CN104109689B - Vaccine expression system, and vector and bacterial strain included in system - Google Patents
Vaccine expression system, and vector and bacterial strain included in system Download PDFInfo
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- CN104109689B CN104109689B CN201310556652.9A CN201310556652A CN104109689B CN 104109689 B CN104109689 B CN 104109689B CN 201310556652 A CN201310556652 A CN 201310556652A CN 104109689 B CN104109689 B CN 104109689B
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Abstract
The invention relates to a vaccine expression system, a vector and a bacterial strain included in the system, and a method for secreting protein into fermentation broth by using signal peptide. A promoter used in the invention is originated from the glucoamylase gene of Aspergillus niger; a terminator is originated from an indole glycerophosphoric acid synthase coding gene in the tryptophan operon of Aspergillus nidulans; the signal peptide is originated from the signal peptide gene (IPU) of isopullulanase gene (ipuA) of Aspergillus niger; the expression vector is constructed, and the fusarium strain containing the expression vector is obtained. According to the invention, the strong promoter PglaA of Aspergillus niger is used to express coe, the signal peptide IPU of Aspergillus niger is used to secrete target protein into the fermentation broth, so tedious cell disruption and protein separation and purification are eliminated and cost is reduced to a certain extent.
Description
Technical field
The invention belongs to technical field of bioengineering, relate to structure and the heterogenous vaccine of the integrated expression vector of filamentous fungi
Expression, carrier that a kind of vaccine expression system and this system comprise and bacterial strain.
Background technology
Vaccine expression is the important component part in the fields such as modern industry, medical treatment and basic research, is also current biological skill
The difficult point of art and focus, the system expressed for recombiant vaccine is broadly divided into:
Prokaryotic protein expression system with escherichia expression system as representative, its have genetic background understand, low cost,
Expression is high and the isolated and purified advantage such as relatively easy of expression product, but lacks processing mechanism after protein translation, such as disulfide bond
Formed, protein glycosylation and correctly folding so that it is the probability obtaining having bioactive albumen is less.
With methanol yeast for representing Yeast protein expression systems and have that expression is high, can induce, glycosylation machinery is close to high
Deng eukaryote, secretory protein easy purification, easily realizing the advantages such as highly dense fermentation, shortcoming is that part protein product is degradable, expresses
Measure uncontrollable.
The major advantage of mammalian cell and insect cell expression system is that protein translation post-treatment mechanism is closest to body
Interior native form, is easiest to retain biological activity, but its expression is the most relatively low, and cultivation cycle is longer, stable cell lines
Establishing techniques difficulty is big, and production cost is high.
Filamentous fungi is increasingly subject to people's attention due to its energy the Production of Heterologous Proteins.Filamentous fungi is expressed different
Source protein has that expression is big, exocytosis rate high, protein molecular folds and modification system is close to features such as higher eucaryotic cells,
And the foreign protein expressed has natural activity.It addition, filamentous fungi can also carry out various post translational processing, as glycosylation is repaiied
The cutting of decorations, protease and the formation etc. of disulfide bond.Filamentous fungi has good safety, many strain such as aspergillus nigers
(Aspergillus niger), aspergillus oryzae (Aspergillus oryzae) and Filamentous fungi (Trichoderma reesei)
Deng prolonged application in food and food processing industry, and belonged to Generally by federal food drug and cosmetic act and drug administration's identification
Recognized as Safe (GRAS) and the bacterial strain of Food and Drug Administration (FDA) scope.Meanwhile, silk
The fermenting procedure of shape fungus is more ripe, and cost is relatively low, puts into industrialized production for it and haves laid a good foundation.
Some key properties of filamentous fungi are the most gradually recognized by people, and these characteristics include that expression is big, outside born of the same parents
Secretion rate height, protein molecule folding and modification system have natural activity close to the foreign protein of higher eucaryotic cells, production
Deng.Therefore, filamentous fungi has quickly become to have much the expressive host system that can be used for producing heterologous recombinant protein of captivation.Thread
Expression and secretion capacity that fungus is remarkable are always the maximum feature attracting people.
Fusarium spp. Fusarium venenatum has following some (1) as filamentous fungi expression strain and is prone to convert；
(2) Protease Levels of low secretion；(3) relatively low secretory protein background；(4) ability of high-caliber heterogenous expression；(5) fermentation
Form；(6) existing or potential GRAS or the state of equivalence.F.venenatum is in the conduct first of the later stage sixties 20th century
Silk-like proteins produces, and it was originated by the albumen that work done in the manner of a certain author is the mankind at that time.Through the intensive test of 12 years, British agriculture in 1984
Portion, fishery and food department approval F.venenatum can be as food sellings.F.venenatum in Britain as human foods
Commercial source 30 years the soonest.With unlike the Fusarium spp. of other kind, this strain Fusarium spp. does not produce mycotoxin and to planting
Thing is also non-pathogenic.Additionally, this strain bacterium also has form of preferably fermenting.These features make F.venenatum become
One attractive heterogenous expression host.
It is used safely in human consumption as food composition after F.venenatum is approved (to sell as " QuornTM" thalline
Albumen).QuornTMFungus is originally confirmed as Fusarium graminearum, but has convictive molecule, phylogeny, form and
The evidences such as mycotoxin, it has been shown that A3/5QuornTMBacterial strain should reclassify referred to as F.venenatum.This work be by
Two independent research groups (O'Donnell et al., 1998；Yoder and Christianson, 1998), after 3 years
Nirenberg (1995) announces and has published deep cultivation and morphological evidence by Fusarium sambucinum sensu
Lato component is three independent species；Fusarium sambucinum sensu stricto,Fusarium
torulosum,and Fusarium venenatum.
The S glycoprotein of Porcine epidemic diarrhea virus (porcine epidemic diarrhea virus, PEDV) is induction
Body produces neutralizing antibody and provides immanoprotection action to divide major structural protein.The partial protection antigen gene of PEDV is (i.e.
Coe gene) the part S glycoprotein that encodes has antigenicity, it is possible to and induction body produces and neutralizes antigen.
Summary of the invention
It is an object of the invention to overcome the deficiency of existing expression system, it is provided that a kind of new vaccine expression system and this be
Unite the carrier pCAMBIA1300 (GenBank:FJ362600.1) and gene coe, the purpose egg that the present invention will express comprised
After white gene pig epidemic diarrhea virus coe gene is connected to signal peptide and promoter, by signal peptide, destination protein is divided
Secrete in fermentation liquid, eliminate the purge process that destination protein is complicated.
The technical scheme that the present invention realizes purpose is as follows:
Containing promoter PglaA, the integrated protein expression vector of fungus of gene coe and terminator Ttrpc
pCAMBIA1300-PglaA-coe-Ttrpc。
Fusarium spp. engineered strain containing protein expression vector pCAMBIA1300-PglaA-coe-Ttrpc, described bacterial strain
Deposit number be CGMCC No.8356, preservation address: in China Committee for Culture Collection of Microorganisms's common micro-organisms
The heart, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, preservation date, on October 18th, 2013, Classification And Nomenclature Fusarium
venenatum。
Fusarium spp. engineered strain containing protein expression vector pCAMBIA1300-PglaA-coe-Ttrpc is expressed at vaccine
In purposes.
Fusarium spp. engineered strain expressing protein containing protein expression vector pCAMBIA1300-PglaA-coe-Ttrpc
Method, it is characterised in that: by inoculation to Vogel maltodextrin fluid medium, 25-30 DEG C, 150-200rpm shakes training
Support 5-7 days, fermentation liquid is collected by filtration.
Fusarium spp. engineered strain expressing protein containing protein expression vector pCAMBIA1300-PglaA-coe-Ttrpc
Method, it is characterised in that: described maltodextrin concentration is 1-2% (g/L).
Advantages of the present invention and good effect are as follows:
1, the present invention utilizes a kind of new heterologous expression system F.venenatum bacterial strain to express the different of non-filamentous originated from fungus
Source vaccine protein encoding gene coe, it has been assert the bacterial strain belonging to GRAS and FDA scope by federal food drug and cosmetic act and drug administration.
2, the present invention expresses coe, and profit first with the strong promoter PglaA of aspergillus niger (Aspergillus niger)
With the signal peptide IPU of aspergillus niger, destination protein is secreted in fermentation liquid, thus eliminates loaded down with trivial details cell breakage and albumen divides
From and purge process, production cost can be reduced to a certain extent.
Accompanying drawing explanation
Fig. 1 is expression vector pCAMBIA1300-PglaA-coe-Ttrpc plasmid construction collection of illustrative plates of the present invention；
Fig. 2 is that expression vector pCAMBIA1300-PglaA-coe-Ttrpc is proceeded to F.venenatum bacterial strain by the present invention
Screening flat board；
Fig. 3 is the PCR proof diagram that expression vector pCAMBIA1300-PglaA-coe-Ttrpc of the present invention converts Fusarium spp.；
Fig. 4 is that F.venenatum-pCAMBIA1300-PglaA-coe-Ttrpc of the present invention expresses coe and secretes to fermentation
The protein electrophoresis figure of liquid.
Detailed description of the invention
Below in conjunction with embodiment, following embodiment is illustrative, is not determinate, it is impossible to limit with following embodiment
Determine protection scope of the present invention.
Present disclosure includes: with strong promoter PglaA, terminator Ttrpc and the expression of pig epidemic virus coe
Plasmid, containing the F.venenatum bacterial strain that can produce destination protein of expression plasmid, the structure of described F.venenatum bacterial strain
The method that construction method and described F.venenatum bacterial strain produce destination protein.
Strong promoter gene PglaA of the present invention derives from aspergillus niger (Aspergillus niger), bacterial strain preservation
Numbered ATCC 1015, its nucleotide sequence is as shown in sequence 7.Terminator Ttrpc derives from aspergillus nidulans (Aspergillus
Nidulans), its nucleotide sequence is as shown in sequence 9.Pig epidemic virus coe is preserved by this laboratory, its nucleotides sequence
Row are as shown in sequence 8.PglaA gene, coe gene and Ttrpc gene are sequentially inserted into initial load by described expression plasmid respectively
In body pCAMBIA1300, construct expression vector pCAMBIA1300-PglaA-coe-Ttrpc, and it is imported
In F.venenatum, it is thus achieved that the F.venenatum bacterial strain containing expression vector pCAMBIA1300-PglaA-coe-Ttrpc
F.venenatum-pCAMBIA1300-PglaA-coe-Ttrpc, after the fermented cultivation of above-mentioned bacterial strains, coe successfully expresses point
Secrete in the fermentation liquid of F.venenatum.
F.venenatum bacterial strain F.venenatum-pCAMBIA1300-PglaA-coe-Ttrpc heterogenous expression pig is popular
The method of property diarrhea virus antigen is: be inoculated into by F.venenatum in Vogel maltodextrin fluid medium, fermented training
Support, filter, collect fermentation liquid, centrifugal, obtain the fermentation liquid containing secretion Porcine epidemic diarrhea virus antigen；Described cultivation temperature
Degree is for 25-30 DEG C；Described incubation time is 5-7d.
The building process of concrete pCAMBIA1300-PglaA-coe-Ttrpc is as follows:
One, the structure of the integrated expression vector of filamentous fungi
1, with the genome of aspergillus niger (A.niger) as template, utilize the primer in table 1 to carry out PCR and expand PglaA.
PCR reaction system is: 2 × PCR Buffer is (containing Mg2+) 10 μ l, dNTP (2.5mM) 2 μ l, forward primer PglaA-F
1 μ l each with downstream primer PglaA-R (10 μMs), template (aspergillus niger complete genome DNA) 1 μ l, KOD Fx DNA is (purchased from TOYOBO
Company, article No. KFX-101) polymerase 0.5 μ l, adding sterilized water to final volume is 20 μ l.
PCR reaction condition is: 94 DEG C of denaturations 2min, 98 DEG C of degeneration 15s, 58 DEG C of annealing 30s, and 68 DEG C extend 60s, reaction
35 circulations, extend 10min after 68 DEG C.
PCR obtains complete purpose fragment nucleotide sequence after terminating, its sequence is shown in sequence 7.
Table 1 the primer sequence
Acquired DNA fragmentation PglaA Kpn I and XbaI is carried out double digestion, after recovery with as restriction endonuclease process
The plasmid fragments pCAMBIA1300 crossed is attached, by connection product conversion escherichia coli jm109 competent cell, and uniformly
Coat on the LB flat board with kalamycin resistance (100 μ g/ml), 37 DEG C of overnight incubation, picking monoclonal, carry out bacterium colony
PCR checking and digestion verification, it is thus achieved that expression vector pCAMBIA1300-PglaA.
2, with the genome of aspergillus nidulans (A.nidulans) as template, the primer in table 2 is utilized to carry out PCR amplification
Ttrpc。
PCR reaction system is: 2 × PCR Buffer is (containing Mg2+) 10 μ l, dNTP (2.5mM) 2 μ l, forward primer Ttrpc-F
1 μ l each with downstream primer Ttrpc-R (10 μMs), template (aspergillus nidulans complete genome DNA) 1 μ l, KOD Fx archaeal dna polymerase 0.5
μ l, adding sterilized water to final volume is 20 μ l.
PCR reaction condition is: 94 DEG C of denaturations 2min, 98 DEG C of degeneration 15s, 58 DEG C of annealing 30s, and 68 DEG C extend 50s, reaction
35 circulations, extend 10min after 68 DEG C.
PCR obtains complete purpose fragment nucleotide sequence after terminating, its sequence is shown in sequence 9.
Table 2 the primer sequence
Acquired genes of interest Ttrpc BssH II and Hind III is carried out double digestion, interior as warp after recovery
Cut the plasmid fragments pCAMBIA1300-PglaA that ferment treatment crosses to be attached, product will be connected and convert e. coli jm109 impression
State cell, and be spread evenly across on the LB flat board with kalamycin resistance (100 μ g/ml), 37 DEG C of overnight incubation, picking Dan Ke
Grand, carry out bacterium colony PCR checking and digestion verification, it is thus achieved that expression vector pCAMBIA1300-PglaA-Ttrpc.
The present invention connects purpose fragment on the basis of the plasmid pCAMBIA1300-PglaA-Ttrpc built and builds
Expression vector.
3, the present invention is addition signal peptide on the forward primer of destination protein, and downstream primer adds his label, lists table in
3, purpose fragment is carried out PCR amplification.
PCR reaction system is: 2 × PCR Buffer is (containing Mg2+) 10 μ l, dNTP (2.5mM) 2 μ l, forward primer coe-F and
The each 1 μ l of downstream primer coe-R (10 μMs), template 1 μ l, KOD Fx archaeal dna polymerase 0.5 μ l, adding sterilized water to final volume is 20 μ
l。
PCR reaction condition is: 94 DEG C of denaturations 2min, 98 DEG C of degeneration 15s, 58 DEG C of annealing 30s, and 68 DEG C extend 60s, reaction
35 circulations, 68 DEG C re-extend 10min.
PCR obtains complete purpose fragment nucleotide sequence after terminating, its sequence is shown in sequence 8.
Table 3 the primer sequence
Acquired genes of interest coe BamHI is carried out single endonuclease digestion, dephosphorylation and the same tail through BamHI after recovery
The plasmid fragments pCAMBIA1300-PglaA-Ttrpc that enzyme BglII single endonuclease digestion processed is attached, and will connect product and convert big
Enterobacteria JM109 competent cell, and be spread evenly across on the LB flat board with kalamycin resistance (100 μ g/ml), 37 DEG C of trainings
Support overnight, picking monoclonal, carry out bacterium colony PCR checking and digestion verification, it is thus achieved that expression vector pCAMBIA1300-PglaA-
coe-Ttrpc。
Two, the structure of the Fusarium spp. bacterial strain containing expression vector
Vogel maltodextrin: Vogel's Salts 20mL, maltodextrin 15g, distilled water dissolves and is settled to
1000ml.115 DEG C of sterilizing 20min.
Vogel sucrose: Vogel's Salts 20mL, sucrose 15g, agar 15g, distilled water dissolves and is settled to
1000ml.115 DEG C of sterilizing 20min.
Vogel's 50X salts:
Sodium citrate 2H20150g, KH2PO4250g, NH4NO3100g, MgSO4·7H2010g, CaCl2·2H205g, micro-
Secondary element 5ml, Biotin Solution 2.5ml, distilled water dissolves and is settled to 1000ml, adds 0.2ml chloroform as anticorrosion
Agent, preserves under room temperature；
Trace element solution:
Citric acid H2O 5.00g, ZnSO4·7H2O 5.00g, Fe (NH4)2(SO4)2·6H2O 1.00g, CuSO4·
5H2O 0.25g, MnSO4·H2O 0.05g, H3BO30.05g, Na2MoO4·2H2O 0.05g, distilled water dissolves and is settled to
100ml, adds 1ml chloroform as preservative, preserves under room temperature；
Biotin solution:
Biotin 5.0mg, distilled water dissolves and is settled to 50ml ,-20 DEG C of preservations.
YPD culture medium:
Yeast extract 1%, tryptone 2%, glucose 2%.115 DEG C of sterilizing 20min.
Yeast extract and glucose at high temperature can occur mailland reaction, and glucose is preferably made into the mother of 20%
Liquid, separately sterilizing, the glucose of 20% is sterilizing 15min under the conditions of 115 DEG C.
Inducing culture IM:
Wherein Ksalts, MNsalts, the formula of trace element etc. is respectively as follows:
K salts
K2HPO4 1.25M
KH2PO4 1.25M
By 1.25mol/L KH2PO4Join 1.25mol/L K2HPO4, adjusting pH is 4.8；121 DEG C of sterilizing 20min；
MN salts
Mg2SO4·7H2O 3%
NaCl 1.5%
121 DEG C of sterilizing 20min；
Trace element
121 DEG C of sterilizing 20min；
1%CaCl2: weigh 1g CaCl2, add water to 100mL, 121 DEG C of sterilizing 20min；
50% glycerol: add in 50mL glycerol to 50mL water, 121 DEG C of sterilizing 20min；
20%NH4NO3: weigh 20g NH4NO3It is dissolved in distilled water and is settled to 100mL, 121 DEG C of sterilizing 20min；
20% glucose: weigh 20g glucose and be dissolved in distilled water and be settled to 100mL, 115 DEG C of sterilizing 20min；
0.01%FeSO4: weigh 0.1g FeSO4To 1L water, filtration sterilization；
1mol/L MES: weigh 195.24g MES, add water to 1L；PH to 5.5 is regulated with NaOH；Filtration sterilization.4 DEG C of guarantors
Deposit；
DNA extraction buffer: NaCl 100mmol/L；EDTA 50mmol/L；Tris-HCl 50mmol/L pH8.5；
SDS 1%.
Phenol mixture: 100mL mixture, containing phenol 48mL, chloroform 48mL, isoamyl alcohol 4mL.
When acquired expression vector is imported Fusarium spp. genome, pass through Agrobacterium_mediated method.First by
The expression vector electricity built proceeds to Agrobacterium, afterwards by co-culturing the genome that purpose fragment imports Fusarium spp..
1, above-mentioned acquired expression vector electricity is turned Agrobacterium AGL1, method particularly includes:
(1) drawing 3 μ L on LB flat board from-80 DEG C of AGL1 glycerol pipes preserved, line, 28 DEG C of cultivations, to list occur
Bacterium colony.
(2) picking list bacterium colony, is inoculated in the LB fluid medium of 3mL, 28 DEG C, 200rpm, and shaken cultivation is overnight.
(3) take 2mL bacterium solution to be inoculated in the LB fluid medium of 100mL, 28 DEG C, 200rpm, activation culture 4～6h, extremely
OD600It is 0.5.
(4) by the bacterium solution ice bath 40min after activation, proceed in aseptic 50mL centrifuge tube.4 DEG C, 5000rpm is centrifuged
10min, abandons supernatant, collects thalline.
(5) fully suspending thalline with the sterilized water of 10mL cooling, 4 DEG C, 5000rpm is centrifuged 10min, abandons supernatant, collects bacterium
Body.This step comes again.
(6) fully suspending thalline with 10% glycerol of 10mL cooling, 4 DEG C, 5000rpm is centrifuged 10min, abandons supernatant, collects
Thalline.This step comes again.
(7) fully suspend thalline with 10% glycerol of 2mL cooling.
(8) take 1.2 μ L recombiant plasmid, join in 70 μ L Agrobacterium competent cells, mix homogeneously, proceed to the electricity of pre-cooling
In revolving cup, it is placed on ice.
(9) putting in electroporation by electricity revolving cup, regulation electroporation parameter is carried out as follows electricity and converts:
(10) taking out electricity revolving cup, be rapidly added the LB culture medium of 1mL pre-cooling, fully mix, sucking-off bacterium solution to aseptic EP is managed
In, in 28 DEG C, 200rpm activation culture 4-6h, draw 100 μ L bacterium solution, coat the LB containing 100 μ g/mL kanamycin and put down
Plate, cultivates 2d for 28 DEG C, it is thus achieved that single bacterium colony.
Picking transformant, with coe-F, coe-R as primer, carries out Porcine epidemic diarrhea virus base to above-mentioned transformant respectively
The bacterium colony PCR checking of cause.
PCR reaction system is: 10 × PCR Buffer 2 μ l, dNTP (10mM) 0.4 μ l, forward primer coe-F and downstream
Primer coe-R (10 μMs) each 0.4 μ l, template 0.4 μ l, Taq archaeal dna polymerase 0.4 μ l, adding sterilized water to final volume is 20 μ l.
PCR reaction condition is: 94 DEG C of denaturations 5min, 94 DEG C of degeneration 30s, 58 DEG C of annealing 30s, and 72 DEG C extend 1min, instead
Answer 35 circulations, after 72 DEG C extend 10min, using plasmid pCAMBIA1300-PglaA-coe-Ttrpc for PCR amplification template as
Positive control, using sterilized water for PCR amplification template as negative control, the whole cell DNA of transformant is that template carries out PCR amplification
Product, compared with negative control bacterial strain, positive transformant can amplify the band of coe gene (951bp), and expression vector is described
PCAMBIA1300-PglaA-coe-Ttrpc the most successfully proceeds in Agrobacterium AGL1.
2, by the above-mentioned Agrobacterium AGL1 mediated transformation Fusarium spp. F.venenatum having contained expression vector, concrete grammar
For:
1, the preparation of F.venenatum spore:
(1) it is connected on Vogel sucrose plate with inoculating loop picking a little spore point from PDA inclined-plane；
(2) go to illumination box 3～4 days again at 30 DEG C of light culture 2～3, produce abundant spore；
(3) it is added on sporogenic Vogel sucrose plate with 5～6mL normal saline, with spreader, spore is washed down, outstanding
With aseptic Miracloth filter-cloth filtering after floating spore, and spore suspension is transferred in aseptic EP pipe；
(4) estimate the spore count of F.venenatum with blood counting chamber, the concentration of regulation F.venenatum spore is extremely
105/mL。
2, the preparation of the Agrobacterium converted
(1) by the Agrobacterium containing recombiant plasmid and Ti mini-plasmids according to the method for three rides be inoculated in containing card that
On the LB flat board of mycin, cultivate 3d at 28 DEG C；
(2) the mono-bacterium colony of picking one ring Agrobacterium AGL1 is in the 5mL LB fluid medium containing kanamycin, 28 DEG C of trainings
Support 24h；
(3) taking the culture fluid of 1.5mL in aseptic EP pipe, 4000r/min is centrifuged 10min, adds 250 after discarding supernatant
μ L IM (being not added with acetosyringone), 4000r/min discard supernatant after being centrifuged 5min.Add 5mL afterwards and contain 0.2mmol/
The IM culture medium of L acetosyringone, at 28 DEG C, 100rpm continuation cultivation 6h；
(4) OD of culture fluid is measured600Value, adjusts OD value and is about 0.8, and now agrobatcerium cell concentration is 4～5 × 108
Individual/mL.
3, the co-culturing of F.venenatum and Agrobacterium AGL1
(1) 100 μ L OD are taken600Being about the conversion Agrobacterium induction broth of 0.8,100 μ L contain 105Individual/mL's
F.venenatum spore suspension mixes with the ratio of 1:1；
(2) nylon membrane of 0.45 μm is taken with sterilized tweezers as the IM solid containing 0.2 μM of acetosyringone
In culture medium；
(3) 200 μ L Agrobacterium AGL1 and F.venenatum spore mixture are drawn to nylon membrane；
Cultivate 2d for (4) 25 DEG C.
4, the screening of transformant
(1) the culture sterilized water on film is washed down by spreader, then transfer to resist containing hygromycin with pipettor
On the PDA plate of property；
(2) flat board is cultivated until the bacterium colony converted grows at 30 DEG C；
(3) the single bacterium colony on picking PDA plate in YPD shaking flask 30 DEG C, 200rpm cultivates 2-3d；
(4) use filtered through gauze mycelium, and clean twice with distilled water；
(5) by liquid nitrogen grinding method, mycelium is ground into powder；
(6) powder obtained is added in 2mL centrifuge tube, by 950 μ L Extraction buffer suspended powder, places at 65 DEG C
30min；
(7) adding 350 μ L 5mol/L potassium acetate mixings in suspension makes it be acidified then at-20 DEG C of placement 15min；
(8) 4 DEG C, under the conditions of 12000rpm after centrifugal 15min, supernatant is transferred in the centrifuge tube of 2mL；
(9) add equal-volume phenol mixture, acutely mix and 12000rpm is centrifuged 15min at 4 DEG C；
(10) aqueous phase containing genome is transferred in new 1.5mL centrifuge tube, is added dropwise over 350 μ L isopropanols and 4
At DEG C, 12000rpm is centrifuged 10min；
(11) abandon supernatant and collect precipitation；
(12) with 70% ethanol purge precipitation and 4 DEG C, 8000rpm be centrifuged 5min, collect genome；
(13) uncovered air-drying is dissolved in 70 μ L 1 × TE buffer (pH 8.0) afterwards, and places about 1h in room temperature.
(14) with extract DNA as template, with coe-F, coe-R as primer, respectively the DNA of above-mentioned transformant is carried out
PCR verifies.
PCR reaction system is: 10 × PCR Buffer 2 μ l, dNTP (10mM) 0.4 μ l, forward primer coe-F and downstream
Primer coe-R (10 μMs) each 0.4 μ l, template 0.4 μ l, Taq archaeal dna polymerase 0.4 μ l, adding sterilized water to final volume is 20 μ l.
PCR reaction condition is: 94 DEG C of denaturations 5min, 94 DEG C of degeneration 30s, 58 DEG C of annealing 30s, and 72 DEG C extend 1min, instead
Answering 35 circulations, extend 10min after 72 DEG C, result is as it is shown on figure 3, the first swimming lane is positive control, with plasmid
PCAMBIA1300-PglaA-coe-Ttrpc is that PCR expands template, and the second swimming lane is wild type control, expands with sterilized water for PCR
Increase template, rear nine swimming lanes be the whole cell DNA of 9 transformants be the pcr amplification product that template is carried out, with wild type control bacterium
Strain is compared, and positive transformant can amplify the band of coe gene (951bp), and expression vector pCAMBIA1300-is described
PglaA-coe-Ttrpc the most successfully proceeds to Fusarium spp. F.venenatum.
Three, the vaccine expression of the F.venenatum containing expression vector is analyzed
Above-mentioned F.venenatum transformant and wild type are inoculated in Vegol sucrose medium, cultivate 3-4d for 28 DEG C,
Spore is washed down, is seeded in the triangular flask containing 50mLVogel maltodextrin fluid medium, 28 DEG C, 150rpm cultivate 5-
7d.Six layers of filtered through gauze remove mycelia, collect fermentation liquid.Processing fermentation liquid by TCA method, processing procedure is as follows:
(1) bacterium solution centrifugal 10000rpm, 5min at 4 DEG C, collects supernatant.
(2) take 500-1000 μ l supernatant in EP pipe, add the 100%TCA of 1/9 volume, reverse 10 mixings.
(3) more than 0.5 hour during sample is placed in ice bath, overnight effect is more preferable.
(4) 4 DEG C, 15000rpm, centrifugal 10-20 minute, it is seen that have brownish black to precipitate, outwell supernatant, by 1.5mL centrifuge tube
Tip upside down on control gently in absorbent paper several under, remove and remain in the liquid of the mouth of pipe.
(5) add 200 acetone ice-cold for μ l, flick centrifuge tube with finger, wash away at the bottom of pipe and the TCA of tube wall remnants.
(6) 4 DEG C, 15000rpm, centrifugal 10-20 minute, outwell supernatant, centrifuge tube is tipped upside down on control gently in absorbent paper several
Under, remove the liquid remaining in the mouth of pipe.
(7) 1.5mL centrifuge tube is inverted in absorbent paper or places 37 DEG C of baking ovens 5 minutes, confirms do not have liquid at the bottom of tube wall and pipe
Body remains.
(8) 20-50ul Loading buffer is added, 95 DEG C of heating 10min, then loadings；
(9) first turn down voltage 70V to enter after separation gel until sample, then adjust voltage to make bromophenol blue band migrate to separate to 110V
Electrophoresis is stopped bottom glue；
(10) first with coomassie brilliant blue R_250 dyeing liquor dyeing 1-2h after glue being taken out；
(11) outwell dyeing liquor, first rinse with clear water, then decolour extremely at decolorization swinging table with coomassie brilliant blue staining destaining solution
Without background colour (period often changes destaining solution).
Result as shown in Figure 4, is labeled as bar for the purpose of transformant and wild type are than the band having more at 35KDa at maker
Band, and the expression of different transformant different be that the genome of radom insertion Fusarium spp. is relevant with genes of interest.Described bacterial strain
Deposit number be CGMCC No.8356, preservation address: in China Committee for Culture Collection of Microorganisms's common micro-organisms
The heart, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, preservation date, on October 18th, 2013, Classification And Nomenclature Fusarium
venenatum。
Claims (3)
1. contain promoter PglaA, the integrated protein expression vector of fungus of gene coe and terminator Ttrpc
PCAMBIA1300-PglaA-coe-Ttrpc, the sequence of described PglaA is shown in that sequence 7, the sequence of described coe are shown in sequence 8, described
The sequence of Ttrpc is shown in sequence 9.
2. contain the Fusarium spp. engineered strain of protein expression vector pCAMBIA1300-PglaA-coe-Ttrpc, described bacterial strain
Deposit number is CGMCC No.8356, preservation address: China Committee for Culture Collection of Microorganisms's common micro-organisms center,
Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, preservation date, on October 18th, 2013, Classification And Nomenclature Fusarium
venenatum。
3. the purposes in vaccine is expressed of the bacterial strain described in claim 2.
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Non-Patent Citations (3)
| Title |
|---|
| Funcitional characterization of Rho GTPases in Aspergillus niger uncovers conserved and diverged roles of Tho proteins within filamentous fungi;Min Jin Kwon，等;《Molecular Microbiology》;20110331;第79卷(第5期);1151-1167 * |
| 米根霉CICIM F0071 α-淀粉酶：基因克隆、鉴定与异源表达;李松;《中国博士学位论文全文数据库》;20121231;61-63 * |
| 黑曲霉分泌表达载体的构建以及绿色荧光蛋白的表达;郑红玉 等;《华南农业大学学报》;20130909;第34卷(第4期);574-579 * |
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