CN106754921A - Mammalian cell expression promoter and production and preparation method thereof - Google Patents
Mammalian cell expression promoter and production and preparation method thereof Download PDFInfo
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- CN106754921A CN106754921A CN201611139166.7A CN201611139166A CN106754921A CN 106754921 A CN106754921 A CN 106754921A CN 201611139166 A CN201611139166 A CN 201611139166A CN 106754921 A CN106754921 A CN 106754921A
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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
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- 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
- C12N15/63—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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- 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
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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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
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
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- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/101—Plasmid DNA for bacteria
Abstract
The invention discloses a kind of mammalian cell expression promoter and production and preparation method thereof, the method is comprised the following steps：Step one, the synthetic primer by way of chemical synthesis；Step 2, is reacted by PCR, obtains promoter sequence；Step 3, by restriction enzyme site：BglII and Kpn1, above-mentioned promoter sequence insertion includes ori, card and receives in resistant gene, sv40, sv40 polyA, the plasmid backbone of many restriction enzyme site sequences, obtains pHep/L1 plasmids；Step 4, being transfected into E.Coli Escherichia coli carries out plasmid amplification, extracts plasmid, is sequenced；Step 5, SEAP and IL10 genes of interest is inserted by the new plasmid for obtaining, and pure plasmid is extracted by CsCL density-gradient centrifugation methods.The present invention improves the expression efficiency of existing plasmid and AAV viruses.
Description
Technical field
The present invention relates to a kind of mammalian cell expression promoter, more particularly to a kind of mammalian cell expression is opened
Mover and production and preparation method thereof.
Background technology
Mammalian promoter sequence has important application in plasmid and viral vectors, is widely used in life science, doctor
The gene expression aspect in etc. field.Controlled in protein expression, plasmid construction, virus formulation, destination gene expression and transmission, gene
The fields such as treatment have important application.The expression vector for commonly using at present includes what plasmid vector and viral vectors were used
Promoter sequence is generally viral promoter, such as CMV, and expression quantity of these carriers in mammalian cell is higher, but
Belong to transient expression, expression time is shorter.For these problems, scientist develops the promoter of some mammalian sources,
Such as human serum albumins Human Serum Albumin promoter sequences, the animal derived promoter sequence of these eucaryons is bright
It is aobvious to extend expression time of the foreign vector in cell and animal body, but they also have two shortcomings, first, expression quantity it is bright
It is aobvious lower than viral promoter carrier, second, the promoter sequence of this class directly replicated from mammalian cell, one
As it is long, applying in some viral vectors can be subject to limitation, and such as AAV carriers can only at most accommodate the external source of 4.7KB
DNA fragmentation, long promoter sequence limits the length of genes of interest and reduces the application range of AAV carriers.For disease
The limitation of poison and mammalian promoter, we utilize bioinformatics means, by substantial amounts of calculating simulation, cell and dynamic
Thing is screened, and obtains a promoter sequence that expression quantity is high, expression time is long.The sequence can be as the startup of plasmid vector
Son, is applied to expression of the foreign gene in mammalian cell, adult animals and human body.
The content of the invention
The technical problems to be solved by the invention are to provide a kind of mammalian cell expression promoter and its manufacture and make
With method, it obtains one can be long-acting in mammalian cell, a large amount expression alien gene and comprising less base number
Purpose promoter sequence, its length is 226bp.This promoter can provide a high-quality for the gene therapy based on plasmid vector
Promoter, greatly improve the expression efficiency of existing plasmid.
The present invention is to solve above-mentioned technical problem by following technical proposals：A kind of mammalian cell expression starts
Son, it is characterised in that it uses enough long-acting in mammalian cell, a large amount expression alien gene and comprising less base number
Purpose nucleic acid sequence.
The present invention also provides a kind of method of manufacture and use thereof of mammalian cell expression promoter, it is characterised in that institute
The method of manufacture and use thereof for stating long-acting mammalian cell expression nucleic acid sequence of promoter is comprised the following steps：
Step one, synthetic primer one by way of chemical synthesis：5’-ACGGGGTACCTCACTCTTGGCACGGGGAAT-3’；
Primer two：5’-GGAAGATCTCATTGACGTCAATCAAAATGTCGTAACAACTCC-3’ ；
Step 2, is reacted by PCR, obtains mammalian cell expression promoter sequence；
Step 3, by restriction enzyme site：BglII and Kpn1, above-mentioned mammalian cell expression promoter sequence insertion
Include ori, card to receive in resistant gene, sv40, sv40 polyA, the plasmid backbone of many restriction enzyme site sequences, obtain plasmid；
Step 4, being transfected into E.Coli Escherichia coli carries out plasmid amplification, extracts plasmid, is sequenced；
Step 5, SEAP and IL10 genes of interest is inserted by the new plasmid for obtaining, and extracts pure by CsCL density-gradient centrifugation methods
Net plasmid；
Step 6, during 10ug plasmids are incorporated into physiological saline, by surging, gene delivery mode is imported in the liver of kunming mice；
Step 7, at the 12nd hour, 1 day, 2 days, 3 days, 5 days, 7 days, 14 days, 21 days, 28 days, 42 days, 56 days, 90 days, passes through
Mouse tail vein takes blood, extracts blood plasma；
Step 8, by Alkaline Phosphatase Kit, the SEAP contents in detection blood.
Preferably, the mammalian cell expression promoter sequence is suitable for the plasmid of all mammalian cell expressions
And viral vectors.
Preferably, the mammalian cell expression promoter behaviour source sequence.
Preferably, the mammalian cell expression promoter is short Binding site for transcription factor.
Positive effect of the invention is：One, this promoter (pHep/L1) is suitable for mammalian cell expression
Plasmid and viral vectors, for the gene therapy in exogenous gene expression, human body in Ex vivo cell transfection, animal body have weight
Apply；Two, the promoter (pHep/L1) is people's source sequence, is suitable for the gene therapy for human diseases；Three, the startup
Sub (pHep/L1) is short Binding site for transcription factor, comprising less base quantity, advantageous as the startup of viral vectors
Subsequence；Four, the promoter has tissue specificity higher, is suitable for the target gene expression of liver；Five, the promoter energy
Effectively extend expression time and the expression quantity of foreign gene.
Brief description of the drawings
Fig. 1 represents the expression curve of the mouse IL10 genes in Mice Body of new promoter sequence and CMV promoter guiding
Figure.
Fig. 2 represents the expression curve map of the SEAP genes in Mice Body of new promoter sequence and CMV promoter guiding.
Fig. 3 is that promoter sequence insertion includes ori, card and receives resistant gene, sv40, sv40 polyA, many restriction enzyme sites
The schematic diagram of the plasmid backbone of sequence.
Fig. 4 be the present invention relates to nucleotide sequence figure.
Specific embodiment
Present pre-ferred embodiments are given below in conjunction with the accompanying drawings, to describe technical scheme in detail.
Mammalian cell expression promoter of the present invention, it is characterised in that its use it is enough it is long-acting in mammalian cell,
A large amount expression alien gene and comprising the nucleotide sequence of less base number, the promoter sequence being related to is as follows：
cattgacgtcaat caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt
gtacggtggg aggtctatat aagcagagct cgtttagtga accgtcagat cgcctggaga cgccatccac
gctgttttga cctccataga agacaccggg accgatccag cctccgcggc cgggaacggt gcattggaac
gcggattccc cgtgccaagagtga（As shown in Figure 4）.
The method of manufacture and use thereof of mammalian cell expression promoter of the present invention is comprised the following steps：
Step one, synthetic primer one by way of chemical synthesis：5’-ACGGGGTACCTCACTCTTGGCACGGGGAAT-3’；
Primer two：5’-GGAAGATCTCATTGACGTCAATCAAAATGTCGTAACAACTCC-3’ ；
Step 2, by PCR（Polymerase chain reaction, Polymerase Chain Reaction）Reaction, obtains promoter sequence
Row；
Step 3, by restriction enzyme site：BglII and Kpn1, above-mentioned promoter sequence insertion includes ori, Ka Na and resists
Property gene, sv40, sv40 polyA, the plasmid backbone of many restriction enzyme site sequences（Fig. 3）In, obtain pHep/L1 plasmids；
Step 4, being transfected into E.Coli Escherichia coli carries out plasmid amplification, extracts plasmid, is sequenced.
Step 5, SEAP and IL10 genes of interest is inserted by the new plasmid for obtaining, and is carried by CsCL density-gradient centrifugation methods
Take pure plasmid；
Step 6, during 10ug plasmids are incorporated into physiological saline, by surging, gene delivery mode imports Kunming（KM）The liver of mouse
In dirty；
Step 7, at the 12nd hour, 1 day, 2 days, 3 days, 5 days, 7 days, 14 days, 21 days, 28 days, 42 days, 56 days, 90 days, passes through
Mouse tail vein takes blood, extracts blood plasma；
Step 8, by Alkaline Phosphatase Kit, the SEAP contents in detection blood.
The transcription factor that present invention search can be expressed in humans and animals liver, and screen the special of these transcription factors
Property binding site, sets up Binding site for transcription factor storehouse.The side that these Binding site for transcription factor sequences are passed through into chemical synthesis
Formula synthesizes, and insertion includes ori, card and receives in resistant gene, sv40, sv40 polyA, the carrier framework of many restriction enzyme site sequences,
It is built into new expression vector.Then the genes of interest such as SEAP, IL10 are inserted many restriction enzyme sites of the carrier, by the base that surges
Because the mode transmitted imports Kunming（KM）In Mice Body, blood is taken in different time points, then detected using commercial kit
The foreign genes such as SEAP, IL10 concentration in blood, determines its expression quantity and expression time.
Fig. 1 represents that the expression of the mouse IL10 genes in Mice Body of new promoter sequence and CMV promoter guiding is bent
Line.The expression time of new promoter extends 2 months than CMV promoter, and long-time expression quantity improves 1000 times.
Fig. 2 represents the expression curve of the SEAP genes in Mice Body of new promoter sequence and CMV promoter guiding.Newly open
The expression time of mover extends 2 months than CMV promoter, and long-time expression quantity improves 1000 times.
Particular embodiments described above, technical problem, technical scheme and beneficial effect to solution of the invention are carried out
Further describe, should be understood that and the foregoing is only specific embodiment of the invention, be not limited to
The present invention, all any modification, equivalent substitution and improvements within the spirit and principles in the present invention, done etc., should be included in this
Within the protection domain of invention.
Claims (5)
1. a kind of mammalian cell expression promoter, it is characterised in that it uses enough long-acting in mammalian cell, a large amounts
Expression alien gene and comprising the nucleotide sequence of less base number.
2. a kind of method of manufacture and use thereof of mammalian cell expression promoter, it is characterised in that the long-acting mammal
The method of manufacture and use thereof of cell expression nucleic acid sequence of promoter is comprised the following steps：
Step one, synthetic primer one by way of chemical synthesis：5’-ACGGGGTACCTCACTCTTGGCACGGGGAAT-3’；
Primer two：5’-GGAAGATCTCATTGACGTCAATCAAAATGTCGTAACAACTCC-3’ ；
Step 2, is reacted by PCR, obtains mammalian cell expression promoter sequence；
Step 3, by restriction enzyme site：BglII and Kpn1, above-mentioned mammalian cell expression promoter sequence insertion
Include ori, card to receive in resistant gene, sv40, sv40 polyA, the plasmid backbone of many restriction enzyme site sequences, obtain plasmid；
Step 4, being transfected into E.Coli Escherichia coli carries out plasmid amplification, extracts plasmid, is sequenced；
Step 5, SEAP and IL10 genes of interest is inserted by the new plasmid for obtaining, and extracts pure by CsCL density-gradient centrifugation methods
Net plasmid；
Step 6, during 10ug plasmids are incorporated into physiological saline, by surging, gene delivery mode is imported in the liver of kunming mice；
Step 7, at the 12nd hour, 1 day, 2 days, 3 days, 5 days, 7 days, 14 days, 21 days, 28 days, 42 days, 56 days, 90 days, passes through
Mouse tail vein takes blood, extracts blood plasma；
Step 8, by Alkaline Phosphatase Kit, the SEAP contents in detection blood.
3. the method for manufacture and use thereof of mammalian cell expression promoter as claimed in claim 2, it is characterised in that described
Mammalian cell expression promoter sequence is suitable for the plasmid and viral vectors of all mammalian cell expressions.
4. the method for manufacture and use thereof of mammalian cell expression promoter as claimed in claim 2, it is characterised in that described
Mammalian cell expression promoter behaviour source sequence.
5. the method for manufacture and use thereof of mammalian cell expression promoter as claimed in claim 2, it is characterised in that described
Mammalian cell expression promoter is short Binding site for transcription factor.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1335890A (en) * | 1998-12-31 | 2002-02-13 | 瓦尔欧姆德有限公司 | High efficiency mammalian gene expression vectors that contain exogenous promoter and entire 5' untranslated region in the upstream from start codon for inherent gene as transcription regulatory site |
| US20080112971A1 (en) * | 2003-11-24 | 2008-05-15 | Sonja Leyrer | Promoters for Expression in Modified Vaccinia Virus Ankara |
-
2016
- 2016-12-12 CN CN201611139166.7A patent/CN106754921A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1335890A (en) * | 1998-12-31 | 2002-02-13 | 瓦尔欧姆德有限公司 | High efficiency mammalian gene expression vectors that contain exogenous promoter and entire 5' untranslated region in the upstream from start codon for inherent gene as transcription regulatory site |
| US20080112971A1 (en) * | 2003-11-24 | 2008-05-15 | Sonja Leyrer | Promoters for Expression in Modified Vaccinia Virus Ankara |
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Effective date of registration: 20170524 Address after: 211299, Qinhuai Road, Lishui District, Jiangsu, Nanjing, No. 288 Applicant after: Nanjing seeking Biotechnology Co., Ltd. Address before: 211200, room 9, building five, 1003 garden, Gulou District, Jiangsu, Nanjing Applicant before: Sun Hao |
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Application publication date: 20170531 |