Construction of fusion recombinant protein ABT for preventing brucellosis and application thereof in preparation of protective vaccine

Abstract

The application is suitable for the fields of genetic engineering and biological medicine technology, and provides a fusion recombinant protein ABT for preventing brucellosis and application of the fusion recombinant protein ABT in preparation of a protective vaccine. The fusion recombinant protein ABT is composed of an immune enhancement antigen and a multi-epitope tandem antigen. The immune enhancement antigen (named as A) comprises a Brucella ribosome L7 / L12 protein and a PADRE sequence. The multi-epitope tandem antigen is composed of a dominant B cell epitope part (named as B) derived from Brucella SurA, OMP31, BP26 and Trigger factor protein and a dominant Tc cell epitope and Th cell epitope part (named as T) in series. The ABT Brucella multi-epitope subunit vaccine prepared by mixing the fusion recombinant protein ABT and an immune adjuvant has the characteristics of good purity, high safety and strong immunogenicity, can stimulate the body to produce protective antibodies, and the antibodies can be maintained for a long time.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and biomedicine, and particularly relates to the construction of a fusion recombinant protein ABT for the prevention of brucellosis and its application in the preparation of protective vaccines. Background Technology

[0002] Brucella ( Brucella Brucella is an aerobic, non-spore-forming, Gram-negative intracellular parasite that thrives in slightly acidic environments at room temperature. It can survive for extended periods in contaminated dairy products, meat, soil, water, and feces. Based on host preference, it can be classified into 12 species: Brucella malata, Brucella abortus, Brucella suis, Brucella ovis, Brucella sarinii, Brucella canis, Brucella cetacea, Brucella finnidica, Brucella vole, Brucella kiwi, Brucella spp., Brucella baboniflora, and Brucella spp. Among these, Brucella malata, Brucella abortus, and Brucella suis pose the most serious threats to humans and livestock.

[0003] Brucellosis, caused by Brucella bacteria, is a serious zoonotic infectious disease and is currently subject to mandatory immunization. This disease can cause enormous losses and poses a significant threat to both human health and the healthy development of animal husbandry.

[0004] Currently, the prevention and control of brucellosis mainly relies on vaccination. The brucellosis vaccines available on the market are all attenuated live vaccines. However, these attenuated live vaccines pose a certain risk of infection to humans and animals, and may cause miscarriages in humans and pregnant animals.

[0005] In contrast, subunit vaccines can induce both humoral and cellular immunity and are characterized by high safety. Multi-epitope subunit vaccines, by utilizing dominant epitopes of proteins, can be designed to target both humoral and cellular immunity, thereby expanding the protective effect and improving safety and stability. Therefore, this invention proposes the construction of a fusion recombinant protein ABT for the prevention of brucellosis and its application in the preparation of a protective vaccine. Summary of the Invention

[0006] The purpose of this invention is to provide a method for constructing a fusion recombinant protein ABT for the prevention of brucellosis and its application in the preparation of a protective vaccine, thereby addressing the problems mentioned in the background art.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] A fusion recombinant protein ABT for the prevention of brucellosis, the amino acid sequence of the fusion recombinant protein ABT is shown in SEQ ID NO.1, and the nucleotide sequence of the gene encoding the fusion recombinant protein ABT is shown in SEQ ID NO.2;

[0009] The fusion recombinant protein ABT is composed of an immune-enhancing antigen and a multi-epitope tandem antigen, wherein: the immune-enhancing antigen contains Brucella ribosomal L7 / L12 protein and PADRE sequence, linked by an EAAAK linker; the multi-epitope tandem antigen is composed of dominant B-cell epitope portions and dominant Tc-cell and Th-cell epitope portions derived from Brucella SurA, OMP31, BP26, and Trigger factor proteins, linked by a KK linker; the immune-enhancing antigen and the multi-epitope tandem antigen are linked by a KK linker to form the fusion recombinant protein ABT;

[0010] The immune-enhancing antigen is named A;

[0011] The dominant B cell epitope is named B;

[0012] The dominant Tc cell epitope and Th cell epitope are named T.

[0013] A method for constructing the above-described fusion recombinant protein ABT for the prevention of brucellosis includes the following steps:

[0014] By using the coding DNA of the KK linker, B cell dominant epitopes derived from four Brucella proteins (SurA, OMP31, BP26, and Trigger factor) and T cell dominant epitopes including those from Tc and Th cells were ligated to construct:

[0015] B-gene, nucleotide sequence as shown in SEQ ID NO.2 427-1107bp, corresponding amino acid sequence as shown in SEQ ID NO.1 143-369aa;

[0016] T-gene, nucleotide sequence as shown in SEQ ID NO.2 1108-1572bp, corresponding amino acid sequence as shown in SEQ ID NO.1 370-524aa;

[0017] The coding DNA of ribosomal protein L7 / L12 derived from Brucella was linked with the coding DNA of PADRE sequence using the coding DNA of EAAAK linker to construct A-gene. The nucleotide sequence is shown as 1-426bp of SEQ ID NO.2 and the corresponding amino acid sequence is shown as 1-142aa of SEQ ID NO.1.

[0018] Using overlap PCR technology, ordered tandem sequencing of gene fragments was achieved by designing primers: the upstream primer of gene A contains... Nco The first restriction endonuclease site is shown in SEQ ID NO. 3; the downstream primer of A-gene is shown in SEQ ID NO. 4; the upstream primer of B-gene contains a partially overlapping sequence of A-gene, as shown in SEQ ID NO. 5; the downstream primer of B-gene is shown in SEQ ID NO. 6; the upstream primer of T-gene contains a partially overlapping sequence of B-gene, as shown in SEQ ID NO. 7; the downstream primer of T-gene contains... Xho I. Restriction endonuclease site, as shown in SEQ ID NO.8; finally, A-gene, B-gene, and T-gene are tandemly linked to construct a structure containing Nco I, Xho ABT-gene with restriction endonuclease sites I.

[0019] A recombinant expression system for the aforementioned fusion recombinant protein ABT used to prevent brucellosis, wherein the recombinant expression system is an Escherichia coli prokaryotic expression system.

[0020] A recombinant expression plasmid expressing the aforementioned fusion recombinant protein ABT for the prevention of brucellosis, wherein the base vector of the recombinant expression plasmid is pET-28a, and the plasmid is used... Nco I, Xho After double digestion of pET-28a with restriction endonuclease I, the DNA was recovered. Seamless cloning primers were designed to fuse the recombinant protein ABT-encoding DNA. The upstream primer contained pET-28a... Nco The partially overlapping sequence of the restriction endonuclease site, as shown in SEQ ID NO.9, and the downstream primer containing pET-28a contain Xho Partially overlapping sequences of restriction endonuclease sites, as shown in SEQ ID NO.10; recombinant expression plasmid pET28a-ABT, fusion recombinant protein ABT, was constructed using a seamless cloning reaction procedure.

[0021] A method for inducing expression of the aforementioned recombinant fusion protein ABT for preventing brucellosis includes the following steps:

[0022] The recombinant expression plasmid pET28a-ABT was transformed into E. coli. E. coli In Rosetta(DE3) competent cells, E. coli genetically engineered expression strain Ec-RD-pET28a-ABT, which can express the fusion recombinant protein ABT, was obtained. After expression was induced by IPTG, the recombinant protein in the supernatant was purified to obtain the fusion recombinant protein ABT.

[0023] The application of a fusion recombinant protein ABT for the prevention of brucellosis as described above, or a fusion recombinant protein ABT obtained by the induced expression method of the fusion recombinant protein ABT described above, in the preparation of an ABT brucellosis multi-epitope subunit vaccine.

[0024] A Brucella multi-epitope subunit vaccine comprising an immune adjuvant and the aforementioned fusion recombinant protein ABT for the prevention of brucellosis, or a fusion recombinant protein ABT obtained by the aforementioned induction expression method of the fusion recombinant protein ABT.

[0025] Furthermore, the immune adjuvant and the fusion recombinant protein ABT are mixed at a volume ratio of 1:1, and the immune adjuvant is aluminum hydroxide sol adjuvant (aluminum ion concentration: 1 mg / mL).

[0026] The application of the above-described fusion recombinant protein ABT or ABT Brucella multi-epitope subunit vaccine for the prevention of brucellosis in the preparation of a protective vaccine against brucellosis.

[0027] Compared with the prior art, the beneficial effects of the present invention are:

[0028] This invention selects four highly conserved proteins—SurA, OMP31, BP26, and Trigger factor—from *Brucella malata*, *Brucella abortus*, and *Brucella suis*, which pose the most serious threats to humans and livestock. Multiple software programs were used to predict dominant epitopes, and then multiple software programs were used to screen for the final dominant epitopes. The N-terminus of the fusion recombinant protein contains the *Brucella* ribosomal L7 / L12 protein and the PADRE sequence, enhancing the immunogenicity and protective efficacy of the fusion recombinant protein ABT for preventing brucellosis. The ABT brucellosis multi-epitope subunit vaccine prepared from this protein can stimulate BALB / c mice to produce a large number of protective antibodies. Simultaneously, the codons encoding the DNA of the fusion recombinant protein ABT were optimized based on the codon preference of *E. coli*, significantly increasing the expression level of the fusion recombinant protein ABT in the *E. coli* expression system and achieving soluble expression, ensuring sufficient production of the fusion recombinant protein ABT for subsequent subunit vaccine preparation. In summary, the ABT Brucella multi-epitope subunit vaccine prepared based on the fusion recombinant protein ABT has advantages such as high antigen expression level, long antibody maintenance time, high purity, good safety, strong immunogenicity, and strong protection. Attached Figure Description

[0029] Figure 1 The tertiary structure of the fusion recombinant protein ABT is shown in the following spatial representation: Mesh: candidate Tc and Th cell epitopes; Spheres: immune-enhancing antigens; Sticks: candidate B cell epitopes.

[0030] Figure 2 The image shows the Ramachandran plot results, illustrating the distribution of amino acid residues in the fully allowed, allowed, maximum allowed, and disallowed regions.

[0031] Figure 3 This is a PCR amplification result diagram of the fusion recombinant protein ABT encoding gene obtained by ligating A-gene, B-gene, and T-gene using PCR and Overlap PCR methods in step (2) of Embodiment 2 of the present invention; wherein: lane M is DL 2000 DNA Marker; lane 1 is the encoding gene of the fusion recombinant protein ABT.

[0032] Figure 4 The plasmid map of the recombinant expression plasmid pET28a-ABT.

[0033] Figure 5 The results are for bacterial culture PCR identification; lane M is the DL 2000 DNA Marker; lanes 1-4 are the encoding genes for the fusion recombinant protein ABT; lane 5 is the empty vector control bacteria.

[0034] Figure 6This is an image showing the SDS-PAGE electrophoresis results of Ec-RD-pET28a-ABT induced expression; where lane M is the protein marker and lane 1 contains only the empty pET-28a vector. E. coli The precipitate collected after induction of expression in Rosetta(DE3) control bacteria; lane 2 contains only the empty pET-28a vector. E. coli The supernatant collected after expression was induced by Rosetta(DE3) control bacteria; lane 3 is the precipitate collected after Ec-RD-pET28a-ABT induction; lane 4 is the precipitate collected without Ec-RD-pET28a-ABT induction; lane 5 is the supernatant collected after Ec-RD-pET28a-ABT induction; lane 6 is the supernatant collected without vEc-RD-pET28a-ABT induction.

[0035] Figure 7 The image shows the SDS-PAGE electrophoresis results of the purified fusion recombinant protein ABT in step (2) of Example 3 of this invention; where lane M is the protein marker; lane 1 is the purified fusion recombinant protein ABT induced by the genetically engineered expression bacteria Ec-RD-pET28a-ABT.

[0036] Figure 8 For the immunogenicity analysis of the fusion recombinant protein in step (2) of Example 4 of the present invention, the serum antibody titer was measured at week 1 after the first immunization with the fusion recombinant protein. The results showed that *P<0.05, **P<0.01, ***P<0.001, and the statistical significance was analyzed using the Mann-Whitney test.

[0037] Figure 9 The immunogenicity of the fusion recombinant protein was analyzed in step (2) of embodiment 4 of the present invention. The antibody growth and decline pattern after the second immunization of the fusion recombinant protein is shown in the figure.

[0038] Figure 10 For the analysis of the binding of immune serum to Brucella vaccine strain S2 in step (2) of embodiment 4 of the present invention, the antibody titer of immune serum binding to Brucella vaccine strain S2 is given, where *P<0.05, **P<0.01, ***P<0.001, and the statistical significance analysis is performed using the Mann-Whitney test.

[0039] Figure 11 This is a Dot Blot result of the binding of immune serum to Brucella vaccine strain S2 in step (2) of embodiment 4 of the present invention.

[0040] Figure 12In the antibacterial analysis of immune serum in step (2) of Example 4 of the present invention, the antibacterial rate of immune serum against Brucella vaccine strain S2 is measured.

[0041] Figure 13 The results of bacterial load in the spleens of mice in the PBS group and ABT group in step (2) of Example 4 of this invention are as follows: *P<0.05, **P<0.01, ***P<0.001. The statistical significance analysis was performed using the T-test. Detailed Implementation

[0042] In order to provide a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention will now be described in detail below, but it should not be construed as limiting the scope of implementation of the present invention.

[0043] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0044] Example 1: Design of recombinant protein ABT based on immunoinformatics;

[0045] (1) The amino acid sequences of four proteins of Brucella, namely SurA, OMP31, BP26 and Trigger factor, were determined;

[0046] The amino acid sequences of four proteins—surA (GenBank:XMD05064), OMP31 (GenBank:ACS50328), BP26 (GenBank:AAO39771), and TriggerFactor (GenBank:AIJ68576)—collected from the NCBI database (https: / / www.ncbi.nlm.nih.gov / guide / proteins / ) in *Brucella malata*, *Brucella abortus*, and *Brucella suis* were used as conservation templates for analysis. All protein data were collected in FASTA file format.

[0047] (2) Cell epitope screening;

[0048] MHC-I epitopes (Tc cell epitopes) of four proteins—SurA, OMP31, BP26, and Trigger factor—were predicted using IEDB (https: / / www.iedb.org / ), Rankpep (http: / / imed.med.ucm.es / Tools / rankpep.html), and Syfpeithi (http: / / www.syfpeithi.de / ). MHC-II epitopes (Th cell epitopes) of the same four proteins were predicted using IEDB, Rankpep, and NetMHCII (https: / / services.healthtech.dtu.dk / services / NetMHCIIpan-4.0 / ). Linear B cell epitope predictions of the same four proteins were performed using IEDB and SVMTriP (http: / / sysbio.unl.edu / SVMTriP / ). The predicted linear B-cell epitopes were then scored using DeepLBCEPred (http: / / www.biolscience.cn / DeepLBCEPred / ). Epitopes with scores greater than 0.5 were selected as candidate epitopes. The candidate epitopes were then analyzed for instability index, hydrophilicity index, antigenicity index, toxicity, and allergenicity using Expasy-ProtParam (https: / / web.expasy.org / cgi-bin / protparam / protparam), vaxijen v2.0 (https: / / ddg-pharmfac.net / vaxijen / VaxiJen / VaxiJen.html), ToxinPred2 (https: / / webs.iiitd.edu.in / raghava / toxinpred2 / index.html), and AllerTOPv.2 (https: / / www.ddg-pharmfac.net / AllerTOP). The final dominant linear B-cell epitopes were then selected.

[0049] For Tc cell and Th cell epitopes, the top ten epitopes predicted by three different software programs were selected. If an epitope appeared twice in the top ten predicted by the three software programs, it was selected as a candidate epitope. The candidate epitopes were analyzed for instability index, hydrophilicity index, antigenicity index, toxicity, and allergenicity using Expasy-ProtParam, Vaxijen v2.0, ToxinPred2, and AllerTOPv.2.0 to select the final dominant Tc cell and Th cell epitopes. The final dominant linear B cell epitopes are shown in Table 1, as shown in SEQ ID NO.11-17; the final dominant Tc cell epitopes are shown in Table 2, as shown in SEQ ID NO.18-22; and the final dominant Th cell epitopes are shown in Table 3, as shown in SEQ ID NO.23-28.

[0050] Table 1 Dominant linear B cell epitopes

[0051]

[0052] Table 2 Dominant Tc cell epitopes

[0053]

[0054] Table 3 Dominant Th cell epitopes

[0055]

[0056] (3) Construction of the fusion recombinant protein ABT;

[0057] Based on the dominant epitopes obtained through immunoinformatics screening, this invention uses a KK linker to sequentially link B cell, Tc cell, and Th cell epitopes together. The d-lysine (KK) linker maintains the independent immunogenic activity of each epitope. To enhance the immunogenicity and protective activity of the fusion recombinant protein ABT, Brucella ribosomal L7 / L12 protein was added to the N-terminus of the fusion protein, followed by a PADRE sequence. An EAAAK linker was used to link these two amino acid sequences as the immunostimulatory antigen. The final selected Brucella dominant epitopes were then linked to the PADRE sequence using a KK linker to construct the fusion recombinant protein ABT. The amino acid sequence of the fusion recombinant protein ABT is shown in SEQ ID NO.1, and the nucleotide sequence of the encoding gene for the fusion recombinant protein ABT is shown in SEQ ID NO.2.

[0058] SEQ ID NO.1:

[0059] 。

[0060] SEQ ID NO.2:

[0061]

[0062] (4) Physicochemical properties analysis of the fusion recombinant protein ABT;

[0063] The fusion recombinant protein ABT has a full length of 524 amino acids, a molecular weight of 57.09492 kDa, and a pI of 9.25. At a threshold of 40, the predicted instability index is 17.82, classifying it as a stable antigen. Vaxijen v2.0 predicts a protective antigen value of 0.8673 at a threshold of 0.4, indicating its suitability as an antigen; the predicted protein solubility is 0.776, indicating protein solubility.

[0064] (5) Prediction of the secondary structure of the fusion recombinant protein ABT;

[0065] According to the results of PSIPRED (https: / / bioinf.cs.ucl.ac.uk / psipred / ) prediction of the secondary structure of the fusion recombinant protein ABT, the protein consists of 43.51% helices, 13.55% folds, and 42.94% coils.

[0066] (6) Modeling and validation of the tertiary structure of the fusion recombinant protein ABT;

[0067] Modeling was performed using the Robbetta (https: / / robetta.bakerlab.org / ) online server. The amino acid sequence of the fusion recombinant protein ABT was input into the online server. After modeling was completed, the file was opened with PyMOL software to obtain the tertiary structure of the fusion recombinant protein ABT, as shown below. Figure 1 As shown. The tertiary structure of the fusion recombinant protein ABT was analyzed using a Ramachandran plot with PyMod 3.0, based on... Figure 2 The results show that 99.3% of the amino acid residues in the tertiary structure of the fusion recombinant protein ABT fall within the fully permissible, permissible, and maximum permissible regions, which is greater than 90%. This indicates that the tertiary structure of the fusion recombinant protein ABT predicted by Robbetta conforms to stereochemical rules.

[0068] Example 2: Construction of an engineered bacterium expressing the ABT gene fused with the recombinant protein;

[0069] (1) Based on the results of cell epitope screening, the coding DNA of the selected dominant epitope protein fragments was linked using the coding DNA nucleotide sequence of the KK linker, successfully constructing two multi-antigen epitope tandem protein coding DNAs, namely B-gene (nucleotide sequence as shown in SEQ ID NO.2 427-1107bp, corresponding amino acid sequence as shown in SEQ ID NO.1 143-369aa) and T-gene (nucleotide sequence as shown in SEQ ID NO.2 1108-1572bp, corresponding amino acid sequence as shown in SEQ ID NO.1 370-524aa). Using the coding DNA nucleotide sequence of the EAAAK linker, the coding DNA of Brucella ribosome L7 / L12 protein and PADRE sequence was linked, successfully constructing the coding DNA of the immune-enhancing antigen, namely A-gene (nucleotide sequence as shown in SEQ ID NO.2 1-426bp, corresponding amino acid sequence as shown in SEQ ID NO.1 1-142aa). The DNA encoding these three antigen proteins was directly synthesized chemically after codon optimization based on the codon usage preference of the E. coli expression system. The DNA synthesis was completed by Shanghai Sangon Biotech Co., Ltd.

[0070] (2) Using the overlap PCR method, the A-gene, B-gene, and T-gene were tandemly linked in sequence to construct a gene containing... Nco I and Xho The ABT-gene is located at the sticky end of the I restriction site. The specific experimental steps are as follows:

[0071] SnapGene designed overlap PCR primers for A-gene and B-gene, as well as overlap PCR primers for AB-gene and T-gene. Using A-gene as a template and A-gene-F and A-gene-R from Table 4 as primers, PCR amplification was performed according to the PCR amplification system and conditions in Table 5, amplifying genes containing... Nco The A-gene' at the sticky end of the I restriction site. Using the B-gene as a template, and B-gene-F and B-gene-R from Table 4 as primers, amplification was performed according to the PCR amplification system and conditions in Table 5 to amplify the B-gene' containing part of the A-gene. Then, using the A-gene' and B-gene' as templates, and A-gene-F and B-gene-R from Table 4 as primers, amplification was performed according to the Overlap PCR amplification system and conditions in Table 6 to amplify the B-gene' containing part of the A-gene. Nco AB-gene at the sticky end of the I restriction site.

[0072] Using T-gene as a template and T-gene-F and T-gene-R from Table 7 as primers, PCR amplification was performed according to the PCR amplification system and conditions in Table 5. This amplified genes containing part of the AB-gene and those containing... Xho The T-gene' at the sticky end of the I restriction site. Then, using AB-gene and T-gene' as templates, and A-gene-F from Table 4 and T-gene-R from Table 7 as primers, amplification was performed according to the Overlap PCR amplification system and conditions in Table 6 to amplify the gene containing... Nco I and Xho The ABT-gene at the sticky terminus of the I restriction site. The PCR product was subjected to 1% agarose gel electrophoresis, and the results are as follows... Figure 3 As shown, lane 1 showed a clear, bright band of the expected size at a position greater than 1000 bp, followed by gel recovery.

[0073] Table 4 Information on all primers

[0074]

[0075] Table 5 PCR amplification system and conditions

[0076]

[0077] Note: The PCR reaction program is 95℃ for 5 min; 35 cycles (95℃ for 30 s; 60℃ for 30 s; 72℃ for 1 min); 72℃ for 10 min; store at 4℃.

[0078] Table 6 Overlap PCR amplification system and conditions

[0079]

[0080] Note: The Overlap PCR reaction program is 95℃ for 5 min; 35 cycles (95℃ for 30 s; 60℃ for 30 s; 72℃ for 1 min); 72℃ for 10 min; store at 4℃.

[0081] Table 7 Information on all primers

[0082]

[0083] (3) Using the seamless cloning method, the ABT-gene and the pET-28a vector were combined to construct pET28a-ABT. The specific steps are as follows:

[0084] SnapGene designed seamless cloning primers for the ABT-gene. Using the ABT-gene as a template, Table 8 shows ABT-gene-F (containing pET-28a). NcoPartially overlapping sequence of restriction endonuclease site I), ABT-gene-R (containing pET-28a containing Xho Using primers (partially overlapping sequences of restriction endonuclease sites), PCR amplification was performed according to the PCR amplification system and conditions in Table 9 to amplify the ABT-gene' suitable for seamless cloning. Nco I and Xho pET-28a was double-digested with enzyme I, according to the digestion system in Table 10. After digestion, the target DNA fragment was recovered using a DNA gel extraction kit, yielding a target DNA fragment with sticky ends at different restriction enzyme sites, suitable for seamless cloning. The ABT-gene' and... (The sentence is incomplete and requires further context to be fully translated.) Nco I and Xho The pET-28a fragment, after double digestion with enzyme I, was seamlessly cloned according to the system and conditions in Table 11 to obtain the pET28a-ABT ligation product, the chromatogram of which is shown below. Figure 4 As shown. The pET28a-ABT ligation product was transformed into E. coli. E. coli In Rosetta(DE3), bacterial culture PCR was used to identify whether the recombinant expression plasmid was successfully transformed into E. coli. E. coli The PCR amplification results of the DNA encoding the recombinant protein ABT in Rosetta(DE3) are as follows: Figure 5 As shown, bacteria with a clear and bright single band greater than 1000 bp were selected as positive clones, and these positive clones were sent to a biotechnology company for sequencing. The plasmid with correct sequencing was the prokaryotic expression plasmid pET28a-ABT used to express the recombinant fusion protein ABT. [The text then abruptly shifts to a different topic:] ...containing this recombinant expression plasmid... E. coli The Rosetta(DE3) expression strain was named Ec-RD-pET28a-ABT.

[0085] Table 8 Information on all primers

[0086]

[0087] Table 9 PCR amplification system and conditions

[0088]

[0089] Note: The PCR reaction program is 95℃ for 5 min; 35 cycles (95℃ for 30 s; 65℃ for 30 s; 72℃ for 1 min); 72℃ for 10 min; store at 4℃.

[0090] Table 10 pET-28a vector double enzyme digestion system

[0091]

[0092] Table 11 Seamless Cloning System and Conditions

[0093]

[0094] Note: The seamless cloning reaction procedure is 50°C for 15 minutes.

[0095] Example 3: Expression and purification of the fusion recombinant protein ABT;

[0096] (1) The engineered strain Ec-RD-pET28a-ABT was inoculated into 5 mL of liquid LB medium containing 100 μg / mL kanamycin and cultured at 37℃ and 180 rpm for 4 h. Then, IPTG was added to bring the final concentration to 0.5 mmol / L, and the culture was transferred to 16℃ and cultured at 160 rpm for 20 h. The induced expression strain was sonicated and the sonicated bacterial solution was centrifuged at 8000 rpm and 4℃ for 10 min. The supernatant and precipitate were collected. The expression level and soluble state of the target protein ABT in the engineered strain were detected by SDS-PAGE electrophoresis.

[0097] The SDS-PAGE electrophoresis results of the engineered strain Ec-RD-pET28a-ABT are as follows: Figure 6 As shown, lanes 3 and 5 show distinct specific bands compared to lanes 4 and 6; at the same time, differential bands can also be seen when comparing lanes 1 and 2. This indicates that the fusion recombinant protein ABT can be successfully induced to express, and is expressed in both the supernatant and the precipitate.

[0098] (2) Purification of the fusion recombinant protein ABT;

[0099] After inducing expression of the engineered strain Ec-RD-pET28a-ABT with IPTG, the precipitate and supernatant were collected. Given that SDS-PAGE analysis showed expression of the recombinant fusion protein in both the supernatant and precipitate, the supernatant was collected, and the target protein was purified using Ni-NTA affinity chromatography medium. The specific procedures are as follows:

[0100] ① Expanded culture and induced expression: 5 mL of Ec-RD-pET28a-ABT bacterial culture that had been cultured overnight at 37℃ was inoculated into 1 L of sterile LB medium, 100 μg / mL kanapenem was added, and the culture was carried out at 37℃ and 180 rpm for 4 h. Then IPTG was added to make the final concentration 0.5 mmol / L, and the culture was induced at 16℃ and 160 rpm for 20 h.

[0101] ② Bacterial cell washing and supernatant collection: After induction, the bacterial solution was centrifuged at 8000 rpm and 4℃ for 15 min, and the bacterial cell precipitate was collected. The collected bacterial cells were resuspended in Buffer A (Tris 6.058 g, glycerol 100 mL, NaCl 29.25 g, imidazole 0.68 g to 1 L, pH adjusted to 7.2-7.4), and the bacterial cells were sonicated to disrupt the cell structure. The sonicated bacterial solution was centrifuged at 8000 rpm and 4℃ for 50 min, and the supernatant was collected.

[0102] ③ Protein binding and elution on the Ni-NTA column: The column was washed with elution buffer B (Tris 6.058 g, glycerol 100 mL, NaCl 29.25 g, imidazole 34.04 g, adjusted to 1 L, pH 7.2-7.4) to remove contaminating proteins. The collected supernatant was thoroughly bound to the Ni-NTA affinity chromatography medium. The column was then eluted with elution buffers containing 40 mM imidazole (prepared by mixing 28.164 mL of Buffer A with 1.836 mL of Buffer B), 60 mM imidazole (prepared by mixing 26.94 mL of Buffer A with 3.06 mL of Buffer B), 80 mM imidazole (prepared by mixing 25.713 mL of Buffer A with 4.287 mL of Buffer B), and 150 mM imidazole (prepared by mixing 21.429 mL of Buffer A with 8.571 mL of Buffer B). Elution buffers containing different imidazole concentrations were prepared as follows: 18.366 mL Buffer A and 11.634 mL Buffer B were mixed and prepared; 220 mM imidazole elution buffers were prepared by mixing 17.142 mL Buffer A and 12.858 mL Buffer B; 250 mM imidazole elution buffers were prepared by mixing 15.306 mL Buffer A and 14.694 mL Buffer B; and 300 mM imidazole elution buffers were prepared by mixing 12.246 mL Buffer A and 17.754 mL Buffer B. Buffer B was used to elute the target protein. Protein eluates were collected from these buffers.

[0103] ④ Protein dialysis and concentration: The protein eluent, eluted with 80-200 mM imidazole elution buffer, was placed in a 20000 Dal molecular weight cutoff dialysis bag to remove imidazole. The dialysis bag containing the protein eluent was immersed in PBS for dialysis 3-4 times, with fresh PBS replaced every 3 hours. After dialysis, the protein was concentrated with PEG20000 for 3 hours. The concentrated liquid obtained was the purified target protein ABT. The concentration of the purified protein was determined using the BCA protein concentration assay kit from Beyotime Biotechnology. The concentration of the purified recombinant fusion protein ABT was determined to be 1.007 mg / mL. The SDS-PAGE electrophoresis results of the purified recombinant fusion protein ABT are shown below. Figure 7 As shown, the purified fused recombinant protein ABT exhibits the target protein band at the expected size.

[0104] Example 4: Preparation and immunoprotective analysis of ABT Brucella multi-epitope subunit vaccine;

[0105] (1) Preparation of subunit vaccines;

[0106] The fusion recombinant protein ABT obtained in Example 3 was diluted to 1.0 mg / mL and mixed with aluminum hydroxide sol adjuvant (aluminum ion concentration: 1 mg / mL) at a volume ratio of 1:1 to prepare an ABT Brucella multi-epitope subunit vaccine.

[0107] (2) Subunit vaccine immunization effect analysis experiment;

[0108] ① Immunogenicity analysis;

[0109] Seven-week-old specific pathogen-free (SPF) BALB / c mice were selected and divided into two groups of six each. One group was the immunization group, immunized with an ABT Brucella multi-epitope subunit vaccine prepared from the recombinant protein ABT; the other group was the PBS control group, injected with the same volume of PBS containing aluminum hydroxide sol as adjuvant. The immunization program consisted of two immunizations, with the second immunization administered intraperitoneally 14 days after the first. One week after the first immunization, serum samples were collected from the immunized mice via tail vein, and antibody titers were detected using an indirect ELISA method. The results are as follows: Figure 8 As shown in the figure. Serum samples were collected from immunized mice via tail vein sampling at weeks 1, 2, 6, 8, 10, 14, and 18 after the second immunization to detect changes in antibody titer levels and monitor whether the vaccine could provide long-term protection to mice. The antibody titer in the serum was detected using an indirect ELISA method, and the results are shown in the figure. Figure 9 As shown. By Figure 8As can be seen, the antibody titer in the ABT-immunized group was significantly higher than that in the PBS control group, indicating that the ABT Brucella multi-epitope subunit vaccine prepared by fusing recombinant protein ABT can significantly stimulate antibody production in the body. Figure 9 It is evident that the antibody titer in the ABT immunization group remained at a high level, and the antibody duration in vivo was long, while the antibody titer in the PBS group remained consistently low. These results indicate that the prepared recombinant fusion protein ABT has good immunogenicity, and that the vaccine composed of ABT and adjuvant can stimulate the body to produce antibodies with strong binding ability and long duration of action.

[0110] ② Analysis of the binding affinity of immune serum to Brucella vaccine strain S2;

[0111] Immune serum collected eight weeks after the second immunization was used to verify the binding effect of the immune serum with Brucella vaccine strain S2 (produced by Harbin Pharmaceutical Group Biological Vaccine Co., Ltd.) using indirect ELISA and Dot Blot methods.

[0112] The specific steps for indirect ELISA are as follows: Coat the ELISA plate with Brucella vaccine strain S2, 100 μL / well, incubate overnight at 4°C. After washing three times with PBST, block with 1% BSA blocking buffer at 37°C for 2 hours, 300 μL / well. After washing three times with PBST, add 100 μL / well of 1:300 serially diluted immune serum sample, incubate at 37°C for 1 hour. After washing three times with PBST, add 100 μL / well of 1:10000 diluted HRP-labeled goat anti-mouse secondary antibody, incubate at 37°C for 1 hour. After washing three times with PBST, add 100 μL / well of TMB chromogenic solution, react at room temperature in the dark for 20 minutes, stop the reaction by adding 50 μL / well of 2M H2SO4, and read the OD value using an ELISA reader. 450 The specific antibody titer of immune serum against Brucella vaccine strain S2 was calculated, and the results are as follows: Figure 10 As shown in the figure, the antibody titer in the ABT group (immunized with ABT Brucella multi-epitope subunit vaccine group) was significantly higher than that in the PBS control group. This indicates that after immunization with the ABT Brucella multi-epitope subunit vaccine, the antibodies in the immune serum produced by mice can specifically bind to the Brucella vaccine strain S2.

[0113] The Dot Blot experimental procedure was as follows: Brucella vaccine strain S2 was ultrasonically disrupted using a non-contact ultrasonic disruptor. After centrifugation at 8000 rpm and 4℃ for 15 min, the supernatant protein was collected. The PVDF membrane was activated with methanol for 5 s, and the supernatant protein was spotted onto the PVDF membrane at a rate of 1-2 μL per spot, for a total of 6 spots. The membrane was then air-dried and immersed in 1% BSA blocking buffer at 37℃ for 2 h. The membrane was washed three times with TBST for 5 min each time. Immune serum was diluted 1:1000 with TBST, and the membrane was immersed in the diluted immune serum and incubated at 37℃ for 1 h. The membrane was washed three times with TBST for 5 min each time. HRP-labeled goat anti-mouse secondary antibody was diluted 1:10000 with TBST, and the membrane was immersed in the diluted secondary antibody solution and incubated at 37℃ for 1 h. The membrane was washed three times with TBST for 5 min each time. ECL chemiluminescent substrate was evenly dropped onto the membrane, and the signal was detected using a chemiluminescence imaging system. The results are as follows: Figure 11 As shown in the figure, after incubation and exposure with the ECL chemiluminescent substrate, obvious black specific signal spots were detected at the spotting locations, further demonstrating that the antibodies in the immune serum produced by mice after immunization with the ABT Brucella multi-epitope subunit vaccine can specifically bind to the Brucella vaccine strain S2.

[0114] ③ Antibacterial analysis of immune serum;

[0115] Serum samples collected two weeks after the second immunization with the ABT Brucella multi-epitope subunit vaccine were used to conduct a serum antibacterial experiment to investigate the antibacterial effect of the immune serum on Brucella vaccine strain S2. The specific steps were as follows: The positive control group consisted of Brucella-positive infected bovine serum, and the negative control group consisted of serum from unimmunized mice. Serum was diluted at dilutions of 1:20, 1:40, 1:80, 1:160, 1:320, and 1:640. 20 μL of mouse serum at different dilutions was added to each PCR tube, and the tubes were incubated at 56°C for 30 min to inactivate complement. Then, 1 × 10⁻⁶ spores of Brucella vaccine strain S2 were added to each PCR tube. 4 CFU / mL), 20 μL per tube, incubated at 37°C for 1 h; then guinea pig serum (complement activity 100 U / mL), 10 μL per tube, incubated at 37°C for 1 h; the reaction solution in each tube was diluted 1:2 and spread on TSA plates, incubated at 37°C for 72 h, the colony count of each group was counted, and the inhibition rate of each serum group was calculated using the formula (1 - colony count / colony count of negative control group) × 100%. The results are as follows. Figure 12As shown in the figure, both the positive control group and the ABT group (immunized with ABT Brucella multi-epitope subunit vaccine) serum exhibited a certain antibacterial rate. Furthermore, the antibacterial rate of the ABT group serum gradually decreased with increasing serum dilution, but remained significantly higher than that of the negative control group overall. This indicates that the immune serum produced by mice after immunization with the ABT Brucella multi-epitope subunit vaccine has a relatively significant antibacterial effect against Brucella vaccine strain S2.

[0116] ④ Determination of bacterial load in mouse spleen;

[0117] Seven-week-old specific pathogen-free (SPF) BALB / c mice were selected and divided into two groups of six each. One group was the immunization group, immunized with an ABT Brucella multi-epitope subunit vaccine prepared from the recombinant protein ABT; the other group was the PBS control group, injected with the same volume of PBS containing aluminum hydroxide sol as adjuvant. The immunization procedure consisted of two immunizations. The mice were immunized again 14 days after the first immunization via intraperitoneal injection. Fourteen days after the second immunization, both groups of mice were challenged with Brucella vaccine strain A19 (produced by Harbin Pharmaceutical Group Biological Vaccine Co., Ltd.) at a dose of 1×10⁻⁶. 7 (CFU / mouse) On day 14 post-challenge, mice were euthanized by cervical dislocation. Spleens were collected and weighed from both groups of mice. The spleen tissue was ground and homogenized into 10-fold serial dilutions, then spread onto TSA plates. To minimize error, each spleen homogenate was repeated once. Colony counts were performed, and the bacterial load per gram of spleen was calculated for the PBS and ABT groups. Results are as follows: Figure 13 As shown, the bacterial load in the spleen of mice immunized with the ABT Brucella multi-epitope subunit vaccine was significantly lower than that in the PBS group.

[0118] Conclusion: The recombinant protein ABT designed based on immunoinformatics in this invention exhibits good immunogenicity. The ABT Brucella multi-epitope subunit vaccine composed of ABT and adjuvant can significantly stimulate the body to produce antibodies with strong binding ability. After two immunizations, the high level of antibodies can persist in the body. The serum produced after immunization can bind to Brucella vaccine strain S2 and has a significant antibacterial effect. Furthermore, after two immunizations, the ABT Brucella multi-epitope subunit vaccine shows a significant protective effect against challenge with Brucella vaccine strain A19.

[0119] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent.

Claims

1. A fusion recombinant protein ABT for the prevention of brucellosis, characterized in that, The amino acid sequence of the fusion recombinant protein ABT is shown in SEQ ID NO.1, and the nucleotide sequence of the gene encoding the fusion recombinant protein ABT is shown in SEQ ID NO.2; The fusion recombinant protein ABT is composed of an immune-enhancing antigen and a multi-epitope tandem antigen, wherein: the immune-enhancing antigen contains Brucella ribosomal L7 / L12 protein and PADRE sequence, linked by an EAAAK linker; the multi-epitope tandem antigen is composed of dominant B-cell epitope portions and dominant Tc-cell and Th-cell epitope portions derived from Brucella SurA, OMP31, BP26, and Trigger factor proteins, linked by a KK linker; the immune-enhancing antigen and the multi-epitope tandem antigen are linked by a KK linker to form the fusion recombinant protein ABT; The immune-enhancing antigen is named A; The dominant B cell epitope is named B; The dominant Tc cell epitope and Th cell epitope are named T.

2. A method for constructing the fusion recombinant protein ABT for the prevention of brucellosis according to claim 1, characterized in that, Includes the following steps: By using the coding DNA of the KK linker, B cell dominant epitopes derived from four Brucella proteins (SurA, OMP31, BP26, and Trigger factor) and T cell dominant epitopes including those from Tc and Th cells were ligated to construct: B-gene, nucleotide sequence as shown in SEQ ID NO.2 427-1107bp, corresponding amino acid sequence as shown in SEQ ID NO.1 143-369aa; T-gene, nucleotide sequence as shown in SEQ ID NO.2 1108-1572bp, corresponding amino acid sequence as shown in SEQ ID NO.1 370-524aa; The coding DNA of ribosomal protein L7 / L12 derived from Brucella was linked with the coding DNA of PADRE sequence using the coding DNA of EAAAK linker to construct A-gene. The nucleotide sequence is shown as 1-426bp of SEQ ID NO.2 and the corresponding amino acid sequence is shown as 1-142aa of SEQ ID NO.

1. Using overlap PCR technology, ordered tandem sequencing of gene fragments was achieved by designing primers: the upstream primer of gene A contains... Nco The first restriction endonuclease site is shown in SEQ ID NO. 3; the downstream primer of A-gene is shown in SEQ ID NO. 4; the upstream primer of B-gene contains a partially overlapping sequence of A-gene, as shown in SEQ ID NO. 5; the downstream primer of B-gene is shown in SEQ ID NO. 6; the upstream primer of T-gene contains a partially overlapping sequence of B-gene, as shown in SEQ ID NO. 7; the downstream primer of T-gene contains... Xho I. Restriction endonuclease site, as shown in SEQ ID NO.8; finally, A-gene, B-gene, and T-gene are tandemly linked to construct a structure containing Nco I, Xho ABT-gene with restriction endonuclease sites I.

3. A recombinant expression system for the fusion recombinant protein ABT for the prevention of brucellosis according to claim 1, characterized in that, The recombinant expression system is an Escherichia coli prokaryotic expression system.

4. A recombinant expression plasmid expressing the fusion recombinant protein ABT for the prevention of brucellosis as described in claim 1, characterized in that, The recombinant expression plasmid was based on the pET-28a vector, and utilized... Nco I, Xho After double digestion of pET-28a with restriction endonuclease I, the DNA was recovered. Seamless cloning primers were designed to fuse the recombinant protein ABT-encoding DNA. The upstream primer contained pET-28a... Nco The partially overlapping sequence of the restriction endonuclease site, as shown in SEQ ID NO.9, and the downstream primer containing pET-28a contain Xho Partially overlapping sequences of restriction endonuclease sites, as shown in SEQ ID NO.10; recombinant expression plasmid pET28a-ABT, fusion recombinant protein ABT, was constructed using a seamless cloning reaction procedure.

5. A method for inducing expression of the recombinant fusion protein ABT for the prevention of brucellosis according to claim 1, characterized in that, Includes the following steps: The recombinant expression plasmid pET28a-ABT was transformed into E. coli. E. coli In Rosetta(DE3) competent cells, E. coli genetically engineered expression strain Ec-RD-pET28a-ABT, which can express the fusion recombinant protein ABT, was obtained. After expression was induced by IPTG, the recombinant protein in the supernatant was purified to obtain the fusion recombinant protein ABT.

6. The use of the fusion recombinant protein ABT for the prevention of brucellosis according to claim 1, or the fusion recombinant protein ABT obtained by the induction expression method of the fusion recombinant protein ABT according to claim 5, in the preparation of ABT Brucella multi-epitope subunit vaccine.

7. An ABT Brucella multi-epitope subunit vaccine, characterized in that, The ABT Brucella multi-epitope subunit vaccine comprises an immune adjuvant and the fusion recombinant protein ABT for the prevention of brucellosis as described in claim 1, or the fusion recombinant protein ABT obtained by the induction expression method of the fusion recombinant protein ABT as described in claim 5.

8. The ABT Brucella multi-epitope subunit vaccine according to claim 7, characterized in that, The immune adjuvant and the fusion recombinant protein ABT are mixed at a volume ratio of 1:1; the immune adjuvant is aluminum hydroxide sol adjuvant, wherein the aluminum ion content is 1 mg / mL.

9. The use of the fusion recombinant protein ABT for the prevention of brucellosis according to claim 1 or the ABT Brucella multiepitope subunit vaccine according to claim 7 in the preparation of a protective vaccine against brucellosis.

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