CN106344919A - I-group 4-type aviadenovirus genetic engineering subunit vaccine and preparation method thereof - Google Patents
I-group 4-type aviadenovirus genetic engineering subunit vaccine and preparation method thereof Download PDFInfo
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- 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
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
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- C12N2710/10011—Adenoviridae
- C12N2710/10211—Aviadenovirus, e.g. fowl adenovirus A
- C12N2710/10222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- C12N2710/00011—Details
- C12N2710/10011—Adenoviridae
- C12N2710/10211—Aviadenovirus, e.g. fowl adenovirus A
- C12N2710/10234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Abstract
The invention provides an I-group 4-type aviadenovirus genetic engineering subunit vaccine and a preparation method thereof. According to the technical scheme, the preparation method comprises the following steps: cloning an encoding gene of fibrous protein C-terminal from an I-group 4-type aviadenovirus genome according to a PCR technology and performing sequence analysis; cloning the gene to an expression vector pET-32a, transforming escherichia coli, constructing engineering bacteria, and inducing the engineering bacteria by isopropyl-beta-D-thiogalactopyranoside to express the fibrous protein C-terminal; performing lysis on an engineering bacterial cell, performing centrifugal separation on an inclusion body of the engineering bacterial cell, dissolving urea and diluting for renaturation; preparing the vaccine according to the conventional preparation method of a mineral oil adjuvant inactivated vaccine. According to the I-group 4-type aviadenovirus genetic engineering subunit vaccine prepared by the method, the immune effect of the vaccine is evaluated by a serological method and an immunity challenge method, and the result indicates that the aviadenovirus inactivated vaccine prepared by the method can provide effective immunoprotection and has a good commercialized development prospect.
Description
Technical field
The invention belongs to technical field of vaccines, be related to the preparation of recombinant bacterium and the expression of recombiant protein simultaneously and in particular to
One population 4 type aviadenovirus genetic engineering subunit vaccine and preparation method thereof.
Background technology
Group I fowl adenovirus constitute the Aviadenovirus of Adenoviridae.Group's (haemorrhagic enteritis of turkey and correlated virus)
Clear and definite with the relation of disease with group's (egg drop syndrome) adenoviruss, by contrast, the aviadenovirus of most of groups are to birdss
Pathogenic effects also do not determine completely.But fadv-1 and fadv-4 substantially belongs to exception, wherein fadv-1 can cause Carnis Coturnicis japonicae bronchus
Inflammation, fadv-4 is the Etiological of hydropericardium hepatitis syndrome.When the health of chicken suffers damage, such as accompanying infection chicken is infected
Other cause of diseases such as property anemia virus (ciav) and infectious bursal disease viruses (ibdv), other adenovirus strain can make condition
Property primary life of causing a disease quickly is infected.Group I fowl adenovirus have identified 5 aviadenovirus, and its title letter a～e represents.Each
Virus in kind is mainly further divided into different serotypes according to cross-neutralization experimental result.Virion is a diameter of 70～
90nm, no cyst membrane, in 20 face body symmetrical structures.Viral nucleic acid be bifilar dna, account for whole virion 11.3%～
13.5%, remainder is protein.Hexonmer is main capsid protein, determines containing type, group and group specific antigen
Determine cluster, thus the specific antibody of corresponding type, group and group after infection, can be produced.
Group I fowl adenovirus are in worldwide distribution, and each age group poultry is all susceptible.Can be by horizontal and vertical two kinds of approach
Propagate.Although the virulence between the 12 of infected chicken serotypes and within serotype is had nothing in common with each other, 12 serotypes all can lure
Send out inclusion body hepatitis (ibh).The main clinic symptoms of the hydropericardium hepatitis syndrome (hhs) that fadv-4 causes are egg drop reduction
With cause dead (mortality rate 20%～80%), main pathological change is that have faint yellow limpid hydrops, liver in pericardial cavity
Multiple focal necrosis in enlargement, pale, kidney enlargement, heart and liver, see intranuclear inclusion in hepatocyte.fav-4
There is the age bracket that significantly causes a disease, the strongest to the pathogenicity of chickling, can be propagated and vertical transmission by exposure level.1987
Angola that Karachi is closed on by Pakistan reports primary disease first, 1989 in Mexico, thereafter Iraq, India several
The states such as nation, Ecuador, Peru, Chile, Central and South America, Russia and Bangladesh find this disease in succession.Nowadays, all over the world
There is the trace of hhs, throughout some countries in Latin America-Middle East and Asia.
By the Epidemiological study to group I fowl adenovirus, this disease sickness rate in China chicken group is higher, and is in go up year by year
The trend of liter.The host range of morbidity is more and more wider, and white meat-type chickens, Breeder hens, laying hen, yellow plumage chicken all can infection morbidity.Particularly
Increase trend was assumed with sequela in 2010, all have popular in China.Clinical manifestation inclusion body hepatitis (ibh), pericardium
Hydrops hepatitis syndrome (hhs).With developing rapidly of China's poultry husbandry, the sickness rate of group I fowl adenovirus causes to animal husbandry
Serious economic loss.With regard to the preventing and treating of this disease, abroad prevent the commercialized vaccine of group I fowl adenovirus, domestic so far
There is no vaccine can use, lead to group I fowl adenovirus prevention and control to there is leak.
At present, fav-4 inoculated into chick embryo or primary chicken kidney cellular hepatocyte prepare vaccine to inoculate.Embryo Gallus domesticus propagative viruseses poison
Valency is low, and virus needs high power to concentrate, with high costs；And primary liver and kidney cells are prepared loaded down with trivial details, easy pollution, are difficult to transfect, and in system
Easily it is mixed into chick embryo fibroblast (cef) during detailed born of the same parents, be unfavorable for experimental implementation.So being badly in need of new antigen preparation way
Footpath.
Group I fowl adenovirus have a typical adenovirion form, a diameter of 70-90nm of virion, and no cyst membrane is spherical in shape,
Icosahedral symmetry structure.Virion has 252 capsomeres, and 240 non-vertex capsomers are six adjacent bodies, and 12 vertex capsomers are
Penton, each penton has 2 fiber initiations.Research shows that fiber initiation has good prototype of exempting from service.
Content of the invention
It is contemplated that for the technological deficiency of prior art, providing a population 4 type aviadenovirus gene engineered subunit
Vaccine and preparation method thereof, to solve the difficult technical problem of group 4 type aviadenovirus inactivated vaccine preparation in prior art.
The invention solves the problems that another technical problem be in prior art, to lack a kind of Asia for group 4 type aviadenovirus
Subunit vaccine.
The invention solves the problems that another technical problem be group 4 type aviadenovirus in prior art subunit vaccine due to
Selection of antigen is unreasonable and leads to immune effect poor.
The invention solves the problems that another technical problem be this albumen when using albumen provided by the present invention as antigen
Acquisition less efficient.
For realizing above technical purpose, the present invention employs the following technical solutions
One population 4 type aviadenovirus genetic engineering subunit vaccine, this vaccine is with aminoacid sequence for seq id no:1
Albumen be antigen.
A kind of preparation method of above-mentioned group 4 type aviadenovirus genetic engineering subunit vaccine, comprises the following steps:
1) preparation sequence is the expressing gene of the albumen of seq id no:1；
2) by step 1) gene cloning of gained, on expression vector pet-32a, obtains recombiant plasmid；
3) by step 2) described recombinant plasmid transformed, to escherichia coli, obtains recombinant bacterium；
4) expression induction step 1) described albumen；
5) crack described recombinant bacterium, then collect and the described albumen in rapid 1) of purification；
6) utilizing step 5) albumen of gained prepares vaccine.
Preferably, step 1) described in expressing gene be that the template of described pcr is fav-4 using the preparation of pcr method
Virus, the upstream and downstream primer of described pcr is dna that sequence is seq id no:3 respectively and sequence is seq id no:4
dna.
Preferably, the reaction condition of described pcr is: 94 DEG C of degeneration 45s, 56 DEG C of annealing 45s, 72 DEG C of extension 1min, altogether
Carry out 30 circulations.
Preferably, step 2) described in be cloned on expression vector pet-32a, be first by step 1) pcr of gained produces
Thing is cloned on pmd-18t carrier, obtains recombinant vector, then utilizes the method for double digestion by described recombinant vector and pet-
32a plasmid is connected, that is, obtain described recombiant plasmid.
Preferably, enzyme used by described double digestion is respectively ecor i and hind iii, digestion products carry out 1% agarose
Gel electrophoresiss, are separately recovered purpose fragment and pet-32a, and then 16 DEG C connect overnight.
Preferably, step 4) described in abduction delivering, be to be realized using the iptg of final concentration of 0.5mm.
Preferably, step 5) described in cracking include following operation: 20min is centrifuged with the rotating speed of 8000rpm, it is heavy to collect
Form sediment and add combination buffer 100ml suspension thalline, then carry out 750w ultrasonic disruption, and with 8000rpm under the conditions of 4 DEG C
Rotating speed centrifugation 30min, collect precipitation, that is, obtain inclusion body；Wherein said combination buffer comprises 20mmol/l
Tris.hcl, 0.5mol/l nacl, 5mmol/l imidazoles；The ph of described combination buffer is 8.0；The each work of described ultrasonication
Make time 10s, interval time, 20s, crushed 120 times altogether.
Preferably, step 5) described purification includes following operation: by the solubilization of inclusion bodies of gained in urea liquid, and
Cross ni Ao's resin post of 8mol/l carbamide balance afterwards, then use the 8mol/l carbamide eluting mesh that 30ml elution buffer solution is prepared
Albumen, collect effluent, after 4 DEG C of dialysed overnight, lyophilization；Wherein said elution buffer solution comprises 20mmol/l
Tris.hcl, 0.5mol/lnacl, 300mmol/l imidazoles；The ph of described elution buffer solution is 8.0.
Preferably, the described solubilization of inclusion bodies by gained is in urea liquid, operate including following: by 5g occlusion body
Add 2% (w/v) tritonx-100 of 120ml, stir 30min, ultrasonication 40 times with agitator, agitator continues stirring
30min, then freezing centrifugation 8000rpm, 30min.Again respectively with 2%, 2.2%, 2.4%, 2.5% (w/v's)
The each 120ml of tritonx-100 washs as stated above, washs 4 times altogether, adds 100ml in the occlusion body precipitation of last washing
Concentration is the urea liquid of 8mol/l, uses magnetic stirrer 40min, frozen centrifugation 8000rpm, 30min, retains supernatant.
Vaccine of the present invention can be used for preventing chicken hydropericardium hepatitis syndrome.
In above technical scheme, step 6) described prepare vaccine, be with step 5) albumen of gained prepares epidemic disease for antigen
Seedling, concrete operation method can be according to the conventional vaccine preparation method execution in this area that is to say, that with step 1)～5) institute
On the basis of the albumen of preparation is antigen, using this area, any one conventional vaccine preparation method all can reach present invention institute in advance
The effect of phase, and specifically adopt which kind of vaccine preparation technology, can carry out adapting to Sexual behavior mode according to specific technological requirement.
The invention provides a population 4 type aviadenovirus genetic engineering subunit vaccine and preparation method thereof, this technical side
Case is cloned the encoding gene of fibrin c- end from group 4 type aviadenovirus genome first with pcr technology and is carried out sequence
Row analysis；Then by this gene cloning to expression vector pet-32a, after conversion escherichia coli, engineering bacteria, isopropyl-β-d- are built
Thiogalactoside induces engineering bacterium expression fibrin c- end；Finally crack engineering bacteria cell, centrifugation bag therein
Containing body, after carbamide dissolving, dilution refolding；Routinely mineral oil adjuvant inactivated vaccine preparation method is prepared into vaccine again.The present invention
The method that the aviadenovirus genetic engineering subunit vaccine vaccine of research employs genetic engineering fermentation prepares antigen, low cost,
Antigen is pure, using safety, and has good immune effect.The group 4 type aviadenovirus subunit vaccine of present invention preparation,
Assess the immune effect of vaccine using serological method and Immunization method, result shows, the aviadenovirus of preparation in the present invention
Inactivated vaccine can provide effective immunoprotection to fowl, has good commercialized development prospect.
Specific embodiment
The specific embodiment of the present invention will be described in detail below.In order to avoid excessively unnecessary details,
Will not be described in detail to belonging to known structure or function in following examples.
Approximating language used in following examples can be used for quantitative expression, shows in the feelings not changing basic function
Quantity can be allowed under condition to have certain variation.Therefore, it is not limited to this with the numerical value that the language such as " about ", " left and right " is revised accurate
Numerical value itself.In certain embodiments, " about " represent the numerical value allowing its correction in positive and negative 10 (10%) scope
Interior change, such as, what " about 100 " represented can be any numerical value between 90 to 110.Additionally, " the about first numerical value arrives
In the statement of second value ", at about revise the first and second numerical value two values.In some cases, approximating language
May be relevant with the precision of measuring instrument.
In addition to being defined, in following examples, technology used and scientific terminology have and art technology people of the present invention
The identical meanings that member is commonly understood by.In following examples, term used " first ", " second " etc. are not offered as any order, number
Amount or importance, and be only used for distinguishing a kind of material and another kind of material.
Embodiment 1 (fibrin c- terminal gene clone and sequence analysis)
Design synthesis pair of primers:
Primer 1:5 '-gcgaattcatggctatgctacagat-3 '；
Primer 2: 5 '-ggaagcttttacgggagggagcc-3 '.
Fav-4 kind poison is purchased from China Veterinery Drug Inspection Office.
With fav-4 virus as template, according to 94 DEG C of degeneration 45s, 56 DEG C of annealing 45s, 72 DEG C of extension 1min, carry out 30 altogether and follow
Ring carries out pcr amplification；By on pcr product Direct Cloning to pmd-18t carrier (takara company), build pmd-18t-hhs；Profit
Carry out sequencing with dideoxy chain termination, result shows, clone the coding of fibrin c- end from fav-4 genome
Gene has the nucleotide sequence shown in seq id no:2, and the aminoacid sequence of its coding is as shown in seq id no:1.
Embodiment 2 (structure of expression vector and engineering bacteria)
By pmd-18t-hhs and pet-32a ecor i and hind iii double digestion, digestion products carry out 1% agarose
Gel electrophoresiss, are separately recovered purpose fragment and pet-32a, and then 16 DEG C connect overnight；Connection product is transformed into e.coli
Dh5 α competent cell, picking single bacterium colony is inoculated into the lb culture medium that 2ml contains 100 μ g/ml ampicillin, 37 DEG C of shaken cultivation
12h, is identified with double digestion after extracting plasmid, obtains expression plasmid pet-32a-hhs.By recombinant plasmid transformed to e.coli
Bl21 (de3), picking single bacterium colony, it is inoculated into the lb culture medium that 200ml contains 100 μ g/ml ampicillin, 37 DEG C of shaken cultivation
12h, after be transferred in 4l same medium, after 37 DEG C of shaken cultivation 3h, add iptg to final concentration of 0.5mm, continue culture
4h, then sds-page electrophoretic analysiss.
Embodiment 3 (purification of restructuring group 4 type aviadenovirus fibrin c- end)
Picking engineering bacteria single bacterium colony is in 200ml lb fluid medium (0.1g/l containing ampicillin concentration), 37 DEG C of constant temperature
Shaken overnight is cultivated；By overnight culture, the ratio for 1: 20 pours lb fluid medium (the ammonia benzyl green grass or young crops amounting to 4l into by volume
Mycin concentration 0.1g/l), 37 DEG C of constant-temperature shaking culture 3h；Being subsequently adding iptg makes its final concentration of 5mm, 37 DEG C of constant temperature oscillation trainings
Foster 4h.
Culture is centrifuged with large-scale low-temperature refrigerated centrifuger, 8000rpm, 20min；Abandon supernatant, add combination buffer
100ml (20mmol/ltris.hcl, ph 8.0,0.5mol/l nacl, 5mmol/l imidazoles) suspension thalline, then carries out 750w
Ultrasonic disruption (working time 10s, interval time, 20s, crushed 120 times).Then 4 DEG C, 8000rpm, 30min, retain precipitation
(occlusion body).
2% (w/v) tritonx-100 of 120ml will be added in 5g occlusion body, stir 30min, ultrasonication with agitator
40 times, agitator continues stirring 30min, then freezing centrifugation 8000rpm, 30min.Again respectively with 2%, 2.2%,
The each 120ml of tritonx-100 of 2.4%, 2.5% (w/v) washs as stated above, washs 4 times altogether, to comprising of last washing
Add, in body precipitation, the carbamide (in conjunction with buffer preparation) that 100ml concentration is 8mol/l, use magnetic stirrer 40min,
Frozen centrifugation 8000rpm, 30min, retain supernatant.
Carbamide is dissolved ni Ao's resin that on the supernatant of occlusion body, 8mol/l carbamide (in conjunction with buffer preparation) balances
Post (2 × 10cm), first rinses chromatographic column with the 8mol/l carbamide (in conjunction with buffer preparation) of 10 times of bed volumes, then with 2 times
The washing buffer solution (20mmol/l tris.hcl, ph8.0,0.5mol/l nacl, 100mmol/l imidazoles) of bed volume is joined
System 8mol/l urea washes chromatographic column, finally use 30ml elution buffer solution (20mmol/l tris.hcl, ph8.0,
0.5mol/lnacl, 300mmol/l imidazoles) the 8mol/l carbamide eluting destination protein prepared, collect effluent.Effluent is in 4
After DEG C to 1l distilled water dialysed overnight, lyophilization.
Embodiment 4 (restructuring group 4 type aviadenovirus fibrin c- end antigenicity analysis)
After the edsv fibrin c- end of the restructuring of 0.1 μ g purification is separated through sds-page, electrotransfer to pvdf is (poly-
Vinylidene) on film, the defatted milk powder with 5% closes 2h, pbs vibration washing 3 times, and it is separately added into one anti-(chicken antiserum) 1: 250 and make
With 2h, vibration washing 3 times, add two anti-1: 1000 (rabbit anti-chicken igg of hrp labelling) effect 2h, vibration washing develops the color in nitrite ion
(20mmol/ltris.hcl, ph8.0,50mmol/lnacl, 0.03%nicl2,0.01% diaminobenzidine, 0.3%
h2o2).Result shows that recombinant fiber albumen c- end has good antigenicity.
Embodiment 5 (preparation of group 4 type aviadenovirus genetic engineering subunit vaccine)
Oil phase: marcol 52 import white oil (volume ratio), span-80 (volume ratio), aluminium stearate.Various materials are joined
Than after, mix, cool down standby after autoclaving.
Aqueous phase: pet32a-hhs recombinant protein, protein concentration is 0.4mg/ml, plus appropriate tween-80.
Take 2 parts of oil phase to import in emulsion tank, start motor stirring at low speed, after slowly adding 1 part of aqueous phase simultaneously, 3600r/
Min, emulsifying 30 minutes.Vaccine 10ml is taken to add in centrifuge tube, 3000r/min is centrifuged 15 minutes, observing tube bottom has or not aqueous phase analysis
Go out, separate out if any aqueous phase, metering separates out the volume of aqueous phase.
Quantitative separating, seals, 2～8 DEG C of preservations.
Embodiment 6 (vaccine safety effect detection)
With 21 age in days spf chicken 10, the subcutaneous branch of every cervical region vaccinates 1.0ml (the subcutaneous each injection in nape both sides
0.5ml), observe 14, see whether any locally and systemically untoward reaction being caused by vaccine.Result shows, vaccine immunity
All test chickens spirit, searching for food and drinking water is showed no exception, all no red and swollen reaction in injection site.
Embodiment 7 (immune effect of vaccine detection)
1 serological method takes 21 age in days spf chicken 20, and 10 each cervical regions subcutaneously or intramuscularly vaccinate 0.3ml, another 10
Compare.Inoculate latter 7 days to 5 months, every chicken is taken a blood sample respectively, separate serum, detect immune chicken and comparison chicken with agar diffusion test
Group 4 type aviadenovirus antibody, reporter antibody result.
2 Immunization methods take 21 age in days spf chicken 20, and 10 each cervical regions subcutaneously or intramuscularly vaccinate 0.3ml, another 10
Compare.Immunity after 21～28 days, all immunity chickens and comparison chicken, intramuscular injection group 4 type aviadenovirus virus liquid (containing about
105.5tcid50/ 0.1ml), every 0.2ml.Observe and itemized record immunity chicken and the incidence compareing chicken.
Result shows: 2 vaccine immunity groups 21 days and later fine jade expand antibody all >=8/10 positives, all the moon of matched group chicken
Property；Counteracting toxic substances protect result, observe 14 after 2 batches of vaccine immune chicken counteracting toxic substances, and protection number is no less than 9；In comparison chicken counteracting toxic substances 7 days
All fall ill, dead 8, dead chicken (cut open inspection after death) and morbidity chicken (cut open inspection on the 7th after counteracting toxic substances) cut open inspection pathological change: all occur
Typical hydropericardium, liver enlargement pathological changes.
Above embodiments of the invention are described in detail, but described content have been only presently preferred embodiments of the present invention,
Not in order to limit the present invention.All any modification, equivalent and improvement made in the application range of the present invention etc., all should
It is included within protection scope of the present invention.
Claims (10)
1. a population 4 type aviadenovirus genetic engineering subunit vaccine is it is characterised in that this vaccine is with aminoacid sequence as seq
The albumen of id no:1 is antigen.
2. group 4 type aviadenovirus genetic engineering subunit vaccine described in a kind of claim 1 preparation method it is characterised in that
Comprise the following steps:
1) preparation sequence is the expressing gene of the albumen of seq id no:1；
2) by step 1) gene cloning of gained, on expression vector pet-32a, obtains recombiant plasmid；
3) by step 2) described recombinant plasmid transformed, to escherichia coli, obtains recombinant bacterium；
4) expression induction step 1) described albumen；
5) crack described recombinant bacterium, then collect and the described albumen in rapid 1) of purification；
6) utilizing step 5) albumen of gained prepares vaccine.
3. preparation method according to claim 2 is it is characterised in that step 1) described in expressing gene be using pcr method
Preparation, the template of described pcr is fav-4 virus, and the upstream and downstream primer of described pcr is sequence respectively is seq id no:3
Dna and sequence are the dna of seq id no:4.
4. preparation method according to claim 3 is it is characterised in that the reaction condition of described pcr is: 94 DEG C of degeneration 45s,
56 DEG C of annealing 45s, 72 DEG C of extension 1min, carry out 30 circulations altogether.
5. preparation method according to claim 4 is it is characterised in that step 2) described in be cloned into expression vector pet-32a
On, it is first by step 1) the pcr product cloning of gained, to pmd-18t carrier, obtains recombinant vector, then utilizes double digestion
Described recombinant vector is connected by method with pet-32a plasmid, that is, obtain described recombiant plasmid.
6. preparation method according to claim 5 it is characterised in that enzyme used by described double digestion be respectively ecor i and
Hind iii, digestion products carry out 1% agarose gel electrophoresiies, are separately recovered purpose fragment and pet-32a, then 16 DEG C of connections
Overnight.
7. preparation method according to claim 6 is it is characterised in that step 4) described in abduction delivering, be to utilize final concentration
Iptg for 0.5mm realizes.
8. preparation method according to claim 7 is it is characterised in that step 5) described in cracking include following operation: with
The rotating speed centrifugation 20min of 8000rpm, collects precipitation and adds combination buffer 100ml suspension thalline, then carry out 750w ultrasound wave
Broken, and under the conditions of 4 DEG C, 30min is centrifuged with the rotating speed of 8000rpm, collect precipitation, that is, obtain inclusion body；Wherein said knot
Close buffer and comprise 20mmol/l tris.hcl, 0.5mol/l nacl, 5mmol/l imidazoles；The ph of described combination buffer is
8.0；The each working time 10s of described ultrasonication, interval time, 20s, crushed 120 times altogether.
9. preparation method according to claim 8 is it is characterised in that step 5) described purification includes following operation: by gained
Solubilization of inclusion bodies in urea liquid, then cross 8mol/l carbamide balance ni Ao's resin post, then use 30ml eluting delay
Rush the 8mol/l carbamide eluting destination protein of solution preparation, collect effluent, after 4 DEG C of dialysed overnight, lyophilization；Wherein institute
State elution buffer solution and comprise 20mmol/l tris.hcl, 0.5mol/lnacl, 300mmol/l imidazoles；Described elution buffer is molten
The ph of liquid is 8.0.
10. preparation method according to claim 9 is it is characterised in that the described solubilization of inclusion bodies by gained is in urea liquid
In, operate including following: 2% (w/v) tritonx-100 of 120ml will be added in 5g occlusion body, stir 30min with agitator,
Ultrasonication 40 times, agitator continues stirring 30min, then freezing centrifugation 8000rpm, 30min.Again respectively with 2%,
The each 120ml of tritonx-100 of 2.2%, 2.4%, 2.5% (w/v) washs as stated above, washs 4 times altogether, washs to last
Occlusion body precipitation in add 100ml concentration be 8mol/l urea liquid, use magnetic stirrer 40min, frozen centrifugation
8000rpm, 30min, retain supernatant.
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| CN201610777561.1A CN106344919A (en) | 2016-08-31 | 2016-08-31 | I-group 4-type aviadenovirus genetic engineering subunit vaccine and preparation method thereof |
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| CN201610777561.1A CN106344919A (en) | 2016-08-31 | 2016-08-31 | I-group 4-type aviadenovirus genetic engineering subunit vaccine and preparation method thereof |
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| CN106946995A (en) * | 2017-04-05 | 2017-07-14 | 苏州米迪生物技术有限公司 | The I group of type aviadenovirus genetic engineering subunit vaccine of serum 4, its preparation method and application |
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| CN107365365A (en) * | 2017-08-30 | 2017-11-21 | 广州医科大学附属第医院 | The adenovirus cilia protein peptide of recombination expression, adenovirus subunit vaccine and preparation method thereof |
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| CN107602672B (en) * | 2017-08-30 | 2021-06-18 | 广州医科大学附属第一医院 | Recombinant expression adenovirus cilia protein peptide, adenovirus subunit vaccine and preparation method thereof |
| CN108558989A (en) * | 2018-04-17 | 2018-09-21 | 扬州优邦生物药品有限公司 | 4 type aviadenovirus subunit vaccines of a kind of prevention and its preparation method and application |
| CN108558989B (en) * | 2018-04-17 | 2019-07-02 | 扬州优邦生物药品有限公司 | 4 type aviadenovirus subunit vaccines of a kind of prevention and its preparation method and application |
| CN114149493A (en) * | 2021-12-08 | 2022-03-08 | 山东滨州沃华生物工程有限公司 | Group I4 avian adenovirus fiber-2 protein antigen and method for preparing genetic engineering subunit vaccine and application thereof |
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