CN102250224B

CN102250224B - Duck plague virus gG truncated recombinant protein and preparation method thereof

Info

Publication number: CN102250224B
Application number: CN 201110177128
Authority: CN
Inventors: 程安春; 杨晓圆; 汪铭书; 陈孝跃
Original assignee: ENGINEERING AND TECHONLOGY CENTER FOR LABORATORY ANIMALS OF SICHUAN AGRICULTURAL UNIVERSITY
Current assignee: Sichuan Agricultural University
Priority date: 2011-06-28
Filing date: 2011-06-28
Publication date: 2013-06-05
Anticipated expiration: 2031-06-28
Also published as: CN102250224A

Abstract

The invention relates to the field of bioengineering, in particular to a duck plague virus gG truncated recombinant protein and a preparation method thereof. The amino acid sequence of the protein is shown as SEQIDNO:1, and the nucleotide sequence is shown as SEQIDNO:2; on the basis of cloning the nucleotide sequence of the protein by a gene engineering technology, the protein is connected with a prokaryotic expression vector pET-32a(+) to successfully transform gene engineering products induced and expressed by an Escherichia coli BL21(DE3) expression system; the protein is gGM/His fusion protein in an expression form, the molecular weight of the expressed fusion protein is 50kDa, and the product is subjected to fusion expression to facilitate purification and increase the immunogenicity of expressed protein; and after a product expressed by a prokaryotic expression system is identified by Western blot, the surface has immunological reaction activity similar to that of natural protein.

Description

Duck plague virus gG truncated recombinant protein and preparation method thereof

Technical field

The present invention relates to bioengineering field, particularly the test kit of duck plague virus antibody and detection method.

Background technology

Duck plague (Duck Plague, DP) is the septic transmissible disease of a kind of acute, hot, the contagious infection of the aquatic birds such as duck, goose and swan that caused by the duck plague virus in herpetoviridae (Duck plague virus, DPV).This disease can cause commodity aquatic bird egg productivity to descend and be dead, and wildfowl is also had different lethality rates.The clinical manifestation of DP is that spirit is depressed, and high heat is delaied, and two leg paralysis, diarrhea, sheds tears, and the enlargement of the sick duck neck of part is a kind of transmissible disease of extensively having a liking for systemic infection, and is clinical take the acute sepsis process as feature; It is blood vessel injury that pathology is analysed feature, and histoorgan is hemorrhage, digestive tract hemorrhage, inflammation and necrosis, and some privileged site of gastrointestinal mucosal has rash shape infringement, and the impaired and degeneration organa parenchymatosum of lymphoid organ changes, and its M ﹠ M is all very high.In recent years, along with foster duck industry is produced to future development intensive, mass-producing, duck plague is supported one of the most serious contagious disease of duck industry as threatening, and has caused huge financial loss.

Past is mainly to utilize totivirus to detect as antigen to the detection method of duck plague virus serum antibody, but traditional Virus Isolation method approximately needs 4～5 days, method is loaded down with trivial details, waste time and energy, and can doped with many host cell compositions, cause false positive results to occur in the virus after purifying.

Summary of the invention

One of purpose of the present invention is to provide a kind of duck plague virus gG truncated recombinant protein, and this albumen can be as the trapping agent of duck plague virus antibody.

For realizing such scheme, technical scheme of the present invention is:

Duck plague virus gG truncated recombinant protein, its aminoacid sequence is as shown in SEQ ID NO:1.

The DNA sequence dna of described duck plague virus gG truncated recombinant protein is as shown in SEQ ID NO:2.

Two of purpose of the present invention is to provide the preparation method of duck plague virus gG truncated recombinant protein, and the method can be used for duck plague virus gG truncated recombinant protein scale operation.

For achieving the above object, technical scheme of the present invention is:

The preparation method of described duck plague virus gG truncated recombinant protein specifically comprises the following steps:

A, take the DPV genomic dna as template, adopt the nucleotide sequence duck plague virus gG truncated gene fragment that increases to get of the upstream and downstream primer PCR as shown in SEQ ID No.3 and SEQ ID No.4 respectively, with being connected of pMD18-T, get pMD18-gGM;

B, restriction enzyme BamH I and Xho I be double digestion pMD18-gGM plasmid and prokaryotic expression carrier pET32a (+) respectively, and connects, and gets recombined pronucleus expression plasmid pET32a-gGM;

C also is transformed into expressive host bacterium abduction delivering with the recombinant plasmid pET32a-gGM of said extracted, gets the duck plague virus gG truncated recombinant protein crude product.

Further, step C gained duck plague virus gG truncated recombinant protein crude product after nickel sepharose affinitive layer purification, is got duck plague virus gG truncated recombinant protein;

Further, the Host Strains in step C is bacterial strain BL21 (DE3), and at IPTG concentration 〉=0.2mmol/L, inducing temperature is to carry out abduction delivering 2 hours under 30 ℃ of conditions, gets duck plague virus gG truncated recombinant protein.

Beneficial effect of the present invention is: duck plague virus gG truncated recombinant protein is the Main Antigenic Region (68aa-377aa) of choosing duck plague virus (DPV) gG gene coded protein, and called after gGM; Then utilize genetic engineering technique, on its basis of cloning, it is connected with prokaryotic expression carrier pET-32a (+), successfully transform the gene engineering product that e. coli bl21 (DE3) expression system is induced, expressed.This protein expression form is the gGM/His fusion rotein, and the fusion protein molecule amount of expression is 50kDa, is that amalgamation and expression one is to consider to be convenient to purifying with product design, the 2nd, can increase the immunogenicity of expressing protein.Product after prokaryotic expression identifies that through Western blot the rear surface has the immune response activity similar to native protein.

The product that adopts method of the present invention to produce can be used for:

1. this product can be used as the detectable antigens of the indirect ELISA method that detects duck plague virus antibody.

2. can be used as the genetic engineering subunit vaccine of preventing duck pestivirus infection.

Product advantage applies of the present invention exists:

1. because this detectable antigens is duck plague virus gG truncated recombinant protein, be not totivirus, dangerous without loose poison when using this detectable antigens and detect with this.

2. as ELISA Detection of antigen duck plague virus antibody, high specificity, no cross reaction.

3. stable performance, production cost are low, are fit to batch production production, and market application foreground is wide.

More beneficial effects will embody in an embodiment.

Fig. 1 is the agarose gel electrophoresis figure of the pcr amplification of gG intercept gene; The 1 specific DNA band that obtains through agarose gel electrophoresis for gG intercept gene PCR amplified production wherein, M is DNA Marker.

Fig. 2 is the agarose gel electrophoresis figure of recombinant plasmid pMD18-gGM; Wherein, M is DNA Marker, and 1 is recombinant plasmid pMD18-gGM BamH I and Xho I double digestion, and enzyme is cut two specific bands that product obtains through agarose gel electrophoresis.

Fig. 3 is the agarose gel electrophoresis figure that the enzyme of pET32a-gGM is cut evaluation; Wherein, M is DNA Marker, 1 is recombinant expression plasmid pET32a-gGM Xho I single endonuclease digestion, enzyme is cut the specific band that product obtains through agarose gel electrophoresis, 2 are recombinant expression plasmid pET32a-gGM BamH I and Xho I double digestion, and enzyme is cut two specific bands that product obtains through agarose gel electrophoresis.

Fig. 4 is the SDS-PAGE electrophorogram of the duck plague virus gG truncated recombinant protein of recombinant expression plasmid pET32a-gGM expression, wherein M is protein Marker, 1 induces with IPTG for recombinant expression plasmid pET32a-gGM, the bacterium liquid that obtains carries out ultrasonic disruption, after centrifugal, the gained precipitation is carried out the result of SDS-PAGE electrophoresis, 2 induce with IPTG for recombinant expression plasmid pET32a-gGM, the bacterium liquid that obtains carries out ultrasonic disruption, after centrifugal, the gained supernatant carries out the result of SDS-PAGE electrophoresis, 3 do not induce with IPTG for recombinant expression plasmid pET32a-gGM, through SDS-PAGE electrophoresis acquired results, 4 induce by SDS-PAGE electrophoresis acquired results with IPTG for recombinant expression plasmid pET32a-gGM, 5 do not induce with IPTG for empty carrier pET-32a (+), through SDS-PAGE electrophoresis acquired results, 6 induce by SDS-PAGE electrophoresis acquired results with IPTG for empty carrier pET-32a (+).

Fig. 5 is the SDS-PAGE electrophorogram that contains Host Strains E.coli BL21 (DE3) the expression duck plague virus gG truncated recombinant protein of recombinant plasmid pET32a-gGM under different IP TG concentration; Wherein, M is protein Marker, 1 for the final concentration of IPTG be 1.2 mmol/L, 2 for the final concentration of IPTG be 1.0 mmol/L, 3 for the final concentration of IPTG be 0.8 mmol/L, 4 for the final concentration of IPTG is 0.5 mmol/L, and 5 for the final concentration of IPTG is 0.2 mmol/L, 6 for the final concentration of IPTG be 0mmol/L.

Fig. 6 is the SDS-PAGE electrophorogram that contains Host Strains E.coli BL21 (DE3) the expression duck plague virus gG truncated recombinant protein of recombinant plasmid pET32a-gGM under differing temps; Wherein, M is protein Marker, and 1 for inducing temperature is 25 ℃, and 2 for inducing temperature is 30 ℃, 3 for inducing temperature be 37 ℃.

Fig. 7 is the SDS-PAGE electrophorogram that contains Host Strains E.coli BL21 (DE3) the expression duck plague virus gG truncated recombinant protein of recombinant plasmid pET32a-gGM under different induction times; Wherein M is protein Marker, and 1 for inducing 12h, and 2 for inducing 6h, and 3 for inducing 5h, and 4 for inducing 4h, and 5 for inducing 3h, and 6 for inducing 2h, and 7 for inducing 0h.

Fig. 8 is nickel sepharose (Ni ²⁺-NAT) the duck plague virus gG truncated recombinant protein SDS-PAGE electrophorogram after affinitive layer purification, wherein M is protein Marker, 1 is the gG intercept recombinant protein after nickel sepharose affinitive layer purification.

Embodiment

In order to make the purpose, technical solutions and advantages of the present invention clearer, the below is described in detail the preferred embodiments of the present invention.

Hereinafter with reference to accompanying drawing, the preferred embodiments of the present invention are described in detail.The experimental technique of unreceipted actual conditions in preferred embodiment, usually according to normal condition, the molecular cloning experiment guide (third edition for example, J. the work such as Pehanorm Brooker, Huang Peitang etc. translate, Science Press, 2002) described in condition, or carry out according to the condition that manufacturer advises.

The corresponding abbreviation of partial amino-acid is as follows:

Glutamine gln Q; Glycine gly G; Serine ser S; L-Ala ala A; Threonine thr T; α-amino-isovaleric acid val V; Isoleucine ile I; Leucine leu L; Tyrosine tyr Y; Phenylalanine phe F; Histidine his H; Proline(Pro) pro P; L-asparagine asn N; Methionine(Met) met M; L-glutamic acid glu E; Tryptophane trp W; Methionin lys K; Halfcystine cys C; Arginine arg R; Aspartic acid Asp D.

The preparation of embodiment 1 duck plague virus gG truncated recombinant protein

One material is prepared

1 bacterial strain, plasmid and strain

Plasmid pMD18-T is available from Dalian precious biotechnology company limited; Prokaryotic expression plasmid pET32a (+), Novagen company product; Cloning host bacterium E.coli DH5 α, expressive host bacterium E.coli BL21 (DE3) and DPV CHv virulent strain are provided by Sichuan Agricultural University's poultry diease research centre.Goat-anti duck IgG-HRP (the goat-anti duck IgG of horseradish peroxidase mark) and tetramethyl benzidine (TMB) are all available from U.S. KPL company, and goat-anti duck IgG-HRP's is freeze-dried, is packed as the 0.1mg/ bottle, and during use, by specification is dissolved as 1mL.

2 experimental animals

10 age in days duck embryos, its kind duck DPV and antibody are all negative.

3 experimental techniques

3.1 DPV gG gene cloning

3.1.1 design of primers

Aminoacid sequence Main Antigenic Region (68aa-377aa and called after gGM) according to the gG genes encoding utilizes Primer Premier5.0 software design pair of primers.Upstream primer as shown in SEQ ID NO:3: 5 '- GgatccCacacaggacgatatgag-3 ' (the line part is BamH I site); Downstream primer as shown in SEQ ID NO:4: 5 '- CtcgagAtgagggtgattgtggtag-3 ' (the line part is Xho I site).After primer was synthetic, with appropriate sterilization deionized water dissolving, making its final concentration was 20mmol/L, and-20 ℃ save backup.

3.1.2 the extraction of DPV genomic dna

The making method of a, normal DEF (DEF): getting the healthy duck embryo of 10d age in days, is that 5% tincture of iodine and volume fraction are 75% alcohol disinfecting eggshell surface with volume fraction respectively.Under aseptic technique, idiosome taken out and with PBS, idiosome cleaned, cutting off head, wing, leg and internal organ, PBS is cut into 1mm with idiosome after rinsing ³The fritter of size adds PBS appropriate, is placed in afterwards in triangular flask, adds cell dispersion agent (volume fraction is 2.5% trypsinase) 150 μ L/ embryos, digests 3min in 37 ℃ of water-baths.Immediately with cell suspension with the centrifugal 5min of 4000r/min, the tipping supernatant, after cell precipitation suspends with appropriate MEM, with 6 layers of filtered through gauze, add in the filtrate volume fraction be 10% calf serum and 100IU/mL two anti-after, be sub-packed in the 100mL Tissue Culture Flask, the 7mL/ bottle, level is statically placed in 37 ℃ of cell culture incubators and cultivates.

B, DPV propagation: get the DEF that just grows up to fine and close individual layer, abandon growth nutrient solution, after sterilization PBS cleaning cell surface 2 times, adding DPV virus liquid 2-3mL to cover cell surface adsorbs, abandon virus liquid after 37 ℃ of absorption 1h, then add and contain the two anti-MEM of 3% calf serum and 100IU/mL and keep nutritive medium, 37 ℃ of cultivations afterwards.Do simultaneously the DEF contrast that does not connect poison.

C, DNA extraction method: directly extract the DPV genomic dna from the DEF that infects, concrete steps are as follows: (1) is chosen with DPV kind poison infected cell pathology (CPE) and is reached 80% DEF; Choosing simultaneously the normal DEF of cellular form compares; (2) cell culture fluid that inclines adds the cell pyrolysis liquid of 500 μ L, and adding simultaneously Proteinase K (10mg/mL) to final concentration is 200 μ g/mL, after mixing, hatches 10 minutes for 37 ℃ gently; (3) cell suspending liquid is poured in the EP centrifuge tube, and remained in the interior lysate of cell bottle with the saturated phenol washing of 500 μ L, pour in centrifuge tube; (4) use saturated phenol: chloroform and chloroform extracting 2 times, then process 2 times with the water saturation ether; (5) add 1/10 times of volume 3mol/L NaAC, after mixing, add 2 times of cold dehydrated alcohols of volume, placed 30-60 minute for-20 ℃; (6) 13000r/ minute centrifugal 20 minutes, precipitation was twice of 70% washing with alcohol with the volume fraction of precooling; (7) after vacuum is drained, be dissolved in appropriate TE damping fluid, add 1 μ L RNA enzyme, 37 ℃ act on 30 minutes, and-20 ℃ save backup.

3.1.3 PCR amplification duck plague virus gG truncated gene

The PCR reaction system is:

Mixing gently, the instantaneous centrifugal laggard performing PCR of 2000r/min.

Reaction parameter: 95 ℃ of 5min, then enter 94 ℃ of 50s, 58 ℃ of 50s, 72 ℃ of 90s, totally 30 circulations, last 72 ℃ are extended 10 min, save backup in 4 ℃.Get 6 μ L PCR products electrophoresis on 1% sepharose, observe the length of amplified fragments.

3.1.4 the recovery of duck plague virus gG truncated gene PCR product

Reclaim the test kit specification sheets by Beijing match Parkson DNA of gene engineering company limited and carry out, the DNA after recovery is stored in-20 ℃ and saves backup.

3.1.5 the duck plague virus gG truncated gene of purifying and being connected of pMD18-T

The ligation system is as follows:

Mentioned reagent is added in the EP pipe of 0.2mL, careful mixing, instantaneous centrifugal after, spend the night in 16 ℃ of connections.

3.1.6 the preparation of DH5 α competent cell

Adopt Calcium Chloride Method to prepare fresh DH5 α strain competent escherichia coli cell, be summarized as follows: on (1) aseptic picking flat board, the DH5 alpha monoclonal colony inoculation of fresh culture in 5mL LB nutrient solution, spends the night in 37 ℃ of shaking culture; (2) get the above-mentioned nutrient solution of 1mL and be inoculated in 100mL LB nutrient solution, 37 ℃ of 200r/min shaking culture 2.5～3h make the OD600=0.5 left and right; (3) bacterial cultures is poured in the rear ice-cold centrifuge tube of sterilization into ice bath 10min; (4) in 4 ℃ of centrifugal 8min of 4000r/min, abandon supernatant; (5) add the CaCl of the 0.1mol/L of 10mL ice precooling ₂, the gentle bacterial precipitation, ice bath 30min of having hanged; (6) in 4 ℃ of centrifugal 8min of 4000r/min, abandon supernatant, add the CaCl of the 0.1mol/L of 4mL ice precooling ₂Resuspended precipitation again, adding final concentration is 15% sterile glycerol, is packed as 200 μ L/ pipes after mixing, preserves in-4 ℃ of refrigerators and spends the night to improve transformation efficiency.

3.1.7 transformed competence colibacillus cell

The whole taking-up of 10 μ l linked systems is added in 200 μ L DH5 α competent cells ice bath 30min; Be placed in again 42 ℃ of temperature and bathe 90s, afterwards ice bath 2min again; Then add immediately 0.8mL LB substratum, 37 ℃ of water-bath shaking culture 45min get 200 μ L and are applied on the substratum that contains Amp, put overnight incubation in 37 ℃ of incubators.The single white colony of picking next day is inoculated in the LB liquid nutrient medium that contains Amp, carries out plasmid extraction after 37 ℃ of water-bath shaking culture 18h.

3.1.8 the extracting of plasmid

Undertaken by Beijing match Parkson gene engineering company limited plasmid extraction test kit specification sheets, reclaim product and be stored in-20 ℃ and save backup.

3.1.9 PCR and the enzyme of recombinant plasmid are cut evaluation

PCR and the enzyme of recombinant plasmid are cut evaluation

With the recombinant plasmid called after pMD18-gGM of previous step extracting, respectively with BamH I/Xho I double digestion digestion and the digestion of Xho I single endonuclease digestion, 1.0% gel electrophoresis observations.Do simultaneously the pcr amplification goal gene.

3.1.10 gG intercept gene sequencing

Send Shanghai English fine horse biology company limited and check order identifying correct plasmid.

3.2 the optimization of the structure of prokaryotic expression plasmid pET32a-gGM, abduction delivering and expression condition

3.2.1 the Construction and identification of prokaryotic expression plasmid pET32a-gG

The enzyme of a purpose fragment is cut and is connected: restriction enzyme BamH I and Xho I be double digestion pMD18-gGM plasmid and prokaryotic expression carrier pET32a (+) respectively, and the enzyme system of cutting is:

37 ℃ of water-bath 4 h after reclaiming respectively the purpose fragment by DNA recovery test kit operation instruction, spend the night according to 16 ℃ of connections of following linked system.

The conversion of b recombinant plasmid: adopt Calcium Chloride Method to prepare DH5 α competent cell.Afterwards, get connecting fluid 15 μ L and be added in the centrifuge tube that contains 200 μ L competence DH5 α, ice bath 30min after mixing; Be placed in 42 ℃ of water-bath 90sec, then rapid ice bath 2min; Add the LB liquid nutrient medium 800 μ l that do not contain Amp, 1～1.5 h is cultivated in 37 ℃ of joltings (150r/min); Get 200 μ l cultures and coat the LB flat board that contains Amp, 37 ℃ of overnight incubation, the single colony inoculation of picking next day is in the LB of 5mL liquid nutrient medium, cultivate 12～16h for 37 ℃, set up simultaneously empty carrier conversion group (empty carrier 10 μ L+competence DH5 α 200 μ L), carrier free control group (sterilization ultrapure water 10 μ L+competence DH5 α 200 μ L).

The enzyme of c recombinant plasmid is cut with PCR and is identified: clone's bacterial classification of above-mentioned preservation is inoculated in the LB liquid nutrient medium that 5mL contains Amp, 37 ℃ of water-bath jolting overnight incubation, extract according to a conventional method recombinant plasmid next day, then identify this recombinant plasmid with Xho I single endonuclease digestion, BamH I and Xho I double digestion, it is as follows that its enzyme is cut system:

Then, take recombinant plasmid as template, the upstream primer as shown in SEQ ID NO:3: 5 '- GgatccCacacaggacgatatgag-3 ' (the line part is BamH I site); Downstream primer as shown in SEQ ID NO:4: 5 '- CtcgagAtgagggtgattgtggtag-3 ' (the line part is Xho I site) carried out the PCR reaction, and its method and amplification condition are the same, get PCR product electrophoresis detection on 1% sepharose.Cut with PCR through enzyme and identify, obtain recombined pronucleus expression plasmid pET32a-gGM.

3.2.2 the abduction delivering of recombinant expression plasmid pET32a-gGM

The extraction of a recombinant plasmid pET32a-gGM: picking has identified that the DH5 α bacterial classification streak inoculation that contains positive recombinant plasmid pET32a-gGM is on the LB agar plate that contains Amp, 37 ℃ of overnight incubation, get single colony inoculation next day on the LB liquid nutrient medium, thermal agitation was cultivated 10～16 hours, centrifugal collection bacterium liquid extracts by the U1traPureTM plasmid DNA extraction and purification that recombinant plasmid is carried out in the test kit explanation in a small amount.

B recombinant plasmid pET32a-gGM transforms and expresses bacterium: adopt Calcium Chloride Method to prepare E.coli BL21 (DE3) competent cell, and the recombinant plasmid pET32a-gGM of said extracted is transformed in expressive host bacterium E.coli BL21 (DE3).

the abduction delivering of c recombinant plasmid pET32a-gGM: from above-mentioned LB solid medium, picking positive colony bacterium, inoculation LB liquid nutrient medium, 37 ℃ of overnight incubation, getting bacterium liquid next day contains in the LB liquid nutrient medium of Amp in the access of the ratio of 1:50, when thermal agitation is cultured to OD600=0.4, add respectively the IPTG inducing culture, collect 1mL and cultivate bacterium liquid, 4 ℃ of centrifugal 2 min of 13000r/min, abandon supernatant, add 40 μ L ultrapure waters and 10 μ L 10 * SDS sample-loading buffers in precipitation, 100 ℃ of heating in water bath sex change 10min, carry out the 12%SDS-PAGE gel electrophoresis, observe expression of results.

The soluble analysis of d recombinant plasmid pET32a-gGM expression product: with the bacterium liquid of abduction delivering and the bacterium liquid of abduction delivering not, press respectively step process: 4 ℃, the centrifugal 5min of 10000r/min, bacterial sediment 20mL 20mmol Tris-HCl(pH8.0) suspend; Put-20 ℃ spend the night after, adding N,O-Diacetylmuramidase to final concentration is 1mg/mL, 4 ℃ are stirred 30min, ultrasonic wave (ice bath) is broken thalline (600w, 30sec/ time, 10 times) intermittently, 4 ℃, 1. the centrifugal 10min of 10000r/min get supernatant standby; Precipitation suspends with 10mL washing lotion (10mmol/L PBS+2mol/L urea+0.2% Triton X-100), 4 ℃, after the centrifugal 10min of 10000r/min, precipitation suspends with the 10mL washing lotion again, after repeated washing three times, with appropriate urea soln (10mmol/L PBS+8mol/L urea) dissolution precipitation 2., cryopreservation is standby.Get respectively appropriate supernatant 1. with the precipitation of urea soln dissolving 2., add wherein 40 μ L ultrapure waters and 10 μ L10 * SDS sample-loading buffer, 100 ℃ of heating in water bath sex change 10min carry out the 12%SDS-PAGE gel electrophoresis, with gel with coomassie brilliant blue staining after, observations.And will dye lustful gel and induce in bacterium liquid recombinant protein relative percentage composition of (precipitating 2. the inclusion body form) in endochylema (supernatant 1., solubility) and precipitation through full automatic gel imaging analysis system scan and Quantity One software analysis.

3.2.3 the optimization of recombinant plasmid pET32a-gGM expression condition

The concentration optimization of a inductor IPTG: get the expressive host bacterium BL21 (DE3) that contains recombinant plasmid pET32a-gGM, be inoculated in the LB liquid nutrient medium that contains Amp 37 ℃ of jolting overnight incubation.Turn by 1:50 next day and be inoculated in the LB liquid nutrient medium that contains Amp, 37 ℃ of cultivations were cultured to the OD600 value at approximately 0.4 o'clock, get wherein 6 test tubes, add respectively IPTG to final concentration be after 30 ℃ of 0mmol/L, 0.2mmol/L, 0.5mmol/L, 0.8mmol/L, 1.0mmol/L, 1.2mmol/L induce 2h, as stated above sample is processed, the 12%SDS-PAGE electrophoresis, observations.

The b temperature condition is optimized: get the expressive host bacterium BL21 (DE3) that contains recombinant plasmid pET32a-gGM, be inoculated in the 5mL LB liquid nutrient medium that contains Amp 37 ℃ of jolting overnight incubation.Turn by 1:50 next day and be inoculated in the LB liquid nutrient medium that contains Amp, 37 ℃ of cultivations were cultured to the OD600 value at approximately 0.4 o'clock, get wherein 3 test tubes, add respectively IPTG to final concentration be 0.2mmol/L, be placed in respectively 25 ℃, 30 ℃, 37 ℃ and induce 2h, by above-mentioned ordinary method, sample is processed 12%SDS-PAGE electrophoresis, observations.

The c induction time is optimized: get the expressive host bacterium BL21 (DE3) that contains recombinant plasmid pET32a-gGM, be inoculated in the LB liquid nutrient medium that contains Amp 37 ℃ of jolting overnight incubation.Turn by 1:50 next day and be inoculated in the LB liquid nutrient medium that contains Amp, continued to be cultured to the OD600 value at approximately 0.4 o'clock, add IPTG to final concentration be 0.2mmol/L, 30 ℃ induce respectively 0,2,3,4,5,6h, spend the night after, draw the 1mL nutrient solution, as stated above sample is processed 12%SDS-PAGE electrophoresis, observations.

3.3 a large amount of preparations, purifying and the renaturation of duck plague virus gG truncated recombinant protein

3.3.1 a large amount of preparations of duck plague virus gG truncated recombinant protein (inclusion body processing)

With the 500mL bacterium liquid of abduction delivering in 4 ℃ of centrifugal 10min of 10000r/min, with bacterial sediment 40mL 20mmolTris-HCl(pH8.0) suspend; Put-20 ℃ and add N,O-Diacetylmuramidase by 1mg/mL after spending the night, 4 ℃ are stirred 30min, and ultrasonic wave (ice bath) is broken thalline (200w, 30sec/ time, 5～10 times) intermittently, 4 ℃ of centrifugal 10min of 10000r/min.To precipitate with 20mL washing lotion (10mmol/L PBS+2mol/L urea+0.2% Triton X-100) suspension, after 4 ℃ of centrifugal 10min of 10000r/min, with appropriate urea soln (10mmol/L PBS+8mol/L urea) dissolution precipitation, 4 ℃ save backup.

3.3.2 the purifying of duck plague virus gG truncated recombinant protein

Nickel sepharose (Ni ²⁺-NAT) affinitive layer purification duck plague virus gG truncated recombinant protein: the affinity interaction special to nickel ion according to the 6-His label of fusion rotein, fusion rotein with label can be incorporated on the nickel sepharose, change the condition of elutriant with its wash-out, and reach the purpose of purifying.Concrete operation step is:

(1) dress post: nickel sepharose dress post, column volume is about 40mL;

(2) balance: use approximately 5 bed volume balance chromatography columns of level pad I, flow velocity is 1mL/min;

(3) loading: with the about 5mL of the solubility inclusion body sample of 0.45 μ m membrane filtration, add in chromatography column, flow velocity is 0.5mL/min;

(4) washing: wash 2-5 bed volume with the level pad II, flow velocity is 1mL/min again;

(5) wash-out: respectively with contain 50,300, the elution buffer III of 500mmol imidazoles carries out gradient elution, flow velocity is 1mL/min, collects the elution peak of each gradient, detects molecular size range and the purity of fusion rotein with SDS-PAGE;

(6) clean: with 5 column volumes of ultrapure washing, then wash 3 column volumes with 25% ethanol, flow velocity is 1mL/min, reclaims nickel sepharose post, preserves in 4 ℃.

4 experimental results

4.1 the amplification of duck plague virus gG truncated gene, T-cloning and identification result

4.1.1 the pcr amplification result of duck plague virus gG truncated gene

Take DPV CHv strain genomic dna as template, gG intercept gene is carried out pcr amplification, its product has obtained the specific DNA band (Fig. 1) of treaty 930 bp through 1.0% agarose gel electrophoresis.

4.1.2 gG intercept gene T clone identification result

The PCR product is connected with the pMD18-T carrier and transformed competence colibacillus cell DH5 α after glue reclaims purifying, the T that obtains clone called after pMD18-gGM.PMD18-gGM is carried out PCR, enzyme cut that (Fig. 2: recombinant plasmid pMD18-gGM obtains two fragments with BamH I and Xho I double digestion, the molecular size range of small segment is about 930 bp) and order-checking identify, result shows that the gG intercept gene DNA sequence (shown in SEQ ID NO:2) that the T clone obtains is in full accord with known duck plague virus gG truncated gene DNA sequence.

4.2 the Construction and identification of prokaryotic expression plasmid pET32a-gGM, abduction delivering and optimum result thereof

4.2.1 structure and the enzyme of recombinant expression plasmid pET32a-gGM are cut evaluation

With reclaiming the purpose fragment after BamH I and Xho I double digestion T cloned plasmids, be connected with the pET-32a expression vector of cutting through same enzyme and transform DH5 α, obtain recombinant expression plasmid pET32a-gGM, obtain two bar segment after BamH I and Xho I double digestion, obtain a bar segment after Xho I single endonuclease digestion, and size conforms to theoretical value, shows recombined pronucleus expression plasmid construction success (Fig. 3).

4.2.2 the abduction delivering of recombinant plasmid pET32a-gGM

Inducible strain does not have the specific proteins band to occur; Inducing empty carrier pET-32a (+) to transform bacterial strain and a specific proteins band occurs at about 20kDa place, is that carrier itself is big or small; The recombination fusion protein that recombinant expression plasmid pET32a-gGM expresses conforms to theoretical value at about 50KDa place; In addition, expressing protein soluble analysis result shows that it mainly is present in precipitation, illustrates that there be (Fig. 4) in recombinant expression protein with insoluble inclusion body form in thalline.

4.2.3 the optimization of recombinant plasmid pET32a-gGM expression condition

The optimization of a IPTG concentration: under 30 ℃ of conditions, add IPTG to make its final concentration be respectively 0 mmol/L, 0.2 mmol/L, 0.5 mmol/L, 0.8 mmol/L, 1.0 mmol/L, 1.2 mmol/L inducing culture 2h, result shows: do not add the control tube of inductor without the specific proteins band; With increasing of IPTG concentration, protein induced amount does not have considerable change (Fig. 5).Therefore, consider the saving reagent material, select the IPTG concentration of 0.2 mmol/L as abduction delivering concentration.

The optimization of b inducing temperature condition: the IPTG final concentration is 0.2mmol/L, induces 2h, the result demonstration respectively at 25 ℃, 30 ℃, 37 ℃, during 25 ℃ and 37 ℃, the inducible protein amount is less, and the highest in the time of 30 ℃ when temperature, therefore, select 30 ℃ of best inducing temperatures of conduct of temperature (Fig. 6).

The optimization of c induction time: be 0.2 mmol/L in IPTG concentration, under 30 ℃ of conditions, induce respectively 0h, 2h, 3h, 4h, 5h, 6h, spend the night, a large amount of protein expressions is arranged during 2 h as a result, and induce 2-6 h and induce the expression of recombinant proteins amount of spending the night to compare and change little (Fig. 7), therefore, select 2h as best induction time.

The purifying of embodiment 2 duck plague virus gG truncated recombinant proteins

The expression product that contains duck plague virus gG truncated recombinant protein that embodiment 1 is made through a series of pre-treatments such as lysozyme lysis, ultrasonic disruption, urea washings after, nickel sepharose affinitive layer purification restructuring gG albumen.The UV graphic representation that protein sample is crossed after column purification demonstrates 3 peaks, and peak 1 is for penetrating the peak, and peak 2 is 50mmol imidazoles elution peak, and peak 3 is 300mmol imidazoles elution peak.Collect simultaneously different concns imidazoles elution peak, carry out the SDS-PAGE electrophoresis, check purity and concentration, result shows: only have and contain a large amount of highly purified gG intercept recombinant proteins (Fig. 8) in 300mmol imidazoles elution peak.After gG intercept recombinant protein dialysis renaturation with purifying, measure the final concentration of albumen with the Bradford method, and its dilution is 1mg/mL, standby after packing.

Embodiment 3 duck plague virus gG truncated recombinant proteins are as the indirect ELISA reagent kit of duck plague virus antibody capture agent

Duck plague virus gG truncated recombinant protein mainly describes by indirect ELISA reagent kit as the application of duck plague virus antibody capture agent.

Solid phase carrier: solid phase carrier as sorbent material and container, does not participate in chemical reaction in ELISA mensuration process.Can make in ELISA the material of solid phase carrier a lot, as polystyrene, it must satisfy the performance with stronger adsorbed proteins, still keeps original immunologic competence after antibody or proteantigen adsorb on it, can be made into various forms.The shape of ELISA carrier mainly contains three kinds: microtiter plate, globule and small test tube.The most commonly used with microtiter plate, the product that is exclusively used in EILSA is called elisa plate, and the microtiter plate of standard is 8 * 12 96 cellular types in the world.For ease of doing the detection of a small amount of sample, have to make 8 hole bars or 12 hole bars, after putting into mounting, size is identical with the standard ELISA plate.The characteristics of elisa plate are to carry out simultaneously the detection of a large amount of samples, and can read rapidly result on special colorimeter.The ELISA that now existing multiple self-reacting device is used for the microtitration template detects, and comprises the steps such as application of sample, washing, insulation, colorimetric, and is very favourable to the stdn of operation.

Antibody capture agent: because complicacy and the purity of the totivirus purification process of duck plague virus are not ideal enough, hindered the totivirus of duck plague virus as the large-scale application of antibody capture agent as the ELISA method (DPV-ELISA) of coated totivirus.The antibody capture agent of adopting in the present invention is the duck plague virus gG truncated recombinant protein after embodiment 2 purifying.

ELIAS secondary antibody: the ELIAS secondary antibody that adopts in the present invention is goat-anti duck IgG-HRP (the goat-anti duck IgG of horseradish peroxidase mark), also can adopt the anti-duck IgG-HRP of other non-duck animals, except with the horseradish peroxidase mark, also can adopt other enzyme labellings, as phosphoesterase.

Substrate: substrate can be selected tetramethyl benzidine (TMB), O-Phenylene Diamine, and 2,2-azino-two-(3-ethyl benzo thiazole phenanthroline-6-sulfonic acid) diamino salt (ABTS) and the coloured binding substances of other HRP are as substrate.To adopt binding substances coloured with it as substrate when adopting other enzyme labellings.

Confining liquid: sealing is the process of using the irrelevant protein solution of high density to be coated with again after coated.Concentration used when antigen or antibody sandwich is lower, and after absorbing, surface of solid phase carriers still has the space that is not occupied, and sealing is exactly to allow a large amount of incoherent protein fillings these spaces, thereby repels the absorption again of interfering substance in ELISA step thereafter.The formality of sealing is with coated similar.The most frequently used encapsulant is that volume fraction is the bovine serum albumin of 0.05%-0.5%, and also useful 10% calf serum or 1% gelatin are as encapsulant, and skim-milk is also a kind of good encapsulant.

One experimental technique

1 indirect ELISA step

1. the duck plague virus gG truncated recombinant protein of purifying being diluted to final concentration with coating buffer is 2.5 μ g/100 μ L, adds successively in enzyme plate l00 μ L/hole, 4 ℃ of coated spending the night;

2. wash three times, abandon liquid, add 200 μ L confining liquids in every hole, 37 ℃ of sealing 30min;

3. wash three times, abandon liquid, with after each serum to be checked dilution and loading, hatched 30 minutes for 37 ℃ in 100 μ L/ holes;

4. wash three times, abandon liquid, add the goat-anti duck IgG-HRP ELIAS secondary antibody of dilution in every hole, hatched 30 minutes for 37 ℃ in 100 μ L/ holes;

5. wash three times, abandon liquid, add TMB100 μ L in every hole, then lucifuge colour developing 10 minutes adds stop buffer 50 μ L/ holes, pats mixing, surveys OD450 with microplate reader _nmValue.

The 2 best coated concentration of antigens and antibody is determining of the suitableeest extension rate

With duck plague virus gG truncated recombinant protein with coating buffer doubling dilution and coated elisa plate, 100 μ L/holes; DPV positive serum and negative serum are used respectively antibody diluent volume doubling dilution (1:40,1:80,1:160,1:320,1:640), then add in enzyme plate, 100 μ L/holes.Test with the square formation method, select P/N (positive serum OD _450nmValue/negative serum OD _450nmWhen value) maximum, and P at the coated concentration of the antigen (antibody capture agent) of 1.0 left and right and serum diluting multiple as the antigen coated concentration of the best and the suitableeest antibody dilution multiple.

3 enzyme labelled antibody best effort concentration determinations

Coated concentration best according to fixed antigen and the suitableeest extension rate of antibody, goat-anti duck IgG-HRP ELIAS secondary antibody (available from U.S. KPL company) is made different volumes multiple (1:1000,1:2000,1:4000) dilution is gone forward side by side and is connect the ELISA test in the ranks, each extent of dilution repeats 3 times, obtain mean P value and N value, thereby determine the best effort concentration of goat-anti duck IgG-HRP ELIAS secondary antibody.

Determining of 4 yin and yang attribute threshold value criterion

Get the negative duck serum of 20 parts of DPV, detect according to the indirect ELISA method of above-mentioned foundation.OD with 20 parts of negative samples _450nmThe mean value (X) of value and 3 times of standard variances (SD) sum are as the upper limit of negative sample value, the i.e. OD of sample to be checked _450nmValue＞negative sample OD _450nmBe judged to be the positive during threshold value of value (X+3SD), on the contrary negative.

5 specific tests

with the duck plague virus gG truncated recombinant protein of the purifying of embodiment 2 preparation as envelope antigen, by the indirect ELISA method set up respectively with itself and duck plague virus positive serum, duck viral hepatitis (Duck virus hepatitis, DHV) positive serum, avian influenza virus (AIV) positive serum, infectious serositis of duck (Riemerella anatipestifer infectious, RA) positive serum, Salmonella anatis (Salmonella bacillus, SB) positive serum, the reaction of E. coli isolated from ducks (E.coli) positive serum, to detect its specificity.

6 stability tests

With the duck plague virus gG truncated recombinant protein coated elisa plate with the purifying of a collection of preparation, get respectively three parts of serum to be checked, detect according to the indirect ELISA method of setting up, every duplicate samples repeats 3 holes, duplicate detection 4 times, repeatability in judging batch.Get the gG intercept recombinant protein of the purifying of different time preparation again, after coated, sealing, three parts of serum to be checked are detected, every part of serum detects 3 holes, and duplicate detection 4 times is repeated between judging batch.

6 sensitivity tests

Duck plague virus gG truncated recombinant protein with the purifying of embodiment 2 preparation is coated with as antigen (antibody capture agent) and by the coated concentration of the best, the DPV positive serum is carried out the volume doubling dilution by 1:80,1:160,1:320,1:640,1:1280, l:2560,1:5120 respectively, then carry out the indirect ELISA test.

Two experiment conclusion

Determining of the coated concentration of 1 antigen (antibody capture agent) and serum dilution

By the square formation burette test, the detected result of standard positive serum and standard female serum respectively as shown in Table 1 and Table 2, when the coated concentration of antibody capture agent is 2.5 μ g/100 μ L, when serum dilution volume multiple is 1:80, positive serum OD _450nmValue is 1.238, corresponding negative serum OD _450nmValue is 0.157, P/N value maximum (7.885).Therefore, the best coated concentration of antibody capture agent is 2.5 μ g/100 μ L, and antibody is the most fit, and long-pending extension rate is 1:80.

Determining of 2 enzyme labelled antibody optimal concentrations

The results are shown in Table 3, when enzyme mark goat-anti duck IgG antibody was made the 1:2000 volume dilution, the P/N value was 8.301 to the maximum, determined that therefore best enzyme labelled antibody volume dilution multiple is 1:2000.

Determining of 3 yin and yang attribute threshold value criterion

By 20 parts of DPV negative serum samples are detected, acquired results is as shown in table 4.The X value is that 0.124, SD value is 0.032, determines that the yin and yang attribute stagnation point is X+3SD=0.124+3 * 0.032=0.22, therefore, and as the OD450 of testing sample _nmValue was judged to be the positive greater than 0.22 o'clock, was judged to be feminine gender when being less than or equal to.

4 specific tests

By the indirect ELISA method of having set up, 6 kinds of positive serums that comprise duck plague are measured respectively, result is as shown in table 5, except the duck plague virus positive serum, and the OD450 of all the other 5 kinds of positive serums _nmValue is all less than 0.22, show except the duck plague virus positive serum, this antigen not with above-mentioned other 5 kinds of positive serum generation cross reactions, what explanation was set up thus has specificity preferably with duck plague virus gG truncated recombinant protein as the ELISA method of Detection of antigen DPV antibody.

5 stability tests

Respectively with the duck plague virus gG truncated recombinant protein coated elisa plate of the purifying of same batch of preparation and different batches preparation, then with these two kinds of albumen, three parts of serum to be checked are carried out the ELISA detection respectively, by the 0D450 of every part of serum of gained _nmValue calculates average 0D450nm value and standard variance (SD), then calculates batch interior and interassay coefficient of variation (CV) of every part of serum, and result is as shown in table 6.In batch, three groups of serum variation lines number averages of revision test are lower than 10%, between batch, the variation coefficient of three groups of serum of revision test does not all surpass 10% yet, illustrates that thus the indirect ELISA method based on duck plague virus gG truncated recombinant protein that this research is set up has stability preferably.

6 sensitivity tests

Result is as shown in table 7, along with the dilution increase of DPV positive serum, OD450 _nmValue also obviously descends thereupon, and when extension rate was 1:2560, the serum detected result was worth lower than feminine gender, and therefore, the duck plague virus gG truncated recombinant protein of the purifying for preparing with this research is 1:1280 as the minimum positive serum extension rate that detects of antigen.

Dilution in above-described embodiment is all carried out with the volume multiple proportions.

Explanation is at last, above embodiment is only unrestricted in order to technical scheme of the present invention to be described, although by invention has been described with reference to the preferred embodiments of the present invention, but those of ordinary skill in the art is to be understood that, can make various changes to it in the form and details, and not depart from the spirit and scope of the present invention that appended claims limits.

＜110〉Sichuan Agricultural University laboratory animal engineering center

＜120〉duck plague virus gG truncated recombinant protein and preparation method thereof

<160> 4

<210> 1

<211> 310

<212> PRT

＜213〉duck (duck)

<220>

＜223〉duck plague virus gG truncated recombinant protein

<400> 1

His Thr Gly Arg Tyr Glu Asp Ile Gly Thr Met Asp Ile Ala Val

1 5 10 15

Qln Trp Cys Asp Leu Thr Leu Leu Ala Pro Ile Ser Ser Val Ser

20 25 30

Ala Gly Asn Gly Ala Pro Tyr Asp Met Ser Val Thr Trp Tyr His

35 40 45

Ile Thr Ala Asn Cys Thr Met Pro Val Gly Tyr Val Glu Tyr Tyr

50 55 60

Asn Cys Ser Gly Asp Qln Pro Ser Val Trp Thr Cys Gly Gly Tyr

65 70 75

Ser His Ile Tyr Ser Tyr Tyr Ile Asp Gly Qln Met Asp Thr Leu

80 85 90

Pro Tyr Gly Thr Gly Leu Ile Ile Ser Ser Gly Met Tyr Asn Ser

95 100 105

Gly Leu Tyr Lys Phe Ile Leu Arg Thr Asp Asn Asn Thr Lys Thr

110 115 120

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

125 130 135

Val Asn Lys Phe Gly Arg Thr Ser Gly Ala Asp Leu Asp Ile Val

140 145 150

Asp Ile Gly Thr Trp Met Ser Pro Asp Gly Leu Pro His Pro Asn

155 160 165

Thr Tyr Asn Ile Ser Thr Lys Asp Gly Lys Ile Arg Phe Ser Phe

170 175 180

Qln Asn Gly Thr Glu Thr Val Cys Qln Leu Val Lys Ser Leu Glu

185 190 195

Glu Ile Arg Asp Glu Ser Glu Ser Trp Asp Glu Arg Asn Val Asp

200 205 210

Pro Ala Met Leu Gly Phe Pro Arg Cys Gly Tyr Glu Asp Tyr Gly

215 220 225

Ser Thr Asp His Gly Ser Glu Cys Tyr Asp Asp Ile Asp Asn Glu

230 235 240

Cys Val Asn Glu Thr Asp Ile Trp Thr Val Pro Lys Thr Asn Ala

245 250 255

Asn Phe Arg Asn Qln Thr Gly Gly Tyr Leu Leu Leu Ala Ser Phe

260 265 270

Met Val His Ser Thr Gly Arg Met Pro Arg Ile Gly Leu Lys Met

275 280 285

Ala Asn Leu Ser Thr Cys Thr Thr Ile Asn Thr Thr Ser Thr His

290 295 300

Ser His Gly Ser Tyr His Asn His Pro His

305 310

<210> 2

<211> 930

<212> DNA

＜213〉duck (duck)

<220>

＜223〉duck plague virus gG truncated gene

<400> 2

cacacaggac gatatgagga cattggaacg atggatattg cagttcaatg gtgcgacctt 60

acactcctcg cgcctatttc cagtgtttcc gccgggaatg gtgcgccata tgacatgtct 120

gttacttggt atcacatcac cgctaattgc acgatgcctg ttggctacgt ggaatactac 180

aactgctctg gggatcagcc gtctgtatgg acctgtggcg gctattcgca tatttattcc 240

tattatattg atggacaaat ggacacgcta ccatatggta ctggcttgat tatatctagt 300

gggatgtata atagtggact atataagttt attctaagaa cagacaataa tacgaagact 360

ggaacactaa atgtaacttt tgaaacatca gacgatcctt attgcgttaa taaatttggc 420

agaacgtctg gcgctgacct ggacattgtt gatataggta cgtggatgtc tccagatggt 480

ttgccccatc ccaatacgta taatattagt acgaaagacg ggaagatacg atttagtttc 540

caaaatggca cagagaccgt ttgtcaactg gtgaagagtc tcgaagaaat cagggacgag 600

agtgagtctt gggatgaacg caatgtcgat ccggccatgc ttggtttccc ccggtgtggt 660

tatgaagact acggctctac agatcatgga tctgaatgct atgatgacat agataatgaa 720

tgtgtaaatg agacggacat atggacagta cctaaaacca atgccaattt cagaaatcaa 780

actggtgggt atttgttact cgccagtttc atggtacatt ctactggaag gatgccgcgg 840

attggtctta aaatggcgaa cctatcgaca tgtacaacta ttaataccac ctcaacacat 900

tcgcatggaa gctaccacaa tcaccctcat 930

<210> 3

<211> 24

<212> DNA

＜213〉artificial sequence

<220>

＜223〉primer DPV-gGM F

<400> 3

ggatc ccaca cagga cgata tgag 24

<210> 4

<211> 25

<212> DNA

＜213〉artificial sequence

<220>

＜223〉primer DPV-gGM R

<400> 4

ctcga gatga gggtg attgt ggtag 25

Claims

1. duck plague virus gG truncated recombinant protein, it is characterized in that: its aminoacid sequence is as shown in SEQ ID NO:1.

2. duck plague virus gG truncated recombinant protein according to claim 1, it is characterized in that: its DNA sequence dna is as shown in SEQ ID NO:2.

3. the preparation method of duck plague virus gG truncated recombinant protein claimed in claim 1 is characterized in that:

A, take the DPV genomic dna as template, adopt the nucleotide sequence duck plague virus gG truncated gene fragment that increases to get of the upstream and downstream primer PCR as shown in SEQ ID No.3 and SEQ ID No.4 respectively, be connected with pMD18-T, get pMD18-gGM;

4. the preparation method of duck plague virus gG truncated recombinant protein according to claim 3, it is characterized in that: step C gained duck plague virus gG truncated recombinant protein crude product after nickel sepharose affinitive layer purification, is got duck plague virus gG truncated recombinant protein.

5. the preparation method of duck plague virus gG truncated recombinant protein according to claim 3, it is characterized in that: the Host Strains in step C is bacterial strain BL21 (DE3), at IPTG concentration 〉=0.2mmol/L, inducing temperature is to carry out abduction delivering 2 hours under 30 ℃ of conditions, gets duck plague virus gG truncated recombinant protein.