CN109082444A - A kind of Pichia pastoris high efficiency gene knockout technique - Google Patents
A kind of Pichia pastoris high efficiency gene knockout technique Download PDFInfo
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- CN109082444A CN109082444A CN201810853635.4A CN201810853635A CN109082444A CN 109082444 A CN109082444 A CN 109082444A CN 201810853635 A CN201810853635 A CN 201810853635A CN 109082444 A CN109082444 A CN 109082444A
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- pichia pastoris
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
- C12N15/905—Stable introduction of foreign DNA into chromosome using homologous recombination in yeast
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
- C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
Abstract
The present invention provides a kind of Pichia pastoris high efficiency gene knockout technique, which is characterized in that comprises the steps of: that S1. knocks out plasmid pGE1203-ADE-URA3 by plasmid pYES2 building；S2. plasmid will be knocked out and knock in target gene in the Pichia pastoris group respectivelyoch1 upstream, downstream；S3. there is the protein identification for knocking out plasmid activity, the shearing nucleotide sequence, knock out the target geneoch1, it obtains and knocks out the target geneoch1 Pichia pastoris.The present invention is effectively improved Pichia pastoris to the glycosylation modified of albumen, fusion protein excessive glycosylation is solved the problems, such as, to Pichia pastorisoch1 gene has carried out efficient knockout.
Description
Technical field
The invention belongs to technical field of bioengineering, and in particular to a kind of Pichia pastoris high efficiency gene knockout technique.
Background technique
In recent years, with the development of genetic and cell engineering technology, numerous target gene are in protokaryon and eukaryocyte
In be cloned and express, so as to improve the ability to express of foreign protein.For a long time, Escherichia coli have been used as expressing
The host strain of foreign gene, and a variety of foreign proteins are successfully expressed, but it is unable to the protein of expression structure complexity, and
Secretion type expression yield is lower.And a kind of host strain of the yeast as expression alien gene, both have prokaryotes growth characteristics,
It is easy to operate, and the posttranslational modification processing characteristics with eukaryocyte.When expressing certain gene engineering products, can advise greatly
Mould production,
To be effectively reduced cost.Wherein, saccharomyces cerevisiae is to be used as heterologous protein gene expression system earliest and be subject to
The Yeast system of application, however, studies have shown that, it is not the ideal host strain of expression alien gene.Until 1969,
Ogata etc. find for the first time a kind of thermophilic methanol yeast-pichia pastoris yeast (Pichia pastoris), it can be in methanol
To be grown under conditions of sole carbon source, wherein alcohol oxidase (Alcoholoxidase, AOX) is in methanol metabolic pathway
One enzyme, methanol can be oxidized to formaldehyde and hydrogen peroxide by it, and under conditions of using methanol as sole carbon source, AOX1 can be accounted for
It is total to cell secretion
The 30% of albumen.By the development of recent decades, currently, pichia pastoris yeast has become widest foreign protein table
Up to one of system.
Pichia pastoris yeast (Pichia pastoris)With the unrivaled superiority of other expression systems institute: raw
Long speed is fast, and expression quantity is high, can foreign gene correctly be translated and be modified, and overcomes Bacillus coli expression protein structure letter
Single, the low disadvantage of expression quantity also overcomes Saccharomyces cerevisiae system and lacks strong promoter, and secernment efficiency is poor, expresses bacterial strain
It is not sufficiently stable, expression plasmid is easy to the disadvantages of losing and the background proteins of itself secretion are few, and degree of glycosylation is low, wants to nutrition
It asks low, cheap culture medium can be used, it can be achieved that high density fermentation.Some researches show that produce recombinant protein using Pichia pastoris
A, after the induction of 68 h, rSPA yield accounts for 80% or more of total protein in fermentation liquid, compared with Escherichia coli up to 8.8 g/L
Expression system is significantly improved；By contrasting to the characteristic from Escherichia coli and the rLTB of Pichia pastoris, result table
Bright, rLTB exists
Expression quantity in Pichia pastoris is its 6 in Escherichia coli times and has higher activity, has significant advantage.
Just because of these advantages, so that pichia pastoris yeast, which rapidly develops, becomes the neck such as molecular biology, medicine, biotechnology
Research hotspot in domain.
But simultaneously the study found that the deficiency of pichia pastoris yeast heterogenous expression system.When expressing foreign protein, bar
Often there is more serious protein degradation phenomenon in this moral Pichia pastoris recombinant bacterium.It can be to albumen when Yeast expression external source glycoprotein
It is glycosylation modified to carry out excessive N-, generates high mannose type sugar chain, protein active is influenced, to limit pichia pastoris yeast
Express application of the source of people type protein expression in terms of medicine.Only by its transcriptional control, expression regulation and posttranslational modification
Etc. mechanism have further understanding, can search out and a kind of influence expression albumen effectively be reached on the way by related gene knockout
The method of diameter realizes the building of the Pichia pastoris recombinant bacterium of improvement.
Summary of the invention
In view of this, the present invention provides a kind of Pichia pastoris high efficiency gene knockout technique, Pichia pastoris is effectively improved to egg
White is glycosylation modified, fusion protein excessive glycosylation is solved the problems, such as, to Pichia pastorisoch 1 gene efficiently strike
It removes.
The technical solution of the present invention is as follows: a kind of Pichia pastoris high efficiency gene knockout technique, which is characterized in that include following step
It is rapid:
S1. plasmid pGE1203-ADE-URA3 is knocked out by plasmid pYES2 building；
S2. plasmid will be knocked out and knock in target gene in the Pichia pastoris group respectivelyoch1 upstream, downstream；
S3. there is the protein identification for knocking out plasmid activity, the shearing nucleotide sequence, knock out the target geneoch
1, it obtains and knocks out the target geneoch1 Pichia pastoris.
Further, in step S1, the construction method for knocking out plasmid pGE1203-ADE-URA3 are as follows:
A. using plasmid pYES2 as template, PCR reaction is carried out by amplimer P1 and P2, generates pGE1201；
It b. is template, design object gene by known Pichi strainoch1 homology arm sequence P3 and P4；
C. Oligonucleolide primers P5 and P6 are designed by Pichia pastoris URA3 gene；
D. Oligonucleolide primers P7 and P8 are designed by saccharomyces cerevisiae ADE gene；
E. willoch15 ' end homology arm withBam H I/NotI digestion, is cloned into useBam H I/NotI digestion
On pGE1201 plasmid, formed pGE1202, then by 3 ' end homology arms withNotI andKpn I digestion, is cloned into useNotI andKpn
On the pGE1202 of I digestion, pGE1203 is formed；Again by URA3 gene withPst I/Kpn I digestion is cloned into same usePst I/Kpn On the pGE1203 of I digestion, pGE1203- URA3 is formed；By pGE1203- URA3 withNotI digestion, then gone with CIAP
Phosphorylation is used with sameNotThe ADE gene of I digestion connects, and is formed and knocks out plasmid pGE1203-ADE-URA3.
Further, the plasmid pYES2 amplimer sequence as: shown in SEQ ID NO:1 and SEQ ID NO:2.
Further, the target geneoch1 homology arm sequence such as SEQ ID NO:3 and SEQ ID NO:4 institute
Show.
Further, the Pichia pastoris URA3 gene designs Oligonucleolide primers sequence such as: SEQ ID NO:5 and SEQ
Shown in ID NO:6.
Further, the saccharomyces cerevisiae ADE gene designs Oligonucleolide primers sequence such as: SEQ ID NO:7 and SEQ
Shown in ID NO:8.
Further, in step S3, specific method is homologous with 5 ' ends including that will knock out plasmid pGE1203-ADE-URA3
On armBgl ⅡRestriction enzyme site linearisation, electric shock are transferred in the JC308 competent cell prepared, are coated on containing arginine
On the MD culture medium of histidine, 30 DEG C are cultivated 1-2.5 days.
Compared with fusion DNA vaccine construction method in the prior art: the present invention, which need to only carry out a PCR reaction, can reach
The purpose that genetic fragment disposably merges eliminates annealing temperature cumbersome when segment composition two-by-two without adding any primer
Grope process, it is time saving and energy saving, purpose band can be enriched with effectively to obtain high specific high-purityoch 1 gene knockout group
Part, fusion efficiencies are high, have the advantages that easy, fast and efficiently, are more advantageous to gene knockout, saccharomyces cerevisiae monoploid separates etc.
The progress of follow-up test, the directional transformation for carrying out metabolic engineering for yeast are laid a good foundation so as to improve its fermenting property.
Meanwhile it is rightoch The Pichi strain of 1 gene delection can be with the humanization modified of further progress glycosyl, will
The enzyme of the synthesis complexity glycoprotein lacked in yeast is introduced into Engineering Yeast, is more conducive to preparing medicinal recombination sugar to obtain
The saccharomyces neoformans bacterial strain of albumen.
Specific embodiment
Technical solution of the present invention is clearly and completely described below in conjunction with embodiment, it is clear that described reality
Applying example is only a part of the embodiment of the present invention, instead of all the embodiments.
Embodiment
A kind of Pichia pastoris high efficiency gene knockout technique, which is characterized in that comprise the steps of:
S1. plasmid pGE1203-ADE-URA3 is knocked out by plasmid pYES2 building；
S2. plasmid will be knocked out and knock in target gene in the Pichia pastoris group respectivelyoch1 upstream, downstream；
S3. there is the protein identification for knocking out plasmid activity, the shearing nucleotide sequence, knock out the target geneoch
1, it obtains and knocks out the target geneoch1 Pichia pastoris.
Further, in step S1, the construction method for knocking out plasmid pGE1203-ADE-URA3 are as follows:
F. using plasmid pYES2 as template, PCR reaction is carried out by amplimer P1 and P2, generates pGE1201；
It g. is template, design object gene by known Pichi strainoch1 homology arm sequence P3 and P4；
H. Oligonucleolide primers P5 and P6 are designed by Pichia pastoris URA3 gene；
I. Oligonucleolide primers P7 and P8 are designed by saccharomyces cerevisiae ADE gene；
J. willoch15 ' end homology arm withBam H I/NotI digestion, is cloned into useBam H I/NotI digestion
On pGE1201 plasmid, formed pGE1202, then by 3 ' end homology arms withNotI andKpn I digestion, is cloned into useNotI andKpn
On the pGE1202 of I digestion, pGE1203 is formed；Again by URA3 gene withPst I/Kpn I digestion is cloned into same usePst I/Kpn On the pGE1203 of I digestion, pGE1203- URA3 is formed；By pGE1203- URA3 withNotI digestion, then gone with CIAP
Phosphorylation is used with sameNotThe ADE gene of I digestion connects, and is formed and knocks out plasmid pGE1203-ADE-URA3.
Further, the plasmid pYES2 amplimer sequence as: shown in SEQ ID NO:1 and SEQ ID NO:2.
Further, the target geneoch1 homology arm sequence such as SEQ ID NO:3 and SEQ ID NO:4 institute
Show.
Further, the Pichia pastoris URA3 gene designs Oligonucleolide primers sequence such as: SEQ ID NO:5 and SEQ
Shown in ID NO:6.
Further, the saccharomyces cerevisiae ADE gene designs Oligonucleolide primers sequence such as: SEQ ID NO:7 and SEQ
Shown in ID NO:8.
Further, in step S3, specific method is homologous with 5 ' ends including that will knock out plasmid pGE1203-ADE-URA3
On armBgl ⅡRestriction enzyme site linearisation, electric shock are transferred in the JC308 competent cell prepared, are coated on containing arginine
On the MD culture medium of histidine, 30 DEG C are cultivated 1-2.5 days.
Compared with fusion DNA vaccine construction method in the prior art: the present invention, which need to only carry out a PCR reaction, can reach
The purpose that genetic fragment disposably merges eliminates annealing temperature cumbersome when segment composition two-by-two without adding any primer
Grope process, it is time saving and energy saving, purpose band can be enriched with effectively to obtain high specific high-purityoch 1 gene knockout group
Part, fusion efficiencies are high, have the advantages that easy, fast and efficiently, are more advantageous to gene knockout, saccharomyces cerevisiae monoploid separates etc.
The progress of follow-up test, the directional transformation for carrying out metabolic engineering for yeast are laid a good foundation so as to improve its fermenting property.
Meanwhile it is rightoch The Pichi strain of 1 gene delection can be with the humanization modified of further progress glycosyl, will
The enzyme of the synthesis complexity glycoprotein lacked in yeast is introduced into Engineering Yeast, is more conducive to preparing medicinal recombination sugar to obtain
The saccharomyces neoformans bacterial strain of albumen.
It is obvious to a person skilled in the art that invention is not limited to the details of the above exemplary embodiments, Er Qie
In the case where without departing substantially from spirit or essential attributes of the invention, the present invention can be realized in other specific forms.Therefore, no matter
From the point of view of which point, the present embodiments are to be considered as illustrative and not restrictive, and the scope of the present invention is by appended power
Benefit requires rather than above description limits, it is intended that all by what is fallen within the meaning and scope of the equivalent elements of the claims
Variation is included within the present invention.
In addition, it should be understood that although this specification is described in terms of embodiments, but not each embodiment is only wrapped
Containing an independent technical solution, this description of the specification is merely for the sake of clarity, and those skilled in the art should
It considers the specification as a whole, the technical solutions in the various embodiments may also be suitably combined, forms those skilled in the art
The other embodiments being understood that.It is noted that the technical characteristic being not described in detail in the present invention, can pass through this
Field any prior art is realized.
Sequence table
<110>Huizhou health professional technical college
<120>a kind of Pichia pastoris high efficiency gene knockout technique
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 28
<212> DNA
<213>plasmid pYES2
<400> 1
atagatctag aacatgtgag caaaaggc 28
<210> 2
<211> 68
<212> DNA
<213>plasmid pYES2
<400> 2
acagatctgg cccgataggc catcccgggc gcggccgcgg taccctagct tttcaattca 60
attcatca 68
<210> 3
<211> 44
<212> DNA
<213>Pichia pastoris target gene och 1
<400> 3
acgcggccgc ggatcctaat gaggccaagg aattggagct ggct 44
<210> 4
<211> 30
<212> DNA
<213>Pichia pastoris target gene och 1
<400> 4
agggtacctg ggaagagatg tcttgtgcac 30
<210> 5
<211> 34
<212> DNA
<213>Pichia pastoris URA3 gene
<400> 5
aaccaactgc agtggggaga taaccacctt tgac 34
<210> 6
<211> 33
<212> DNA
<213>Pichia pastoris URA3 gene
<400> 6
tttcggtacc ttgctggcta ctccttgagt ctg 33
<210> 7
<211> 35
<212> DNA
<213>saccharomyces cerevisiae ADE gene
<400> 7
atttgcggcc gctattcacg agtcagtctg actct 35
<210> 8
<211> 35
<212> DNA
<213>saccharomyces cerevisiae ADE gene
<400> 8
atttgcggcc gcaatcctcg agaagcaagc tattg 35
Claims (7)
1. a kind of Pichia pastoris high efficiency gene knockout technique, which is characterized in that comprise the steps of:
S1. plasmid pGE1203-ADE-URA3 is knocked out by plasmid pYES2 building；
S2. plasmid will be knocked out and knock in target gene in the Pichia pastoris group respectivelyoch1 upstream, downstream；
S3. there is the protein identification for knocking out plasmid activity, the shearing nucleotide sequence, knock out the target geneoch
1, it obtains and knocks out the target geneoch1 Pichia pastoris.
2. a kind of Pichia pastoris high efficiency gene knockout technique according to claim 1, which is characterized in that in step S1, institute
State the construction method for knocking out plasmid pGE1203-ADE-URA3 are as follows:
Using plasmid pYES2 as template, PCR reaction is carried out by amplimer P1 and P2, generates pGE1201；
It is template, design object gene by known Pichi strainoch1 homology arm sequence P3 and P4；
Oligonucleolide primers P5 and P6 are designed by Pichia pastoris URA3 gene；
Oligonucleolide primers P7 and P8 are designed by saccharomyces cerevisiae ADE gene；
It willoch15 ' end homology arm withBam H I/NotI digestion, is cloned into useBam H I/NotThe pGE1201 of I digestion
On plasmid, formed pGE1202, then by 3 ' end homology arms withNotI andKpn I digestion, is cloned into useNotI andKpn I digestion
PGE1202 on, formed pGE1203；Again by URA3 gene withPst I/Kpn I digestion is cloned into same usePst I/Kpn I
On the pGE1203 of digestion, pGE1203- URA3 is formed；By pGE1203- URA3 withNotI digestion, then phosphoric acid is removed with CIAP
Change, is used with sameNotThe ADE gene of I digestion connects, and is formed and knocks out plasmid pGE1203-ADE-URA3.
3. a kind of Pichia pastoris high efficiency gene knockout technique according to claim 2, which is characterized in that the plasmid
PYES2 amplimer sequence as: shown in SEQ ID NO:1 and SEQ ID NO:2.
4. a kind of Pichia pastoris high efficiency gene knockout technique according to claim 2, which is characterized in that the target geneoch1 homology arm sequence as: shown in SEQ ID NO:3 and SEQ ID NO:4.
5. a kind of Pichia pastoris high efficiency gene knockout technique according to claim 2, which is characterized in that the Pichia pastoris
URA3 gene design Oligonucleolide primers sequence as: shown in SEQ ID NO:5 and SEQ ID NO:6.
6. a kind of Pichia pastoris high efficiency gene knockout technique according to claim 2, which is characterized in that the saccharomyces cerevisiae
ADE gene design Oligonucleolide primers sequence as: shown in SEQ ID NO:7 and SEQ ID NO:8.
7. a kind of Pichia pastoris high efficiency gene knockout technique according to claim 1, which is characterized in that in step S3,
Specific method includes that will knock out plasmid pGE1203-ADE-URA3 on 5 ' end homology armsBgl ⅡRestriction enzyme site linearisation, electricity
It hits and is transferred in the JC308 competent cell prepared, be coated on the MD culture medium containing arginine and histidine, 30 DEG C of cultures
1-2.5 it.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108779470A (en) * | 2015-12-17 | 2018-11-09 | 赢创德固赛(中国)投资有限公司 | The box gene knocked out for homologous recombination in yeast cells |
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| CN101195809A (en) * | 2006-12-07 | 2008-06-11 | 中国人民解放军军事医学科学院生物工程研究所 | Pichia pastoris strain with deletion of alpha-1,6-mannose transferase and construction method thereof |
| CN102120967A (en) * | 2010-12-09 | 2011-07-13 | 江南大学 | Preparation and application of OCH1 genetic flaw type P. pastoris X-33 bacterial strain |
| US20150267212A1 (en) * | 2012-10-23 | 2015-09-24 | Research Corporation Technologies, Inc. | Pichia pastoris strains for producing predominantly homogeneous glycan structure |
| CN105779490A (en) * | 2014-12-16 | 2016-07-20 | 北京集智新创科技有限公司 | Construction method of Pichia pastoris expressed by OCH1 defect anti-CD20 tetravalent antibody |
| CN108004264A (en) * | 2017-12-14 | 2018-05-08 | 华中科技大学 | Pichia pastoris gene knockout and resistant gene recycling carrier and construction method and utilization |
-
2018
- 2018-07-30 CN CN201810853635.4A patent/CN109082444A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101195809A (en) * | 2006-12-07 | 2008-06-11 | 中国人民解放军军事医学科学院生物工程研究所 | Pichia pastoris strain with deletion of alpha-1,6-mannose transferase and construction method thereof |
| CN102120967A (en) * | 2010-12-09 | 2011-07-13 | 江南大学 | Preparation and application of OCH1 genetic flaw type P. pastoris X-33 bacterial strain |
| US20150267212A1 (en) * | 2012-10-23 | 2015-09-24 | Research Corporation Technologies, Inc. | Pichia pastoris strains for producing predominantly homogeneous glycan structure |
| US20170166910A1 (en) * | 2012-10-23 | 2017-06-15 | Research Corporation Technologies, Inc. | Pichia pastoris strains for producing predominantly homogeneous glycan structure |
| CN105779490A (en) * | 2014-12-16 | 2016-07-20 | 北京集智新创科技有限公司 | Construction method of Pichia pastoris expressed by OCH1 defect anti-CD20 tetravalent antibody |
| CN108004264A (en) * | 2017-12-14 | 2018-05-08 | 华中科技大学 | Pichia pastoris gene knockout and resistant gene recycling carrier and construction method and utilization |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108779470A (en) * | 2015-12-17 | 2018-11-09 | 赢创德固赛(中国)投资有限公司 | The box gene knocked out for homologous recombination in yeast cells |
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