CN106381290A - Recombinant European avian-borne H1N1 subtype swine influenza vaccine strain, and preparation method and application thereof - Google Patents
Recombinant European avian-borne H1N1 subtype swine influenza vaccine strain, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a recombinant European avian-borne H1N1 subtype swine influenza vaccine strain. The recombinant vaccine strain comprises the HA and NA genes of an European avian-borne H1N1 subtype swine influenza virus strain SH1 and six inner genes consisting of PA, PB1, PB2, M, NP and NS of a PR8 virus. The invention also discloses a preparation method and application thereof of the recombinant swine influenza vaccine strain. The recombinant swine influenza vaccine strain has high virus titer on MDCK cells; and a recombinant high-yield inactivated vaccine prepared through inactivation of the recombinant swine influenza vaccine strain can provide complete-response immunoprotection against attack by European and Asian avian-borne H1N1 subtype swine influenza viruses.
Description
Technical field
The present invention relates to technical field of bioengineering, the European fowl source H1N1 hypotype swine flue vaccine strain of more particularly, to a kind of restructuring and
Its preparation method and application.
Background technology
Swine flue (Swine influenza, SI) is the swine influenza viruses (Swine being belonged to by orthomyxoviridae family's influenza A
Influenza virus, SIV) infection, cause the high degree in contact respiratory tract that the symptoms such as hyperpyrexia, cough, dyspnea in pig to pass
Catch an illness, there is sickness rate height, spread speed is fast, the low feature of mortality rate.Have been reported that and show swine influenza virus infection swinery simultaneously
Also easily cause other antibacterials and the mixed infection of virus, such as pig syndrome of breeding and respiration disorders, contagious pleuropneumonia, pig
Streptococcus etc., make epidemic situation become more complicated, cause great difficulty to disease control.Cause the SIV hypotype master of pig morbidity at present
H1N1, H1N2 and H3N2 to be, main inclusion European fowl source H1N1 hypotype SIV, classic H1N1 hypotype, restructuring
H1N2 hypotype SIV, people source H3N2 hypotype SIV and restructuring H3N2 hypotype SIV etc..Research shows the airway epithelial of pig
There is human influenza viruses receptor SA α -2,3-Gal (sialic acid α -2,3 galactosides) and bird flu viruss (Avian in cell surface simultaneously
Influenza virus, AIV) receptor SA α -2,6-Gal (sialic acid α -2,3 galactosides), in transmission of influenza virus, pig quilt
It is considered " couveuse " that potential " blender " of viral gene restructuring and new epidemic isolates produce, be also influenza virus leap
One of important intermediate host that species barrier is propagated.Therefore prevention and control SIV is to prevention swinery outburst influenza virus and other antibacterials of secondary
While significant with virus infection, equally with the influenza virus of fowl, positive assosting effect is played to guidance and prevention and control people.
At present, technically ripe and widely used SI vaccine is mainly the oily H1N1 hypotype of assistant type and the unit price of H3N2 hypotype
Or multivalence inactivated virus vaccine, although SI inactivated virus vaccine has the advantages that efficient, simple to operate, cost is relatively low,
But there is also the deficiencies such as generation antibody is relatively slow, stress is strong.Particularly vaccine kind poison comes from epidemic isolates mostly, once
There is virus leakage in vaccine preparation process, easily induce new epidemic situation, bring very big inconvenience to control and prevention of disease.Additionally, current state
Inside and outside production influenza vaccines still mainly take egg inoculation culture method, although egg inoculation is simple to operate, deposit is experienced,
It is inevitably to bring time-consuming amount greatly, consumption is many, production process is also easy to produce pollution, difficult quality guarantee, receives the chicken after poison
The shortcomings of embryo is difficult, in addition influenza virus pass in Embryo Gallus domesticus and antigenic variation often can occur in inoculation, affect vaccine effect.There is report
Road, Hong Kong in 1997 infect people H5N1 bird flu viruss Embryo Gallus domesticus can be made to produce giant cell pathological changes so that be difficult to Embryo Gallus domesticus Lai
Propagation produces vaccine.And influenza virus adaptability on common mdck cell is generally relatively low, this hinders it to develop further and make
With.
Content of the invention
The technical problem to be solved in the present invention is to provide a kind of restructuring European fowl source H1N1 hypotype swine flue vaccine strain, and it comprises Europe
HA the and NA gene of continent fowl source H1N1 hypotype swine influenza viruses SH1 strain and PR8 virus PA, PB1, PB2,
Six internal gene of M, NP and NS, this Recombinant Swine influenza vaccines strain can not only be produced in a large number by cell culture, Er Qieyou
The inactivated vaccine of this recombinant vaccine strain preparation can stimulate and produces high-caliber antibody in the body short time.
In addition, it is also desirable to provide a kind of preparation method and application of above-mentioned restructuring Europe fowl source H1N1 hypotype swine flue vaccine strain.
In order to solve above-mentioned technical problem, the present invention is achieved through the following technical solutions:
In one aspect of the invention, there is provided a kind of Recombinant Swine influenza vaccines strain, this recombinant vaccine strain comprises：European fowl source H1N1
HA the and NA gene of hypotype swine influenza viruses SH1 strain and PA, PB1, PB2, M, NP and NS of PR8 virus
Six internal gene,
The nucleotide sequence of the HA gene of described swine influenza viruses SH1 strain is selected from：
(1) nucleotide sequence of aminoacid sequence shown in coding SEQ ID NO.1；
(2) coding and aminoacid sequence shown in SEQ ID NO.1 have the nucleotide sequence of the aminoacid sequence of more than 98% homology；
The nucleotide sequence of the NA gene of described swine influenza viruses SH1 strain is selected from：
(1) nucleotide sequence of aminoacid sequence shown in coding SEQ ID NO.2；
(2) coding and aminoacid sequence shown in SEQ ID NO.2 have the nucleotide sequence of the aminoacid sequence of more than 98% homology.
Preferably, the HA gene of described swine influenza viruses SH1 strain has the nucleotide sequence shown in SEQ ID NO.3.
Preferably, the NA gene of described swine influenza viruses SH1 strain has the nucleotide sequence shown in SEQ ID NO.4.
In another aspect of this invention, additionally provide a kind of recombinant vector, comprise European fowl source H1N1 hypotype swine influenza viruses SH1
HA the or NA gene of strain, the nucleotide sequence of described HA gene is selected from：
(1) nucleotide sequence of aminoacid sequence shown in coding SEQ ID NO.1；
(2) coding and aminoacid sequence shown in SEQ ID NO.1 have the nucleotide sequence of the aminoacid sequence of more than 98% homology；
The nucleotide sequence of described NA gene is selected from：
(1) nucleotide sequence of aminoacid sequence shown in coding SEQ ID NO.2；
(2) coding and aminoacid sequence shown in SEQ ID NO.2 have the nucleotide sequence of the aminoacid sequence of more than 98% homology.
Described recombinant vector is recombinant plasmid vector, and its empty carrier is PBD carrier.
In another aspect of this invention, additionally provide a kind of preparation method of Recombinant Swine influenza vaccines strain, comprise the following steps：
Build and comprise the HA gene of European fowl source H1N1 hypotype swine influenza viruses SH1 strain and the recombinant vector of NA gene respectively；
By the described recombinant vector containing HA gene and containing NA gene recombinant vector, with comprise respectively PR8 virus PA, PB1,
Six plasmids of PB2, M, NP, NS internal gene transfect 293T cell, the cell after culture transfection together；
The cell conditioned medium of culture is inoculated in mdck cell, occurs collecting virus liquid after pathological changes after mdck cell, detection should
The hemagglutinative titer of virus liquid, if there are hemagglutination activity, and does not have through sequence analysis determination after unexpected mutation, that is, obtains restructuring
Swine flue vaccine strain.
In another aspect of this invention, additionally provide a kind of vaccine combination, comprise：The inactivation of above-mentioned Recombinant Swine influenza vaccines strain
Vaccine, and adjuvant or pharmaceutical carrier.
In another aspect of this invention, additionally provide a kind of above-mentioned Recombinant Swine influenza vaccines strain and in preparation prevention or treat swine flue
Application in vaccine.
Present invention restructuring European fowl source H1N1 hypotype swine flue vaccine strain, compared with wild strain virus, has higher on cell
Replication capacity, can reach higher virus titer, and confirms, prepared by this recombinant vaccine strain by mouse immune and challenge viral dosage
Inactivated vaccine head exempt from after can obtain higher antibody titer within the 2nd week, and can be for Eurasian fowl source H1N1 hypotype swine flue
The attack of virus provides fully effective immunoprotection.
Brief description
The present invention is further detailed explanation with reference to the accompanying drawings and detailed description.
Fig. 1 is NA the and HA base of the domestic epidemic strain SH1 of the embodiment of the present invention 1 European fowl source H1N1 hypotype swine influenza viruses
Because of pcr amplification product electrophoretogram；
Fig. 2 is NA the and HA base of the domestic epidemic strain SH1 of the embodiment of the present invention 1 European fowl source H1N1 hypotype swine influenza viruses
Because of bacterium solution PCR qualification result figure；
Fig. 3 is that the embodiment of the present invention 2 virus inoculates the growth curve chart after mdck cell with 0.001MOI；
Fig. 4 is that the embodiment of the present invention 2 virus inoculates the growth curve chart after SPF Embryo Gallus domesticus with 0.001MOI；
Fig. 5 is the embodiment of the present invention 2 virus plaques testing result figure；
Fig. 6 is the embodiment of the present invention 3 blood clotting suppression (HI) antibody test result figure；
Fig. 7 is the embodiment of the present invention 3 NAT figure；
Fig. 8 is the lgG antibody titer figure of the embodiment of the present invention 3；
Fig. 9 is the 3rd, 5 days mouse lung tissue virus titer block diagrams after the embodiment of the present invention 3 counteracting toxic substances；
Figure 10 is the body weight change figure of the embodiment of the present invention 3 counteracting toxic substances mice after 14 days；
Figure 11 is the survival rate figure of 14 days mices after the embodiment of the present invention 3 counteracting toxic substances.
Specific embodiment
In the following example, the experimental technique of unreceipted actual conditions, generally routinely condition, such as《Fine works molecular biology is real
Test guide》(F.M. Ao Sibai, R.E. James Kingston, J.G. Sai Deman etc. edits, Ma Xuejun, and Su Yuelong translates. north
Capital：Science Press, 2004) method described in is carried out.
The present invention is with the domestic epidemic strain A/swine/Shanghai/1/2014 (H1N1) of European fowl source H1N1 hypotype swine influenza viruses
(SH1) be material, HA and NA gene expanded using RT-PCR, and is cloned in PBD carrier, build HA and
The recombiant plasmid of NA gene.By this HA and NA gene recombination plasmid and from strains of influenza viruses A/Puerto Rico/8/1934
(H1N1) 6 internal gene (PB2, PB1, PA, NP, M and NS) recombiant plasmid cotransfection 293T of (PR8) are thin
Born of the same parents, successfully construct the European fowl source H1N1 hypotype swine flue vaccine strain SH/PR8 of restructuring.This recombinant vaccine strain SH/PR8 exists
Higher virus titer is had on mdck cell or Embryo Gallus domesticus, the restructuring high yield cell inactivation vaccine of preparation can flow to pig after inactivation
Influenza Virus SH1 attacks the immunoprotection providing 100%.
The structure of the European fowl source H1N1 hypotype SIV recombinant strain SH/PR8 of embodiment 1
According to the description on TRizol test kit, extract the domestic epidemic strain of European fowl source H1N1 hypotype swine influenza viruses
The viral RNA of A/swine/Shanghai/1/2014 (SH1), and reverse transcription synthesis cDNA, carry out purpose piece by RT-PCR
The amplification of section HA and NA, PCR reaction condition：95 DEG C, 1min；95 DEG C, 20s；48 DEG C, 20s；72 DEG C, 1min；
72 DEG C, 10min, 30 circulations.HA and NA nucleic acid product after amplification identify through 1% agarose gel electrophoresiies (see Fig. 1,
In Fig. 1, M：DNA molecular quality standard (DL2000)；1：The pcr amplification product (1410bp about) of NA gene；
2：The pcr amplification product (1701bp about) of HA gene), and product reclaims.Respectively the glue containing HA and NA is returned
Receive product and carry out endonuclease reaction through restricted enzyme BspQ Ι, carry out glue reclaim afterwards, then with BspQ Ι enzyme action after PBD
Carrier connects.Product conversion after connecting enters in DH5 α competent escherichia coli cell.Product after conversion is coated in and contains
Have in the LB culture plate of ampicillin, after 37 DEG C of culture 12-14h, choose monoclonal bacterium colony and put into containing ammonia benzyl green grass or young crops
In the LB culture medium of mycin, through 37 DEG C, the horizontal shaker of 180rpm/min is cultivated 14-16h.Reflected by bacterium solution PCR
The method of determining filters out positive colony (see Fig. 2, in Fig. 2, M：DNA molecular quality standard (DL2000)；A figure is HA bacterium
PCR liquid qualification result, wherein 1-6 qualification result are the positive；B figure is NA bacterium solution PCR qualification result, wherein Isosorbide-5-Nitrae, 5,
7 qualification results are the positive), and positive colony is sent to handsome biotech firm carry out sequencing.Sequencing result is correctly positive
Plasmid is named as PBD-HA and PBD-NA, and extracts sun according to the description of QIANGEN Plasmid Purification Kit
Property grain.Take 8 pUC pUCs of 1ug PBD-HA and 1ug PBD-NA and A/Puerto Rico/8/1934 (H1N1) (PR8)
Remaining 6 plasmid (PBD-PR8M, PBD-PR8PB1, PBD-PR8PB2, PBD-PR8PA, PBD-PR8NS,
PBD-PR8NP) each 1ug mixing, is transfected with reference to liposome Lipofectamine2000 transfection reagent product description.Turn
Dye 293T cell, after 48h, is collected supernatant and is simultaneously inoculated in mdck cell, obvious pathological changes (CPE) after cell and receives afterwards
Collection virus liquid.The virus liquid collected is carried out with the detection of hemagglutinative titer, extracts viral RNA, after reverse transcription synthesis cDNA, with
Eight genetic fragments of the virus that this cDNA saves for template PCR amplifications, and carry out mensure and the comparison of gene fragment order.
The supernatant that after collecting inoculation 72h, MDCK breeds, detects its hemagglutinative titer with hemagglutination test, and blind passage three generations continues to detect its blood
Solidifying potency.Show successfully to save out recombinant viruses SH/PR8 by sequencing result and blood coagulation tests result.
Embodiment 2 recombinant strain SH/PR8 and the comparison of original wild strain SH1 replication capacity on cell and Embryo Gallus domesticus
Prepare influenza infection liquid, add TPCK (protease).Virus dilution, according to 10 times of dilution methods, with virus sense
Dye liquor doubling dilution virus, fully mixes, and by virus from 10-1It is diluted to 10-11, the virus having diluted is added be covered with successively
In 96 orifice plates of mdck cell, put into 37 DEG C, 5% CO2Cell culture incubator cultivates 72h, observation of cell pathological changes (CPE) feelings
Condition, and record result, sucking-off cell supernatant carries out Blood coagulation test experiment and checks experimental result, calculates TCID50Value.
Prepare influenza infection liquid, by the virus infection liquid dilution of SH/P8 and original strain SH1 virus, according to the inoculation of every hole
The virus quantity aseptic inoculation of 0.001MOI enters in 12 porocyte culture plates, and 12 porocyte culture plates cover with density about 90
Mdck cell, is spaced 12h, difference sterile collection cell supernatant after 24h, 36h, 48h, 60h, 72h, detects different time sections HA
Hemagglutinative titer.Result shows, the restructuring SH/PR8 virus prime strain SH1 that compares has higher duplication energy on mdck cell
Power, after 60h, HA potency reaches 29(see Fig. 3).
With viral dilution liquid, viral dilution is become 100TCID50, according to 100TCID50Virus titer virus is sterilely inoculated into
In the SPF Embryo Gallus domesticus of 9-11 age in days, Embryo Gallus domesticus are put in 37 DEG C of incubator, keep suitable humidity and across same time period
Stir Embryo Gallus domesticus, be spaced 12h, aseptic respectively after 24h, 36h, 48h, 60h, 72h collect chick embryo allantoic liquid, after 3000rpm, 5min centrifugation
Collect supernatant, it is carried out with HA Blood coagulation test experiment.Result shows, restructuring SH/PR8 virus is with respect to original wild strain SH1
Higher replication capacity (see Fig. 4) is had on SPF Embryo Gallus domesticus.
Prepare (LMP) agarose gel of 2% low melting point, put into after autoclaving in 37 DEG C of cell culture incubators.In advance will
Mdck cell inoculate 6 porocyte culture plates in, when cell culture to cover with 6 porocyte culture plates when, configuration virus infection liquid simultaneously
Add TPCK protease, according to 10 times of size doubling dilution virus liquids.Clean mdck cell both sides with PBS phosphate buffer,
Clean one time with DMEM culture medium again.The virus liquid having diluted is separately added in 6 well culture plates according to every hole 1ml, by virus
Liquid puts into 37 DEG C, in 5% CO2 cell culture incubator, allows viruses adsorption enter in mdck cell, middle mixed at interval of 30min
Even viral dilution liquid, allows viruses adsorption 1.5h.Virus liquid, DMEM culture medium cleaning one is discarded from 6 porocyte culture plates
Time.Amount according to the 2%LMP of every orifice plate 4ml adds in 6 well culture plates.Standing 2h under room temperature, until 2%LMP is complete
After solidification, 6 porocyte culture plates are tipped upside down on 37 DEG C, in 5% CO2 cell culture incubator.Cultivate to 48h about observation of cell disease
Change situation.Until occurring substantially and after the plaque of suitable size, after terminating cultivating and add 4% formaldehyde to fix 1.5h-2h.Discard
Formalin, the aseptic agarose gel choosing low melting point, every hole adds the Gentian Violet dyeing liquor of about 2ml to fix, after about 15min
Reclaim dyeing liquor, flowing water observes plaque form after cleaning up.Result shows, restructuring SH/PR8 strain is with respect to original wild strain
The virus plaques size that SH1 is formed is bigger, shows that restructuring SH/PR8 has higher replication capacity (see Fig. 5).In Figure 5,
A:Normal control；B:Original wild strain SH1；C:Recombinant strain SH/PR8.
The embodiment 3 European fowl source H1N1 hypotype SIV restructuring preparation of high yield inactivated vaccine and its evaluation of immune protection effectiveness
By original wild strain SH1 and recombinant strain SH/PR8 according to 0.001MOI dose inoculation in mdck cell culture bottle
In, after a large amount of amplicon virus, collect cell supernatant, 3000rpm, 10min centrifugal separating cell fragment simultaneously collects supernatant.
The original wild strain SH1 that amplification is obtained and recombinant viruses SH/PR8 carries out HA Blood coagulation test test, detects its viral hemoagglutination
Potency.
Choose the water in oil mineral adjuvant of Montanide ISA 61VG and the Montanide ISA 15A that match BIC Corp of France produces
VG oil-in-water mineral adjuvant has done two groups and has been compared.By virus through 37 DEG C of inactivation 24-48h of 0.1% formalin, after inactivation
Virus blind passage 3 generation on mdck cell after, respectively detect HA hemagglutinative titer.By the virus after inactivation according to emulsifying description
Requirement emulsifying virus, the adjuvant of 61VG according to the virus of weight 40% antigen and 60% adjuvant ratio emulsifying restructuring, 15AVG's
Adjuvant is according to the virus of the part by weight emulsifying restructuring of weight 15% and 85%.
Choose the SPF level female BAl BIc/c mice of 6-8 week old, be randomly divided into 5 groups, first group is that selection is different with second group respectively
The water in oil 61VG of vaccine of adjuvant and oil-in-water 15VG, the 3rd group and the 4th group is to choose to be not added with going out of adjuvant emulsion respectively
Live recombinant viruses, the 5th group be immune DMEM matched group.Immune programme for children is：Subcutaneous injection one is exempted from, interval subcutaneous note after 2 weeks
Penetrate two to exempt from, interval counteracting toxic substances after 2 weeks.Immunization wayses are subcutaneous multi-point injection inoculation, according to the inoculum concentration difference of every mice 200ul
Immune 5 groups of mices, first group is SH/PR8+61VG group；Second group is SH/PR8+15VG group；3rd group is
(emulsifying according to 61VG requires SH/PR8+DMEM group, and antigen and DMEM mass ratio are 40：60 are mixed)；The
Four groups is that (emulsifying according to 15AVG requires SH/PR8+DMEM group, and antigen and DMEM mass ratio are 15：85 are mixed
Even；5th group is DMEM matched group.
Mice is carried out with the blood sampling of eye socket clump and collects each group mice serum, in surrounding after head exempts from for each group mice at interval of one week respectively
Collection once, will be collected serum and carry out blood clotting Inhibition test and neutralization experiment respectively.By each group mice serum and RDE (receptor
Destructive enzyme) according to volume ratio 1：3 ratio mixes, 37 DEG C of incubation 18-20h, 56 DEG C of inactivation 30min-60min.Reference《Country
Influenza central standard rule of operation》Carry out blood clotting suppression (HI) experiment, record experimental result.Result shows, when exempting from head
Between prolongation, HI antibody titer gradually steps up, 2 exempt from after the 2nd week antibody titer is up to 29Left and right, wherein organizes one
SH/PR8+61VG water-in-oil adjuvant group produces HI antibody titer highest.Organize two SH/PR8+15A oil-in-waters and exempt from the 3rd week in head
Higher blood clotting suppression (HI) antibody titer (see Fig. 6) just can be reached.In figure 6, exempt from rear interval time (all) headed by abscissa,
Vertical coordinate is blood clotting suppression potency (Log2)；SH/PR8+61VG is the immune group adding water-in-oil adjuvant；SH/PR8+15AVG
For adding the immune group of oil-in-water adjuvant；SH/PR8+DMEM is the immune group without adjuvant；DMEM is nonimmune group.
Prepare virus infection liquid and add dual anti-(penicillin and streptomycin mixed liquor), be 1000 according to virus infection liquor ratio TPCK volume ratio：
2 ratio adds TPCK in infection liquid.Configuration virus infection liquid simultaneously adds dual anti-(penicillin and streptomycin mixed liquor), will collect
56 DEG C of serum heat inactivation 30min, by the serum after inactivation according to 1：10,1：20,1：40,1：80,1：160,1：
320,1：640,1:1280 ratio infection liquid is diluted, and each sample does 2 repetitions, dilutes original influenza virus SH1
To 100TCID50Virus titer, in 96 porocyte culture plates, every hole adds the serum after 50ul dilution and 50ul
(100TCID50) virus liquid, 37 DEG C incubation 2h.Serum and viral mixed liquor are added drop-wise to and cover with the 96 of mdck cell
In well culture plate, set up the positive control of a virus inoculation and the negative control of not virus inoculation.Observation of cell disease after 48h
Change situation, detects serum NAT, and sucking-off cell supernatant carries out blood coagulation tests and checks experimental result and record.Result
Show, with the prolongation of First immune time, NAT gradually steps up, 2 exempt from rear 2nd week NAT is up to
103More than, second group of SH/PR8+15A oil-in-water is exempted from the 2nd week NAT in head and just can be reached 103Left and right (see Fig. 7).
By the original wild strain SH1 of 30%-60% sucrose gradient ultracentrifugation purification, reference《National influenza central standard operation rule
Journey》, carry out lgG antibody test with ELISA, comprise the following steps that.
1st, it is coated：The viral SH1 carbonate buffer solution of purification is coated ELISA ELISA Plate, every hole package amount is
200ng/100 μ L, 4 DEG C overnight.
2nd, wash：Washed with the PBST containing 5 ‰ tween-20 3 times, 200 μ L/ holes.5 minutes every time.
3rd, close：37 DEG C of 5% skimmed milk (BSA) is incubated 2 hours, 200 μ L/ holes.
4th, wash：Washed with the PBST containing 5 ‰ Tween-20 3 times, 200 μ L/ holes.5 minutes every time.
5th, Jia one anti-：With 5% skimmed milk (BSA) gradient dilution Sample serum, 1：300,1：600,1：1200,2400,
1：4800,1：9600,1；19200,37 DEG C of incubation 2h.
6th, wash：Washed with the PBST containing 5 ‰ Tween-20 3 times, 200 μ L/ holes.5 minutes every time.
7th, two are added to resist：Press 1 with 5% skimmed milk：After 10000 times of dilution HRP sheep anti-mouse iggs, every hole 100 μ L adds enzyme
Target, 37 DEG C are incubated 1 hour.
8th, wash：Washed with the PBST containing 5 ‰ Tween-20 3 times, 200 μ L/ holes.5 minutes every time.
9th, develop the color：Every hole adds 50 μ L TMB nitrite ions, lucifuge incubated at room 15 minutes.
10th, terminate：Every hole adds 50 μ L 2M H2SO4.
11st, OD value detection：Microplate reader measures OD450Value IgG antibody potency.Criterion reference《National influenza central standard behaviour
Make code》
Result shows, with the prolongation of First immune time, IgG antibody potency gradually steps up, and 2 exempt from rear 2nd week antibody titer is up to
105More than, wherein organize two SH/PR8+15A oil-in-waters and exempt from the 3rd week to reach 10 in head5Left and right (see Fig. 8).
Original wild strain SH1 after amplification is carried out virus titer TCID50Detection, according to every mice 1x105TCID50
Virus inoculation amount counteracting toxic substances, wherein matched group choose 6 not counteracting toxic substances as normal mouse compare.Mice spirit shape is observed after counteracting toxic substances
State, takes mouse lung to carry out titration of virus (result is shown in Fig. 9) after counteracting toxic substances 3d and 5d.Weigh 14 days body weight situations of change of mice
(see Figure 10) and survival rate (see Figure 11).Result shows, after counteracting toxic substances, nonimmune group of continued weight declines, and after 6 days, mice goes out
Existing 100% is dead, and immune group, death does not occur, shows that the inactivated vaccine using recombinant vaccine strain preparation can be for Europe
Fowl source H1N1 hypotype swine influenza viruses wild strain SH1 attacks the immunoprotection providing 100%.
Embodiment described above only have expressed embodiments of the present invention, and its description is more concrete and in detail, but can not therefore and
It is interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, do not taking off
On the premise of present inventive concept, some deformation can also be made and improve, these broadly fall into protection scope of the present invention.Therefore,
The protection domain of patent of the present invention should be defined by claims.
Claims (10)
1. a kind of Recombinant Swine influenza vaccines strain is it is characterised in that this recombinant vaccine strain comprises：European fowl source H1N1 hypotype swine flue
HA the and NA gene of viral SH1 strain and six inside of PA, PB1, PB2, M, NP and NS of PR8 virus
Gene,
The nucleotide sequence of the HA gene of described swine influenza viruses SH1 strain is selected from：
(1) nucleotide sequence of aminoacid sequence shown in coding SEQ ID NO.1；
(2) coding and aminoacid sequence shown in SEQ ID NO.1 have the nucleotide sequence of the aminoacid sequence of more than 98% homology；
The nucleotide sequence of the NA gene of described swine influenza viruses SH1 strain is selected from：
(1) nucleotide sequence of aminoacid sequence shown in coding SEQ ID NO.2；
(2) coding and aminoacid sequence shown in SEQ ID NO.2 have the nucleotide sequence of the aminoacid sequence of more than 98% homology.
2. Recombinant Swine influenza vaccines strain according to claim 1 is it is characterised in that described swine influenza viruses SH1 strain
HA gene has the nucleotide sequence shown in SEQ ID NO.3.
3. Recombinant Swine influenza vaccines strain according to claim 1 is it is characterised in that described swine influenza viruses SH1 strain
NA gene has the nucleotide sequence shown in SEQ ID NO.4.
4. a kind of recombinant vector is it is characterised in that comprise HA or NA of European fowl source H1N1 hypotype swine influenza viruses SH1 strain
Gene, the nucleotide sequence of described HA gene is selected from：
(1) nucleotide sequence of aminoacid sequence shown in coding SEQ ID NO.1；
(2) coding and aminoacid sequence shown in SEQ ID NO.1 have the nucleotide sequence of the aminoacid sequence of more than 98% homology；
The nucleotide sequence of described NA gene is selected from：
(1) nucleotide sequence of aminoacid sequence shown in coding SEQ ID NO.2；
(2) coding and aminoacid sequence shown in SEQ ID NO.2 have the nucleotide sequence of the aminoacid sequence of more than 98% homology.
5. recombinant vector according to claim 4 is it is characterised in that the HA gene of described swine influenza viruses SH1 strain has
There is the nucleotide sequence shown in SEQ ID NO.3.
6. recombinant vector according to claim 4 is it is characterised in that the NA gene of described swine influenza viruses SH1 strain has
There is the nucleotide sequence shown in SEQ ID NO.4.
7. recombinant vector according to claim 4 is it is characterised in that the empty carrier of described recombinant vector is PBD carrier.
8. a kind of preparation method of Recombinant Swine influenza vaccines strain is it is characterised in that comprise the following steps：
Build and comprise the HA gene of European fowl source H1N1 hypotype swine influenza viruses SH1 strain and the recombinant vector of NA gene respectively；
By the described recombinant vector containing HA gene and containing NA gene recombinant vector, with comprise respectively PR8 virus PA, PB1,
Six plasmids of PB2, M, NP, NS internal gene transfect 293T cell, the cell after culture transfection together；
The cell conditioned medium of culture is inoculated in mdck cell, occurs collecting virus liquid after pathological changes after mdck cell, detection should
The hemagglutinative titer of virus liquid, if there are hemagglutination activity, and does not have through sequence analysis determination after unexpected mutation, that is, obtains restructuring
Swine flue vaccine strain.
9. a kind of vaccine combination is it is characterised in that comprise：Recombinant Swine influenza vaccines strain described in any one of claims 1 to 3
Inactivated vaccine, and adjuvant or pharmaceutical carrier.
10. the answering in the vaccine of preparation prevention or treatment swine flue of Recombinant Swine influenza vaccines strain described in any one of claims 1 to 3
With.
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| CN201510477308.XA CN106381290B (en) | 2015-08-06 | 2015-08-06 | Recombinate European fowl source H1N1 hypotype swine flu vaccine strain and its preparation method and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112220922A (en) * | 2020-10-19 | 2021-01-15 | 浙江美保龙生物技术有限公司 | Porcine pseudorabies live vaccine immunity-enhancing diluent and use method thereof |
| CN112877300A (en) * | 2021-02-10 | 2021-06-01 | 中国农业大学 | Preparation of G4 avian European subtype H1N1 inactivated vaccine for swine influenza virus |
| CN116665779A (en) * | 2023-04-07 | 2023-08-29 | 南京农业大学 | Method for screening poultry RNA virus source siRNA and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102304495A (en) * | 2011-09-08 | 2012-01-04 | 中国农业科学院上海兽医研究所 | Recombinant influenza virus capable of expressing HA (hemagglutinin) protein with high efficiency and preparation method and application thereof |
-
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| CN102304495A (en) * | 2011-09-08 | 2012-01-04 | 中国农业科学院上海兽医研究所 | Recombinant influenza virus capable of expressing HA (hemagglutinin) protein with high efficiency and preparation method and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| LIANG,H.等: "AIE50784.1", 《GENBANK》 * |
| ZU,R.等: "AGN69292.1", 《GENBANK》 * |
| 闰丽萍等: "中国流行的2009甲型H1N1猪源流感病毒HA和NA基因的分子和遗传特征", 《中国动物传染病学报》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112220922A (en) * | 2020-10-19 | 2021-01-15 | 浙江美保龙生物技术有限公司 | Porcine pseudorabies live vaccine immunity-enhancing diluent and use method thereof |
| CN112877300A (en) * | 2021-02-10 | 2021-06-01 | 中国农业大学 | Preparation of G4 avian European subtype H1N1 inactivated vaccine for swine influenza virus |
| CN116665779A (en) * | 2023-04-07 | 2023-08-29 | 南京农业大学 | Method for screening poultry RNA virus source siRNA and application thereof |
| CN116665779B (en) * | 2023-04-07 | 2024-05-03 | 南京农业大学 | Method for screening poultry RNA virus source siRNA and application thereof |
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