CN110184287A - A kind of method and its application of preparation and reorganization virus - Google Patents
A kind of method and its application of preparation and reorganization virus Download PDFInfo
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- CN110184287A CN110184287A CN201910443328.3A CN201910443328A CN110184287A CN 110184287 A CN110184287 A CN 110184287A CN 201910443328 A CN201910443328 A CN 201910443328A CN 110184287 A CN110184287 A CN 110184287A
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Abstract
The present invention provides a kind of methods of preparation and reorganization virus, it is using viral vectors as matrix, it first passes through Red/ET recombination and introduces bidirectional screening gene, further recombination replaces bidirectional screening gene with target gene, it obtains containing target gene and virus genomic recombinant plasmid, and then saves and obtain recombinant virus.The beneficial effect is that this method step is simple, the building of recombinant virus can be quickly and accurately realized, and the successful recombinant virus of building can accommodate foreign gene, realize how site-directed insertion, no trace insertion, the convenient advantage of selection markers, the recombinant virus that can express a variety of antigen genes is ultimately formed, carrier bacterin is applied to, also has host range wide, preparation method is easy, safety is good, while panimmunity being induced to react, the advantages of without adding adjuvant.
Description
Technical field
The invention belongs to gene engineering technology fields, a kind of method more particularly, to preparation and reorganization virus and its answer
With.
Background technique
Vaccine inoculation is to completely cut off one of the important way of disease infections channel between animals and human beings class.Currently, China's needle
A variety of vaccines are had developed to different epidemic disease types, wherein just including recombinant vaccine.With the fast development of recombinant technique,
Recombinant vaccine also has obtained more being widely applied in zoonosis prevention and treatment, and establishes to promote Prevention of Infectious Diseases effect
Solid foundation is determined.
In recent years, recombinant viral vector vaccine receives extensive attention, it is to be inserted into external source protective antigen gene
It is obtained in viral genome, recombinant virus gives expression to corresponding destination protein after body is immunized, to induce immune response
A kind of carrier bacterin.Recombinant viral vector vaccine has insertion foreign gene long, and route of inoculation is more, can induce humoral immunity and thin
The advantages that born of the same parents' immune response and preparation easy to produce, and certain drawbacks of traditional vaccine can be overcome, the pre- of disease can be greatlyd improve
Anti- property, is widely used in vaccine development.
In terms of main modification technique currently used for viral genetic engineering has following four:
RecA protein mediated homologous recombination the disadvantage is that: firstly the need of longer homology arm, can only be grasped using Escherichia coli
More limit to, operating procedure complexity is cumbersome, and false positive rate is higher etc.；The modification technique that Cre/loxp is mediated: advantage is space-time
Specifically, high efficiency (not influenced by target gene size and location), accuracy (non-specificity recombination is few), rapidity, the disadvantage is that step
It is rapid cumbersome, it is at high cost；Tn transposons can mediate gene mutation and radom insertion, the disadvantage is that insertion cannot be pinpointed, insertion is copied
Shellfish number is not easy to control, low efficiency.As it can be seen that common homologous recombination needs the homology arm up to several hundred or even thousands of a bases, and
It is easy the limitation of conditionality restriction enzyme site, is used inflexible.
Summary of the invention
The present invention is directed to problem above, and it is a kind of quick, accurate to provide, a kind of side of preparation and reorganization virus easy to operate
Method, which is characterized in that the described method comprises the following steps:
S1: by the genome of virus to be selected and acceptor carrier homologous recombination, viral vectors plasmid is obtained；
S2: the plasmid containing bidirectional screening gene is chosen, is expanded, is obtained for the bidirectional screening gene design primer
The amplified production of bidirectional screening gene containing homology arm, wherein the bidirectional screening gene includes resistance screening gene and anti-
To screening-gene, the resistance screening gene include kalamycin resistance gene (Knr), benzyl mycin resistant gene (Amp), tide it is mould
Element one of (Hyg) or chloramphenicol resistance gene (Cmr) or a variety of, and the plasmid of the gene containing bidirectional screening and step S1
In acceptor carrier resistance it is different, the reversed screening-gene includes CcdB, ScaB, Rpsl, TetR, Phes, Galk, ThyA
Or one of TolC or a variety of；
S3: amplified production described in viral vectors plasmid described in step S1 and step S2 is transferred to competent cell 1 jointly, is resisted
Property screen to obtain the recombinant plasmid containing bidirectional screening gene and viral vector gene, wherein the competent cell 1 be can hold
It is receiving reversed screening-gene in step S2 and the engineering bacteria of Red α/β recombinase can be expressed；
S4: recombinant plasmid described in the expression casette containing homology arm target gene and step S3 is transferred to competence jointly
Cell 2, screening obtain the recombinant plasmid that bidirectional screening gene is replaced with HA, are simultaneously to virus to be selected and load through rescue
The immune recombinant virus of antigen, wherein competent cell 2 is that can express Red α/β recombinase, but cannot accommodate anti-in step S2
To the engineering bacteria of screening-gene.
This method is reversely to be sieved based on the viral vectors of infection clones using RED/ET recombinant technique and ccdB
Selecting technology obtains the recombinant virus containing target gene by two step homologous recombinations.This recombination system is safe and reliable, and can realize
Stablize expression.
In the above method, the genome of virus to be selected is known, tradition with acceptor carrier methods of homologous recombination in step S1
Homologous recombination method be by the insertion point both ends of acceptor carrier introduce with the homologous homology arm of target gene, containing
There is the homologous recombination completed in the engineering bacteria body of recombinase.
Further, genome is DNA or cDNA in the step S1.
This method is suitable for the virus with DNA for main inhereditary material, is also applied for the disease with RNA for main inhereditary material
RNA reverse transcription only need to can be carried out above-mentioned steps at cDNA by poison.
Further, acceptor carrier is in plasmid vector, phage vector or artificial chromosome vector in the step S1
It is a kind of.
Further, bidirectional screening gene is ccdB-amp in the step S2, and competent cell 1 is in the step S3
GBred gyrA462 E.coli。
Ccd operon is present on the F plasmid of Escherichia coli, is a kind of toxin-antitoxin system, the system by ccdA and
CcdB two parts composition, wherein ccdB is toxalbumin, and the expression of only ccdB gene will lead to general host cell (as felt
By state cell 2) it is dead, by two kinds of competent cells, to the difference of the containment of ccdB, (such as ccdB gene can be in the present invention
Express and survive in competent cell 1) recombinant plasmid containing screening-gene ccdB is filtered out with the plasmid for replacing ccdB
Come, simplify screening step, positive rate is high, using being more simple and efficient.
Further, homology arm is located at viral insertion point both ends to be selected, the nucleotide of the homology arm in the step S3
Sequence is 35~50bp.
A kind of preparation and reorganization viral methods are preparing the application in vaccine.
It is to be selected viral for by immune host in step S1 when the method for above-mentioned preparation and reorganization virus is applied to vaccines arts
Safety, can cause effective immune response and can accommodate the virus of foreign gene, such as HVT, IBV, NDV, PRV or ADV.
Further, the vaccine is the infectious bronchitis virus for expressing H9N2 Avian Influenza Virus HA Gene.
A kind of infectious bronchitis virus rH120- Δ 5a/H9HA expressing HA gene, the sequence of the HA gene is such as
It shown in SEQ ID No.1, is preserved in China typical culture collection center (CCTCC), deposit number is CCTCC NO:
V201931。
Further, the rH120- Δ 5a/H9HA, is prepared by the following method:
S1: the recombinant viral plasmid vector of plasmid vector p15A building infectious bronchitis virus vaccine strain H120 is utilized
p15A-H120；
S2: using pBR322 plasmid-kanR-amp-ccdB-rpsLneo as template, the amp-ccdB containing homology arm is expanded
Bi-directional selection markers expression casette；
S3: by amp-ccdB bi-directional selection markers gene in recombinant viral plasmid vector p15A-H120 in step S1 and step S2
Expression cassette is transferred to jointly in the GBred gyrA462 somatic cells of competence, and homologous recombination obtains recombinant plasmid p15A-H120-
amp-ccdB；
S4: by plasmid p15A- obtained by the expression casette containing the HA gene as shown in SEQ ID No.1 and step S3
H120-amp-ccdB is transferred to jointly in competence GBdir E.coli, and screening obtains recombinant plasmid p15A-H120-HA；
S5: building helper plasmid pVAX1-H120 N, by recombinant plasmid p15A-H120-HA described in step S4 and auxiliary matter
Grain pVAX1-H120 N transfects BSR-T7 cell jointly, collects transfection supernatants and is transferred in chicken embryo again, and passage obtains expression HA base
The infectious bronchitis virus rH120- Δ 5a/H9HA of cause.
To achieve the goals above, the present invention specifically uses following technical scheme:
The infection clones carrier containing H120 full-length genome cDNA is constructed first, and the first step is in expression recombinant protein Red
It in the GBred gyrA462 Escherichia coli of α/Red β, is recombinated by " line-ring ", is replaced with riddled basins amp-ccdB
5a gene in p15A-H120, obtains p15A-H120- Δ 5a/amp-ccdB, and digestion prepares linearized vector p15A-H120-
Δ5a/amp-ccdB.Second step utilizes " line-ring " to recombinate in GBred Escherichia coli, target gene and linear carrier p15A-
H120- Δ 5a/amp-ccdB is recombinated, and is realized that foreign gene replaces amp-ccdB gene, is obtained insertion point at 5a
p15A-H120-ΔSa/H9HA.Third step saves to obtain recombinant virus rH120- Δ 5a/H9HA.
Compared with the existing technology, the present invention has the advantage that and effect:
Method and step used in the present invention is simple, can quickly and accurately realize the building of recombinant virus, and construct successfully
Recombinant virus have and can accommodate multiple large fragment foreign genes, the how site-directed insertion of realization, no trace is inserted into, selection markers
Convenient advantage ultimately forms the recombinant virus that can express a variety of antigen genes, is applied to carrier bacterin, also has host
Range is wide, and preparation method is easy, and safety is good, while panimmunity being induced to react, without adding adjuvant, to also reduce
The probability of adjuvant initiation adverse reaction.
Biomaterial preservation information
Classification naming: avian infectious bronchitis virus IBV rH120- Δ 5a/H9HA, Classification system: Infectious
Bronchitis virus, depositary institution: China typical culture collection center, preservation address: Wuhan, China Wuhan University is protected
It hides the date: on May 13rd, 2019, deposit number is CCTCC NO:V201931.
Detailed description of the invention
Fig. 1 is that PCR amplification prepares the riddled basins containing homology arm, wherein 1: Δ 5a/amp-ccdB；2: blank pair
According to；M:DNA Marker DL2,000.
Fig. 2 is recombinant plasmid p15A-H120- Δ 5a/amp-ccdB digestion identification, wherein A:p15A-H120- Δ 5a/
Amp-ccdB digestion SnapGene software prediction, B:p15A-H120- Δ 5a/amp-ccdB is through XhoI and Bstz17I digestion；M:
NEB 1Kb DNA Ladder。
Fig. 3 is reversed riddled basins functional verification.
Fig. 4: the H9 HA gene containing homology arm is prepared for PCR amplification, wherein 1: Δ 5a/H9 HA；2: blank control；M:
DNA Marker DL2,000.
Fig. 5 is recombinant plasmid p15A-H120- Δ 5a/H9 HA digestion identification, wherein A:p15A-H120- Δ 5a/H9 HA
Digestion SnapGene software prediction, B:p15A-H120- Δ 5a/H9 HA is through XhoI and Bstz17I digestion；M:NEB 1Kb DNA
Ladder。
Fig. 6 is F5 plants of PCR amplification rH120 and H120- Δ F5 plants of S1, M and 5ab genes of 5a/H9 HA, wherein 1-3:
F5 plants of S1, M and 5ab genes of rH120；F5 plants of S1, M of 4-6:H120- Δ 5a/H9 HA and Δ 5a/H9 HA gene；7: negative right
According to；M:DNA Marker DL5,000.
Fig. 7 is the exogenous gene expression wherein A of indirect immunofluorescene assay rH120- Δ 5a/H9 HA F5 recombinant virus:
The CK cell of recombinant virus rH120- Δ 5a/H9 HA F5 infection；The CK cell of B:rH120 F5 infection；C: blank CEF cell)
Fig. 8 is recombinant virus rH120- Δ 5a/H9 HA F5 chicken embryo growth curve.
Fig. 9 is pBR322-kanR-amp-ccdB-rpsLneo structural schematic diagram.
Figure 10 is p15A structural schematic diagram.
Specific embodiment
Further illustrate the present invention below in conjunction with specific embodiment, but embodiment the present invention is not done it is any type of
It limits.Unless stated otherwise, the present invention uses reagent, method and apparatus is the art conventional reagents, method and apparatus.
Unless stated otherwise, agents useful for same and material of the present invention are commercially available.
Embodiment 1
1) building of infection clones p15A-H120
It is announced three complete genome sequences (including 5 '-UTR, 3 '-UTR and Ploy (A)) according to Genbank: FJ888351,
H120 complete genome sequence is divided into 4 sections and carries out cDNA clones to the pBR322 load containing homology arm by FJ807652 and GU393335
On body, pBR322-H120 A~D is formed, the overlapping region 50-70b β is contained between adjacent two sections as recombination homology arm.5 ' ends the
One section of H120-A adds T7 promoter by PCR in cloning procedure.Each segment clone need to carry out digestion identification and sequence verification
Then correct sequence obtains four segment DNAs (H120-A~D) by digestion.Using plasmid pUC57-HDVR-T7 ter as template
PCR amplification contains the HDVR-T7 ter sequence of 3 ' homology arm of genome as the 5th section of sequence of H120 infection clones.It utilizes
H120-A~D, HDVR-T7 ter and linear carrier p15A structure containing homology arm are shown in Figure 10 by RED/ET recombinant technique,
It is assembled in E.coli, forms infection clones p15A-cm-T7 promotor-H120 genome-HDVribozyme-T7
Terminator (abbreviation p15A-H120).
2) PCR amplification of riddled basins Δ 5a/amp-ccdB
Fig. 9 is seen as template using pBR322 plasmid-kanR-amp-ccdB-rpsLneo structure, and PCR amplification contains homology arm
Riddled basins Δ 5a/amp-ccdB.The primer is as follows:
Δ 5a/amp-ccdB-F:
5‘ACCTACACTACTTACTTGTAATAAGGGCGTTTGGACTTACAAGCGCTTAACAAATACAGACGGCGA
TCGCTTTGTTTATTTTTCTAAATAC3’
Δ 5a/amp-ccdB-R:
5‘CTGGCTTTTTTTTGAACAAAGCGATCGCGCATATACGCCCACCCAATCGCTGGTATGAATAATAGT
AAAGATAATCCTTTTCGCGGAGCAAT’3
Reaction system is shown in Table 1, and PCR amplification parameter is 98 DEG C of 2min；98℃10s；55℃5s；72 DEG C, 20s；Amplification 35
Circulation；Finally in 72 DEG C of extension 5min.PCR product takes 5uL to observe amplification through 1% agarose gel electrophoresis, as a result sees figure
1, remaining PCR product is recycled and is purified with PCR product QIAquick Gel Extraction Kit, and measures nucleic acid concentration, sequence verification correct sequence.
1 PrimeSTAR Max DNA Polymerase polymerase PCR reaction system of table
Electrophoresis result shows that PCR obtains the riddled basins ccdB-amp containing homology arm.PCR product is through 1% agar
Sugared gel electrophoresis, primer size 1.5Kb or so, with expected consistent (Fig. 1).
The PCR product restriction enzyme DpnI of recovery purifying is digested to remove plasmid template, and digestion system is pressed
Table 2 is prepared, 37 DEG C of reaction 2h, and digestion products observe amplification through 1% agarose gel electrophoresis, and use gel reclaims kit
Recycling and purifying digestion products, measure nucleic acid concentration.
2 digestion of table identification
3) riddled basins and p15A--H120 electricity turn GBred gyrA462 competence
GBred gyrA462 electricity turns competence preparation: sterile 2ml centrifuge tube is taken, lid is punched with 2mL syringe needle,
The LB culture medium that 1.8mL contains streptomysin is added, picking GBred gyrA462 single colonie is seeded in 2mL centrifuge tube, and 30 DEG C,
260rpm shakes overnight incubation.Second day, sterile 1.5mL centrifuge tube is taken, lid is punched with 2mL syringe needle, and 1.4mL is added
The LB culture medium of antibiotic-free adds 40 μ L and stays overnight bacterium solution, and 30 DEG C, then 35 μ L 10% are added in 260rpm shaking culture 2h
L-arabinose solution, 37 DEG C, 260rpm shaking culture 50min inducing expression Red α/Red β recombinant protein.Hereafter bacterium solution operates
It should be placed on ice, sterilizing pure water is pre-chilled in advance, and centrifuge should shift to an earlier date is cooled to 2 DEG C in advance, and 2 DEG C of bacterium solution are centrifuged, 9000rpm, 30s；It abandons
Supernatant adds precooling pure water 1ml, and thallus is resuspended in piping and druming, and 2 DEG C of bacterium solution are centrifuged second, 10000rpm, 30s；Supernatant is abandoned, pre-cooling is added
Thallus is resuspended in pure water 1ml, piping and druming, and 2 DEG C of centrifugation third times of bacterium solution, 11000rpm, 30s abandon supernatant, add 30 μ L of precooling pure water weight
It is outstanding.It should be placed on ice after competence preparation, use immediately.
First time regrouping process: the electric shock cup of cleaning sterile is placed in aeration-drying in super-clean bench, is placed in and is pre-chilled on ice.
The GBred gyrA462 electricity just prepared is added in each 500ng of riddled basins ccdB-amp and p15A-H120 and turns competence
In, 1.5mL centrifuge tube tube bottom is dialed with hand and is mixed gently, and is transferred in 1mm electric shock cup, electric shock cup is placed in electroporation,
It carries out electricity by the RED/ET electricity carryover sequence of setting to turn, electricity turns parameter 1350V, 10 μ F, 600 Ohms.Electricity is added immediately after the completion of turning
Thallus is resuspended and is transferred in 2mL centrifuge tube into electric shock cup by 1mL SOC culture medium.Bacterium solution after electricity is turned is placed in 37
DEG C shaking table, 260rpm shaking culture 60min complete recombination and resistance recovery.Bacterium solution is placed in centrifuge, 3000rpm centrifugation
1min precipitates thallus, inhales and abandons most of supernatant, retains 200 μ L supernatants and blows and beats resuspension, is all coated on containing ammonia benzyl mycin
LB plate, plate is placed in 37 DEG C of cultures, is inverted culture 12-16h after first just setting 1h.
Recombinant plasmid screening containing riddled basins: bacterium colony is picked from the plate, being inoculated in 500 μ L, to contain ammonia benzyl mould
In the LB liquid medium of element, 37 DEG C, after 6h is cultivated in 260rpm shaking, takes 100 μ L bacterium solutions to be inoculated in 10mL and contain ammonia benzyl mycin
LB liquid medium in expand culture.37 DEG C, after 6h is cultivated in 260rpm shaking, the small extraction reagent kit of 5mL bacterium solution plasmid is taken to take out
Upgrading grain simultaneously carries out digestion with restriction enzyme identification, and digestion system is prepared by table 3, and 37 DEG C of reaction 2h, digestion products are through 1% fine jade
Sepharose electrophoresis observes amplification, identifies that correct plasmid send Huada gene company to be sequenced, and correctly recombination is sequenced
Plasmid is named as p15A-H120- Δ 5a/amp-ccdB.
3 digestion identification system of table
The results show that linear riddled basins ccdB-amp and ring-type p15A-H120 plasmid are in expression Red α/Red β weight
Line-ring recombination occurs in the GBred gyrA462 Escherichia coli of histone, riddled basins are replaced with 5a gene, produced
Raw recombinant plasmid, by Amp resistance screening, plasmid enzyme restriction identification (Fig. 2) and sequencing evaluation and screening go out correct recombinant plasmid.Weight
Group plasmid is respectively designated as p15A-H120-ccdB-amp.
Riddled basins functional verification: electricity turns GBred to the recombinant plasmid p15A-H120-ccdB-amp of preparation respectively
GyrA462 and GBred electricity turns competence (not needing inducing expression recombinant protein).Bacterium solution is coated on containing ammonia after conversion recovery
Plate is placed in 37 DEG C of overnight incubations, observes Escherichia coli Growth situation by the LB plate of benzyl mycin.
Recombinant plasmid p15A-H120- Δ 5a/amp-ccdB electricity is recovered after turning GBred and GBred gyrA462 competence,
Plated overnight culture, GBred gyrA462 can grow a large amount of bacterium colonies, and GBred cannot or grow minute quantity bacterium colony (Fig. 3),
Illustrate that reversed riddled basins ccdB can be with normal expression.
Electricity turns the GBred gyrA462 normal growth of p15A-H120- Δ 5a/amp-ccdB, can grow a large amount of bacterium colonies,
And GBred cannot or only grow minute quantity bacterium colony.
4) recombination of antigen gene replacement screening-gene
Expand the antigen gene Δ 5a/H9 HA containing homology arm:
Avian influenza virus A/chicken/Guangdong/YF/ is extracted according to the specification of RNeasy plus Mini Kit
2018 RNA.
It takes above-mentioned RNA to illustrate to carry out reverse transcription by reverse transcription reagent box, obtains cDNA as template, amplification is obtained containing same
The HA gene of source arm.
Primer: Δ 5a/H9 HA-F:
ACCACCTACACTACTTACTTGTAATAAGGGCGTTTGGACTTACAAGCGCTTAACAAATACAGACGATGG
AGACAGTATCACTAATAAC
Δ 5a/H9 HA-R:
CTCTAATCCTTCTCTCAGATAAATTCGCGCTTTTCTTGCTATTGCTCCGCGAAAAGGTTATTATATACA
AATGTTGCATCTG
Reaction system is 98 DEG C of initial denaturation 5min with table 1:PCR reaction condition, 98 DEG C of denaturation 10s, 55 DEG C of annealing 5s, 72 DEG C
Extend 20s, completes 35 circulations；72 DEG C of extension 10min.
Amplification is shown in Fig. 4
GBred electricity turns competence preparation: preparation process turns competence preparation with GBred gyrA462 electricity, it is also desirable to use L-
Arabinose inducing expression Red α/β recombinant protein.It should be placed on ice after competence preparation, use immediately.
Second of regrouping process: the electric shock cup of cleaning sterile is placed in aeration-drying in super-clean bench, is placed in and is pre-chilled on ice.
Each 500ng of HA gene Δ 5a/H9 HA containing homology arm and linear carrier p15A-H120- Δ 5a/amp-ccdB is added rigid
The GBred electricity of preparation turns in competence, and the method and program recombinated by first time carries out electricity and turn and recover.By recovery bacterium solution
3000rpm is centrifuged 1min and precipitates thallus, inhales and abandons most of supernatant, retains 200 μ L supernatants and blows and beats resuspension, is all coated on and contains
There is the LB plate of chloramphenicol, plate is placed in 37 DEG C of cultures, is inverted culture 12-16h after first just setting 1h.
Recombinant plasmid screening
Bacterium colony is picked from the plate, expansion shakes bacterium solution, extracts plasmid progress digestion identification.Digestion result is shown in Fig. 5, and identification is correct
Bacterium solution take 5mL be added in the LB liquid medium for containing chloramphenicol to 200mL expand culture, 37 DEG C, 260rpm shaking culture
After 16h, plasmid is extracted by a large amount of extracts kit specifications of endotoxin-free plasmid, -20 DEG C is placed in and freezes.A small amount of plasmid is taken to send China
Big genome company carries out sequence verification to recombination Gene Partial.It verifies correct recombinant plasmid and is named as p15A-H120- Δ 5a/
H9 HA。
5) rescue of recombinant virus
BSR-T7 cell is passaged to six orifice plates by the day before transfection, changes culture medium into when cell grows to 80% or so
2% serum, without dual anti-and G418 GMEM culture medium.Take recombinant plasmid p15A-H120-HA and helper plasmid pVAX1-
Each 1ug of H120N (the every hole DAN transfection amount of six orifice plates), according to 3000 Reagent of lipofectamine Lipofectamine
(invitrogen) specification is prepared rotaring redyeing system and is added in cell culture fluid, and cell is placed in 37 DEG C, the training of 5%CO2 incubator
Culture medium is changed into 2% serum after feeding 4h, and the GMEM culture medium containing G418 continues to cultivate.After 48h, culture dish is placed in -80 and is taken the photograph
The freezing of family name's degree, room temperature melt, and smudge cells, harvest cell conditioned medium are named as F0 to multigelation twice.Cell conditioned medium is through allantoic cavity
10 5 pieces of age in days SPF chicken embryos are inoculated with, 0.2ml/ pieces, 37 DEG C hatch.24 hours observation chicken embryos and dead chicken embryo is discarded after inoculation.It connects
Kind after 48h harvest chick embryo allantoic liquid and continue chicken embryo passage, 5 generation of blind passage and harvest the 5th generation chick embryo allantoic liquid be named as rH120
F5, packing is stored in -80 DEG C after 0.22 μM of membrane filtration.
Experimental example 1
The identification of Revive virus
RT-PCR detection
Take 200 F5 plants of μ L rH120 and rH120- Δ 5a/H9 HA F5 virus liquid by Axyprep body fluid viral DNA/RNA
A small amount of extraction agent box specifications extract viral RNA, and the RNA for extracting acquisition is dissolved in 40 μ L RNase-free TE buffer, takes
10 μ L RNA remove DNA by Recombinant DNase I (RNase-free) (Takara) specification.
Using above-mentioned RNA as template, primer
M-F:5 '-CCTAAGAACGGTTGGAAT-3 '；
M-R:5 '-TACTCTCTACACACACAC-3 '；
S1-F:5 '-AAGACTGAACAAAAGACCGACT-3 '；
S1-R:5 '-CAAAACCTGCCATAACTAACATA
- 3 ' 5ab-F:ACCACCTACACTACTTACTTG；
5ab-R:ATTATCTGTGTGTTCCTCACAAG
By One step RT-PCR kit PrimeScriptTMThe amplification of One Step RT-PCR Kit Ver.2 specification
Expand M, S1 and 5ab gene.PCR product is shown in Fig. 6 through 1% agarose gel electrophoresis observation amplification, and it is raw to serve the raw work in sea
The sequencing of object engineering company.
Indirect immunofluorescence (IFA)
In order to identify the expression of recombinant virus foreign protein, by the rH120 F5 of rescue and recombinant virus H120- Δ 5a/H9
HA is connected to by 100MOI poison amount to be grown up on the primary CK of freshly prepared single layer, while setting sky CK control, connects cell culture fluid before poison
It changes into containing 2% fetal calf serum DMEM culture solution, is incubated at 37 DEG C, 5%CO2Cell incubator in.Malicious 36h is met, by indirect
The exogenous gene expression of the recombinant virus H120- Δ 5a/H9 HA of immunofluorescence (IFA) detection rescue.Cell culture medium is inhaled
Fall, is washed twice with hot PBS；15-20min is fixed with 4% paraformaldehyde room temperature；PBST is washed cell 3 times, 5min/ times；Punching: make
With the Triton-100 (PBS) of 0.4%-0.5%, i.e., Triton-100: PBS=0.4: 100, incubation 15-20min；PBST is washed
Cell 3 times, 5min/ times；Closing: 30min or 4 DEG C is closed at room temperature with 5%BSA overnight；Primary antibody be (anti-H9 hypotype HA gene
Monoclonal antibody), it is incubated at room temperature 1h；PBST is washed cell 3 times, 5min/ times；It is protected from light, secondary antibody (goat anti-rabbit igg of FITC label), room temperature is incubated
Educate 1h；It is protected from light, PBST is washed 3 times, 5min/ times each；It is protected from light, nuclear staining: DAPI: PBS=1: 200, it is incubated for 15min；It is protected from light,
PBST is washed 3 times, 5min/ times each.Fig. 7 is seen with fluorescence microscope result of taking pictures.
The measurement of rH120- Δ 5a/H9 HA viral growth curves
By rH120- Δ 5a/H9 HA and rH120 normal saline dilution, 10 age in days SPF chicken embryos each 30 are inoculated with through allantoic cavity
Piece, 100EID50/ embryo.6h, 12h after inoculation, for 24 hours, 36h and 48h respectively harvest the viral allantoic fluid of 5 pieces of chicken embryos, mixing
After dispense, be placed in -80 DEG C and freeze.Different time points virus EID50 is measured using Reed-Muench method.
Claims (10)
1. a kind of method of preparation and reorganization virus, which is characterized in that the described method comprises the following steps:
S1: by the genome of virus to be selected and acceptor carrier homologous recombination, viral vectors plasmid is obtained；
S2: the plasmid containing bidirectional screening gene is chosen, is expanded, is contained for the bidirectional screening gene design primer
The amplified production of the bidirectional screening gene of homology arm, wherein the bidirectional screening gene includes resistance screening gene and reversed sieve
Gene is selected, the resistance screening gene includes kalamycin resistance gene (Knr), benzyl mycin resistant gene (Amp), hygromycin
(Hyg) or one of chloramphenicol resistance gene (Cmr) or a variety of, and in the plasmid of the gene containing bidirectional screening and step S1
Acceptor carrier resistance it is different, the reversed screening-gene includes CcdB, ScaB, Rpsl, TetR, Phes, Galk, ThyA or
One of TolC or a variety of；
S3: amplified production described in viral vectors plasmid described in step s1 and step S2 is transferred to competent cell 1, resistance sieve jointly
Choosing obtains the recombinant plasmid containing bidirectional screening gene and viral vector gene, wherein the competent cell 1 is that can accommodate step
In rapid S2 reversed screening-gene and the engineering bacteria of Red α/β recombinase can be expressed；
S4: being transferred to competent cell 2 for recombinant plasmid described in destination gene expression box and step S3 containing homology arm jointly, sieve
Choosing obtains the recombinant plasmid that bidirectional screening gene is replaced with HA, is immune to virus to be selected and antigen loaded simultaneously through rescue
Recombinant virus, wherein competent cell 2 is that can express Red α/β recombinase, but cannot accommodate in step S2 and reversely screen base
The engineering bacteria of cause.
2. the method for preparation and reorganization virus according to claim 1, which is characterized in that genome is DNA in the step S1
Or cDNA.
3. the method for preparation and reorganization virus according to claim 1, which is characterized in that acceptor carrier is matter in the step S1
One of grain carrier, phage vector or artificial chromosome vector.
4. the method for preparation and reorganization virus according to claim 1, which is characterized in that bidirectional screening gene in the step S2
For ccdB-amp, competent cell 1 is GBred gyrA462 E.coli in the step S3.
5. the method for preparation and reorganization virus according to claim 1, which is characterized in that in the step S3 homology arm be located to
Viral genome insertion point both ends are selected, the nucleotides sequence of the homology arm is classified as 35~50bp.
6. preparation and reorganization viral methods described in a kind of Claims 1 to 5 any one are preparing the application in vaccine.
7. vaccine is the infectious bronchitis virus for expressing H9N2 Avian Influenza Virus HA Gene according to claim 6.
8. expressing the infectious bronchitis virus of H9N7 Avian Influenza Virus HA Gene according to claim 7, feature exists
In the sequence of the HA gene is as shown in SEQ ID No.1.
9. the infectious bronchitis virus rH120- Δ 5a/H9HA of HA gene is expressed described in a kind of expression claim 7, it is special
Sign is that for the sequence of the HA gene as shown in SEQ ID No.1, oneself is preserved in China typical culture collection center, preservation
Number is CCTCC NO:V201931.
10. rH120- Δ 5a/H9HA according to claim 9, is prepared by the following method:
SI: the recombinant viral plasmid vector p15A- of plasmid vector p15A building infectious bronchitis virus vaccine strain H120 is utilized
H120；
S2: using pBR322 plasmid-kanR-amp-ccdB-rpsLneo as template, amp-ccdB of the amplification containing homology arm is two-way
Riddled basins expression cassette；
S3: by amp-ccdB bi-directional selection markers gene expression in recombinant viral plasmid vector p15A-H120 in step S1 and step S2
Box is transferred to jointly in the GBred gyrA462 somatic cells of competence, and homologous recombination obtains recombinant plasmid p15A-H120-amp-
ccdB；
S4: by plasmid p15A-H120- obtained by the expression casette containing the HA gene as shown in SEQ ID No.1 and step S3
Amp-ccdB is transferred to jointly in competence GBdir E.coli, and screening obtains recombinant plasmid p15A-H120-HA；
S5: building helper plasmid pVAX1-H120N, by recombinant plasmid p15A-H120-HA and helper plasmid described in step S4
PVAX1-H120N transfects BSR-T7 cell jointly, collects transfection supernatants and is transferred in chicken embryo again, and passage obtains expression HA gene
Infectious bronchitis virus rH120- Δ 5a/H9HA.
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| CN110713989A (en) * | 2019-11-22 | 2020-01-21 | 华南农业大学 | Method for rapidly preparing epidemic infectious bronchitis vaccine |
| CN114561367A (en) * | 2022-02-25 | 2022-05-31 | 华南农业大学 | Recombinant duck adenovirus type 3, preparation method and application thereof |
| CN114807061A (en) * | 2021-12-22 | 2022-07-29 | 华南农业大学 | Chicken infectious bronchitis marker vaccine strain, preparation method thereof and vaccine |
Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6355412B1 (en) * | 1999-07-09 | 2002-03-12 | The European Molecular Biology Laboratory | Methods and compositions for directed cloning and subcloning using homologous recombination |
| EP1241177A1 (en) * | 2001-03-15 | 2002-09-18 | Akzo Nobel N.V. | Recombinant infectious laryngotracheitis virus vaccine |
| CN101220374A (en) * | 2007-01-11 | 2008-07-16 | 华南农业大学 | Fowl pox virus double-gene expression carrier (PG7.5N) |
| CN102533674A (en) * | 2010-12-08 | 2012-07-04 | 中国农业科学院哈尔滨兽医研究所 | Recombinant duck virus enteritis virus vaccine strain (rDEVul41HA) for expressing avian influenza virus hemagglutinin (HA) genes and construction method as well as application thereof |
| US20130210681A1 (en) * | 2010-06-10 | 2013-08-15 | Youming Zhang | Direct Cloning |
| US20140287938A1 (en) * | 2013-03-15 | 2014-09-25 | The Broad Institute, Inc. | Recombinant virus and preparations thereof |
| CN104250637A (en) * | 2014-08-29 | 2014-12-31 | 乾元浩生物股份有限公司 | Avian flu H9N2 subtype virus strain and application thereof |
| CN105695422A (en) * | 2016-03-15 | 2016-06-22 | 中国农业科学院哈尔滨兽医研究所 | Recombined chicken Marek's disease virus vaccine strain for expressing Gag and Env genes of avian leukosis virus subgroup J and construction method and application of recombined chicken Marek's disease virus vaccine strain |
| CN106047823A (en) * | 2016-07-01 | 2016-10-26 | 四川大学 | Bivalent vaccine strain for recombining and expressing NCV HN gene by virtue of IBV H120 strain based on reverse genetic technique |
| CN107296956A (en) * | 2017-06-29 | 2017-10-27 | 青岛易邦生物工程有限公司 | A kind of genetic recombination live vector vaccine |
| CN108324937A (en) * | 2018-01-17 | 2018-07-27 | 华中农业大学 | The herpes turkey virus live vector vaccine of 4 type penton protein of expression aviadenovirus serum and its preparation and application |
| CN108841866A (en) * | 2018-06-04 | 2018-11-20 | 云舟生物科技（广州）有限公司 | A kind of adenovirus vector and its construction method |
-
2019
- 2019-05-24 CN CN201910443328.3A patent/CN110184287B/en active Active
Patent Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6355412B1 (en) * | 1999-07-09 | 2002-03-12 | The European Molecular Biology Laboratory | Methods and compositions for directed cloning and subcloning using homologous recombination |
| EP1241177A1 (en) * | 2001-03-15 | 2002-09-18 | Akzo Nobel N.V. | Recombinant infectious laryngotracheitis virus vaccine |
| CN101220374A (en) * | 2007-01-11 | 2008-07-16 | 华南农业大学 | Fowl pox virus double-gene expression carrier (PG7.5N) |
| US20130210681A1 (en) * | 2010-06-10 | 2013-08-15 | Youming Zhang | Direct Cloning |
| CN102533674A (en) * | 2010-12-08 | 2012-07-04 | 中国农业科学院哈尔滨兽医研究所 | Recombinant duck virus enteritis virus vaccine strain (rDEVul41HA) for expressing avian influenza virus hemagglutinin (HA) genes and construction method as well as application thereof |
| US20140287938A1 (en) * | 2013-03-15 | 2014-09-25 | The Broad Institute, Inc. | Recombinant virus and preparations thereof |
| CN104250637A (en) * | 2014-08-29 | 2014-12-31 | 乾元浩生物股份有限公司 | Avian flu H9N2 subtype virus strain and application thereof |
| CN105695422A (en) * | 2016-03-15 | 2016-06-22 | 中国农业科学院哈尔滨兽医研究所 | Recombined chicken Marek's disease virus vaccine strain for expressing Gag and Env genes of avian leukosis virus subgroup J and construction method and application of recombined chicken Marek's disease virus vaccine strain |
| CN106047823A (en) * | 2016-07-01 | 2016-10-26 | 四川大学 | Bivalent vaccine strain for recombining and expressing NCV HN gene by virtue of IBV H120 strain based on reverse genetic technique |
| CN107296956A (en) * | 2017-06-29 | 2017-10-27 | 青岛易邦生物工程有限公司 | A kind of genetic recombination live vector vaccine |
| CN108324937A (en) * | 2018-01-17 | 2018-07-27 | 华中农业大学 | The herpes turkey virus live vector vaccine of 4 type penton protein of expression aviadenovirus serum and its preparation and application |
| CN108841866A (en) * | 2018-06-04 | 2018-11-20 | 云舟生物科技（广州）有限公司 | A kind of adenovirus vector and its construction method |
Non-Patent Citations (7)
| Title |
|---|
| DÖRTELÜSCHOW等: "Protection of chickens from lethal avian influenza A virus infection by live-virus vaccination with infectious laryngotracheitis virus recombinants expressing the hemagglutinin (H5) gene", 《VACCINE》 * |
| ERICA BICKERTON等: "Recombinant infectious bronchitis viruses expressing heterologous S1 subunits: potential for a new generation of vaccines that replicate in Vero cells", 《JOURNAL OF GENERAL VIROLOGY》 * |
| 兰德松等: "利用Rde/ET技术构建表达H5亚型禽流感病毒HA基因的重组火鸡疱疹病毒", 《微生物学报》 * |
| 李奇蒙 * |
| 李奇蒙: "表达IBDVvp2蛋白的rHVT的构建及其免疫保护评价", 《中国优秀硕士学位论文全文数据库（农业科技辑）》 * |
| 王海龙: "DNA直接克隆的优化和ccdB反向筛选在Red/ET重组工程中的应用", 《中国博士学位论文全文数据库（基础科学辑）》 * |
| 谢青梅等: "H9N2禽流感病毒HA全长基因的高效可溶表达及免疫原性研究", 《华南农业大学学报(自然科学版)》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110713989A (en) * | 2019-11-22 | 2020-01-21 | 华南农业大学 | Method for rapidly preparing epidemic infectious bronchitis vaccine |
| CN110713989B (en) * | 2019-11-22 | 2023-11-17 | 华南农业大学 | Method for rapidly preparing epidemic infectious bronchitis vaccine |
| CN114807061A (en) * | 2021-12-22 | 2022-07-29 | 华南农业大学 | Chicken infectious bronchitis marker vaccine strain, preparation method thereof and vaccine |
| CN114561367A (en) * | 2022-02-25 | 2022-05-31 | 华南农业大学 | Recombinant duck adenovirus type 3, preparation method and application thereof |
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