CN109943590A - A kind of replication defect type pig circular ring virus, preparation method and application - Google Patents
A kind of replication defect type pig circular ring virus, preparation method and application Download PDFInfo
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Abstract
The invention belongs to gene engineering technology fields, and in particular to replication defect type pig circular ring virus, preparation method and application.The normal expression function of REP gene or cap gene that the present invention knocks out wild type PCV using artificial DNA mutation technology first obtains replication defect type PCV, then corresponding PCV trans-complementation gene cell strain is infected using replication defect type PCV, rescue obtains replication defect type pig circular ring virus.The replication-defective virus is up to 99.77% genomic DNA with the identical and sequence homology with the identical virus protein primary structure of wild type PCV strain and higher structure and size, with wild-type virus immunogenicity having the same and antigen submission efficiency, therefore it is as preventing or PCV vaccine, the PCV antibody test antigen and as the recombinant vaccine vector of expression alien gene for the treatment of PCV infection.
Description
Technical field
The invention belongs to gene engineering technology fields, and in particular to a kind of replication defect type pig circular ring virus (PCV), its system
Preparation Method and application.
Background technique
Pig circular ring virus and its related disease: pig circular ring virus (Porcine circovirus, PCV) is found so far
A kind of the smallest animal virus.PCV virus is single-stranded DNA viruses, has known that PCV has 3 hypotypes, i.e. PCV1, PCV2 and PCV3,
Wherein PCV1 is the virus of non-pathogenic, and PCV3 is the novel PCV virus occurred just now, pathogenic unknown at present, and PCV2
It is postweaning multisystemic wasting syndrome (postweaning m μ Ltisystemic wasting syndrome, PMWS)
Main pathogen.PMWS caused by PCV2 is found in Canadian (1991) earliest, includes quickly me in some countries in American-European and Asia
State occurs and prevalence.It is a large amount of later the study found that in addition to PMWS, PDNS(pigskin is scorching and nephrotic syndrome), PNP(it is Hypertrophic
Necrotizing pneumonia), PRDC(porcine respiratory disease syndrome), breeding difficulty, it is congenital tremble, the diseases such as enteritis are also felt with PCV2
It is infected with important association.PCV2 infects the relevant swine disease death rate to be differed for 10%~30%, is infected serious pig farm and is even as high as
40%, it is resulted in significant economic losses to pig breeding industry.In addition, a large number of studies show that PCV2 infection can cause the serious immune barrier of pig
Hinder, so as to cause other secondary sexually transmitted diseases (such as swine fever, blue otopathy, streptococcosis).Therefore PCV2 is known as by various countries
One of maximum virus is threatened to pig breeding industry.
The immune protection technology of Infection of Porcine circovirus: as most of virosis, Infection of Porcine circovirus does not have at present
The special therapeutic agent having, vaccine are the control most effective means of Infection of Porcine circovirus.There are many PCV2 is entirely sick now
Malicious inactivated vaccine, the PCV2-CAP subunit vaccine of prokaryotic expression and sub- by the eukaryotic expression PCV2-CAP of carrier of baculoviral
Subunit vaccine is used for production practices.But Infection of Porcine circovirus is still to pig breeding industry worldwide, particularly in China
The infectious disease for causing maximum economic loss illustrates that the clinical control effect of these existing PCV2 vaccines is not still good enough.
The deficiency of existing virus vaccine technologies: existing viral vaccine mainly includes 3 classes according to the difference of technical principle, i.e.,
Inactivated virus vaccine, attenuated live vaccines and genetic engineering subunit vaccine.Inactivated vaccine is the epidemic disease with the pathogen preparation of inactivation
Seedling, safety is very high, but since the immunogenicity that inactivation process will lead to vaccine changes, and antigen submission low efficiency, therefore immune
Control efficiency is not good enough.Attenuated live vaccines are the vaccines prepared with the lower Strain of virulence, although it is because of preferable immunogenicity
With antigen submission efficiency and have preferable immune effect, but due to attenuated live vaccines in animal body can still provide for duplication pass
Generation, and in succeeding generations virus may occur unpredictable mutation, evolve to restore virulence even generate virulence it is stronger
Strain, therefore the vaccine safety of attenuated live vaccines and biological safety are lower.Genetic engineering subunit vaccine is with gene work
The part proteantigen of the pathogen of journey technological expression, although safety is preferably, its immunogenicity is imperfect, therefore its immune effect
Fruit is also undesirable.
Summary of the invention
To solve the above problems, the present invention provides a kind of preparation method of replication defect type pig circular ring virus, the preparation side
The replication defect type PCV virus that method obtains has and the identical virus protein primary structure of wild type PCV virus and advanced
Structure and the identical virion of size and shape, therefore have identical sense completely with having with wild type PCV virus
Dye ability and immunogenicity, and the obtained replication defect type PCV virus of the preparation method cannot normal host cell (including
Living animal cell) in duplication passage.
The preparation method of replication defect type pig circular ring virus provided by the invention the following steps are included:
(1) building of PCV trans-complementation transgenosis cell strain: by stable transfection or lentiviruses transduction technology by wild type PCV
Cap gene or REP gene the eukaryon expression plasmid cell strain that is inserted into any reproducible PCV genome in obtain the final product, preferably
Ground, the cell strain of any reproducible PCV are PK-15 cell strain or Vero cell strain；
(2) building of replication defect type PCV infection clones: by the full-length genome DNA of PCV strain selected by PCR amplification, gram
It is grand enter plasmid vector in, then by DNA site-directed mutagenesis technique (site-directed mutagenesis) construct replication defective
Type PCV infection clones.Preferably, the DNA site-directed mutagenesis technique is any one in following three kinds: 1. in wild type
One or more Premature stop codons (TAG, TAA or TGA) are introduced in the cap gene of PCV or the ORF sequence of REP gene；②
The insertion mutation of single base or double alkali yl is introduced at 5 ' ends of the ORF sequence of the cap gene or REP gene of wild type PCV or is deleted
Except mutation；3. being inserted into displacement some or all of cap gene or REP gene ORF sequence with the whole code of foreign gene (in-frame)；
(3) transfection of replication defect type PCV infection clones and virus rescue and culture: with infectious gram of replication defect type PCV
It is grand to transfect above-mentioned corresponding PCV trans-complementation transgenosis cell strain by liposomal transfection teclmiques, acquisition duplication can be saved and lacked
Swaged PCV virus, and by the PCV trans-complementation transgenosis cell strain culture and prepare replication defect type PCV.
When replication defect type infection clone is obtained by DNA rite-directed mutagenesis CAP, corresponding PCV trans-complementation
Gene cell strain is that the eukaryon expression plasmid of CAP is at least transferred to reproducible PCV by stable transfection or lentiviruses transduction technology
Cell strain obtain；When replication defect type infection clone is obtained by DNA rite-directed mutagenesis REP, corresponding PCV is anti-
Formula complementary genes cell strain is at least to be transferred to the eukaryon expression plasmid of REP by stable transfection or lentiviruses transduction technology and can answer
What the cell strain of PCV processed obtained.
Above-mentioned replication defect type PCV virus can replicate passage simultaneously in above-mentioned corresponding PCV trans-complementation transgenosis cell strain
It is able to largely prepare multiple.
The genome of above-mentioned replication defect type PCV is functional defect type genome (REP base necessary to its genome duplication
Because or progeny virus packaging necessary to cap gene can not normal expression), (refer to that above-mentioned PCV is anti-when it infects normal host cell
Other host cells other than formula complementation transgenosis cell strain) after cannot complete viral genome duplication or progeny viral particles
It packs and progeny virus cannot be generated, so it, which is that a kind of scarce type of duplication is viral for normal host cell, (cannot form
There is the progeny virus of infection ability).But the replication defect type PCV has and the identical viral clothing of wild type PCV
Shell, to have and the identical infection ability of wild type PCV and immunogenicity.Therefore, the replication defect type PCV can be with
As the novel live-virus vaccine for preventing or treating Infection of Porcine circovirus, the detection for being alternatively arranged as being used as detection PCV antibody is anti-
It is former.
Replication defect type pig circular ring virus of the present invention is as follows as the possible mechanism of therapeutic vaccine: (1) described multiple
After defect PCV cell entry processed has infected the cell of wild type PCV, the genome of functional defect by with wild-type virus just
Normal genome is competed in genomic DNA duplication link, thus the duplicating efficiency of Reverse transcriptase wild-type virus genome.
(2) genome of its functional defect will be competed with the normal gene group of wild-type virus in progeny virus packaging link, from
And the progeny virus packaging efficiency of Reverse transcriptase wild-type virus.
If knocking out CAP with the part ORF sequence construct that the whole code of foreign antigen genes (in-frame) is inserted into displacement cap gene
Replication defective PCV virus；After replication defective PCV virus infection enters normal host cell, although filial generation disease cannot be formed
Malicious particle, but its genomic DNA remains to be normally carried out duplication and expresses the foreign antigen genes of insertion, to be presented to siberian crabapple
System, excitation are directed to the immune response of exogenous antigen, therefore, have as the recombination for expressing other pathogenic microorganism antigen genes
The potentiality of vaccine carrier.
Replication defective pig circular ring virus of the present invention is as the recombinant vaccine for expressing other pathogenic microorganism antigen genes
The possible mechanism of carrier is as follows: with some or all of the whole code of foreign antigen genes (in-frame) displacement cap gene ORF sequence
Building knocks out the replication defective PCV virus of CAP；After replication defective PCV virus infection enters normal host cell, although cannot
Progeny virion is formed, but its genomic DNA remains to be normally carried out duplication, and expresses the foreign antigen genes of insertion, thus
It is presented to immune system, excitation is directed to the immune response of exogenous antigen.
The preparation method of replication defect type pig circular ring virus provided by the invention also can be applied to its of circovirus section
Its circovirus is to prepare corresponding replication defect type circovirus vaccine.
Heretofore described pig circular ring virus (PCV) includes PCV1, PCV2, PCV3 or other Asias that future is likely to occur
The PCV virus of type.
The beneficial effects of the present invention are:
Compared with above-mentioned 3 kinds traditional viral vaccines, replication defective PCV of the present invention has following skills when being used as preventative vaccine
Art advantage: (1) it has infection ability identical with wild-type virus, the antigen presenting cell (APCs) of energy efficient infection animal
To efficiently excite the immune response of host, therefore has than inactivated virus vaccine and genetic engineering subunit vaccine and preferably exempt from
Epidemic disease protecting effect.(2) its have with the identical immunogenicity of wild-type virus, can excite animal generate it is complete, be directed to
The immunity of wild type infection, therefore there is more preferable immunoprotection than inactivated virus vaccine and genetic engineering subunit vaccine
Effect.(3) it is replication-defective virus, can not replicate, pass on after entering in animal body as vaccine, i.e., can not be in animal body
It is interior to evolve, therefore there is higher vaccine safety and biological safety compared with traditional attenuated live vaccines.
The advantage of detection antigen that replication defective PCV virus of the present invention is used as detection PCV antibody is: (1) its
With viral capsid (capsid) albumen completely the same with wild-type virus, when being used as PCV antibody test antigen, with recombination table
The detection antigen reached is more complete compared to immunogenicity, therefore it is truer and accurate to the result of PCV antibody test.(2) it is
Replication defective virus, therefore there is high bio-safety compared with being used as detection antigen with wild-type virus or low virulent strain virus
Property.
The preparation method of replication defect type pig circular ring virus provided by the invention is easy to operate, can quick and large batch of life
Replication defect type pig circular ring virus is produced, the industrialization production of replication defect type pig circular ring virus is helped to realize.
Specific embodiment
The present invention is further detailed below in conjunction with embodiment.
If not reagent of the present invention and material specified otherwise, can be obtained by commercial channel.
Embodiment 1: based on CAP trans-complementation transgenosis cell strain preparation PCV2 replication defective virus and its immune efficacy
Verifying
1, CAP trans-complementation transgenosis PK-15 cell strain constructs
1) PCV2 extracting genome DNA: with commercially available PCV2 inactivated vaccine (DBN-SX07 plants) 1mL, being added isometric chloroform, high
Speed concussion 4 minutes, 12000rpm is centrifuged 10 minutes, draws upper strata aqueous phase, extracts PCV2 genome with viral DNA extracts kit
DNA。
2) CAP construction of eukaryotic expression vector: the PCV2 genomic DNA obtained using previous step designs upstream primer as template
Primer pair (the 5'GCTAGCGCCACC of the sequence containing KozakATGACGTATCCAAGGAGGCGT3', 5'
GGATCCTTAAGGGTTAAGTGGGGGGTCT3', underscore part are template binding region sequence), pass through PCR amplification, T-A grams
The cap gene complete encoding sequence of PCV2 is cloned into generalized eukaryotic expression plasmid by grand, NheI and BamHI double digestion subclone
pCDNA3.1+, obtain pCDNA-CAP plasmid.
3) the transient transfection verifying of CAP carrier for expression of eukaryon: turned with pCDNA-CAP plasmid by liposomal transfection teclmiques
PK-15 cell strain is contaminated, is carried out indirect immunofluorescence assay (IFA) after 36 hours with PCV2 positive serum, the cell of transfection is presented
Green fluorescence illustrates that pCDNA-CAP plasmid can correctly express CAP albumen in PK-15 cell.
4) stable transfection of PK-15 cell: with restriction endonuclease MfeI by 40 μ g pCDNA-CAP plasmid linearizations, then lead to
Liposomal transfection teclmiques transfection PK-15 cell strain is crossed, is passed within 24 hours after transfection, the replacement when cell fusion degree reaches 50-70%
Pressurization screening is carried out for the culture medium of the G418 containing suitable concentration, changes once within every 3 days the culture medium of the G418 containing suitable concentration, Zhi Daoyou
The cell cluster of G418 resistance is grown.
5) CAP trans-complementation transgenosis cell strain monoclonal screens: the G418 obtained by infinite dilution method to previous step
Resistance PK-15 cell cluster carries out monoclonal screening, and the CAP that can stablize expression PCV2-CAP in conjunction with above-mentioned IFA test acquisition is anti-
Formula complementation transgenosis PK-15 monoclonal cell strain.
2, the building of PCV2 infection clones and its CAP knockout mutations
1) PCV2 infection clones construct: according to DBN-SX07-2 pnca gene group sequence in its REP gene and cap gene tail end
Between area's design primer to (5 ' TTTTTTATCACTTCGTAATGGTTTT3 ', 5 '
GACTCAGTAATTTATTTCATATGGAAAT3 '), using the PCV2 genomic DNA of aforementioned extraction as template, with Q5 high-fidelity DNA
Then polymeric enzymatic amplification PCV2 full-length genome DNA is cloned into pUC-T carrier using T-A clone technology to get the wild of PCV2
Type infection clones pUC-PCV2.
2) CAP knockout mutations: using the wild type infectious clone pUC-PCV2 of PCV2 as template, design primer is to (5'TAGTGATAAACTATCAAGCGAACCACAGTC3', 5'GGTGCGGGAGAGGCGGGT3', the underscore part of upstream primer
For 3 continuous terminator codons of corresponding CAP 53-55 amino acids), using the DNA mutation kit of the precious biology in Dalian
MutanBest Kit is simultaneously operated by its specification to get the PCV2 infection clones pUC-PCV2 of CAP is knocked outΔCAP。
, PCV2 wild-type virus and CAP knock out the rescue of virus
1) normal PK-15 cell strain, 3 Dai Houyong of blind passage are transfected with the 20 μ g of wild type infectious clone pUC-PCV2 plasmid of PCV2
IFA measures virus titer (TCID50), and harvest wild type PCV2 virus.
2) with the PCV2 infection clones pUC-PCV2 for knocking out CAPΔCAP20 μ g of plasmid transfects above-mentioned CAP trans-complementation and turns base
Because of PK-15 cell strain, virus titer (TCID is measured with IFA after 3 generation of blind passage50), and harvest the mutated viruses PCV2 for knocking out CAPΔCAP。
4, mutated viruses PCV2ΔCAPReplication defective verifying
1) Cells In Vitro is verified: with 104.0 TCID50Mutated viruses PCV2ΔCAPNormal PK-15 cell is infected, is set simultaneously
The homophyletic wild type infection of equal infective dose compares, and IFA detection is carried out after 3 generation of blind passage, the results showed that PCV2ΔCAPSense
The normal PK-15 cell of dye can not detect virus after passing on 3 times, and the normal PK-15 cell of homophyletic wild type infection
With the presence of a large amount of viruses after passage 3 times, illustrate the mutated viruses PCV2 that CAP is knocked outΔCAPIt cannot be passed in normal PK-15 cellular replication
Generation.
2) mouse living animal is verified: under SPF experimental situation, distinguishing abdomen with two groups of each 6 SPF grades of BALB/c mouses
Intracavitary inoculation 0.2*105.0 TCID50Mutated viruses PCV2ΔCAPWith homophyletic wild-type virus, vaccinization once a day 3 days.Most
Venous blood, spleen, the lymph node tissue sample of two groups of mouse of aseptic collection are distinguished in primary inoculation afterwards after 2 weeks, extract viral DNA,
With REP gene specific primer (the 5'TGTTGGCGAGGAGGGTAATGAG 3', 5' of PCV2
TCCCACCACTTGTTTCTAGGCG 3') carry out PCR detection.The result shows that: 6 are vaccinated with PCV2ΔCAPThree kinds of tissues of mouse
The specific PCR detection of sample is feminine gender, and three kinds of tissue samples that 6 are vaccinated with homophyletic wild type PCV2 virus mouse
Specific PCR detection is the positive, illustrates the mutated viruses PCV2 that CAP is knocked outΔCAPIt cannot be replicated in Mice Body.
3) piglet living animal is verified: under cleaning grade zoopery environment, with two groups of each 6 PCV2 antigens and antibody
Detection is negative 3 week old piglets difference intranasal vaccination mutated viruses PCV2ΔCAPWith homophyletic wild-type virus, dosage of inoculation
It is 2*106.0 TCID50, vaccinization once a day 3 days.Last time inoculation distinguished two groups of piglets of aseptic collection after 2 weeks
Venous blood, spleen, lymph node tissue sample extract viral DNA, carry out PCR detection with the REP gene specific primer of PCV2.Knot
Fruit shows: 6 are vaccinated with mutated viruses PCV2ΔCAPThe specific PCR detection of three kinds of tissue samples of piglet is feminine gender, and 6
The specific PCR detection for being only vaccinated with three kinds of tissue samples of homophyletic wild type PCV2 virus piglet is the positive, illustrates that CAP strikes
The mutated viruses PCV2 removedΔCAPIt cannot be replicated in piglet body.
PK-15 cell infection, mouse and the experiment of piglet live infection confirm constructed mutated viruses PCV2ΔCAPIt cannot
Passage is replicated in its normal host cell, therefore it is a kind of replication defective virus for normal host cell.
, replication defective virus PCV2ΔCAPImmune efficacy verifying
1) PCV2ΔCAPThe preparation of experimental vaccine: replication defective virus PCV2 is usedΔCAPInfect above-mentioned CAP trans-complementation transgenosis
PK-15 cell strain, culture to virus titer reach 106.5 TCID50Three times, 4000rpm is centrifuged 10 minutes multigelation when above；
After supernatant containing virus passes through 0.45um membrane filtration, with PEG-6000 concentrating virus.The disease after concentration is resuspended with appropriate PBS
Poison makes virus titer not less than 107.0 TCID50To get PCV2ΔCAPExperimental vaccine.
2) PCV2ΔCAPThe immune efficacy of experimental vaccine measures: under cleaning grade zoopery environment, by PCV2 antigen
It is that negative 3 week old piglet 16 is randomly divided into 2 groups with antibody test, every group 8, test group musculi colli injects 107.0
TCID50 PCV2ΔCAPExperimental vaccine, control group musculi colli inject commercially available homophyletic PCV2 inactivated vaccine 1mL(107.0
TCID50).The venous blood sample that two groups of piglets are acquired after 6 weeks immune is detected each with commercially available PCV2 ELISA antibody assay kit
The PCV2 antibody of serum sample.The result shows that: 8 serum samples of test group are the positive, and 5 in 8 serum samples of control group
A is the positive, and 3 are feminine gender.Prove the PCV2 replication defective Viral experiment vaccine of the CAP knockout in single immunization situation
Under immune efficacy be apparently higher than homophyletic wild type PCV2 inactivated vaccine.
Embodiment 2: based on REP trans-complementation transgenosis cell strain preparation PCV2 replication defective virus
1. REP trans-complementation transgenosis PK-15 cell strain constructs
1) REP construction of eukaryotic expression vector: using the PCV2 genomic DNA extracted in embodiment 1 as template, design upstream is drawn
Primer pair (the 5'GCTAGCGCCACC of object sequence containing KozakATGCCCAGCAAGAAGAATGGA3', 5'GGATCCTCAGTAA TTTATTTCATATGGAAATTCAGG3', underscore part are template binding region sequence), cloned by PCR amplification, T-A,
The REP gene complete encoding sequence of PCV2 is cloned into generalized eukaryotic expression plasmid by NheI and BamHI double digestion subclone
pCDNA3.1+, obtain pCDNA-REP plasmid.
2) the transient transfection verifying of REP carrier for expression of eukaryon: turned with pCDNA-REP plasmid by liposomal transfection teclmiques
PK-15 cell strain is contaminated, is carried out indirect immunofluorescence assay (IFA) after 36 hours with PCV2 positive serum, the cell of transfection is presented
Green fluorescence, confirmation pCDNA-REP plasmid can correctly express REP albumen in PK-15 cell.
3) stable transfection of PK-15 cell: with restriction endonuclease MfeI by 40 μ g pCDNA-REP plasmid linearizations, then lead to
Liposomal transfection teclmiques transfection PK-15 cell strain is crossed, is passed within 24 hours after transfection, the replacement when cell fusion degree reaches 50-70%
Pressurization screening is carried out for the culture medium of the G418 containing suitable concentration, changes once within every 3 days the culture medium of the G418 containing suitable concentration, Zhi Daoyou
The cell cluster of G418 resistance is grown.
4) REP trans-complementation transgenosis cell strain screens: the G418 resistance obtained by infinite dilution method to previous step
PK-15 cell cluster carries out monoclonal screening, and the REP that can stablize expression PCV2-REP in conjunction with above-mentioned IFA test acquisition is trans- mutually
Mend transgenosis PK-15 cell strain monoclonal.
The PCV2 infection clones of knockout construct and mutated viruses rescue
1) the REP knockout mutations of PCV2 infection clones: with the wild type infectious clone of the PCV2 constructed in embodiment 1
PUC-PCV2 is template, and design primer is to (5'TAATGATAGACACCTCACCTCCAGGGGTTC3', 5'
CTCATTACCCTCCTCGCCAAC3', the underscore part of upstream primer are that 3 of corresponding REP 52-54 amino acids are continuous
Terminator codon), using the precious biology in Dalian DNA mutation kit MutanBest Kit and by its specification operate to get
The PCV2 infection clones pUC-PCV2 that REP is knocked outΔREP。
2) rescue of REP knockout mutations virus: the PCV2 infection clones pUC-PCV2 knocked out with REPΔREPIt transfects above-mentioned
REP trans-complementation transgenosis PK-15 cell strain measures virus titer (TCID with IFA after 3 generation of blind passage50), and harvest REP knockout
Replication defective PCV2 mutated viruses PCV2ΔREP。
Mutated viruses PCV2ΔREPReplication defective verifying:
1) Cells In Vitro is verified: with 104.0 TCID50Mutated viruses PCV2ΔREPNormal PK-15 cell is infected, is set simultaneously
The homophyletic wild type infection of equal infective dose compares, and IFA detection is carried out after 3 generation of blind passage, the results showed that mutated viruses
PCV2ΔREPThe normal PK-15 cell of infection can not detect virus after passing on for 3 generations, and homophyletic wild type infection is normal
With the presence of a large amount of viruses after the passage of PK-15 cell 3 times, illustrate the mutated viruses PCV2 that REP is knocked outΔREPIt cannot be in normal PK-15
Cellular replication.
2) mouse living animal is verified: under SPF experimental situation, distinguishing abdomen with two groups of each 6 SPF grades of BALB/c mouses
Intracavitary inoculation 0.2*105.0 TCID50Mutated viruses PCV2ΔREPWith homophyletic wild-type virus, vaccinization once a day 3 days.Most
Venous blood, spleen, the lymph node of two groups of mouse of aseptic collection are distinguished in primary inoculation afterwards after 3 weeks, extract viral in each tissue samples
DNA, with cap gene special primer (the 5'GGAAAAATGGCATCTTCAACACC 3', 5' of PCV2
CGGGAGGAGTAGTTTACATAGGGG PCR detection) is carried out.The result shows that: 6 are vaccinated with mutated viruses PCV2Δ(REP)Mouse
The PCV2 specific PCR detection of three kinds of tissue samples is feminine gender, and three kinds of groups of mouse that 6 are vaccinated with homophyletic wild-type virus
The PCV2 specific PCR detection for knitting sample is the positive, illustrates the mutated viruses PCV2 that REP is knocked outΔREPIt cannot be in intravital mouse
Duplication in vivo.
3) piglet living animal is verified: under cleaning grade zoopery environment, with two groups of each 6 PCV2 antigens and antibody
Detection is negative three week old piglets difference intranasal vaccination mutated viruses PCV2ΔREPWith homophyletic wild-type virus, Inoculant
Amount is 2*106.0 TCID50, vaccinization once a day 3 days.Last time inoculation distinguished two groups of piglets of aseptic collection after 3 weeks
Venous blood, spleen, lymph node, extract tissue samples in viral DNA, carry out PCR inspection with the cap gene special primer of PCV2
It surveys.The result shows that: 6 are vaccinated with mutated viruses PCV2ΔREPThree kinds of tissue samples of piglet PCV2 specific PCR detection be
Feminine gender, and 6 PCV2 specific PCR detections for being vaccinated with three kinds of tissue samples of piglet of homophyletic wild-type virus are the positive,
Illustrate the PCV2 mutated viruses PCV2 that REP is knocked outΔREPIt cannot be replicated in living piglet body.
PK-15 cell infection, mouse and the experiment of piglet live infection confirm mutated viruses PCV2ΔREPIt cannot be normal at it
Duplication passage in host cell, therefore it is a kind of replication defective virus for normal host cell.
Embodiment 3: replication defect type PCV2 virus is used as PCV2 antibody test antigen
1) prepared by PCV2 replication defective virus envelope antigen
The replication defective virus PCV2 of CAP trans-complementation transgenosis PK-15 cell strain proliferation, harvest will be used in embodiment 1ΔCAP
It is concentrated through PEG-6000, is resuspended with appropriate 50mM carbonate buffer solution (pH 9.6), makes virus titer 107.0 TCID50To get
PCV2 antigen coat liquid.
2) PCV2 replication defective viral antigen is coated with
Above-mentioned PCV2 antigen coat liquid is added in elisa plate item, every hole 100uL, sets 4 DEG C overnight, outwell, exhaust coating buffer.
3) PCV2 ELISA antibody test is verified
It (is detection with the PCV2 CAP albumen of recombinant expression with the PCV2 ELISA antibody assay kit of certain commercially available import brand
Antigen) as control, the PCV2 replication defective virus respectively with the kit antigen coat lath carried and previous step preparation is anti-
Primordial covering lath carries out ELISA antibody test, positive mark to the positive standard serum provided in kit and negative standards' serum
Quasi- serum and negative standards' serum are all provided with 3 repeating holes, remaining detecting step presses kit specification progress.As a result table
Bright: the included envelope antigen of kit is respectively 2.3 ± 0.25 to the detection OD value of positive standard serum and negative standards' serum
It is with detection OD value of 0.31 ± 0.08, the PCV2 replication defective virus envelope antigen to positive standard serum and negative standards' serum
2.8 ± 0.28 and 0.32 ± 0.10.PCV2 replication defective virus envelope antigen compares reagent to the detection OD value of positive standard serum
The included envelope antigen of box is high by 21.7% to the detection OD value of positive standard serum, illustrates PCV2 replication defective virus envelope antigen
It is higher than the detection sensitivity for recombinantly expressing envelope antigen that the kit carries；Meanwhile inspection of the two to negative standards' serum
Survey OD value it is substantially suitable, illustrate PCV2 replication defective virus as envelope antigen while improving ELISA detection sensitivity not
The accuracy of detection can be substantially reduced.
Claims (9)
1. a kind of preparation method of replication defect type pig circular ring virus knocks out wild type PCV's using artificial DNA mutation technology
The normal expression function of REP gene or cap gene obtains replication defect type PCV infection clones, is felt using replication defect type PCV
Metachromia clone transfects corresponding PCV trans-complementation gene cell strain, and rescue obtains replication defect type PCV.
2. preparation method as described in claim 1, which is characterized in that the PCV trans-complementation gene cell strain is will be wild
What the cell strain that the cap gene of type PCV or the eukaryon expression plasmid of REP gene are inserted into any reproducible PCV virus obtained.
3. preparation method as described in claim 1, which is characterized in that comprising the following three steps:
(1) it the building of PCV trans-complementation transgenosis cell strain: is transfected by lentiviruses transduction or plasmid stabilisation by wild type PCV
Cap gene or REP gene the eukaryon expression plasmid cell strain that is inserted into reproducible PCV genome in, it is trans- to obtain PCV
Complementary transgenosis cell strain；
(2) replication defective PCV infection clones construct: the full length DNA of wild type PCV being cloned into plasmid vector, is then led to
The normal expression function that artificial DNA mutation technology knocks out cap gene or REP gene is crossed, obtains infectious gram of replication defective PCV
It is grand；
(3) it the transfection of replication defect type PCV infection clones and virus rescue and culture: is infected using the replication defective PCV
Property clone transfect corresponding PCV trans-complementation transgenosis cell strain, rescue obtains replication defect type PCV, and passes through the cell strain
Cultivate and prepare replication defect type PCV.
4. preparation method as described in any one of claims 1-3, which is characterized in that artificial DNA mutation technology is following three kinds
Any one: one or more Premature stop codons are 1. introduced in the ORF sequence of cap gene or REP gene；2.
5 ' ends of cap gene or REP gene ORF sequence introduce the insertion mutation of single base or double alkali yl or delete mutation；3. using external source
Some or all of the whole code displacement cap gene of gene or REP gene ORF sequence.
5. the preparation method as described in claim 1, which is characterized in that the cell strain of the reproducible PCV is PK-15 cell strain
Or Vero cell strain.
6. the replication defect type pig circular ring virus of any one of the claim 1-5 preparation method preparation.
7. any one of the claim 1-5 preparation method or replication defect type pig circular ring virus as claimed in claim 6 are being made
Application in standby preventative or therapeutic PCV vaccine.
8. any one of the claim 1-5 preparation method or replication defect type pig circular ring virus as claimed in claim 6 are being made
Application in the recombinant vaccine of standby expression foreign antigen genes.
9. any one of the claim 1-5 preparation method or replication defect type pig circular ring virus as claimed in claim 6 are being made
Application in the detection antigen of standby detection PCV antibody.
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| CN113355292A (en) * | 2021-06-04 | 2021-09-07 | 天康制药(苏州)有限公司 | Porcine circovirus gene modified attenuated strain, construction method and application thereof |
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| CN113355292A (en) * | 2021-06-04 | 2021-09-07 | 天康制药(苏州)有限公司 | Porcine circovirus gene modified attenuated strain, construction method and application thereof |
| CN113355292B (en) * | 2021-06-04 | 2024-02-06 | 天康制药股份有限公司 | Porcine circovirus gene modified attenuated strain, construction method and application thereof |
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