CN116063409A - Influenza B Mosaic recombinant protein, recombinant plasmid, construction method and application thereof - Google Patents
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Abstract
The invention relates to the technical field of biology, and particularly discloses a influenza B Mosaic recombinant protein, a recombinant plasmid, a construction method and application thereof. Influenza B mosaics recombinant proteins comprising influenza viruses of the Victoria subfamily B and the Yamagata subfamily covered with a maximum of T cell epitopes are designed. The invention also adopts an insect cell-baculovirus expression system to construct a plurality of recombinant baculoviruses containing a mosaicHA sequence and influenza B virus B M1 protein genes, and then the recombinant baculoviruses are infected with Sf9 cells to obtain IBV VLPs capable of simulating the surface space conformation of the influenza virus in a natural state, and the IBV VLPs have better immunogenicity and safety and provide a new technical support for the research and development of novel influenza B vaccines and the effective prevention and control of the influenza B virus.
Description
Technical Field
The invention relates to the technical field of biology, in particular to influenza B Mosaic recombinant protein, recombinant plasmid, a construction method and application thereof.
Background
Currently, influenza b poses a serious threat to human public health safety, and some serious complications (such as viral myocarditis, encephalopathy, etc.) are also closely related to influenza b virus infection. Influenza vaccines are the most effective means of preventing influenza, and currently commercially available influenza vaccines are trivalent or tetravalent influenza vaccines. Trivalent inactivated influenza vaccine (TIV) was first introduced in the late 1970 s, since which it was widely used in many countries to prevent influenza infection. Trivalent vaccines include two types of A strains (A/H1N 1, A/H3N 2) and one type B strain (B/Victoria or B/Yamagata). The World Health Organization (WHO) scrutinizes circulating influenza strains twice a year and recommends the inclusion of these strains into TIVs to cope with the upcoming northern and southern hemisphere influenza seasons. Although influenza B viruses are classified as a single influenza type, but the two antigenically and genetically distinct lineages co-propagate, trivalent influenza vaccines include only one B-line and thus may not match the major circulatory line. In many countries, the mismatch between the B-type influenza pedigree and the circulating B-pedigree of TIV occurs approximately once every two to four years, with 2011-2019 of 8 years, the B-type influenza epidemic strain in China is mismatched with the B-type strain contained in the trivalent influenza vaccine in two years 2015-2016, 2017-2018, so that the effectiveness of the vaccine is greatly reduced. WHO proposed a proposal for seasonal influenza vaccine comprising two strains of strain B in month 2 2012, and began recommending tetravalent vaccines from the northern hemisphere influenza season of 2013-2014, comprising two strains of type a (a/H1N 1, a/H3N 2) and two strains of type B (B/Victoria, B/Yamagata) to improve vaccine matching. China's tetravalent vaccine is marketed in batches in 2018-2019, but the annual epidemic strain B is not matched with the antigenicity of the vaccine strain, which suggests that antigen drift may occur. Influenza b viruses exhibit less antigenic drift than influenza a viruses, but they effectively evade recognition by virus neutralizing antibodies present in the human population. Although the existing influenza vaccine on the market protects the masses from seasonal influenza to a certain extent, the effectiveness of the influenza vaccine depends on the matching degree of vaccine components and epidemic strains, and only the strains matched with the influenza vaccine can be protected, so that the influenza vaccine is far insufficient for coping with continuously mutated novel strains. Therefore, the development of new influenza vaccines is urgent. Currently, the general influenza vaccine (universal influenza vaccine) being developed is mainly studied as follows: the conservative B cell or T cell epitope on the viral protein is stimulated more by developing new adjuvants, new antigens, new vectors, new immunization strategies, etc.
Hemagglutinin (HA) is a glycoprotein on the surface of the viral membrane, HAs good immunogenicity, and the HA-induced neutralizing antibody can act on the virus at the first time of infection, combine with the virus, and inhibit virus invasion into cells. HA HAs therefore been a key antigen in the development of influenza vaccines, and researchers have performed various modifications on HA as immunogens, such as deletion of the head of HA, or removal of the stem segment of HA, or design of chimeric HA, or modification of the HA oligosaccharide group; the modified unmodified HA is expressed and modified by various living virus vectors, DNA vectors, VLPs and other systems, and different immune strategies such as prime-boost and the like are adopted, even the modified HA is combined with influenza virus or traditional influenza vaccine to stimulate the organism to generate broad-spectrum neutralizing antibodies, so that a broad-spectrum immune protection effect is obtained. The above attempts lay a solid foundation for the development of novel influenza vaccines and further determine the direction of research for inducing broad-spectrum and high-titer neutralizing antibodies as novel influenza vaccines.
The original purpose of Mosaic (mosaics) vaccine design is for the control of human immunodeficiency virus type 1 (HIV-1). Although scientists are developing treasures against AIDS all the day around, due to the rapid evolutionary ability and diversity of HIV-1, no good vaccine and medicine for preventing and treating AIDS exists so far. The mosaics vaccine is based on the diversity of virus genes, a Mosaic protein is combined from a large number of natural sequences to replace or represent all natural sequences, and the vaccine prepared after artificial optimization can induce organisms to generate wider protection. 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. Kamlangdee et al constructed a recombinant Ankara poxvirus vaccine (MVA-H5M) expressing H5N1Mosaic HA, and evaluated the protective effect on humans using mammals (mice and rhesus monkeys), which showed that the vaccine could protect H5 AIV from different clades and caused cross-protective effects on HIN1 in the body. Therefore, the antigen sequence which is designed by taking the Mosaic method as a core and covers the most T cell epitopes has the potential of preparing novel vaccines, and can induce organisms to generate wider protection.
Virus-like particles (VLPs) developed in the early century are a type of nanoscale scaffold produced using viral structural proteins, similar to viruses in many respects, the most important difference being that VLPs contain no genetic material and cannot replicate in host cells, overcoming the safety limitations of conventional vaccines and being a novel vaccine platform. The VLPs platform has made considerable progress in developing vaccines against human papillomaviruses, hepatitis b viruses, hepatitis e viruses, and malaria, and has been approved for commercial production. VLPs can self-assemble into spherical nanostructures of 20nm to 200nm, contain no viral genetic material inside, and have no infectivity and self-replication capacity, so that the VLPs are high in safety. The appearance morphologically maintains the structure of the native viral particles and displays epitopes in a high density manner, depending on the surface, size and shape of the VLPs, thereby effectively stimulating the immune system. The process of binding to cellular receptors after entry into the body is similar to that of viral infection: on the one hand, B cells are stimulated to mediate immune responses, and antibodies are generated to resist viruses; on the one hand, the CD4+ T cells are excited by the exogenous pathogenic way, and the pathogenic is presented to the CD8+ T cells through cross presentation to mediate the cytotoxicity reaction, so that the cellular immunity is induced, and the vaccine is a safe vaccine form. The vaccine prepared by the VLP expression system HAs the advantages of high expression level, strong immunogenicity, mature process, relatively simple preparation, high safety, small side effect and the like, so the VLP system expresses modified HA and induces a broad-spectrum neutralizing antibody by a prime-boost immune mode, which is probably the most promising general influenza vaccine development strategy at present.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a influenza B Mosaic recombinant protein, a recombinant plasmid, a construction method and application thereof.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the first object of the invention is to provide a influenza B Mosaic recombinant protein, which is:
(a) A protein consisting of the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No. 2; or alternatively, the process may be performed,
(b) The amino acid sequence in (a) is a protein formed by substituting, deleting or adding one or more amino acids.
The invention applies the design method of the Mosaic vaccine to the construction of the influenza B vaccine, combines a genetic algorithm to optimize a protein sequence, synthesizes the influenza B virus isolates which are 35811 strains in total after analysis, and designs the influenza B Mosaic recombinant protein (Mosaic HA sequence) which contains B type Victoria sub-line and Yamagata sub-line and HAs the maximum T cell epitope covered by the influenza virus, wherein the immune response effect of the influenza B Mosaic recombinant protein is better, the cross protection is good, and the safety is also better.
The second object of the invention is to provide a coding gene, wherein the coding gene comprises a coding gene for the influenza B Mosaic recombinant protein and a coding gene for the influenza B virus type M1 protein according to claim 1, and the nucleotide sequences of the coding gene are shown in SEQ ID NO. 3-5 respectively.
The third object is to provide a recombinant plasmid comprising the above influenza B mosaics recombinant protein.
As a preferred embodiment of the recombinant plasmid of the present invention, the above-mentioned influenza B Mosaic recombinant protein and influenza B virus type M1 protein are respectively ligated into expression vectors.
As a preferred embodiment of the recombinant plasmid of the present invention, the expression vector includes a pFastBac-Dual vector.
As a preferred embodiment of the recombinant plasmid of the present invention, the above influenza B Mosaic recombinant proteins are ligated into the multicloning sites before the p10 promoter of the pFastBac-Dual vector, respectively; the influenza B virus type B M1 protein was ligated into the pFastBac-Dual vector in the multiple cloning site after the pH promoter.
The fourth object of the present invention is to provide a cell comprising the recombinant plasmid described above.
As a preferred embodiment of the cell of the present invention, the cell is obtained by transferring the above recombinant plasmid into a host cell of E.coli. The E.coli host cell is an E.coli DH10Bac competent cell.
Fifth, the invention provides a construction method of influenza B Mosaic recombinant protein, comprising the following steps:
1) Inserting the influenza B Mosaic recombinant protein according to claim 1 into the multiple cloning site before the p10 promoter of the pFastBac-Dual vector, inserting the influenza B virus type B M1 protein into the multiple cloning site after the pH promoter of the pFastBac-Dual vector, and transforming by E.coli DH5 alpha competent cells to obtain pFastBac-Dual-HAM-YAM 1, pFastBac-Dual-HAM-VIC-M1 recombinant plasmids;
2) Transforming pFastBac-Dual-HAM-YAM-M1 and pFastBac-Dual-HAM-VIC-M1 recombinant plasmids respectively by escherichia coli DH10Bac competent cells to obtain recombinant baculovirus shuttle plasmids containing influenza B Mosaic recombinant proteins as defined in claim 1 and influenza B Mosaic recombinant proteins as defined in claim 1, namely Bacmid-HAM-YAM-M1 and Bacmid-HAM-VIC-M1 respectively;
3) And 2) respectively transfecting the Bacmid-HAM-YAM 1, the Bacmid-HAM-VIC-M1 and the empty stem grains in the step 2) into insect cells, collecting cell supernatant, harvesting P0 generation recombinant baculovirus, and then continuously subculturing to collect cell supernatant to obtain the influenza B Mosaic recombinant protein.
The invention adopts an insect cell-baculovirus expression system to construct a plurality of recombinant baculoviruses containing Mosaic HA (influenza B Mosaic recombinant protein) and M1 protein genes, and then the recombinant baculoviruses are infected with insect cells to obtain IBV VLPs capable of simulating the surface space conformation of the influenza viruses in a natural state. The IBV VLPs have better immunogenicity and safety, and provide new technical support for research and development of novel influenza B vaccines and effective prevention and control of the disease.
As a preferred embodiment of the method for constructing influenza B mosaics recombinant protein of the invention, the insect cells in the step 3) include Sf9 insect cells.
The sixth object is to provide a virus-like particle comprising the above influenza b mosaics recombinant protein or the recombinant plasmid.
The virus-like particles (VLPs) prepared by the invention have a spherical structure and a particle size of 100nm, and are similar to natural influenza viruses; the virus-like particles (VLPs) including YAM-VLP and VIC-VLP were striped at 100bp and 35-40bp, showing successful expression of both influenza B Mosaic recombinant protein and M1 protein.
The virus-like particles (VLPs) of the invention are capable of producing 2 10 In the control group (Mock), the generation of the blood coagulation phenomenon was not observed.
In different immunization formats, mosaic VLPs produce high levels of serum IgG-specific antibodies detectable against homologous BV or BY strains.
The seventh object of the invention is to provide the application of the above-mentioned influenza B Mosaic recombinant protein in preparing universal influenza B vaccine.
In an eighth aspect, the present invention provides a vaccine formulation comprising the influenza b Mosaic recombinant protein, the encoding gene, or the recombinant plasmid as described above.
The vaccine preparation composed of the influenza B Mosaic recombinant protein does not generate abnormal toxic reaction on mice, and has better safety. Vaccine formulations also have better immunogenicity.
As a preferred embodiment of the vaccine formulation according to the invention, the vaccine formulation further comprises an immunologically and pharmaceutically acceptable carrier or adjuvant.
The ninth object is to provide the application of the vaccine preparation in preparing a medicament for preventing and/or treating influenza B.
The influenza B VLP vaccine based on the mosaic strategy has the advantages of high yield and low production cost, and the produced influenza B virus-like particles have good immunogenicity and cross protection, can be used for preparing influenza vaccines in a large scale, have no biological safety risk and other problems, and can excite organisms to immunize against various influenza viruses. And animal experiments initially prove that the universal influenza B virus-like particle vaccine can provide safe and effective protection effects under a plurality of different vaccination modes.
Compared with the prior art, the invention has the following beneficial effects:
according to the invention, through a Mosaic vaccine design method and combining a genetic algorithm to optimize a protein sequence, a Mosaic HA sequence (namely the influenza B Mosaic recombinant protein) with maximum T cell epitopes covered by influenza viruses comprising a type B Victoria subfamily and a type Yamagata subfamily is designed. The invention also adopts an insect cell-baculovirus expression system to construct a plurality of recombinant baculoviruses containing a Mosaic HA sequence and influenza B virus B type M1 protein genes, and then the recombinant baculoviruses are infected with Sf9 cells to obtain IBV VLPs capable of simulating the surface space conformation of the influenza virus in a natural state. Animal experiments are carried out through various immunization approaches such as mucosal immunization (nasal cavity), systemic immunization (intramuscular injection, subcutaneous injection) and the like, so that the preliminary evaluation of the immunogenicity and the safety of IBV VLPs is finished, and a novel technical support is provided for the research and the development of novel influenza B vaccines and the effective prevention and control of the diseases.
Drawings
FIG. 1 is a schematic diagram showing the construction of recombinant plasmids pFastBac-Dual-HAM-YAM-M1, pFastBac-Dual-HAM-VIC-M1;
FIG. 2 is a diagram showing the electrophoretically identified pFastBac-Dual-HAM-YAM-M1 and pFastBac-Dual-HAM-VIC-M1 recombinant plasmids (left side shows the electrophoretically identified pFastBac-Dual-HAM-YAM-M1 recombinant plasmid; right side shows the electrophoretically identified pFastBac-Dual-HAM-VIC-M1 recombinant plasmid), wherein 1 represents plasmid, 2 represents plasmid digested with BamHI and EcoRI, and M represents KB labder);
FIG. 3 is a graph showing the result of Western Blot detection of recombinant protein expression (using Bacmid-HAM-YAM-M1) of the P2-generation baculovirus (in the figure, 1 represents the cell supernatant of the P1-generation baculovirus, 2 represents the cell supernatant of the P2-generation baculovirus, 3 represents the cell lysate of the P2-generation baculovirus, 4 represents the blank control, and M is the protein ladder);
FIG. 4 is a graph showing the result of Western Blot detection of recombinant protein expression (using Bacmid-HAM-VIC-M1) of the P2-generation baculovirus (in the figure, 1 represents the cell supernatant of the P1-generation baculovirus, 2 represents the cell supernatant of the P2-generation baculovirus, 3 represents the cell lysate of the P2-generation baculovirus, 4 represents a blank control, and M is a protein ladder);
FIG. 5 is a graph showing the expression of proteins in VLPs detected by Western Blot (1 represents YAM-VLP,2 represents YAM blank, 3 represents VIC-VLP,4 represents VIC blank, and M is protein ladder);
FIG. 6 is a morphology of YAM-VLPs viewed by transmission electron microscopy;
FIG. 7 is a morphology of the VIC-VLP as observed by transmission electron microscopy;
FIG. 8 is a graph showing the results of the hemagglutination assay for VLPs;
FIG. 9 is a graph showing the results of different immunization of mice against the production of antibodies against B/Phuket/3073/2013 strain hemagglutination inhibition;
FIG. 10 is a graph showing the results of various immunization of mice against the production of antibodies against hemagglutination inhibition of the B/Washington/02/2019 strain;
FIG. 11 is a graph showing the results of different immunization of mice against the production of antibodies against hemagglutination inhibition of the B/Colorado/06/2017 strain;
FIG. 12 is a graph showing the results of different immunization of mice against B/Brisbane/60/2008 strain hemagglutination inhibition antibodies;
FIG. 13 is a graph showing the results of various immunization of mice against B/Massachusetts/2/2012 strain hemagglutination inhibition antibodies;
FIG. 14 is a graph showing the results of different immunization of mice against the production of antibodies against hemagglutination inhibition of B/Wisconsin/1/2010 strain;
FIG. 15 is a graph I showing the results of different immunization of mice with specific antibodies;
FIG. 16 is a graph II showing the results of different immunization of mice with specific antibodies.
Detailed Description
For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments.
In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are commercially available.
Example 1, sequence design, optimization and screening of influenza B Mosaic recombinant protein
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 BV HA amino acid sequences and 16614 BY HA amino acid sequences respectively, and removing sequences with poor repeated sequence and sequencing quality BY using computer algorithm and software to obtain 4122 BV HA amino acid sequences and 2745 BY HA amino acid sequences. Uploading the amino acid sequence obtained by screening to Mosaic Vaccine Designer website (https:// www.hiv.lanl.gov/content/sequence/mosai/makevaccine. Html) in FAS format, and setting parameters: the Cocktail Size is set to "1" to obtain 1 mosaics sequence for the next step; epitope length was set to "12" to obtain a Mosaic sequence covering more cd4+ Th 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 12 amino acids are finally obtained; and (3) optimizing each population sequentially by using a genetic algorithm, wherein new recombinants are generated, the epitope coverage rate of the new recombinants is calculated and tested, and finally 2 optimal Mosaic HA amino acid sequences (influenza B Mosaic recombinant proteins) are obtained after epitope prediction, genetic evolution analysis and spatial conformation analysis, wherein the amino acid sequences are respectively shown as SEQ ID NO. 1-2.
Example 2 construction method of influenza B Mosaic recombinant protein
The construction method of the influenza B Mosaic recombinant protein comprises the following steps:
(1) After optimizing according to insect cell preference codons, obtaining HAM-VIC and HAM-YAM proteins (namely influenza B Mosaic recombinant proteins with amino acid sequences of SEQ ID NO:1 and SEQ ID NO: 2) and encoding genes of M1 proteins of influenza B virus B/Anhui-Baohe/127/2015 (AH 127/15) by a gene synthesis technology, wherein the nucleotide sequences of the encoding genes are respectively shown as SEQ ID NO: 3-5;
(2) And (2) carrying out multiple cloning site analysis on the coding genes of the HAM-VIC, HAM-YAM and M1 proteins in the step (1) and the genes of the pFastBac-Dual vector respectively, selecting two restriction endonuclease sites (BamHI and EcoRI) which are provided on the pFastBac-Dual vector and are not provided on the target fragment, amplifying and recycling the target fragment, and then utilizing the characteristics of the pFastBac-Dual bicistronic baculovirus to insert the target fragments HAM-VIC and HAM-YAM into the multiple cloning sites before the p10 promoter of the pFastBac-Dual vector respectively, and inserting the target fragment M1 protein into the multiple cloning sites after the pH promoter of the pFastBac-Dual vector, and transforming the pFastBac-Dual-HAM-YAM 1 and pFastBac-Dual-YAM-1 plasmid through escherichia coli DH5 alpha competent cells to obtain 2 recombinant plasmids. The construction schematic diagram of the recombinant plasmid is shown in figure 1, and the electrophoresis of the recombinant plasmid is shown in figure 2;
(3) Transforming the recombinant plasmids (pFastBac-Dual-HAM-YAM-M1 and pFastBac-Dual-HAM-VIC-M1) obtained in the step (2) by using escherichia coli DH10Bac competent cells to obtain recombinant baculovirus shuttle plasmids containing HAM-VIC, HAM-YAM and M1 proteins, namely Bacmid-HAM-YAM-M1 and Bacmid-HAM-VIC-M1;
(4) Respectively transfecting the Bacmid-HAM-YAM-M1, the Bacmid-HAM-VIC-M1 and the empty stem grains (serving as blank control) in the step (3) into Sf9 insect cells with good growth state, and collecting cell supernatant after the cells show signs of infection, thus obtaining the P0 generation recombinant baculovirus; and (3) inoculating the P0 generation recombinant baculovirus according to 3-4% of the volume of the cell supernatant, culturing the virus in a cell culture box at the temperature of 27 ℃ for about 4 days, continuously carrying out passage under the same culture conditions until the P2 generation recombinant baculovirus is harvested, collecting the cell supernatant, and carrying out Western Blot detection on the expression of the recombinant protein after the cell is lysed.
The cell supernatant of the P1 generation recombinant baculovirus, the cell supernatant of the P2 generation recombinant baculovirus and the cells thereof are subjected to Western Blot detection after being lysed, and the result is shown in figures 3-4, the protein expression amount of the P1 generation recombinant baculovirus is less, and the P2 generation recombinant baculovirus can effectively express the corresponding recombinant protein.
EXAMPLE 3 construction methods of mosaics VLPs
The method for constructing the mosaicvlps of the embodiment comprises the following steps:
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; concentrating the obtained supernatant by a vivaflow2000 membrane bag, centrifuging at 10000rpm for 10min at 4deg.C, collecting supernatant, ultracentrifugating at 30000rpm for 2h at 4deg.C, discarding supernatant, adding 2mL PBS, re-suspending the precipitate, and standing at 4deg.C overnight; 20%,30% and 60% sucrose solutions were prepared. Sucrose was slowly added to the ultracentrifuge tube with a puncture needle in order of 20%,30% and 60%, and finally 20% sucrose solution was suspended to the top, and then the resuspended pellet solution was added to the top layer, and ultracentrifugation was performed at 30000rpm for 2 hours at 4 ℃. The milky liquid between 30% and 60% sucrose solution was slowly aspirated with a 1mL pipette, the collected milky liquid diluted with sterile PBS and ultracentrifuged at 30,000rpm for 2 hours at 4 ℃. The centrifugate supernatant containing sucrose was discarded, the centrifugated pellet was resuspended in 2mL of sterile PBS, and the resuspension was the purified mosaic VLPs (i.e., YAM-VLPs and VIC-VLPs), and the suspension was sub-packaged to-80℃for storage.
1. And (3) transmission electron microscope observation:
purified YAM-VLPs and VIC-VLPs were diluted 20-fold and then added dropwise to the prepared copper mesh, and incubated at room temperature for 5 minutes. And then, lightly sucking the excessive liquid on the copper mesh by using water absorbing paper, dripping 1% phosphotungstic acid dye liquor for dyeing for 3 minutes after drying, then, slowly absorbing the excessive phosphotungstic acid on the copper mesh by using the water absorbing paper, drying at room temperature, and finally, observing the morphology of VLPs by using a transmission electron microscope JEM-1400. As shown in FIGS. 6-7, IBV VLPs with spherical structures and sizes around 100nm similar to the natural influenza virus were observed, indicating successful assembly of YAM-VLPs and VIC-VLPs.
2. Western Blot detection:
further using Western Blot to detect the expression of each protein in YAM-VLP and VIC-VLP, 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) The HA antibody against Influenza B Virus (available from GeneTex under the designation GTX 128543) and the M1 antibody against Influenza B Virus (available from Santa under the designation SC-57886) were each prepared according to a 1:5000 and 1:800, the membrane is cut off due to different antibodies, and primary antibodies are respectively added at 4 ℃ overnight; 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 and goat anti-rabbit IgG antibodies (both purchased from Friedel creatures, the product numbers are FDM007 and FDR007 respectively) with 1:10000 times dilution 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. Western Blot results are shown in FIG. 5, YAM-VLP and VIC-VLP are banded at 100bp and 35-40bp, and HA protein and M1 protein in both mosaics VLP are identified to be successfully expressed.
3. Erythrocyte agglutination assay detection:
hemagglutination titers of 1% chicken erythrocytes were measured on cell culture supernatants of purified YAM-VLPs and VIC-VLPs with a blank control (blank control used in step 4 of example 2) as follows:
preparing 1% chicken erythrocyte suspension, adding 50 mu L of PBS into a 96-well plate, adding 50 mu L of YAM-VLP or VIC-VLP into a first well, blowing uniformly, sucking out 50 mu L to a 2 nd well, diluting to an 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. 8, VLPs were observed to be capable of producing 2 10 While no hemagglutination was observed in the blank (Mock), further demonstrating that the purified virus-like particles could be subjected to immunogenicity studies.
EXAMPLE 4 immune Effect and safety experiments of mosaics VLPs
BALB/c mice were immunized with two mosaic VLPs (YAM-VLP and VIC-VLP) obtained by expression and purification in example 3 as immunogens, and the immunization effect of the obtained mosaic VLPs was examined.
Immunized mice:
1. immune effect evaluation group:
protein concentrations of the two mosaic VLPs were detected separately using a BCA detection kit (purchased from Biyundian Co., ltd., cat. No. P0012), and uniformly mixed at a concentration ratio of 1:1, and the mixed VLPs were uniformly mixed with 10 ten thousand units/mL of IL-2 and 0.1% chitosan, to obtain mosaic VLPs vaccine.
20 BALB/c female mice with the age of 6-8 weeks are randomly divided into 4 groups, namely blank control groups: intramuscular injection of 100 μl PBS; VLP nasal drop immunization group (IN): nasal drip immunization 50 μl of vaccine containing 15 μg of mosaicvlps; VLP intramuscular injection group (IM): intramuscular injection of 100 μl of vaccine containing 15 μg of mosaicvlps; VLP intraperitoneal injection group (IP): 100. Mu.L of vaccine containing 15. Mu.g of mosaicVLPs was intraperitoneally injected;
2. safety evaluation group:
vaccine preparation and immunization protocol grouping were the same as the immunization efficacy evaluation group, but the immunization dose was raised to 100 μg of mosaicvlps in the injection volume, and safety of the vaccine was tested by overdose. All mice were weighed before and after immunization and survival was calculated.
As shown in Table 1, the vaccine of mosaicVLPs did not produce abnormal toxic reaction to mice, and had good safety.
TABLE 1
3. Hemagglutination inhibition (hemagglutination inhibiion, HAI) experiments:
preparation of RDE treated mouse serum: the receptor destroying enzyme (RDE, purchased from Japanese Kogyo under the trade designation 340122) was mixed with serum of each group of mice in a volume ratio of 3:1 in a test tube, and placed in a 37℃water bath for 16 hours; taking out the test tube, and placing the test tube in a water bath at 56 ℃ for 30min to inactivate RDE; PBS was added to the tube to achieve a serum dilution of 1:5; cooling to room temperature, adding 1/2 volume of chicken red blood cells of original serum, mixing, standing at 4deg.C for 1 hr, and mixing again every 15min; centrifuging at 1200rpm for 1min, absorbing supernatant to obtain RDE treated mouse serum, and standing at 4deg.C for use.
Preparation of four standard antigens: HA titers of B/phyket/3073/2013, B/Massachusetts/2/2012, B/Wisconsin/1/2010 (3 types of B-type Yamagata sublines strains presented by the chinese disease prevention control center), B/Washington/02/2019, B/Colorado/06/2017, B/Brisbane/60/2008 (3 types of B-type Victoria sublines presented by the chinese disease prevention control center) were detected respectively, each antigen was diluted with PBS to 8 hemagglutination units, HA titer confirmation was performed again, and further diluted to 4 hemagglutination units, to obtain four-unit standard antigens.
Hemagglutination inhibition assay: 25. Mu.L of PBS was added to each of columns 2-10 and 12 of the 96-well plate, and 50. Mu.L of PBS was added to each of column 11 of the 96-well plate; adding 25 mu L of mouse serum treated by RDE into each of the column 1 and the column 2, and uniformly mixing; sucking 25 mu L of the mixed solution in the 2 nd column, adding the mixed solution into the 3 rd column, and uniformly mixing; repeating the above operation until column 10, and discarding 25 μl of the solution mixed in column 10; adding 25 mu L of four-unit standard antigen into the 1 st to 10 th columns and the 12 th column, wherein the 12 th column is used as a virus control column, and simultaneously adding positive serum (mouse serum obtained in the earlier stage of a laboratory) into the 11 th column to make a standard positive control; after fully and evenly mixing, standing the 96-well plate at room temperature for 45min; 50 mu L of 1% chicken erythrocyte suspension is added into each hole, the mixture is kept stand at room temperature for 25min, a 96-well plate is inclined at 45 ℃, and whether erythrocyte flows in a teardrop shape or not is observed.
The standards of the European Union drug evaluation and U.S. food and drug administration require that after influenza vaccination: (1) Hemagglutinin inhibition (Hemagglutination inhibition, HI) antibody is not less than 1:40; (2) Serum positive transfer rate, i.e., pre-immunization HI antibody <1:10, post-immunization HI antibody > 1:40, or pre-immunization HI antibody > 1:10, post-immunization HI antibody Geometric Mean Titers (GMT) increased 4-fold or more.
According to the HI experiment, the effect of different immunization modes on the positive transfer rate of serum HI antibodies after immunization of the divalent virus-like particle vaccine of the seasonal influenza B virus was calculated.
As shown in FIGS. 9-14, the immune response effects of the mosaicVLPs vaccine in different immunization modes are even far superior to those of the influenza vaccine.
4. Specific IgG antibody detection:
the purified inactivated B/Phuket/3073/2013 (BY strain), B/Washington/02/2019 (BV strain) viruses were diluted to 5. Mu.g/mL with ELISA coating solution (purchased from Soy pal under the trade designation C1050), added to 100ul of each well in a 96-well plate, and incubated overnight at 4 ℃; taking out 96-well plates the next day, discarding the solution in each well, washing with PBST for 6 times, each time for 3min; PBST containing Tween 20 at 0.05v/v% and BSA at 1w/v% was added to the 96-well plate, 200. Mu.L per well, and blocked at room temperature for 2 hours; the solution in each well was discarded and washed 6 times with PBST for 3min each time; 100 mu L of diluted serum samples (the serum obtained at the 4 th week after the first immunization is respectively 1000 times, 4000 times, 16000 times, 64000 times, 256000 times and 1024000 times, PBS is used as a diluent) are added into a 96-well plate, 3 duplicate wells are made for each serum sample, and the mixture is incubated for 2 hours at room temperature; the solution in the 96-well plate was discarded, and washed with PBST 6 times for 3min each time; mu.L of HRP-labeled goat anti-mouse IgG (purchased from southern Biotech, cat# 1036-05) diluted 1:8000 with PBST containing 2w/v% BSA was added and incubated for 1h at room temperature; washing with PBST for 6 times, each time for 3min, adding 100 mu LTMB staining solution into each hole, and standing at room temperature for 30min; mu.L of 2M H was added to each well 2 SO 4 The reaction was stopped and immediately the absorbance at 450nm (OD 450 nm)。
As a result, as shown in FIGS. 15-16, the mosaicVLPs produced detectable high levels of serum IgG-specific antibodies against homologous BV or BY strains in various immunization formats after 4 weeks of initial immunization.
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 the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.
Claims (15)
1. The influenza B Mosaic recombinant protein is characterized in that the influenza B Mosaic recombinant protein is:
(a) A protein consisting of the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No. 2; or alternatively, the process may be performed,
(b) The amino acid sequence in (a) is a protein formed by substituting, deleting or adding one or more amino acids.
2. The coding gene is characterized by comprising a coding gene for the influenza B Mosaic recombinant protein and a coding gene for the influenza B virus type M1 protein according to claim 1, wherein the nucleotide sequences of the coding gene are shown in SEQ ID NO. 3-5.
3. A recombinant plasmid comprising the influenza b Mosaic recombinant protein of claim 1.
4. The recombinant plasmid according to claim 3, wherein the influenza B Mosaic recombinant protein and the influenza B M1 protein according to claim 1 are each ligated into an expression vector.
5. The recombinant plasmid of claim 4 wherein the expression vector comprises a pFastBac-Dual vector.
6. The recombinant plasmid according to claim 4, wherein the influenza b Mosaic recombinant protein according to claim 1 is ligated into the multicloning site before the p10 promoter of the pFastBac-Dual vector; the influenza B virus type B M1 protein was ligated into the pFastBac-Dual vector in the multiple cloning site after the pH promoter.
7. A cell comprising the recombinant plasmid of any one of claims 3-6.
8. The cell of claim 7, wherein the cell is obtained by transferring the recombinant plasmid of any one of claims 3-6 into an e.
9. The construction method of the influenza B Mosaic recombinant protein is characterized by comprising the following steps of:
1) Inserting the influenza B Mosaic recombinant protein according to claim 1 into the multiple cloning site before the p10 promoter of the pFastBac-Dual vector, inserting the influenza B virus type B M1 protein into the multiple cloning site after the pH promoter of the pFastBac-Dual vector, and transforming by E.coli DH5 alpha competent cells to obtain pFastBac-Dual-HAM-YAM 1, pFastBac-Dual-HAM-VIC-M1 recombinant plasmids;
2) Transforming pFastBac-Dual-HAM-YAM-M1 and pFastBac-Dual-HAM-VIC-M1 recombinant plasmids respectively by escherichia coli DH10Bac competent cells to obtain recombinant baculovirus shuttle plasmids containing influenza B Mosaic recombinant proteins as defined in claim 1 and influenza B Mosaic recombinant proteins as defined in claim 1, namely Bacmid-HAM-YAM-M1 and Bacmid-HAM-VIC-M1 respectively;
3) And 2) respectively transfecting the Bacmid-HAM-YAM 1, the Bacmid-HAM-VIC-M1 and the empty stem grains in the step 2) into insect cells, collecting cell supernatant, harvesting P0 generation recombinant baculovirus, and then continuously subculturing to collect cell supernatant to obtain the influenza B Mosaic recombinant protein.
10. The method of claim 9, wherein the insect cells in step 3) comprise Sf9 insect cells.
11. A virus-like particle comprising the influenza b Mosaic recombinant protein of claim 1 or the recombinant plasmid of any one of claims 3-6.
12. The use of the influenza b Mosaic recombinant protein of claim 1 for preparing an influenza b universal vaccine.
13. A vaccine formulation comprising the influenza b Mosaic recombinant protein of claim 1, the encoding gene of claim 2, or the recombinant plasmid of any one of claims 3-6.
14. The vaccine formulation of claim 13, further comprising an immunologically and pharmaceutically acceptable carrier or adjuvant.
15. Use of a vaccine formulation according to claim 13 or 14 in the manufacture of a medicament for the prophylaxis and/or treatment of influenza b.
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