CN108371710A - Cat infectiousness nose conjunctivitis and feline panleukopenia bigeminy vaccine and preparation method - Google Patents

Cat infectiousness nose conjunctivitis and feline panleukopenia bigeminy vaccine and preparation method Download PDF

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CN108371710A
CN108371710A CN201810089278.9A CN201810089278A CN108371710A CN 108371710 A CN108371710 A CN 108371710A CN 201810089278 A CN201810089278 A CN 201810089278A CN 108371710 A CN108371710 A CN 108371710A
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virus
feline
particle
cat
feline panleukopenia
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CN108371710B (en
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夏振强
金宏丽
石晶
殷玉和
赵健
刘冰
刘伟
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CHANGCHUN SR BIOLOGICAL TECHNOLOGY Co Ltd
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CHANGCHUN SR BIOLOGICAL TECHNOLOGY Co Ltd
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Abstract

The present invention provides cat infectiousness nose conjunctivitis and feline panleukopenia bigeminy vaccine and preparation methods, the bigeminy vaccine of the present invention is to produce the virus-like particle of feline calicivirus and the virus-like particle of Feline Panleukopenia Virus respectively using baculoviral/insect cell expression system, aforementioned two kinds of viral virus-like particles are mixed with preservative and adjuvant, cat infectiousness nose conjunctivitis and feline panleukopenia bigeminy vaccine is prepared.The bigeminy vaccine of the present invention have many advantages, such as it is safe efficient, easy to produce, at low cost, to cat infectiousness nose conjunctivitis and feline panleukopenia with good immunoprophylaxis effect.

Description

Cat infectiousness nose conjunctivitis and feline panleukopenia bigeminy vaccine and preparation method
Technical field
The present invention relates to microorganism field, genetic engineering field and veterinary biological pharmaceutical fields, specifically, being related to one kind The side of cat infectiousness nose conjunctivitis and feline panleukopenia bigeminy vaccine is produced with insect cell-baculovirus expression system Method.
Background technology
Cat infectiousness nose conjunctivitis is caused by feline calicivirus (Feline calicivirus, FCV), clinically with nose Inflammation, conjunctivitis, Acute oral ulcer, pneumonia and chronic gastritis are characterized.It is now recognized that FCV is in worldwide distribution, and may feel Contaminate all felids.Feline panleukopenia is by Feline Panleukopenia Virus (Feline Panleukopenia virus, FPV) cause, clinical manifestation is to suffer from cat burst high fever, vomiting, diarrhea, dehydration and cycle blood flow Middle Neuroleptic Leukocytopenia is characterized.The many animals such as FPV main infections cat family and Mustelidae, especially with 6 monthly ages following brood Morbidity and mortality higher is the most important infectious disease of felid.
In order to which cat is from the threat of infectious disease, its physical and mental health is kept, is crucial to its vaccine inoculation.Need the core being inoculated with The antigen that heart vaccine includes has：FPV, FCV, cat infectious bovine rhinotracheitis viral (Feline herpesvirus, FHV), mad dog Virus (Rabies, RABV).Currently, relevant domestic vaccine has cat bigeminy vaccine, including FPV, RABV, but in online, city It can not find the specifying information about this vaccine on field, it is likely to the pilot product of certain R＆D institution.Commercial available vaccines be into Mouth vaccine, predominantly cat triple vaccine, including FPV, FCV, FHV.These vaccines are attenuated vaccine or inactivated vaccine, and weak poison Vaccine has potential security risk, inactivated vaccine to be unable to the shortcomings of effective stimulus body generates cellular immunity.In addition, virus variation It is the main reason for leading to current vaccines immuning failure.Therefore novel, efficient, safe new generation vaccine is prepared for popular strain It is of great significance to the prevention and control of cat infectious disease.
The present invention selects domestic popular strain, prepares virus-like particle antigen using genetic engineering means, carries out novel cat The preparation of bigeminy recombinant vaccine can prevent cat infectiousness nose conjunctivitis, feline panleukopenia simultaneously.Due to virus-like Particle shape and structure are similar to natural viral particle, therefore body can effectively be induced to generate cellular immunity and humoral immunity； Since virus-like particle does not contain nucleic acid, it is unable to autonomous replication, therefore safety higher.In addition, it is also easy to produce, potency is high, The advantages that at low cost, while having filled up the domestic blank about feline vaccines development, commercialization.
Invention content
The object of the present invention is to provide a kind of cat infectiousness nose conjunctivitis and feline panleukopenia bigeminy vaccine and its Preparation method.
A kind of cat infectiousness nose conjunctivitis provided by the invention and feline panleukopenia bigeminy vaccine contain cat cup-shaped Virus-virus like particles and Feline Panleukopenia Virus virus-like particle.
In the feline calicivirus virus-like particle, the gene order of feline calicivirus virus as shown in SEQ ID NO.1, Amino acid sequence is as shown in SEQ ID NO.2.
In the Feline Panleukopenia Virus virus-like particle, the gene order such as SEQ ID of feline calicivirus virus Shown in NO.3, amino acid sequence is as shown in SEQ ID NO.4.
In order to realize that the object of the invention, the present invention produce cat cup-shaped disease using insect cell-baculovirus expression system Poison and Feline Panleukopenia Virus virus-like particle.
Present invention firstly provides a kind of feline calicivirus VP1 genes of optimization, a kind of feline panleukopenias of optimization Virus VP 2 gene, i.e., the gene optimized according to insect cell preference codon.Feline calicivirus VP1 genes after optimization As shown in SEQ ID No.1, amino acid sequence is as shown in SEQ ID No.2；Feline Panleukopenia Virus VP2 after optimization Gene is as shown in SEQ ID No.3, and amino acid sequence is as shown in SEQ ID No.4.
The present invention provides the preparation method of above-mentioned cat infectiousness nose conjunctivitis and feline panleukopenia bigeminy vaccine, Including preparing feline calicivirus virus-like particle and Feline Panleukopenia Virus virus-like particle, by two kinds of virus-like particles It is mixed, adds preservative and adjuvant, and before mixing, feline calicivirus virus-like particle content is not less than 4mg/ml, and cat is general Leukopenia viral hemoagglutination potency is not less than 1:214。
In the feline calicivirus virus-like particle, the gene order of feline calicivirus virus as shown in SEQ ID NO.1, Amino acid sequence is as shown in SEQ ID NO.2；In the Feline Panleukopenia Virus virus-like particle, feline calicivirus disease The gene order of poison is as shown in SEQ ID NO.3, and amino acid sequence is as shown in SEQ ID NO.4.
Specifically, the system of a kind of feline calicivirus and Feline Panleukopenia Virus virus-like particle provided by the invention Preparation Method includes the following steps：(1) feline calicivirus VP1 genes, cat whiting cell are subtracted according to insect cell preference codon Few syndrome virus VP2 genes optimize, and the gene of optimization is inserted respectively into insect cell expression vector, convert DH10Bac Competence extracts recombinant baculovirus genome；(2) by recombinant baculovirus genome transfection insect cell, rescue is weighed Group baculoviral is inoculated with insect cell by MOI=0.1-1, mixture is harvested after 4-5 days, i.e., it is anti-to obtain feline calicivirus respectively Former, Feline Panleukopenia Virus antigen；(3) supernatant will be harvested after feline calicivirus antigen multigelation 1 time, that is, obtains cat Calicivirus virus-like particle；Supernatant is harvested after Feline Panleukopenia Virus antigen is handled cell using lysate, i.e., Obtain Feline Panleukopenia Virus virus-like particle.
In a preferred embodiment, a kind of described feline calicivirus and Feline Panleukopenia Virus virus-like The preparation method of particle, wherein the lysate for handling cell, is NaHCO3Solution, more preferably be 25mM concentration NaHCO3Solution.
It is a further object of the present invention to provide a kind of cat infectiousness nose conjunctivitis and feline panleukopenia bigeminy gene Engineered vaccine, wherein including at least one feline calicivirus antigen and at least one Feline Panleukopenia Virus antigen.
In order to realize that two kinds of virus-like particles of above-mentioned preparation are mixed in a certain ratio by the object of the invention, the present invention, and It is aided with preservative and adjuvant is prepared.Wherein, two kinds of virus-like particle mixed volume ratios are 1:1-5:1, it is preferably in a proportion of 3:1, the preservative is thimerosal etc., preferably final concentration of 0.005%；The adjuvant is aluminum phosphate etc., preferably final concentration of 0.4%.
The cat cup cat infectiousness nose conjunctivitis and feline panleukopenia bigeminy recombinant vaccine of the present invention is muscle Vaccinate or be subcutaneously injected vaccine.
The present invention further provides the cat infectiousness nose conjunctivitis of above-mentioned preparation and feline panleukopenia bigeminy genes Application of the engineered vaccine in preventing cat infectiousness nose conjunctivitis and feline panleukopenia.
The present invention is using baculoviral as expression vector, using Sf9 cells as bioreactor, the cat infectiousness nose of preparation Conjunctivitis and feline panleukopenia bigeminy recombinant vaccine are high with titre, immunogenicity is high, safety is good, convenient for big The advantages that large-scale production, can induce body and generate high-caliber specific antibody after being immunized, general to cat infectiousness nose conjunctivitis, cat Leukopenia has good immunoprophylaxis effect.
Description of the drawings
Fig. 1 is the recombinate shape virus infection Sf9 cells that feline calicivirus VP1 albumen can be expressed in the embodiment of the present invention 1 Indirect immunofluorescene assay result afterwards；Wherein, A is virus-infected wells, and B is normal cell hole.
Fig. 2 is the recombinate shape virus infection Sf9 cells that feline calicivirus VP1 albumen can be expressed in the embodiment of the present invention 1 SDS-PAGE, the Western Blot qualification results of antigen are harvested afterwards；Wherein, A is SDS-PAGE qualification results, and swimming lane 1 is attached most importance to Antigen centrifuged deposit of freeze thawing is harvested after group baculovirus infection Sf9 cells, swimming lane 2 is recombinate shape virus infection Sf9 The antigen harvested after cell, swimming lane 3 are supernatant after harvest antigen freeze thawing after recombinate shape virus infection Sf9 cells once centrifugation, Swimming lane 4 is the cell toxicant of F81 cell culture, and swimming lane 5 is albumen rainbow Marker, and B is Western Blot qualification results, swimming lane 1 is harvests antigen centrifuged deposit of freeze thawing after recombinate shape virus infection Sf9 cells, swimming lane 2 is recombinant baculovirus sense Supernatant after antigen freeze thawing once centrifuges is harvested after contaminating Sf9 cells, swimming lane 3 is the cell toxicant of F81 cell culture, and swimming lane 4 is albumen Exposure Marker, swimming lane 5 are albumen rainbow Marker.
Fig. 3 is the recombinate shape virus infection Sf9 cells that feline calicivirus VP1 albumen can be expressed in the embodiment of the present invention 1 The Electronic Speculum for harvesting antigen observes result.
Fig. 4 is the feline calicivirus monoclonal antibody indirect immunofluorescene assay result prepared in the embodiment of the present invention 1；Its In, A is the Sf9 cells for the recombinate shape virus infection for expressing feline calicivirus VP1 albumen, and B is normal Sf9 cells, and C is cat cup The F81 cells of shape virus infection, D are normal F81 cells.
Fig. 5 is the feline calicivirus polyclonal antibody indirect immunofluorescene assay result prepared in the embodiment of the present invention 1；Its In, A is the Sf9 cells for the recombinate shape virus infection for expressing feline calicivirus VP1 albumen, and B is normal Sf9 cells, and C is cat cup The F81 cells of shape virus infection, D are normal F81 cells.
Fig. 6 is the recombinant baculovirus that Feline Panleukopenia Virus VP2 albumen can be expressed in the embodiment of the present invention 2 Indirect immunofluorescene assay result after the recombinate shape virus infection Sf9 cells of VP1；Wherein, A is virus-infected wells, and B is normal Cell hole.
Fig. 7 is the recombinant baculovirus sense that Feline Panleukopenia Virus VP2 albumen can be expressed in the embodiment of the present invention 2 SDS-PAGE, the Western Blot qualification results of antigen are harvested after dye Sf9 cells；Wherein, A is SDS-PAGE qualification results, B For Western Blot qualification results.
Fig. 8 is the recombinant baculovirus sense that Feline Panleukopenia Virus VP2 albumen can be expressed in the embodiment of the present invention 2 The Electronic Speculum for contaminating Sf9 cells harvest antigen observes result.
Fig. 9 is the recombinant baculovirus sense that Feline Panleukopenia Virus VP2 albumen can be expressed in the embodiment of the present invention 2 Contaminate the Blood coagulation test result of Sf9 cells harvest antigen；Wherein, A is the antigen prepared after sequence optimisation, and B is after sequence is not optimised The antigen of preparation.
Specific implementation mode
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment Used in the conventional means that are well known to those skilled in the art of technological means, raw materials used is commercial goods.
The preparation and identification of 1 feline calicivirus virus-like particle of embodiment
After FCV-JL2 strains extraction RNA, reverse transcription, PCR are sequenced, VP1 sequences are obtained, according to insect NEW II cells favors optimize, and codon-bias index is adjusted by 0.47 to 0.98, G/C content by 42.6% adjust to 57.6%, and unfavorable cis-acting elements is removed, mRNA is easily at neck-ring region etc. for removal.By sequence after optimization send company into Row gene chemical synthesis.NotI-HindIII, SmaI-XhoI are passed through respectively using primer amplification VP1 genes to the sequence after optimization It is connected into pFastBac Dual carriers, obtains the transfer vector for carrying double copy exogenous sequences.Transfer vector is converted DH10Bac competence is recombinated, and the recombinant baculovirus genome Bacmid for carrying double copy exogenous sequences is obtained.It will After Bacmid transfects Sf9 cells, rescue obtains recombinant baculovirus.By recombinate shape virus infection Sf9 cells, can generate apparent Cytopathy, as volume increase, cell detachment, intracellular granular increase.To the Sf9 of long single layer is inoculated with recombination in 24 orifice plates Baculoviral carries out indirect immunofluorescene assay after 48h, it is seen that have specificity using the monoclonal antibody for feline calicivirus Green fluorescence, see Fig. 1 A figure, showing can successful expression destination protein.
The Sf9 cells of poison amount inoculation suspension culture are connect with MOI=0.1 to recombinant baculovirus, 4 days harvests are anti-after connecing poison Original, after primary to antigen freeze thawing, 3000rpm 30min centrifugations, separation supernatant precipitation carries out SDS-PAGE detections, thin with F81 The cell toxicant of born of the same parents' culture is as a contrast.As a result as it can be seen that the purposeful protein bands of VLPs (Fig. 2A, 1-3 swimming lanes) of harvest, cell The thick band that poison control swimming lane is shown is the serum composition in culture solution, non-destination protein.Use the list for feline calicivirus Clonal antibody (being purchased from MyBioSourc companies) carries out Western Blot detections, it is seen that VLPs sample lanes have specific purpose Band (Fig. 2 B, 1-2 swimming lanes), and after freeze thawing is primary, destination protein primarily enters in supernatant.And the purpose egg of the VLPs of harvest It is significantly more than cytotoxic control (Fig. 2 B, the 3rd swimming lane) in vain.After antigen negative staining, Electronic Speculum observation is carried out, it is as a result visible to have diameter The virus-like particle of 38nm or so, form is similar to natural viral (Fig. 3), shows that the destination protein of expression can successfully be assembled into disease Malicious sample particle.
The Sf9 cells of poison amount inoculation suspension culture are connect with MOI=0.1 to recombinant baculovirus, 4 days harvests are anti-after connecing poison Original, after freeze thawing is primary, 3000rpm 30min harvest supernatant.Through ultracentrifugation 30000rpm 1.5h, the precipitation of acquisition is molten with PBS Solution.Through sucrose gradient centrifugation, (sucrose density is followed successively by from top to bottom again：10%, 20%, 30%, 40%), harvest 10-20% it Between band virus, be dissolved in PBS, then through 30000rpm 1.5h, the precipitation of acquisition is dissolved with PBS.Egg is measured with BCA methods After white concentration, mouse is immunized, prepares monoclonal antibody.Finally screening obtains one plant of monoclonal cell strain, is 13#, a concentration of 2.5mg/ml, hypotype G2b.Using 13# monoclonal antibodies respectively to the Sf9 cells (figure of the recombinate shape virus infection of expression FCV-VP1 4 A, B figure), live F81 cells C, the D of Fig. 4 (scheme) of poison infection of FCV carry out indirect IF staining, virus inoculation hole is aobvious It is shown as the positive (A, C of Fig. 4 schemes) of green, cell control well is negative (B, D of Fig. 4 schemes).
After the virus liquid freeze thawing twice for poison infection F81 cells harvest of living to FCV, 3000rpm30min harvests supernatant.By 1/ 1.3M zinc acetates are added in 50 ratios, and 4 DEG C of precipitations 1h, 10000rpm30min are suspended with saturation EDTA and precipitated, then through saccharose gradient (sucrose density is followed successively by from top to bottom for centrifugation：20%, 30%, 40%), the virus for harvesting band between 20-30%, is dissolved in In PBS, then through 30000rpm 1.5h, the precipitation of acquisition is dissolved with PBS.It is more using back after measuring albumen concentration with BCA methods Rabbit is immunized in point, 500ug/ times, is immunized four times altogether, prepares polyclonal antibody, a concentration of 9.8mg/ml.Distinguished using polyclonal antibody F81 cells (Fig. 5 that Sf9 cells (A, B of Fig. 5 schemes), the FCV work poison of the recombinate shape virus infection for expressing FCV-VP1 are infected C, D figure) carry out indirect IF staining, virus inoculation hole be illustrated as green the positive (Fig. 5 A, C), cell control well For negative (B, D of Fig. 5 schemes).
Using the 13# monoclonal antibodies of preparation as capture antibody, coating elisa plate, peridium concentration is the holes 1ug/, adds and waits for Antigen (VLPs of preparation) is examined, then using the mostly anti-10000 times of dilutions of the rabbit of preparation as detection antibody, HRP- anti-rabbit antibody 20000 It is diluted to secondary antibody again, is developed the color with developing solution, OD values are read using microplate reader after color development stopping.As a result (table 1) is shown, through sequence The VLPs OD values of recombinant baculovirus expression prepared by row optimization are not optimised the recombinant baculovirus expression of preparation higher than sequence The cell positive poison of VLPs, F81 cell culture, shows FCV VP1 after sequence optimisation, changed dramatically destination protein expression Amount, and it is higher than the cell toxicant expression quantity of routine culture.
1 double-antibody sandwich Elisa results of table
The preparation and identification of 2 Feline Panleukopenia Virus virus-like particle of embodiment
After FPV strains extraction genome is sequenced, VP2 gene orders are obtained, it is inclined according to insect NEW II cells Love property optimizes, and codon-bias index is adjusted by 0.37 to 0.87, and G/C content is adjusted by 35.1% to 53.0%.It will be excellent Sequence send company to carry out gene chemical synthesis after change.NotI- is passed through respectively using primer amplification VP2 genes to the sequence after optimization HindIII, XhoI-NheI are connected into pFastBac Dual carriers, obtain the transfer vector for carrying double copy exogenous sequences. Transfer vector conversion DH10Bac competence is recombinated, the recombinant baculovirus base for carrying double copy exogenous sequences is obtained Because of a group Bacmid.After Bacmid is transfected Sf9 cells, rescue obtains recombinant baculovirus.Recombinate shape virus infection Sf9 is thin Born of the same parents can generate apparent cytopathy, as volume increase, cell detachment, intracellular granular increase.To long single layer in 24 orifice plates Sf9 be inoculated with recombinant baculovirus, carry out indirect immunofluorescence inspection using the monoclonal antibody for feline panleucopenia virus after 48h It surveys, it is seen that have the green fluorescence (A of Fig. 6) of specificity, showing can successful expression destination protein.
Poison amount inoculation suspension Sf9 cells are connect with MOI=0.1 to recombinant baculovirus, connect 4 days harvest antigen after poison, confrontation Original carries out SDS-PAGE detections, as a result visible purposeful protein band (A of Fig. 7 schemes).Use the Dan Ke for feline panleucopenia virus Grand antibody needle carries out Western Blot detections, it is seen that has specific purpose band (B of Fig. 7 schemes).After antigen negative staining, carry out Electronic Speculum is observed, and the as a result visible virus-like particle for having diameter 20nm or so, form is similar to natural viral (Fig. 8), shows to express Destination protein can succeed assembling assembly virus-like particle.The measurement of hemagglutinative titer is carried out using 15mM pH6.5PBS, it is excellent through sequence The FPV antigen valences of the expression of recombinant virus harvested after change are up to 1:218The A of Fig. 9 (scheme), and without being harvested after sequence optimisation The FPV antigen valences of expression of recombinant virus are only capable of up to 1:212(B of Fig. 9 schemes), the results showed that, after sequence optimisation, FPV VP2 expression Showed increased is measured, and the antigen of hemagglutination activity can be assembled into.
3 cat immunity test of embodiment
Above-mentioned recombinant baculovirus is inoculated with to suspension Sf9 cells respectively by MOI=0.1, culture harvests after 4 days.It will inoculation After the Sf9 cells freeze thawing 1 time of FCV antigens, 3000rpm centrifuges 30min, supernatant is obtained, as containing FCV virus-like particles Harvest liquid.Albumen concentration is measured using BCA methods, is 4.9mg/ml.To be vaccinated with the direct 3000rpm of Sf9 cells of FPV antigens from Heart 30min abandons supernatant, the isometric 25mM NaHCO of cell3It suspends, acts on 30min on ice, 3000rpm centrifuges 30min, obtains Obtain supernatant, the as harvest liquid containing FPV virus-like particles.The measurement of hemagglutinative titer, HA 1 are carried out using pig blood:216。
The FCV virus-like particles of above-mentioned preparation, FPV virus-like particles are pressed 3:1 volume mixture, then be added in 1/5 ratio 20% aluminium glue adjuvant (breathing out six factory of medicine), 0.005% thimerosal, are made cat bigeminy vaccine.Prepare cat FCV respectively as stated above again Vaccine, cat FPV vaccines.The vaccine of preparation is in 4 DEG C of preservations.
By 8-9 week old childrens cat, (FCV neutralize antibody titers are not higher than 1:4, FPV HI potency is not higher than 1:4) it is divided into 4 groups, point A groups, immune B groups, immune C groups and control group Wei not be immunized, 3/group, cat bigeminy vaccine, cat FCV vaccines, cat FPV are immunized respectively Vaccine and PBS, immunizing dose are 1.0ml/, and the 4th week booster immunization is primary after head exempts from.It takes a blood sample every 2 weeks after immune, detaches blood Clearly, its FCV neutralize antibody titers, FPV HI antibody titers are measured.
The results show that 2 weeks after immune, immune A groups can stimulate the anti-feline calicivirus neutralizing antibody (table 2) of body generation and Anti- Feline Panleukopenia Virus hemagglutination inhibition antibody (table 3), and antibody increases after booster immunization, and may persist to the 8th week. And immune group and control group diet, the state of mind, body temperature are normal, also without other adverse reactions.In addition, for the blood clotting of FPV For inhibiting antibody titer, the effect of A groups and immune C groups is immunized without significant difference, when HI antibody titer highest Reach 1:211.And for the neutralize antibody titers of FCV, immune A groups antibody titer is higher than immune B groups, and good immune effect is exempted from The neutralize antibody titers of epidemic disease A groups reach as high as 1：39.81.It is immunized after illustrating two kinds of virus-like particle mixing, stimulation body generates Potency on the basis of original be used alone, can further improve.
Table 2FCV neutralize antibody titers (1：n)
Table 3FPV HI antibody titers (1：2n)
Although above the present invention is described in detail with a general description of the specific embodiments, On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Sequence table
<110>Xinuo Biological Science ＆ Technology Co., Ltd., Changchun
<120>Cat infectiousness nose conjunctivitis and feline panleukopenia bigeminy vaccine and preparation method
<160> 4
<170> SIPOSequenceListing 1.0
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atggctgacg acggtagcgt gacagcccct gagcagggta cccctgtcgg tggtgtgatc 60
gctgaaccct ccagccagat ggctgctgcc gctgacatgg ccactggaaa gagcgtggac 120
agcgaatggg aagccttctt cagcttccac acaagcgtca actggagcac ttcagagacc 180
caaggcaaga tcctcttcaa gcagtctctg ggtcccctgc tgaaccccta cctggaacac 240
ctgagcaagc tctacgtcgc ttggtccggc agcgttgacg tgcgtttcag catcagcggt 300
agcggcgtct tcggaggcaa gctcgctgct atcgtcgtgc ccccaggtat cgaccccgtt 360
cagagcacat ccatgctcca atacccccac gtgctgttcg acgcccgtca ggttgaaccc 420
gtgatcttca gcatccctga cctcaggagc acactgtacc acctgatggc tgacactgac 480
accacctccc tggtcatcat ggtgtacaac gacctgatca acccatacgc taacgactcc 540
aacagctccg gctgtatcgt gaccgtggag acaaagcctg gtccagactt ccgcttccac 600
ctcctgaagc caccaggaag catgctgacc cacggttccg tgccatccga cctgatccct 660
aagtcttcct ctctgtggat cggcaaccgt cactggacag acatcacaga attcgtgatc 720
cgtcctttcg tgttccaggc caaccgtcac ttcgacttca accaggagac agccggttgg 780
tccacccctc gcttccgtcc tatgacagtg accgtctccc agaagaacgg tgagaagctg 840
ggcatcggtg tcgctaccga ctacatcgtt cccggcatcc ctgacggctg gcctgacacc 900
acaatccctg aggagctgac ccctgccggc gactacgcca tcacttctag caacggtaac 960
gacatcacaa ccctggccga ctacaacagc gctgacgtga tcaagaacaa cacaaacttc 1020
cgcggcatgt acatctgcgg ctccctgcag cgcgcctggg gtgacaagaa gatctccaac 1080
acagctttca tcacaatcgg agacgtggaa ggcagcaaga tcaagcctag ctctgtgatc 1140
agccaggcca agatcgctat cttccaggac aaccacgtca accacgacgt gcagacttcc 1200
gacatcacct acgctctgct gggttacacc ggtatcggtg aggaggccat cggcgctacc 1260
cgcgaccgcg tggctaggat ctccatcctg cctgaaaccg gtgctcgtgg cggcaaccac 1320
cctatcttct acaagaactc catgaagctg ggatacgtga tcaagagcat cgacgtgttc 1380
aacagccaga tcctccacac tagccgccag ctgagcctga acaactacct gctcgcccct 1440
gactccttcg ccgtgtaccg tatcacagac agcaacggta gctggttcga catcggtatc 1500
gaccacgacg gtttcagctt cgtgggtgtg agcaacatcc caaagctgga attccccctc 1560
acttcctcct acatgggtat ccagctggct aagatccgcc tggccagcaa catccgtagc 1620
atgatgacta agatctaa 1638
<210> 2
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<212> PRT
<213>Artificial sequence (Artificial Sequence)
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Met Ala Asp Asp Gly Ser Val Thr Ala Pro Glu Gln Gly Thr Pro Val
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Gly Gly Val Ile Ala Glu Pro Ser Ser Gln Met Ala Ala Ala Ala Asp
20 25 30
Met Ala Thr Gly Lys Ser Val Asp Ser Glu Trp Glu Ala Phe Phe Ser
35 40 45
Phe His Thr Ser Val Asn Trp Ser Thr Ser Glu Thr Gln Gly Lys Ile
50 55 60
Leu Phe Lys Gln Ser Leu Gly Pro Leu Leu Asn Pro Tyr Leu Glu His
65 70 75 80
Leu Ser Lys Leu Tyr Val Ala Trp Ser Gly Ser Val Asp Val Arg Phe
85 90 95
Ser Ile Ser Gly Ser Gly Val Phe Gly Gly Lys Leu Ala Ala Ile Val
100 105 110
Val Pro Pro Gly Ile Asp Pro Val Gln Ser Thr Ser Met Leu Gln Tyr
115 120 125
Pro His Val Leu Phe Asp Ala Arg Gln Val Glu Pro Val Ile Phe Ser
130 135 140
Ile Pro Asp Leu Arg Ser Thr Leu Tyr His Leu Met Ala Asp Thr Asp
145 150 155 160
Thr Thr Ser Leu Val Ile Met Val Tyr Asn Asp Leu Ile Asn Pro Tyr
165 170 175
Ala Asn Asp Ser Asn Ser Ser Gly Cys Ile Val Thr Val Glu Thr Lys
180 185 190
Pro Gly Pro Asp Phe Arg Phe His Leu Leu Lys Pro Pro Gly Ser Met
195 200 205
Leu Thr His Gly Ser Val Pro Ser Asp Leu Ile Pro Lys Ser Ser Ser
210 215 220
Leu Trp Ile Gly Asn Arg His Trp Thr Asp Ile Thr Glu Phe Val Ile
225 230 235 240
Arg Pro Phe Val Phe Gln Ala Asn Arg His Phe Asp Phe Asn Gln Glu
245 250 255
Thr Ala Gly Trp Ser Thr Pro Arg Phe Arg Pro Met Thr Val Thr Val
260 265 270
Ser Gln Lys Asn Gly Glu Lys Leu Gly Ile Gly Val Ala Thr Asp Tyr
275 280 285
Ile Val Pro Gly Ile Pro Asp Gly Trp Pro Asp Thr Thr Ile Pro Glu
290 295 300
Glu Leu Thr Pro Ala Gly Asp Tyr Ala Ile Thr Ser Ser Asn Gly Asn
305 310 315 320
Asp Ile Thr Thr Leu Ala Asp Tyr Asn Ser Ala Asp Val Ile Lys Asn
325 330 335
Asn Thr Asn Phe Arg Gly Met Tyr Ile Cys Gly Ser Leu Gln Arg Ala
340 345 350
Trp Gly Asp Lys Lys Ile Ser Asn Thr Ala Phe Ile Thr Ile Gly Asp
355 360 365
Val Glu Gly Ser Lys Ile Lys Pro Ser Ser Val Ile Ser Gln Ala Lys
370 375 380
Ile Ala Ile Phe Gln Asp Asn His Val Asn His Asp Val Gln Thr Ser
385 390 395 400
Asp Ile Thr Tyr Ala Leu Leu Gly Tyr Thr Gly Ile Gly Glu Glu Ala
405 410 415
Ile Gly Ala Thr Arg Asp Arg Val Ala Arg Ile Ser Ile Leu Pro Glu
420 425 430
Thr Gly Ala Arg Gly Gly Asn His Pro Ile Phe Tyr Lys Asn Ser Met
435 440 445
Lys Leu Gly Tyr Val Ile Lys Ser Ile Asp Val Phe Asn Ser Gln Ile
450 455 460
Leu His Thr Ser Arg Gln Leu Ser Leu Asn Asn Tyr Leu Leu Ala Pro
465 470 475 480
Asp Ser Phe Ala Val Tyr Arg Ile Thr Asp Ser Asn Gly Ser Trp Phe
485 490 495
Asp Ile Gly Ile Asp His Asp Gly Phe Ser Phe Val Gly Val Ser Asn
500 505 510
Ile Pro Lys Leu Glu Phe Pro Leu Thr Ser Ser Tyr Met Gly Ile Gln
515 520 525
Leu Ala Lys Ile Arg Leu Ala Ser Asn Ile Arg Ser Met Met Thr Lys
530 535 540
Ile
545
<210> 3
<211> 1755
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
atgtccgatg gagctgtgca accagatggt ggtcagccag ctgtccgtaa cgagagagct 60
acaggatctg gcaacggcag cggtggtggt ggcggcggcg gaagtggagg tgttggaatt 120
agcacaggaa cattcaacaa ccagactgaa ttcaagttcc tggagaacgg ttgggtggaa 180
attaccgcca atagctctcg tttggtccac ctcaacatgc ccgaaagtga gaactacaag 240
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 (10)

1. a kind of cat infectiousness nose conjunctivitis and feline panleukopenia bigeminy vaccine, which is characterized in that contain cat cup-shaped disease Malicious virus-like particle and Feline Panleukopenia Virus virus-like particle.
2. cat infectiousness nose conjunctivitis as described in claim 1 and feline panleukopenia bigeminy vaccine, which is characterized in that In the feline calicivirus virus-like particle, the gene order of feline calicivirus virus is as shown in SEQ ID NO.1, amino acid sequence Row are as shown in SEQ ID NO.2.
3. cat infectiousness nose conjunctivitis as described in claim 1 and feline panleukopenia bigeminy vaccine, which is characterized in that In the Feline Panleukopenia Virus virus-like particle, the gene order such as SEQ ID NO.3 institutes of feline calicivirus virus Show, amino acid sequence is as shown in SEQ ID NO.4.
4. cat infectiousness nose conjunctivitis as described in any one of claims 1-3 and feline panleukopenia bigeminy vaccine, special Sign is that feline calicivirus virus-like particle and Feline Panleukopenia Virus virus-like particle are prepared by the following method It arrives：
(1) according to insect cell preference codon to feline calicivirus VP1 genes, Feline Panleukopenia Virus VP2 genes It optimizes, the feline calicivirus VP1 genes of optimization and Feline Panleukopenia Virus VP2 genes is inserted respectively into insect In fibrocyte expression vector, DH10Bac competence is converted, extracts recombinant baculovirus genome；The feline calicivirus of the optimization The nucleotide sequence of VP1 genes and Feline Panleukopenia Virus VP2 genes is respectively as shown in SEQ ID NO.1,3；
(2) by recombinant baculovirus genome transfection insect cell, rescue obtains recombinant baculovirus, is inoculated with by MOI=0.1-1 Insect cell harvests mixture after 4-5 days, i.e., obtain feline calicivirus antigen, Feline Panleukopenia Virus antigen respectively；
(3) supernatant will be harvested after feline calicivirus antigen multigelation 1 time, that is, obtains feline calicivirus virus-like particle；By cat Panleucopenia virus antigen harvests supernatant after handling cell using lysate, that is, obtains Feline Panleukopenia Virus Virus-like particle.
5. cat infectiousness nose conjunctivitis as claimed in claim 4 and feline panleukopenia bigeminy vaccine, which is characterized in that Step (3) described lysate is NaHCO3Solution, the preferably NaHCO of 25mM concentration3Solution.
6. cat infectiousness nose conjunctivitis according to any one of claims 1 to 5 and feline panleukopenia bigeminy vaccine, special Sign is, also contains preservative and adjuvant.
7. cat infectiousness nose conjunctivitis as claimed in claim 6 and feline panleukopenia bigeminy vaccine, which is characterized in that The preservative be thimerosal, final concentration of 0.005%.
8. cat infectiousness nose conjunctivitis as claimed in claim 6 and feline panleukopenia bigeminy vaccine, which is characterized in that The adjuvant be aluminum phosphate, final concentration of 0.4%.
9. the preparation side of any cat the infectiousness nose conjunctivitis and feline panleukopenia bigeminy vaccine of claim 1-8 Method, which is characterized in that including preparing feline calicivirus virus-like particle and Feline Panleukopenia Virus virus-like particle, inciting somebody to action Two kinds of virus-like particles are mixed, and add preservative and adjuvant, and before mixing, feline calicivirus virus-like particle content is not low In 4mg/ml, Feline Panleukopenia Virus hemagglutinative titer is not less than 1:214。
10. preparation method as claimed in claim 9, which is characterized in that in the feline calicivirus virus-like particle, cat cup-shaped The gene order of virus-virus is as shown in SEQ ID NO.1, and amino acid sequence is as shown in SEQ ID NO.2；
In the Feline Panleukopenia Virus virus-like particle, the gene order such as SEQ ID of feline calicivirus virus Shown in NO.3, amino acid sequence is as shown in SEQ ID NO.4.
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CN109096393A (en) * 2018-08-31 2018-12-28 广西壮族自治区兽医研究所 The preparation method of cat distemper heat cure antibody
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CN110240648A (en) * 2019-06-17 2019-09-17 长春西诺生物科技有限公司 Cat infectiousness nose conjunctivitis and feline panleukopenia bigeminy freeze-dried yolk antibody and preparation and application
CN111632137A (en) * 2020-06-19 2020-09-08 郑州爱科生物科技有限公司 Triple vaccine for feline calicivirus disease, feline infectious rhinotracheitis and feline panleukopenia as well as preparation method and application thereof
CN111632137B (en) * 2020-06-19 2022-12-13 郑州爱科生物科技有限公司 Triple vaccine for feline calicivirus disease, feline infectious rhinotracheitis and feline panleukopenia as well as preparation method and application thereof
CN113896773A (en) * 2021-10-09 2022-01-07 苏州世诺生物技术有限公司 Recombinant FCV antigen and feline calicivirus genetic engineering subunit vaccine
CN113896773B (en) * 2021-10-09 2023-12-22 苏州沃美生物有限公司 Recombinant FCV antigen and feline calicivirus genetic engineering subunit vaccine
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