CN103088043A - Construction method of colon bacillus genetic engineering strain for secretory expression of Metchnikowin antibacterial peptide - Google Patents
Construction method of colon bacillus genetic engineering strain for secretory expression of Metchnikowin antibacterial peptide Download PDFInfo
- Publication number
- CN103088043A CN103088043A CN2013100049530A CN201310004953A CN103088043A CN 103088043 A CN103088043 A CN 103088043A CN 2013100049530 A CN2013100049530 A CN 2013100049530A CN 201310004953 A CN201310004953 A CN 201310004953A CN 103088043 A CN103088043 A CN 103088043A
- Authority
- CN
- China
- Prior art keywords
- metchnikowin
- antibacterial peptide
- gene
- plasmid
- construction process
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a construction method of a colon bacillus genetic engineering strain for secretory expression of antibacterial peptide Metchnikowin. The construction method comprises the steps of: genetically connecting a Metchnikowin antibacterial peptide gene, constructing a recombinant pET-22b plasmid, and transforming a colon bacillus BL21 competent cell. The construction method disclosed by the invention has the beneficial effects that the colon bacillus genetic engineering strain has efficient and stable secretory expression, and the protein cleavage is low in cost, and safe due to a downstream DP hydrolysis locus.
Description
Technical field
The present invention relates to a kind of construction process of secreting, expressing antibacterial peptide Metchnikowin Recombinant organism.
Background technology
Antibacterial peptide is under inductive condition, and the small active peptides class that organism immunity defence system produces is distributed widely in plant, animal and the mankind.Antibacterial peptide is by genes encoding, by Ribosome biogenesis.The antibacterial peptide that has been found that at present, identifies surpasses thousand kinds, the large clean positive charge of multi-band.From structure can be divided into alpha-helix type, beta sheet type, be rich in disulfide linkage, the broad variety such as proline rich.The antibacterial peptide antimicrobial spectrum is wide, and some antibacterial peptide can also suppress the growth of some tumour cell specifically.Antibacterial peptide can be used for the aspects such as medical and health, foodstuff additive, fodder additives.Antibacterial peptide is different from traditional microbiotic antifungal mechanism, and the mode of action of antibacterial peptide is mainly optionally to attack the cytolemma of pathogen, and correspondingly pathogen is difficult for producing the resistance for antibacterial peptide, therefore uses antibacterial peptide can solve the bacterial drug resistance problem.Simultaneously again because it is a kind of peptide, can the digestion of digested road, absorb harmless, noresidue, therefore, antibacterial peptide has application prospect very widely at aspects such as medicine, livestock industry, agriculturals.Antibacterial peptide content in vivo is extremely low, and production cost is high, has a strong impact on its widespread use; Research and develop its large scale production method and have very positive meaning.
At present, the method of producing antibacterial peptide mainly contains three kinds: directly extract natural antibacterial peptide from organism 1.: antibacterial peptide extensively is present in the particular organization of low higher animal by the time, but content is considerably less, and extraction process is complicated, cost is expensive, is difficult to satisfy the requirement of scale operation; 2. chemical process synthetic antibacterial peptide: the chemical synthesis cost is high, is difficult to simultaneously guarantee natural structure and the biological activity of synthetic peptide class; 3 gene engineering method: adopting the method is the focus of nowadays studying, and is also one of effective way that obtains antibacterial peptide.
Metchnikowin is a kind of little peptide of proline rich.The mechanism of action that contains the proline(Pro) antibacterial peptide is commonly considered as directly passing bacterial cell membrane, enter after cell with intracellular biomacromolecule for example nucleic acid molecule interact, thereby reach the effect of bactericidal.This type of contains the proline(Pro) antibacterial peptide mainly for Gram-negative bacteria great majority, but the Metchnikowin that derives from fruit bat works mainly for gram-positive microorganism and fungi.Due in vivo, the antibacterial peptide of different structure characteristics cooperatively interacts and plays a role, and therefore correspondingly need to study different antibacterial peptides external.At present few for the recombinant expressed report that contains the proline(Pro) antibacterial peptide, the recombinant expressed report that also has no of Metchnikowin.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of construction process of secreting, expressing antibacterial peptide Metchnikowin Recombinant organism.
The present invention is achieved through the following technical solutions.
A kind of method that builds above-mentioned secreting, expressing antibacterial peptide Metchnikowin Recombinant organism comprises antibacterial peptide Metchnikowin gene is connected with fusion gene, builds restructuring pET-22b plasmid, transforms the e. coli bl21 competent cell.
Further, above-mentioned antibacterial peptide Metchnikowin gene contains nucleotide sequence SEQ ID: NO 1, SEQ ID: NO2, contains aminoacid sequence SEQ ID NO3:SEQ ID NO4.
Further, above-mentioned antibacterial peptide Metchnikowin gene obtains by amplification, and take the plasmid that contains the Metchnikowin gene order as template, at its downstream design EcoRI restriction enzyme site, its primer is:
MF:5’-CATCGTCATCAGGGTCCG-3’;
MR:5’-GGAATTCTTAATAAATCGG?-3’。
Further, the amplification of above-mentioned fusion gene, take the plasmid that contains antigen-4 fusion protein gene as template, at sequence upstream design Nco I restriction enzyme site, its primer:
CF:5’-CATGCCATGGATCACCATCATCATCAT-3’?;
CR:?5’-AAGGGTTTCCGAAGGCTTGG-3’。
Further, in above-mentioned structure restructuring pET-22b plasmid, be connected with the T4DNA ligase enzyme after the antibacterial peptide Metchnikowin gene phosphorylation with the antigen-4 fusion protein gene of above-mentioned amplification and above-mentioned amplification.
Further, above-mentioned abduction delivering: the shaking culture genetic engineering bacterium adds inductor to induce 24 hours, culture temperature: 25 ℃ to logarithmic phase.
Beneficial effect of the present invention:
The secreting, expressing of efficient stable, downstream DP are hydrolyzed the site and make the scinderin cost low, safer.
Description of drawings
Fig. 1 is electrophoresis result schematic diagram of the present invention;
Fig. 2 is active schematic diagram of the present invention.
Embodiment
The below is described in further detail the present invention according to drawings and embodiments.
1, gene amplification:
(1) amplification of Metchnikowin gene: take the plasmid that contains the Metchnikowin gene order as template, at its downstream design EcoRI restriction enzyme site, primer MF:5 '-CATCGTCATCAGGGTCCG-3 '; MR:5 '-GGAATTCTTAATAAATCGG-3 ', reaction conditions: 94 ℃ of 45s, 50 ℃ of 45s, 72 ℃ of 30s, 30 circulations, 72 ℃ of 10min.Reaction product detects with 1.5% agarose gel electrophoresis, and gel reclaims test kit and reclaims.
(2) amplification of antigen-4 fusion protein gene: take the plasmid that contains antigen-4 fusion protein gene as template, at sequence upstream design Nco I restriction enzyme site, primer: CF:5 '-CATGCCATGGATCACCATCATCATCAT-3 ', CR::5 '-AAGGGTTTCCGAAGGCTTGG-3 '.
Reaction conditions: 94 ℃ of 45s, 50 ℃ of 45s, 72 ℃ of 30s, 30 circulations, 72 ℃ of 10min.Reaction product detects with 1.5% agarose gel electrophoresis, and gel reclaims test kit and reclaims.(3) link of Metchnikowin gene and fusion rotein: Metchnikowin gene PCR product and antigen-4 fusion protein gene PCR product are pressed the 1:1 mixing, carry out connecting with the T4DNA ligase enzyme after phosphorylation with T4PNK.The primer MetchnikowinF and the primer CR that re-use in (1) and (2) carry out pcr amplification, reaction conditions: 94 ℃ of 45s, 50 ℃ of 45s, 72 ℃ of 30s, 30 circulations, 72 ℃ of 10min.Reaction product detects with 1.5% agarose gel electrophoresis, and gel reclaims test kit and reclaims.
2, the clone transforms: PCR product and carrier are all cut with EcoRI and NcoI enzyme, purified rear the connection with the T4DNA ligase enzyme of product obtains recombinant plasmid 1:pET-22b-MetchnikowinC, transform BL21(DE3) competent cell, the penbritin screening positive clone, a small amount of plasmid that extracts, and order-checking.
3, abduction delivering: the engineering bacteria that will contain recombinant plasmid 1 is seeded to 37 ℃ of overnight shakings cultivations in the LB substratum that contains the 50ug/ml penbritin, by the 1% fresh LB substratum of amount access, 37 ℃ of shaking culture are during to OD600 ≈ 0.5, the IPTG that adds final concentration 1mmol/L, induce 24h for 25 ℃, the centrifugal 1min of 9000r/min collects supernatant liquor.
4, chemical chop and purifying
Get the 20ml supernatant liquor, slowly drip 50% acetic acid solution until pH value of solution=2.5.Encloses container, as for constant temperature in 50 degrees centigrade of water-baths 72 hours, the 12000 centrifugal 10min of r/min obtained containing the Metchnikowin supernatant liquor, adopted cation-exchange chromatography, purifying Metchnikowin.
Above-described embodiment only is explanation technical conceive of the present invention and characteristics, and its purpose is to allow the personage who is familiar with this art can understand content of the present invention and be implemented, and can not limit protection scope of the present invention with this.All equivalences that spirit is done according to the present invention change or modify, and all should be encompassed in protection scope of the present invention.
Claims (5)
1. a method that builds described secreting, expressing antibacterial peptide Metchnikowin Recombinant organism, is characterized in that, comprises antibacterial peptide Metchnikowin gene is connected, and builds restructuring pET-22b plasmid, transforms the e. coli bl21 competent cell.
2. construction process according to claim 1, is characterized in that, described antibacterial peptide Metchnikowin gene contains nucleotide sequence SEQ ID: NO 1, SEQ ID: NO2, contains aminoacid sequence SEQ ID NO3:SEQ ID NO4.
3. construction process according to claim 1, is characterized in that, described antibacterial peptide Metchnikowin gene obtains by amplification, and take the plasmid that contains the Metchnikowin gene order as template, at its downstream design EcoRI restriction enzyme site, its primer is:
MF:5’-CATCGTCATCAGGGTCCG-3’;
MR:5’-GGAATTCTTAATAAATCGG?-3’。
4. construction process according to claim 1, is characterized in that, the amplification of described antibacterial peptide Metchnikowin gene, and take the plasmid that contains antigen-4 fusion protein gene as template, at sequence upstream design Nco I restriction enzyme site, its primer:
CF:5’-CATGCCATGGATCACCATCATCATCAT-3’?;
CR:?5’-AAGGGTTTCCGAAGGCTTGG-3’。
5. construction process according to claim 1, is characterized in that, in described structure restructuring pET-22b plasmid, is connected with the T4DNA ligase enzyme after the antibacterial peptide Metchnikowin gene phosphorylation with the antigen-4 fusion protein gene of described amplification and described amplification.
Construction process according to claim 1 is characterized in that, described abduction delivering: the shaking culture genetic engineering bacterium adds inductor to induce 24 hours, culture temperature: 25 ℃ to logarithmic phase.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2013100049530A CN103088043A (en) | 2013-01-07 | 2013-01-07 | Construction method of colon bacillus genetic engineering strain for secretory expression of Metchnikowin antibacterial peptide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2013100049530A CN103088043A (en) | 2013-01-07 | 2013-01-07 | Construction method of colon bacillus genetic engineering strain for secretory expression of Metchnikowin antibacterial peptide |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103088043A true CN103088043A (en) | 2013-05-08 |
Family
ID=48201172
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2013100049530A Pending CN103088043A (en) | 2013-01-07 | 2013-01-07 | Construction method of colon bacillus genetic engineering strain for secretory expression of Metchnikowin antibacterial peptide |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103088043A (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102618551A (en) * | 2012-03-15 | 2012-08-01 | 安徽希普生物科技有限公司 | Method for using brewer's yeast to express antibacterial peptide G13 |
-
2013
- 2013-01-07 CN CN2013100049530A patent/CN103088043A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102618551A (en) * | 2012-03-15 | 2012-08-01 | 安徽希普生物科技有限公司 | Method for using brewer's yeast to express antibacterial peptide G13 |
Non-Patent Citations (6)
| Title |
|---|
| ELENA A. LEVASHINA ET AL.: "Metchnikowin, a novel immune-inducible proline-rich peptide from DrosophiZa with antibacterial and antifungal properties", 《EUR. J. BIOCHEM》 * |
| KRISHNA MOHAN PADMANABHA DAS ET AL.: "A Novel Thermostability Conferring Property of Cherry Tag and its Application in Purification of Fusion Proteins", 《J MICROBIAL BIOCHEM TECHNOL》 * |
| YANNICK GUEGUEN ET AL.: "Oyster hemocytes express a proline-rich peptide displaying synergistic antimicrobial activity with a defensin", 《MOLECULAR IMMUNOLOGY》 * |
| 余曜: "黑腹果蝇抗菌肽基因Metchnikowin在毕赤酵母中分泌型表达及活性检测", 《湖北大学学位论文》 * |
| 刘杨: "阳离子抗菌肽G13分泌表达系统的构建", 《中国优秀硕士学位论文全文数据库基础科学辑》 * |
| 邓小娟等: "果蝇抗微生物肽基因的免疫诱导模式", 《昆虫学报》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Rasala et al. | Photosynthetic biomanufacturing in green algae; production of recombinant proteins for industrial, nutritional, and medical uses | |
| CN105061603B (en) | Antibacterial peptide-lysozyme fusion protein preparation method and applications | |
| An et al. | Expression of a functional recombinant human basic fibroblast growth factor from transgenic rice seeds | |
| CA2780540A1 (en) | Novel bacillus thuringiensis gene with lepidopteran activity | |
| Wang et al. | Stable expression of basic fibroblast growth factor in chloroplasts of tobacco | |
| RU2020105862A (en) | TOXIN GENES AND WAYS OF THEIR APPLICATION | |
| WO2007118790A3 (en) | Active potassium channel transporters (akt) and their use to create stress tolerant plants | |
| CN107312094A (en) | A kind of heterozygous antibacterial peptide and its preparation method and application | |
| CN104004803A (en) | Method for producing antimicrobial peptide through fermentation of brevibacillus laterosporu | |
| CN104263709A (en) | Egg-white lysozyme and preparation method thereof | |
| Vaseva et al. | Heat-Stress-Mitigating effects of a protein-Hydrolysate-Based biostimulant are linked to changes in protease, DHN, and HSP gene expression in maize | |
| Lucena et al. | Similarities and differences in the acquisition of Fe and P by dicot plants | |
| CN102337288A (en) | Gene engineering preparation method of tilapia hepcidin | |
| CN103555739A (en) | Recombined microorganism glutamine transaminase gene and preparation method thereof | |
| CN102586262B (en) | Defensin gene of antimicrobial peptide of bemisia tabaci (Gennadius), antimicrobial peptide encoded by defensin gene and preparation method for defensin gene | |
| CN104131021A (en) | Antibacterial peptide coexpression vector, construction and expression method thereof | |
| CN103088043A (en) | Construction method of colon bacillus genetic engineering strain for secretory expression of Metchnikowin antibacterial peptide | |
| Li et al. | Expression and Characterization of a GH16 Family β-Agarase Derived from the Marine Bacterium Microbulbifer sp. BN3 and Its Efficient Hydrolysis of Agar Using Raw Agar-Producing Red Seaweeds Gracilaria sjoestedtii and Gelidium amansii as Substrates | |
| CN103641525B (en) | A kind of tobacco leaf tailored version biological organic fertilizer and preparation method thereof | |
| CN102212541A (en) | Construction of gene engineering bacteria for expressing recombinant cationic antimicrobial peptides (AMPs) G13 escherichia coli | |
| CN102532325A (en) | Method for preparing cecropins AD and frog Buforin II fusion antimicrobial peptide by using hydroxylamine cutting method, and uses thereof | |
| CN105274128A (en) | Method for utilizing genetic engineering high-efficiency expression to amidate EC07 | |
| CN104988170A (en) | Fused antibacterial peptide, and preparation method and application thereof | |
| CN102241756B (en) | High expression of tenebrio molitor antibacterial peptide TmAMP3m in escherichia coli and application of TmAMP3m | |
| CN103194442A (en) | Novel polypeptide and hydrolyzed antibacterial peptide and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20130508 |