CN107375918A - Recombinate H1N1 hypotype swine flu bivalent inactivated vaccines and its preparation method and application - Google Patents
Recombinate H1N1 hypotype swine flu bivalent inactivated vaccines and its preparation method and application Download PDFInfo
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Abstract
The invention discloses one kind to recombinate H1N1 hypotype swine flu bivalent inactivated vaccines, includes European fowl source H1N1 hypotypes swine influenza virus and classic H1N1 hypotypes swine flue antigen.The invention also discloses the preparation method and application of above-mentioned swine flu bivalent inactivated vaccine.The restructuring H1N1 hypotype swine flu bivalent inactivated vaccines of the present invention; body can be induced to produce higher serum blood clotting and suppress (HI) antibody, neutralizing antibody and specific IgG antibody titer, and the immunoprotection of the attack offer 100% of European fowl source and classic H1N1 hypotypes swine influenza virus can be provided.
Description
Technical field
The present invention relates to technical field of bioengineering, more particularly to a kind of restructuring H1N1 hypotype swine flu bivalent inactivated vaccines and its system
Preparation Method and application.
Background technology
Swine influenza abbreviation swine flu (Swine Influenza, SI), is the influenza belonged to by orthomyxoviridae family's influenza A
Virus infection, causes pig high fever, cough, expiratory dyspnea, high degree in contact respiratory infectious disease occur, have the incidence of disease it is high,
The features such as spread speed is fast, the death rate is low, not only seriously endanger China's pig industry development, also endanger the mankind health (Shinde,
Bridges et al., 2009), thus trigger the sanitarian concern in the whole world.Because porcine respiratory surface epithelial cell is present simultaneously
Human influenza virus's acceptor and avian influenza virus acceptor, therefore be considered as that influenza virus producer is matched somebody with somebody and across the important of kind of propagation again
Intermediate host (Ma, Kahn et al., 2008).At present both at home and abroad with H1N1, H1N2 and H3N2 hypotype swine influenza virus (Swine
Influenza Virus, SIV) be main epidemic strain, and different strains can producer restructuring cause variant generation (Yang Shuai etc.,
2003), therefore the threat of vigilant and close attention SIV to public safety becomes extremely important.
Along with Spain break out the big influenza of the mankind, 1918 the U.S., Hungary and first Chinese report SI (Webster et al.,
2002).Nineteen thirty, H1N1 hypotypes SIV (Shope et al., 1931) is separated to from the swinery in the U.S. first, then in people
Also isolated similar virus, this H1N1 hypotypes SIV and similar related virus are named as classic H1N1 in group
Hypotype SIV.In nearly 50 years afterwards, SIV is nearly all mainly worldwide popular with classic H1N1 hypotypes.Directly
By 1979, European fowl source H1N1 hypotypes SIV appearance, classic H1N1 hypotypes SIV gradually disappeared from European swinery
(Pensaert et al., 1981).Before 1998, classic H1N1 hypotypes SIV is still most important in north America region swinery
SIV (Hinshaw et al., 1978；Chambers et al., 1991；Olsen et al., 2000).1979, in European swinery
There is the H1N1 subtype influenza virus in fowl source.This virus antigenicity and gene order and classic H1N1 hypotypes SIV not phase
With (Scholtissek et al., 1983；Brown et al., 1998), and the classic H1N1 hypotypes SIV in swinery is replaced rapidly
As Major Epidemic strain.Currently, Europe swinery in fowl source H1N1 hypotype SIV and H3N2 and H1N2 hypotypes SIV
And deposit simultaneously common popular.Before 2005, classic H1N1 hypotypes SIV is SIV hypotypes main in Chinese commodity swinery, mesh
It is preceding still popular in swinery.In recent years, European fowl source H1N1 hypotypes SIV is separated and is increasingly becoming in the swinery in China
Main popular strain.
Vaccine inoculation at present is still one of major way for controlling influenza.Although traditional swine flu inactivated virus vaccine has height
Effect, the advantages that simple to operate, cost is relatively low, technological reserve is ripe, but there is also produce antibody is relatively slow, stress reaction is strong etc.
Deficiency.Particularly vaccine kind poison comes from popular strain mostly, once virus leakage occurs for vaccine preparation process, easily induces new
Epidemic situation, bring very big inconvenience to control and prevention of disease.
In addition, the preparation method of influenza vaccines mainly still uses cultivation in the chick embryo.Because chicken embryo is frequently as main Jie of culture virus
Matter, thus it is higher to the quality requirement of chicken embryo, and SPF level chicken embryos are typically primary selection.But due to SPF kind fowl raising difficulty
Greatly, cost is high, the expensive increase for usually bringing vaccine cost.The great outburst of influenza virus and global season in the past few years
The wilderness demand of property influenza vaccines, the large-scale production for awarding chicken embryo vaccine bring immense pressure, and the vaccine based on chicken embryo
Production process is labor intensive process, it is necessary to long-term plan and annual long-term production cycle process (Gerdil C et al.,
2003).Egg inoculation production vaccine is although simple to operate, and deposit is experienced, but inevitably brings time-consuming amount big, disappears
Consumption is more, and production process is also easy to produce pollution, and quality is difficult to ensure, receives the shortcomings of chicken embryo after poison is difficult, in addition influenza virus
Antigenic variation can often occur in chicken embryo passage inoculation, influence vaccine effect.If popular virus is HPAI (High Pathogenic AI) virus,
Inoculated into chick embryo can then cause large quantities of death to have a strong impact on the production (Takada A et al., 1999) of normal vaccine.
The content of the invention
The invention solves the technical problem of the current swine flu vaccine for lacking highly effective and safe, there is provided one kind restructuring H1N1 hypotypes pig stream
Feel bivalent inactivated vaccine, it includes the immunoprotection antigen of European fowl source and classic H1N1 hypotypes swine influenza virus, can be two kinds to this
The attack of virus provides good immune protection effectiveness.
A kind of in addition, it is also desirable to provide preparation method and application of above-mentioned restructuring H1N1 hypotypes swine flu bivalent inactivated vaccine.
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, there is provided a kind of swine flu bivalent inactivated vaccine, include European fowl source H1N1 hypotype swine flus
Viral and classic two immunoprotection antigens of H1N1 hypotypes swine influenza virus.
Preferably, the European fowl source H1N1 hypotypes swine flue antigen flows for the restructuring Europe fowl source H1N1 hypotypes pig of inactivation
Influenza virus vaccine strain SH1/PR8.The SH1/PR8 Recombinant Swine influenza vaccines strains contain European fowl source H1N1 hypotype SIV epidemic strains
A/swine/Shanghai/1/2014 (H1N1) (abbreviation SH1) HA and NA genes, wherein, the amino acid sequence of HA genes
As shown in SEQ ID NO.1, nucleotide sequence as shown in SEQ ID NO.3, the amino acid sequence such as SEQ ID NO.2 of NA genes
Shown, nucleotide sequence is as shown in SEQ ID NO.4.
Preferably, the classic H1N1 hypotypes swine flue antigen is the restructuring allusion H1N1 hypotype swine influenza virus epidemic diseases of inactivation
Seedling strain G11/PR8.The G11/PR8 Recombinant Swine influenza vaccines strains contain classic H1N1 hypotypes SIV epidemic strains A/swine/
Guangdong/1/2011 (H1N1) (abbreviation G11) HA and NA genes, wherein, the amino acid sequence such as SEQ of HA genes
Shown in ID NO.5, nucleotide sequence as shown in SEQ ID NO.7, the amino acid sequences of NA genes as shown in SEQ ID NO.6,
Nucleotide sequence is as shown in SEQ ID NO.8.
The vaccine also includes oil emulsion adjuvant.
In another aspect of this invention, the preparation method of a swine flu bivalent inactivated vaccine is additionally provided, is comprised the following steps：
After European fowl source H1N1 hypotypes swine influenza virus and classic H1N1 hypotypes swine influenza virus are bred on cell respectively,
Inactivator inactivation is separately added into, adds immunologic adjuvant, swine flu bivalent inactivated vaccine is made in emulsification.
Preferably, the European fowl source H1N1 hypotypes swine influenza virus is the European fowl source H1N1 hypotype swine influenza virus epidemic diseases of restructuring
Seedling strain SH1/PR8；The classic H1N1 hypotypes swine influenza virus is the classic H1N1 hypotypes swine influenza virus vaccine strain of restructuring
G11/PR8。
Preferably, the cell is mdck cell.Mdck cell is a kind of epithelial cell of adherent growth, is by Madin
With Darby in the Madin-Darby canine for separating and setting up from the kidney of a bull cocker sleuth in 1958
Kidney cell lines (abbreviation mdck cell).Present invention preferably employs mdck cell culture swine influenza virus, chicken can be overcome
Embryo produces the shortcomings that vaccine.
The inactivator includes formaldehyde, beta-propiolactone (BPL), binary ethylenimine (BEI) or Polyhaxemethylenguanidine Hydrochloride (PHMG).
Preferably inactivator of the invention is formaldehyde.
In another aspect of this invention, additionally provide a kind of swine flu bivalent inactivated vaccine and prepare the medicine of prevention or treatment swine flu
Application in thing.
The restructuring H1N1 hypotype swine flu bivalent inactivated vaccines of the present invention, include European fowl source and classic H1N1 hypotypes swine flu disease
The immunoprotection antigen of poison, is shown by mouse immune and challenge test result, and mouse is after the restructuring bivalent inactivated vaccine is immunized
The serum blood clotting that can reach higher for 4 weeks suppresses (HI) antibody, neutralizing antibody and specific IgG antibody titer, and can pin
Attack to European fowl source and classic H1N1 hypotypes swine influenza virus provides 100% immunoprotection.Moreover, the restructuring swine flu
Bivalent inactivated vaccine can largely be produced by cell culture, be advantageous to industrialization and market application.
Brief description of the drawings
The present invention is further detailed explanation with reference to the accompanying drawings and detailed description.
Fig. 1 is the testing result figure of HI antibody titers in the mice serum of the embodiment of the present invention 3；
Fig. 2 is the testing result figure of the mice serum neutralize antibody titers of the embodiment of the present invention 3；
Fig. 3 is the titration of virus result figure that the embodiment of the present invention 3 attacks the 3rd, 5 day mouse lung tissue after poison；
Fig. 4 is Pathologic changes (200 ×) figure that the embodiment of the present invention 3 attacks the 5th day each group mouse lung tissue after poison；
Fig. 5 is the changes of weight figure that the embodiment of the present invention 3 attacks 14 days each group mouse after poison；
Fig. 6 is the survival results figure that the embodiment of the present invention 3 attacks poison each group mouse after 14 days.
Embodiment
In the following example, the experimental method of unreceipted actual conditions, generally routinely condition, such as《Fine works molecular biology is real
Test guide》(F.M. Ao Sibai, R.E. James Kingstons, J.G. Sai Deman etc. are edited, Ma Xuejun, and Su Yuelong's translates north
Capital：Science Press, 2004) method described in is carried out.
In research before, this laboratory utilizes high yield and the human influenza virus A/Puerto Rico/8/34 (H1N1) of Attenuation
(abbreviation PR8) is skeleton, with reference to the popular strain HA in current place and NA Protection of antigen genes, passes through reverse genetic manipulation skill
Art, successfully construct respectively containing European fowl source H1N1 hypotype SIV epidemic strains A/swine/Shanghai/1/2014 (H1N1) (referred to as
SH1 the restructuring SIV vaccine strain SH1/PR8 of HA and NA genes), and contain classic H1N1 hypotypes SIV epidemic strains A/swine/
The restructuring SIV vaccine strain G11/PR8 of Guangdong/1/2011 (H1N1) (abbreviation G11) HA and NA genes, the two plant weights group base
Because of engineered vaccine strain, there is stronger virus multiplication ability on mdck cell compared to more original strain, can obtain higher
Hemagglutinative titer and virus titer.SH/PR8 and G11/PR8 two strain virus vaccine strain of the invention by restructuring, on mdck cell
After propagation, bivalent inactivated vaccine is prepared into by oil emulsion adjuvant emulsification.The evaluation of immune protection effectiveness is carried out in Mice Body, is ground
Study carefully as shown by data：Immune restructuring bivalent inactivated vaccine can effective stimulus animal body produce higher antibody titer, can effectively support
The anti-attack from European fowl source and classic H1N1 hypotypes SIV simultaneously provides good immunoprotection.
The European fowl source H1N1 hypotypes SIV recombinant strains SH1/PR8 of embodiment 1 and classic H1N1 hypotypes SIV restructuring poison
Strain G11/PR8 structure
With European fowl source H1N1 hypotype swine influenza viruses country epidemic strain A/swine/Shanghai/1/2014 (H1N1) (SH1) for material
Material, HA and NA genes (SEQ ID NO.3 and SEQ ID NO.4) are expanded using RT-PCR, and be cloned into PBD
In carrier, the recombinant plasmid of HA and NA genes is built.By HA the and NA gene recombination plasmids and from influenza virus
Strain A/Puerto Rico/8/1934 (H1N1) (PR8) 6 internal genes (PB2, PB1, PA, NP, M and NS) weight
Group plasmid co-transfection 293T cells, successfully construct the European fowl source H1N1 hypotype swine flu vaccine strains SH1/PR8 of restructuring.This is heavy
Group vaccine strain SH1/PR8 has higher virus titer and hemagglutinative titer on mdck cell or chicken embryo.
With classic H1N1 hypotypes swine influenza virus country epidemic strain A/swine/Guangdong/1/2011 (H1N1) (G11) for material
Material, HA and NA genes (SEQ ID NO.7 and SEQ ID NO.8) are expanded using RT-PCR, and be cloned into PBD
In carrier, the recombinant plasmid of HA and NA genes is built.By HA the and NA gene recombination plasmids and from influenza virus
Strain A/Puerto Rico/8/1934 (H1N1) (PR8) 6 internal genes (PB2, PB1, PA, NP, M and NS) weight
Group plasmid co-transfection 293T cells, successfully construct the classic H1N1 hypotypes swine flu vaccine strain G11/PR8 of restructuring.The restructuring epidemic disease
Seedling strain G11/PR8 has higher virus titer and hemagglutinative titer on mdck cell or chicken embryo.
Embodiment 2 recombinates the preparation of H1N1 hypotype swine flu bivalent inactivated vaccines
The Gu of the European fowl source H1N1 hypotypes swine influenza virus (SH/PR8) of a large amount of propagation restructuring and restructuring on mdck cell
Allusion quotation H1N1 hypotypes swine influenza virus (G11/PR8).By the virus of propagation respectively by 0.1% formalin, 37 DEG C of inactivation 48h, go out
HA hemagglutinative titers are detected after the generation of viral blind passage 3 after work respectively and (if inspection does not measure HA hemagglutinative titers, illustrate that inactivation of viruses is
Safety, the emulsification procedure of next step can be entered).Restructuring inactivation of viruses SH/PR8 and G11/PR8 are passed through into oil emulsion adjuvant respectively
(MONTANIDETMISA 61VG, match BIC Corp, France) emulsified, the virus after inactivation is with reference to adjuvant explanation
The requirement of book, using adjuvant and antigen weight ratio as 60：40 emulsify and are prepared into inactivated vaccine, draw the vaccine drop after a drop emulsification
Enter to detect the effect of its emulsification in water, be not easy to scatter if dropped in water, the state slowly discharged is presented, then shows emulsifying effectiveness
It is good.
Embodiment 3 recombinates the immune protection effectiveness evaluation of H1N1 hypotype swine flu bivalent inactivated vaccines
1st, bivalent inactivated vaccine is carried out as model using mouse and is immunized and attacks the malicious program evaluated
(1) choose SPF levels female BAl BIc/c mouse of 6-8 week old health, be randomly divided into 5 groups, group one, group two, group three,
Each 11 of group four, organize 5 22 (wherein 11 are served only for challenge viral dosage, and 11 are used as environmental control).According to the requirement of SPF levels
Feeding management mouse, the normal 3d that raises allows mouse to be familiar with feeding environment, and orbital vein blood sampling collects mice serum as blank pair
According to；
(2) the specific immune programme for children of mouse of experimental group (group one, group two, group three, group four, group five) is as shown in table 1 below.It will exempt from
The μ l of epidemic disease 200 inactivated vaccine (SH1/PR8+G11/PR8)+61VG is used as immune group one；By immune 200 μ l inactivated vaccine
(SH1/PR8+61VG) it is used as immune group two；Using immune 200 μ l inactivated vaccine G11/PR8+61VG as immune group three；
Using 200 μ l DMEM as immune group four；Mock is group five not attack the normal control of poison.All small mouse's heads of immune group are exempted from
Same dose and the secondary immunity of same way are carried out after 2nd week.
The packet of the immunoprotection experiment of table 1
(3) gather weekly after small mouse's head is exempted from and separate serum in 4 weeks, Blood collection is orbital vein from blood sampling.Suppressed by blood clotting
Experiment (HI) detection head exempt from after the 1st, 2 week and two exempt from after HI antibody levels in serum are immunized within the 1st, 2 week in mouse；Using end
Specific IgG antibodies are horizontal in point dilution ELISA method detection mice serum；Immunized mice serum is detected on mdck cell
Neutralize antibody titers.
(4) the 4th week after head exempts from, collunarium is carried out to every group of mouse with original strain SH1 and mouse adapted strain G11 and attacks poison, attacks toxic agent
Measure as 1 × 105TCID50/ only.
(5) the mouse state of mind is observed daily after attacking poison, records mouse weight situation of change and death state in 14 days.After attacking poison
Take out every group of mouse lung tissue within 3rd day and the 5th day, for titration of virus and make tissue pathological slice.
2nd, mice serum blood clotting suppresses the detection of HI antibody titers
(1) every group after immune 4 weeks of collection of mouse blood room is stood into 2h in temperature, then blood is put into 4 DEG C of processing overnight, will
Blood is put into 4000rpm in 4 DEG C of centrifuges and centrifuges 15min, and the supernatant collected after centrifugation is mice serum sample；
(2) receptor destroying enzyme (RDE) processing will be added, according to volume ratio 3 in blood serum sample：1 fully mixes, 37 DEG C of incubations
18-24h, 56 DEG C of incubation 30-60min, to remove nonspecific agglutination element.
Reference《National influenza central standard operational procedure》Carry out blood clotting and suppress HI experiments, log (see Fig. 1).
As a result show：Immune restructuring bivalent inactivated vaccine SH1/PR8+G11/PR8+61VG can induce body to produce high level and be directed to
Protovirus Europe fowl source virus SH1 and classic viral G11 HI antibody, HI antibody titers gradually increase in 4 weeks after immune,
And reached 2 at immune 4th week10Left and right.
In Fig. 1, exempt from headed by abscissa rear interval time (week), ordinate is that blood clotting suppresses potency (log2)。SH1/PR8+
G11/PR8+61VG is the restructuring bivalent inactivated vaccine of addition adjuvant；SH1/PR8+61VG is the monovalent inactivated vaccine of addition adjuvant；
G11/PR8+61VG is the monovalent inactivated vaccine of addition adjuvant；NV/NC is nonimmune and nonimmune control group.
3rd, the detection of mice serum neutralize antibody titers
Serum neutralize antibody titers are detected on mdck cell, concrete operations are as follows：
(1) mdck cell is inoculated with 96 porocyte culture plates, treats that cell covers with 96 well culture plates；
(2) configuration virus infects liquid and added dual anti-(penicillin and streptomycin mixed liquor)；
(3) the serum of collection is inactivated into 30min by 56 DEG C of heat；
(4) by the serum after inactivation according to 1：20,1：40,1：80,1：160,1：320,1：640,1：1280,
1:2560 ratio carries out gradient dilution with infection liquid, and each sample does 2 repetitions；
(5) original influenza virus SH1 and G11 to 100TCID is diluted50Virus titer；
(6) the serum and 50 μ l (100TCID added in 96 porocyte culture plates per hole after 50 μ l dilutions50) virus liquid,
37 DEG C of cell culture incubators act on 2h；
(7) serum and viral mixed liquor are added drop-wise in 96 well culture plates for covering with mdck cell, set up positive control and feminine gender
Control wells, 48h is acted in cell culture incubator；
(8) observe cytopathy situation (CPE) and record result, suction out cell supernatant and carry out HA experiment review experimental results.
Neutralizing antibody testing result is shown (see Fig. 2)：The bivalent vaccine SH1/PR8+G11/PR8+61VG of immune restructuring inactivation
Induced animal body produces the neutralizing antibody for European fowl source virus SH1 and classic viral G11 of higher level, after immune
Neutralize antibody titers gradually increase in 4 weeks, and reach 1 × 10 at immune 4th week3Left and right.
In fig. 2, rear interlude (week) is exempted from headed by abscissa, ordinate is neutralize antibody titers (Log10)。SH1/PR8+
G11/PR8+61VG is the restructuring bivalent inactivated vaccine of addition adjuvant；SH1/PR8+61VG is the monovalent inactivated vaccine of addition adjuvant；
G11/PR8+61VG is the monovalent inactivated vaccine of addition adjuvant；NV/NC is nonimmune and nonimmune control group.
4th, in mice serum specific IgG antibodies detection
Original strain SH1 and G11, detailed process reference are purified by 30%-60% sucrose gradient ultracentrifugations《National influenza center
Standard practice instructions》.
Collection head exempts from each group mice serum detection specific IgG antibody titer in 4 weeks, passes through the A/swine/Shanghai/1 of purifying
/ 2014 (H1N1) and A/swine/Guangdong/1/2011 (H1N1) viruses are antigen, using end dilution indirect ELISA method
Detect 4 groups of mice serum antibody titers.
(1) it is coated with：The protovirus SH1 and G11 of purifying is coated with ELISA ELISA Plates with carbonate buffer solution, wrapped per hole
Measured as 200ng/100 μ L, 4 DEG C overnight.
(2) wash：Washed 3 times with the PBST containing 5 ‰ tween-20,200 μ L/ holes.5 minutes every time.
(3) close：37 DEG C of 5% skimmed milk (BSA) is incubated 2 hours, 200 μ L/ holes.
(4) wash：Washed 3 times with the PBST containing 5 ‰ Tween-20,200 μ L/ holes.5 minutes every time.
(5) primary antibody is added：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) wash：Washed 3 times with the PBST containing 5 ‰ Tween-20,200 μ L/ holes.5 minutes every time.
(7) secondary antibody is added：1 is pressed with 5% skimmed milk：After 10000 times of dilution HRP sheep anti-mouse iggs, added per the μ L of hole 100
ELISA Plate, 37 DEG C are incubated 1 hour.
(8) wash：Washed 3 times with the PBST containing 5 ‰ Tween-20,200 μ L/ holes.5 minutes every time.
(9) develop the color：50 μ L TMB nitrite ions are added per hole, lucifuge is incubated at room temperature 15 minutes.
(10) terminate：50 μ L 2M H are added per hole2SO4。
(11) OD values detect：ELIASA determines OD450The IgG antibody potency of value.Criterion reference《National influenza center mark
Quasi- operational procedure》, positive findings：(mean OD values of mean OD value-blank control wells of Positive control wells)/(negative control
The mean OD value of the mean OD value-blank control wells in hole) >=2.1.
IgG antibody testing result shows and (is shown in Table 2, table 3)：The bivalent vaccine SH1/PR8+G11/PR8+61VG of immune restructuring inactivation
Also body can be induced to produce the IgG antibody for protovirus SH1 and G11, after immune in 4 weeks in serum IgG antibody potency by
Cumulative height, and reached 10 at immune 4th week4.7Left and right.
Table 2 detects the IgG antibody potency in serum with European fowl source H1N1 swine influenza viruses SH1
Table 3 detects the IgG antibody potency in serum with classic H1N1 swine influenza viruses G11
Note：* represent and control group significant difference (P ＜ 0.05).
5th, mouse attacks lung tissue viral level detection in the 3rd, 5 day after poison
According to 1 × 106TCID50Virus titer collunarium attack malicious every mouse, dosage of inoculation be 0.1ml/ only.Wherein control group is chosen
Poison is not attacked for 6 to compare as normal mouse.The mouse state of mind is observed after attacking poison, takes mouse lung to carry out virus after attacking malicious 3d and 5d
Titration.Specific steps：
(1) 3d and 5d after poison is attacked, passes through carbon dioxide asphyxia in super-clean bench and puts to death mouse, the sterile taking-up left lungs of mouse
And weigh, operated on ice；
(2) complete right lung is placed in 4% sterile paraformaldehyde solution and fixed, the lung tissue fixed is sent into Google
Biotech firm carries out the making of pathological section, finally carries out statistics and analysis to pathological examination；
(3), according to the μ l of weight (g) × 9 × 1000 of mouse lung, the addition of virus infection liquid is calculated, configures good virus infection
Liquid, corresponding sample is added to the amount of corresponding infection liquid；
(4) 2 sterile small steel balls are put into the EP pipes equipped with lung tissue, lung tissue is ground by tissue grinder；
(5) ground lung tissue is put into 6000rpm in 4 DEG C of centrifuge, centrifuges 10min；
(6) draw the lung tissue supernatant after centrifugation and carry out virus titer TCID50Detection.
As a result display is (see Fig. 3)：Immune bivalent inactivated vaccine group SH1/PR8+G11/PR8+61VG for protovirus SH1 and
Virus is detected during mouse adapted strain G11 attack with being all not apparent from.Rather than immune group is for protovirus SH1 and mouse adapted strain
G11 attack can detect very high viral level.
In figure 3, abscissa is the time for attacking poison, and ordinate is viral titre (TCID50/ml,log10).Sum in A figures
Virus is protovirus SH1；The virus of sum is that protovirus G11SH1/PR8+G11/PR8+61VG is the restructuring for adding adjuvant in B figures
Bivalent inactivated vaccine；SH1/PR8+61VG is the monovalent inactivated vaccine of addition adjuvant；G11/PR8+61VG is the unit price of addition adjuvant
Inactivated vaccine；NV/NC is nonimmune and nonimmune control group.
6th, mouse attacks the histopathology of the 5th day lungs after poison
To each group mouse collunarium inoculation 1 × 105TCID50/ protovirus SH1 and G11, it is small to take out each group within the 5th day after poison is attacked
Mouse lung tissue, pathological examination and microscopy observation are done to lung tissue, as a result sees Fig. 4, control group (A) lung tissue structure is just
Often；Bivalent vaccine group (B and C) lung tissue structure when passing through the attack of SH1 and G11 viruses respectively is immunized to show normally,
Have no obvious pathological change.Rather than there are obvious pathological characters and shows different degrees of inflammatory activity in immune group (F and I).
In Fig. 4, A:Control group；B：Immune SH1/PR8+G11/PR8+61VG (attacking malicious SH1)；C：Immune SH1/PR8+
G11/PR8+61VG (attacks malicious G11)；D：Immune SH1/PR8+61VG (attacking malicious SH1)；E：Immune SH1/PR8+61VG (is attacked
Malicious G11)；F：Nonimmune group (attacking malicious SH1)；G：Immune G11/PR8+61VG (attacking malicious SHI)；H：Immune G11/PR8+61VG
(attacking malicious G11)；I：Nonimmune group (attacking malicious G11).
7th, mouse attacks the change of 14 days mouse weights and survival rate after poison
Attack after poison in 14d, weigh each group mouse weight in same time period daily, and record the result of changes of weight.Record
Each group dead mouse result in lower 14d, and mouse of the Body weight loss more than 25% can regard as death, count simultaneously processing data.
14 days body weight situations of change (see Fig. 5) of mouse and survival rate (see Fig. 6).
As a result show：It is being inoculated with respectively after protovirus SH1 and mouse adapted strain G11 in the change of each group mouse weight, control group mice
Body weight slowly increases, the upper and lower slight fluctuations of immune divalence restructuring inactivated vaccine body weight normal type size before poison is attacked, and with
Time passage body weight gradually increases.
It is being inoculated with respectively after protovirus SH1 and mouse adapted strain G11 in each group mouse survival rate, it is small that divalence restructuring inactivated vaccine is immunized
Mouse survival rate is 100%, rather than immune group after 6 days mouse it is all dead, survival rate 0%.
In fig. 5 and fig., SH1/PR8+G11/PR8+61VG is the restructuring bivalent inactivated vaccine of addition adjuvant；SH1/PR8+61VG
To add the monovalent inactivated vaccine of adjuvant；G11/PR8+61VG is the monovalent inactivated vaccine of addition adjuvant；NV/NC is nonimmune and non-
Immune control group.
Embodiment described above only expresses embodiments of the present invention, its describe it is more specific and in detail, but can not therefore and
It is interpreted as the limitation 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, various modifications and improvements can be made, these belong to protection scope of the present invention.Therefore,
The protection domain of patent of the present invention should be determined by the appended claims.
Claims (10)
1. a kind of swine flu bivalent inactivated vaccine, flowed comprising European fowl source H1N1 hypotypes swine influenza virus and classic H1N1 hypotypes pig
Influenza Virus antigen.
2. swine flu bivalent inactivated vaccine according to claim 1, it is characterised in that the European fowl source H1N1 hypotype pigs
Influenza antigen is the restructuring Europe fowl source H1N1 hypotype swine influenza virus vaccine strains SH1/PR8 of inactivation.
3. swine flu bivalent inactivated vaccine according to claim 1, it is characterised in that the classic H1N1 hypotypes swine flu
Viral antigen is the restructuring allusion H1N1 hypotype swine influenza virus vaccine strains G11/PR8 of inactivation.
4. swine flu bivalent inactivated vaccine according to any one of claim 1 to 3, it is characterised in that the vaccine is also
Include oil emulsion adjuvant.
5. a kind of preparation method of swine flu bivalent inactivated vaccine, it is characterised in that comprise the following steps：
After European fowl source H1N1 hypotypes swine influenza virus and classic H1N1 hypotypes swine influenza virus are bred on cell respectively,
Inactivator inactivation is separately added into, adds immunologic adjuvant, swine flu bivalent inactivated vaccine is made in emulsification.
6. preparation method according to claim 5, it is characterised in that the European fowl source H1N1 hypotype swine influenza viruses
To recombinate European fowl source H1N1 hypotype swine influenza virus vaccine strains SH1/PR8.
7. preparation method according to claim 5, it is characterised in that the classic H1N1 hypotypes swine influenza virus is attached most importance to
The classic H1N1 hypotypes swine influenza virus vaccine strain G11/PR8 of group.
8. preparation method according to claim 5, it is characterised in that the cell is mdck cell.
9. preparation method according to claim 5, it is characterised in that the inactivator is formaldehyde.
10. any one of Claims 1-4 swine flu bivalent inactivated vaccine is in the medicine for preparing prevention or treatment swine flu
Using.
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| CN102166352A (en) * | 2011-04-13 | 2011-08-31 | 北京华威特生物科技有限公司 | Preparation method of swine influenza bivalent inactivated vaccine and product of swine influenza bivalent inactivated vaccine |
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| CN102166352A (en) * | 2011-04-13 | 2011-08-31 | 北京华威特生物科技有限公司 | Preparation method of swine influenza bivalent inactivated vaccine and product of swine influenza bivalent inactivated vaccine |
Non-Patent Citations (3)
| Title |
|---|
| CONSTANTINOS S. KYRIAKIS ET AL.: "Serologic Cross-Reactivity with pandemic(H1N1)2009 virus in pigs, europe", 《EMERGING INFECTIOUS DISEASES》 * |
| 汪秀会等: "古典H1N1亚型猪流感病毒反向遗传操作平台的建立", 《中国动物传染病学报》 * |
| 阮宝阳等: "重组高产欧亚禽源H1N1亚型猪流感疫苗株的构建与保护效力评价", 《中国畜牧兽医学会动物传染病学分会第十六次学术研讨会论文集》 * |
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