CN105671064B - Recombinant antigen gene, recombinant antigen protein and the subunit vaccine composition of anti-porcine reproductive and respiratory syndrome virus infection - Google Patents
Recombinant antigen gene, recombinant antigen protein and the subunit vaccine composition of anti-porcine reproductive and respiratory syndrome virus infection Download PDFInfo
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Abstract
The present invention relates to a kind of recombination fusion antigen genes of anti-porcine reproductive and respiratory syndrome viral (PRRSV) infection, recombinate fusion antigen protein and the subunit vaccine composition containing it, it is that PRRSV is had the glycoprotein GP5 for truncating the end N ' decoy epitopes, after catenation sequence and memebrane protein M are fused to recombination sequence and carry out codon optimization, resulting recombination fusion antigen gene carries out vivoexpression using recombinant baculovirus expression system, its yield for recombinating fusion antigen protein can not only be promoted, resulting recombination fusion antigen protein is when being applied to subunit vaccine composition, preferable vaccine protection can be provided to immunized animal not have toxin expelling again and return malicious risk.
Description
Technical field
The present invention relates to a kind of recombination fusion antigen gene of animal, recombination fusion antigen protein and contain its Asia list
Position vaccine composition, more particularly to a kind of recombination fusion antigen gene, again of anti-porcine reproductive and respiratory syndrome virus infection
Group fusion antigen protein and its application in subunit vaccine composition.
Background technique
Porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome；
PRRS it) was found for the first time in North America in 1987, the several years is broken out in global pig farm successively later, and pig raising industry is caused greatly
Loss, therefore PRRS how is prevented and treated as global pig raising industry urgent problem to be solved.The pig of all ages all may be infecteds
PRRS, other than causing pig breeding difficulty, it also occur that respiratory symptom and death.PRRS is bred by pig and breathes synthesis
Syndrome virus (Porcine reproductive and respiratory syndrome virus；PRRSV caused by) infecting,
This virus is subordinate to artery Viraceae (Arteriviridae), containing positive (+) single stranded RNA genome, has coating, virus is straight
Diameter is about 50-70nm.The genome overall length of PRRSV is 15kb, shares 10 opening code-reading frames (ORFs).Wherein ORF5 can
The glycated protein GP5 of 26kDa is translated, and ORF6 can then translate the both the non-glycated memebrane protein (unglycosylated of 19kDa
membrane protein；M).
Presently commercially available PRRSV vaccine mainly has a three classes, including malicious attenuated vaccine living, dead malicious vaccine and is given birth to by Escherichia coli
The subunit vaccine of production.Though poison attenuated vaccine living can induce simultaneously cellularity and react with humoral immune, its neutralizing antibody power
Valence is relatively low, and Vaccine effectiveness is insufficient and has toxin expelling and returns malicious risk.Though dead poison vaccine nothing returns malicious risk but can only induce antibody mediated exempt from
Epidemic disease reaction, need to be immunized and just can reach protecting effect twice, and lose protection completely to heterologous virus.In addition, having no at present
Definite data, which are shown, has Vaccine effectiveness by the subunit vaccine of Escherichia coli production.
It is RNA virus in view of PRRSV, evolution mutating speed is quite rapid, needs to develop a kind of safety, does not have toxin expelling
With return malicious risk and have immune protective efficiency vaccine, to overcome the various shortcoming of currently available vaccines, existing vaccines.
Summary of the invention
It is an aspect of the present invention to provide a kind of isolated nucleic acid, and it includes such as sequence number (SEQ ID NO.): shown in 1
Recombination fusion antigen gene, wherein this recombination fusion antigen gene be by porcine reproductive and respiratory syndrome virus (porcine
reproductive and respiratory syndrome virus；Having PRRSV) truncates the end N ' decoy epitopes
Glycoprotein GP5, catenation sequence and the memebrane protein M fusion of (truncated N '-terminal decoy epitope) are attached most importance to
Group sequence simultaneously carries out codon optimization.
Secondly, another aspect of the present invention is to provide a kind of recombinant viral vector, it includes as shown in SEQ ID NO.:1
Recombination fusion antigen gene.
Furthermore it is a further aspect of the present invention to provide a kind of recombination fusion antigen proteins, and it includes such as SEQ ID NO.:1
Shown in recombination recombination fusion antigen protein of the fusion antigen gene through recombinant baculovirus expression system expression, thus promote it
Recombinate the expression quantity of fusion antigen protein.
It is another aspect of the present invention to provide a kind of subunit vaccine compositions of anti-PRRSV infection, and it includes such as SEQ
Recombination fusion antigen protein of the recombination fusion antigen gene through recombinant baculovirus expression system expression shown in ID NO.:1 and
Pharmaceutically acceptable carrier, to provide preferable vaccine protection and not have toxin expelling and return malicious risk.
Above-mentioned aspect according to the present invention proposes a kind of isolated nucleic acid, and it includes such as sequence number (SEQ ID NO.):
Fusion antigen gene is recombinated shown in 1.
Above-mentioned aspect according to the present invention, separately proposes a kind of recombinant viral vector, and it includes as shown in SEQ ID NO.:1
Recombination fusion antigen gene.
Above-mentioned aspect according to the present invention, and propose a kind of recombination fusion antigen protein, it includes such as SEQ ID NO.:1
Shown in recombination recombination fusion antigen protein of the fusion antigen gene through recombinant baculovirus expression system expression.
Above-mentioned aspect according to the present invention reintroduces a kind of subunit vaccine composition of anti-PRRSV infection, and it includes such as
Recombination fused antigen egg of the fusion antigen gene through recombinant baculovirus expression system expression is recombinated shown in SEQ ID NO.:1
The pharmaceutically acceptable carrier of bletilla.
An embodiment according to the present invention, above-mentioned pharmaceutically acceptable carrier include adjuvant and/or immunopotentiator.
In illustrating at one, foregoing immune synergist may be, for example, CpG synergist, and sequence is as shown in SEQ ID NO.:2.
Using anti-PRRSV infection of the invention recombination fusion antigen gene and its recombination fusion antigen protein, be by
PRRSV's there is the glycoprotein GP5, catenation sequence and the memebrane protein M that truncate the end N ' decoy epitopes to be fused to recombination fused antigen
Gene and by this sequence carry out codon optimization after, recycle recombinant baculovirus expression system carry out vivoexpression, can be promoted
Its yield for recombinating fusion antigen protein.Resulting recombination fusion antigen protein, can when being applied to subunit vaccine composition
Preferable vaccine protection is provided and does not have toxin expelling and returns malicious risk, thus be effectively improved existing PRRSV Yield of Antigen it is low,
Immune protective efficiency is bad and has toxin expelling and returns the variety of problems such as malicious risk.
Detailed description of the invention
In order to enable above and other objects of the present invention, feature, advantage and embodiment to be clearer and more comprehensible, it is detailed to provide attached drawing
Carefully it is described as follows:
Figure 1A is painted the Vector map of recombinant viral vector according to an embodiment of the invention.
Figure 1B shows electrophoretic analysis photo of the recombinant vector according to an embodiment of the invention after limiting enzyme effect.
Fig. 2 shows the electrophoretic analysis of product of the recombinant viral vector according to an embodiment of the invention after PCR reacts photographs
Piece.
Fig. 3 shows SDS-PAGE points of Hi-5 cell expression recombination fusion antigen protein according to an embodiment of the invention
Analyse photo.
Fig. 4 shows the western blot of Hi-5 cell expression recombination fusion antigen protein according to an embodiment of the invention
Analyze photo.
Specific embodiment
Described in brought forward, the present invention provides the recombination fusion antigen gene and its recombination fused antigen of a kind of anti-PRRSV infection
Albumen is that there is the glycoprotein GP5 for truncating the end N ' decoy epitopes, catenation sequence and memebrane protein M fusion to attach most importance to PRRSV
After organizing fusion antigen gene and this sequence being carried out codon optimization, recombinant baculovirus expression system is recycled to carry out external table
It reaches, its yield for recombinating fusion antigen protein can be promoted.
The present invention " recombination fusion antigen gene " referred to herein refers to such as sequence number (SEQ ID NO.): shown in 1
Recombinate fusion antigen gene sequence.In one embodiment, the base after the end the N ' decoy epitopes of the glycoprotein GP5 of PRRSV being truncated
Because of sequence, recombination sequence (or being 6LdN5) is fused to using the gene order of connection (linker) sequence and memebrane protein M.
In specific words, the glycoprotein GP5 and memebrane protein M of PRRSV is the main component of peplos (envelope), the two
The dimer combined with cystine linkage is formed, content at least accounts for the 1/2 of total virus albumen.Glycoprotein GP5 and memebrane protein M can be lured
Hair protective immunological reaction simultaneously induces host's generation neutralizing antibody, but the decoy epitopes positioned at the end N- of glycoprotein GP5
(decoy epitope) about 30 amino acid can lower the generation and its reactivity of neutralizing antibody, thus feature of the invention it
One is exactly to clip N ' end the 1st amino acid of decoy epitopes of glycoprotein GP5 to the 10th amino acid, is formed and " truncates N ' and hold bait
The glycoprotein GP5 of epitope " is fused to recombination sequence using catenation sequence and memebrane protein M, and carries out codon to this recombination sequence
Thus optimization not only increases the subsequent yield in recombinant baculovirus expression system, resulting recombination fusion antigen protein warp
After crossing the rear translation modification of eukaryotic expression system, it may have correct tertiary structure and folding.
For the present invention " catenation sequence " referred to herein to connect glycoprotein GP5 and memebrane protein M, length is unlimited, can
For example, 6 bases are preferable with 9 bases to 24 bases, and be with 9 bases to 15 bases to 30 bases
More preferably.In one example, the catenation sequence can be 12 bases.
The present invention " codon optimization " referred to herein refers under the premise of not changing original acid sequence, according to recombination bar
Shape virus expression systems carry out codon optimization, carry out recombinant protein correctly to the hobby of each amino acid codes
Translation modification afterwards plays immune protective effect.The reason for this is that although Escherichia coli are current most common protein expression systems
System, has many advantages, such as that growth cycle is fast, yield is high and at low cost, but can not carry out the posttranslational modifications such as correct saccharification,
So that the probability for forming insoluble endosome is higher.In comparison, recombinant baculovirus expression system can produce a large amount of recombination eggs
It is white, and the posttranslational modifications such as correct saccharification can be carried out.In one embodiment, resulting recombination fusion is thus expressed
Antigen protein has correct glycosyl and foldable structure.
Recombination fusion antigen gene sequence shown in above-mentioned SEQ ID NO.1 (or being 6LdN5), utilizes commercially available sequence point
It analyses software (such as DNAStar), after comparing with original gene (or wild-type), comparison result is as shown in table 1.
Table 1
As the comparison result of table 1 it is found that recombination fusion antigen gene sequence shown in SEQ ID NO.1 (or is
6LdN5) after codon optimization, the similarity with original gene (or wild type) is only 89.5%.
The present invention " recombinant baculovirus expression system " referred to herein refers to rhabdovirus expression vector (baculovirus
expression vector；) and its gene expression system that is constituted of host insect cell BEV.Baculoviral is a kind of infection
The large-scale DNA virus of invertebrate, main host plants are insect and part arthropod.All due to the mankind and other vertebrates
It is not the host of baculoviral, therefore the rod string design (baculovirus developed by baculoviral
expression vector system；BEVS) very safe.So-called rhabdovirus expression vector, which refers to, is loaded with foreign gene
Recombinant baculovirus (recombinant baculovirus).Once rhabdovirus expression vector infects host insect cell, i.e.,
Using the expression of the strong promoter control foreign gene in rhabdovirus expression vector, and produce a large amount of recombinant protein.
By the posttranslational modification (post-translational of the resulting recombinant protein of rod string design
Modifications), for example, it is similar with the institute producer in mammalian cells, therefore utilize rhabdovirus expression vector
System recombinant protein produced, no matter in antigenicity, immunogenicity and bioactivity, generally with original protein phase
Like even almost consistent.
In one example, aforementioned recombination fusion antigen gene sequence is after being implemented in baculovirus vector, recombination fusion
The end 5' of antigen gene sequences is more optionally with baculoviral membrane glycoprotein (membrane glycoprotein) gp64's
The gene order of signal peptide connects.Thus the recombination fusion antigen protein of signal peptide of the expression with gp64, passing in the cell
During sending, the signal peptide of gp64 can be removed.
In use, recombination fusion antigen protein of the invention and pharmaceutically acceptable carrier can be made sub- single
Position vaccine composition, is inoculated in pig.In one embodiment, aforementioned pharmaceutically acceptable carrier includes adjuvant and/or exempts from
Epidemic disease synergist, wherein the type of aforementioned adjuvant is unlimited, specific example may include but be not limited to aluminium glue adjuvant, oily adjuvant (example
Such as Freund's complete adjuvant, incomplete Freund's adjuvant) or above-mentioned any combination.Foregoing immune synergist can be for example with CpG
The immunostimulatory oligonucleotide (or CpG synergist) on island etc..In illustrating one, aforementioned CpG synergist can such as SEQ ID
Sequence shown in NO.:2.In one embodiment, the immune pig of the subunit vaccine composition through above-mentioned containing CpG synergist
Only, the protection of PRRSV infection is fought up to 100%.
Since recombination fusion antigen protein of the invention is by the end the N- decoy epitopes (decoy of the glycoprotein GP5 of PRRSV
Epitope) truncate and with memebrane protein M Gene Fusion, centre be added catenation sequence, then by this recombination fusion antigen gene sequence into
Row codon optimization can strengthen it in the expression of insect cell.Secondly, when the recombinant virus with this recombination fusion antigen gene
Carrier is transfected to recombinant baculovirus expression system when being expressed, and can effectively improve the yield of recombination fusion antigen protein.
Secondly, the present invention is produced using recombinant baculovirus expression system, suspension culture can be carried out, is not only operated
Simply, yield is high, production scale is easy amplification, and host insect cell there is translation similar with mammalian cell after repair
Decorations effect, can produce the antigen protein for possessing original biological function.The present invention is through subsequent animal immune it is experimentally confirmed that being made with this
The Vaccine effectiveness of subunit vaccine composition be up to 100%, it is possible to provide do not had again by the preferable vaccine protection of immune animal
Toxin expelling and malicious risk is returned, so technology is expected to apply the PRRSV vaccine composition in novel form, to prevent and treat pig breeding and respiratory tract
Syndrome.
Illustrate application of the invention following with several embodiments, however it is not intended to limit the invention, skill of the present invention
Technical staff in art field without departing from the spirit and scope of the present invention, can various modifications may be made with change.
The foundation of one: pBacF-gp-6LdN5 recombinant viral vector of embodiment
In this embodiment, contain PRRSV ORF5 (glycoprotein GP5) and ORF6 disclosed in foundation NCBI Genbank
The sequence AF035409 of (M albumen), design 6LdN5 recombinate fusion antigen gene.
Refering to the sequence of SEQ ID No.:1, firstly, ORF6 (the 145th base to the 681st base) is designed at
The front end of ORF5 (the 694th base to the 1266th base), catenation sequence is added in centre, and (the 682nd base is to the 693rd
Base), and design has Sal I (the 121st base to the 126th base), multiple limitation in ORF5 and ORF6 gene both ends
Restriction enzyme site sequence (the 127th base to the 144th base) and Not I's (the 1288th base to the 1295th base)
Restriction site.At 5 ' ends of above-mentioned Sal I restriction site, subsequent converted by pBacF-gp can be further attached to and carried
The gene order (the 1st base to the 120th base) for the gp64 signal peptide that body (transfer vector) provides, makes PRRSV
N ' the terminal sequence of ORF6 is merged with gp64 signal sequence.The another connection after ORF5 (the 694th base to the 1266th base)
His label (the 1267th base to the 1284th base) and terminator codon (the 1285th base to the 1287th alkali
Base), and with commercially available gene design software (such as the Codon that Genomics BioSci & Tech, Ltd. are provided
The commercially available software of Optimization Services or other, as EnCor Biotechnology Inc. is provided
Codon Optimization Calculator, what DNA2.0, Inc. were providedExpression
The Codon Optimization and that Optimization Technology, ProteinCT Biotech is provided
Verification Services etc.) carry out codon optimization after, synthesis recombination simultaneously be named as 6LdN5, size is
1295bp。
After the nucleic acid fragment of above-mentioned 6LdN5 recombination fusion antigen gene is purified, (i.e. with pBacPAK8-gp conversion carrier
Baculovirus vector, the gene order with gp64 signal peptide) it is engaged, to construct recombinant viral vector.
The overall length size of pBacPAK8 conversion carrier above-mentioned is about 5.5kb, including Autographa californica nuclear polyhedral body
Virus (Autographa californica multiple nuclear polyhedrosis virus；AcMNPV) gene,
It can provide the sequence recombinated with the DNA converted.PBacPAK8 conversion carrier separately contains the polyhedral body starting of height expression
Son (polyhedrin promoter；PPH), ampicillin resistant (ampicillin；Amp selected marker and SV40)
Polyadenylation signal (polyadenylation signal).Recombination and the pBacPAK8-gp virus of 6LdN5 above-mentioned
After carrier (gene order with gp64 signal peptide) engagement, as recombinant viral vector (or pBacPAK8-gp-6LdN5),
Its overall length size is about 6.5bp, as shown in Figure 1A.
Above-mentioned resulting recombinant viral vector (or pBacPAK8-gp-6LdN5) is converted again to Escherichia coli
(Escherichia coli XL-1 Blue；Invitrogen, California, USA) competent cell, with antibiotic-screening
Target gene is inserted into the bacterial strain of expression vector really, recycles restriction enzyme Xba I (on pBacPAK8-gp viral vectors, tightly
The upstream that adjacent gp34 gene order 5 ' is held) and Not I be cut into target gene and through DNA electrophoretic analysis, result such as Figure 1B
It is shown.
B refering to fig. 1 shows the electricity of product of the recombinant viral vector according to an embodiment of the invention after limiting digestion
Swimming analysis photo, wherein the road M is DNA marker, the 1st for pBacPAK8-gp viral vectors using restriction enzyme Xba I and
DNA fragmentation after Not I cutting, the 2nd utilizes restriction enzyme Xba I for recombinant viral vector (pBacPAK8-gp-6LdN5)
(restriction enzyme site is on viral vectors) and Not I cutting after DNA fragmentation, and the 2nd 1295bp at as 6LdN5 weight
The DNA fragmentation of group gene and partial vector sequences.
In addition, above-mentioned resulting transformant, recycling PCR reacts and through DNA electrophoretic analysis, and result is as shown in Figure 2.On
State PCR reaction can using 5 μ L 2x PCR Mastermix (0.5U SuperTherm DNA polymerase mix, 200 μM
DNTPs, 1.5mM MgCl2, 1x buffer) (Bertec, Taiwan), 1 μ L 2.5mM 6L5 (SalI) forward primer, 1 μ L
2.5mM 5his (NotI) reverse primer and 3 μ L secondary deionized water be configured to reactant after, first 94 DEG C react 5 points
Clock, then sequentially 94 DEG C carry out 1 minute DNA denaturing step, 56 DEG C carry out 90 seconds DNA and primer bind step and
In three steps such as the DNA cloning steps that 72 DEG C carry out 1 minute, 30 circulations are carried out, after circulation terminates, carry out 7 at 72 DEG C
The DNA cloning step of minute.After reaction, 4 μ L PCR products is taken to inspect result using 1% agarose gel electrophoresis.It is aforementioned
The sequence of 6L5 (SalI) forward primer is as shown in SEQ ID NO:3, and the sequence of 5his (NotI) reverse primer is then such as SEQ ID
Shown in NO:4.With the DNA sequence dna that the primer pair amplifies of above-mentioned 6L5 (SalI) and 5his (NotI) go out, bait is held comprising truncating N '
Gene order, the gene order of catenation sequence and memebrane protein M of the glycoprotein GP5 of epitope, but be free of the gene of gp64 signal peptide
Sequence.
Referring to Fig.2, it shows the aggregated enzyme chain reaction of recombinant viral vector according to an embodiment of the invention
(polymerase chain reaction；PCR the electrophoretic analysis photo of the product after) reacting, wherein the road M is DNA marker,
1st is recombinant viral vector (pBacPAK8-gp-6LdN5) after PCR reaction and 1% agar-agar electrophoretic analysis, is confirmed resulting
DNA fragmentation is 1151bp.
Separately entrust Taibei Yuan Zi international bio Science and Technology Co., Ltd. with nucleic acid sequence the above-mentioned transformant primarily determined
After column automatic sequencer (ABI 3730, CD Genomics, USA) carries out nucleic acid sequencing, confirm that the sequence of insertion is correct.
Aforementioned building conversion carrier, cell transformation, screening aimed strain, plastid DNA extraction, limitation enzyme reaction, PCR reaction, DNA
Electrophoretic analysis, DNA sequencing etc., should be any in the technical field of the invention has known to ordinary skill, herein no longer
It repeats.
Embodiment two: the foundation of recombinant baculovirus expression system
1. Insect cellculture
This embodiment is with autumn armyworm pupa Sf9 cell strain (Spodoptera frugiperda) (BCRC 60011；ATCC
) or cabbage looper (Trichoplusia ni) ovum High Five (hereinafter referred to as Hi-5) cell strain (BTI-TN- CRL-1711
5B1-4) (such as Invitrogen Co.) establishes recombinant baculovirus expression system.
The Sf-9 cell culture medium or Hi-5 cell culture medium of 9mL are taken, is added in 10cm culture dish.By Sf9 cell strain or
Cell liquid after quickly thawing into 37 DEG C of water baths, is homogeneously dispersed in the training of Sf-9 cell by the freezing tubule of Hi-5 cell strain
It supports in base or Hi-5 cell culture medium.Later, culture dish is placed in 28 DEG C of incubators and is cultivated, and grown in a manner of attaching, this
When cell kenel be smooth rounded shapes.
Above-mentioned Sf-9 cell culture medium is Grace ' s Insect cell culture medium, every 1000mL NaHCO containing 0.35g3、
3g milk protein hydrolysate, 20mL yeast extract and 10% FBS, separately add 10,000 unit/mL penicillin/streptomycin.
Such as Sf-900II TM SFM (Invitrogen/GIBCO, USA) can be used in above-mentioned Hi-5 cell culture medium.
The transfection of 2.Sf-9 cell
Take 3 × 105/ mL Sf9 cell is cultivated in 12 hole tissue culture dishes, at least 1 hour at room temperature is stood, to thin
Born of the same parents attach, then distinctly take 1 μ g recombinant viral vector DNA and 2 μ L flashBAC DNA and 6 μ LReagentWith Unsupplemented Grace ' s MediumIt is uniformly mixed, stands 45 at room temperature
Minute, remove the cell supernatant in 12 hole tissue culture dishes.Later, after with Sf-900II SFM cleaning 2 times, culture is removed
Base, and said mixture 1mL is added in the tissue culture dishes of celliferous 12 hole, in 27 DEG C after stationary culture 5 hours, in removal
Clear liquid adds 1mL growth medium (TNM-FH；Sigma-Aldrich Co.LLC., USA), it is statically placed in 27 DEG C of incubators 5
It is above or until observing that infection symptoms occurs in cell.
3. the separation of recombinant baculovirus and viral power valence increase spoke
By the Sf9 cell after above-mentioned transfection through culture in 3 to 5 days, if there is the cell being infected with the virus in observation cell
Lesion kenel (cytopathic effect；When CPE), that is, it may be recovered recombinant baculovirus.
Firstly, supernatant is moved into 1.5mL centrifuge tube with 1,000xg, it is centrifuged 5 minutes, then will be recombinated containing zero generation (P0)
The kind poison inventory liquid (virus stock) of baculoviral, packing move into new brown 1.5mL centrifuge tube, are protected from light and are stored in -80 DEG C
It is spare.
Take 3 × 105The Sf9 cell of/mL uniformly returns kind into 12 hole tissue culture dishes, stands to cell paste at room temperature
It is attached, then zero generation (P0) the recombinant baculovirus liquid of 15 μ L is taken to be added in culture medium, 27 DEG C of incubators are statically placed in after 5 days, Ji Kejin
Row separates (P1) recombinant baculovirus primary.When separation, cell supernatant is moved into centrifuge tube, is centrifuged 5 with the revolving speed of 1,000xg
After minute, then kind poison inventory's liquid of (P1) recombinant baculovirus primary will be contained, packing moves into new brown centrifugation tubule, keeps away
Light be stored in -80 DEG C it is spare.
It repeats the above steps, after the yield for amplifying the recombinant baculovirus of the second generation (P2), recycles virus liquid and by second
Generation (P2) recombinant baculovirus kind poison inventory's liquid be stored in -80 DEG C it is spare.
Embodiment three: the expression assessment of the recombination fusion antigen protein of recombinant baculovirus expression system
1. separation recombination fusion antigen protein
Take the 1 × 10 of 25mL6/ mL Hi-5 cell returns kind in 125mL triangle shake bottle, be added after recombinant baculovirus in
27 DEG C are collected cell after culture 3-5 days, and in 4 DEG C 10,000xg, centrifugation removes supernatant after ten minutes, and cellular portions are then to surpass
Sound wave crusher breaks cell, and the extraction of recombination fusion antigen protein (or BV-gp6LdN5) is completed after Quantitative Western,
And it is analyzed with SDS-PAGE and western blot method.
2.SDS-PAGE analysis
By the sample protein of quantitative mistake that converts, 4x Sample Buffer is added and is uniformly mixed, and boils 5 points in boiling water
Clock, using SDS make protein denaturation and uniformly it is negatively charged.The glass plate for making colloid (gel) is set up, and is filled
It is appropriate whether pure water test equipment is installed.Reagent shown in table 2, production separation colloid is sequentially added in small beaker.
Table 2
It is ready for after above-mentioned separation colloid, separation colloid is slowly injected into glass plate, and at the interface of separation colloid
On slowly add ddH2O separates colloid condensation polymerization after 25 minutes, with filter paper by remaining ddH2O is blotted.It is sequentially added again
Reagent shown in table 3 makes lamination colloid (stacking gel).
Table 3
After about 15 minutes, lamination gel polymerisation is completed, and can inject sample.By glass plate in device on Vertial electrophorestic tank
It is appropriate, 1x Running buffer is added.It places a sample into colloid, sample sets 60V when lamination colloid, waits and goes to separation gel
Structural reform is with 120V protein isolate matter.After about 3 hours, achievable electrophoretic procedures, result is as shown in Figure 3.
Refering to Fig. 3, the SDS- of Hi-5 cell expression recombination fusion antigen protein according to an embodiment of the invention is shown
PAGE analyzes photo, wherein the band of BSA of 1 μ g, 2 μ g, 4 μ g, 8 μ g are used to establish standard curve, the road M is protein mark
Note, the 1st is the band of Hi-5 cell protein, the 2nd band to express recombination fusion antigen protein by Hi-5 cell.By
The result of the 2nd band of Fig. 3 is it is found that the size for recombinating fusion antigen protein expressed by Hi-5 cell is 41kDa.
3.Western Northern blot analysis
After the completion of above-mentioned SDS-PAGE protein electrophorese, sheet glass excision lamination colloid is removed, separation colloid is soaked in
It transfers in buffer (Transfer buffer) 10 minutes.It is another to prepare to turn stain folder, layer overlay is pressed from both sides in turning stain to transfer buffer
The Whatman 3M filter paper (Advantec, Japan) impregnated.Cut appropriately sized PVDF transfer film (Immobilon TM-
P Transfer Membrane, Millipore, Ireland) to be soaked in for 100% methanol time be about 5 minutes or so, then with
Transfer buffer is dipped to surface, is placed on filter paper.The film for being soaked in transfer buffer is placed on pvdf membrane, finally
A filter paper is spread, the stain that turns for clipping glue and film is folded up and turns in stain slot.There is PDVF film that need to be placed in towards anode, and film
Towards cathode, under the conditions of 250mA, carry out turning stain 75 minutes.
Pvdf membrane is removed with 1x PBST cleaning 5 minutes, Block buffer is added, is shaken 1 hour in 4 DEG C.It is anti-that level-one is added
Body (Mouse anti-His Monoclonal Antibody, Rural Technologies, Inc.；Extension rate: 1:2,
000) 4 DEG C, are placed in shake 18 hours.After removing Primary antibodies, after cleaning pvdf membrane for several times with 1xPBST, it is anti-to add second level
Body (Goat Anti-mouse IgG-HRP, Chemicon, USA；Extension rate: 1:15,000), it is placed in 4 DEG C and shakes 60 points
Clock.After removing secondary antibody, for several times with 1x PBST cleaning pvdf membrane.Then, PBST buffer is removed, it is aobvious that ECL Plus is added
Toner (ECL Plus Western Blotting Detection Reagents, GE Healthcare, UK) reacts 1 point
Clock.Later, pvdf membrane is placed in the platform middle of cold light instrument photographic system (SYNGENE G, Bock science and technology, Taiwan), with
After commercially available image analysis software is analyzed, the molecular weight of target protein can be calculated, result is as shown in Figure 4.
Refering to Fig. 4, it is anti-that Hi-5 cell expression recombination fusion antigen protein utilization according to an embodiment of the invention is shown
The Western blot analysis photo of His monoclonal antibody, wherein the road M is protein labeling, the road C is Hi-5 cell protein
Band, the 1st expresses the band of the 2nd day recombination fusion antigen protein for Hi-5 cell, and the 2nd is Hi-5 cell expression the 3rd
The band of it recombination fusion antigen protein, the 3rd expresses the band of the 4th day recombination fusion antigen protein for Hi-5 cell,
4th expresses the band of the 5th day recombination fusion antigen protein for Hi-5 cell.It can by the result of the road the 1-4 band of Fig. 4
Know, the yield for recombinating fusion antigen protein is incremented by successively with the increase of expression number of days, and fused antigen egg was recombinated at the 5th day
White yield is 533 μ g/mL.
4. virulence test
Choose the rigid weanling pig of 3-4 week old, PRRSV and swine fever (classical swine fever；CSF) antigen and
Pig 8 of antibody all feminine genders.3 days before attack poison, all test pigs are measured body temperature 3 days, the normal person of body temperature just carries out
Attack poison.When carrying out attacking poison, with 105.0TCID50The PRRSV virus power valence of/mL, in every incidence intramuscular injection 3mL.After attacking poison, often
Its measurement body temperature is simultaneously observed 14 days.After observation period, dissect observation clinical symptoms and pathological change, while progress of taking a blood sample are carried out
PCR detection.
5. Immunization is tested
The rigid weanling pig of 3-4 week old is chosen, it is pig 15 of PRRSV and CSF antigen, antibody feminine gender, random to divide three groups,
First group: be immunized aforementioned extraction recombination fusion antigen protein and commercially available oily adjuvant (such as incomplete Freund's adjuvant etc.) 5,
The subunit vaccine composition (containing 50 μ g antigen proteins and 1.2mL oil adjuvant) of every incidence intramuscular injection 2mL.The
Two groups: the recombination fusion antigen protein of aforementioned extraction, commercially available oily adjuvant (such as incomplete Freund's adjuvant etc.) and CpG synergist
(sequence as shown in SEQ ID NO.:2), subunit vaccine composition (the 50 μ g antigen eggs of every incidence intramuscular injection 2mL
White, the oily adjuvant of 1.2mL and 50 μ g CpG synergist).Remaining nonvaccinated pig is only inoculated with High 5 as negative control
Cell liquid and oily adjuvant (containing 125 μ L cell extraction liquid and 1.2mL oil adjuvant), isolated rearing.It will after 28 days immune
All test pigs carry out PRRSV virus liquid and attack poison, musculi colli injection 105.0TCID50/ mL, every injection 3mL.Observation 21 days.
Dissect is carried out after phase to be seen, is determined according to clinical symptoms, pathological change and antigen, antibody test.
After the test of above-mentioned Immunization, the Vaccine effectiveness of first group of pig is up to 75%, the guarantor of second group of pig
Shield effect is more up to 100%, and the Vaccine effectiveness of the pig of third group is 0%.Thus it is proved really with subunit made from this
Vaccine composition can provide the Vaccine effectiveness for filling part really, and add immunopotentiator in subunit vaccine composition, protection
Effect is more preferably, it is expected to the PRRSV subunit vaccine composition in novel form is applied, to prevent and treat pig breeding and respiratory complication.
It need to supplement, though the present invention is with specific recombinant plasmid, expression system, separation method, cultural method, analysis side
Method or particular instrument illustrate the recombination fused antigen base of anti-porcine reproductive and respiratory syndrome virus infection of the invention as illustrating
Cause, recombination fusion antigen protein and the subunit vaccine composition containing it, it is any with general in the technical field of the invention
The knowledgeable is notified it is found that the present invention is not limited thereto, without departing from the spirit and scope of the present invention, anti-pig breeding of the invention with
Recombination fusion antigen gene, recombination fusion antigen protein and the subunit vaccine containing it of breathing syndrome virus infection combine
Other recombinant plasmids, other eukaryotic expression systems, other separation methods, other cultural methods, other analyses also can be used in object
Method or Other Instruments carry out.
By the embodiments of the present invention it is found that the recombination of anti-porcine reproductive and respiratory syndrome virus infection of the invention is merged
Antigen gene, recombination fusion antigen protein and the subunit vaccine composition containing it the advantage is that and cut having for PRRSV
Glycoprotein GP5, catenation sequence and the memebrane protein M of the end short N ' decoy epitopes are fused to recombination fusion antigen gene and by this sequences
After carrying out codon optimization, recombinant baculovirus expression system is recycled to carry out vivoexpression, it can be promoted and recombinate fused antigen
The yield of albumen.Resulting recombination fusion antigen protein is when being applied to subunit vaccine composition, it is possible to provide preferable vaccine
Protection does not have again toxin expelling and returns malicious risk, thus be effectively improved existing PRRSV Yield of Antigen is low, immune protective efficiency not
Good and tool and returns the variety of problems such as malicious risk at toxin expelling.
Although the present invention is disclosed above with several embodiments, it is not intended to limit the invention, in institute of the present invention
Belonging to any in technical field has ordinary skill, without departing from the spirit and scope of the present invention, can various modifications may be made and change
Become, therefore protection scope of the present invention is subject to the appended claims.
Claims (7)
1. a kind of isolated nucleic acid, to recombinate fusion antigen gene shown in sequence number SEQ ID NO.:1.
2. a kind of recombinant viral vector, it includes recombinate fusion antigen gene shown in SEQ ID NO.:1.
3. a kind of recombination fusion antigen protein, it is rod-shaped through recombinating that it includes recombination fusion antigen genes shown in SEQ ID NO.:1
The recombination fusion antigen protein of virus expression systems expression.
4. a kind of subunit vaccine composition of anti-porcine reproductive and respiratory syndrome virus PRRSV infection, it includes SEQ ID
Recombination fusion antigen protein and medicine of the fusion antigen gene through recombinant baculovirus expression system expression are recombinated shown in NO.:1
Acceptable carrier on.
5. the vaccine composition of anti-PRRSV infection according to claim 4, the wherein pharmaceutically acceptable carrier packet
Containing adjuvant/or immunopotentiator.
6. the vaccine composition of anti-PRRSV infection according to claim 5, wherein the immunopotentiator includes CpG synergy
Agent.
7. the vaccine composition of anti-PRRSV infection according to claim 6, wherein the CpG synergist is SEQ ID NO.:
Sequence shown in 2.
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Non-Patent Citations (3)
| Title |
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| immune response of pigs inoculated with mycobacterium bovis bcg expressing a truncated form of GP5 and M protein of porcine reproductive and respiratory syndrome virus;reginaldo g. bastos et al.;《vaccine》;20041231;467-474 * |
| the immunogenicity of dna constructs co-expressing GP5 and M protein of porcine reproductive and respiratory syndrome virus conjugated by gpgp linker in pigs;min yuan chia et al.;《veterinary microbiology》;20101230;189-199 * |
| 共表达猪繁殖与呼吸综合征病毒E、M、和N基因重组腺病毒的构建;徐娜;《中国优秀硕士学位论文全文数据库 农业科技辑》;20111115(第11期);D050-61 * |
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