CN110078802A - A kind of feline panleucopenia virus VP2 albumen and the virus-like particle of preparation - Google Patents
A kind of feline panleucopenia virus VP2 albumen and the virus-like particle of preparation Download PDFInfo
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Abstract
The present invention provides the virus-like particle of a kind of this feline panleucopenia virus VP2 albumen and preparation, and wherein the amino acid sequence of VP2 albumen is SEQ ID NO:2；The nucleotides sequence of its encoding gene is classified as SEQ ID NO:1.Another aspect of the present invention provides a kind of recombinant baculovirus, wherein including the nucleotide fragments for encoding above-mentioned VP2 albumen；Recombinant baculovirus constructed by the present invention is used to prepare the virus-like particle of feline panleucopenia virus in insect cell.Include antigen and vaccine adjuvant the present invention also provides a kind of feline panleucopenia virus subunit vaccine, used in antigen be virus-like particle prepared by the present invention.The antibody titer of cat can be improved after vaccine immunity cat prepared by the present invention, can effectively prevent the infection of feline panleucopenia virus.
Description
Technical field
The invention belongs to gene engineering technology field, relate more specifically to a kind of feline panleucopenia virus VP2 albumen, and uses elder brother
The virus-like particle (VLP) of worm cell expression system expression feline panleucopenia virus；Recombinant virus sample particle obtained is further related to, and
Its application in feline panleucopenia virus vaccine development.
Background technique
The tiny reduction disease of cat whiting is caused by feline panleucopenia virus (Feline panleukopenia virus, FPV)
A kind of impatient sexually transmitted disease of high degree in contact has very high morbidity and mortality.Infected animal shows as high fever, vomiting, abdomen
It rushes down, number of white blood cells seriously reduces and the symptoms such as enteritis.The virus infects cat family and Mustelidae many animals under field conditions (factors), such as
Tiger, leopard, lion and racoon, but it is the most susceptible with the lesser felid of figure, including mink.The disease is every year to animal farming industry
Bring heavy economic losses.
Feline panleucopenia virus category Parvoviridae parvovirus category is sub-thread minus strand linear DNA virus, no cyst membrane, to physics
Chemical factor has very strong resistance.2 kinds of structural proteins (VP1, VP2) of FPV viral genome codes and 2 kinds of non-structural proteins
(NS1, NS2), wherein VP2 structural proteins are the important antigen proteins that virus stimulation body generates protection antibody.
Baculoviral is a kind of large-scale rod-shaped togavirus, and genome is circular double stranded DNA, and size is about 80-
180kbp.Baculoviral parasitizes arthropod as pathogenic microorganism, and the host specificity with height, host mainly has
Lepidoptera Diptera and hymenopteran, not yet the baculoviral host other than discovery arthropod.In the numerous of baculoviral
In member, studying and utilize at most at present is more embedding nuclear polyhedrosis virus (AcMNPV) of autographa california.
Double-stranded DNA of the AcMNPV viral genome for virus covalently closed circular supercoil, about 130kb, genome sequence have been measured at present.
In recent years, rhabdovirus expression vector accounts for leading status with the advantage of itself on expression vector.Baculoviral table
Up to system compared with other expression systems, baculovirus expression system have it is easy to operate, highly-safe, big mesh can be accommodated
Gene, expression foreign protein effect is high, plays the role of posttranslational modification, expresses the immunogenicity, bioactivity and day of albumen
The advantages that right albumen is similar (Anderson et al., 1995；Wang et al.,2001；Ribeiro et al.,
2001)。
Currently on the market there are no the specific drug of reply FPV and effective treatment method, clinically puted prevention first with immune,
But the domestic FPV vaccine that really approval lists not yet.So needing to develop a kind of safe and effective subunit vaccine to fill out
Mend the blank in market.
Summary of the invention
The present invention provides the virus-like particle of a kind of feline panleucopenia virus VP2 albumen and preparation, to make up the prior art
It is insufficient.
Present invention firstly provides the novel VP2 albumen for screening acquisition in a kind of feline panleucopenia virus, amino acid sequence is
SEQ ID NO:2；
The present invention also optimizes above-mentioned VP2 albumen, and the amino acid sequence of the albumen after optimization is SEQ ID NO:
4；
The gene of above-mentioned VP2 albumen is encoded, nucleotides sequence is classified as SEQ ID NO:3；
The present invention also provides a kind of recombinant baculovirus, wherein including the nucleotide fragments for encoding VP2 albumen；
Recombinant baculovirus constructed by the present invention is used to prepare the virus-like particle of feline panleucopenia virus in insect cell；
Another aspect of the present invention provides a kind of virus-like particle of feline panleucopenia virus, is using the above-mentioned rod-shaped disease of recombination
After malicious infected insect cell, acquisition is collected in culture.
The insect cell is Insect cells Sf9；
Virus-like particle prepared by the present invention is used to prepare vaccine；
Include antigen and vaccine adjuvant the present invention also provides a kind of feline panleucopenia virus subunit vaccine, used in it is anti-
It originally was virus-like particle prepared by the present invention；
The antibody titer that cat can be improved after vaccine immunity cat prepared by the present invention, prevents the infection of feline panleucopenia virus.
Specific embodiment
The present invention is screened and is analyzed respectively to the cDNA complete sequence of the VP2 of feline panleucopenia virus, has chosen wherein one section
Epitope is relatively abundant, can in insect cell high efficient expression cDNA sequence, construct rod granule carrier, and in insect cell
In successfully give expression to soluble recombinant protein, the albumen automatic assembling assembly virus-like particle (VLPS) in vivo, VLP is in sky
Between conceive upper more similar and natural viral, body can be stimulated to generate cellular immunity and humoral immunity simultaneously, good immune effect, and by
Viral nucleic acid is not contained in VLP, potential viral pathogenesis gene is not present, safety is higher.In addition, utilizing insect cell-bar
The FPV-VLP yield of shape virus expression systems preparation is significantly higher than the virus of feline kidney cells culture, and production cost is substantially reduced.
The present invention is described in detail combined with specific embodiments below.Method applied by the present invention can use epidemic disease
Common method in seedling preparation field is not limited solely to the specific record of the embodiment of the present invention, those skilled in the art
The present invention can be realized with other conventional methods.
The amplification of embodiment 1:VP2 gene and sequence analysis
It collects within 2018 the tiny pathological material of disease of doubtful cat to be handled, pathological material of disease is subjected to conventional separation, identification, is determined as that cat is tiny
HA detection, HA-HI test 7log2 are carried out with swine erythrocyte.HI test is carried out with the tiny serum of known cat, as a result cross reaction
Difference；Prove that pathogenic feline panleucopenia virus strain is mutated.
1, the tiny VP2 gene of cat is expanded
According to the tiny VP2 gene order of the cat delivered in NCBI, primer is designed and synthesized, the sequence information of primer is such as
Under:
Primer1:5 '-ATGAGTGATGGAGCAGTTCAACC-3 '；
Primer2:5 '-TTAATATAATTTTCTAGGTGCTAGTTG-3 '.
The isolated tiny nucleic acid of cat is extracted as template, carries out PCR amplification purpose piece with primer primer1 and primer2
Section, through sequencing, nucleotides sequence is classified as SEQ ID NO:1, and the amino acid sequence of coding is SEQ ID NO:2.
It is compared with the tiny VP2 albumen (AAA47152.1) of cat announced in NCBI, finds multiple site hairs
Variation has been given birth to, wherein the 80th (K becomes S), the 87th (M becomes V), the 232nd (I becomes V), the 267th (F becomes C), the 564th
(N becomes R), the 568th (A becomes G) site are made a variation.The result shows that isolated strain is new parvovirus, containing new
VP2 antigen protein.
2, the shearing, optimization of VP2 gene
The antigen site of the structural protein gene VP2 of isolated strain is analyzed, its N-terminal 32aa is deleted, M is added
Amino acid, C-terminal 9aa is deleted, while codon being optimized；Sequence after optimization modification is that can give expression to antigen position well
Point, and it can be assembled into VLPS automatically.Amino acid sequence after optimization modification is SEQ ID NO:4；Encoding gene has carried out password
Son optimization, eliminates gene rare codon, so as to express VP2 albumen more preferably in baculovirus expression system；After optimization
The nucleotides sequence of gene be classified as SEQ ID NO:3.
Embodiment 2, express VP2 gene rod granule building
2.1 endonuclease reaction
2.1.1 label needs well the 1.5mL EP used to manage, and sample-adding is carried out according to the following table in 1.5mL EP pipe, mixes:
Reaction system is 50 μ L, and sample-adding is as shown in the table:
2.1.2 the 1.5mL EP pipe in step 2.2.1 is placed in 37 DEG C of thermostat water baths, water-bath 2-3h.
2.1.3 double enzyme digestion product glue recycles
Above-mentioned double digestion system is taken out, carries out agarose gel electrophoresis to recycle DNA fragmentation therein.
(1) sample collection EP pipe, adsorption column and collecting pipe have been marked.
(2) the empty EP pipe weight marked is weighed, and records numerical value.
(3) single target DNA band is carefully cut from Ago-Gel with scalpel on bale cutting instrument be put into it is dry
In net 1.5mL centrifuge tube.
(4) 600 μ L PC buffer50 DEG C water-baths are added in the 1.5mL centrifuge tube in step (3) and place 5min or so,
Centrifuge tube is mildly constantly spun upside down therebetween, to ensure that blob of viscose sufficiently dissolves.
(5) column equilibration: into adsorption column CB2,500 μ L equilibrium liquid BL, centrifugation is added in (adsorption column is placed in advance in collecting pipe)
12,000rpm, 1min outwell the waste liquid in collecting pipe, adsorption column are placed back in collecting pipe.
(6) step (5) acquired solution is added in adsorption column CB2, stand 2min, 10,000rpm, be centrifuged 30s, outwell receipts
Waste liquid in collector, then adsorption column CB2 is put into collecting pipe.
(7) 600 μ L rinsing liquid PW buffer are added into adsorption column, stand 3min, is centrifuged 10,000rpm, 30s, outwells
Adsorption column CB2 is put into collecting pipe by the waste liquid in collecting pipe.
(8) step (7) are repeated.
(9) suction attached column is centrifuged, 12,000rpm, 2min, as far as possible removing rinsing liquid.Adsorption column is placed in and is placed at room temperature for
10min thoroughly dries.
(10) adsorption column CB2 is put into collecting pipe, 50 μ LElutionbuffer is vacantly added dropwise to adsorbed film middle position
(65 DEG C of preheatings), stands 3min, is centrifuged 12,000rpm, 2min.
(11) from centrifuge tube in step (10) is taken out in centrifuge, intermediate adsorption column CB2 is abandoned, centrifuge tube lid is covered
Son retains the DNA sample in centrifuge tube.
(12) DNA sample in step 11 is placed in 4 DEG C of preservations, prepares agarose gel electrophoresis identification glue and recycles DNA piece
Section.
2.2 connection reactions
(1) label needs the 0.2mL centrifuge tube used.
(2) it is loaded in marking complete 0.2mL pipe according to 20 μ L reaction systems of following table:
Wherein insertion DNA fragmentation is the nucleotide fragments that the sequence after optimization is SEQ ID NO:3 and the SEQ being not optimised
The nucleotide fragments of ID NO:1.
(3) it after completing sample-adding, is gently blown and beaten with pipettor and mixes each component several times.
(4) 0.2mL centrifuge tube is placed in 37 DEG C of reaction 30min, to after the reaction was completed, reaction tube is placed in ice-water bath immediately
Middle cooling 5min.
(5) reaction product of step (4) can directly carry out transformation experiment, can also be stored in -20 DEG C, thaw turn when needed
Change.
2.3 conversion reaction
(1) 10 μ L connection reaction solutions are rapidly joined in 100 μ L competent cells, and blows and beats mixing, ice bath 30min.
(2) after the completion of step (1), sample cell is taken out, is placed in 42 DEG C of water-bath 100s, immediately after ice bath 2min.
(3) it after the completion of step (2), takes out sample cell and 600 μ L liquid LB is added into sample cell in superclean bench
Culture medium, is then placed in 37 DEG C of constant-temperature tables for sample cell, and 220rpm cultivates 1h.
(4) preparation conversion plate, according to plasmid resistance preparation conversion LB resistant panel.
(5) coated plate: taking out sample cell in step (3), and room temperature is centrifuged 8,000rpm, 2min, removes 600 μ L supernatant fluids,
The thallus of bottom of the tube is resuspended in remaining supernatant, and the bacterium solution of resuspension is put into corresponding conversion plate center, will be converted with bacteria stick is applied
The bacterium solution of plate center is uniformly spread out.
(6) step (5) plate is just being placed in biochemical constant incubator, after 37 DEG C of culture 1h, will conversion plate be inverted into
Row culture 15h.
(7) conversion results are observed and recorded.
2.4 plasmid extractions and PCR are identified
2.4.1 plasmid extraction
(1) with 10 μ L liquid transfer gun heads from conversion plate in picking monoclonal to the 5ml resistance of benzyl containing ammonia LB liquid medium
In, 37 DEG C, 220rpm shakes bacterium and stays overnight.
(2) bacterium solution is drawn into 1.5mL EP pipe, and room temperature centrifugation, 12,000rpm, 2min abandon supernatant.
(3) 250 μ L plasmids are added in the EP pipe in step (2) and extract reagent P1buffer, thorough suspension thalline.
(4) 250 μ L P2buffer are added into step (3) solution, immediately mildly 5-10 mixing of reverse centrifuge tube.Room
Temperature stands 2-4min.
(5) 350 μ L P3buffer are added into step (4) solution, immediately mildly 5-10 mixing of reverse centrifuge tube.Room
Temperature stands 2-4min.
(6) by step (5) solution, room temperature centrifugation, 14,000rpm, 10min.
(7) supernatant solution in step (6) is moved into adsorption column center, room temperature centrifugation, 12,000rpm, 30s outwell collection
Liquid in pipe.
(8) 500 μ L Buffer DW1 are added to adsorption column center, room temperature centrifugation, 12,000rpm, 30s outwell collecting pipe
Middle liquid.
(9) 500 μ L wash solution are added to adsorption column center, room temperature centrifugation, 12,000rpm, 30s outwell receipts
Liquid in collector.It is repeated once.
(10) suction attached column, room temperature centrifugation, 12,000rpm, 2min.
(11) adsorption column is put into a clean 1.5ml centrifuge tube, 30 μ L is added to absorption center membrane
Elutionbuffer is stored at room temperature 5min, and room temperature is centrifuged, 12,000rpm, 2min, DNA solution in 4 DEG C of preservation pipes.
2.4.2 PCR is identified
(1) PCR pipe for needing to use well is marked, sample-adding is carried out according to the following table, mixes, reaction system is 25 μ L:
(2) PCR amplification program:
(3) it is sequenced: sending sequencing company to be sequenced the positive plasmid of pcr identification, determine positive plasmid.
2.5 conversion
The positive plasmid of sequencing is converted into DH10bac competent cell, the same 2.3. of method
2.6 picking rod granules and PCR are identified
2.6.1 picking rod granule
(1) from 2.5 conversion plate in, with 10 μ L liquid transfer gun heads from conversion plate in picking white monoclonal colonies to
5ml containing kalamycin resistance, tetracyclin resistance, gentamicin resistance LB liquid medium in, 37 DEG C, 220rpm shakes bacterium mistake
Night.
(2) bacterium solution is drawn into 1.5mL EP pipe, and room temperature centrifugation, 12,000rpm, 2min abandon supernatant.
(3) 250 μ L plasmids are added in the EP pipe in step (2) and extract reagent P1buffer, thorough suspension thalline.
(4) 250 μ L P2buffer are added into step (3) solution, immediately mildly 5-10 mixing of reverse centrifuge tube.Room
Temperature stands 2-4min.
(5) 350 μ L P3buffer are added into step (4) solution, immediately mildly 5-10 mixing of reverse centrifuge tube.Room
Temperature stands 2-4min.
(6) by step (5) solution, room temperature centrifugation, 14,000rpm, 10min.
(7) supernatant solution in step (6) is moved into adsorption column center, room temperature centrifugation, 12,000rpm, 30s outwell collection
Liquid in pipe.
(8) 500 μ L Buffer DW1 are added to adsorption column center, room temperature centrifugation, 12,000rpm, 30s outwell collecting pipe
Middle liquid.
(9) 500 μ L wash solution are added to adsorption column center, room temperature centrifugation, 12,000rpm, 30s outwell receipts
Liquid in collector.It is repeated once.
(10) suction attached column, room temperature centrifugation, 12,000rpm, 2min.
(11) adsorption column is put into a clean 1.5ml centrifuge tube, 30 μ L is added to absorption center membrane
Elutionbuffer is stored at room temperature 5min, and room temperature is centrifuged, 12,000rpm, 2min, DNA solution in 4 DEG C of preservation pipes.
2.6.2 PCR identifies the DNA that will be extracted in 2.6.1, carries out pcr identification, identifies that positive plasmid serves marine growth
Engineering Co., Ltd is sequenced, and the positive is sequenced is used for SF9 cell transfecting.
Embodiment 3SF9 cell transfecting
(1) prepare: Biohazard Safety Equipment ultraviolet sterilization 30min；TNM-FH culture solution is placed in 27 DEG C of water-baths and is preheated to 27 DEG C.
(2) 2 μ g recombinant DNAs are added in 100 μ l serum-frees and dual anti-TNM-FH culture solution, are mixed.By 9 μ l
Cellfectin Reagent is added in 100 μ l serum-frees and dual anti-TNM-FH culture solution, is mixed.By liposome and recombination
DNA mixing, the static 40min of room temperature.
(3) 6 orifice plates cell is taken out from 27 DEG C of incubators, is discarded supernatant culture medium, is washed with the TNM-FH culture solution of pre-temperature
Cell three times, and discards TNM-FH culture solution.
(4) the TNM-FH culture solution of 10% fetal calf serum of 2ml is added in each cell hole.
(5) mixture of recombinant DNA and liposome is gently added in the cell of every hole, is mixed gently, in 27 DEG C of conditions
5~6h of lower static gas wave refrigerator.
(6) liquid in hole is discarded, is added the complete TNM-FH culture solution of 2ml (containing dual anti-and 10% serum), 27 DEG C
Under the conditions of static gas wave refrigerator 5~6 days.
(7) to cellular swelling, volume becomes larger, and after falling off, collects supernatant, labeled as P1 for recombinant baculovirus, name
For FPV-VLP-P1.
(8) the Sf9 cell newly cultivated is infected with FPV-VLP-P1, improves recombinant baculovirus content, was inoculated with for 2 generations repeatedly
Afterwards, cell supernatant is collected, 4 DEG C or -80 DEG C save backup.
4 protein purification of embodiment and detection
Expression and identification of the 4.1 recombination vp2 albumen in Insect cells Sf9:
By recombinant baculovirus FPV-VLP-P1 infected insect cell Sf9,27 DEG C of culture 72h, while with uninfecting virus
27 DEG C of culture 48h of normal Insect cells Sf9 as control, harvest cell, culture supernatant frozen spare.Cell pH7.4
PBS washing after, be added 1 × SDS-PAGE sample loading buffer [50mM Tris-HCl (pH6.8), 100mM
Dithiothreitol (DTT), 2%SDS, 0.05%Bromophemol blue, 10%Glycerol], 5min is boiled, is used
12% separation gel, 5% concentration glue carry out polyacrylamide gel electrophoresis, 100 volts of about 2.5h, coomassie brilliant blue R250 dyeing.
The result shows that the recombinant baculovirus for the VP2 gene being not optimised expression quantity in insect cell can't detect, and it is excellent
There is VP2 protein expression in the Insect cells Sf9 lysate of the VP2 recombinate shape virus infection of change.
The chromatographic purifying of 4.2 recombinant VP 2 albumen:
Recombinant baculovirus FPV-VLP is inoculated with Insect cells Sf9, after 27 DEG C are cultivated 72 hours, it is heavy that cell is collected by centrifugation
Form sediment, appropriate physiological saline be added in cell precipitation, cell precipitation is resuspended, through ultrasonic treatment cell, with 4 DEG C of 1000r/min from
Heart 10min, collecting supernatant is, carries out the VLP protein liquid that chromatographic purifying obtains VP2 albumen with Ni2+ column.
4.3 Western blot:
Albumen prepared by the tiny totivirus of cat and 4.2 is subjected to SDS-PAGE simultaneously, is transferred using 20 volts of semidry method
Destination protein band is transferred to pvdf membrane by 30min, and transfer film is closed overnight with confining liquid, and PBST is washed 3 times, 1: 500 dilution
37 DEG C of feline panleucopenia virus positive serum effect 1.5h, PBST is washed 3 times, with the rabbit-anti cat enzyme mark of 1: 2000 diluted HRP label
37 DEG C of antibody effect 1.5h, PBST washing 3 times, substrate solution acts on 5min, develops the color in chemiDOC, as a result totivirus item
Band is very weak, and the nucleotides sequence after optimizing is classified as the VP2 albumen one that the gene of SEQ ID NO:3 is expressed in Insect cells Sf9
Band is very bright, and the vp2 protein immunogenic after illustrating optimization is more preferable.
The albumen of 4.2 preparations is carried out HA detection with Swine serum by 4.4 HA detection, as a result the HA-HI test difference of FPV-VLP
It is 11log2 higher than totivirus for 18log2.Illustrate that the VP2 protein immunogenic of expression is more preferable.
The feline panleucopenia virus of screening is concentrated 100 times.By the totivirus liquid of concentration and recombinant baculovirus in SF9 cell
Expression product carry out Electronic Speculum after negative staining simultaneously and observe.Results expression product is assembled into many virus-like particles, size 20nm,
And the totivirus liquid for being concentrated 100 times only has micro particle.
Embodiment 5: the preparation of vaccine
(1) prepared by water phase: VLP protein liquid and sterile saline proper proportion that the step 4.2 of embodiment 2 purifies are mixed
It closes, HA is not less than 6log2 in water phase；
(2) the gel01 adjuvant for 15% volume of phase of fetching water is emulsified with water phase, and with 1000r/min, emulsification is made for 5 minutes
Subunit vaccine.
The safety of vaccine and efficacy test
1) safety verification:
The susceptible cat (the tiny neutralizing antibody of cat is not higher than 1:4) 20 of health is taken, is randomly divided into 2 groups.One group is exempted from for vaccine
Every group 10, the subunit vaccine of the invention of 5 parts (5ml) is subcutaneously injected in epidemic disease group；Another 10 not immune to compare.It is immune
After be observed continuously 14, whether record body temperature, the state of mind, appetite, excrement situation of change and injection site have adverse reaction.In
It tests to cut open last day and kill, check that whether there is or not pathological changes for injection site.Within the observation period, immune group and control group cat body temperature,
The state of mind, appetite, excrement are normal, and injection site does not go out without the adverse reactions such as swelling and inflammation, injection site dissect inspection
Existing anomalous variation, vaccine inoculation group cat growing state and control group difference is not significant.The above results show subunit's epidemic disease of preparation
Seedling meets safety detection requirement.
2) efficacy test
The susceptible cat (the tiny neutralizing antibody of cat is not higher than 1:4) 30 of health is taken, is randomly divided into 3 groups, every group 10.One group
It is immunized homemade subunit vaccine, one group of immune tiny vaccine of market cat, one group not immune to compare.21 days after exempting from, blood sampling inspection
Survey HI.And feline panleucopenia virus strong virus attack is carried out simultaneously, take orally 8ml (106.5TCID50/ ml) and intraperitoneal injection 2ml, i.e., every is attacked
Toxic dose is 107.5TCID50.The results show that the HI average value of subunit's immune group is 1:891.4, the HI of market vaccine immunity group
Average value is 1:111.4, and control group HI < 2log2, illustrate that the tiny subunit vaccine of cat can generate good antibody, and compare
Market vaccine titre is much higher.Attack poison the result shows that, subunit vaccine immune group 10/10 protect, market vaccine immunity group 8/10
Protection, and control group 10/10 is fallen ill.Illustrate that the tiny subunit vaccine of cat can resist the virulent attack of feline panleucopenia virus well,
Protecting effect is good.See Table 1 for details.
The efficacy test result of the tiny subunit vaccine of 1 cat of table
Note: antibody is the geometric average value for antibody of this group of cat.
The above results show that the subunit vaccine of virus-like particle preparation can prevent the infection of feline panleucopenia virus well,
And the antibody that subunit vaccine generates is much higher than market vaccine group, so having a good application prospect.
To sum up, the present invention is by the VP2 albumen being not optimised and the vp2 albumen of optimization while building is used to express antigen protein
Rod granule carrier, as a result not optimized overall length vp2 gene cannot express in insect cell, and the vp2 albumen optimized is in elder brother
Soluble recombinant protein is successfully given expression in worm cell.Totivirus strain, the vp2 supernatant of expression of being concentrated 100 times is same
The observation of Shi Jinhang Electronic Speculum, the totivirus for being as a result concentrated 100 times only has a small amount of VLP, and the vp2 supernatant of not diluted expression is in body
Interior automatic assembling assembly virus-like particle (VLPS), and there are many virion amount.VLP is more similar with natural disease on Space Idea
Poison can stimulate body to generate cellular immunity and humoral immunity simultaneously, good immune effect, and since VLP does not contain viral nucleic acid, no
There are potential viral pathogenesis gene, safety is higher.
Sequence table
<110>YEBIO Bioengineering Co., Ltd of Qingdao
<120>a kind of feline panleucopenia virus VP2 albumen and the virus-like particle of preparation
<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 (10)
1. a kind of feline panleucopenia virus VP2 albumen, which is characterized in that the amino acid sequence of the VP2 albumen is SEQ ID NO:
2。
2. feline panleucopenia virus VP2 albumen as described in claim 1, which is characterized in that the amino acid sequence of the VP2 albumen
For SEQ ID NO:4.
3. a kind of gene, which is characterized in that the gene is tiny for encoding cat described in claim 1 or claim 2
Virus VP 2 albumen.
4. gene as claimed in claim 3, which is characterized in that for encoding feline panleucopenia virus VP2 egg described in claim 1
White gene, nucleotides sequence are classified as SEQ ID NO:1.
5. gene as claimed in claim 3, which is characterized in that for encoding feline panleucopenia virus VP2 egg as claimed in claim 2
White gene, nucleotides sequence are classified as SEQ ID NO:3.
6. a kind of recombinant baculovirus, which is characterized in that include coding claim 1 or power in the recombinant baculovirus
Benefit require 2 described in feline panleucopenia virus VP2 albumen nucleotide fragments.
7. recombinant baculovirus as claimed in claim 6, which is characterized in that the sequence of the nucleotide fragments is SEQ ID
NO:1 or SEQ ID NO:3.
8. recombinant baculovirus described in claim 6 or 7 is prepared in insect cell in the virus-like particle of feline panleucopenia virus
Application.
9. a kind of virus-like particle of feline panleucopenia virus, which is characterized in that the virus-like particle of the feline panleucopenia virus is to make
After the recombinate shape virus infection insect cell described in claim 6 or 7, acquisition is collected in culture.
10. a kind of feline panleucopenia virus subunit vaccine, which is characterized in that the subunit vaccine includes antigen and vaccine assistant
Agent, used in antigen be feline panleucopenia virus as claimed in claim 9 virus-like particle.
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| CN111876391A (en) * | 2020-07-23 | 2020-11-03 | 中国农业科学院北京畜牧兽医研究所 | Feline panleukopenia virus FPV BJ05 strain and application thereof |
| CN112661818A (en) * | 2020-12-28 | 2021-04-16 | 广东省农业科学院动物卫生研究所 | Canine and cat parvovirus VP2350-420Protein and preparation method and application thereof |
| CN113337478A (en) * | 2021-06-02 | 2021-09-03 | 华中农业大学 | Cat parvovirus strain and application thereof |
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| CN114703204A (en) * | 2022-02-24 | 2022-07-05 | 深圳赫兹生命科学技术有限公司 | Feline parvovirus VP2 protein and resulting self-assembled virus-like particles |
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