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

Abstract

The invention is applicable to the technical field of genetic engineering and biological medicine, and provides construction of a fusion recombinant protein ABT for preventing brucellosis and application of the fusion recombinant protein ABT in preparation of protective vaccines. The fusion recombinant protein ABT is composed of an immunological enhancement antigen and a multi-epitope tandem antigen, wherein the immunological enhancement antigen (named as A) comprises a Brucella ribosome L7 / L12 protein and a PADRE sequence; the multi-epitope tandem antigen is formed by connecting a dominant B cell epitope part (named as B), a dominant Tc cell epitope part and a dominant Th cell epitope part (named as T) in series, wherein the dominant B cell epitope part (named as B) is derived from Brucella SurA, OMP31, BP26 and Trigger factor proteins. The ABT Brucella multi-epitope subunit vaccine prepared by mixing the fusion recombinant protein ABT and an immunologic adjuvant has the characteristics of good purity, high safety and strong immunogenicity, can stimulate an organism to generate a protective antibody, and has long antibody maintenance time.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering and biomedicine technology, and in particular relates to the construction of a fusion recombinant protein ABT for preventing brucellosis and its application in preparing a protective vaccine. Background Art

[0002] Brucella ( Brucella ) is an aerobic, non-spore-forming, Gram-negative intracellular parasite that grows well in room temperature, slightly acidic environments and can survive for a long time in contaminated dairy products, meat, soil, water and feces. It can be divided into 12 species based on host preference, namely: Brucella malta, Brucella abortus, Brucella suis, Brucella ovis epididymis, Brucella sarinus, Brucella canis, Brucella cetacea, Brucella fin, Brucella vole, Brucella divaricata, Brucella baboon and Brucella red fox. Among them, Brucella malta, Brucella abortus and Brucella suis pose the most serious threat to humans and animal husbandry.

[0003] Brucellosis, caused by the bacterium Brucella, is a serious zoonotic disease that currently requires compulsory vaccination. It causes significant losses and poses a significant threat to both human health and the healthy development of the livestock industry.

[0004] At present, the prevention and control of brucellosis mainly relies on vaccine immunization. The existing brucellosis vaccines on the market are all attenuated vaccines. However, this type of attenuated vaccine has a certain risk of infection for humans and animals, and there is a safety hazard of causing miscarriage in humans and pregnant animals.

[0005] In contrast, subunit vaccines can elicit both humoral and cellular immunity and are highly safe. Multi-epitope subunit vaccines, on the other hand, can leverage dominant protein epitopes to design subunit vaccines that target both humoral and cellular immunity, thereby expanding the vaccine's protective efficacy and improving safety and stability. To this end, the present invention proposes the construction of a fusion recombinant protein, ABT, for preventing brucellosis and its use in the preparation of a protective vaccine. Summary of the Invention

[0006] The purpose of the present invention is to provide a construction of a fusion recombinant protein ABT for preventing brucellosis and its application in preparing a protective vaccine, aiming to solve the problems raised in the above background technology.

[0007] The purpose of the present invention is achieved through the following technical solutions: A fusion recombinant protein ABT for preventing brucellosis, wherein 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 comprises Brucella ribosomal L7 / L12 protein and PADRE sequence, connected by an EAAAK linker; the multi-epitope tandem antigen is composed of a dominant B cell epitope portion derived from Brucella SurA, OMP31, BP26, and Trigger factor protein, and a dominant Tc cell epitope and a Th cell epitope portion in series, connected by a KK linker; the immune-enhancing antigen and the multi-epitope tandem antigen are connected in series via a KK linker to form the fusion recombinant protein ABT; The immune enhancing antigen is named A; The dominant B cell epitope portion is named B; The dominant Tc cell epitope and Th cell epitope portion are named T.

[0008] A method for constructing the above-mentioned fusion recombinant protein ABT for preventing brucellosis comprises the following steps: The coding DNA of KK linker was used to connect the B cell dominant epitopes of four proteins derived from Brucella SurA, OMP31, BP26, and Trigger factor with the coding DNA of T cell dominant epitopes containing Tc cells and Th cells to construct: B-gene, the nucleotide sequence is shown as 427-1107 bp of SEQ ID NO. 2, and the corresponding amino acid sequence is shown as 143-369 aa of SEQ ID NO. 1; T-gene, the nucleotide sequence is shown as 1108-1572 bp of SEQ ID NO. 2, and the corresponding amino acid sequence is shown as 370-524aa of SEQ ID NO. 1; The coding DNA for the ribosomal protein L7 / L12 from Brucella and the coding DNA for the PADRE sequence were linked using the EAAAK linker to construct an A-gene. The nucleotide sequence is shown in 1-426 bp of SEQ ID NO. 2, and the corresponding amino acid sequence is shown in 1-142 aa of SEQ ID NO. 1. Using Overlap PCR technology, the ordered concatenation of gene fragments is achieved by designing primers: the upstream primer of A-gene contains NcoI restriction endonuclease site, as 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 were connected in series to construct a gene containing Nco I. Xho I restriction endonuclease site ABT-gene.

[0009] A recombinant expression system of the above-mentioned fusion recombinant protein ABT for preventing brucellosis, wherein the recombinant expression system is an Escherichia coli prokaryotic expression system.

[0010] A recombinant expression plasmid expressing the above-mentioned fusion recombinant protein ABT for preventing brucellosis, wherein the basic vector of the recombinant expression plasmid is pET-28a, and the recombinant expression plasmid is expressed by Nco I. Xho After double digestion of pET-28a with restriction endonuclease I, DNA was recovered and seamless cloning primers were designed to fuse the recombinant protein ABT encoding DNA. The upstream primer contained the Nco I restriction endonuclease site partially overlapping sequence, as shown in SEQ ID NO.9, the downstream primer contains pET-28a containing Xho I restriction endonuclease site, as shown in SEQ ID NO.10; through seamless cloning reaction procedures, the recombinant expression plasmid pET28a-ABT of the fusion recombinant protein ABT was constructed.

[0011] A method for inducing expression of the above-mentioned fusion recombinant protein ABT for preventing brucellosis comprises the following steps: The recombinant expression plasmid pET28a-ABT was transformed into Escherichia coli E. coli In Rosetta (DE3) competent cells, E. coli genetically engineered expression bacteria Ec-RD-pET28a-ABT capable of expressing the fusion recombinant protein ABT were obtained. After induction of expression with IPTG, the recombinant protein in the supernatant was purified to obtain the fusion recombinant protein ABT.

[0012] A use of the above-mentioned fusion recombinant protein ABT for preventing brucellosis or the fusion recombinant protein ABT obtained by the above-mentioned induced expression method of the fusion recombinant protein ABT in preparing an ABT Brucella multi-epitope subunit vaccine.

[0013] The invention discloses an ABT Brucella multi-epitope subunit vaccine, which comprises an immune adjuvant and the above-mentioned fusion recombinant protein ABT for preventing brucellosis or the fusion recombinant protein ABT obtained by the induced expression method of the above-mentioned fusion recombinant protein ABT.

[0014] Furthermore, the immune adjuvant and the fusion recombinant protein ABT are mixed in a volume ratio of 1:1, and the immune adjuvant is an aluminum hydroxide sol adjuvant (aluminum ion concentration: 1 mg / mL).

[0015] A use of the above-mentioned fusion recombinant protein ABT or ABT Brucella multi-epitope subunit vaccine for preventing brucellosis in the preparation of a protective vaccine for preventing brucellosis.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The present invention selects four highly conserved proteins, SurA, OMP31, BP26, and Trigger factor, from Brucella malta, Brucella abortus, and Brucella suis, which are the most serious threats to humans and the livestock industry. Multiple software programs are used to predict dominant epitopes, and the final dominant epitopes are screened using multiple software programs. The N-terminus of the fusion recombinant protein contains the Brucella ribosomal L7 / L12 protein and the PADRE sequence, which enhances the immunogenicity and protective properties of the fusion recombinant protein ABT for preventing brucellosis. The ABT Brucella multi-epitope subunit vaccine prepared from this protein can stimulate BALB / c mice to produce a large amount of protective antibodies. At the same time, the codons of the DNA encoding the fusion recombinant protein ABT are optimized based on the codon preference of Escherichia coli, significantly increasing the expression level of the fusion recombinant protein ABT in the Escherichia coli expression system and achieving soluble expression, ensuring the production of sufficient 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 the advantages of high antigen expression, long antibody maintenance time, high purity, good safety, strong immunogenicity, and strong protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the spatial display of the tertiary structure of the fusion recombinant protein ABT: Mesh: candidate Tc cell and Th cell epitopes; Spheres: immune enhancement antigens; Sticks: candidate B cell epitopes.

[0018] Figure 2 The Ramachandran plot results show the distribution of amino acid residues in the fully allowed, allowed, maximum allowed, and unallowed regions.

[0019] Figure 3 This is a diagram showing the PCR amplification results of the fusion recombinant protein ABT encoding gene obtained by connecting A-gene, B-gene, and T-gene by PCR and Overlap PCR in step (2) of Example 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.

[0020] Figure 4 This is the plasmid map of the recombinant expression plasmid pET28a-ABT.

[0021] Figure 5 The results of bacterial liquid PCR identification are shown below. Lane M is DL 2000 DNA Marker; Lanes 1-4 are the coding genes of the fusion recombinant protein ABT; Lane 5 is the empty control bacteria.

[0022] Figure 6 The results of SDS-PAGE electrophoresis of the induced expression of Ec-RD-pET28a-ABT are shown in Figure 1. Lane M is the protein marker; Lane 1 is the empty vector containing only pET-28a. E. coli Rosetta (DE3) control bacteria induced expression after the collection of precipitation; Lane 2 contains only pET-28a empty vector E. coli The supernatant was collected after induction of expression with the Rosetta (DE3) control bacteria; lane 3 is the precipitate collected after induction with Ec-RD-pET28a-ABT; lane 4 is the precipitate collected without induction with Ec-RD-pET28a-ABT; lane 5 is the supernatant collected after induction with Ec-RD-pET28a-ABT; lane 6 is the supernatant collected without induction with vEc-RD-pET28a-ABT.

[0023] Figure 7 This is a graph showing the results of SDS-PAGE electrophoresis of the purified fusion recombinant protein ABT in step (2) of Example 3 of the present invention; wherein: lane M is a protein marker; lane 1 is the purified fusion recombinant protein ABT induced by the genetically engineered expression bacteria Ec-RD-pET28a-ABT.

[0024] Figure 8The immunogenicity of the fusion recombinant protein was analyzed in the immunogenicity experiment ① in step (2) of Example 4 of the present invention. The serum antibody titer at week 1 after the first immunization with the fusion recombinant protein was as follows: *P<0.05, **P<0.01, ***P<0.001. The Mann-Whitney test was used for statistical significance analysis.

[0025] Figure 9 This is the immunogenicity experiment ① in step (2) of Example 4 of the present invention to analyze the immunogenicity of the fusion recombinant protein, and the antibody growth and decline pattern after the second immunization of the fusion recombinant protein.

[0026] Figure 10 This is the binding analysis of ② immune serum and Brucella vaccine strain S2 in step (2) of Example 4 of the present invention, the antibody titer of the immune serum binding to Brucella vaccine strain S2, where *P<0.05, **P<0.01, ***P<0.001, and the statistical significance analysis was performed using the Mann-Whitney test.

[0027] Figure 11 This is the Dot Blot result of the binding between the immune serum and the Brucella vaccine strain S2 in step (2) of Example 4 of the present invention.

[0028] Figure 12 This is the antibacterial rate of the immune serum against Brucella vaccine strain S2 in the antibacterial analysis of the immune serum in step (2) of Example 4 of the present invention.

[0029] Figure 13 These are the bacterial load results of the spleens of mice in the PBS group and the ABT group in the mouse spleen bacterial load experiment in step (2) of Example 4 of the present invention, where *P<0.05, **P<0.01, ***P<0.001. T-test was used for statistical significance analysis. DETAILED DESCRIPTION

[0030] In order to have a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention is now described in detail below, but it should not be understood as limiting the scope of implementation of the present invention.

[0031] The specific implementation of the present invention is described in detail below with reference to specific embodiments.

[0032] Example 1: Design of fusion recombinant protein ABT based on immunoinformatics; (1) Determination of the amino acid sequences of four Brucella proteins: SurA, OMP31, BP26, and Trigger factor; The amino acid sequences of four proteins, surA (GenBank: XMD05064), OMP31 (GenBank: ACS50328), BP26 (GenBank: AAO39771), and TriggerFactor (GenBank: AIJ68576), collected from the NCBI (https: / / www.ncbi.nlm.nih.gov / guide / proteins / ) database, in Brucella malta, Brucella abortus, and Brucella suis were selected as conservation templates for analysis. All protein data were collected in FASTA file format.

[0033] (2) Cell epitope screening; MHC-I epitopes (i.e., T cell epitopes) were predicted for the four proteins SurA, OMP31, BP26, and Triggerfactor using IEDB (https: / / www.iedb.org / ), Rankpep (http: / / imed.med.ucm.es / Tools / rankpep.html), and Syfpeithi (http: / / www.syfpeithi.de / ). MHC-II epitopes (i.e., Th cell epitopes) were predicted for the four proteins SurA, OMP31, BP26, and Triggerfactor using IEDB, Rankpep, and NetMHCII (https: / / services.healthtech.dtu.dk / services / NetMHCIIpan-4.0 / ). Linear B cell epitopes were predicted for the four proteins SurA, OMP31, BP26, and Triggerfactor using IEDB and SVMTriP (http: / / sysbio.unl.edu / SVMTriP / ). DeepLBCEPred (http: / / www.biolscience.cn / DeepLBCEPred / ) was then used to score the predicted linear B cell epitopes. Epitopes with scores greater than 0.5 were screened as candidate epitopes. 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) were used to analyze the instability index, hydrophilicity index, antigenic index, toxicity, and allergenicity of the candidate epitopes, and the final dominant linear B cell epitopes were selected.

[0034] For Tc cell and Th cell epitopes, the top ten scoring epitopes predicted by the three software were selected. If the top ten scoring epitopes predicted by the three software appeared twice, they were screened as candidate epitopes. Expasy-ProtParam, vaxijen v2.0, ToxinPred2, and AllerTOPv.2.0 were used to analyze the candidate epitopes for instability index, hydrophilicity index, antigenic index, toxicity, and allergenicity, and the final dominant Tc cell and Th cell epitopes were selected. The final dominant linear B cell epitopes are shown in Table 1, as shown in SEQ ID NOs. 11-17; the final dominant Tc cell epitopes are shown in Table 2, as shown in SEQ ID NOs. 18-22; the final dominant Th cell epitopes are shown in Table 3, as shown in SEQ ID NOs. 23-28.

[0035] Table 1 Predominant linear B cell epitopes

[0036] Table 2 Predominant Tc cell epitopes

[0037] Table 3 Dominant Th cell epitopes

[0038] (3) Construction of fusion recombinant protein ABT; Based on the dominant epitopes identified through the aforementioned immunoinformatics screening, the present invention uses a KK linker to sequentially link the B cell epitopes, Tc cell epitopes, and Th cell epitopes. The dual lysine (KK) linker maintains the independent immunogenic activity of each epitope. To enhance the immunogenicity and protective properties of the fusion recombinant protein ABT, the Brucella ribosomal L7 / L12 protein was added to the N-terminus of the fusion protein, followed by a PADRE sequence. These two amino acid sequences were connected using an EAAAK linker to form the immune-enhancing antigen. The PADRE sequence was followed by a KK linker to link the final selected Brucella dominant epitopes 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 gene encoding the fusion recombinant protein ABT is shown in SEQ ID NO. 2.

[0039] SEQ ID NO.1: 。

[0040] SEQ ID NO.2:

[0041] (4) Analysis of the physical and chemical properties of the fusion recombinant protein ABT; The fusion recombinant protein ABT is 524 amino acids long, has a molecular weight of 57.09492 kDa, and a pI of 9.25. Its predicted instability index is 17.82 at a threshold of 40, classifying it as a stable antigen. Vaxijen v2.0 predicts a protective antigen value of 0.8673 at a threshold of 0.4, indicating compatibility as an antigen. The predicted protein solubility is 0.776, indicating that the protein is soluble.

[0042] (5) Prediction of the secondary structure of the fusion recombinant protein ABT; The secondary structure prediction results of the fusion recombinant protein ABT based on PSIPRED (https: / / bioinf.cs.ucl.ac.uk / psipred / ) showed that the protein was composed of 43.51% helices, 13.55% folded sheets and 42.94% coils.

[0043] (6) Modeling and verification of the tertiary structure of the fusion recombinant protein ABT; The modeling was performed using the Robetta (https: / / robetta.bakerlab.org / ) online server. The amino acid sequence of the fusion recombinant protein ABT was input into the online server. After the modeling was completed, the file was opened with PyMOL software to obtain the tertiary structure of the fusion recombinant protein ABT, as shown in the following figure: Figure 1 The tertiary structure of the fusion recombinant protein ABT was analyzed by Ramachandran plot using PyMod 3.0. Figure 2 The results show that the proportion of amino acid residues in the tertiary structure of the fusion recombinant protein ABT falling in the fully allowed, allowed, and maximum allowed regions is 99.3%, which is greater than 90%, indicating that the tertiary structure of the fusion recombinant protein ABT predicted by Robetta conforms to the stereochemical rules.

[0044] Example 2: Construction of an engineered bacterium expressing the gene encoding the fusion recombinant protein ABT; (1) Based on the results of cell epitope screening, the coding DNA nucleotide sequence of the selected dominant epitope protein fragments was connected using the coding DNA nucleotide sequence of the KK linker, and two multi-antigen epitope tandem protein coding DNAs were successfully constructed, namely B-gene (nucleotide sequence as shown in 427-1107 bp of SEQ ID NO.2, corresponding amino acid sequence as shown in 143-369 aa of SEQ ID NO.1) and T-gene (nucleotide sequence as shown in 1108-1572 bp of SEQ ID NO.2, corresponding amino acid sequence as shown in 370-524 aa of SEQ ID NO.1). Using the coding DNA nucleotide sequence of the EAAAK linker, the coding DNA of the Brucella ribosomal L7 / L12 protein and the PADRE sequence was connected, and the coding DNA of the immune enhancement antigen, namely A-gene (nucleotide sequence as shown in 1-426 bp of SEQ ID NO.2, corresponding amino acid sequence as shown in 1-142 aa of SEQ ID NO.1) was successfully constructed. The DNA encoding the three antigenic proteins was directly synthesized chemically after being codon-optimized in E. coli according to the codon usage preference of the E. coli expression system. DNA synthesis was performed by Shanghai Sangon Biotechnology Co., Ltd.

[0045] (2) Using the Overlap PCR method, A-gene, B-gene and T-gene were connected in sequence to construct Nco I and Xho I enzyme cutting site sticky end of ABT-gene. The specific experimental steps are as follows: 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 template and A-gene-F and A-gene-R in Table 4 as primers, the PCR amplification system and conditions in Table 5 were used to amplify the gene containing Nco I enzyme cutting site sticky end of A-gene'. Using B-gene as template, B-gene-F and B-gene-R in Table 4 as primers, according to the PCR amplification system and conditions in Table 5, amplify B-gene' containing part of A-gene. Then using A-gene' and B-gene' as templates, A-gene-F and B-gene-R in Table 4 as primers, according to the OverlapPCR amplification system and conditions in Table 6, amplify Nco I restriction enzyme cutting site sticky end AB-gene.

[0046] Using T-gene as template and T-gene-F and T-gene-R in Table 7 as primers, amplify according to the PCR amplification system and conditions in Table 5 to amplify the fragments containing part of AB-gene and part of Xho I restriction enzyme cutting site sticky end T-gene'. Then use AB-gene and T-gene' as templates, A-gene-F in Table 4 and T-gene-R in Table 7 as primers, and amplify according to the Overlap PCR amplification system and conditions in Table 6 to amplify the Nco I and Xho I enzyme cutting site sticky end ABT-gene. PCR products were subjected to 1% agarose gel electrophoresis. The results are as follows Figure 3 As shown, a clear bright band with the expected size appeared in lane 1 at a size greater than 1000 bp, and gel recovery was subsequently performed.

[0047] Table 4 All primer information

[0048] Table 5 PCR amplification system and conditions

[0049] Note: The PCR reaction program was 95°C for 5 min; 35 cycles (95°C for 30 s; 60°C for 30 s; 72°C for 1 min); 72°C for 10 min; and storage at 4°C.

[0050] Table 6 Overlap PCR amplification system and conditions

[0051] Note: 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; and storage at 4℃.

[0052] Table 7 All primer information

[0053] (3) Using the seamless cloning method, ABT-gene and pET-28a vector were constructed to form pET28a-ABT. The specific steps are as follows: SnapGene designed seamless cloning primers for ABT-gene. Using ABT-gene as template, ABT-gene-F in Table 8 (containing pET-28a containing Nco I restriction endonuclease site), ABT-gene-R (containing pET-28a containing XhoI restriction endonuclease site) as primers, and amplified according to the PCR amplification system and conditions in Table 9 to amplify ABT-gene' suitable for seamless cloning. Nco I and Xho I enzyme double enzyme digestion of pET-28a, double enzyme digestion system according to Table 10. After enzyme digestion, use DNA gel recovery kit to recover target DNA fragments, obtain target DNA fragments with different enzyme cutting sites and sticky ends, suitable for seamless cloning. Nco I and Xho The pET-28a fragment after double digestion with I enzyme was seamlessly cloned according to the system and conditions in Table 11 to obtain the pET28a-ABT ligation product, the map of which is shown in FIG. Figure 4 The pET28a-ABT ligation product was transformed into Escherichia coli E. coli In Rosetta (DE3), PCR was used to identify whether the recombinant expression plasmid was successfully transformed into E. coli. E. coli In Rosetta (DE3), the PCR amplification results of the coding DNA of the fusion recombinant protein ABT are as follows: Figure 5 As shown, bacteria with a clear and bright single band greater than 1000bp were screened as positive clones and sent to a biological company for sequencing. The plasmid with the correct sequencing result was the prokaryotic expression plasmid pET28a-ABT for expressing the fusion recombinant protein ABT. E. coli The Rosetta (DE3) expression strain was named Ec-RD-pET28a-ABT.

[0054] Table 8 All primer information

[0055] Table 9 PCR amplification system and conditions

[0056] Note: The PCR reaction program was 95°C for 5 min; 35 cycles (95°C for 30 s; 65°C for 30 s; 72°C for 1 min); 72°C for 10 min; and storage at 4°C.

[0057] Table 10 pET-28a vector double enzyme digestion system

[0058] Table 11 Seamless cloning system and conditions

[0059] Note: The seamless cloning reaction procedure is 50°C, 15 min.

[0060] Example 3: Expression and purification of fusion recombinant protein ABT; (1) The expression engineered strain Ec-RD-pET28a-ABT was inoculated into 5 mL of liquid LB medium containing 100 μg / mL kanamycin and cultured at 37°C and 180 rpm for 4 h. IPTG was then added to a final concentration of 0.5 mmol / L and cultured at 16°C and 160 rpm for 20 h. The induced expression strain was ultrasonically disrupted, and the sonicated bacterial solution was centrifuged at 8000 rpm and 4°C for 10 min. The supernatant and precipitate were collected. The expression level and solubility of the target protein ABT in the genetically engineered expression strain were detected by SDS-PAGE electrophoresis.

[0061] The results of SDS-PAGE electrophoresis of the expression engineering bacteria Ec-RD-pET28a-ABT are as follows Figure 6 As shown, lanes 3 and 5 have obvious specific bands compared with lanes 4 and 6; at the same time, by comparing lanes 1 and 2, differential bands can also be seen, indicating that the fusion recombinant protein ABT can be successfully induced to express and is expressed in both the supernatant and the precipitate.

[0062] (2) Purification of the fusion recombinant protein ABT; After the expression of the engineered bacteria Ec-RD-pET28a-ABT was induced with IPTG, the precipitate and supernatant were collected. Since the SDS-PAGE test results showed that the fusion recombinant protein was expressed in both the supernatant and the precipitate, the supernatant was collected and the target protein was purified using the affinity chromatography medium Ni-NTA. The specific operation is as follows: ① Expand the culture and induce expression: inoculate 5 mL of Ec-RD-pET28a-ABT cultured overnight at 37°C into 1 L of sterilized LB medium, add 100 μg / mL kanamycin, and culture at 37°C, 180 rpm, and shake for 4 h. Then add IPTG to a final concentration of 0.5 mmol / L, and induce at 16°C, 160 rpm, and shake for 20 h.

[0063] ② Washing the cells and collecting the supernatant: After induction, centrifuge the bacterial suspension at 8000 rpm and 4°C for 15 minutes to collect the bacterial pellet. Resuspend the collected cells in Buffer A (Tris 6.058 g, glycerol 100 mL, NaCl 29.25 g, imidazole 0.68 g, dilute to 1 L, pH 7.2-7.4) and disrupt the cells by ultrasonication. Centrifuge the sonicated bacterial suspension at 8000 rpm and 4°C for 50 minutes, and collect the supernatant.

[0064] ③ Protein nickel column binding and elution: Use elution buffer B (Tris 6.058g, glycerol 100mL, NaCl 29.25g, imidazole 34.04g to 1L, adjust pH 7.2-7.4) to wash the column to remove impurities; after the collected supernatant is fully combined with the affinity chromatography medium Ni-NTA; use elution buffer containing 40mM imidazole (prepared by mixing 28.164mL Buffer A with 1.836mL Buffer B), elution buffer containing 60mM imidazole (prepared by mixing 26.94mL Buffer A with 3.06mL Buffer B), elution buffer containing 80mM imidazole (prepared by mixing 25.713mL Buffer A with 4.287mL Buffer B), elution buffer containing 150mM imidazole (prepared by mixing 21.429mL Buffer A with 8.571mL Buffer B), and elution buffer containing 150mM imidazole. The target protein was eluted with elution buffer containing 200 mM imidazole (prepared by mixing 18.366 mL of Buffer A and 11.634 mL of Buffer B), elution buffer containing 220 mM imidazole (prepared by mixing 17.142 mL of Buffer A and 12.858 mL of Buffer B), elution buffer containing 250 mM imidazole (prepared by mixing 15.306 mL of Buffer A and 14.694 mL of Buffer B), elution buffer containing 300 mM imidazole (prepared by mixing 12.246 mL of Buffer A and 17.754 mL of Buffer B), and Buffer B. The protein eluates eluted with different imidazole concentrations were collected.

[0065] ④ Protein dialysis and concentration: The protein eluate eluted with elution buffer containing 80-200mM imidazole was loaded into a dialysis bag with a molecular weight cutoff of 20,000 Da1 to remove the imidazole in the protein eluate. The dialysis bag containing the protein eluate was immersed in PBS for 3-4 times, and new PBS was replaced every 3 hours for dialysis. After the dialysis, it was concentrated with PEG20000 for 3 hours. The concentrated liquid obtained was the purified target protein ABT. The BCA protein concentration determination kit of Biyuntian was used to determine the concentration of the purified protein. After determination, the concentration of the purified fusion recombinant protein ABT was 1.007 mg / mL. The results of SDS-PAGE electrophoresis detection of the purified fusion recombinant protein ABT are as follows: Figure 7 As shown, after purification, the fusion recombinant protein ABT has a target protein band at the expected size.

[0066] Example 4: Preparation of ABT Brucella multi-epitope subunit vaccine and immune protection analysis experiment; (1) Preparation of subunit vaccines; The fusion recombinant protein ABT purified 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 ABT Brucella multi-epitope subunit vaccine.

[0067] (2) Experiments analyzing the immune effects of subunit vaccines; ① Immunogenicity analysis; Seven-week-old specific pathogen-free (SPF) BALB / c mice were selected and divided into two groups, with 6 mice in each group. One group was the immunization group, which was immunized with the ABT Brucella multi-epitope subunit vaccine prepared with the fusion recombinant protein ABT; the other group was the PBS control group, which was injected with the same volume of PBS, in which the adjuvant was aluminum hydroxide sol. The immunization program was divided into two immunizations. The mice were immunized for the second time 14 days after the first immunization, and the immunization method was intraperitoneal injection. In the first week after the first immunization, the serum samples of the immunized mice were collected by tail vein blood sampling, and the antibody titer was detected by indirect ELISA method. The results are as follows Figure 8 Serum samples of the immunized mice were collected by tail vein blood collection at 1, 2, 6, 8, 10, 14, and 18 weeks after the second immunization for the detection of changes in antibody titer levels to monitor whether the vaccine can provide long-term protection for the mice. The antibody titer in the serum was detected by indirect ELISA method. The results are shown in the figure. Figure 9 As shown. Figure 8 It can be seen that the antibody titer of mice in the ABT immunization group was significantly higher than that in the PBS control group, indicating that the ABT Brucella multi-epitope subunit vaccine prepared by fusion recombinant protein ABT can significantly stimulate the body to produce antibodies. Figure 9 As can be seen, the antibody titer in the ABT immunization group not only remained at a high level but also persisted for a long time in the body, while the antibody titer in the PBS group remained low. These results indicate that the prepared fusion recombinant protein ABT has good immunogenicity and that the vaccine composed of it and adjuvant can stimulate the body to produce antibodies with strong binding ability and long-lasting effect.

[0068] ②Analysis of the binding between immune serum and Brucella vaccine strain S2; The immune serum collected eight weeks after the second immunization was selected, and the binding effect between the immune serum and Brucella vaccine strain S2 (produced by Harbin Pharmaceutical Group Biological Vaccine Co., Ltd.) was verified by indirect ELISA and Dot Blot methods.

[0069] The specific steps of indirect ELISA are as follows: coat the enzyme-labeled plate with Brucella vaccine strain S2, 100uL / well, overnight at 4℃, wash three times with PBST, block with 1% BSA blocking solution at 37℃ for 2h, 300uL / well, wash three times with PBST, add 1:300 diluted immune serum sample, 100uL / well, incubate at 37℃ for 1h, wash three times with PBST, add 1:10000 diluted HRP-labeled goat anti-mouse secondary antibody, 100uL / well, incubate at 37℃ for 1h, wash three times with PBST, add TMB colorimetric solution, 100uL / well, react at room temperature in the dark for 20min, add 2M H2SO4, 50uL / well to terminate the reaction, and read the OD with a microplate reader. 450 , calculate the specific antibody titer of immune serum against Brucella vaccine strain S2, the results are as follows Figure 10 As shown in the figure, the antibody titer of the ABT group (immunized with ABT Brucella multi-epitope subunit vaccine group) was significantly higher than that of the PBS control group, indicating that after immunization with ABT Brucella multi-epitope subunit vaccine, the antibodies in the immune serum produced by mice can specifically bind to Brucella vaccine strain S2.

[0070] The Dot Blot experimental steps are as follows: use a non-contact ultrasonic disruptor to ultrasonically disrupt Brucella vaccine strain S2, centrifuge at 8000 rpm and 4°C for 15 minutes, collect supernatant protein, activate the PVDF membrane with methanol for 5 seconds, spot the supernatant protein on the PVDF membrane, 1-2uL per spot, spot 6 spots and let dry, immerse the membrane in 1% BSA blocking solution, block at 37°C for 2 hours, wash the membrane with TBST 3 times, 5 minutes each time, dilute the immune serum with TBST at 1:1000, immerse the membrane in the diluted immune serum, incubate at 37°C for 1 hour, wash the membrane with TBST 3 times, 5 minutes each time, dilute HRP-labeled goat anti-mouse secondary antibody with TBST at 1:10000, immerse the membrane in the diluted secondary antibody solution, incubate at 37°C for 1 hour, wash the membrane with TBST 3 times, 5 minutes each time, evenly drop ECL chemiluminescent substrate on the membrane, and detect the signal with a chemiluminescent imager. The results are as follows Figure 11 As shown in the figure, after incubation with ECL chemiluminescent substrate and exposure, obvious black specific signal spots were detected at the sample location, further proving that after immunization with ABT Brucella multi-epitope subunit vaccine, the antibodies in the immune serum produced by mice can specifically bind to Brucella vaccine strain S2.

[0071] ③ Antibacterial analysis of immune serum; The serum antibacterial experiment was conducted on immune serum collected two weeks after the second immunization with the ABT Brucella multi-epitope subunit vaccine to explore the antibacterial effect of the immune serum on Brucella vaccine strain S2. The specific steps were as follows: the positive control group selected serum from Brucella-positive infected cattle, and the negative control group selected serum from unimmunized mice. The serum was diluted at 1:20, 1:40, 1:80, 1:160, 1:320, and 1:640 dilutions. The mouse serum of different dilutions was added to PCR tubes, 20uL per tube, and treated at 56°C for 30 minutes to inactivate the complement in the serum. Then, Brucella vaccine strain S2 bacterial solution (1×10 4 CFU / mL), 20uL per tube, incubated at 37℃ for 1h; then guinea pig serum (complement activity 100U / mL) was added, 10uL per tube, incubated at 37℃ for 1h; the reaction solution in each tube was diluted 1:2 and spread on TSA plates, incubated at 37℃ for 72h, the number of colonies in each group was counted, and the antibacterial rate of each group of serum was calculated using the formula (1-number of colonies / number of colonies in the negative control group) × 100%. The results are shown in the figure. Figure 12 As shown in the figure, both the positive control group and the ABT group (immunized with the ABT Brucella multi-epitope subunit vaccine) exhibited a certain degree of bacteriostasis. Furthermore, the bacteriostasis rate of the ABT group decreased with increasing serum dilution, but remained significantly higher than that of the negative control group overall. This indicates that the immune sera produced by mice immunized with the ABT Brucella multi-epitope subunit vaccine exhibited a significant antibacterial effect against the Brucella vaccine strain S2.

[0072] ④ Determination of bacterial load in mouse spleen; Seven-week-old specific pathogen-free (SPF) BALB / c mice were selected and divided into two groups, with six mice in each group. One group was the immunization group, which was immunized with the ABT Brucella multi-epitope subunit vaccine prepared with the fusion recombinant protein ABT; the other group was the PBS control group, which was injected with the same volume of PBS, in which the adjuvant was aluminum hydroxide sol. The immunization procedure was divided into two immunizations. The mice were immunized for the second time 14 days after the first immunization, and the immunization method was intraperitoneal injection. 14 days after the second immunization, the two groups of mice were challenged with Brucella vaccine strain A19 (produced by Harbin Pharmaceutical Group Bio-Vaccine Co., Ltd.) (1×10 7 CFU / mouse). On the 14th day after challenge, mice were killed by cervical dislocation. The spleens of the two groups of mice were collected and weighed. The spleen tissue was ground and then homogenized in a 10-fold gradient dilution. The homogenate was then spread on TSA plates. To reduce error, the operation was repeated once for each spleen homogenate. The number of colonies was counted and the bacterial load per gram of spleen in the PBS and ABT groups was calculated. The results are shown in the figure. Figure 13 As shown in the figure, the bacterial load in the spleen of mice immunized with ABT Brucella multi-epitope subunit vaccine was significantly lower than that in the PBS group.

[0073] Conclusion: The recombinant protein ABT designed based on immunoinformatics in the present invention has good immunogenicity. The ABT Brucella multi-epitope subunit vaccine composed of it and adjuvant can significantly stimulate the body to produce antibodies with strong binding ability. After two immunizations, the high-efficiency antibody level can persist in the body. The serum produced after immunization can bind to the Brucella vaccine strain S2 and has a significant antibacterial effect. After two immunizations, the ABT Brucella multi-epitope subunit vaccine showed obvious protective effect in the face of the Brucella vaccine strain A19.

[0074] The above are only preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, several variations and improvements can be made without departing from the concept of the present invention. These should also be regarded as the scope of protection of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent.

Claims

1. A fusion recombinant protein ABT for preventing 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 comprises Brucella ribosomal L7 / L12 protein and PADRE sequence, connected by an EAAAK linker; the multi-epitope tandem antigen is composed of a dominant B cell epitope portion derived from Brucella SurA, OMP31, BP26, and Trigger factor protein, and a dominant Tc cell epitope and a Th cell epitope portion in series, connected by a KK linker; the immune-enhancing antigen and the multi-epitope tandem antigen are connected in series via a KK linker to form the fusion recombinant protein ABT; The immune enhancing antigen is named A; The dominant B cell epitope portion is named B; The dominant Tc cell epitope and Th cell epitope portion are named T.

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

1. Using Overlap PCR technology, the ordered concatenation of gene fragments is achieved by designing primers: the upstream primer of A-gene contains Nco I restriction endonuclease site, as 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 were connected in series to construct a gene containing Nco I. Xho I restriction endonuclease site ABT-gene.

3. A recombinant expression system of the fusion recombinant protein ABT for preventing 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 preventing brucellosis according to claim 1, characterized in that: The basic vector of the recombinant expression plasmid is pET-28a. Nco I. Xho After double digestion of pET-28a with restriction endonuclease I, DNA was recovered and seamless cloning primers were designed to fuse the recombinant protein ABT encoding DNA. The upstream primer contained the Nco I restriction endonuclease site partially overlapping sequence, as shown in SEQ ID NO.9, the downstream primer contains pET-28a containing Xho I restriction endonuclease site, as shown in SEQ ID NO.10; through seamless cloning reaction procedures, the recombinant expression plasmid pET28a-ABT of the fusion recombinant protein ABT was constructed.

5. A method for inducing expression of a fusion recombinant protein ABT for preventing brucellosis according to claim 1, characterized in that: The following steps are involved: The recombinant expression plasmid pET28a-ABT was transformed into Escherichia coli E. coli In Rosetta (DE3) competent cells, E. coli genetically engineered expression bacteria Ec-RD-pET28a-ABT capable of expressing the fusion recombinant protein ABT were obtained. After induction of expression with IPTG, the recombinant protein in the supernatant was purified to obtain the fusion recombinant protein ABT.

6. Use of the fusion recombinant protein ABT for preventing brucellosis according to claim 1 or the fusion recombinant protein ABT obtained by the inducible expression method of the fusion recombinant protein ABT according to claim 5 in preparing an 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 preventing 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.

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

9. Use of the fusion recombinant protein ABT for preventing brucellosis according to claim 1 or the ABT Brucella multi-epitope subunit vaccine according to claim 7 in preparing a protective vaccine for preventing brucellosis.

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