CN108342414A - A kind of expression African swine fever virus B646L gene recombinant adenovirus vectors, construction method and recombined adhenovirus preparation method - Google Patents

A kind of expression African swine fever virus B646L gene recombinant adenovirus vectors, construction method and recombined adhenovirus preparation method Download PDF

Info

Publication number
CN108342414A
CN108342414A CN201810263507.4A CN201810263507A CN108342414A CN 108342414 A CN108342414 A CN 108342414A CN 201810263507 A CN201810263507 A CN 201810263507A CN 108342414 A CN108342414 A CN 108342414A
Authority
CN
China
Prior art keywords
swine fever
african swine
fever virus
expression
cell
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201810263507.4A
Other languages
Chinese (zh)
Inventor
张泉
孙靖谕
钱淼
王涛
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Yangzhou University
Original Assignee
Yangzhou University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Yangzhou University filed Critical Yangzhou University
Priority to CN201810263507.4A priority Critical patent/CN108342414A/en
Publication of CN108342414A publication Critical patent/CN108342414A/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10041—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/12011—Asfarviridae
    • C12N2710/12022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Virology (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The present invention provides a kind of expression African swine fever virus B646L gene recombinant adenovirus vectors, construction method and recombined adhenovirus preparation methods, belong to gene engineering technology field.This method obtains recombined adhenovirus expression plasmid pAD B646L using adenovirus shuttle vector pKO FH, pAD EF1a GFP through a series of pilot process.The linearisation recombinant adenoviral vector plasmid transfection HEK293 cells that will be finally obtained；The cytopathy recombinant celo virus formed according to adenovirus infection, it is final to realize adenovirus packaging process, and the recombined adhenovirus for capableing of direct infection eukaryocyte is obtained by amplification, concentration and identification, to realize the purpose of B646L genes normal expression in eukaryocyte, lay the foundation to study the adenovirus carrier vaccine based on expression B646L.

Description

A kind of expression African swine fever virus B646L gene recombinant adenovirus vectors, construction method And recombined adhenovirus preparation method
Technical field
The invention belongs to gene engineering technology fields, and in particular to B646L gene overexpressions adenovirus vector and its structure Method.
Background technology
African swine fever (African Swine Fever, ASF) is by African swine fever virus (African Swine Fever Virus, ASFV) caused by pig a kind of acute, deadly infectious disease.It is clinically dead with high fever, general hemorrhage and height The rate of dying is characterized.The disease is huge to pig breeding industry harm, and A class epidemic diseases are classified as by OIE.Although China there is no the incoming of the disease, when Incoming risk is faced in facet.Therefore, deposit Journal of Sex Research is carried out to the disease, is of great significance for the strick precaution of the disease.
ASFV is positive 20 face body, diameter about 175nm~215nm.By peplos, viral capsid, inner investment, nucleocapsid, virus 5 component parts of nucleic acid.Viral nucleic acid is double-stranded DNA, and length is 170kb~190kb.More than 200 kinds of egg of viral gene codified White matter, wherein structural proteins have 54 kinds.P72 is a kind of major structural protein of ASFV, by B646L gene codes.It is viral clothing The chief component of shell results from viral later period of infection, be located at intracellular virus particle middle level or surface layer and content most More virus proteins accounts for the 32% of virus protein total amount.P72 is a kind of very conservative antigenic protein, is had to natural infection There is immunogenicity, diagnosis index and vaccine development can be used as.So far, prevent African swine fever there has been no any in the world The vaccine of virus infection lacks the research of the vaccine or other precautionary measures prevented epidemic to African swine fever in the prior art.
Invention content
In view of this, the purpose of the present invention is to provide a kind of expression African swine fever virus B646L gene recombinant adenovirus Carrier, construction method and recombined adhenovirus preparation method, to prepare the recombined adhenovirus for being overexpressed B646L genes, for studying ASFV candidate vaccines.
Adenovirus is one of current most efficiently reliable expression of recombinant virus system, be can be used for high in mammalian cell Flatly express express target protein.Because adenovirus infection does not depend on the cell cycle, therefore it can also both be felt with infection development cell Contaminate non-proliferative cell.While its host range that can be infected is extensive, can infect the almost all kinds of cell in addition to people, wraps Include the species such as rat, mouse, rabbit, pig, sheep, monkey, chicken.And it is high with virus titer, it can be used for animal experiment and have preferably Biological safety the features such as.Therefore, the structure and again of expression B646L gene recombinant adenovirus vectors is carried out using adenovirus The packaging of group adenovirus is of great significance to studying the candidate vaccine based on expression African swine fever p72 albumen.
In order to achieve the above-mentioned object of the invention, the present invention provides following technical scheme：
A kind of expression African swine fever virus B646L gene recombinant adenovirus vectors, including pAD-EF1 α-GFP adenovirus carry Body, the African swine fever virus B646L genes for being inserted into pAD-EF1 α-GFP adenovirus vector multiple cloning sites.The sequence of B646L genes Row are as shown in SEQ No.1.The multiple cloning sites are the restriction enzyme site of AsisI and MluI.
A kind of construction method of expression African swine fever virus B646L gene recombinant adenovirus vectors, includes the following steps：
1) B646L sequences are synthesized in gene chemical synthesis company, the sequence both ends enzyme enzyme site Asis I and MLu I；
2) gene chemical synthesis sequence B 646L and pKO-FH carrier is carried out respectively with restriction enzyme A sis I and MLu I double Digestion respectively obtains B646L genetic fragments and pKO linearized vector segments；By obtained B646L genetic fragments and pKO carriers Segment is attached, and obtains pKO-B646L；
3) pKO-B646L and pAD- that the step 2) is obtained respectively with restriction enzyme PI-SCE and I-CEU EF1 α-GFP adenovirus vectors carry out double digestion, respectively obtain linearisation B646L genetic fragments and adenovirus pAD carrier segments；
4) linearisation B646L genetic fragments and adenovirus pAD carrier segments that the step 3) obtains are attached, are obtained To pAD-B646L, it is sequenced using CMV-F and FH-R；
5) the plasmid pAD-B646L that the step 4) obtains is subjected to single endonuclease digestion with restriction enzyme Pac I, selects weight Group positive plasmid carries out subsequent experimental.
Preferably, endonuclease reaction system such as the following table 1 that double digestion reacts in the step 2)：
1 double digestion system of table
B646L genetic fragments or pKO-FH carriers 20uL
10×Buffer 3μL
AsisⅠ 1μL
MLuⅠ 1μL
ddH2O 5μL
It amounts to 30μL
The temperature of the double digestion is 37 DEG C, and the time of the double digestion is 1 hour.
Preferably, linked system such as the following table 2 in the step 2)：
2 linked system of table
Target gene B646L segments 2~6 μ L
PKO carrier segments 2~4 μ L
10×T4Buffer 1μL
T4DNA ligases (10U/ μ L) 1μL
It amounts to 10μL
The temperature of the connection is 22 DEG C, and the time of the connection is 2 hours.
Preferably, further include after connection in the step 2)：Recombinant plasmid is obtained to the connection and carries out positive bacterium colony Structure and identification.
Preferably, in the step 3) double digestion reaction system such as the following table 3：
3 double digestion reaction system of table
Digestion program such as the following table 4 of the double digestion：
4 double digestion response procedures of table
Preferably, linked system such as the following table 5 in the step 4)：
5 linked system of table
Target gene B646L segments 2~6 μ L
Adenovirus pAD carrier segments 2~4 μ L
10×T4Buffer 1μL
T4DNA ligases (10U/ μ L) 1μL
It amounts to 10μL
The temperature of the connection is 22 DEG C, and the time of the connection is 2 hours.
Preferably, in the step 5) single endonuclease digestion endonuclease reaction system such as the following table 6：
6 single endonuclease digestion reaction system of table
Recombinant plasmid pAD-B646L 20 μ L (about 2-3 μ g)
10×Buffer 5μL
ddH2O 24.5μL
PacⅠ 0.5μL
It amounts to 50μL
The single endonuclease digestion program is 37 DEG C, 3hr；95 DEG C, 5min.
The present invention provides the preparation methods of the recombined adhenovirus of expression African swine fever virus B646L genes, by following step Suddenly it is prepared：
A) the polyetherimide PEI of 8 μ L and 250 μ L DMEM culture mediums are configured to mixed liquor Mix1, stand 5 minutes with On.By abovementioned steps 5) in obtained recombination positive plasmid and 250 μ L DMEM culture mediums mixed liquor Mix2 is made.By mixed liquor Mix1 and Mix2 uniformly mixes and shakes mixing, centrifuges and stands 30 minutes.
B) cell number 0.3-0.5 × 10 are paved on cell plates6The HEK293 cells in a/hole.
C) cell plates are added dropwise in the mixed liquor after standing that step a) is obtained, cross mixing is placed on CO2Incubator Culture 2-3 days.
D) cell with CPE effects is collected, smudge cells are transferred in centrifuge tube, and supernatant cell is collected after centrifugation The recombined adhenovirus of the gene expression containing B646L is obtained by filtration in precipitation, supernatant.
The present invention provides the adenovirus amplifies containing B646L genes to detect with concentration and titre with receipts poison, purifying, is special Opposite sex detection.
Step 4) is specially：
The cell with CPE effects is collected, electricity consumption liquid-transfering gun moves in centrifuge tube, and carries out corresponding label, and centrifugation is abandoned Supernatant, precipitation obtain back dissolving liquid with A195 back dissolvings；First dry ice freezes 6-10min after EP pipes packing back dissolving liquid, then is placed in 37 DEG C, waits for Take out and shake up when pipe medium floe will melt, after multigelation 4 times, 12000g centrifuges 2min, takes supernatant spare, 4 DEG C preserve or- 80 DEG C of preservations；
The pretreatment of virus：
A) viral supernatants are handled：The supernatant preserved under the conditions of 4 DEG C is centrifuged, 3500g, 4 DEG C, centrifuges 30min；After centrifugation Supernatant is abandoned, viral pellet is collected；Viral pellet is resuspended with A195, is collected into pipe；
B) cell precipitation is handled：Cell precipitation is resuspended with A195, mixing, multigelation four times；Sample is placed in dry when freeze thawing Freeze 12-15 minutes in ice, is then transferred in 37 DEG C of water-baths and melts 2-3 minutes, repeatedly four times；After freeze thawing, 3500g 4 DEG C, centrifuges 30min, collects supernatant cell precipitation respectively；Supernatant is mixed with the viral pellet after being resuspended；Cell is broken 5mol/L NaCl to final concentration of 1moL/L are added after being resuspended with A195 in piece；
C) sonicated cells:Cell fragment ultrasonication after resuspension 3-4 times, AMPL values be 30%, 30s/ times, often Minor tick 20-30s, until liquid is not sticky；After ultrasound, 12000rpm, 4 DEG C of centrifugation 10min；After centrifugation, with viral supernatants It is mixed with sample after cell precipitation processing；
The Iodixanol of various concentration is successively added in centrifuge tube with electric pipettor；60% Iodixanol is first added Solution 4.2mL forms first layer, adds 40% Iodixanol solution 5mL and forms the second layer, 25% Iodixanol is secondly added Solution 6mL forms third layer, is eventually adding 15% Iodixanol solution 9mL and forms the 4th layer；
The virus liquid of pre-treatment step by virus is added to centrifuge tube top layer, 48000rpm is centrifuged 2 hours 30 Minute；Should be by corresponding centrifuge tube trim before centrifugation, control errors are within 0.1g；
Centrifugation finishes, and abandons preceding 5mL, collects 6-10mL liquid to 15mL centrifuge tubes and is diluted with the mixed liquor of PBS, PF68 To volume 15mL, then with 0.20 μm of membrane filtration, filtered liquid is placed in super filter tube, 3500g centrifuges 50min.
It is drawn in viral memotron after remaining liquid in super filter tube is blown and beaten repeatedly, is eventually adding 5 × A195 storing liquids To storing liquid end solubility be 1 ×；
5 × the A195 is the DMEM culture mediums containing 30% fetal calf serum, and PF68 is containing 10% glycerine, 10%DMSO DMEM, percentage are mass fraction；In the mixed liquor of PBS, PF68, PBS, PF68 volume ratio are 1：1, wherein PBS solution： NaCl 137mmol/L, KCl 2.7mmol/L, Na2HPO410mmol/L, KH2PO4 2mmol/L。
Compared with the existing technology, the present invention provides it is a kind of expression B646L recombined adhenovirus and its construction method, with Based on pAD-EF1a-GFP adenovirus expression carriers, ASFV B646L genes are introduced, which carries CMV strong promoters, energy The gene of its carrying is overexpressed in eukaryocyte, while the carrier can be used for screening positive cell with green fluorescent protein, Through screening, the adenovirus for capableing of direct infection eukaryocyte is finally obtained, to realize B646L normal tables in eukaryocyte The purpose reached is laid a solid foundation for further research based on expression B646L recombinant adenoviral vector vaccines.
Description of the drawings
Fig. 1 is the PacI linearization for enzyme restriction figures of recombinant adenoviral vector pAD-B646L in embodiment 5,1% Ago-Gel Electrophoresis detection, two band of electrophoresis showed, one is 3kb or 5kb or so, 30kb or so, according to the difference of recombination mechanism, Stripe size is different after digestion；
Fig. 2 is the luciferase expression figure of plasmid transfection cell after a week in the embodiment of the present invention 5；
Fig. 3 is that cell CPE schemes plasmid transfection after a week in the embodiment of the present invention 5；
Fig. 4 is the pKO-FH carrier structure figures of the present invention；
Fig. 5 is the pAD-EF1 α-GFP carrier structure figures of the present invention.
Specific implementation mode
The present invention is not particularly limited the method for the mixing, and the culture scheme of use is not particularly limited.
In the present invention, the method for the filtering preferably uses micro-filtration.The aperture of the micro-filtration is preferably 0.2 μm.
In the present invention, the method for the centrifugation is not particularly limited, and is using cell centrifugation protocol known in the art It can.
In the present invention, the type of the cell line is preferably HEK293 cells.
The present invention provides a kind of African swine fever virus B646L gene recombinant adenovirus and its construction methods, with pAD- Based on EF1a-GFP adenovirus expression carriers, B646L genes are introduced；The B646L genes have such as Seq ID in sequence table Nucleotide sequence shown in No.1.
Embodiment 1
African swine fever virus B646L gene Gene ID:KJ195685.1, African swine fever virus B646L genes, by Shanghai Sheng Gong Co., Ltds synthesize and are added restriction enzyme site XhoI, AsisI, and the B646L genes of synthesis are connected in carrier T, obtain band There is the carrier T of B646L segments.
Embodiment 2
With restriction enzyme A sis I and MLu I, to gene chemical synthesis sequence B 646L, (T for carrying B646L segments is carried respectively Body) and adenovirus shuttle plasmid carrier pKO-FH (Fig. 4) progress double digestions, respectively obtain linearisation B646L genetic fragments and pKO Carrier segments.Endonuclease reaction system such as the following table 1 of double digestion reaction：
1 double digestion system of table
B646L genetic fragments or pKO-FH carriers 20uL
10×Buffer 3μL
AsisⅠ 1μL
MLuⅠ 1μL
ddH2O 5μL
It amounts to 30μL
The temperature of the double digestion is 37 DEG C, and the time of the double digestion is 1 hour.
Digestion purpose band size is detected with 1% agarose gel electrophoresis after reaction, blend compounds QIAquick Gel Extraction Kit is returned Receive target fragment.Carrier pKO-FH does same digestion, and glue recycles carrier sequence.
Embodiment 3
B646L genetic fragments will be linearized with T4 ligases and pKO carrier segments are attached, and obtain pKO-B646L.Even Junctor system such as the following table 2：
2 linked system of table
Target gene B646L segments 2~6 μ L
PKO carrier segments 2~4 μ L
10×T4Buffer 1μL
T4DNA ligases (10U/ μ L) 1μL
It amounts to 10μL
Micro- centrifugation after mixing, 22 DEG C of connection 2h.
Connection product is converted into bacillus coli DH 5 alpha competent cell.The LB tablets containing kalamycin resistance are coated on to carry out Screening, picking single bacterium colony extract plasmid after expanding culture, and Asis I and I double digestions of MLu identify positive colony, and are sequenced and test Card obtains correct pKO-B646L clones, uses CMV-F (SEQ No.2:5’CAATGGGAGTTTGTTTTGGCACCA-3’) With FH-R (SEQ No.3:5 ' CTTATTAGTGGTGGTGGTGGTGGTGCTCG-3 ') it is sequenced.
Conversion process is as follows：
(1) the DH5a competence prepared in advance is taken out from -80 DEG C to be placed in ice bath.
(2) after the thawing of DH5a competent cells, take 1 μ L connection products in 20 μ L DH5a competent cells, it is fully mixed It is even, stand 30 minutes in ice bath.
(3) centrifuge tube is put into 42 DEG C of water-baths 40 seconds (period not shake centrifuge tube), is then quickly moved to ice bath In, stand 2 minutes.
(4) the sterile LB culture mediums (not added with antibiotic) of 200 μ L are added into centrifuge tube, mixing is placed on 37 in shaking table DEG C, 200rpm shakes 1 hour.It is applied in the solid medium plate of kalamycin resistance.
Overnight incubation in (5) 37 DEG C of incubators.
Embodiment 4
With restriction enzyme PI-SCE and I-CEU respectively to connection product pKO-B646L and adenovirus pAD-EF1 α- GFP carriers (Fig. 5) double digestion linearizes, and glue recycles respective segments.Ligase connects B646L genetic fragments and pAD carrier segments, By connection product conversion Escherichia coli amplification, plasmid is then extracted, pAD-B646L is obtained after identifying correctly.The reaction of double digestion System such as the following table 3：
3 double digestion reaction system of table
Digestion program such as the following table 4 of the double digestion：
4 double digestion response procedures of table
When ligase connects B646L genetic fragments and pAD carrier segments, linked system is as shown in table 5：
5 linked system of table
Target gene B646L segments 2~6 μ L
Adenovirus pAD carrier segments 2~4 μ L
10×T4Buffer 1μL
T4DNA ligases (10U/ μ L) 1μL
It amounts to 10μL
After the plasmid (pAD-B646L) of extraction I single endonuclease digestions of Pac (linearized enzyme digestion), recombinant plasmid and PEI are linearized Mixing centrifuges, and stands, and DMEM culture mediums are added.Single endonuclease digestion reaction system such as table 6.
6 single endonuclease digestion reaction system of table
Recombinant plasmid pAD-B646L 20 μ L (about 2-3 μ g)
10×Buffer 5μL
ddH2O 24.5μL
PacⅠ 0.5μL
It amounts to 50μL
Single endonuclease digestion reaction condition：37 DEG C, 3hr；95 DEG C, 5min, 50 μ l systems.
By recombinant adenovirus plasmid carrier mixed liquor transfected HEK 293, virus packaging is completed to get to containing B646L Gene recombinant adenovirus (Fig. 2 and Fig. 3).By Fig. 2 and Fig. 3 it is found that recombinant virus is packed successfully.Transfection process is as follows：
1. 250 μ l DMEM and 8 μ l PEI are made into Mix1,5min or more is stood.
2. the recombinant plasmid after 250 μ l DMEM and single endonuclease digestion is made into Mix2.
3. Mix1 and Mix2 is mixed, mixing is shaken, 30min is stood after centrifugation.
4. spreading 6 orifice plates HEK293 cells, cell number about 0.3-0.5 × 10 during standing6A/hole.
5. the mixed liquor after standing is added dropwise in the cell of 6 orifice plates.Cross mixing is placed on CO2It is trained in incubator It supports.
In above technical scheme, the stripe size recycled after the pKO-B646L plasmids double digestion is purpose gene B646L gene order sizes add 1.2kb.
In above technical scheme, the ligase is T4DNA ligases, and the Escherichia coli are DH5 α Escherichia coli；
In above technical scheme, the HEK293 cells are loaded in 10cm cell dish, and cross, which shakes up, is placed on CO2Culture Case culture 2-3 days.
Embodiment 5
There is situation in the CPE of observation Transfected cells, and the cell for CPE occur is expanded, and receives poison.Steps are as follows：
1) cell for having CPE in six orifice plates is blown down together with culture medium and is added in 10cm disks, shaken up and be placed on CO2Incubator Poison is received in middle culture after 2-3 days.
2) the 10cm disk cells with CPE effects are collected, are moved in 15ml centrifuge tubes with electronic liquid-transfering gun, and carry out phase The label answered；3500rpm centrifuges 7min；
3) after the completion of centrifuging, centrifuge tube is taken out, supernatant is outwelled and (virus of large amplification is needed to need supernatant pouring into one Spare in new 15ml pipes, short time preservation can be placed in 4 DEG C of refrigerators, preserve need to be placed in -80 DEG C of refrigerators for a long time), Nubbin is blotted only with 200 μ l rifles.Then the precipitation 1 ╳ A195 back dissolvings of 1ml；
4) precipitation is constantly blown and beaten with A195, ensures complete back dissolving, is then drawn onto respectively in 1.5ml EP pipes, and carry out phase It should mark；
5) ready EP pipes are put into dry ice and freeze 6-10min, be then placed in dry-type thermostat, setting temperature is 37.0 DEG C, until taking-up overturns mixing with hand when remaining some ice cubes in pipe, freeze thawing four times repeatedly；
6) after the completion of freeze thawing, EP pipes 12000g centrifuges 2min；
7) supernatant is moved on to 1ml rifles in pipe, is then deposited in -80 DEG C of refrigerator.
Embodiment 6
Collected viral supernatants are subjected to large amplification and receive poison, steps are as follows：
1) collected viral supernatants are averagely added drop-wise in 10 10cm disks, mixing is placed on CO2It is trained in incubator It supports, poison is received after 2-3 days.
2) 10 10cm disk cells with CPE effects are collected, are moved to respectively in 50ml centrifuge tubes with electronic liquid-transfering gun, and Carry out corresponding label；
3) 3500rpm centrifuges 7min, collects supernatant cell precipitation respectively；
4) it is shaken up after NaCl and PEG8000 being added in viral supernatants, shakes up once, shaken altogether three times per 30min, 4 DEG C upright It stands overnight.Cell precipitation is placed in -80 DEG C of preservations, and subsequent experimental is used.
Embodiment 7
By the processing before purification of viral supernatants and cell precipitation.
1. viral supernatants are handled
(1) 4 DEG C of overnight supernatants are centrifuged, 3500g, 4 DEG C, centrifuges 30min；
(2) supernatant is discarded after centrifuging, and collects viral pellet；
(3) viral pellet is resuspended with 2ml A195, is collected into 15ml pipes.
2. cell precipitation is handled
(1) cell precipitation is resuspended with 6ml A195, mixing, multigelation four times.Sample is placed in dry ice when freeze thawing and is frozen It 12-15 minutes, then takes and melts in 37 DEG C of water-baths 2-3 minutes, repeatedly four times；
(2) after freeze thawing, 3500g 4 DEG C, centrifuges 30min, collects supernatant precipitation (cell fragment) respectively；
(3) supernatant is mixed with the viral pellet after being resuspended；0.5ml 5mol/ are added after being resuspended with 2ml A195 in cell fragment L NaCl to final concentration of 1mol/L.
3. sonicated cells
(1) cell fragment ultrasonication 3-4 time (AMPL values are 30%, 30s/ times, often minor tick 20-30s) after being resuspended, It is not sticky to liquid；
(2) after ultrasound, by liquid subpackage to 2 EP pipes, 12000rpm, 4 DEG C centrifuge 10min；
(3) after having centrifuged, sample and the viral supernatants (production that viral supernatants processing obtains that sonicated cells step obtains The supernatant obtained in object, cell precipitation processing step) and cell precipitation treated sample (cell precipitation processing step obtains Product after cell fragment is resuspended) mixing.
Embodiment 8
Viral purification and concentration, are as follows：
Purifying：
1) Iodixanol of various concentration is successively added in 50mL centrifuge tubes with electric pipettor.60% iodine gram is first added Husky alcohol layer 4.2mL adds 40% Iodixanol layer 5mL, and 25% Iodixanol layer 6mL is secondly added, is eventually adding 15% iodine Gram husky alcohol layer 9mL.A concentration of mass concentration of Iodixanol solution, solvent be 150mmol/L KCl, 30mmol/L MgCl2, The KOH solution of 120mmol/L trimethylglycines, pH7.8.
2) virus liquid handled well is added to top layer, 48000rpm is centrifuged 30 minutes 2 hours.It should will be right before centrifugation The centrifuge tube trim answered, control errors are within 0.1g.
Concentration：
(1) after centrifuging, super filter tube bottom is punctured with syringe needle, discards preceding 5ml, collect 6ml to 10ml solution extremely In 15ml pipes.
(2) the 5ml liquid of collection is diluted to volume 15ml with PBS+PF68, then with 0.20 μm of membrane filtration.
(3) filtered liquid is placed in the super filter tube of a 15ml, 3500g centrifuges 50min.If remained in super filter tube Under liquid volume be less than volume of ideal, need to discard the liquid that down of centrifugation, it is dilute that PBS+PF68 be added into super filter tube It releases, centrifuges again.Time length can be depending on the sliminess of solution.
(4) it is drawn in viral memotron after blowing and beating remaining liquid in super filter tube repeatedly, is eventually adding 5 × A195 storages Liquid to storing liquid end solubility be 1 ×, indicate title and date.
(5) collected virus is vortexed after concussion mixing and is centrifuged, inhaled 10 μ l virus liquids and carry out titre detection.
5 × the A195 is the DMEM culture mediums containing 30% fetal calf serum, and PF68 is containing 10% glycerine, 10%DMSO DMEM, percentage are mass fraction；In the mixed liquor of PBS, PF68, PBS, PF68 volume ratio are 1：1, wherein PBS solution： NaCl 137mmol/L, KCl 2.7mmol/L, Na2HPO410mmol/L, KH2PO42mmol/L.A195, PF68 of selection Formula so that small to viral damage.
Embodiment 9
Titre detection is carried out to the virus prepared, is as follows：
1) virus coat is removed, the following table of each compositional system 7：
7 titre of table detects constituent
Constituent Volume
Virus liquid 5μL
Proteinase K (5 μ g/ μ L) 1μL
Ultra-pure water 4μL
37 DEG C of incubation 30min, then maintain 5min that enzyme is made to inactivate for 95 DEG C.
2) 12,000rpm centrifuges 2min, takes supernatant.
3) 7 gradient dilution concentration are arranged is respectively：2.58╳1012Vp/ μ l, 2.58 ╳ 1011Vp/ μ l, 2.58 ╳ 1010Vp/ μ l, 2.58 ╳ 109Vp/ μ l, 2.58 ╳ 108Vp/ μ l, 2.58 ╳ 107Vp/ μ l, and using ultra-pure water as negative control.
4) PCR is detected, in total 20 μ L systems, specific reaction system such as the following table 8：
8 titre of table detects specific reaction system
The following 95 DEG C of 5min of PCR in the step 4)；95℃ 15sec；60℃ 15sec； 72℃ 15sec；40 are followed Ring.
5) virion number (a/mL)=and standard items relative value × 1000.Virion number
Testing result is 3.1 ╳ 109pfu/μl。
Embodiment 10
Virus-specific detects, and is as follows：
1) virus coat is removed, the following table of each compositional system 9：
9 specific detection constituent of table
Constituent Volume
Virus liquid 5μL
Proteinase K (5 μ g/ μ L) 1μL
Ultra-pure water 4μL
37 DEG C of incubation 30min, then maintain 5min that enzyme is made to inactivate for 95 DEG C.
2) 12,000rpm centrifuges 2min, takes supernatant.
3) PCR reacts, in total 20 μ L systems, specific reaction system such as the following table 10：F, R primers have specific primer respectively With universal primer, the two PCR system is the same；
10 PCR reaction systems of table
PCR response procedures are as follows in the step 3)：
95℃ 4min；95℃ 30sec；64℃ 45sec；72℃ 1min 1cycles
95℃ 30sec；62℃ 45sec；72℃ 1min 2cycles
95℃ 30sec；60℃ 45sec；72℃ 1min 3cycles
95℃ 30sec；58℃ 45sec；72℃ 1min 26cycle
72℃ 10min；4 DEG C unlimited.
4) PCR product runs glue, glue recycling sequencing.PCR product runs glue, sweeps glue, compares stripe size, if specific primer PCR product band unobvious, dilute the PCR product of universal primer：Add 3 times of sequencings of ultra-pure water, aligned sequences.By PCR product with B646L sequences carry out sequence alignment, and result is homology 100%.
The above display describes the basic principles and main features and advantage of the present invention.The technical staff of the industry should Understand, the present invention is not limited to the above embodiments, what is described in the above embodiment and the description is only saying the principle of the present invention, Without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, these changes and improvements are all It drops into the claimed scope of the invention.The scope of the present invention is defined by the appended claims and its equivalents.
Sequence table
<110>Yangzhou University
<120>A kind of expression African swine fever virus B646L gene recombinant adenovirus vectors, construction method and recombined adhenovirus system Preparation Method
<130> xhx2018032701
<141> 2018-03-27
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1938
<212> DNA
<213> African swine fever virus
<400> 1
atggcatcag gaggagcttt ttgtcttatt gctaacgatg ggaaggccga caagattata 60
ttggcccaag acttgctgaa tagcaggatc tctaacatta aaaatgtgaa caaaagttat 120
gggaaacccg atcccgaacc cactttgagt caaatcgaag aaacacattt ggtgcatttt 180
aatgcgcatt ttaagcctta tgttccagta gggtttgaat acaataaagt acgcccgcat 240
acgggtaccc ccaccttggg aaacaagctt acctttggta ttccccagta cggagacttt 300
ttccatgata tggtgggcca tcatatattg ggtgcatgtc attcatcctg gcaggatgct 360
ccgattcagg gcacgtccca gatgggggcc catgggcagc ttcaaacgtt tcctcgcaac 420
ggatatgact gggacaacca aacaccctta gagggcgccg tttacacgct tgtagatcct 480
tttggaagac ccattgtacc cggcacaaag aatgcgtacc gaaacttggt ttactactgc 540
gaataccccg gagaacgact ttatgaaaac gtaagattcg atgtaaatgg aaattcccta 600
gacgaatata gttcggatgt cacaacgctt gtgcgcaaat tttgcatccc aggggataaa 660
atgactggat ataagcactt ggttggccag gaggtatcgg tggagggaac cagtggccct 720
ctcctatgca acattcatga tttgcacaag ccgcaccaaa gcaaacctat tcttaccgat 780
gaaaatgata cgcagcgaac gtgtagccat accaacccga aatttctttc acagcatttt 840
cccgagaact ctcacaatat ccaaacagca ggtaaacaag atattactcc tatcacggac 900
gcaacgtatc tggacataag acgtaatgtt cattacagct gtaatggacc tcaaacccct 960
aaatactatc agccccctct tgcgctctgg attaagttgc gcttttggtt taatgagaac 1020
gtgaaccttg ctattccctc agtatccatt cccttcggcg agcgctttat caccataaag 1080
cttgcatcgc aaaaggattt ggtgaatgaa tttcctggac tttttgtacg ccagtcacgt 1140
tttatagctg gacgccccag tagacgcaat atacgcttta aaccatggtt tatcccagga 1200
gtcattaatg aaatctcgct cacgaataat gaactttaca tcaataacct gtttgtaacc 1260
cctgaaatac acaacctttt tgtaaaacgc gttcgctttt cgctgatacg tgtccataaa 1320
acgcaggtga cccacaccaa caataaccac cacgatgaaa aactaatgtc tgctcttaaa 1380
tggcccattg aatatatgtt tataggatta aaacctacct ggaacatctc cgatcaaaat 1440
cctcatcaac accgagattg gcacaagttc ggacatgttg ttaacgccat tatgcagccc 1500
actcaccacg cagagataag ctttcaggat agagatacag ctcttccaga cgcatgttca 1560
tctatatctg atattagccc cgttacgtat ccgatcacat tacctattat taaaaacatt 1620
tccgtaactg ctcatggtat caatcttatc gataaatttc catcaaagtt ctgcagctct 1680
tacataccct tccactacgg aggcaatgcg attaaaaccc ccgatgatcc gggtgcgatg 1740
atgattacct ttgctttgaa gccacgggag gaataccaac ccagtggtca tattaacgta 1800
tccagagcaa gagaatttta tattagttgg gacacggatt acgtggggtc tatcactacg 1860
gctgatcttg tggtatcggc atctgctatt aactttcttc ttcttcagaa cggttcagct 1920
gtgctgcgtt acagtacc 1938
<210> 2
<211> 24
<212> DNA
<213> African swine fever virus
<400> 2
caatgggagt ttgttttggc acca 24
<210> 3
<211> 29
<212> DNA
<213> African swine fever virus
<400> 3
cttattagtg gtggtggtgg tggtgctcg 29

Claims (10)

1. a kind of expression African swine fever virus B646L gene recombinant adenovirus vectors, which is characterized in that including pAD-EF1 α-GFP Adenovirus vector, the African swine fever virus B646L genes for being inserted into pAD-EF1 α-GFP adenovirus vector multiple cloning sites；It is described B646L genes have nucleotide sequence shown in Seq ID No.1.
2. a kind of expression African swine fever virus B646L gene recombinant adenovirus vectors according to claim 1, feature exist In the multiple cloning sites are the restriction enzyme site of AsisI and MluI.
3. a kind of construction method of expression African swine fever virus B646L gene recombinant adenovirus vectors described in claim 1, It is characterized in that, includes the following steps：
1) artificial synthesized African swine fever virus B646L gene orders, the gene order both ends enzyme enzyme site Asis I and MLu I；
2) double digestion is carried out to gene order B646L and pKO-FH carrier respectively with restriction enzyme A sis I and MLu I, respectively Obtain linearisation B646L genetic fragments and pKO-FH carrier segments；Obtained linearisation B646L genetic fragments and pKO-FH are carried Body segment is attached, and obtains pKO-B646L；
3) pKO-B646L the and pAD-EF1 α-that the step 2) is obtained respectively with restriction enzyme PI-SCE and I-CEU GFP adenovirus vectors carry out double digestion, respectively obtain linearisation B646L genetic fragments and pAD-EF1 α-GFP carrier segments；
4) linearisation B646L genetic fragments and pAD-EF1 α-GFP carrier segments that the step 3) obtains are attached, are obtained To pAD-B646L, sequencing；
5) the plasmid pAD-B646L that the step 4) obtains is subjected to single endonuclease digestion with restriction enzyme Pac I, selects recombination sun Property grain is used for subsequent experimental.
4. a kind of structure side of expression African swine fever virus B646L gene recombinant adenovirus vectors according to claim 3 Method, which is characterized in that further include following steps after being connected in the step 2)：Sun is carried out to the obtained recombinant plasmid that connects The structure of property bacterium colony and identification.
5. a kind of structure side of expression African swine fever virus B646L gene recombinant adenovirus vectors according to claim 3 Method, which is characterized in that the endonuclease reaction system that double digestion reacts in the step 2) is as follows：
The temperature of the double digestion reaction is 37 DEG C, and the time of the double digestion reaction is 1 hour；
Linked system is as follows in the step 2)：
The temperature of connection reaction is 22 DEG C, and the time for connecting reaction is 2 hours.
6. a kind of structure side of expression African swine fever virus B646L gene recombinant adenovirus vectors according to claim 3 Method, which is characterized in that the reaction system that double digestion reacts in the step 3) is as follows：
The digestion program of the double digestion reaction is as follows：
37 DEG C 2.5 hours
65 DEG C 20 minutes.
7. a kind of structure side of expression African swine fever virus B646L gene recombinant adenovirus vectors according to claim 3 Method, which is characterized in that linked system is as follows in the step 4)：
The temperature of the connection is 22 DEG C, and the time of the connection is 2 hours.
8. a kind of structure side of expression African swine fever virus B646L gene recombinant adenovirus vectors according to claim 3 Method, which is characterized in that the endonuclease reaction system of single endonuclease digestion is as follows in the step 5)：
Single endonuclease digestion response procedures are as follows：
37 DEG C 3 hours
95 DEG C 5 minutes.
9. a kind of preparation method of expression African swine fever virus B646L gene recombinant adenovirus, which is characterized in that by following steps It is prepared：
A) the polyetherimide PEI of 8 μ L and 250 μ L DMEM culture mediums are configured to mixed liquor Mix1, stand 5 minutes or more；It will Mixed liquor Mix2 is made in recombination positive plasmid and 250 μ L DMEM culture mediums described in claim 2；By mixed liquor Mix1 and Mix2 uniformly mixes and shakes mixing, centrifuges and stands 30 minutes；
B) cell number 0.3-0.5 × 10 are paved on cell plates6The HEK293 cells in a/hole；
C) cell plates are added dropwise in the mixed liquor after standing that step a) is obtained, cross mixing is placed on CO2It is cultivated in incubator 2-3 days；
D) cell with CPE effects is collected, smudge cells are transferred in centrifuge tube, and supernatant cell precipitation is collected after centrifugation, The recombined adhenovirus of the gene expression containing B646L is obtained by filtration in supernatant.
10. a kind of preparation method of expression African swine fever virus B646L gene recombinant adenovirus according to claim 9, It is characterized in that, step 4) is specially：
The cell with CPE effects is collected, electricity consumption liquid-transfering gun moves in centrifuge tube, and carries out corresponding label, and supernatant is abandoned in centrifugation, Precipitation 1*A195 back dissolvings；First dry ice freezes 6-10min after EP pipes packing back dissolving liquid, then is placed in 37 DEG C, waits for that pipe medium floe will It takes out and shakes up when thawing, after multigelation 4 times, 12000g centrifuges 2min, takes supernatant spare, and 4 DEG C preserve or -80 DEG C of preservations；
The pretreatment of virus：
A) viral supernatants are handled：The supernatant preserved under the conditions of 4 DEG C is centrifuged, 3500g, 4 DEG C, centrifuges 30min；It is abandoned after centrifugation Clearly, viral pellet is collected；Viral pellet is resuspended with A195, is collected into pipe；
B) cell precipitation is handled：Cell precipitation is resuspended with A195, mixing, multigelation four times；Sample is placed in dry ice when freeze thawing Freeze 12-15 minutes, is then transferred in 37 DEG C of water-baths and melts 2-3 minutes, repeatedly four times；After freeze thawing, 3500g, 4 DEG C, 30min is centrifuged, collects supernatant cell precipitation respectively；Supernatant is mixed with the viral pellet after being resuspended；Cell fragment A195 5mol/L NaCl to final concentration of 1moL/L are added after resuspension；
C) sonicated cells:Cell fragment ultrasonication after resuspension 3-4 times, AMPL values be 30%, 30s/ time, often minor tick 20-30s, until liquid is not sticky；After ultrasound, 12000rpm, 4 DEG C of centrifugation 10min；After centrifugation, with viral supernatants and cell Sample mixes after precipitation process；
The Iodixanol of various concentration is successively added in centrifuge tube with electric pipettor；60% Iodixanol solution is first added 4.2mL forms first layer, adds 40% Iodixanol solution 5mL and forms the second layer, 25% Iodixanol solution is secondly added 6mL forms third layer, is eventually adding 15% Iodixanol solution 9mL and forms the 4th layer；
The virus liquid of pre-treatment step by virus is added to centrifuge tube top layer, 48000rpm is centrifuged 30 minutes 2 hours； Should be by corresponding centrifuge tube trim before centrifugation, control errors are within 0.1g；
Centrifugation finishes, and abandons preceding 5mL, collects 6-10mL liquid and is diluted to volume to 15mL centrifuge tubes and with the mixed liquor of PBS, PF68 Filtered liquid is placed in super filter tube by 15mL then with 0.20 μm of membrane filtration, and 3500g centrifuges 50min；
It is drawn in viral memotron after remaining liquid in super filter tube is blown and beaten repeatedly, is eventually adding 5 × A195 storing liquids to storage Liquid storage end solubility be 1 ×；
5 × the A195 is the DMEM culture mediums containing 30% fetal calf serum, and PF68 is containing 10% glycerine, 10%DMSO DMEM, percentage are mass fraction；In the mixed liquor of PBS, PF68, PBS, PF68 volume ratio are 1：1, wherein PBS solution：NaCl 137mmol/L, KCl 2.7mmol/L, Na2HPO410mmol/L, KH2PO4 2mmol/L。
CN201810263507.4A 2018-03-28 2018-03-28 A kind of expression African swine fever virus B646L gene recombinant adenovirus vectors, construction method and recombined adhenovirus preparation method Pending CN108342414A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201810263507.4A CN108342414A (en) 2018-03-28 2018-03-28 A kind of expression African swine fever virus B646L gene recombinant adenovirus vectors, construction method and recombined adhenovirus preparation method

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201810263507.4A CN108342414A (en) 2018-03-28 2018-03-28 A kind of expression African swine fever virus B646L gene recombinant adenovirus vectors, construction method and recombined adhenovirus preparation method

Publications (1)

Publication Number Publication Date
CN108342414A true CN108342414A (en) 2018-07-31

Family

ID=62957450

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201810263507.4A Pending CN108342414A (en) 2018-03-28 2018-03-28 A kind of expression African swine fever virus B646L gene recombinant adenovirus vectors, construction method and recombined adhenovirus preparation method

Country Status (1)

Country Link
CN (1) CN108342414A (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113150079A (en) * 2021-01-21 2021-07-23 中国检验检疫科学研究院 Eukaryotic expression African swine fever virus p72 antigen and application thereof
CN114107389A (en) * 2021-11-26 2022-03-01 中国农业科学院北京畜牧兽医研究所 Recombinant adenovirus expressing African swine fever virus B602L-B646L protein and construction method thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101870987A (en) * 2010-06-25 2010-10-27 西北农林科技大学 Construction method for goat SCD gene recombinant adenovirus vector
CN104962581A (en) * 2015-04-07 2015-10-07 中国农业科学院兰州兽医研究所 Recombined new castle disease virus vaccine strain for expressing African swine fever virus p72 proteins
CN107475298A (en) * 2017-09-11 2017-12-15 扬州大学 CdtB gene overexpressions slow virus carrier and its construction method and the slow virus comprising cdtB genes and its application

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101870987A (en) * 2010-06-25 2010-10-27 西北农林科技大学 Construction method for goat SCD gene recombinant adenovirus vector
CN104962581A (en) * 2015-04-07 2015-10-07 中国农业科学院兰州兽医研究所 Recombined new castle disease virus vaccine strain for expressing African swine fever virus p72 proteins
CN107475298A (en) * 2017-09-11 2017-12-15 扬州大学 CdtB gene overexpressions slow virus carrier and its construction method and the slow virus comprising cdtB genes and its application

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
SHEHNAZ LOKHANDWALA等: "Induction of Robust Immune Responses in Swine by Using a Cocktail of Adenovirus-Vectored African Swine Fever Virus Antigens", 《CLINICAL AND VACCINE IMMUNOLOGY》 *
VLASOVA,N. N.等: "African swine fever virus p72(B646L) gene, complete cds", 《GENBANK》 *
维真生物: "腺病毒技术手册", 《维真生物官方网站》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113150079A (en) * 2021-01-21 2021-07-23 中国检验检疫科学研究院 Eukaryotic expression African swine fever virus p72 antigen and application thereof
CN114107389A (en) * 2021-11-26 2022-03-01 中国农业科学院北京畜牧兽医研究所 Recombinant adenovirus expressing African swine fever virus B602L-B646L protein and construction method thereof

Similar Documents

Publication Publication Date Title
CN108504686A (en) A kind of expression African swine fever virus EP153R gene recombinant adenovirus vectors, construction method and recombined adhenovirus preparation method
CN108441514A (en) A kind of expression African swine fever virus CP204L gene recombinant adenovirus vectors, construction method and recombined adhenovirus preparation method
CN111218459A (en) Recombinant novel coronavirus vaccine taking human replication-defective adenovirus as vector
CN108504687A (en) A kind of expression African swine fever virus EP402R gene recombinant adenovirus vectors, construction method and recombined adhenovirus preparation method
CN104846013B (en) A kind of 55 type adenovirus vector of replication deficient human and its preparation method and application
CN108342362A (en) A kind of stable cell lines MDCK and its construction method for expanding recombination hepatitis infectiosa canis virus CAV2
CN107012261A (en) Ana 1 aviadenovirus A types and the dual EvaGreen real-time fluorescence quantitative PCRs detection primer of 2 types
CN106967748B (en) Goat pox virus recombination system without plaque cloning and screening label and construction of double-expression PPRV H/F protein vaccine
CN112458064A (en) Gatasavir full-length infectious clone, replicon system, preparation and application thereof
CN104152416A (en) Pseudorabies virus gene-deleted attenuated strain as well as preparation method and application thereof
CN108342414A (en) A kind of expression African swine fever virus B646L gene recombinant adenovirus vectors, construction method and recombined adhenovirus preparation method
CN114164184A (en) Newcastle disease virus gene VI type vaccine strain and application thereof
CN108531511A (en) A kind of expression African swine fever virus E183L gene recombinant adenovirus vectors, construction method and recombined adhenovirus preparation method
CN113564133A (en) Coxsackie virus A16 type strain and immunogenic composition and application thereof
CN105713866A (en) Human cytomegalovirus (HCMV) infectious clone as well as construction method and applications of HCMV infectious clone
Chen et al. Construction of an infectious clone of human adenovirus type 41
CN106929487A (en) A kind of foot and mouth disease virus Hybrid virus like particles and preparation method thereof
CN113248577B (en) Coronavirus vaccine using adenovirus as carrier and its preparing method
CN114214338B (en) Porcine PIV5 full-length infectious clone and preparation method and application thereof
CN107663523A (en) A kind of chicken alpha interferon gene Eukaryotic expression recombinant plasmid of optimization and preparation method and application
CN105274089B (en) A kind of construction method for the REV viral infectivities clone for expressing green fluorescence cyst membrane fusion protein
CN108130340A (en) Express the application of the method and this method of duck source avian influenza virus NS1 albumen
CN114457042A (en) Peste des petits ruminants virus virulent reverse genetic system and animal infection model
CN113416713A (en) Construction and application of recombinant adenovirus
CN105749272B (en) Vaccine for expressing panda canine distemper virus H, F gene recombinant goat pox virus, preparation method and immune application method thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication
RJ01 Rejection of invention patent application after publication

Application publication date: 20180731