CN108517331A - A kind of engineering bacteria construction method of amalgamation and expression antibacterial peptide and red fluorescent protein - Google Patents
A kind of engineering bacteria construction method of amalgamation and expression antibacterial peptide and red fluorescent protein Download PDFInfo
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- CN108517331A CN108517331A CN201810223031.1A CN201810223031A CN108517331A CN 108517331 A CN108517331 A CN 108517331A CN 201810223031 A CN201810223031 A CN 201810223031A CN 108517331 A CN108517331 A CN 108517331A
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- antibacterial peptide
- fluorescent protein
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- C—CHEMISTRY; METALLURGY
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
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- C07K2319/00—Fusion polypeptide
- C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
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Abstract
The invention discloses the engineering bacteria construction methods of a kind of amalgamation and expression antibacterial peptide and red fluorescent protein, during genetic engineering produces cationic antibacterial peptide LfcinB, mApple fusion tau genes are connect with antibacterial peptide gene, construction recombination plasmid, electrotransformation saccharomyces cerevisiae W303 competent cells；The technology used is the technique for gene engineerings such as PCR, digestion with restriction enzyme, ligase connection and conversion；Cationic antibacterial peptide refers to the polypeptide with antibacterial activity with net positive charge；Used vector plasmid is the plasmid pYES2 α for carrying saccharomyces cerevisiae alpha signal peptide gene.The saccharomyces cerevisiae engineered yeast has merged expression antibacterial peptide and red fluorescent protein; allow saccharomyces cerevisiae engineered yeast that there is selective immune activation and regulatory function; there are good prevention and protective effect to septicemia, the different colours of wine can also be adjusted by red fluorescent protein.
Description
Technical field
The present invention relates to a kind of saccharomyces cerevisiae engineered yeasts, and in particular to a kind of amalgamation and expression antibacterial peptide and red fluorescent protein
Engineering bacteria construction method.
Background technology
Saccharomyces cerevisiae is a kind of unicellular microorganism, simple in structure, belongs to Mycophyta.Saccharomyces cerevisiae has individual greatly, egg
White matter content is high, and omnivorousness is strong, easily separated, easily cultivates, and metabolite is more, comprehensively utilizes the features such as wide, in brewing industry
Extensive research and application are arrived.Also known as Saccharomyces cerevisiae or budding yeast.Saccharomyces cerevisiae is most widely a kind of with human relation
Yeast, not only due to traditionally it is for making the food such as bread and steamed bun and wine brewing, in modern molecular and cell biology
As eukaryon model organism, the model organism Escherichia coli of protokaryon are functioned as.Saccharomyces cerevisiae is most common in fermentation
Biological species.The cell of saccharomyces cerevisiae is spherical or oval, 5-10 μm of diameter.Its method bred is gemmation.But
Existing saccharomyces cerevisiae is unable to amalgamation and expression antibacterial peptide and red fluorescent protein, cannot there is good prevention and protection to septicemia
Effect can not adjust the different colours of wine by red fluorescent protein.
Invention content
The purpose of the present invention is that provides a kind of amalgamation and expression antibacterial peptide and red fluorescent protein to solve the above-mentioned problems
Engineering bacteria construction method, to solve the above problems.
In order to solve the above technical problem, the present invention provides the following technical solutions：
The present invention provides the engineering bacteria construction methods of a kind of amalgamation and expression antibacterial peptide and red fluorescent protein, are given birth in genetic engineering
During producing cationic antibacterial peptide LfcinB, mApple fusion tau genes are connect with antibacterial peptide gene, structure recombination matter
Grain, electrotransformation saccharomyces cerevisiae W303 competent cells；The technology used be PCR, digestion with restriction enzyme, ligase connection and
The technique for gene engineerings such as conversion.
As a preferred technical solution of the present invention, cationic antibacterial peptide refers to net positive charge there is antibacterial to live
The polypeptide of property.
As a preferred technical solution of the present invention, used vector plasmid is to carry saccharomyces cerevisiae alpha signal peptide
The plasmid pYES2- α of gene.
As a preferred technical solution of the present invention, structure expression recombined cationic antibacterial peptide LfcinB saccharomyces cerevisiae bases
Because engineering bacteria is characterized in：
（1）Contain nucleotide sequence：
SEQ ID ：NO 1：
TTTAAATGTAGAAGATGGCAATGGAGAATGAAAAAATTGGGTGCTCCATCTATTACTTGTGTTAGAAGAGCTT
TT
SEQ ID ：NO2：
ATGGTTTCCAAAGGAGAAGAAAATAATATGGCAATCATTAAAGAATTTATGCGATTCAAAGTTCATATGGAAG
GCAGTGTTAACGGCCACGAGTTTGAAATCGAAGGCGAAGGGGAAGGAAGACCCTACGAAGCATTCCAGACAGCCAAG
CTGAAAGTTACAAAAGGCGGCCCGCTGCCTTTCGCCTGGGATATATTATCACCTCAATTTATGTACGGTTCAAAGGT
TTATATCAAACATCCTGCGGACATACCGGACTATTTCAAATTAAGTTTCCCAGAAGGTTTTCGCTGGGAGCGCGTGA
TGAATTTTGAAGACGGCGGTATCATTCACGTTAATCAGGACTCCTCGCTTCAAGATGGGGTGTTTATCTACAAGGTC
AAGCTGCGCGGCACTAACTTCCCCTCTGACGGTCCAGTCATGCAAAAAAAAACCATGGGCTGGGAAGCGTCTGAAGA
GCGGATGTACCCGGAAGACGGTGCACTGAAAAGTGAAATCAAAAAAAGATTAAAGTTAAAAGATGGCGGTCATTATG
CGGCCGAGGTGAAAACGACCTACAAAGCAAAGAAGCCAGTGCAACTACCTGGTGCTTACATTGTTGACATCAAGCTG
GACATCGTGAGTCACAATGAAGATTACACCATTGTTGAACAGTATGAACGAGCTGAAGGACGTCACTCCACCGGCGG
GATGGATGAACTATATAAATAA。
（2）The amino acid sequence of coding is respectively：
SEQ ID NO3：
FKCRRWQWRMKKLGAPSITCVRRAF；
SEQ ID NO4：
VSKGEENNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEAFQTAKLKVTKGGPLPFAWDILSPQFMYGS
KVYIKHPADIPDYFKLSFPEGFRWERVMNFEDGGIIHVNQDSSLQDGVFIYKVKLRGTNFPSDGPVMQKKTMGWEAS
EERMYPEDGALKSEIKKRLKLKDGGHYAAEVKTTYKAKKPVQLPGAYIVDIKLDIVSHNEDYTIVEQYERAEGRHST
GGMDELYK。
The advantageous effect that is reached of the present invention is：The device is a kind of work of amalgamation and expression antibacterial peptide and red fluorescent protein
Journey bacterium construction method, the saccharomyces cerevisiae engineered yeast have merged expression antibacterial peptide and red fluorescent protein, allow saccharomyces cerevisiae engineered yeast
With selective immune activation and regulatory function, there are good prevention and protective effect to septicemia, it can also be by red glimmering
Photoprotein adjusts the different colours of wine.
Specific implementation mode
Hereinafter, preferred embodiments of the present invention will be described, it should be understood that preferred embodiment described herein is only used
In the description and interpretation present invention, it is not intended to limit the present invention.
Embodiment 1
The present invention provides a kind of engineering bacteria construction method of amalgamation and expression antibacterial peptide and red fluorescent protein, is produced in genetic engineering
During cationic antibacterial peptide LfcinB, mApple fusion tau genes are connect with antibacterial peptide gene, construction recombination plasmid,
Electrotransformation saccharomyces cerevisiae W303 competent cells；The technology used is PCR, digestion with restriction enzyme, ligase connection and turns
The technique for gene engineerings such as change.
Cationic antibacterial peptide refers to the polypeptide with antibacterial activity with net positive charge.
Used vector plasmid is the plasmid pYES2- α for carrying saccharomyces cerevisiae alpha signal peptide gene.
Structure expression recombined cationic antibacterial peptide LfcinB Saccharomyces cerevisiae gene engineering bacterias are characterized in：
（1）Contain nucleotide sequence：
SEQ ID ：NO 1：
TTTAAATGTAGAAGATGGCAATGGAGAATGAAAAAATTGGGTGCTCCATCTATTACTTGTGTTAGAAGAGCTT
TT
SEQ ID ：NO2：
ATGGTTTCCAAAGGAGAAGAAAATAATATGGCAATCATTAAAGAATTTATGCGATTCAAAGTTCATATGGAAG
GCAGTGTTAACGGCCACGAGTTTGAAATCGAAGGCGAAGGGGAAGGAAGACCCTACGAAGCATTCCAGACAGCCAAG
CTGAAAGTTACAAAAGGCGGCCCGCTGCCTTTCGCCTGGGATATATTATCACCTCAATTTATGTACGGTTCAAAGGT
TTATATCAAACATCCTGCGGACATACCGGACTATTTCAAATTAAGTTTCCCAGAAGGTTTTCGCTGGGAGCGCGTGA
TGAATTTTGAAGACGGCGGTATCATTCACGTTAATCAGGACTCCTCGCTTCAAGATGGGGTGTTTATCTACAAGGTC
AAGCTGCGCGGCACTAACTTCCCCTCTGACGGTCCAGTCATGCAAAAAAAAACCATGGGCTGGGAAGCGTCTGAAGA
GCGGATGTACCCGGAAGACGGTGCACTGAAAAGTGAAATCAAAAAAAGATTAAAGTTAAAAGATGGCGGTCATTATG
CGGCCGAGGTGAAAACGACCTACAAAGCAAAGAAGCCAGTGCAACTACCTGGTGCTTACATTGTTGACATCAAGCTG
GACATCGTGAGTCACAATGAAGATTACACCATTGTTGAACAGTATGAACGAGCTGAAGGACGTCACTCCACCGGCGG
GATGGATGAACTATATAAATAA。
（2）The amino acid sequence of coding is respectively：
SEQ ID NO3：
FKCRRWQWRMKKLGAPSITCVRRAF；
SEQ ID NO4：
VSKGEENNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEAFQTAKLKVTKGGPLPFAWDILSPQFMYGS
KVYIKHPADIPDYFKLSFPEGFRWERVMNFEDGGIIHVNQDSSLQDGVFIYKVKLRGTNFPSDGPVMQKKTMGWEAS
EERMYPEDGALKSEIKKRLKLKDGGHYAAEVKTTYKAKKPVQLPGAYIVDIKLDIVSHNEDYTIVEQYERAEGRHST
GGMDELYK。
The invention is a kind of engineering bacteria construction method of amalgamation and expression antibacterial peptide and red fluorescent protein, is given birth in genetic engineering
During producing cationic antibacterial peptide LfcinB, mApple fusion tau genes are connect with antibacterial peptide gene, structure recombination matter
Grain, electrotransformation saccharomyces cerevisiae W303 competent cells；The technology used be PCR, digestion with restriction enzyme, ligase connection and
The technique for gene engineerings such as conversion；Cationic antibacterial peptide refers to the polypeptide with antibacterial activity with net positive charge；It is used
Vector plasmid is the plasmid pYES2- α for carrying saccharomyces cerevisiae alpha signal peptide gene.
The advantageous effect that is reached of the present invention is：The device is a kind of work of amalgamation and expression antibacterial peptide and red fluorescent protein
Journey bacterium construction method, the saccharomyces cerevisiae engineered yeast have merged expression antibacterial peptide and red fluorescent protein, allow saccharomyces cerevisiae engineered yeast
With selective immune activation and regulatory function, there are good prevention and protective effect to septicemia, it can also be by red glimmering
Photoprotein adjusts the different colours of wine.
Finally it should be noted that：The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention,
Although the present invention is described in detail referring to the foregoing embodiments, for those skilled in the art, still may be used
With technical scheme described in the above embodiments is modified or equivalent replacement of some of the technical features.
All within the spirits and principles of the present invention, any modification, equivalent replacement, improvement and so on should be included in the present invention's
Within protection domain.
Claims (4)
1. the engineering bacteria construction method of a kind of amalgamation and expression antibacterial peptide and red fluorescent protein, which is characterized in that in genetic engineering
During producing cationic antibacterial peptide LfcinB, mApple fusion tau genes are connect with antibacterial peptide gene, structure recombination matter
Grain, electrotransformation saccharomyces cerevisiae W303 competent cells；The technology used be PCR, digestion with restriction enzyme, ligase connection and
The technique for gene engineerings such as conversion.
2. the engineering bacteria construction method of a kind of amalgamation and expression antibacterial peptide and red fluorescent protein according to claim 1,
It is characterized in that, cationic antibacterial peptide refers to the polypeptide with antibacterial activity with net positive charge.
3. the engineering bacteria construction method of a kind of amalgamation and expression antibacterial peptide and red fluorescent protein according to claim 1,
It is characterized in that, used vector plasmid is the plasmid pYES2- α for carrying saccharomyces cerevisiae alpha signal peptide gene.
4. the engineering bacteria structure side of a kind of amalgamation and expression antibacterial peptide and red fluorescent protein according to claim 1,2 or 3
Method, which is characterized in that structure expression recombined cationic antibacterial peptide LfcinB Saccharomyces cerevisiae gene engineering bacterias are characterized in：
（1）Contain nucleotide sequence：
SEQ ID ：NO 1：
TTTAAATGTAGAAGATGGCAATGGAGAATGAAAAAATTGGGTGCTCCATCTATTACTTGTGTTAGAAGAGCTT
TT
SEQ ID ：NO2：
ATGGTTTCCAAAGGAGAAGAAAATAATATGGCAATCATTAAAGAATTTATGCGATTCAAAGTTCATATGGAAG
GCAGTGTTAACGGCCACGAGTTTGAAATCGAAGGCGAAGGGGAAGGAAGACCCTACGAAGCATTCCAGACAGCCAAG
CTGAAAGTTACAAAAGGCGGCCCGCTGCCTTTCGCCTGGGATATATTATCACCTCAATTTATGTACGGTTCAAAGGT
TTATATCAAACATCCTGCGGACATACCGGACTATTTCAAATTAAGTTTCCCAGAAGGTTTTCGCTGGGAGCGCGTGA
TGAATTTTGAAGACGGCGGTATCATTCACGTTAATCAGGACTCCTCGCTTCAAGATGGGGTGTTTATCTACAAGGTC
AAGCTGCGCGGCACTAACTTCCCCTCTGACGGTCCAGTCATGCAAAAAAAAACCATGGGCTGGGAAGCGTCTGAAGA
GCGGATGTACCCGGAAGACGGTGCACTGAAAAGTGAAATCAAAAAAAGATTAAAGTTAAAAGATGGCGGTCATTATG
CGGCCGAGGTGAAAACGACCTACAAAGCAAAGAAGCCAGTGCAACTACCTGGTGCTTACATTGTTGACATCAAGCTG
GACATCGTGAGTCACAATGAAGATTACACCATTGTTGAACAGTATGAACGAGCTGAAGGACGTCACTCCACCGGCGG
GATGGATGAACTATATAAATAA
（2）The amino acid sequence of coding is respectively：
SEQ ID NO3：
FKCRRWQWRMKKLGAPSITCVRRAF；
SEQ ID NO4：
VSKGEENNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEAFQTAKLKVTKGGPLPFAWDILSPQFMYGS
KVYIKHPADIPDYFKLSFPEGFRWERVMNFEDGGIIHVNQDSSLQDGVFIYKVKLRGTNFPSDGPVMQKKTMGWEAS
EERMYPEDGALKSEIKKRLKLKDGGHYAAEVKTTYKAKKPVQLPGAYIVDIKLDIVSHNEDYTIVEQYERAEGRHST
GGMDELYK。
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109734815A (en) * | 2019-02-22 | 2019-05-10 | 武汉巴菲尔生物技术服务有限公司 | A kind of enhanced fluorescin |
| CN111440827A (en) * | 2020-05-22 | 2020-07-24 | 苏州泓迅生物科技股份有限公司 | Information storage medium, information storage method and application |
| CN116375818A (en) * | 2023-02-28 | 2023-07-04 | 华南农业大学 | Construction and application of recombinant H5N8 subtype avian influenza virus carrying mApple fluorescent reporter gene |
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| CN102154260A (en) * | 2010-12-31 | 2011-08-17 | 湖北大学 | Novel method for efficiently cloning antibacterial peptide encoding gene |
| CN103421844A (en) * | 2013-06-14 | 2013-12-04 | 广州格拉姆生物科技有限公司 | Silkworm bioreactor for producing fodder antimicrobial peptides and construction method thereof |
| CN103555756A (en) * | 2013-10-29 | 2014-02-05 | 湖北希普生物科技有限公司 | Method for secretory expression of antibacterial peptide LfcinB by use of saccharomyces cerevisiae |
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2018
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Patent Citations (3)
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|---|---|---|---|---|
| CN102154260A (en) * | 2010-12-31 | 2011-08-17 | 湖北大学 | Novel method for efficiently cloning antibacterial peptide encoding gene |
| CN103421844A (en) * | 2013-06-14 | 2013-12-04 | 广州格拉姆生物科技有限公司 | Silkworm bioreactor for producing fodder antimicrobial peptides and construction method thereof |
| CN103555756A (en) * | 2013-10-29 | 2014-02-05 | 湖北希普生物科技有限公司 | Method for secretory expression of antibacterial peptide LfcinB by use of saccharomyces cerevisiae |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109734815A (en) * | 2019-02-22 | 2019-05-10 | 武汉巴菲尔生物技术服务有限公司 | A kind of enhanced fluorescin |
| CN111440827A (en) * | 2020-05-22 | 2020-07-24 | 苏州泓迅生物科技股份有限公司 | Information storage medium, information storage method and application |
| CN116375818A (en) * | 2023-02-28 | 2023-07-04 | 华南农业大学 | Construction and application of recombinant H5N8 subtype avian influenza virus carrying mApple fluorescent reporter gene |
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Application publication date: 20180911 |