CN105368791A - Porcine pseudorabies virus gene-deleted strain, vaccine composition and their preparation method and application - Google Patents
Porcine pseudorabies virus gene-deleted strain, vaccine composition and their preparation method and application Download PDFInfo
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Abstract
The invention provides a vaccine composition, comprising porcine pseudorabies virus gE gene-deleted strain of immunizing dose, or an attenuated live totivirus antigen or inactivated totivirus antigen of the culture thereof. The vaccine composition is effective in inducing generation of antibodies to provide effective protection for swine and can be used as a marker vaccine to effectively distinguish wild strains and vaccine strains.
Description
The application is the divisional application of patent application 201410059931.9
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, belong to animal virology field.
Background technology
Pseudoabies, sick also known as AujeszkyShi, be the multiple domestic animal such as pig, ox, sheep caused by herpesvirus suis I type (Suidherpesvirus1strain) in herpetoviridae (Herpesviridae) α subfamily, the one of poultry and wildlife with heating, very to itch (except pig) and encephalomyelitis is 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 pregnant sow miscarriage, stillborn foetus or mummy tire and piglet to occur nervous symptoms, paralysis, and mortality ratio is high.PRV has stronger pantropic, neurotropism and latent infection characteristic, can latent infection for a long time in peripheral nervous system, becomes infectious virus, will be fallen ill by the host of latent infection when latent virus is activated.
Research shows that subunit vaccine can provide corresponding protection to immune animal, and subunit vaccine utilizes gene engineering method to be cloned in protokaryon or eukaryotic expression system by cause of disease protective antigen gene, makes its high expression and the vaccine made.GB, gC, gD in current discovery Pseudorabies virus glycoprotein all can make body produce neutralizing antibody, these antibody be no matter in vivo, external, or deposit with or without complement have in case in 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 the 11 kinds of glycoprotein found at present pseudorabies virus, gB, gC, gD all can induce body to produce neutralizing antibody.In the presence not having complement, in the monoclonal anti physical efficiency of gB, gC, gD and PRV.The monoclonal antibody of pig and injected in mice anti-gB, gC, gD all can resist 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, better protecting can be induced to react.US6858385 and US6521231 discloses and utilizes porcine pseudorabies virus gD albumen can prepare the prevention of vaccine for porcine pseudorabies.
The factor important for of vaccine of the plan of eradicating is Serological markers, and in PRV Serology tests all at present, gE-ELISA is the method the most effectively distinguishing street strain and vaccine strain.Therefore gE deletion of vaccine is conducive to the elimination plan of pseudo-rabies.The developed country such as the U.S. and the European Community clear stipulaties only can use pseudo-rabies gE gene-deleted vaccine., because it can not identify that street strain and vaccine strain infect, 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 polynucleotide SEQIDNO.1, and not containing the nucleotide sequence of coding gE glycoprotein.
Selectively, the aminoacid sequence that described porcine pseudorabies virus has SEQIDNO.1 or the gD glycoprotein that the aminoacid sequence having more than 98% homology with it represents, and not containing gE glycoprotein.
Selectively, described porcine pseudorabies virus can comprise the gB albumen that the aminoacid sequence with SEQIDNO.2 or its fragment have more than 95% nucleotide homology.
Selectively, described porcine pseudorabies virus can comprise the gC albumen that the aminoacid sequence with SEQIDNO.3 or its fragment have more than 95% nucleotide homology.
Term of the present invention " gD glycoprotein ", being that porcine pseudorabies virus carries out infecting required structural protein, is one of the primary glycoproteins 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 be calculated by any appropriate means well known in the art, such as, target amino acid (or Nucleotide) sequence and reference amino acid (or Nucleotide) sequence can be carried out sequence alignment, vacancy can be introduced if desired, make amino acid (or Nucleotide) number identical between the sequence of two comparisons reach optimization, and calculate the per-cent of same amino acid (or Nucleotide) between two amino acid (or Nucleotide) sequences on this basis.The comparison of amino acid (or Nucleotide) sequence and the calculating of homology can pass through software simulating well known in the art, such as, but be not limited to, BLAST software (can obtain: http://blast.ncbi.nlm.nih.gov/Blast.cgi in the network address at US National Biotechnology Information center (NCBI), or see, such as, AltschulS.F.etal, J.Mol.Biol., 215:403-410 (1990); StephenF.etal, NucleicAcidsRes., 25:3389-3402 (1997)), ClustalW2 software (can obtain: http://www.eji.ac.uk/Toolsa/clustalw2/ in European Bioinformatics institute network address, separately see, such as, HigginsD.G.etal, MethodsinEnzymology, 266:383-402 (1996); LarkinM.A.etal, Bioinformatics (Oxford, England), 23 (21): 2947-8 (2007)); (can obtain on the website of information biology institute of Sweden: http://tcoffee.vital-it.ch/cgi-bin/Tcoffee/tcoffee_cgi/index.cg i with TCoffee software etc., separately see, such as, PoirotO.etal, NucleicAcidsRes., 31 (13): 3503-6 (2003); NotredameC.etal, J.Mol.Boil., 302 (1): 205-17 (2000)).When using software to carry out sequence alignment, can use the default parameters that software provides, or also can adjust the parameter that software provides according to practical situation, these 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 is not containing the nucleotide sequence of coding gE glycoprotein.
Preferably, described porcine pseudorabies virus strain is for remove gE gene HN1201 strain (Pseudorabiesvirus by genetic engineering means, strainHN1201) or its culture, described PRV (Pseudorabies virus) HN1201 strain (PseudorabiesvirusstrainHN1201) preserving number is CCTCCNO.V201311; Be preserved in China typical culture collection center; Preservation address is Wuhan, China Wuhan University, and preservation date is on May 20th, 2013.
Described term " culture " is the different generation subcultures of virus, and those skilled in the art know its gene order between different generation only small variation may occur, and preferably, described culture is the culture within 5-35 generation.
Preferably, the gE gene-deleted strain karyomit(e) gE gene locus nucleotides sequence of described HN1201 strain is classified as the nucleotide sequence of the aminoacid sequence shown in polynucleotide SEQIDNO.10.
Another 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.The aminoacid sequence that porcine pseudorabies virus for the composition of vaccine has SEQIDNO.1 or the gD glycoprotein that the aminoacid sequence having more than 98% homology with it represents, and not containing gE glycoprotein.
Refer to the antigen part of virus component for antigen of the present invention, it causes immunne response, and can comprise the aminoacid sequence had with SEQIDNO.1.
Selectively, antigen can comprise the gB albumen that the aminoacid sequence with SEQIDNO.2 or its fragment have more than 95% nucleotide homology.
Selectively, antigen can comprise the gC albumen that the aminoacid sequence with SEQIDNO.3 or its fragment have more than 95% 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 as antigen to produce the suspension of the inactivation of viruses of immunizing power.The example of inactivated vaccine comprises whole virus vaccine and cracking type vaccine.Use currently known methods can produce inactivated vaccine easily.Such as, by can inactivated virus vaccine be obtained by formaldehyde solution process virus.Cracking type vaccine can be prepared by peplos after with ether process.
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 use time, vaccine can not effective stimulus antibody produce.On the other hand, the amount exceeded may be uneconomic.
Preferably, described vaccine composition comprises >=the described porcine pseudorabies virus strain gE gene-deleted strain of 25 μ g/ml or the gD proteantigen of its culture.
In addition, pseudorabies vaccine of the present invention can combinationally use to prepare with other inactivation pathogenic agent or antigen the combined vaccine or combination vaccine of resisting the various diseases comprising porcine pseudorabies.Term used herein " combined vaccine " is used in reference to the vaccine prepared 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 prepared from virus 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.Example 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 thereto.In practice, all substances prepared 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 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) porcine pseudorabies virus strain gE gene-deleted strain or its culture of described propagation is gathered in the crops, deactivation; (3) adjuvant is added, mixing.
Particularly, described method is: porcine pseudorabies virus vaccine strain is inoculated in respective permissive cell by (1), and cultivates described postvaccinal permissive cell; Harvested cell culture;
(2) by the virus of formaldehyde solution, BPL (beta-propiolactone) or BEI (binary ethylenimine) process from step (1);
Described permissive cell can be continuous cell line, also can be primary cell.The permissive cell being suitable for porcine pseudorabies virus includes but not limited to, (ATCC numbers ST clone: CRL-1746), (ATCC numbers PK-15 clone: CCL-33), (ATCC numbers African green monkey kidney cell Marc-145 clone: CRL-12219), (ATCC numbers bovine kidney cells MDBK clone: CCL-22), (ATCC numbers bull testis passage cell BT clone: CRL-1390), (ATCC numbers Vero cell line: CCL-81), (ATCC numbers BHK-21 cells: CCL-10), porcine kidney cell system (as: IBRS-2, see such as, DECASTRO, M.P.1964.Behavioroffootandmouthdiseasevirusincellculture: susceptibilityoftheIB-RS-2swinecellline.ArquivosInstitut oBiologica31:63-78), rabbit kidney continuous cell line (RK, CCL-106) as: ATCC numbers: the continuous cell line such as, or the primary cell such as chick embryo fibroblast and porcine kidney cell.Primary cell can by means commonly known in the art, carry out being separated and preparing with the tissue in animal body.
Vaccine composition according to the present invention can be prepared into oral dosage form or non-oral dosage forms.Preferably by intracutaneous, muscle, intraperitoneal, intravenously, subcutaneous, nose or the non-oral dosage forms that gives of epidural pathways.
An also object of the present invention is to provide a kind of method 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) the gD proteantigen of described expression is gathered in the crops; (3) adjuvant is added, mixing.
Of the present inventionly additionally provide the application of described vaccine composition in the medicine preparing prevention and therapy porcine pseudorabies virus relative disease.
Term used herein " porcine pseudorabies virus relative disease " is used in reference to and infects by pseudorabies poison the disease caused.Its example comprise morbidity piglet show obvious nervous symptoms, lethargic sleep, toot cry, vomit, have loose bowels, fervescence, once fall ill, farrowing sow can be miscarried, produce mummy fetus or stillborn foetus or breeding difficulty, but is not limited thereto.
Term used herein " prevention " refers to by giving suppress pseudorabies to infect according to vaccine composition of the present invention or postpone all behaviors of seizure of disease.Term " treatment " refers to all behaviors making porcine pseudorabies virus infect the symptom caused to alleviate or take a turn for the better by giving vaccine composition according to the present invention.
Accompanying drawing explanation
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 deleted strain by PCR method, wherein, from left to right: the first swimming lane is the first-generation virus vPRV-gE of gE disappearance
-p1 genome primer carries out the electrophoresis result after pcr amplification; Second swimming lane is the s-generation passaged virus vPRV-gE of gE disappearance
-p2 genome primer carries out the electrophoresis result after pcr amplification; 3rd swimming lane is the third generation passaged virus vPRV-gE of gE disappearance
-p3 genome primer carries out the electrophoresis result after pcr amplification; 4th swimming lane is the electrophoresis result after porcine pseudorabies virus HN1201 strain virus genome primer carries out pcr amplification; The rightest swimming lane is Marker.
in sequence table:
Sequence 1 is porcine pseudorabies virus HN1201 strain gE deleted strain gD aminoacid sequence;
Sequence 2 is porcine pseudorabies virus HN1201 strain gE deleted strain gB aminoacid sequence;
Sequence 3 is porcine pseudorabies virus HN1201 strain gE deleted strain gC aminoacid sequence;
Sequence 4 is porcine pseudorabies virus HN1201 strain gE deleted strain gD nucleotide sequence;
Sequence 5 is porcine pseudorabies virus HN1201 strain gE deleted strain gB nucleotide sequence;
Sequence 6 is porcine pseudorabies virus HN1201 strain gE deleted strain gC nucleotide sequence;
Sequence 7 does not lack gE nucleotide sequence before gE for porcine pseudorabies virus HN1201 strain gE deleted 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 lacks;
Sequence 10 is remaining gE aminoacid sequence after porcine pseudorabies virus HN1201 strain part gE lacks.
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 description.But these embodiments are only exemplary, do not form any restriction to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, 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%tissuecultureinfectivedose) refer to half cell culture infective amount, is a kind of representation representing virus infectivity.
MEM liquid nutrient medium (liquid) the MEM dehydrated medium of purchased from American LifeTechnologies company is prepared according to its specification sheets.
DMEM substratum of the present invention is with reference to the preparation of GB/T18641-2002 appendix A compound method.
" PBS " described in the present invention refers to the english abbreviation of phosphate buffered saline buffer (PhosphateBufferSaline), uses the PBS of the pH7.4 of 0.01mM in the present invention, prepares by described in " molecular cloning " third edition.
The collection of embodiment 1, virus is separated
Separating sample aseptic collection pig brain tissue from the sample that the doubtful pseudorabies from Henan infects, MEM nutrient solution is added with 1: 10 (volume ratio), grinding, prepares tissue suspension, after 3 freeze thawing repeatedly, the centrifugal 15min of 2000r/min, collect supernatant liquor, then through 0.2 μm of filter membrane frit, PK-15 cell goes down to posterity 37 DEG C and cultivate 1h, change the MEM nutrient solution added 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 added containing 2% calf serum.Adopt porcine pseudorabies virus PCR detection kit (Anheal Laboratories Co., Ltd) to detect porcine pseudorabies virus, result is positive; PCR kit is utilized 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 be separated, PCR detected result is feminine gender, shows that seed culture of viruses is pure.
Submit the Pseudorabies virus be separated to preservation, described porcine pseudorabies strain is HN1201 strain (Pseudorabiesvirus, strainHN1201), and preserving number is CCTCCNO.V201311; Be preserved in China typical 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
The hereditary property of the virus be separated in embodiment 1 is determined by genetic analysis.Utilize the porcine pseudorabies virus genomic dna be separated on PK15 cell to do template, the primer shown in use table 1 carries out PCR.PrimerPremier5.0 is used to be designed for the primer sequence of amplification gB, gC, gD gene respectively.
With the genomic dna extracted for template, preparation PCR amplification system is as follows: template DNA 100 μ g, PrimerSTARHSDNAPolymerase (2.5U/ μ l) 0.5 μ l, 2 × PrimerSTARGCBuffer25 μ l, the each 1 μ l of upstream and downstream primer (10pmol/ μ l), dNTPMix (2.5mMeach) 4 μ l, and with distilled water, volume is supplied 50 μ l.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.PCR reaction is stopped at 4 DEG C.By containing ethidium bromide 1% sepharose on carry out the PCR primer of electrophoretic analysis gained.PCR primer carries out sequencing.With the sequence data that Lasergene software analysis obtains.
Table 1PCR primer sequence
The pathogenicity of embodiment 3, virus
3.1 different days piglets pathogenic
34 ~ 35 age in days porcine pseudorabies negative antibody piglets 6 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 cultivating the strong malicious HN1201 strain in 3 generations after 4 49 age in days piglets 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.
Pathogenic to different days piglet of the strong malicious HN1201 strain of table 2 pseudorabies
Result shows, and the piglet of porcine pseudorabies virus HN1201 strain inoculation different days all can cause piglet to fall ill, and the piglet of more than 3/4 can be caused dead.
Pathogenic to piglet of 3.2 various dose
Negative for 49 age in days pseudorabies antibodies piglet 8 is divided into 2 groups at random, often organizes 4, separately get 2 piglets in contrast.Experimental group is collunarium inoculation 2 × 10 respectively
7.0tCID
50/ head and 2 × 10
8.0tCID
50/ head 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.
Pathogenic to piglet of table 3 various dose porcine pseudorabies virus HN1201 strain
Result shows, and the clinical separation HN1201 of porcine pseudorabies virus inoculates the piglet of 49 ages in days with various dose, piglet all can be caused to fall ill, and the piglet of 4/4 is dead.
The preparation of embodiment 4, PRVHN1201 strain gE deleted strain
The structure of 4.1PRVHN1201GFP recombinant virus transfer vector
According to the sequence of the gE gene (sequence table SEQ IDNO.7) that will lack, at its two ends design homology arm, be respectively gEA and gEB.GEA and gEB is cloned on pUC19 carrier, called after pUCgEAB.Then by GFP gene clone on pUCgEAB, obtain recombinant virus transfer vector, name pUCgEA-GFP-B.In transfer vector, homology arm is gE both sides sequences, so the recombinant virus obtained 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
Two pairs of primers are designed, the homology arm for gE gene both sides of increasing according to the gene order of HN1201 virus:
The upstream and downstream primer of left side homology arm gEA is respectively:
GEAF:CCG
gAATTCaAAAGCGCAGCCCGGTCCGTAG (underscore is EcoRI restriction enzyme site)
GEAR:CTAG
tCTAGAataacttcgtataatgtatgctatacgaagttatCAGAAAGGGCCGCATGGTCTCA AC (underscore is XbaI enzyme cutting site, and lowercase is loxp site)
The upstream and downstream primer of right side homology arm gEB is respectively:
GEBF:ACAT
gCATGCataacttcgtatagcatacattatacgaagttatCCCGCCCCGCTTAAATACCG (underscore is SphI restriction enzyme site, and lowercase is loxp site)
GEBR:CCC
aAGCTTcCAGGAGCACCTGGTCGCAGA (underscore is HindIII restriction enzyme site)
Get PRVHN1201 and infect vero cell, after pathology occurs cell more than 80%, get part supernatant, adopt GeneaidViralNucleicAcidExtraction 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 as follows:
| PRV HN1201 DNA | 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 gEA with the gEB fragment that pcr amplification goes out is separated by agarose gel electrophoresis, reclaims test kit with sky root glue and reclaim object fragment.After gEA fragment and pUC19 carrier are used EcoRI and XbaI double digestion respectively, reclaim object fragment, after T4DNAligase connects, transform DH5 α, coating ammonia benzyl plate 37 DEG C of overnight incubation.Picking mono-clonal enzyme identifies correct plasmid called after pUCgEA after cutting qualification correctly.PUCgEA and gEB is reclaimed object fragment with after SphI and HindIII double digestion respectively, after T4DNAligase connects, transforms DH5 α, coating ammonia benzyl plate 37 DEG C of overnight incubation.Picking mono-clonal extracts plasmid, cuts and checks order 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 BglII and SmaI double digestion, reclaim linearizing carrier, fill through DNAPolymeraseILarge (Klenow) Fragment, after T4DNALigase connects, transformed competence colibacillus cell DH5 α obtains the GFP plasmid deleting MCS, called after: pAcGFP Δ MCS.
4.1.2.2GFP the amplification of gene
Primer according to pAcGFP-C1 carrier sequence design amplification GFP:
Upstream primer
CMVU:ACGC
gTCGACtAGTTATTAATAGTAATCAATTACG (underscore is SalI restriction enzyme site)
Downstream primer
SV40R:ACAT
gCATGCcTAGAATGCAGTGAAAAAAATGC (underscore is SphI restriction enzyme site)
With plasmid pAcGFP Δ MCS for template amplification GFP gene, system and condition 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 the connection that object band carries out next step.
4.1.3GFP the connection of marker gene and pUCgEAB
After GFP SalI and SphI double digestion, reclaim object fragment, be connected, transformed competence colibacillus cell DH5 α with the pUCgEAB plasmid through same double digestion, picking mono-clonal extracts plasmid, the plasmid called after pUCgEA-GFP-B that enzyme is cut and qualification of checking order is correct.
4.2 acquisitions of recombinant virus containing GFP
4.2.1 transfer vector and HN1201DNA cotransfection Vero cell obtain recombinant virus
With liposome method cotransfection Vero cell, by 3ugPRV-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 in 37 DEG C of incubators containing 5%CO2.36-48h after transfection, cytopathy to appear, and after lesion has fluorescence, harvested cell supernatant, is P0 for recombinant virus, called after rPRV-GFP-gE
-(in figure 3).
4.2.2 the plaque purification of recombinant virus
By the P0 of acquisition for recombinant virus rPRV-GFP-gE
-after vero cells infection, spread the low melting-point agarose of 2%, occur after obvious pathology and fluorescence until cell after 48h, 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 plate completed in advance, continue picking green fluorescence plaque and carry out purifying, through the plaque purification that 8 take turns, obtain purifying not containing the recombinant virus rPRV-GFP-gE that the gE of wild-type virus HN1201 lacks
-.
4.3gE lacks the deletion of GFP marker gene in recombinant virus
PBS185 plasmid (buy the loxP site from addgene, Cre enzyme identification homology arm gEA downstream and gEB upstream, the sequence in the middle of two loxp sites and GFP gene order are deleted) and the recombinant virus rPRV-GFP-gE of Cre enzyme will be expressed
-genomic dna cotransfection vero cell, after result transfection there is obvious pathology in 24h, and single fluorescence is many.The P0 of results carries out plaque select for inoculation after viral doubling dilution, and picking does not have the plaque of fluorescence to carry out next round purifying.Take turns screening purifying through 2, obtain the virus not having fluorescence, called after vPRV-gE
-.Extraction purification virus genom DNA, PCR qualification shows gE genetically deficient, and GFP marker gene is also deleted.Illustrate not containing the gE deleted virus purifying success of GFP marker gene.
4.5PRVHN1201 strain gE deleted strain confirms
Extract the viral genome of gE deleted virus and wild-type virus, carry out PCR qualification, primer is as follows:
gEDCF：acgaagaggaggaggacgaggaggg
gEDCR：tcgtgcgtctcggtggtgatgtagaa
The PCR primer size of wild-type virus amplification is the PCR fragment size that 3109bp, gE deleted virus increases is 1398bp, PCR qualification result Fig. 3.
The preparation of embodiment 5 pseudorabies gD albumen
The amplification of 1.PRVgD gene
Well-grown PK15 cell is inoculated the culture of PRVHN1201 virus gE gene-deleted strain or its different generation, the culture of this different generation is the culture within 5-35 generation, extracts PRV genomic dna after results virus with TAKARA company MiniBESTViralRNA/DNAExtractionKitVer.3.0 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
hSDNAPolymerasewithGCBuffer, 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 primer called after gD.Its nucleotide sequence is shown in SEQNO.2, and its aminoacid sequence of deriving is SEQNO.1
2. to recombinate the acquisition of Bacmid and qualification
The PCR primer gD that high-fidelity enzymatic amplification obtains is cloned into pFastBac/HBM-TOPO carrier (purchased from Invitrogen company, article No. A11339), clone's system is as follows: PCR primer gD4 μ l, Saltsolution (salts solution) 1 μ l, TOPOvector1 μ l, totally 6 μ l.Mix, incubated at room 5min, transform OneShotRMach1
tMt1R competent cell, coating amicillin resistance is dull and stereotyped, the direction of insertion of picking mono-clonal qualification gD gene, and the plasmid that direction of insertion is correct send Invitrogen company to check order, the exactness of qualification gD sequence.The plasmid called after pFastBac/HBM-TOPO-gD checking order correct.
PFastBac/HBM-TOPO-gD Plastid transformation DH10Bac competent cell (source), shuttle plasmid Bacmid in pFastBac/HBM-TOPO-gD and competent cell carries out swivel base, the recombinant plasmid obtained is extracted with the PureLinkHiPurePlasmidDNAMiniprepKit of Invitrogen, and identify the insertion of gD with pUCM13Forward/pUCM13Reverse primer, positive Bacmid called after Bacmid-gD.
3. transfection obtains recombinant baculovirus
The method provided according to the specification sheets of Invitrogen company Bac-to-BacHBMTOPOSecretedExpressionSystem is carried out.6 orifice plate every hole pavings 8 × 10
5individual sf9 cell, transfection is carried out according to the specification sheets of Cellfectin II transfection reagent: dilute in 8 μ lCellfectin II and 1 μ gBacmid-gDDNA to 100ulSF-900 II substratum respectively after cell attachment, Vortex mixes, DNA after mixed diluting and the Cellfectin II (cumulative volume ~ 210 μ l) after diluting, mix incubated at room 15 ~ 30min, to be one after another drop ofly added in cell.After transfection after 72h cytopathy to appear, collecting cell culture supernatant, is designated as P0 for recombinant virus vBac-gD.P0 infects sf9 cell for recombinant virus vBac-gD, and after 3 generation enlarged culturing, the P3 of acquisition is used for expression of recombinant proteins for vBac-gD.
4. recombinate shape virus infection High-five cell obtains recombinant protein
P3 is inoculated High-five cell (purchased from Invitrogen, article No. B85502) for recombinant baculovirus vBac-gD.In 500ml triangular flask, suspension culture High-five cell, 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 that volume concentration is original volume by the tangential flow filtration system of Millipore 1/10.With TritonX-100 (purchased from sigma) the deactivation baculovirus of 1% (volume ratio), it is 200 μ g/ml that SDS-PAGE optical densitometric method measures protein content.
The preparation of the sick deactivation vaccine of embodiment 6, porcine pseudorabies virus
First seed lot is formed by the culture of the different generations of be separated strain being inoculated in PK-15 cell culture according to table 4, then formed in the PK cell culture of individual layer by 1% (V/V) access of virus culture liquid measure, put 37 DEG C of rotating and culturing, when pathology reaches 80%, gather in the crops toxic cell culture fluid, after 2 freeze thawing, receive poison, measure malicious valency.The final concentration of formaldehyde is made to be 0.2% (V/V) respectively to adding 10% (v/v) formaldehyde solution in different generation virus liquid, 37 DEG C of deactivations 18 hours, within every 4 hours, stir 1 time, each stirring 10min, after deactivation, the PBS liquid of virus liquid PH7.4 is diluted to the viral level shown in table 3 and then mixes according to volume ratio 54:46 with 206 adjuvants (French SEPPIC Products), and under 30 DEG C of conditions, 120 revs/min are stirred 15 minutes.
The preparation of table 4 each group pseudorabies vaccine
The preparation of embodiment 7, porcine pseudorabies virus sick gE gene-deleted strain deactivation vaccine
Sick for the porcine pseudorabies virus of preparation gE gene-deleted strain is inoculated in PK-15 cell culture by embodiment 4 and first forms seed lot, then formed in the PK cell culture of individual layer by 1% (V/V) access of virus culture liquid measure, put 37 DEG C of rotating and culturing, when pathology reaches 80%, gather in the crops toxic cell culture fluid, after 2 freeze thawing, receive poison, measure malicious valency.The final concentration of formaldehyde is made to be 0.2% (V/V) respectively to adding 10% (v/v) formaldehyde solution in different generation virus liquid, 37 DEG C of deactivations 18 hours, within every 4 hours, stir 1 time, each stirring 10min, after deactivation, the PBS liquid of virus liquid PH7.4 is diluted to the viral level shown in table 5 and then mixes according to volume ratio 54:46 with 206 adjuvants (French SEPPIC Products), and under 30 DEG C of conditions, 120 revs/min are stirred 15 minutes.
The preparation of table 5 each group pseudorabies vaccine
Embodiment 8, inactivated vaccine immunogenicity experiments
21 age in days PRV negative antibody piglets 24 are divided into 6 groups at random, 4/group, vaccine immunity pseudorabies inactivated vaccine prepared by the vaccine immunity pseudorabies inactivated vaccine 2ml/ head prepared 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 attacks 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, every day measures piglet body temperature, and 3 kinds of ELISA kit (IDEXX) of getting the porcine blood serum PRV (Pseudorabies virus) gE antibody of test group and control group before observation clinical symptom and death condition (the results are shown in Table 6) attack poison respectively carry out gE antibody test according to its specification sheets.
Table 6 Study On Immunogenicity animal divides into groups
| 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, weekly with reference to the NAT of the method mensuration inactivated vaccine group of 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 display of table 7, after pseudorabies inactivated vaccine immunity piglet, can produce higher neutralizing antibody, and raise gradually with immunization time.
Immunity attacks 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, observation clinical symptom and death condition are in table 8, and after attacking poison, every day measures piglet body temperature in table 9.
Table 8 pseudorabies inactivated vaccine immunity piglet after attack malicious situation
The piglet of table 9 pseudorabies vaccine immunity attacks the rear piglet Temperature changing of poison
The result display of table 8 and table 9; after pseudorabies inactivated vaccine immunity piglet; although (occurring clinical symptom) can not be infected by blocking virus; but 100% (4/4) can be provided to protect for piglet; and contrasting piglet, to attack latter 4 days of poison all dead afterwards; therefore, pseudorabies inactivated vaccine has good protection.In addition relative to control group living vaccine, the piglet fervescence time of inactivated vaccine immune group is shorter, and vaccine group C and group D does not rise to 41 degree, and appetite is normal, and does not substantially have clinical symptom, demonstrates good immunoprotection.
Pseudorabies gEElisa 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, are negative.
Embodiment 9, pseudorabies are to the preparation of gD subunit vaccine
By subunit antigen prepared by embodiment 5, the volume being diluted to table 10 with PBS liquid (pH7.4) mixes according to volume ratio 54:46 with 206 adjuvants (French SEPPIC Products), and under 30 DEG C of conditions, 120 revs/min are stirred 15 minutes.
The preparation of the pseudo-mad subunit vaccine of table 10 each group pig
| Group | Protein content | Vaccine proportioning (inactivation of viruses liquid: 206 adjuvants) |
| A | 25μg/ml | 54:46 |
| B | 100μg/ml | 54:46 |
Embodiment 10, subunit vaccine are to the immunogenicity experiments of pig
21 age in days PRV negative antibody piglets 12 are divided into 3 groups, 4/group at random, inject the vaccine of embodiment 9 preparation according to table 2, immune swine pseudorabies virus subunit vaccine 2ml/ head.Control group inoculation DMEM substratum 2ml/ head.Immunity attacks poison after latter 28 days, and attacking toxic agent amount is porcine pseudorabies virus HN1201 strain 2 × 10
8.0tCID
50/ head, after attacking poison, every day measures piglet body temperature, observes clinical symptom and death condition (the results are shown in Table 12).
Table 11 Study On Immunogenicity animal divides into groups
| 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 attacks poison after latter 28 days, and attacking toxic agent amount is porcine pseudorabies virus HN1201 strain 2 × 10
8.0tCID
50/ head, observation clinical symptom and death condition are in table 12, and after attacking poison, every day measures piglet body temperature in table 13.
Table 12 porcine pseudorabies virus subunit vaccine immunity piglet after attack malicious situation
The piglet of table 13 porcine pseudorabies virus subunit vaccine immunity attacks the rear piglet Temperature changing of poison
The result display of table 12 and table 13; after porcine pseudorabies virus subunit vaccine immunity piglet; although (occurring clinical symptom) can not be infected by blocking virus; but 100% (4/4) can be provided to protect for piglet; and contrasting piglet, to attack latter 4 days of poison all dead afterwards, demonstrate good immunoprotection.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within 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 polynucleotide SEQIDNO.1, and not containing 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 is not containing 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 as the nucleotide sequence of aminoacid sequence shown in polynucleotide SEQIDNO.10.
4. a vaccine composition, wherein, described vaccine composition comprises totivirus antigen, inactivated whole virus antigen, the subunit antigen of the porcine pseudorabies virus strain gE gene-deleted strain described in any one of claims 1 to 3 of immunity amount or the attenuated live of its culture.
5. vaccine composition according to claim 4, wherein, described vaccine composition comprises>=and 10
6.0tCID
50totivirus antigen, the inactivated whole virus antigen of the porcine pseudorabies virus strain gE gene-deleted strain described in any one of claims 1 to 3 of/ml or the attenuated live of its culture.
6. vaccine composition according to claim 4, wherein, described vaccine composition comprises >=the porcine pseudorabies virus strain gE gene-deleted strain described in any one of claims 1 to 3 of 25 μ g/ml 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 according to claim 4, described method comprises:
(1) the described porcine pseudorabies virus strain gE gene-deleted strain of propagation or its culture is cultivated;
(2) porcine pseudorabies virus strain gE gene-deleted strain or its culture of described propagation is gathered in the crops, deactivation;
(3) adjuvant is added, mixing.
9. prepare a method for vaccine composition according to claim 4, described method comprises:
(1) the gD proteantigen of described porcine pseudorabies virus strain gE gene-deleted strain or its culture is expressed;
(2) the gD proteantigen of described expression is gathered in the crops;
(3) adjuvant is added, mixing.
10. according to the application of the vaccine composition described in claim 4 ~ 7 in the medicine preparing prevention and therapy porcine pseudorabies virus relative disease.
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| WO2019149265A1 (en) * | 2018-02-01 | 2019-08-08 | 厦门大学 | Pseudorabies virus for treating tumors |
| CN110327461A (en) * | 2019-07-17 | 2019-10-15 | 苏州世诺生物技术有限公司 | A kind of preparation method and applications of porcine pseudorabies virus subunit vaccine |
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| CN103923884B (en) * | 2014-02-21 | 2017-03-29 | 普莱柯生物工程股份有限公司 | A kind of porcine pseudorabies virus gene-deleted strain, vaccine combination and its preparation method and application |
| CN104250640A (en) | 2014-08-22 | 2014-12-31 | 普莱柯生物工程股份有限公司 | Porcine pseudorabies virus gene deletion strain, vaccine composition, preparation method and application thereof |
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