A bovine group a rotavirus polyepitope fusion protein and application thereof

Abstract

The present application belongs to the technical field of genetic engineering, and particularly relates to a bovine rotavirus group A multi-epitope fusion protein and application thereof. The present application provides a bovine rotavirus group A multi-epitope peptide, fusion protein based on ferritin nanocarriers and application thereof. The multi-epitope peptide is based on bovine rotavirus group A VP4 and VP7 protein sequences, and highly conserved and strong immunogenicity cytotoxic T lymphocyte epitopes, helper T cell epitopes and B cell epitopes are screened by using immunoinformatics technology, and are spliced to form a multi-epitope fusion antigen with good conformational stability. Immunological evaluation shows that the multi-epitope fusion antigen has good antigen specificity and neutralizing activity. Animal experiment results show that the epitope peptide and fusion protein can induce the body to produce high-level neutralizing antibodies against multiple genotypes of BRVA G6, G8 and G10, and significantly improve the broad spectrum and durability of immunoprotection.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of genetic engineering, and particularly relates to a bovine rotavirus A group polypeptide fusion protein and application thereof. BACKGROUND

[0002] Rotavirus A (RVA) belongs to the family Reoviridae and the genus Rotavirus, is a non-enveloped double-stranded RNA virus, and is one of the important pathogenic factors of diarrhea in infants and young animals around the world. According to statistics, diarrhea related to RVA causes more than 200,000 deaths in children around the world each year, accounting for about 5% of the total number of deaths in children under the age of 5. In animals, bovine rotavirus A (BRVA) is one of the main pathogens of neonatal calf diarrhea, which can cause severe dehydration, electrolyte imbalance and even death, and cause significant economic losses to the livestock industry. BRVA mainly infects small intestinal villus epithelial cells, causing clinical symptoms such as fever, vomiting, and diarrhea. In rare cases, the virus can spread to extra-intestinal tissues such as the respiratory tract, liver, kidney, and central nervous system.

[0003] More and more molecular epidemiological evidence shows that there is cross-species transmission and genetic reassortment between human and animal RVA, forming new recombinant virus strains. Dogs, cats, pigs, cattle and other domestic animals show a higher frequency of genetic recombination in cross-species transmission, which not only expands the genetic diversity of RVA, but also brings challenges to vaccine prevention and control. Therefore, the molecular epidemiological study of BRVA and the development of broad-spectrum vaccines have become an important direction for the prevention and control of new and recurring diarrhea.

[0004] The RVA genome consists of 11 segments, encoding 6 structural proteins (VP1-VP4, VP6, VP7) and 6 non-structural proteins, with a total length of about 18.5 kb. Among them, the outer shell protein VP7 and the spike protein VP4 are the key proteins that determine the virus serotype and host receptor recognition, and are also the main antigens that induce the production of neutralizing antibodies. According to the sequence differences of VP7 and VP4, RVA adopts a G / P binary typing system, and 36 G types and 51 P types have been identified. In human RVA, 12 G types and 15 P types are commonly seen, while in BRVA, genotypes such as G6, G8, and G10 are mainly included. The prevalent types detected in China are mainly G6P[1], G6P[5], G10P

[11] , G6P

[11] , and G8P[1] types.

[0005] Current RVA prevention and control strategies mainly rely on vaccine immunization. Six human oral attenuated live vaccines have been marketed globally, which significantly reduce the infection rate of RVA in infants. However, vaccines for animal RVA (especially BRVA) are still scarce. Existing veterinary vaccines are mostly multi-component inactivated vaccines or attenuated live vaccines, but they have narrow immune spectrum, poor stability and safety risks. The diversity, gene reassortment and cross-species transmission characteristics of RVA further weaken the cross-protection ability of existing vaccines.

[0006] The existing BRVA prevention and control methods also face the following problems: (1) the epidemic strains are diverse and frequently reassort, and the existing vaccines have limited cross-protection; (2) the safety and stability of traditional vaccines are insufficient, making it difficult to meet the industrialization needs; (3) the epitope analysis and verification of BRVA VP4 and VP7 are still insufficient, which limits the development of multi-epitope vaccines.

[0007] Therefore, it is of great significance to develop a broad-spectrum multi-epitope vaccine based on BRVA VP4 and VP7 key antigen epitopes to improve the prevention and control level of calf viral diarrhea and reduce the loss of the livestock industry. SUMMARY

[0008] To solve the above problems, the purpose of the present application is to provide a bovine group A rotavirus multi-epitope fusion protein and its application.

[0009] The present application provides a bovine group A rotavirus multi-epitope peptide, which comprises cytotoxic T lymphocyte, helper T cell and B cell epitopes of bovine group A rotavirus VP4 and VP7 proteins, and the epitopes are spliced by a connecting peptide; wherein,

[0010] The cytotoxic T lymphocyte epitope is selected from at least one of the amino acid sequences shown in SEQ ID NO. 1-4;

[0011] The helper T cell epitope is selected from at least one of the amino acid sequences shown in SEQ ID NO. 5-7;

[0012] The B cell epitope is selected from at least one of the amino acid sequences shown in SEQ ID NO. 8-15.

[0013] The sequence of the connecting peptide is AAY, GPGPG and / or KK.

[0014] Preferably, the cytotoxic T lymphocyte (CTL) epitopes are connected by AAY connecting peptides to form transport sites that facilitate binding to TAP transporters and promote efficient presentation of epitopes through the MHC-I pathway.

[0015] The HTL epitopes are connected by GPGPG connecting peptides, so as to maintain the conformation-dependent antigen structure and enhance the immune activation effect through the MHC-II pathway.

[0016] The B cell epitopes are connected by KK connecting peptides, so as to improve the flexibility and exposure between epitopes, thereby enhancing the humoral immune response.

[0017] Further, the immunomodulatory sequence beta-defensin-3 is further included, preferably, the beta-defensin-3 is connected at the N terminal of the polyepitope polypeptide.

[0018] The amino acid sequence of the polyepitope peptide is shown in SEQ ID NO. 16 or SEQ ID NO. 17.

[0019] The application further provides a fusion protein comprising the polyepitope peptide and ferritin connected.

[0020] The ferritin is connected at the N terminal of the polyepitope peptide and connected with the polyepitope peptide through the EAAAK sequence; preferably, the ferritin is derived from Helicobacter pylori.

[0021] The application further provides a coding sequence of the polyepitope polypeptide and the fusion protein; preferably, the sequence is shown in SEQ ID NO. 18.

[0022] The application further provides a nanoparticle assembled based on the fusion protein.

[0023] The application further provides an application of the polyepitope polypeptide, the fusion protein and the nanoparticle in preparing a bovine rotavirus A group prevention or treatment drug.

[0024] The drug is a vaccine or a diagnostic reagent.

[0025] The application utilizes the methods of immunoinformatics and structural biology to screen the cell toxicity CTL epitope, the HTL epitope and the B cell epitope with high conservation, high antigenicity, no toxicity and no sensitization risk from the BRVA VP4 and VP7 proteins. According to the immune response characteristics, the epitope connection strategy is optimized, the AAY, GPGPG and KK connecting peptides are adopted to realize the ordered splicing of different types of epitopes, the beta-defensin-3 (BD-3) immune enhancement domain is introduced at the N terminal of the polyepitope sequence, and the ferritin (Ferritin) carrier is connected through the EAAAK connecting peptide to form the polyepitope fusion antigen rBRVA-Ferritin.

[0026] The rBRVA-Ferritin fusion protein is efficiently and solubly expressed by an E. coli expression system and is purified by affinity chromatography, and shows good water solubility and structural stability. Bioinformatics analysis shows that the molecular weight of the fusion protein is about 37.4 kDa, the theoretical isoelectric point is 9.78, and the instability index is 30.51, which is less than 40, and belongs to stable protein; the aliphatic index is 73.93, and the average hydrophilicity index is -0.51, indicating that it is a stable and hydrophilic antigen molecule; the VaxiJen2.0 prediction antigenicity score is 0.7059, which is higher than the threshold value of 0.4, showing strong antigenicity.

[0027] Animal immunization results show that the rBRVA-Ferritin vaccine can induce the host to produce high-titer neutralizing antibodies, and at the same time activate cytotoxic T cells (CTL) and helper T cell (HTL) immune responses, significantly improve the cross-protection level of different genotypes of BRVA G6, G8, G10 and other strains. The present application combines the self-assembly of ferritin nanocarriers and the immunoinformatics epitope screening strategy, and provides a new technical approach and theoretical basis for developing new, safe, efficient and broad-spectrum BRVA subunit vaccines.

[0028] The specific embodiments in the form of the description, drawings and examples are further described below, but the examples do not make any form of limitation on the present application. Any changes, modifications, substitutions, combinations, simplifications made by those skilled in the art without departing from the spirit and principles of the present application shall be equivalent replacement methods, and shall be included in the protection scope of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 rBRVA sequence schematic diagram, N' end green font sequence is beta-defensin-3, connected by EAAAK and blue font CTLs, CTLs are connected by AAY, red font is HTL, connected by GPGPG, yellow font is linear B cell epitope, connected by KK, C' end is His-tag.

[0030] Figure 2 is a secondary structure schematic diagram of rBRVA.

[0031] Figure 3 is a schematic diagram of the tertiary structure of rBRVA.

[0032] Figure 4 ERRAT evaluation of rBRVA, the right Ramachandran diagram shows that 96.5% of the residues of the rBRVA model are in the reasonable area, indicating that the structure is stable.

[0033] Figure 5Results of the simulated immune response of rBRVA, a, b: B cells; c-f: T cells; g: dendritic cells; h: antibodies; i: cytokines.

[0034] Figure 6 Molecular interaction sites (red) formed between rBRVA molecules (yellow) and integrin α2 subunits (green).

[0035] Figure 7 rBRVA recombinant plasmid, the gray part is the inserted fragment, and the 5' and 3' ends are Nco I and Xho I enzyme digestion sites, respectively.

[0036] Figure 8 Different concentrations of IPTG were used to induce rBRVA expression at 16℃ for 16 h, M: protein marker; 1: 0.1 mmol; 2: 0.3 mmol; 3: 0.5 mmol; 4: 0.8 mmol; 5: 1.0 mmol.

[0037] Figure 9 rBRVA Western blot verification, M: protein marker; 1: rBRVA appeared a single band at 37 kDa; 2: no band appeared in the empty control.

[0038] Figure 10 The level of specific antibodies targeting VP4 and VP7 proteins in the serum of the immunized rabbits was significantly increased.

[0039] Figure 11 Cross-neutralization test of G6, G8 and G10 type BRVA, a~c: G6, G8 and G10 neutralization test 48 h non-disease wells; e~g: G6, G8 and G10 neutralization test 48 h disease wells; d: negative well 48 h cell morphology; h: positive well 48 h cell lesion morphology. DETAILED DESCRIPTION

[0040] The application will be further described in detail below in combination with examples, but the application is not limited thereto.

[0041] The reagents and materials used in the examples are as follows:

[0042] MA104 cells were preserved by the Animal Medicine Laboratory of Southwest University of Nationalities; E. coli BL21 (DE3) competent cells were purchased from Beijing Genbioke Technology Co., Ltd.; a plasmid extraction kit was purchased from OMEGA Bio-Company, His Cap 6FF nickel ion purification column was purchased from Changzhou Tiandihuan Bio-Technology Co., Ltd., hypersensitive ECL chemiluminescence substrate was purchased from Sibo Biological Company, HRP labeled goat anti-rabbit IgG, HRP labeled goat anti-mouse IgG were purchased from Beijing Boao Sun Biological Technology Co., Ltd., FITC labeled goat anti-mouse IgG was purchased from Beijing Boao Sun Biological Technology Co., Ltd., BCA protein concentration determination kit was purchased from Dr. De Biological Engineering Co., Ltd., isopropyl thiogalactoside (IPTG) and imidazole (Imidazole) were purchased from Sigma Company, Montanide ISA 201 adjuvant was purchased from SEPPIC Company.

[0043] Example 1 Design of epitope vaccine

[0044] 1 Experimental method

[0045] 1.1 Target protein sequence acquisition

[0046] All P[1], P[5], P

[11] type BRVA VP4 protein full sequences and G6, G8, G10 type VP7 protein full sequences were downloaded from GenBank database. The antigenicity of each protein was predicted by using VaxiJen v2.0.1 online tool, and the antigen score greater than 0.4 was considered to have antigenicity. The amino acid sequences with the highest antigenicity in P[1], P[5], P

[11] type VP4 and G6, G8, G10 type VP7 were selected as template sequences for subsequent epitope prediction and vaccine design.

[0047] 1.2 T cell epitope prediction and screening

[0048] CTL epitopes were predicted using NetMHCpan 4.0 (https: / / services.healthtech.dtu.dk / services / NetMHCpan-4.0 / ) and HTL epitopes were predicted by NetMHCIIpan 4.0 (https: / / services.healthtech.dtu.dk / services / NetMHCIIpan-4.0 / ). The top 10 epitopes with the highest scores were selected for toxicity, allergenicity and antigenicity evaluation using AllerTOP (https: / / www.ddg-pharmfac.net / AllerTOP / ), ToxinPred (http: / / crdd.osdd.net / raghava / toxinpred / ) and VaxiJen v2.0. Epitopes with no toxicity, no allergenicity and antigenicity score greater than 0.4 were reserved for subsequent vaccine design.

[0049] 1.3 Linear B cell epitope prediction and screening

[0050] Linear B cell epitope prediction was performed using the ABCPred server with a threshold of 0.51, and epitopes with scores higher than this value were considered to have binding ability. The predicted results were further screened by AllerTOP, ToxinPred and VaxiJen v2.0, and candidate epitopes with no toxicity, no allergenicity and antigenicity score greater than 0.4 were reserved for vaccine construction.

[0051] 1.4 Conservation analysis of candidate epitopes

[0052] MEGA Align was used for multiple sequence alignment of BRVA VP4 and VP7 amino acid sequences. Biopython scripts were used to analyze the conservation of each candidate epitope in all reported strains in GenBank, and epitopes with a conservation greater than 0.9 were selected for final vaccine design.

[0053] 1.5 Multi-epitope vaccine design

[0054] According to the screening of CTL, HTL and B cell epitopes, a multi-epitope fusion protein was constructed according to the immunological design principle. Specifically, a beta-defensin-3 (BD-3, 45 aa) was introduced at the N-terminus as an immune-enhancing sequence, and connected to the downstream epitopes through an EAAAK linker peptide to enhance the overall immunogenicity and maintain the sequence structure independence; AAY linker peptide was used between CTL epitopes to promote the binding of epitopes to TAP transporters and enhance CTL response; GPGPG linker peptide was used between HTL epitopes to maintain the conformational integrity required for helper T cell response; linear B cell epitopes were connected to each other through KK linker to increase antibody recognition and immunogenicity. This design strategy can achieve efficient immune recognition of multi-epitope fusion protein while maintaining the spatial independence and conformational integrity of each epitope. The final multi-epitope fusion protein is named rBRVA.

[0055] 1.6 rBRVA secondary and tertiary structure prediction

[0056] The secondary structure composition of rBRVA was predicted using the SOPMA online tool. Subsequently, the three-dimensional structure of rBRVA was simulated by trRosetta (https: / / yanglab.qd.sdu.edu.cn / trRosetta / ). The obtained model was comprehensively evaluated by ERRAT, Procheck, 3D Refine and MolProbity online platforms to confirm its structure reliability, stereochemical rationality and overall stability.

[0057] 1.7 rBRVA stability, solubility and antigenicity evaluation

[0058] The physicochemical properties of rBRVA were analyzed by ProtParam (https: / / web.expasy.org / protparam / ). When the instability index is less than 40, the protein is considered stable, and the average hydrophilicity index is negative, indicating that the protein has good hydrophilicity and strong solubility. At the same time, VaxiJen v2.0 was used to predict the antigenicity score to evaluate the potential immunogenicity of rBRVA.

[0059] 1.8 Immune simulation

[0060] The C-ImmSim Server (https: / / 150.146.2.1 / C-IMMSIM / ) was used to simulate the immune response process induced by rBRVA. The total step size was set to 1050, corresponding to time steps of 1, 63 and 126 (each step corresponding to 8 h), to simulate the long-term response of the body with three immunization processes. The dynamic changes of B cell and T cell populations and the levels of IgG, IFN-γ and other cytokines were output.

[0061] 1.9 Molecular docking

[0062] The integrin alpha 2 subunit plays a crucial role in stimulating immune responses. The protein sequence of the integrin alpha 2 subunit (PDB ID: 2VDN) was retrieved from the RCSB database, the interaction between the integrin alpha 2 subunit and rBRVA was simulated using the AlphaFold Server, and visualized using PyMOL.

[0063] 2 Result analysis

[0064] 2.1 Amino acid sequence of VP7 / VP4 protein and comparison analysis results

[0065] After predicting the BRVA VP4 and VP7 protein sequences downloaded from NCBI by VaxiJen v2.0.1, it was found that the antigenicity scores of the 6 strains selected were the highest, and the sequence numbers were MN928491.1, ACM47532.1, BAB18912.1, BCM78495.1, BCM78517.1, and QSL97064.1. The results suggest that the VP7 / VP4 proteins of these strains have strong antigenic potential, providing a basis for subsequent epitope screening.

[0066] 2.2 CTLs prediction results

[0067] Based on the analysis results of NetMHCpan 4.0, combined with toxicity, allergenicity and conservation screening, a total of 4 CTL epitopes were screened (Table 1). The antigen scores of these epitopes were all higher than 1.0, and they were non-toxic, non-allergenic, and had a conservation of more than 94%, showing good immune recognition potential.

[0068] Table 1 CTLs obtained by screening

[0069]

[0070] 2.3 HTLs epitope prediction results

[0071] After prediction by NetMHCIIpan and comprehensive evaluation, 3 preferred HTL epitopes were obtained (Table 2). The antigenicity scores of these epitopes were all greater than 0.7 and the conservation was more than 90%, which could effectively induce helper T cell response.

[0072] Table 2 HTLs obtained by screening

[0073]

[0074] 2.4 Linear B cell epitope prediction results

[0075] After ABCPred prediction and multi-index screening, 8 B cell epitopes with high antigenicity, non-toxicity and non-sensitization were screened (Table 3). The conservation of these epitopes was all above 0.85, showing good antigen recognition potential.

[0076] Table 3 B cell epitopes screened

[0077]

[0078] 2.5 Design of rBRVA

[0079] The finally constructed rBRVA contains 4 CTL, 3 HTL and 10 B cell epitopes.

[0080] In addition, 2 B cell epitopes are 87 IEASNEIADTEWKNT 101 and 208 LTTDPNTFETVATT 221 .

[0081] AAY, GPGPG and KK linkers are used to connect the epitopes, respectively to enhance epitope presentation, maintain structural stability and improve immunogenicity. The β-defensin-3 (BD-3) sequence is introduced at the N-terminus and connected by the EAAAK linker, which can further enhance the immune stimulating ability. The overall design is shown in Figure 1 The rBRVA fusion protein structure has good continuity and immunorecognition region exposure.

[0082] The rBRVA sequence after connecting each epitope is as follows (SEQ ID NO. 16):

[0083] KSGGLGYKWAAYRMMRVNWKKAAYNPMDITLYYAAYAIIDFKTLKGPGPGTGSVYFKEYADIAAGPGPGLGPRENVAVIQVGGANGPGPGMVVILAAVTNAQNYGKKIEASNEIADTEWKNTKKLTTDPNTFETVATTKKPVSIVSRNIVYTRAQPKKIQEIGSTKTQDVTVNPKKGIEYTTILIFLISITLKKLCNPMDITLYYYQQTDKKFSLISLYPSNDDYQTPKKAQEAKCTKYINNGLPPKKMSKRSRSLNSSAFYYRKKGGANLDITADPTTAP

[0084] The rBRVA sequence after connecting each epitope and introducing the BD-3 sequence is as follows (SEQ ID NO. 17):

[0085] GIINTLQKYYCRVRGGRCAVLSCLPKEEQIGKCSTRGRKCCRRKKEAAAKKSGGLGYKWAAYRMMRVNWKKAAYNPMDITLYYAAYAIIDFKTLKGPGPGTGSVYFKEYADIAAGPGPGLGPRENVAVIQVGGANGPGPGMVVILAAVTNAQNYGKKIEASNEIADTEW KNTKKLTTDPNTFETVATTKKPVSIVSRNIVYTRAQPKKIQEIGSTKTQDVTVNPKKGIEYTTILIFLISITLKKLCNPMDITLYYYQQTDKKFSLISLYPSNDDYQTPKKAQEAKCTKYINNGLPPKKMSKRSRSLNSSAFYYRKKGGANLDITADPTTAPHHHHHH*

[0086] 2.6 rBRVA Level 2 and Level 3 Structural Assessment

[0087] SOPMA predictions show that in rBRVA, α-helices account for 20.78%, extended chains 29.52%, and random coils 49.70%. The optimal model obtained through trRosetta modeling ( Figures 2-3 The ERRAT score was 93.33. Amino acid exposure analysis showed that 21% of residues were buried, 26% were moderately exposed, and 53% were exposed on the surface. Figure 4 This shows that rBRVA has good spatial accessibility and structural stability.

[0088] 2.7 Results of stability, solubility and antigenicity assessment of rBRVA

[0089] rBRVA consists of 338 amino acids, with a theoretical molecular weight of 37.4 kDa and a pI of 9.78. Its instability index is 30.51 (<40), indicating it is a stable protein; its aliphatic index is 73.93, and its average hydrophilicity index is -0.51, suggesting good solubility. The VaxiJen predictive antigenicity is 0.7059, higher than the threshold of 0.4, suggesting that rBRVA possesses strong antigenicity and potential immunogenicity.

[0090] 2.8 Simulated immunization results

[0091] C-ImmSim simulation results ( Figure 5 The results showed that rBRVA could induce significant expansion of B cells, CTLs, and HTLs, and promote the secretion of cytokines such as IgG, IgM, and IFN-γ. Furthermore, the number of memory T cells increased significantly, suggesting that rBRVA can induce a durable immune memory response.

[0092] 2.9 Results of molecular docking

[0093] Through AlphaFold modeling and PyMOL visualization analysis, it was found that rBRVA could form a stable complex with integrin α2 subunit Figure 6 , and there were multiple hydrogen bonds and hydrophobic interactions between them, indicating that rBRVA had good host receptor binding potential, providing structural basis for its induction of immune recognition and protective effect.

[0094] Example 2 Preparation and application of polyepitope vaccine

[0095] 1 Experimental method

[0096] 1.1 Codon optimization and expression vector construction of rBRVA

[0097] In order to improve the expression level of polyepitope vaccine in E. coli, the target fusion gene sequence was codon optimized using ExpOptimizer online tool. According to the codon usage bias of E. coli BL21 (DE3), the synonymous codons of recombinant BRVA polyepitope fusion gene (rBRVA) were optimized to avoid the influence of rare codons and mRNA secondary structure formation on expression. Based on the Helicobacter pylori ferritin gene sequence (GenBank No. WP_000949190.1), the designed rBRVA was connected to the N terminus of Helicobacter pylori ferritin sequence through GGGGS. pET-28a (+) was selected as the prokaryotic expression vector, and Nco I and Xho I restriction enzyme sites were introduced at the N and C termini of the nucleotide sequence, respectively, to facilitate subsequent directional cloning. After connecting ferritin and target rBRVA, the fragment was inserted between Nco I and Xho I of pET-28a (+) as the prokaryotic expression vector.

[0098] SnapGene software was used for sequence splicing and cloning simulation to ensure that the reading frame of the inserted fragment was correct and that it was fused with His tag for expression. The optimized sequence length was about 1028 bp. Finally, the designed recombinant plasmid sequence was synthesized and verified by Shenguo Bioengineering (Shanghai) Co., Ltd.

[0099] The amino acid sequence of the ferritin vector is:

[0100] MLSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS

[0101] 1.2 Transformation and verification of rBRVA recombinant plasmid

[0102] The synthetic rBRVA recombinant plasmid was transformed into E. coli BL21 (DE3) competent cells. 100 μL of competent cells were taken, 5 μL of plasmid solution was added, and ice bath was performed for 30 min. After 42 ℃ heat shock for 90 s, ice bath was performed for 2 min immediately. Then 900 μL of SOC medium was added, and recovery was performed at 37 ℃, 200 rpm for 1 h. Subsequently, it was coated on LB plate containing 50 μg / mL kanamycin, and cultured at 37 ℃ for 12-16 h. A single positive clone was picked for colony PCR verification, and positive strains were screened and subcultured for 5 times to detect the genetic stability of the plasmid. The positive bacterial solution was sent to Shengong Biotechnology Co., Ltd. for sequencing, and pET28a (+) universal primers T7-F (5'-TAATACGACTCACTATAGGG-3') and T7-R (5'-GCTAGTTATTGCTCAGCGG-3') were used for sequencing. The strain with consistent sequencing results and designed sequence was the successfully constructed rBRVA engineering bacteria.

[0103] 1.3 Optimization of rBRVA expression conditions

[0104] In order to obtain high expression amount and mainly soluble protein form of rBRVA, the induction conditions were optimized. The induction time was set to 8 h and 16 h at 16 ℃ and 37 ℃, respectively, and different concentrations of IPTG (0.1, 0.3, 0.5, 0.8, 1.0 mmol / L) were set for induction expression. After induction, the bacterial cells were collected, washed with PBS, and then ultrasonically broken. The supernatant and precipitate were centrifuged, and SDS-PAGE detection was performed to analyze the expression product.

[0105] The solubility of rBRVA under different induction conditions was analyzed by 12% SDS-PAGE electrophoresis. If there is a clear target protein band in the supernatant and the band in the precipitate is weak, it indicates that the target protein mainly exists in the soluble form. The protein concentration was determined by BCA method.

[0106] 1.5 Purification of BRVA

[0107] rBRVA protein was purified by His Cap 6FF nickel ion affinity purification column. The purification process included three steps: lysis loading, washing (containing 20 mmol / L imidazole), and elution (containing 250 mmol / L imidazole). After each elution component was collected, the imidazole was removed by dialysis, and the purity was verified by SDS-PAGE.

[0108] 1.6 Western blot verification of rBRVA

[0109] The purified rBRVA protein sample was subjected to SDS-PAGE electrophoresis and membrane transfer, using rabbit anti-BRVA whole virus serum as the primary antibody (1:1000) and HRP-labeled goat anti-rabbit IgG as the secondary antibody (1:5000), and color development detection was performed using a supersensitive ECL chemiluminescence substrate. The appearance of positive bands indicates that the recombinant protein has good antigenicity and immunoreactivity.

[0110] 1.7 Vaccine preparation and animal immunization

[0111] The purified rBRVA protein was emulsified with Montanide ISA 201 adjuvant at a volume ratio of 1:1 to prepare an oil-in-water emulsion vaccine. Three healthy New Zealand rabbits were selected, with each receiving a subcutaneous multi-point injection of 200 μg of the vaccine. After 14 days, the rabbits were given a booster immunization at the same dose and route. Blood was collected from the ear artery every 7 days after the first immunization, and the serum was separated and stored at -20 ℃ for use. This process was repeated until 14 days after the second immunization.

[0112] 1.8 Indirect ELISA detection of rabbit serum antibody titer

[0113] The levels of anti-BRVA VP7 and VP4 specific antibodies in rabbit serum were detected by indirect ELISA. The rVP7 and rVP4 proteins were used to coat the enzyme-labeled plates (1 μg / mL), and after blocking, sample addition, incubation, plate washing, secondary antibody addition, and color development, the OD values were measured at 450 nm. A positive / negative (P / N) ratio of ≥2.1 was considered positive, and the antibody titer was defined as the highest dilution factor at which the OD450 value was equal to the positive limit.

[0114] 1.9 Neutralization test to detect serum neutralization titer

[0115] The neutralizing antibody titer of rabbit serum 14 days after the second immunization was detected by the fixed virus dilution serum method (virus amount of 100 TCID 50 ). The neutralizing activity of the serum against BRVA G6, G8, and G10 was detected. The serum was serially diluted by 2-fold, mixed with an equal amount of virus at 37 ℃ for 1 h, and then inoculated into MA104 cells. Cell pathology (CPE) was observed after 96 h. The neutralizing antibody titer was calculated according to the Reed-Muench method.

[0116] 2 Result analysis

[0117] 2.1 rBRVA codon optimization and cloning vector design

[0118] The recombinant gene sequence with a length of 1028 bp was obtained after ExpOptimizer codon optimization, and the GC content was adjusted from 42.7% to 53.2%, avoiding the use of low-frequency codons of E. coli. After simulation verification by SnapGene software, the rBRVA fragment was successfully cloned into the pET-28a(+) vector ( Figure 7 ). The sequencing results were completely consistent with the designed sequence, indicating that the construction was correct and could be used for subsequent expression.

[0119] The optimized rBRVA DNA sequence is as follows (SEQ ID NO. 18):

[0120]

[0121] 2.2 Optimization of rBRVA expression conditions

[0122] After transforming the expression plasmid into the BL21 (DE3) strain, the expression amount was detected under different induction conditions. The results showed that when the induction temperature was 16 ℃, the induction time was 16 h, and the IPTG concentration was 0.5 mmol / L, the target protein band was the clearest and mainly existed in a soluble form ( Figure 8 ), and the condition was determined as the optimal induction expression condition of rBRVA.

[0123] 2.3 rBRVA solubility verification and purification

[0124] After induction expression under the optimized conditions, SDS-PAGE detection after ultrasonic disruption and centrifugation showed that rBRVA appeared an obvious band of about 37 kDa in the supernatant, suggesting that it mainly existed in a soluble form. After purification by a nickel ion affinity column, a single target band appeared in the eluate, with a purity higher than 90% and consistent with the expected molecular weight.

[0125] 2.4 Western blot verification of rBRVA

[0126] Western blot results showed that a clear positive signal appeared at 37 kDa with BRVA whole virus rabbit serum as the primary antibody ( Figure 9 ), indicating that rBRVA protein had good immunoreactivity and could be specifically recognized.

[0127] 2.5 Immunogenicity of rBRVA

[0128] The antibody titers of rabbit serum after immunization were detected by indirect ELISA, and the results showed that rBRVA immunization could induce specific antibodies against BRVA VP7 and VP4 proteins. The serum antibody titers reached 1:1,000,000 (VP7) and 1:900,000 (VP4) at 14 days after the second immunization ( Figure 10 ), suggesting that rBRVA had significant humoral immunogenicity.

[0129] 2.6 Neutralizing activity of rBRVA

[0130] The neutralization experiment results showed that the neutralizing antibody titers of rabbit serum at 14 days after the second immunization against BRVA G6, G8, and G10 were 1:360, 1:280, and 1:360, respectively ( Figure 11 ). The results showed that rBRVA vaccine could induce broad-spectrum cross-neutralizing antibodies and had strong immunoprotective potential.

[0131] In conclusion, the polypeptide fusion protein with good conformation stability is obtained, and immunological evaluation shows that the polypeptide fusion protein has good antigen specificity and neutralization activity. Animal experiment results show that the polypeptide fusion protein can induce the body to produce high-level neutralizing antibodies against multiple genotypes of BRVA G6, G8 and G10, and significantly improve the broad spectrum and durability of immune protection. The polypeptide fusion protein has a wide development and application prospect.

Claims

1. A bovine group A rotavirus multi-epitope peptide fusion protein, characterized in that, It is obtained by linking a multi-epitope peptide with an amino acid sequence as shown in SEQ ID NO. 17 with ferritin; wherein, ferritin is linked to the N-terminus of the multi-epitope peptide through an EAAAK sequence, and the ferritin is derived from Helicobacter pylori.

2. A nucleic acid molecule encoding the fusion protein of claim 1, characterized in that, Its nucleotide sequence is shown in SEQ ID NO:

18.

3. A nanoparticle assembled based on the fusion protein of claim 1.

4. The use of the fusion protein of claim 1 or the nanoparticles of claim 3 in the preparation of drugs for the prevention or treatment of bovine group A rotavirus.

5. The application according to claim 4, characterized in that, The drug is a vaccine or a diagnostic reagent.

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