CN106822887A - A kind of influenza virus tetravalence subunit vaccine and its application - Google Patents
A kind of influenza virus tetravalence subunit vaccine and its application Download PDFInfo
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Abstract
The invention discloses a kind of influenza virus tetravalence subunit vaccine and its application, belong to pharmaceutical technology field., using baculovirus expression system come the quick trimer protein matter vaccine for preparing high-purity, obtained vaccine immunogenicity is strong, with short production cycle, and in the absence of any potential safety hazard, can prevent the influenza virus of various hypotypes for the present invention.
Description
Technical field
The present invention relates to a kind of influenza virus tetravalence subunit vaccine and its application, belong to pharmaceutical technology field.
Background technology
Influenza virus (influenzavirus), abbreviation influenza virus is that one kind causes the mankind and animal to suffer from stream
The RNA virus that row sexuality emits, influenza virus belongs to Orthomyxoviridae family (Orthomyxoviridae).Influenza virus can cause urgency
The property infection of the upper respiratory tract, and rapidly propagated by air, often had all over the world and be periodically very popular.Influenza
Virus can cause more serious symptom in the patient of the weaker old man of immunity or child and some immune disorders, such as pneumonia or
Cardiopulmonary exhaustion etc..
Influenza virus is mainly made up of cyst membrane and nucleocapsid, and cyst membrane is by fine prominent, BLM and stromatin (M) structure
Into.Fibre is prominent to be divided into two classes：One class is made up of in bar-shaped the tripolymer of hemagglutinin (HA) molecule；It is another kind of in mushroom, by nerve
The tetramer of propylhomoserin enzyme (NA) molecule is constituted.BLM from host cell membrane to obtain when virion is sprouted.Matrix
Albumen is arranged closely in cyst membrane inner side, is the structural proteins for maintaining morphology of virus.Nucleocapsid, core clothing are surrounded inside stromatin
Shell is made up of 8 RNA- nucleoprotein complexs (RNP) for twist arranging, and each RNP is by the outer bread of a sub-thread strand RNA
Formed by nucleoprotein (NP), 8 strand RNAs of influenza virus gene group are formed 8 RNP.According to NP and base
The antigenic differences of matter albumen M, influenza virus is divided into the type of A, B, C tri-, also there is scholar referred to as first, second, the third three types.They do not have
Common antigen, but people can be infected, wherein with the popular largest of influenza A.Various influenza virus is again according to it
The hemagglutinin (HA is abbreviated as H) and neuraminidase (NA is abbreviated as N) of virion surface are antigenic different and be divided into
The H hypotypes being had been found that in various hypotypes, wherein influenza A have 18 kinds, and N hypotypes have 11 kinds, H and N hypotypes can be formed
Different combinations, such as H1N1, H5N1, H3N2, H9N2 hypotype.
Four times being broken out for big influenza seriously threaten human health with the propagation of annual seasonal influenza and cause great warp
Ji loss, the highly pathogenic bird flu moment for bursting frequently warns the potentially possible of the big Influenza Outbreak of mankind's new round.Influenza disease
The hemagglutinin (hemagglutinin, HA) of poison is the main antigen protein in one, virus envelope surface.HA albumen is most basic
Function be identification receptor and film fusion, certain effect is also functioned in virus particle package and Budding process.HA molecules with
The host range of influenza virus, virulence, transmission capacity, and the new prevalence strain of the mankind or the strain that is very popular have it is close
Relation, is also medicine and the main target molecule of vaccine design.
Influenza virus vaccine inoculation is the first-selected measure of current mankind flu-prevention.However, due to influenza virus serotype
It is numerous, once the antigenicity of influenza virus vaccine strain and epidemic strain is mismatched, may result in vaccine failure, it is impossible to provide corresponding
Protection, it is impossible to provide cross protection to all types of influenza viruses.The vaccine for having been approved by listing at present is all virolysis
Vaccine, i.e. inactivated vaccine, such as the production of GSK companiesThe production of Quadrivalent and Sai Nuofei Pasteur S.A.All it is the influenza virus cracking vaccine of multivalence.Such vaccine needs first to prepare influenza virus live virus, therefore raw
There is security risk in product process, and production cost is also greatly increased.The influenza virus subunit epidemic disease that there is no approval to list at present
Seedling.
The content of the invention
The present invention is for the H1N1 of seasonal current Influenza Virus H3N2, big influenza in 2009 is viral and the and of Type B influenza virus B 35
B51, we devise tetravalence influenza virus subunit vaccine, and strains of influenza viruses A/California/04/2009 is chosen respectively
(H1N1), A/HongKong/2014 (H3N2), B/Brisbane/60/2008 (B35) and B/Massachusetts/2/2012
(B51) the baculovirus expression plasmid of its HA extracellular region, is built respectively, is obtained respectively by insect cell expression system solvable
Property albumen H1HA, H3HA, B35HA and B51HA, and obtain the tripolymer mesh of high-purity using molecular sieve and ion column purifying
Albumen, by immune mouse test and cell in and test, demonstrate the validity of the tetravalent vaccine.
First purpose of the invention is to provide a kind of tetravalence protein vaccine, including four kinds of albumen, the protein point
H1HA, H3HA, B35HA and B51HA are not named as, its amino acid sequence part is SEQ ID NO:1、SEQ ID NO:2、SEQ ID
NO:3、SEQ ID NO:Shown in 4；This four albumen are respectively in H1N1 subtype influenza virus strain hemagglutinin, H3N2 subtype influenzas
Viral strain hemagglutinin, Type B strains of influenza viruses B35 hypotypes hemagglutinin, the extracellular region of Type B strains of influenza viruses B51 hypotype hemagglutinin
Above add GP67 signal peptide sequences, behind add fibrin ferment restriction enzyme site, the tripolymer label and 6 × His of fibritin
Label.
The protein vaccine also includes adjuvant, including aluminium adjuvant, MF59 adjuvant.
The preparation method of the protein vaccine, comprises the following steps：
(1) construction recombination plasmid
Amplification coding strains of influenza viruses A/California/04/2009 (H1N1), A/HongKong/2014 (H3N2), B/
The nucleotide sequence of the extracellular region of Brisbane/60/2008 (B35) and B/Massachusetts/2/2012 (B51), adds
GP67 secreting signal peptides, guiding destination protein is effectively secreted into extracellular；And encode fibrin ferment (Thrombin) plus one end
The nucleotides sequence of restriction enzyme site, the tripolymer label for coming from T4 bacteriophage minority fibers albumen fibritin and 6 × His labels
Row so that the C-terminal of destination protein carries fibrin ferment (Thrombin) restriction enzyme site, comes from T4 bacteriophage minority fibers albumen
The tripolymer label and 6 × His labels of fibritin；Gained fragment is connected into pFastBacTMOn 1 carrier, restructuring matter is formed
Grain (see Fig. 1)；
(2) convert
By recombinant plasmid transformed DH10BACTME.coli competence, obtains positive restructuring bacterium；
(3) Bacmid is extracted
Bacmid is extracted from positive restructuring bacterium；
(4) transfect
Bacmid is transfected into Sf9 or Sf21 cells, P1 is obtained for recombinant baculovirus；
(5) poison is expanded
P1 is expanded into poison for recombinant baculovirus and obtains P3 for recombinant baculovirus；
(6) protein expression and purification
P3 is isolated and purified from cell and is obtained destination protein for recombinate shape virus infection Sf9 or Sf21 cell；
(7) 4 kinds of destination proteins are mixed according to special ratios with adjuvant, tetravalence subunit vaccine.
Second object of the present invention is to provide application of the tetravalent vaccine in terms of flu-prevention virus.
Third object of the present invention is to provide the gene order for encoding four kinds of albumen.
Fourth object of the present invention is to provide expression four kinds of plasmids and cell system of albumen.
Beneficial effects of the present invention：
Present invention obtains a kind of influenza virus tetravalence subunit vaccine, the vaccine on mouse has good protective effect,
There is the application value of flu-prevention virus.The present invention is using baculovirus expression system come the quick trimerization for preparing high-purity
Body protein vaccine, obtained vaccine immunogenicity is strong, with short production cycle, and in the absence of any potential safety hazard, can prevent
The influenza virus of various hypotypes.
Brief description of the drawings
The structure schematic diagram of Fig. 1, expression plasmid.
The molecular sieve purification result of Fig. 2, influenza virus H1HA albumen；Albumen numbering is from 4 to 23 in peak figure.
The molecular sieve purification result of Fig. 3, influenza virus H3HA albumen；In figure on abscissa after 40ml, secondary graduation mark
The numbering of the albumen of mark is from left to right 1 to 20.
The molecular sieve purification result of Fig. 4, influenza virus B 35HA albumen；In figure on abscissa after 40ml, secondary graduation mark
The numbering of the albumen of mark is from left to right 1 to 40.
The molecular sieve purification result of Fig. 5, influenza virus B 51HA albumen；In figure on abscissa between 40~70ml, secondary quarter
The numbering for spending the albumen of wire tag is from left to right 1 to 19.
In Fig. 2~5, precipitation represents the cell precipitation that centrifugation is obtained when collecting albumen；M represents molecular weight protein marker；
Numeral represents the protein component of relevant position in peak figure respectively.
The neutralization of Fig. 6, mouse immune serum to H1N1 viruses；
The neutralization of Fig. 7, mouse immune serum to H3N2 viruses；
The neutralization of Fig. 8, mouse immune serum to B35 viruses；
The neutralization of Fig. 9, mouse immune serum to B51 viruses；
In Fig. 6~9, abscissa is the serum of different group immune mouses, and Al-5-3 represents four kinds of HA extracellular region proteins each 5
The serum that μ g behind the 0th, 14 and 28 days three immune mouses separate totally after being mixed with aluminium adjuvant, Al-PBS-3 represents PBS and aluminium
The serum that adjuvant behind the 0th, 14 and 28 days three Mice Inoculateds separate totally after mixing, MF-10-3 represents four kinds of HA extracellular region eggs
The serum that white each 10 μ g behind the 0th, 14 and 28 days three immune mouses separate totally after being mixed with MF59 adjuvant, MF-PBS-3 is represented
The serum that PBS behind the 0th, 14 and 28 days three Mice Inoculateds separate totally after being mixed with MF59 adjuvant, NC represents negative control, is
The serum for from the mouse without any immune treatment separate, PC-3 is represented and split using the influenza virus of Sai Nuofei Pasteur S.A.
The serum for after solution three immune mouses of vaccine separate.
Specific embodiment
Embodiment 1：The expression and purifying of Influenza virus HA protein
1st, the structure of expression plasmid
Strains of influenza viruses A/California/04/2009 (H1N1), A/HongKong/2014 (H3N2), B/ are chosen respectively
Brisbane/60/2008 (B35) and B/Massachusetts/2/2012 (B51), by company's synthetic gene sequence, builds it
The baculovirus expression plasmid of HA extracellular regions.
Specifically, the HA full length sequences of A/California/04/2009 (H1N1) are by Dalian treasured biotinylated biomolecule engineering finite
Company synthesizes.On this basis, the extracellular region 11-505 nucleosides of (being sorted by H3) amino acid of coding H1N1 albumen is amplified
Acid sequence, adds GP67 secreting signal peptides before it, can effectively be secreted into destination protein extracellular.To promote HA albumen
Trimerizing, improve the immunogenicity of albumen, and facilitate follow-up purifying, we add the preceding paragraph to modify in the C-terminal of HA extracellular regions
Sequence：This sequence includes fibrin ferment (Thrombin) restriction enzyme site, comes from T4 bacteriophage minority fibers albumen fibritin
Tripolymer label and end 6 × His labels, obtain fragment of the sequence as shown in SEQ ID NO.1, the fragment is connected into
pFastBacTMOn 1 carrier, pFastBac is formedTM1-H1HA recombinant plasmids.The sequence of GP67 signal peptides such as SEQ ID NO.5 institutes
Show, the sequence of Thrombin restriction enzyme sites as shown in SEQ ID NO.6, the sequence of tripolymer label as shown in SEQ ID NO.7,
His labels are 6 histidines.
The HA extracellular regions of H3N2 and GP67 signal peptides above, Thrombin restriction enzyme sites below, tripolymer label
It is directly synthesized by Shanghai Jierui Biology Engineering Co., Ltd with His labels, clone's structure is consistent with the extracellular region of H1N1, is named as
pFastBacTM1-H3HA。
The HA extracellular regions of B35 and B51 are synthesized by Suzhou Jin Weizhi bio tech ltd, clone build with H1N1 and
The extracellular region of H3N2 is consistent, is also connected into pFastBacTMOn 1 carrier.PFastBac is formed respectivelyTM1-B35HA recombinant plasmids and
pFastBacTM1-B51HA recombinant plasmids.
2nd, convert
Take the DH10BAC of 100 μ lTME.coli competent cells are placed on ice, add to few 1ng pFastBacTM1-H1HA
Recombinant plasmid is to 100 μ l DH10BacTMIn E.coli competent cells, gently mix, 30min is placed on ice.42 DEG C of heat shocks
45s, turns back on ice immediately, stands 2min.In super-clean bench, often pipe adds 800 μ l nonreactive LB culture mediums, 37 DEG C of culture 4h.4 is small
When culture after, take 100 μ l bacterium solutions apply three anti-flat boards (LB flat boards, contain：50 μ g/ml kanamycins (kanamycin), 7 μ g/ml are celebrated
Big mycin (gentamicin), 10 μ g/ml tetracyclines (tetracycline), 300 μ g/ml Bluo-gal, 40 μ g/ml
IPTG), 4 DEG C of remaining bacterium solution is kept in, and flat board cultivates 24h in being inverted in 37 DEG C of baking ovens.pFastBacTM1-H3HA、
pFastBacTM1-B35HA and pFastBacTMThe method for transformation and pFastBac of 1-B51HA recombinant plasmidsTM1-H1HA complete one
Cause.
3rd, Bacmid is extracted
The hickie bacterium solution for being accredited as the positive by PCR bacterium solutions is forwarded to LB culture mediums of the 4ml containing tri- kinds of antibiotic of KGT and relays
Continuous 37 DEG C of cultures 16-24h, extracts Bacmid (S.N.A.P.TMMidiPrep Kit, Catalog no.K1910-01 or different
The propyl alcohol precipitation method are extracted).Plasmid concentration is surveyed, OD is selected260/OD280Between 1.8-2.0, concentration it is higher (500ng/ μ l or with
On) Bacmid do transfection it is relatively good.And enter performing PCR identification again.
4th, transfect
Be prepared in advance suspend culture Sf9 or Sf21 cells, cell be in exponential phase, density about 1.5-2.5 ×
106Individual cell/ml.Cell counting count board number cell, determines cell density.Six orifice plates (bore dia 35mm) are spread, per hole 8 × 105It is individual thin
Born of the same parents, cell is shaken up.Treat that plate floor cells reaches proper density, draw nutrient solution, 1ml Grace ' s Medium are added per hole.
Cellfectin II 6-8 μ l (4 DEG C of preservations, plus preceding mixing) add the μ l of Grace ' s Medium 100, gently mix, and are labeled as
Pipe 1, stands 5min.It is that the positive μ g of restructuring Bacmid 2 add the μ l of Grace ' s Medium 100 by PCR qualification results, gently
Mix, labeled as pipe 2.The solution of pipe 1 is transferred in pipe 2 (μ l of cumulative volume about 210), is gently mixed, and stands 20min.By six holes
Culture medium in plate is suctioned out with pipettor, adds 2ml Grace ' s Medium to wash per hole 2 times, is washed and is discarded culture medium.Plus
0.8ml Grace ' s Medium in the pipe 1 and the mixed liquor of pipe 2 of step 4,1ml altogether, it is light it is mixed after slowly add along six orifice plate walls
Enter.Six orifice plates are put into 5h in 27 DEG C of incubators.Change liquid：The mixed liquor in six orifice plates is discarded, 2ml Sf-900 are added per holeTM III
SFM culture mediums (contain three kinds of antibiotic：100 units/ml penicillin, 100 units/ml streptomycin and 0.25 μ g/
mlAntimycotic, plays antibacterium and antifungic action).It is incubated in 27 DEG C of incubators.After about 72h, carefully
Cellular lysate, cell monolayer floating.2ml supernatants are collected, clean centrifuge tube, masking foil parcel 4 are transferred to after 500g, 5min centrifugation
DEG C keep in dark place, (plus hyclone to final concentration 2% can prevent protease from being broken to virus for recombinant baculovirus to obtain P1
It is bad).If need to preserve for a long time, -80 DEG C are stored in after P1 viruses are frozen in liquid nitrogen.
5th, poison is expanded
The titre general about 1 × 10 of recombinant baculovirus amplification P1 generation viruses6-1×107pfu/ml.pfu：plaque
Forming units (plaque forming unit).The MOI for expanding poison should be controlled in 0.05-0.1.MOI：multiplicity
Ofinfection (infection multiplicity), inoculation volume (ml)=MOI (pfu/cell) × number of cells/virus titer (pfu/ml).
It is as follows that P1 expands P2 experimental procedures：Each 10cm disk paving 1 × 107Individual Sf9 or Sf21 cells.Required P1 volumes are calculated, virus is added
Enter in disk, 27 DEG C of incubators are incubated.48-72h receives poison.The titre of general P2 viruses is 107-108pfu/ml.P2 viruses can be straight
Expressing protein is connect, or continues to expand P3 viruses, with P3 for expressing viral albumen.The P2 viruses of general 200 μ l can infect 100ml
Suspend Sf9 (1.5-2.0 × 10 cultivated6) or Sf21 (1.0-1.5 × 106) cell, it is viral that 48-72h receives P3.General P3 viruses
Titre reach 108pfu/ml。
6th, protein expression and purification
By the expression of Western blot testing goal albumen, then P3 is viral infects High FiveTMCell density is about
1.5-2.0×106(Sf9 density 2.0-3.0 × 106；Sf21 density 1.0-2.0 × 106), 27 DEG C of shaking table culture 48h, microscopy is thin
The death rate of born of the same parents about 50% receives cell, and cell is poured into centrifugal barrel, and 7,000g centrifugation 45min collect supernatant, and 0.22 μm of filter membrane is taken out
Filter.Using HisTrapTMHP 5ml prepacked columns (GE Healthcare), buffer3 (20mM Tris, 150mM NaCl,
300mM imidazoles, pH 8.0) wash-out purpose HA albumen, it is concentrated into about 10ml by dialysis.HA albumen after concentration is used
The GL column of SuperdexTM200 10/300 carry out molecular sieve purification (20mM Tris-HCl, 50mM NaCl's, pH 8.0
Buffer solution), the final HA albumen for obtaining purpose.And purity of protein is identified by SDS-PAGE, purity of protein reaches more than 95%.
Four kinds of albumen H1HA, H3HA, B35HA and B51HA are prepared using same method.H1HA, H3HA, B35HA and
The extracellular domain sequence of B51HA and GP67 signal peptides, fibrin ferment (Thrombin) restriction enzyme site, T4 bacteriophage minority fibers albumen
The fusion fragment of the tripolymer label of fibritin and 6 × His labels of end is respectively such as SEQ ID NO.2, SEQ ID
Shown in NO.3, SEQ ID NO.4.
Embodiment 2：The performance detection of tetravalence subunit vaccine
Tetravalence subunit vaccine is immunized to mouse, mouse inoculation scheme is see table 1.Respectively at the 0th day, 14 days
Three times were carried out with 28 days to be inoculated with mouse leg, the type and quantity one of every group of each albumen being inoculated with of mouse and adjuvant
Cause.12 days tail blood of collection mouse after exempting to exempt from three two respectively, separate serum.The method that blood was taken using eyeball at the 0th day
The sample that collection separates 6 mouse does negative control.
It is immunized using different albumen dosage, different adjuvants during immune mouse.Albumen dosage is divided into 30 μ g, 10 μ
Tetra- gradients of g, 5 μ g and 2.5 μ g, adjuvant is divided into two kinds of aluminium and MF59.For example in immunization protocol first group is by four kinds of albumen
Each 10 μ g of H1HA, H3HA, B35HA and B51HA are mixed so that cumulative volume is 600 μ l.The MF59 adjuvant of 600 μ l is subsequently adding, is made
Blown and beaten repeatedly with 1ml syringes, fully mix albumen and adjuvant, then take the 150 μ l mixed liquor Mice Inoculated huckles, connect altogether
Plant 6 mouse.
Table 1, mouse immunization protocol
Then, we are by the neutralization in influenza virus with experiment detection mice serum infected by influenza.Adopt respectively
Strains of influenza viruses is：A/California/04/2009 (H1N1), A/Texas/50/2012 (H3N2), B/Brisbane/
60/2008 and B/Massachusetts/2/2012.After serum is processed with RDE, 2 times of doubling dilutions are carried out, it is and isometric
Viral (200TCID50/100ul) is incubated 1.5 hours.Take 100 μ l to be added in 96 orifice plates for covering with mdck cell, 37 DEG C of cultures 72
Hour, period routine observation cytopathy situation.Experimental result is neutralized by Hemagglutination Method detection.
Result finds that mouse can significantly neutralize A/California/04/ by the serum after three secondary aluminium adjuvant immunities
2009 (H1N1) viruses (5 μ g HA albumen/aluminium adjuvant group, TICD50=10E-1.6) and B/Massachusetts/2/2012
(B51) it is viral (5 μ g HA albumen/aluminium adjuvant group, TICD50=10E-2.58).Mouse is by the blood after three secondary aluminium adjuvant immunities
The clear neutralization to B/Brisbane/60/2008 (B35) viruses and A/Beijing Huairou/1178/2014 (H3N2) viruses
Potency is relatively low, but the serum plus MF59 adjuvant after immune can significantly neutralize B/Brisbane/60/2008 (B35) viruses
(10 μ g HA albumen/MF59 adjuvant group, TCID50=10E-2.36), and can to a certain extent neutralize H3N2 viruses (A/
Beijing Huairou/1178/2014) (2.5 μ g HA albumen/MF59 adjuvant group TCID50=10E-1.76).
We pass through mouse immunization protocol and virus neutralizes experiment, and the tetravalent vaccine that can be added after adjuvant with preliminary proof can
To effectively improve animal to H1N1 and H3N2 viruses and Type B influenza virus (B35, B51) immunocompetence.
To sum up, we have invented a kind of influenza virus tetravalence subunit vaccine, the vaccine include four kinds of HA albumen, H1HA,
H3HA, B35HA and B51HA, after adding aluminium or MF59 adjuvant, the tetravalent vaccine can effectively improve mouse to A (H 1 N 1) virus
With Type B influenza virus (B35, B51) immunocompetence, also there is certain immune effect to H3N2 viruses.
Although the present invention is disclosed as above with preferred embodiment, it is not limited to the present invention, any to be familiar with this skill
The people of art, without departing from the spirit and scope of the present invention, can do various changes with modification, therefore protection model of the invention
Enclose being defined of being defined by claims.
Claims (7)
1. a kind of tetravalence subunit vaccine, it is characterised in that the tetravalence subunit vaccine includes four kinds of albumen, its amino acid sequence
Row are respectively SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.
2. the tetravalence subunit vaccine described in claim 1, it is characterised in that also including adjuvant.
3. the tetravalence subunit vaccine described in claim 2, it is characterised in that adjuvant is aluminium adjuvant or MF59 adjuvant.
4. application of the tetravalence subunit vaccine described in claim 1 in terms of flu-prevention virus.
5. a kind of gene order, it is characterised in that the albumen in coding claim 1.
6. the carrier or cell of gene order described in claim 5 are carried.
7. application of the gene order described in claim 5 in flu-prevention virus.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2020035609A3 (en) * | 2018-08-17 | 2020-05-28 | Glaxosmithkline Biologicals Sa | Immunogenic compositions and uses thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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