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

Abstract

The present invention provides a kind of expression equipment for secreting and expressing exogenous protein in Aspergillus oryzae cells and the Aspergillus oryzae genetically engineered bacterium thereof, and the expression equipment comprises from 5' to 3' in sequence: (1) controlling exogenous gene in Aspergillus oryzae The promoter of transcription; (2) the coding sequence of the signal peptide that controls the secretion and expression of foreign protein in Aspergillus oryzae; (3) the coding sequence of the tag used for affinity purification of foreign protein; (4) multiple cloning site; (5) the terminator that controls exogenous gene to terminate transcription in Aspergillus oryzae; (6) is used for the expression box of the Aspergillus oryzae orotate phosphoribosyltransferase gene of plasmid transformation screening marker, its coding sequence is as shown in SEQ ID: 2 Show. The expression equipment uses auxotrophic gene screening markers to increase the positive rate of transformation, shorten operation time and workload, and is suitable for the secretion and expression of most foreign genes; the Aspergillus oryzae genetically engineered bacteria can efficiently express acidic and neutral amylases. The paper and textile industries have important applications.

Description

A kind of expression device and its rice for secreting and expressing exogenous protein in Aspergillus oryzae cells Aspergillus genetically engineered bacteria

technical field

The invention belongs to the field of biotechnology, and more specifically relates to an expression device for exogenous protein secreted and expressed in Aspergillus oryzae cells and an Aspergillus oryzae genetically engineered bacterium.

Background technique

Aspergillus oryzae (Aspergillus oryzae) is a commonly used strain in the fermentation industry. Mycelium is generally yellow-green, then yellowish brown; Aspergillus oryzae mycelium is composed of multi-cells and is a strain that produces complex enzymes. In addition to protease, it can also be used to secrete amylase, cellulase, pectinase, glycoside enzymes, lipases, peptidases, and phytases; among them, the industrially common peak amylase (TAA) is the α-amylase derived from Aspergillus oryzae. Aspergillus oryzae is a safe microorganism (GRAS) recognized by the FDA. It is widely used in brewing, condiment, feed manufacturing, textile, paper, pulp and other industrial fields, and has been used safely for more than 1,000 years. At present, the entire gene of Aspergillus oryzae RIB40 has been sequenced, and the expression system of filamentous fungi based on Aspergillus oryzae has attracted more and more attention.

In addition to its excellent ability to synthesize and secrete proteins, A.oryzae also has the protein secretion mechanism of eukaryotic microorganisms and the protein post-translational modification and processing performance similar to that of mammalian systems; its expression system has more advantages than prokaryotic expression systems Great advantages, such as protein modification after foreign protein expression, intron recognition, signal peptide clipping and correct folding of peptide chains and protein secretion. Due to its many advantages of being suitable for protein production and its non-toxicity to humans, Aspergillus oryzae has also become an important biological material for relevant researchers to carry out fungal cytology, genetics, and physiological and biochemical studies. Japan has funded and supported the sequencing of the Aspergillus oryzae genome, and the entire genome sequence has been completed and published. The completion of the complete genome sequencing of Aspergillus oryzae laid the foundation for the study of functional genes with important biological significance by using functional genomics (Machida M et al. Genome sequencing and analysis of Aspergillus oryzae. Nature, 2005, 438: 1157-1161).

Since the 1980s, the expression system of filamentous fungi has been reported successively, and the high-yield expression of homologous proteins has been proved. There is no natural plasmid, the genome data is large, and the secretion and expression process is complicated. As a result, there is no expression system (including expression host and expression plasmid) for industrial production like Escherichia coli and Pichia pastoris. By screening Aspergillus oryzae-related auxotrophs, constructing corresponding anaplerotic expression vectors, and achieving heterologous expression of genes from fungi, animals and plants through strong promoters, fusion expression, etc. have been reported (Minetoki T et al.Improvement of Promoter activity by the introduction of multiple copies of the conserved region III sequence, involved in the efficient expression of Aspergillus oryzae amylase-encodinggene.Appl Microbilo Biotechnol,1998,50(4):459～467.Shiba Y et al.High level of secretory production phospholipase A1 by Saccharomyces cerevisiae and Aspergillus oryzae. Biosci Biotechnol Biochem, 2001, 65(1):94-101). In 1997, RandyM.Berka et al. used PamyB and TamyB to control the heterologous expression of the laccase gene of the mesophilic fungus Myceliophthora thermophila in the Aspergillus oryzae pyrG-affected deficient system, but the expression level was only ~10mg/l (Novo NBiotec.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); in 2011, Nobuo Yamashita et al used sC as a screening marker in Aspergillus oryzae, The protease inhibitor gene from Aspergillus fumigatus (Aspergillus fumigatus) was placed between the Aspergillus oryzae TAA promoter and the terminator, and the heterologous expression of the gene was realized after induction (Nobuo Yamashita et al. High-yields heterologous production of the novel Aspergillus fumigatus elastase inhibitor AFUEI in Aspergillusoryzae, J biosci bioeng, 2011, 112(2):114～117). Nevertheless, purifiable universal expression vectors for the corresponding universal replaceable elements of these auxotrophic systems have not been reported so far.

Contents of the invention

The purpose of the present invention is to provide a kind of expression equipment and its Aspergillus oryzae genetically engineered bacteria for secreting and expressing exogenous protein in Aspergillus oryzae cells, thereby solving the problem of low foreign protein expression in the Aspergillus oryzae filamentous fungus expression system in the prior art The defect of separation and purification difficulties.

In order to solve the above technical problems, the present invention adopts the following technical solutions:

The first aspect of the present invention is to provide a kind of expression equipment that is used for secreting and expressing exogenous protein in Aspergillus oryzae cell, and described expression equipment comprises following element successively from 5 ' to 3 ': (1) control exogenous gene in Aspergillus oryzae (2) the coding sequence of the signal peptide that controls the secretion and expression of the foreign protein in Aspergillus oryzae; (3) the coding sequence of the tag used for the affinity purification of the foreign protein; (4) the multiple cloning site (5) the terminator that controls exogenous gene to terminate transcription in Aspergillus oryzae; (6) is used for the expression cassette of the Aspergillus oryzae orotate phosphoribosyltransferase gene of plasmid transformation screening marker, the coding sequence of described expression cassette is as follows Shown in SEQ ID: 2.

In the present invention, the promoter is a promoter capable of controlling the transcription of foreign genes in Aspergillus oryzae, preferably the Aspergillus oryzae α-amylase promoter (PamyB), and the sequence of the more preferred promoter is SEQ ID No. 7 to No. 623 of NO:1.

In the present invention, the signal peptide can be any signal peptide (secretory peptide) that can control the secretion and expression of foreign proteins in Aspergillus oryzae in the art, and the coding sequence of the preferred signal peptide is the first sequence of SEQ ID NO:1 Positions 624 to 695 are shown.

In the present invention, the tag used for affinity purification of exogenous protein can be any tag that can be used for affinity purification of protein in the art, preferably His-Tag tag, and the coding sequence of a better tag is as SEQ No. 696 to No. 713 of the sequence shown in ID NO:1.

In the present invention, the multiple cloning site can be various conventional multiple cloning sites in the art, preferably, the coding sequence of the multiple cloning site is as shown in SEQ ID NO: 1 from the 714th to the 1st 784 bits.

In the present invention, the terminator is a terminator capable of controlling exogenous genes to terminate transcription in Aspergillus oryzae, preferably Aspergillus oryzae α-amylase terminator (TamyB), and the sequence of the more preferred terminator is SEQ ID No. 785 to 1681 of NO:1.

Most preferably, in the expression equipment provided by the present invention, the coding sequence of the Aspergillus oryzae α-amylase promoter is as shown in SEQ ID NO: 1 from the 7th to the 623rd; the coding of the signal peptide The sequence is as the 624th to the 695th of the sequence shown in SEQ ID NO: 1; the coding sequence of the His-Tag tag used for the affinity purification of foreign proteins is as the 696th to the 696th of the sequence shown in SEQ ID NO: 1 The 713th position; the coding sequence of the multiple cloning site is as shown in SEQ ID NO:1 from the 714th to the 784th position of the sequence; the coding sequence of the Aspergillus oryzae α-amylase terminator is as SEQ ID NO:1 Positions 785 to 1681 of the sequence shown.

Preferably, the expression device further includes a coding sequence of a foreign protein, and the coding sequence of the foreign protein is located in, before or after the multiple cloning site.

The second aspect of the present invention also provides a recombinant expression vector comprising the above-mentioned expression equipment.

Preferably, the vector plasmid is a filamentous fungal expression vector, such as pBC-Hygro, pBARGPE1, pAN7-1, pRTRI, pBC-phleo, but not limited to these vectors, more preferably pBC-Hygro.

The third aspect of the present invention also provides a genetically engineered Aspergillus oryzae strain expressing a foreign protein, wherein the genome of the genetically engineered Aspergillus oryzae strain contains the expression device as described above.

The host bacteria of the Aspergillus oryzae genetically engineered bacteria are preferably Aspergillus oryzae RIB40, 3.042, AS3.863, AS3.951, HBR9, RIB326, etc., more preferably Aspergillus oryzae uracil auxotroph strain RIB40-PF1. The bacterial strain RIB40-PF1 used in the present invention is obtained by the inventor's own screening. This bacterial strain has been preserved in the General Microorganism Center (CGMCC, located in Beijing, China) of the China Microbiological Culture Collection Management Committee. The preservation number is CGMCC NO: 9129, and the preservation date is 2014.05.08.

The fourth aspect of the present invention also provides a preparation method of Aspergillus oryzae genetically engineered bacteria expressing foreign proteins as described above, comprising: transforming the recombinant expression vector containing the expression equipment as described above into Aspergillus oryzae uracil auxotroph Strain RIB40-PF1, positive clones were selected to obtain the Aspergillus oryzae genetically engineered bacteria expressing foreign proteins.

The fifth aspect of the present invention also provides a method for producing a protein, comprising culturing the Aspergillus oryzae genetically engineered bacteria expressing a foreign protein, and obtaining the foreign protein from the culture.

The sixth aspect of the present invention also provides an application of the above-mentioned Aspergillus oryzae genetically engineered bacteria expressing foreign proteins in the preparation of industrial enzyme preparations, feed additives or protein drugs. The enzyme preparation is α-amylase, glucoamylase, β-glucosidase, lactase, glucose oxidase, glucanase, xylanase, pullulanase, phytase, lipase, Protease, lipoxygenase, endocellulase, exocellulase, cellobiase, etc.

Compared with the prior art, the present invention has the following beneficial effects:

1. The expression device of the present invention is on a circularized plasmid, which is easy to preserve and operate on DNA.

2. The expression equipment of the present invention uses the Aspergillus oryzae orotate phosphoribosyltransferase gene plasmid for the first time to transform the screening marker, which can increase the positive rate of transformation and shorten the operation time and workload.

3. The expression device of the present invention has optimized secretion expression ability, which is suitable for the secretion expression of most exogenous genes.

4. The multi-cloning site of the expression device of the present invention contains a rare interface and a flat-end interface, which is compatible with the connection of most foreign genes, saving operation time and improving work efficiency.

5. The expression device of the present invention can realize efficient secretion and expression of exogenous protein genes derived from animals, plants and fungi in the filamentous multicellular eukaryotic microorganism Aspergillus oryzae. Due to the extensive culture conditions of Aspergillus oryzae, it is suitable for solid culture and liquid submerged fermentation; Aspergillus oryzae itself is not toxic, and will not produce mycotoxins and antibiotics under the condition of enzyme production, which meets international food safety certification; the extracellular protein produced by Aspergillus oryzae is easy to separate Purification and low cost. Therefore, the present invention can provide genetic engineering production strains for enzyme preparation industries such as washing, textile, feed, food, papermaking, medicine, etc., improve the production efficiency of enzyme preparations and reduce production costs.

6. The expression equipment (including the promoter terminator) of the present invention is shortened, and the final constructed expression cassette is smaller, which is more conducive to the connection operation of constructing the expression plasmid and the improvement of the transformation efficiency. The expression level is more stable and will not fluctuate greatly due to the influence of culture conditions. If the expression cassette fluctuates greatly depending on the culture conditions, the requirements for the operator are high. For the expression of the expression device of the present invention, the condition of the cell culture medium can be more rough and cheap. Bacteria grow faster, and reducing time means reducing costs. The expression cassette of the present invention can be used in combination with other expression cassettes to simultaneously express two foreign proteins.

7. The Aspergillus oryzae genetically engineered bacteria of the present invention uses the Aspergillus oryzae uracil auxotrophic strain RIB40-PF1 obtained by screening for the first time, combined with the Aspergillus oryzae orotate phosphoribosyltransferase gene plasmid transformation screening marker, which can efficiently express acidic, neutral Amylase has very important applications in the starch industry, papermaking and textile industries.

Description of drawings

Figure 1 is the physical map of the Aspergillus oryzae expression device pBC12FA2 and its construction process. Each restriction site is shown in the figure, wherein, PamyB: α-amylase (TAA) promoter; TamyB: α-amylase (TAA) terminator; pyrF: Aspergillus oryzae orotate phosphoribosyltransferase (ORPTase) genetic selection markers;

Figure 2 is a construction diagram of the general expression vector pBC2FNH of Aspergillus oryzae;

Fig. 3 is the expression of green fluorescent protein in Aspergillus oryzae, wherein, A: the hyphae of transformant PF1-pBC12FNHEGFP-6 induction culture are observed under green fluorescent light source (left) and common light source (right); B: wild Observation results of mycelia induced by strain RIB40 under green fluorescent light source (left) and normal light source (right).

detailed description

The present invention will be further described below in conjunction with specific embodiments. It should be understood that the following examples are only used to illustrate the present invention but not to limit the scope of the present invention.

The raw materials or reagents used in the present invention are commercially available unless otherwise specified.

1. Expression equipment

The invention provides an expression device for secreting and expressing foreign protein in Aspergillus oryzae cells. Among them, the Aspergillus oryzae α-amylase promoter is at the 5' end of the gene, consisting of about 617 nucleotides; the Aspergillus oryzae α-amylase terminator is at the 3' end of the gene, consisting of about 897 nucleotides Composition; After the Aspergillus oryzae α-amylase promoter, there is also a signal peptide sequence, which encodes a secretory peptide of 21 amino acid sequences. The present invention takes the promoter, signal peptide and terminator of the Aspergillus oryzae α-amylase, and adds an affinity purification tag and a multiple cloning site for expressing the protein after the signal peptide, so as to facilitate the smooth insertion and expression of foreign genes. And after the terminator, the gene coding sequence of Aspergillus oryzae orotate phosphoribosyltransferase used for plasmid transformation screening marker is connected, and the final structure is promoter-signal peptide-purification tag-MCS-terminator-expression of screening marker equipment. The expression equipment can secrete and express various exogenous proteins in Aspergillus oryzae, with high expression level, easy separation and purification, and low cost.

The preparation method of the expression device of the present invention is as follows:

(1) Through PCR amplification, the promoter of Aspergillus oryzae α-amylase (PamyB+signal peptide, PaS) containing the secretory peptide sequence, an exogenous amylase gene (GpA2), and the terminator of Aspergillus oryzae α-amylase were respectively obtained. The respective DNA sequences of the child (TamyB) and orotate phosphoribosyltransferase gene screening marker expression cassette.

(2) The fusion sequence (PaSA2) obtained by fusion PCR of the promoter, the signal peptide sequence (PaS) and the amylase gene (GpA2), the terminator (TamyB), orotate phosphoribosyltransferase gene screening marker expression cassette , PaSA2 were sequentially connected to the expression vector pBC-Hygro, and the recombinant expression vector 1 was constructed, namely pBC12FA2; the expression vector 2, namely pBC12NHA2, was obtained by inserting the 6×His-Tag purification tag through site-directed mutagenesis, and the exogenous protein was deleted by reverse PCR. The gene GpA2 thus obtains a general expression vector 3 containing the expression device but not foreign genes, namely pBC12FNH.

(3) Inserting the exogenous target gene into the above expression vector 3 so that the exogenous gene is located between the promoter and the terminator to obtain the recombinant expression plasmid 4, namely pBC12FNH-EGFP.

The multiple cloning sites used for connection in the above step (1) are carried by the backbone vector pBC-Hygro; the promoter, terminator and screening markers are connected, and the methods of restriction endonuclease digestion and ligase connection can be used to connect each The element is connected into the expression vector, and the restriction endonuclease digestion and ligase connection are common practice in the field.

In the above-mentioned step (3), inserting the foreign gene into the above-mentioned expression vector 3 can be performed by restriction endonuclease digestion and ligase connection, and the restriction endonuclease digestion and ligase connection are both common practice in the field.

2. Aspergillus oryzae genetically engineered bacteria

The present invention also provides a genetic engineering bacterium of Aspergillus oryzae, which is prepared by integrating the expression device of the present invention into Aspergillus oryzae RIB40-PF1 cells through PEG-mediated protoplast transformation, and culturing and screening transformants. The preparation steps are as follows:

(1) the carrier of the present invention containing expression equipment is transformed into Aspergillus oryzae RIB40-PF1 cell with the protoplast transformation method mediated by PEG;

(2) Screening and identifying the above-mentioned transformed cells to obtain the Aspergillus oryzae genetically engineered bacteria expressing the foreign protein.

Experimental methods not indicating specific conditions in the following examples are usually carried out according to conventional conditions, such as the conditions described in "Molecular Cloning: A Laboratory Manual" (NewYork: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's instructions. suggested conditions.

The buffer and culture medium used in the embodiment are as follows:

(1) TE (1000ml):

ZnSO ₄ 7H ₂ O 0.35g, MnCl ₂ 4H ₂ O 0.5g, H ₃ BO ₃ 0.03g, CoCl ₂ 6H ₂ O 0.945g, CuCl ₂ 2H ₂ O 0.01g, NiCl ₂ 6H ₂ O0. 12g, NaMoO ₄ ·2H ₂ O 0.18g, 6M HCl 13ml, add deionized water to 500mL.

(2) Sodium phosphate buffer (100ml, pH5.8):

Na ₂ HPO ₄ ·12H ₂ O 0.5788g, NaH ₂ PO ₄ ·2H ₂ O 2.8996g.

(3) Tris-HCl buffer solution (100ml, pH7.5):

Tris 0.6057g, adjusted to pH 7.5 with HCl.

(4) Solution I:

NaCl 0.7M, sucrose 1.0g, dissolved in 50ml of sodium phosphate buffer, and dilute (add water) to 100ml.

(5) Solution II:

22.3085g of sorbitol, 20.578g of CaCl, and 0.2045g of NaCl were dissolved in 20ml of Tri-HCl buffer solution, and the volume was adjusted to 100ml by adding water.

(6) Solution III:

PEG4000 30g, CaCl ₂ 0.289g, dissolved in 10ml Tri-HCl buffer solution, dilute (add water) to 50ml.

(7) CM medium (100ml):

NaNO ₃ 0.6g, KCl 0.05g, KH ₂ PO ₄ 0.08g, K ₂ HPO ₄ 0.104g, Glucose 1g, MgSO ₄ 0.052g, Peptone 0.2g, Yeast extract 0.1g, Uridine 0.1221g, TE100ul; For inclined planes, add 1.5-2.0 g of agar powder.

(8) MM medium (100ml):

NaNO ₃ 0.6g, KCl 0.05g, KH ₂ PO ₄ 0.08g, K ₂ HPO ₄ 0.104g, glucose 1g, MgSO ₄ 0.052g, TE100ul, sucrose 20.54g.

Agar powder: 0.8g for the upper layer and 1.2g for the lower layer.

(9) Fermentation medium (100ml):

Dextrin 2g, sucrose 0.3g, peptone 0.1g, yeast extract 0.5g, NaNO ₃ 0.1g, MgSO ₄ ·7H ₂ O 0.05g, FeSO ₄ ·7H ₂ O 0.001g.

Embodiment 1. comprises the construction of the expression vector of Aspergillus oryzae expression equipment of the present invention

1.1. Extraction of Aspergillus oryzae genome

Aspergillus oryzae RIB40 (National Research Institute of Brewing Stock Culture, ATCC42149) was inoculated on CM medium, and incubated at 30°C for 7 days until the spores matured. Prepare an appropriate amount of spore suspension and inoculate it in CM medium liquid seed medium, and cultivate it at 30°C and 200rpm for 2 days until the mycelium concentration reaches 4-5g/L for genome extraction. The method refers to the instructions of the Omega fungal genome extraction kit. .

1.2. Isolation of Aspergillus oryzae α-amylase promoter (PamyB) and its signal peptide sequence

According to the sequence of the amyB promoter released on GeneBank, the above-mentioned extracted Aspergillus oryzae genomic DNA was used as a template, and the upstream primer PaXf and the downstream primer Pamr were used for specific PCR amplification to isolate PaS (PamyB+signalpeptide). in:

The upstream primer PaXf sequence is:

CCG CTCGAG GAATTCATGGTGTTTTGATCATTTT, which contains the XhoI restriction site (the primer was synthesized by Shanghai Jierui Bioengineering Co., Ltd., the same below);

The downstream primer Pamr sequence is:

A GGCGCGCCGCTAGC AGGCGTTGCAGCCAAAGC, which contains AscI and NheI restriction sites.

1.3. Isolation of Aspergillus oryzae α-amylase terminator (TamyB)

According to the sequence of the TamyB terminator released on GeneBank, using the above-mentioned extracted Aspergillus oryzae genomic DNA as a template, specific PCR amplification was performed with the upstream primer TaNf and the downstream primer TaNr to isolate PaS (PamyB+signalpeptide). in:

The upstream primer TaNf sequence is:

ATAAGAAT GCGGCCGC GGGTGGAGAGTATATG, which contains the NotI restriction site; the downstream primer TaNr sequence is:

ATAAGAAT GCGGCCGC AATTCTTGAGGACCATTAC, which contains the NotI restriction site.

1.4. Isolation of exogenous auxiliary amylase gene

Using pMD19Tsimple linked to GpA2 (NCBI GenBank acceleration number: KJ093844) as a template, the GpA2 gene was amplified by PCR using primers 12A2mf and 12A2Hr. in

The sequence of the upstream primer 12A2mf is:

GCCT GCTAGCGGCGCGCC T AAAACCGCCGCGGAATGG, which contains AscI and NheI restriction sites;

The downstream primer 12A2Hr sequence is:

CCC AAGCTT TTAAGCACATAAACTGCCCT, which contains a HindIII restriction site.

1.5. Isolation of Aspergillus oryzae orotidine phosphoribosyltransferase screening marker gene

According to the sequence of pyrF in the whole genome of Aspergillus oryzae RIB40 released on GeneBank, the above-mentioned extracted Aspergillus oryzae genomic DNA was used as a template, and the pyrF expression cassette was isolated by specific PCR amplification with upstream primer FsNf and downstream primer FxNr. in:

The upstream primer FsNf sequence is:

GGAATTC CATATG TGAAAGACTGCTGCAAAGCC, which contains the NdeI restriction site;

The downstream primer FxNr sequence is:

GGAATTC CATATG AAGCAGTCGTACATACATGG, which contains the NdeI restriction site.

All the above PCR amplification reaction conditions and reaction system refer to the instructions of Vazyme high-fidelity enzyme kit, and the gel cutting and purification refer to the instructions of Axygen kit. The PCR product was subjected to agarose gel electrophoresis, recovered, connected to pEASY-Bluntcloning vector (Transgen, USA), and the positive clones verified by colony PCR were sent for sequencing. The sequencing was completed by Ruidi Biological Sequencing Company.

1.6. Construction of PaSA2 fusion fragment

Using PaS and GpA2 obtained after separation and purification by PCR as templates, the upstream primer PaXf and the downstream primer 12A2Hr were used for fusion PCR amplification to obtain the PaSA2 fragment, and the restriction sites NheI and AscI were introduced. Its reaction system is:

For fusion PCR reaction conditions, refer to the instructions of the Vazyme high-fidelity enzyme kit, and for gel cutting and purification, refer to the instructions of the Axygen kit. The PCR product was subjected to agarose gel electrophoresis, recovered, connected to pEASY-Blunt clonning vector, and the positive clones verified by colony PCR were sent for sequencing, and the sequencing was completed by Ruidi Biological Sequencing Company.

1.7. Construction of Aspergillus oryzae pBC12FA2 expression vector

Using pBC-Hygro as the original plasmid, the above fragments were ligated sequentially to construct pBC12FA2 by using the enzyme cutting sites on it. The sequence of restriction endonuclease digestion and ligation is as follows: NotI single-digests TamyB and pBC-Hygro respectively, and pBC12 is obtained after ligation; NdeI single-digests pyrF and pBC12 respectively, and pBC12F is obtained after ligation; XhoI and HindIII double-digest pBC12F respectively and PaSA2, pBC12FA2 was obtained after connection. After the DNA ligation reaction was completed, the ligation solution was transferred to Escherichia coli competent DH5α (Tiangen Company), and then coated with LB plates containing chloramphenicol (50 mg/ml). Single clones were picked out, inserted into LB liquid medium containing chloramphenicol (50 mg/ml), and positive clones verified by colony PCR were sent for sequencing. After the expanded culture, the plasmid was extracted with reference to the Tiangen small extraction plasmid kit, and the expression vector pBC12FA2 was obtained, that is, the Aspergillus oryzae expression vector capable of expressing the exogenous gene GpA2 was obtained.

1.8. Construction of Aspergillus oryzae pBC12FNHA2 affinity-purified expression vector

Using pBC12FA2 as a template, the KOD site-directed mutagenesis kit was used to insert a His-Tag tag commonly used for affinity purification after the signal peptide and before NheI and AscI. The tag sequence is: CATCATCACCATCACCAT. The primers used for site-directed insertion were FA2NHf and FA2NHr. The sequences are:

FA2NHf: CATCACCAT GCTAGCGGCGCGCCTAAAAC;

FA2NHr: GTGATGATG AGGCGTTGCAGCCAAAGCAG.

The operating steps refer to the instructions of the KOD site-directed mutagenesis kit (ToYoBo, Japan). The subsequent steps are as described in 1.6. The expression vector pBC12FNHA2 capable of purifying foreign protein GpA2 was obtained.

1.9. Construction of general expression vector for affinity purification of Aspergillus oryzae pBC12FNH

Using pBC12FNHA2 as a template, the KOD site-directed mutagenesis kit was used to delete the GpA2 sequence, and the primers for site-directed insertion were 12FNHf and 12FNHr. The sequences are:

12FNHf:AAGCTTGATATCGAATTCCTGCAGCC;

12FNHr: AGGCGCGCCGCTAGCATGGTGATG.

The steps are as described in 1.7. A general expression vector pBC12FNH capable of purifying foreign proteins was obtained.

Embodiment 2. Utilize Aspergillus oryzae expression equipment to express green fluorescent protein

2.1. Amplification of the green fluorescent protein gene

Using the plasmid pMD19-T simple vector linked to the green fluorescent protein GFP (NCBI GenBank accession number: AY013824) as a template, the 717bp (including restriction site) enhanced green was amplified by PCR using primers GFPNf/GFPHr fluorescent protein gene. The primer sequences are:

GFPNf: CACTA GCTAGC ATGAGTAAAGGAGAAGAAC, which contains NheI restriction site;

GFPHr: CCC AAGCTT TTATTTGTATAGTTCATCCATG, which contains a HindIII restriction site.

The length of the amplified product and the results of sequence analysis were in line with expectations.

2.2. Connection between GFP and expression equipment

The above-mentioned amplified product (GFP gene) was digested with NheI and HindIII restriction enzymes, and connected to the expression vector pBC12FNH with NheI and HindIII restriction enzymes using T4 ligase to obtain the expression vector containing PaS+His-Tag+GFP+TamyB The recombinant vector pBC12FNHGFP expresses the device. The follow-up method is described in 1.6.

2.3. Preparation of Aspergillus oryzae uracil auxotrophic RIB40-PF1 protoplasts

(1) Inoculate the spores of A.oryzae RIB40-PF1 (CGMCC No: 9129) slant into CM liquid medium, culture overnight (12-16h) at 30°C and 200rpm, and form dense and small fungal spheres (d<2mm) After washing with sterile water, filter the culture medium with sterile Microcloth to obtain mycelia, and wash with solution I for 2 to 3 times. During the washing process, centrifuge at 6000 rpm at 4°C for 1 min, and remove the supernatant. Finally a clean mycelium (approximately 200 mg) was obtained.

(2) Enzyme solution system (5ml): 0.1g cellulase, 0.1g helicase, 0.025g lysozyme. Filter sterilize.

(3) Add 1 ml of the enzyme solution prepared in step (2) to the mycelium prepared in step (1), and perform enzymatic hydrolysis at 30° C. for 3 hours at 150 rpm.

(4) After the enzymatic hydrolysis is completed, centrifuge at 3500 rpm at 4°C for 5 minutes, remove the supernatant, and wash with solution II for 1-2 times.

(5) Add an appropriate amount of solution II (about 1 ml) to suspend the protoplasts.

2.4 Transformation of Aspergillus oryzae uracil auxotrophic RIB40-PF1 protoplasts into pBC12FNHGFP

(1) Mix 200 ul of protoplasts prepared in 2.2 with 1 ng (about 20 ul) of plasmid pBC12FNHGFP, add 50 ul of solution III, and ice-bath for 30 min.

(2) Add 2ml of solution III to the medium system in step (1), and let it stand at room temperature for 5 minutes.

(3) Add 4ml of solution II to the system in (2), centrifuge at 5000rpm at 4°C for 5min, remove the supernatant, and suspend in 400ul of solution II.

(4) Pour the MM lower medium on the plate and solidify; the MM upper medium is lowered to about 40 ° C, mixed with the protoplast transformation solution in (3), shake the plate quickly, spread evenly, and place at 30 ° C for 3 to 5 days.

2.5. Identification of transformants and expression of target genes

Extract the genomic DNA of the positive transformant obtained in step 2.4 of Example 2 as a template, and perform PCR amplification with primers GFPf and GFPr. Its sequence is:

GFPf: ATGAGTAAAGGAGAAGAAC;

GFPr: TTATTTGTATAGTTCATCCATG.

The results showed that the amplified product of the transformant obtained a specific band of the size of the target gene, while the same PCR reaction was performed using the genomic DNA of the original strain A.oryzaeRIB40-PF1 as a template, and no amplified product appeared.

The obtained recombinant Aspergillus oryzae strain was inoculated in a liquid fermentation medium, and after shaking culture at 30°C and 200rpm for 72 hours, the fermentation broth was taken and spotted on a glass slide, observed under a fluorescent microscope, and green fluorescent protein was observed by excitation with blue light. The results are shown in Figure 4. The recombinant A. oryzae mycelia emitted green fluorescence under a fluorescence microscope, while the original A. oryzae mycelia had no green fluorescence under a fluorescence microscope.

What is described above is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. Various changes can also be made to the above embodiments of the present invention. That is to say, all simple and equivalent changes and modifications made according to the claims and description of the application for the present invention fall within the protection scope of the claims of the patent of the present invention. What is not described in detail in the present invention is conventional technical contents.

Claims (4)

1. an aspergillus oryzae genetically engineered bacterium expressing foreign protein, is characterized in that, described aspergillus oryzae genetically engineered bacterium comprises host bacterium and expression vector, and described host bacterium is aspergillus oryzae uracil auxotroph strain RIB40-PF1, The preservation number of the bacterial strain RIB40-PF1 is CGMCC NO: 9129, and the expression vector includes the following elements in sequence from 5' to 3': (1) Aspergillus oryzae α-amylase promoter that controls the transcription of foreign genes in Aspergillus oryzae , the sequence is as the 7th to the 623rd of the sequence shown in SEQ ID NO:1; (2) the sequence of the signal peptide that controls the secretion and expression of the foreign protein in Aspergillus oryzae, such as the sequence of the sequence shown in SEQ ID NO:1 Position 624 to position 695; (3) the sequence of the His-Tag tag used for affinity purification of foreign proteins, such as the sequence from position 696 to position 713 of the sequence shown in SEQ ID NO: 1; (4) multiple cloning positions Point, the sequence is as the 714th to the 784th of the sequence shown in SEQ ID NO:1; (5) Aspergillus oryzae α-amylase terminator that controls exogenous gene to terminate transcription in Aspergillus oryzae, the sequence is as SEQ IDNO:1 The 785th to the 1681st of the shown sequence; (6) the expression cassette of the Aspergillus oryzae orotate phosphoribosyltransferase gene that is used for the plasmid transformation screening marker, and the Aspergillus oryzae orotic acid that is used for the plasmid transformation selection marker The sequence of the expression cassette of the phosphoribosyltransferase gene is shown in SEQ ID:2. 2. Aspergillus oryzae genetically engineered bacterium according to claim 1, is characterized in that, described expression vector further comprises the coding sequence of foreign protein, and the coding sequence of described foreign protein is positioned among the multiple cloning site. 3. a method for preparing the Aspergillus oryzae genetically engineered bacterium expressing foreign protein according to any one of claims 1-2, characterized in that, comprising: said expression of the coding sequence comprising foreign protein Transform the Aspergillus oryzae uracil auxotroph strain RIB40-PF1 with the vector, and select positive clones to obtain the Aspergillus oryzae genetically engineered strain expressing the foreign protein. 4. An application of the Aspergillus oryzae genetically engineered bacterium expressing foreign protein according to any one of claims 1-2 in the preparation of industrial enzyme preparations, feed additives or protein medicines.