Application of flagellin hypervariable region h as intramolecular adjuvant in preparation of vaccine

Abstract

This invention relates to the field of biological protein application technology, and discloses the application of flagellin hypervariable region H as an intramolecular adjuvant in vaccine preparation. The amino acid sequence of flagellin hypervariable region H is shown in SEQ ID NO.3, and the nucleotide sequence of the gene encoding flagellin hypervariable region H is shown in SEQ ID NO.4. This invention also discloses an expression cassette, recombinant vector, recombinant cells, or recombinant bacteria containing the gene encoding flagellin hypervariable region H. This invention uses the aforementioned flagellin hypervariable region H as an intramolecular adjuvant in vaccine preparation. This invention connects flagellin hypervariable region H to an antigenic epitope to obtain a fusion protein. Compared with existing technologies, the solubility and activity of the fusion protein are improved. Simultaneously, this invention overcomes the expression form of inclusion bodies in existing E. coli expression systems, eliminating the need for subsequent refolding and assembly, thus simplifying the vaccine preparation process.

Description

Technical Field

[0001] This invention relates to the field of biological protein application technology, and in particular to the application of the hypervariable region H of flagellar protein as an intramolecular adjuvant in vaccine preparation. Background Technology

[0002] Flagellin is a structural protein that constitutes bacterial flagella, the filamentous appendages on the outside of bacteria that assist in bacterial motility. In different strains of *E. coli* and some Gram-negative bacteria, the N-terminus and C-terminus of flagellin are highly conserved, together forming the filamentous structure at the center of the flagellum. The central region of the flagellin structure is a highly variable region, presenting as a β-sheet structure on the outer surface of the flagellum filament. Flagellin possesses a number of characteristics that make it suitable as a vaccine adjuvant. For example, flagellin can exert its effects at very low doses, typically 1-10 μg in non-human primates; flagellin does not elicit an IgE response; the initial immunization against flagellin does not diminish its efficacy as an immune adjuvant; exogenous antigen sequences can be inserted into the highly variable region of the central flagellin sequence; no detectable toxicity was observed 28 days after intranasal or intravenous injection of twice the recommended dose (100-500 μg) into rabbits; and it is easily mass-produced under Good Manufacturing Practices (GMP). However, whether fusion protein vaccines using flagellin as an intramolecular adjuvant will cause side effects, such as systemic cytokine storms or local inflammatory reactions during immunization, requires further investigation in specific vaccine applications. These side effects may limit the application of this type of fusion protein as a vaccine adjuvant.

[0003] Flagellin can bind to Toll-like receptor 5 (TLR5). The TLR5 binding region is located in the conserved regions of the N-terminus and C-terminus of flagellin, activating innate immunity and, through dendritic cell (DC) mediation, further activating antigen-specific immune responses. This has led to the current trend of designing fusion proteins as vaccines, often containing the N-terminus and C-terminus of flagellin and an exogenous antigenic epitope. The flagellin-derived structure acts as an adjuvant, effectively stimulating both innate and specific immunity. Past research has yielded numerous successful designs using flagellin as an adjuvant in animals. However, the hypervariable region H of flagellin, considered highly variable, is often removed during vaccine preparation, retaining only the N-terminus and C-terminus, and then inserting an exogenous antigenic epitope between them. However, this construction method often results in inclusion body expression in E. coli expression systems. Inclusion body expression requires subsequent refolding and reassembly, which makes subsequent processes very cumbersome. Furthermore, the refolded protein has a different spatial structure than the native protein and is likely to lose its original function. Therefore, further research is needed to develop adjuvants that achieve soluble expression without masking the activity of recombinant proteins. Summary of the Invention

[0004] The purpose of this invention is to provide the application of the hypervariable region H of flagellin as an intramolecular adjuvant in vaccine preparation. This invention connects the hypervariable region H of flagellin to an antigenic epitope to obtain a fusion protein. Compared with the prior art, the solubility and activity of the fusion protein are improved. At the same time, this invention overcomes the expression form of inclusion bodies in the existing E. coli expression system, and does not require subsequent refolding and assembly, thus simplifying the vaccine preparation process.

[0005] To achieve the above objectives, the present invention provides the application of flagellin hypervariable region H as an intramolecular adjuvant in the preparation of vaccines, wherein the amino acid sequence of flagellin hypervariable region H is as shown in SEQ ID NO.3.

[0006] To achieve the above objectives, the present invention also provides the application of a gene encoding the hypervariable region H of flagellin as an intramolecular adjuvant in the preparation of vaccines, and the nucleotide sequence encoding the hypervariable region H of flagellin is shown in SEQ ID NO.4.

[0007] To achieve the above objectives, the present invention also provides the application of expression cassettes, recombinant vectors, recombinant cells or recombinant bacteria as intramolecular adjuvants in the preparation of vaccines, wherein the expression cassettes, recombinant vectors, recombinant cells or recombinant bacteria contain a gene encoding the hypervariable region H of flagellin, and the nucleotide sequence encoding the hypervariable region H of flagellin is shown in SEQ ID NO.4.

[0008] Furthermore, it is applied to: ① Improve the soluble expression of the fusion protein; ②Enhance the activity of fusion proteins.

[0009] To achieve the above objectives, the present invention also provides a fusion protein, which is obtained by linking the hypervariable region H of flagellin and an antigenic epitope, wherein the amino acid sequence of the hypervariable region H of flagellin is shown in SEQ ID NO.3.

[0010] Furthermore, the antigenic epitopes include the B epitope and T epitope of the foot-and-mouth disease virus (FMDV) capsid protein.

[0011] Furthermore, the present invention also provides the application of the above-mentioned fusion protein as an intramolecular adjuvant in the preparation of vaccines.

[0012] To achieve the above objectives, the present invention also provides a vaccine adjuvant comprising flagellin hypervariable region H or a gene encoding flagellin hypervariable region H. The amino acid sequence of the hypervariable region H of flagellin is shown in SEQ ID NO.3; The nucleotide sequence encoding the hypervariable region H of flagellin is shown in SEQ ID NO.4.

[0013] The advantages and positive effects of using the hypervariable region H of flagellin as an intramolecular adjuvant in vaccine preparation as described in this invention are: The hypervariable region H of flagellin is considered a highly variable region and is often removed during vaccine preparation, retaining only the N-terminus and C-terminus. An exogenous antigenic epitope is then inserted between the N-terminus and C-terminus to create the vaccine. However, this construction method often results in inclusion body expression in E. coli expression systems. Inclusion body expression requires subsequent refolding and reassembly, making subsequent processes very cumbersome. Furthermore, the refolded protein has a different spatial structure than the native protein and is likely to lose its original function.

[0014] In this invention, the hypervariable region H of flagellar protein is linked to an antigenic epitope to obtain a fusion protein. Compared with the prior art, the solubility and activity of the fusion protein are improved. At the same time, this invention overcomes the expression form of inclusion bodies in the existing E. coli expression system, and does not require refolding and reassembly in the later stage, thus simplifying the vaccine preparation process.

[0015] Therefore, the hypervariable region H of flagellin can be used as an intramolecular adjuvant in the development of vaccines for various diseases, including bacterial, viral, and parasitic diseases.

[0016] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0017] Figure 1The following are the construction results of the H fusion protein soluble expression system platform in this embodiment of the invention: A is the agarose gel electrophoresis result of the H gene fragment, where M is the DNA marker and H is the H gene fragment; B is the agarose gel electrophoresis result of pMD18-TH plasmid enzyme digestion identification, where M is the DNA marker and H is the H gene fragment; C is the agarose gel electrophoresis result of pET28a-H plasmid PCR, where M is the DNA marker and 1-4 are four different plasmids selected; D is the agarose gel electrophoresis result of pET28a-H plasmid double enzyme digestion identification, where M is the DNA marker and 1-4 are four different plasmids selected. Figure 2 The following are the results of VP1 epitope design and expression analysis in the embodiments of the present invention, wherein A is a schematic diagram of VP1 epitope design; B is an SDS-PAGE analysis of VP1 expression in Escherichia coli, in which M is a DNA marker, S is the supernatant, and P is the precipitate; C is an SDS-PAGE analysis of VP1 soluble expression promoted by different inducers and solutes, in which M is a DNA marker, S is the supernatant, and P is the precipitate; Figure 3 The following are the results of VP1-H epitope design and expression analysis in the embodiments of the present invention. A is a schematic diagram of VP1-H epitope design, B is an SDS-PAGE analysis of VP1-H expression in Escherichia coli, and in B, 0 represents before induction, 1 represents after induction, M represents DNA marker, S represents supernatant, and P represents precipitate. Figure 4 The above are the analytical results of VP1-H purification in the embodiments of the present invention. A is the SDS-PAGE analysis of VP1-H purification. In A, M is DNA marker, F is flow-through, S is before purification, E is elution, and D is desalting. B is the detection of serum antibodies induced by VP1-H. Figure 5 In this embodiment of the invention, VP1-H induces mice to produce antibodies that recognize inactivated FMDV. A represents the immunization process, and B represents the absorbance result. Figure 6 The following are the predicted structures of the B1T-H, B1TB1B2-H, (B1TB1B2)2-H, and (B1TB1B2)3-H fusion proteins in this embodiment of the invention, wherein A is a schematic diagram of the connection; B is the predicted structure of B1T-H, C is the predicted structure of B1TB1B2-H, D is the predicted structure of (B1TB1B2)2-H, and E is the predicted structure of (B1TB1B2)3-H. Figure 7SDS-PAGE analysis of Escherichia coli expression of fusion protein in this embodiment of the invention, wherein A is B1T-H, B is B1TB1B2-H, C is (B1TB1B2)2-H, D is (B1TB1B2)3-H; 0 is before induction, 1 is after induction, M is DNA marker, S is supernatant, and P is precipitate; Figure 8 The SDS-PAGE analysis of the fusion protein purification in this embodiment of the invention is shown in the figure. In the figure, A is B1T-H, B is B1TB1B2-H, C is (B1TB1B2)2-H, D is (B1TB1B2)3-H; M is DNA marker, F is flow-through, S is before purification, E is elution, D is desalting, and L is washing. Figure 9 Serum antibody detection for blank group, VP1, B1T-H, B1TB1B2-H, (B1TB1B2)2-H, and (B1TB1B2)3-H induced in the embodiments of the present invention; Figure 10 In this embodiment of the invention, B1T-H, B1TB1B2-H, (B1TB1B2)2-H, and (B1TB1B2)3-H induce mice to produce antibodies that recognize inactivated FMDV. Detailed Implementation

[0018] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0019] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0020] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available materials.

[0021] The restriction endonucleases used in this invention are all commercially available.

[0022] The plasmids used in this invention, namely the bacterial strain BL21(DE3) and the pET28a plasmid, were both obtained from the Molecular Biology Laboratory of the School of Basic Medical Sciences, Jilin University (the public can contact us to obtain them if needed).

[0023] The flagellin gene nucleotide sequence (SEQ ID NO.2) used in this invention was obtained from Salmonella Typhi. Salmonella enterica subsp. enterica serovar Typhimurium str.The genome of *Salmonella enterica* subspecies *Salmonella typhimurium* ATCC 50087 was obtained. ATCC 50087 was obtained from the Pathogenic Microbiology Laboratory of the School of Basic Medical Sciences, Jilin University (available to the public upon request). Guinea pig serum and rabbit serum were purchased from Sigma-Aldrich, USA. The mice used in the examples were purchased from the Experimental Animal Center of the School of Basic Medical Sciences, Jilin University, ethical number 2023031.

[0024] Example The amino acid sequence of flagellin is shown in SEQ ID NO.1, SEQ ID NO.1: MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANGTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKEISSKTLGLDKLNVQDAYTPKETAVTVD KTTYKNGTDPITAQSNTDIQTAIGGGATGVTGADIKFKDGQYYLDVKGGASAGVYKATY DETTKKVNIDTTDKTPLATAEATAIRGTATITHNQIAEVTKEGVDTTTVAAQLAAAGVTGADKDNTSLVKLSFEDK NGKVIDGGYAVKMGDDFYAATYDEKTGAITAKTTTYTDGTGVAQTGAVKFGGANGKSEVVTATDGKTYLASDL DKH NFRTGGELKEVNTDKTENPLQKIDAALAQVDTLRSDLGAVQNRFNSAITNLGNTVNNLSSARSRIEDSDYATEVSN MSRAQILQQAGTSVLAQANQVPQNVLSLLRLEHHHHHH .

[0025] In the amino acid sequence of flagellin shown in SEQ ID NO.1, the bolded part is the N-terminus of flagellin, the part with a single underline is the hypervariable region of flagellin, and the bolded slanted part is the C-terminus of flagellin.

[0026] The nucleotide sequence of flagellin gene is shown in SEQ ID NO.2, SEQ ID NO.2: CCATGGCACAAGTCATTAATACAAACAGCCTGTCGCTGTTGACCCAGAATAACCTGAACAAATCCCAGTCCGCACTGGGCACTGCTATCGAGCGTTTGTCTTCCGGTCTGCGTATCAACAGCGCGAAAGACGATGCGG CAGGACAGGCGATTGCTAACCGTTTTACCGCGAACATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGCGCCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTG AACTGGCGGTTCAGTCTGCGAATGGTACTAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTGAACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAGGAC AACACCCTGACCATCCAGGTTGGTGCCAACGACGGTGAAACTATCGATATTGATTTAAAAGAAATCAGCTCTAAAACACTGGGACTTGATAAGCTTAATGTCCAAGATGCCTACACCCCGAAAGAAACTGCTGTAACCGTTGAT AAAACTACCTATAAAAATGGTACAGATCCTATTACAGCCCAGAGCAATACTGATA TCCAAACTGCAATTGGCGGTGGTGCAACGGGGGTTACTGGGGCTGATATCAAATTTAAAGATGGTCAATACTATTT AGATGTTAAAGGCGGTGCTTCTGCTGGTGTTTATAAAGCCACTTATGATGAAACTACAAAGAAAGTTAATATTGAT ACGACTGATAAAACTCCGTTGGCAACTGCGGAAGCTACAGCTATTCGGGGAACGGCCACTATAACCCACAACCAAA TTGCTGAAGTAACAAAAGAGGGTGTTGATACGACCACAGTTGCGGCTCAACTTGCTGCAGCAGGGGTTACTGGCGC CGATAAGGACAATACTAGCCTTGTAAAACTATCGTTTGAGGATAAAAACGGTAAGGTTATTGATGGTGGCTATGCA GTGAAAATGGGCGACGATTTCTATGCCGCTACATATGATGAGAAAACAGGTGCAATTACTGCTAAAACCACTACTT ATACAGATGGTACTGGCGTTGCTCAAACTGGAGCTGTGAAATTTGGTGGCGCAAATGGTAAATCTGAAGTTGTTAC TGCTACCGATGGTAAGACTTACTTAGCAAGCGACCTT GACAAACTAACTTCAGAACAGGCGGTGAGCTTAAAGAG GTTAATACAGATAAGACTGAAAACCCACTGCAGAAAATTGATGCTGCCTTGGCACAGGTTGATACACTTCGTTCTG ACCTGGGTGCGGTTCAGAACCGTTTCAACTCCGCTATCACCAACCTGGGCAATACCGTAAATAACCTGTCTTCTGC CCGTAGCCGTATCGAAGATTCCGACTACGCAACCGAAGTCTCCAACATGTCTCGCGCGCAGATTCTGCAGCAGGCC GGTACCTCCGTTCTGGCGCAGGCGAACCAGGTTCCGCAAAACGTCCTCTCTTTACTGCGTCTCGAG CACCACCACCACCACCACTGA.

[0027] In the flagellin gene nucleotide sequence shown in SEQ ID NO.2, the bolded part is the nucleotide sequence encoding the N-terminus of flagellin, the single underlined part is the nucleotide sequence encoding the hypervariable region of flagellin, and the bolded slanted part is the nucleotide sequence encoding the C-terminus of flagellin.

[0028] The amino acid sequence of the hypervariable region H of flagellin is shown in SEQ ID NO.3.

[0029] The nucleotide sequence encoding the hypervariable region H of flagellin is shown in SEQ ID NO.4.

[0030] Using the nucleotide sequence SEQ ID NO.4 encoding the hypervariable region of flagellin as a template, restriction enzyme sites were designed, and upstream and downstream primers were designed to retrieve the nucleotide sequence encoding the hypervariable region of flagellin.

[0031] The nucleotide sequence of the upstream primer is SEQ ID NO.5: CGCCGCCGG GGATCCGAATTCA AAACTACCTATAAAAATGG (where the underlined part is the enzyme cleavage site).

[0032] The nucleotide sequence of the downstream primer is SEQ ID NO.6: TTACTTAGCAAGCGACCTT AAGCTT CCGCGGGAATTCC (where the underlined part is the enzyme cleavage site).

[0033] The nucleotide sequence of the complementary sequence of the downstream primer is SEQ ID NO.7: GGAATTCCCGCGGAAGCTTAAGGTCGCTTGCTAAGTAA.

[0034] Primers need to be analyzed using Oligo software and optimized based on the analysis results. The final designed primers are synthesized by Shanghai Bioengineering Co., Ltd.

[0035] After obtaining the gene sequence by PCR, the experiment proceeded through the following steps: TA cloning, recombinant plasmid transformation, construction of the pET28-H recombinant plasmid (ligating epitope fragments), expression of the recombinant plasmid in E. coli, purification of the recombinant protein, and functional detection of the protein. The experimental procedure is as follows: The H peptide from the hypervariable region of flagellar protein was selected as the protein antigen fusion peptide (with the N and C ends removed) and fused with VP1 (Zhao Tiesuo. Purification and Immunological Activity Detection of Recombinant Proteins with B and T Epitopes of Swine O-type Foot-and-Mouth Disease Virus [D]. Jilin University, 2009), which has been confirmed to be expressed as inclusion bodies in the laboratory, for fusion expression (referred to as VP1-H). To explore the effect of the H peptide on the soluble expression and activity of VP1 in *E. coli*, the H-encoding gene was first obtained by PCR, and the pET28a-H plasmid was constructed through TA cloning and subcloning. VP1-H was expressed in *E. coli*, and then VP1-H was isolated and purified by nickel affinity chromatography. Mice were immunized with VP1-H to detect its activity. The results are as follows: 1. Obtaining the H gene fragment: (1) PCR amplification of the H gene fragment: Primers containing multiple restriction sites (shown in SEQ ID NO. 5-7) were designed to extract the 208-amino acid encoding H fragment from plasmid ATCC-flg (a full-length flagellin gene constructed by our laboratory at Beihua University, which can be obtained from the public upon request). The H gene fragment is 624 bp in length. The PCR-amplified H fragment gene was identified by 2% agarose gel electrophoresis. Figure 1 (A)

[0036] (2) TA cloning of the H gene fragment: The PCR-amplified H gene fragment was recovered by agarose gel electrophoresis, ligated into the pMD18-T vector, transformed, and the plasmid was extracted and identified. Double digestion with restriction enzyme sites designed in the fragment was used for identification, and positive clones containing the pMD18-TH plasmid were screened. After enzyme digestion, the pMD18-TH plasmid released a 620bp gene fragment, as shown below. Figure 1 As shown in Figure B, the identified positive clones were sent to Shanghai Bioengineering Co., Ltd. for sequencing. The sequencing results showed that the pMD18-TH plasmid sequence was completely consistent with the expected sequence, and the pMD18-TH plasmid was successfully constructed.

[0037] 2. Construction of the H fusion protein soluble expression system platform: To enable soluble expression of exogenous proteins using the H short peptide, the H short peptide gene was subcloned into the pET-28a expression vector to construct the pET28a-H expression plasmid. An NcoⅠ restriction site (containing the start codon ATG) was introduced upstream of the H short peptide gene, and two restriction sites, NdeⅠ and EcoRI, were introduced downstream. The pMD18-TH plasmid and the empty pET28a plasmid were double-digested with NdeⅠ and EcoRI. The released H gene fragment and the linearized pET28a plasmid were recovered, ligated, transformed, cultured, and the plasmid was extracted. Identification was performed using restriction enzyme digestion and PCR to obtain the pET28a-H plasmid. Figure 1 (C) Figure 1 The four plasmids of C were digested and identified by enzymes, and the results are as follows: Figure 1 As shown in D, 1-4 are four different plasmids selected. Only plasmid No. 3 was successfully digested with enzymes, and the target fragment appeared.

[0038] Positive clones identified by enzyme digestion and PCR were sent to Shanghai Bioengineering Co., Ltd. for sequencing. The results showed that the H fragment gene was fused with the pET28a vector, and the base sequence of the recombinant plasmid pET28a-H matched the prediction. Thus, the soluble expression plasmid of the H fusion protein was successfully constructed. Next, the effect of the VP1 protein, which is expressed as inclusion bodies in *E. coli*, on the soluble expression plasmid of the H fusion protein was verified.

[0039] 3. Construction, induction of expression, and activity assay of the VP1-H fusion protein: (1) Construction and induced expression of VP1-H fusion protein: The results showed that the flagellin H region is crucial for the soluble expression of recombinant proteins. Therefore, the next step is to combine the flagellin H region with viral epitopes to form recombinant proteins and observe their solubility and activity. A VP1 epitope recombinant protein, constructed and repeatedly validated in our laboratory, was selected (Zhao Tiesuo. Purification and Immunological Activity Detection of Recombinant Proteins of B and T Epitopes of Swine O-type Foot-and-Mouth Disease Virus [D]. Jilin University, 2009). Figure 2 (A) is a recombinant VP1 epitope protein vaccine that can prevent bovine O-type foot-and-mouth disease virus infection. It is constructed by tandemly linking T-cell and B-cell antigenic epitopes on VP1, which can effectively activate the body's immune response. The epitope arrangement is B1TB1B2 B1TB1B2 B1TB1B2. However, the biggest drawback of the VP1 epitope recombinant protein is that it is expressed as inclusion bodies in the E. coli expression system. Figure 2 Even with the use of two different inducers (specifically lactose and IPTG), the expression of its inclusion body form could not be altered (in Chinese B). Figure 2 To verify whether the hypervariable region H of flagellin can act as an adjuvant to help the fragment achieve soluble expression, a fusion protein VP1-H was constructed using the H fusion protein soluble expression plasmid and VP1. Figure 3 (A), induced expression in Escherichia coli. Results showed ( Figure 3 The VP1-H region (B) was found in the supernatant of the ultrasonically lysed culture. This indicates that the hypervariable region H plays a role in increasing the soluble expression of recombinant proteins in *E. coli*.

[0040] (2) Activity assay of VP1-H fusion protein: To detect the activity of the constructed VP1-H, purified VP1-H ( Figure 4 Mice were immunized twice, with immunizations every 14 days. Blood samples were collected before immunization and 28 days after immunization. First, the serum from VP1-H-immunized mice was analyzed; serum samples from different time points were diluted 1:100 before testing. An indirect ELISA method was used, with VP1-H as the coating antigen, to determine whether VP1-H could induce antibody production in mice. Results are as follows... Figure 4 As shown in Figure B: VP1-H can induce mice to produce antibodies.

[0041] To determine whether antibodies generated from VP1-H immunized guinea pig and rabbit serum could recognize inactivated foot-and-mouth disease virus (FMDV), inactivated FMDV was used as the coating antigen. VP1-H immunized guinea pig and rabbit serum was diluted 1:100 to serve as the detection antibody, and detection was performed using an indirect ELISA method. Figure 5 The recognition effect of VP1-immunized guinea pig and rabbit serum on inactivated foot-and-mouth disease virus was compared to explore the influence of the H fragment on protein activity. VP1-immunized guinea pig and rabbit serum (1:50 dilution) were used. The results showed that VP1-H immunized mouse serum better recognized inactivated foot-and-mouth disease virus. This indicates that after VP1 is fused with the H fragment, not only is soluble expression achieved in E. coli, but the activity is also enhanced.

[0042] In summary, a fusion protein VP1-H was constructed using a soluble expression plasmid of the H fusion protein and VP1, and its expression was induced in *E. coli*. The results showed that the H fragment could carry the insoluble VP1 fragment, achieving partial soluble expression in *E. coli*. After purifying the soluble VP1-H and immunizing mice, ELISA analysis revealed a significant increase in VP1-H-specific antibodies in mouse serum compared to pre-immunization levels. Furthermore, compared to VP1, the serum of mice immunized with VP1-H showed better recognition of inactivated foot-and-mouth disease virus. This result indicates that the H hypervariable region of flagellin has the potential to promote the soluble expression of exogenous proteins in *E. coli*, and the expressed fusion protein exhibits good immunomodulatory activity. Therefore, a soluble expression plasmid platform for the H fusion protein will be constructed to further validate the length and function of its carrier fragment.

[0043] 4. Validation of the H fusion protein soluble expression system: To further verify the promoting effect of the H fusion protein soluble expression system on the soluble expression of exogenous proteins in Escherichia coli, four fragments of different lengths composed of B and T epitopes in VP1 were selected to verify the length and function of the H-carrying fragments. The recombinant proteins formed were: B1T-H, B1TB1B2-H, B1TB1B2 B1TB1B2-H (abbreviated as (B1TB1B2)2-H), and B1TB1B2 B1TB1B2 B1TB1B2-H (abbreviated as (B1TB1B2)3-H).

[0044] (1) Three-dimensional structure prediction of viral epitope-H fusion protein: To achieve soluble expression of recombinant fusion proteins centered on epitope peptides in *E. coli*, the amino acid sequences of four viral epitope-H fusion proteins were submitted to the SWISS-MODLE database for homology modeling to predict tertiary structures. Two main principles were followed during the prediction and screening process: ① the overall protein structure resembled the native structure of flagellin; ② the epitope peptide was located on the molecular surface. The results showed ( Figure 6The H moiety carries exogenous epitopes, forming a spatial structure similar to the native structure of flagellin. The epitope polypeptides exist relatively independently in the recombinant protein, and their structure is not excessively affected by the H moiety. They exhibit more α-helical structures with strong hydrophilicity, which may help increase their solubility.

[0045] (2) Induction, expression and identification of viral epitope-H fusion protein in Escherichia coli: The plasmids pET28a-B1T-H, pET28a-B1TB1B2-H, pET28a-(B1TB1B2)2-H, and pET28a-(B1TB1B2)3-H were transformed into competent *E. coli* BL21 cells, respectively. Two single colonies of each plasmid were inoculated into 5 ml of LB medium and cultured. When the OD value of the bacterial culture reached 0.6-0.8, IPTG was added to the culture to induce expression. Samples were collected before and after induction. The bacterial culture after induction was lysed by sonication, and the supernatant and precipitate were separated and retained. After sample processing, the expression products and expression mode were identified by SDS-PAGE electrophoresis. Figure 7 As shown, almost all four recombinant proteins appeared in the bacterial supernatant, indicating that the H fusion protein can carry viral epitopes and is expressed in soluble form in E. coli.

[0046] (3) Purification and quantification of viral epitope-H fusion protein: To obtain high-purity B1T-H, B1TB1B2-H, (B1TB1B2)2-H, and (B1TB1B2)3-H, soluble recombinant proteins were purified using nickel affinity chromatography. After eluting the recombinant proteins with a high concentration of imidazole, the high concentration of imidazole and other small molecule salts in the eluted sample was removed using Sephadex G-25. The purified proteins were identified by 12% SDS-PAGE, and the results are shown below. Figure 8 As shown, B1T-H, B1TB1B2-H, (B1TB1B2)2-H and (B1TB1B2)3-H with high purity were obtained.

[0047] (4) Activity detection of viral epitope-H fusion protein: To investigate the activities of B1T-H, B1TB1B2-H, (B1TB1B2)2-H, and (B1TB1B2)3-H, purified recombinant proteins were mixed with an equal volume of oil-in-water emulsion at a 1:1 ratio and then used to immunize guinea pigs and rabbits. Immunization was performed twice, every 14 days. Blood samples were collected before the first immunization and 14 days after the second immunization.

[0048] First, the sera from guinea pigs and rabbits immunized with the four groups of recombinant proteins were tested. Serum samples from different time points were diluted 1:100 before detection. An indirect ELISA method was used, with B1T-H, B1TB1B2-H, (B1TB1B2)2-H, and (B1TB1B2)3-H as coating antigens, to detect whether B1T-H, B1TB1B2-H, (B1TB1B2)2-H, and (B1TB1B2)3-H could induce antibody production in mice. The results are as follows: Figure 9 As shown, B1T-H, B1TB1B2-H, (B1TB1B2)2-H and (B1TB1B2)3-H can all induce antibody production in guinea pigs and rabbits.

[0049] To determine whether antibodies produced from mouse serum immunized with B1T-H, B1TB1B2-H, (B1TB1B2)2-H, and (B1TB1B2)3-H could recognize inactivated foot-and-mouth disease virus (FMDV), inactivated FMDV was used as the coating antigen. Mouse serum immunized with B1T-H, B1TB1B2-H, (B1TB1B2)2-H, and (B1TB1B2)3-H was diluted 1:100 to serve as the detection antibody, and detection was performed using an indirect ELISA method. Figure 10 The test results showed that B1TB1B2-H, (B1TB1B2)2-H, and (B1TB1B2)3-H could induce the production of foot-and-mouth disease virus-specific antibodies in mice, and the longer the epitope, the better the antigenicity of the recombinant protein. Therefore, the flagellin H fusion protein soluble expression platform improved the solubility and activity of the protein expressed in E. coli, and the fusion protein with a longer epitope showed better antigenicity.

[0050] Therefore, this invention uses the aforementioned hypervariable region H of flagellin as an intramolecular adjuvant in vaccine preparation. By linking the hypervariable region H of flagellin to an antigenic epitope, a fusion protein is obtained. Compared with the prior art, the solubility and activity of the fusion protein are improved. At the same time, this invention overcomes the expression form of inclusion bodies in the existing E. coli expression system, eliminating the need for subsequent refolding and assembly, thus simplifying the vaccine preparation process.

[0051] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. The application of flagellin hypervariable region H as an intramolecular adjuvant in vaccine preparation, characterized by: The amino acid sequence of the hypervariable region H of flagellin is shown in SEQ ID NO.

3.

2. The application of the gene encoding the hypervariable region H of flagellin as an intramolecular adjuvant in vaccine preparation, characterized by: The nucleotide sequence of the gene encoding the hypervariable region H of flagellin is shown in SEQ ID NO.

4.

3. The application of expression cassettes, recombinant vectors, recombinant cells, or recombinant bacteria as intramolecular adjuvants in vaccine preparation, characterized in that: The expression cassette, recombinant vector, recombinant cell, or recombinant bacterium contains a gene encoding the hypervariable region H of flagellin, and the nucleotide sequence of the gene encoding the hypervariable region H of flagellin is shown in SEQ ID NO.

4.

4. The application according to any one of claims 1-3, characterized in that, Applied to: ① Improve the soluble expression of the fusion protein; ②Enhance the activity of fusion proteins.

5. A fusion protein, characterized in that: It is obtained by linking the hypervariable region H of flagellin and the antigenic epitope, wherein the amino acid sequence of the hypervariable region H of flagellin is shown in SEQ ID NO.

3.

6. The fusion protein according to claim 5, characterized in that: The antigenic epitopes include the B epitope and T epitope of the foot-and-mouth disease virus (FMDV) capsid protein.

7. The use of the fusion protein according to claim 5 or 6 as an intramolecular adjuvant in the preparation of vaccines.

8. A vaccine adjuvant, characterized in that: Including the flagellin hypervariable region H or the gene encoding the flagellin hypervariable region H; The amino acid sequence of the hypervariable region H of flagellin is shown in SEQ ID NO.3; The nucleotide sequence of the gene encoding the hypervariable region H of flagellin is shown in SEQ ID NO.4.

Images