CN106381290B - Recombinate European fowl source H1N1 hypotype swine flu vaccine strain and its preparation method and application - Google Patents
Recombinate European fowl source H1N1 hypotype swine flu vaccine strain and its preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of recombination European fowl source H1N1 hypotype swine flu vaccine strains, which includes: HA the and NA gene of SH1 plants of hypotype swine influenza virus of European fowl source H1N1 and six internal genes of PA, PB1, PB2, M, NP and NS of PR8 virus.The invention also discloses the preparation method and applications of above-mentioned Recombinant Swine influenza vaccines strain.Recombinant Swine influenza vaccines strain of the invention, the virus titer with higher on mdck cell, the recombination high yield cell inactivation vaccine prepared after inactivating can provide fully effective immunoprotection to the attack of Eurasian fowl source H1N1 hypotype swine influenza virus.
Description
Technical field
The present invention relates to technical field of bioengineering more particularly to a kind of recombination European fowl source H1N1 hypotype swine flu vaccines
Strain and its preparation method and application.
Background technique
Swine flu (Swine influenza, SI) is the swine influenza virus belonged to by orthomyxoviridae family's influenza A
(Swine influenza virus, SIV) infection, causes pig the high degree in contact of the symptoms such as high fever, cough, expiratory dyspnea occur
Property respiratory infectious disease, have disease incidence high, the features such as spread speed is fast, and the death rate is low.It has been reported that and shows swine influenza virus sense
Dye swinery also easily causes the mixed infection of other bacteriums and virus simultaneously, such as pig breeding and disordered breathing syndrome, infectiousness
Pleuropneumonia, Streptococcus suis etc., become more complicated epidemic situation, cause great difficulty to disease control.Pig is caused to fall ill at present
SIV hypotype be mainly H1N1, H1N2 and H3N2, main includes European fowl source H1N1 hypotype SIV, classic H1N1 hypotype, again
Group H1N2 hypotype SIV, source of people H3N2 hypotype SIV and recombination H3N2 hypotype SIV etc..Research shows that the airway epithelial cell table of pig
Face exists simultaneously human influenza virus receptor SA α -2,3-Gal (sialic acid α -2,3 galactosides) and avian influenza virus (Avian
Influenza virus, AIV) receptor SA α -2,6-Gal (sialic acid α -2,3 galactosides), in transmission of influenza virus, pig
Be considered as potential " mixer " of viral gene recombination and " couveuse " of new popular strain generation and influenza virus across
More one of the important intermediate host of species barrier propagation.Therefore prevention and control SIV to prevention swinery outburst influenza virus and it is secondary other
While bacterium and virus infection are of great significance, equally the influenza virus of guidance and prevention and control people and fowl is played positive auxiliary
Help effect.
Currently, technically maturation and widely used SI vaccine are mainly the H1N1 hypotype of oily assistant type and the list of H3N2 hypotype
Valence or multivalence inactivated virus vaccine, although to have that efficient, easy to operate, cost is relatively low etc. excellent for SI inactivated virus vaccine
Point, but there is also generate antibody compared with slow, stress reaction is strong the deficiencies of.Especially vaccine kind poison mostly from popular strain,
Once virus leakage occurs for vaccine preparation process, it is easy to induce new epidemic situation, brings very big inconvenience to control and prevention of disease.In addition, mesh
Preceding domestic and international production influenza vaccines still mainly take egg inoculation cultivation, and egg inoculation is although easy to operate, and deposit experience is rich
Richness, but be inevitable and bring time-consuming amount big, consumption is more, and production process is also easy to produce pollution, and quality is difficult to ensure, after receiving poison
Chicken embryo it is difficult the disadvantages of, in addition influenza virus chicken embryo passage inoculation in antigenic variation can often occur, influence vaccine effect.
It has been reported that, the H5N1 avian influenza virus of Hong Kong infection people in 1997 can make chicken embryo generate giant cell lesion so that being difficult to use chicken
Embryo is proliferated generation vaccine.And influenza virus adaptability on common mdck cell is generally lower, this hinders it further to develop
With use.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of recombination European fowl source H1N1 hypotype swine flu vaccine strain, packets
Containing SH1 plants of hypotype swine influenza virus of European fowl source H1N1 of HA and NA gene and PA, PB1, PB2, M, NP of PR8 virus and
Six internal genes of NS, the strain of Recombinant Swine influenza vaccines can not only be by cell culture mass productions, and by the recombination epidemic disease
The inactivated vaccine of seedling strain preparation, which can stimulate in the body short time, generates high-caliber antibody.
In addition, it is also desirable to provide a kind of preparation method of above-mentioned recombination Europe fowl source H1N1 hypotype swine flu vaccine strain and answering
With.
In order to solve the above-mentioned technical problem, the present invention is achieved through the following technical solutions:
In one aspect of the invention, a kind of Recombinant Swine influenza vaccines strain is provided, which includes: European fowl
In HA the and NA gene of SH1 plants of source H1N1 hypotype swine influenza virus and PA, PB1, PB2, M, NP and NS six of PR8 virus
Portion's gene,
The nucleotide sequence of described swine influenza virus SH1 plants of HA gene is selected from:
(1) nucleotide sequence of amino acid sequence shown in SEQ ID NO.1 is encoded；
(2) core for the amino acid sequence that there is 98% or more homology with amino acid sequence shown in SEQ ID NO.1 is encoded
Nucleotide sequence；
The nucleotide sequence of described swine influenza virus SH1 plants of NA gene is selected from:
(1) nucleotide sequence of amino acid sequence shown in SEQ ID NO.2 is encoded；
(2) core for the amino acid sequence that there is 98% or more homology with amino acid sequence shown in SEQ ID NO.2 is encoded
Nucleotide sequence.
Preferably, described swine influenza virus SH1 plants of HA gene has nucleotide sequence shown in SEQ ID NO.3.
Preferably, described swine influenza virus SH1 plants of NA gene has nucleotide sequence shown in SEQ ID NO.4.
In another aspect of this invention, a kind of recombinant vector is additionally provided, European fowl source H1N1 hypotype swine flu disease is included
The nucleotide sequence of malicious SH1 plants of HA or NA gene, the HA gene is selected from:
(1) nucleotide sequence of amino acid sequence shown in SEQ ID NO.1 is encoded；
(2) core for the amino acid sequence that there is 98% or more homology with amino acid sequence shown in SEQ ID NO.1 is encoded
Nucleotide sequence；
The nucleotide sequence of the NA gene is selected from:
(1) nucleotide sequence of amino acid sequence shown in SEQ ID NO.2 is encoded；
(2) core for the amino acid sequence that there is 98% or more homology with amino acid sequence shown in SEQ ID NO.2 is encoded
Nucleotide sequence.
The recombinant vector is recombinant plasmid vector, and empty carrier is PBD carrier.
In another aspect of this invention, a kind of preparation method of Recombinant Swine influenza vaccines strain, including following step are additionally provided
It is rapid:
The recombination that building separately includes the HA gene and NA gene of SH1 plants of hypotype swine influenza virus of European fowl source H1N1 carries
Body；
By the recombinant vector of the recombinant vector of the gene containing HA and the gene containing NA, with separately include 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, virus liquid is collected after lesion occurs in mdck cell, detection should
The hemagglutinative titer of virus liquid is recombinated if there is hemagglutination activity, and after sequence analysis determines no unexpected mutation
Swine flu vaccine strain.
In another aspect of this invention, a kind of vaccine composition is additionally provided, includes: above-mentioned Recombinant Swine influenza vaccines strain
Inactivated vaccine and adjuvant or pharmaceutical carrier.
In another aspect of this invention, a kind of above-mentioned Recombinant Swine influenza vaccines strain is additionally provided in preparation prevention or treatment pig
Application in the vaccine of influenza.
The present invention recombinates European fowl source H1N1 hypotype swine flu vaccine strain to be had more on cell compared with wild strain virus
Strong replication capacity can reach higher virus titer, and be confirmed by mouse immune and challenge viral dosage, the recombinant vaccine strain
The inactivated vaccine head of preparation exempt from after can obtain within the 2nd week higher antibody titer, and can be for Eurasian fowl source H1N1 hypotype pig
The attack of influenza virus provides fully effective immunoprotection.
Detailed description of the invention
The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.
Fig. 1 is NA the and HA gene of the European fowl source H1N1 hypotype swine influenza virus country epidemic strain SH1 of the embodiment of the present invention 1
Pcr amplification product electrophoretogram；
Fig. 2 is NA the and HA gene of the European fowl source H1N1 hypotype swine influenza virus country epidemic strain SH1 of the embodiment of the present invention 1
Bacterium solution PCR qualification result figure；
Fig. 3 is that 2 virus of the embodiment of the present invention is inoculated with the growth curve chart after mdck cell with 0.001MOI；
Fig. 4 is that 2 virus of the embodiment of the present invention is inoculated with the growth curve chart after SPF chicken embryo with 0.001MOI；
Fig. 5 is 2 virus plaques testing result figure of the embodiment of the present invention；
Fig. 6 is that 3 blood clotting of the embodiment of the present invention inhibits (HI) antibody test result figure；
Fig. 7 is 3 neutralize antibody titers figure of the embodiment of the present invention；
Fig. 8 is the lgG antibody titer figure of the embodiment of the present invention 3；
Fig. 9 is that the embodiment of the present invention 3 attacks the 3rd, 5 day mouse lung tissue virus titer histogram after poison；
Figure 10 is the changes of weight figure that the embodiment of the present invention 3 attacks poison mouse after 14 days；
Figure 11 is the survival rate figure that the embodiment of the present invention 3 attacks 14 days mouse after poison.
Specific embodiment
In the following example, test method without specific conditions, usually routinely condition, such as " fine works molecular biosciences
Learn experiment guide " (F.M. Ao Sibai, R.E. James Kingston, the chief editor such as J.G. Sai Deman, Ma Xuejun, Su Yuelong's translates Beijing: section
Learn publishing house, 2004) described in method carry out.
The present invention is with European fowl source H1N1 hypotype swine influenza virus country epidemic strain A/swine/Shanghai/1/2014
(H1N1) (SH1) is material, expands HA and NA gene using RT-PCR, and be cloned into PBD carrier, constructs HA and NA base
The recombinant plasmid of cause.By HA the and NA gene recombination plasmid and derive from strains of influenza viruses A/Puerto Rico/8/1934
(H1N1) 6 internal gene (PB2, PB1, PA, NP, M and NS) recombinant plasmid cotransfection 293T cells of (PR8), successfully construct
Recombinate European fowl source H1N1 hypotype swine flu vaccine strain SH/PR8.Recombinant vaccine strain SH/PR8 is on mdck cell or chicken embryo
Virus titer with higher, the recombination high yield cell inactivation vaccine prepared after inactivating can be to the attack of swine influenza virus SH1
100% immunoprotection is provided.
The building of the European fowl source H1N1 hypotype SIV recombinant strain SH/PR8 of embodiment 1
According to the specification on TRizol kit, European fowl source H1N1 hypotype swine influenza virus country epidemic strain A/ is extracted
The viral RNA of swine/Shanghai/1/2014 (SH1), and reverse transcription synthesizes cDNA, carries out target fragment HA by RT-PCR
With the amplification of 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
Circulation.HA and NA nucleic acid product after amplification identify through 1% agarose gel electrophoresis (see Fig. 1, in Fig. 1, M:DNA molecular mass
Standard (DL2000)；The pcr amplification product (1410bp or so) of 1:NA gene；(1701bp is left for the pcr amplification product of 2:HA gene
It is right)), and product recycles.It is anti-that the glue recovery product containing HA and NA is subjected to digestion by restriction enzyme BspQ Ι respectively
It answers, carries out glue recycling later, then connect with the PBD carrier after BspQ Ι digestion.Product conversion after connection is entered into DH5 α large intestine
In bacillus competent cell.Product after conversion is coated in the LB culture plate containing ampicillin, is cultivated by 37 DEG C
After 12-14h, chooses monoclonal colonies and be put into the LB culture medium containing ampicillin, by 37 DEG C, the water of 180rpm/min
14-16h is cultivated on yawing bed.By bacterium solution PCR identification method filter out positive colony (see Fig. 2, in Fig. 2, M:DNA molecular mass
Standard (DL2000)；A figure is HA bacterium PCR liquid qualification result, and wherein 1-6 qualification result is the positive；B figure is NA bacterium solution PCR identification
As a result, wherein Isosorbide-5-Nitrae, 5,7 qualification results are the positive), and positive colony is sent into handsome biotech firm and carries out sequencing.It will survey
The correct positive plasmid of sequence result is named as PBD-HA and PBD-NA, and according to QIANGEN Plasmid Purification
The specification of Kit extracts positive plasmid.Take 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- of 8 pUC pUCs
PR8NS, PBD-PR8NP) each 1ug mixing, turned referring to liposome Lipofectamine2000 transfection reagent product description
Dye.293T cell is transfected after 48h, collect supernatant and is inoculated into mdck cell, after obvious lesion (CPE) occurs in cell
Collect virus liquid.The detection of hemagglutinative titer is carried out to the virus liquid of collection, viral RNA is extracted, after reverse transcription synthesizes cDNA, with this
CDNA is eight genetic fragments of the virus of template PCR amplifications rescue, and carries out the measurement and comparison of gene fragment order.It collects
The supernatant that MDCK is proliferated after inoculation 72h detects its hemagglutinative titer with hemagglutination test, and blind passage three generations continues to test its blood clotting effect
Valence.By sequencing result and blood coagulation tests the result shows that successfully having saved out recombinant virus SH/PR8.
2 recombinant strain SH/PR8 of embodiment is with original wild strain SH1 on cell and chicken embryo compared with replication capacity
Influenza infection liquid is prepared, is added TPCK (protease).Dilution virus, according to 10 times of dilution methods, with virus
Liquid doubling dilution virus is infected, is mixed well, and by virus from 10-1It is diluted to 10-11, paving successively is added in the virus diluted
Have in 96 orifice plates of mdck cell, is put into 37 DEG C, 5% CO2Cell incubator culture 72h observes cytopathy (CPE) feelings
Condition, and record and check experimental result as a result, cell supernatant is sucked out and carries out Blood coagulation test experiment, calculate TCID50Value.
Influenza infection liquid is prepared, SH/P8 and original strain SH1 virus virus infection liquid are diluted, according to every
The virus quantity aseptic inoculation that hole is inoculated with 0.001MOI enters in 12 porocyte culture plates, and 12 porocyte culture plates cover with density about
The mdck cell of 90 ﹪ is spaced 12h, for 24 hours, 36h, 48h, sterile collection cell supernatant respectively after 60h, 72h, when detecting different
Between section HA hemagglutinative titer.The result shows that recombination SH/PR8 virus compares, prime strain SH1 has higher multiple on mdck cell
Ability processed, HA potency reaches 2 after 60h9(see Fig. 3).
With viral dilution by viral dilution at 100TCID50, according to 100TCID50Virus titer by virus sterilely
It is inoculated into the SPF chicken embryo of 9-11 age in days, chicken embryo is put into 37 DEG C of incubator, keep suitable humidity and across phase
Chicken embryo is stirred with the period, is spaced 12h, for 24 hours, 36h, 48h are sterile respectively after 60h, 72h to collect chick embryo allantoic liquid, 3000rpm,
Supernatant is collected after centrifugation in 5min, and HA Blood coagulation test experiment is carried out to it.The results show that recombination SH/PR8 virus is relative to original
Wild strain SH1 has higher replication capacity in SPF chicken embryo (see Fig. 4).
(LMP) Ago-Gel of 2% low melting point is prepared, is put into 37 DEG C of cell incubators after high pressure sterilization.It mentions
It is preceding that mdck cell is inoculated in 6 porocyte culture plates, when cell culture is to when covering with 6 porocyte culture plates, configure virus infection
Simultaneously TPCK protease is added in liquid, according to 10 times of size doubling dilution virus liquids.Mdck cell two is cleaned with PBS phosphate buffer
Side, then cleaned one time with DMEM culture medium.The virus liquid diluted is separately added into 6 well culture plates according to every hole 1ml, it will be sick
Venom is put into 37 DEG C, in 5% CO2 cell incubator, viruses adsorption is allowed to enter in mdck cell, intermediate mixed at interval of 30min
Even viral dilution, allows viruses adsorption 1.5h or so.Virus liquid, DMEM culture medium cleaning one are discarded from 6 porocyte culture plates
Time.It is added in 6 well culture plates according to the amount of the 2%LMP of every orifice plate 4ml.2h or so is stood at room temperature, until 2%LMP is completely solidifying
After Gu, 6 porocyte culture plates are tipped upside down on 37 DEG C, in 5% CO2 cell incubator.It cultivates to 48h or so observation cytopathy
Situation.Until after there is the obvious and plaque of suitable size, after the 4% fixed 1.5h-2h of formaldehyde is cultivated and is added in termination.It discards
Formalin, the sterile Ago-Gel for choosing low melting point, the gentian violet dyeing liquor that about 2ml is added in every hole is fixed, after about 15min
Dyeing liquor is recycled, flowing water observes plaque form after cleaning up.The results show that recombination SH/PR8 strain is relative to original wild strain
The virus plaques size that SH1 is formed is bigger, shows that recombinating SH/PR8 has higher replication capacity (see Fig. 5).In Fig. 5, A:
Normal control；B: original wild strain SH1；C: recombinant strain SH/PR8.
3 Europe fowl source H1N1 hypotype SIV of embodiment recombinates the preparation of high yield inactivated vaccine and its commenting for immune protection effectiveness
Valence
By original wild strain SH1 and recombinant strain SH/PR8 according to the dose inoculation of 0.001MOI in mdck cell culture bottle
In, after massive amplification virus, cell supernatant, 3000rpm are collected, 10min centrifugal separating cell fragment simultaneously collects supernatant.It is right
It expands obtained original wild strain SH1 and recombinant virus SH/PR8 and carries out the test of HA Blood coagulation test, detect its viral hemoagglutination potency.
Choose the mineral adjuvant and Montanide of the Montanide ISA 61VG Water-In-Oil of match BIC Corp, France production
The oil-in-water mineral adjuvant of ISA 15A VG has done two groups and has been compared.Virus is passed through into 0.1% formalin, 37 DEG C of inactivation 24-
48h, the virus after inactivation after 3 generation of blind passage, detect HA hemagglutinative titer on mdck cell respectively.By the virus after inactivation according to cream
The requirement for changing specification emulsifies virus, the disease that the adjuvant of 61VG is recombinated according to 40% antigen of weight and the emulsification of 60% adjuvant ratio
Poison, the virus that the adjuvant of 15AVG is recombinated according to the weight ratio emulsification of weight 15% and 85%.
The SPF grade female BAl BIc/c mouse for choosing 6-8 week old, being randomly divided into 5 groups, first group and second group is to choose respectively
The 61VG and oil-in-water 15VG of the vaccine Water-In-Oil of different adjuvants, third group and the 4th group are to choose that adjuvant emulsion is not added respectively
Inactivation recombinant virus, the 5th group be immune DMEM control group.Immune programme are as follows: subcutaneous injection one is exempted from, and interval is subcutaneous after 2 weeks
Injection two is exempted from, and poison was attacked after 2 weeks in interval.Immunization ways are the inoculation of subcutaneous multi-point injection, according to the inoculum concentration point of every mouse 200ul
Not Mian Yi 5 groups of mouse, first group be SH/PR8+61VG group；Second group is SH/PR8+15VG group；Third group is SH/PR8+DMEM
Group (being that 40:60 is mixed by antigen and DMEM mass ratio according to the emulsification requirement of 61VG)；4th group is SH/PR8+DMEM
Group (according to the emulsification requirement of 15AVG, antigen is mixed with DMEM mass ratio for 15:85；5th group is DMEM control group.
The blood sampling of eye socket clump is carried out to mouse and collects each group mice serum, the surrounding of each group mouse after the first exemption is interior at interval of one
Week, acquisition was primary respectively, will be collected into serum and carries out blood clotting Inhibition test respectively and neutralize experiment.By each group mice serum and RDE
(receptor destroying enzyme) is mixed according to the ratio of volume ratio 1:3,37 DEG C of incubations 18-20h, 56 DEG C of inactivation 30min-60min.Reference
" national influenza central standard operating instruction " carries out blood clotting inhibition (HI) experiment, records experimental result.The result shows that as head exempts from
The extension of time, HI antibody titer are gradually increased, and 2, which exempt from rear 2nd week antibody titer, is up to 29Left and right, wherein organizing a SH/PR8
+ 61VG water-in-oil adjuvant group generates HI antibody titer highest.Two SH/PR8+15A oil-in-waters of group head exempt to reach for the 3rd week compared with
High blood clotting inhibits (HI) antibody titer (see Fig. 6).Exempt from rear interval time (week) in Fig. 6, headed by abscissa, ordinate is blood
It is solidifying to inhibit potency (Log2)；SH/PR8+61VG is the immune group for adding water-in-oil adjuvant；SH/PR8+15AVG is addition oil-in-water
The immune group of adjuvant；SH/PR8+DMEM is the immune group for not adding adjuvant；DMEM is nonimmune group.
It prepares virus infection liquid and is added dual anti-(penicillin and streptomycin mixed liquor), according to virus infection liquor ratio TPCK volume ratio
TPCK is added into infection liquid for the ratio of 1000:2.Configuration virus infection liquid is simultaneously added dual anti-(penicillin and streptomycin mixed liquor), will
56 DEG C of the serum heat inactivation 30min of collection, by the serum after inactivation according to 1:10,1:20,1:40,1:80,1:160,1:320,
The ratio of 1:640,1:1280 are diluted with infection liquid, and each sample does 2 repetitions, are diluted original influenza virus SH1 and are arrived
100TCID50Virus titer, serum and 50ul after 50ul dilution is added in every hole in 96 porocyte culture plates
(100TCID50) virus liquid, 37 DEG C of incubation 2h.Serum and viral mixed liquor are added drop-wise to the 96 holes culture for covering with mdck cell
In plate, the positive control and the not negative control of virus inoculation of a virus inoculation are set up.Cytopathy feelings are observed after 48h
Condition detects serum neutralize antibody titers, and cell supernatant is sucked out and carries out blood coagulation tests review experimental result and records.As a result table
Bright, with the extension of First immune time, neutralize antibody titers are gradually increased, and 2, which exempt from rear 2nd week neutralize antibody titers, is up to 103
More than, second group of SH/PR8+15A oil-in-water, which exempts from the 2nd week neutralize antibody titers in head, can reach 103Left and right (see Fig. 7).
Original wild strain SH1 is purified by 30%-60% sucrose gradient ultracentrifugation, referring to " national influenza central standard
Operating instruction ", lgG antibody test is carried out with ELISA, specific step is as follows.
1, it is coated with: the viral SH1 of purifying carbonate buffer solution being coated with ELISA ELISA Plate, every hole package amount is
200ng/100 μ L, 4 DEG C overnight.
2, it washs: being washed 3 times with the PBST containing 5 ‰ tween-20,200 holes μ L/.5 minutes every time.
3, close: 37 DEG C of 5% skimmed milk (BSA) are incubated for 2 hours, 200 holes μ L/.
4, it washs: being washed 3 times with the PBST containing 5 ‰ Tween-20,200 holes μ L/.5 minutes every time.
Plus primary antibody 5: 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.
6, it washs: being washed 3 times with the PBST containing 5 ‰ Tween-20,200 holes μ L/.5 minutes every time.
7, secondary antibody is added: after diluting HRP sheep anti-mouse igg by 1:10000 times with 5% skimmed milk, enzyme mark is added in every 100 μ L of hole
Plate, 37 DEG C are incubated for 1 hour.
8, it washs: being washed 3 times with the PBST containing 5 ‰ Tween-20,200 holes μ L/.5 minutes every time.
9, develop the color: 50 μ L TMB developing solutions are added in every hole, are protected from light incubation at room temperature 15 minutes.
10, terminate: 50 μ L 2M H are added in every hole2SO4。
11, OD value detects: microplate reader measures OD450It is worth IgG antibody potency.Judgment criteria is referring to " national influenza central standard
Operating instruction "
The results show that IgG antibody potency is gradually increased with the extension of First immune time, 2 exempt from rear 2nd week antibody titer most
Up to 105More than, exempt to reach 10 on the 3rd week in head wherein organizing two SH/PR8+15A oil-in-waters5Left and right (see Fig. 8).
Original wild strain SH1 after amplification is subjected to virus titer TCID50Detection, according to every mouse 1x105TCID50
Virus inoculation amount attack poison, wherein control group choose 6 do not attack poison as normal mouse compare.Mouse spirit shape is observed after attacking poison
State takes mouse lung to carry out titration of virus after attacking malicious 3d and 5d (result is shown in Fig. 9).Weigh 14 days weight situations of change of mouse (see
Figure 10) and survival rate (see Figure 11).The result shows that nonimmune group of continued weight decline after attacking poison, mouse occurs 100% after 6 days
, there is not death in death, and immune group, and showing can be for European fowl source using the inactivated vaccine of recombinant vaccine strain preparation
The attack of H1N1 hypotype swine influenza virus wild strain SH1 provides 100% immunoprotection.
Embodiments of the present invention above described embodiment only expresses, the description thereof is more specific and detailed, but can not
Therefore limitations on the scope of the patent of the present invention are interpreted as.It should be pointed out that for those of ordinary skill in the art,
Without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection model of the invention
It encloses.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Claims (4)
1. a kind of Recombinant Swine influenza vaccines strain, which is characterized in that the recombinant vaccine strain includes: European fowl source H1N1 hypotype swine flu
Viral SH1 plants of HA and NA gene and six internal genes of PA, PB1, PB2, M, NP and NS of PR8 virus, the pig stream
Amino acid sequence shown in SH1 plants of Influenza Virus of HA gene coding SEQ ID NO.1, nucleotide sequence such as SEQ ID NO.3 institute
Show；Amino acid sequence shown in described swine influenza virus SH1 plants of NA gene coding SEQ ID NO.2, nucleotide sequence such as SEQ
Shown in ID NO.4.
2. a kind of preparation method of Recombinant Swine influenza vaccines strain, which comprises the following steps: building separately includes Europe
The HA gene of SH1 plants of fowl source H1N1 hypotype swine influenza virus and the recombinant vector of NA gene, the HA gene encode SEQ ID
Amino acid sequence shown in NO.1, nucleotide sequence is as shown in SEQ ID NO.3；The NA gene encodes SEQ ID NO.2 institute
Show amino acid sequence, nucleotide sequence is as shown in SEQ ID NO.4；The empty carrier of the recombinant vector is PBD carrier；
By the recombinant vector of the recombinant vector of the gene containing HA and the gene containing NA, with separately include PR8 virus PA, PB1, PB2,
M, six plasmids of NP, NS internal gene transfect 293T cell, the cell after culture transfection together；
The cell conditioned medium of culture is inoculated in mdck cell, collects virus liquid after lesion occurs in mdck cell, detects the virus
The hemagglutinative titer of liquid obtains Recombinant Swine stream if there is hemagglutination activity, and after sequence analysis determines no unexpected mutation
Influenza vaccine strain.
3. a kind of vaccine composition, characterized by comprising: Recombinant Swine influenza vaccines described in claim 1 strain inactivated vaccine,
And adjuvant or pharmaceutical carrier.
4. application of the strain of Recombinant Swine influenza vaccines described in claim 1 in the vaccine of preparation prevention or treatment swine flu.
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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 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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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 |
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| AGN69292.1;Zu,R.等;《GenBank》;20130930;全文 * |
| AIE50784.1;Liang,H.等;《GenBank》;20140821;全文 * |
| 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;闰丽萍等;《中国动物传染病学报》;20100831;第18卷(第4期);第13-19页 * |
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