CN114230671B - Zika virus recombinant protein vaccine and preparation method thereof - Google Patents

Zika virus recombinant protein vaccine and preparation method thereof Download PDF

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CN114230671B
CN114230671B CN202111374523.9A CN202111374523A CN114230671B CN 114230671 B CN114230671 B CN 114230671B CN 202111374523 A CN202111374523 A CN 202111374523A CN 114230671 B CN114230671 B CN 114230671B
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zika virus
recombinant protein
zediii
rhf
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崔宗强
荣恒
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Wuhan Institute of Virology of CAS
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Abstract

The invention discloses a Zika virus recombinant protein vaccine and a preparation method thereof, the constructed Zika virus recombinant protein vaccine is zEDIII-rHF recombinant protein with an amino acid sequence shown as SEQ ID NO.2, and the gene sequence is shown as SEQ ID NO. 1. The third domain (zEDIII) of the envelope protein (envelop) of the Zika virus is displayed on the surface of a cage structure of recombinant human heavy chain ferritin (rHF). The zEDIII-rHF recombinant protein nanoparticle provided by the invention can induce high-strength humoral immunity and cellular immunity in mice, protect the mice from being infected by the lethal dose of Zika virus, has simple preparation process, does not involve live virus culture, has good safety and high product yield, is suitable for large-scale production, and provides a new technical means and technical strategy for the development of Zika virus vaccines.

Description

Zika virus recombinant protein vaccine and preparation method thereof
Technical Field
The invention relates to the technical field of vaccines, in particular to a recombinant protein specifically induced to Zika virus, and a coding gene, an expression vector and a preparation method thereof.
Background
Zika Virus belongs to the Flaviviridae, genus Flaviviridae, a arbovirus transmitted by mosquitoes. Reported studies indicate that zika virus infection can lead to neurological and autoimmune system complications and is associated with neonatal small head deformity.
Zika virus was first found in Uganda Zika jungle in 1947, and subsequently has been reported in Africa, american, asia and Pacific areas. In 2013 and 2015, the farnesian brinesian and brazil outbreaks, respectively, caused about 44-150 ten thousand infections in total. So far, more than 80 countries and regions worldwide have been reported with the Zika virus cases. Zika virus has become a pathogen that seriously jeopardizes human health and poses a significant threat to global public health.
At present, no effective vaccine against the Zika virus exists, and no specific medicine can prevent and treat the Zika virus infection. The research and development of Zika virus vaccine is mainly in preclinical and clinical experimental research stage. The Zhai Ka virus candidate vaccine types mainly comprise full-inactivated vaccine, attenuated live vaccine, nucleic acid vaccine and virus vector vaccine, and the organism is protected from Zhai Ka virus infection mainly by inducing the organism to generate Zhai Ka virus specific neutralizing antibodies.
However, the existing Zika virus candidate vaccine still has a plurality of defects, such as the need of large-scale culture of highly pathogenic viruses in the preparation process of inactivated vaccines and attenuated live vaccines, high preparation environment requirements and potential infection risks; attenuated live vaccines cause transient hypoviremia, and it is reported in the literature that congenital village-card syndrome is thought to occur even in asymptomatic infected pregnant women with hypoviremia, so the safety of attenuated live vaccines needs to be carefully assessed before they can be used in women of childbearing age; the nucleic acid vaccine and the viral vector vaccine have complicated preparation process and high technological requirements, and have strict requirements on transportation and storage conditions of products. If the Zhai Ka virus epidemic outbreaks are encountered, it is difficult to timely produce sufficient vaccine to cope with epidemic spread.
In addition, patent document CN111514286a discloses a "zhai ka virus E protein combined vaccine and a preparation method thereof", which adopts the technical route that beta-glucan and zhai ka virus E protein are covalently combined through a connecting bridge, and the treatment through chemical reaction is needed, so that the process is relatively complex.
Disclosure of Invention
The invention provides a Zika virus recombinant vaccine and a preparation method thereof, which utilize the third structural domain (zEDIII) of Zika virus envelope protein (envelop) to display on the surface of the cage structure of recombinant human heavy chain ferritin (rHF), and can effectively interact with human lymphocytes such as B cells and CD4 + T lymphocytes, CD8 + The rHF specific binding site on the surface of the T lymphocyte is identified, a virus vector or a virus inactivation mode is not utilized, the safety is good, the preparation process requirement is low, and the transportation and the preservation of recombinant proteins are convenient.
The invention provides the following technical scheme:
the invention provides a Zika virus recombinant protein vaccine, wherein the recombinant protein is the peptide of a third structural domain of Zika virus E protein and is displayed on the surface of recombinant human heavy chain ferritin.
Further, the protein is a zEDIII peptide segment of a third structural domain of a recombinant human heavy chain ferritin protein E protein which is connected in series at the amino terminus, and the zEDIII peptide segment of the third structural domain of the Zika virus E protein is expressed on the surface of the recombinant human heavy chain ferritin protein in a linear mode; the Zika virus recombinant protein vaccine specifically induces and generates IgG antibodies to the zEDIII antigen of the third structural domain of the Zika virus E protein.
Further, the recombinant protein self-assembles into a cage-shaped hollow protein nanoparticle structure; further, the cage-shaped hollow protein nano-particles have an outer diameter of 12nm and an inner diameter of 6-8 nm.
Further, the amino acid sequence of the protein is shown as SEQ ID NO. 2.
The nucleotide sequence of the gene for encoding the Zika virus recombinant protein vaccine is shown as SEQ ID NO. 1.
An expression vector comprising the gene encoding the recombinant protein vaccine of the Zika virus as described above.
Further, the construction method adopts a forward primer sequence shown as SEQ ID NO.3 and a reverse primer sequence shown as SEQ ID NO. 4:
SEQ ID NO.3：5'-CGCGGATCCATGGATAAACTTAGATTGAAGGG-3'；
SEQ ID NO.4：5'-CCCAAGCTTTTAGCTTTCATTATCACTGTCTCC-3'。
further, the expression vector skeleton is pET28a escherichia coli expression plasmid.
The invention also provides a preparation method of the recombinant protein specifically induced to the Zika virus, which comprises the following steps:
1) Inserting a nucleotide sequence shown in SEQ ID NO.1 into an escherichia coli expression plasmid to obtain a recombinant plasmid pET28a/zEDIII-rHF;
2) Expressing the recombinant plasmid pET28a/zEDIII-rHF in an escherichia coli prokaryotic expression system to obtain the recombinant protein which is specifically induced to be directed against the Zika virus, namely the Zika virus nano vaccine containing zEDIII-rHF recombinant protein nano particles.
Furthermore, the recombinant plasmid pET28a/zEDIII-rHF in the step 2) is expressed in a prokaryotic expression system of escherichia coli, and is subjected to first purification by a nickel column and then second purification by a molecular sieve.
The invention also provides a combined vaccine containing the Zika virus recombinant protein vaccine, which comprises the preparation of the Zika virus vaccine by combining the zEDIII-rHF recombinant protein vaccine, namely the zEDIII-rHF recombinant protein nanoparticle and other adjuvants, or the preparation of the multi-linked vaccine by combining the zEDIII-rHF recombinant protein nanoparticle and other active ingredients.
The beneficial effects of the invention are as follows:
1. compared with the traditional method for preparing the vaccine by using cell amplified virus, the method for preparing the vaccine by using the escherichia coli prokaryotic system has the advantages of simple operation and low cost, is suitable for large-scale production and culture without involving live virus, has high overall operation safety and does not have potential infection risk.
2. The recombinant human heavy chain ferritin is selected as an antigen display carrier, has low self-immunogenicity and good biocompatibility, and the ferritin nanocage display mode greatly increases the display quantity of antigen epitopes, thereby being beneficial to inducing and generating strong T cell dependent antibody reaction. In addition, the ferritin has the function of an adjuvant, so that the immunogenicity of the zEDIII antigen is obviously improved, and no additional adjuvant is needed in the immunization process, thereby simplifying the vaccine preparation process and reducing the risk of anaphylactic reaction caused by the adjuvant.
3. Under physiological conditions, the zEDIII-rHF recombinant protein nanoparticle provided by the invention can induce high-strength humoral immunity and cellular immunity by immunizing mice through an intramuscular injection way, and protect the mice from being infected by lethal dose of Zika virus. The invention adopts the expression of the zEDIII peptide segment of the third structural domain of the Zika virus E protein in a linear mode and displays the zEDIII peptide segment on the surface of the recombinant human heavy chain ferritin, and can effectively interact with human lymphocytes such as B cells and CD4 + T lymphocytes, CD8 + The rHF specific binding site on the surface of the T lymphocyte is identified, self-assembled ferritin is adopted to display conservative antigen peptide, and a new technical means and a new technical strategy are provided for the development of Zika virus vaccines.
Drawings
The invention will be described in more detail hereinafter on the basis of embodiments and with reference to the accompanying drawings. Wherein:
FIG. 1 is a schematic diagram of the construction of the expression sequence of the zEDIII-rHF recombinant protein in example 1;
FIG. 2 is a schematic diagram of the assembly of zEDIII-rHF recombinant protein nanoparticles in example 1;
FIG. 3 is a SDS-PAGE electrophoresis of the purified zEDIII-rHF recombinant protein of test example 1;
FIG. 4 is a western blot identification chart of the purified zEDIII-rHF recombinant protein of test example 1;
FIG. 5 is a graph showing transmission electron microscopy characterization of zEDIII-rHF assembled nanoparticles and rHF nanoparticles of test example 1;
FIG. 6 is a graph showing the statistics of the change in the titers of the zEDIII-specific IgG antibodies in the immune serum of test example 2;
FIG. 7 is a statistical plot of the percentage of CD4+ and CD8+ T cells in spleens of mice post immunization in test example 2;
FIG. 8 is a statistical plot of the number of lymphocytes secreted by IL-4-and IFN-gamma in the spleen of mice after immunization in test example 2;
FIG. 9 is a chart showing statistics of virus titers in various tissues after infection with Zika virus (SZ-WIV 01) in test example 3;
FIG. 10 is a statistical graph showing the weight change of mice infected with Zika virus (SZ-WIV 01) in test example 4;
FIG. 11 is a statistical plot of survival of mice infected with Zika virus (SZ-WIV 01) in test example 4.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
In the examples of the present invention, all raw material components are commercially available products well known to those skilled in the art unless specified otherwise; in the embodiments of the present invention, unless specifically indicated, all technical means used are conventional means well known to those skilled in the art.
Example 1
The embodiment provides a preparation method of zEDIII-rHF recombinant protein nano-particles serving as a Zika virus nano-vaccine, wherein the zEDIII-rHF recombinant protein nano-particles can also be called as zEDIII-rHF nano-vaccine, are recombinant proteins specifically induced to the Zika virus, and comprise the following steps:
1) Constructing zEDIII-rHF recombinant protein expression plasmid pET28a/zEDIII-rHF;
PCR amplification rHF (Gene ID: 2495) sequence: plasmid pET-rHF (offered by Dr Paolo Santambrogio (Milan, italy)) encoding the H chain sequence of human ferritin (rHF) was used as template (nanoscales, 2012,4,188)
sense primer：5'C GAGCCACCCCCTCCTGAGCCACCCCCTCCTATGACGACCGCGTCCACCTC C 3'，reverse primer：5'CCCAAGCTTTTAGCTTTCATTATCACTGTCTCC 3'；
PCR amplification of the zEDIII sequence: the nucleotide sequence of zEDIII was found in NCBI (Gene ID: KU 963796.1), and the complete genome of the Zika virus was extracted from the strain deposited with the Wuhan Virus research Virus Bank of China department (SZ-WIV 01). Using the extracted whole genome sequence as a template, sense primer:5'CGCGGATCCATGGATAAACTTAGATTGAAGGG 3', reverse primer:5'AGGAGGGGGTGGCTCAGGAGGGGGTGGCTCGGTGCTGCCACTCCTGTGCC 3'.
The amino acid sequence of zEDIII is: DKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGST, and the amino acid sequence of the zEDIII is identical with that of a virus strain of a virus library (SZ-WIV) of the Wuhan virus of China academy of sciences.
2) The primer of zEDIII-rHF is amplified by using PCR products rHF and zEDIII as templates, as shown in figure 1, and by using overlay-PCR: 5'CGCGGATCCATGGATAAACTTAGATTGAAGGG 3', reverse primer:5 'CCCAAGCTTTTTAGCTTTATTATCATGTCTCC 3' to obtain the zEDIII-rHF nucleotide sequence shown in SEQ ID No. 1.
SEQ ID NO.1：
ATGGATAAACTTAGATTGAAGGGCGTGTCATACTCCTTGTGTACCGCAGCGTTCACATTCACCAAGATCCCGGCTGAAACACTGCACGGGACAGTCACAGTGGAGGTACAGTACGCAGGGACAGATGGACCTTGCAAGGTTCCAGCTCAGATGGCGGTGGACATGCAAACTCTGACCCCAGTTGGGAGGCTGATAACCGCTAACCCCGTAATCACTGAAAGCACTGAGAACTCCAAGATGATGCTGGAACTTGATCCACCATTTGGGGACTCTTACATTGTCATAGGAGTCGGGGAGAAGAAGATCACCCACCACTGGCACAGGAGTGGCAGCACCGAGCCACCCCCTCCTGAGCCACCCCCTCCTATGACGACCGCGTCCACCTCGCAGGTGCGCCAGAACTACCACCAGGACTCAGAGGCCGCCATCAACCGCCAGATCAACCTGGAGCTCTACGCCTCCTACGTTTACCTGTCCATGTCTTACTACTTTGACCGCGATGATGTGGCTTTGAAGAACTTTGCCAAATACTTTCTTCACCAATCTCATGAGGAGAGGGAACATGCTGAGAAACTGATGAAGCTGCAGAACCAACGAGGTGGCCGAATCTTCCTTCAGGATATCAAGAAACCAGACTGTGATGACTGGGAGAGCGGGCTGAATGCAATGGAGTGTGCATTACATTTGGAAAAAAATGTGAATCAGTCACTACTGGAACTGCACAAACTGGCCACTGACAAAAATGACCCCCATTTGTGTGACTTCATTGAGACACATTACCTGAATGAGCAGGTGAAAGCCATCAAAGAATTGGGTGACCACGTGACCAACTTGCGCAAGATGGGAGCGCCCGAATCTGGCTTGGCGGAATATCTCTTTGACAAGCACACCCTGGGAGACAGTGATAATGAAAGCTAA。
The amplification conditions were: denaturation at 98℃for 30s, annealing at 60℃for 30s, extension at 72℃for 30s, amplification for 30 cycles, and storage at 16 ℃. The amplified product is subjected to BamHI and HindIII double enzyme digestion, and then the zEDIII-rHF sequence is inserted into an expression vector pET28a, so that a zEDIII-rHF recombinant protein expression plasmid pET28a/zEDIII-rHF is constructed. The sequence of the target gene is verified to be correct by sequencing.
3) pET28a/zEDIII-rHF is constructed in E.coli BL21 (lambda DE 3) expression system to express zEDIII-rHF recombinant protein. As shown in FIG. 2, the assembly of the zEDIII-rHF recombinant protein nanoparticle is schematically shown.
Culture conditions: volume percentage 1:100 is inoculated into thalli, LB culture is carried out for 4-6 hours until OD600 is between 0.4 and 0.6 based on 37 ℃, then the thalli is placed at 20 ℃ and is further induced for 18 hours by using IPTG with the final concentration of 1mM, and then the thalli are centrifugally collected and crushed under high pressure. The inclusion bodies were subjected to renaturation dialysis using 8M urea-denatured neutral buffer. The first purification step was performed using a nickel column that can bind a 6-his tag, and Superose 6 molecular sieve column SEC (GE Healthcare, uppsala, sweden) was further purified. The purified protein was quantified using BCA protein assay kit (Beyotime, china) and endotoxin concentrations were measured using ToxinSensor Single Tests kit (GenScript, US).
The amino acid sequence of the zEDIII-rHF recombinant protein (specifically inducing recombinant protein aiming at Zika virus) is shown as SEQ ID NO. 2. After induction purification, 1L of the thalli can be purified to obtain 0.5mg of zEDIII-rHF recombinant protein.
SEQ ID NO.2：
MDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTEPPPPEPPPPMTTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGRIFLQDIKKPDCDDWESGLNAMECALHLEKNVNQSLLELHKLATDKNDPHLCDFIETHYLNEQVKAIKELGDHVTNLRKMGAPESGLAEYLFDKHTLGDSDNES。
Zika virus is a single-stranded positive-strand RNA virus, whose genome is about 11kb in length, and encodes 3 structural proteins (capsid protein capsidp protein-C, membrane protein-M or precursor Membrane protein prM, envelope protein-E) and 7 nonstructural proteins (NSl, NS2A, NS2B, NS, NS4A, NS4B, NS). Current research on the zhai card virus vaccine is mainly focused on the E protein region. The E protein is used as a main epitope region, contains a plurality of neutralizing epitopes and specific epitopes, and participates in a plurality of life processes such as virus adsorption, penetration, pathopoiesia and the like. The E protein contains 3 recognizable binding domains (DI, DII, DIII), wherein the third binding domain (zEDIII) of the E protein is regarded as a main neutralizing epitope, has high sequence conservation and good immunogenicity, can induce generation of specific neutralizing antibodies of the Zika virus, and therefore, becomes a potential target for developing the Zika vaccine with high protective efficacy.
The ferritin nanocages are 24-polymer symmetrical protein cage structures formed by self-assembly of 8 ferritin 3 polymers, and can resist changes caused by temperature, PH and chemical reagents in a certain range. The ferritin nano-cage is about 12nm in size, a cavity of 6-8nm is formed in the inner part, and the N end of the encoded ferritin sequence is positioned on the outer surface of the nano-cage, so that gene editing, protein fusion expression and polypeptide modification can be facilitated. The fusion expression of ferritin and other proteins does not affect the self-assembly and assembly properties of ferritin and other proteins, and the zEDIII-rHF recombinant protein nanoparticle serving as the Zika virus nano vaccine provided by the embodiment is characterized in that the N-terminal of ferritin is connected in series with a protein sequence to be expressed, and after the protein is expressed, the zEDIII peptide of the third structural domain of Zika virus E protein in the fusion protein obtained after the self-assembly is displayed on the outer surface of a ferritin cage in a linear mode at intervals of about 5-10nm. The expression mode greatly increases the display quantity of the antigen epitope, and is favorable for inducing the generation of strong T cell dependent antibody response. In addition, ferritin itself may act as an adjuvant and be effective against human lymphocytes such as B cells, CD4 + T lymphocytes, CD8 + The rHF specific binding site on the surface of T lymphocytes recognizes, facilitating recognition of the displayed antigen by immune cells.
The zEDIII-rHF recombinant protein nanoparticle serving as the Zika virus nano vaccine provided by the invention has the advantages that ferritin is used as an antigen presenting nano carrier with excellent properties, the third structural domain (zEDIII) peptide of the Zika virus E protein is displayed on the surface of the recombinant human heavy chain ferritin cage structure, the immunogenicity of the zEDIII is greatly improved, the Zika virus nano vaccine with high protection efficacy is prepared, the Zika virus nano vaccine has the potential of being a novel Zika virus vaccine, and a novel preparation thought, a preparation way, a realization technical means and a method are provided for the preparation of the Zika virus immune vaccine.
Test example 1 recombinant protein expression results detection
The size, purity and specificity of the recombinant zEDIII-rHF recombinant protein were detected by SDS-PAGE and Western Blot.
SDS-PAGE of the purified zEDIII-rHF recombinant protein is shown in FIG. 3, and the single band of the zEDIII-rHF protein can be seen at 35-45 kDa by SDS-PAGE.
The antibody used in Western Blot detection is anti-Zika virus envelope protein domain III antibody (Sino Biological Cat: 40543-R101), the Western Blot identification chart of the purified zEDIII-rHF recombinant protein is shown in figure 4, and the result shows that the purified zEDIII-rHF recombinant protein retains the special domain of E protein domainIII.
As shown in fig. 5, transmission Electron Microscope (TEM) characterization was performed on rHF nanoparticles and zEDIII-rHF recombinant protein nanoparticles obtained by self-assembly. As can be seen from the transmission electron microscope characterization, rHF protein subunits can self-assemble in ferritin assembly buffer solution to form a hollow protein cage structure with an outer diameter of 12nm and an inner diameter of 8nm, TEM images show that EDI-rHF protein is the same as rHF protein subunits, and can self-assemble to form a protein cage structure, and the assembled nano particles have better dispersibility (figure 5). The zEDIII peptide of the third structural domain of the Zika virus E protein in the fusion protein is displayed on the outer surface of a ferritin cage in a linear mode, and the expression mode greatly increases the display quantity of antigen epitopes, is favorable for inducing and generating strong T cell dependent antibody reaction, and can effectively induce the generation of antibody proteins. In addition, ferritin itself may act as an adjuvant and be effective against lymphocytes such as B cells, CD4 + T lymphocytes, CD8 + The rHF specific binding site on the surface of the T lymphocyte is recognized, which is favorable for the recognition of the displayed antigen by immune cells and further enhances the immune response.
Test example 2 detection of immune antibody to immune Effect
The test example tests the immune effect of the zEDIII-rHF recombinant protein nanoparticle Zika vaccine prepared in the embodiment 1, and the immune effect is verified in a mouse body, and the method comprises the following steps:
(1) A129 (B6.129S2-Ifnar 1tm1 Agt) mice 6-8 weeks old were randomly grouped, immunized with 10. Mu.g of the zEDIII-rHF recombinant protein nanoparticle, 3.75. Mu.g of the zEDIII polypeptide (containing equimolar amounts of zEDIII as compared to the zEDIII-rHF recombinant protein nanoparticle), 6.25. Mu.g of rHF nanoparticle (containing equimolar amounts of rHF as compared to the zEDIII-rHF recombinant protein nanoparticle), and PBS, respectively, by the leg intramuscular route, the A129 mice were immunized three times with a total of 100 ul/time immunization application volume for the mice, with a 14 day interval between adjacent two immunizations. Mouse serum was collected 7 days after each immunization and stored at-20 ℃ and ELISA was used to detect ediii-specific IgG antibodies.
The antibody detection results show that no zEDIII specific IgG antibodies are detected in serum of mice immunized by the rHF nano-particles and PBS control mice after three times of immunization, the low-titer zEDIII specific antibodies can be detected by the zEDIII polypeptide group immunized mice, and as shown in FIG. 6, a large amount of zEDIII specific IgG antibodies are induced by the zEDIII-rHF recombinant protein nano-particles, so that strong humoral immune response is proved to be induced by the zEDIII-rHF recombinant protein nano-particles.
(2) After three times of immunization, the spleen of the mouse is taken, and the spleen is aseptically treated for in vitro spleen lymphocyte culture. The spleen is ground to separate single cells, the mouse lymphocyte separation medium is adopted for centrifugation at 800rpm for 30min, the middle layer cloud-like cells are collected, and the spleen lymphocytes are obtained through separation. Detection of CD4 in spleen lymphocytes using LSRFortessa flow cytometer + And CD8 + T positive cells, positive cells were labeled with fluorescently labeled antibodies, including anti-CD 3 (APC/Cy 7), anti-CD 4 (FITC) and anti-CD 8 (PerCP). Data analysis using Flow Jo software, data CD3 + /CD4 + Or CD3 + /CD8 + Double positive loop gate selection to finally obtain CD4 + And CD8 + Percentage of T positive cells, 3 replicates per sample.
As shown in FIG. 7, CD4 in spleen of immunized mice + CD8 + T cell percentage statistical plot, CD4 of zEDIII-rHF group + And CD8 + The T cell percentage was significantly higher than the other groups. CD4 in mice immunized by zEDIII-rHF recombinant protein nano-particles prepared in real-time example 1 + And CD8 + The T positive cells are obviously increased, so that the antigen recognition effect of the third domain of the Zika virus E protein is obviously improvedSpecific antibodies are effectively generated, and the immunity of the immunized mice to the Zika virus is improved.
IL-4 and IFN-gamma secreting cell numbers in spleen lymphocytes were detected using the cytokine enzyme-linked immunospot (ELISPOT) method, and pre-coated mouse IL-4 and IFN-gamma ELISPOT kits were purchased from Daidae as Bio Inc. 1-3×10 of each well is added to the well plate 5 The spleen lymphocytes were stimulated by adding 1. Mu.g of zEDIII, and incubated in an incubator at 37℃for 16-24 hours. Pouring cell culture solution in the hole, adding ice-cold deionized water, standing in a refrigerator at 4 ℃ for 10min, repeatedly washing, adding diluted enzyme-labeled avidin working solution at 37 ℃ for incubation for 1h, standing in a dark place at room temperature for 30min after washing, stopping color development by using the AEC color development solution, and performing spot statistical analysis after airing.
ELISPOT test further evaluates the lymphocyte number of zEDIII specific interleukin in spleen as shown in figure 8, which is a statistical chart of lymphocyte numbers secreted by IL-4-and IFN-gamma in spleen of immunized mice, and the lymphocyte numbers secreted by IL-4-and IFN-gamma in spleen of mice immunized with zEDIII-rHF recombinant protein nanoparticle prepared by the invention in real-time example 1 are obviously increased, and rHF, zEDIII group and PBS group have no obvious difference. In the zEDIII-rHF group, the number of IFN-gamma secreting lymphocytes was much higher than the number of IL-4 secreting lymphocytes. The result shows that the zEDIII-rHF recombinant protein nano-particles induce strong cellular immune response, and the immune type is mainly Th1 type cellular immunity. Therefore, the mice immunized by the zEDIII-rHF recombinant protein nano particles provided by the embodiment 1 of the invention are proved to have improved disease resistance to the Zika virus.
Test example 3 Virus titre detection of immune Effect
Mice were infected with Zika virus SZ-WIV01, serum from 5 mice was collected on days 3 and 10 after virus infection, and samples of visceral tissue (brain, liver, spleen, kidney, testis) from mice were collected 7 days after infection for virus titer detection. Tissue virus titer was determined using a real-time quantitative PCR (qRT-PCR) method. The tissue was homogenized and centrifuged at 5000rpm for 5min to collect the supernatant. Total RNA was extracted using an RNA extraction kit (Magen Cat: R4173-03).
ZIKV RNA was amplified using a pair of universal primers (sense primer:5'AGGATCATAGGTGATGAAGAAAAGT 3' and reverse primer:5'CCTGACAACACTAAGATTGGTGC 3'). All real-time quantitative PCR was performed for sample processing using One step TB Green PrimeScriptTM PLUS RT-PCR kit (TaKaRa Code No. RR096A). RT-PCR assays were all performed on a CFX96 Touchreal-time PCR detection system (Bio-Rad).
The result of the viral RNA assay shown in FIG. 9 shows that the virus titer in blood and tissues of mice of an immunized group adopting the zEDIII-rHF recombinant protein nanoparticles is obviously reduced, and the virus titer in blood and tissues of other groups is not obviously different from that in PBS group, thus the result shows that the zEDIII-rHF recombinant protein nanoparticles effectively inhibit the amplification of various tissues of Zika virus in animal bodies.
Test example 4 survival detection of immune Effect
Mice were infected with Zika virus SZ-WIV01 and immunized three times for 14 days with 20 μl 10LD 50 Zika virus strain (SZ-WIV 01) infected mice, the mice were observed for 21 days, and the weight change and survival rate of the mice were counted. Mice with weight loss of more than 20% were considered dead. Euthanasia treatments were performed according to animal ethics. After Zika infection, zEDIII-rHF immunized mice begin to recover after undergoing a premature decline in body weight, with a maximum percent weight decline of no more than 20%, and eventually all survives.
As shown in fig. 10 and 11, the body weight of other immunized mice was continuously reduced, and finally all the mice died, indicating that the zEDIII-rHF recombinant protein nanoparticles can protect the mice from the infection of the lethal dose of the zika virus.
It should be noted that the above examples are only for further illustrating and describing the technical solution of the present invention, and are not intended to limit the technical solution of the present invention, and the method of the present invention is only a preferred embodiment and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
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Claims (8)

1. The Zika virus recombinant protein vaccine is characterized in that the recombinant protein is a third structural domain peptide of Zika virus E protein and is displayed on the surface of recombinant human heavy chain ferritin;
the recombinant protein self-assembles into a cage-shaped hollow protein nanoparticle structure;
the amino acid sequence of the recombinant protein is shown as SEQ ID NO. 2.
2. A gene encoding the recombinant protein vaccine of zika virus according to claim 1, wherein the nucleotide sequence of the gene is shown in SEQ ID No. 1.
3. An expression vector comprising the gene encoding the recombinant protein vaccine of zika virus of claim 2.
4. The expression vector of claim 3, wherein the construction method comprises the use of a forward primer sequence as set forth in SEQ ID NO.3 and a reverse primer sequence as set forth in SEQ ID NO. 4:
SEQ ID NO.3：5'-CGCGGATCCATGGATAAACTTAGATTGAAGGG-3'；
SEQ ID NO.4：5'-CCCAAGCTTTTAGCTTTCATTATCACTGTCTCC-3'。
5. the expression vector of claim 3 or 4, wherein the expression vector backbone is pET28a escherichia coli expression plasmid.
6. The preparation method of the Zika virus recombinant protein is characterized by comprising the following steps:
1) Inserting a nucleotide sequence shown in SEQ ID NO.1 into an escherichia coli expression plasmid to obtain a recombinant plasmid pET28a/zEDIII-rHF;
2) Expressing the recombinant plasmid pET28a/zEDIII-rHF in an escherichia coli prokaryotic expression system, and separating and purifying to obtain the Zika virus recombinant protein containing the Zika virus E protein third structural domain peptide and the human heavy chain ferritin.
7. The method for preparing the recombinant protein of Zika virus according to claim 6, wherein in the step 2), the recombinant plasmid pET28a/zEDIII-rHF is purified for the second time by a molecular sieve after being expressed in a prokaryotic expression system of escherichia coli and being subjected to the first purification by a nickel column.
8. A combination vaccine comprising the zika virus recombinant protein vaccine of claim 1.
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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108699561A (en) * 2015-11-19 2018-10-23 创嘉生物科技有限公司 Recombinant lactic acid bacteria and its purposes in oral universal influenza vaccine
CN109081868A (en) * 2017-06-14 2018-12-25 中国科学院上海巴斯德研究所 Target the monoclonal antibody and its application of zika virus envelope protein conserved epitope
CN111328346A (en) * 2017-11-06 2020-06-23 西纳生物技术有限公司 Fusion proteins based on human ferritin and protease cleavable peptides and their use as chemotherapeutic carriers
CN112321718A (en) * 2019-08-05 2021-02-05 中国农业科学院生物技术研究所 Self-assembly ferritin-based nano antigen particle, peste des petits ruminants vaccine and preparation method and application thereof
CN112876570A (en) * 2021-02-09 2021-06-01 中国农业科学院生物技术研究所 African swine fever virus vaccine and preparation method thereof
KR20210082306A (en) * 2019-12-24 2021-07-05 강원대학교산학협력단 Development of recombinant subunit Zika virus vaccine and preparing method thereof
CN113121704A (en) * 2020-03-25 2021-07-16 昆山新蕴达生物科技有限公司 Nanoparticle-based coronavirus vaccines

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017132210A1 (en) * 2016-01-25 2017-08-03 Iogenetics, Llc Interventions for flavivirus infections
US10961283B2 (en) * 2016-06-27 2021-03-30 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Self-assembling insect ferritin nanoparticles for display of co-assembled trimeric antigens

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108699561A (en) * 2015-11-19 2018-10-23 创嘉生物科技有限公司 Recombinant lactic acid bacteria and its purposes in oral universal influenza vaccine
CN109081868A (en) * 2017-06-14 2018-12-25 中国科学院上海巴斯德研究所 Target the monoclonal antibody and its application of zika virus envelope protein conserved epitope
CN111328346A (en) * 2017-11-06 2020-06-23 西纳生物技术有限公司 Fusion proteins based on human ferritin and protease cleavable peptides and their use as chemotherapeutic carriers
CN112321718A (en) * 2019-08-05 2021-02-05 中国农业科学院生物技术研究所 Self-assembly ferritin-based nano antigen particle, peste des petits ruminants vaccine and preparation method and application thereof
KR20210082306A (en) * 2019-12-24 2021-07-05 강원대학교산학협력단 Development of recombinant subunit Zika virus vaccine and preparing method thereof
CN113121704A (en) * 2020-03-25 2021-07-16 昆山新蕴达生物科技有限公司 Nanoparticle-based coronavirus vaccines
CN112876570A (en) * 2021-02-09 2021-06-01 中国农业科学院生物技术研究所 African swine fever virus vaccine and preparation method thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
"铁蛋白纳米载体及其在生物医学领域的应用潜力";苏恺等;《畜牧兽医学报》;第52卷(第4期);第909-919页 *

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