CN110078802B

CN110078802B - Cat parvovirus VP2 protein and prepared virus-like particle

Info

Publication number: CN110078802B
Application number: CN201910379471.0A
Authority: CN
Inventors: 郭伟伟; 刘大卫; 向银辉; 陈俭梅; 范根成; 杜元钊
Original assignee: Qingdao Yebio Bioengineering Co Ltd
Current assignee: Qingdao Yebio Bioengineering Co Ltd
Priority date: 2019-05-08
Filing date: 2019-05-08
Publication date: 2022-07-22
Anticipated expiration: 2039-05-08
Also published as: CN110078802A

Abstract

The invention provides a feline parvovirus VP2 protein and a prepared virus-like particle, wherein the amino acid sequence of the VP2 protein is SEQ ID NO. 2; the nucleotide sequence of the coding gene is SEQ ID NO. 1. In still another aspect, the present invention provides a recombinant baculovirus, which comprises a nucleotide fragment encoding the VP2 protein; the recombinant baculovirus constructed in the invention is used for preparing virus-like particles of feline parvovirus in insect cells. The invention also provides a feline parvovirus subunit vaccine which comprises an antigen and a vaccine adjuvant, wherein the antigen is the virus-like particle prepared by the invention. The vaccine prepared by the invention can improve the antibody titer of cats after the cats are immunized by the vaccine, and can effectively prevent the infection of feline parvovirus.

Description

Cat parvovirus VP2 protein and prepared virus-like particle

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a feline parvovirus VP2 protein and a virus-like particle (VLP) for expressing the feline parvovirus by using an insect cell expression system; also relates to the obtained recombinant virus-like particles and application thereof in development of feline parvovirus vaccines.

Background

Feline panleukopenia is a highly contagious acute infectious disease caused by Feline Parvovirus (FPV), with high morbidity and mortality. The affected animals show symptoms of high fever, vomiting, diarrhea, severe decrease in white blood cell count, and enteritis. The virus naturally infects a variety of animals in the families felidae and ferrets, such as tigers, leopards, lions and raccoons, but is most susceptible to smaller felidae, including minks. The disease causes great economic loss to animal breeding industry every year.

The feline parvovirus genus is parvovirus genus of parvoviridae family, is a single-stranded negative-strand linear DNA virus, has no envelope, and has strong resistance to physicochemical factors. The FPV genome encodes 2 structural proteins (VP1, VP2) and 2 non-structural proteins (NS1, NS2), wherein VP2 structural protein is an important antigen protein for stimulating the body to produce protective antibodies.

Baculovirus is a large baculovirus envelope, and the genome is circular double-stranded DNA, and the size is about 80-180 kbp. Baculovirus parasitizes arthropods as a pathogenic microorganism and has high host specificity, and the host mainly comprises lepidopteran diptera and hymenoptera insects, and baculovirus hosts other than arthropods have not been found. Among the numerous members of baculoviruses, most currently studied and utilized is Autographa californica multigrain embedded nuclear polyhedrosis virus (AcMNPV). The AcMNPV viral genome is a double-stranded DNA with covalently closed circular supercoils, about 130kb, whose genomic sequence has been determined so far. In recent years, baculovirus expression vectors have dominated expression vectors by themselves. Compared with other expression systems, the baculovirus expression system has the advantages of simple operation, high safety, capacity of accommodating large target genes, high foreign protein expression efficiency, post-translational modification effect, similarity of immunogenicity and biological activity of expressed proteins with natural proteins and the like (Anderson et al, 1995; Wang et al, 2001; Ribeiro et al, 2001).

At present, no specific medicine and effective treatment method for FPV exist in the market, the immunization prevention is mainly used clinically, but no FPV vaccine which is truly approved to be on the market exists in China. Therefore, a safe and effective subunit vaccine needs to be developed to fill the gap of the market.

Disclosure of Invention

The invention provides a feline parvovirus VP2 protein and a prepared virus-like particle, thereby making up the defects of the prior art.

The invention firstly provides a novel VP2 protein obtained by screening from cat parvovirus, the amino acid sequence of the protein is SEQ ID NO. 2;

the invention also optimizes the VP2 protein, and the amino acid sequence of the optimized protein is SEQ ID NO. 4;

the nucleotide sequence of the gene for coding the VP2 protein is SEQ ID NO. 3;

the invention also provides a recombinant baculovirus, which comprises a nucleotide fragment for encoding the VP2 protein;

the recombinant baculovirus constructed by the invention is used for preparing virus-like particles of feline parvovirus in insect cells;

in still another aspect, the present invention provides a feline parvovirus-like particle, which is obtained by infecting insect cells with the recombinant baculovirus described above and then culturing and collecting the infected insect cells.

The insect cell is an insect cell Sf 9;

the virus-like particles prepared by the invention are used for preparing vaccines;

the invention also provides a feline parvovirus subunit vaccine, which comprises an antigen and a vaccine adjuvant, wherein the antigen is the virus-like particle prepared by the invention;

the vaccine prepared by the invention can improve the antibody titer of cats after the cats are immunized, and prevent the infection of feline parvovirus.

Detailed Description

The complete cDNA sequence of VP2 of feline parvovirus is screened and analyzed, one cDNA sequence with rich antigen epitope capable of being expressed in insect cell efficiently is selected, bacmid carrier is constructed, soluble recombinant protein is expressed successfully in insect cell and assembled into virus-like particle (VLPS) automatically in vivo, VLP is similar to natural virus in spatial conception, can stimulate organism to produce cell immunity and humoral immunity simultaneously, and has good immune effect. In addition, the yield of the FPV-VLP prepared by the insect cell-baculovirus expression system is obviously higher than that of viruses cultured by the feline kidney cells, and the production cost is obviously reduced.

The present invention will be described in detail with reference to specific examples. The method applied in the present invention can adopt the method commonly used in the field of vaccine preparation, and is not limited to the specific description of the embodiments of the present invention, and the person skilled in the art can implement the present invention by other conventional methods.

Example 1: amplification and sequence analysis of VP2 Gene

And collecting suspected cat litter material in 2018, treating the suspected cat litter material, performing conventional separation and identification on the material, and judging that the cat litter material is subjected to HA (hemagglutinin) detection by using pig red blood cells, wherein the blood coagulation price is 7log 2. The HI test was performed with known cat fine serum and the cross-reactivity was poor; the pathogenic feline parvovirus strains were demonstrated to be mutated.

1. Amplification of cat fine VP2 Gene

Primers were designed and synthesized based on the cat fine VP2 gene sequence published in NCBI, and the sequence information of the primers is as follows:

primer1：5′-ATGAGTGATGGAGCAGTTCAACC-3′；

primer2：5′-TTAATATAATTTTCTAGGTGCTAGTTG-3′。

extracting separated cat tiny nucleic acid as a template, performing PCR amplification on a target fragment by using primers 1 and 2, and determining the sequence, wherein the nucleotide sequence is SEQ ID NO. 1, and the coded amino acid sequence is SEQ ID NO. 2.

Comparison with the published feline parvovp 2 protein (AAA47152.1) in NCBI revealed mutations at multiple positions, including position 80 (K to S), position 87 (M to V), position 232 (I to V), position 267 (F to C), position 564 (N to R), and position 568 (a to G). The result shows that the separated strain is a new parvovirus and contains a new VP2 antigen protein.

2. Splicing and optimization of VP2 gene

Analyzing the antigenic site of the structural protein gene VP2 of the separated strain, deleting 32aa at the N end, adding M amino acid, deleting 9aa at the C end, and optimizing codons; the modified sequence is optimized to express antigen sites well and can be automatically assembled into VLPS. The optimized and modified amino acid sequence is SEQ ID NO. 4; the encoding gene is subjected to codon optimization, and rare codons of the gene are eliminated, so that VP2 protein can be better expressed in a baculovirus expression system; the optimized nucleotide sequence of the gene is SEQ ID NO. 3.

Example 2 construction of bacmid expressing VP2 Gene

2.1 enzyme digestion

2.1.1 labeling the required 1.5mL EP tube, loading and mixing in 1.5mL EP tube according to the following table: the reaction system was 50. mu.L, and the samples were loaded as shown in the following table:

2.1.2 the 1.5mL EP tube from step 2.2.1 was placed in a 37 deg.C thermostat water bath for 2-3 h.

2.1.3 recovery of product gels from double digestion

Taking out the double enzyme digestion system, and carrying out agarose gel electrophoresis to recover the DNA fragment.

(1) Marked sample collection EP tube, adsorption column and collection tube.

(2) The marked empty EP tube was weighed and the value recorded.

(3) The single DNA band of interest was carefully excised from the agarose gel on a gel cutter with a scalpel into a clean 1.5mL centrifuge tube.

(4) And (3) adding 600 mu L of PC buffer50 ℃ into the 1.5mL centrifuge tube in the step (3), standing for about 5min, and turning the centrifuge tube up and down continuously and gently to ensure that the gel block is fully dissolved.

(5) Column balancing: 500. mu.L of the equilibrium solution BL was added to the adsorption column CB2 (the adsorption column was previously placed in the collection tube), centrifuged at 12,000rpm for 1min, the waste liquid in the collection tube was discarded, and the adsorption column was replaced in the collection tube.

(6) And (3) adding the solution obtained in the step (5) into an adsorption column CB2, standing for 2min at 10,000rpm, centrifuging for 30s, pouring waste liquid in a collecting pipe, and putting the adsorption column CB2 into the collecting pipe.

(7) Adding 600 μ L of rinsing liquid PW buffer into the adsorption column, standing for 3min, centrifuging at 10,000rpm for 30s, pouring off waste liquid in the collection tube, and placing adsorption column CB2 into the collection tube.

(8) And (5) repeating the step (7).

(9) The column was centrifuged at 12,000rpm for 2min to remove the rinse as much as possible. The column was left at room temperature for 10min and air dried completely.

(10) Placing adsorption column CB2 into a collecting tube, suspending and dripping 50 μ L of Lulitionbuffer (preheated at 65 deg.C) into the middle position of the adsorption film, standing for 3min, and centrifuging at 12,000rpm for 2 min.

(11) And (4) taking the centrifuge tube in the step (10) out of the centrifuge, discarding the middle adsorption column CB2, and covering the cover of the centrifuge tube to keep the DNA sample in the centrifuge tube.

(12) And (3) storing the DNA sample in the step 11 at 4 ℃, and preparing an agarose gel electrophoresis identification gel to recover the DNA fragment.

2.2 ligation reaction

(1) Labeling required 0.2mL centrifuge tubes.

(2) Samples were loaded in a 0.2mL tube labeled intact according to the 20. mu.L reaction system of the following table:

wherein the inserted DNA fragments are nucleotide fragments with optimized sequences of SEQ ID NO. 3 and nucleotide fragments with unoptimized SEQ ID NO. 1.

(3) After the sample addition was completed, the components were mixed by gently pipetting several times.

(4) Placing a 0.2mL centrifuge tube at 37 ℃ for reaction for 30min, and immediately placing the reaction tube in an ice water bath for cooling for 5min after the reaction is finished.

(5) The reaction product of step (4) can be directly used for conversion experiment, or can be stored at-20 ℃ and thawed and converted when required.

2.3 conversion reaction

(1) Add 10. mu.L of ligation reaction rapidly to 100. mu.L of competent cells and blow-stir well and ice-wash for 30 min.

(2) After the step (1) is finished, taking out the sample tube, placing the sample tube in a water bath at 42 ℃ for 100s, and immediately carrying out ice bath for 2 min.

(3) And (3) after the step (2) is finished, taking out the sample tube, adding 600 mu L of liquid LB culture medium into the sample tube in a super-clean workbench, and then placing the sample tube in a constant-temperature shaking table at 37 ℃ and at 220rpm for culturing for 1 h.

(4) Transformation plates were prepared, and LB resistant plates for transformation were prepared based on the plasmid resistance.

(5) Coating a plate: and (4) taking out the sample tube in the step (3), centrifuging at room temperature for 2min at 8,000rpm, removing 600 mu L of supernatant liquid, re-suspending the bacteria at the bottom of the tube by the residual supernatant liquid, putting the re-suspended bacteria liquid into the center of a corresponding transformation plate, and uniformly spreading the bacteria liquid in the center of the transformation plate by a bacteria coating rod.

(6) And (3) placing the plate in the step (5) in a biochemical constant temperature incubator, culturing for 1h at 37 ℃, and then, inverting the transformation plate for culturing for 15 h.

(7) The transformation results were observed and recorded.

2.4 plasmid extraction and PCR identification

2.4.1 plasmid extraction

(1) Single clones were picked from the transformation plates with a 10. mu.L pipette tip into 5ml of LB liquid medium containing benzyl-amine resistance, shaken at 37 ℃ and 220rpm overnight.

(2) The resulting suspension was aspirated into a 1.5mL EP tube, centrifuged at 12,000rpm for 2min at room temperature, and the supernatant was discarded.

(3) To the EP tube in the step (2), 250. mu.L of a plasmid extraction reagent P1buffer was added, and the cells were completely suspended.

(4) 250 μ L P2buffer was added to the solution from step (3) and the tube was immediately mixed by gentle inversion 5-10 times. Standing at room temperature for 2-4 min.

(5) 350 mu L P3buffer was added to the solution of step (4) and the tube was immediately mixed by gentle inversion 5-10 times. Standing at room temperature for 2-4 min.

(6) The solution of step (5) was centrifuged at room temperature at 14,000rpm for 10 min.

(7) Transferring the supernatant solution in the step (6) to the center of an adsorption column, centrifuging at room temperature for 30s at 12,000rpm, and pouring off the liquid in a collection tube.

(8) 500 μ L Buffer DW1 was added to the center of the adsorption column, centrifuged at room temperature for 30s at 12,000rpm, and the collection tube was decanted.

(9) Add 500. mu.L wash solution to the center of the adsorption column, centrifuge at room temperature, 12,000rpm for 30s, and pour off the liquid in the collection tube. And repeating the steps once.

(10) Empty adsorption column, centrifuge at room temperature, 12,000rpm, 2 min.

(11) The adsorption column was placed in a clean 1.5ml centrifuge tube, 30. mu.L of Elutionbuffer was added to the center of the adsorption membrane, allowed to stand at room temperature for 5min, centrifuged at room temperature, 12,000rpm, 2min, and the DNA solution in the tube was stored at 4 ℃.

2.4.2 PCR identification

(1) And marking a PCR tube which needs to be used, loading and mixing uniformly according to the following table, wherein the reaction system is 25 mu L:

(2) PCR amplification procedure:

(3) sequencing: and (3) sequencing the plasmid with pcr positive identification by a sequencing company to determine the positive plasmid.

2.5 transformation

The positive plasmid was transformed into DH10bac competent cells as described in 2.3.

2.6 rod grain picking and PCR identification

2.6.1 picking the Stem grain

(1) From the 2.5 transformed plates, white monoclonal colonies were picked from the transformed plates with a 10. mu.L pipette tip into 5ml of LB liquid medium containing kanamycin resistance, tetracycline resistance, gentamicin resistance, and shaken at 220rpm at 37 ℃ overnight.

(4) 250 μ L P2buffer was added to the solution from step (3) and the tube was immediately gently inverted 5-10 times and mixed. Standing at room temperature for 2-4 min.

(7) And (4) transferring the supernatant solution in the step (6) to the center of the adsorption column, centrifuging at room temperature for 30s at 12,000rpm, and pouring out the liquid in the collection tube.

(10) Empty adsorption column, centrifuge at room temperature, 12,000rpm, 2 min.

2.6.2 PCR identification the DNA extracted from 2.6.1 was subjected to PCR identification, and positive plasmids were sequenced by Shanghai bioengineering, Inc. and used for transfection of SF9 cells.

Example 3 transfection of SF9 cells

(1) Preparing: sterilizing the biological safety cabinet for 30min by ultraviolet; the TNM-FH culture solution was preheated to 27 ℃ in a 27 ℃ water bath.

(2) Mu.g of recombinant DNA was added to 100. mu.l of serum-free and double-antibody TNM-FH culture medium and mixed well. Add 9. mu.l Cellffectin Reagent to 100. mu.l serum free and double antibody TNM-FH culture medium, mix well. The liposomes were mixed with the recombinant DNA and allowed to stand at room temperature for 40 min.

(3) The 6-well plate cells were removed from the 27 ℃ incubator, the supernatant medium was discarded, the cells were washed three times with the pre-warmed TNM-FH culture medium, and the TNM-FH culture medium was discarded.

(4) 2ml of 10% fetal bovine serum TNM-FH culture medium was added to each well.

(5) And gently adding the mixture of the recombinant DNA and the liposome into each hole of the cell, gently mixing uniformly, and performing static culture at the temperature of 27 ℃ for 5-6 h.

(6) The liquid in the wells was discarded, 2ml of complete TNM-FH culture medium (containing diabody and 10% serum) was added, and the mixture was incubated at 27 ℃ for 5-6 days.

(7) After the cells were swollen, enlarged in volume and fallen off, the supernatant was collected and labeled as recombinant baculovirus of P1 generation, named FPV-VLP-P1.

(8) Newly cultured Sf9 cells are infected by FPV-VLP-P1, the content of the recombinant baculovirus is increased, after repeated inoculation for 2 generations, cell supernatant is collected and stored at 4 ℃ or-80 ℃ for later use.

Example 4 protein purification and detection

4.1 expression and identification of recombinant vp2 protein in insect cell Sf 9:

the recombinant baculovirus FPV-VLP-P1 is used for infecting insect cells Sf9, the cells are cultured for 72h at 27 ℃, meanwhile, the normal insect cells Sf 927 ℃ which are not infected with the virus are used as a control, the cells are harvested, and culture supernatants are frozen for standby. After the cells were washed with PBS at pH7.4, 1 XSDS-PAGE sample buffer [50mM Tris-HCl (pH6.8), 100mM Dithiothreitol (DTT), 2% SDS, 0.05% Bromopheol blue, 10% Glycerol ] was added, boiled for 5min, subjected to polyacrylamide gel electrophoresis using 12% separation gel, 5% concentration gel, 100V for about 2.5h, and stained with Coomassie Brilliant blue R250.

The results show that the expression level of the non-optimized VP2 gene recombinant baculovirus in insect cells is not detected, and the VP2 protein is expressed in the optimized VP2 recombinant baculovirus-infected insect cell Sf9 lysate.

4.2 chromatographic purification of recombinant VP2 protein:

inoculating the recombinant baculovirus FPV-VLP to insect cell Sf9, culturing at 27 ℃ for 72 hours, centrifuging to collect cell precipitate, adding a proper amount of normal saline into the cell precipitate, resuspending the cell precipitate, lysing cells by ultrasonic wave, centrifuging at 4 ℃ for 10min at 1000r/min, collecting supernatant, and performing chromatographic purification by using a Ni2+ column to obtain VLP protein liquid of VP2 protein.

4.3 Western blot：

SDS-PAGE is carried out on the cat parvovirus and the protein prepared by 4.2 at the same time, a semidry method is adopted for 20V transfer printing for 30min, a target protein band is transferred to a PVDF membrane, a transfer printing membrane is sealed by a sealing liquid overnight, PBST is washed for 3 times, cat parvovirus positive serum diluted by 1: 500 is acted for 1.5h at 37 ℃, PBST is washed for 3 times, rabbit anti-cat enzyme labeled antibody diluted by 1: 2000 and marked by HRP is acted for 1.5h at 37 ℃, PBST is washed for 3 times, a substrate solution is acted for 5min, and color development is carried out on chemiDOC, so that the whole virus band is very weak, and a VP2 protein band expressed in insect cell Sf9 by the gene with the optimized nucleotide sequence of SEQ ID NO:3 is very bright, which shows that the optimized VP2 protein has better immunogenicity.

4.4 HA assay was performed on the protein prepared in 4.2 using pig serum, and as a result, the hemagglutination values of FPV-VLPs were 18log2, respectively, which was 11log2 higher than that of the whole virus. Indicating that the expressed VP2 protein has better immunogenicity.

The screened feline parvovirus was concentrated 100-fold. The concentrated whole virus liquid and the expression product of the recombinant baculovirus in SF9 cells are simultaneously subjected to negative staining and then observed by an electron microscope. As a result, the expression product was assembled into many virus-like particles with a size of 20nm, and the whole virus solution concentrated 100-fold contained only a trace amount of particles.

Example 5: preparation of vaccines

(1) Preparation of an aqueous phase: the purified VLP protein solution of example 2, step 4.2, was mixed with sterile physiological saline in appropriate proportions, all with HA in the aqueous phase not below 6log 2;

(2) the water phase is emulsified with 15% volume of gel01 adjuvant and then emulsified at 1000r/min for 5min to obtain subunit vaccine.

Safety and efficacy testing of vaccines

1) And (4) safety inspection:

20 healthy susceptible cats (all the small neutralizing antibodies of the cats are not higher than 1:4) are taken and randomly divided into 2 groups. One group was a vaccine immunization group of 10 per group, 5 parts (5ml) of the subunit vaccine of the invention were injected subcutaneously; another 10 immunization controls were not run. After immunization, the patient is continuously observed for 14 days, and the body temperature, the mental state, the appetite, the change condition of the feces and whether adverse reactions exist on the injection part or not are recorded. The injection site was examined for pathological changes on the last day of the experiment. In the observation period, the body temperature, the mental state, the appetite and the feces of the cats in the immunization group and the cats in the control group are normal, adverse reactions such as swelling and inflammation do not exist at the injection part, abnormal changes do not occur in the autopsy of the injection part, and the differences between the growth conditions of the cats in the vaccination group and the cats in the control group are not obvious. The results show that the prepared subunit vaccine meets the safety detection requirements.

2) Efficacy test

30 healthy susceptible cats (all the small neutralizing antibodies of the cats are not higher than 1:4) are taken and randomly divided into 3 groups of 10 cats. One group immunizes self-made subunit vaccines, one group immunizes market cat parvo vaccines, and the other group does not immunize as a control. And (3) collecting blood and detecting HI 21 days after immunization. Simultaneously, the cat parvovirus virulent challenge is carried out, and 8ml (10 ml) is orally taken^6.5TCID₅₀Per ml) and 2ml for intraperitoneal injection, i.e. each toxin is 10^7.5TCID₅₀. The results showed that the HI of the subunit-immunized group was 1:891.4 on average, the HI of the market vaccine-immunized group was 1:111.4 on average, and the HI of the control group was all<2log2, indicating that the feline small subunit vaccine produced very good antibodies and was much higher in titer than the commercial vaccine. The challenge results show that 10/10 in the subunit vaccine immunization group protected, 8/10 in the market vaccine immunization group protected, and 10/10 in the control group suffered from diseases. The cat parvovirus subunit vaccine can well resist the attack of cat parvovirus virulent virus and has good protection effect. See table 1 for details.

TABLE 1 efficacy test results for feline parvosubunit vaccines

Note: the antibodies are the geometric mean antibody values for this group of cats.

The results show that the subunit vaccine prepared from the virus-like particles can well prevent the infection of the feline parvovirus, and the antibody generated by the subunit vaccine is far higher than that of a market vaccine group, so the subunit vaccine has good application prospect.

In conclusion, the invention constructs a bacmid vector for expressing the antigen protein by using the non-optimized VP2 protein and the optimized VP2 protein at the same time, so that the non-optimized full-length VP2 gene cannot be expressed in insect cells, and the optimized VP2 protein successfully expresses soluble recombinant protein in the insect cells. When the 100-fold concentrated whole virus strain and the expressed vp2 supernatant are simultaneously subjected to electron microscope observation, the result shows that the 100-fold concentrated whole virus only has a small amount of VLPs, and the undiluted expressed vp2 supernatant automatically assembles into virus-like particles (VLPS) in vivo, and the quantity of virus particles is large. VLP is similar to natural virus in spatial conception, can stimulate the organism to generate cellular immunity and humoral immunity at the same time, has good immune effect, does not contain virus nucleic acid, has no potential virus pathogenic gene and has higher safety.

Sequence listing

<110> Qingdao Yibang bioengineering Co., Ltd

<120> feline parvovirus VP2 protein and prepared virus-like particle

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1755

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atgagtgatg gagcagttca accagacggt ggtcaacctg ctgtcagaaa tgaaagagct 60

acaggatctg ggaacgggtc tggaggcggg ggtggtggtg gttctggggg tgtggggatt 120

tctacgggta ctttcaataa tcagacggaa tttaaatttt tggaaaacgg gtgggtggaa 180

atcacagcaa actcaagcag acttgtacat ttaaatatgc cagaaagtga aaattattct 240

agagtagttg taaataatgt ggataaaact gcagttaaag gaaacatggc tttagatgat 300

actcatgtac aaattgtaac accttggtca ttggttgatg caaatgcttg gggagtttgg 360

tttaatccag gagattggca actaattgtt aatactatga gtgagttgca tttagttagt 420

tttgaacaag aaatttttaa tgttgtttta aagactgttt cagaatctgc tactcagcca 480

ccaactaaag tttataataa tgatttaact gcatcattga tggttgcatt agatagtaat 540

aatactatgc catttactcc agcagctatg agatctgaga cattgggttt ttatccatgg 600

aaaccaacca taccaactcc atggagatat tattttcaat gggatagaac attaatacca 660

tctcatactg gaactagtgg cacaccaaca aatgtgtatc atggtacaga tccagatgat 720

gttcaatttt atactattga aaattctgtg ccagtacact tactaagaac aggtgatgaa 780

tttgctacag gaacattttg ctttgattgc aaaccatgta gactaacaca tacatggcaa 840

acaaatagag cattgggctt accaccattt ttaaattctt tgcctcaatc tgaaggagct 900

actaactttg gtgatatagg agttcaacaa gataaaagac gtggtgtaac tcaaatggga 960

aatacagact atattactga agctactatt atgagaccag ctgaggttgg ttatagtgca 1020

ccatattatt cttttgaagc gtctacacaa gggccattta aaacacctat tgcagcagga 1080

cgggggggag cgcaaacaga tgaaaatcaa gcagcagatg gtgatccaag atatgcattt 1140

ggtagacaac atggtcaaaa aactactaca acaggagaaa cacctgagag atttacatat 1200

atagcacatc aagatacagg aagatatcca gaaggagatt ggattcaaaa tattaacttt 1260

aaccttcctg taacaaatga taatgtattg ctaccaacag atccaattgg aggtaaaaca 1320

ggaattaact atactaatat atttaatact tatggtcctt taactgcatt aaataatgta 1380

ccaccagttt atccaaatgg tcaaatttgg gataaagaat ttgatactga cttaaaacca 1440

agacttcatg taaatgcacc atttgtttgt caaaataatt gtcctggtca attatttgta 1500

aaagttgcgc ctaatttaac gaatgaatat gatcctgatg catctgctaa tatgtcaaga 1560

attgtaactt attcagattt ttggtggaaa ggtaaattag tatttaaagc taaactaaga 1620

gcatctcata cttggaatcc aattcaacaa atgagtatta atgtagataa ccaatttaac 1680

tatgtaccac gtaatattgg aggtatgaaa attgtatatg aaaaatctca actagcacct 1740

agaaaattat attaa 1755

<210> 2

<211> 584

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Ser Asp Gly Ala Val Gln Pro Asp Gly Gly Gln Pro Ala Val Arg

1 5 10 15

Asn Glu Arg Ala Thr Gly Ser Gly Asn Gly Ser Gly Gly Gly Gly Gly

20 25 30

Gly Gly Ser Gly Gly Val Gly Ile Ser Thr Gly Thr Phe Asn Asn Gln

35 40 45

Thr Glu Phe Lys Phe Leu Glu Asn Gly Trp Val Glu Ile Thr Ala Asn

50 55 60

Ser Ser Arg Leu Val His Leu Asn Met Pro Glu Ser Glu Asn Tyr Ser

65 70 75 80

Arg Val Val Val Asn Asn Val Asp Lys Thr Ala Val Lys Gly Asn Met

85 90 95

Ala Leu Asp Asp Thr His Val Gln Ile Val Thr Pro Trp Ser Leu Val

100 105 110

Asp Ala Asn Ala Trp Gly Val Trp Phe Asn Pro Gly Asp Trp Gln Leu

115 120 125

Ile Val Asn Thr Met Ser Glu Leu His Leu Val Ser Phe Glu Gln Glu

130 135 140

Ile Phe Asn Val Val Leu Lys Thr Val Ser Glu Ser Ala Thr Gln Pro

145 150 155 160

Pro Thr Lys Val Tyr Asn Asn Asp Leu Thr Ala Ser Leu Met Val Ala

165 170 175

Leu Asp Ser Asn Asn Thr Met Pro Phe Thr Pro Ala Ala Met Arg Ser

180 185 190

Glu Thr Leu Gly Phe Tyr Pro Trp Lys Pro Thr Ile Pro Thr Pro Trp

195 200 205

Arg Tyr Tyr Phe Gln Trp Asp Arg Thr Leu Ile Pro Ser His Thr Gly

210 215 220

Thr Ser Gly Thr Pro Thr Asn Val Tyr His Gly Thr Asp Pro Asp Asp

225 230 235 240

Val Gln Phe Tyr Thr Ile Glu Asn Ser Val Pro Val His Leu Leu Arg

245 250 255

Thr Gly Asp Glu Phe Ala Thr Gly Thr Phe Cys Phe Asp Cys Lys Pro

260 265 270

Cys Arg Leu Thr His Thr Trp Gln Thr Asn Arg Ala Leu Gly Leu Pro

275 280 285

Pro Phe Leu Asn Ser Leu Pro Gln Ser Glu Gly Ala Thr Asn Phe Gly

290 295 300

Asp Ile Gly Val Gln Gln Asp Lys Arg Arg Gly Val Thr Gln Met Gly

305 310 315 320

Asn Thr Asp Tyr Ile Thr Glu Ala Thr Ile Met Arg Pro Ala Glu Val

325 330 335

Gly Tyr Ser Ala Pro Tyr Tyr Ser Phe Glu Ala Ser Thr Gln Gly Pro

340 345 350

Phe Lys Thr Pro Ile Ala Ala Gly Arg Gly Gly Ala Gln Thr Asp Glu

355 360 365

Asn Gln Ala Ala Asp Gly Asp Pro Arg Tyr Ala Phe Gly Arg Gln His

370 375 380

Gly Gln Lys Thr Thr Thr Thr Gly Glu Thr Pro Glu Arg Phe Thr Tyr

385 390 395 400

Ile Ala His Gln Asp Thr Gly Arg Tyr Pro Glu Gly Asp Trp Ile Gln

405 410 415

Asn Ile Asn Phe Asn Leu Pro Val Thr Asn Asp Asn Val Leu Leu Pro

420 425 430

Thr Asp Pro Ile Gly Gly Lys Thr Gly Ile Asn Tyr Thr Asn Ile Phe

435 440 445

Asn Thr Tyr Gly Pro Leu Thr Ala Leu Asn Asn Val Pro Pro Val Tyr

450 455 460

Pro Asn Gly Gln Ile Trp Asp Lys Glu Phe Asp Thr Asp Leu Lys Pro

465 470 475 480

Arg Leu His Val Asn Ala Pro Phe Val Cys Gln Asn Asn Cys Pro Gly

485 490 495

Gln Leu Phe Val Lys Val Ala Pro Asn Leu Thr Asn Glu Tyr Asp Pro

500 505 510

Asp Ala Ser Ala Asn Met Ser Arg Ile Val Thr Tyr Ser Asp Phe Trp

515 520 525

Trp Lys Gly Lys Leu Val Phe Lys Ala Lys Leu Arg Ala Ser His Thr

530 535 540

Trp Asn Pro Ile Gln Gln Met Ser Ile Asn Val Asp Asn Gln Phe Asn

545 550 555 560

Tyr Val Pro Arg Asn Ile Gly Gly Met Lys Ile Val Tyr Glu Lys Ser

565 570 575

Gln Leu Ala Pro Arg Lys Leu Tyr

580

<210> 3

<211> 1635

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atgggtggta gtggaggcgt gggcataagt actggcactt ttaataacca aaccgagttt 60

aaattccttg agaatggttg ggtggaaatt accgctaatt catccagact tgtgcatctc 120

aacatgcccg agtccgagaa ttatagtcgc gtcgtcgtta ataatgtcga caaaacggcg 180

gtaaagggaa acatggctct tgatgacacc catgtccaga tagtcactcc ttggagcctc 240

gtggatgcta acgcctgggg cgtatggttt aaccctggtg attggcagct catcgttaat 300

actatgtcag aattgcatct tgtatctttc gagcaagaga tttttaacgt agttctgaaa 360

acggtatcgg aaagtgcaac tcaaccccct acgaaggtgt acaataatga cttgactgcc 420

tcgttgatgg ttgccttgga ttctaacaac actatgccgt ttaccccagc tgcgatgcgt 480

tctgagacac tgggcttcta tccgtggaaa cctaccatcc caacgccctg gcgctactac 540

tttcaatggg acaggaccct gatccccagc cataccggca cttcgggaac gcccaccaac 600

gtgtatcatg gtaccgaccc cgacgacgta cagttttata cgattgaaaa ctcagtcccc 660

gtgcacctct tgcgtaccgg cgatgaattt gccacgggta ccttctgttt cgattgtaaa 720

ccgtgtcgtt tgacccatac ttggcaaact aaccgcgctc ttggattgcc ccctttcctg 780

aattcattgc ctcagtctga aggagcgact aactttggcg atattggcgt ccaacaggac 840

aaaaggcgtg gtgtcaccca aatgggtaat acagactaca taaccgaggc gacgatcatg 900

cgcccagctg aggtgggcta tagcgccccc tactactcat ttgaggcatc aacacaaggc 960

ccattcaaaa ctccgatcgc cgcaggacgc ggcggcgcac aaacggacga aaatcaggcg 1020

gccgacggtg atccccgtta cgcatttggc agacagcatg gacaaaagac gacaacaacg 1080

ggtgaaacac ctgaaagatt tacgtacatt gcgcatcagg atacgggtag gtacccggaa 1140

ggcgattgga tccaaaacat taattttaac ctgcctgtga ccaatgacaa tgtgttgctt 1200

cccacagacc ctattggagg taagacggga attaattata caaacatctt taacacttat 1260

ggaccgctga cggctcttaa taacgtgcca cccgtatacc ctaatggaca aatctgggat 1320

aaagaattcg acacggacct gaagccaaga ttgcacgtta atgctccatt tgtttgtcaa 1380

aataattgtc ccggtcaact gtttgtgaag gttgcgccaa atcttaccaa tgaatatgat 1440

ccagatgcat cagcgaacat gagtaggata gtcacgtact ccgacttctg gtggaaaggt 1500

aagctcgtgt tcaaagcgaa actgagggcg tcgcatacgt ggaaccctat ccagcagatg 1560

tcaattaatg tggacaatca attcaattat gttccgcgta atattggtgg tatgaaaatc 1620

gtgtacgaaa aataa 1635

<210> 4

<211> 544

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Met Gly Gly Ser Gly Gly Val Gly Ile Ser Thr Gly Thr Phe Asn Asn

1 5 10 15

Gln Thr Glu Phe Lys Phe Leu Glu Asn Gly Trp Val Glu Ile Thr Ala

20 25 30

Asn Ser Ser Arg Leu Val His Leu Asn Met Pro Glu Ser Glu Asn Tyr

35 40 45

Ser Arg Val Val Val Asn Asn Val Asp Lys Thr Ala Val Lys Gly Asn

50 55 60

Met Ala Leu Asp Asp Thr His Val Gln Ile Val Thr Pro Trp Ser Leu

65 70 75 80

Val Asp Ala Asn Ala Trp Gly Val Trp Phe Asn Pro Gly Asp Trp Gln

85 90 95

Leu Ile Val Asn Thr Met Ser Glu Leu His Leu Val Ser Phe Glu Gln

100 105 110

Glu Ile Phe Asn Val Val Leu Lys Thr Val Ser Glu Ser Ala Thr Gln

115 120 125

Pro Pro Thr Lys Val Tyr Asn Asn Asp Leu Thr Ala Ser Leu Met Val

130 135 140

Ala Leu Asp Ser Asn Asn Thr Met Pro Phe Thr Pro Ala Ala Met Arg

145 150 155 160

Ser Glu Thr Leu Gly Phe Tyr Pro Trp Lys Pro Thr Ile Pro Thr Pro

165 170 175

Trp Arg Tyr Tyr Phe Gln Trp Asp Arg Thr Leu Ile Pro Ser His Thr

180 185 190

Gly Thr Ser Gly Thr Pro Thr Asn Val Tyr His Gly Thr Asp Pro Asp

195 200 205

Asp Val Gln Phe Tyr Thr Ile Glu Asn Ser Val Pro Val His Leu Leu

210 215 220

Arg Thr Gly Asp Glu Phe Ala Thr Gly Thr Phe Cys Phe Asp Cys Lys

225 230 235 240

Pro Cys Arg Leu Thr His Thr Trp Gln Thr Asn Arg Ala Leu Gly Leu

245 250 255

Pro Pro Phe Leu Asn Ser Leu Pro Gln Ser Glu Gly Ala Thr Asn Phe

260 265 270

Gly Asp Ile Gly Val Gln Gln Asp Lys Arg Arg Gly Val Thr Gln Met

275 280 285

Gly Asn Thr Asp Tyr Ile Thr Glu Ala Thr Ile Met Arg Pro Ala Glu

290 295 300

Val Gly Tyr Ser Ala Pro Tyr Tyr Ser Phe Glu Ala Ser Thr Gln Gly

305 310 315 320

Pro Phe Lys Thr Pro Ile Ala Ala Gly Arg Gly Gly Ala Gln Thr Asp

325 330 335

Glu Asn Gln Ala Ala Asp Gly Asp Pro Arg Tyr Ala Phe Gly Arg Gln

340 345 350

His Gly Gln Lys Thr Thr Thr Thr Gly Glu Thr Pro Glu Arg Phe Thr

355 360 365

Tyr Ile Ala His Gln Asp Thr Gly Arg Tyr Pro Glu Gly Asp Trp Ile

370 375 380

Gln Asn Ile Asn Phe Asn Leu Pro Val Thr Asn Asp Asn Val Leu Leu

385 390 395 400

Pro Thr Asp Pro Ile Gly Gly Lys Thr Gly Ile Asn Tyr Thr Asn Ile

405 410 415

Phe Asn Thr Tyr Gly Pro Leu Thr Ala Leu Asn Asn Val Pro Pro Val

420 425 430

Tyr Pro Asn Gly Gln Ile Trp Asp Lys Glu Phe Asp Thr Asp Leu Lys

435 440 445

Pro Arg Leu His Val Asn Ala Pro Phe Val Cys Gln Asn Asn Cys Pro

450 455 460

Gly Gln Leu Phe Val Lys Val Ala Pro Asn Leu Thr Asn Glu Tyr Asp

465 470 475 480

Pro Asp Ala Ser Ala Asn Met Ser Arg Ile Val Thr Tyr Ser Asp Phe

485 490 495

Trp Trp Lys Gly Lys Leu Val Phe Lys Ala Lys Leu Arg Ala Ser His

500 505 510

Thr Trp Asn Pro Ile Gln Gln Met Ser Ile Asn Val Asp Asn Gln Phe

515 520 525

Asn Tyr Val Pro Arg Asn Ile Gly Gly Met Lys Ile Val Tyr Glu Lys

530 535 540

Claims

1. The feline parvovirus VP2 protein is characterized in that the amino acid sequence of the VP2 protein is SEQ ID NO. 4.

2. A gene encoding the feline parvovirus VP2 protein of claim 1.

3. The gene of claim 2 having the nucleotide sequence of SEQ ID NO 3.

4. A recombinant baculovirus comprising a nucleotide fragment encoding the feline parvovirus VP2 protein of claim 1; the sequence of the nucleotide fragment is SEQ ID NO. 3.

5. Use of the recombinant baculovirus of claim 4 for the preparation of virus-like particles of feline parvovirus in insect cells.

6. A feline parvovirus-like particle obtained by infecting insect cells with the recombinant baculovirus of claim 4 and collecting the feline parvovirus-like particle by culture.

7. A feline parvovirus subunit vaccine comprising an antigen and a vaccine adjuvant, wherein the antigen is the feline parvovirus virus-like particle of claim 6.