CN103421794A - Expression element composed of aspergillus niger and expression vector composed of expression element, and recombined aspergillus niger and construction method and application thereof - Google Patents
Expression element composed of aspergillus niger and expression vector composed of expression element, and recombined aspergillus niger and construction method and application thereof Download PDFInfo
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Abstract
The invention discloses an expression element composed of an aspergillus niger and an expression vector composed of the expression element, and a recombined aspergillus niger and a construction method and an application thereof. The expression element is prepared from an aspergillus niger transcription elongation factor gene promoter and an aspergillus niger transcription elongation factor gene terminator, wherein the aspergillus niger transcription elongation factor gene promoter is composed of 1490, 990 or 690 basic groups of which sequence is as shown in SEQID NO.1, SEQID NO.2 and SEQID NO.3; the aspergillus niger transcription elongation factor gene terminator is composed of 1022 basic groups of which sequence is as shown in SEQID NO.5. The expression element can start protein expression without adding an inductor, so that the production cost is saved; the expression element can be used for preparation of pharmaceutical protein of eukaryotes and microbial sources and enzymes for industrial purposes.
Description
Technical field
The invention belongs to the genetically engineered research field, be specifically related to a kind of aspergillus niger constitutive expression carrier and application thereof.
Background technology
Filamentous fungus has powerful protein excretion ability, and the outer total protein concentration of the born of the same parents of some thread funguss secretion reaches 40g/L, and this efficient protein excretion ability is that the prokaryotic expression host such as bacterium can not compare.Filamentous fungus also possesses range gene translation post-treatment ability, as glycosylation modified, and the formation of signal peptide cutting and disulfide linkage etc.Aspergillus oryzae, the filamentous funguss such as aspergillus niger and Trichodermareesei are all the aliment security level bacterial strains, by FDA Food and Drug Administration, are regarded as GRAS(Generally recognized as safe) bacterial strain.In the production of industrial enzymes, the thread fungus fermentation occupies the status of core, and 40% the enzyme production nearly of the zymin of world market comes from the filamentous fungus fermentation.
Aspergillus niger is a kind of important industrial microorganism, can prepare for plurality of enzymes preparation fermentations such as amylase, aspartic protease, cellulase, polygalacturonase, glucose oxidases.Along with completing of the gene order-checking work of aspergillus niger, in order to further investigate, the aspergillus niger protein excretion is expressed and regulatory mechanism is had laid a good foundation, and also for the heterologous protein expression ability that improves aspergillus niger by genetic engineering means, provides new thinking and method simultaneously.
Aspergillus niger is the superior strain of glucase, show that the promotor of glucoamylase gene has the effect of efficiently transcribing, so the promotor of this gene is developed the promotor for high-expression vector.In addition, Sucrase A,, α-Amylase, Alcohol dehydrogenase I and the isogenic promoter sequence of Arabinanase be developed as the promoter element of expression vector in succession.But these expression vectors, when expression alien gene, need to add the materials such as maltose, starch, sucrose to be induced.For the industrial production of recombinant protein, increased production cost on the one hand, likely increase on the other hand the generation of background albumen in fermented liquid, increased the difficulty of recombinant protein purification.
With respect to the promotor of induction type, the promotor of composing type does not need to add the startup expression that inductor can carry out gene, in actual production, can reduce production cost, therefore has more due value.In other expression systems, as pichia spp, the aspergillus oryzae expression system, wooden mould expression system is all developed efficient constitutive expression carrier in succession.Transcriptional elongation factor gene (Transcription elongation factor1, tef1) be house-keeping gene important in aspergillus niger, control lots of genes transcription in cell, its promoter sequence is likely the promotor of composing type for the structure of efficient carrier.People (the Storms R et al.Plasmid vectors for protein production such as Storms R, gene expression and molecular manipulations in Aspergillus niger.2005, Plasmid53:191 – 204) reported the expression vector of the aspergillus niger constitutive promoter that contains aspergillus niger pyruvate kinase gene promoter.Up to the present, also do not utilize the bibliographical information of aspergillus niger Tef promotor construction of expression vector.
Summary of the invention
The object of the present invention is to provide a kind of aspergillus niger constitutive expression element that can carry out exogenous gene expression without the interpolation inductor.
Second purpose of the present invention is to provide a kind of expression vector and construction process that contains described aspergillus niger constitutive expression element.
The 3rd purpose of the present invention is to provide recombinant aspergillus niger and the construction process that contains described expression vector.
The 4th purpose of the present invention is to provide the application of above-mentioned recombinant aspergillus niger.
Purpose of the present invention is achieved through the following technical solutions:
A kind of aspergillus niger constitutive expression element, described Expression element is comprised of aspergillus niger transcriptional elongation factor gene promoter and aspergillus niger transcriptional elongation factor gene terminator; Described aspergillus niger transcriptional elongation factor gene promoter is by 1490,990 or 690 based compositions, and sequence is as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3; Described aspergillus niger transcriptional elongation factor gene terminator is by 1022 based compositions, and sequence is as shown in SEQ ID NO.5.
The method of above-mentioned aspergillus niger constitutive expression element construction of expression vector comprises the following steps:
1) extract the genomic dna of aspergillus niger, the design primer, utilize round pcr, and take the aspergillus niger genomic dna as template, increase and clone the promotor of the aspergillus niger transcriptional elongation factor gene that length is 1490,990 or 690 bases and the terminator that length is 1022 bases, called after P respectively
tef1490, P
tef990, P
tef690 and T
tef
2) the Amds gene is entered in the pbluescript carrier by kpnI and XhoI restriction endonuclease clone, obtain intermediate carrier one; By P
tefGene fragment utilizes XhoI and HindIII restriction endonuclease clone to enter in above-mentioned intermediate carrier one, obtains intermediate carrier two; By T
tefGene fragment utilizes XbaI and NotI clone to enter intermediate carrier two, obtains aspergillus niger expression vector pTef.
Described primer sequence is as follows:
The method of the expression vector establishment recombinant aspergillus niger of utilizing aforesaid method to build comprises the following steps:
1) by lipase calb gene clone in aspergillus niger expression vector pTef, using restriction endonuclease is EcoRI and XbaI, obtains calb expression vector pTef-calb; 2) pTef-calb is transformed to the aspergillus niger protoplastis, screen positive recombinant bacterial strain, obtain recombinant aspergillus niger GAP-calb.The Aspergillus niger strain GAP3-calb expression product of restructuring is analyzed, and by protein electrophoresis, detects and the enzyme substrates detection shows that recombinant bacterial strain can constitutive expression lipase calb.
The application of recombinant aspergillus niger in preparing industrial enzyme preparation or medical protein that aforesaid method builds.
Described industrial enzyme preparation is lipase, proteolytic enzyme, phytase or zytase.
Compared with prior art, the present invention has following beneficial effect:
The constitutive expression carrier of the Expression element that the promotor that contains the aspergillus niger transcriptional elongation factor that the present invention builds and transcription termination sequence form, without the interpolation of inductor, can start protein expression, saves production cost.Promoter transcription efficiency than other length when the promotor length of aspergillus niger transcriptional elongation factor is 990bp is higher, and, than the high 5 times of left and right of pyruvate kinase gene promoter exogenous gene expression ability of bibliographical information, can be used for eukaryote and microbe-derived pharmaceutical protein and the preparation of industrial enzyme.
The accompanying drawing explanation
Fig. 1 is aspergillus niger constitutive expression carrier collection of illustrative plates.
Fig. 2 is the recombinant expressed electrophorogram of recombinant expressed CALB; Swimming lane one: molecular weight of albumen marker; Swimming lane two: the Aspergillus niger strain culture supernatant that does not contain the calb gene; Swimming lane three: the aspergillus niger recombinant bacterium GAP3/pTef990-calb supernatant that ferments.
Fig. 3 is that different recombinant aspergillus niger are expressed the active comparison diagram of CALB.
Fig. 4 is continuous passage recombinant aspergillus niger enzymic activity comparison diagram.
Embodiment
Below in conjunction with specific embodiment, the present invention is more specifically described in detail, but embodiments of the present invention are not limited to this, for not dated especially processing parameter, can carry out with reference to routine techniques.
Obtaining of aspergillus niger transcriptional elongation factor gene promoter and terminator sequence
Aspergillus niger strain (purchased from microbial strains preservation center, Guangdong Microbes Inst) is inoculated on the PDA solid medium, cultivate until the spore maturation for 28 ℃, get appropriate spore and be prepared into suspension inoculation in liquid PDA, in 28 ℃, under the 200rpm condition, cultivate 3 days, and the results mycelium is for extracting the aspergillus niger genomic dna.
The aspergillus niger genome adopts Benzyl chloride method to extract: mycelium and SDS and Benzyl Chloride are mixed, 50 ℃ of heating, after add the 3M sodium acetate soln to mix, ice-water bath 15min, centrifugal 15min, get supernatant, adds equal-volume Virahol precipitation at room temperature 20min.Room temperature high speed centrifugation 15min, precipitation is washed once with 70% ethanol, is dissolved in Tris-EDTA damping fluid (TE) after drying, after RNAase digestion, for follow-up test.
Aspergillus niger genomic information (the accession number: AM270408.1) of announcing according to Genbank, gene fragment and the transcription termination sequence of design aspergillus niger transcriptional elongation factor gene promoter different lengths, the aspergillus niger transcriptional elongation factor gene promoter Ptef1490 of different lengths, Ptef990, Ptef690, Ptef390 is by 1490, 990, 690 and 390 based compositions, the sequence of amplification is as SEQ ID NO.1, EQ ID NO.2, shown in EQ ID NO.3 and EQ ID NO.4, aspergillus niger transcriptional elongation factor gene terminator Ttef is by 1022 based compositions, as shown in SEQ ID NO.5.Primer sequence is as follows:
Table one primer information list
Use the PrimeSTAR high-fidelity DNA polymerase of Takara company, take the aspergillus niger genome as template, the PCR reaction conditions: 98 ℃ of 3min of denaturation; Carry out again 25 circulations: 98 ℃ of sex change 5sec, 55 ℃ are extended 10sec, and 72 ℃ are extended 1min; Last 72 ℃ are extended 5min, PCR product utilization agarose nucleic acid electrophoresis detected magnitude, and carry out glue and reclaim purifying target DNA sheet degree, and the PCR product is sent to Hua Da genome company and carries out sequence verification.
Obtaining of aspergillus niger pyruvate kinase gene promoter and terminator sequence
(accession number: S38698) the design primer amplification obtains its promotor and transcription termination sequence to the aspergillus niger pyruvate kinase gene information of announcing according to Genbank, aspergillus niger pyruvate kinase gene promoter element sequences is as shown in SEQ ID NO.7, and the terminator element sequences is as shown in SEQ ID NO.8.Primer sequence is as follows:
Table two primer information list
Use the PrimeSTAR of Takara company high-fidelity DNA polymerase, the aspergillus niger genome of said extracted of take is template, according to polysaccharase specification sheets condition, is increased, and PCR product size utilizes the agarose nucleic acid electrophoresis to detect, and carries out sequence verification.
The aspergillus niger constitutive expression carrier builds
According to Amds gene order design specific amplification primer, sequence is as follows: Amds-F:
GGGGTACCTTTTGAATAGCTCGCCCGCT（KpnI）;Amds-R:
CCGCTCGAGCTAGACTGGAAACGCAACCC(XhoI), use Amds-F and Amds-R, and obtaining the Amds gene fragment with commercialization pKLAC1 carrier (the U.S. New England Biolabs company) amplification that contains the Amds gene, the Amds gene order is as shown in SEQ ID NO.9.Utilize KpnI and XhoI enzyme to cut the Amds gene fragment, the clone enters commercialization carrier pblusecript(U.S. stratagene company) middle intermediate carrier one pblusecript-Amds that obtains.In order to obtain the expression vector contained the transcriptional elongation factor gene expression element, by its promoter gene fragment P
tef1490, P
tef990, P
tef690 and P
tef390 utilize respectively XhoI and HindIII enzyme to cut, and the clone enters in intermediate carrier pbluescript-Amds and obtains pbluescript-P
tef1490, pbluescript-P
tef990, pbluescript-P
tef690pbluescript-P
tef390, further by T
tefGene fragment utilizes XbaI and NotI to be cloned in middle above-mentioned carrier, obtains aspergillus niger constitutive expression carrier pTef1490, pTef990, pTef690 and pTef390.
In order to obtain the expression vector contained the pyruvate kinase gene expression element, promoter gene fragment Ppki and transcription terminator fragment Tpki that embodiment 2 is obtained, successively be cloned in pbluescript-Amds and obtain the pPki expression vector.
Lipase calb aspergillus niger constitutive expression carrier builds and aspergillus niger transforms
Use calb forward:GGAATTCATGAAGCTACTCTCTCTGAC, calb reverse:GCTCTAGATCAGGGGGTGACGATGCCGG primer amplification lipase calb gene, using the genome of antarctic candida as template, and the pcr amplification program is: 98 ℃ of 3min of denaturation; Carry out again 25 circulations: 98 ℃ of sex change 5sec, 55 ℃ are extended 10sec, and 72 ℃ are extended 40sec; Last 72 ℃ are extended 5min and, by EcoRI and XbaI enzyme cutting, clone and enter in pTef serial carrier and pPki carrier respectively, obtaining calb expression vector pTef1490-calb, pTef990-calb, pTef690-calb, pTef390-calb and pPki-calb.Lipase calb gene order is as shown in SEQ ID NO.6.
Adopt CaCl
2-PEG method in aspergillus niger, and is coated on recombinant plasmid transformed ethanamide by the bacterium of conversion and selects substratum to exist, and 30 ℃ of cultivations, until grow the clone on flat board.To the recombinant clone grown on flat board, cultivate and extract its genome, and utilize calb forward and calb reverse primer to carry out PCR and identify that the calb gene integration is in the aspergillus niger genome.Thereby obtain recombinant aspergillus niger calb genetic expression bacterial strain GAP3/pTef1490-calb, GAP3/pTef990-calb, GAP3/pTef690-calb, GAP3/pTef390-calb and GAP3/pPki-calb.
Recombinant aspergillus niger calb gene copy number detects and the expression activity of recombinant bacterial strain lipase CALB detects
Extract the genomic dna of recombinant aspergillus niger and wild-type aspergillus niger, the glyceraldehyde 3-phosphate gene (GADPH) of take is reference gene, and the calb gene integration is detected to the genomic copy number of aspergillus niger.Adopt two calibration curve methods, utilize respectively the plasmid that contains GADPH gene and calb gene, carry out the real-time fluorescence quantitative PCR reaction, the Criterion curve.Utilize calb gene starting template copy number and GADPH gene starting template copy number in fluorescence quantitative PCR detection recombinant bacterial strain genome, both ratio is the copy number of calb gene in the aspergillus niger genome, and result shows that the recombinant bacterial strain of selecting all contains the calb gene of single copy.The quantitative fluorescent PCR response procedures is: stage1: 95 ℃ of denaturations, 30sec; Stage2:PCR reaction: 95 ℃ of 5sec, 60 ℃ of 20sec(40Cycles); Stage3: solubility curve is analyzed 65 ℃, 15sec.
Table three fluorescent quantitation primer information table
Different recombinant bacterium GAP3/pTef1490-calb, GAP3/pTef990-calb, GAP3/pTef690-calb, GAP3/pTef390-calb and GAP3/pPki-calb are cultivated in the PDA nutrient solution, cultivated 4 days for 30 ℃.10000rpm, 4 ℃ of centrifugal 15min get supernatant.Utilize oil of mirbane phenolic ester lipase artificial substrates (pnp-C8) to carry out the lipase activity detection: at 10 μ l50mM Tris-HCl(pH8.0), add 10 μ l fermented liquid supernatant in the reaction system of 1mM pNPC, 40 ℃ the reaction 10min, after add the Virahol termination reaction.1 unit enzyme activity unit is defined as the per minute hydrolysis substrate and produces the required enzyme amount of 1 μ mol nitrophenols.
Found that GAP3/pTef990-calb strain fermentation supernatant is all higher than the activity of other recombinant bacteriums, result as shown in Figure 4, activity is GAP3/pTef990-calb successively > GAP3/pTef1490-calb > GAP3/pTef690-calb > GAP3/pPki-calb > GAP3/pTef390-calb, wherein, GAP3/pTef990-calb is than about 5 times of the active height of GAP3/pPki-calb.
Embodiment 6
The genetic stability checking of recombinant bacterial strain GAP3/pTef990-calb
Recombinant bacterial strain GAP3/pTef990-calb is carried out to 30 ℃ of cultivations at the dull and stereotyped enterprising row of the PDA of acetamide-containing not, continuous passage five times, and the recombinant bacterial strain that each time gone down to posterity cultivated, and detect the enzymic activity of fermentation supernatant.Result shows that the ability of the GAP3/pTef990-calb expression lipase that each time gone down to posterity does not have significant difference, illustrates that constructed recombinant bacterial strain has good genetic stability.
Claims (8)
1. an aspergillus niger constitutive expression element, is characterized in that, described Expression element is comprised of aspergillus niger transcriptional elongation factor gene promoter and aspergillus niger transcriptional elongation factor gene terminator; Described aspergillus niger transcriptional elongation factor gene promoter is by 1490,990 or 690 based compositions, and sequence is as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3; Described aspergillus niger transcriptional elongation factor gene terminator is by 1022 based compositions, and sequence is as shown in SEQ ID NO.5.
2. by the method for the described aspergillus niger constitutive expression of claim 1 element construction of expression vector, it is characterized in that, comprise the following steps:
1) extract the genomic dna of aspergillus niger, the design primer, utilize round pcr, and take the aspergillus niger genomic dna as template, increase and clone the promotor of the aspergillus niger transcriptional elongation factor gene that length is 1490,990 or 690 bases and the terminator that length is 1022 bases, called after P respectively
tef1490, P
tef990, P
tef690 and T
tef
2) the Amds gene is entered in the pbluescript carrier by kpnI and XhoI restriction endonuclease clone, obtain intermediate carrier one; By P
tefGene fragment utilizes XhoI and HindIII restriction endonuclease clone to enter in above-mentioned intermediate carrier one, obtains intermediate carrier two; By T
tefGene fragment utilizes XbaI and NotI clone to enter intermediate carrier two, obtains aspergillus niger expression vector pTef.
3. method according to claim 2, is characterized in that, described primer sequence is as follows:
4. the expression vector that the described method of claim 2 or 3 builds.
5. by the method for the described expression vector establishment recombinant aspergillus niger of claim 4, it is characterized in that, comprise the following steps:
1) by lipase calb gene clone in aspergillus niger expression vector pTef, using restriction endonuclease is EcoRI and XbaI, obtains calb expression vector pTef-calb;
2) pTef-calb is transformed to the aspergillus niger protoplastis, screen positive recombinant bacterial strain, obtain recombinant aspergillus niger GAP3-calb.
6. the recombinant aspergillus niger that the described method of claim 5 builds.
7. the application of the described recombinant aspergillus niger of claim 6 in preparing industrial enzyme preparation or medical protein.
8. application according to claim 7, is characterized in that, described industrial enzyme preparation is lipase, proteolytic enzyme, phytase or zytase.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104480028A (en) * | 2014-12-16 | 2015-04-01 | 青岛蔚蓝生物集团有限公司 | Aspergillus niger mutant strain of stable high-yield lipase |
| CN106191096A (en) * | 2016-07-21 | 2016-12-07 | 华南理工大学 | The recombiant plasmid of constitutive expression aspergillus niger surface display lipase and application thereof |
| CN107129996A (en) * | 2016-02-26 | 2017-09-05 | 浙江大学 | One plasmid vector and its construction method and application |
| CN113584023A (en) * | 2021-07-13 | 2021-11-02 | 江西省农业科学院园艺研究所 | Aspergillus oryzae gene promoter induced by metal ions, and preparation method and application thereof |
| CN115074361A (en) * | 2021-03-12 | 2022-09-20 | 中国科学院天津工业生物技术研究所 | Strong promoter from fungus and application thereof |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104480028A (en) * | 2014-12-16 | 2015-04-01 | 青岛蔚蓝生物集团有限公司 | Aspergillus niger mutant strain of stable high-yield lipase |
| CN104480028B (en) * | 2014-12-16 | 2018-02-23 | 青岛蔚蓝生物集团有限公司 | A kind of aspergillus niger mutant strain of stable, high-yielding lipase |
| CN107129996A (en) * | 2016-02-26 | 2017-09-05 | 浙江大学 | One plasmid vector and its construction method and application |
| CN107129996B (en) * | 2016-02-26 | 2020-05-12 | 浙江大学 | Plasmid vector and construction method and application thereof |
| CN106191096A (en) * | 2016-07-21 | 2016-12-07 | 华南理工大学 | The recombiant plasmid of constitutive expression aspergillus niger surface display lipase and application thereof |
| CN115074361A (en) * | 2021-03-12 | 2022-09-20 | 中国科学院天津工业生物技术研究所 | Strong promoter from fungus and application thereof |
| CN113584023A (en) * | 2021-07-13 | 2021-11-02 | 江西省农业科学院园艺研究所 | Aspergillus oryzae gene promoter induced by metal ions, and preparation method and application thereof |
| CN113584023B (en) * | 2021-07-13 | 2023-07-28 | 江西省农业科学院园艺研究所 | Metal ion induced aspergillus oryzae gene promoter, preparation method and application thereof |
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