CN108118067A - A kind of composing type in Escherichia coli, the carrier of secretion type expression - Google Patents
A kind of composing type in Escherichia coli, the carrier of secretion type expression Download PDFInfo
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- CN108118067A CN108118067A CN201810185847.XA CN201810185847A CN108118067A CN 108118067 A CN108118067 A CN 108118067A CN 201810185847 A CN201810185847 A CN 201810185847A CN 108118067 A CN108118067 A CN 108118067A
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- 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
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C12N2800/101—Plasmid DNA for bacteria
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Abstract
The present invention relates to the present invention relates to foreign protein prokaryotic expression fields, specifically a kind of composing type in Escherichia coli, the carrier of secretion type expression, it is characterised in that：Constitutive expression carrier framework construction is linked to by coding sequence of secretory signal peptide double-stranded sequence to form, the advantage of the invention is that：In Bacillus coli expression special bacterial strain can be not required in any one expression in escherichia coli in foreign gene；It need not be induced, can independently be expressed；Expression product is directly secreted into culture medium, is existed with soluble form, and expression quantity is high and facilitates later separation, purifying, and some expression in Escherichia coli body to the virose albumen of Escherichia coli, can also be expressed.
Description
Technical field
The present invention relates to foreign protein prokaryotic expression fields, are specifically one kind composing type, secreting type table in Escherichia coli
The carrier reached.
Background technology
Importing Escherichia coli are commonly referred to as expressing the method for a large amount of protein after foreign gene is inserted into suitable carrier
Prokaryotic expression.This method has application in protein purification, positioning and functional analysis etc..Escherichia coli recombinate for expressing
Albumen has following characteristics：It is easy to grow and controls；It is held high for the material of Bacteria Culture not as good as the material of mammalian cell system
It is expensive；There are various coli strains and the plasmid of matching tool various characteristics available.But existing side
Method has the disadvantages that：1st, need plus derivant induces, foreign protein, and the inductive condition of each foreign protein could be expressed
It differs, so to devote a tremendous amount of time, energy goes to grope the condition for inducing exogenous protein expression；2nd, in Bacillus coli cells
Expression, expressing quantity compares, and relatively low, easily formation inclusion body, some albumen toxic to Escherichia coli cannot express；3、
Need specific coli strain that could express foreign protein.
The content of the invention
It is an object of the invention to provide a kind of composing type in Escherichia coli, the carrier of secretion type expression, in solution
State the problem of being proposed in background technology.
In order to solve the above technical problems, technical solution provided by the invention is：One kind composing type, secretion in Escherichia coli
The carrier of type expression, is linked to constitutive expression carrier framework construction by coding sequence of secretory signal peptide double-stranded sequence and forms.
The making step of the constitutive expression carrier is：
1) constitutive promoter positive segment is dissolved into 100 μM of combined positive segment solution with 1xTE buffer solutions, by group
Constitutive promoter negative film section is dissolved into 100 μM of combined negative film section solution；
2) the combined positive segment solution of 1 μ l and combined negative film section solution is respectively taken to be added to the 0.5xTE buffer solutions of 98 μ l,
It is mixed into combined positive and negative segment mixed liquor；
3) combined positive and negative segment mixed liquor is placed in PCR instrument, temperature is kept for 95 DEG C, is taken out after 5min and is positioned over experiment
Platform, cooled to room temperature, forward and reverse segment are annealed into double-strand, form constitutive promoter；
4) pET-22b carriers are formed into pET-22b carrier frameworks by Bgl II and Xba I double digestions；
5) constitutive promoter double-stranded sequence is linked to pET-22b carrier frameworks, forms pET-22b composing type carriers；
6) pET-22b composing types carrier is formed into constitutive expression carrier by Nde I and Nco I double digestions.
The making step of the coding sequence of secretory signal peptide is：
1) coding sequence of secretory signal peptide positive segment with 1xTE buffer solutions is dissolved into 100 μM of secreting type positive segment solution, will be divided
Secrete the secreting type negative film section solution that type signal peptide negative film section is dissolved into 100 μM；
2) the secreting type positive segment solution of 1 μ l and secreting type negative film section solution is respectively taken to be added to the 0.5xTE buffer solutions of 98 μ l,
It is mixed into the positive and negative segment mixed liquor of secreting type；
3) the positive and negative segment mixed liquor of secreting type is placed in PCR instrument, temperature is kept for 95 DEG C, is taken out after 5min and is positioned over experiment
Platform, cooled to room temperature, forward and reverse segment are annealed into double-strand, form coding sequence of secretory signal peptide.
Preferably, the constitutive promoter positive segment is CEPr-F：5’-
GATCTAACATTGAGGCAACGACAAGGTCGATTCTGCTATCCTTGAGGAGGCTCCTC T-3 ', the group
Constitutive promoter negative film section is CEPr-R：5’-
CTAGAGAGGAGCCTCCTCAAGGATAGCAGAATCGACCTTGTCGTTGCCTCAATGTTA-3’。
Preferably, the coding sequence of secretory signal peptide positive segment is OmpA-F：5’-
TAATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTCGCTACCGTA GCGCAGGCCGCTGC-3 ', institute
The coding sequence of secretory signal peptide negative film section stated is OmpA-R：5’-
CATGGCAGCGGCCTGCGCTACGGTAGCGAAACCAGCCAGTGCCACTGCAATCGCGATAGCTGTCTTTTTCAT-3’。
The advantage of the invention is that：Foreign gene in Bacillus coli expression, can in any one Escherichia coli table
It reaches, special bacterial strain is not required；It need not be induced, can independently be expressed；Expression product is directly secreted into culture medium, with
Soluble form exists, and expression quantity is high and facilitates later separation, purifying, and some expression in Escherichia coli body are to large intestine bar
The virose albumen of bacterium, can also express.
Description of the drawings
Fig. 1 is the WB detection figures of the embodiment of the present invention.
Specific embodiment
Illustrate the present invention with specific embodiment below, be not limitation of the present invention.
Embodiment
1) with Bgl II and Xba I double digestion pET-22b carriers, GFP encoding genes are recycled；
2) with Nde I and Nco I double digestion composing type secreted expression carriers, carrier framework is recycled；
3) GFP encoding genes are linked to composing type secreted expression carrier skeleton, build GFP composing type secretion type expressions
Carrier；
4) GFP composing type secreted expression carriers are transformed into e.colistraindh5α, random 5 positives gram of picking
Grand, 37 DEG C in 4ml LB fluid nutrient mediums, when 200rpm cultures about 16 are small, then room temperature 12,000rpm, centrifuge 5 minutes, receive
Collect supernatant；20ul supernatants is taken to carry out WB detections, obtain WB detection figures.
According to shown in Fig. 1,5 positive colonies of GFP composing type secreted expression carriers in e.colistraindh5α,
Without special bacterial strain, without induction, accomplish independently to express.
The foregoing is only a preferred embodiment of the present invention, but protection scope of the present invention be not limited thereto,
Any one skilled in the art in the technical scope disclosed by the present invention, technique according to the invention scheme and its
Inventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.
Claims (5)
1. a kind of composing type in Escherichia coli, the carrier of secretion type expression, it is characterised in that：By coding sequence of secretory signal peptide double-strand sequence
Row are linked to constitutive expression carrier framework construction and form.
2. a kind of composing type in Escherichia coli according to claim 1, the carrier of secretion type expression, which is characterized in that
The making step of the constitutive expression carrier is：
1) constitutive promoter positive segment is dissolved into 100 μM of combined positive segment solution with 1xTE buffer solutions, by composing type
Promoter negative film section is dissolved into 100 μM of combined negative film section solution；
2) the combined positive segment solution of 1 μ l and combined negative film section solution is respectively taken to be added to the 0.5xTE buffer solutions of 98 μ l, is mixed
Uniformly into combined positive and negative segment mixed liquor；
3) combined positive and negative segment mixed liquor being placed in PCR instrument, temperature is kept for 95 DEG C, is taken out after 5min and is positioned over experimental bench, from
It is so cooled to room temperature, forward and reverse segment is annealed into double-strand, forms constitutive promoter；
4) pET-22b carriers are formed into pET-22b carrier frameworks by Bgl II and Xba I double digestions；
5) constitutive promoter double-stranded sequence is linked to pET-22b carrier frameworks, forms pET-22b composing type carriers；
6) pET-22b composing types carrier is formed into constitutive expression carrier by Nde I and Nco I double digestions.
3. a kind of composing type in Escherichia coli according to claim 1, the carrier of secretion type expression, which is characterized in that
The making step of the coding sequence of secretory signal peptide is：
1) coding sequence of secretory signal peptide positive segment is dissolved into 100 μM of secreting type positive segment solution with 1xTE buffer solutions, by secreting type
Signal peptide negative film section is dissolved into 100 μM of secreting type negative film section solution；
2) the secreting type positive segment solution of 1 μ l and secreting type negative film section solution is respectively taken to be added to the 0.5xTE buffer solutions of 98 μ l, is mixed
Non-uniform components secrete the positive and negative segment mixed liquor of type；
3) the positive and negative segment mixed liquor of secreting type being placed in PCR instrument, temperature is kept for 95 DEG C, is taken out after 5min and is positioned over experimental bench, from
It is so cooled to room temperature, forward and reverse segment is annealed into double-strand, forms coding sequence of secretory signal peptide.
4. a kind of composing type in Escherichia coli according to claim 2, the carrier of secretion type expression, it is characterised in that：
The constitutive promoter positive segment is CEPr-F：5’
GATCTAACATTGAGGCAACGACAAGGTCGATTCTGCTATCCTTGAGGAGGCTCCTC T-3 ', the composing type
Promoter negative film section is CEPr-R：5’
CTAGAGAGGAGCCTCCTCAAGGATAGCAGAATCGACCTTGTCGTTGCCTCAATGTTA-3’。
5. the carrier of a kind of composing type in Escherichia coli stated according to claim 3, secretion type expression, it is characterised in that：Institute
The coding sequence of secretory signal peptide positive segment stated is OmpA-F：5’-
TAATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTCGCTACCGTAGCGCAGGCCGCTGC-
3 ', the coding sequence of secretory signal peptide negative film section is OmpA-R：5’-
CATGGCAGCGGCCTGCGCTACGGTAGCGAAACCAGCCAGTGCCACTGCAATCGCGATAGCTGTCTTTTTCAT-
3’。
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| CN201810185847.XA CN108118067A (en) | 2018-03-07 | 2018-03-07 | A kind of composing type in Escherichia coli, the carrier of secretion type expression |
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| CN201810185847.XA CN108118067A (en) | 2018-03-07 | 2018-03-07 | A kind of composing type in Escherichia coli, the carrier of secretion type expression |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1908175A (en) * | 2006-08-22 | 2007-02-07 | 中山大学 | Prokaryotic secretion expression carrier and application thereof |
| CN101560516A (en) * | 2009-01-19 | 2009-10-21 | 广西大学 | Constitutive expression promoter in escherichia coli and applications in escherichia coli |
| CN104342445A (en) * | 2013-07-24 | 2015-02-11 | 中国科学院海洋研究所 | Vector for efficiently secreting and expressing heterogenous protein, and its application |
| CN107287225A (en) * | 2017-06-27 | 2017-10-24 | 深圳劲宇生物科技有限公司 | Gold ion is detected and adsorption system and its Host Strains, gold ion recovery method |
-
2018
- 2018-03-07 CN CN201810185847.XA patent/CN108118067A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1908175A (en) * | 2006-08-22 | 2007-02-07 | 中山大学 | Prokaryotic secretion expression carrier and application thereof |
| CN101560516A (en) * | 2009-01-19 | 2009-10-21 | 广西大学 | Constitutive expression promoter in escherichia coli and applications in escherichia coli |
| CN104342445A (en) * | 2013-07-24 | 2015-02-11 | 中国科学院海洋研究所 | Vector for efficiently secreting and expressing heterogenous protein, and its application |
| CN107287225A (en) * | 2017-06-27 | 2017-10-24 | 深圳劲宇生物科技有限公司 | Gold ion is detected and adsorption system and its Host Strains, gold ion recovery method |
Non-Patent Citations (5)
| Title |
|---|
| DUFFAUD,G.D. ET AL.: ""Artificial DNA; cloning vector pIN-III-ompA2"，Accession Number：AJ223122.1", 《GENBANK》 * |
| JOHN GHRAYEB ET AL.: ""Secretion cloning vectors in Escherichia coli"", 《THE EMBO JOURNAL》 * |
| LI, SHENGNAN ET AL.: ""Cloning and Expression of cDNA Encoding a Cysteine Protease Inhibitor from Clamworm and Its Possible Use in Managing Anoplophora glabripennisMotschulsky (Coleoptera: Cerambycidae)"", 《J. MICROBIOL. BIOTECHNOL.》 * |
| 叶兼菱 等: ""半胱氨酸蛋白酶抑制剂防治桑天牛的室内生测"", 《林业科技开发》 * |
| 李盛楠 等: ""表达荧光假单胞菌鞭毛蛋白工程菌的构建及其对黑松的毒性"", 《青岛大学学报(工程技术版)》 * |
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