A Salmonella antigen fusion protein and its recombinant Lactococcus lactis

Abstract

The present invention provides a Salmonella - specific antigen fusion protein and its recombinant Lactococcus lactis. The antigen fusion protein is a Salmonella OmpC and Flic antigen fusion protein, and its amino acid sequence is SEQ ID NO: 1. The fusion polypeptide provided by the present invention can be used as an antigen to prepare a vaccine, and the vaccine prepared can provide highly efficient immune protection after immunizing animals. Moreover, the provided fusion polypeptide has no allergenicity and will not cause allergy in immunized animals, ensuring its safety as a vaccine.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering drugs and vaccine manufacturing, and specifically relates to a Salmonella antigen fusion protein and its recombinant Lactococcus lactis. Background Art

[0002] Salmonella is one of the important pathogenic bacteria causing foodborne diseases, which can be transmitted to humans through various animals and pose a serious threat to human health. At present, more than 2,600 serotypes of Salmonella have been discovered, among which Salmonella enteritidis and Salmonella typhimurium are the most common serotypes. Salmonella typhimurium has a wide range of host specificity and can infect animals such as pigs, cattle, and chickens. Therefore, preventing and controlling Salmonella typhimurium has an important economic protection effect on food safety and the livestock industry.

[0003] Compared with traditional vaccines, epitope vaccines, as an emerging vaccine R & D technology, can improve the safety and effectiveness of vaccines. Probiotics are a class of active microorganisms that colonize the animal intestine and reproductive system, and can produce definite health effects to improve the host microecological balance and play a beneficial role. Lactobacillus is an important probiotic. In recent years, the development of vaccines using Lactobacillus as a carrier has received increasing attention. Lactobacillus oral vaccines can be taken orally directly without injection, avoiding the risk of injury and infection caused by injection; they can mimic the natural infection route, induce mucosal immune responses, and activate the immune system; they have an immune adjuvant effect, and the surface of Lactobacillus can enhance the immune function of the body and strengthen the vaccine effect. Summary of the Invention

[0004] The object of the present invention is to provide a Salmonella - specific antigen fusion protein and its recombinant Lactococcus lactis, wherein the antigen fusion protein is a Salmonella OmpC and Flic antigen fusion protein, and a recombinant Lactococcus lactis for recombinantly expressing the above - mentioned fusion protein.

[0005] The present invention first provides a fusion polypeptide, which comprises:

[0006] 1) A polypeptide with the amino acid sequence of SEQ ID NO: 1;

[0007] 2) A polypeptide derived from 1) by substituting, deleting, or adding one or several amino acids on the polypeptide of 1).

[0008] Another aspect of the present invention also provides a nucleic acid fragment encoding the above - mentioned fusion polypeptide;

[0009] As a specific record of an embodiment, the sequence of the nucleic acid fragment is SEQ ID NO: 2.

[0010] In another aspect, the present invention also provides a recombinant expression vector inserted with the above nucleic acid fragment;

[0011] As a specific record of the embodiment, the recombinant expression vector is the pNZ8149 plasmid expression vector;

[0012] The present invention also provides a recombinant strain, which is used for recombinantly expressing the above fusion polypeptide;

[0013] The recombinant strain, as a specific record of the embodiment, is Lactococcus lactis.

[0014] Another aspect of the present invention also provides a use of the above fusion polypeptide or recombinant strain, which is an application in the preparation of a vaccine.

[0015] In yet another aspect, the present invention also provides a vaccine, wherein the antigen of the vaccine is the fusion polypeptide or the recombinant strain.

[0016] The fusion polypeptide provided by the present invention can be used as an antigen to prepare a vaccine. After immunizing animals with the prepared vaccine, it will provide highly efficient immune protection. Moreover, the provided fusion polypeptide has no allergenicity and will not cause allergy in immunized animals, ensuring its safety as a vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 : Amino acid sequence diagram of the multi-epitope fusion peptide (SEQ ID NO: 1), where yellow: linker, green: B cell epitope, blue: Th cell epitope, purple: CTL cell epitope.

[0018] Figure 2 : Three-dimensional model diagram of the multi-epitope fusion protein, where A is the tertiary structure, B is the Ramachandran plot, and C is the Z-score plot.

[0019] Figure 3 : Growth curve of the recombinant strain;

[0020] Figure 4 : Detection result diagram of specific sIgA antibody in immunized mice, where the blank group is the non-immunized group; the pNZ8149 group is the group fed with the empty vector pNZ8149 / NZ3900 lactic acid bacteria feed; the NZ8149-OF group is the group fed with the NZ8149-OF / NZ3900 lactic acid bacteria feed. ** indicates P < 0.01%, with extremely significant difference.

[0021] Figure 5:Detection results of OMPC antibodies in immunized mice. The blank group was the non-immunized group; the pNZ8149 group was the group fed with the immunized empty vector pNZ8149 / NZ3900 lactic acid bacteria feed; the NZ8149-OF group was the group fed with the NZ8149-OF / NZ3900 feed. ** indicates P < 0.01%, with extremely significant differences.

[0022] Figure 6 :Detection results of Flic antibodies in immunized mice. The blank group was the non-immunized group; the pNZ8149 group was the group fed with the immunized empty vector pNZ8149 / NZ3900 lactic acid bacteria feed; the NZ8149-OF group was the group fed with the NZ8149-OF / NZ3900 feed. ** indicates P < 0.01%, with extremely significant differences. Detailed implementation manners

[0023] Multiepitope vaccines, also known as cocktail vaccines, are a type of subunit vaccine designed based on the amino acid sequences of target antigen epitopes. Multiepitope vaccines can be classified into forms such as linear tandem multiepitope vaccines, multivalent antigen peptide epitope vaccines, and virus-like particle vaccines. According to different antigen epitope types, they can be divided into B cell epitope vaccines, T cell epitope vaccines, and mixed epitope vaccines.

[0024] Salmonella OmpC is a porin of its outer membrane protein and has good immunogenicity. Flic can promote the activation of immune cells and the expression of pro-inflammatory cytokines, and can also induce the production of IgA in the intestinal mucosa locally against pathogen infection.

[0025] Based on the OmpC of Salmonella typhimurium in GenBank (accession number: NP_461210.1), the OmpC protein contains a total of 378 amino acids, while the Flic protein sequence (accession number: NP_460912.1) contains a total of 495 amino acids.

[0026] After molecular sequence analysis of the cellular epitopes of OmpC and Flic of Salmonella in the present invention, the selected antigen epitope polypeptides were conjugated to form a fusion protein, and the nucleic acid fragment encoding it was cloned into the Escherichia coli-lactic acid bacteria shuttle plasmid pNZ8149, electrotransformed into Lactococcus lactis NZ3900, made into feed, and used to immunize mice for preparing a clinical candidate vaccine for Salmonella.

[0027] The present invention will be described in detail below in combination with specific embodiments and drawings.

[0028] Example 1: Screening antigen epitopes to construct a fusion protein

[0029] 1. Analysis and screening of B cell antigen epitope polypeptides of Salmonella OmpC protein

[0030] Analyze the B-cell epitopes of the Salmonella OmpC gene. The IEDB prediction results of OmpC are shown in Table 1, and the ABCpred prediction results are shown in Table 2.

[0031] Table 1: IEDB analysis table of OmpC B-cell epitopes

[0032]

[0033] Table 2: ABC pred analysis table of OmpC B-cell epitopes

[0034]

[0035] Take the overlapping fragments of the IEDB prediction results and ABCpred prediction results of the epitope polypeptides as target fragments, preferably select the sequences with longer fragments among them, and merge adjacent sequences.

[0036] Based on the results obtained from the two prediction methods, the dominant B-cell epitope amino acid regions of OmpC are: 121 - 136, 156 - 171, 196 - 211; the related amino acid sequences of the dominant B-cell amino acid regions of Flic are 304 - 320, 214 - 229, 269 - 284.

[0037] 2. Predict Th cell epitopes

[0038] Use the MHC-II Binding Predictions online tool to predict the Th cell epitopes of OmpC and Flic. Tables 3 and 4 show the Th cell epitope prediction results of the OmpC and Flic amino acid sequences. For OmpC, the first two peptide segments with higher scores in the Th antigen epitopes are selected and merged, and the leading bases that are B-cell epitopes are removed to obtain the Th cell epitope of OmpC as TSYGANKANVVAYKANVVAY at positions 212 - 232. For Flic, the peptide segment with the highest score is selected as the candidate Th antigen epitope, and the Th cell epitope of Flic is obtained as GHNFKAQPDLAEAAA at positions 388 - 402.

[0039] Table 3: Th antigen epitope analysis table of OmpC

[0040]

[0041] Table 4: Th antigen epitope analysis table of Flic

[0042]

[0043] 3. Prediction of CTL cell epitopes

[0044] The CTL epitopes of OmpC and Flic were predicted using the MHC-I Binding Predictions online tool. Tables 5 and 6 show the prediction results of the CTL epitopes of the OmpC and Flic amino acid sequences. For OmpC, two peptide segments with higher scores were selected as its dominant CTL epitopes, and the CTL epitopes of OmpC were found to be WTDVGGDTY at positions 93 - 101 and YVDVGATYY at positions 250 - 258. For Flic, two peptide segments with higher scores were selected as its dominant CTL epitopes, and the CTL epitopes of Flic were found to be DTAATVTGY at positions 183 - 191 and QNRFNSAITNLGNTVNNLTSAR at positions 430 - 451.

[0045] Table 5: Analysis Table of CTL Epitopes of OmpC

[0046]

[0047] Table 6: Analysis Table of CTL Epitopes of Flic

[0048]

[0049] 4. Linkage of multi-epitope peptides

[0050] Based on the above bioinformatics analysis and the B and T cell epitopes obtained by merging overlapping amino acid sequences (see Table 7), the B cell epitope, Th and CTL epitopes were linked together in sequence using GKK, AAY and PGPG linkers. Through different arrangements, three different amino acid sequences were obtained, named Linkage I peptide, Linkage II peptide and Linkage III peptide.

[0051] The AllerTop v.2.0 online tool was used for allergenicity detection, and the results showed that Linkage I peptide, Linkage II peptide and Linkage III peptide were all non-allergens. The Vaxijen online software was used to predict the antigenicity of Linkage I, II and III peptides. The score of Linkage I peptide was 1.1328 when the set threshold was 0.4; the score of Linkage II peptide was 1.1422 when the set threshold was 0.4; the score of Linkage III peptide was 1.1347 when the set threshold was 0.4. Predicting the antigenicity values, Linkage II peptide (1.1422) > Linkage III peptide (1.1347) > Linkage I peptide (1.1328), and the results showed that Linkage II peptide ( Figure 1 ) had the highest antigenicity.

[0052] Table 7: Polypeptide Information Table of B and T Cell Epitopes of OmpC and Flic

[0053]

[0054]

[0055] Therefore, the linker peptide II was used as the sequence for constructing the Salmonella typhimurium OmpC-Flic multi-epitope vaccine. The linker peptide II consists of 228 amino acids, with a molecular weight of 23178.18, an isoelectric point of 9.38, and an instability index of 8.10, indicating that the linker peptide II has a stable structure.

[0056] 5. Tertiary structure modeling of the multi-epitope fusion peptide

[0057] The three-dimensional (3D) modeling of the Salmonella typhimurium OmpC-Flic multi-epitope fusion peptide was performed using the Swiss-Model online tool. The global model quality estimation (GMQE) value of the multi-epitope vaccine was 0.66, indicating good quality of the 3D modeling. The Ramachandran plot was used to further evaluate the quality of the model. The allowed region is the dark green area in the figure, the most favored region is the light green area, and the disallowed region is the blank area. It can be seen that most of the dot structures in the figure fall within the range. The Z-score plot represents the quality of the entire model. The Z-score of the multi-epitope peptide was 0.32, indicating within the characteristic range of native proteins. Therefore, the credibility of this tertiary structure model is the highest ( Figure 2 )

[0058] 6. Codon optimization and reverse translation of the multi-epitope peptide

[0059] The amino acids of the above multi-epitope vaccine were optimized according to the codon preference of Lactobacillus, a signal peptide sequence was added at its front end, and sequences such as dendritic cell-targeting peptides were added at its rear end. The finally determined base sequence of the multi-epitope peptide segment is as follows:

[0060] The finally determined base sequence of the multi-epitope peptide segment (SEQ ID NO: 2) is as follows:

[0061] ATGGTGATGAAAAAAAAGATTATCTCAGCTATTTTAATGTCTACAGTGATTCTTTCTGCTGCAGCCCCATTGTCAGGTGTTTACGCTGCATGTGAAGCTGCTGCTAAAGATGGTGATGCTTATGGTAAAAATGGTTCAGTTTCAGGTAATACAAATGGTAAAAAAGGTGGTGCTACAACATCAAAACGTACAGCTGATAATAATACAGCTAATGGTAAAAAAGATCAAAAAATTGATGGTGATTTAAAATTTGATGATACAACAGGTAAAGGTAAAAAAGCTGGTGGTGCTACATCACCATTAACAGGTGGTTTACCAGCTACAGCTACAGAAGATGTTAAAAATGTTCAAGTTGGTAAAAAAAC

[0062] AGCTTCAGTTGTTAAAATGTCATATACAGATAATAATGGTAAAACAATTGGTAAAAAATATT

[0063] CAACATATAATGCTACACGTGGTACATCAAATGGTTCAAATTCAACATCATATGGTGCTAATA

[0064] AAGCTAATGTTGTTGCTTATAAAGCTAATGTTGTTGCTTATCCAGGTCCAGGTGGTCATAATT

[0065] TTAAAGCTCAACCAGATTTAGCTGAAGCTGCTGCTCCAGGTCCAGGTCAAAATCGTTTTAA

[0066] TTCAGCTATTACAAATTTAGGTAATACAGTTAATAATTTAACATCAGCTCGTCCAGGTCCAG

[0067] GTTATGTTGATGTTGGTGCTACATATTATGCTGCTTATTGGACAGATGTTGGTGGTGATACAT

[0068] ATGCTGCTTATGATACAGCTGCTACAGTTACAGGTTATGGTGGTGGCGGCTCTGGTGGTGGC

[0069] GGCGGCTCAGGTGGCGGTGGCTCAGGTGGCGGTGGCTCATTTTACCCATCATACCACTCAA

[0070] CGCCACAACGTCCAGGTGGCGGTGGCTCAGGTGGCGGTGGCTCAGGTGGCGGTGGCTCAGGTGGCGGTGGCTCATACGGTCGTCGGGCTCGTCGGCGTCGGCGTCGGTAA;

[0071] The amino acid sequence of the encoded fusion polypeptide is as follows:

[0072] MVMKKKIISAILMSTVILSAAAPLSGVYAACEAAAKDGDAYGKNGSVSGNT

[0073] NGKKGGATTSKRTADNNTANGKKDQKIDGDLKFDDTTGKGKKAGGATSPLTGG

[0074] LPATATEDVKNVQVGKKTASVVKMSYTDNNGKTIGKKYSTYNATRGTSNGSNST

[0075] SYGANKANVVAYKANVVAYPGPGGHNFKAQPDLAEAAAPGPGQNRFNSAITNL

[0076] GNTVNNLTSARPGPGYVDVGATYYAAYWTDVGGDTYAAYDTAATVTGYGGGGS

[0077] GGGGGSGGGGSGGGGSFYPSYHSTPQRPGGGGSGGGGSGGGGSGGGGSYGRRA

[0078] RRRRRR(SEQ ID NO: 1).

[0079] The above base sequence (SEQ ID NO: 2) was synthesized by Shanghai Sangon Biological Engineering Co., Ltd. and cloned into the pET28a plasmid, and this plasmid was named pET28a-OF.

[0080] Example 2: Construction of recombinant Lactococcus lactis

[0081] 1. Amplification of the target fragment.

[0082] The plasmid was extracted from the strain pET28a-OF using a plasmid extraction kit and used as a template. With the primers OF-F (5'-AATAAATTATAAGGAGGCACTCACCATGGTGATGA AAAAAAAGATTATCTCA-3') and OF-R (5'-CGGGCTCGTCGGCGTCGGCG TCGGTAAAGAGCTCAAGCTTTCTTTGAACCAAAAT-3'), the nucleic acid sequence of the multivalent epitope peptide was amplified (amplification fragment 1480 bp). The PCR reaction system and conditions were as follows: The PCR reaction system was 20 μL, including 10 μL of 2× Taq Master Mix, 1 μL of Primer F, 1 μL of Primer R, 1 μL of Vector, and 7 μL of ddH2O. Reaction procedure: Pre-denaturation at 95 °C for 5 min; denaturation at 95 °C for 1 min, annealing at 46 °C for 1 min, extension at 72 °C for 90 s, for a total of 35 cycles; final extension at 72 °C for 10 min.

[0083] 2. Extraction and digestion of pNZ8149 plasmid

[0084] The pNZ8149 / NZ3900 strain (preserved in the laboratory) was inoculated into GM17 medium and cultured statically at 30 °C overnight. Take 2 mL of the bacterial solution, and extract the pNZ8149 plasmid using a plasmid miniprep kit for Gram-positive bacteria (Solarbio). Use the restriction endonucleases SacⅠ and NcoⅠ for double digestion. The digestion system was 2 μL of NEB buffer, 8 μL of pNZ8149 plasmid, 1 μL of Sac1, 1 μL of Nco1, and 8 μL of ddH20. After water bath heating at 37 °C for 3 h, the digestion products were detected by agarose gel electrophoresis, and the target band was recovered and purified using a gel recovery kit (Omega) for subsequent vector construction.

[0085] 3. Construction of recombinant lactic acid bacteria strains

[0086] After recovering the amplified OF sequence fragment with a gel extraction kit, it was mixed with the pNZ8149 plasmid fragment that had been double-digested and purified. 5 μL of seamless cloning premix (Monad), 3 μL of the OF fragment, and 2 μL of the recovered pNZ8149 plasmid were added, and the mixture was incubated in a water bath at 37 °C for 25 min. 5 μL of the above ligation product was mixed with 50 μL of NZ3900 competent cells, incubated on ice for 15 min, then transferred to an electroporation cuvette (1 mm) pre-cooled for 15 min, and electroporated at 1250 V. Then, 950 μL of recovery medium was quickly added, and the mixture was incubated at 30 °C for 1.5 - 2 h. 100 μL was taken and inoculated on an ELIKER plate, cultured at 30 °C for 2 days. Yellow single colonies were picked and transferred to 5 mL of liquid medium, and statically cultured overnight at 30 °C. The recombinant plasmid pNZ8149-OF was extracted using a positive bacterial plasmid extraction kit. The sequencing of the recombinant plasmid showed that the sequence was correct, confirming the successful construction of the pNZ8149-OF / NZ3900 strain.

[0087] 4. Stability test of the recombinant strain

[0088] The recombinant strain pNZ8149-OF / NZ3900 was passaged in M17 (0.5% lactose) medium. After 20 passages, it was inoculated on an Eliker screening medium, and more than 98% of the colonies were still positive colonies. After plasmid extraction, it was identified as correct by sequencing. It shows that more than 98% of the strains can still remain stable after 20 passages of the recombinant strain.

[0089] Using the empty vector bacteria as the control group and the recombinant bacteria as the experimental group, both were inoculated in M17 (0.5% lactose) medium, and the OD was measured every half hour. 600 The growth curves of the two were plotted and compared, and it was found that the growth curves of the recombinant bacteria and the parental bacteria were basically the same ( Figure 3 ); at the same time, through t-test, there was no significant difference in the growth curves of the two. It was confirmed that the growth performance of the recombinant bacteria was not affected compared with the parental bacteria.

[0090] 5. Preparation of lactic acid bacteria feed for immunization

[0091] Take the identified lactic acid bacteria pNZ8149-OF / NZ3900 and inoculate it into 5 mL of GM17 liquid medium, and statically culture overnight at a constant temperature of 30 °C. The next day, take 5 mL of the activated bacterial liquid and inoculate it into 200 mL of GM17 medium for further culture. Culture until OD 600When it is 0.3 - 0.4, add Nisin solution with a final concentration of 10 ng / mL. After standing and inducing at 30 °C for 6 h, mix the bacterial liquid with 2% sodium alginate solution (the mixing ratio is 1:2); add starch with a final concentration of 4%, fructose with a final concentration of 4%, glucose with a final concentration of 1%, and whole egg liquid at 200 mL / L. After fully mixing, drop it into 2% CaCl₂ solution to form gel beads. After filtration, wash with pure water 3 times, and solidify and shape for 30 min - 60 min. After filtration, wash with pure water 3 times, add it to 1% chitosan solution (the ratio to lactic acid bacteria is 1:1), stir, and film for 30 min - 60 min. After filtration, wash 3 times and air dry at room temperature.

[0092] 6. Immunization of lactic acid bacteria feed

[0093] Take the air-dried feed, add M17 liquid medium, let it stand for 10 min, and then homogenize it with a beating homogenizer. After homogenization, use the plate colony counting method (or turbidimetry) for counting. According to the counting results, adjust the concentration of the original bacterial liquid to ensure that the immunization concentration of the feed is 5×10 9 cfu / g, and immunize mice by quantitative feeding.

[0094] Randomly divide 18 BABL / c mice into 3 groups, with 6 mice in each group. Among them, the first group is set as the blank control, without immunizing any vaccine; the second group is set as the lactic acid bacteria strain control, and quantitatively feeds and immunizes the empty vector pNZ8149 / NZ3900 lactic acid bacteria feed; the third group is set as the NZ8149 - OF / NZ3900 lactic acid bacteria feed experimental group, and quantitatively feeds and immunizes the NZ8149 - OF / NZ3900 feed. The immunization procedure is as follows: Primarily immunize the mice on the 1st, 2nd, 3rd, 4th, and 5th days, continuously immunize for 5 days; give the second immunization to the mice on the 30th, 31st, 32nd, 33rd, and 34th days, continuously immunize for 5 days; give the third booster immunization to the mice on the 59th, 60th, 61st, 62nd, and 63rd days, continuously immunize for 5 days. The immunization dose is: Each mouse is quantitatively fed 2 g of feed with 5×10 9 cfu / g. 25 days after the last immunization, sacrifice all 18 mice and collect serum; also cut back 5 cm of the ileum of the small intestine, rinse the intestinal mucosa with 200 μL of small intestine lavage fluid, and collect intestinal fluid.

[0095] Using the prokaryotically expressed OmpC and Flic proteins as antigens, establish mouse-specific sIgA antibody, mouse OmpC antibody, and mouse Flic antibody ELISA detection methods, and detect the collected serum and small intestine lavage fluid samples. The results show that the sIgA, OmpC, and Flic antibodies of the experimental group mice all increased significantly (see Figure 4 、 Figure 5 、 Figure 6 ) respectively.

[0096] In summary, the present invention performs bioinformatics analysis on the antigenic epitope sequences of OmpC and Flic of Salmonella typhimurium, combines the B- and T-cell dominant epitopes of both, designs its multi-epitope vaccine, and transfers it into Lactococcus lactis to construct a Salmonella typhimurium OmpC-Flic multi-epitope recombinant Lactococcus lactis, laying a foundation for the development of Salmonella vaccines for clinical application.

Claims

1. A fusion polypeptide, characterized in that: The amino acid sequence of the fusion polypeptide is SEQ ID NO:

1.

2. A nucleic acid fragment, characterized in that The nucleic acid fragment is used to encode the fusion polypeptide according to claim 1.

3. The nucleic acid fragment according to claim 2, characterized in that The sequence of the nucleic acid fragment is SEQ ID NO:

2.

4. A recombinant expression vector, characterized in that: The nucleic acid fragment according to claim 2 is inserted into the recombinant expression vector.

5. The recombinant expression vector according to claim 4, characterized in that The recombinant expression vector is a pNZ8149 plasmid expression vector.

6. A recombinant engineering strain, characterized in that: The recombinant engineering strain carries the recombinant expression vector according to claim 4.

7. The recombinant engineered strain according to claim 6, characterized in that The recombinant engineering strain is Lactococcus lactis.

8. Use of the fusion polypeptide according to claim 1 or the recombinant engineered strain according to claim 6 as an antigen in the preparation of a Salmonella vaccine.

9. A Salmonella vaccine, characterized in that: The vaccine contains the fusion polypeptide according to claim 1 as an antigen.

10. The vaccine according to claim 9, characterized in that The vaccine also contains the recombinant engineered strain according to claim 6.

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