CN113461788B - Cat coronavirus recombinant antigen, genetic engineering subunit vaccine thereof and application - Google Patents
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Abstract
The invention discloses a feline coronavirus recombinant antigen, a genetic engineering subunit vaccine and application thereof. The feline coronavirus recombinant antigen comprises a polypeptide having the sequence of SEQ ID NO:2 or an increased or truncated sequence thereof. The vaccine comprises the recombinant protein and a pharmaceutically acceptable carrier. The feline coronavirus recombinant antigen provided by the invention has high immunogenicity, can generate high-concentration neutralizing antibody, and cannot generate ADE effect. Meanwhile, the expression of the feline coronavirus recombinant antigen is carried out by adopting a baculovirus insect cell expression system and using a suspension culture Sf9 cell mode, so that the expression level is high, the protein immunogenicity is good, and the prepared vaccine also has the advantages of easiness in quality control, stability among batches, low production cost and the like.
Description
Technical Field
The invention relates to a genetic engineering vaccine, in particular to a feline coronavirus recombinant antigen, a genetic engineering subunit vaccine and application thereof, belonging to the technical field of animal immunity drugs.
Background
Feline Coronavirus (FCoV) is a common pathogen in felines, and mainly includes Feline Enteric Coronavirus (FECV) and Feline Infectious Peritonitis Virus (FIPV). The infection of the FECV is limited in intestinal tract, has high infectivity, can cause symptoms of body temperature rise, inappetence, dehydration and the like of cats, and has low fatality rate. FIPV is a highly virulent mutant of FECV that acquires the ability to replicate within macrophages, breaking the intestinal tract and causing more organs to become infected. The pathogenic mechanism of FIPV mainly depends on the Antibody-dependent enhancement (ADE) induced in the body after viral infection. FIPV infection can be classified into the presence of fluid accumulation in the abdominal cavity (wet type) and the absence of fluid accumulation in the abdominal cavity (dry type), and is mainly characterized by peritonitis, pyogenic granulomatous vasculitis, large ascites accumulation, high infection rate, high mortality rate, etc. After part of cats are infected with FcoV, the initial asymptomatic or mild intestinal infection is converted into peritonitis symptoms, and the development of cat breeding and related industries is seriously influenced.
FCoV belongs to the genus coronavirus of the family Coronaviridae, is an enveloped, non-segmented, single-stranded RNA virus with a typical coronavirus genomic structure that encodes a viral polymerase (Pol), 4 structural proteins (mainly including the capsid protein N, the fiber glycoprotein S, the membrane protein M, the secondary transmembrane protein E) and 5 specific accessory proteins (3a-c, 7a and 7 b). The S protein is a type I transmembrane protein, has a long N-terminal functional region and a short C-terminal cytoplasmic tail region, the N terminal (S1 region) is responsible for a Receptor-binding Domain (RBD) and is an important protein for determining a virus antigen and inducing a neutralizing antibody, and a specific antibody aiming at the coronavirus RBD can be combined with the S protein RBD to generate a neutralizing effect. The fusion peptide contained in the C-terminal (S2 region) is an essential element for fusion, can mediate fusion with target cell membrane, and plays an important role in virus invasion, replication and collective immune response.
With the increasing number of cats kept in humans year by year, there is an increasing clinical need for a FcoV therapeutic and prophylactic agent. Antiviral, immunostimulating, anti-inflammatory and interferon drugs have been used experimentally for FcoV treatment, but with little success. Some conventional vaccines also have poor control of FcoV infection, mainly due to ADE phenomenon of FIPV, and FcoV antibodies previously present in the host enhance FIPV infection against surface proteins, resulting in increased disease.
Disclosure of Invention
The invention mainly aims to provide a feline coronavirus recombinant antigen, a genetic engineering subunit vaccine thereof and application thereof, so as to overcome the defects in the prior art.
In order to achieve the purpose, the technical scheme adopted by the invention comprises the following steps:
the embodiment of the invention provides a recombinant protein which has the amino acid sequence shown in SEQ ID NO:2 or a sequence similar to SEQ ID NO:2, a sequence which is 95% or more identical to the full-length sequence of the polypeptide. That is, the amino acid sequence of the recombinant protein provided by the embodiments of the present invention may be an original sequence, an added sequence or a truncated sequence.
The embodiment of the invention also provides a gene for coding the recombinant protein.
In some embodiments, the gene has the sequence of SEQ ID NO:1 or a sequence corresponding to SEQ ID NO:1 is 95% or more identical to the full-length sequence of the above-mentioned polypeptide.
The embodiment of the invention also provides a feline coronavirus recombinant antigen which comprises the recombinant protein.
The embodiment of the invention also provides a recombinant vector containing the encoding gene of the recombinant protein.
In some embodiments, the recombinant vector includes, but is not limited to, pFastBac1, pVL1393, pFastBac dual, etc., and preferably pFastBac1 is employed.
The embodiment of the invention also provides a host cell containing the coding gene of the recombinant protein.
In some embodiments, the host cell is selected from insect cells, such as the Sf9 cell line, preferably the Sf9 cell line includes but is not limited to Sf9, High Five or Sf21 cells, more preferably Sf9 cells.
The embodiment of the invention also provides an immune composition, which is characterized by comprising the following components: said recombinant protein or said feline coronavirus recombinant antigen; and a pharmaceutically acceptable carrier.
In some embodiments, the pharmaceutically acceptable carrier includes, but is not limited to, one or a combination of two or more of MONTANIDE ISA 206VG, MONTANIDE ISA 201 VG, MONTANIDE GEL 01 ST, freund's adjuvant, aluminum hydroxide GEL adjuvant, vegetable oil, cytokines, and preferably aluminum hydroxide GEL adjuvant is used.
The embodiment of the invention also provides a preparation method of the recombinant protein, which comprises the following steps: culturing said host cell under suitable conditions, and isolating said recombinant protein from the culture broth and/or said host cell.
In some embodiments, the preparation method specifically comprises:
cloning the encoding gene of the recombinant protein into a shuttle vector to obtain a recombinant shuttle vector containing a target gene;
transforming the recombinant shuttle vector into competent cells, and separating to obtain a recombinant baculovirus genome plasmid containing a target gene expression frame;
transfecting insect cells by using the recombinant baculovirus genome plasmid, and separating to obtain recombinant baculovirus;
inoculating insect cells with the recombinant baculovirus, culturing, and then separating and purifying to obtain the recombinant protein.
Wherein, the cultured insect cells can be cracked by various methods known in the field, and then the recombinant protein can be obtained by separating and purifying from the lysate.
Among the methods used to isolate, purify the recombinant protein include, but are not limited to, chromatography, dialysis, or other methods known in the art.
In some embodiments, the shuttle vector includes, but is not limited to, pFastBac1, pVL1393, pFastBac dual, etc., preferably pFastBac1 is employed.
In some embodiments, the insect cells include, but are not limited to Sf9, High Five or Sf21 cells, and the like, preferably Sf9 cells.
The embodiment of the invention also provides a preparation method of the feline coronavirus genetic engineering subunit vaccine, which comprises the following steps: recombinant proteins are prepared using any of the methods described above and are admixed with a pharmaceutically acceptable carrier.
The embodiment of the invention also provides application of the recombinant protein or the immune composition in preparing a feline coronavirus detection reagent, in producing a medicament for inducing an immune response to a feline coronavirus antigen in a test animal or in producing a medicament for preventing the animal from being infected by the feline coronavirus.
The embodiment of the invention also provides application of the recombinant protein or the immune composition in preparing a feline coronavirus genetic engineering subunit vaccine.
The embodiment of the invention provides a feline coronavirus genetically engineered subunit vaccine which comprises any one of the immune compositions. Further, the vaccine may further comprise a pharmaceutically acceptable carrier.
The embodiment of the invention also provides the application of the recombinant vector or the host cell containing the coding gene of the recombinant protein in the production of a reagent for detecting the infection of animals by the feline coronavirus.
The embodiments of the present invention also provide the use of a recombinant vector or host cell comprising a gene encoding the recombinant protein in the manufacture of a medicament for inducing an immune response against a feline coronavirus antigen in a test animal.
The embodiment of the invention also provides the application of the recombinant vector or the host cell containing the coding gene of the recombinant protein in the production of a medicament for preventing animals from being infected by feline coronavirus.
The embodiments also provide a method of inducing an immune response against a feline coronavirus antigen, the method comprising administering the feline coronavirus genetically engineered subunit vaccine to a subject animal.
The embodiments also provide a method of protecting a subject animal from feline coronavirus infection, the method comprising administering to the subject animal the feline coronavirus genetically engineered subunit vaccine.
Embodiments of the invention also provide a vaccine suitable for generating an immune response in a test animal against feline coronavirus infection, the vaccine comprising: the recombinant protein and an adjuvant of the invention. An "adjuvant" as described in the present specification means any molecule added to the vaccine described in the present specification to enhance the immunogenicity of the antigen encoded by the gene. Preferably, the adjuvant can be related adjuvants produced by Suzhou Shino biotechnology, Inc. to improve the effect of the vaccine.
The embodiment of the invention also provides a kit which comprises the feline coronavirus recombinant antigen, the gene, the recombinant vector, the recombinant cell or the immune composition. Further, the kit may further comprise a container or a device, such as a syringe, for packaging or administering the feline coronavirus recombinant antigen, the gene, the recombinant vector, the recombinant cell, or the immunological composition to an animal.
Compared with the prior art, the technical scheme provided by the embodiment of the invention has at least the following advantages:
(1) through selecting 256N to 566D positions of main antigen regions RBD of fiber glycoprotein S (S protein) of FCoV, connecting the two RBD regions by using a flexible linker and then performing tandem expression, the recombinant protein with obviously improved immunogenicity can be obtained; preferably, by performing two point mutations in one of the RBD regions, i.e., 515Q and 516Q to PP, a dimeric protein with higher immunogenicity can be formed, high concentrations of neutralizing antibodies can be produced, and no ADE effect can be produced; more preferably, the immunogenicity of the tandem two RBD regions is further improved by mutating both 515Q and 516Q into PP, which is also beneficial to the folding of the dimeric protein.
(2) By adopting a baculovirus insect cell expression system and using suspension culture Sf9 cells and the like for expression and large-scale serum-free suspension culture preparation, the obtained product has the antigenicity, immunogenicity and functions similar to those of natural proteins, has higher expression level and strong immunogenicity, can provide good immune effect only by a small amount, has no pathogenicity to animals, is suitable for being widely applied as a cat coronavirus recombinant genetic engineering subunit vaccine, and greatly reduces the production cost of the vaccine.
Drawings
FIG. 1 is codon optimized S in step 1 of example 1RBDGel electrophoresis of the PCR amplification product of the dimer gene, in which the band of interest appears at the 2.0kbp position.
FIG. 2 is a gel electrophoresis chart of the colony PCR amplification product in example 1, in which the band of interest appears at the 2.0kbp position.
FIG. 3 is the transfer vector pF-S of example 1RBDSchematic representation of the structure of dimer.
FIG. 4 shows the results of SDS-PAGE in example 4.
FIG. 5 shows the result of Western Blot detection in example 5.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
As mentioned previously, the S protein is one of the major structural proteins of FCoV, which plays a crucial role in determining viral antigens and inducing neutralizing antibodies. The major antigenic region of the S protein, RBD, is 231I through 604Q. According to the embodiment of the invention, the recombinant protein with better immunogenicity can be obtained by selecting 256N to 566D positions of main antigen regions RBD of the S protein, connecting the two RBD regions by using a flexible linker and carrying out tandem expression.
Preferably, the sequence of the flexible linker connecting the two RBD regions can be a short peptide sequence unit, such as GGGGGS, or can be formed by repeating and connecting two, three, four or more short peptide sequence units, preferably three short peptide sequence units, so that the protein has high renaturation efficiency.
Preferably, by performing two point mutations in one of the RBD regions, i.e., 515Q and 516Q to PP, the resulting dimeric protein is more immunogenic, produces high concentrations of neutralizing antibodies, and does not produce ADE effects.
More preferably, the dimeric protein (which may be designated as S) is also favored by mutating both 515Q and 516Q of the two RBD regions in tandem to PPRBDDimer protein) to further increase its immunogenicity.
Furthermore, the embodiment of the invention adopts a baculovirus insect cell expression system, and uses suspension culture Sf9 cells to express the recombinant protein, so that the expression level is high, and the protein immunogenicity is good.
Furthermore, the recombinant protein can be used for preparing a feline coronavirus genetic engineering subunit vaccine.
For example, in one embodiment of the present invention, a method for preparing a feline coronavirus genetically engineered subunit vaccine can specifically comprise:
(1) preparation of a recombinant protein (S) encoding the aforementionedRBD-dimer protein);
(2) cloning the nucleic acid molecules which are prepared in the step (1) and code the recombinant proteins into shuttle vectors respectively to obtain recombinant shuttle vectors containing target genes;
(3) transforming the recombinant shuttle vector obtained in the step (2) into DH10Bac bacteria, selecting recombinant bacteria, extracting genome to transfect Sf9 cells (or other insect cells) to obtain recombinant baculovirus;
(4) incubating said Sf9 cell (or other insect cell as described above) for recombinant expression to produce a recombinant protein;
(5) and adding the obtained recombinant protein into an adjuvant to obtain the vaccine.
In this embodiment, Sf9 cells are used to express SRBDThe product has antigenicity, immunogenicity and function similar to those of natural protein, high expression level, strong immunogenicity, no pathogenicity to animal, and may be prepared through large scale serum-free suspension culture in bioreactor, and has greatly reduced vaccine contentAnd (5) the production cost.
The feline coronavirus genetic engineering subunit vaccine provided by the embodiment of the invention has no toxicity, high safety and good immunogenicity, can generate stronger humoral immunity in an animal body, and the immunized animal can resist strong toxicity attack, and also has a series of advantages of large-scale batch production, easy quality control, stable batch-to-batch, low production cost and the like.
When the feline coronavirus genetic engineering subunit vaccine provided by the embodiment of the invention is applied, only an effective amount of the vaccine needs to be inoculated to animals. As used herein, the term "effective amount" refers to an amount sufficient to obtain, or at least partially obtain, a desired effect. For example, a disease-preventing effective amount refers to an amount sufficient to prevent, or delay the onset of disease; a therapeutically effective amount for a disease is an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. It is well within the ability of those skilled in the art to determine such effective amounts.
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The reagents and starting materials used in the following examples are commercially available, and the test methods in which specific conditions are not specified are generally carried out under conventional conditions or conditions recommended by the respective manufacturers. Further, unless otherwise indicated, the assays, detection methods, and preparations disclosed herein are performed using molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA techniques, and techniques conventional in the art. These techniques are well described in the literature.
Example 1 transfer vector pF-SRBDConstruction and characterization of-dimer
1.SRBDAmplification and purification of the-dimer Gene
The codon-optimized S was synthesized in Nanjing Kingsrei Biotechnology LtdRBDThe dimer gene (SEQ ID NO: 1) and cloned into pUC17 vector to obtain pUC-SRBD-dimer plasmid vector. In pUC-SRBD-dimer plasmid as template, SRBD-dimer-F、SRBDPCR amplification with-dimer-R as upstream and downstream primers (S)RBD-dimer-F、SRBD-dimer-R has the gene sequence as shown in SEQ ID NO: 3. 4) and the amplification system is shown in Table 1.
TABLE 1SRBD-dimer gene amplification system
The reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 45 seconds, annealing at 54 ℃ for 45 seconds, extension at 72 ℃ for 1 minute, 35 cycles; extension at 72 ℃ for 10 min.
The PCR product was subjected to gel electrophoresis to verify the size of the target gene, and as shown in FIG. 1, a band of interest appeared at a position of 2.0kbp, and the target gene was successfully amplified and recovered and purified using a gel recovery and purification kit.
2. Digestion and purification
pFastBac1 plasmid and SRBDThe PCR amplification product of the-dimer gene is subjected to double enzyme digestion at 37 ℃ for 3 hours by using BamH I and Hind III, and the specific enzyme digestion reaction system is shown in tables 2 and 3.
Performing gel electrophoresis on the enzyme digestion product, and respectively purifying the enzyme digestion pFastBac1 plasmid and S by using a gel recovery and purification kitRBD-dimer gene fragment.
TABLE 2SRBD-dimer gene enzyme digestion reaction system
TABLE 3 pFastBac1 plasmid digestion reaction System
3. Connection of
The digested pFastBac1 plasmid and SRBDThe product of the digestion of the-dimer gene is ligated with T4 DNA ligase in a water bath at 16 ℃ overnight, and the linkerSee table 4.
TABLE 4SRBD-dimer gene and pFastBac1 plasmid connecting system
4. Transformation of
Mu.l of the ligation product was added to 100. mu.l of DH 5. alpha. competent cells, mixed well, heat-shocked at 42 ℃ for 90 seconds, ice-bathed for 2 minutes, added to 900. mu.l of LB medium without Amp, and cultured at 37 ℃ for 1 hour. 1.0mL of the cell suspension was concentrated by centrifugation to 100. mu.l, and the concentrated solution was applied to LB solid medium containing Amp and cultured at 37 ℃ for 16 hours.
5. Colony PCR and sequencing identification
Selecting single colony on the plate, inoculating LB liquid culture medium, culturing at 37 deg.C for 2 hr, using bacterial liquid as template, SRBD-dimer-F and SRBDColony PCR was performed with-dimer-R as a primer. The size of the target gene was confirmed by subjecting the PCR product to gel electrophoresis, and as shown in FIG. 2, a sample showing a band of approximately 2.0kbp was positive. And (4) sending the bacterial liquid with positive colony PCR identification to a sequencing company for sequencing, and selecting the bacterial liquid with correct sequencing for storage. Constructed transfer vector pF-S containing target geneRBDSchematic representation of the-dimer is shown in FIG. 3.
Example 2 recombinant baculovirus genome Bac-SRBDConstruction of dimer
Transformation of DH10Bac bacteria
Mu.l of pF-S obtained in example 1 was takenRBDThe-dimer plasmid was added to 100. mu.l of DH10Bac competent cells and mixed well, ice-bathed for 30 minutes, water-bathed heat shock at 42 ℃ for 90 seconds, ice-bathed for 2 minutes, 900. mu.l of LB liquid medium without Amp was added, and cultured at 37 ℃ for 5 hours. Then 100. mu.l of the diluted bacterial solution was diluted 81 times, and 100. mu.l of the diluted bacterial solution was applied to LB solid medium containing gentamicin, kanamycin, tetracycline, X-gal and IPTG, and cultured at 37 ℃ for 48 hours.
2. Selection of monoclonal
Large white colonies were picked using an inoculating needle and placed on L containing gentamicin, kanamycin, tetracycline, X-gal and IPTGAnd B, streaking on a solid culture medium, culturing for 48 hours at 37 ℃, then selecting a single colony, inoculating an LB liquid culture medium containing gentamicin, kanamycin and tetracycline, culturing, preserving strains, and extracting plasmids. Obtaining recombinant plasmid Bacmid-SRBD-dimer。
Example 3 recombinant baculovirus transfection
Six well plates were seeded 0.8X 10 per well6The confluency of Sf9 cells is 50-70%. The following complexes were prepared for each well: diluting 4. mu.l of Cellffectin transfection reagent with 100. mu.l of transfection medium T1, and shaking briefly with vortex; mu.g of recombinant Bacmid-S from example 2 was diluted with 100. mu.l of transfection medium T1RBD-dimer plasmid, mixing diluted transfection reagent and plasmid, gently blowing up to prepare transfection mixture. And adding the transfection compound after the cells adhere to the wall, incubating for 5 hours at 27 ℃, removing the supernatant, adding 2mLSF-SFM fresh culture medium, and culturing for 4-5 days at 27 ℃ to obtain the supernatant. Obtaining recombinant baculovirus rBac-SRBDDimer, detection of the virus content, rBac-S, of the harvested F1-generation recombinant baculovirus using indirect immunofluorescenceRBDThe content of-dimer F1 generation seed virus is 1.81 x 108TCID50. Amplification of recombinant baculovirus rBac-SRBD-dimer as seed virus for use.
Recombinant baculoviruses expressing the control group listed in table 5 below were additionally constructed as per the above example methods:
table 5 collation group setup packet
Note: the sequence of the control group Srbd-Srbd protein is shown as SEQ ID NO: and 6.
Example 4SDS-PAGE detection
The rBac-S harvested in example 3 was usedRBDNon-reducing SDS-PAGE detection of cell cultures of-dimer group and control group, and simultaneous taking of rBac-SRBDCell cultures of the dimer group were subjected to a reducing SDS-PAGE assay and Sf9 cells infected with empty baculovirus were used as a negative control (reducing SDS-PAGE assay)The difference between the detection by non-reducing SDS-PAGE and the detection by non-reducing SDS-PAGE is whether a reducing agent beta-mercaptoethanol is added or not during sample treatment). The specific operation is as follows: and (3) adding 10 mul of 5 × loading buffer into 40 mul of harvested cell culture, adding 25 mul of beta-mercaptoethanol into each tube before using the cell culture for reducibility detection, uniformly mixing, carrying out boiling water bath for 5 minutes, centrifuging at 12000r/min for 1 minute, taking supernatant, carrying out SDS-PAGE gel (12% concentration gel) electrophoresis, taking gel after electrophoresis, dyeing and decoloring, and observing a target band.
As shown in FIG. 4, rBac-S was detected by non-reducing SDS-PAGERBDThe dimer group showed the desired band at a molecular weight of about 74kDa, the control group showed the desired band at a molecular weight of about 35kDa, and rBac-S was detected by reducing SDS-PAGERBDThe dimer group showed bands around 35kDa, and the negative control showed no band at the corresponding position.
Example 5Western Blot assay
The product after SDS-PAGE electrophoresis in example 4 was transferred to an NC (nitrocellulose) membrane, blocked with 5% skim milk for 2 hours, incubated with a cat-derived anti-FCoV positive serum as a primary antibody for 2 hours, rinsed, incubated with a goat anti-cat IgG labeled with HRP as a secondary antibody for 2 hours, rinsed, then added dropwise with an enhanced chemiluminescent fluorescent substrate, and photographed using a chemiluminescent imager. The results are shown in FIG. 5, where the recombinant baculovirus expression sample had a band of interest, and the negative control had no band of interest, indicating that the protein of interest (S)RBDDimer protein) was correctly expressed in Sf9 cells.
EXAMPLE 6 protein purification
Collection of the rBac-S harvested in example 3RBDCarrying out ultrasonic disruption on the dimer cell culture, centrifuging at 12000r/min for 30 minutes, taking the supernatant, filtering by using a 0.22-micron filter membrane, removing impurities, and concentrating by 10 times by using an ultrafiltration tube with the molecular weight cutoff of 100kDa to obtain the purified target protein. Quantifying the purified target protein by using BCA total protein, then determining the purity of the target protein by combining gray scanning, and finally obtaining SRBDDimer protein concentration 850. mu.g/mL, purity 95%.
Example 7 vaccine preparation
The purified protein stock solution harvested in example 6 was added to an alumina-hydroxide gel adjuvant such that the final concentration of recombinant antigenic protein after mixing was 100. mu.g/ml. Sampling after emulsification, inspecting, subpackaging after being qualified, and storing at 4 ℃ to obtain the experimental group vaccine. The control group was prepared as a vaccine and stored in the same manner.
Example 8 immunization experiment
15 Balb/c mice of 6-8 weeks old were selected and randomly divided into 3 groups and 5 mice per group, and the experimental groups are shown in Table 6. Each mouse in the first 2 groups received 3 immunizations on day 0, day 21 and day 108, respectively, each group was injected with 0.1ml of vaccine intraperitoneally, and the negative control group was injected with 0.1ml of physiological saline in the same manner. The orbital blood collection is carried out on each group of mice before immunization, 10 days after the first immunization, 10 days after the second immunization and 10 days after the third immunization respectively, serum is separated and subjected to a neutralization test, and the average neutralization titer of the serum of each group of mice is calculated.
TABLE 6 mean neutralization titers of the sera of the groups of mice
It is to be understood that the above-described embodiments are part of the present invention, and not all embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Sequence listing
<110> Soy Mildy Biotechnology Ltd
<120> feline coronavirus recombinant antigen, and genetic engineering subunit vaccine and application thereof
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1986
<212> DNA
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<400> 1
atggaaaccg ataccctgct gctgtgggtg ctgctgctgt gggtgccggg cagcaccggc 60
aacagcaccg cgggcagcaa ctatacccat ctgcagctga aagaatgcca tagcgattat 120
tgcgcgggct atgcgaaaaa cgtgtttgtg ccgattgatg gcaaaattcc ggaaagcttt 180
agctttagca actggtttct gctgagcgat aaaagcaccc tggtgcaggg ccgcgtgctg 240
agccgccagc cggtgtttgt gcagtgcctg cgcccggtgc cgacctggag caacaacagc 300
gcggtggtgt tttttaaaaa cgatgcgttt tgcccgaacg tgaccgcgga tgtgctgcgc 360
tttaacctga actttagcga taccgatgtg tataccgaaa gcaccaacga tgatcagctg 420
tattttacct ttgaagataa caccaccgcg agcattgcgt gctatagcag cgcgaacgtg 480
accgattttc agccggcgaa caacagcgtg agccatgtgc cgtttggcaa aaccgaacat 540
agctattttt gctttgcgaa ctttagccat gcggtggtga gccgccagtt tctgggcatt 600
ctgccgccga ccgtgcgcga atttgcgttt ggccgcgatg gcagcatttt tgtgaacggc 660
tataaatatt ttagcctgcc gccgattaaa agcgtgaact ttagcattag cagcgtggaa 720
cagtatggct tttggaccat tgcgtatacc aactataccg atgtgatggt ggatgtgaac 780
ggcaccagca ttacccgcct gttttattgc gatagcccgc tgaaccgcat taaatgcccg 840
ccgctgaaac atgaactgcc ggatggcttt tatagcgcga gcatgctggt gaaaaaagat 900
ctgccgaaaa cctttgtgac catgccgcag ttttataact ggatgaacgt gaccctgcat 960
gtggtgctga acgataccga aaaaaaagcg gatggcggcg gcggcggcag cggcggcggc 1020
ggcggcagcg gcggcggcgg cggcagcaac agcaccgcgg gcagcaacta tacccatctg 1080
cagctgaaag aatgccatag cgattattgc gcgggctatg cgaaaaacgt gtttgtgccg 1140
attgatggca aaattccgga aagctttagc tttagcaact ggtttctgct gagcgataaa 1200
agcaccctgg tgcagggccg cgtgctgagc cgccagccgg tgtttgtgca gtgcctgcgc 1260
ccggtgccga cctggagcaa caacagcgcg gtggtgtttt ttaaaaacga tgcgttttgc 1320
ccgaacgtga ccgcggatgt gctgcgcttt aacctgaact ttagcgatac cgatgtgtat 1380
accgaaagca ccaacgatga tcagctgtat tttacctttg aagataacac caccgcgagc 1440
attgcgtgct atagcagcgc gaacgtgacc gattttcagc cggcgaacaa cagcgtgagc 1500
catgtgccgt ttggcaaaac cgaacatagc tatttttgct ttgcgaactt tagccatgcg 1560
gtggtgagcc gccagtttct gggcattctg ccgccgaccg tgcgcgaatt tgcgtttggc 1620
cgcgatggca gcatttttgt gaacggctat aaatatttta gcctgccgcc gattaaaagc 1680
gtgaacttta gcattagcag cgtggaacag tatggctttt ggaccattgc gtataccaac 1740
tataccgatg tgatggtgga tgtgaacggc accagcatta cccgcctgtt ttattgcgat 1800
agcccgctga accgcattaa atgcccgccg ctgaaacatg aactgccgga tggcttttat 1860
agcgcgagca tgctggtgaa aaaagatctg ccgaaaacct ttgtgaccat gccgcagttt 1920
tataactgga tgaacgtgac cctgcatgtg gtgctgaacg ataccgaaaa aaaagcggat 1980
taatga 1986
<210> 2
<211> 660
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 2
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly Asn Ser Thr Ala Gly Ser Asn Tyr Thr His Leu Gln
20 25 30
Leu Lys Glu Cys His Ser Asp Tyr Cys Ala Gly Tyr Ala Lys Asn Val
35 40 45
Phe Val Pro Ile Asp Gly Lys Ile Pro Glu Ser Phe Ser Phe Ser Asn
50 55 60
Trp Phe Leu Leu Ser Asp Lys Ser Thr Leu Val Gln Gly Arg Val Leu
65 70 75 80
Ser Arg Gln Pro Val Phe Val Gln Cys Leu Arg Pro Val Pro Thr Trp
85 90 95
Ser Asn Asn Ser Ala Val Val Phe Phe Lys Asn Asp Ala Phe Cys Pro
100 105 110
Asn Val Thr Ala Asp Val Leu Arg Phe Asn Leu Asn Phe Ser Asp Thr
115 120 125
Asp Val Tyr Thr Glu Ser Thr Asn Asp Asp Gln Leu Tyr Phe Thr Phe
130 135 140
Glu Asp Asn Thr Thr Ala Ser Ile Ala Cys Tyr Ser Ser Ala Asn Val
145 150 155 160
Thr Asp Phe Gln Pro Ala Asn Asn Ser Val Ser His Val Pro Phe Gly
165 170 175
Lys Thr Glu His Ser Tyr Phe Cys Phe Ala Asn Phe Ser His Ala Val
180 185 190
Val Ser Arg Gln Phe Leu Gly Ile Leu Pro Pro Thr Val Arg Glu Phe
195 200 205
Ala Phe Gly Arg Asp Gly Ser Ile Phe Val Asn Gly Tyr Lys Tyr Phe
210 215 220
Ser Leu Pro Pro Ile Lys Ser Val Asn Phe Ser Ile Ser Ser Val Glu
225 230 235 240
Gln Tyr Gly Phe Trp Thr Ile Ala Tyr Thr Asn Tyr Thr Asp Val Met
245 250 255
Val Asp Val Asn Gly Thr Ser Ile Thr Arg Leu Phe Tyr Cys Asp Ser
260 265 270
Pro Leu Asn Arg Ile Lys Cys Pro Pro Leu Lys His Glu Leu Pro Asp
275 280 285
Gly Phe Tyr Ser Ala Ser Met Leu Val Lys Lys Asp Leu Pro Lys Thr
290 295 300
Phe Val Thr Met Pro Gln Phe Tyr Asn Trp Met Asn Val Thr Leu His
305 310 315 320
Val Val Leu Asn Asp Thr Glu Lys Lys Ala Asp Gly Gly Gly Gly Gly
325 330 335
Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Asn Ser Thr
340 345 350
Ala Gly Ser Asn Tyr Thr His Leu Gln Leu Lys Glu Cys His Ser Asp
355 360 365
Tyr Cys Ala Gly Tyr Ala Lys Asn Val Phe Val Pro Ile Asp Gly Lys
370 375 380
Ile Pro Glu Ser Phe Ser Phe Ser Asn Trp Phe Leu Leu Ser Asp Lys
385 390 395 400
Ser Thr Leu Val Gln Gly Arg Val Leu Ser Arg Gln Pro Val Phe Val
405 410 415
Gln Cys Leu Arg Pro Val Pro Thr Trp Ser Asn Asn Ser Ala Val Val
420 425 430
Phe Phe Lys Asn Asp Ala Phe Cys Pro Asn Val Thr Ala Asp Val Leu
435 440 445
Arg Phe Asn Leu Asn Phe Ser Asp Thr Asp Val Tyr Thr Glu Ser Thr
450 455 460
Asn Asp Asp Gln Leu Tyr Phe Thr Phe Glu Asp Asn Thr Thr Ala Ser
465 470 475 480
Ile Ala Cys Tyr Ser Ser Ala Asn Val Thr Asp Phe Gln Pro Ala Asn
485 490 495
Asn Ser Val Ser His Val Pro Phe Gly Lys Thr Glu His Ser Tyr Phe
500 505 510
Cys Phe Ala Asn Phe Ser His Ala Val Val Ser Arg Gln Phe Leu Gly
515 520 525
Ile Leu Pro Pro Thr Val Arg Glu Phe Ala Phe Gly Arg Asp Gly Ser
530 535 540
Ile Phe Val Asn Gly Tyr Lys Tyr Phe Ser Leu Pro Pro Ile Lys Ser
545 550 555 560
Val Asn Phe Ser Ile Ser Ser Val Glu Gln Tyr Gly Phe Trp Thr Ile
565 570 575
Ala Tyr Thr Asn Tyr Thr Asp Val Met Val Asp Val Asn Gly Thr Ser
580 585 590
Ile Thr Arg Leu Phe Tyr Cys Asp Ser Pro Leu Asn Arg Ile Lys Cys
595 600 605
Pro Pro Leu Lys His Glu Leu Pro Asp Gly Phe Tyr Ser Ala Ser Met
610 615 620
Leu Val Lys Lys Asp Leu Pro Lys Thr Phe Val Thr Met Pro Gln Phe
625 630 635 640
Tyr Asn Trp Met Asn Val Thr Leu His Val Val Leu Asn Asp Thr Glu
645 650 655
Lys Lys Ala Asp
660
<210> 3
<211> 39
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<213> Artificial sequence (Artificial sequence)
<400> 3
ataggatcca tggaaaccga taccctgctg ctgtgggtg 39
<210> 4
<211> 39
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 4
ataaagctta tccgcttttt tttcggtatc gttcagcac 39
<210> 5
<211> 1986
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 5
atggaaaccg ataccctgct gctgtgggtg ctgctgctgt gggtgccggg cagcaccggc 60
aacagcaccg cgggcagcaa ctatacccat ctgcagctga aagaatgcca tagcgattat 120
tgcgcgggct atgcgaaaaa cgtgtttgtg ccgattgatg gcaaaattcc ggaaagcttt 180
agctttagca actggtttct gctgagcgat aaaagcaccc tggtgcaggg ccgcgtgctg 240
agccgccagc cggtgtttgt gcagtgcctg cgcccggtgc cgacctggag caacaacagc 300
gcggtggtgt tttttaaaaa cgatgcgttt tgcccgaacg tgaccgcgga tgtgctgcgc 360
tttaacctga actttagcga taccgatgtg tataccgaaa gcaccaacga tgatcagctg 420
tattttacct ttgaagataa caccaccgcg agcattgcgt gctatagcag cgcgaacgtg 480
accgattttc agccggcgaa caacagcgtg agccatgtgc cgtttggcaa aaccgaacat 540
agctattttt gctttgcgaa ctttagccat gcggtggtga gccgccagtt tctgggcatt 600
ctgccgccga ccgtgcgcga atttgcgttt ggccgcgatg gcagcatttt tgtgaacggc 660
tataaatatt ttagcctgcc gccgattaaa agcgtgaact ttagcattag cagcgtggaa 720
cagtatggct tttggaccat tgcgtatacc aactataccg atgtgatggt ggatgtgaac 780
ggcaccagca ttacccgcct gttttattgc gatagcccgc tgaaccgcat taaatgccag 840
cagctgaaac atgaactgcc ggatggcttt tatagcgcga gcatgctggt gaaaaaagat 900
ctgccgaaaa cctttgtgac catgccgcag ttttataact ggatgaacgt gaccctgcat 960
gtggtgctga acgataccga aaaaaaagcg gatggcggcg gcggcggcag cggcggcggc 1020
ggcggcagcg gcggcggcgg cggcagcaac agcaccgcgg gcagcaacta tacccatctg 1080
cagctgaaag aatgccatag cgattattgc gcgggctatg cgaaaaacgt gtttgtgccg 1140
attgatggca aaattccgga aagctttagc tttagcaact ggtttctgct gagcgataaa 1200
agcaccctgg tgcagggccg cgtgctgagc cgccagccgg tgtttgtgca gtgcctgcgc 1260
ccggtgccga cctggagcaa caacagcgcg gtggtgtttt ttaaaaacga tgcgttttgc 1320
ccgaacgtga ccgcggatgt gctgcgcttt aacctgaact ttagcgatac cgatgtgtat 1380
accgaaagca ccaacgatga tcagctgtat tttacctttg aagataacac caccgcgagc 1440
attgcgtgct atagcagcgc gaacgtgacc gattttcagc cggcgaacaa cagcgtgagc 1500
catgtgccgt ttggcaaaac cgaacatagc tatttttgct ttgcgaactt tagccatgcg 1560
gtggtgagcc gccagtttct gggcattctg ccgccgaccg tgcgcgaatt tgcgtttggc 1620
cgcgatggca gcatttttgt gaacggctat aaatatttta gcctgccgcc gattaaaagc 1680
gtgaacttta gcattagcag cgtggaacag tatggctttt ggaccattgc gtataccaac 1740
tataccgatg tgatggtgga tgtgaacggc accagcatta cccgcctgtt ttattgcgat 1800
agcccgctga accgcattaa atgccagcag ctgaaacatg aactgccgga tggcttttat 1860
agcgcgagca tgctggtgaa aaaagatctg ccgaaaacct ttgtgaccat gccgcagttt 1920
tataactgga tgaacgtgac cctgcatgtg gtgctgaacg ataccgaaaa aaaagcggat 1980
taatga 1986
<210> 6
<211> 660
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 6
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly Asn Ser Thr Ala Gly Ser Asn Tyr Thr His Leu Gln
20 25 30
Leu Lys Glu Cys His Ser Asp Tyr Cys Ala Gly Tyr Ala Lys Asn Val
35 40 45
Phe Val Pro Ile Asp Gly Lys Ile Pro Glu Ser Phe Ser Phe Ser Asn
50 55 60
Trp Phe Leu Leu Ser Asp Lys Ser Thr Leu Val Gln Gly Arg Val Leu
65 70 75 80
Ser Arg Gln Pro Val Phe Val Gln Cys Leu Arg Pro Val Pro Thr Trp
85 90 95
Ser Asn Asn Ser Ala Val Val Phe Phe Lys Asn Asp Ala Phe Cys Pro
100 105 110
Asn Val Thr Ala Asp Val Leu Arg Phe Asn Leu Asn Phe Ser Asp Thr
115 120 125
Asp Val Tyr Thr Glu Ser Thr Asn Asp Asp Gln Leu Tyr Phe Thr Phe
130 135 140
Glu Asp Asn Thr Thr Ala Ser Ile Ala Cys Tyr Ser Ser Ala Asn Val
145 150 155 160
Thr Asp Phe Gln Pro Ala Asn Asn Ser Val Ser His Val Pro Phe Gly
165 170 175
Lys Thr Glu His Ser Tyr Phe Cys Phe Ala Asn Phe Ser His Ala Val
180 185 190
Val Ser Arg Gln Phe Leu Gly Ile Leu Pro Pro Thr Val Arg Glu Phe
195 200 205
Ala Phe Gly Arg Asp Gly Ser Ile Phe Val Asn Gly Tyr Lys Tyr Phe
210 215 220
Ser Leu Pro Pro Ile Lys Ser Val Asn Phe Ser Ile Ser Ser Val Glu
225 230 235 240
Gln Tyr Gly Phe Trp Thr Ile Ala Tyr Thr Asn Tyr Thr Asp Val Met
245 250 255
Val Asp Val Asn Gly Thr Ser Ile Thr Arg Leu Phe Tyr Cys Asp Ser
260 265 270
Pro Leu Asn Arg Ile Lys Cys Gln Gln Leu Lys His Glu Leu Pro Asp
275 280 285
Gly Phe Tyr Ser Ala Ser Met Leu Val Lys Lys Asp Leu Pro Lys Thr
290 295 300
Phe Val Thr Met Pro Gln Phe Tyr Asn Trp Met Asn Val Thr Leu His
305 310 315 320
Val Val Leu Asn Asp Thr Glu Lys Lys Ala Asp Gly Gly Gly Gly Gly
325 330 335
Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Asn Ser Thr
340 345 350
Ala Gly Ser Asn Tyr Thr His Leu Gln Leu Lys Glu Cys His Ser Asp
355 360 365
Tyr Cys Ala Gly Tyr Ala Lys Asn Val Phe Val Pro Ile Asp Gly Lys
370 375 380
Ile Pro Glu Ser Phe Ser Phe Ser Asn Trp Phe Leu Leu Ser Asp Lys
385 390 395 400
Ser Thr Leu Val Gln Gly Arg Val Leu Ser Arg Gln Pro Val Phe Val
405 410 415
Gln Cys Leu Arg Pro Val Pro Thr Trp Ser Asn Asn Ser Ala Val Val
420 425 430
Phe Phe Lys Asn Asp Ala Phe Cys Pro Asn Val Thr Ala Asp Val Leu
435 440 445
Arg Phe Asn Leu Asn Phe Ser Asp Thr Asp Val Tyr Thr Glu Ser Thr
450 455 460
Asn Asp Asp Gln Leu Tyr Phe Thr Phe Glu Asp Asn Thr Thr Ala Ser
465 470 475 480
Ile Ala Cys Tyr Ser Ser Ala Asn Val Thr Asp Phe Gln Pro Ala Asn
485 490 495
Asn Ser Val Ser His Val Pro Phe Gly Lys Thr Glu His Ser Tyr Phe
500 505 510
Cys Phe Ala Asn Phe Ser His Ala Val Val Ser Arg Gln Phe Leu Gly
515 520 525
Ile Leu Pro Pro Thr Val Arg Glu Phe Ala Phe Gly Arg Asp Gly Ser
530 535 540
Ile Phe Val Asn Gly Tyr Lys Tyr Phe Ser Leu Pro Pro Ile Lys Ser
545 550 555 560
Val Asn Phe Ser Ile Ser Ser Val Glu Gln Tyr Gly Phe Trp Thr Ile
565 570 575
Ala Tyr Thr Asn Tyr Thr Asp Val Met Val Asp Val Asn Gly Thr Ser
580 585 590
Ile Thr Arg Leu Phe Tyr Cys Asp Ser Pro Leu Asn Arg Ile Lys Cys
595 600 605
Gln Gln Leu Lys His Glu Leu Pro Asp Gly Phe Tyr Ser Ala Ser Met
610 615 620
Leu Val Lys Lys Asp Leu Pro Lys Thr Phe Val Thr Met Pro Gln Phe
625 630 635 640
Tyr Asn Trp Met Asn Val Thr Leu His Val Val Leu Asn Asp Thr Glu
645 650 655
Lys Lys Ala Asp
660
Claims (17)
1. A feline coronavirus recombinant antigen characterized by: the sequence of the recombinant antigen is shown as SEQ ID NO. 2.
2. A gene, characterized by: the gene is used for recombinant protein with a coding sequence shown as SEQ ID NO. 2.
3. The gene according to claim 2, characterized in that: the sequence of the gene is shown as SEQ ID NO. 1.
4. A recombinant vector comprising the gene of any one of claims 2-3.
5. A host cell comprising the gene of any one of claims 2-3.
6. The host cell of claim 5, wherein: the cells are selected from Sf9, High Five or Sf21 cells.
7. The host cell of claim 6, wherein: the cells were Sf9 cells.
8. An immunological composition characterized by comprising: the sequence is as shown in SEQ ID NO. 2; and a pharmaceutically acceptable carrier.
9. The immunogenic composition of claim 8, wherein: the pharmaceutically acceptable carrier is selected from any one or a combination of more than two of MONTANIDE ISA 206VG, MONTANIDE ISA 201 VG, MONTANIDE GEL 01 ST, Freund's adjuvant, aluminum hydroxide GEL adjuvant, vegetable oil and cell factor.
10. The immunogenic composition of claim 9, wherein: the pharmaceutically acceptable carrier is an alumina gel adjuvant.
11. A method for preparing a feline coronavirus recombinant antigen, comprising: culturing the host cell of any one of claims 5 to 7 under suitable conditions, and isolating the recombinant protein having the sequence shown in SEQ ID NO. 2 from the culture broth and/or the host cell.
12. The preparation method according to claim 11, which specifically comprises:
cloning the encoding gene of the recombinant protein into a shuttle vector to obtain a recombinant shuttle vector containing a target gene;
transforming the recombinant shuttle vector into competent cells, and separating to obtain a recombinant baculovirus genome plasmid containing a target gene expression frame;
transfecting insect cells by using the recombinant baculovirus genome plasmid, and separating to obtain recombinant baculovirus;
inoculating insect cells with the recombinant baculovirus, culturing, and then separating and purifying to obtain the recombinant protein.
13. The method of claim 12, wherein: the shuttle vector is selected from pFastBac1, pVL1393 or pFastBac dual.
14. The method of claim 13, wherein: the shuttle vector is pFastBac 1.
15. Use of a feline coronavirus recombinant antigen according to claim 1 or an immunological composition according to any one of claims 8-10 in the preparation of a feline coronavirus detection reagent in the manufacture of a medicament for inducing an immune response against a feline coronavirus antigen in a subject animal or in the manufacture of a medicament for preventing infection of an animal by a feline coronavirus.
16. Use of the feline coronavirus recombinant antigen of claim 1 or the immunogenic composition of any one of claims 8-10 in the preparation of a feline coronavirus genetically engineered subunit vaccine.
17. A kit comprising the feline coronavirus recombinant antigen of claim 1, the gene of any one of claims 2 to 3, the recombinant vector of claim 4, the recombinant cell of any one of claims 5 to 7, or the immunogenic composition of any one of claims 8 to 10.
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| CN113896773B (en) * | 2021-10-09 | 2023-12-22 | 苏州沃美生物有限公司 | Recombinant FCV antigen and feline calicivirus genetic engineering subunit vaccine |
| CN114015702A (en) * | 2022-01-04 | 2022-02-08 | 北京科牧丰生物制药有限公司 | mRNA vaccine for preventing feline infectious peritonitis and preparation method thereof |
| CN114835804B (en) * | 2022-05-19 | 2023-04-25 | 安徽中起生物科技有限公司 | Egg yolk antibody composition for cat infectious peritonitis as well as preparation method and application thereof |
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