CN116284432A - Influenza B virus recombinant protein vaccine and preparation method thereof - Google Patents

Influenza B virus recombinant protein vaccine and preparation method thereof Download PDF

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CN116284432A
CN116284432A CN202211099880.3A CN202211099880A CN116284432A CN 116284432 A CN116284432 A CN 116284432A CN 202211099880 A CN202211099880 A CN 202211099880A CN 116284432 A CN116284432 A CN 116284432A
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influenza
recombinant
virus
ham
antigen
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舒跃龙
赵天旖
刘雪洁
罗楚铭
孙彩军
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Sun Yat Sen University
Sun Yat Sen University Shenzhen Campus
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Sun Yat Sen University Shenzhen Campus
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Abstract

The invention provides a recombinant protein vaccine of influenza B virus and a preparation method thereof, belonging to the technical field of biology. The invention discloses a mosaic strategy-based construction method of influenza B virus recombinant HA antigen, which comprises the following steps: (1) Analyzing the influenza B virus, and screening to obtain an influenza B virus HA amino acid sequence; (2) And (3) analyzing the HA amino acid sequence of the influenza B virus by adopting a mosaic strategy, removing rare epitopes, and optimizing by adopting a genetic algorithm to obtain the recombinant HA antigen of the influenza B virus assembled by the short peptides. According to the invention, through a Mosaic vaccine design method and combining a genetic algorithm to optimize a protein sequence, a Mosaic HA sequence with maximum T cell epitopes covered by influenza viruses comprising a B type Victoria subline and a Yamagata subline is designed.

Description

Influenza B virus recombinant protein vaccine and preparation method thereof
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an influenza B virus recombinant protein vaccine and a preparation method thereof.
Background
Influenza virus (influenzavirus) is a type of envelope virus belonging to the orthomyxoviridae (orthomyxoviridae) and is classified into four types A, B, C, D according to antigenicity and genotype of Nucleoprotein (NP) and matrix protein (M). Influenza A (IAV) and Influenza B (IBV) of H1N1 and H3N2 subtypes each year cause outbreaks of human respiratory disease. To date, influenza b has received less attention, however, influenza b virus infection has a major impact on morbidity and mortality in the population. Influenza b becomes a dominant strain in europe about every 7 epidemic seasons. Influenza b outbreaks occur in different regions of the world and in people of different age groups, and although influenza b virus causes disease in all age groups, the burden of influenza b virus infection is highest among children and young people. Studies have shown that influenza A and B infections result in similar morbidity and mortality in hospitalized adults, and similar clinical characteristics in outpatients. These results indicate that influenza b virus can cause as severe an infection as influenza a virus. In addition, the excessive death burden caused by influenza B epidemic is heavy, and the average excessive death rate of influenza B per year in China is 2.3/10 ten thousand (95% CI: 2.1-2.5) in 2005-2015. Some serious complications (such as viral myocarditis, encephalopathy, etc.) are also closely related to influenza b virus infection.
The influenza b genome consists of 8 single negative strand RNA segments. Wherein RNA segment 4 encodes hemagglutinin HA, which is capable of binding to sialic acid receptors on the cell surface, mediating virus contact with the cell, is the first step in virus infection of the cell. HA HAs a better immunogenicity, and HA-induced neutralizing antibodies can act on the virus at the first time of infection, bind to the virus, and inhibit virus invasion into cells, so HA HAs been a key antigen in developing influenza vaccines. Fischer et al "assembled" a Mosaic protein from a natural HIV sequence by using computer-optimized methods, so that the protein contained the most fully covered T cell epitopes, and compared to the HIV natural protein, found that the coverage of the Mosaic protein was significantly increased for diverse HIV virus populations, both as a readily mutated protein and as a conserved protein. The Mosaic sequence designed by the method is not only suitable for HIV, but also suitable for other pathogens with relatively rapid variation, such as influenza virus.
Subunit vaccine refers to a vaccine prepared by extracting substances with immunogenicity from virus particles and adding an adjuvant. Hemagglutinin protein is the major surface antigen and protective antigen of influenza virus, and is also the main target antigen for research of influenza subunit vaccine. Along with the development of genetic engineering technology, one clones the HA gene onto an expression vector, then introduces the HA gene into an expression system, and performs amplification and expression in a large amount in the expression system, so that a large amount of HA protein can be obtained. The HA protein produced by the method HAs high yield, low cost, good safety and no problems of strong virulence, virus diffusion, environmental pollution and the like. Influenza vaccines are the most effective means of preventing influenza, however, studies in various countries worldwide show that in recent years the epidemic type b strain and the vaccine contained type b strain or vaccine strain are antigenically mismatched. Meanwhile, it takes several months to produce multivalent influenza vaccines that match the current year influenza virus subtype, and large financial and labor investment is required. Therefore, there is an urgent need to develop a universal vaccine capable of preventing seasonal influenza b.
Disclosure of Invention
Aiming at the problems, the invention aims to provide the influenza B virus recombinant protein vaccine and the preparation method thereof, wherein the influenza B virus recombinant HA protein core antigen is efficiently expressed, and the recombinant HA protein HAs higher hemagglutination value.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: the construction method of the influenza B virus recombinant HA antigen based on the mosaic strategy comprises the following steps:
(1) Analyzing the influenza B virus, and screening to obtain an influenza B virus HA amino acid sequence;
(2) And (3) analyzing the HA amino acid sequence of the influenza B virus by adopting a mosaic strategy, removing rare epitopes, and optimizing by adopting a genetic algorithm to obtain the recombinant HA antigen of the influenza B virus assembled by the short peptides.
Research shows that the existing vaccine has poor cross protection, longer production period and high cost. The inventor of the application aims at the problem, successfully and efficiently expresses the recombinant HA protein core antigen of the influenza B virus through an insect cell-baculovirus expression system based on a mosaic vaccine strategy synthesized by a genetic algorithm, and the recombinant HA protein HAs higher hemagglutination value, so that the recombinant HA protein can be proved to be further subjected to immunogenicity research, and a theoretical basis and a material basis are provided for the subsequent research and development of the recombinant influenza B protein vaccine.
As a preferred embodiment of the construction method of the mosaic strategy-based recombinant HA antigen of the influenza B virus, the influenza B virus comprises a Victoria subline and a Yamagata subline.
As a preferred implementation mode of the mosaic strategy-based construction method of the influenza B virus recombinant HA antigen, the frequency of occurrence of the rare epitope in the HA amino acid sequence is less than 3.
As a preferred implementation mode of the mosaic strategy-based construction method of the influenza B virus recombinant HA antigen, the short peptide consists of 9 amino acids.
The invention also provides an influenza B virus recombinant HA antigen, which is prepared by adopting the construction method.
As a preferred embodiment of the recombinant HA antigen of the influenza B virus according to the present invention, the recombinant HA antigen of the influenza B virus comprises the recombinant HA antigen HAM-VIC of the subfamily Victoria and the recombinant HA antigen HAM-YAM of the subfamily Yamagata.
As a preferred embodiment of the recombinant HA antigen of the influenza B virus, the amino acid sequence of the HAM-VIC is shown as SEQ ID NO. 1; the amino acid sequence of the HAM-YAM is shown as SEQ ID NO. 2.
The invention also provides a recombinant pFastBac Dual-HAM plasmid, which is obtained by retaining the extracellular region of the recombinant HA antigen of the influenza B virus, removing the signal peptide of the recombinant HA antigen of the influenza B virus, adding the gp67 signal peptide and optimizing according to the preferred codons of insect cells.
The invention also provides a recombinant shuttle vector Bacmid, which comprises the recombinant pFastBac Dual-HAM plasmid.
The invention also provides a preparation method of the recombinant baculovirus for expressing the HAM recombinant protein of the influenza B virus, which comprises the following steps:
(1) Obtaining pFastBac Dual-HAM plasmid for expressing HAM recombinant protein of influenza B virus by gene synthesis technology;
(2) Transforming the pFastBacDual-HAM plasmid obtained in the step (1) into DH10Bac competent cells to obtain a recombinant shuttle vector Bacmid;
(3) Transfecting the recombinant shuttle vector Bacmid obtained in the step (2) into insect cells to obtain baculovirus;
(4) And (3) further purifying the baculovirus in the step (3) to obtain the recombinant baculovirus expressing the influenza B virus HAM recombinant protein.
As a preferred embodiment of the method for preparing recombinant baculovirus expressing influenza B virus HAM recombinant protein, the purification in the step (4) is to inoculate baculovirus into insect cells with good growth state continuously, and culture the baculovirus until obtaining fourth generation baculovirus.
The invention also provides a recombinant baculovirus for expressing the HAM recombinant protein of the influenza B virus, which is prepared by adopting the preparation method of the recombinant baculovirus for expressing the HAM recombinant protein of the influenza B virus.
The invention also provides application of the recombinant baculovirus expressing the HAM recombinant protein of the influenza B virus in preparing influenza B vaccine and medicines for preventing or treating influenza B.
The invention also provides a recombinant protein vaccine of the influenza B virus, which comprises the recombinant baculovirus.
As a preferred embodiment of the influenza b virus recombinant protein vaccine of the present invention, the influenza b virus recombinant protein vaccine further comprises an adjuvant.
The beneficial effects of the invention are as follows: according to the invention, through a Mosaicvaccine design method and combining a genetic algorithm to optimize a protein sequence, a MosaicHA sequence with maximum T cell epitopes covered by influenza viruses comprising a B type Victoria subfsystem and a Yamagata subfsystem is designed; the recombinant HA protein is expressed and purified by utilizing an insect cell-baculovirus expression system, so that the influenza B virus recombinant HA protein core antigen is successfully and efficiently expressed, the recombinant HA protein HAs higher hemagglutination value, the recombinant HA protein can be further subjected to immunogenicity research, and a theoretical basis and a material basis are provided for the subsequent research and development of influenza B recombinant protein vaccines.
Drawings
FIG. 1 is an average of the coverage of HAM-VIC for the whole epitope.
FIG. 2 is an epitope coverage per amino acid of HAM-VIC.
FIG. 3 shows the descending epitope coverage of HAM-VIC.
FIG. 4 is a drawing of a potential HAM-VIC epitope.
FIG. 5 is an electrophoretically identified map of recombinant plasmid, wherein A is an electrophoretically identified map of recombinant pFastBac-Dual-HAM-YAM, B is an electrophoretically identified map of recombinant plasmid pFastBac-Dual-HAM-VIC, 1 represents plasmid, 2 represents plasmid digested with EcoRI and HindIII, and M represents KBlader.
FIG. 6 is a diagram showing the result of WesternBlot detection of recombinant protein expression of P2-generation baculovirus, wherein A is Bacmid-HAM-YAM; b is Bacmid-HAM-VIC; in the figure, 1 represents the cell supernatant of the P1-generation recombinant baculovirus, 2 represents the cell supernatant of the P2-generation recombinant baculovirus, 3 represents the cell lysate of the P2-generation recombinant baculovirus, 4 represents the blank control, and M is the protein ladder.
FIG. 7 is an IFA identification of recombinant proteins; wherein A is Bacmid-HAM-YAM infected Sf9 cells; b is Bacmid-HAM-VIC infected Sf9 cells.
FIG. 8 is a SDS-PAGE analysis of the effluent fractions after passing through the NI column, wherein A is HAM-YAM; b is HAM-VIC; in the figures, 1, 2, 3, 4, 5 and 6 represent different outflow components, and M is a protein ladder.
FIG. 9 is a graph showing the results of the measurement of the hemagglutination activity of recombinant proteins by the hemagglutination assay.
Detailed Description
In order to more clearly demonstrate the technical scheme, objects and advantages of the present invention, the technical scheme of the present invention is described in detail below with reference to the specific embodiments and the accompanying drawings.
EXAMPLE 1 construction of HAM recombinant protein sequence based on mosaic strategy
1. HAM protein sequence design and acquisition
Downloading all humanized B-type Victoria subfsystem and Yamagata subfsystem in 2000-2021 years from the GISAID database and the NCBI database to obtain 19197 BVHA amino acid sequences and 16614 BYHA amino acid sequences, and removing sequences with poor repeated sequence and sequencing quality by using computer software and algorithm such as BioAider, MAFFT, aliview, spyder to obtain 4122 BVHA amino acid sequences and 2745 BYHA amino acid sequences. Uploading the amino acid sequence obtained by screening to Mosaic VaccineDesigner website (https:// www.hiv.lanl.gov/content/sequence/mosai/makevaccine. Html) in FAS format, and setting parameters: the CocktailSize is set to "1" to obtain 1 mosaic sequence for the next step; the epitope length was set to "9" to obtain a mosaic sequence covering more cd8+ CTL cell epitopes; the threshold is set to "3" to reduce the number of rare epitopes; the fixed sequence was not added. After genetic algorithm operation, a series of mosaics sequences assembled by short peptides composed of 9 amino acids are finally obtained; and optimizing each population sequentially by using a genetic algorithm, wherein new recombinants are generated, the epitope coverage rate of the recombinants is calculated and tested, and finally 2 optimal mosaic amino acid sequences are obtained, wherein the amino acid sequences are respectively shown as SEQ ID NO. 1-2.
2. Analysis of HAM protein sequences
The antigen epitope coverage of HAM proteins was evaluated using EpitopeCoverageAssessmentTool (Epicover) (https:// www.hiv.lanl.gov/content/sequence/MOSAIC/epicap. Html) and strain amino acid sequences (3206 BVHA amino acid sequences and 2198 BVHA amino acid sequences) aligned prior to setting in the GISAID database and NCBI database download analysis were used as a natural strain background protein set. Taking HAM-VIC as an example, 3206 HA sequences aligned by the above analysis were used as a background protein set of natural strains. The 9 epitope length was set to analyze the coverage of Tc cell epitopes, respectively, and the final results were expressed as an average of the epitope coverage, and the results were shown in fig. 1, with a precise matching rate (9/9) of 94.02% for the 9 epitope length of HAM-VIC sequence to 3206 natural strain sequences, a matching rate of 99.20% for 8/9, and a matching rate of 99.78% for 7/9.
PositionalEpitopeCoverageAssessmentTool (Posicover) (https:// www.hiv.lanl.gov/content/sequence/MOSAIC/posicover. Html) was used to analyze the epitope coverage of HAM proteins at each amino acid and the background protein set used was as above. Setting parameters: the epitope length is set to 9 and the other parameters select default values. The tool can specifically calculate the epitope coverage of the natural strain HA concentrated on the background protein at each amino acid position, and the final result is expressed as the position average epitope coverage. Taking HAM-VIC as an example, 3206 HA sequences which are analyzed and aligned as described above are used as a background protein set of a natural strain, and the epitope length is set to be 9.
FIGS. 2 and 3 are the percent match/deletion of the 9-mers of the HAM-VIC sequence to the test protein sequence, wherein FIG. 3 is arranged in descending order of the percent amino acids and FIG. 2 is arranged in the natural order of amino acids. FIG. 4 shows potential epitopes of HAM sequences in each test sequence. Each row represents a natural strain sequence, each column represents an alignment position, and each pixel represents an amino acid. If the 9-mers of the HAM sequence are perfectly matched in the corresponding positions in the test protein sequence, the amino acid is yellow in color; the less the corresponding position matches, the darker the red; if the amino acid residues are not included in the 9-mer at all, it is shown to be black. The results show that most of the color of HAM-VIC is yellow, which indicates better matching with the test protein set and higher epitope coverage.
EXAMPLE 2 expression of recombinant HAM proteins by insect cell baculovirus System
1. Synthesis of HAM protein sequences
The HAM-VIC and HAM-YAM sequences have the same synthetic design concept, taking HAM-VIC as an example. Protein sequence transmembrane analysis was performed using TMHMM-2.0 (https:// services. Healthcare. Dtu. Dk/services. PhpTMHMM-2.0) with the extracellular domain of HAM-VIC retained; signal peptide prediction was performed using SignalP-6.0 (https:// services. Heathtech. Dtu. Dk/services. PhpSignalP), the HAM-VIC original signal peptide was removed, and gp67 signal peptide was added. After codon optimization according to insect cell preference (optimized species: inseneswii), recombinant pFastBacDual-HAM-VIC plasmid was obtained by gene synthesis technique, and electrophoresis of the recombinant plasmid was shown in fig. 5.
2. Construction of recombinant shuttle vector Bacmid
Taking DH10Bac competent cells, melting on ice, adding 50ng recombinant pFastBac1-HAM plasmid (plasmid not exceeding competent 10%), stirring by hand to EP tube bottom, and standing in ice for 25-30min. And (3) carrying out heat shock for 45s in a water bath at the temperature of 42 ℃, quickly putting the mixture back into ice, and standing the mixture for 2 to 5min (shaking can reduce the conversion efficiency). Add 500. Mu.l of SOC liquid medium, resuscitate at 37℃and 225rpm for 3-4h. Without centrifugation, 80ul of the bacterial liquid was applied to a three-antibody LB plate (containing 7. Mu.g/ml gentamicin, 50. Mu.g/ml kanamycin, 10. Mu.g/ml tetracyclomycin, 40. Mu.g/ml IPTG and 100. Mu.g/ml X-gal). Plates were placed in an incubator at 37℃for 48h (higher antibiotic content, long incubation at 37℃was required to pick the appropriate clone, the single clone was small, and it was difficult to distinguish whether it was blue clone or not before 24 h). Several white and blue colonies were picked separately, and streaked on a new triple antibody LB agar plate to further determine positive clones as white colonies. A white colony of the monoclonal antibody was selected and added to LB liquid medium (7. Mu.g/ml gentamicin, 50. Mu.g/ml kanamycin, 10. Mu.g/ml tetracyclomycin) containing three antibiotics, and cultured at 37℃for 12-16 hours at 225rpm to give recombinant bacmid.
3. Bacmid is carried out
Extracting recombinant Bacmid according to alkaline lysis method by taking 1.5mL of bacterial liquid, centrifuging at 4deg.C for 3min at 12000 Xg, discarding supernatant, adding 220 uLresolution I to gently resuspend precipitated bacteria, adding 300uL of now-prepared solution II, closing the nozzle, gently inverting up and down for 5-6 times, making the solution transparent, ice-bathing for 3-5min, slowly adding 220uL of pre-chilled solution III at 4deg.C, gently inverting up and down for 5-6 times, precipitating white thick protein, ice-bathing for 5min, adding equal volume of phenol: chloroform (1:1), mixing upside down for 10 times, standing at room temperature for 3min, centrifuging at 4deg.C for 12,500Xg for 10min, transferring the supernatant to another sterilized EP tube, adding chloroform of equal volume to the supernatant, mixing gently upside down for 5-10 times, standing at room temperature for 3min, centrifuging at 4deg.C for 12,500Xg for 10min, taking carefully, slowly sucking the supernatant, adding 700pL isopropanol, mixing gently upside down for several times, standing at room temperature for 3min, centrifuging at 4deg.C for 12,500Xg for 20min; carefully discard the supernatant and add 1mL of 70% ethanol to wash the pellet; centrifuging at 7500 Xg for 10min at 4deg.C, and discarding supernatant carefully on a super clean bench; repeating the washing for one time; air-drying for 5-10min, volatilizing ethanol, and dissolving the precipitate with 20uL of sterilized deionized water; note that in order to prevent gene disruption, it is not possible to blow dry mechanically, it should be naturally dried and stored at 4 ℃ after DNA dissolution.
4. sf9 cell culture and cryopreservation
Sf9 cell passage density: 0.5-4OD (i.e., 0.5-4X 10) 6 ) cells/ml; complete medium: SF900 III+1% PS (for passaging), or SF921+1% PS (for use in experiments with expanded culture); frozen stock solution: SF900 III (containing 1% PS) +10% DMSO.
5. Transfection of bacmid
Cell plating: the cells are at an OD of 1-2.5 (i.e., 1-2.5X10) 6 ) Plating was performed in the cell/ml state, and a six-well plate was plated at 0.8OD per well (i.e., 0.8X10) 6 ) cells/ml,2.5mL, and incubated at 27℃for 1-2h to allow them to adhere. The medium in the six well plate was aspirated and replaced with PSfree medium.
Post transfection: 100ul SF900 III medium (PSfree) was added to two empty 1.5ml EP tubes; tube 1:8ul cellfcctin+100ul SF900 III, and uniformly blowing by a yellow gun head; tube 2: 14-15ulbacmid+100ul SF900 III, and uniformly blowing by using a yellow gun head; adding the tube 2bacmid into the tube 1cellfectin, dripping, and flicking with fingers; incubating for 15min or more; dripping the mixture into a six-hole plate uniformly; changing the solution into SF900 III+1% PS for 3-5h, culturing at 27 ℃ for 72h, and observing every day.
6. Receiving P1 generation baculovirus, receiving P2
Prepared 50ml of 1.5-2.5X10 6 The cells/ml sf9 cells were used for grafting P2. Firstly, P2: 500ul of liquid near the top of the well was aspirated with a gun and added to 50ml of sf9 cells without blowing. The remaining supernatant was collected: 100ul of samples were taken for subsequent WB and the remaining aliquots were frozen at-80 ℃. On day 3 of P2 culture, 100ul of supernatant was collected, centrifuged at 3000g for 6min, and the supernatant and cell pellet were used as WB, respectively. Counting every day after P2 culture until day 3, calculating cell death rate (dead cells/living cells), collecting P2 when cell death rate reaches 90% (generally 5/6 days), centrifuging at 2000rpm for 10min, collecting clear supernatant, and sub-packaging 6ml tube for freezing at-80deg.C.
7. WesternBlot validation of P2 generation baculovirus
P1 supernatant, P2 cells. The target band indicates that the P2 generation virus is available, and the specific steps are as follows:
(1) Preparation of SDS-PAGE electrophoresis samples: mixing 35 μl purified VLPs and baculovirus solution (blank) containing no target protein with 5×protein loading buffer at a ratio of 4:1, boiling for 10min, and centrifuging;
(2) SDS-PAGE electrophoresis: according to the size of the target protein, preparing a reagent kit gel by adopting a Yazyme PAGE gel, sampling by using a sampling needle, taking the standard molecular mass of a 180KD protein as a reference, and taking out the gel after running at a constant voltage of 80V for 2 hours;
(3) Cutting the adhesive tape to a proper size, cutting the PVDF film and the filter paper to be as large as the adhesive tape, sensitizing the PVDF film in methanol for 15s, and transferring the adhesive tape, the PVDF film and the filter paper into a transfer buffer solution for soaking for 15min;
(4) When the membrane is turned, the anode carbon plate, the filter paper, the PVDF membrane, the gel, the filter paper and the cathode carbon plate are placed and aligned in sequence from bottom to top, air bubbles are removed, a power supply is turned on, the current is regulated to 400mA, constant current running is carried out for 40min, and an electrophoresis tank is placed in ice;
(5) After the transfer is finished, taking out the membrane, adding a sealing solution, incubating for 2 hours at 37 ℃ by a shaking table, discarding the sealing solution, and washing the membrane by PBST for 3 times for 15 minutes each time;
(6) Anti-6XHIS was performed with PBST containing 5w/v% BSA
Figure BDA0003839896830000091
antibody[HIS.H8](ex abcam, cat No. ab 18184) according to 1: diluting at 5000, and standing overnight at 4 ℃; discarding the primary antibody, and washing the membrane with PBST for 3 times for 15min each time;
(7) Adding PVDF membrane into HRP-labeled goat anti-mouse IgG antibody (purchased from Friedel-crafts, with the product number of FDM 007) diluted by 1:10000 times respectively, incubating for 1h at 37 ℃, taking out PVDF membrane, and washing the membrane with PBST for 3 times each for 15min;
(8) And (3) dropwise adding the newly prepared ECL chemiluminescent working solution on the PVDF film, and developing at room temperature in a dark place.
As shown in FIG. 6, the WesternBlot results show that the Bacmid-HAM-YAM and the Bacmid-HAM-VIC have bands at 100bp, and the successful expression of both recombinant HA proteins is identified.
8. Indirect immunofluorescence assay
The adherent Sf cells were passaged up to 100% and diluted to a concentration of 4×10 with adherent Sf cell medium (containing 8% bi serum and 1% diabodies) 5 cells/mL. mu.L of cells were spread evenly in each well of a 24-well plate cell culture plate. Then, it was cultured in a thermostatic incubator at 27℃for 10-12 hours, the 24-well plate was removed, and Sf9 cells in the 24-well plate were inoculated with the recombinant virus at a ratio of 3%, and cultured in a thermostatic incubator at 27℃for 48 hours. The 24-well plate was removed, the cell supernatant broth was discarded, and 80% chilled acetone was slowly added at room temperature and allowed to act for 30 minutes in each well of the cell culture plate. The cold acetone solution was slowly discarded with a 1mL micropipette and PBST was slowly added to the 24-well plate at room temperature and washed 3 times for 5 minutes on a shaker. The primary antibody against the HA protein was incubated at room temperature for 30 minutes by adding to each well a primary antibody diluted with 1% Bovine Serum Albumin (BSA). Incubated primary antibodies were slowly discarded with micropipettes and were then removed from the reaction vesselPBST was slowly added to the 24-well plate at room temperature and washed 3 times for 5 minutes in a shaker. Fluorescein Isothiocyanate (FITC) -labeled secondary antibody (with PBST containing 1% evans) was diluted in the dark and then added to 24-well plates for 60 min incubation at room temperature. The incubated secondary antibody was slowly discarded with a micropipette and PBST was slowly added to the 24-well plate at room temperature and washed 3 times for 5 minutes on a shaker. The expression of the green fluorescent protein stained on each well was observed under a fluorescent microscope in a dark room.
The results are shown in fig. 7, further verifying that recombinant HA proteins are capable of large soluble expression in Sf9 cells.
9. After the recombinant protein expression identification is successful, the obtained P2 generation recombinant baculovirus is continuously inoculated to Sf9 insect cells with good growth state, and the like is pushed until the P4 generation recombinant baculovirus is obtained; centrifuging Sf9 insect cell suspension for culturing the P4-generation recombinant baculovirus at 5000rpm for 30min at 4 ℃ and collecting a supernatant; the obtained supernatant was concentrated by passing through a vivaflow2000 membrane pack. Ni column purification by equilibrating the Ni column (HistrapeHP, 5 mL) with solution A at a flow rate of 5.0mL/min for about 15 column volumes; then, the collected solution flows through a Ni column at a flow rate of 5.0 mL/min; balancing the column volume by 15 times by using the solution A; then, carrying out gradient elution by using the solution A and the solution B, collecting all outflow components, analyzing the outflow components by using 10% SDS-PAGE, and collecting proper outflow components; molecular sieves A protein sample was concentrated by equilibrating a molecular sieve column (Superdex 7510/300 gelfiltrationcolumn) with liquid C at a flow rate of 1.0 mLmin. All the effluent fractions were sampled and collected for the electrophoresis analysis described above, and the results are shown in FIG. 8. Mixing all components with purity of above 98%, concentrating by superionization, detecting protein concentration by using Nanodrop, packaging, quick freezing with liquid nitrogen, and storing at-80deg.C.
10. Erythrocyte agglutination assay
The hemagglutination titer of the purified recombinant proteins HAM-YAM and HAM-VIC and the cell culture supernatant of the blank control was measured for 1% chicken erythrocytes by the following steps:
preparing 1% chicken erythrocyte suspension, adding 50 mu L of PBS into a 96-well plate, adding 50 mu L of HAM-YAM and HAM-VIC into a first well, blowing uniformly, sucking out 50 mu L to a 2 nd well, diluting to a 11 th well by a ratio of 2 times in sequence, taking the 12 th well as a negative control, adding 50 mu L of 1% erythrocyte suspension, standing for 25min at room temperature after uniform mixing, and observing the result.
As shown in FIG. 9, it can be observed that HAM-YAM can reach 1: 128. HAM-VIC can reach 1:64, while no hemagglutination was observed in the blank (Mock), the recombinant protein was shown to have similar hemagglutination activity and immunogenicity to the inactivated virus antigen.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention.

Claims (15)

1. The construction method of the influenza B virus recombinant HA antigen based on the mosaic strategy is characterized by comprising the following steps:
(1) Analyzing the influenza B virus, and screening to obtain an influenza B virus HA amino acid sequence;
(2) And (3) analyzing the HA amino acid sequence of the influenza B virus by adopting a mosaic strategy, removing rare epitopes, and optimizing by adopting a genetic algorithm to obtain the recombinant HA antigen of the influenza B virus assembled by the short peptides.
2. The method of claim 1, wherein the influenza b virus comprises a Victoria sub-line and a Yamagata sub-line.
3. The method of claim 1, wherein the rare epitope is present in the HA amino acid sequence less than 3 times.
4. The method of claim 1, wherein the short peptide consists of 9 amino acids.
5. An influenza b virus recombinant HA antigen, characterized in that it is prepared by the construction method of any one of claims 1 to 4.
6. The recombinant HA antigen of influenza b virus of claim 5, wherein said recombinant HA antigen of influenza b virus comprises the recombinant HA antigen HAM-VIC of the Victoria sub-line and the recombinant HA antigen HAM-yac of the Yamagata sub-line.
7. The recombinant HA antigen of influenza b virus of claim 6 wherein said HAM-VIC HAs the amino acid sequence shown in SEQ ID No. 1; the amino acid sequence of the HAM-YAM is shown as SEQ ID NO. 2.
8. The recombinant pFastBac Dual-HAM plasmid is characterized in that the recombinant pFastBac Dual-HAM plasmid is obtained by retaining the extracellular region of the recombinant HA antigen of the influenza B virus, removing the signal peptide of the recombinant HA antigen of the influenza B virus, adding the gp67 signal peptide and optimizing according to the preferred codons of insect cells.
9. A recombinant shuttle vector Bacmid, comprising the recombinant pFastBac Dual-HAM plasmid of claim 8.
10. A method for preparing recombinant baculovirus expressing influenza b virus HAM recombinant protein, comprising the steps of:
(1) Obtaining pFastBac Dual-HAM plasmid for expressing HAM recombinant protein of influenza B virus by gene synthesis technology;
(2) Transforming the pFastBac Dual-HAM plasmid obtained in the step (1) into DH10Bac competent cells to obtain a recombinant shuttle vector Bacmid;
(3) Transfecting the recombinant shuttle vector Bacmid obtained in the step (2) into insect cells to obtain baculovirus;
(4) And (3) further purifying the baculovirus in the step (3) to obtain the recombinant baculovirus expressing the influenza B virus HAM recombinant protein.
11. The method according to claim 10, wherein the purification in the step (4) is performed by inoculating baculovirus into insect cells with good growth status, and culturing until the fourth generation baculovirus is obtained.
12. A recombinant baculovirus expressing influenza b virus HAM recombinant protein, prepared by the method of any one of claims 10 to 11.
13. The use of the recombinant baculovirus expressing the HAM recombinant protein of influenza b virus of claim 12 in the preparation of influenza b vaccine, medicament for preventing or treating influenza b.
14. An influenza b virus recombinant protein vaccine comprising the recombinant baculovirus of claim 12.
15. The influenza b virus recombinant protein vaccine of claim 14, further comprising an adjuvant.
CN202211099880.3A 2022-09-09 2022-09-09 Influenza B virus recombinant protein vaccine and preparation method thereof Pending CN116284432A (en)

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