CN104004760A - Foreign protein expression device used for secretory expression in Aspergillus oryzae cells and Aspergillus oryzae genetically engineered bacteria - Google Patents
Foreign protein expression device used for secretory expression in Aspergillus oryzae cells and Aspergillus oryzae genetically engineered bacteria Download PDFInfo
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Abstract
The invention provides a foreign protein expression device used for secretory expression in Aspergillus oryzae cells and Aspergillus oryzae genetically engineered bacteria. From 5' to 3', the expressin device sequentially comprises a promoter for controlling foreign genes to transcribe in Aspergillus oryzae, a coding sequence of signal peptides for controlling secretory expression of foreign genes in the Aspergillus oryzae, a coding sequence of labels used for performing affinity purification on the foreign genes , a multiple-cloning site, a terminator for controlling the foreign genes to terminate transcribing in the a Aspergillus oryzae and an expression box of Aspergillus oryzae ribose phosphate transferase genes for performing plasmid transformation on selection markers. The coding sequence is as shown by SEQ ID No:2. According to the expression device, the auxotroph gene selection markers are used, so that the transformation positive rate is increased, operation time is shortened, workloads are reduced, and the expression device is suitable for secretory expression of most of foreign genes; the Aspergillus oryzae genetically engineered bacteria can efficiently express acid and neutral amylase and is important in application to the paper industry and the textile industry.
Description
Technical field
The invention belongs to biological technical field, relate more specifically to a kind of for express expression equipment and the aspergillus oryzae genetic engineering bacterium thereof of foreign protein in aspergillus oryzae emiocytosis.
Background technology
Aspergillus oryzae (Aspergillus oryzae) is the conventional bacterial strain of fermentation industry, is a kind of aerobic property fungi, belongs to Deuteromycotina, Aspergillus.Mycelia is generally yellow-green colour, is tawny afterwards; Aspergillus oryzae mycelia is comprised of many cells, is the bacterial strain that a class is produced prozyme, except proteolytic enzyme, also can be used to secreting amylase, cellulase, polygalacturonase, Glycosylase, lipase, peptase and phytase etc.; Wherein, industrial common taka-diastase (TAA) comes from the α-amylase of aspergillus oryzae.The safe microorganisms that aspergillus oryzae is assert by FDA (GRAS), is widely used in the industrial circles such as Brewing industry, seasonings, feed production, weaving, papermaking, slurrying, and by safe handling more than 1000 year.At present, aspergillus oryzae RIB40 is gene sequencing entirely, and the filamentous fungus expression system based on aspergillus oryzae is also more and more subject to extensive concern.
Except having the ability of fabulous synthetic and secretory protein, A.oryzae also has the protein excretion mechanism and protein post-translational modification, the processing characteristics similar to mammlian system of eukaryotic microorganisms; Its expression system is compared with prokaryotic expression system has very large advantage, such as protein modified, intron after exogenous protein expression are identified, signal peptide is wiped out and the correct folding and protein excretion of peptide chain.Because it is suitable for the plurality of advantages of protein production, and the mankind are not had to toxicity, aspergillus oryzae has also become the important biomolecule material that related researcher carries out fungal cell, genetics and physiological and biochemical research.Japan subsidizes and has supported the genomic examining order of aspergillus oryzae, and whole genome sequence has completed and announced at present.The functional gene of meaning (the Machida M et al.Genome sequencing and analysis of Aspergillus oryzae.Nature that lays a good foundation is learned in completing as utilizing the research of functional genomics method to have important biomolecule of aspergillus oryzae genome sequencing, 2005,438:1157-1161).
Since the eighties in last century, filamentous fungus expression system is reported in succession, the high-yield expression of homologous protein is verified, but the expression great majority of foreign protein are still in～mg/l under the same conditions, and because filamentous fungus does not exist natural plasmid, genomic data is larger, and secreting, expressing process is complicated, causes and not yet has the expression system (comprising expressive host and expression plasmid) as intestinal bacteria and the same suitability for industrialized production of pichia spp at present.By the relevant auxotroph of screening aspergillus oryzae, build corresponding covering expression vector, pass through strong promoter, the means such as amalgamation and expression realize existing relevant report (the Minetoki T et al.Improvement of promoter activity by the introduction of multiple copies of the conserved region III sequence of heterogenous expression that derives from fungi and animal-plant gene, involved in the efficient expression of Aspergillus oryzae amylase-encoding gene.Appl Microbilo Biotechnol, 1998, 50 (4): 459～467.Shiba Y et al.High level secretory production of phospholipase A1by Saccharomyces cerevisiae and Aspergillus oryzae.Biosci Biotechnol Biochem, 2001, 65 (1): 94～101).1997, Randy M.Berka etc. affects in defective type system by have a liking for the heterogenous expression of warm fungi Myceliophthora thermophila laccase gene with the control of PamyB and TamyB at aspergillus oryzae pyrG, but expression amount is only～10mg/l (Novo N Biotec.Characterization of the gene encoding an extracellular laccase of Myceliophthora thermophile and analysis of the recombinant enzyme expressed in Aspergillus oryzae.Appl Environ Microbiol, 1997, 63 (8): 3151-3157), Nobuo Yamashita in 2011 etc. be take sC as selection markers in aspergillus oryzae, the protease inhibitor gene that comes from Aspergillus fumigatus (Aspergillus fumigatus) is placed between aspergillus oryzae TAA promotor and terminator, by realizing heterogenous expression (the Nobuo Yamashita et al.High-yields heterologous production of the novel Aspergillus fumigatus elastase inhibitor AFUEI in Aspergillus oryzae of this gene after inducing culture, J biosci bioeng, 2011, 112 (2): 114～117).However, up to now not yet relevant for the report of the purifiable universal expression vector of the corresponding general interchangeable elements of these auxotroph systems.
Summary of the invention
The object of this invention is to provide a kind of for express expression equipment and the aspergillus oryzae genetic engineering bacterium thereof of foreign protein in aspergillus oryzae emiocytosis, thereby solve the defect that in prior art, aspergillus oryzae filamentous fungus expression system is low to foreign protein expression amount and separation and purification is difficult.
In order to solve the problems of the technologies described above, the present invention by the following technical solutions:
A first aspect of the present invention is to provide a kind of for express the expression equipment of foreign protein in aspergillus oryzae emiocytosis, and described expression equipment from 5 ' to 3 ' comprises following element successively: (1) controls foreign gene in the promotor of aspergillus oryzae transcription; (2) control the encoding sequence of foreign protein signal peptide of secreting, expressing in aspergillus oryzae; (3) for the encoding sequence of the label of foreign protein affinity purification; (4) multiple clone site; (5) control foreign gene and in aspergillus oryzae, stop the terminator of transcribing; (6) for the expression cassette of the aspergillus oryzae orotate phosphoribosyl transferase gene of plasmid transformation and selection mark, the encoding sequence of described expression cassette is as shown in SEQ ID:2.
In the present invention, described promotor is can control foreign gene in the promotor of aspergillus oryzae transcription, is preferably aspergillus oryzae α-amylase promotor (PamyB), and the sequence of better promotor is shown in the 7th to the 623rd of SEQ ID NO:1.
In the present invention, described signal peptide can be any signal peptide (secretion peptide) that can control foreign protein secreting, expressing in aspergillus oryzae in this area, and preferably the encoding sequence of signal peptide is shown in the 624th to the 695th of SEQ ID NO:1.
In the present invention, the described label for foreign protein affinity purification can be any label that this area can be used in affinity purification albumen, be preferably His-Tag label, the encoding sequence of better label the 696th to the 713rd of sequence as shown in SEQ ID NO:1.
In the present invention, described multiple clone site can be the multiple clone site of the various routines in this area, preferably, and the encoding sequence of described multiple clone site the 714th to the 784th of sequence as shown in SEQ ID NO:1.
In the present invention, described terminator is can control foreign gene in aspergillus oryzae, to stop the terminator of transcribing, preferably aspergillus oryzae α-amylase terminator (TamyB), the sequence of better terminator is shown in the 785th to the 1681st of SEQ ID NO:1.
Most preferably, in expression equipment provided by the invention, the encoding sequence of described aspergillus oryzae α-amylase promotor the 7th to the 623rd of sequence as shown in SEQ ID NO:1; The encoding sequence of described signal peptide the 624th to the 695th of sequence as shown in SEQ ID NO:1; The encoding sequence of the described His-Tag label for foreign protein affinity purification the 696th to the 713rd of sequence as shown in SEQ ID NO:1; The encoding sequence of described multiple clone site the 714th to the 784th of sequence as shown in SEQ ID NO:1; The encoding sequence of described aspergillus oryzae α-amylase terminator the 785th to the 1681st of sequence as shown in SEQ ID NO:1.
Preferably, described expression equipment further comprises the encoding sequence of foreign protein, the encoding sequence of described foreign protein is positioned among multiple clone site, before or after.
A second aspect of the present invention also provides a kind of recombinant expression vector that comprises expression equipment as above.
Preferably, vector plasmid is filamentous fungus expression vector, as pBC-Hygro, pBARGPE1, pAN7-1, pRTRI, pBC-phleo, but is not limited to these carriers, the better pBC-Hygro that is selected from.
A third aspect of the present invention also provides a kind of aspergillus oryzae genetic engineering bacterium of expressing foreign protein, contains expression equipment as above in the genome of described aspergillus oryzae genetic engineering bacterium.
The Host Strains of described aspergillus oryzae genetic engineering bacterium is aspergillus oryzae RIB40,3.042, AS3.863, AS3.951, HBR9, RIB326 etc. preferably, is more preferably aspergillus oryzae uracil auxotrophy bacterial strain RIB40-PF1.Bacterial strain RIB40-PF1 used in the present invention is that contriver oneself screening obtains, this bacterial strain has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC, be positioned at BeiJing, China), preserving number is CGMCC NO:9129, preservation day is 2014.05.08.
A fourth aspect of the present invention also provides a kind of preparation method of aspergillus oryzae genetic engineering bacterium of expression foreign protein as above, comprise: the recombinant expression vector that contains expression equipment as above is transformed to aspergillus oryzae uracil auxotrophy bacterial strain RIB40-PF1, select positive colony, make the aspergillus oryzae genetic engineering bacterium of described expression foreign protein.
A fifth aspect of the present invention also provides a kind of life albuminiferous method, comprises the aspergillus oryzae genetic engineering bacterium of cultivating described expression foreign protein, obtains this foreign protein from culture.
The application of the aspergillus oryzae genetic engineering bacterium that a sixth aspect of the present invention also provides a kind of expression foreign protein as above in preparing industrial enzyme preparation, fodder additives or pharmaceutical grade protein.Described zymin be α-amylase, glucoamylase, beta-glucosidase, Sumylact L, glucose oxidase, dextranase, zytase, Pullulanase,, phytase, lipase, proteolytic enzyme, lipoxygenase, Mierocrystalline cellulose restriction endonuclease, Mierocrystalline cellulose excision enzyme, cellobiase etc.
Compared with prior art, the present invention has following beneficial effect:
1, expression equipment of the present invention, on a cyclisation plasmid, is easy to preserve and DNA operation.
2, expression device first of the present invention is utilized aspergillus oryzae orotate phosphoribosyl transferase gene plasmid transformation and selection mark, can improve conversion positive rate, shortens operating time and workload.
3, expression equipment of the present invention has the secreting, expressing ability of optimization, is applicable to the secreting, expressing of most of foreign genes.
4, in the multiple clone site of expression equipment of the present invention, contain rare interface peace end interface, the connection of compatible most foreign genes, saves the operating time and increases work efficiency.
5, expression equipment of the present invention can be realized the efficient secretory expression of foreign protein plasmagene in thread many cells eukaryotic microorganisms aspergillus oryzae that derives from animal, plant and fungi.Because aspergillus oryzae culture condition is extensive, be applicable to solid culture and liquid submerged fermentation; Itself does not have toxicity aspergillus oryzae, under condition of enzyme production, can not produce mycotoxins and microbiotic, meets international food safety certification; The exoprotein that aspergillus oryzae produces is easy to separation and purification, with low cost.Therefore the present invention can produce bacterial strain for the zymin industries such as washing, weaving, feed, food, papermaking, medicine provide genetically engineered, and the production efficiency that improves zymin reduces production costs.
6, expression equipment of the present invention (comprising promotor terminator) shortens, and the final expression cassette building is less, is more conducive to the attended operation of construction expression plasmid and the raising of transformation efficiency.Expression amount is more steady, not affected by culture condition and have larger fluctuation.If it is larger that expression cassette depends on the fluctuation of culture condition, operator are had relatively high expectations.The expression of expression equipment of the present invention, the condition of yeast culture base can be more rough, cheap.Thalli growth is very fast, and the minimizing time reduces costs exactly.Expression cassette of the present invention can be used in combination with other expression cassettes, expresses two exogenous protein simultaneously.
7, aspergillus oryzae genetic engineering bacterium of the present invention adopts the aspergillus oryzae uracil auxotrophy bacterial strain RIB40-PF1 that screening obtains first, and in conjunction with aspergillus oryzae orotate phosphoribosyl transferase gene plasmid transformation and selection mark, can be high acid, the neutral starch enzyme of efficient expression, in starch industry, paper-making industry and textile industry, there is very important application.
Accompanying drawing explanation
Fig. 1 is physical map and the building process thereof that aspergillus oryzae is expressed equipment pBC12FA2.Each restriction enzyme site as shown in the figure, wherein, PamyB: α-amylase (TAA) promotor; TamyB: α-amylase (TAA) terminator; PyrF: aspergillus oryzae orotate phosphoribosyl transferase (ORPTase) genescreen mark;
Fig. 2 is aspergillus oryzae universal expression vector pBC2FNH design of graphics;
Fig. 3 is the expression of green fluorescent protein in aspergillus oryzae, wherein, and A: the observations of the mycelia of transformant PF1-pBC12FNHEGFP-6 inducing culture under green fluorescence light source (left side) and ordinary light source (right side); B: the observations of the mycelia of wild mushroom RIB40 inducing culture under green fluorescence light source (left side) and ordinary light source (right side).
Embodiment
Below in conjunction with specific embodiment, the present invention will be further described.Should be understood that following examples are only for the present invention is described but not for limiting the scope of the invention.
The raw material that the present invention is used or reagent except special instruction, equal commercially available obtaining.
One, expression equipment
The invention provides a kind of for express the expression equipment of foreign protein in aspergillus oryzae emiocytosis.Wherein, aspergillus oryzae α-amylase promotor, at 5 ' end of this gene, is approximately comprised of 617 Nucleotide; Aspergillus oryzae α-amylase terminator, at 3 ' end of this gene, is approximately comprised of 897 Nucleotide; After this aspergillus oryzae α-amylase promotor, also connecting a segment signal peptide sequence, the secretion peptide of 21 aminoacid sequences of coding.The present invention gets promotor, signal peptide and the terminator of this aspergillus oryzae α-amylase, and after signal peptide, add for expressing protein affinity purification label and multiple clone site, thereby being convenient to foreign gene embeds and is expressed smoothly, and on connecting after terminator for the gene coded sequence of the aspergillus oryzae orotate phosphoribosyl transferase of plasmid transformation and selection mark, finally forming structure is the expression equipment of promotor-signal peptide-purification tag-MCS-terminator-selection markers.This expression equipment can be in aspergillus oryzae the various foreign proteins of secreting, expressing, expression amount is high, is easy to separation and purification, and with low cost.
The preparation method of expression equipment of the present invention is as follows:
(1) pass through pcr amplification, promotor (PamyB+signal peptide, PaS), certain external source amylase gene (GpA2) of obtaining respectively comprising the aspergillus oryzae α-amylase of secreting peptide sequence, aspergillus oryzae α-amylase terminator (TamyB), orotate phosphoribosyl transferase genescreen marker expression box be DNA sequence dna separately.
(2) promoter-signal sequence (PaS) and amylase gene (GpA2) are merged to the fusion sequence (PaSA2) that PCR obtains, terminator (TamyB), orotate phosphoribosyl transferase genescreen marker expression box, PaSA2 are connected in expression vector pBC-Hygro successively, structure obtains recombinant expression vector 1, i.e. pBC12FA2; By rite-directed mutagenesis, insert 6 * His-Tag purification tag, obtain expression vector 2, pBC12NHA2, fixes a point to delete foreign gene GpA2 by inverse PCR and obtains comprising the universal expression vector 3 that this expression equipment does not comprise foreign gene, i.e. pBC12FNH.
(3) external source goal gene is inserted in above-mentioned expression vector 3, makes foreign gene between promotor and terminator, obtain recombinant expression plasmid 4, i.e. pBC12FNH-EGFP.
In above-mentioned steps (1), for the multiple clone site connecting, be that skeleton carrier pBC-Hygro carries; Promotor, terminator and selection markers are connected, and the method that available constraints endonuclease digestion is connected with ligase enzyme is connected into expression vector by each element, and it is all the conventional way in this area that described digestion with restriction enzyme is connected with ligase enzyme.
In above-mentioned steps (3), foreign gene is inserted in above-mentioned expression vector 3, the method that available constraints endonuclease digestion is connected with ligase enzyme, it is all the conventional way in this area that described digestion with restriction enzyme is connected with ligase enzyme.
Two, aspergillus oryzae genetic engineering bacterium
The present invention also provides a kind of aspergillus oryzae genetic engineering bacterium, and this genetic engineering bacterium is that the protoplast transformation that expression equipment of the present invention is mediated by PEG is incorporated into aspergillus oryzae RIB40-PF1 cell, cultivates screening transformant and prepares and obtain, and concrete preparation process is as follows:
(1) carrier that contains expression equipment of the present invention is transformed into aspergillus oryzae RIB40-PF1 cell by the protoplast transformation method of PEG mediation;
(2) cell after above-mentioned conversion is screened, identified, obtain expressing the aspergillus oryzae genetic engineering bacterium of foreign protein.
The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition, as < < molecular cloning: laboratory manual > > (NewYork:Cold Spring Harbor Laboratory Press, 1989) condition described in is carried out, or the condition of advising according to manufacturer.
Damping fluid and the substratum in embodiment, used are as follows:
(1)TE(1000ml)：
ZnSO
47H
2o0.35g, MnCl
24H
2o0.5g, H
3bO
30.03g, CoCl
26H
2o0.945g, CuCl
22H
2o0.01g, NiCl
26H
2o0.12g, NaMoO
42H
2o0.18g, 6M HCl13ml, adds deionized water and is settled to 500mL.
(2) sodium phosphate buffer (100ml, pH5.8):
Na
2HPO
4·12H
2O0.5788g，NaH
2PO
4·2H
2O2.8996g。
(3) Tris-HCl damping fluid (100ml, pH7.5):
Tris0.6057g, regulates pH to 7.5 with HCl.
(4) solution I:
NaCl0.7M, sucrose 1.0g, is dissolved in 50ml sodium phosphate buffer, and constant volume (adding water) is to 100ml.
(5) solution II:
Sorbyl alcohol 22.3085g, CaCl20.578g, NaCl0.2045g, is dissolved in 20ml Tri-HCl damping fluid, and constant volume (adding water) is to 100ml.
(6) solution III:
PEG400030g, CaCl
20.289g, is dissolved in 10ml Tri-HCl damping fluid, and constant volume (adding water) is to 50ml.
(7) CM substratum (100ml):
NaNO
30.6g, KCl0.05g, KH
2pO
40.08g, K
2hPO
40.104g, glucose 1g, MgSO
40.052g, Peptone0.2g, Yeast extract0.1g, uridine 0.1221g, TE100ul; If be made into inclined-plane, add 1.5～2.0g agar powder.
(8) MM substratum (100ml):
NaNO
30.6g, KCl0.05g, KH
2pO
40.08g, K
2hPO
40.104g, glucose 1g, MgSO
40.052g, TE100ul, sucrose 20.54g.
Agar powder: upper strata 0.8g, the 1.2g of lower floor.
(9) fermention medium (100ml):
Dextrin 2g, sucrose 0.3g, peptone 0.1g, yeast extract 0.5g, NaNO
30.1g, MgSO
47H
2o0.05g, FeSO
47H
2o0.001g.
Embodiment 1. comprises the structure that aspergillus oryzae of the present invention is expressed the expression vector of equipment
1.1, the genomic extraction of aspergillus oryzae
Aspergillus oryzae Aspergillus oryzae RIB40 (National Research Institute of Brewing StockCulture, ATCC42149) is inoculated on CM substratum, and at 30 ℃, constant temperature culture 7 days is ripe to spore.Prepare appropriate spore suspension and be inoculated in CM medium liquid seed culture medium, cultivate and reach 4～5g/L for extracting genome to mycelium concentration in 2 days under 30 ℃ of 200rpm conditions, method is extracted test kit specification sheets with reference to Omega fungal gene group.
1.2, the separation of aspergillus oryzae α-amylase promotor (PamyB) and signal peptide sequence thereof
According to the sequence of the amyB promotor of the upper issue of GeneBank, the aspergillus oryzae genomic dna of said extracted of take is template, with upstream primer PaXf and downstream primer Pamr, carries out the separated PaS of special pcr amplification (PamyB+signal peptide).Wherein:
Upstream primer PaXf sequence is:
CCG
cTCGAGgAATTCATGGTGTTTTGATCATTTT, wherein contains XhoI restriction enzyme site (this primer is synthesized by Shanghai Jierui Biology Engineering Co., Ltd, lower same);
Downstream primer Pamr sequence is:
A
gGCGCGCCGCTAGCaGGCGTTGCAGCCAAAGC, wherein contains AscI and NheI restriction enzyme site.
1.3, the separation of aspergillus oryzae α-amylase terminator (TamyB)
According to the sequence of the TamyB terminator of the upper issue of GeneBank, the aspergillus oryzae genomic dna of said extracted of take is template, with upstream primer TaNf and downstream primer TaNr, carries out the separated PaS of special pcr amplification (PamyB+signal peptide).Wherein:
Upstream primer TaNf sequence is:
ATAAGAAT
gCGGCCGCgGGTGGAGAGTATATG, wherein contains NotI restriction enzyme site; Downstream primer TaNr sequence is:
ATAAGAAT
gCGGCCGCaATTCTTGAGGACCATTAC, wherein contains NotI restriction enzyme site.
1.4, the separation of the auxiliary amylase gene of external source
The pMD19Tsimple that has connected GpA2 (NCBI GenBank accension number:KJ093844) of take is template, uses primer 12A2mf and 12A2Hr to obtain GpA2 gene through pcr amplification.Wherein
Upstream primer 12A2mf sequence is:
GCCT
gCTAGCGGCGCGCCt AAAACCGCCGCGGAATGG, wherein contains AscI and NheI restriction enzyme site;
Downstream primer 12A2Hr sequence is:
CCC
aAGCTTtTAAGCACATAAACTGCCCT, wherein contains HindIII restriction enzyme site.
1.5, the separation of aspergillus oryzae orotidine phosphoribosyltransferase selection markers gene
According to the sequence of pyrF in the full genome of aspergillus oryzae RIB40 of the upper issue of GeneBank, the aspergillus oryzae genomic dna of said extracted of take is template, with upstream primers F sNf and downstream primer FxNr, carries out the separated pyrF expression cassette of special pcr amplification.Wherein:
Upstream primer FsNf sequence is:
GGAATTC
cATATGtGAAAGACTGCTGCAAAGCC, wherein contains NdeI restriction enzyme site;
Downstream primer FxNr sequence is:
GGAATTC
cATATGaAGCAGTCGTACATACATGG, wherein contains NdeI restriction enzyme site.
All pcr amplification reaction conditions and reaction system, with reference to Vazyme high-fidelity enzyme reagent kit specification sheets, are cut glue purification with reference to Axygen test kit specification sheets above.PCR product is carried out to agarose gel electrophoresis, reclaim, connect pEASY-Blunt clonning vector (Transgen, USA), bacterium colony PCR checking positive colony send order-checking, and order-checking is completed by the biological order-checking of farsighted enlightening company.
1.6, PaSA2 merges the structure of fragment
PaS and the GpA2 obtaining after PCR separation and purification of take is template, with upstream primer PaXf and downstream primer 12A2Hr, merges pcr amplification, obtains PaSA2 fragment, and introduces restriction enzyme site NheI and AscI.Its reaction system is:
Merge PCR reaction conditions with reference to Vazyme high-fidelity enzyme reagent kit specification sheets, cut glue purification with reference to Axygen test kit specification sheets.PCR product is carried out to agarose gel electrophoresis, reclaim, connect pEASY-Blunt clonning vector, bacterium colony PCR checking positive colony send order-checking, and order-checking is completed by the biological order-checking of farsighted enlightening company.
1.7, the structure of aspergillus oryzae pBC12FA2 expression vector
Take pBC-Hygro as original plasmid, utilize the restriction enzyme site that its top carries that above-mentioned fragment is connected successively and builds pBC12FA2.Digestion with restriction enzyme and the order of connection are as follows: NotI is single endonuclease digestion TamyB and pBC-Hygro respectively, obtain pBC12 after connection; NdeI is single endonuclease digestion pyrF and pBC12 respectively, obtains pBC12F after connection; XhoI and HindIII be double digestion pBC12F and PaSA2 respectively, obtains pBC12FA2 after connection.DNA ligation completes connecting fluid is proceeded to intestinal bacteria competence DH5 α (Tian Gen company), and the LB that then coating contains paraxin (50mg/ml) is dull and stereotyped.Choose mono-clonal, the LB liquid nutrient medium that access contains paraxin (50mg/ml), bacterium colony PCR checking positive colony send order-checking.After enlarged culturing, plasmid extraction is with reference to the little plasmid kit of carrying of sky root, obtains expression vector pBC12FA2, obtain can expression alien gene GpA2 aspergillus oryzae expression vector.
1.8, aspergillus oryzae pBC12FNHA2 can affine purification Table reaches the structure of carrier
Take pBC12FA2 as template, utilize KOD rite-directed mutagenesis test kit after signal peptide, insert the His-Tag label that is usually used in affinity purification before NheI and AscI, this sequence label is: CATCATCACCATCACCAT.Primer for the insertion of fixing a point is FA2NHf and FA2NHr.Its sequence is respectively:
FA2NHf：
CATCACCATGCTAGCGGCGCGCCTAAAAC；
FA2NHr：
GTGATGATGAGGCGTTGCAGCCAAAGCAG。
Its operation steps is with reference to KOD rite-directed mutagenesis test kit (ToYoBo, Japan) specification sheets.Its subsequent step is as described in 1.6.Obtain can purifying foreign protein GpA2 expression vector pBC12FNHA2.
1.9, the structure that aspergillus oryzae pBC12FNH can affinity purification universal expression vector
Take pBC12FNHA2 as template, utilize KOD rite-directed mutagenesis test kit to delete GpA2 sequence, is 12FNHf and 12FNHr for the primer of fixing a point to insert.Its sequence is respectively:
12FNHf:AAGCTTGATATCGAATTCCTGCAGCC；
12FNHr:AGGCGCGCCGCTAGCATGGTGATG。
Its step is as described in 1.7.Obtain can purifying foreign protein universal expression vector pBC12FNH.
Embodiment 2. utilizes aspergillus oryzae to express equipment expressing green fluorescent protein
2.1, the amplification of green fluorescence protein gene
The plasmid pMD19-T simple vector that is connected with green fluorescent protein GFP (NCBI GenBank accession number:AY013824) of take is template, uses primer GFPNf/GFPHr through pcr amplification, to obtain the enhanced green fluorescent protein gene of 717bp (comprising restriction enzyme site interior).Primer sequence is:
GFPNf:CACTA
gCTAGCaTGAGTAAAGGAGAAGAAC, wherein contains NheI restriction enzyme site;
GFPHr:CCC
aAGCTTtTATTTGTATAGTTCATCCATG, wherein contains HindIII restriction enzyme site.
The length of amplified production and the result of sequential analysis all conform to expection.
2.2, GFP and expression equipment docks
By above-mentioned amplified production (GFP gene) process NheI, HindIII restriction enzyme digestion, use T4 ligase enzyme to be connected to through NheI, in the expression vector pBC12FNH of HindIII restriction enzyme digestion, obtained expressing containing PaS+His-Tag+GFP+TamyB the recombinant vectors pBC12FNHGFP of equipment.Its follow-up method is with reference to described in 1.6.
2.3, the preparation of aspergillus oryzae uracil auxotrophy RIB40-PF1 protoplastis
(1) by the spore inoculating on A.oryzae RIB40-PF1 (CGMCC No:9129) inclined-plane in CM liquid nutrient medium, 30 ℃ of 200rpm incubated overnight (12～16h), form fine and close very little bacterium ball (d<2mm) with under aseptic washing, with aseptic Microcloth, filter substratum and obtain mycelium, with solution I washing 2～3 times.In washing process, 4 ℃ of centrifugal 1min of 6000rpm, remove supernatant.Finally obtain clean mycelium (approximately 200mg).
(2) configuration enzyme liquid system (5ml): cellulase 0.1g, helicase 0.1g, lywallzyme 0.025g.Filtration sterilization.
(3) in the prepared mycelium of step (1), add the enzyme liquid of configuration in 1ml step (2), 150rpm, 30 ℃ of enzymolysis 3h.
(4) after enzymolysis, 4 ℃ of centrifugal 5min of 3500rpm, remove supernatant, solution II washing 1～2 time.
(5) add appropriate solution II (about 1ml) suspension protoplastis.
2.4, aspergillus oryzae uracil auxotrophy RIB40-PF1 protoplast transformation pBC12FNHGFP
(1) the 200ul protoplastis making in 2.2 is mixed with 1ng (about 20ul) plasmid pBC12FNHGFP, add 50ul solution III, ice bath 30min.
(2) in the middle system of step (1), add 2ml solution III, room temperature is placed 5min.
(3) in (2), in system, add 4ml solution II, 4 ℃ of centrifugal 5min of 5000rpm, remove supernatant, and 400ul solution II suspends.
(4) JiangMM lower floor substratum is down flat plate, solidifies; MM upper strata substratum is down to 40 ℃ of left and right, sneaks into protoplast transformation liquid in (3), rocks rapidly flat board, and evenly tiling is placed 3～5d for 30 ℃.
2.5, the evaluation of transformant and the expression of goal gene
The genomic dna of the positive transformant of extraction embodiment 2 step 2.4 gained, as template, carries out pcr amplification with primer GFPf and GFPr.Its sequence is:
GFPf：ATGAGTAAAGGAGAAGAAC；
GFPr：TTATTTGTATAGTTCATCCATG。
Result demonstration, the amplified production of transformant obtains the special band of target gene size, and is that template is carried out same PCR reaction with the genomic dna of original strain A.oryzaeRIB40-PF1, does not occur amplified production.
The restructuring aspergillus oryzae strain of acquisition is inoculated in liquid fermentation medium, after 30 ℃, 200rpm shaking culture 72h, gets fermented liquid point to slide glass, be put into fluorescence microscopy Microscopic observation, with blue-light excited observation green fluorescent protein.As shown in Figure 4, restructuring aspergillus oryzae filament sends green fluorescence to result under fluorescent microscope, and original aspergillus oryzae filament does not have green fluorescence under fluorescent microscope.
Above-described, be only preferred embodiment of the present invention, not in order to limit scope of the present invention, the above embodiment of the present invention can also make a variety of changes.Be that simple, the equivalence that every claims according to the present patent application and description are done changes and modify, all fall into the claim protection domain of patent of the present invention.The present invention not detailed description be routine techniques content.
Claims (9)
1. for express an expression equipment for foreign protein in aspergillus oryzae emiocytosis, it is characterized in that, described expression equipment from 5 ' to 3 ' comprises following element successively: (1) controls foreign gene in the promotor of aspergillus oryzae transcription; (2) control the encoding sequence of foreign protein signal peptide of secreting, expressing in aspergillus oryzae; (3) for the encoding sequence of the label of foreign protein affinity purification; (4) multiple clone site; (5) control foreign gene and in aspergillus oryzae, stop the terminator of transcribing; (6) for the expression cassette of the aspergillus oryzae orotate phosphoribosyl transferase gene of plasmid transformation and selection mark, the encoding sequence of the expression cassette of the described aspergillus oryzae orotate phosphoribosyl transferase gene for plasmid transformation and selection mark is as shown in SEQ ID:2.
2. expression equipment according to claim 1, is characterized in that, described promotor is aspergillus oryzae α-amylase promotor; The described label for foreign protein affinity purification is His-Tag label; Described terminator is aspergillus oryzae α-amylase terminator.
3. expression equipment according to claim 2, is characterized in that, the encoding sequence of described aspergillus oryzae α-amylase promotor the 7th to the 623rd of sequence as shown in SEQ ID NO:1; The encoding sequence of described signal peptide the 624th to the 695th of sequence as shown in SEQ ID NO:1; The encoding sequence of the described His-Tag label for foreign protein affinity purification the 696th to the 713rd of sequence as shown in SEQ ID NO:1; The encoding sequence of described multiple clone site the 714th to the 784th of sequence as shown in SEQ ID NO:1; The encoding sequence of described aspergillus oryzae α-amylase terminator the 785th to the 1681st of sequence as shown in SEQ ID NO:1.
4. expression equipment according to claim 3, is characterized in that, described expression equipment further comprises the encoding sequence of foreign protein, the encoding sequence of described foreign protein is positioned among multiple clone site, before or after.
5. the recombinant expression vector containing the expression equipment described in any one in good grounds claim 1-4.
6. an aspergillus oryzae genetic engineering bacterium of expressing foreign protein, is characterized in that, contains the expression equipment described in any one in good grounds claim 1-4 in the genome of described aspergillus oryzae genetic engineering bacterium.
7. aspergillus oryzae genetic engineering bacterium according to claim 6, is characterized in that, the Host Strains of described aspergillus oryzae genetic engineering bacterium is aspergillus oryzae uracil auxotrophy bacterial strain RIB40-PF1 (CGMCC NO:9129).
8. the preparation method of the aspergillus oryzae genetic engineering bacterium of an expression foreign protein according to claim 7, it is characterized in that, comprise: the recombinant expression vector of the expression equipment containing described in any one in good grounds claim 1-4 is transformed to aspergillus oryzae uracil auxotrophy bacterial strain RIB40-PF1, select positive colony, make the aspergillus oryzae genetic engineering bacterium of described expression foreign protein.
9. the application of the aspergillus oryzae genetic engineering bacterium of an expression foreign protein according to claim 6 in preparing industrial enzyme preparation, fodder additives or pharmaceutical grade protein.
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