Salmonella antigen fusion protein and recombinant lactococcus lactis thereof

Abstract

The invention provides a salmonella specific antigen fusion protein and recombinant lactococcus lactis thereof, the antigen fusion protein is a salmonella OmpC and Flic antigen fusion protein, and the amino acid sequence of the antigen fusion protein is SEQ ID NO: 1. The fusion polypeptide provided by the invention can be used as an antigen to prepare vaccines, and the prepared vaccines can provide efficient immune protection after immunizing animals. The fusion polypeptide provided by the invention has no anaphylaxis, does not cause allergy of immune animals, and ensures the safety of the fusion polypeptide as a vaccine.

Description

Technical Field

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

[0002] Salmonella is one of the important pathogens that cause foodborne diseases. It can be transmitted to humans through a variety of animals, posing a serious threat to human health. At present, more than 2,600 serotypes of Salmonella have been discovered, of which Salmonella Enteritidis and Salmonella Typhimurium are the most common serotypes. Salmonella Typhimurium is pantropic and can infect pigs, cattle, chickens and other animals. Therefore, the prevention and control of Salmonella Typhimurium plays an important economic protective role in food safety and the livestock industry.

[0003] Compared with traditional vaccines, epitope vaccines, as an emerging vaccine development technology, can improve the safety and effectiveness of vaccines. Probiotics are a type of active microorganisms, which are colonized in the intestines and reproductive systems of animals, can produce definite health effects, thereby improving the host's microecological balance and playing a beneficial role. Lactic acid bacteria are an important probiotic. In recent years, the development of vaccines using lactic acid bacteria as carriers has received more and more attention. Lactic acid bacteria oral vaccines can be taken directly orally without injection, avoiding the risk of damage and infection caused by injection; they can mimic natural infection routes, induce mucosal immune responses, and activate the immune system; they have an immune adjuvant effect, and the surface of lactic acid bacteria can enhance the body's immune function and strengthen the vaccine effect. Summary of the invention

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

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

[0006] 1) a polypeptide having an amino acid sequence of SEQ ID NO: 1;

[0007] 2) Substituting, deleting or adding one or more amino groups to the polypeptide of 1) to obtain a polypeptide derived from 1);

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

[0009] As a specific description of the 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 into which the above nucleic acid fragment is inserted;

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

[0012] The present invention also provides a recombinant strain, which is used to recombinantly express the above-mentioned fusion polypeptide;

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

[0014] Another aspect of the present invention provides a use of the fusion polypeptide or recombinant strain in the preparation of a vaccine.

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

[0016] The fusion polypeptide provided by the present invention can be used as an antigen to prepare a vaccine, and the prepared vaccine can provide efficient immune protection after immunizing animals. In addition, the fusion polypeptide provided is non-allergenic and will not cause allergies in the immunized animals, thus 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), wherein yellow: linker, green: B cell epitope, blue: Th cell epitope, purple: CTL cell epitope.

[0018] Figure 2 : Three-dimensional model diagram of 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 recombinant strain;

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

[0021] Figure 5: OMPC antibody detection results of immunized mice, where the blank group is the non-immunized group; the pNZ8149 group is the group fed with the immune empty vector pNZ8149 / NZ3900 lactic acid bacteria feed; the NZ8149-OF group is the group fed with the NZ8149-OF / NZ3900 feed. ** indicates P < 0.01%, and the difference is extremely significant.

[0022] Figure 6 : The results of Flic antibody detection in immunized mice, where the blank group is the non-immunized group; the pNZ8149 group is the group fed with the immune empty vector pNZ8149 / NZ3900 lactic acid bacteria feed; the NZ8149-OF group is the group fed with the NZ8149-OF / NZ3900 feed. ** indicates P < 0.01%, and the difference is extremely significant. DETAILED DESCRIPTION

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

[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 to fight against pathogen infection.

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

[0026] The present invention performs molecular sequence analysis on the cell epitopes of OmpC and Flic of Salmonella, couples the selected antigen epitope polypeptides to form a fusion protein, clones the encoding nucleic acid fragment into the Escherichia coli-lactic acid bacteria shuttle plasmid pNZ8149, and electrotransforms it into Lactococcus lactis NZ3900 to prepare bacterial feed, immunize mice, and is used to prepare a Salmonella clinical candidate vaccine.

[0027] The present invention is described in detail below in conjunction with specific embodiments and drawings.

[0028] Example 1: Screening of antigenic epitopes to construct fusion proteins

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

[0030] The B cell antigen epitopes of the Salmonella OmpC gene were analyzed, wherein 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 of OmpC B cell antigen epitopes

[0032]

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

[0034]

[0035] The fragments of the antigen epitope polypeptide whose IEDB prediction results and ABCpred prediction results overlap are taken as target fragments, and the sequences with longer fragments are preferred, and the adjacent sequences are merged.

[0036] Combining the results of the two prediction methods, the dominant B cell epitope amino acid segments of OmpC were obtained as follows: 121-136, 156-171, 196-211; the dominant B cell amino acid segments of Flic were related to amino acid sequences of 304-320, 214-229, 269-284.

[0037] 2. Prediction of Th cell epitopes

[0038] The MHC-II Binding Predictions online tool was used to predict the Th cell antigen epitopes of OmpC and Flic. Tables 3 and 4 show the prediction results of the Th cell antigen epitopes of the amino acid sequences of OmpC and Flic. The Th antigen epitope of OmpC selected the first two peptides with higher scores and merged them, and the head base as the B cell epitope was removed. The Th cell antigen epitope of OmpC was TSYGANKANVVAYKANVVAY at positions 212 to 232. The peptide with the highest score was selected as the candidate Th antigen epitope of Flic, and the Th cell antigen epitope of Flic was GHNFKAQPDLAEAAA at positions 388 to 402.

[0039] Table 3: Analysis of Th antigen epitopes of OmpC

[0040]

[0041] Table 4: Analysis of Th antigen epitopes of Flic

[0042]

[0043] 3. Prediction of CTL cell epitopes

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

[0045] Table 5: OmpC CTL antigen epitope analysis table

[0046]

[0047] Table 6: Analysis of CTL antigen epitopes of Flic

[0048]

[0049] 4. Connection of multi-epitope peptides

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

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

[0052] Table 7: Information of B and T cell antigen epitope peptides of OmpC and Flic

[0053]

[0054]

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

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

[0057] The swiss-model online tool was used to perform three-dimensional modeling on the OmpC-Flic multi-epitope fusion peptide of Salmonella typhimurium. The GMQE value of the multi-epitope vaccine was 0.66, and the quality of the three-dimensional modeling was good. The Ramachandran plot was used to further evaluate the quality of the model. The allowed area is the dark green area in the figure, the maximum allowed area is the light green area in the figure, and the unallowed area is the blank area in the figure. It can be seen that most of the point structures in the figure fall within the interval. The Z-score graph represents the quality of the entire model. The Z-score of the multi-epitope peptide is 0.32, which means it is within the characteristic range of natural proteins. Therefore, the credibility of this tertiary structure model is the highest ( Figure 2 ).

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

[0059] The lactic acid bacteria codon preference of the above-mentioned multi-epitope vaccine amino acids was optimized, and a signal peptide sequence was added to the front end, and a dendritic cell targeting peptide sequence was added to the back. The final multi-epitope peptide base sequence is as follows:

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

[0061] ATGGTGATGAAAAAAAAGATTATCTCAGCTATTTTAATGTCTACAGTGATTCTTTCTGCTGCAGCCCCATTGTCAGGTGTTTACGCTGCATGTGAAGCTGCTGCTAAAGATGGTGATGCTTATGGTAAAAATGGTTCAGTTTCAGGTAATACAAATGGTAAAAAAGGTGGTGCTACAACATCAAAACGTACAGCTGATAATAATACAGCTAATGGTAAAAAAGATCAAAAAAATTGATGGTGATTTAAAATTTGATGATACAACAGGTAAAGGTAAAAAAGCTGGTGGTGCTACATCACCATTAACAGGTGGTTTACCAGCTACAGCTACAGAAGATGTTAAAAATGTTCAAGTTGGTAAAAAAAC

[0062] AGCTTCAGTTGTTAAAATGTCATATACAGATAATAATGGTAAAACAATTGGTAAAAAATATT

[0063] CAACATATAATGCTACACGTGGTACATCAAATGGTTCAAATTCAACATCATATGGTGCTAATA

[0064] AAGCTAATGTTGTTGCTTATAAAGCTAATGTTGTTGCTTATCCAGGTCCAGGTGGTCATAATT

[0065] TTAAAGCTCAACCAGATTTAGCTGAAGCTGCTGCTCCAGGTCCAGGTCAAAATCGTTTTAA

[0066] TTCAGCTATTACAAATTTAGGTAATACAGTTAATAATTTAACATCAGCTCGTCCAGGTCCAG

[0067] GTTATGTTGATGTTGGTGCTACATATTATGCTGCTTATTGGACAGATGTTGGTGGTGATACAT

[0068] ATGCTGCTTATGATACAGCTGCTACAGTTACAGGTTATGGTGGTGGCGGCTCTCTGGTGGTGGC

[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 Biotechnology Co., Ltd. and cloned into pET28a plasmid, which was named pET28a-OF.

[0080] Example 2: Construction of recombinant Lactococcus lactis

[0081] 1. Amplification of target fragments.

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

[0083] 2. Extraction and restriction digestion of pNZ8149 plasmid

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

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

[0086] After the amplified OF sequence fragment was recovered with a gel recovery kit, it was mixed with the pNZ8149 plasmid fragment after double enzyme digestion and purification, and 5 μL of seamless cloning premix PreMix (Monad), 3 μL of OF fragment, and 2 μL of recovered pNZ8149 plasmid were added, and the mixture was incubated at 37°C for 25 minutes. 5 μL of the above ligation product was mixed with 50 μL of NZ3900 competent cells, incubated on ice for 15 minutes, and then moved to an electric shock cup (1 mm) precooled for 15 minutes, and then electroporated at 1250V, and then 950 μL of recovery medium was quickly added, and the mixture was incubated at 30°C for 1.5 to 2 hours. Take 100 μL and inoculate it on the ELIKER plate, culture it at 30°C for 2 days, pick up a single yellow colony and put it into 5 mL of liquid culture medium, culture it at 30°C overnight, use the positive bacteria plasmid extraction kit to extract the recombinant plasmid pNZ8149-OF, and the sequencing of the recombinant plasmid showed that the sequence was correct, confirming that the pNZ8149-OF / NZ3900 strain was successfully constructed.

[0087] 4. Stability test of recombinant strains

[0088] The recombinant strain pNZ8149-OF / NZ3900 was subcultured in M17 (0.5% lactose) medium and inoculated in Eliker screening medium after 20 generations. More than 98% of the colonies were still positive colonies, and the plasmid was extracted and sequenced to identify that it was correct. This shows that after 20 subcultures, more than 98% of the recombinant strains can still remain stable.

[0089] The empty bacteria were used as the control group and the recombinant bacteria were used as the test group. They were inoculated into M17 (0.5% lactose) medium and the OD was measured every half an hour. 600 , and the growth curves of the two were plotted and compared, and it was found that the growth curves of the recombinant bacteria and the parent bacteria were basically the same ( Figure 3 ); at the same time, the t test showed that there was no significant difference in the growth curves of the two. This confirmed that the growth performance of the recombinant bacteria was not affected compared with that of the parent bacteria.

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

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

[0092] 6. Immunity of lactic acid bacteria feed

[0093] Take the dried bacterial material, add M17 liquid culture medium, let it stand for 10 minutes, then use a beating homogenizer to homogenize, and then use the plate colony counting method (or turbidimetry) to count. According to the counting results, adjust the original bacterial liquid concentration to ensure that the immune concentration of the bacterial material is 5×10 9 cfu / g, by quantitative feeding of immunized mice.

[0094] Eighteen BABL / c mice were randomly divided into three groups, with six mice in each group. The first group was set as a blank control without any vaccine; the second group was set as a lactic acid bacteria strain control, and was quantitatively fed with immune empty vector pNZ8149 / NZ3900 lactic acid bacteria feed; the third group was set as the NZ8149-OF / NZ3900 lactic acid bacteria feed test group, and was quantitatively fed with immune NZ8149-OF / NZ3900 feed. The immunization procedure was as follows: the mice were primed at 1d, 2d, 3d, 4d, and 5d, and immunized for 5 consecutive days; the mice were immunized twice at 30d, 31d, 32d, 33d, and 34d, and immunized for 5 consecutive days; the mice were given a third booster immunization at 59d, 60d, 61d, 62d, and 63d, and immunized for 5 consecutive days. The immunization dose was: each mouse was quantitatively fed 5×10 9 cfu / g bacterial feed 2g. 25 days after the last immunization, all 18 mice were killed and serum was collected; 5 cm of the ileum segment of the small intestine was cut, and the intestinal mucosa was washed with 200 μL of small intestinal lavage fluid to collect intestinal fluid.

[0095] Using prokaryotic OmpC and Flic proteins as antigens, mouse-specific sIgA antibody, mouse OmpC antibody, and mouse Flic antibody ELISA detection methods were established, and the collected serum and small intestinal lavage fluid samples were tested. Results The sIgA, OmpC, and Flic antibodies of mice in the experimental group were significantly increased (see Table 1). Figure 4 , Figure 5 , Figure 6 ).

[0096] In summary, the present invention conducts bioinformatics analysis on the antigenic epitope sequences of OmpC and Flic of Salmonella typhimurium, combines the B and T cell dominant epitopes of the two, designs their multi-epitope vaccines, and transforms them into Lactococcus lactis to construct Salmonella typhimurium OmpC-Flic multi-epitope recombinant Lactococcus lactis, laying the foundation for the development of Salmonella vaccines for clinical application.

Claims

1. A fusion polypeptide, characterized in that: The fusion polypeptide comprises: 1) a polypeptide having an amino acid sequence of SEQ ID NO: 1; 2) Substitution, deletion or addition of one or more amino groups in the polypeptide of 1) to obtain a polypeptide derived from 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 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 vaccine.

9. A vaccine, characterized in that The antigen in the vaccine is the fusion polypeptide according to claim 1.

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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