CN104004760B - A kind of expression equipment and its aspergillus oryzae genetic engineering bacterium being used in Aspergillus oryzae cell secreting, expressing foreign protein - Google Patents
A kind of expression equipment and its aspergillus oryzae genetic engineering bacterium being used in Aspergillus oryzae cell secreting, expressing foreign protein Download PDFInfo
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Abstract
The present invention provides a kind of expression equipment and its aspergillus oryzae genetic engineering bacterium being used in Aspergillus oryzae cell secreting, expressing foreign protein, and the expression equipment from 5 ' to 3 ' includes successively：(1) promoter that control foreign gene is transcribed in aspergillus oryzae；(2) coded sequence of foreign protein signal peptide of secreting, expressing in aspergillus oryzae is controlled；(3) it is used for the coded sequence of the label of foreign protein affinity purification；(4) multiple cloning sites；(5) control foreign gene terminates the terminator of transcription in aspergillus oryzae；(6) it is used for the expression cassette of the aspergillus oryzae deic acid phosphate ribose transferase gene of plasmid transformation and selection mark, its coded sequence such as SEQ ID:Shown in 2.The expression equipment utilization auxotroph genescreen marks, and improves conversion positive rate, shortens operating time and workload, is adapted to the secreting, expressing of most of foreign genes；The aspergillus oryzae genetic engineering bacterium can high efficient expression be acid, neutral starch enzyme, has important application in paper-making industry and textile industry.
Description
Technical field
The invention belongs to biological technical field, relates more specifically to a kind of be used in Aspergillus oryzae cell secreting, expressing external source egg
White expression equipment and its aspergillus oryzae genetic engineering bacterium.
Background technology
Aspergillus oryzae (Aspergillus oryzae) is the conventional bacterial strain of fermentation industry, is a kind of aerobic fungi, belongs to
Deuteromycotina, aspergillus.Mycelia is afterwards typically yellowish-brown in yellow green；Aspergillus oryzae mycelia is made up of many cells, is a kind of production
The bacterial strain of complex enzyme, outside removing protein enzyme, may further be used to secreting amylase, cellulase, pectase, glycosidase, lipase, peptase and
Phytase etc.；Wherein, industrial common taka-diastase (TAA) comes from the alpha-amylase of aspergillus oryzae.Aspergillus oryzae is by FDA
The safe microorganisms (GRAS) of identification, are widely used in the works such as brewing industry, flavouring, feed manufacturing industry, weaving, papermaking, slurrying
Industry field, and by safe handling more than 1000 years.At present, full genome is sequenced aspergillus oryzae RIB40, thread true based on aspergillus oryzae
Bacterium expression system also increasingly receives significant attention.
In addition to the ability with fabulous synthesis and secretory protein, the A.oryzae albumen also with eukaryotic microorganisms point
Secrete mechanism and the protein post-translational modification similar to mammlian system, processing characteristics；Its expression system and prokaryotic expression system
System compared to having a very big advantage, such as after exogenous protein expression it is protein modified, introne identifies, signal peptide is wiped out and peptide chain
Correct folding and protein secretion.Because it is suitable to the plurality of advantages of protein production, and there is no toxicity, aspergillus oryzae to the mankind
The important biomolecule material that related researcher carries out fungal cell, science of heredity and physiological and biochemical research is turned into.Japan subsidizes
The examining order of aspergillus oryzae genome is supported, whole genome sequence has been completed and announced at present.Aspergillus oryzae full-length genome is surveyed
The completion of sequence is laid a good foundation for the functional gene for having important biomolecule meaning using functional genomics technique study
(Machida M et al.Genome sequencing and analysis of Aspergillus oryzae.Nature,
2005,438:1157-1161)。
Since the eighties in last century, filamentous fungi expression system is reported in succession, and the high-yield expression of homologous protein is
It is verified, but most of expression of foreign protein under the same conditions is still in~mg/l, and because filamentous fungi is not deposited
In natural plasmid, genomic data is larger, and secreting, expressing process is complicated, causes at present that there has been no such as Escherichia coli and finish red ferment
The expression system (including expressive host and expression plasmid) of female the same industrialized production.By screening aspergillus oryzae related nutritional defect
Type, corresponding covering expression vector is built, is realized by means such as strong promoter, amalgamation and expressions and derives from fungi and animals and plants base
Existing relevant report (the Minetoki T et al.Improvement of promoter activity by of the heterogenous expression of cause
the introduction of multiple copies of the conserved region III sequence,
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. is in aspergillus oryzae pyrG influences deficiency system by with the mesophilic fungi of PamyB and TamyB control
The heterogenous expression of Myceliophthora thermophila laccase genes, but expression quantity 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)；2011
Nobuo Yamashita etc., using sC as selection markers, will come from aspergillus fumigatus (Aspergillus in aspergillus oryzae
Fumigatus protease inhibitor gene) is placed between aspergillus oryzae TAA promoters and terminator, by real after Fiber differentiation
Now heterogenous expression (the Nobuo Yamashita et al.High-yields heterologous production of the gene
of the novel Aspergillus fumigatus elastase inhibitor AFUEI in Aspergillus
oryzae,J biosci bioeng,2011,112(2):114~117).Nevertheless, there has been no on these so far
The report of the purifiable universal expression vector of the corresponding general interchangeable elements of auxotroph system.
The content of the invention
It is an object of the invention to provide it is a kind of be used for Aspergillus oryzae cell secreting, expressing foreign protein expression equipment and its
Aspergillus oryzae genetic engineering bacterium, so as to solve aspergillus oryzae filamentous fungi expression system in the prior art it is low to foreign protein expression quantity with
And the defects of isolating and purifying difficulty.
In order to solve the above-mentioned technical problem, the present invention uses following technical scheme：
The first aspect of the present invention is to provide a kind of expression equipment being used in Aspergillus oryzae cell secreting, expressing foreign protein,
The expression equipment from 5 ' to 3 ' includes elements below successively：(1) promoter that control foreign gene is transcribed in aspergillus oryzae；
(2) coded sequence of foreign protein signal peptide of secreting, expressing in aspergillus oryzae is controlled；(3) for foreign protein affinity purification
The coded sequence of label；(4) multiple cloning sites；(5) control foreign gene terminates the terminator of transcription in aspergillus oryzae；(6) use
In the expression cassette of the aspergillus oryzae deic acid phosphate ribose transferase gene of plasmid transformation and selection mark, the code sequence of the expression cassette
Row such as SEQ ID:Shown in 2.
In the present invention, described promoter is the promoter that foreign gene can be controlled to be transcribed in aspergillus oryzae, preferably
For oryzae alpha-amylase promoter (PamyB), the sequence of more preferably promoter is SEQ ID NO:The 7th to the 623rd of 1
It is shown.
In the present invention, described signal peptide can be that foreign protein secreting, expressing in aspergillus oryzae can be controlled in this area
Any signal peptide (secretion peptide), the coded sequence of preferable signal peptide is SEQ ID NO:The 624th of 1 is to the 695th institute
Show.
In the present invention, the label for foreign protein affinity purification can be used in affinity purification in this area
Any label of albumen, preferably His-Tag labels, the more preferably coded sequence of label such as SEQ ID NO:Sequence shown in 1
The 696th to the 713rd.
In the present invention, the multiple cloning sites can be the various conventional multiple cloning sites in this area, it is preferable that described more
The coded sequence of cloning site such as SEQ ID NO:The 714th to the 784th of sequence shown in 1.
In the present invention, the terminator is the terminator that foreign gene can be controlled to terminate transcription in aspergillus oryzae, preferably
Be oryzae alpha-amylase terminator (TamyB), the sequence of more preferably terminator is SEQ ID NO:The 785th of 1 is to
Shown in 1681.
Most preferably, in expression equipment provided by the invention, the coded sequence of the oryzae alpha-amylase promoter
Such as SEQ ID NO:The 7th to the 623rd of sequence shown in 1；The coded sequence of the signal peptide such as SEQ ID NO:Sequence shown in 1
The 624th to the 695th of row；The coded sequence such as SEQ ID of the His-Tag labels for foreign protein affinity purification
NO:The 696th to the 713rd of sequence shown in 1；The coded sequence of the multiple cloning sites such as SEQ ID NO:Sequence shown in 1
The 714th to the 784th；The coded sequence of the oryzae alpha-amylase terminator such as SEQ ID NO:Sequence shown in 1
785th to the 1681st.
Preferably, the expression equipment further comprises the coded sequence of foreign protein, the code sequence of the foreign protein
Row be located at multiple cloning sites among, before or after.
The second aspect of the present invention also provides a kind of recombinant expression carrier for including expression equipment as described above.
Preferably, vector plasmid is filamentous fungi expression vector, as pBC-Hygro, pBARGPE1, pAN7-1, pRTRI,
PBC-phleo, but these carriers are not limited to, more preferably selected from pBC-Hygro.
The third aspect of the present invention also provides a kind of aspergillus oryzae genetic engineering bacterium for expressing foreign protein, the aspergillus oryzae base
Because containing expression equipment as described above in the genome of engineering bacteria.
The Host Strains of the aspergillus oryzae genetic engineering bacterium be preferably aspergillus oryzae RIB40,3.042, AS3.863,
AS3.951, HBR9, RIB326 etc., more preferably it is aspergillus oryzae uracil auxotrophy bacterial strain RIB40-PF1.The present invention is made
Bacterial strain RIB40-PF1 is that inventor oneself screens acquisition, and the bacterial strain has been preserved in Chinese microorganism strain preservation management committee
Member's meeting common micro-organisms center (CGMCC, positioned at BeiJing, China), preserving number is CGMCC NO：9129, preservation day is
2014.05.08。
The fourth aspect of the present invention also provides a kind of aspergillus oryzae genetic engineering bacterium of expression foreign protein as described above
Preparation method, including：Recombinant expression carrier containing expression equipment as described above is converted into aspergillus oryzae uracil auxotrophy
Type bacterial strain RIB40-PF1, selects positive colony, and the aspergillus oryzae genetic engineering bacterium of the expression foreign protein is made.
The fifth aspect of the present invention also provides a kind of raw albuminiferous method, includes the rice of the culture expression foreign protein
Aspergillus genetic engineering bacterium, the foreign protein is obtained from culture.
The sixth aspect of the present invention also provides a kind of aspergillus oryzae genetic engineering bacterium of expression foreign protein as described above and existed
Prepare the application in industrial enzyme preparation, feed addictive or pharmaceutical grade protein.The enzyme preparation is alpha-amylase, glucoamylase
Enzyme, beta-glucosidase, lactase, glucose oxidase, dextranase, zytase, Pullulanase, phytase, fat
Enzyme, protease, lipoxygenase, cellulose restriction endonuclease, cellulose excision enzyme, cellobiase etc..
Compared with prior art, the present invention has the advantages that：
1st, expression equipment of the invention is easy to preserve and DNA is operated on a circularized plasmid.
2nd, expression equipment of the invention utilizes aspergillus oryzae deic acid phosphate ribose transferase gene plasmid transformation and selection first
Mark, conversion positive rate can be improved, shorten operating time and workload.
3rd, expression equipment of the invention has the secreting, expressing ability of optimization, is adapted to the secretion table of most of foreign genes
Reach.
4th, rare interface peace end interface is contained in the multiple cloning sites of expression equipment of the invention, it is compatible most
The connection of foreign gene, save the operating time and improve operating efficiency.
5th, expression equipment of the invention can realize the exogenous proteins gene from animal, plant and fungi thread more
Efficient secretory expression in cell eukaryotic microorganisms aspergillus oryzae.Because aspergillus oryzae condition of culture is extensive, suitable for solid culture and
Liquid deep layer fermenting；Aspergillus oryzae does not have toxicity in itself, and mycotoxin and antibiotic will not be produced under condition of enzyme production, meets the world
Food security certification；Exoprotein caused by aspergillus oryzae is easily isolated purifying, and cost is cheap.Therefore the present invention can be to wash
Wash, weave, feed, food, papermaking, the enzyme preparation industry such as medicine provide genetic engineering production bacterial strain, improve the production of enzyme preparation
Efficiency reduces production cost.
6th, expression equipment (including promoter terminator) of the invention shortens, and the expression cassette finally built is smaller, is more conducive to
The attended operation of construction expression plasmid and the raising of transformation efficiency.Expression quantity is more steady, will not be influenceed and had by condition of culture
Larger fluctuation.If expression cassette is larger dependent on the fluctuation of condition of culture, higher are required to operating personnel.The table of the present invention
Up to the expression of equipment, the condition of thalline culture medium can be more rough, cheap.Thalli growth is very fast, and it is exactly to reduce to reduce the time
Cost.Expression cassette of the present invention can be applied in combination with other expression cassettes, while express two exogenous proteins.
7th, the aspergillus oryzae uracil auxotrophy bacterium that aspergillus oryzae genetic engineering bacterium of the invention is obtained using screening first
Strain RIB40-PF1, and aspergillus oryzae deic acid phosphate ribose transferase gene plasmid transformation and selection mark is combined, can high efficient expression acid
Property, neutral starch enzyme, have critically important application in starch industry, paper-making industry and textile industry.
Brief description of the drawings
Fig. 1 is the physical map and its building process that aspergillus oryzae expresses equipment pBC12FA2.Each restriction enzyme site as illustrated,
Wherein, PamyB:Alpha-amylase (TAA) promoter；TamyB:Alpha-amylase (TAA) terminator；pyrF:Aspergillus oryzae orotic acid phosphorus
Sour phosphoribosynltransferase (ORPTase) genescreen mark；
Fig. 2 is aspergillus oryzae universal expression vector pBC2FNH structure figures；
Fig. 3 is expression of the green fluorescent protein in aspergillus oryzae, wherein, A：Transformant PF1-pBC12FNHEGFP-6 is induced
Observation result of the mycelia of culture under green fluorescence light source (left side) and ordinary light source (right side)；B：Wild mushroom RIB40 Fiber differentiations
Observation result of the mycelia 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.It should be understood that following examples are merely to illustrate this
Invention is not for limitation the scope of the present invention.
Raw material or reagent used in the present invention is commercially available in addition to special instruction.
First, equipment is expressed
The invention provides a kind of expression equipment being used in Aspergillus oryzae cell secreting, expressing foreign protein.Wherein, meter Qu
Mould alpha-amylase promoter is held the 5 ' of the gene, is about made up of 617 nucleotides；Oryzae alpha-amylase terminator is in the base
3 ' ends of cause, are about made up of 897 nucleotides；A segment signal peptide sequence is also connected with after the oryzae alpha-amylase promoter
Row, encode the secretion peptide of 21 amino acid sequences.The present invention takes promoter, signal peptide and the termination of the oryzae alpha-amylase
Son, and added after signal peptide for expressing protein affinity purification label and multiple cloning sites, consequently facilitating foreign gene is smooth
Insertion is expressed, and is used for the aspergillus oryzae orotic acid ribose phosphate transfer of plasmid transformation and selection mark in connection after terminator
The gene coded sequence of enzyme, finally constitute the table that structure is promoter-signal peptide-purification tag-MCS- terminators-selection markers
Up to equipment.The expression equipment can the various foreign proteins of secreting, expressing, expression quantity be high in aspergillus oryzae, are easily isolated purifying, and
Cost is cheap.
The preparation method of the expression equipment of the present invention is as follows：
(1) expanded by PCR, respectively obtain the promoter (PamyB+ of the oryzae alpha-amylase comprising secretion peptide sequence
Signal peptide, PaS), certain exogenous amylases gene (GpA2), oryzae alpha-amylase terminator (TamyB), orotic acid
Phosphoribosyltransferase genescreen marks expression cassette each DNA sequence dna.
(2) the fusion sequence for obtaining promoter-signal sequence (PaS) and amylase gene (GpA2) progress fusion DNA vaccine
Arrange (PaSA2), mark expression cassette, PaSA2 to connect successively terminator (TamyB), deic acid phosphate ribose transferase genescreen
It is connected in expression vector pBC-Hygro, structure obtains recombinant expression carrier 1, i.e. pBC12FA2；By rite-directed mutagenesis insertion 6 ×
His-Tag purification tags, expression vector 2, i.e. pBC12NHA2 are obtained, it is to obtain to be pinpointed by inverse PCR and delete foreign gene GpA2
The universal expression vector 3 of foreign gene, i.e. pBC12FNH are not included comprising the expression equipment.
(3) external source target gene is inserted into above-mentioned expression vector 3, make foreign gene be located at promoter and terminator it
Between, obtain recombinant expression plasmid 4, i.e. pBC12FNH-EGFP.
The multiple cloning sites for being used to connect in above-mentioned steps (1) carry for skeleton carrier pBC-Hygro；By promoter, end
Only son connects with selection markers, each element can be connected into expression with the method that digestion with restriction enzyme connects with ligase and carried
Body, it is all this area Normal practice that described digestion with restriction enzyme connects with ligase.
In above-mentioned steps (3), foreign gene is inserted into above-mentioned expression vector 3, can use digestion with restriction enzyme and
The method of ligase connection, it is all this area Normal practice that described digestion with restriction enzyme connects with ligase.
2nd, aspergillus oryzae genetic engineering bacterium
Present invention also offers a kind of aspergillus oryzae genetic engineering bacterium, the genetic engineering bacterium is to lead to the expression equipment of the present invention
The protoplast transformation for crossing PEG mediations is incorporated into aspergillus oryzae RIB40-PF1 cells, and culture screening transformant is prepared, specifically
Preparation process is as follows：
(1) carrier containing expression equipment of the present invention is transformed into meter Qu with the PEG protoplast transformation methods mediated
Mould RIB40-PF1 cells；
(2) cell after above-mentioned conversion is screened, identified, obtain expressing the aspergillus oryzae genetic engineering of foreign protein
Bacterium.
The experimental method of unreceipted actual conditions in the following example, generally according to normal condition, such as《Molecular cloning：It is real
Test room handbook》(NewYork：Cold Spring Harbor Laboratory Press, 1989) condition described in is carried out,
Or according to the condition proposed by manufacturer.
The buffer solution and culture medium used in embodiment are as follows：
(1)TE(1000ml)：
ZnSO4·7H2O0.35g, MnCl2·4H2O0.5g, H3BO30.03g, CoCl2·6H2O0.945g, CuCl2·
2H2O0.01g, NiCl2·6H2O0.12g, NaMoO4·2H2O0.18g, 6M HCl13ml, add deionized water to be settled to 500mL.
(2) sodium phosphate buffer (100ml, pH5.8)：
Na2HPO4·12H2O0.5788g, NaH2PO4·2H2O2.8996g。
(3) Tris-HCl buffer solutions (100ml, pH7.5)：
Tris0.6057g, pH to 7.5 is adjusted with HCl.
(4) solution I：
NaCl0.7M, sucrose 1.0g, it is dissolved in 50ml sodium phosphate buffers, constant volume (adding water) to 100ml.
(5) solution II：
Sorbierite 22.3085g, CaCl20.578g, NaCl0.2045g, it is dissolved in 20ml Tri-HCl buffer solutions, constant volume
(adding water) is to 100ml.
(6) solution III：
PEG400030g, CaCl20.289g, it is dissolved in 10ml Tri-HCl buffer solutions, constant volume (adding water) to 50ml.
(7) CM culture mediums (100ml)：
NaNO30.6g, KCl0.05g, KH2PO40.08g, K2HPO40.104g, glucose 1g, MgSO40.052g,
Peptone0.2g, Yeast extract0.1g, uridine 0.1221g, TE100ul；If being made into inclined-plane, 1.5~2.0g is added
Agar powder.
(8) MM culture mediums (100ml)：
NaNO30.6g, KCl0.05g, KH2PO40.08g, K2HPO40.104g, glucose 1g, MgSO40.052g,
TE100ul, sucrose 20.54g.
Agar powder：Upper strata 0.8g, lower floor 1.2g.
(9) fermentation medium (100ml)：
Dextrin 2g, sucrose 0.3g, peptone 0.1g, yeast extract 0.5g, NaNO30.1g, MgSO4·7H2O0.05g,
FeSO4·7H2O0.001g。
Embodiment 1. includes the structure of the expression vector of the aspergillus oryzae expression equipment of the present invention
1.1st, the extraction of aspergillus oryzae genome
By aspergillus oryzae Aspergillus oryzae RIB40 (National Research Institute of
Brewing StockCulture, ATCC42149) be inoculated on CM culture mediums, at 30 DEG C incubated 7 days to spore into
It is ripe.Prepare appropriate spore suspension to be inoculated in CM medium liquid seed culture mediums, 2 days are cultivated under the conditions of 30 DEG C of 200rpm extremely
Mycelia bulk concentration reaches 4~5g/L and is used to extract genome, and method is with reference to Omega fungal gene group extracts kit specifications.
1.2nd, the separation of oryzae alpha-amylase promoter (PamyB) and its signal peptide sequence
According to the sequence for the amyB promoters issued on GeneBank, using the aspergillus oryzae genomic DNA of said extracted as mould
Plate, special PCR amplification separation PaS (PamyB+signal are carried out with sense primer PaXf and anti-sense primer Pamr
peptide).Wherein：
Sense primer PaXf sequences are：
CCGCTCGAGGAATTCATGGTGTTTTGATCATTTT, wherein (primer is by Shanghai containing XhoI restriction enzyme sites
JaRa bioengineering Co., Ltd synthesizes, similarly hereinafter)；
Anti-sense primer Pamr sequences are：
AGGCGCGCCGCTAGCAGGCGTTGCAGCCAAAGC, wherein containing AscI and NheI restriction enzyme sites.
1.3rd, the separation of oryzae alpha-amylase terminator (TamyB)
According to the sequence for the TamyB terminators issued on GeneBank, using the aspergillus oryzae genomic DNA of said extracted as mould
Plate, special PCR amplification separation PaS (PamyB+signal are carried out with sense primer TaNf and anti-sense primer TaNr
peptide).Wherein：
Sense primer TaNf sequences are：
ATAAGAATGCGGCCGCGGGTGGAGAGTATATG, wherein containing NotI restriction enzyme sites；Anti-sense primer TaNr sequences
It is classified as：
ATAAGAATGCGGCCGCAATTCTTGAGGACCATTAC, wherein containing NotI restriction enzyme sites.
1.4, external source auxiliary amylase gene separation
To have connected GpA2 (NCBI GenBank accension number:KJ093844 pMD19Tsimple) is
Template, expand through PCR to have obtained GpA2 genes using primer 12A2mf and 12A2Hr.Wherein
Sense primer 12A2mf sequences are：
GCCTGCTAGCGGCGCGCCT AAAACCGCCGCGGAATGG, wherein containing AscI and NheI restriction enzyme sites；
Anti-sense primer 12A2Hr sequences are：
CCCAAGCTTTTAAGCACATAAACTGCCCT, wherein containing HindIII restriction enzyme sites.
1.5th, the separation of aspergillus oryzae orotidine phosphoribosyltransferase riddled basins
According to the sequence of pyrF in the aspergillus oryzae RIB40 full-length genomes issued on GeneBank, with the meter Qu of said extracted
Mould genomic DNA is template, and special PCR amplification separation pyrF expression is carried out with upstream primers F sNf and anti-sense primer FxNr
Box.Wherein：
Sense primer FsNf sequences are：
GGAATTCCATATGTGAAAGACTGCTGCAAAGCC, wherein containing NdeI restriction enzyme sites；
Anti-sense primer FxNr sequences are：
GGAATTCCATATGAAGCAGTCGTACATACATGG, wherein containing NdeI restriction enzyme sites.
All of above pcr amplification reaction condition and reaction system are cut with reference to Vazyme high-fidelity enzyme reagent kit specifications
Glue purification is with reference to Axygen kit specifications.PCR primer is entered into row agarose gel electrophoresis, reclaimed, even pEASY-Blunt
Clonning vector (Transgen, USA), bacterium colony PCR checking positive colony send sequencing, are sequenced public by farsighted enlightening biological order-checking
Department completes.
1.6th, PaSA2 merges the structure of fragment
The PaS and GpA2 obtained after being isolated and purified using PCR is entered as template with sense primer PaXf and anti-sense primer 12A2Hr
Row fusion DNA vaccine expands, and obtains PaSA2 fragments, and introduce restriction enzyme site NheI and AscI.Its reaction system is：
Fusion DNA vaccine reaction condition cuts glue purification with reference to Axygen reagents with reference to Vazyme high-fidelity enzyme reagent kit specifications
Box specification.PCR primer is entered into row agarose gel electrophoresis, reclaimed, even pEASY-Blunt clonning vector, bacterium colony
PCR checking positive colonies send sequencing, are sequenced and are completed by Rui Di biological order-checkings company.
1.7th, the structure of aspergillus oryzae pBC12FA2 expression vectors
Using pBC-Hygro as original plasmid, above-mentioned fragment is sequentially connected structure using the restriction enzyme site that its top carries
pBC12FA2.Digestion with restriction enzyme and the order of connection are as follows：NotI distinguishes single endonuclease digestion TamyB and pBC-Hygro, after connection
Obtain pBC12；NdeI distinguishes single endonuclease digestion pyrF and pBC12, and pBC12F is obtained after connection；XhoI and HindIII difference double digestions
PBC12F and PaSA2, pBC12FA2 is obtained after connection.DNA coupled reactions are completed connection liquid being transferred to E. coli competent DH5 α
(Tiangeng company), then coating contain the LB flat boards of chloramphenicol (50mg/ml).Choose monoclonal, access contains chloramphenicol
The LB fluid nutrient mediums of (50mg/ml), bacterium colony PCR checking positive colonies send sequencing.Plasmid extraction is with reference to Tiangeng after expanding culture
It is small to carry plasmid kit, expression vector pBC12FA2 is obtained, that is, obtains to express carrying with expression alien gene GpA2 aspergillus oryzae
Body.
1.8th, aspergillus oryzae pBC12FNHA2 can affinity purification expression vector structure
Using pBC12FA2 as template, using KOD site-directed mutagenesis kits after signal peptide, inserted before NheI and AscI
It is usually used in the His-Tag labels of affinity purification, the sequence label is：CATCATCACCATCACCAT.For pinpointing drawing for insertion
Thing is FA2NHf and FA2NHr.Its sequence is respectively：
FA2NHf：CATCACCATGCTAGCGGCGCGCCTAAAAC；
FA2NHr：GTGATGATGAGGCGTTGCAGCCAAAGCAG。
Its operating procedure is with reference to KOD site-directed mutagenesis kits (ToYoBo, Japan) specification.The institute of its subsequent step such as 1.6
State.Obtain purifying foreign protein GpA2 expression vector pBC12FNHA2.
1.9th, aspergillus oryzae pBC12FNH can affinity purification universal expression vector structure
Using pBC12FNHA2 as template, GpA2 sequences are deleted using KOD site-directed mutagenesis kits, for pinpointing drawing for insertion
Thing is 12FNHf and 12FNHr.Its sequence is respectively：
12FNHf:AAGCTTGATATCGAATTCCTGCAGCC；
12FNHr:AGGCGCGCCGCTAGCATGGTGATG。
Its step is as described in 1.7.Obtain purifying the universal expression vector pBC12FNH of foreign protein.
Embodiment 2. utilizes aspergillus oryzae expression equipment expressing green fluorescent protein
2.1st, the amplification of green fluorescence protein gene
To be connected with green fluorescent protein GFP (NCBI GenBank accession number:AY013824 plasmid)
PMD19-T simple vector are template, expand to have obtained 717bp through PCR using primer GFPNf/GFPHr and (include digestion
Including site) enhanced green fluorescent protein gene.Primer sequence is：
GFPNf：CACTAGCTAGCATGAGTAAAGGAGAAGAAC, wherein containing NheI restriction enzyme sites；
GFPHr：CCCAAGCTTTTATTTGTATAGTTCATCCATG, wherein containing HindIII restriction enzyme sites.
The length of amplified production and the result of sequence analysis are consistent with expection.
2.2nd, GFP is with expressing docking for equipment
Above-mentioned amplified production (GFP genes) is passed through into NheI, the restricted digestions of HindIII, is connected to using T4 ligases
By NheI, in the expression vector pBC12FNH of the restricted digestions of HindIII, obtain containing PaS+His-Tag+GFP+TamyB
Express the recombinant vector pBC12FNHGFP of equipment.Its subsequent processes is with reference to described in 1.6.
2.3rd, the preparation of aspergillus oryzae uracil auxotrophy RIB40-PF1 protoplasts
(1) by A.oryzae RIB40-PF1 (CGMCC No：9129) spore inoculating on inclined-plane is into CM fluid nutrient mediums,
30 DEG C of 200rpm are incubated overnight (12~16h), form the bacterium ball (d of fine and close very little<2mm) with sterile washing, with sterile
Microcloth filtering culture mediums obtain mycelium, are washed 2~3 times with solution I.4 DEG C of 6000rpm centrifugation 1min in washing process,
Remove supernatant.Finally give clean mycelium (about 200mg).
(2) enzyme liquid system (5ml) is configured：Cellulase 0.1g, glusulase 0.1g, lywallzyme 0.025g.Filtration sterilization.
(3) enzyme liquid of configuration in 1ml steps (2), 150rpm, 30 DEG C of enzymes are added in the mycelium prepared by step (1)
Solve 3h.
(4) after digesting, 4 DEG C of 3500rpm centrifuge 5min, remove supernatant, and solution II is washed 1~2 time.
(5) appropriate solution II (about 1ml) suspension protoplast is added.
2.4th, aspergillus oryzae uracil auxotrophy RIB40-PF1 protoplast transformations pBC12FNHGFP
(1) obtained 200ul protoplasts in 2.2 are mixed with 1ng (about 20ul) plasmids pBC12FNHGFP, added
50ul solution IIIs, ice bath 30min.
(2) 2ml solution IIIs are added in the middle system of step (1), room temperature places 5min.
(3) 4ml solution IIs are added in system in (2), 4 DEG C of 5000rpm centrifuge 5min, remove supernatant, 400ul solution IIs are hanged
It is floating.
(4) MM lower floors culture medium is down flat plate, solidified；MM upper stratas culture medium is down to 40 DEG C or so, is mixed into plasm in (3)
Body conversion fluid, flat board is rocked rapidly, evenly laid out, 30 DEG C of 3~5d of placement.
2.5th, the identification of transformant and the expression of target gene
The genomic DNA for extracting the positive transformant obtained by embodiment 2 step 2.4 is used as template, with primer GFPf with
GFPr enters performing PCR amplification.Its sequence is：
GFPf：ATGAGTAAAGGAGAAGAAC；
GFPr：TTATTTGTATAGTTCATCCATG.
As a result show, the amplified production of transformant obtains the special band of target gene size, and uses original strain
A.oryzaeRIB40-PF1 genomic DNA is that template carries out same PCR reactions, amplified production does not occur.
The restructuring aspergillus oryzae strain of acquisition is inoculated in liquid fermentation medium, 30 DEG C, 200rpm shaken cultivations 72h
Afterwards, take zymotic fluid point to be put into fluorescence microscopy Microscopic observation to slide, observation green fluorescent protein is excited with blue light.As a result
As shown in figure 4, restructuring aspergillus oryzae filament sends green fluorescence under fluorescence microscope, and original aspergillus oryzae filament is glimmering
There is no green fluorescence under light microscope.
Above-described, only presently preferred embodiments of the present invention is not limited to the scope of the present invention, of the invention is upper
Stating embodiment can also make a variety of changes.What i.e. every claims and description according to the present patent application were made
Simply, equivalent changes and modifications, the claims of patent of the present invention are fallen within.The not detailed description of the present invention is
Routine techniques content.
Claims (4)
1. a kind of aspergillus oryzae genetic engineering bacterium for expressing foreign protein, it is characterised in that the aspergillus oryzae genetic engineering bacterium includes
Host Strains and expression vector, the Host Strains are aspergillus oryzae uracil auxotrophy bacterial strain RIB40-PF1, bacterial strain RIB40-
PF1 preserving number is CGMCC NO：9129, the expression vector from 5 ' to 3 ' includes elements below successively：(1) external source base is controlled
Because of the oryzae alpha-amylase promoter transcribed in aspergillus oryzae, sequence such as SEQ ID NO:The 7th of sequence shown in 1 is to
623；(2) sequence of foreign protein signal peptide of secreting, expressing in aspergillus oryzae is controlled, such as SEQ ID NO:Sequence shown in 1
624th to the 695th；(3) it is used for the sequence of the His-Tag labels of foreign protein affinity purification, such as SEQ ID NO:Shown in 1
The 696th to the 713rd of sequence；(4) multiple cloning sites, sequence such as SEQ ID NO:The 714th of sequence shown in 1 is to
784；(5) control foreign gene terminates the oryzae alpha-amylase terminator of transcription, sequence such as SEQ ID in aspergillus oryzae
NO:The 785th to the 1681st of sequence shown in 1；(6) it is used for the aspergillus oryzae orotic acid ribose phosphate of plasmid transformation and selection mark
The expression cassette of transferase gene, the aspergillus oryzae deic acid phosphate ribose transferase gene for plasmid transformation and selection mark
The sequence of expression cassette such as SEQ ID:Shown in 2.
2. aspergillus oryzae genetic engineering bacterium according to claim 1, it is characterised in that the expression vector further comprises outer
The coded sequence of source protein, the coded sequence of the foreign protein are located among multiple cloning sites.
A kind of 3. preparation of the aspergillus oryzae genetic engineering bacterium of the expression foreign protein in 1-2 according to claim described in any one
Method, it is characterised in that including：The expression vector for the coded sequence for including foreign protein is converted into aspergillus oryzae uracil
Auxotrophic strain RIB40-PF1, selects positive colony, and the aspergillus oryzae genetic engineering bacterium of the expression foreign protein is made.
4. prepared by a kind of aspergillus oryzae genetic engineering bacterium of the expression foreign protein in 1-2 according to claim described in any one
Application in industrial enzyme preparation, feed addictive or pharmaceutical grade protein.
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