CN103923884A - Porcine pseudorabies virus gene deletion strain, vaccine composition, and preparation method and application of vaccine composition - Google Patents
Porcine pseudorabies virus gene deletion strain, vaccine composition, and preparation method and application of vaccine composition Download PDFInfo
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Abstract
The invention provides a porcine pseudorabies virus gene deletion strain, a vaccine composition, and a preparation method and an application of the vaccine composition. The vaccine composition comprises an immunizing dose of an attenuated livetotivirus antigen and an inactivated totivirus antigen of the porcine pseudorabies virus gene deletion strain or its culture. The vaccine composition can effectively induce the antibody production, can effectively protect pigs, and can be used as a marking vaccine to effectively differentiate wild strains and vaccine strains.
Description
Technical field
The present invention relates to the vaccine composition of a kind of porcine pseudorabies virus gene-deleted strain and preparation thereof, and preparation method and application, animal virology field belonged to.
Background technology
Pseudoabies, claim again AujeszkyShi disease, be by the one of multiple domestic animal, poultry and the wildlifes such as the caused pig of herpesvirus suis I type (Suid herpesvirus1strain) in herpetoviridae (Herpesviridae) α subfamily, ox, sheep taking heating, very itch (except pig) and encephalomyelitis as the acute infectious disease of primary symptom.The pseudoabies of pig extensively exists in China, and harm is serious, is one of main epidemic disease of restriction large-scale pig farm production.It can cause that nervous symptoms, paralysis appear in pregnant sow miscarriage, stillborn foetus or mummy tire and piglet, and mortality ratio is high.PRV has stronger pantropic, neural preferendum and latent infection characteristic, at peripheral nervous system latent infection for a long time, becomes infectious virus when latent virus is activated, and will be fallen ill by the host of latent infection.
Research shows that subunit vaccine can provide corresponding protection to immune animal, and subunit vaccine is to utilize gene engineering method that cause of disease protective antigen gene is cloned in protokaryon or eukaryotic expression system, makes its high efficient expression and the vaccine made.Find that at present gB, gC, gD in Pseudorabies virus glycoprotein all can make body produce neutralizing antibody, these antibody be no matter in vivo, external, or in having in the situation that having or not complement to exist and the ability of PRV." pseudoabies subunit vaccine progress " (Yang Chenghuai, Lou Gaoming, Chen Nanhui " Jiangxi animal and veterinary magazine " the 3rd phase in 2004) disclose in 11 kinds of glycoprotein having found at present pseudorabies virus, gB, gC, gD all can induce body to produce neutralizing antibody.In the presence that there is no complement, in the monoclonal anti physical efficiency of gB, gC, gD and PRV.The monoclonal antibody of the anti-gB of pig and injected in mice, gC, gD all can be resisted the attack of the strong poison of PRV.Therefore, gB, gC, gD are the first-selected albumen of development PRV subunit vaccine.Glycoprotein gd a kind of important in and antigen, be also the major objective of protection antibody, can induce better protecting reaction.US6858385 and US6521231 disclose and have utilized porcine pseudorabies virus gD albumen can prepare the prevention of vaccine for porcine pseudorabies.
An important factor that is used for the vaccine of the plan of eradicating is serology mark, and at present all PRV Serology tests, gE-ELISA is the method for the most effectively distinguishing street strain and vaccine strain.Therefore gE deletion of vaccine is conducive to the elimination plan of pseudo-rabies.Clearly specify only can use pseudo-rabies gE gene-deleted vaccine in the developed country such as the U.S. and the European Community., because it can not identify street strain and vaccine strain infection, therefore in production practice, there is suitable limitation in the variant inactivated vaccine of the pseudorabies that patent application CN103305474A provides.
Summary of the invention
For solving the deficiencies in the prior art, main purpose of the present invention is to provide a kind of porcine pseudorabies virus strain, wherein, described porcine pseudorabies virus strain has the nucleotide sequence of the aminoacid sequence gD glycoprotein shown in code sequence list SEQ ID NO.1, and does not contain the nucleotide sequence of coding gE glycoprotein.
Selectively, described porcine pseudorabies virus has the aminoacid sequence of SEQ ID NO.1 or has the gD glycoprotein that the aminoacid sequence of 98% above homology represents with it, and containing gE glycoprotein.
Selectively, described porcine pseudorabies virus can comprise the gB albumen that has the aminoacid sequence of SEQ ID NO.2 or its fragment and have 95% above nucleotide homology.
Selectively, described porcine pseudorabies virus can comprise the gC albumen that has the aminoacid sequence of SEQ ID NO.3 or its fragment and have 95% above nucleotide homology.
Term of the present invention " gD glycoprotein ", is that porcine pseudorabies virus infects essential structural protein, is one of the main glycoprotein on ripe virus particle cyst membrane surface, also referred to as gp50 albumen.
Term of the present invention " homology " refers to the similarity degree of two aminoacid sequences or two nucleotide sequences in this application.The homology of aminoacid sequence or nucleotide sequence can calculate by any appropriate means well known in the art, for example, target amino acid (or Nucleotide) sequence and reference amino acid (or Nucleotide) sequence can be carried out to sequence alignment, can introduce if desired vacancy, make amino acid identical between the sequence of two comparisons (or Nucleotide) number reach optimization, and calculate on this basis the per-cent of same amino acid (or Nucleotide) between two amino acid (or Nucleotide) sequence.The comparison of amino acid (or Nucleotide) sequence and the calculating of homology can realize by software well known in the art, for example, but be not limited to, BLAST software (can obtain: http://blast.ncbi.nlm.nih.gov/Blast.cgi in the network address of state-run biotechnology information center of the U.S. (NCBI), or see, for example, Altschul S.F.et al, J.Mol.Biol., 215:403-410 (1990); Stephen F.et al, Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 software (can obtain: http://www.eji.ac.uk/Toolsa/clustalw2/ in European bioinformation institute network address, separately see, for example, Higgins D.G.et al, Methods in Enzymology, 266:383-402 (1996); Larkin M.A.et al, Bioinformatics (Oxford, England), 23 (21): 2947-8 (2007)); (on the website of information biology institute of Sweden, can obtain: http://tcoffee.vital-it.ch/cgi-bin/Tcoffee/tcoffee_cgi/index.cg i with TCoffee software etc., separately see, for example, Poirot O.et al, Nucleic Acids Res., 31 (13): 3503-6 (2003); Notredame C.et al, J.Mol.Boil., 302 (1): 205-17 (2000)).Use software while carrying out sequence alignment, the default parameters that can use software to provide, or the parameter that also can provide software according to practical situation adjusts, and these are all in the knowledge of those skilled in the range.
Another object of the present invention is to provide the gE gene-deleted strain of a kind of porcine pseudorabies virus HN1201 strain, and wherein, the gE gene-deleted strain of described HN1201 strain does not contain the nucleotide sequence of coding gE glycoprotein.
Preferably, described porcine pseudorabies virus strain is for to remove gE gene HN1201 strain (Pseudorabies virus by genetic engineering means, strain HN1201) or its culture, described PRV (Pseudorabies virus) HN1201 strain (Pseudorabies virus strain HN1201) preserving number is CCTCC NO.V201311; Be preserved in Chinese Typical Representative culture collection center; Preservation address is Wuhan, China Wuhan University, and preservation date is on May 20th, 2013.
Described term " culture " is viral different generation subcultures, and those skilled in the art know its gene order between different generations, and small variation only may occur, and preferably, described culture is that 5-35 generation is with interior culture.
Preferably, the gE gene-deleted strain karyomit(e) gE gene locus nucleotides sequence of described HN1201 strain is classified the nucleotide sequence of the aminoacid sequence shown in encoding sequence table SEQ ID NO.10 as.
A further object of the present invention is to provide a kind of vaccine composition, wherein, described vaccine composition comprises totivirus antigen, inactivated whole virus antigen, the subunit antigen of the described porcine pseudorabies virus strain gE gene-deleted strain of immunity amount or the attenuated live of its culture.
Preferably, vaccine composition of the present invention comprises described porcine pseudorabies virus or its antigen as activeconstituents.There is the aminoacid sequence of SEQ ID NO.1 or have the gD glycoprotein that the aminoacid sequence of 98% above homology represents with it for the porcine pseudorabies virus of the composition of vaccine, and do not contain gE glycoprotein.
The antigen part that refers to virus component for antigen of the present invention, it causes immunne response, and can comprise the aminoacid sequence having with SEQ ID NO.1.
Selectively, antigen can comprise the gB albumen that has the aminoacid sequence of SEQ ID NO.2 or its fragment and have 95% above nucleotide homology.
Selectively, antigen can comprise the gC albumen that has the aminoacid sequence of SEQ ID NO.3 or its fragment and have 95% above nucleotide homology.
Preferably, described vaccine composition comprise>=10
6.0tCID
50totivirus antigen, the inactivated whole virus antigen of the described porcine pseudorabies virus strain gE gene-deleted strain of/ml or the attenuated live of its culture.
Term " inactivated vaccine " used, also referred to as inactivated vaccines, refers to the suspension with the inactivation of viruses of generation immunizing power as antigen.The example of inactivated vaccine comprises whole virus vaccine and cracking type vaccine.Use currently known methods can produce easily inactivated vaccine.For example, can obtain inactivated virus vaccine by processing virus by formaldehyde solution.Cracking type vaccine can be prepared by peplos after processing with ether.
Preferably, vaccine composition of the present invention can comprise every part 10
6.0tCID
50the porcine pseudorabies virus of amount.When porcine pseudorabies virus is to be less than 10
6.0tCID
50amount while using, vaccine can not effective stimulus antibody produces.On the other hand, the amount exceeding may be uneconomic.
Preferably, the described porcine pseudorabies virus strain gE gene-deleted strain of comprise >=25 μ g/ml of described vaccine composition or the gD proteantigen of its culture.
In addition, pseudorabies vaccine of the present invention can be used in combination to prepare combined vaccine or the combination vaccine that opposing comprises the various diseases of porcine pseudorabies with other inactivation pathogenic agent or antigen.Term used herein " combined vaccine " is used in reference to the vaccine of preparing from the virus mixture of porcine pseudorabies virus of the present invention and at least one different virus.Term " combination vaccine " refers to the vaccine of preparing from viral and bacterium.
Preferably, described vaccine composition also comprises medium, adjuvant, vehicle.
Vaccine composition of the present invention also can comprise medium, adjuvant and/or vehicle.Physiological saline or distilled water can be used as medium.The example that is used for the adjuvant of vaccine composition comprises Freund's incomplete adjuvant or Freund's complete adjuvant, aluminum hydroxide gel, vegetables oil or the mineral wet goods of Fu Shi.The example of vehicle comprises aluminum phosphate, aluminium hydroxide and potassium aluminium sulfate, but is not limited to this.In practice, all substances of preparing for vaccine well known by persons skilled in the art are all applicable to vaccine composition of the present invention.
Another object of the present invention is to provide a kind of method of preparing described vaccine composition, and described method comprises: (1) cultivates the described porcine pseudorabies virus strain gE gene-deleted strain of propagation or its culture; (2) gather in the crops porcine pseudorabies virus strain gE gene-deleted strain or its culture of described propagation, deactivation; (3) add adjuvant, mix.
Particularly, described method is: porcine pseudorabies virus vaccine strain is inoculated in permissive cell separately by (1), and cultivates described postvaccinal permissive cell; Harvested cell culture;
(2) process the virus from step (1) with formaldehyde solution, BPL (beta-propiolactone) or BEI (divinyl imines);
Described permissive cell can be continuous cell line, can be also primary cell.The permissive cell that is suitable for porcine pseudorabies virus includes but not limited to, ST clone (ATCC numbering: CRL-1746), PK-15 clone (ATCC numbering: CCL-33), African green monkey kidney cell Marc-145 clone (ATCC numbering: CRL-12219), bovine kidney cells MDBK clone (ATCC numbering: CCL-22), bull testis passage cell BT clone (ATCC numbering: CRL-1390), Vero clone (ATCC numbering: CCL-81), BHK-21 cells (ATCC numbering: CCL-10), (as: the IBRS-2 of porcine kidney cell system, for example see, DECASTRO, M.P.1964.Behavior offoot and mouth disease virus in cell culture:susceptibility of the IB-RS-2swine cell line.Arquivos Instituto Biologica31:63-78), rabbit kidney continuous cell line (RK, CCL-106) as: ATCC numbering: the continuous cell line such as, or the primary cell such as chick embryo fibroblast and porcine kidney cell.Primary cell can be by means commonly known in the art, separates and prepare with the tissue in animal body.
Vaccine composition according to the present invention can be prepared into oral dosage form or parenteral formulation.Preferably can be by intracutaneous, muscle, intraperitoneal, intravenously, subcutaneous, nose or the parenteral formulation that gives of exterior dura approach.
An also object of the present invention is to provide a kind of method of preparing described vaccine composition, and described method comprises: (1) expresses the gD proteantigen of described porcine pseudorabies virus strain gE gene-deleted strain or its culture; (2) gather in the crops the gD proteantigen of described expression; (3) add adjuvant, mix.
Of the present inventionly also provide the application in the medicine of preparation prevention and treatment porcine pseudorabies virus relative disease of described vaccine composition.
Term used herein " porcine pseudorabies virus relative disease " is used in reference to by pseudorabies poison and infects the disease causing.Its example comprise morbidity piglet show obvious nervous symptoms, lethargic sleep, toot cry, vomit, have loose bowels, fervescence, once fall ill, mummy fetus or stillborn foetus or breeding difficulty can be miscarried, be produced to farrowing sow, but be not limited to this.
Term used herein " prevention " refers to all behaviors by giving to suppress according to vaccine composition of the present invention pseudorabies infection or postponement seizure of disease.Term " treatment " refers to make porcine pseudorabies virus infect all behaviors that the symptom causing alleviates or takes a turn for the better by giving vaccine composition according to the present invention.
Brief description of the drawings
Fig. 1 porcine pseudorabies virus HN1201 strain recombinant transfer vector builds schematic diagram;
Fig. 2 porcine pseudorabies virus HN1201 strain gE gene elmination and recombinable site;
Fig. 3 confirms porcine pseudorabies virus HN1201 strain gE disappearance strain by PCR method, wherein, and from left to right: the first swimming lane is that first-generation virus vPRV-gE-P1 genome primer of gE disappearance carries out the electrophoresis result after pcr amplification; The second swimming lane is that the s-generation of the gE disappearance viral vPRV-gE-P2 genome primer that goes down to posterity carries out the electrophoresis result after pcr amplification; The 3rd swimming lane is that the third generation of the gE disappearance viral vPRV-gE-P3 genome primer that goes down to posterity carries out the electrophoresis result after pcr amplification; The 4th swimming lane is that porcine pseudorabies virus HN1201 strain virus genome primer carries out the electrophoresis result after pcr amplification; The rightest swimming lane is Marker.
in sequence table:
Sequence 1 is porcine pseudorabies virus HN1201 strain gE disappearance strain gD aminoacid sequence;
Sequence 2 is porcine pseudorabies virus HN1201 strain gE disappearance strain gB aminoacid sequence;
Sequence 3 is porcine pseudorabies virus HN1201 strain gE disappearance strain gC aminoacid sequence;
Sequence 4 is porcine pseudorabies virus HN1201 strain gE disappearance strain gD nucleotide sequence;
Sequence 5 is porcine pseudorabies virus HN1201 strain gE disappearance strain gB nucleotide sequence;
Sequence 6 is porcine pseudorabies virus HN1201 strain gE disappearance strain gC nucleotide sequence;
Sequence 7 does not lack the front gE nucleotide sequence of gE for porcine pseudorabies virus HN1201 strain gE lacks strain;
Sequence 8 is the gE aminoacid sequence of porcine pseudorabies virus HN1201 strain;
Sequence 9 is remaining gE nucleotide sequence after porcine pseudorabies virus HN1201 strain part gE disappearance;
Sequence 10 is remaining gE aminoacid sequence after porcine pseudorabies virus HN1201 strain part gE disappearance.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with describing.But these embodiment are only exemplary, scope of the present invention are not formed to any restriction.It will be understood by those skilled in the art that lower without departing from the spirit and scope of the present invention and can the details of technical solution of the present invention and form be modified or be replaced, but these amendments and replacement all fall within the scope of protection of the present invention.
Term " head part " in the present invention refers to the amount of vaccine of every pig injection.
" TCID described in the present invention
50" (50%tissue culture infective dose) refer to half cell culture infective dose, is that the one that represents virus infectivity represents mode.
MEM liquid nutrient medium (liquid) is used purchased from the MEM dehydrated medium of Life Technologies company of the U.S. and is prepared according to its specification sheets.
DMEM substratum of the present invention is prepared with reference to GB/T18641-2002 appendix A compound method.
" PBS " described in the present invention refers to the english abbreviation of phosphate buffered saline buffer (Phosphate Buffer Saline), and that in the present invention, use is the PBS of the pH7.4 of 0.01mM, prepares by described in " molecular cloning " third edition.
The collection of embodiment 1, virus separates
Separating sample aseptic collection pig brain tissue from the sample infecting from the doubtful pseudorabies in Henan, with 1: 10(volume ratio) add MEM nutrient solution, grind, prepare tissue suspension, after 3 freeze thawing repeatedly, the centrifugal 15min of 2000r/min, collect supernatant liquor, then filter through 0.2 μ m filter membrane filter, on PK-15 cell, go down to posterity 37 DEG C and cultivate 1h, change the MEM nutrient solution adding containing 2% calf serum, cultivate 5 for 37 DEG C.Gather in the crops toxic nutrient solution, after 2 freeze thawing, receive poison, change the MEM nutrient solution adding containing 2% calf serum.Adopt porcine pseudorabies virus PCR detection kit (Anheal Laboratories Co., Ltd) to detect porcine pseudorabies virus, result is positive; Utilize PCR test kit to carry out the detection (porcine reproductive and respiratory syndrome virus detection kit, pig parvoviral PCR detection kit Pestivirus suis RT-PCR detection kit are the limited public property product of the Beijing Century prosperous animal epidemic prevention technology of unit) of exogenous virus to the virus utilization separating, PCR detected result is all negative, shows that seed culture of viruses is pure.
Submit the Pseudorabies virus being separated to preservation, described porcine pseudorabies strain is HN1201 strain (Pseudorabies virus, strain HN1201), and preserving number is CCTCC NO.V201311; Be preserved in Chinese Typical Representative culture collection center; Preservation address is Wuhan, China Wuhan University, and preservation date is on May 20th, 2013.
The hereditary property of embodiment 2, isolated viral
Determine the viral hereditary property separating in embodiment 1 by genetic analysis.Utilize the porcine pseudorabies virus genomic dna separating on PK15 cell to do template, the primer shown in use table 1 carries out PCR.Be designed for respectively the primer sequence of amplification gB, gC, gD gene with Primer Premier5.0.
Taking extract genomic dna as template, prepare pcr amplification system as follows: template DNA 100 μ g, (2.5U/ μ is 0.5 μ l l) for PrimerSTAR HS DNA Polymerase, 2 × PrimerSTAR GC Buffer25 μ l, the each 1 μ l(10pmol/ μ of upstream and downstream primer l), dNTP Mix(2.5mM each) 4 μ l, and volume is supplied to 50 μ l with distilled water.Carry out two-step pcr reaction: at 98 DEG C of sex change 10sec, then in 68 DEG C of annealing and extension (calculating required time according to 1kb/min), totally 30 circulations.Stop PCR reaction at 4 DEG C.By carry out the PCR product of electrophoretic analysis gained on 1% the sepharose that contains ethidium bromide.PCR product carries out sequencing.The sequence data obtaining with Lasergene software analysis.
Table 1PCR primer sequence
The pathogenicity of embodiment 3, virus
3.1 different days piglets pathogenic
34～35 6 of age in days porcine pseudorabies negative antibody piglets are divided into 2 groups at random, 4/group (test group) and 2/group (control group), (attacking toxic agent amount is 2 × 10 in collunarium Pigs Inoculated pseudorabies virus HN1201 strain
8.0tCID
50/ head), control group inoculation DMEM substratum; By the strong malicious HN1201 strain of cultivating for 3 generations after 4 49 age in days piglet inoculation preservations, (attacking toxic agent amount is 2 × 10 simultaneously
8.0tCID
50/ head), still with 35 age in days piglets in contrast.After virus inoculation, measure piglet body temperature every day, observe clinical symptom and death condition, concrete outcome is in table 2.
The strong malicious HN1201 strain of table 2 pseudorabies is pathogenic to different days piglet
Result demonstration, the piglet of porcine pseudorabies virus HN1201 strain inoculation different days all can cause piglet morbidity, and can cause more than 3/4 piglet death.
3.2 various dose are pathogenic to piglet
Negative 49 age in days pseudorabies antibodies 8 of piglets are divided into 2 groups at random, 4 every group, separately get 2 piglets in contrast.Experimental group is collunarium inoculation 2 × 10 respectively
7.0tCID
50/ head and 2 × 10
8.0tCID
50/ porcine pseudorabies virus HN1201 strain, control group inoculation DMEM substratum.After virus inoculation, measure piglet body temperature every day, observe clinical symptom and death condition, concrete outcome is in table 3.
Table 3 various dose porcine pseudorabies virus HN1201 strain is pathogenic to piglet
Result demonstration, the clinical separation HN1201 of porcine pseudorabies virus inoculates the piglet of 49 ages in days with various dose, all can cause piglet morbidity, and 4/4 piglet death.
The preparation of embodiment 4, PRV HN1201 strain gE disappearance strain
The structure of 4.1PRV HN1201GFP recombinant virus transfer vector
According to the sequence of the gE gene that will lack (sequence table SEQ ID NO.7), at its two ends design homology arm, be respectively gEA and gEB.GEA and gEB are cloned on pUC19 carrier to called after pUCgEAB.Then GFP gene clone is upper to pUCgEAB, obtain recombinant virus transfer vector, name pUCgEA-GFP-B.In transfer vector, homology arm is gE both sides sequences, so the recombinant virus obtaining after restructuring is gE genetically deficient.Recombinant transfer vector builds schematic diagram and sees Fig. 1, and Fig. 2 is gEA and gEB homology arm position on genome.
4.1.1, the amplification of homologous recombination arm and clone
4.1.1.1 design of primers and template preparation
According to two pairs of primers of gene order design of HN1201 virus, the homology arm for the gE gene both sides of increasing:
The upstream and downstream primer of left side homology arm gEA is respectively:
GEAF:CCG
gAATTCaAAAGCGCAGCCCGGTCCGTAG(underscore is EcoR I restriction enzyme site)
GEAR:CTAG
tCTAGAataacttcgtataatgtatgctatacgaagttatCAGAAAGGGCCGCATGGTCTCA AC(underscore is Xba I restriction enzyme site, and lowercase is loxp site)
The upstream and downstream primer of right side homology arm gEB is respectively:
GEBF:ACAT
gCATGCataacttcgtatagcatacattatacgaagttatCCCGCCCCGCTTAAATACCG(underscore is Sph I restriction enzyme site, and lowercase is loxp site)
GEBR:CCC
aAGCTTcCAGGAGCACCTGGTCGCAGA(underscore is Hind III restriction enzyme site)
Get PRV HN1201 and infect vero cell, more than 80% occur until cell, after pathology, to get part supernatant, adopt Geneaid Viral Nucleic Acid Extraction test kit to extract virus genom DNA, as the template of homology arm amplification.
4.1.1.2 the amplification of homology arm gEA, gEB and clone
Utilize the PrimeSTAR of TAKARA to carry out pcr amplification gEA, gEB, system and condition are as follows:
| PRV?HN1201DNA | 1μL |
| primSTAR | 0.5μL |
| 2*primSTAR?GC?buffer | 25μL |
| dNTP(25mM) | 4μL |
| Upstream primer | 0.5μL |
| Downstream primer | 0.5μL |
| Water | Supply 50 μ L |
After the gEA that pcr amplification goes out separates by agarose gel electrophoresis with gEB fragment, reclaim test kit with day root glue and reclaim object fragment.GEA fragment and pUC19 carrier are used respectively after EcoRI and XbaI double digestion, reclaimed object fragment, after T4DNA ligase connects, transform DH5 α, 37 DEG C of overnight incubation of coating ammonia benzyl plate.Picking mono-clonal enzyme is cut after qualification correctly, identifies correct plasmid called after pUCgEA.PUCgEA and gEB, respectively with reclaiming object fragment after SphI and HindIII double digestion, after T4DNA ligase connects, are transformed to DH5 α, 37 DEG C of overnight incubation of coating ammonia benzyl plate.Picking mono-clonal extracts plasmid, cuts and checks order and identify correct plasmid called after pUCgEAB through enzyme.
4.1.2 the amplification of marker gene GFP
4.1.2.1GFP the removal of carrier pAcGFP-C1 multiple clone site
By pAcGFP-C1 plasmid (purchased from Clontech, article No. 632470) with after Bgl II and Sma I double digestion, reclaim linearizing carrier, fill through DNA Polymerase I Large (Klenow) Fragment, after T4DNA Ligase connects, transformed competence colibacillus cell DH5 α obtains the GFP plasmid of deleting MCS, called after: pAcGFP Δ MCS.
4.1.2.2GFP the amplification of gene
Primer according to pAcGFP-C1 carrier sequences Design amplification GFP:
Upstream primer
CMVU:ACGC
gTCGACtAGTTATTAATAGTAATCAATTACG (underscore is SalI restriction enzyme site)
Downstream primer
SV40R:ACAT
gCATGCcTAGAATGCAGTGAAAAAAATGC (underscore is Sph I restriction enzyme site)
Taking plasmid pAcGFP Δ MCS as template amplification GFP gene, system and condition are as follows:
| pAcGFPΔMCS | 1μL |
| primSTAR | 0.5μL |
| 2*primSTAR?GC?buffer | 25μL |
| dNTP(25mM) | 4μL |
| Upstream primer CMVU | 0.5μL |
| Downstream primer SV40R | 0.5μL |
| Water | Supply 50 μ L |
Electrophoresis reclaims object band and carries out next step connection.
4.1.3GFP being connected of marker gene and pUCgEAB
GFP, with after Sal I and Sph I double digestion, is reclaimed to object fragment, be connected with the pUCgEAB plasmid through same double digestion, transformed competence colibacillus cell DH5 α, picking mono-clonal extracts plasmid, and enzyme is cut and is checked order and identify correct plasmid called after pUCgEA-GFP-B.
The acquisition of 4.2 recombinant viruses that contain GFP
4.2.1 transfer vector and HN1201DNA cotransfection Vero cell obtain recombinant virus
With liposome method cotransfection Vero cell, by 3ug PRV-HN1201 virus genom DNA and 5ug transfer vector pUCgEA-GFP-B, carry out transfection according to the step on Lipofectamine2000 (Invitrogen, article No. 11668030) specification sheets.Cultivate containing in the incubator of 5%CO2 at 37 DEG C.36-48h after transfection, cytopathy to be occurred, and lesion has after fluorescence, and harvested cell supernatant, is P0 for recombinant virus, called after rPRV-GFP-gE-(is in Fig. 3).
4.2.2 the plaque purification of recombinant virus
By obtain P0 for recombinant virus rPRV-GFP-gE-vero cells infection after, spread 2% low melting-point agarose, after 48h after there is obvious pathology and fluorescence in cell, picking with the plaque of green fluorescence after-70 DEG C of freeze thawing 3 times, 10 times of doubling dilutions are inoculated in Vero cell six orifice plates of completing in advance, continue picking green fluorescence plaque and carry out purifying, through 8 plaque purifications of taking turns, obtain the recombinant virus rPRV-GFP-gE-not lacking containing the gE of wild-type virus HN1201 of purifying.
The deletion of GFP marker gene in 4.3gE disappearance recombinant virus
The pBS185 plasmid of expressing Cre enzyme (is bought from addgene, the loxP site of Cre enzyme identification homology arm gEA downstream and gEB upstream, be that GFP gene order is deleted by the sequence in the middle of two loxp sites) and recombinant virus rPRV-GFP-gE-genomic dna cotransfection vero cell, after result transfection there is obvious pathology in 24h, and single fluorescence is many.Results P0 for viral doubling dilution after inoculation carry out plaque select, picking does not have the plaque of fluorescence to carry out next round purifying.Take turns screening purifying through 2, obtain not having the virus of fluorescence, called after vPRV-gE-.Extract purified virus genomic dna, PCR qualification shows gE genetically deficient, and GFP marker gene is also deleted.The not gE disappearance viral purification success containing GFP marker gene is described.
4.5PRV HN1201 strain gE disappearance strain is confirmed
Extract the viral genome of gE disappearance virus and wild-type virus, carry out PCR qualification, primer is as follows:
gEDCF：acgaagaggaggaggacgaggaggg
gEDCR：tcgtgcgtctcggtggtgatgtagaa
The PCR product size of wild-type virus amplification is 3109bp, and the PCR clip size of gE disappearance virus amplification is 1398bp, PCR qualification result Fig. 3.
The preparation of embodiment 5 pseudorabies gD albumen
The amplification of 1.PRV gD gene
On well-grown PK15 cell, inoculate the culture of PRV HN1201 virus gE gene-deleted strain or its different generations, the culture of this difference generation be 5-35 generation with interior culture, after results virus, extract PRV genomic dna with the MiniBEST Viral RNA/DNA Extraction Kit Ver.3.0 of TAKARA company test kit.Get 1 μ l genomic dna as template, utilize gD Auele Specific Primer:
GDSF:5 ' ATGCTGCTCGCAGCGCTATTGGC3 ' and
gDSR:5′CTACGGACCGGGCTGCGCTTTTAG3′
Carry out pcr amplification, utilize the high-fidelity enzyme of TAKARA
hS DNA Polymerase with GC Buffer, amplification condition is: 94 DEG C of 3min; 94 DEG C of 30s, 68 DEG C of 90s, 30cycles; 72 DEG C of 5min.PCR product called after gD.Its nucleotide sequence is shown in SEQNO.2, and its aminoacid sequence of deriving is SEQNO.1
2. restructuring the obtaining and identifying of Bacmid
The PCR product gD that high-fidelity enzymatic amplification is obtained is cloned into pFastBac/HBM-TOPO carrier (purchased from Invitrogen company, article No. A11339), clone's system is as follows: PCR product gD4 μ l, Salt solution (salts solution) 1 μ l, TOPO vector1 μ l, totally 6 μ l.Mix, incubated at room 5min, transforms One ShotR Mach1
tMt1R competent cell, coating amicillin resistance flat board, the direction of insertion of picking mono-clonal qualification gD gene, the plasmid that direction of insertion is correct send the order-checking of Invitrogen company, the exactness of qualification gD sequence.The plasmid called after pFastBac/HBM-TOPO-gD that checks order correct.
PFastBac/HBM-TOPO-gD plasmid transforms DH10Bac competent cell (source), shuttle plasmid Bacmid in pFastBac/HBM-TOPO-gD and competent cell carries out swivel base, extract the recombinant plasmid of acquisition with the PureLink HiPure Plasmid DNA Miniprep Kit of Invitrogen, and with the insertion of pUCM13Forward/pUCM13Reverse primer qualification gD, positive Bacmid called after Bacmid-gD.
3. transfection obtains recombinant baculovirus
The method providing according to the specification sheets of the Bac-to-Bac HBM TOPO Secreted Expression System of Invitrogen company is carried out.The 6 every hole of orifice plate pavings 8 × 10
5individual sf9 cell, after cell attachment, carry out transfection according to the specification sheets of Cellfectin II transfection reagent: dilute respectively 8 μ lCellfectin II and 1 μ g Bacmid-gD DNA in 100ul SF-900 II substratum, Vortex mixes, (cumulative volume～210 μ l) for Cellfectin II after DNA after mixed diluting and dilution, mix incubated at room 15～30min, one after another drop of being added in cell.After transfection in 72h after there is cytopathy, and collecting cell culture supernatant, is designated as P0 for recombinant virus vBac-gD.P0 infects sf9 cell for recombinant virus vBac-gD, after 3 generation enlarged culturing, the P3 of acquisition for vBac-gD for expression of recombinant proteins.
4. recombinate shape virus infection High-five cell obtains recombinant protein
P3 is inoculated to High-five cell (purchased from Invitrogen, article No. B85502) for recombinant baculovirus vBac-gD.Suspension culture High-five cell in 500ml triangular flask, reaches 7.0 × 10 to cell density
5after cell/ml, according to the amount virus inoculation of 1MOI, after infecting, 72h collects cells and supernatant.Utilize the tangential flow filtration system of Millipore by 1/10 of volume simmer down to original volume.Use 1%(volume ratio) Triton X-100(purchased from sigma) deactivation baculovirus, SDS-PAGE optical densitometric method measure protein content be 200 μ g/ml.
The preparation of embodiment 6, the sick deactivation vaccine of porcine pseudorabies virus
First form seed lot by the culture of the different generations of separated strain being inoculated in to PK-15 cell culture according to table 4, then by the 1%(V/V of virus culture liquid measure) access in the PK cell culture that forms individual layer, put 37 DEG C of rotating and culturing, in the time that pathology reaches 80%, gather in the crops toxic cell culture fluid, after 2 freeze thawing, receive poison, measure malicious valency.In different generation virus liquids, adding 10%(v/v respectively) to make the final concentration of formaldehyde be 0.2% (V/V) to formaldehyde solution, 37 DEG C of deactivations 18 hours, within every 4 hours, stir 1 time, each 10min that stirs, after deactivation, virus liquid is diluted to the viral level shown in table 3 with the PBS liquid of PH7.4 and then mixes according to volume ratio 54:46 with 206 adjuvants (French SEPPIC company product), and under 30 DEG C of conditions, 120 revs/min are stirred 15 minutes.
The preparation of the each group pseudorabies of table 4 vaccine
The preparation of embodiment 7, the sick gE gene-deleted strain of porcine pseudorabies virus deactivation vaccine
Embodiment 4 is inoculated in to PK-15 cell culture by sick the porcine pseudorabies virus of preparation gE gene-deleted strain and first forms seed lot, then by the 1%(V/V of virus culture liquid measure) access in the PK cell culture that forms individual layer, put 37 DEG C of rotating and culturing, in the time that pathology reaches 80%, gather in the crops toxic cell culture fluid, after 2 freeze thawing, receive poison, measure malicious valency.In different generation virus liquids, adding 10%(v/v respectively) to make the final concentration of formaldehyde be 0.2% (V/V) to formaldehyde solution, 37 DEG C of deactivations 18 hours, within every 4 hours, stir 1 time, each 10min that stirs, after deactivation, virus liquid is diluted to the viral level shown in table 5 with the PBS liquid of PH7.4 and then mixes according to volume ratio 54:46 with 206 adjuvants (French SEPPIC company product), and under 30 DEG C of conditions, 120 revs/min are stirred 15 minutes.
The preparation of the each group pseudorabies of table 5 vaccine
Embodiment 8, inactivated vaccine immunogenicity experiments
21 24 of age in days PRV negative antibody piglets are divided into 6 groups at random, 4/group, vaccine immunity pseudorabies inactivated vaccine prepared by the vaccine immunity pseudorabies inactivated vaccine 2ml/ head of preparing according to table 6 vaccinate deactivation vaccine group injection embodiment 6 and embodiment 7, the pseudorabies living vaccine SA215 strain that control vaccine adopts CN101186902 method to prepare, according to the use of its patent specification immunogenicity determining method, control group inoculation DMEM substratum 2ml/ head.Immunity is attacked poison after latter 28 days, and attacking toxic agent amount is HN1201 strain porcine pseudorabies virus 2 × 10
8.0tCID
50/ head, after attacking poison, measure piglet body temperature every day, observes clinical symptom and death condition (the results are shown in Table 6) and attack 3 kinds of ELISA test kits (IDEXX) of getting respectively the porcine blood serum PRV (Pseudorabies virus) gE antibody of test group and control group before poison and carry out gE antibody test according to its specification sheets.
The grouping of table 6 Study On Immunogenicity animal
| Group | Vaccinate | Immunizing dose |
| Deactivation vaccine A | Vaccine group A prepared by embodiment 6 | 2ml/ head |
| Deactivation vaccine B | Vaccine group B prepared by embodiment 6 | 2ml/ head |
| Deactivation vaccine C | Vaccine group A prepared by embodiment 7 | 2ml/ head |
| Deactivation vaccine D | Vaccine group B prepared by embodiment 7 | 2ml/ head |
| Living vaccine SA215 | Porcine pseudorabies virus living vaccine | 10 6.0TCID 50/ head |
| Control group | DMEM substratum | 2ml/ head |
After vaccine immunity, measure weekly the NAT of inactivated vaccine group with reference to the method for GB/T18641-2002 method serum neutralization test, the results are shown in Table 7.
The antibody situation of different time after table 7 pseudorabies inactivated vaccine immunity piglet
The result of table 7 shows, after pseudorabies inactivated vaccine immunity piglet, can produce higher neutralizing antibody, and raises gradually with the immune time.
Immunity is attacked poison after latter 28 days, and attacking toxic agent amount is porcine pseudorabies virus HN1201 strain porcine pseudorabies virus 2 × 10
8.0tCID
50/ head, observes clinical symptom and death condition in table 8, attacks poison and measures piglet body temperature in table 9 rear every day.
After table 8 pseudorabies inactivated vaccine immunity piglet, attack malicious situation
After the piglet of table 9 pseudorabies vaccine immunity is attacked poison, piglet body temperature changes
The result of table 8 and table 9 shows; after pseudorabies inactivated vaccine immunity piglet; although can not infect (occurring clinical symptom) by blocking virus; but can provide 100%(4/4 for piglet) protection; and contrast piglet is attacked poison all death afterwards in latter 4 days; therefore, pseudorabies inactivated vaccine has good protection.With respect to control group living vaccine, the piglet fervescence time of inactivated vaccine immune group is shorter in addition, and vaccine group C and group D do not rise to 41 degree, and appetite normal, and substantially there is no clinical symptom, demonstrates good immunoprotection.
Pseudorabies gE Elisa antibody test: vaccine A group and vaccine B group detect gE antibody, are positive; And vaccine C group and vaccine D group and living vaccine group do not detect gE antibody, be negative.
Embodiment 9, pseudorabies are to the preparation of gD subunit vaccine
Subunit antigen prepared by embodiment 5, the volume that is diluted to table 10 with PBS liquid (pH7.4) and 206 adjuvants (French SEPPIC company product) mix according to volume ratio 54:46, and under 30 DEG C of conditions, 120 revs/min are stirred 15 minutes.
The preparation of the pseudo-mad subunit vaccine of the each group pig of table 10
| Group | Protein content | Vaccine proportioning (inactivation of viruses liquid: 206 adjuvants) |
| A | 25μg/ml | 54:46 |
| B | 100μg/ml | 54:46 |
Embodiment 10, the immunogenicity experiments of subunit vaccine to pig
21 12 of age in days PRV negative antibody piglets are divided into 3 groups at random, 4/group, inject vaccine prepared by embodiment 9 according to table 2, immune swine pseudorabies virus subunit vaccine 2ml/ head.Control group inoculation DMEM substratum 2ml/ head.Immunity is attacked poison after latter 28 days, and attacking toxic agent amount is porcine pseudorabies virus HN1201 strain 2 × 10
8.0tCID
50/ head, attacks poison and measures piglet body temperature rear every day, observes clinical symptom and death condition (the results are shown in Table 12).
The grouping of table 11 Study On Immunogenicity animal
| Group | Vaccinate | Immunizing dose |
| Subunit vaccine A | Vaccine group A prepared by embodiment 9 | 2ml/ head |
| Subunit vaccine B | Vaccine group B prepared by embodiment 9 | 2ml/ head |
| Control group | DMEM substratum | 2ml/ head |
Immunity is attacked poison after latter 28 days, and attacking toxic agent amount is porcine pseudorabies virus HN1201 strain 2 × 10
8.0tCID
50/ head, observes clinical symptom and death condition in table 12, attacks poison and measures piglet body temperature in table 13 rear every day.
After table 12 porcine pseudorabies virus subunit vaccine immunity piglet, attack malicious situation
After the piglet of table 13 porcine pseudorabies virus subunit vaccine immunity is attacked poison, piglet body temperature changes
The result of table 12 and table 13 shows; after porcine pseudorabies virus subunit vaccine immunity piglet, although can not infect (occurring clinical symptom) by blocking virus, can provide 100%(4/4 for piglet) protection; and contrast piglet is attacked poison all death afterwards in latter 4 days, demonstrate good immunoprotection.
The foregoing is only preferred embodiment of the present invention, in order to limit the present invention, within the spirit and principles in the present invention not all, any amendment of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Claims (10)
1. a porcine pseudorabies virus strain, wherein, described porcine pseudorabies virus strain has the nucleotide sequence of the aminoacid sequence gD glycoprotein shown in code sequence list SEQ ID NO.1, and does not contain the nucleotide sequence of coding gE glycoprotein.
2. a gE gene-deleted strain for porcine pseudorabies virus HN1201 strain, wherein, the gE gene-deleted strain of described HN1201 strain does not contain the nucleotide sequence of coding gE glycoprotein.
3. the gE gene-deleted strain of porcine pseudorabies virus HN1201 according to claim 2 strain, wherein, the gE gene-deleted strain karyomit(e) gE gene locus nucleotides sequence of described HN1201 strain is classified the nucleotide sequence of aminoacid sequence shown in encoding sequence table SEQ ID NO.10 as.
4. a vaccine composition, wherein, described vaccine composition comprises totivirus antigen, inactivated whole virus antigen, the subunit antigen of the attenuated live of porcine pseudorabies virus strain gE gene-deleted strain described in claim 1～3 any one of immunity amount or its culture.
5. vaccine composition according to claim 4, wherein, described vaccine composition comprises>=10
6.0tCID
50totivirus antigen, the inactivated whole virus antigen of the porcine pseudorabies virus strain gE gene-deleted strain described in claim 1～3 any one of/ml or the attenuated live of its culture.
6. vaccine composition according to claim 4, wherein, the porcine pseudorabies virus strain gE gene-deleted strain described in claim 1～3 any one of comprise >=25 μ g/ml of described vaccine composition or the gD proteantigen of its culture.
7. vaccine composition according to claim 4, wherein, described vaccine composition also comprises medium, adjuvant, vehicle.
8. prepare a method for vaccine composition claimed in claim 4, described method comprises:
(1) cultivate the described porcine pseudorabies virus strain gE gene-deleted strain of propagation or its culture;
(2) gather in the crops porcine pseudorabies virus strain gE gene-deleted strain or its culture of described propagation, deactivation;
(3) add adjuvant, mix.
9. prepare a method for vaccine composition claimed in claim 4, described method comprises:
(1) express the gD proteantigen of described porcine pseudorabies virus strain gE gene-deleted strain or its culture;
(2) gather in the crops the gD proteantigen of described expression;
(3) add adjuvant, mix.
10. the application in the medicine of preparation prevention and treatment porcine pseudorabies virus relative disease according to the vaccine composition described in claim 4～7.
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