CN108371710B

CN108371710B - Feline infectious rhinoconjunctivitis and feline panleukopenia bivalent vaccine and preparation method thereof

Info

Publication number: CN108371710B
Application number: CN201810089278.9A
Authority: CN
Inventors: 夏振强; 金宏丽; 石晶; 殷玉和; 赵健; 刘冰; 刘伟
Original assignee: Changchun Sr Biological Technology Co ltd
Current assignee: Changchun Sr Biological Technology Co ltd
Priority date: 2018-01-30
Filing date: 2018-01-30
Publication date: 2021-09-21
Anticipated expiration: 2038-01-30
Also published as: CN108371710A

Abstract

The bivalent vaccine is prepared by respectively producing virus-like particles of feline calicivirus and virus-like particles of feline panleukopenia virus by utilizing a baculovirus/insect cell expression system, and mixing the virus-like particles of the feline calicivirus and the feline panleukopenia virus with a preservative and an adjuvant. The bivalent vaccine has the advantages of safety, high efficiency, easy production, low cost and the like, and has good immune prevention effect on feline infectious rhinoconjunctivitis and feline panleukopenia.

Description

Feline infectious rhinoconjunctivitis and feline panleukopenia bivalent vaccine and preparation method thereof

Technical Field

The invention relates to the fields of microorganisms, genetic engineering and veterinary biomedicine, in particular to a method for producing a feline infectious rhinoconjunctivitis and feline panleukopenia bivalent vaccine by using an insect cell-baculovirus expression system.

Background

Feline infectious rhinoconjunctivitis is caused by Feline Calicivirus (FCV), and is clinically characterized by rhinitis, conjunctivitis, acute oral ulceration, pneumonia, and chronic gastritis. FCVs are currently believed to be distributed worldwide and may infect all felines. Feline panleukopenia is caused by Feline Panleukopenia Virus (FPV), and is characterized clinically by a sudden high fever, vomiting, diarrhea, dehydration, and leukopenia in the circulating blood stream in a diseased cat. FPV primarily infects many animals, especially young animals below 6 months of age, with higher morbidity and mortality, and is the most important infectious disease in felines.

Vaccination of cats is critical in order to protect them from infectious diseases and to maintain their physical and mental health. The core vaccine to be vaccinated includes antigens such as: FPV, FCV, Feline infectious rhinotracheitis virus (FHV), Rabies virus (Rabies, RABV). At present, the related domestic vaccines comprise feline bivalent vaccines including FPV and RABV, but specific information about the vaccines cannot be found on the internet and the market, and the vaccines are probably pilot products of some scientific research units. The commercial vaccines are imported vaccines, mainly feline triple vaccines, including FPV, FCV, FHV. The vaccines are all attenuated vaccines or inactivated vaccines, and the attenuated vaccines have potential safety hazards and have the defects that the inactivated vaccines cannot effectively stimulate organisms to generate cellular immunity and the like. In addition, viral variation is the leading cause of immune failure in current vaccines. Therefore, the preparation of novel, efficient and safe vaccines aiming at the epidemic strains has great significance for the prevention and control of the infectious diseases of cats.

The invention selects domestic epidemic strains, uses genetic engineering means to prepare virus-like particle antigen, and carries out the preparation of novel feline bivalent genetic engineering vaccine, and can simultaneously prevent feline infectious rhinoconjunctivitis and feline panleukopenia. Because the shape and the structure of the virus-like particles are similar to those of natural virus particles, the virus-like particles can effectively induce the organism to generate cellular immunity and humoral immunity; since the virus-like particle does not contain nucleic acid and cannot autonomously replicate, the safety is higher. In addition, the method has the advantages of easy production, high titer, low cost and the like, and fills the blank of the development and commercialization of the cat vaccine in China.

Disclosure of Invention

The invention aims to provide a feline infectious rhinoconjunctivitis and feline panleukopenia bivalent vaccine and a preparation method thereof.

The invention provides a feline infectious rhinoconjunctivitis and feline panleukopenia bivalent vaccine which contains feline calicivirus virus-like particles and feline panleukopenia virus-like particles.

In the feline calicivirus virus-like particle, the gene sequence of the feline calicivirus virus is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2.

In the feline panleukopenia virus-like particle, the gene sequence of the feline calicivirus is shown as SEQ ID NO.3, and the amino acid sequence is shown as SEQ ID NO. 4.

To achieve the objects of the present invention, the present invention utilizes an insect cell-baculovirus expression system to produce feline calicivirus and feline panleukopenia virus like particles.

The invention firstly provides an optimized feline calicivirus VP1 gene and an optimized feline panleukopenia virus VP2 gene, namely genes optimized according to insect cell preferred codons. The optimized gene of the feline calicivirus VP1 is shown as SEQ ID No.1, and the amino acid sequence is shown as SEQ ID No. 2; the optimized gene of the feline panleukopenia virus VP2 is shown as SEQ ID No.3, and the amino acid sequence is shown as SEQ ID No. 4.

The invention provides a preparation method of the feline infectious rhinoconjunctivitis and feline panleukopenia bivalent vaccine, which comprises the steps of preparing feline calicivirus virus-like particles and feline panleukopenia virus-like particles, mixing the feline calicivirus virus-like particles and the feline panleukopenia virus-like particles, adding a preservative and an adjuvant, and before mixing, the content of the feline calicivirus virus-like particles is not less than 4mg/ml, and the hemagglutination titer of the feline panleukopenia virus is not less than 1:2¹⁴。

In the feline calicivirus virus-like particles, the gene sequence of the feline calicivirus virus is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2; in the feline panleukopenia virus-like particle, the gene sequence of the feline calicivirus is shown as SEQ ID NO.3, and the amino acid sequence is shown as SEQ ID NO. 4.

Specifically, the preparation method of the feline calicivirus and feline panleukopenia virus-like particles provided by the invention comprises the following steps: (1) optimizing a feline calicivirus VP1 gene and a feline panleukopenia VP2 gene according to insect cell preferred codons, respectively inserting the optimized genes into insect cell expression vectors, transforming DH10Bac competence, and extracting a recombinant baculovirus genome; (2) transfecting insect cells with recombinant baculovirus genomes, rescuing to obtain recombinant baculovirus, inoculating the insect cells according to MOI (molar equivalent of 1-0.1), and harvesting a mixture after 4-5 days to obtain feline calicivirus antigen and feline panleukopenia virus antigen respectively; (3) repeatedly freezing and thawing the feline calicivirus antigen for 1 time, and then harvesting the supernatant to obtain feline calicivirus virus-like particles; and (3) treating the feline panleukopenia virus antigen with a lysis solution, and then harvesting supernatant to obtain the feline panleukopenia virus-like particles.

In a preferred embodiment, the feline calicivirus and feline panleukopenia virus-like particle preparation method is that the lysate used to process the cells is NaHCO₃Solutions, more preferably NaHCO, at 25mM concentration₃And (3) solution.

Another objective of the invention is to provide a feline infectious rhinoconjunctivitis and feline panleukopenia bivalent genetically engineered vaccine comprising at least one feline calicivirus antigen and at least one feline panleukopenia virus antigen.

In order to achieve the purpose of the invention, the invention is prepared by mixing the two virus-like particles prepared in the way mentioned above according to a certain proportion and adding a preservative and an adjuvant. Wherein, the mixing volume ratio of the two virus-like particles is 1:1-5:1, the preferred ratio is 3:1, the preservative is thimerosal and the like, and the preferred final concentration is 0.005%; the adjuvant is aluminum phosphate and the like, and the optimal final concentration is 0.4%.

The feline calix feline infectious rhinoconjunctivitis and feline panleukopenia bivalent genetic engineering vaccine is an intramuscular injection vaccine or a subcutaneous injection vaccine.

The invention further provides application of the prepared feline infectious rhinoconjunctivitis and feline panleukopenia bivalent genetic engineering vaccine in prevention of feline infectious rhinoconjunctivitis and feline panleukopenia.

The baculovirus is used as an expression vector, the Sf9 cells are used as a bioreactor, and the prepared feline infectious rhinoconjunctivitis and feline panleukopenia bivalent genetic engineering vaccine has the advantages of high titer, high immunogenicity, good safety, convenience for large-scale production and the like, can induce an organism to generate high-level specific antibodies after immunization, and has a good immunoprophylaxis effect on the feline infectious rhinoconjunctivitis and the feline panleukopenia.

Drawings

FIG. 1 shows the results of indirect immunofluorescence assay after Sf9 cells are infected with recombinant baculovirus capable of expressing feline calicivirus VP1 protein in example 1 of the present invention; wherein, A is a virus infected hole, and B is a normal cell hole.

FIG. 2 shows the results of SDS-PAGE and Western Blot analysis of antigen harvest after Sf9 cells are infected with the recombinant baculovirus capable of expressing feline calicivirus VP1 protein in example 1 of the present invention; wherein, A is an SDS-PAGE identification result, lane 1 is an antigen obtained after recombinant baculovirus infects Sf9 cells and is frozen and thawed once and centrifuged to obtain a precipitate, lane 2 is an antigen obtained after recombinant baculovirus infects Sf9 cells, lane 3 is an antigen obtained after recombinant baculovirus infects Sf9 cells and is frozen and thawed once and centrifuged to obtain a supernatant, lane 4 is a cell poison obtained after F81 cell culture, lane 5 is a protein rainbow Marker, B is a Western Blot identification result, lane 1 is an antigen obtained after recombinant baculovirus infects Sf9 cells and is frozen and thawed once and centrifuged to obtain a precipitate, lane 2 is an antigen obtained after recombinant baculovirus infects Sf9 cells and is frozen and centrifuged once and supernatant, lane 3 is a cell poison obtained after F81 cell culture, lane 4 is a protein exposure Marker, and lane 5 is a protein rainbow Marker.

FIG. 3 is the result of electron microscope observation of antigen harvest from Sf9 cells infected with recombinant baculovirus expressing feline calicivirus VP1 protein in example 1 of the present invention.

FIG. 4 shows the results of indirect immunofluorescence assay of feline calicivirus monoclonal antibodies prepared in example 1 of the present invention; wherein, A is Sf9 cell infected by recombinant baculovirus expressing feline calicivirus VP1 protein, B is normal Sf9 cell, C is feline calicivirus infected F81 cell, and D is normal F81 cell.

FIG. 5 shows the results of indirect immunofluorescence assay of feline calicivirus polyclonal antibodies prepared in example 1 of the present invention; wherein, A is Sf9 cell infected by recombinant baculovirus expressing feline calicivirus VP1 protein, B is normal Sf9 cell, C is feline calicivirus infected F81 cell, and D is normal F81 cell.

FIG. 6 shows the results of indirect immunofluorescence assay after Sf9 cells were infected with the recombinant baculovirus of the recombinant baculovirus VP1 that can express the protein of feline panleukopenia VP2 in example 2 of the present invention; wherein, A is a virus infected hole, and B is a normal cell hole.

FIG. 7 shows the results of SDS-PAGE and Western Blot analysis of antigen harvest after Sf9 cells are infected with the recombinant baculovirus capable of expressing the protein VP2 of feline panleukopenia virus in example 2 of the present invention; wherein, A is the result of SDS-PAGE identification, and B is the result of Western Blot identification.

FIG. 8 is the result of electron microscope observation of antigen harvest by Sf9 cells infected with recombinant baculovirus expressing the protein VP2 of feline panleukopenia virus in example 2 of the present invention.

FIG. 9 shows the hemagglutination test results of Sf9 cell harvest antigen infected by recombinant baculovirus capable of expressing the protein VP2 of feline panleukopenia virus in example 2 of the present invention; wherein, A is an antigen prepared after sequence optimization, and B is an antigen prepared after sequence optimization.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.

Example 1 preparation and characterization of feline calicivirus virus-like particles

After the FCV-JL2 strain is subjected to RNA extraction, reverse transcription and PCR sequencing, a VP1 sequence is obtained, optimization is performed according to the preference of an instct NEW II cell, the codon preference index is adjusted from 0.47 to 0.98, the GC content is adjusted from 42.6% to 57.6%, adverse cis-acting elements are removed, and the region of mRNA which is easy to form a neck-loop region is removed. The optimized sequence is sent to a company for gene synthesis. For the optimized sequence, the VP1 gene is amplified by using primers and linked into a pFastBac Dual vector through NotI-HindIII and SmaI-XhoI respectively to obtain a transfer vector carrying double copies of the exogenous fragment. And transforming the transfer vector into DH10Bac competence for recombination to obtain the recombinant baculovirus genome Bacmid carrying the double-copy exogenous segment. After Bacmid is transfected into Sf9 cells, recombinant baculovirus is obtained through rescue. Infection of Sf9 cells with recombinant baculovirus can produce significant cytopathic effects such as increased volume, cell shedding, and increased intracellular granules. After a single layer of Sf9 in a 24-well plate is inoculated with recombinant baculovirus and subjected to indirect immunofluorescence detection by using a monoclonal antibody aiming at feline calicivirus 48 hours later, specific green fluorescence can be seen, and the specific green fluorescence is shown in a picture A of figure 1, which indicates that the target protein can be successfully expressed.

Recombinant baculovirus was inoculated with suspension cultured Sf9 cells at MOI ═ 0.1 inoculation amount, antigen was harvested 4 days after inoculation, freeze-thawed once for antigen, centrifuged at 3000rpm for 30min, supernatant and pellet were separated and subjected to SDS-PAGE assay, and cytotoxicity cultured with F81 cells was used as control. As a result, the VLPs were harvested as bands containing the target protein (FIG. 2A, lanes 1-3), and the crude band shown in the cytotoxic control lane was the serum component in the culture, which was not the target protein. Western Blot using monoclonal antibodies against feline calicivirus (purchased from MyBioSourc Inc.) revealed that VLPs sample lanes had specific bands of interest (FIG. 2B, lanes 1-2) and that the protein of interest was predominantly in the supernatant after one freeze-thaw. And the harvested VLPs had significantly more protein of interest than the cytotoxic control (fig. 2B, lane 3). After negative antigen staining, the result of electron microscope observation shows that virus-like particles with the diameter of about 38nm exist, the shape is similar to that of natural viruses (figure 3), and the expressed target protein can be successfully assembled into the virus-like particles.

Recombinant baculovirus was inoculated with suspension cultured Sf9 cells at MOI ═ 0.1 inoculation amount, antigen was harvested 4 days after inoculation, and supernatant was harvested 30min at 3000rpm after one freeze-thaw. The obtained pellet was dissolved in PBS by ultracentrifugation at 30000rpm for 1.5 h. Then, the virus with bands of 10-20% is harvested by sucrose gradient centrifugation (sucrose density is 10%, 20%, 30% and 40% from top to bottom in sequence), dissolved in PBS, and then dissolved in PBS at 30000rpm for 1.5h to obtain a precipitate, which is then dissolved in PBS. After the protein concentration was measured by the BCA method, mice were immunized to prepare monoclonal antibodies. Finally, a monoclonal cell strain is obtained by screening, wherein the monoclonal cell strain is 13#, the concentration is 2.5mg/ml, and the subtype is G2 b. The 13# mab was used to indirectly immunofluorescent stain the FCV-VP 1-expressing recombinant baculovirus-infected Sf9 cells (fig. 4, A, B) and FCV live virus-infected F81 cells (fig. 4, C, D), and the inoculated virus wells all showed green positive (fig. 4, A, C) and the cell control wells negative (fig. 4, B, D).

After freezing and thawing the virus liquid harvested from FCV live virus infected F81 cells twice, the supernatant was harvested at 3000rpm for 30 min. Adding 1.3M zinc acetate according to the proportion of 1/50, precipitating for 1h at 4 ℃, performing 10000rpm for 30min, suspending and precipitating by saturated EDTA, performing sucrose gradient centrifugation (the sucrose density is 20%, 30% and 40% in sequence from top to bottom), harvesting 20-30% of virus with bands, dissolving in PBS, and dissolving the obtained precipitate by PBS for 1.5h at 30000 rpm. After the protein concentration was measured by BCA method, the polyclonal antibody was prepared by immunizing rabbits with multiple back spots at 500 ug/time for four times in total, and the concentration was 9.8 mg/ml. Indirect immunofluorescence staining was performed using polyclonal antibodies on recombinant baculovirus-infected Sf9 cells (fig. 5, panel A, B) and FCV live virus-infected F81 cells (fig. 5, panel C, D) expressing FCV-VP1, respectively, and the inoculated virus wells all showed green positive (fig. 5A, C) and the cell control wells negative (fig. 5, panel B, D).

Using the prepared 13# monoclonal antibody as a capture antibody, coating an ELISA plate, adding the antigen to be detected (prepared VLPs) with the coating concentration of 1 ug/hole, diluting the prepared rabbit polyclonal antibody by 10000 times to be used as a detection antibody, diluting the HRP-anti-rabbit antibody by 20000 times to be used as a secondary antibody, developing by using a developing solution, and reading the OD value by using an enzyme-labeling instrument after the color development is stopped. The results show (table 1), the OD value of VLPs expressed by the recombinant baculovirus prepared by sequence optimization is higher than that of VLPs expressed by the recombinant baculovirus prepared by sequence non-optimization and positive cell toxicity cultured by F81 cells, which indicates that after the sequence optimization of FCV VP1, the expression level of the target protein is significantly changed, and is higher than that of the cell toxicity cultured conventionally.

TABLE 1 double antibody sandwich Elisa results

Example 2 preparation and characterization of feline panleukopenia Virus-like particles

After the FPV strain extracted genome is sequenced, a VP2 gene sequence is obtained, optimization is carried out according to the preference of an instct NEW II cell, the codon preference index is adjusted from 0.37 to 0.87, and the GC content is adjusted from 35.1% to 53.0%. The optimized sequence is sent to a company for gene synthesis. For the optimized sequence, VP2 gene is amplified by using primers and is respectively linked into a pFastBac Dual vector through NotI-HindIII and XhoI-NheI to obtain a transfer vector carrying double-copy foreign fragments. And transforming the transfer vector into DH10Bac competence for recombination to obtain the recombinant baculovirus genome Bacmid carrying the double-copy exogenous segment. After Bacmid is transfected into Sf9 cells, recombinant baculovirus is obtained through rescue. Infection of Sf9 cells with recombinant baculovirus can produce significant cytopathic effects such as increased volume, cell shedding, and increased intracellular granules. After a single layer of Sf9 in a 24-well plate is inoculated with recombinant baculovirus and subjected to indirect immunofluorescence detection by using a monoclonal antibody aiming at feline parvovirus 48 hours later, specific green fluorescence (A in figure 6) can be seen, and the target protein can be successfully expressed.

The recombinant baculovirus was inoculated with suspended Sf9 cells at an MOI of 0.1, and the antigen was harvested 4 days after inoculation and subjected to SDS-PAGE detection, resulting in a band of the desired protein being visible (fig. 7, panel a). Western Blot detection using a monoclonal antibody needle against feline parvovirus revealed a specific band (FIG. 7, panel B). After negative antigen staining, the result of electron microscope observation shows that virus-like particles with the diameter of about 20nm exist, the shape is similar to that of natural viruses (figure 8), and the expressed target protein can be successfully assembled into the virus-like particles. The hemagglutination titer is measured by using 15mM pH6.5PBS, and the FPV antigen titer expressed by the recombinant virus harvested after sequence optimization can reach 1:2¹⁸(Panel A of FIG. 9), whereas the FPV antigen titer expressed by the recombinant virus harvested without sequence optimization only reached 1:2¹²(FIG. 9B), the results showed that the expression of FPV VP2 was significantly increased after sequence optimization, and that it was able to assemble into an antigen with hemagglutinating activity.

Example 3 feline immunoassay

The recombinant baculovirus was inoculated with suspended Sf9 cells at MOI of 0.1, respectively, and harvested after 4 days of culture. Freezing and thawing the Sf9 cells inoculated with the FCV antigen for 1 time, and centrifuging at 3000rpm for 30min to obtain a supernatant, namely the harvest solution containing the FCV virus-like particles. The protein concentration was measured using BCA method and was 4.9 mg/ml. The FPV antigen inoculated Sf9 cells were centrifuged directly at 3000rpm for 30min, the supernatant was discarded and the cells were treated with an equal volume of 25mM NaHCO₃Suspending, acting on ice for 30min, and centrifuging at 3000rpm for 30min to obtain supernatant, namely the harvest solution containing FPV virus-like particles. Hemagglutination titer was determined using pig blood with an HA of 1:2¹⁶。

Mixing the FCV virus-like particles and the FPV virus-like particles according to the volume ratio of 3:1, and adding 20% of alumina gel adjuvant (Hayao Liu Shi works) and 0.005% of thimerosal according to the proportion of 1/5 to prepare the feline bivalent vaccine. Then preparing cat FCV vaccine and cat FPV vaccine respectively according to the above-mentioned method. The prepared vaccines are all stored at 4 ℃.

8-9 weeks old kittens (FCV neutralizing antibody titer not higher than 1:4, FPV HI titer not higher than 1:4) were divided into 4 groups, i.e., immunization group A, immunization group B, immunization group C and control group, and 3 cats/group, i.e., feline bivalent vaccine, feline FCV vaccine, feline FPV vaccine and PBS, respectively, at an immunization dose of 1.0 ml/cat, and boosted once at 4 weeks after the first immunization. Blood was collected every 2 weeks after immunization, and serum was separated and its FCV neutralizing antibody titer and FPV hemagglutination inhibiting antibody titer were measured.

The results show that 2 weeks after immunization, group a stimulated the production of neutralizing antibodies against feline calicivirus (table 2) and hemagglutination-inhibiting antibodies against feline panleukopenia virus (table 3), and that the antibodies increased after booster immunization and persisted for 8 weeks. And the immune group and the control group are normal in diet, mental state and body temperature and have no other adverse reactions. In addition, the hemagglutination inhibition antibody titer of FPV has no significant difference between the immune group A and the immune group C, and the hemagglutination inhibition antibody titer can reach 1:2 when the antibody titer is highest¹¹. And aiming at the neutralizing antibody titer of FCV, the neutralizing antibody titer of the immune group A is higher than that of the immune group B, the immune effect is good, and the neutralizing antibody titer of the immune group A can reach 1: 39.81. the two virus-like particles are mixed and then are immunized, and the titer generated by the body is further improved on the basis of the original single use.

TABLE 2FCV neutralizing antibody titers (1: n)

TABLE 3FPV hemagglutination inhibition antibody titers (1: 2)ⁿ)

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

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cgtgtggtcg ttaacaacat ggacaagaca gctgtgaagg gcaacatggc tctggacgac 300

acacatgttc agatcgtgac accttggtct ttggtcgacg ctaacgcttg gggcgtgtgg 360

ttcaatcccg gagattggca gctgatcgtg aacaccatgt ccgaactgca cttggtctcc 420

ttcgaacaag aaatctttaa cgttgtcctg aagactgttt cagagtccgc tacacaaccc 480

ccaacaaagg tgtacaacaa cgatctcact gcctctctga tggttgctct ggacagcaac 540

aacacaatgc ccttcacccc tgccgccatg cgttcagaga cactcggatt ttacccatgg 600

aagcctacta tcccaacacc ttggaggtac tacttccaat gggataggac actcatccct 660

tcacacacag gcacctcagg aacacctact aacgtttacc acggcactga tcccgacgat 720

gttcaattct acaccattga gaactccgtg cccgtgcatc tcttgcgcac aggagacgag 780

ttcgccaccg gcaccttctt cttcgattgc aaaccctgtc gcctgaccca cacatggcaa 840

accaaccgtg ccctgggctt gccacctttc ctcaacagcc tgccacaatc cgagggtgct 900

actaacttcg gagatatcgg agtccagcaa gacaagcgcc gcggcgttac tcaaatgggc 960

aataccgact acatcactga agccactatc atgaggccag ctgaggttgg ctacagcgcc 1020

ccctactact cctttgaggc ctccactcag ggcccattca agaccccaat cgccgctggt 1080

aggggaggcg cccagactga cgaaaaccag gccgccgacg gagatcccag atacgccttc 1140

ggacgccagc acggtcagaa gactaccact accggtgaaa cccctgagcg ttttacttat 1200

atcgctcacc aagacaccgg tcgctatccc gaaggtgact ggatccagaa catcaacttc 1260

aatctgcctg tgactaacga caacgtgctc ctccccacag acccaatcgg tggcaagacc 1320

ggtatcaatt acaccaatat ttttaatacc tacggtcctt tgacagctct caataacgtc 1380

ccacctgtct accctaacgg ccagatctgg gacaaagaat tcgacaccga tttgaagcca 1440

cgcctccacg tgaacgcccc tttcgtgtgt cagaacaact gccctggtca actgttcgtg 1500

aaggtcgctc ccaacctgac caacgaatac gaccccgacg cttcagccaa tatgagccgc 1560

atcgtcactt acagcgactt ctggtggaag ggtaaactcg tgtttaaggc taagctgcgt 1620

gcctcacaca cttggaaccc aattcaacag atgtctatca acgttgacaa ccagttcaac 1680

tacgtcccca ataacatcgg agccatgaag atcgtttacg agaagtctca gctcgctcct 1740

cgtaaactgt actaa 1755

<210> 4

<211> 584

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

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 Lys

65 70 75 80

Arg Val Val Val Asn Asn Met 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 Phe 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 Asn Asn Ile Gly Ala Met Lys Ile Val Tyr Glu Lys Ser

565 570 575

Gln Leu Ala Pro Arg Lys Leu Tyr

580

Claims

1. A feline infectious rhinoconjunctivitis and feline panleukopenia bivalent vaccine comprising feline calicivirus-like particles and feline panleukopenia virus-like particles;

in the feline calicivirus virus-like particles, the gene sequence of the feline calicivirus is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2;

in the feline panleukopenia virus-like particle, the gene sequence of the feline panleukopenia virus is shown as SEQ ID NO.3, and the amino acid sequence is shown as SEQ ID NO. 4;

wherein, the preparation of the vaccine comprises the following steps: firstly, preparing feline calicivirus virus-like particles and feline panleukopenia virus-like particles, then mixing the two virus-like particles, adding a preservative and an adjuvant, wherein the content of the feline calicivirus-like particles is not less than 4mg/ml and the hemagglutination titer of the feline panleukopenia virus is not less than 1:2 before mixing¹⁴。

2. The feline infectious rhinoconjunctivitis and feline panleukopenia bivalent vaccine of claim 1 wherein the vaccine is prepared with a combined volume ratio of the two virus-like particles of from 1:1 to 5: 1.

3. The feline infectious rhinoconjunctivitis and feline panleukopenia bivalent vaccine of claim 1 wherein the vaccine is prepared by adding thimerosal as a preservative and aluminum phosphate as an adjuvant.

4. The feline infectious rhinoconjunctivitis and feline panleukopenia bivalent vaccine of any one of claims 1-3, wherein the feline calicivirus virus-like particle and the feline panleukopenia virus-like particle are prepared by the process of:

(1) optimizing a feline calicivirus VP1 gene and a feline panleukopenia virus VP2 gene according to insect cell preferred codons, respectively inserting the optimized feline calicivirus VP1 gene and the optimized feline panleukopenia virus VP2 gene into insect cell expression vectors, converting DH10Bac competence, and extracting a recombinant baculovirus genome; the nucleotide sequences of the optimized feline calicivirus VP1 gene and the optimized feline panleukopenia virus VP2 gene are respectively shown as SEQ ID No.1 and SEQ ID No. 3;

(2) transfecting insect cells with recombinant baculovirus genomes, rescuing to obtain recombinant baculovirus, inoculating the insect cells according to MOI (molar equivalent of 1-0.1), and harvesting a mixture after 4-5 days to obtain feline calicivirus antigen and feline panleukopenia virus antigen respectively;

(3) repeatedly freezing and thawing the feline calicivirus antigen for 1 time, and then harvesting the supernatant to obtain feline calicivirus virus-like particles; and (3) treating the feline panleukopenia virus antigen with a lysis solution, and then harvesting supernatant to obtain the feline panleukopenia virus-like particles.

5. The feline infectious rhinoconjunctivitis and feline panleukopenia bivalent vaccine of claim 4, wherein the lysate of step (3) is 25mM NaHCO₃And (3) solution.

6. The feline infectious rhinoconjunctivitis and feline panleukopenia bivalent vaccine of any one of claims 1-3, wherein the preservative is thimerosal at a final concentration of 0.005%.

7. The feline infectious rhinoconjunctivitis and feline panleukopenia bivalent vaccine of any one of claims 1-3, wherein the adjuvant is aluminum phosphate at a final concentration of 0.4%.

8. The method for preparing the feline infectious rhinoconjunctivitis and feline panleukopenia bivalent vaccine as claimed in any one of claims 1 to 7, comprising preparing feline calicivirus virus-like particles and feline panleukopenia virus-like particles, mixing the two viruslike particles, adding a preservative and an adjuvant, wherein the feline calicivirus-like particle content is not less than 4mg/ml and the feline panleukopenia virus hemagglutination titer is not less than about 4mg/ml before mixing1:2¹⁴。

9. The preparation method according to claim 8, wherein in the feline calicivirus virus-like particle, the gene sequence of the feline calicivirus is represented by SEQ ID No.1, and the amino acid sequence is represented by SEQ ID No. 2;

in the feline panleukopenia virus-like particle, the gene sequence of the feline panleukopenia virus is shown as SEQ ID NO.3, and the amino acid sequence is shown as SEQ ID NO. 4.