CN104195119B - Virus-like particle and vaccine of a kind of infectivity resistant spleen and kidney necrosis virus and preparation method thereof - Google Patents
Virus-like particle and vaccine of a kind of infectivity resistant spleen and kidney necrosis virus and preparation method thereof Download PDFInfo
- Publication number
- CN104195119B CN104195119B CN201410381379.5A CN201410381379A CN104195119B CN 104195119 B CN104195119 B CN 104195119B CN 201410381379 A CN201410381379 A CN 201410381379A CN 104195119 B CN104195119 B CN 104195119B
- Authority
- CN
- China
- Prior art keywords
- virus
- particle
- spleen
- kidney necrosis
- orf24
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention discloses a kind of virus-like particle of infectivity resistant spleen and kidney necrosis virus and vaccine and preparation method thereof, the virus-like particle of the infectivity resistant spleen and kidney necrosis virus is made up of the Influenza matrix albumen M1 and fusion protein HA 24 expressed simultaneously；The fusion protein HA 24 is built-up by Influenza virus HA protein and fishes infectious spleen and kidney necrosis virus ORF24, the nucleotide sequence such as SEQ ID NO of the fusion protein HA 24:3rd, shown in 5,7,9 or 11.The preparation method of the virus-like particle of the infectivity resistant spleen and kidney necrosis virus, comprises the following steps：Prepare fusion HA 24；Prepare restructuring baculovirus shuttle vector rBacmid；Prepare restructuring insect baculovirus；Obtain the virus-like particle of expression of influenza viral matrix protein M1 and fusion protein HA 24 simultaneously.The vaccine includes the virus-like particle described in any of the above-described.Hybrid virus sample particle vaccines of the present invention can cause body immune system to produce strong type response, and immunity is strong, duration length, can prevent fishes infectious spleen and kidney necrosis virus disease, and very safe.
Description
Technical field
The present invention relates to virus-like particle field, more particularly to a kind of virus-like particle of infectivity resistant spleen and kidney necrosis virus
With vaccine and preparation method thereof.
Background technology
Infectious spleen and kidney necrosis virus (Infectious Spleen and Kidney Necrosis Virus, ISKNV)
Belong to Iridoviridae, a variety of fish can be made to obtain infectious spleen renal necrosis disease, cause mandarin fish, lithosporic, egg-shaped pompano etc. a large amount of
Death, it is dead up to more than 90% in 10 days after infection, very big economic loss is brought to cultured fishes.
It is vaccine immunity to control the sick best method at present, but there is no active drug both at home and abroad.Inactivated virus vaccine
Effect it is undesirable, protective rate is usually no more than 50%.Japan, South Korea have been reported that similar to ISKNV using DNA vaccination control
Other irido virus, and obtain certain effect.But the use of DNA vaccination has danger, it is impossible to is directly entered the food of the mankind
Thing chain.The structural proteins for having scholar to use Bacillus coli expression ISKNV, prepare subunit vaccine, obtain certain effect, but
Requirement of the industry production to vaccine is not reached.Therefore, the immune efficacy of ISKNV recombinant vaccines is improved, develops new vaccine
With urgency.
Scientific research proves that virus-like particle (VLPs) has good immune efficacy and application prospect.VLPs is to contain certain
One or more major structural proteins of kind virus, the sky not comprising viral nucleic acid being assembled into automatically in expression system in vitro
Heart particle.The surface membrane protein HA of influenza virus is the main evocator for causing body to produce immune response, and matrix prote m1
It is the main body to form virus coat, can act also as the evocator of tectotype immune response.Relevant research has shown that, matrix prote m1
Correct expression, be ensure virus coat formed important step.M1 albumen and this 2 major structural proteins of HA are expressed just simultaneously
It can be assembled into automatically hollow without virus genomic virus-like particle.VLPs form and size and real virion
Son is same or similar, so can effectively induce body immune system to produce immunoprotection reaction, shows as a kind of new generation vaccine
Show good application prospect.
The content of the invention
In view of this, it is necessary to the problems such as above-mentioned vaccine potency difference, there is provided a kind of infectious spleen and kidney necrosis virus
Virus-like particle and vaccine and preparation method thereof.
To achieve these goals, the present invention adopts the following technical scheme that：
A kind of virus-like particle of infectivity resistant spleen and kidney necrosis virus, by the Influenza matrix albumen M1 that expresses simultaneously and
Fusion protein HA-24 is formed；The fusion protein HA-24 is by Influenza virus HA protein and fishes infectious spleen and kidney necrosis virus
ORF24 is built-up, the nucleotide sequence such as SEQ ID NO of the fusion protein HA-24:3rd, shown in 5,7,9 or 11.
A kind of preparation method of the virus-like particle of infectivity resistant spleen and kidney necrosis virus, comprises the following steps：
1. infectious spleen and kidney necrosis virus major structural protein ORF24 genes are replaced one of influenza virus HA genes
Point, the two forms fusion HA-24, fusion HA-24 nucleotide sequence such as SEQ ID NO:3rd, shown in 5,7,9 or 11；
2. the matrix prote m1 gene of influenza virus and fusion protein HA-24 genes are inserted into insect baculovirus
On pFastBac-Dual carriers, identify and be sequenced through digestion, filter out positive recombinant vector, and convert and worn containing baculoviral
The DH10Bac competent cells of shuttle carrier, obtain recombinant baculovirus shuttle vector rBacmid；
3. utilizing lipofectamine, the recombinant baculovirus shuttle vector rBacmid containing foreign gene is transfected into
In host insect cell strain sf-9, recombinant baculovirus is obtained；
4. cultivating the host insect cell transfected by recombinant baculovirus, enable its efficiently expression of influenza virus
Structural proteins M1 and fusion protein HA-24, and be assembled into automatically while expression of influenza viral matrix protein M1 and fusion protein
HA-24 virus-like particle.
Preferably, step 1. in HA-24 genes be by infectious spleen and kidney necrosis virus ORF24 and influenza virus HA genes
Fusion is first synthesized, is then formed with PCR amplifications.
Preferably, step 3. at 27 DEG C cultivate 4-5 days by middle sf-9 cells, collects cell culture supernatant, then infect new
Sf-9 cells, the recombinant baculovirus of high titre after being amplified.
Preferably, step 4. in by recombinant baculovirus transfection host insect cell 27 DEG C cultivate 3 days, expression
Influenza virus M1 albumen and fusion protein HA-24, be assembled into VLPs automatically.
A kind of fishes infectious spleen and kidney necrosis virus gene engineering epidemic disease of the virus-like particle comprising described in any of the above-described
Seedling.
Virus-like particle and vaccine of infectivity resistant spleen and kidney necrosis virus provided by the invention and preparation method thereof have bright
The advantages of aobvious and effect：
(1) VLPs vaccines can cause body immune system to produce strong type response, and immunity is strong, duration length, can prevent
Fishes infectious spleen and kidney necrosis virus disease.
(2) virus-like particle is free of viral genome, and the VLPs of this hollow shell structure would not be sent out in immune animal body
Sick virus gene and host chromosome gene integration, and whole production process does not contact infectious virus living, therefore
It is very safe.
(3) compare with general genetic engineering subunit vaccine, it is substantial amounts of to infect in this new virus-like particle
Property spleen and kidney necrosis virus structural proteins be located at particle surface, body can be stimulated to produce more preferable immune response, immune effect is more preferable.
Brief description of the drawings
Fig. 1 is pcr amplification product electrophoretogram in the embodiment of the present invention 1.Wherein, swimming lane 1 is marker, and swimming lane 2 is HA-
24, swimming lane 3 is M1, and swimming lane 4 is blank control.
Fig. 2 is that M1 and HA-24 genes are expressed in the insect cell sf-9 for suspending culture in the embodiment of the present invention 1
Western blots scheme.Wherein, swimming lane 1 is marker, and swimming lane 2 is negative control, and swimming lane 3 is experimental group sample.
Fig. 3 is the Western blots figures of the virus-like particle in the embodiment of the present invention 1 after purification.Wherein, swimming lane 1 is
Marker, swimming lane 2 are negative control, and swimming lane 3 is experimental group sample.
Embodiment
The present invention is by influenza virus VLPs platforms, by the major structural protein of fishes infectious spleen and kidney necrosis virus
ORF24 and influenza virus HA a part, which merges, forms a new fusion protein HA-24, then in insect cell while table
Up to HA-24 and M1 albumen, VLPs is assembled into automatically.Using expressed fusion protein HA-24 VLPs as antigen, a kind of prevention is prepared
The novel gene engineered vaccine of fishes infectious spleen and kidney necrosis virus.The new fishes infectious spleen and kidney necrosis virus base of the present invention
Because engineered vaccine can also include adjuvant.
The present invention is passed Influenza virus HA protein and fish based on Bac-to-Bac insect baculovirus expression systems
Metachromia spleen and kidney necrosis virus ORF24 is built into fusion protein HA-24, while expression of influenza viral matrix protein M1 and fusion protein
The VLP ghost particles of similar influenza virus are constructed in HA-24, the two common automatic assembling.
First, fishes infectious spleen and kidney necrosis virus ORF24 genes are replaced to a part for influenza virus HA genes, the two
Fusion (HA-24) is formed, ORF24 genes are located at fusion HA-24 5 ' ends, replace the 5 ' of the HA genes of equal length
Terminal sequence, to ensure that fusion H A-24 length and HA mrna lengths are roughly equal.
Then, according to the method for forming influenza virus-like particles, by the matrix prote m1 of influenza virus and fusion protein HA-
24 DNA segment, is implemented in the vector plasmid of insect baculovirus, and it is recombined into the inhereditary material of insect baculovirus
In DNA, these foreign proteins are expressed using host insect cell, are assembled into the virus-like without influenza virus inhereditary material automatically
Particle, virus-like particle will be released into cell culture fluid after being formed.The VLPs so formed has infectious spleen renal necrosis sick
Malicious ORF24 albumen.
The virus-like particle of the infectivity resistant spleen and kidney necrosis virus of embodiment 1
1st, M1 genes and HA-24 amplification
1.1 fusion HA-24 synthesis
Infectious spleen and kidney necrosis virus major structural protein gene ORF24 is replaced into H1N1 Influenza virus HA protein genes
A part, the two forms fusion (HA-24), and ORF24 genes are located at fusion HA-24 5 ' ends, replace equivalent length
HA genes 5 ' terminal sequences, to ensure that fusion HA-24 length and HA mrna lengths are roughly equal.In fusion sequence
The length of ORF24 and HA sequences can do corresponding adjustment.The nucleotide sequence of fusion such as SEQ ID NO:Shown in 3, its amino
Acid sequence such as SEQ ID NO:Shown in 4.SEQ ID NO:3 1-936 bit bases be ORF24 gene, 937-1701 positions
Base is HA gene；SEQ ID NO:4 1-312 amino acids are ORF24 amino acid, and 313-566 bit bases are
HA amino acid.Fusion is artificial synthesized according to its nucleotide sequence.
1.2 Influenza Virus RNAs extract and RT-PCR
The use of GibcoBRL company's T RIzol LS Reagent RNA extracts kits is pressed in the extraction of Influenza Virus RNA
Specification (method) is carried out.250 μ L influenza virus H1N1 hypotypes virus strain infection's allantoic fluids and 750 μ L TRIzol LS are taken respectively,
Add in 1.5 mL microcentrifugal tubes, blown and beaten with suction pipe and fully mixed, room temperature places 10min；200 μ L chloroforms are added, are acutely shaken
15s is swung, after being stored at room temperature 5 minutes, 12000rpm centrifuges 15min at 4 DEG C；Supernatant is taken in a new sterilizing 1.5mL centrifuge tubes,
500 μ L isopropanols are added, are fully mixed, room temperature places 10min, and 12000r/m centrifuges 10min at 4 DEG C；Incline supernatant, precipitation
The μ L of ethanol 750 of middle addition 70%, gently mix, washed once, and 12000r/m centrifuges 15min at 4 DEG C, supernatant discarding, air-dries；
The tri-distilled water lytic virus RNA (precipitation) without RNase of 10 μ L DEPC water process is added, is directly used in RT-PCR or -80 DEG C
Save backup.
RT-PCR is carried out with reference to the operation instruction of TaKaRa AMV reverse transcriptase, is separately added into 20 μ L reaction systems
Following components：RNA：3μL；5×RT buffer：4μL；dNTPs：4μL；RNase inhibitor：0.5μL；Primer UP：1μL；Primer
DN:1μL；AMV：2μL；DEPC water：Mend to 20 μ L.After mixing, room temperature places 10min, 42 DEG C of insulation 1h, ice bath 2min, RT production
Thing is directly used in PCR amplifications or -20 DEG C of preservations.
1.3HA-24 expanded with the PCR of M1 genes
According to M1 gene orders (SEQ ID NO:1, its protein sequence is SEQ ID NO:2) 1 pair of primer of design, is used for
M1 genes are expanded, the restriction enzyme site that its both ends adds Sal I and Hind III respectively is located at PPHUnder promoter, this 2 primer sequences
(SEQ ID NO:13-14) it is respectively：
M1SalⅠ：5’-GCCGTCGACATGAGTCTTCTAACCGAG-3’
M1HindⅢ：5’-GCCAAGCTTTCACTTGAATCGTTG-3’
According to fusion HA-24 sequence (SEQ ID NO:3) 1 pair of primer of design, for expanding fusion HA-
24, its both ends adds Xho I and Sph I restriction enzyme sites respectively, under P10 promoters, primer sequence (SEQ ID NO:15-
16) it is as follows：
H Xhol I：5'-GG CTCGATATGGATAAGTATGTGCTCAG-3'
H Sph I：5'-ACAT GCATGCTTAAATACATATTCTGCACT-3'
Using the respective specific primer of M1 and HA-24, the DNA segment of reverse transcription product or synthesis is directly used as PCR and expanded
Increase the template of M1 and HA-24 genes.PCR reaction conditions are 94 DEG C of pre-degenerations 3min, 94 DEG C of denaturation 40S, 56 DEG C of 90s that anneal, 72
DEG C extension 90s, circulates 30 times, finally extends 10min, PCR primer with 1.5% Ago-Gel (ethidium bromide containing 0.5ug/mL,
EB) electrophoresis detection.Electrophoresis result is as shown in figure 1, the length about 0.75Kb of the M1 genes of PCR amplifications, fusion HA-24 length
Degree is about 1.7Kb.
All PCR primer samples obtain M1 the and HA-24 gene DNA fragments of purifying after running gel extracts, through limit
After I/Hind of property restriction endonuclease Sal processed III (digestion M1 fragments), Xho I and 37 DEG C of Sph I (digestion HA-24 segments) digest respectively, then
It is further purified, in case construction recombination plasmid is used.Concrete operations are as follows：Prepare 1% Ago-Gel bromination containing 0.5ug/mL second
Ingot, by all PCR samples add gel sample cell in, set voltage 100V, electrophoresis time 40min under long-wave ultra violet lamp,
The gel strips containing sample band are cut, are fitted into small plastic centrifuge tube.Said with reference to glue reclaim kit (Qiagen Products)
Bright book, extracting and purifying M1 and HA-24 gene DNA fragment.After 37 DEG C of restriction enzyme is digested overnight, with glue reclaim kit
(Qiagen Products), are instructed to specifications, centrifuge post, M1 the and HA-24 fragments after digestions are reclaimed in purifying.
2nd, the baculovirus expression plasmid and the synthesis restructuring in insect cell of construction expression M1 and HA-24 genes
Insect baculovirus
2.1 construction expression M1 recombinant plasmid
Insect baculovirus plasmid PFastBac-dual (Invitrogen Products) through restriction enzyme Sal I/
After III 37 DEG C of Hind digestions 3 hours, with the plasmid PFastBac-dual after glue reclaim kit recovery purifying digestion.In T4DNA
In the presence of ligase, the M1DNA fragments after plasmid and digestion after digestion are stayed overnight in 16 DEG C of connections.Reaction system is as follows：10
DNA fragmentation 3mL, the μ L of digestion PFastBac-dual plasmids recovery product 1 of × T4 connection buffer solution 1mL, M1 digestion recovery,
T4DNA ligases 1 μ L, ddH2O are mended to 10 μ L.Connection product is transduceed in Top10 competent cells using heat shock procedures
It is added in a small plastic centrifuge tube, tubule is placed in 30min on ice after being gently mixed, is transferred to heat shock in 42 DEG C of water-baths
90s, put back to rapidly 5 minutes on ice, add 200 μ LLB nutrient solutions thereto.37 DEG C of shaking table cultures 1 hour.100 μ L bacterium solutions are taken to apply
It is distributed on LB solid mediums (containing two kinds of antibiotic of ammonia benzyl and gentamicin), 37 DEG C of culture 16h, picking is positive from flat board
Colonies, bacterium solution PCR is carried out, with I/Hind of Sal III single or double digestion identification is carried out after extracting plasmid.After determined dna sequence,
Obtain recombinant plasmid PFastBac-dualM1.
The recombinant plasmid of 2.2 construction expression HA-24 and M1 genes
Recombinant plasmid PFastBac-dualM1 is after the digestions of I/Nco of restriction enzyme Xho I, in T4DNA ligases
Under effect, the lower section of P10 promoters will be inserted into by the HA-24 gene DNA fragments after same endonuclease digestion, this connection production
Thing is transduceed in Top10 competent cells after through hot body gram method, cultivates, extracts the recombinant plasmid of several positive colony bacterium colonies
After the digestion identifications of I/Nco of DNA, bacterium solution PCR and Xho I and DNA sequence dna sequencing determination are errorless, the plasmids of two degree of restructuring are obtained.DNA
After sequencing, as required recombinant baculovirus expression plasmid PFastBac-dual-M1-HA-24.
The synthesis and extraction of 2.3 recombinant baculovirus genomes
The restructuring PFastBac-dual-M1-HA-24 plasmid transductions of purifying are entered into special E.coli competent cell strains
In DH10Bac cells (U.S.'s Invitrogen Products).DH10Bac cells contain a special macromolecular plasmid
Bacmid, it contains insect baculovirus AcMNPV full gene group.Once the expression plasmid of restructuring is integrated into big point
Behind sub- plasmid Bacmid special site, screening and IPIG through 3 kinds of antibiotic (gentamicin, tetracycline and kanamycins)
Induction and X-Gal substrate reactions carry out blue hickie screening, and positive colony bacterium colony is white, and the wild bacterium colony of non-recombinant is in orchid
Color.The laboratory manual that experiment condition provides according to Invitrogen companies is instructed and set.The positive colony selected is placed in 3mL
LB nutrient solutions in (contain above-mentioned 3 kinds of antibiotic), through 37 DEG C of shaking table cultures 24 hours, the macromolecular matter indicated according to laboratory manual
Grain Bacmid mini prep methods, restructuring macromolecular plasmid Bacmid of the extraction purification with M1 genes and HA-24 genes.
The preparation of 2.4 recombinant baculovirus
Insect cell line sf-9 cells (U.S.'s Invitrogen Products) are incubated at the sf-900II elder brothers of serum-free
In worm cell culture fluid (Invitrogen Products), temperature setting is 27 DEG C, the reality provided according to Invitrogen companies
Test handbook, cell density 5x105Individual/mL, using lipofection, by the restructuring macromolecular plasmid Bacmid1 μ g of purifying
200 μ L serum-free is mixed into without dual anti-culture medium with the μ L of lipid soln cellfectin (Invitrogen Products) 6
In, sf-9 cells are transfected, after 27 DEG C are cultivated 4 to 5 days, collect cell culture supernatant, 3000r/m is centrifuged 10 minutes and collected supernatant
Liquid removes cell fragment, obtains the recombinant baculovirus of low titre, then the sf-9 cells newly cultivated with the infection of this supernatant
(every milliliter of supernatant infects 4x106Individual sf-9 cells)；Cell culture supernatant, as required amplification culture are collected after 3 days
High concentration recombinant baculovirus afterwards, is named as Bac-M1-HA-24.
Plaque experiment is carried out to the recombinant baculovirus for being used to amplify culture and protein expression of acquisition, it is determined that viral
Plaque forming unit (plaque forming units, PFU), concretely comprise the following steps：
1) Sf-9 passages are inoculated into six well culture plates with Grace ' the s culture mediums containing 10%FBS, cell density
It is about 1 × 106Individual cells/mL, adds 2mL (6 orifice plate) per hole, and gently mixing room temperature makes cell attachment more than 1 hour.
2) by P3 generations kind venom, with Grace ' the s culture mediums for being free of FBS, to do 10 times of doubling dilutions stand-by.
3) culture medium in six orifice plates is discarded, cleans cell 3 times with the culture medium without serum, then by above-mentioned dilution well
Recombinant virus liquid adding hole in, each dilution factor does two multiple holes, at room temperature infect 1 hour.
4) covering liquid (the following amount for one piece of six orifice plate) is prepared：7mL2 × Grace ' s medium+140 μ l it is dual anti-+
7mL2% autoclaving agarose glue+1.4mL FBS, are lightly mixed, then bottle is placed to 42 DEG C of water-baths, virus sense again
Virus liquid in being exhausted after dye 1h per hole, and quickly cover cell with the covering liquid of above-mentioned preparation.
5) six orifice plates are wrapped with preservative film after agarose solidification and is placed in cultivating 3-5 days in 27 DEG C of incubators.
6) dimethyl diaminophenazine chloride that 1mL concentration is 1mg/mL is added, incubation at room temperature sucks dye liquor after 2 hours, it was observed that recombinant virus
The dot of the similar transparent of formation is plaque.Counting statistics observes the formational situation (PFU) of virus plaque, and viral titre is about
For 4.5 × 109(PFU/mL)。
3rd, expression of the M1 and HA-24 genes in the insect cell sf-9 for suspending culture
200mL sf-9 cell mixtures are suspended and are incubated in the triangle shaking flask of 1 liter of volume, cell culture fluid is without blood
Clear sf-900II (or Grace insect medium of Invitrigen companies), it is 100r/m that shaking table, which shakes speed, and temperature is constant
In 27 DEG C.When cell concentration reaches 2 × 1 cells/mL, sf-9 cells are transfected with Bac-M1-HA-24 insect baculovirus DNA,
The MOI=1 of virus.The cell of transfection collects all samples after constant temperature shakes culture 3 days, and 4 DEG C centrifuge 30 minutes, centrifugation speed
Spend for 3000r/m, collection cell culture supernatant.After the cell pellet centrifuged is handled with cell pyrolysis liquid, 4 DEG C of centrifugations
10 minutes, centrifugal speed 10,000r/m, retain the cell cracking extracting supernatant after centrifugation.Structure while experiment is carried out
The wild insects baculoviral of synthesis is used for the sf-9 cells of Transfection ofsuspension culture, MOI=1, as the negative control of setting,
The condition of the collection of cell culture, sample after transfection and cell cracking is with step as above-mentioned experiment.
The all samples of collection are analyzed for Western blots.Its experimental implementation is as follows：
1) each sample (including negative control) takes 10 μ L cell to crack extracting supernatant respectively, then each adds 10 μ L
2 × SDS sample-loading buffers.After 100 DEG C of processing 5min, all 20 μ L biased sample is added into 4%-12%SDS polypropylene
In the loading wells of acrylamide gel.Constant voltage 120V is set, and temperature is 4 DEG C, the 3 hours time, when blue indicator bromjophenol blue is complete
When leaning on entirely into gel bottom, stop electrophoresis, take out gel.
2) nitrocellulose filter (pvdf membrane) and two filter paper, pvdf membrane for cutting one and gel formed objects are soaked with methanol
Bubble is immersed in the transfering buffering liquid of precooling more than 2 hours after 5 minutes together with gel, filter paper, --- gel --- nitre by filter paper
The order of acid cellulose film --- filter paper is installed presss from both sides into transfer.By the side joint negative pole in folder close to gel, constant pressure 25V transfer electricity
Swimming 1h. grip out nitrocellulose filter with tweezers, washed with PBS-T rinsing liquids, be then transferred to 5% skimmed milk power/PBS it is molten
In liquid, closing 1 hour is swayed.
3) washed 3 times, every time 3 minutes with PBS-T rinsing liquids；Then nitrocellulose filter is transferred in a polybag, added
Enter 3mL with 1:500 are diluted in 5% skimmed milk power/PBS moderate resistance H1N1 influenza virus chickens source polyclonal antibodies (primary antibody), are placed in 4 DEG C
Gentle shaken over night.On next day, cellulose membrane is taken out, 3 times are washed with PBS-T rinsing liquids, 10 minutes every time, by this nitrocellulose
Film is reloaded into another new polybag, adds 5mL with 1:10000 are diluted in the horseradish peroxidating in 5% skimmed milk power/PBS
The donkey anti-chicken IgG (secondary antibody) of thing enzyme mark, shake incubate 1h at room temperature.Abandon secondary antibody, PBS-T rinsings cellulose membrane 3 times, every time 10
Minute, the nitrocellulose filter after rinsing is moved in a plate, adds the nitrite ions of DAB the 5th.
As shown in Fig. 2 there are M1 (about 25KDa) and HA-24 (about 70KDa) specificity on corresponding molecular weight
Band.Illustrate that M1 and HA-24 are effectively expressed in the SF-9 insect cells of the suspension culture of transfection.
4th, the purifying of virus-like particle
The above-mentioned cell culture supernatant being collected by centrifugation is loaded in 13mL ultracentrifugation pipe, weighs, balance, tube sealing
Afterwards, it is put into ultracentrifuge (Bechmem Products), 4 DEG C of 100,000rpm are centrifuged 1 hour, then take out centrifuge tube, small
The heart outwells supernatant, retains the deep thing at centrifuge tube bottom.5mL PBS is added, is put into 4 DEG C of refrigerators, is dissolved 24 hours.Next day,
In another 13mL ultracentrifugation pipe, 1mL 60% multitudinous sugar juice is first carefully added into, then sequentially adds 1mL
30% and 3mL20% multitudinous sugar juice, it is finally that the sample liquid after 5mL dissolving is placed on it.After being precisely weighed, balancing, envelope
Ultracentrifuge on pipe.4 DEG C of 100,000rpm are centrifuged 1 hour.Centrifuge tube is taken out, collection is located at 30% and 60% concentration intersection
Band, that is, the virus-like particle purified.
Virus-like particle sample after Western blots analysis purifying concentrations.In its operating procedure and above-mentioned steps 3
Western blots analyses are the same, simply diluted the sample taken, i.e. 1 μ L virus-like particle sample, add 9 μ
L water, add 10 μ L 2 × SDS sample-loading buffers.After 100 DEG C of denaturation 5min, loading enters 4%-12% polyacrylamides and coagulated
In glue sample cell.Shown by Fig. 3 Western blots results, the macromolecular particle obtained, really by M1 and HA-24 structures
Into virus-like particle.After illustrating transfection, virus-like particle effectively self assembly, and be released into cell training in host cell
Support in supernatant.
The virus-like particle of the infectivity resistant spleen and kidney necrosis virus of embodiment 2
Infectious spleen and kidney necrosis virus major structural protein gene ORF24 is replaced into H1N1 Influenza virus HA protein genes
A part, the two forms fusion (HA-24), and ORF24 genes are located at fusion HA-24 5 ' ends, replace equivalent length
HA genes 5 ' terminal sequences, to ensure that fusion HA-24 length and HA mrna lengths are roughly equal.In fusion sequence
The length of ORF24 and HA sequences can do corresponding adjustment.The nucleotide sequence of fusion such as SEQ ID NO:Shown in 5, its amino
Acid sequence such as SEQ ID NO:Shown in 6.SEQ ID NO:5 1-906 bit bases be ORF24 gene, 907-1701 positions
Base is HA gene；SEQ ID NO:6 1-302 amino acids are ORF24 amino acid, and 303-566 bit bases are
HA amino acid.Fusion is artificial synthesized according to its nucleotide sequence.Other experimental procedures and experiment condition and the phase of embodiment 1
Together.
The virus-like particle of the infectivity resistant spleen and kidney necrosis virus of embodiment 3
Infectious spleen and kidney necrosis virus major structural protein gene ORF24 is replaced into H1N1 Influenza virus HA protein genes
A part, the two forms fusion (HA-24), and ORF24 genes are located at fusion HA-24 5 ' ends, replace equivalent length
HA genes 5 ' terminal sequences, to ensure that fusion HA-24 length and HA mrna lengths are roughly equal.In fusion sequence
The length of ORF24 and HA sequences can do corresponding adjustment.The nucleotide sequence of fusion such as SEQ ID NO:Shown in 7, its amino
Acid sequence such as SEQ ID NO:Shown in 8.SEQ ID NO:7 1-876 bit bases be ORF24 gene, 877-1701 positions
Base is HA gene；SEQ ID NO:8 1-292 amino acids are ORF24 amino acid, and 293-566 bit bases are
HA amino acid.Fusion is artificial synthesized according to its nucleotide sequence.Other experimental procedures and experiment condition and the phase of embodiment 1
Together.
The virus-like particle of the infectivity resistant spleen and kidney necrosis virus of embodiment 4
Infectious spleen and kidney necrosis virus major structural protein gene ORF24 is replaced into H1N1 Influenza virus HA protein genes
A part, the two forms fusion (HA-24), and ORF24 genes are located at fusion HA-24 5 ' ends, replace equivalent length
HA genes 5 ' terminal sequences, to ensure that fusion HA-24 length and HA mrna lengths are roughly equal.In fusion sequence
The length of ORF24 and HA sequences can do corresponding adjustment.The nucleotide sequence of fusion such as SEQ ID NO:Shown in 9, its amino
Acid sequence such as SEQ ID NO:Shown in 10.SEQ ID NO:9 1-846 bit bases be ORF24 gene, 847-1701 positions
Base is HA gene；SEQ ID NO:10 1-282 amino acids are ORF24 amino acid, and 283-566 bit bases are
HA amino acid.Fusion is artificial synthesized according to its nucleotide sequence.Other experimental procedures and experiment condition and the phase of embodiment 1
Together.
The virus-like particle of the infectivity resistant spleen and kidney necrosis virus of embodiment 5
Infectious spleen and kidney necrosis virus major structural protein gene ORF24 is replaced into H1N1 Influenza virus HA protein genes
A part, the two forms fusion (HA-24), and ORF24 genes are located at fusion HA-24 5 ' ends, replace equivalent length
HA genes 5 ' terminal sequences, to ensure that fusion HA-24 length and HA mrna lengths are roughly equal.In fusion sequence
The length of ORF24 and HA sequences can do corresponding adjustment.The nucleotide sequence of fusion such as SEQ ID NO:Shown in 11, its amino
Acid sequence such as SEQ ID NO:Shown in 12.SEQ ID NO:11 1-816 bit bases be ORF24 gene, 817-1701
Bit base is HA gene；SEQ ID NO:12 1-272 amino acids be ORF24 amino acid, 273-566 bit bases
For HA amino acid.Fusion is artificial synthesized according to its nucleotide sequence.Other experimental procedures and experiment condition and the phase of embodiment 1
Together.
The vaccine immunity of embodiment 6 is tested
Virus-like particle (VLPs) that embodiment 1-5 is purified and adjuvant (MONTANIDE ISA 763A, seppic,
France VLPs vaccines) are mixed and made into equal volume, 4 DEG C save backup.
Avette Chang Ajigasawa (golden pomfret) the average weight 100g of 280 tails, raises in cement pit, is randomly divided into 7 groups.1st~5 group
For test group, VLPs vaccines prepared by 1~embodiment of embodiment 5 are immunized respectively, 0.1mL (containing antigen 50ug) VLPs is penetrated per endnote
Vaccine；The negative control vaccine (being free of antigen) that 6th group of injection PBS solution mixes with equivalent adjuvant；7th group is not injected epidemic disease
Seedling.Immunization wayses are injected for muscle of back.28 days tail vein bloods after immune, every group of 5 tails are conventional to separate serum, with purifying
Infectious spleen and kidney necrosis virus is antigen, and Serum Antibody is detected using ELISA method.
As a result show, the 6th, 7 group of antibody is feminine gender, and the 1st~5 group of all positive of sample, averagely ELISA titres are distinguished
For：1st group 2700 ± 360, the 2nd group 2693 ± 425, the 3rd group 2599 ± 410, the 4th group 2812 ± 393, the 5th group 2581 ±
403, illustrate that VLPs vaccines can stimulate and produce high titre antibody.30 days after immune, 1x10 is used5TCID50ISKNV viruses attack poison, see
Examine 3 weeks.As a result show, the 1st group of dead 8 tail, 32 tails of surviving；2nd group of dead 8 tail, 32 tails of surviving；3rd group of dead 9 tail, survival
31 tails；4th group of dead 7 tail, 33 tails of surviving；5th group of dead 10 tail, 30 tails of surviving；6th, 7 group all dead in 14 days, table
Bright VLPs vaccines have preferable protective effect to egg-shaped pompano, can be with infection prevention spleen and kidney necrosis virus to egg-shaped pompano
Infection.
Embodiment described above only expresses the several embodiments of the present invention, and its description is more specific and detailed, but simultaneously
Therefore the limitation to the scope of the claims of the present invention can not be interpreted as.It should be pointed out that for one of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the guarantor of the present invention
Protect scope.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Claims (6)
1. a kind of virus-like particle of infectivity resistant spleen and kidney necrosis virus, it is characterised in that the virus-like particle is by table simultaneously
The Influenza matrix albumen M1 and fusion protein HA-24 reached is formed；The fusion protein HA-24 by Influenza virus HA protein with
Fishes infectious spleen and kidney necrosis virus ORF24 is built-up, the nucleotide sequence such as SEQ ID NO of the fusion protein HA-24:3、
5th, shown in 7,9 or 11；
The sequence SEQ ID NO:3 1-936 bit bases are ORF24 gene, and 937-1701 bit bases are HA base
Cause；
The sequence SEQ ID NO:5 1-906 bit bases are ORF24 gene, and 907-1701 bit bases are HA base
Cause；
The sequence SEQ ID NO:7 1-876 bit bases are ORF24 gene, and 877-1701 bit bases are HA base
Cause；
The sequence SEQ ID NO:9 1-846 bit bases are ORF24 gene, and 847-1701 bit bases are HA base
Cause；
The sequence SEQ ID NO:11 1-816 bit bases are ORF24 gene, and 817-1701 bit bases are HA base
Cause.
2. a kind of preparation method of the virus-like particle of infectivity resistant spleen and kidney necrosis virus, it is characterised in that comprise the following steps：
1. infectious spleen and kidney necrosis virus major structural protein ORF24 genes are replaced to a part for influenza virus HA genes, two
Person forms fusion HA-24, fusion HA-24 nucleotide sequence such as SEQ ID NO:3rd, shown in 5,7,9 or 11；
2. the matrix prote m1 gene of influenza virus and fusion protein HA-24 genes are inserted into insect baculovirus pFastBac-
On Dual carriers, identify and be sequenced through digestion, filter out positive recombinant vector, and convert and contain baculovirus shuttle vector
DH10Bac competent cells, obtain recombinant baculovirus shuttle vector rBacmid；
3. utilizing lipofectamine, the recombinant baculovirus shuttle vector rBacmid containing foreign gene is transfected into host
In insect cell line sf-9, recombinant baculovirus is obtained；
4. cultivating the host insect cell transfected by recombinant baculovirus, enable its efficiently viral knot of expression of influenza
Structure albumen M1 and fusion protein HA-24, and be assembled into automatically while expression of influenza viral matrix protein M1 and fusion protein HA-24
Virus-like particle.
3. the preparation method of the virus-like particle of infectivity resistant spleen and kidney necrosis virus according to claim 2, its feature exist
In, step 1. in HA-24 genes be first to be synthesized to merge base with influenza virus HA genes by infectious spleen and kidney necrosis virus ORF24
Cause, then formed with PCR amplifications.
4. the preparation method of the virus-like particle of infectivity resistant spleen and kidney necrosis virus according to claim 2, its feature exist
In step 3. at 27 DEG C cultivate 4-5 days by middle sf-9 cells, collects cell culture supernatant, then infects new sf-9 cells, obtains
The recombinant baculovirus of high titre after amplification.
5. the preparation method of the virus-like particle of infectivity resistant spleen and kidney necrosis virus according to claim 2, its feature exist
In, step 4. in cultivated 3 days at 27 DEG C by the host insect cell of recombinant baculovirus transfection, the influenza virus M1 of expression
Albumen and fusion protein HA-24, are assembled into VLPs automatically.
A kind of 6. preparation method for including any one of the claim 1-5 virus-like particle of the infectivity resistant spleen and kidney necrosis virus
The fishes infectious spleen and kidney necrosis virus gene engineering vaccine of prepared virus-like particle.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410381379.5A CN104195119B (en) | 2013-08-06 | 2014-08-05 | Virus-like particle and vaccine of a kind of infectivity resistant spleen and kidney necrosis virus and preparation method thereof |
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2013103403546 | 2013-08-06 | ||
| CN201310340354.6 | 2013-08-06 | ||
| CN201310340354 | 2013-08-06 | ||
| CN201410381379.5A CN104195119B (en) | 2013-08-06 | 2014-08-05 | Virus-like particle and vaccine of a kind of infectivity resistant spleen and kidney necrosis virus and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104195119A CN104195119A (en) | 2014-12-10 |
| CN104195119B true CN104195119B (en) | 2018-04-10 |
Family
ID=52080488
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410381379.5A Active CN104195119B (en) | 2013-08-06 | 2014-08-05 | Virus-like particle and vaccine of a kind of infectivity resistant spleen and kidney necrosis virus and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104195119B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108220252B (en) * | 2018-03-14 | 2022-02-11 | 中山大学 | Infectious spleen and kidney necrosis virus ORF022 gene deletion strain and preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101168054A (en) * | 2006-10-23 | 2008-04-30 | 中山大学 | Fishes infectious spleen and kidney necrosis virus gene engineering vaccine and preparation method thereof |
| CN102373183A (en) * | 2010-08-09 | 2012-03-14 | 中山大学 | Mixed virus-like particle (VLP) of avian influenza and Newcastle disease, preparation method thereof and application thereof |
-
2014
- 2014-08-05 CN CN201410381379.5A patent/CN104195119B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101168054A (en) * | 2006-10-23 | 2008-04-30 | 中山大学 | Fishes infectious spleen and kidney necrosis virus gene engineering vaccine and preparation method thereof |
| CN102373183A (en) * | 2010-08-09 | 2012-03-14 | 中山大学 | Mixed virus-like particle (VLP) of avian influenza and Newcastle disease, preparation method thereof and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| Genotype and host range analysis of infectious spleen and kidney;Xiaozhe Fu;《Virus Genes》;20111231(第42期);第97-109页 * |
| H5亚型禽流感病毒样颗粒的构建及生物学活性鉴定;张超林;《中国预防兽医学报》;20120731;第34卷(第7期);第515-517页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104195119A (en) | 2014-12-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103122353B (en) | Porcine O-type foot-and-mouth disease virus recombinant baculovirus as well as preparation method and application thereof | |
| CN106867975B (en) | Newcastle disease virus chimeric virus-like particle, vaccine and preparation method | |
| CN104328222B (en) | The test kit of reverse transcription PCR detection and somatotype dengue virus and detection method thereof | |
| CN103387996B (en) | Canine parvovirus-like particles and preparation method and application thereof | |
| CN103642761B (en) | A kind of respiratory syncytial virus sample granule and its preparation method and application | |
| CN104962581A (en) | Recombined new castle disease virus vaccine strain for expressing African swine fever virus p72 proteins | |
| CN104059927B (en) | Preparation method of newcastle disease glycoprotein viral antigen and products thereof | |
| CN109321535A (en) | A kind of heat-staple newcastle disease virus attenuated vaccine Candidate Strain | |
| CN105087645A (en) | Building and application of M protein three-amino acid site-mutated vesicular stomatitis virus (VSV) carrier for pigs | |
| CN104353070B (en) | Genetic engineering subunit vaccine of chicken infectious bronchitis virus and preparation method thereof | |
| US11607448B2 (en) | Whole avian-origin reverse genetic system and its use in producing H7N9 subtype avian influenza vaccine | |
| CN106244602A (en) | A kind of carry two G genes and the recombinant rabies virus of VP2 gene and application thereof | |
| CN104152418B (en) | Virus-like particle and vaccine of a kind of anti-SVCV and preparation method thereof | |
| CN105349562A (en) | Recombinant vector and recombinant strain for expressing PPV (porcine parvovirus) VP2 protein and applications of recombinant vector and recombinant strain | |
| CN106754765A (en) | A kind of NDV sample particle, preparation method and applications | |
| CN102533677B (en) | Malaria vaccine and preparation method thereof | |
| CN101979502B (en) | Recombinant brucella expressing VP1 gene of O-type foot-and-mouth disease virus and method for producing vaccines thereof | |
| CN102373183A (en) | Mixed virus-like particle (VLP) of avian influenza and Newcastle disease, preparation method thereof and application thereof | |
| CN106085970B (en) | The heat-resisting vaccine strain of recombinant Newcastle disease and preparation method of the H5 subtype avian influenza HA albumen of expression signal peptide replacement | |
| CN104195119B (en) | Virus-like particle and vaccine of a kind of infectivity resistant spleen and kidney necrosis virus and preparation method thereof | |
| CN104403006B (en) | Mink Parvovirus virus-like particle and preparation method and application | |
| CN101560522A (en) | Method for expressing IBV-HN99 membrane protein gene in insect-rhabdovirus system | |
| CN109943576A (en) | A kind of recombinant rabies virus of chimeric canine distemper virus principal immune gene and its application | |
| CN112494643B (en) | Porcine CSFV-PCV bivalent DNA vaccine and preparation method thereof | |
| CN101979503A (en) | Recombinant brucella for expressing Asia type-I foot and mouth disease virus VP1 genes and method for producing vaccines thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |