CN108504686A - A kind of expression African swine fever virus EP153R gene recombinant adenovirus vectors, construction method and recombined adhenovirus preparation method - Google Patents
A kind of expression African swine fever virus EP153R gene recombinant adenovirus vectors, construction method and recombined adhenovirus preparation method Download PDFInfo
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Abstract
The present invention provides a kind of expression African swine fever virus EP153R 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 EP153R 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 EP153R genes normal expression in eukaryocyte, lay the foundation to study the adenovirus carrier vaccine based on expression EP153R.
Description
Technical field
The invention belongs to gene engineering technology fields, and in particular to EP153R 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 bleeding heat, high degree in contact communicable disease, clinical symptoms and pathology
Variation shows as high fever, dermohemia, oedema and internal organs extensive bleeding and the change of respiratory system and nervous function,
Lethality is up to 100%.In June, 2007, ASF is confirmed in Georgia's Caucasus region, and has spread to neighbouring country.From
Since 2009, this disease travels to the Russian some areas bordered on China through states such as Georgia and repeatedly breaks out, at present
Grave danger is constituted to the sustainable development of China's pig breeding industry.World Organization for Animal Health (OIE), which is classified as, must report class animal
Epidemic disease, the disease are paid much attention to by countries in the world, this disease is defined as a kind of Animal diseases by China.
ASFV is the only known representative species of African swine fever virus section, is a kind of double-stranded DNA virus that is big, having cyst membrane；
The unimolecule threadiness double-stranded DNA that its genome is closed for terminal covalent, size 170-190kb, whole gene group contain 150
Open reading frame can encode 150-200 kind albumen, and to identify 86 kinds of virus proteins more for separation from ASFV infection cells
Peptide.
The P30 albumen of ASFV is a kind of critically important structural proteins, by EP153R gene codes.Research finds that P30 can
Inducing host cell generates the neutralizing antibody for inhibiting cell internalizing, so that seizure of disease delay even protection cell resistance disease
Poison infection, therefore P30 has important role in blocking virus and cell interaction.Early proteins of the P30 as virus,
It is distributed mainly in cytoplasm after infection cell, can be detected in endochylema within 4 hours after infection；P30 is also most anti-
One of ASFV albumen of originality has very strong immunogenicity, and body is can induce in infection animal body and generates virucidin,
Therefore usually it is used as diagnostic antigen.In addition, P30 albumen and the P54 albumen receptor or knot different from two on permissive cell
Site interaction is closed, the course of disease can be mitigated.Therefore, the candidate vaccine for developing the anti-ASFV based on P30, for preventing ASF to have
Important meaning.
Invention content
In view of this, the purpose of the present invention is to provide a kind of expression African swine fever virus EP153R genetic recombination adenopathies
Poisonous carrier, construction method and recombined adhenovirus preparation method, to prepare the recombined adhenovirus for being overexpressed EP153R genes, for grinding
Study carefully 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 EP153R 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 EP153R gene recombinant adenovirus vectors, including pAD-EF1 α-GFP adenovirus
Carrier, the African swine fever virus EP153R genes for being inserted into pAD-EF1 α-GFP adenovirus vector multiple cloning sites.EP153R genes
Sequence 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 EP153R gene recombinant adenovirus vectors, includes the following steps：
1) EP153R 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 EP153R and pKO-FH carrier is carried out respectively with restriction enzyme A sis I and MLu I
Double digestion respectively obtains EP153R genetic fragments and pKO linearized vector segments；The EP153R genetic fragments and pKO that will be obtained
Carrier segments are attached, and obtain pKO-EP153R；
3) pKO-EP153R 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 EP153R genetic fragments and adenovirus pAD carrier segments；
4) linearisation EP153R genetic fragments and adenovirus pAD carrier segments that the step 3) obtains are attached,
PAD-EP153R is obtained, is sequenced using CMV-F and FH-R；
5) the plasmid pAD-EP153R that the step 4) obtains is subjected to single endonuclease digestion with restriction enzyme Pac I, selected
It recombinates positive plasmid and 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
| EP153R 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 EP153R 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
| Volume | 50ul |
| 37℃ | 2.5h |
| 65℃ | 20min |
| 4℃ | It keeps |
Preferably, linked system such as the following table 5 in the step 4)：
5 linked system of table
| Target gene EP153R 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
The single endonuclease digestion program is 37 DEG C, 3hr；95 DEG C, 5min.
The method for expressing the recombined adhenovirus of EP153R genes is prepared by following steps：
PAD-EP153R recombinant plasmids are used PEI transfection reagent transfected HEK 293s by I,.
II, blows down the cell for CPE occur together with culture medium, expands culture, and culture receives poison after 3 days.
The present invention provides expression African swine fever virus EP153R genes recombined adhenovirus preparation method, specifically by with
Lower step 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 for being overexpressed EP153R genes is obtained by filtration in precipitation, supernatant.
The present invention provides the adenovirus amplifies containing EP153R genes to detect with receipts poison, purifying with concentration and titre,
Specific 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 a kind of recombinant adenoviral vector pAD- of expression EP153R genes
EP153R introduces EP153R genes based on pAD-EF1a-GFP adenovirus expression carriers at multiple cloning sites.The present invention
By the way that foreign gene EP153R is replaced non-essential sequence in multiple cloning sites, to build the recombination gland of expression EP153R genes
Viral vectors, the described carrier carry CMV strong promoters, and the gene of its carrying, while the load can be overexpressed in eukaryocyte
Body is used to screen positive cell after polyclonal carrier with green fluorescence sequence, makes P30 albumen table directly in eukaryocyte
It reaches.The present invention also provides the recombined adhenovirus of expression EP153R genes, realize recombined adhenovirus packaging process, obtaining can be straight
The adenovirus for connecing infection of eukaryotic cells, to realize EP153R genes normal expression in eukaryocyte, for further research
Expression EP153R gene recombinant adenovirus candidate vaccines provide tool.
Description of the drawings
Fig. 1 is the PacI linearization for enzyme restriction figures of recombinant adenoviral vector pAD-EP153R in embodiment 5, and 1% agarose is solidifying
Gel electrophoresis detect, two band of electrophoresis showed, one be 3kb or 5kb or so, 30kb or so, not according to recombination mechanism
Together, stripe size is different after digestion；
Fig. 2 is that cell CPE schemes plasmid transfection after a week in the embodiment of the present invention 5；
Fig. 3 is the luciferase expression figure of plasmid transfection cell 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 specific implementation mode of the present invention will be described in detail below.In order to avoid excessive unnecessary details,
It will not be described in detail in following embodiment to belonging to well known structure or function.
In addition to being defined, technical and scientific term used in following embodiment has and fields technology people of the present invention
The identical meanings that member is commonly understood by.
Test reagent consumptive material used in following embodiment is unless otherwise specified conventional biochemical reagent；The experiment
Method is unless otherwise specified conventional method；Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result
It is averaged；% in following embodiment is unless otherwise instructed mass percentage.
In the following example, test method without specific conditions, usually routinely condition, such as《Fine works molecular biosciences
Learn experiment guide》(F.M. Ao Sibai, R.E. James Kingstons, the chief editors such as J.G. Sai Deman, Ma Xuejun, Su Yuelong's translates the Beijing：Section
Learn publishing house, 2004) described in method carry out.
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 EP153R gene recombinant adenovirus and its construction methods, with pAD-
Based on EF1a-GFP adenovirus expression carriers, EP153R genes are introduced；The EP153R genes have such as Seq in sequence table
Nucleotide sequence shown in ID No.1.
Embodiment 1
EP153R genes (the GenBank that inquiry NCBI is included website:KU752796.1), target gene piece is directly synthesized
Section, and I/MLu of enzyme enzyme site Asis I.African swine fever virus EP153R genes are synthesized and are added by Shanghai Sheng Gong Co., Ltds
Enter restriction enzyme site XhoI, AsisI.Segment is connected to carrier T, convenient for amplification and digestion.
Embodiment 2
With restriction enzyme A sis I and MLu I respectively to gene chemical synthesis sequence EP153R and adenovirus shuttle plasmid carrier
PKO-FH (Fig. 4) carries out double digestion, respectively obtains linearisation EP153R genetic fragments and pKO carrier segments.Double digestion reaction
Endonuclease reaction system such as the following table 1：
1 double digestion system of table
| EP153R genetic fragments or pKO-FH carriers | 20uL |
| 10×Buffer | 3μL |
| AsisⅠ | 1μL |
| MLuⅠ | 1μL |
| ddH2O | 5μL |
| It amounts to | 30μL |
Wherein EP153R genetic fragments are preferably 100ng/ul, and the temperature of the double digestion is 37 DEG C, the double digestion
Time 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
EP153R genetic fragments will be linearized with T4 ligases and pKO carrier segments are attached, and obtain pKO-EP153R.
Linked system such as the following table 2：
2 linked system of table
| Target gene EP153R 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-EP153R 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-EP153R and adenovirus pAD-EF1 α-
GFP carriers (Fig. 5) double digestion linearizes, and glue recycles respective segments.Ligase connects EP153R genetic fragments and pAD carrier-pellets
Connection product conversion Escherichia coli amplification is then extracted plasmid, pAD-EP153R is obtained after identifying correctly by section.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
| Volume | 50ul |
| 37℃ | 2.5h |
| 65℃ | 20min |
| 4℃ | It keeps |
When ligase connects EP153R genetic fragments and pAD carrier segments, linked system is as shown in table 5：
5 linked system of table
By the plasmid (pAD-EP153R) of extraction with after I single endonuclease digestions of Pac (linearized enzyme digestion), linearisation recombinant plasmid with
PEI mixings centrifuge, and stand, 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-EP153R | 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 complete to get to containing
EP153R 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-EP153R plasmids double digestion is purpose gene
EP153R 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
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
| Constituent | Volume |
| 2X SYBRGreen buffer solutions | 10μL |
| F, R primer mixtures | 0.8μL |
| Ultra-pure water | 7.2μL |
| Viral sample or standard items | 2μL |
| It amounts to | 20μL |
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 2.7 ╳ 1010pfu/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
Primer has specific primer and universal primer, the two PCR system the same respectively；
10 PCR reaction systems of table
| Gained supernatant in step 2) | 1.5μL |
| 10 × Taq enzyme buffer | 2μL |
| dNTP | 0.4μL |
| Taq enzyme | 0.3μL |
| DMSO | 1μL |
| F, R primer mixture | 4μL |
| Ultra-pure water | 10.8μL |
| It amounts to | 20μL |
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
EP153R 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
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Claims (10)
1. a kind of expression African swine fever virus EP153R gene recombinant adenovirus vectors, which is characterized in that including pAD-EF1 α-
GFP adenovirus vectors, the African swine fever virus EP153R genes for being inserted into pAD-EF1 α-GFP adenovirus vector multiple cloning sites；Institute
EP153R genes are stated with nucleotide sequence shown in Seq ID No.1.
2. a kind of expression African swine fever virus EP153R gene recombinant adenovirus vectors according to claim 1, feature
It is, 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 EP153R gene recombinant adenovirus vectors described in claim 1,
It is characterised in that it includes following steps：
1) artificial synthesized African swine fever virus EP153R gene orders, the gene order both ends enzyme enzyme site Asis I and MLu I；
2) double digestion is carried out to gene order EP153R and pKO-FH carrier respectively with restriction enzyme A sis I and MLu I, point
Linearisation EP153R genetic fragments and pKO-FH carrier segments are not obtained；The linearisation EP153R genetic fragments and pKO- that will be obtained
FH carrier segments are attached, and obtain pKO-EP153R；
3) pKO-EP153R 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 EP153R genetic fragments and pAD-EF1 α-GFP carrier segments；
4) linearisation EP153R genetic fragments and pAD-EF1 α-GFP carrier segments that the step 3) obtains are attached, are obtained
To pAD-EP153R, sequencing；
5) the plasmid pAD-EP153R that the step 4) obtains is subjected to single endonuclease digestion with restriction enzyme Pac I, selects recombination
Positive plasmid is used for subsequent experimental.
4. a kind of structure side of expression African swine fever virus EP153R 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 EP153R 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 EP153R 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 EP153R 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 EP153R 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 EP153R gene recombinant adenovirus, which is characterized in that 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 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 EP153R is obtained by filtration in supernatant.
10. a kind of preparation method of expression African swine fever virus EP153R 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。
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| CN109652449A (en) * | 2018-12-07 | 2019-04-19 | 扬州大学 | A kind of EP153R and EP402R gene co-expressing recombinant adenoviral vector constructs and adenovirus packing method |
| CN109735567A (en) * | 2019-01-18 | 2019-05-10 | 扬州大学 | A kind of the recombinant adenoviral vector building and adenovirus packing method of African swine fever EP153R and P54 gene co-expressing |
| CN110862436A (en) * | 2019-12-11 | 2020-03-06 | 浙江鼎持生物制品有限公司 | CHO cell strain for efficiently expressing African swine fever EP153R protein and construction method thereof |
| CN113897394A (en) * | 2020-07-06 | 2022-01-07 | 嘉兴安宇生物科技有限公司 | Recombinant adenovirus vaccine for African swine fever and construction method thereof |
| CN113897394B (en) * | 2020-07-06 | 2023-09-12 | 嘉兴安宇生物科技有限公司 | Recombinant adenovirus vaccine for African swine fever and construction method thereof |
| CN111925996A (en) * | 2020-08-27 | 2020-11-13 | 军事科学院军事医学研究院军事兽医研究所 | African swine fever gene deletion attenuation and live vaccine thereof |
| CN112626101A (en) * | 2021-01-06 | 2021-04-09 | 佛山科学技术学院 | African swine fever virus I177L gene recombinant plasmid, expression vector and preparation method |
| CN113913462A (en) * | 2021-11-26 | 2022-01-11 | 中国农业科学院北京畜牧兽医研究所 | Recombinant adenovirus expressing African swine fever virus EP153R protein and construction method thereof |
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Application publication date: 20180907 |
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| RJ01 | Rejection of invention patent application after publication |