CN113912738A - Novel feline coronavirus subunit vaccine and preparation method and application thereof - Google Patents

Novel feline coronavirus subunit vaccine and preparation method and application thereof Download PDF

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CN113912738A
CN113912738A CN202110812101.9A CN202110812101A CN113912738A CN 113912738 A CN113912738 A CN 113912738A CN 202110812101 A CN202110812101 A CN 202110812101A CN 113912738 A CN113912738 A CN 113912738A
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sars
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金梅林
赵亚
余世曼
杨丽
吴超
胡长敏
张强
张宇飞
钟鸣
孙小美
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Huazhong Agricultural University
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Abstract

The invention belongs to the field of biotechnology, and particularly relates to a novel feline coronavirus subunit vaccine and a preparation method and application thereof, wherein the RBD structure domain of the novel coronavirus S protein provided by the invention is fused with the cat IgG FC structure domain to express RBD-cat Fc protein, the subunit protein has good immunogenicity, high-level antibodies can be induced after cats are immunized, the generated antibodies have the effect of neutralizing SARS-CoV-2 live viruses, and the RBD-cat Fc recombinant protein can be used for preventing cats from being infected by novel coronavirus and has important significance in preventing the spread among novel coronavirus species compared with the RBD-his protein of SARS-CoV-2 virus S protein which is directly expressed, the activity of the RBD protein is not influenced, and the protein stability can be increased.

Description

Novel feline coronavirus subunit vaccine and preparation method and application thereof
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a novel feline coronavirus subunit vaccine, and a preparation method and application thereof.
Background
Extensive studies have shown that SARS-CoV-2 exists in a broad host range and that hamsters, ferrets, cats, dogs and some non-human primates (rhesus, cynomolgus) can be infected with SARS-CoV-2. Laboratory studies have shown that the new coronavirus is able to replicate efficiently in cats and to spread between cats via respiratory droplets, and further that our pre-laboratory studies have shown that SARS-CoV-2 infects cat populations, and that 14.7% of cats in the sample investigated can be serologically positive by indirect enzyme-linked immunosorbent assay (E LISA), these infections possibly being caused by exposure of cats to SARS-CoV-2 contaminated environments or patients in close contact with cats. Although the intermediate host of SARS-CoV-2 is still unclear at present, immune protection of animals, particularly cats, which are in close contact with humans and susceptible to infection is imminent.
The Spike protein (Spike protein) of SARS-CoV-2 is a large type I transmembrane protein comprising two subunits, S1 and S2. S1 contains primarily a receptor binding domain, RBD, responsible for recognizing cell surface receptors. S2 contains essential elements required for membrane fusion. The S protein plays a key role in neutralizing antibody and T cell responses and in the induction of protective immunity. The main functions of the S protein are summarized as: mediate receptor binding and membrane fusion; defining the scope of the host and the specificity of the virus; a primary component that binds to neutralizing antibodies; is a major goal of vaccine design. Spike protein (S) is currently widely recognized as a key factor in coronavirus invasion and cross-species transmission. Researchers have found that recombinant vaccines comprising SARS-CoV-2-RBD can induce effective functional antibodies in immunized mice, rabbits and non-human primates (macaques) 7 or 14 days after single immunization and can prevent infection with SARS-CoV-2, and have also demonstrated that S-RBD as a target sequence for recombinant subunit vaccine design can effectively prevent infection with SARS-CoV-2. Therefore, obtaining recombinant proteins with high stability, good immunogenicity, and high induced antibody levels is a central issue in the preparation of subunit vaccines. The recombinant subunit vaccine prepared by the eukaryotic expression system has the advantages of post-translational modification, good activity of expressed protein and the like. Among them, CHO (Chinese hamster ovary) has the advantages of suitability for high-density culture, high expression level and the like, so that the CHO is widely applied to the field of antigen detection and vaccine production. The yield of the secretion expression of the CHO expression system is often determined by factors such as protein size, selection of signal peptide, codon optimization and the like. Different codon-optimized sequences for the same protein and the suitability of different signal peptides for the protein of interest are decisive for their production. Therefore, the selection of the target sequence most suitable for CHO expression and the signal peptide most suitable for the sequence are important for preparing stable cell lines with high yield.
A series of SARS-CoV-2 vaccines for human beings including inactivated vaccines, nucleic acid vaccines, viral vector vaccines, recombinant subunit vaccines, etc. are now in urgent approval for use, but no novel coronavirus vaccines for susceptible animals such as cats have been reported. Therefore, the development of a vaccine aiming at SARS-CoV-2 virus for protecting susceptible animals cats and blocking the spread of the virus from humans to cats is of great importance, an effective prevention and control strategy can be provided for coping with potential novel coronavirus cross-species spread events, and the vaccine has great significance for the public health practice of SA RS-CoV-2 prevention and control.
Disclosure of Invention
Based on the problems, the invention provides a recombinant subunit vaccine for preventing cats from being infected by novel coronavirus, wherein the subunit vaccine is recombinant fusion protein SARS-CoV-2-RBD-cat Fc, and the sequence is shown in SEQ ID NO. 14. The protein has good immunogenicity, induces stronger immune response in cats, generates antibodies with neutralizing activity, can neutralize novel coronavirus, and has important effect on preventing cats from being infected with the novel coronavirus.
Another objective of the invention is to provide a monoclonal CHO cell strain of high expression recombinant fusion protein SARS-CoV-2-RBD-cat Fc, the preservation number of the cell strain is: CCTCC NO: C2021190.
another purpose of the invention is to provide an expression vector suitable for a CHO expression system, wherein the expression vector is obtained by constructing a sequence shown in SEQ ID NO.7 into a pCMV-GS vector.
The last purpose of the invention is to provide the application of the recombinant fusion protein SARS-CoV-2-RBD-cat Fc in preparing the recombinant subunit vaccine for preventing cats from being infected with the novel coronavirus.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a recombinant subunit vaccine for preventing cats from being infected by novel coronavirus, wherein the subunit vaccine is recombinant fusion protein SARS-CoV-2-RBD-cat Fc, the sequence is shown in SEQ ID NO.14, and the subunit vaccine is R BD-cat Fc in the invention or in short.
The invention also provides an expression vector suitable for a CHO expression system, wherein the expression vector is obtained by constructing a sequence shown in SEQ ID NO.7 into a pCMV-GS vector, transfecting the expression vector into the CHO expression system, and finally obtaining a monoclonal cell strain for efficiently expressing the recombinant fusion protein SARS-CoV-2-RBD-cat Fc by screening the protein expression quantity, wherein the cell strain is delivered to the China center for type culture collection (CCCCTCC) at 7 and 16 months 2021 for collection, and is classified and named: c HO-K1/RBD-cat Fc-1, accession number: CCTCC NO: c2021190, address: wuhan university in Wuhan, China.
Application of recombinant fusion protein SARS-CoV-2-RBD-cat Fc in preparing recombinant subunit vaccine for preventing cat from infecting new type coronavirus includes utilizing conventional mode in field, including but not limited to SE Q ID NO.14 protein prepared by utilizing above-mentioned monoclonal cell, for preparing recombinant subunit vaccine for preventing cat from infecting new type coronavirus
When the recombinant subunit vaccine is used for immunizing cats, the optimal adjuvant is an aluminum hydroxide gel adjuvant.
Compared with the prior art, the invention has the beneficial effects that:
(1) the subunit protein has good immunogenicity, can induce high-level antibodies after immunizing cats, and the generated antibodies have the effect of neutralizing SARS-CoV-2 live viruses, can be used for preventing cats from infecting novel coronavirus, and have important significance in preventing the spread among novel coronavirus species.
(2) Compared with the directly expressed SARS-CoV-2 virus S protein RBD-his protein, the RBD-cat Fc recombinant protein has the advantages that the protein stability can be increased while the activity of the RBD protein is not influenced by a cat IgG Fc label, the cat IgG Fc label is easier to purify, and the purification and recovery efficiency is higher.
(3) Compared with the directly expressed SARS-CoV-2 virus S protein RBD-his target sequence, the fusion expression of RBD and cat IgG Fc can obviously increase the stability of the protein, prolong the half life of plasma and enhance the humoral immunity and cellular immunity effect in the body of a cat.
(4) Compared with the S protein RBD sequence of SARS-CoV-2 virus, the RBD-cat Fc sequence obtained by codon optimization and signal peptide screening has the advantage of high expression level.
(5) The CHO stable expression cell strain constructed by the invention has the advantages of high yield, good stability, easy large-scale production and the like.
Drawings
FIG. 1 shows protein expression conditions (immunoblot detection) of different optimized sequences in fermentation supernatants of 72h transfected CHO cells under cat Fc tag and His tag after pCMV-RBD-cat Fc-GS4 optimized sequences (cat Fc # 1-4 #) and pCMV-RBD-His-GS4 optimized sequences (His # 1-4 #) are shown.
FIG. 2 is a 96h cell supernatant expression immunoblot of SARS-CoV-2-RBD-cat Fc and RBD-His monoclonal strains.
FIG. 3 is an SDS-PAGE electrophoresis of purified SARS-CoV-2-RBD-cat Fc and RBD-His proteins.
FIG. 4 shows the results of the binding force and half-effective concentration determination of SARS-CoV-2-RBD-cat Fc and RBD-His protein and cat angiotensin ACE2 protein.
FIG. 5 shows the ELISA test results of the antibody titer in the serum of cats after different dosages of the subunit vaccines prepared from SARS-CoV-2-RBD-cat Fc and SARS-CoV-2-RBD-His recombinant protein are used for immunizing cats respectively.
FIG. 6 shows the result of detecting the neutralization activity of SARS-CoV-2 virus by the serum of an immunized cat after the cat is immunized with the subunit vaccines prepared from SARS-CoV-2-RBD-cat Fc and SARS-CoV-2-RBD-His recombinant proteins with different dosages.
Detailed Description
The invention is described in further detail below with reference to the figures and specific embodiments. The following examples are carried out on the premise of the technical scheme of the invention, detailed embodiments and specific operating procedures are given, and materials and reagents for the following examples which are not described in detail are all commercially available, but the scope of the invention is not limited to the following examples.
Example 1: construction of SARS-CoV-2-RBD-cat Fc and RBD-His recombinant plasmid
1. Codon optimization of novel coronavirus RBD coding gene
In order to realize the high-efficiency expression of RBD protein (NCBI accession number of RBD-original sequence: NC-045512, shown in SEQ ID NO. 13), the invention optimizes the sequence of SARS-CoV-2-RBD-cat Fc and S ARS-CoV-2-RBD-His coding gene according to the codon preference of CHO eukaryotic expression system. Codon optimization is a key technical means for realizing high-efficiency expression of heterologous proteins, and protein expression is a system engineering, so that codon optimization is carried out by only replacing codons with the highest usage frequency in a certain species, and the optimal effect is possibly not achieved.
The invention discovers that the expression level and purity of the obtained coding gene sequence can not meet the requirement of high-purity large-scale preparation in vaccine production by optimizing by adopting the conventional codon preference in the codon optimization process, and the expression level difference of the protein of different optimization sequences is found by detecting the expression level of target protein and comparing different optimization sequences, and no obvious rule exists between the optimization position and degree of the codon and the expression level.
The applicant designs a large number of codon optimized sequences aiming at the coding sequences of novel coronavirus RBD recombinant fusion proteins SARS-CoV-2-RBD-cat Fc and SARS-CoV-2-RBD-His, and respectively selects 2 optimized sequences, wherein 2 sequences of SARS-CoV-2-RBD-cat Fc are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2; 2 sequences of SARS-Co V-2-RBD-His are shown as SEQ ID NO.3 and SEQ ID NO.4, respectively, and the codon optimization screening process is exemplified by these sequences. The specific sequence optimization and recombinant plasmid construction method comprises the following steps:
2. construction of SARS-CoV-2-RBD-cat Fc and SARS-CoV-2-RBD-His recombinant plasmid
In order to obtain recombinant proteins with high expression quantity and secreted outside cells, two optimized sequences of SARS-CoV-2-RBD-cat Fc and S ARS-CoV-2-RBD-His are respectively synthesized and two signal peptide sequences are adapted at the 5' end so as to promote the expressed RBD-ca t Fc and RBD-His proteins to be effectively secreted outside cells. Wherein, 2 codon optimized sequences aiming at SARS-CoV-2-RBD-cat Fc are combined with signal peptide to respectively obtain sequences shown as SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, and the rest two signal peptides aiming at 2 codon optimized sequences of SARS-CoV-2-RBD-His are combined to respectively obtain SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO. 12.
SARS-CoV-2-RBD-cat Fc sequence SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and S EQ ID NO.8 and SARS-CoV-2-RBD-His sequence SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12 were added, respectively. The recombinant plasmid is constructed into a pCMV-GS vector plasmid, and is entrusted to Beijing Okagaku biotechnology limited company for synthesis to obtain a recombinant expression plasmid, wherein the recombinant plasmid is named as pCMV-RBD-catFc-GS-1#, pCMV-RBD-cat Fc-GS-2#, pCMV-RBD-cat Fc-GS-3#, pCMV-RBD-catFc-GS-4#, pCMV-RBD-His-GS-1#, pCMV-RBD-Hi-GS-2 #, pCMV-RBD-His-GS-3#, and pCMV-RBD-His-GS-4#, and the result is consistent with the target gene sequence after sequencing comparison.
Example 2: eukaryotic expression of pCMV-RBD-cat Fc and pCMV-RBD-His recombinant plasmid and screening, cloning and domesticating of stable and high-efficiency expression cell strain
Adopting a CHO eukaryotic expression system, inoculating cells into a fresh culture medium at the density of 2x10^6 cells/mL one day before transfection, and culturing in a constant-temperature shaking table at the rotating speed of 150-; on the day of transfection, samples were taken for cell density and viability. The cell density should be 3-5x10^6 cells/mL, and the activity rate is higher than 90%. Adjusting the cell density to 3x10^6 cells/mL, placing the cells in a 125mL cell shake flask, and ensuring that the total volume is 20 mL; respectively diluting 20 mu g of plasmids (p CMV-RBD-catFc-GS-1#, pCMV-RBD-catFc-GS-2 #, pCMV-RBD-catFc-GS-3 #, pCMV-RB D-catFc-GS-4#, pCMV-RBD-His-GS-1#, pCMV-RBD-His-GS-2#, pCMV-RBD-His-GS-3#, p CMV-RBD-His-GS-4 #) by using an optim culture medium until the total volume is 0.5mL, and lightly blowing and uniformly mixing by using a pipette; diluting 40 mu L of lip 8000 transfection reagent by optim culture medium to the total volume of 0.5mL, and gently blowing and mixing by a pipette; and (2) slightly and uniformly mixing the diluted DNA and a transfection reagent, dropwise adding the mixture into a cell culture solution, slightly shaking a cell bottle while dropwise adding the mixture, uniformly shaking the mixture, then putting the mixture back to a shaking table for continuous culture, collecting culture supernatant for 96 hours of culture for detecting cell expression, and detecting the expression conditions of proteins of different optimized sequences of R BD-cat Fc and RBD-His through immunoblotting (western-Blot). As shown in FIG. 1, pCMV-RBD-cat Fc-GS-3# and p CMV-RBD-His-GS-4# show stronger secretion expression capability, which indicates that the lgKappa signal peptide and the optimized sequence are more suitable for expression in the CHO system. Cells transferred into plasmids pCMV-RBD-cat Fc-GS-3# and pCMV-RBD-His-GS-4# were subjected to low-speed centrifugation at 1500 rpm for 5 minutes to change the medium to a fresh medium containing 25. mu.M of MSX (methionine maple imide), and the medium was uniformly plated in 96-well plates at a density of 0.5 to 1 cell per well per 150. mu.L per well, and each plate was plated in 5 plates. After 3-4 weeks of static culture, cell culture supernatant was aspirated weekly and supplemented with fresh medium to 150 μ L per well. Detecting the protein expression quantity of each clone by an immunoblot (western-Blot) of the sucked 100ul cell culture supernatant, selecting 20 clones for subsequent subcloning, finding that the expression quantity does not rise and fall along with the increase of the cloning times in the process of subcloning, even no protein expression is detected in the subsequent clones of some subclones, transferring 5 clones with higher expression quantity to a 6-pore plate step by step after three rounds of subcloning, expanding the monoclonal cell strain with the highest expression quantity to a cell shake flask for feed culture, determining the cell yield of the monoclonal strain after clarification and centrifugal filtration purification, and screening the screened high-efficiency expression pCMV-RBD-cat F c-GS-3-01 strain and pCMV-RBD-His-GS # -4 strain, wherein the results are shown in Table 1, the results of western-Blot detection of protein expressed by the cell strain are shown in FIG. 2, and the selected CHO-RBD-cat Fc-GS-3# -01 monoclonal cell strain with the highest expression level is delivered to the China center for type culture Collection at 7/16 of 2021 for storage, and is classified and named: CHO-K1/RBD-cat Fc-1, accession number: c2021190, address: wuhan university in Wuhan, China.
TABLE 1 determination of protein expression amount of clone
Figure BDA0003168758960000051
Figure BDA0003168758960000061
Example 3: the purification and activity identification of SARS-CoV-2-RBD-cat Fc and SARS-CoV-2-RBD-his recombinant protein.
1. Expression and purification of SARS-CoV-2-RBD-cat Fc and SARS-CoV-2-RBD-his recombinant protein
The stable high-efficiency expression cell strain selected in the example 2 is enlarged to a 1L cell bottle of 250mL culture solution, the strain is continuously cultured for 14 days at 37 ℃ under 5% carbon dioxide at 120 r/min, after the supplementary culture, the collected supernatant is purified by protein A and N i excel filler using AKTA purifier, RBD-cat Fc recombinant protein is eluted by 0.1Imol/L glycine (pH3.0), the collected eluent is neutralized by 1mol/L Tris-HCl (pH8.8) for standby, the optimal impurity and imidazole concentrations of RBD-His are respectively 50mmol and 300mmol, the collected eluent is desalted for standby, the effective concentration of the purified RBD-cat Fc recombinant protein is 195 mug/mL, the effective concentration of the RBD-recombinant His protein is 191 mug/mL, and the purified S ARS-CoV-2-RBD-cat Fc and the SARS-CoV-2-RBD-His are obtained The purification efficiency of the recombinant protein is shown in Table 2, as shown by SDS-PAGE in FIG. 3.
TABLE 2 purification and recovery efficiency of two recombinant proteins
Sample name Concentration in culture supernatant (. mu.g/mL) Concentration after purification (μ g/mL) Recovery (%)
RBD-cat Fc 213 195 91.5
RBD-His 246 191 77.7
2. SARS-CoV-2-RBD-cat Fc and SARS-CoV-2-RBD-his purified protein activity and stability identification
The activity of the purified protein was verified by detecting the binding force of SARS-CoV-2-RBD-cat Fc and SARS-CoV-2-RBD-his to cat ACE2 by ELISA method. The specific operation steps are as follows:
ACE2-His protein was coated onto an ELISA plate at 1. mu.g/mL 4 ℃ overnight, blocked with 5% BSA at 37 ℃ and blown dry before being stored in a refrigerator at 4 ℃ for further use. Taking out the ELISA plate, balancing for 5 minutes at room temperature for later use, carrying out gradient dilution on RBD-cat Fc and RBD-His protein from 2 mu g/mL initial concentration by 10 times, adding each protein with dilution gradient into the coated ELSI A plate by 100 mu L/hole, incubating for 1h at room temperature, and repeating each sample for three times; removing the supernatant, washing the mixture for five times by using TBST, and inversely arranging the ELISA plate on water-absorbent paper for drying; adding HRP-labeled goat anti-cat IgG and goat anti-mouse His monoclonal antibodies respectively, incubating at 37 ℃ for 60min, taking T MB color development A B liquid, adding 50 mu L of the T MB color development A B liquid into each hole, developing in the dark for 10-15 min, and adding 50 mu L of stop solution into each hole to stop the reaction. And (3) determining the OD value of each hole at the wavelength of 630nm on an enzyme-labeling instrument, and drawing a RBD-cat Fc and RBD-His recombinant protein and recombinant ACE2 protein binding curve by taking the concentration of the expressed and purified protein as an abscissa and the OD630 as an ordinate. The results are shown in FIG. 4, and the half effective concentration EC50 of the two proteins SARS-Co V-2-RBD-cat Fc and SARS-CoV-2-RBD-his is 120ng, which proves that the recombinant proteins RBD and cat Fc exhibit higher biological activity, and the two proteins can be correctly folded respectively without causing steric hindrance.
The purified protein may have flocculation reaction under the influence of temperature and concentration, thereby affecting the spatial structure stability of the protein and further affecting the immunogenicity of the antigen protein. The stability of the RBD-cat Fc and the RBD-His protein is compared by determining whether the RBD-cat Fc and the RBD-His recombinant protein with different concentrations have flocculation phenomenon at 4 ℃ within 35 days. The specific operation steps are as follows:
RBD-cat Fc and RBD-His recombinant protein solution are diluted by 0.85% sterilized normal saline to protein concentrations of 1.0, 2.0, 3.0, 4.0, 5.0 and mg/ml respectively, and placed in 1.5ml EP tubes, and 6 tubes are prepared at each concentration, and each tube is 1.0 ml. All the dilutions of 5 dilutions were stored at 4 ℃, sampled every 7 days to test the stability of the protein, and tested 5 times in total, with the following specific operations: taking 1 tube of protein solution and stock solution with different concentrations every 7 days, and observing whether precipitates are generated or not by naked eyes; centrifuging the EP tube at 6000rpm for 15min, and observing whether a precipitate is generated; and (3) taking 100 mu l of supernatant after centrifugation, and determining the protein concentration according to a BSA colorimetric method, wherein the results are shown in tables 3 and 4, and the results show that the RBD-cat Fc purified protein with the protein concentration of 1.0-5.0 mg/ml does not generate visible precipitates within 35 days, does not generate precipitates after centrifugation, and does not change the protein concentration. While RBD-his purified protein at protein concentrations of 4.0 and 5.0mg/ml was centrifuged at day 35 to produce a small amount of precipitate and a drop in protein concentration. Therefore, compared with RBD-His recombinant protein, the RBD-cat Fc has more stable performance under the same storage condition, which indicates that the cat Fc tag is beneficial to the stable structure and long-term storage of the protein and the subsequent preparation of subunit vaccine.
TABLE 3 stability test results of RBD-cat Fc protein at the same concentration
Figure BDA0003168758960000071
TABLE 4 stability test results of RBD-His protein at the same concentration
Figure BDA0003168758960000081
Note "-": indicates no visible precipitation; "+: "there is a visible precipitate with naked eyes;
×) c: indicates that the centrifugation is free of sediment; ↓: indicating that the centrifugation had sedimented.
Example 4: preparation of SARS-CoV-2-RBD-cat Fc and RBD-His recombinant protein subunit vaccine
And (3) fully and uniformly mixing the RBD-cat Fc and the RBD-His recombinant proteins obtained by expression and purification with an aluminum hydroxide gel normal saline solution according to a volume ratio of 7:1 to prepare the genetic engineering subunit vaccine, wherein the antigen contents of the 2 recombinant protein vaccines are respectively 20 mu g, 30 mu g and 50 mu g.
Example 5: immune and antibody titer evaluation of SARS-CoV-2-RBD-cat Fc and SARS-CoV-2-RBD-His recombinant protein subunit vaccine in cat body
The subunit vaccine prepared in example 4 was used to immunize cats, while a PBS control was established; 21 three-month-old female cats were selected, randomly divided into 7 groups of 3 animals each, and immunized by subcutaneous multipoint injection. 1-3 groups of RBD-cat Fc with antigen content of 20 μ g, 30 μ g and 50 μ g, 4-6 groups of RBD-His with antigen content of 20 μ g, 30 μ g and 50 μ g, and 7 groups of PBS control (aluminum hydroxide gel adjuvant); measuring body temperature every day after immunization, periodically collecting blood from brachial cephalic vein, enhancing immunization once after one month, detecting antibody level by ELIS A, and continuously monitoring dynamic change rule of antibody two months after the first immunization.
The results are shown in fig. 5 and table 5, the RBD-cat Fc and RBD-His recombinant protein antigens can stimulate the body to produce specific antibodies, three dose groups of RBD-cat Fc and 50 μ g of RBD-His antibodies are positive after the third week of immunization, and all antibodies in the 6 th immunization group are positive after the fourth week of immunization; the antibody level reached the highest level at the fifth week after immunization (i.e., the first week after the second immunization), and no adverse reaction occurred. The RBD-cat Fc three doses of immunization induced higher levels of specific antibodies compared to RBD-His, and the two month antibodies still maintained higher levels, whereas the PBS group failed to produce antibodies. Sera were selected at the fifth week after the first immunization and were tested for virus neutralization to determine the serum neutralization titer. Each serum sample was diluted with medium (DMEM) in 2-fold or 3-fold volume depending on OD value and mixed with an equivalent dilution of SARS-CoV-2 virus and incubated at 37 ℃ for 1 hour in an incubator. Vero E6 cells cultured in a 24-well plate were added with the treated serum virus mixture and allowed to stand at 37 ℃ for 1 hour, and then the serum virus mixture was replaced with DMEM containing 2.5% serum FBS and 0.8% carboxymethylcellulose and allowed to stand at 37 ℃ in an incubator for 72 hours. The plaque number was calculated by fixing the plates with 8% paraformaldehyde, staining with 0.5% crystal violet, photographing after drying, and calculating the neutralization titer by serum dilution, which is defined as the reduction of plaque by at least 50% due to serum dilution. All samples were tested in duplicate and negative serum and complete virus controls were set up. The results are shown in FIG. 6, where the serum dilution of neutralizing antibodies generated by the two-component recombinant vaccine was between 120-1080, which inhibited SARS-CoV-2 infection of VERO E6 cells. Moreover, the neutralizing antibodies generated by the RBD-cat Fc are higher than those generated by the RBD-His group, which indicates that the RBD-cat Fc subunit vaccine has good immunogenicity, can stimulate the body to generate high-level protective neutralizing antibodies, and can be used for preparing high-efficiency subunit vaccines.
TABLE 5 serum antibody titer ELISA assay results after recombinant protein subunit vaccines have been administered to cats
Figure BDA0003168758960000091
Sequence listing
<110> university of agriculture in Huazhong
<120> novel feline coronavirus subunit vaccine and preparation method and application thereof
<160> 14
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1368
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
agggtgcagc ccaccgagag catcgtgagg ttccccaaca tcaccaacct gtgccccttc 60
ggcgaggtgt tcaacgccac caggttcgcc agcgtgtacg cctggaacag gaagaggatc 120
agcaactgcg tggccgacta cagcgtgctg tacaacagcg ccagcttcag caccttcaag 180
tgctacggcg tgagccccac caagctgaac gacctgtgct tcaccaacgt gtacgccgac 240
agcttcgtga tcaggggcga cgaggtgagg cagatcgccc ccggccagac cggcaacatc 300
gccgactaca actacaagct gcccgacgac ttcaccggct gcgtgatcgc ctggaacagc 360
aacaacctgg acagcaaggt gggcggcaac tacaactacc tgtacaggct gttcaggaag 420
agcaacctga agcccttcga gagggacatc agcaccgaga tctaccaggc cggcagcacc 480
ccctgcaacg gcgtgaaggg cttcaactgc tacttccccc tgcagagcta cggcttccag 540
cccacctacg gcgtgggcta ccagccctac agggtggtgg tgctgagctt cgagctgctg 600
cacgcccccg ccaccgtgtg cggccccaag aagagcacca acctggtgaa gaacaagtgc 660
gtgaacttcg gcggcggcgg cagcgtgccc agggacagcg gctgcaagcc ctgcatctgc 720
accgtgcccg aggtgagcag cgtgttcatc ttccccccca agcccaagga cgtgctgacc 780
atcaccctga cccccaaggt gacctgcgtg gtggtggaca tcagcaagga cgaccccgag 840
gtgcagttca gctggttcgt ggacgacgtg gaggtgcaca ccgcccagac ccagcccagg 900
gaggagcagt tcaacagcac cttcaggagc gtgagcgagc tgcccatcat gcaccaggac 960
tggctgaacg gcaaggagtt caagtgcagg gtgaacagcg ccgccttccc cgcccccatc 1020
gagaagacca tcagcaagac caagggcagg cccaaggccc cccaggtgta caccatcccc 1080
ccccccaagg agcagatggc caaggacaag gtgagcctga cctgcatgat caccgacttc 1140
ttccccgagg acatcaccgt ggagtggcag tggaacggcc agcccgccga gaactacaag 1200
aacacccagc ccatcatgga caccgacggc agctacttcg tgtacagcaa gctgaacgtg 1260
cagaagagca actgggaggc cggcaacacc ttcacctgca gcgtgctgca cgagggcctg 1320
cacaaccacc acaccgagaa gagcctgagc cacagccccg gcaagtag 1368
<210> 2
<211> 1368
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
cgcgtgcagc ccaccgagag catcgtgcgc ttccccaaca tcaccaacct gtgccccttc 60
ggcgaggtgt tcaacgccac ccgcttcgcc agcgtgtacg cctggaaccg caagcgcatc 120
agcaactgcg tggccgacta cagcgtgctg tacaacagcg ccagcttcag caccttcaag 180
tgctacggcg tgagccccac caagctgaac gacctgtgct tcaccaacgt gtacgccgac 240
agcttcgtga tccgcggcga cgaggtgcgc cagatcgccc ccggccagac cggcaacatc 300
gccgactaca actacaagct gcccgacgac ttcaccggct gcgtgatcgc ctggaacagc 360
aacaacctgg acagcaaggt gggcggcaac tacaactacc tgtaccgcct gttccgcaag 420
agcaacctga agcccttcga gcgcgacatc agcaccgaga tctaccaggc cggcagcacc 480
ccctgcaacg gcgtgaaggg cttcaactgc tacttccccc tgcagagcta cggcttccag 540
cccacctacg gcgtgggcta ccagccctac cgcgtggtgg tgctgagctt cgagctgctg 600
cacgcccccg ccaccgtgtg cggccccaag aagagcacca acctggtgaa gaacaagtgc 660
gtgaacttcg gcggcggcgg cagcgtgccc cgcgacagcg gctgcaagcc ctgcatctgc 720
accgtgcccg aggtgagcag cgtgttcatc ttccccccca agcccaagga cgtgctgacc 780
atcaccctga cccccaaggt gacctgcgtg gtggtggaca tcagcaagga cgaccccgag 840
gtgcagttca gctggttcgt ggacgacgtg gaggtgcaca ccgcccagac ccagccccgc 900
gaggagcagt tcaacagcac cttccgcagc gtgagcgagc tgcccatcat gcaccaggac 960
tggctgaacg gcaaggagtt caagtgccgc gtgaacagcg ccgccttccc cgcccccatc 1020
gagaagacca tcagcaagac caagggccgc cccaaggccc cccaggtgta caccatcccc 1080
ccccccaagg agcagatggc caaggacaag gtgagcctga cctgcatgat caccgacttc 1140
ttccccgagg acatcaccgt ggagtggcag tggaacggcc agcccgccga gaactacaag 1200
aacacccagc ccatcatgga caccgacggc agctacttcg tgtacagcaa gctgaacgtg 1260
cagaagagca actgggaggc cggcaacacc ttcacctgca gcgtgctgca cgagggcctg 1320
cacaaccacc acaccgagaa gagcctgagc cacagccccg gcaagtag 1368
<210> 3
<211> 669
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
cgcgtgcagc ccaccgagag catcgtgcgc ttccccaaca tcaccaacct gtgccccttc 60
ggcgaggtgt tcaacgccac ccgcttcgcc agcgtgtacg cctggaaccg caagcgcatc 120
agcaactgcg tggccgacta cagcgtgctg tacaacagcg ccagcttcag caccttcaag 180
tgctacggcg tgagccccac caagctgaac gacctgtgct tcaccaacgt gtacgccgac 240
agcttcgtga tccgcggcga cgaggtgcgc cagatcgccc ccggccagac cggcaacatc 300
gccgactaca actacaagct gcccgacgac ttcaccggct gcgtgatcgc ctggaacagc 360
aacaacctgg acagcaaggt gggcggcaac tacaactacc tgtaccgcct gttccgcaag 420
agcaacctga agcccttcga gcgcgacatc agcaccgaga tctaccaggc cggcagcacc 480
ccctgcaacg gcgtgaaggg cttcaactgc tacttccccc tgcagagcta cggcttccag 540
cccacctacg gcgtgggcta ccagccctac cgcgtggtgg tgctgagctt cgagctgctg 600
cacgcccccg ccaccgtgtg cggccccaag aagagcacca acctggtgaa gaacaagtgc 660
gtgaacttc 669
<210> 4
<211> 669
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
agggtgcagc ccaccgagag catcgtgagg ttccccaaca tcaccaacct gtgccccttc 60
ggcgaggtgt tcaacgccac caggttcgcc agcgtgtacg cctggaacag gaagaggatc 120
agcaactgcg tggccgacta cagcgtgctg tacaacagcg ccagcttcag caccttcaag 180
tgctacggcg tgagccccac caagctgaac gacctgtgct tcaccaacgt gtacgccgac 240
agcttcgtga tcaggggcga cgaggtgagg cagatcgccc ccggccagac cggcaacatc 300
gccgactaca actacaagct gcccgacgac ttcaccggct gcgtgatcgc ctggaacagc 360
aacaacctgg acagcaaggt gggcggcaac tacaactacc tgtacaggct gttcaggaag 420
agcaacctga agcccttcga gagggacatc agcaccgaga tctaccaggc cggcagcacc 480
ccctgcaacg gcgtgaaggg cttcaactgc tacttccccc tgcagagcta cggcttccag 540
cccacctacg gcgtgggcta ccagccctac agggtggtgg tgctgagctt cgagctgctg 600
cacgcccccg ccaccgtgtg cggccccaag aagagcacca acctggtgaa gaacaagtgc 660
gtgaacttc 669
<210> 5
<211> 1419
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
atggtgagga ccgccgtgct gatcctgctg ctggtgaggt tcagcgagcc cagggtgcag 60
cccaccgaga gcatcgtgag gttccccaac atcaccaacc tgtgcccctt cggcgaggtg 120
ttcaacgcca ccaggttcgc cagcgtgtac gcctggaaca ggaagaggat cagcaactgc 180
gtggccgact acagcgtgct gtacaacagc gccagcttca gcaccttcaa gtgctacggc 240
gtgagcccca ccaagctgaa cgacctgtgc ttcaccaacg tgtacgccga cagcttcgtg 300
atcaggggcg acgaggtgag gcagatcgcc cccggccaga ccggcaacat cgccgactac 360
aactacaagc tgcccgacga cttcaccggc tgcgtgatcg cctggaacag caacaacctg 420
gacagcaagg tgggcggcaa ctacaactac ctgtacaggc tgttcaggaa gagcaacctg 480
aagcccttcg agagggacat cagcaccgag atctaccagg ccggcagcac cccctgcaac 540
ggcgtgaagg gcttcaactg ctacttcccc ctgcagagct acggcttcca gcccacctac 600
ggcgtgggct accagcccta cagggtggtg gtgctgagct tcgagctgct gcacgccccc 660
gccaccgtgt gcggccccaa gaagagcacc aacctggtga agaacaagtg cgtgaacttc 720
ggcggcggcg gcagcgtgcc cagggacagc ggctgcaagc cctgcatctg caccgtgccc 780
gaggtgagca gcgtgttcat cttccccccc aagcccaagg acgtgctgac catcaccctg 840
acccccaagg tgacctgcgt ggtggtggac atcagcaagg acgaccccga ggtgcagttc 900
agctggttcg tggacgacgt ggaggtgcac accgcccaga cccagcccag ggaggagcag 960
ttcaacagca ccttcaggag cgtgagcgag ctgcccatca tgcaccagga ctggctgaac 1020
ggcaaggagt tcaagtgcag ggtgaacagc gccgccttcc ccgcccccat cgagaagacc 1080
atcagcaaga ccaagggcag gcccaaggcc ccccaggtgt acaccatccc cccccccaag 1140
gagcagatgg ccaaggacaa ggtgagcctg acctgcatga tcaccgactt cttccccgag 1200
gacatcaccg tggagtggca gtggaacggc cagcccgccg agaactacaa gaacacccag 1260
cccatcatgg acaccgacgg cagctacttc gtgtacagca agctgaacgt gcagaagagc 1320
aactgggagg ccggcaacac cttcacctgc agcgtgctgc acgagggcct gcacaaccac 1380
cacaccgaga agagcctgag ccacagcccc ggcaagtag 1419
<210> 6
<211> 1419
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
atggtgagga ccgccgtgct gatcctgctg ctggtgaggt tcagcgagcc ccgcgtgcag 60
cccaccgaga gcatcgtgcg cttccccaac atcaccaacc tgtgcccctt cggcgaggtg 120
ttcaacgcca cccgcttcgc cagcgtgtac gcctggaacc gcaagcgcat cagcaactgc 180
gtggccgact acagcgtgct gtacaacagc gccagcttca gcaccttcaa gtgctacggc 240
gtgagcccca ccaagctgaa cgacctgtgc ttcaccaacg tgtacgccga cagcttcgtg 300
atccgcggcg acgaggtgcg ccagatcgcc cccggccaga ccggcaacat cgccgactac 360
aactacaagc tgcccgacga cttcaccggc tgcgtgatcg cctggaacag caacaacctg 420
gacagcaagg tgggcggcaa ctacaactac ctgtaccgcc tgttccgcaa gagcaacctg 480
aagcccttcg agcgcgacat cagcaccgag atctaccagg ccggcagcac cccctgcaac 540
ggcgtgaagg gcttcaactg ctacttcccc ctgcagagct acggcttcca gcccacctac 600
ggcgtgggct accagcccta ccgcgtggtg gtgctgagct tcgagctgct gcacgccccc 660
gccaccgtgt gcggccccaa gaagagcacc aacctggtga agaacaagtg cgtgaacttc 720
ggcggcggcg gcagcgtgcc ccgcgacagc ggctgcaagc cctgcatctg caccgtgccc 780
gaggtgagca gcgtgttcat cttccccccc aagcccaagg acgtgctgac catcaccctg 840
acccccaagg tgacctgcgt ggtggtggac atcagcaagg acgaccccga ggtgcagttc 900
agctggttcg tggacgacgt ggaggtgcac accgcccaga cccagccccg cgaggagcag 960
ttcaacagca ccttccgcag cgtgagcgag ctgcccatca tgcaccagga ctggctgaac 1020
ggcaaggagt tcaagtgccg cgtgaacagc gccgccttcc ccgcccccat cgagaagacc 1080
atcagcaaga ccaagggccg ccccaaggcc ccccaggtgt acaccatccc cccccccaag 1140
gagcagatgg ccaaggacaa ggtgagcctg acctgcatga tcaccgactt cttccccgag 1200
gacatcaccg tggagtggca gtggaacggc cagcccgccg agaactacaa gaacacccag 1260
cccatcatgg acaccgacgg cagctacttc gtgtacagca agctgaacgt gcagaagagc 1320
aactgggagg ccggcaacac cttcacctgc agcgtgctgc acgagggcct gcacaaccac 1380
cacaccgaga agagcctgag ccacagcccc ggcaagtag 1419
<210> 7
<211> 1434
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
atggagaccg acaccctgct gctgtgggtg ctgctgctgt gggtgcccgg cagcaccggc 60
gacgccaggg tgcagcccac cgagagcatc gtgaggttcc ccaacatcac caacctgtgc 120
cccttcggcg aggtgttcaa cgccaccagg ttcgccagcg tgtacgcctg gaacaggaag 180
aggatcagca actgcgtggc cgactacagc gtgctgtaca acagcgccag cttcagcacc 240
ttcaagtgct acggcgtgag ccccaccaag ctgaacgacc tgtgcttcac caacgtgtac 300
gccgacagct tcgtgatcag gggcgacgag gtgaggcaga tcgcccccgg ccagaccggc 360
aacatcgccg actacaacta caagctgccc gacgacttca ccggctgcgt gatcgcctgg 420
aacagcaaca acctggacag caaggtgggc ggcaactaca actacctgta caggctgttc 480
aggaagagca acctgaagcc cttcgagagg gacatcagca ccgagatcta ccaggccggc 540
agcaccccct gcaacggcgt gaagggcttc aactgctact tccccctgca gagctacggc 600
ttccagccca cctacggcgt gggctaccag ccctacaggg tggtggtgct gagcttcgag 660
ctgctgcacg cccccgccac cgtgtgcggc cccaagaaga gcaccaacct ggtgaagaac 720
aagtgcgtga acttcggcgg cggcggcagc gtgcccaggg acagcggctg caagccctgc 780
atctgcaccg tgcccgaggt gagcagcgtg ttcatcttcc cccccaagcc caaggacgtg 840
ctgaccatca ccctgacccc caaggtgacc tgcgtggtgg tggacatcag caaggacgac 900
cccgaggtgc agttcagctg gttcgtggac gacgtggagg tgcacaccgc ccagacccag 960
cccagggagg agcagttcaa cagcaccttc aggagcgtga gcgagctgcc catcatgcac 1020
caggactggc tgaacggcaa ggagttcaag tgcagggtga acagcgccgc cttccccgcc 1080
cccatcgaga agaccatcag caagaccaag ggcaggccca aggcccccca ggtgtacacc 1140
atcccccccc ccaaggagca gatggccaag gacaaggtga gcctgacctg catgatcacc 1200
gacttcttcc ccgaggacat caccgtggag tggcagtgga acggccagcc cgccgagaac 1260
tacaagaaca cccagcccat catggacacc gacggcagct acttcgtgta cagcaagctg 1320
aacgtgcaga agagcaactg ggaggccggc aacaccttca cctgcagcgt gctgcacgag 1380
ggcctgcaca accaccacac cgagaagagc ctgagccaca gccccggcaa gtag 1434
<210> 8
<211> 1434
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
atggagaccg acaccctgct gctgtgggtg ctgctgctgt gggtgcccgg cagcaccggc 60
gacgcccgcg tgcagcccac cgagagcatc gtgcgcttcc ccaacatcac caacctgtgc 120
cccttcggcg aggtgttcaa cgccacccgc ttcgccagcg tgtacgcctg gaaccgcaag 180
cgcatcagca actgcgtggc cgactacagc gtgctgtaca acagcgccag cttcagcacc 240
ttcaagtgct acggcgtgag ccccaccaag ctgaacgacc tgtgcttcac caacgtgtac 300
gccgacagct tcgtgatccg cggcgacgag gtgcgccaga tcgcccccgg ccagaccggc 360
aacatcgccg actacaacta caagctgccc gacgacttca ccggctgcgt gatcgcctgg 420
aacagcaaca acctggacag caaggtgggc ggcaactaca actacctgta ccgcctgttc 480
cgcaagagca acctgaagcc cttcgagcgc gacatcagca ccgagatcta ccaggccggc 540
agcaccccct gcaacggcgt gaagggcttc aactgctact tccccctgca gagctacggc 600
ttccagccca cctacggcgt gggctaccag ccctaccgcg tggtggtgct gagcttcgag 660
ctgctgcacg cccccgccac cgtgtgcggc cccaagaaga gcaccaacct ggtgaagaac 720
aagtgcgtga acttcggcgg cggcggcagc gtgccccgcg acagcggctg caagccctgc 780
atctgcaccg tgcccgaggt gagcagcgtg ttcatcttcc cccccaagcc caaggacgtg 840
ctgaccatca ccctgacccc caaggtgacc tgcgtggtgg tggacatcag caaggacgac 900
cccgaggtgc agttcagctg gttcgtggac gacgtggagg tgcacaccgc ccagacccag 960
ccccgcgagg agcagttcaa cagcaccttc cgcagcgtga gcgagctgcc catcatgcac 1020
caggactggc tgaacggcaa ggagttcaag tgccgcgtga acagcgccgc cttccccgcc 1080
cccatcgaga agaccatcag caagaccaag ggccgcccca aggcccccca ggtgtacacc 1140
atcccccccc ccaaggagca gatggccaag gacaaggtga gcctgacctg catgatcacc 1200
gacttcttcc ccgaggacat caccgtggag tggcagtgga acggccagcc cgccgagaac 1260
tacaagaaca cccagcccat catggacacc gacggcagct acttcgtgta cagcaagctg 1320
aacgtgcaga agagcaactg ggaggccggc aacaccttca cctgcagcgt gctgcacgag 1380
ggcctgcaca accaccacac cgagaagagc ctgagccaca gccccggcaa gtag 1434
<210> 9
<211> 768
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
atggtgagga ccgccgtgct gatcctgctg ctggtgaggt tcagcgagcc ccgcgtgcag 60
cccaccgaga gcatcgtgcg cttccccaac atcaccaacc tgtgcccctt cggcgaggtg 120
ttcaacgcca cccgcttcgc cagcgtgtac gcctggaacc gcaagcgcat cagcaactgc 180
gtggccgact acagcgtgct gtacaacagc gccagcttca gcaccttcaa gtgctacggc 240
gtgagcccca ccaagctgaa cgacctgtgc ttcaccaacg tgtacgccga cagcttcgtg 300
atccgcggcg acgaggtgcg ccagatcgcc cccggccaga ccggcaacat cgccgactac 360
aactacaagc tgcccgacga cttcaccggc tgcgtgatcg cctggaacag caacaacctg 420
gacagcaagg tgggcggcaa ctacaactac ctgtaccgcc tgttccgcaa gagcaacctg 480
aagcccttcg agcgcgacat cagcaccgag atctaccagg ccggcagcac cccctgcaac 540
ggcgtgaagg gcttcaactg ctacttcccc ctgcagagct acggcttcca gcccacctac 600
ggcgtgggct accagcccta ccgcgtggtg gtgctgagct tcgagctgct gcacgccccc 660
gccaccgtgt gcggccccaa gaagagcacc aacctggtga agaacaagtg cgtgaacttc 720
ggcggcggcg gcagccacca ccaccaccac caccaccacc accactag 768
<210> 10
<211> 768
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
atggtgagga ccgccgtgct gatcctgctg ctggtgaggt tcagcgagcc cagggtgcag 60
cccaccgaga gcatcgtgag gttccccaac atcaccaacc tgtgcccctt cggcgaggtg 120
ttcaacgcca ccaggttcgc cagcgtgtac gcctggaaca ggaagaggat cagcaactgc 180
gtggccgact acagcgtgct gtacaacagc gccagcttca gcaccttcaa gtgctacggc 240
gtgagcccca ccaagctgaa cgacctgtgc ttcaccaacg tgtacgccga cagcttcgtg 300
atcaggggcg acgaggtgag gcagatcgcc cccggccaga ccggcaacat cgccgactac 360
aactacaagc tgcccgacga cttcaccggc tgcgtgatcg cctggaacag caacaacctg 420
gacagcaagg tgggcggcaa ctacaactac ctgtacaggc tgttcaggaa gagcaacctg 480
aagcccttcg agagggacat cagcaccgag atctaccagg ccggcagcac cccctgcaac 540
ggcgtgaagg gcttcaactg ctacttcccc ctgcagagct acggcttcca gcccacctac 600
ggcgtgggct accagcccta cagggtggtg gtgctgagct tcgagctgct gcacgccccc 660
gccaccgtgt gcggccccaa gaagagcacc aacctggtga agaacaagtg cgtgaacttc 720
ggcggcggcg gcagccacca ccaccaccac caccaccacc accactag 768
<210> 11
<211> 783
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
atggagaccg acaccctgct gctgtgggtg ctgctgctgt gggtgcccgg cagcaccggc 60
gacgcccgcg tgcagcccac cgagagcatc gtgcgcttcc ccaacatcac caacctgtgc 120
cccttcggcg aggtgttcaa cgccacccgc ttcgccagcg tgtacgcctg gaaccgcaag 180
cgcatcagca actgcgtggc cgactacagc gtgctgtaca acagcgccag cttcagcacc 240
ttcaagtgct acggcgtgag ccccaccaag ctgaacgacc tgtgcttcac caacgtgtac 300
gccgacagct tcgtgatccg cggcgacgag gtgcgccaga tcgcccccgg ccagaccggc 360
aacatcgccg actacaacta caagctgccc gacgacttca ccggctgcgt gatcgcctgg 420
aacagcaaca acctggacag caaggtgggc ggcaactaca actacctgta ccgcctgttc 480
cgcaagagca acctgaagcc cttcgagcgc gacatcagca ccgagatcta ccaggccggc 540
agcaccccct gcaacggcgt gaagggcttc aactgctact tccccctgca gagctacggc 600
ttccagccca cctacggcgt gggctaccag ccctaccgcg tggtggtgct gagcttcgag 660
ctgctgcacg cccccgccac cgtgtgcggc cccaagaaga gcaccaacct ggtgaagaac 720
aagtgcgtga acttcggcgg cggcggcagc caccaccacc accaccacca ccaccaccac 780
tag 783
<210> 12
<211> 783
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
atggagaccg acaccctgct gctgtgggtg ctgctgctgt gggtgcccgg cagcaccggc 60
gacgccaggg tgcagcccac cgagagcatc gtgaggttcc ccaacatcac caacctgtgc 120
cccttcggcg aggtgttcaa cgccaccagg ttcgccagcg tgtacgcctg gaacaggaag 180
aggatcagca actgcgtggc cgactacagc gtgctgtaca acagcgccag cttcagcacc 240
ttcaagtgct acggcgtgag ccccaccaag ctgaacgacc tgtgcttcac caacgtgtac 300
gccgacagct tcgtgatcag gggcgacgag gtgaggcaga tcgcccccgg ccagaccggc 360
aacatcgccg actacaacta caagctgccc gacgacttca ccggctgcgt gatcgcctgg 420
aacagcaaca acctggacag caaggtgggc ggcaactaca actacctgta caggctgttc 480
aggaagagca acctgaagcc cttcgagagg gacatcagca ccgagatcta ccaggccggc 540
agcaccccct gcaacggcgt gaagggcttc aactgctact tccccctgca gagctacggc 600
ttccagccca cctacggcgt gggctaccag ccctacaggg tggtggtgct gagcttcgag 660
ctgctgcacg cccccgccac cgtgtgcggc cccaagaaga gcaccaacct ggtgaagaac 720
aagtgcgtga acttcggcgg cggcggcagc caccaccacc accaccacca ccaccaccac 780
tag 783
<210> 13
<211> 672
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
atgagagtcc aaccaacaga atctattgtt agatttccta atattacaaa cttgtgccct 60
tttggtgaag tttttaacgc caccagattt gcatctgttt atgcttggaa caggaagaga 120
atcagcaact gtgttgctga ttattctgtc ctatataatt ccgcatcatt ttccactttt 180
aagtgttatg gagtgtctcc tactaaatta aatgatctct gctttactaa tgtctatgca 240
gattcatttg taattagagg tgatgaagtc agacaaatcg ctccagggca aactggaaag 300
attgctgatt ataattataa attaccagat gattttacag gctgcgttat agcttggaat 360
tctaacaatc ttgattctaa ggttggtggt aattataatt acctgtatag attgtttagg 420
aagtctaatc tcaaaccttt tgagagagat atttcaactg aaatctatca ggccggtagc 480
acaccttgta atggtgttga aggttttaat tgttactttc ctttacaatc atatggtttc 540
caacccacta atggtgttgg ttaccaacca tacagagtag tagtactttc ttttgaactt 600
ctacatgcac cagcaactgt ttgtggacct aaaaagtcta ctaatttggt taaaaacaaa 660
tgtgtcaatt tc 672
<210> 14
<211> 455
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 14
Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
1 5 10 15
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val
20 25 30
Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
35 40 45
Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val
50 55 60
Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp
65 70 75 80
Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
85 90 95
Thr Gly Asn Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
100 105 110
Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly
115 120 125
Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys
130 135 140
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr
145 150 155 160
Pro Cys Asn Gly Val Lys Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser
165 170 175
Tyr Gly Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val
180 185 190
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
195 200 205
Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Gly
210 215 220
Gly Gly Gly Ser Val Pro Arg Asp Ser Gly Cys Lys Pro Cys Ile Cys
225 230 235 240
Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys
245 250 255
Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val
260 265 270
Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp
275 280 285
Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe
290 295 300
Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp
305 310 315 320
Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe
325 330 335
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys
340 345 350
Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys
355 360 365
Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp
370 375 380
Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys
385 390 395 400
Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser
405 410 415
Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr
420 425 430
Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser
435 440 445
Leu Ser His Ser Pro Gly Lys
450 455

Claims (4)

1. A recombinant subunit vaccine for preventing cats from being infected by novel coronavirus is shown in SEQ ID No. 14.
2. The expression vector is applicable to a CHO expression system for expressing the recombinant subunit vaccine of claim 1, and is obtained by constructing a sequence shown in SEQ ID NO.7 into a pCMV-GS vector.
3. A monoclonal CHO cell line highly expressing the recombinant subunit vaccine of claim 1, having a deposit number of: CCTCC NO: C2021190.
4. use of the recombinant subunit vaccine of claim 1 or the protein expressed by the expression vector of claim 2 for the preparation of a recombinant subunit vaccine for the prevention of feline infection with a novel coronavirus.
CN202110812101.9A 2021-07-19 2021-07-19 Novel feline coronavirus subunit vaccine and preparation method and application thereof Pending CN113912738A (en)

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