGGAAACGCAAAGAAAAAGGCATTAAGCTTAACTATGTGGAAGCGGTAGCTTTGATTAGTGCCCATATTATGGAAGAAGCGAGAGCTGGTAAAAAGACTGCGGCTGAATTGATGCAAGA AGGGCGCACTCTTTTAAAACCGGATGATGTGATGGATGGTGTGGCAAGCATGATCCATGAAGTGGGTATTGAAGCGATGTTTCCTGATGGGACCAAACTCGTAACCGTGCATACCCCTATTGAGGCTAATGGTAAATTGGTTCCTGGTGAGTTGTTCTTAAAAAATGAAGACATCACTA TCAACGAAGGCAAAAAAGCCGTTAGCGTGAAAGTTAAAAACGTGGGCGACAGACCGGTTCAAATCGGTTCACACTTCCATTTCTTTGAAGTGAATAGATGCCTAGACTTTGACAGAGAAAAAACTTTCGGCAAACGCTTAGACATTGCGAGCGGGACAGCGGTAAGGTTTGAGCCTGGC GAAAAAAAATCCGTAGAATTGATTGACATTGGTGGCAACAGAAGAATCTTTGGATTTAACGCATTGGTTGATAGGCAAGCAGACAACGAAAGCAAAAAAATTGCTTTACACAGAGCTAAAGAGCGTGGTTTTCATGGTGCTAAAAGCGATGACAACTATGTAAAAACAATTAAGGAGTAA

[0015] The plasmid expression vector used for ligating the urease subunit UreA gene is pVE5523. The urease subunit UreA gene and the plasmid expression vector are ligated by SalI / EcoRV double digestion.

[0016] The lactococcus lactis uses L. Lactis NZ9000 as the host cell, but is not limited to L. Lactis NZ9000.

[0017] This invention also provides a method for preparing the above-mentioned recombinant lactococcus live vector vaccine, comprising the following steps:

[0018] S1, constructing plasmids

[0019] The urease subunit UreA with the nucleic acid sequence SEQ ID NO:2 and the plasmid expression vector were ligated by SalI / EcoRV double digestion, and the ligation product was then transformed into E. coli DH5α competent cells and cultured overnight to obtain the recombinant Escherichia coli-Lactococcus lactis shuttle plasmid.

[0020] S2, Electroporated Lactococcus lactis competent cells

[0021] Recombinant Escherichia coli-Lactococcus lactis shuttle plasmid was transduced into host Lactococcus lactis competent cells by electroporation.

[0022] S3. Cultivation of recombinant strains and expression of recombinant proteins

[0023] The colonies obtained in step S2 are cultured, and the cultured bacterial cells are collected, which are the recombinant lactococcus live vector vaccine strains; the recombinant protein is expressed and secreted into the extracellular culture medium.

[0024] In step S1 above, the plasmid expression vector can be pVE5523. This is because the inserted nucleotide sequence consists of the constitutive promoter P59 and the signal peptide SP on the vector. Usp45 The expression of recombinant proteins is controlled, therefore, no induction is required, and they are secreted into the extracellular environment after intracellular expression.

[0025] In step S2 above, the host lactococcus competent cells are L. Lactis NZ9000.

[0026] In step S3 above, GM17 is used to culture colonies containing the recombinant Escherichia coli-Lactococcus lactis shuttle plasmid. The recombinant protein is expressed in the vaccine strain and secreted into the extracellular environment during the culture process, without the need for induction.

[0027] The present invention also provides the application of the above-mentioned recombinant lactococcus live vector vaccine in the preparation of anti-Helicobacter pylori vaccines, especially in the preparation of anti-Helicobacter pylori oral vaccines.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] (1) The present invention obtains a novel recombinant live vector vaccine of Lactococcus lactis by transferring the UreA gene of urease subunit into Lactococcus lactis.

[0030] (2) The recombinant live vector vaccine of Lactococcus lactis can be used as an oral vaccine against Helicobacter pylori; after oral immunization of Balb / c mice with the live vector vaccine, it can effectively stimulate the body to produce specific sIgA antibodies, proving that the recombinant live bacterial vaccine (such as L. Lactis UreA-NZ9000 / pVE5523) prepared by the present invention can effectively stimulate the body to produce a mucosal immune response; the immunoprotective efficacy evaluation experiment confirmed that the oral live bacterial vaccine can achieve an immunoprotective rate of more than 60% in mice. Attached Figure Description

[0031] Figure 1 This diagram illustrates the construction of the recombinant plasmid UreA / pVE5523. SP represents the signal peptide SPUsp45; UreA represents the Helicobacter pylori urease A subunit; P59 represents the promoter; MCS represents the multiple cloning site; and T (Terminator) represents the terminator.

[0032] Figure 2 The image shows the recombinant plasmid UreA / pVE5523 and the plasmid double enzyme digestion gel electrophoresis image. M: Marker; 1: plasmid UreA / pVE5523 / DH5α; 2: double enzyme digestion plasmid UreA / pVE5523 / DH5α.

[0033] Figure 3 The image shows the recombinant plasmid UreA / pVE5523 and the plasmid double enzyme digestion gel electrophoresis image. M: Marker; 1: plasmid UreA / pVE5523 / NZ9000; 2: double enzyme digestion plasmid UreA / pVE5523 / NZ9000.

[0034] Figure 4 SDS-PAGE expression identification of recombinant proteins. M: Marker; 1: UreA-NZ9000 / pVE5523; 2: NZ9000 / pVE5523.

[0035] Figure 5 The results show the colonization ability of recombinant lactococcus in the gastrointestinal tract of mice.

[0036] Figure 6 This is the result of the detection of fecal-specific antibody sIgA.

[0037] Figure 7 The results of the evaluation of the protective efficacy of recombinant lactococcal oral vaccine after challenge. Detailed Implementation

[0038] The technical solutions of various embodiments of the present invention will be clearly and completely described in conjunction with 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 part of the present invention.

[0039] All strains used were commercially available plasmids and strains: shuttle expression vector pVE5523 (purchased from Fenghui Biotechnology), L. Lactis NZ9000 (purchased from HonorGene), E. coli DH5α (purchased from Shanghai Chaoyan Biotechnology Co., Ltd.), and H. pylori J99 (purchased from ATCC, USA).

[0040] Example 1: Construction of recombinant Escherichia coli-Lactococcus lactis shuttle plasmid

[0041] Plasmids UreA / pMD19 (purchased from Wuhan Jinkairui Biotechnology Co., Ltd., UreA nucleic acid sequence shown in ID NO. 1) and pVE5523 (purchased from Fenghui Biotechnology) were double-digested with SalI / EcoRV, and then ligated through the SalI / EcoRV restriction sites. Specific dosages and operating procedures were performed according to the SalI (purchased from Takara) and EcoRV (purchased from Takara) instruction manuals. The construction diagram is shown below. Figure 1 As shown in the figure. The ligation product was transformed into E. coli DH5α competent cells and then evenly spread on LB agar plates containing ampicillin (100 μg / mL) (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar powder) and incubated overnight at 37°C. Positive transformants were picked for plasmid extraction and SalI / EcoRV double digestion verification. The results are shown in the figure. Figure 2 As shown in the figure, the recombinant plasmid was sent to BGI Genomics for sequencing, and the nucleotide sequence was correct, indicating that the recombinant E. coli-Lactococcus lactis shuttle plasmid was successfully constructed.

[0042] Example 2: Electroporation of Lactococcus lactis competent cells using recombinant plasmids

[0043] One μL of the recombinant plasmid constructed in Example 1 (i.e., the recombinant Escherichia coli-Lactococcus lactis shuttle plasmid) was transduced into 40 μL of host Lactococcus lactis NZ9000 competent cells via electroporation. The electroporation conditions were as follows: voltage 1150 V, resistance 200 Ω. The pulse frequency was 25 uF for 4.7 ms. Immediately after electroporation, 950 μL 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) was added, and the mixture was incubated on ice for 10 min, followed by static incubation at 30°C for 2 h. 100 μL of the bacterial culture was then evenly spread onto M17 plates (containing 10 μg / mL erythromycin) and incubated at 30°C for approximately 48 h. Positive transformants were validated by plasmid extraction and SalI / EcoRV double digestion. The results are as follows: Figure 3 As shown, the recombinant plasmid was sequenced by BGI Genomics, and the nucleotide sequence was correct. The recombinant Lactococcus lactis UreA-NZ9000 / pVE5523 was successfully constructed.

[0044] Example 3: Expression of UreA protein in recombinant Lactococcus lactis

[0045] Single colonies were picked and inoculated into 5 mL of GM17 liquid medium (containing 10 μg / mL erythromycin) and incubated overnight at 30°C. 400 μL of the overnight colonies were then transferred to 20 mL of GM17 liquid medium (containing 10 μg / mL erythromycin) and incubated at 30°C for approximately 12 h. The supernatant was then collected by centrifugation at 4000 g for 20 min. The supernatant was concentrated using the trichloroacetic acid (TCA)-acetone protein concentration method. SDS-PAGE electrophoresis was then performed using a 12% PAGE separating gel to identify the expression status. The results are shown below. Figure 4 As shown in the figure, UreA-NZ9000 / pVE5523 is expressed normally, and the identification results show that the molecular weight of the UreA gene is approximately 26 kDa, indicating successful secretion and expression.

[0046] Example 4: Animal experiments for evaluating immunization efficacy

[0047] The immunization was divided into two groups, totaling 20 animals, as shown in Table 1. Oral immunization was administered three times, with each administration 10 days apart, and the dose of bacterial cells for each immunization was 2 × 10⁻⁶. 10 CFU.

[0048] Table 1. Oral Immunization Groups of Recombinant Lactococcus lactis

[0049]

[0050] (I) Determination of the colonization ability of recombinant Lactococcus lactis in the gastrointestinal tract of mice

[0051] Ten days after the third immunization of mice, feces were collected and weighed. The feces were thoroughly shaken and allowed to separate into layers at a concentration of 0.1 g feces / mL sterile PBS. 200 µL of the upper fecal resuspension was spread on an antibiotic selection plate and incubated at 30°C for 48 h. The growth of colonies on the plate was then observed.

[0052] The results of the planting ability test are as follows Figure 5 As shown, *Lactococcus lactis* strains used for oral immunization were detected in the feces of mice in the experimental group, while they were not detected in the feces of mice in the control group. This indicates that the recombinant live bacterial oral vaccine UreA-NZ9000 / pVE5523 constructed in this invention successfully achieved short-term colonization in the mouse intestine, and that the Balb / C mice used in the experiment did not originally have the same type of *Lactococcus lactis* strains in their gastrointestinal tract.

[0053] (II) Detection of fecal-specific antibody sIgA

[0054] Fifteen days after the third immunization, feces from Balb / C mice were collected, weighed, and mixed with 0.2 g feces / mL PBS (containing a final concentration of 0.1 mM PMSF). The mixture was thoroughly shaken and centrifuged at 12000g for 5 min. The supernatant was collected as the test sample. UreA protein (patent application number: CN202111571620.7) was diluted to 4 μg / mL in coating buffer (0.05 mol / L carbonate buffer (pH 9.6)). 100 μL / well was used to coat the microplate, incubated at 37°C for 2 h, and washed four times with PBST. 300 μL / well of blocking buffer (10 mM PBS (pH 7.4) + 1% BSA) was added, and the plate was incubated overnight at 4°C. After washing with PBST, 100 μL / well of the test sample was added to the microplate, and the plate was incubated at 37°C for 2 h. The plate was washed four times with PBST. Horseradish enzyme-labeled goat anti-mouse IgA was diluted 1:10000 with antibody dilution buffer (10 mM PBS + 0.05% Tween-20 + 0.5% BSA). 100 μL / well was added to the microplate and incubated at 37°C for 30 min. The plate was washed four times with PBST. 100 μL of chromogenic buffer (TMB stock solution: substrate buffer: 3% hydrogen peroxide = 10:90:1, prepared fresh before use) was added, and the plate was incubated at 37°C for 15 min. The reaction was terminated by adding 50 μL / 2M H2SO4. The antibody dilution buffer was used as a blank control, and the absorbance was measured at 450 nm. Result interpretation: A ratio of A sample / A negative ≥ 2.1 was considered positive. The TMB stock solution was 1 mg / mL TMB (dissolved in DMSO); the substrate buffer consisted of 0.53 mM citric acid (pH 5.0) and 100 mM Na2HPO4.

[0055] Test results are as follows Figure 6As shown, the fecal sIgA positivity rate of the experimental group mice was approximately 60%. This indicates that oral immunization with the recombinant strain UreA-NZ9000 / pVE5523 induced a mucosal immune response in the mice, producing specific antibodies against the UreA antigen protein.

[0056] (III) Evaluation of the protective efficacy of recombinant lactococcal oral vaccine after challenge.

[0057] Fifteen days after the last immunization, mice were challenged with live Helicobacter pylori J99 via oral gavage. The bacterial dose per mouse was 2.0 × 10⁻⁶. 7 CFU. Two weeks later, mice were sacrificed, and stomach tissue was minced and placed in PBS buffer, vortexed for 3 min to obtain the washing stock solution. The washing stock solution was diluted 10-fold with PBS, and the resulting bacterial suspension was plated on Skirrow agar (15 g / L styrax peptone, 2.5 g / L tryptone, 5 g / L yeast extract, 5 g / L sodium chloride, 15 g / L agar powder, 5% defibrinated sheep blood, 0.5% compound antibiotics (1.67 mg / mL vancomycin, 0.0694 mg / mL polymyxin, 0.5 mg / mL trimethoprim, 0.2 mg / mL amphotericin B)) and incubated at 37°C microaerophilic (5% O2, 10% CO2, 85% N2) for 3 days. The presence of *H. pylori* on the plates was detected using *H. pylori* colony characteristics, rapid urease reagent, and microscopic examination to determine whether the mice were infected with *H. pylori*.

[0058] Vaccine protection rate = (Positive infection rate in control group - Positive infection rate in experimental group) / Positive infection rate in control group × 100%.

[0059] The results showed that the positive rate of *Helicobacter pylori* infection in mice in the experimental group immunized with recombinant lactococcus was 30%, while that in the control group was 90%. Figure 7 This demonstrates that the recombinant live lactococcus vaccine UreA-NZ9000 / pVE5523 constructed in this invention can achieve an immunoprotection rate of 66.7% in mice.

[0060] The recombinant live lactococcus oral vaccine prepared by this invention exhibits good immunogenicity, inducing a strong mucosal immune response in mice and effectively inhibiting the colonization of Helicobacter pylori in the mouse stomach. Those skilled in the art will recognize that the embodiments described herein are for the purpose of helping the reader understand the principles of the invention and should be understood as not limiting the scope of protection of the invention 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 recombinant live vector vaccine of Lactococcus lactis, characterized in that, The urease subunit UreA gene was ligated to a plasmid expression vector and then electroporated into *Lactococcus lactis*. The amino acid sequence of the urease subunit UreA gene is shown in SEQ ID NO.

1. The plasmid expression vector used to ligate the urease subunit UreA gene was pVE5523, and the expression of the recombinant protein did not require induction. The plasmids UreA / pMD19 and pVE5523 were digested with SalI / EcoRV and ligated through the SalI / EcoRV restriction sites. The nucleic acid sequence encoding the urease subunit UreA gene is shown in SEQ ID NO.

2. *Lactococcus lactis* NZ9000 was used as the host cell.

2. The method for preparing the recombinant lactococcus live vector vaccine according to claim 1, characterized in that, Includes the following steps: S1, constructing plasmids Plasmids UreA / pMD19 and pVE5523 were digested with SalI / EcoRV and ligated through the SalI / EcoRV restriction sites. The ligation products were transformed into E. coli DH5α competent cells and evenly spread on LB agar plates containing ampicillin and incubated overnight at 37°C. Positive transformants were picked for plasmid extraction and SalI / EcoRV double digestion verification to obtain the recombinant Escherichia coli-Lactococcus lactis shuttle plasmid. S2, Electroporated Lactococcus lactis competent cells Recombinant Escherichia coli-Lactococcus lactis shuttle plasmid was transduced into host Lactococcus lactis competent cells by electroporation. S3. Cultivation of recombinant strains and expression of recombinant proteins The colonies obtained in step S2 are cultured, and the cultured bacterial cells are collected, which are the recombinant lactococcus live vector vaccine strains; the recombinant protein is expressed and secreted into the extracellular culture medium.

3. The method for preparing the recombinant lactococcus live vector vaccine according to claim 2, characterized in that, In step S3, colonies containing the recombinant Escherichia coli-Lactococcus lactis shuttle plasmid are cultured using GM17.

4. The use of the recombinant lactococcus live vector vaccine of claim 1 in the preparation of an oral vaccine against Helicobacter pylori.

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