CN107326025A - A kind of crocea interferon IFN1 promoters, nucleic acid construct, cell and its production and use - Google Patents
A kind of crocea interferon IFN1 promoters, nucleic acid construct, cell and its production and use Download PDFInfo
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- C07K14/461—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from fish
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- A—HUMAN NECESSITIES
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Abstract
The invention discloses a kind of crocea interferon IFN1 promoters, nucleic acid construct, carrier, cell and its production and use.The nucleotide sequence of the crocea interferon IFN1 promoters such as SEQ ID NO：Shown in 1, or it is and SEQ ID NO：1 complementary nucleotide sequence.The present invention also protects described promoter or nucleic acid construct or carrier or recombinant cell or the purposes of fish or mammal in regulation and control destination gene expression or genetically engineered fish containing promoter or nucleic acid construct or carrier or recombinant cell.
Description
Technical field
The present invention relates to gene engineering technology field, more particularly to a kind of crocea interferon IFN1 promoters, nucleic acid
Construct, cell and its production and use.
Background technology
Interferon (interferon, IFN) is the important cell factor of a class, can pass through acceptor corresponding on cell membrane
With reference to active cell signal transduction, with various biological function, such as activation antiviral gene participates in body disease-resistant poison and is immunized, adjusts
Save (Samuel CE.Antiviral actions of interferons [J] .Clin such as immune response, antitumor
Microbiol Rev,2001；14:778–809).Research in mammal is according to gene order, functional characteristic and acceptor class
IFN is divided into three classes by the difference of type：I type interferon, is mainly made up of IFN-α and IFN-β, and II types interferon is IFN-γ, III
Type interferon is IFN- λ (Pestka S, Krause CD, Walter MR.Interferons, interferon-like
cytokines,and their receptors[J].Immunol Rev,2004；202:8–32).Wherein, I types interferon has
There is the function of significant suppressing virus replication, and can induce the expression of a variety of antiviral genes, including protein kinase R (Protein
Kinase R, PKR), 2 ', 5 '-oligoadenylate synthetase (2 ', 5 '-Oligoadenylate synthetase, OAS) and Mx eggs
In vain (Mx protein), played a significant role in the antiviral immunity reaction of body.In view of I types interferon is exempted from host is disease-resistant
Importance in epidemic disease reaction, studies its transcription regulation mechanism and has for the immune response and intracellular signal transduction pathway for disclosing body
It is significant.People's IFN-β promoter luciferase reporting genic system has been developed in correlative study in mammal, research
The function of target gene and its associated signal paths (Sabbah A, Chang TH, the Harnack R, et of mediation
al.Activation of innate immune antiviral responses by Nod2[J].Nat Immunol,
2009；10:1073–80).
At present, I types interferon is cloned in a variety of bony fishes, including zebra fish (Danio rerio), spot
Cha Wei Channel-catfish (Ictalurus punctatus), Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss)
Deng.From unlike the naming rule of interferon in mammal, bony fish I type interferon is the position in its genome
Put, named according to Arabic numerals order (Robertsen B, Bergan V, T,et al.Atlantic
salmon interferon genes:cloning,sequence analysis,expression,and biological
activity[J].J Interferon Cytokine Res,2003；23:601–12).In addition, in research in zebra fish
The promoter sequence of I type interferon has been cloned, luciferase reporter plasmid has been constructed, and be used successfully to immune correlation
Research (Sun F, Zhang YB, Liu TK, the et al.Fish MITA serves as a mediator for of gene function
distinct fish IFN gene activation dependent on IRF3or IRF7[J].J Immunol,2011；
187:2531–9)。
Large yellow croaker is the important marine fish of China, and the research to its anti pathologic immunity basis can not only enrich fish
Immunologic understanding, and can be laid the foundation for control and prevention of disease with disease-resistant molecular modules breeding, thus obtain extensive concern.It is right at present
The expression and regulation mechanism of fish interferon IFN1 genes, especially about the intracellular signal transduction pathway involved by interferon IFN1
The correlative study of regulated and control network is still deficienter.
The content of the invention
It is an object of the invention to provide a kind of crocea interferon IFN1 promoters.
To achieve the above object, the present invention provides a kind of crocea interferon IFN1 promoters, it is characterised in that its nucleosides
Acid sequence such as SEQ ID NO：Shown in 1, or it is and SEQ ID NO：1 complementary nucleotide sequence.
The present invention provides a kind of nucleic acid construct, comprising described IFN1 promoters, is operatively connected with IFN1 promoters
Gene order, wherein the IFN1 promoters are identical with the gene order source or difference.
The present invention provides a kind of carrier, it is characterised in that：The carrier contains described IFN1 promoters or described core
Acid con-struct.
Further, the carrier is described IFN1 promoters or described nucleic acid construct and carrier for expression of eukaryon through weight
The recombinant vector that group is obtained；It is preferred that, carrier for expression of eukaryon is pGL4.17 plasmids.
The present invention provides a kind of recombinant cell, it is characterised in that：The cell contains described IFN1 promoters or described
Nucleic acid construct or described carrier, the recombinant cell be fish cell or mammalian cell；It is preferred that, fish cell
For EPC cells.
The present invention provides one group of primer pair, and the primer pair, which is used to expand, obtains described IFN1 promoters, and its feature exists
In：Two primers of the primer pair are SEQ ID NO：4 and SEQ ID NO：Sequence shown in 5；It is preferred that, the primer pair
Two primers in SEQ ID NO：4 and SEQ ID NO：5 ' ends of sequence shown in 5 are also respectively connected with restrictive restriction enzyme site
And/or protection base；It is preferred that, two primers of the primer pair are respectively SEQ ID NO：2 and SEQ ID NO：Shown in 3
Sequence.
The present invention provides a kind of method for preparing described IFN1 promoters, including：Using large yellow croaker genomic DNA as mould
Plate, is expanded using pair for amplification primer, and the amplimer is according to SEQ ID NO：1 in large yellow croaker genomic DNA
Sequence is designed for head and the tail respectively.
Further, the amplimer is described primer pair.
The present invention provides a kind of method of efficiently expressing exogenous gene in regulation and control fish or mammal, methods described bag
Include, described promoter or described nucleic acid construct or described carrier or described recombinant cell are imported into fish or lactation
Animal；It is preferred that, the fish are large yellow croaker.
The present invention also provides described promoter, described nucleic acid construct or described carrier or described restructuring
Cell or fish or the food in one's mouth containing the promoter or described nucleic acid construct or described carrier or described recombinant cell
Purposes of the newborn animal in regulation and control destination gene expression or genetically engineered fish；It is preferred that, the fish are large yellow croaker.
Gene order (Li Chan, Yao for the crocea interferon IFN1 that present invention applicant has obtained according to early-stage Study
The feature of emerald green mythical bird like the phoenix large yellow croakers (Larimichthys crocea) I type interferon genes and expression analysis Oceanologia et Limnologia Sinicas,
2013；2:499-506) primer is designed based on and has cloned a kind of crocea interferon IFN1 gene promoter region sequences, is built
The luciferase reporter gene system of the promoter sequence, and confirm that the large yellow croaker disturbs by luciferase assay experiment
Plain IFN1 gene promoters have stronger promoter activity.The clone of crocea interferon IFN1 gene promoters and its glimmering
The successful structure of light element enzyme Reporter System, in theory by for the expression regulation machine of research crocea interferon IFN1 genes
System and cell signalling regulated and control network provide good experimental system, are to be carried using the promoter construction expression in application aspect
Body efficiently expressing exogenous gene, the cultivation of genetically engineered fish and the screening of immunopotentiator or disease-resistant drug create condition, tool
There is important theoretical and practical significance.
A kind of crocea interferon IFN1 gene promoter sequences of the present invention are：
TTCCCTCATCTGGCATCAAGGAATAAAAATTTGTTATTTTTTAAAACTACAATGTTATATATTATTAGAAGTTTGAT
TTACAACACATTTGTTATGTTTGTGAAAGAGATATAAAACATATTAATTTTAATTAAGTATGGAGCTGTGATTTTTC
TTATTACAGTCTGAAGATGTCACTGTGACGTAACGTAGGCTGCATTCCCTTTCAGTTTCACTTCGGGGAAACACTTT
CTACTTACTACGTGTTTGTGATGAGGACACACTGAAGTTATATCCACGGTTTGTAGTTGCATAATAATCATGAACAA
TTACGTTATTAAAACACACAGTAGCAATGATGGCAATCCAAACAAAGAGAGTCATCGTTTGTTGACACAGTTTTATG
GCATCGATCTGCTGCGGACTAAATACAATGAATAAAACTGAATAAATAAATAAAGTTTCTTGCGTGAAATCTCCAAA
ATACAAAACAAGTAGCATAAAATCACAGATCAAATAATTCAATGTCTAAATAATTAAATTAAACTCTCAATCAGCGA
CACTGATGTAATTCCAGAAAAAACTTTACTTTGAAAATCATAAAAGAAAGCGTCTTTTATATTTAAATGTGACATCA
TCTTATTCTAGGATAAATATATATTATGGGGACAGTAATTCAAATCCGAAATGTGTGGCCTGGGTTTGAACCGGGTA
AGCCACAGTCCACGTCGCGATTCCCACAGGTGGATGTGAGGAAAATGAAATAGGTGCGTCCTGCTACAGTATAAATG
AGCCGCTGCAGGTGAGTTTGAACACAACACCTGGACACATCCGACTTTTGCCACTCAAAGACACTTTGTCTGTTTGT
AAAGATGCTCAGCAGGATCTTGTTT SEQ ID NO:1。
One kind of the present invention detects crocea interferon IFN1 promoters using dual-luciferase reporter system
The analysis method of activity, comprises the following steps：
1) large yellow croaker IFN1 promoter region sequences are amplified from large yellow croaker genome using PCR method, by the PCR of amplification
Product is connected to TaKaRa companies pMD19-T carriers and sequencing identification；
2) large yellow croaker IFN1 gene promoter region sequences are inserted into Promega companies luciferase reporter gene carrier
In pGL4.17, firefly luciferase gene is set to be located under the control of the promoter, structure obtains recombinant vector pGL4-
IFN1-pro and sequencing identification；
3) promoter activity is analyzed using dual-luciferase reporter system：By the pGL4-IFN1- Jing Guo sequencing identification
Pro recombinant vectors and renilla luciferase control Reporter gene vector pRL-TK cotransfection EPC cell lines, transfection are received after 48 hours
Collect cell, and firefly luciferase and sea pansy are detected by the dual-luciferase reporter system of Promega companies respectively
The enzymatic activity value of luciferase, the ratio for calculating the two draws the relative luciferase activity of transfectional cell；Meanwhile, with cotransfection
The relative luciferase activity of pGL4.17 empty carriers and pRL-TK carrier EPC cells is control, calculates large yellow croaker IFN1 startups
The relative activity of son；
4) using LPS the and poly I of various concentrations:C stimulates pGL4-IFN1-pro recombinant vectors and pRL-TK common respectively
The EPC cells of transfection, collect cell after stimulating 24 hours, the relative activity of IFN1 promoters are detected and calculate, by comparing thorn
Swash the change of front and rear large yellow croaker IFN1 promoter relative activities, determine large yellow croaker IFN1 gene promoters in LPS and poly I:
Activity change under C immunostimulations.
5) by the antiviral signal protein MAVS Eukaryotic expression recombinant vectors p3xFLAG-MAVS and pGL4- of large yellow croaker mitochondria
The EPC cells of IFN1-pro, pRL-TK cotransfection, with cotransfection empty carrier p3xFLAG-CMV-14, pGL4-IFN1-pro,
PRL-TK EPC cells are control, and transfection collects cell after 48 hours, detects and calculate the relative activity of IFN1 promoters, leads to
Cross and compare the big of cotransfection MAVS Eukaryotic expression recombinant vectors p3xFLAG-MAVS and cotransfection empty carrier p3xFALG-CMV-14
The change of yellow croaker IFN1 promoter relative activities, determines whether large yellow croaker IFN1 gene promoter activities can be by large yellow croaker mitochondrias
Antiviral signal protein MAVS is induced.
The application of crocea interferon IFN1 gene promoters of the present invention is as follows：
1) clone of the crocea interferon IFN1 gene promoters described in and promoter activity checking, for research large yellow croaker
The expression regulation and cell signalling regulated and control network of interferon IFN1 genes provide good experimental system；
2) crocea interferon IFN1 gene promoters described in can be used for building carrier for expression of eukaryon in fish cell or
The cultivation of efficiently expressing exogenous gene and genetically engineered fish in mammalian cell；
3) the crocea interferon IFN1 gene promoters described in can be used for the screening of immunopotentiator or disease-resistant drug.
Brief description of the drawings
Fig. 1 is the building process schematic diagram of pGL4-IFN1-pro recombinant vectors.
Fig. 2 is that the ordinary optical microscope observation of pGL4-IFN1-pro recombinant vectors and pRL-TK cotransfection EPC cells is schemed
(100 times).
Fig. 3 is the work that crocea interferon IFN1 gene promoters are analyzed using luciferase reporter gene detecting system
Property figure.
Fig. 4 is to analyze crocea interferon IFN1 gene promoters not using luciferase reporter gene detecting system
With concentration poly I:Activity under C and LPS is stimulated is schemed.
Fig. 5 is using the antiviral signal protein MAVS of luciferase reporter gene detecting system analysis large yellow croaker mitochondria
To the inducing action figure of crocea interferon IFN1 gene promoters.
Embodiment
Embodiments of the invention are described below in detail, the example of the embodiment is shown in the drawings, wherein from beginning to end
Same or similar label represents same or similar element or the element with same or like function.Below with reference to attached
The embodiment of figure description is exemplary, it is intended to for explaining the present invention, and be not considered as limiting the invention.Embodiment
In unreceipted particular technique or condition person, according to the technology or condition described by document in the art or according to the description of product
Book is carried out.Agents useful for same or the unreceipted production firm person of instrument, being can be by the conventional products of acquisition purchased in market.
EPC cells, can purchase and preserve center in Chinese Typical Representative culture, also can purchase in other commercial undertakings.
Embodiment 1：The clone of crocea interferon IFN1 gene promoters
Carried out based on the following Fig. 1 process step.
Its large yellow croaker (by large yellow croaker on the market according to normal Extraction Methods of Genome, is extracted with large yellow croaker genome
Genomic DNA) it is template, primer is designed, primer sequence is as follows；
Forward primer：-3′SEQ ID NO:2；Wherein lower stroke straight line
For KpnI restriction enzyme sites, lower stroke of wave is SEQ ID NO:4；
Reverse primer：-3′SEQ ID NO:3；Wherein lower stroke straight
Line is Bgl II restriction enzyme sites, and lower stroke of wave is SEQ ID NO:5.
Using TaKaRa ExTaq enzymes, PCR amplification crocea interferon IFN1 gene promoters, PCR reaction systems are as follows：
PCR reaction conditions are as follows：
PCR primer is detected through 1% agarose gel electrophoresis, PCR primer is carried out using Omega companies glue reclaim kit
Reclaim；
PCR primer is connected to pMD19-T cloning vectors, Escherichia coli Top10 competent cells, positive gram of screening is converted
Grand and send Sangon Biotech's sequencing identification, the correct clone of sequencing is referred to as pMD19-T-IFN1-
Pro recombinant vectors.
Embodiment 2：Crocea interferon IFN1 gene promoter recombinant vectors pGL4-IFN1-pro structure
1) the Top10 bacterial strains of above-mentioned pMD19-T-IFN1-pro recombinant vectors are expanded and cultivated, extract plasmid, and this is heavy
Group plasmid uses Kpn I/Bgl II double digestions, through agarose gel electrophoresis, the IFN1promoter after gel extraction digestion；
2) pGL4.17 luciferase reporter vectors (promega companies) are used into Kpn I/Bgl II double digestion blend compounds
QIAquick Gel Extraction Kit reclaims the carrier after digestion；
3) IFN1 promoter sequences fragment and carrier through double digestion are connected using the T4DNA ligases of TaKaRa companies
PGL4.17, builds recombinant vector pGL4-IFN1-pro, and linked system is as follows：
Reaction condition：16 DEG C of overnight incubations；
4) above-mentioned connection product is converted into Escherichia coli Top10 competent cells, screening positive clone simultaneously send raw work biological
The sequencing identification of engineering (Shanghai) limited company；
5) it will be trained by the Escherichia coli Top10 cell expansions containing recombinant vector pGL4-IFN1-pro of sequencing identification
Support, extract plasmid, detection plasmid concentration and purity are standby.
Embodiment 3：Luciferase reporter gene detecting system analyzes the work of crocea interferon IFN1 gene promoters
Property
1. EPC cells are inoculated with 24 porocyte culture plates, per hole 2 × 105Individual cell, plus 500 μ l MEM culture mediums, in 25
DEG C biochemical cultivation case overnight incubation；
2. the Lipofectamine of Invitrogen companies is used per hole EPC cellsTM3000 transfection reagent cotransfections
Recombinant vector pGL4-IFN1-pro and 10ng renilla luciferase control the Reporter gene vector pRL- of the gained of 100ng embodiments 2
TK；Meanwhile, compare Reporter gene vector pRL-TK's with cotransfection 100ng empty carrier pGL4.17 and 10ng renilla luciferase
EPC cells are control；Each processing sets 3 repetitions.Cell transfecting is comprised the following steps that：
A) by carrier (100ng pGL4-IFN1-pro+10ng pRL-TK or 100ng pGL4.17+ to be transfected
10ng pRL-TK) mixed respectively with 25 μ l Opti-MEM culture mediums (being purchased from GIBCO)；
B) 0.75 μ l Lipofectamine are takenTM3000 liposomes add 25 μ l Opti-MEM culture mediums and mixed；
C) the above-mentioned carrier to be transfected diluted by Opti-MEM culture mediums and liposome are mixed, is incubated at room temperature 5 points
Clock；
D) 50 μ l mixtures of above-mentioned mixing are carefully added into the culture hole of 24 porocyte culture plates, jiggled thin
Born of the same parents' plate is mixed, and is placed in 25 DEG C of biochemical cultivation cases and is cultivated.
It is thin that pGL4-IFN1-pro recombinant vectors compare Reporter gene vector pRL-TK cotransfections EPC with renilla luciferase
Born of the same parents' result is shown in Fig. 2, it is observed that cell growth state is good.
3. transfection collects transfectional cell after 48 hours, firefly is read respectively with luciferase reporter gene detecting system
The enzymatic activity value of luciferase and renilla luciferase, fluorescence in transfectional cell is drawn by the ratio for calculating the two enzymatic activity value
The relative activity of plain enzyme；Meanwhile, with empty carrier pGL4.17 and renilla luciferase control Reporter gene vector pRL-TK cotransfections
EPC cells in luciferase relative activity as control, as a result see Fig. 3.Fig. 3 is using luciferase reporter gene inspection
The activity figure of examining system analysis crocea interferon IFN1 gene promoters.PGL4.17 represents that empty carrier pGL4.17 is transfected in figure
The relative luciferase activity (as compareing) of cell；IFN1-pro is glimmering for recombinant vector pGL4-IFN1-pro transfectional cells
Light element enzyme relative activity；* significant difference P is represented<0.05.It can be seen that recombinant vector pGL4-IFN1-pro is transfected
The relative luciferase activity of cell compared with the control, with significant difference；That is, the weight containing IFN1 promoter genes
Group carrier is compared with the empty carrier without IFN1 promoter genes, with the obvious effect for starting luciferase.
Luciferase activity determination method refers to Promega companies luciferase reporter gene detecting system specification
Carry out, concretely comprise the following steps：
A) transfection EPC cells washed once after 48 hours with phosphate buffer；
B) per hole add 100 μ l PLB lysates (offer of Passive Lysis Buffer, Promega kit) and
Cell lysis 15 minutes, rock culture plate at room temperature, make its cracking complete, of short duration that cell lysate supernatant is collected by centrifugation；
C) by 100 μ l Luciferase Assay Reagents II (LAR II) and the 20 above-mentioned cell lysate supernatants of μ l in detection pipe
Mixing, firefly luciferase activity is detected with chemiluminescence detector (Promega GloMax 20/20) immediately；
D) 100 μ l Stop＆Glo reagents are added into detection pipe, above-mentioned reaction is quenched, while starting sea pansy fluorescein
Enzyme reaction, detection renilla luciferase activity；
E) the enzymatic activity value of firefly luciferase and renilla luciferase is read respectively, calculates the ratio of the two enzymatic activity value
It is worth the relative activity of luciferase in transfectional cell, meanwhile, compareed with transfecting empty carrier pGL4.17 and renilla luciferase
The relative activity of luciferase is used as control in the EPC cells of Reporter gene vector pRL-TK cotransfections.
4. tested for immunostimulation, in above-mentioned recombinant vector pGL4-IFN1-pro and renilla luciferase control report
Genophore pRL-TK cotransfection EPC cells add final concentration of 100ng/ml poly in the medium after 24 hours, respectively
I:C、1000ng/ml poly I:C and 100ng/ml LPS, 1000ng/ml LPS continue to cultivate 24 hours, collect cell, use
Above-mentioned luciferase reporter gene detecting system is calculated in various concentrations LPS and poly I respectively:Transfectional cell under C incentive conditions
In relative luciferase activity, and be compared with relative luciferase activity in the transfectional cell without stimulation, experiment knot
Fruit is the average value of 3 independent experiment results, as a result sees Fig. 4.Fig. 4 is using the analysis of luciferase reporter gene detecting system
Crocea interferon IFN1 gene promoters are in various concentrations poly I:Activity under C and LPS stimulations.Empty carrier is represented in figure
PGL4.17 transfectional cells (control) and recombinant vector pGL4-IFN1-pro transfectional cells are respectively in non-stimulated or 100ng/ml
poly I:C、1000ng/ml poly I:Luciferase under C, 100ng/ml LPS, 1000ng/ml LPS are stimulated is lived relatively
Property；* significant difference P is represented<0.05.From the result in Fig. 4 it can be found that the poly I of various concentrations:C is stimulated can not be obvious
Change the relative activity of luciferase in transfectional cell, and 1000ng/ml LPS stimulate luciferase in lower transfectional cell relative
Activity is significantly reduced, and illustrates that large yellow croaker IFN1 promoter activities can be suppressed by LPS immunostimulations.
For detect other molecules whether have activation large yellow croaker IFN1 promoter activities experiment, can by build other
The Eukaryotic expression recombinant vector of molecule and above-mentioned gained recombinant vector pGL4-IFN1-pro and pRL-TK cotransfection EPC cells, inspection
Whether survey the expression of other molecules can activate large yellow croaker IFN1 promoters.With the antiviral signal egg of large yellow croaker mitochondria in this experiment
Detected exemplified by white MAVS recombinant vector p3xFLAG-MAVS, wherein recombinant vector p3xFLAG-MAVS construction step is such as
Under：
(large yellow croaker on the market is extracted into total serum IgE with large yellow croaker cDNA according to the TRIZOL methods of standard, takes 1 μ g total serum IgEs to make
It is cDNA with the cDNA synthetic agent box reverse transcriptions of ThermoFisher companies) it is template, primer is designed, primer sequence is as follows；
Forward primer：-3′SEQ ID NO:6；Its
In lower stroke of straight line be Hind III digestions site, lower stroke of wave is SEQ ID NO:8；
Reverse primer：-3′SEQ ID NO:7；Wherein lower stroke
Straight line is BamH I restriction enzyme sites, and lower stroke of wave is SEQ ID NO:9.
1) TaKaRa ExTaq enzymes, the antiviral signal protein MAVS of PCR amplification large yellow croaker mitochondrias Complete Open are used
Reading frame, PCR reaction systems are as follows：
PCR reaction conditions are as follows：
PCR primer is detected through 1% agarose gel electrophoresis, PCR primer is carried out using Omega companies glue reclaim kit
Reclaim；
2) above-mentioned gained PCR primer is used into Hind III/BamH I double digestions, p3xFLAG-CMV-14 carriers is used
Hind III/BamH I double digestions, and respectively through agarose gel electrophoresis, PCR primer and carrier after gel extraction digestion；
3) using MAVS entire open reading frame fragment and load of the connection of T4DNA ligases through double digestion of TaKaRa companies
Body p3xFLAG-CMV-14, builds recombinant vector p3xFLAG-MAVS, and linked system is as follows：
Reaction condition：16 DEG C of overnight incubations；
4) above-mentioned connection product is converted into Escherichia coli Top10 competent cells, screening positive clone simultaneously send raw work biological
The sequencing identification of engineering (Shanghai) limited company；
5) by the Escherichia coli Top10 cell expansions containing recombinant vector p3xFLAG-MAVS of above-mentioned process sequencing identification
Culture, extracts plasmid, and detection plasmid concentration and purity are standby.
By the above-mentioned antiviral signal protein MAVS recombinant vectors p3xFLAG-MAVS of the large yellow croaker mitochondria built
100ng compares Reporter gene vector with above-mentioned gained recombinant vector pGL4-IFN1-pro and the 10ng renilla luciferases of 100ng
PRL-TK cotransfection EPC cell lines, while with cotransfection empty carrier p3xFLAG-CMV-14 100ng and 100ng recombinant vectors
PGL4-IFN1-pro and 10ng pRL-TK EPC cells are control, 48 hours after transfection, collect cell, use above-mentioned fluorescein
Relative luciferase activity in enzyme reporter gene detecting system detection cotransfection p3xFLAG-MAVS recombinant plasmid cells, and with
Relative luciferase activity in transfection empty carrier p3xFLAG-CMV-14 cells is compared, and experimental result is independent real 3 times
The average value of result is tested, Fig. 5 is as a result seen.Fig. 5 is using luciferase reporter gene detecting system analysis large yellow croaker mitochondria
Inducing action figures of the antiviral signal protein MAVS to crocea interferon IFN1 gene promoters.Wherein p3xFLAG-CMV-14
Represent empty carrier p3xFLAG-CMV-14 and the relative luciferase activity after recombinant vector pGL4-IFN1-pro cotransfection cells
(control)；FLAG-MAVS is represented after recombinant vector p3xFLAG-MAVS and recombinant vector pGL4-IFN1-pro cotransfection cells
Relative luciferase activity；* significant difference P is represented<0.05.From the result in Fig. 5 it can be found that p3xFLAG-MAVS with
Relative luciferase activity in pGL4-IFN1-pro cotransfection cells be significantly higher than p3xFLAG-CMV-14 empty carriers with
Relative luciferase activity in pGL4-IFN1-pro cotransfection cells, illustrates that large yellow croaker MAVS can induce IFN1 promoters
Activation, while proving that the pGL4-IFN1-pro recombinant plasmids built can be used for gene functional research and signal path to analyze.
Although embodiments of the invention have been shown and described above, it is to be understood that above-described embodiment is example
Property, it is impossible to limitation of the present invention is interpreted as, one of ordinary skill in the art is not departing from the principle and objective of the present invention
In the case of above-described embodiment can be changed within the scope of the invention, change, replace and modification.
SEQUENCE LISTING
<110>Collects The American University
Xiamen University Tan Kah Kee College
<120>A kind of crocea interferon IFN1 promoters, nucleic acid construct, cell and its production and use
<130> JMDX-17014-CNI
<160> 9
<170> PatentIn version 3.5
<210> 1
<211> 872
<212> DNA
<213>Large yellow croaker
<400> 1
ttccctcatc tggcatcaag gaataaaaat ttgttatttt ttaaaactac aatgttatat 60
attattagaa gtttgattta caacacattt gttatgtttg tgaaagagat ataaaacata 120
ttaattttaa ttaagtatgg agctgtgatt tttcttatta cagtctgaag atgtcactgt 180
gacgtaacgt aggctgcatt ccctttcagt ttcacttcgg ggaaacactt tctacttact 240
acgtgtttgt gatgaggaca cactgaagtt atatccacgg tttgtagttg cataataatc 300
atgaacaatt acgttattaa aacacacagt agcaatgatg gcaatccaaa caaagagagt 360
catcgtttgt tgacacagtt ttatggcatc gatctgctgc ggactaaata caatgaataa 420
aactgaataa ataaataaag tttcttgcgt gaaatctcca aaatacaaaa caagtagcat 480
aaaatcacag atcaaataat tcaatgtcta aataattaaa ttaaactctc aatcagcgac 540
actgatgtaa ttccagaaaa aactttactt tgaaaatcat aaaagaaagc gtcttttata 600
tttaaatgtg acatcatctt attctaggat aaatatatat tatggggaca gtaattcaaa 660
tccgaaatgt gtggcctggg tttgaaccgg gtaagccaca gtccacgtcg cgattcccac 720
aggtggatgt gaggaaaatg aaataggtgc gtcctgctac agtataaatg agccgctgca 780
ggtgagtttg aacacaacac ctggacacat ccgacttttg ccactcaaag acactttgtc 840
tgtttgtaaa gatgctcagc aggatcttgt tt 872
<210> 2
<211> 29
<212> DNA
<213>It is artificial synthesized
<400> 2
cggggtacct tccctcatct ggcatcaag 29
<210> 3
<211> 31
<212> DNA
<213>It is artificial synthesized
<400> 3
ggaagatcta aacaagatcc tgctgagcat c 31
<210> 4
<211> 21
<212> DNA
<213>It is artificial synthesized
<400> 4
cttccctcat ctggcatcaa g 21
<210> 5
<211> 22
<212> DNA
<213>It is artificial synthesized
<400> 5
aaacaagatc ctgctgagca tc 22
<210> 6
<211> 32
<212> DNA
<213>It is artificial synthesized
<400> 6
cccaagctta tggcttcgtt tgccagagac ag 32
<210> 7
<211> 26
<212> DNA
<213>It is artificial synthesized
<400> 7
cgcggatccg ttcttaaact tccacg 26
<210> 8
<211> 23
<212> DNA
<213>It is artificial synthesized
<400> 8
atggcttcgt ttgccagaga cag 23
<210> 9
<211> 17
<212> DNA
<213>It is artificial synthesized
<400> 9
gttcttaaac ttccacg 17
Claims (10)
1. a kind of crocea interferon IFN1 promoters, it is characterised in that its nucleotide sequence such as SEQ ID NO：Shown in 1, or
Person be and SEQ ID NO：1 complementary nucleotide sequence.
2. a kind of nucleic acid construct, comprising the IFN1 promoters described in claim 1, the base being operatively connected with IFN1 promoters
Because of sequence, wherein the IFN1 promoters are identical with the gene order source or difference.
3. a kind of carrier, it is characterised in that：The carrier contains the IFN1 promoters or claim 2 institute described in claim 1
The nucleic acid construct stated.
4. the carrier of claim 3, it is characterised in that：The carrier is that IFN1 promoters or right described in claim 1 will
Ask the nucleic acid construct described in 2 with carrier for expression of eukaryon through recombinating obtained recombinant vector；It is preferred that, carrier for expression of eukaryon is
PGL4.17 plasmids.
5. a kind of recombinant cell, it is characterised in that：The cell contains IFN1 promoters or the claim described in claim 1
The carrier described in nucleic acid construct or claim 3 or 4 described in 2, the recombinant cell is that fish cell or mammal are thin
Born of the same parents；It is preferred that, fish cell is EPC cells.
6. one group of primer pair, the primer pair is used to expand the IFN1 promoters obtained described in claim 1, it is characterised in that：
Two primers of the primer pair are SEQ ID NO：4 and SEQ ID NO：Sequence shown in 5；It is preferred that, the primer pair
Two primers are in SEQ ID NO：4 and SEQ ID NO：Sequence shown in 55 ' end be also respectively connected with restrictive restriction enzyme site and/
Or protection base；It is preferred that, two primers of the primer pair are respectively SEQ ID NO：2 and SEQ ID NO：Sequence shown in 3
Row.
7. one kind prepares SEQ ID NO：The method of IFN1 promoters described in 1, including
Using large yellow croaker genomic DNA as template, expanded using pair for amplification primer, the amplimer is according to SEQ ID
NO：1 sequence in large yellow croaker genomic DNA is designed for head and the tail respectively.
8. the method for claim 7, it is characterised in that the amplimer is any described primer pairs of claim 6-7.
9. a kind of method of efficiently expressing exogenous gene in regulation and control fish or mammal, methods described includes, by claim 1
Carrier described in nucleic acid construct or claim 3 or 4 or claim 5 institute described in described promoter or claim 2
The recombinant cell stated imports fish or mammal；It is preferred that, the fish are large yellow croaker.
10. described in the nucleic acid construct described in promoter or claim 2 or claim 3 or 4 described in claim 1
Carrier or claim 5 described in recombinant cell or containing described in the promoter or claim 2 described in claim 1
The fish or mammal of the recombinant cell described in carrier or claim 5 described in nucleic acid construct or claim 3 or 4 exist
Regulate and control the purposes in destination gene expression or genetically engineered fish；It is preferred that, the fish are large yellow croaker.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108774632A (en) * | 2018-06-20 | 2018-11-09 | 中国海洋大学 | A kind of lefteye flounder fish beta-actin promoter of optimization |
| CN110527683A (en) * | 2019-07-19 | 2019-12-03 | 集美大学 | A kind of Epinephelus coioides IRF10 gene promoter and preparation method |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106754923A (en) * | 2016-12-15 | 2017-05-31 | 国家海洋局第三海洋研究所 | Crocea interferon d gene promoter sequences and its application |
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2017
- 2017-06-16 CN CN201710456729.3A patent/CN107326025A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106754923A (en) * | 2016-12-15 | 2017-05-31 | 国家海洋局第三海洋研究所 | Crocea interferon d gene promoter sequences and its application |
Non-Patent Citations (1)
| Title |
|---|
| 李婵等: "大黄鱼（Larimichthys crocea）I型干扰素基因的特征与表达分析", 《海洋与湖沼》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108774632A (en) * | 2018-06-20 | 2018-11-09 | 中国海洋大学 | A kind of lefteye flounder fish beta-actin promoter of optimization |
| CN110527683A (en) * | 2019-07-19 | 2019-12-03 | 集美大学 | A kind of Epinephelus coioides IRF10 gene promoter and preparation method |
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