CN106755106A - The preparation method and application of Ankara subunit vaccine - Google Patents
The preparation method and application of Ankara subunit vaccine Download PDFInfo
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- CN106755106A CN106755106A CN201710010892.7A CN201710010892A CN106755106A CN 106755106 A CN106755106 A CN 106755106A CN 201710010892 A CN201710010892 A CN 201710010892A CN 106755106 A CN106755106 A CN 106755106A
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Abstract
The invention discloses a kind of preparation method and application of Ankara subunit vaccine,The immunogenic gene Penton that Sf9 cells take restructuring poison Ac Penton expression Ankaras is transfected after obtaining correct restructuring rod granule Bacmid Penton using baculovirus expression system construction recombination plasmid pFastBac HTB Penton conversions DH10Bac E.coli,Fiber2 simultaneously carries out great expression and the purifying of albumen,Expressing quantity is high,Protein active is high,100% protective rate can be reached to Ankara for Ankara subunit vaccine with Penton albumen as antigen and Ankara mixing subunit vaccine with Penton+Fiber2 albumen as antigen and the antibody of two kinds of vaccine generations is high,Hold time length.Ankara disease can be effectively treated, reducing Ankara infection will bring great economic loss to aquaculture.
Description
Technical field
The present invention relates to vaccine preparation technology field, in particular to a kind of preparation method of Ankara subunit vaccine
And application.
Background technology
The ground such as the China of in October, 2015 Shandong, Anhui, Henan outburst Ankara, this disease is caused by adenovirus, main
Cause ephritis, inclusion body hepatitis, hydropericardium, egg drop syndrome etc..Adenovirus is divided into I, II, III hypotype, and I hypotype is not
Common, II hypotype (haemorrhagic enteritis of turkey and correlated virus), III hypotype is exactly ephritis bubbling with noise noisy in the recent period, lays eggs down
Drop syndrome, inclusion body hepatitis and hydropericardium syndrome are (because it breaks out in Pakistani Ankara area, therefore again earliest
It is Ankara disease).The broiler chicken morbidity chicken group that this disease betides 3~6 week old starts death more than 3 week old, and 4~5 week old reach height
Peak, 4~8 days peak durations, 5~6 week old are dead to be reduced.The course of disease 8~15 days, the death rate is up to 20~80%.This disease can be vertical
Propagate and horizontal transmission.Easily with concurrent this disease morbidity hurried of bird flu, ewcastle disease, infectious bursa of Fabricius and infections anaemia
Denier infection will bring one of the great epidemic disease of great economic loss as harm animal husbandry development to aquaculture.Vaccine immunity connects
Plant is to prevent this sick effective measures.
Ankara is a kind of particle for not having tunicary a diameter of 70~90nm, is arranged in twenty face body by 252 capsomeres
Row are constituted.Capsid contains 240 six conjuncted (hexon), 12 pentons (penton) and 12 ciliums (fiber), except this it
There is some other little albumen outward.Penton is one of major structural protein of adenovirus, its polypeptide 452 amino acid long,
12 summit pentons for participating in constituting virion are acted on by itself RGD or LGV motif with the integrin of cell surface,
Contribute to intrusion and the internalization of adenovirus.Its antibody can enter cytoplasm with blocking virus body from acid core endoplasm, so that
Viral infection inactivates penton and the cephalomere area of cilium and can be combined with the virus receptor of cell surface, in virus infected cell mistake
Very important effect is played in journey.Therefore Penton albumen is the first-selection as the subunit vaccine of Ankara.
Baculovirus expression system is exactly a strong instrument, baculovirus insect cell expression system
(Baculovims expression vector system BEVS) be a kind of eukaryotic expression system it with baculoviral be carry
Body by by foreign gene be inserted into Baculovirus Gene group and with recombinant virus infection insect cell or insect larvae come
Expression foreign protein simultaneously carries out a certain degree of packaging modification folding to albumen.To the external source expressed using the expression system at present
Existing thousands of kinds of gene is one of eukaryotic gene efficient expression system of most application prospect.With the development bar of biotechnology
Shape virus develops into expression, the vaccine of present mammalian cell-infecting its application from albumen from Simple infection insect cell
Production be extended to many fields such as surface display, gene therapy and being still within update among development.
The commercialized Ankara vaccine there is no to carry out prevention and control at present as neopathy poison.This disease causes so big warp
Ji loss so that safely and efficiently vaccine put goods on the market it is extremely urgent.And conventional inactivated vaccine is possible in inactivation process
Damage or change effective antigenic determinant；The immune effect of generation is held time short, does not produce local antibody；There may be poison
Property or the potential immune response unfavorable to body；Multiple injection is needed, it is necessary to amount of antigen is than larger, and cost is higher.
The content of the invention
It is an object of the present invention to provide a kind of preparation method and application of Ankara subunit vaccine, the vaccine is with bar
The major antigen Penton expressing quantities of the Ankara of shape virus expression systems expression are high, and protein active is high, Penton
Albumen is Ankara mixing subunit vaccine of antigen for Ankara subunit vaccine and Penton+Fiber2 albumen of antigen
The antibody that the protective rate and two kinds of vaccines that 100% can be reached to Ankara are produced is high, length of holding time.Can be effective
Treatment Ankara disease, reducing Ankara infection will bring great economic loss to aquaculture.
To achieve the above object, a kind of recombinant plasmid pFastBac HTB-Penton that the present invention is provided, the restructuring turns
Moving plasmid pFastBac HTB-Penton is interleave in III two restriction enzyme sites of BamHI and Hind of carrier pFastBac HTB
Enter that Penton is gene constructed to be formed, wherein, the nucleotide sequence of Penton genes is as shown in SEQ IDNO.1.
The invention provides a kind of construction method of above-mentioned recombinant plasmid pFastBac HTB-Penton, including following step
Suddenly：
1) acquisition of Penton genes
Design primer：
Penton-F：5'-AGCGGATCCATGTGGGGGTTGCAGCCG-3',
BamHI
Penton-R：5'-GGTAAGCTTCTACTGCAAGGTCGCGGAACTC-3'；
HindⅢ
Complete genome DNA with Ankara enters performing PCR as template, and amplification recovery obtains containing restriction enzyme site
The Penton genes of BamHI/Hind III；
2) structure of recombinant plasmid pFastBac HTB-Penton
With carrier pFastBac HTB as skeleton, with digestion with restriction enzyme carrier pFastBac HTB and contain digestion
The Penton genes of the Penton genes of site BamHI/Hind III, the pFastBac HTB for being linearized and linearisation, use
PFastBac HTB and the Penton genes connection of linearisation that ligase will be linearized, obtain recombinant plasmid pFastBac
HTB-Penton。
As priority scheme, the step 1) in,
PCR system：
PCR conditions：94 DEG C of predegeneration 5min, PCR cycle is 95 DEG C of 30s, 50 DEG C of annealing 30s, 68 DEG C of 1min；30 circulations
68 DEG C extend 10min afterwards；
The step 2) in, digestion system：
Digestion condition：37 DEG C of endonuclease reaction 3h after above-mentioned system is mixed.
Present invention also offers a kind of recombinant baculovirus Ac-Penton, the base of the recombinant baculovirus Ac-Penton
Because containing the recombinant plasmid pFastBac HTB-Penton described in claim 1 in group.
Present invention also offers a kind of preparation method of above-mentioned recombinant baculovirus Ac-Penton, comprise the following steps：
1) acquisition of Penton genes
Design primer：
Penton-F：5'-AGCGGATCCATGTGGGGGTTGCAGCCG-3',
BamHI
Penton-R：5'-GGTAAGCTTCTACTGCAAGGTCGCGGAACTC-3'；
HindⅢ
Complete genome DNA with OK a karaoke club virus enters performing PCR as template, and amplification recovery obtains containing restriction enzyme site BamHI/
The Penton genes of Hind III；
2) structure of recombinant plasmid pFastBac HTB-Penton
With carrier pFastBac HTB as skeleton, with digestion with restriction enzyme carrier pFastBac HTB and contain digestion
The Penton genes of the Penton genes of site BamHI/Hind III, the pFastBac HTB for being linearized and linearisation, use
PFastBac HTB and the Penton genes connection of linearisation that ligase will be linearized, obtain recombinant plasmid pFastBac
HTB-Penton。
3) restructuring rod granule Bacmid-Penton
Recombinant plasmid pFastBac HTB-Penton are converted into DH10Bac E.coli competent cells, in the training of LB liquid
Culture in base is supported, a small amount of nutrient solution is then taken and is lined and contain three anti-(kanamycins, gentamicin and tetracycline) and IPTG, X-
The high salt LB flat board cultures of gal, screen the single white colony PCR of picking and identify that correct rear inoculation contains antibiotic by blue hickie
LB fluid nutrient mediums in, 37 DEG C of 220rpm/min vibration culture shaking overnight incubations, extraction obtains restructuring rod granule Bacmid-
Penton；
4) recombinant baculovirus Ac-Penton is obtained
Restructuring rod granule Bacmid-Penton is transfected into sf9 cells, observes that cell has obvious lesion after 4-7 days, collect thin
Born of the same parents' nutrient solution, centrifugation removal cell fragment, it is to obtain recombinant baculovirus Ac-Penton to collect supernatant.
As priority scheme, the step 1) in,
PCR system：
PCR conditions：94 DEG C of predegeneration 5min, PCR cycle is 95 DEG C of 30s, 50 DEG C of annealing 30s, 68 DEG C of 1min；30 circulations
Afterwards, 68 DEG C of extension 10min；
The step 2) in, digestion system：
Digestion condition：37 DEG C of endonuclease reaction 3h after above-mentioned system is mixed.
Ankara subunit vaccine is prepared based on recombinant baculovirus Ac-Penton present invention also offers a kind of
Method, comprises the following steps：
1) recombinant baculovirus Ac-Penton inoculations are suspended in the insect cell SF9 of culture, culture is collected by centrifugation thin
Born of the same parents；
2) above-mentioned cell ultrasonication cracking takes supernatant, and affinitive layer purification is reclaimed, and then flushed post wash-out, obtains pure
The albumen of change,
3) by step 2) albumen of purifying is diluted to 300~800 μ g/ml, and albumen and the adjuvant that will be diluted are by weight 2:7
~1:2 are well mixed, and obtain Ankara subunit vaccine.
Preferably, the adjuvant is MontanideTMISA 71 VG、
The above-mentioned Ankara subunit vaccine for preparing is applied in the disease of preventing and treating Ankara.
Ankara is prepared based on recombinant baculovirus Ac-Penton and Ac-Fiber2 present invention also offers one kind
The method for mixing subunit vaccine, comprises the following steps：
1) acquisition of Fiber2 genes
Design primer：
Fiber2-F：5’-AGCTCTAGAATGCTCCGGGCCCCTAAA-3 ',
Xba Ⅰ
Fiber2-R：5’-GGTAAGCTTTTACGGGAGGGAGGCCG-3’；
HindⅢ
Complete genome DNA with OK a karaoke club virus enters performing PCR as template,
PCR system：
PCR conditions：94 DEG C of predegeneration 5min, PCR cycle is 95 DEG C of 30s, 50 DEG C of annealing 30s, 68 DEG C of 1min；30 circulations
Afterwards, 68 DEG C of extension 10min；
Amplification recovery obtains the Fiber2 genes containing I/Hind of restriction enzyme site Xba III；
2) structure of recombinant plasmid pFastBac HTB-Fiber2
With carrier pFastBac HTB as skeleton, with digestion with restriction enzyme carrier pFastBac HTB and contain digestion
The Fiber2 genes of the Fiber2 genes of I/Hind of site Xba III, the pFastBac HTB for being linearized and linearisation, use
PFastBac HTB and the Fiber2 genes connection of linearisation that ligase will be linearized,
Digestion system：
Digestion condition：37 DEG C of endonuclease reaction 3h after above-mentioned system is mixed；Obtain recombinant plasmid pFastBac HTB-
Fiber2。
3) restructuring rod granule Bacmid-Fiber2
Recombinant plasmid pFastBac HTB-Fiber2 are converted into DH10Bac E.coli competent cells, in the training of LB liquid
Culture in base is supported, a small amount of nutrient solution is then taken and is lined and contain three anti-(kanamycins, gentamicin and tetracycline) and IPTG, X-
The high salt LB flat board cultures of gal, screen the single white colony PCR of picking and identify that correct rear inoculation contains antibiotic by blue hickie
LB fluid nutrient mediums in, 37 DEG C of 220rpm/min vibration culture shaking overnight incubations, extraction obtains restructuring rod granule Bacmid-
Fiber2；
4) recombinant baculovirus Ac-Fiber2 is obtained
Restructuring rod granule Bacmid-Fiber2 is transfected into sf9 cells, observes that cell has obvious lesion after 4-7 days, collect thin
Born of the same parents' nutrient solution, centrifugation removal cell fragment, it is to obtain recombinant baculovirus Ac-Fiber2 to collect supernatant；
5) Ankara mixing subunit vaccine is prepared using recombinant baculovirus Ac-Penton and Ac-Fiber2
A., recombinant baculovirus Ac-Penton and Ac-Fiber2 are inoculated in the insect cell SF9 of the culture that suspends respectively
In, culture is collected by centrifugation cell respectively；
B. ultrasonication cracking takes supernatant to above-mentioned cell respectively, and affinitive layer purification is reclaimed, and then flushed post is washed respectively
It is de-, the albumen Penton and Fiber2 of purifying are respectively obtained,
C. albumen Penton and Fiber2 that step b) is purified are diluted to same concentrations 300-800 μ g/ml respectively, will be dilute
The albumen Penton and Fiber2 for releasing by volume 1:Pressed after 1 mixing with adjuvant by weight 2:7~1:2 are well mixed, and obtain
Ankara mixing subunit vaccine.
Preferably, the adjuvant is MontanideTMISA 71 VG。
Applied in the disease of preventing and treating Ankara present invention also offers a kind of above-mentioned Ankara mixing subunit vaccine.
The beneficial effects of the present invention are：
The present invention provides a kind of baculovirus transfer vector pFastBac HTB-Penton and baculoviral restructuring poison Ac-
Penton and using baculovirus expression system expression Penton protein subunits seedling and with Penton albumen and Fiber2
Albumen mixing subunit vaccine.The main antigenic albumen Penton albumen of a large amount of efficient expression Ankaras, this albumen
Expression quantity is high, and protein active is good, vaccine with Penton albumen as antigen and with Penton albumen and Fiber2 albumen as antigen
Mixing seedling Ankara can be reached 100% protective rate.And the antibody of generation is high, length of holding time.
Brief description of the drawings
Fig. 1 is identified for the amplification of purpose gene Fiber2；The amplification of M.DNA marker (DL2000) 1~2.Fiber2；
Fig. 2 is identified for the amplification of purpose gene Penton；The amplification of M.DNA marker (DL2000) 1~2.Penton；
Fig. 3 is identification of the picking hickie to genes of interest Penton and Fiber2 in Bacmid；Genes of interest is used respectively
The sense primer and M13 anti-sense primers of Penton and Fiber2 are expanded and identified, M.DNA marker (DL5000), 1~
The identification of 2.Penton；The identification of 3~4.Fiber2；
Fig. 4 is mirror of the P3 for genes of interest Penton and Fiber2 in recombinant baculovirus Ac-Penton and Ac-Fiber2
Fixed figure；
In figure, M is the identification of DL5000 DNA Marker, 1~3.Penton；The identification of 4~6.Fiber2；
Fig. 5 is P3 for identifications of the recombinant baculovirus Ac-Penton and Ac-Fiber2 without wild poison pollution；
In figure, M is DL5000 DNA Marker, without the identification of wild poison pollution, 3~4.Ac- in 1~2.Ac-Penton
Without the identification of wild poison pollution in Fiber2, the 5. positive control of negative control 6.；
Fig. 6 is to identify that P3 generation restructuring poison Ac-Penton and Ac-Fiber2 infection Sf9 is thin by western-blotting
The expression figure of destination protein Penton and Fiber2 albumen in born of the same parents；
In figure, 1,3 is ProteinMarker, and 2 is Penton albumen, 4.Fiber2 albumen；
Fig. 7 is the expression figure of middle indirect immunofluorescence experiment testing goal albumen Penton, Fiber2；
Fig. 8 is to connect the excellent figure of toxic agent amount to the optimal of the expression of Penton albumen；
In figure, 1 is Protein Marker, 2-7 be various dose (0.1MOI, 0.2MOI, 0.5MOI, 1MOI, 2MOI,
P3 generation restructuring poison Ac-Penton infection Sf9 cells 5MOI) obtain the relative level of destination protein；
Fig. 9 is most preferably to connect malicious injectivity optimizing figure to the expression of Fiber2 albumen；
In figure, 1 is Protein Marker, 2-7 be various dose (0.1MOI, 0.2MOI, 0.5MOI, 1MOI, 2MOI,
P3 generation restructuring poison Ac-Fiber2 infection Sf9 cells 5MOI) obtain the relative level of destination protein；
Figure 10 is that Immunization group and blank attack malicious control group lesion comparison diagram；
Figure 11 is that Immunization group and blank attack malicious control group heart pathology slice map comparison diagram；
Figure 12 is that Immunization group and blank attack malicious control group hepatic pathology section figure comparison diagram；
Figure 13 is that Penton albumen is antibody detection S/P value broken line graphs after the vaccine immunity of antigen；
Figure 14 is that Penton and Fiber2 albumen is the immune rear antibody detection S/P value broken line graphs of the polyvalent vaccine of antigen；
The wing venous blood sampling adenovirus commodity examination in the 7th, 14,21,28,35,42,49,56,63,70 days after head exempts from
Agent box detects serum S/P values rule (S/P value >=0.5 is judged to the positive, and S/P≤0.499 is judged to feminine gender).
Figure 15 is pFastBac HTB plasmid vector collection of illustrative plates；
Figure 16 is pFastBac HTB plasmid vector MCS figures.
Specific embodiment
In order to preferably explain the present invention, below in conjunction with the specific embodiment main contents that the present invention is furture elucidated, but
Present disclosure is not limited solely to following examples.
Embodiment 1：The acquisition of recombinant baculovirus Ac-Penton
The acquisition of 1.Penton genes
The extraction of Ankara DNA：
The extracting of viral DNA uses SDS- Proteinase Ks-phenol/chloroform method, that is, take 600 μ L virus liquids, 4 DEG C,
12000rpm/min is centrifuged 10min, and the Proteinase K and 50 μ L 10%SDS for taking 425 μ supernatants addition, 20 μ L25mg/mL make albumen
Enzyme K and SDS ultimate density are 1mg/ml and 1%.56 DEG C of water-bath 1h and shake frequently；Add isometric phenol:Chloroform:Isoamyl alcohol
(25:24:1) fully mix, 4 DEG C of 10000rpm, 15min is centrifuged.Supernatant is taken, adds isometric chloroform fully to mix in supernatant
Even 4 DEG C of 10000rpm, centrifugation 15min take supernatant and add the NaAc (3M, pH 5.0) of 1/10 volume and anhydrous more than 2 times of volumes
Ethanol is mixed, and -20 DEG C of precipitation 2h, 12000rpm/min centrifugation 15min remove supernatant, and it is pure that precipitation drying is diluted in into 20 μ L sterilizings
In water, -20 DEG C of preservations.
Design primer：
Penton-F：5'-AGCGGATCCATGTGGGGGTTGCAGCCG-3',
BamHI
Penton-R：5'-GGTAAGCTTCTACTGCAAGGTCGCGGAACTC-3'；
HindⅢ
DNA with Ankara enters performing PCR as template, and amplification recovery obtains containing restriction enzyme site BamHI/Hind III
Penton genes；The strain is named as I group I fowl adenovirus strain FAdv-HB, Chinese Wuhan is delivered on January 3rd, 2017 military
The Chinese university China typical culture collection center (CCTCC) preservation, its preserving number is：CCTCC NO：V201703.
PCR system：
Above-mentioned reaction system is mixed into laggard performing PCR amplification, the loop parameter of reaction is:94 DEG C of predegeneration 5min, PCR are followed
Ring is 95 DEG C of 30s, 50 DEG C of annealing 30s, 68 DEG C of 1min；After 30 circulations, 68 DEG C of extension 10min.After total overall reaction terminates, by institute
Some PCR primers carry out 1.0% agarose gel electrophoresis, detect that the amplification of PCR primer obtains correct as shown in Figure 2
Penton gene orders.
2. the structure of recombinant plasmid pFastBac HTB-Penton
With carrier pFastBac HTB as skeleton, with digestion with restriction enzyme carrier pFastBac HTB and contain digestion
The Penton genes of the Penton genes of site BamHI/Hind III, the pFastBac HTB for being linearized and linearisation, use
PFastBac HTB and the Penton genes connection of linearisation that ligase will be linearized, obtain recombinant plasmid pFastBac
HTB-Penton。
Double digestion system：
Double digestion condition：37 DEG C of endonuclease reaction 3h after above-mentioned system is mixed, taking 10 μ L digestion products carries out 1% agarose
Gel electrophoresis is identified.
Linked system
Condition of contact：4 DEG C connect overnight after above-mentioned linked system is mixed.
3. the acquisition and extraction of Bacmid-Penton are recombinated
The acquisition of 3.1 Bacmid-Penton
To identify that correct pFastBac HTB-Penton restructuring donor plasmid conversion DH10Bac E.coli competence is thin
Born of the same parents, cultivate in LB fluid nutrient mediums, 37 DEG C of 220rpm/min vibration culture 4h, with the LB fluid nutrient mediums of non-resistant by bacterium solution
Do 101、102、103Times dilute, be coated on containing gentamicin (7 ㎎/L), kanamycins (50 ㎎/L), tetracycline (10 ㎎/L),
Cultivated on IPTG (40 ㎎/L), the LB agar plates of X-gal (100 ㎎/L), 37 DEG C of culture 8h, the single white colony inoculation of picking
Containing in gentamicin (7 ㎎/L), kanamycins (50 ㎎/L), the LB fluid nutrient mediums of tetracycline (10 ㎎/L), 37 DEG C
220rpm/min vibration culture shaking overnight incubations, extract restructuring rod granule
Under the specific forward primer and M13 of Penton genes primer pair its carry out purpose identification, identification correct result is such as
The restructuring rod granule Bacmid-Penton of gene is obtained shown in Fig. 3 while the single locus coeruleus bacterium colony of picking, is used as negative control.
3.2 Bacmid-Penton extracting methods
1) bacterium solution 1.5mL, 8000rpm/min the centrifugation 5min collects thallines of incubated overnight；
2) the resuspended thalline of 300 μ L solution I is added, is added after 3-5min is placed on ice, 300 μ L solution II are overturned and mixed, ice
Upper standing 2-5min；The solution III of 300 μ L precoolings is added, is overturned and is mixed, 10min is stood on ice；
3) 4 DEG C of centrifugation 15min of 12000rpm/min, take supernatant repeated centrifugation once, then take the supernatant isometric precooling of addition
Isopropanol, overturn mix, be placed in -20 DEG C precipitation 30min；
4) 4 DEG C of centrifugation 15min of 12000rpm/min, abandon supernatant and precipitation are washed into twice rear chamber with the ethanol of 75% precooling
Temperature is dried, and adds 37 DEG C of dissolving 30min of appropriate aseptic RNase water to be saved backup after -80 DEG C；Solution I：50mmol/L grapes
Sugar, 10mmol/LEDTA, 25mmol/LTris-Cl (PH8.0)；
Solution II：0.2M NaOH:1%SDS=1：1 is now with the current；
Solution III：3M potassium acetates, pH is 5.5.
4.Bacmid transfection Sf9 cells obtain recombinating malicious Ac-Penton
The method that 4.1 Bacmid transfect Sf9 cells：
1) take 1 μ gBacmid mixed in 100 μ L Grace culture mediums be named as A liquid stand 10min；
2) gently mixing is named as B liquid standing 10min in 94 μ LGrace culture mediums to take 6 μ L Cellfectin；
3) A liquid and B liquid are mixed, was flicked under 15 every 5 minutes, 15min is stood after 3-4 times, make Cellfectin abundant
Contact parcel Bacmid；
4) to 800 μ LGrace culture mediums are added in mixed liquor, gently piping and druming is mixed；
5) it is 2 × 10 to take culture in six orifice plate Midst densities6The Sf9 cells of cells/ml, discard culture medium, add aseptic
PBS jogs are washed and add 1mLGrace culture mediums afterwards twice；
6) mixed liquor is uniformly added into six orifice plates, makes cell culture medium cumulative volume for 2mL, be put into 26 DEG C of incubators and incubate
Educate 5h；
7) discard cells and supernatant and Grace culture mediums of the 2mL containing 10%FBS is added per hole, wherein containing the μ L of antibiotic 20
It is placed in after gentamicin and 20 μ L streptomysins in 26 DEG C of incubators and cultivates 72-96h until cell 75% occurs lesion；
8) centrifugation discards cell fragment and takes culture supernatant as P1 for recombinant baculovirus.
9) by P1 generation restructuring poison two generations of continuous amplification obtain P3 generation restructuring poison Ac-Penton carry out the identification of genes of interest with
As shown in Figure 4 and Figure 5, we have obtained correct recombinant baculovirus and without wild poison pollution for the identification of wild poison pollution.
Embodiment 2：The expression of recombinant protein Penton, quantitative and purifying
The expression of 1.Western Blotting testing goal albumen Penton
P3 is split for the cell and normal cell cell of the cell life type baculovirus infection of recombinate shape virus infection
Solution liquid cracks 30min and adds 5 × SDS Loading Buffer on ice, and wink from carrying out according to a conventional method after boiling water boiling 5-8min
The SDS-PAGE electrophoretic analysis of 10% separation gel and 5% concentration glue, is transferred to 5% skimmed milk power on pvdf membrane, and 4 DEG C were closed
At night, add 1:The His label primary antibodies of 5000 dilutions, rocked at room temperature 2-3h, TBST wash 3 times additions 1:The AP marks of 5000 dilutions
Rabbit-anti mouse secondary antibody, TBST is washed 3 times after rocked at room temperature 1h, is developed the color using NBT/BCIP systems.Observation specific protein band, shows
Penton albumen has been expressed.(as shown in Figure 6)
2. the expression of indirect immunofluorescence experiment testing goal albumen Penton
It is 1 × 10 that Sf9 cells are cultivated to density on 24 orifice plates6Then this is infected with P3 generation restructuring poison AcPenton thin
Culture supernatant PBS is discarded after 26 DEG C of culture 72h of born of the same parents to wash twice, add 4% paraformaldehyde to fix 10min, discard fixer PBS
Wash 3 times and shake every time and rock 5min, PBS is washed 3 times after adding 1%Triton to change 10min thoroughly, after adding 1%BSA closings 30min
Discard confining liquid PBS and wash 3 times and shake every time and rock 5min, 1%BSA1 is used in addition:The His label primary antibody room temperatures of 1000 dilutions are incubated
Educate supernatant discarded PBS after 2h wash 3 times every time concussion rock 5min, add and dilute the rabbit-anti mouse that FITC is marked with 1%BSA
Supernatant discarded PBS washes 3 times and shakes every time and rocks 5min after IgG lucifuges concussion 30min, adds 500 μ LPBS aobvious in fluorescence per hole
Micro- Microscopic observation, it is control synchronously to set up normal Sf9 cells and wild-type baculovirus infection Sf9 cells.Show destination protein
Penton successes are expressed (as shown in Figure 7) in rhabdovirus system.
3. the optimization of protein expression condition
With the P3 generation restructuring poison Ac- of the difference such as different 0.1MOI, 0.2MOI, 0.5MOI, 1MOI, 2MOI, 5MOI MOI
Penton infect respectively healthy Sf9 cells 27 DEG C be incubated 72-96 hour after observe lesion up to 75% receipts sample 3500rpm/min from
Heart 10min, discards nutrient solution.Collected cell ultrasonication 12000rpm/min centrifugations 10min is taken into cracking supernatant to utilize
Western-blotting determines that optimum expression connects poison amount (as shown in Figure 8) and can show that the minimum of Penton albumen connects toxic agent amount
It is to plant poison 1MOI in P3 generations.
4th, the purifying of the Amplification Culture of recombinant baculovirus Ac-Penton and Penton albumen
With P3 for recombinant baculovirus as kind of a poison, when Sf9 insect cells are in logarithm production period, (cell density is 1-2
×106Cells/ml) virus inoculation, makes viral infection multiplicity MOI be 1.Health Sf9 cells are infected with restructuring poison Ac-Penton
Lesion is observed after being incubated 72-96 hours at 27 DEG C and receives sample 3500rpm/min centrifugation 10min up to 75%, discard nutrient solution.
Collected cell ultrasonication 12000rpm/min centrifugations 10min is taken into cracking supernatant.It is situated between using affinity chromatography
The NTA of matter Ni mono- carry out purifying recovery to destination protein.
Detailed process is by cell liquid BindingBuffer dilutions, upper prop；Again post is rinsed with BingdingBuffer
Son, foreigh protein removing；ElutionBuffer is eluted, and collects eluting peak；Lived again after wash-out pillar.Albumen after purification is diluted to
500μg/ml。
Embodiment 3：The composition of Ankara subunit vaccine oil emu, prepare and detect
Antigen：The destination protein Penton of 500 μ g/ml purifying
Adjuvant：The VG water-in-oil types (W/O) of ISA 71
Antigen and adjuvant qualities ratio：3：7
It is prepared by vaccine：The destination protein Penton of purifying is diluted to 500 μ g/ml and adds container high-ranking officer according to the above ratio again
The VG of agent adjuvant ISA 71 are emulsified in point dripping to container.
Vaccine proterties is detected：During vaccine instills clear water after emulsification, the first drop is scattered, the second oil dripping pearl, rocks liquid level oil
Pearl is not demulsified.
Sterility testing：A little vaccine coating TSA plates are taken, 37 DEG C of incubator cultures grow for 18 hours without bacterium colony.
Stability test：3000rpm/min is centrifuged 15 minutes, vaccine is emulsified after centrifugation and is not layered.
Embodiment 4：The protection detection and antibody variation experiment of the immune SPF chickens of Ankara subunit vaccine
SPF chickens (purchased from Beijing Cimmeria) 40 are taken, 2 groups every group 20 is randomly divided into and is immunized only according to table 1.
1st group：Penton groups；
2nd group：Healthy Sf9 cells crack supernatant (control group).Specific immunization protocol is referring to table 1
2 week old head of chicken group exempt from, and 4 week old two are exempted from, and every group random take 10 and attack poison during 6 week old.Specific immunizing dose, antigen contains
Amount, adjuvant, protective rate is shown in Tables 1 and 2.Every group remaining 10 are exempted from the 7th, 14,21,28,35,42,49,56,63,70 day afterwards in one
Blood sampling BioChek 1 group of (FADV-1) ELISA detection kit (product code of aviadenovirus：CK132) detection antibody growth and decline rule
Rule.S/P value >=0.5 is judged to the positive, and S/P≤0.499 is judged to that negative (as shown in figure 13) visible this vaccine not only protects effect
Fruit is good and produces antibody fast, and the duration is long.
The immune packet of table 1, every group of antigenic content, immunizing dose, mode, number of times etc.
The Immunization protective rate of table 2
According to immunization protocol as shown in table 1, we obtain Ankara subunit vaccine pair with Penton albumen as antigen
Ankara has 100% protective rate.The chicken of malicious control group is attacked to Immunization group and blank and attacks to enter respectively after poison
Row is dissected and finds that blank attacks that malicious control group has obvious hydropericardium and hepatic disease extravasated blood and Immunization group has no substantially different
Often (as shown in Figure 10), malicious control group chicken dirty and liver of coring is attacked to Immunization group and blank in addition to do pathological section and also send out
Existing blank is attacked poison and compareed the agglomerating erythrocyte aggregation of obvious cardiac muscle cell, mainly lymphocyte and fibroblast, on a small quantity
Multinucleate giant cell, central vessel wall smooth muscle cell necrosis (as shown in figure 11).Sinus hepaticus mild hyperaemia, has between portal area and tissue
Assemble pockets of lymphocyte, part cell has cardiac muscle cell and the liver cell of basophilla intranuclear inclusion and Immunization group
Equal Non Apparent Abnormality (as shown in figure 12).
Embodiment 5：The acquisition of recombinant baculovirus Ac-Fiber2
The acquisition of 1.Fiber2 genes
The extraction of Ankara DNA：
The extracting of viral DNA uses SDS- Proteinase Ks-phenol/chloroform method, that is, take 600 μ L virus liquids, 4 DEG C,
12000rpm/min is centrifuged 10min, and the Proteinase K and 50 μ L 10%SDS for taking 425 μ L of supernatant addition, 20 μ L 25mg/mL make egg
White enzyme K and SDS ultimate density is 1mg/ml and 1%.56 DEG C of water-bath 1h and shake frequently；Add isometric phenol:Chloroform:Isoamyl
Alcohol (25:24:1) fully mix, 4 DEG C of 10000rpm, 15min is centrifuged.Supernatant is taken, adds isometric chloroform abundant in supernatant
4 DEG C of 10000rpm are mixed, centrifugation 15min takes supernatant and adds the NaAc (3M, pH 5.0) of 1/10 volume and more than 2 times of nothings of volume
Water-ethanol is mixed, and -20 DEG C of precipitation 2h, 12000rpm/min centrifugation 15min remove supernatant, and precipitation drying is diluted in into 20 μ L sterilizings
In pure water, -20 DEG C of preservations.
Design primer：
Fiber2-F：5’-AGCTCTAGAATGCTCCGGGCCCCTAAA-3 ',
Xba Ⅰ
Fiber2-R：5’-GGTAAGCTTTTACGGGAGGGAGGCCG-3’
HindⅢ
DNA with Ankara enters performing PCR as template, and amplification recovery obtains containing I/Hind of restriction enzyme site Xba III
Penton genes；
PCR system：
Above-mentioned reaction system is mixed into laggard performing PCR amplification, the loop parameter of reaction is:94 DEG C of predegeneration 5min, PCR are followed
Ring is 95 DEG C of 30s, 50 DEG C of annealing 30s, 68 DEG C of 1min；After 30 circulations, 68 DEG C of extension 10min.After total overall reaction terminates, by institute
Some PCR primers carry out 1.0% agarose gel electrophoresis, detect that the amplification (as shown in Figure 1) of PCR primer is obtained correctly
Fiber2 gene orders.
2. the structure of recombinant plasmid pFastBac HTB-Fiber2
With carrier pFastBac HTB as skeleton, with digestion with restriction enzyme carrier pFastBac HTB and contain digestion
The Fiber2 genes of the Penton genes of site BamHI/Hind III, the pFastBac HTB for being linearized and linearisation, use
PFastBac HTB and the Fiber2 genes connection of linearisation that ligase will be linearized, obtain recombinant plasmid pFastBac
HTB-Fiber2。
Double digestion system：
Double digestion condition：37 DEG C of endonuclease reaction 3h after above-mentioned system is mixed, taking 10 μ L digestion products carries out 1% agarose
Gel electrophoresis is identified.
Linked system
Condition of contact：4 DEG C connect overnight after above-mentioned linked system is mixed.
3. the acquisition and extraction of Bacmid-Fiber2 are recombinated
The acquisition of 3.1 Bacmid-Fiber2
To identify that correct pFastBac HTB-Fiber2 restructuring donor plasmid conversion DH10Bac E.coli competence is thin
Born of the same parents, cultivate in LB fluid nutrient mediums, 37 DEG C of 220rpm/min vibration culture 4h, with the LB fluid nutrient mediums of non-resistant by bacterium solution
Do 101、102、103Times dilute, be coated on containing gentamicin (7 ㎎/L), kanamycins (50 ㎎/L), tetracycline (10 ㎎/L),
Cultivated on IPTG (40 ㎎/L), the LB agar plates of X-gal (100 ㎎/L), 37 DEG C of culture 8h, the single white colony inoculation of picking
Containing in gentamicin (7 ㎎/L), kanamycins (50 ㎎/L), the LB fluid nutrient mediums of tetracycline (10 ㎎/L), 37 DEG C
220rpm/min vibration culture shaking overnight incubations, extract restructuring rod granule
Under the specific forward primer and M13 of Fiber2 genes primer pair its carry out purpose identification, identification correct result is such as
The restructuring rod granule Bacmid-Fiber2 of gene is obtained shown in Fig. 3 while the single locus coeruleus bacterium colony of picking, is used as negative control.
3.2 Bacmid-Fiber2 extracting methods
1) bacterium solution 1.5mL, 8000rpm/min the centrifugation 5min collects thallines of incubated overnight；
2) the resuspended thalline of 300 μ L solution I is added, is added after 3-5min is placed on ice, 300 μ L solution II are overturned and mixed, ice
Upper standing 2-5min；The solution III of 300 μ L precoolings is added, is overturned and is mixed, 10min is stood on ice；
3) 4 DEG C of centrifugation 15min of 12000rpm/min, take supernatant repeated centrifugation once, then take the supernatant isometric precooling of addition
Isopropanol, overturn mix, be placed in -20 DEG C precipitation 30min；
4) 4 DEG C of centrifugation 15min of 12000rpm/min, abandon supernatant and precipitation are washed into twice rear chamber with the ethanol of 75% precooling
Temperature is dried, and adds 37 DEG C of dissolving 30min of appropriate aseptic RNase water to be saved backup after -80 DEG C；Solution I：50mmol/L grapes
Sugar, 10mmol/L EDTA, 25mmol/L Tris-Cl (PH8.0)；
Solution II：0.2M NaOH:1%SDS=1：1 is now with the current；
Solution III：3M potassium acetates, pH is 5.5.
4.Bacmid transfection Sf9 cells obtain recombinating malicious Ac-Fiber2
The method that 4.1 Bacmid transfect Sf9 cells：
1) take 1 μ gBacmid mixed in 100 μ L Grace culture mediums be named as A liquid stand 10min；
2) gently mixing is named as B liquid standing 10min in 94 μ LGrace culture mediums to take 6 μ L Cellfectin；
3) A liquid and B liquid are mixed, was flicked under 15 every 5 minutes, 15min is stood after 3-4 times, make Cellfectin abundant
Contact parcel Bacmid；
4) to 800 μ LGrace culture mediums are added in mixed liquor, gently piping and druming is mixed；
5) it is 2 × 10 to take culture in six orifice plate Midst densities6The Sf9 cells of cells/ml, discard culture medium, add aseptic
PBS jogs are washed and add 1mLGrace culture mediums afterwards twice；
6) mixed liquor is uniformly added into six orifice plates, makes cell culture medium cumulative volume for 2mL, be put into 26 DEG C of incubators and incubate
Educate 5h；
7) discard cells and supernatant and Grace culture mediums of the 2mL containing 10%FBS is added per hole, wherein containing the μ L of antibiotic 20
It is placed in after gentamicin and 20 μ L streptomysins in 26 DEG C of incubators and cultivates 72-96h until cell 75% occurs lesion；
8) centrifugation discards cell fragment and takes culture supernatant as P1 for recombinant baculovirus.
9) are obtained into P3 generation restructuring poison P1 generation restructuring poison two generations of continuous amplification carries out the identification and wild poison pollution of genes of interest
Identification as shown in Figure 4 and Figure 5, we have obtained correct recombinant baculovirus Ac-Fiber2 and without wild poison pollution.
The expression of the recombinant protein Fiber2 of embodiment 6, quantitative and purifying
The expression of 1.Western Blotting testing goal albumen Fiber2
P3 is split for the cell and normal cell cell of the cell life type baculovirus infection of recombinate shape virus infection
Solution liquid cracks 30min and adds 5 × SDS Loading Buffer on ice, and wink from carrying out according to a conventional method after boiling water boiling 5-8min
The SDS-PAGE electrophoretic analysis of 10% separation gel and 5% concentration glue, is transferred to 5% skimmed milk power on pvdf membrane, and 4 DEG C were closed
At night, add 1:The His label primary antibodies of 5000 dilutions, rocked at room temperature 2-3h, TBST wash 3 times additions 1:The AP marks of 5000 dilutions
Rabbit-anti mouse secondary antibody, TBST is washed 3 times after rocked at room temperature 1h, is developed the color using NBT/BCIP systems.Observation specific protein band, shows
Fiber2 albumen correctly expresses (as shown in Figure 6).
2. the expression of indirect immunofluorescence experiment testing goal albumen Fiber2
It is 1 × 10 that Sf9 cells are cultivated to density on 24 orifice plates6Then this is infected with P3 generation restructuring poison AcPenton thin
Culture supernatant PBS is discarded after 26 DEG C of culture 72h of born of the same parents to wash twice, add 4% paraformaldehyde to fix 10min, discard fixer PBS
Wash 3 times and shake every time and rock 5min, PBS is washed 3 times after adding 1%Triton to change 10min thoroughly, after adding 1%BSA closings 30min
Discard confining liquid PBS and wash 3 times and shake every time and rock 5min, add and use 1%BSA 1:The His label primary antibody room temperatures of 1000 dilutions are incubated
Educate supernatant discarded PBS after 2h wash 3 times every time concussion rock 5min, add and dilute the rabbit-anti mouse that FITC is marked with 1%BSA
Supernatant discarded PBS washes 3 times and shakes every time and rocks 5min after IgG lucifuges concussion 30min, adds 500 μ LPBS aobvious in fluorescence per hole
Micro- Microscopic observation, it is control synchronously to set up normal Sf9 cells and wild-type baculovirus infection Sf9 cells.Show destination protein
Fiber2 successes are expressed (as shown in Figure 7) in rhabdovirus system.
3. the optimization of protein expression condition
With the P3 generation restructuring poison Ac- of the difference such as different 0.1MOI, 0.2MOI, 0.5MOI, 1MOI, 2MOI, 5MOI MOI
Fiber2 infect respectively healthy Sf9 cells 27 DEG C be incubated 72-96 hour after observe lesion up to 75% receipts sample 3500rpm/min from
Heart 10min, discards nutrient solution.Collected cell ultrasonication 12000rpm/min centrifugations 10min is taken into cracking supernatant to utilize
Western-blotting determines that optimum expression connects malicious amount.(as shown in Figure 9) can show that the minimum of Fiber2 albumen connects toxic agent
Amount is to plant poison 2MOI in P3 generations.
4th, the purifying of the Amplification Culture of recombinant baculovirus Ac-Fiber2 and Fiber2 albumen
With P3 for recombinant baculovirus as kind of a poison, when Sf9 insect cells are in logarithm production period, (cell density is 1-2
×106Cells/ml) virus inoculation, makes viral infection multiplicity MOI be 1.Health Sf9 cells are infected with restructuring poison Ac-Penton
Lesion is observed after being incubated 72-96 hours at 27 DEG C and receives sample 3500rpm/min centrifugation 10min up to 75%, discard nutrient solution.
Collected cell ultrasonication 12000rpm/min centrifugations 10min is taken into cracking supernatant.It is situated between using affinity chromatography
The NTA of matter Ni mono- carry out purifying recovery to destination protein.
Detailed process is by cell liquid BindingBuffer dilutions, upper prop；Again post is rinsed with BingdingBuffer
Son, foreigh protein removing；ElutionBuffer is eluted, and collects eluting peak；Lived again after wash-out pillar.Albumen after purification is diluted to
500μg/ml。
The composition of Ankara mixing subunit vaccine oil emu of embodiment 7, prepare and detect
Antigen：The destination protein Fiber21 of destination protein Penton and 500 μ the g/ml purifying of 500 μ g/ml purifying:1 mixes
Close
Adjuvant：The VG water-in-oil types (W/O) of ISA 71
Antigen and adjuvant qualities ratio：3：7
It is prepared by vaccine：The destination protein Penton and Fiber2 of purifying are diluted to 500 μ g/ml and by 1 respectively:1 adds
Container compares 3 by antigen and adjuvant qualities again：7 the VG of adjuvant adjuvant ISA 71 point are dripped into container in emulsified.
Vaccine proterties is detected：During vaccine instills clear water after emulsification, the first drop is scattered, the second oil dripping pearl, rocks liquid level oil
Pearl is not demulsified.
Sterility testing：A little vaccine coating TSA plates are taken, 37 DEG C of incubator cultures grow for 18 hours without bacterium colony.
Stability test：3000rpm/min is centrifuged 15 minutes, vaccine is emulsified after centrifugation and is not layered.
The protection detection and antibody variation experiment of the immune SPF chickens of Ankara subunit vaccine of embodiment 8
SPF chickens (purchased from Beijing Cimmeria) 40 are taken, 2 groups every group 20 is randomly divided into and is only immunized, specific immunization wayses
Referring to table 1.
1st group：Mixing seedling (Penton+Fiber2) group；
2nd group：Healthy Sf9 cells crack supernatant (control group).Specific immunization protocol is referring to table 1
2 week old head of chicken group exempt from, and 4 week old two are exempted from, and every group random take 10 and attack poison during 6 week old.Specific immunizing dose, antigen contains
Amount, adjuvant, protective rate is shown in Table 3 and table 4.Every group remaining 10 are exempted from the 7th, 14,21,28,35,42,49,56,63,70 day afterwards in one
Blood sampling BioChek 1 group of (FADV-1) ELISA detection kit (product code of aviadenovirus：CK132) detection antibody growth and decline rule
Rule.S/P value >=0.5 is judged to the positive, and S/P≤0.499 is judged to feminine gender.(as shown in figure 13) visible this vaccine not only protects effect
Fruit is good and produces antibody fast, and the duration is long.
The immune packet of table 3, every group of antigenic content, immunizing dose, mode, number of times etc.
The Immunization protective rate of table 4
Ankara that we are obtained with Penton+Fiber2 albumen as antigen according to immunization protocol as shown in table 3 is sub- single
Position polyvalent vaccine has 100% protective rate (as shown in table 4) to Ankara.Malicious control group is attacked to Immunization group and blank
Chicken find that blank is attacked malicious control group and has obvious hydropericardium and hepatic disease extravasated blood with attacking to carry out dissecting respectively after poison
And Immunization group shows no obvious abnormalities (as shown in Figure 10), malicious control group chicken is attacked to Immunization group and blank in addition and is cored
Dirty and liver does pathological section it has also been found that blank attacks poison control the agglomerating erythrocyte aggregation of obvious cardiac muscle cell, mainly lymph
Cell and fibroblast, a small amount of multinucleate giant cell, central vessel wall smooth muscle cell necrosis (as shown in figure 11).Sinus hepaticus is slight
Hyperemia, there is a pockets of lymphocyte of aggregation between portal area and tissue, part cell has a basophilla intranuclear inclusion and Immunization
The cardiac muscle cell and liver cell equal Non Apparent Abnormality (as shown in figure 12) of group.
Other unspecified parts are prior art.Although above-described embodiment is made that to the present invention and retouch in detail
State, but it is only a part of embodiment of the invention, rather than whole embodiments, people can also according to the present embodiment without
Other embodiment is obtained under the premise of creativeness, these embodiments belong to the scope of the present invention.
<110>Hua Zhong Agriculture University
<120>The preparation method and application of Ankara subunit vaccine
<211>1578bp
<212> DNA
<213>Penton genes
<400> 1
atgtgggggttgcagccgccgacgtcgattccgccgcctcctccgccgaccgagttaacgccctcgacctatccggcgat
ggtgaacggctatccgcctccggccgcgtccgcgcagagctgtccctctagcgacggtcagagcgagctgtatatgcccc
ttcagcgggtgatggcccctacggggggacggaacagcattaagtatcgcgattacacgccgtgtcgtaacaccaccaag
ctgttttacgtagacaacaaggctagcgatatcgatacgtataacaaagacgccaaccatagcaatttccgcaccacggt
gatccataaccaggatctggacgcggacacggccgccaccgagtccatccagttggacaaccgctcctgctggggcggcg
acctaaaaacagccgtgcgcaccaactgcccgaacgtgagcagttttttccagagtaacagcgtgcgcgtgcgcatgatg
tggaagcgcgacccgccgactagcacggctcctccgagcgcggtaggcagcggctattcggtgcccggcgcgcagtacaa
gtggtacgacctgacgatacccgagggtaactacgcgctgtgcgaactgatagacctgctcaacgagggcatcgtgcagc
tctacctgagcgaggggcgccagaacaacgtgcaaaaatcggacatcggggtcaagttcgacacgcgcaacttcggcttg
ctccgcgaccccgtgacgggactggtaactccgggcacgtacgtgtacaagggttaccaccccgacatcgtgctgctgcc
cggatgcgcgatcgactttacgtacagccgcctgagcctgctcctgggcatagggaagcgcgagccctactcgaaggggt
tcgttattacctacgaggatctgcagggaggggatatcccggctctgctggacctcgactccgtcgacgtgaacgacgct
gacggtgaagtgatcgagctcgacaacgctgctccccttttacatgacagcgcgggcgtgtcgtataacgtcatttacga
ccaggtgacgggtaaacccgtgacggtgtatcgatcgtggatgttggcttacaacgtgcctaactcgccggccaatcaga
cgaccttgctgacggtgcccgatatggcgggcgggatcggggcgatgtacacgtccctgcccgatacctttatcgcgcct
accgggttcaaggaagataacacgaccaacctttgcccggtcgtcggcatgaacctgttccccacctacaataaagttta
ttaccaggcggcgtccacgtacgtgcagcgcctggaaaattcctgccagtcggccacagccgccttcaaccgctttcccg
aaaacgagattctgaagcaagcgccccccatgaatgtttcgtccgtgtgcgataaccaacccgccgtcgttcagcagggt
gtgttgcctgtgaagacctcgctccccggactgcagcgcgtgctgatcacagacgaccagcgtcgtccgataccctacgt
gtataagtctatcgcgacggttcagccgaccgttctgagttccgcgaccttgcagtag
<210>2
<211> 1440bp
<212> DNA
<213>Fiber2 genes
<400> 2
atgctccgggcccctaaaagaagacattccgaaaacgggaagcccgagaccgaagcgggaccttccccggctccaatcaa
gcgcgccaaacgcatggtgagagcatcccagcttgacctggtttatcctttcgattacgtggccgaccccgtcggagggc
tcaacccgccttttttgggaggctcaggacccctagtggaccagggcggacagcttacgctcaacgtcaccgatcccatc
atcatcaagaacagatcggtggacttggcccacgaccccagtctcgatgtcaacgcccaaggtcaactggcggtggccgt
tgaccccgaaggggccctggacatcacccccgatggactggacgtcaaggtcgacggagtgaccgtaatggtcaacgatg
actgggaactggccgtaaaagtcgacccgtccggcggattggattccaccgcgggtggactgggggtcagcgtggacgac
accttgctcgtggatcagggagaactgggcgtacacctcaaccaacaaggacccatcactgccgatagcagtggtatcga
cctcgagatcaatcctaacatgttcacggtcaacacctcgaccggaagcggagtgctggaactcaacctaaaagcgcagg
gaggcatccaagccgacagttcgggagtgggcgtttccgtggatgaaagcctacagattgtcaacaacactctggaagtg
aaaccggatcccagcggaccgcttacggtctccgccaatggcctagggctgaagtacgacactaataccctagcggtgac
cgcgggcgctttaaccgtggtcggaggggggagcgtctccacacccatcgctacttttgtctcgggaagtcccagcctca
acacctacaatgccacgaccgtcaattccagcgcgaacgccttctcttgcgcctactaccttcaacagtggaacatacag
gggctccttgttacctccctctacttgaaattggacagcgccaccatggggaatcgccctggggacctcaactccgccaa
tgccaaatggttcaccttttgggtgtccgcctatctccagcaatgcaacccctccgggattcaagcgggaacggtcagcc
cctccaccgccaccctcacggactttgaacccatggccaataggagcgtgaccagcccatggacgtactcggccaatgga
tactatgaaccatccatcggggaattccaagtgttcagcccggtggtaacaggtgcctggaacccgggaaacatagggat
ccgcgtcctccccgtgccggtttcggcctccggagagcgatacacccttctatgctatagtctgcagtgcacgaacgcga
gcatttttaatccaaacaacagcggaaccatgatcgtgggacccgtgctctacagctgtccagcggcctccctcccgtaa
Claims (10)
1. a kind of recombinant plasmid pFastBac HTB-Penton, it is characterised in that：The recombinant transfer plasmid pFastBac
HTB-Penton is that insertion Penton is gene constructed between III two restriction enzyme sites of BamHI and Hind of carrier pFastBac HTB
Form, wherein, the nucleotide sequence of Penton genes is as shown in SEQ ID NO.1.
2. the construction method of recombinant plasmid pFastBac HTB-Penton described in a kind of claim 1, it is characterised in that：Including
Following steps：
1) acquisition of Penton genes
Design primer：
Complete genome DNA with Ankara enters performing PCR as template, and amplification recovery obtains containing restriction enzyme site BamHI/
The Penton genes of Hind III；
2) structure of recombinant plasmid pFastBac HTB-Penton
With carrier pFastBac HTB as skeleton, with digestion with restriction enzyme carrier pFastBac HTB and contain restriction enzyme site
The Penton genes of the Penton genes of BamHI/Hind III, the pFastBac HTB for being linearized and linearisation, with connection
PFastBac HTB and the Penton genes connection of linearisation that enzyme will be linearized, obtain recombinant plasmid pFastBac HTB-
Penton。
3. the construction method of recombinant plasmid pFastBac HTB-Penton described in a kind of claim 2, it is characterised in that：It is described
Step 1) in,
PCR system：
PCR conditions：94 DEG C of predegeneration 5min, PCR cycle is 95 DEG C of 30s, 50 DEG C of annealing 30s, 68 DEG C of 1min；After 30 circulations,
68 DEG C of extension 10min；
The step 2) in, digestion system：
Digestion condition：37 DEG C of endonuclease reaction 3h after above-mentioned system is mixed.
4. a kind of recombinant baculovirus Ac-Penton, it is characterised in that：The genome of the recombinant baculovirus Ac-Penton
In contain the recombinant plasmid pFastBac HTB-Penton described in claim 1.
5. the preparation method of recombinant baculovirus Ac-Penton described in a kind of claim 4, it is characterised in that：Including following step
Suddenly：
1) 1) acquisition of Penton genes
Design primer：
Complete genome DNA with OK a karaoke club virus enters performing PCR as template, and amplification recovery obtains containing restriction enzyme site BamHI/Hind
III Penton genes；
2) structure of recombinant plasmid pFastBac HTB-Penton
With carrier pFastBac HTB as skeleton, with digestion with restriction enzyme carrier pFastBac HTB and contain restriction enzyme site
The Penton genes of the Penton genes of BamHI/Hind III, the pFastBac HTB for being linearized and linearisation, with connection
PFastBac HTB and the Penton genes connection of linearisation that enzyme will be linearized, obtain recombinant plasmid pFastBac HTB-
Penton。
3) restructuring rod granule Bacmid-Penton
Recombinant plasmid pFastBac HTB-Penton are converted into DH10Bac E.coli competent cells, in LB fluid nutrient mediums
Middle culture, then takes a small amount of nutrient solution and lines the high salt containing kanamycins, gentamicin and tetracycline and IPTG, X-gal
LB flat board cultures, screen the single white colony PCR of picking and identify correct rear LB liquid of the inoculation containing antibiotic by blue hickie
In body culture medium, 37 DEG C of 220rpm/min vibration culture shaking overnight incubations, extraction obtains restructuring rod granule Bacmid-Penton；
4) recombinant baculovirus Ac-Penton is obtained
Restructuring rod granule Bacmid-Penton is transfected into sf9 cells, observes that cell has obvious lesion after 4-7 days, collect cell training
Nutrient solution, centrifugation removal cell fragment, it is to obtain recombinant baculovirus Ac-Penton to collect supernatant.
6. the preparation method of recombinant baculovirus Ac-Penton according to claim 5, it is characterised in that：The step 1)
In,
PCR system：
PCR conditions：94 DEG C of predegeneration 5min, PCR cycle is 95 DEG C of 30s, 50 DEG C of annealing 30s, 68 DEG C of 1min；After 30 circulations,
68 DEG C of extension 10min；
The step 2) in, digestion system：
Digestion condition：37 DEG C of endonuclease reaction 3h after above-mentioned system is mixed.
7. the method for Ankara subunit vaccine being prepared based on recombinant baculovirus Ac-Penton, it is characterised in that：Including
Following steps：
1) recombinant baculovirus Ac-Penton inoculations are suspended in the insect cell SF9 of culture, culture is collected by centrifugation cell；
2) above-mentioned cell ultrasonication cracking takes supernatant, and affinitive layer purification is reclaimed, and then flushed post wash-out, is purified
Albumen,
3) by step 2) albumen of purifying is diluted to 300~800 μ g/ml, and albumen and the adjuvant that will be diluted are by weight 2:7~1:
2 are well mixed, and obtain Ankara subunit vaccine.
8. the method for preparing Ankara subunit vaccine according to claim 7, it is characterised in that：The adjuvant is
MontanideTMISA 71 VG9。
9. the method that recombinant baculovirus Ac-Penton prepares polyvalent vaccine is obtained using claim 5, it is characterised in that：Bag
Include following steps：
1) acquisition of Fiber2 genes
Design primer：
Complete genome DNA with OK a karaoke club virus enters performing PCR as template,
PCR system：
PCR conditions：94 DEG C of predegeneration 5min, PCR cycle is 95 DEG C of 30s, 50 DEG C of annealing 30s, 68 DEG C of 1min；After 30 circulations,
68 DEG C of extension 10min；
Amplification recovery obtains the Fiber2 genes containing I/Hind of restriction enzyme site Xba III；
2) structure of recombinant plasmid pFastBac HTB-Fiber2
With carrier pFastBac HTB as skeleton, with digestion with restriction enzyme carrier pFastBac HTB and contain restriction enzyme site
The Fiber2 genes of the Fiber2 genes of I/Hind of Xba III, the pFastBac HTB for being linearized and linearisation, use ligase
The pFastBac HTB that will be linearized and the Fiber2 genes connection of linearisation,
Digestion system：
Digestion condition：37 DEG C of endonuclease reaction 3h after above-mentioned system is mixed；Obtain recombinant plasmid pFastBac HTB-Fiber2.
3) restructuring rod granule Bacmid-Fiber2
Recombinant plasmid pFastBac HTB-Fiber2 are converted into DH10Bac E.coli competent cells, in LB fluid nutrient mediums
Middle culture, then takes a small amount of nutrient solution and lines and contain three anti-(kanamycins, gentamicin and tetracycline) and IPTG, X-gal
High salt LB flat board cultures, by blue hickie screen the single white colony PCR of picking identify it is correct after inoculation contain antibiotic
LB fluid nutrient mediums in, 37 DEG C of 220rpm/min vibration culture shaking overnight incubations, extraction obtains restructuring rod granule Bacmid-
Fiber2；
4) recombinant baculovirus Ac-Fiber2 is obtained
Restructuring rod granule Bacmid-Fiber2 is transfected into sf9 cells, observes that cell has obvious lesion after 4-7 days, collect cell training
Nutrient solution, centrifugation removal cell fragment, it is to obtain recombinant baculovirus Ac-Fiber2 to collect supernatant；
5) Ankara mixing subunit vaccine is prepared using recombinant baculovirus Ac-Penton and Ac-Fiber2
A. recombinant baculovirus Ac-Penton and Ac-Fiber2 are inoculated in the insect cell SF9 of the culture that suspends respectively, training
Nutrient is not collected by centrifugation cell；
B. ultrasonication cracking takes supernatant to above-mentioned cell respectively, and affinitive layer purification is reclaimed, and then flushed post is eluted respectively, point
The albumen Penton and Fiber2 not purified,
C. albumen Penton and Fiber2 that step b) is purified are diluted to same concentrations 300-800 μ g/ml respectively, by what is diluted
Albumen Penton and Fiber2 by volume 1:Pressed after 1 mixing with adjuvant by weight 2:7~1:2 are well mixed, and obtain peace card
Virus mixing subunit vaccine is drawn, wherein, the adjuvant is MontanideTMISA 71 VG。
10. a kind of claim 9 prepares Ankara mixing subunit vaccine and is applied in the disease of preventing and treating Ankara.
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| CN201710010892.7A CN106755106A (en) | 2017-01-06 | 2017-01-06 | The preparation method and application of Ankara subunit vaccine |
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