CN107190021A - A kind of cell line of expression EDAR genes and the preparation and application of receptor stimulating agent - Google Patents

Abstract

The present invention provides the cell line and its activator of a kind of stable outer M-band A acceptors (EDAR) of expression recombination human source, discloses the preparation method and application of the activator.The transcriptional activity that the present invention adds NF κ B after activator with detection HEK293 EDAR His cell lines assesses agonist effect, establish it is a kind of can quickly, the method for high frequency zone EDAR activators, with good application prospect.

Description

A kind of cell line of expression EDAR genes and the preparation and application of receptor stimulating agent

Technical field

The present invention relates to the preparation of the structure of human archeocyte system and receptor stimulating agent, and in particular to the stable expression restructuring of one kind The cell line and two of M-band A acceptors (ectodysplasin-A receptor, EDA receptor or EDAR) outside people source Plant EDAR activator.The invention also discloses prepare the method for the cell line, design and prepare activator method and its Utilize cell line selection EDAR activators, the application verified in its effect.

Background technology

Congenital metodontiasis (ectodermal dysplasia, ED) is a kind of heredity disease of tooth development defect Disease, generally permanent teeth fails to develop and formed in tooth bud is formed.It is divided into nonsyndromic agomphosis (non-syndromic Hypodontia, NSH, No. OMIM for 313500) and syndrome and type agomphosis (syndromic hypodontia, SH, No. OMIM is 305100), the former shows as simple agomphosis, and the latter's agomphosis is simultaneously few etc. with oligotrichosis, sweat gland.Congenital metodontiasis meeting The chewing, pronunciation, face that have a strong impact on patient are attractive in appearance, physical and mental development and mental health.

The mutation of outer M-band A (ectodysplasin-A, EDA) gene is to cause nonsyndromic agomphosis and syndrome The deciding factor of type agomphosis, EDA genes (NM_001399) are located at long-armed upper (the Xq 12-13.1) of X chromosome, gene size For 425Kb, different configurations are produced by splicing, the EDA-A1 albumen of most long 391 amino acid of codified belongs to recessive with X Heredity.The bit base of EDA genes the 1061st sports C by T and is mutated c.1061T>C can cause comprehensive agomphosis XLHED.

Outer M-band A acceptor genes (ectodysplasin-A receptor, EDAR) are typically to carry TNF domains Receptor family member.EDAR genes (NM_022336.3) are located at No. 2 chromosomes (2q13), and gene size is 123Kb, there is 13 Individual extron, different configurations are produced by splicing, the EDAR albumen of 448 amino acid of codified and 480 amino acid XEDAR albumen.EDAR albumen includes Death Domain by a signal peptide, three cysteine rich domains, membrane-spanning domain, one Intracellular domain constitute, be a kind of transmembrane protein.Research shows that EDA albumen is by after furin (furin) digestion Extracellular, TNF domains formation tripolymer is secreted into, with the one-to-one combinations of transmembrane protein EDAR, EDAR signaling molecules are activated, and Activate downstream coherent signal such as NF κ B (nuclear transcription factor κ B) path：EDA and EDAR combination can To activate NEMO (NF κ B Essential Modulator)/IKK (NF κ Bkinase) compound, cause I κ B ubiquitinations, make NF κ B enter nucleus, possess transcriptional activity, so that the transcription of activation target gene, and then adjust ectodermic development.EDAR quilts The higher i.e. EDAR activity of degree of activation is higher, then NF κ B transcriptional activity is higher.Therefore, NF κ B transcriptional activity, that is, reflect EDAR activity.

The content of the invention

According to the first aspect of the invention, there is provided a kind of eukaryon recombinant plasmid of people source EDAR genes, the EDAR Gene open reading frame sequence is connected with His sequence labels, and is inserted into the polyclone enzyme of eukaryotic expression vector pcDNA3.1 (+) At enzyme site, obtained recombinant plasmid pcDNA3.1 (+)-EDAR-His；The EDAR gene opens reading frame sequence is SEQ ID No.1。

Preferably, described EDAR genes are obtained by cDNA amplifications.

It is used for stable expression people source EDAR albumen (its amino acid sequence there is provided one kind according to another aspect of the present invention Row are such as SEQ ID No.2) cell line, its deposit number is：CCTCC NO:C201778, Classification And Nomenclature is：Human embryo kidney is thin Born of the same parents HEK293-EDAR-His No.14, preservation date is on May 17th, 2017, and depositary institution's code is：CCTCC, depositary institution Address is：Wuhan University.

A kind of preparation method of stable expression people source EDAR albuminous cells system is provided according to another aspect of the present invention, Characterized in that, this method comprises the following steps：

(1) after people source EDAR gene opens reading frame sequence is connected with His sequence labels, it is inserted into carrier for expression of eukaryon Polyclone enzyme enzyme site at, build the recombinant plasmid containing EDAR genes；

(2) Transfected Recombinant Plasmid for obtaining step (1) enters in eukaryotic, and is screened by drug resistance, obtains Positive cell clone with antibiotic resistance；

(3) the positive cell clone amplification cultivation obtained in step (2) is obtained stablizing expressed fusion protein EDAR- His cell line.

Preferably, the expression vector in the step (1) is pcDNA3.1 (+)；

Preferably, the transfection method in the step (2) is electroporation transfection method, and the eukaryotic is that human embryo kidney is thin Born of the same parents HEK293, the medicine is G418；

According to another aspect of the present invention there is provided a kind of EDAR activator, the activator is EDAR albumen Part is EDA full-length proteins；The nucleotide sequence of the activator such as SEQ ID No.5；Amino acid sequence such as SEQ ID No.6； Expression plasmid is pECFP-C1-EDA recombinant plasmids.

According to another aspect of the present invention there is provided the preparation method of described activator, comprise the following steps：

(1) people source wild type EDA gene open reading frame sequences are inserted into many grams of carrier for expression of eukaryon pECFP-C1 Long Meiqieweidianchu, builds the recombinant plasmid pECFP-C1-EDA containing EDA genes；

(2) Transfected Recombinant Plasmid obtained in step (1) is entered in HEK293-EDAR-His cell lines to express, obtains institute State activator A1.

(3) activity of obtained activator is detected, with cotransfection pECFP-C1-EDA (activator A1) and pECFP-C1- The HEK293-EDAR-His cell lines of EDA-T1061C (saltant type sample) plasmid are experimental group；Individually to transfect pECFP- The HEK293-EDAR-His cell lines of C1-EDA (wild type sample) plasmid are positive control；Individually to transfect pECFP-C1- The HEK293-EDAR-His cell lines of EDA-T1061C (saltant type sample) plasmid are negative control.

As a result show, relative to negative control, be transfected into the activator expressed in HEK293-EDAR-His recombinant cell lines A1 can improve NF κ B transcriptional activity.

According to another aspect of the present invention there is provided second of EDAR activator, the activator is EDA PROTEIN Cs TNF domains are held, EDA albumen is the part of EDAR albumen；The corresponding nucleotide sequence such as SEQ ID No.9 of the activator；Ammonia Base acid sequence such as SEQ ID No.10；Expression vector is pET-28a (+)-TNF plasmids.

According to another aspect of the present invention there is provided the application of second of EDAR activator, comprise the following steps：

(1) nucleotide sequence of people source wild type EDA gene TNF code areas is inserted into prokaryotic expression carrier pET-28a (+) Polyclone enzyme enzyme site at, build recombinant plasmid pET-28a (+)-TNF containing EDA gene TNF code areas；

(2) Transfected Recombinant Plasmid in step (1) is entered in bacterium, induced expression is simultaneously purified, obtains described activator A2。

(3) EDA mutators carrier is transfected into HEK293-EDAR-His cells, while adding step (2) in the medium The activator A2 of middle gained, detection EDAR is activated degree.

(4) activity of second of activator is detected, using electroporation method by recombinant plasmid pECFP-C1-EDA-T1061C (saltant type sample) is transfected into HEK293-EDAR-His cell lines, while adding purifying in the culture medium of this cell line Activator A2 albumen, final concentration of 1 μ g/mL；Individually to transfect pECFP-C1-EDA-T1061C (saltant type sample) plasmid HEK293-EDAR-His cell lines are negative control, individually to transfect the HEK293- of pECFP-C1-EDA (wild type sample) EDAR-His cell lines are positive control.

As a result show, relative to negative control, be added in culture medium, purifying prokaryotic expression protein i.e. second and swash Dynamic agent A2 can improve NF κ B transcriptional activity.

The invention has the advantages that：

(1) present invention sets up cell line using electroporation transfection method, and EDAR expression is stable, is still kept very after multiple passage Good protein expression level；

(2) in recombinant plasmid pcDNA3.1 (+)-EDAR-His that the present invention is built, except encoding EDAR eggs comprising insertion Outside the sequence of white gene, also containing histidine sequence label, it is easy to the detection in later stage；

(3) present invention assesses EDAR activation levels with detection HEK293-EDAR-His cell line NF κ B transcriptional activity, Methods described easily standardizes, reproducible；

(4) present invention is assessed with the transcriptional activity of NF κ B after detection HEK293-EDAR-His cell lines addition activator Agonist effect, establish it is a kind of can quickly, the method for high frequency zone EDAR activators, with good application prospect.

(5) present invention demonstrates two kinds of EDAR activators, and easily prepares, extracts, and has a good application prospect, can portion The influence that the EDA of the recovery mutation divided is caused.

Cell line preparation method of the present invention is simple, with low cost；The cell line of the present invention is by detecting EDAR-His table The effect of EDAR activators is assessed up to level, with low cost, accuracy is high, being capable of high flux, accurately low cost, screening EDAR activators, with good stability and reliability, provide new thinking for EDAR activator screening, have well Market prospects；Two kinds of EDAR activators A1 and A2 of checking are convenient to be extracted, prepares, and has good application value.

Brief description of the drawings

Fig. 1 is stable expression EDAR-His cell line clone Western blot testing results；

Fig. 2 is wild type EDA Transfected Recombinant Plasmid HEK293 cells i.e. activator A1 albumen Western blot detection knots Really；

Fig. 3 is SDS-PAGE testing results after FPLC purifies and separates activator A2 albumen；

Fig. 4 is Western blot testing results after FPLC purifies and separates activator A2 albumen；

Fig. 5 is the result for the transcriptional activity that detection NF κ B are tested with luciferase reporter gene.

Embodiment

In order to make the purpose , technical scheme and advantage of the present invention be clearer, it is right below in conjunction with drawings and Examples The present invention is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, and It is not used in the restriction present invention.As long as in addition, technical characteristic involved in each embodiment of invention described below Not constituting conflict each other can just be mutually combined.

Embodiment 1

The structure of the recombinant cell lines of stable expression EDAR-His albumen and detection

1. encode the gene order design of EDAR albumen and prepare

The gene order design of 1.1 EDAR albumen

With people source EDAR gene opens reading frame sequence (SEQ ID No.1) for template, restructuring is reached designed for coding schedule The specific primer EDAR-F (SEQ ID No.3) and EDAR-R (SEQ ID No.4) of EDAR albumen (SEQ ID No.2). EDAR-F adds Hind III restriction enzyme sites and protectiveness base before initiation codon.EDAR-R adds in EDAR coding ends Have terminator, the restriction enzyme sites of Xba I and a protectiveness base, and add before terminator 6 × His of coding base sequence, with The EDAR PROTEIN Cs end of eukaryotic expression adds 6 × His labels.

It is prepared by the nucleic acid fragment of 1.2 coding EDAR recombinant proteins

CDNA using people source EDAR adds primer EDAR-F and EDAR-R and carries out gene magnification as template.Amplification system is shown in Table 1：

Table 1

2X Taq Mix	20μL
		dNTP	2μL
Primer EDAR-F(10μM)	2μL
		Primer EDAR-R(10μM)	2μL
Template (0.1-1 μ g)	cDNA
		Moisturizing is extremely	40μL

Amplification condition：For 95 DEG C of pre-degeneration 5min, then 35 circulations are carried out, cycling condition is 95 DEG C of denaturation 30s, and 60 DEG C are moved back Fiery 30s, 72 DEG C of extension 60s；Last 72 DEG C of extensions 5min.

2. the structure of recombinant plasmid

Will eukaryotic expression vector pcDNA3.1 (+) with being reclaimed after the digestions of Hind III and Xba I, then with through Hind III Connected with the EDAR-His gene amplification products reclaimed after the digestions of Xba I under T4 connection enzyme effects, conversion DH5 α competence is thin Picking single bacterium colony amplification cultivation is coated on after the LB flat boards of the benzyl containing ammonia, incubated overnight after born of the same parents and plasmid is extracted, with Hind III enzymes Cut and deliver to sequencing company sequencing identification, the positive plasmid of identification is recombinant plasmid needed for the present embodiment, and upgrading grain is preserved simultaneously Strain is preserved, pcDNA3.1 (+)-EDAR-His is named as after sequence verification.

3. recombinate EDAR-His plasmid-transfected cells

To build stable expression EDAR-His eukaryotic cell lines, using electroporation method by recombinant plasmid pcDNA3.1 (+)-EDAR-His is transfected into HEK293 cells, comprises the following steps that shown：

The cells of HEK 293 of passage are taken, culture medium is removed, is washed twice with 5mL PBS solutions, with 1mL's 0.25% Trypsase (Trypsin) vitellophag 5min, adds the washing of 5mL culture mediums with terminating reaction, and repeatedly pressure-vaccum, 1000rpm Centrifuge 5min；Cell is resuspended with 5mL PBS solutions again, 1000rpm centrifugation 5min add appropriate electricity and turn buffer solution suspension cell. Prepare electricity and turn solution, system is as shown in table 2 below,

Table 2

Solution	Volume
		pcDNA3.1(+)-EDAR-His(1λ)	8μL
5X electricity turns buffer solution	40μL
		MgSO4	8μL
Ultra-pure water is added to Total	200μL

Take the cells of 100 μ L HEK 293 to add to 200 μ L electricity respectively to turn in solution, gently mix, be stored at room temperature 10min.Point 300 μ L mixed liquors electric turn trough is not added into, 1000 μ F, 260V electricity turns.Cell is shifted into corresponding 10mL culture mediums, cell is mixed Gently add afterwards in culture dish, be uniformly distributed it, be transferred to CO₂Cultivated in incubator.

4. the screening of positive colony

After transfectional cell culture about 48h, addition G418 (Geneticin, Geneticin) makes its end in cell culture medium Concentration is the fresh culture culture cell that new, the final concentration of 600 μ g/mL of G418 are changed to after 600 μ g/mL, 48h.Every 2 days The fresh culture culture of the G418 containing 600 μ g/mL 2 days is changed, is repeated 6 times.Digestion, collection cell, will be thin with limiting dilution assay Born of the same parents point add the G418 medium cultures containing 600 μ g/mL, culture, wait 10 days or so in 96 orifice plates.In the orifice plate of picking 96 By individual cells propagation Lai a cell into 1 24 orifice plate, after it grows to suitable quantity, digestion, collect cell, pass In generation, is into 12 orifice plates.This process is repeated, cell is cultivated successively in 6 orifice plates, 60mm culture dishes, 100mm culture dishes, and conservation.

5. the identification of positive colony

Clone cell is cultivated 2 days in culture dish, cell is collected and cracks, identified with Western blot, with Obtain the expression of EDAR-His in clone cell.Utilize His specific antibodies, it has been found that select 5, in No. 6 clones There is EDAR-His expression, and expression quantity is variant, we select No. 6 higher cell clones of EDAR-His expression quantity to continue to expand Preserved after big culture, the cell line is named as HEK293-EDAR-His, carry out subsequent experimental.Developing result is as shown in Figure 1.

Embodiment 2

EDAR activators A1 preparation

Using wild type EDA eukaryotic expressions albumen as EDAR activator A1, its preparation process is as described below：

1. encode the gene order design of EDA albumen and prepare

The gene order design of 1.1 EDA albumen

The primer for expanding EDA gene opens reading frame sequence (SEQ ID No.5) is respectively EDA-F (SEQ ID No.7) With EDA-R (SEQ ID No.8).EDA-F nucleotides sequence is listed in before initiation codon plus BamH I restriction enzyme sites and protectiveness Base, EDA-R nucleotides sequence is listed in EDA coding ends added with terminator and the restriction enzyme sites of Xba I and protectiveness base.EDA Protein amino acid sequence is SEQ ID No.6.

It is prepared by the nucleic acid fragment of 1.2 coding EDA wild type recombinant proteins

Using people source EDA gene cDNAs template, add primer EDA-F and EDA-R and carry out gene magnification.Amplification system such as table 3：

Table 3

2X Taq Mix	20μL
		dNTP	2μL
Primer EDA-F(10μM)	2μL
		Primer EDA-R(10μM)	2μL
CDNA templates (0.1-1 μ g)	cDNA
		Moisturizing is extremely	40μL

Amplification condition：For 95 DEG C of pre-degeneration 5min, then 35 circulations are carried out, cycling condition is 95 DEG C of denaturation 30s, and 58 DEG C are moved back Fiery 30s, 72 DEG C of extension 60s；Last 72 DEG C of extensions 5min.

2. recombinate the structure of EDA plasmids

The structure of 2.1 restructuring wild type EDA plasmids

By carrier for expression of eukaryon pECFP-C1 with after the digestion of BamH I and Xba I reclaim, then with through BamH I and Xba The amplification EDA gene outcomes reclaimed after I digestion are connected under T4 connection enzyme effects, are coated on and are contained after conversion DH5 α competent cells Block after that LB flat boards, incubated overnight picking single bacterium colony amplification cultivation and extract plasmid, with BamH I digestions and to deliver to sequencing public Sequencing identification is taken charge of, the positive plasmid of identification is recombinant plasmid needed for the present embodiment, and upgrading grain preserves and preserves strain, ordered Entitled pECFP-C1-EDA.

3. recombinate the detection of wild type EDA plasmid-transfected cells and verify

Normally it can be expressed for detection wild type EDA recombinant plasmids in eukaryotic cell lines, we utilize electroporation method Recombinant plasmid pECFP-C1-EDA is transfected into HEK293 cells, while it is negative control to transfect pECFP-C1 carriers, not turned Contaminate any plasmid for blank control, and detected with Western blot methods.

As a result show, the EDA wild types restructuring being transfected into HEK293 cells can be detected with EDA specific antibodies Plasmid can normal expression EDA recombinant proteins be A1.Developing result is as shown in Figure 2.I.e. by pECFP-C1-EDA Transfected Recombinant Plasmids Enter in HEK293-EDAR-His eukaryotics and express, can obtain activator A1 albumen.

Embodiment 3

EDAR activators A2 preparation

Using EDA albumen TNF domains prokaryotic expression protein as activator A2, its preparation process is as described below：

1. encode the gene order design of activator A2 albumen and prepare

The gene order design of 1.1 activator A2 albumen

The primer for expanding EDA gene TNF code area (SEQ ID No.9) nucleotide sequence is respectively TNF-F (SEQ ID No.11) and TNF-R (SEQ ID No.12), it is EDA albumen 233-391 amino acids to encode obtained amino acid sequence. TNF-F nucleotides sequence is listed in before initiation codon plus BamH I restriction enzyme sites and protectiveness base.TNF-R nucleotide sequence In EDA coding ends added with terminator and Hind III digestions site and protectiveness base.The amino acid sequence bag finally given The His sequences and TEV restriction enzyme sites on carrier are included, sequence is SEQ ID No.10.

It is prepared by the gene order of 1.2 coding EDA albumen

Using pECFP-C1-EDA plasmids as template, add primer TNF-F and TNF-R and carry out gene magnification.Amplification system is such as Table 4：

Table 4

The condition of amplification is：95 DEG C of pre-degeneration 5min, then 25 circulations are carried out, cycling condition is 95 DEG C and is denatured 30s, 52 DEG C Anneal 30s, 72 DEG C of extension 45s；Last 72 DEG C of extensions 5min.

2. the structure of activator A2 expression plasmids

Prokaryotic expression carrier pET-28a (+) is reclaimed with after BamH I and Hind III digestions, then with through BamH I and The amplification activator A2 gene outcomes reclaimed after Hind III digestions are connected under T4 connection enzyme effects, and conversion DH5 α competence is thin It is coated on after born of the same parents containing that LB flat boards of card, picking single bacterium colony amplification cultivation and plasmid is extracted after incubated overnight, with BamH I digestions And sequencing company sequencing identification is delivered to, the positive plasmid of identification is recombinant plasmid needed for the present embodiment, and upgrading grain is preserved and protected Strain is deposited, pET-28a (+)-TNF is named as.

3. prokaryotic expression and the purifying of activator A2 albumen

The prokaryotic expression of 3.1 activator A2 albumen

Recombinant plasmid pET-28a (+)-TNF is transformed into Escherichia coli Rosseta competent cells, dual anti-(card is applied to That mycin 50 μ g/mL, the μ g/mL of chloramphenicol 25) on solid LB media, 37 DEG C of bacteriological incubator overnight incubations, picking whole bacterium Fall to be inoculated in the Double LB liquid mediums of 3L, in 37 DEG C of shaking tables 220rpm shaken cultivations to OD 600 be 1.0 when, add eventually Concentration is 1mM isopropylthiogalactosides (IPTG), and bacterium solution then is placed on into 220rpm in 16 DEG C of shaking tables continues to vibrate training Support 13h.

3.2 affinity chromatographies isolate and purify activator A2 albumen

3.2.1 bacterial sediment is collected：Bacterium solution is transferred in centrifuge tube, 4 DEG C, 4000 × g centrifugation 15min are gone waste and old Culture medium.

3.2.2 pressure breaking：It is heavy with lysate (300mM NaCl, 20mM Tris pH8.0,10% glycerine) suspension thalline Form sediment, final volume is 50mL, add cocktail protease inhibitors, after 500 μ L PMSF (final concentration 1mM) are mixed, add 4 DEG C of high pressure homogenizer (ATS companies AH-1500 types), 900Kpa crushes 2min or so, collects cell pyrolysis liquid, 4 DEG C, 40000 × g centrifuges 40min, takes supernatant to abandon precipitation.

3.2.3 affinity chromatography：The good 1mL nickel column packing (GE companies) of pre-balance is added in the supernatant collected one step up In chromatographic column, the lysate for adding 10mL washes away foreign protein.The lysate containing 20mM imidazoles for adding 10mL is further washed away It is attached to the foreign protein in chromatographic column.

3.2.4 elute activator A2：Add 3mL elution buffers (300mM imidazoles, 300mM NaCl, 20mM Tris pH 8.0th, 10% glycerine), activator A2 eluents are collected, albumen are then surveyed in 280nm absorption values OD280.

3.3 FPLC separate fusion protein

3.3.1 the concentration of protein sample：Take 15mL ultra-filtration centrifuge tubes (Millipore companies Ultra15-MWCO 50kD), activator A2 protein eluates, 4 DEG C, 3000g centrifugations are then first added with the first rinse milipore filter of elution buffer 4min, the protein eluate in even centrifuge tube is blown with pipettor, continues 4 DEG C, 3000g centrifugation 4min, until by whole activators A2 protein eluates are concentrated into 1mL.

3.3.2FPLC protein isolate：By the sample concentrated through 0.45 μm of filtering with microporous membrane, through gel permeation chromatography point From the activator A2 albumen purified.Gel permeation chromatography post is the 10/300GL of Superdex 200 (GE companies), buffer solution For 150mM NaCl, 20mM Tris pH 8.0, flow velocity is 0.5mL/min.Eluting peak at 10mL is collected after loading.

3.3.3 Protein Detection：Separation is collected after terminating and SDS-PAGE analyses, coomassie brilliant blue staining is carried out after sample treatment 30min, destainer is taken off to background transparent, and protein band is clear, observation destination protein pillar location and expression, as a result as schemed It is the destination protein being purified shown in 3, in black surround.The albumen being purified is subjected to Western blot, it is anti-using EDA Experience card, as a result as shown in Figure 4.It is destination protein activator A2 to show obtained purifying protein.

Embodiment 4

The preparation of EDA wild types and EDA-T1061C saltant type recombinant plasmids

Effect for detection of agonist is verified, it is necessary to add activator in saltant type sample.We choose and reported Road, EDA gene mutations T1061C that comprehensive agomphosis can be caused be saltant type sample；The sample of wild type is needed to enter simultaneously Row positive control.Therefore we need to build wild type and saltant type EDA recombinant plasmids.It is detailed preparation process below.

1. encode the gene order design of EDA albumen and prepare

The gene order design of 1.1 EDA albumen

The primer for expanding EDA genes (SEQ ID No.5) is respectively EDA-F (SEQ ID No.7) and EDA-R (SEQ ID No.8).EDA-F nucleotides sequence is listed in before initiation codon plus BamH I restriction enzyme sites and protectiveness base, EDA-R core Nucleotide sequence is in EDA coding ends added with terminator and the restriction enzyme sites of Xba I and protectiveness base.EDA protein amino acid sequences For SEQ ID No.6.

It is prepared by the nucleic acid fragment of 1.2 coding EDA wild type recombinant proteins

Using people source EDA gene cDNAs template, add primer EDA-F and EDA-R and carry out gene magnification.Amplification system such as table 5：

Table 5

2X Taq Mix	20μL
		dNTP	2μL
Primer EDA-F(10μM)	2μL
		Primer EDA-R(10μM)	2μL
Template (0.1-1 μ g)	cDNA
		Moisturizing is extremely	40μL

Amplification condition：For 95 DEG C of pre-degeneration 5min, then 35 circulations are carried out, cycling condition is 95 DEG C of denaturation 30s, and 58 DEG C are moved back Fiery 30s, 72 DEG C of extension 60s；Last 72 DEG C of extensions 5min.

2. recombinate the structure of EDA plasmids

The structure of 2.1 restructuring wild type EDA plasmids

By carrier for expression of eukaryon pECFP-C1 with after the digestion of BamH I and Xba I reclaim, then with through BamH I and Xba The amplification EDA gene outcomes reclaimed after I digestion are connected under T4 connection enzyme effects, are coated on and are contained after conversion DH5 α competent cells Block after that LB flat boards, incubated overnight picking single bacterium colony amplification cultivation and extract plasmid, with BamH I digestions and to deliver to sequencing public Sequencing identification is taken charge of, the positive plasmid of identification is recombinant plasmid needed for the present embodiment, and upgrading grain preserves and preserves strain, ordered Entitled pECFP-C1-EDA.

2.2. the structure of recombination mutation type EDA plasmids

EDA genes T1061C mutation can cause syndrome and type agomphosis, and its sequence is mutated into except the 1061st bit base by T Outside C, remaining sequence is with SEQ ID No.5.The recombinant plasmid of muton is respectively obtained using the method for rite-directed mutagenesis.

Prepare the specific primer EDA-F-M of T1061 mutation EDA genes nucleotide sequence such as SEQ ID No.13 institutes Show, gene-specific primer EDA-R-M nucleotide sequence is as shown in SEQ ID No.14.

Using pECFP-C1-EDA plasmids as template, add primer EDA-F-M and EDA-R-M and carry out gene magnification.Expand body System such as table 6：

Table 6

2X Taq Mix	20μL
		dNTP	2μL
Primer EDA-F-M(10μM)	2μL
		Primer EDA-R-M(10μM)	2μL
The μ g of template 20	pECFP-C1-EDA
		Moisturizing is extremely	40μL

Amplification condition：For 95 DEG C of pre-degeneration 5min, then 35 circulations are carried out, cycling condition is 95 DEG C of denaturation 30s, and 58 DEG C are moved back Fiery 30s, 72 DEG C of extension 60s；Last 72 DEG C of extensions 5min.

It is coated with after above-mentioned T1061C amplified production is digested into 2h, conversion DH5 α competent cells in 37 degree with Dpn I enzymes Picking single bacterium colony amplification cultivation and plasmid is extracted containing that LB flat boards of card, after incubated overnight, with BamH I digestions and deliver to survey The sequencing identification of sequence company, the positive plasmid of identification is recombinant plasmid needed for the present embodiment, and upgrading grain preserves and preserves strain, will It is named as pECFP-C1-EDA-T1061C.

Embodiment 5

EDAR activators A1 and activator A2 checking

Using electroporation method by recombinant plasmid pECFP-C1-EDA (activator A1) and pECFP-C1-EDA-T1061C (saltant type sample) and reporter plasmid pNF κ B-luc cotransfections enter in HEK293-EDAR-His cell lines；Simultaneously with transfection PECFP-C1-EDA-T1061C (saltant type sample) plasmids and reporter plasmid pNF κ B-luc HEK293-EDAR-His are thin Born of the same parents system is negative control, to transfect pECFP-C1-EDA's (wild type sample) and reporter plasmid pNF κ B-luc HEK293-EDAR-His cell lines are positive control, 37 degree, cultivate 48 hours under the conditions of 5%CO2, collect cell and with pair glimmering Light element enzyme Reporter Gene Experiments detection NF κ B transcriptional activities, to verify activator A1 effect.

Using electroporation method by recombinant plasmid pECFP-C1-EDA-T1061C (saltant type sample) and reporter plasmid PNF κ B-luc are transfected into HEK293-EDAR-His cell lines, while adding the excitement of purifying in the culture medium of this cell line Agent A2 albumen, final concentration of 1 μ g/mL；To transfect pECFP-C1-EDA-T1061C (saltant type sample) and reporter plasmid PNF κ B-luc cell line is negative control, to transfect pECFP-C1-EDA (wild type sample) and reporter plasmid pNF κ B-luc cell line is positive control, 37 degree, cultivate 48 hours under the conditions of 5%CO2, and collection cell simultaneously uses Dual-Luciferase report Gene Experiments detection NF κ B transcriptional activities are accused, to verify wild type prokaryotic expression protein activator A2 effect.Report base used Because plasmid is that pNF κ B-luc are purchased from green skies company, production code member is D2206, the luciferase reporter gene kit ForReporter (DLRTM) Assay System (are purchased from Promega companies, article No. is E1960), Experimental procedure is with reference to kit specification.Dual-Luciferase reporter assay testing result is as shown in figure 5, Ratio values represent NF κ B transcriptional activity.

Dual-Luciferase reporter assay result shows, relative to negative control, is transfected into swashing of being expressed in recombinant cell lines Dynamic agent A1 can improve NF κ B transcriptional activity with culture medium, purifying prokaryotic expression protein activator A2 is added to, But do not recover to the expression quantity of EDAR during EDA wild types.

To carry out finer analysis, the above results are quantified, by NF κ B transcriptional activity highest, positive control Group cell line in Ratio be set as 1, as EDAR be activated degree highest when transcriptional activity；It is then relative, negative control Group NF κ B transcriptional activity is 0.29；In activator A1 experimental groups, NF κ B transcriptional activity is 0.53, activator A2 experimental groups In, NF κ B transcriptional activity is 0.42.

Show, relative to negative control, to add after activator A1 or A2, NF κ B transcriptional activity rise, i.e. EDAR activation water Flat rise, illustrates that activator A1 and A2 can serve as EDAR activators.But relative to positive control, NF κ B transcriptional activity, Activation level is low, shows that the two activators can not recover the NF κ B effect of transcriptional activity, i.e. the two activators completely It is not complete.On the other hand, activator A1 effect is better than activator A2.

As it will be easily appreciated by one skilled in the art that the foregoing is merely illustrative of the preferred embodiments of the present invention, it is not used to The limitation present invention, any modifications, equivalent substitutions and improvements made within the spirit and principles of the invention etc., it all should include Within protection scope of the present invention.

Sequence table

<110>The Central China University of Science and Technology

<120>A kind of cell line of expression EDAR genes and the preparation and application of receptor stimulating agent

<130> 14

<141> 2017-06-15

<160> 14

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1347

<212> DNA

<213>Human body

<400> 1

atggcccatg tgggggactg cacgcagacg ccctggctcc ccgtcctggt ggtgtctctg 60

atgtgctcag cccgagcgga atactcaaac tgcggtgaga acgagtacta caaccagact 120

acggggctgt gccaggagtg ccccccgtgt gggccgggag aggagcccta cctgtcctgt 180

ggctacggca ccaaagacga ggactacggc tgcgtcccct gcccggcgga gaagttttcc 240

aaaggaggct accagatatg caggcgtcac aaagactgtg agggcttctt ccgggccacc 300

gtgctgacac caggggacat ggagaatgac gctgagtgtg gcccttgcct ccctggctac 360

tacatgctgg agaacagacc gaggaacatc tatggcatgg tctgctactc ctgcctcctg 420

gcacccccca acaccaagga atgtgtggga gccacttcag gagcttctgc caacttccct 480

ggcacctcgg gcagcagcac cctgtctccc ttccagcacg cccacaaaga actctcaggc 540

caaggacacc tggccactgc cctgatcatt gcaatgtcca ccatcttcat catggccatc 600

gccatcgtcc tcatcatcat gttctacatc ctgaagacaa agccctctgc cccagcctgt 660

tgcaccagcc acccggggaa gagcgtggag gcccaagtga gcaaggacga ggagaagaaa 720

gaggccccag acaacgtggt gatgttctcc gagaaggatg aatttgagaa gctgacagca 780

actccagcaa agcccaccaa gagcgagaac gatgcctcat ccgagaatga gcagctgctg 840

agccggagcg tcgacagtga tgaggagccc gcccctgaca agcagggctc cccggagctg 900

tgcctgctgt cgctggttca cctggccagg gagaagtctg ccaccagcaa caagtcagcc 960

gggattcaaa gccggaggaa aaagatcctc gatgtgtatg ccaacgtgtg tggagtcgtg 1020

gaaggtctta gccccacgga gctgccattt gattgcctcg agaagactag ccgaatgctc 1080

agctccacgt acaactctga gaaggctgtt gtgaaaacgt ggcgccacct cgccgagagc 1140

ttcggcctga agagggatga gattgggggc atgacagacg gcatgcaact ctttgaccgc 1200

atcagcacgg caggctacag catccctgag ctactcacaa aactggtgca gattgagcgg 1260

ctggatgctg tggagtcctt gtgtgcagac atactggagt gggcgggggt tgtgccacct 1320

gcctcccagc cacatgctgc atcctga 1347

<210> 2

<211> 454

<212> PRT

<213>Artificial sequence

<400> 2

Met Ala His Val Gly Asp Cys Thr Gln Thr Pro Trp Leu Pro Val Leu

1 5 10 15

Val Val Ser Leu Met Cys Ser Ala Arg Ala Glu Tyr Ser Asn Cys Gly

20 25 30

Glu Asn Glu Tyr Tyr Asn Gln Thr Thr Gly Leu Cys Gln Glu Cys Pro

35 40 45

Pro Cys Gly Pro Gly Glu Glu Pro Tyr Leu Ser Cys Gly Tyr Gly Thr

50 55 60

Lys Asp Glu Asp Tyr Gly Cys Val Pro Cys Pro Ala Glu Lys Phe Ser

65 70 75 80

Lys Gly Gly Tyr Gln Ile Cys Arg Arg His Lys Asp Cys Glu Gly Phe

85 90 95

Phe Arg Ala Thr Val Leu Thr Pro Gly Asp Met Glu Asn Asp Ala Glu

100 105 110

Cys Gly Pro Cys Leu Pro Gly Tyr Tyr Met Leu Glu Asn Arg Pro Arg

115 120 125

Asn Ile Tyr Gly Met Val Cys Tyr Ser Cys Leu Leu Ala Pro Pro Asn

130 135 140

Thr Lys Glu Cys Val Gly Ala Thr Ser Gly Ala Ser Ala Asn Phe Pro

145 150 155 160

Gly Thr Ser Gly Ser Ser Thr Leu Ser Pro Phe Gln His Ala His Lys

165 170 175

Glu Leu Ser Gly Gln Gly His Leu Ala Thr Ala Leu Ile Ile Ala Met

180 185 190

Ser Thr Ile Phe Ile Met Ala Ile Ala Ile Val Leu Ile Ile Met Phe

195 200 205

Tyr Ile Leu Lys Thr Lys Pro Ser Ala Pro Ala Cys Cys Thr Ser His

210 215 220

Pro Gly Lys Ser Val Glu Ala Gln Val Ser Lys Asp Glu Glu Lys Lys

225 230 235 240

Glu Ala Pro Asp Asn Val Val Met Phe Ser Glu Lys Asp Glu Phe Glu

245 250 255

Lys Leu Thr Ala Thr Pro Ala Lys Pro Thr Lys Ser Glu Asn Asp Ala

260 265 270

Ser Ser Glu Asn Glu Gln Leu Leu Ser Arg Ser Val Asp Ser Asp Glu

275 280 285

Glu Pro Ala Pro Asp Lys Gln Gly Ser Pro Glu Leu Cys Leu Leu Ser

290 295 300

Leu Val His Leu Ala Arg Glu Lys Ser Ala Thr Ser Asn Lys Ser Ala

305 310 315 320

Gly Ile Gln Ser Arg Arg Lys Lys Ile Leu Asp Val Tyr Ala Asn Val

325 330 335

Cys Gly Val Val Glu Gly Leu Ser Pro Thr Glu Leu Pro Phe Asp Cys

340 345 350

Leu Glu Lys Thr Ser Arg Met Leu Ser Ser Thr Tyr Asn Ser Glu Lys

355 360 365

Ala Val Val Lys Thr Trp Arg His Leu Ala Glu Ser Phe Gly Leu Lys

370 375 380

Arg Asp Glu Ile Gly Gly Met Thr Asp Gly Met Gln Leu Phe Asp Arg

385 390 395 400

Ile Ser Thr Ala Gly Tyr Ser Ile Pro Glu Leu Leu Thr Lys Leu Val

405 410 415

Gln Ile Glu Arg Leu Asp Ala Val Glu Ser Leu Cys Ala Asp Ile Leu

420 425 430

Glu Trp Ala Gly Val Val Pro Pro Ala Ser Gln Pro His Ala Ala Ser

435 440 445

His His His His His His

450

<210> 3

<211> 32

<212> DNA

<213>Artificial sequence

<400> 3

cttaagcttc caccatggcc catgtggggg ac 32

<210> 4

<211> 51

<212> DNA

<213>Artificial sequence

<400> 4

gctctagatc aatgatgatg atgatgatgg gatgcagcat gtggctggga g 51

<210> 5

<211> 1176

<212> DNA

<213>Human body

<400> 5

atgggctacc cggaggtgga gcgcagggaa ctcctgcctg cagcagcgcc gcgggagcga 60

gggagccagg gctgcgggtg tggcggggcc cctgcccggg cgggcgaagg gaacagctgc 120

ctgctcttcc tgggtttctt tggcctctcg ctggccctcc acctgctgac gttgtgctgc 180

tacctagagt tgcgctcgga gttgcggcgg gaacgtggag ccgagtcccg ccttggcggc 240

tcgggcaccc ctggcacctc tggcacccta agcagcctcg gtggcctcga ccctgacagc 300

cccatcacca gtcaccttgg gcagccgtca cctaagcagc agccattgga accgggagaa 360

gccgcactcc actctgactc ccaggacggg caccagatgg ccctattgaa tttcttcttc 420

cctgatgaaa agccatactc tgaagaagaa agtaggcgtg ttcgccgcaa taaaagaagc 480

aaaagcaatg aaggagcaga tggcccagtt aaaaacaaga aaaagggaaa gaaagcagga 540

cctcctggac ccaatggccc tccaggaccc ccaggacctc caggacccca gggaccccca 600

ggaattccag ggattcctgg aattccagga acaactgtta tgggaccacc tggtcctcca 660

ggtcctcctg gtcctcaagg accccctggc ctccagggac cttctggtgc tgctgataaa 720

gctggaactc gagaaaacca gccagctgtg gtgcatctac agggccaagg gtcagcaatt 780

caagtcaaga atgatctttc aggtggagtg ctcaatgact ggtctcgcat cactatgaac 840

cccaaggtgt ttaagctaca tccccgcagc ggggagctgg aggtactggt ggacggcacc 900

tacttcatct atagtcaggt agaagtatac tacatcaact tcactgactt tgccagctat 960

gaggtggtgg tggatgagaa gcccttcctg cagtgcacac gcagcatcga gacgggcaag 1020

accaactaca acacttgcta taccgcaggc gtctgcctcc tcaaggcccg gcagaagatc 1080

gccgtcaaga tggtgcacgc tgacatctcc atcaacatga gcaagcacac cacgttcttt 1140

ggggccatca ggctgggtga agcccctgca tcctag 1176

<210> 6

<211> 391

<212> PRT

<213>Artificial sequence

<400> 6

Met Gly Tyr Pro Glu Val Glu Arg Arg Glu Leu Leu Pro Ala Ala Ala

1 5 10 15

Pro Arg Glu Arg Gly Ser Gln Gly Cys Gly Cys Gly Gly Ala Pro Ala

20 25 30

Arg Ala Gly Glu Gly Asn Ser Cys Leu Leu Phe Leu Gly Phe Phe Gly

35 40 45

Leu Ser Leu Ala Leu His Leu Leu Thr Leu Cys Cys Tyr Leu Glu Leu

50 55 60

Arg Ser Glu Leu Arg Arg Glu Arg Gly Ala Glu Ser Arg Leu Gly Gly

65 70 75 80

Ser Gly Thr Pro Gly Thr Ser Gly Thr Leu Ser Ser Leu Gly Gly Leu

85 90 95

Asp Pro Asp Ser Pro Ile Thr Ser His Leu Gly Gln Pro Ser Pro Lys

100 105 110

Gln Gln Pro Leu Glu Pro Gly Glu Ala Ala Leu His Ser Asp Ser Gln

115 120 125

Asp Gly His Gln Met Ala Leu Leu Asn Phe Phe Phe Pro Asp Glu Lys

130 135 140

Pro Tyr Ser Glu Glu Glu Ser Arg Arg Val Arg Arg Asn Lys Arg Ser

145 150 155 160

Lys Ser Asn Glu Gly Ala Asp Gly Pro Val Lys Asn Lys Lys Lys Gly

165 170 175

Lys Lys Ala Gly Pro Pro Gly Pro Asn Gly Pro Pro Gly Pro Pro Gly

180 185 190

Pro Pro Gly Pro Gln Gly Pro Pro Gly Ile Pro Gly Ile Pro Gly Ile

195 200 205

Pro Gly Thr Thr Val Met Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly

210 215 220

Pro Gln Gly Pro Pro Gly Leu Gln Gly Pro Ser Gly Ala Ala Asp Lys

225 230 235 240

Ala Gly Thr Arg Glu Asn Gln Pro Ala Val Val His Leu Gln Gly Gln

245 250 255

Gly Ser Ala Ile Gln Val Lys Asn Asp Leu Ser Gly Gly Val Leu Asn

260 265 270

Asp Trp Ser Arg Ile Thr Met Asn Pro Lys Val Phe Lys Leu His Pro

275 280 285

Arg Ser Gly Glu Leu Glu Val Leu Val Asp Gly Thr Tyr Phe Ile Tyr

290 295 300

Ser Gln Val Glu Val Tyr Tyr Ile Asn Phe Thr Asp Phe Ala Ser Tyr

305 310 315 320

Glu Val Val Val Asp Glu Lys Pro Phe Leu Gln Cys Thr Arg Ser Ile

325 330 335

Glu Thr Gly Lys Thr Asn Tyr Asn Thr Cys Tyr Thr Ala Gly Val Cys

340 345 350

Leu Leu Lys Ala Arg Gln Lys Ile Ala Val Lys Met Val His Ala Asp

355 360 365

Ile Ser Ile Asn Met Ser Lys His Thr Thr Phe Phe Gly Ala Ile Arg

370 375 380

Leu Gly Glu Ala Pro Ala Ser

385 390

<210> 7

<211> 26

<212> DNA

<213>Artificial sequence

<400> 7

atcggatcca tgggctaccc ggaggt 26

<210> 8

<211> 27

<212> DNA

<213>Artificial sequence

<400> 8

gactctagac taggatgcag gggcttc 27

<210> 9

<211> 480

<212> DNA

<213>Human body

<400> 9

ggaccttctg gtgctgctga taaagctgga actcgagaaa accagccagc tgtggtgcat 60

ctacagggcc aagggtcagc aattcaagtc aagaatgatc tttcaggtgg agtgctcaat 120

gactggtctc gcatcactat gaaccccaag gtgtttaagc tacatccccg cagcggggag 180

ctggaggtac tggtggacgg cacctacttc atctatagtc aggtagaagt atactacatc 240

aacttcactg actttgccag ctatgaggtg gtggtggatg agaagccctt cctgcagtgc 300

acacgcagca tcgagacggg caagaccaac tacaacactt gctataccgc aggcgtctgc 360

ctcctcaagg cccggcagaa gatcgccgtc aagatggtgc acgctgacat ctccatcaac 420

atgagcaagc acaccacgtt ctttggggcc atcaggctgg gtgaagcccc tgcatcctag 480

<210> 10

<211> 190

<212> PRT

<213>Artificial sequence

<400> 10

Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro

1 5 10 15

Arg Gly Ser His Met Ala Ser Glu Asn Leu Tyr Phe Gln Gly Ser Gly

20 25 30

Pro Ser Gly Ala Ala Asp Lys Ala Gly Thr Arg Glu Asn Gln Pro Ala

35 40 45

Val Val His Leu Gln Gly Gln Gly Ser Ala Ile Gln Val Lys Asn Asp

50 55 60

Leu Ser Gly Gly Val Leu Asn Asp Trp Ser Arg Ile Thr Met Asn Pro

65 70 75 80

Lys Val Phe Lys Leu His Pro Arg Ser Gly Glu Leu Glu Val Leu Val

85 90 95

Asp Gly Thr Tyr Phe Ile Tyr Ser Gln Val Glu Val Tyr Tyr Ile Asn

100 105 110

Phe Thr Asp Phe Ala Ser Tyr Glu Val Val Val Asp Glu Lys Pro Phe

115 120 125

Leu Gln Cys Thr Arg Ser Ile Glu Thr Gly Lys Thr Asn Tyr Asn Thr

130 135 140

Cys Tyr Thr Ala Gly Val Cys Leu Leu Lys Ala Arg Gln Lys Ile Ala

145 150 155 160

Val Lys Met Val His Ala Asp Ile Ser Ile Asn Met Ser Lys His Thr

165 170 175

Thr Phe Phe Gly Ala Ile Arg Leu Gly Glu Ala Pro Ala Ser

180 185 190

<210> 11

<211> 31

<212> DNA

<213>Artificial sequence

<400> 11

ccggatccgg accttctggt gctgctgata a 31

<210> 12

<211> 26

<212> DNA

<213>Artificial sequence

<400> 12

ggaagcttct aggatgcagg ggcttc 26

<210> 13

<211> 35

<212> DNA

<213>Artificial sequence

<400> 13

tataccgcag gcgtctgtct ccccaaggcc cggca 35

<210> 14

<211> 35

<212> DNA

<213>Artificial sequence

<400> 14

tgccgggcct tggggagaca gacgcctgcg gtata 35

Claims (10)

1. a kind of eukaryon recombinant plasmid of people source EDAR genes, it is characterised in that prepared by following steps：

(1) EDAR gene opens reading frame sequence is connected with His sequence labels, the EDAR gene opens reading frame sequence is SEQ ID No.1；

(2) sequence for obtaining step (1) is inserted at the polyclone enzyme enzyme site of carrier for expression of eukaryon, that is, obtains recombinating matter Grain.

2. the eukaryon recombinant plasmid of people source EDAR genes as claimed in claim 1, it is characterised in that described in step (2) Expression vector is pcDNA3.1 (+).

3. a kind of stabilization expresses the cell line of people source EDAR albumen, its deposit number is：CCTCC NO:C201778.

4. the preparation method of stable expression people source EDAR albuminous cells system as claimed in claim 3, it is characterised in that this method Comprise the following steps：

(1) after people source EDAR gene opens reading frame sequence is connected with His sequence labels, many of carrier for expression of eukaryon are inserted into Clone at restriction enzyme site, build the recombinant plasmid containing EDAR genes；

(2) Transfected Recombinant Plasmid for obtaining step (1) enters in eukaryotic, and is screened by drug resistance, is had The positive cell clone of antibiotic resistance；

(3) the positive cell clone amplification cultivation obtained in step (2) is obtained stablizing expressed fusion protein EDAR-His's Cell line.

5. the preparation method of stable expression people source EDAR albuminous cells system as claimed in claim 4, it is characterised in that

Expression vector in the step (1) is pcDNA3.1 (+)；

Transfection method in the step (2) is electroporation transfection method, and the eukaryotic is human embryonic kidney cell HEK293, institute Medicine is stated for G418.

6. as claimed in claim 3 be used for the application of the stable cell line for expressing people source EDAR albumen, comprise the following steps：

(1) people source wild type EDA gene open reading frame sequences are inserted into carrier for expression of eukaryon pECFP-C1 polyclone enzyme At enzyme site, the recombinant plasmid pECFP-C1-EDA containing EDA genes is built；

(2) saltant type EDA recombinant plasmids are built using the method for rite-directed mutagenesis；

(3) transfection above-mentioned steps (1) and the recombinant plasmid obtained by step (2) enter HEK293-EDAR-His cell lines respectively, logical Luciferase reporter gene method detection NF κ B transcriptional activity is crossed, to assess EDAR activation levels.

7. a kind of EDAR activator, it is characterised in that the activator is EDA full length protein sequences, EDA albumen is EDAR eggs White part；The amino acid sequence of the activator such as SEQ ID No.6；Expression vector is pECFP-C1-EDA plasmids.

8. the preparation method of activator as claimed in claim 7, comprises the following steps：

(1) people source wild type EDA gene open reading frame sequences are inserted into carrier for expression of eukaryon pECFP-C1 polyclone enzyme At enzyme site, the recombinant plasmid pECFP-C1-EDA containing EDA genes is built；

(2) Transfected Recombinant Plasmid obtained in step (1) is entered in HEK293-EDAR-His cell lines to express, obtains described sharp Dynamic agent.

9. EDAR as claimed in claim 7 activator, it is characterised in that the activator is EDA PROTEIN Cs end TNF structures Domain, EDA albumen is the part of EDAR albumen；The amino acid sequence of the activator such as SEQ ID No.10；Expression vector is PET-28a (+)-TNF plasmids.

10. the application of EDAR activators as claimed in claim 9, comprises the following steps：

(1) nucleotide sequence of people source wild type EDA gene TNF code areas is connected with prokaryotic expression carrier pET-28a (+), structure Build recombinant plasmid pET-28a (+)-TNF containing EDA gene TNF code areas；

(2) recombinant plasmid obtained in expression induction step (1), and described activator is obtained after purification；

(3) EDA mutators carrier is transfected into HEK293-EDAR-His cells, while adding institute in step (2) in the medium The activator obtained, to activate EDAR.