CN103014050A - Recombinant porcine amelogenin and preparation method thereof - Google Patents

Abstract

The invention provides a recombinant porcine amelogenin which is encoded by a full-length porcine amelogenin gene. A preparation method comprises the following steps of: synthesizing the full-length porcine amelogenin gene; constructing a recombinant prokaryotic expression plasmid PGEX4T1-pAm including the above gene; converting a strain E.coli.BL21 by the obtained recombinant prokaryotic expression plasmid PGEX4T1-pAm; inducing the obtained recombinant strain to express a fusion protein GST-pAm; and cutting the GST tag protein of the obtained fusion protein GST-pAm, thereby obtaining the recombinant porcine amelogenin pAm. The encoding sequence of the full-length recombinant porcine amelogenin is synthesized for the first time, and the fusion protein is successfully expressed in the escherichia coli by the prokaryotic expression plasmid. The cloned expression strain obtained according to the invention can be stored for a long time and can be copied in quantity at any time, a large number of recombinant porcine amelogenin can be artificially synthesized faster and more conveniently, and the possibility is provided for the mass production and the commercialization of the amelogenin.

Description

A kind of Recombinant Swine amelogenin and preparation method thereof

Technical field

The present invention relates to a kind of albumen, relate in particular to a kind of Recombinant Swine amelogenin and preparation method thereof.

Background technology

ENAMELIN (enamel matrix proteins, EMPs) be tooth development to bell stage, the enameloblast that is come by the inner enamel epithelium cytodifferentiation is synthetic and secrete a series of extracellular matrix proteins.Put forward first after the enamel associated protein of He Teweishi epithelial root sheath can induce the acellular property of tooth root dental cement to form from 1975, experiment in vitro and clinical treatment confirm that extensively EMPs can effectively promote periodontal ligament, dental cement, Regeneration of Alveolar Bone, form the arrangement of similar normal periodontal tissue, reach real periodontal regenerative.The topmost composition of EMPs---amelogenin (amelogenin, Am) in the growth also is the main active ingredient that promotes periodontal regenerative among the EMPs.But the composition of Am is not single, has 9 kinds of compositions at least, molecular weight ranges 5～27kD.Being widely used at present the fundamental research of periodontal regenerative and the EMPs of clinical application is the porcine enamel matrix proteins that extracts substantially, and its main composition is that molecular weight is the Am of 20kD, 13kD, 11kD.

The porcine enamel matrix proteins that extracts mainly is the mixing element of several amelogenins, and its extraction efficiency and component stability influence are to the function of albumen.Secondly the pig EMPs that extracts is heterology albumen, may have immunogenic problem.Unique ENAMELIN commercial goods---the effective constituent of Emdogain also is the derivative of the amelogenin of extraction from pig tooth bud tissue at present.Its complete dependence on import, fetch long price, it is higher to be used for fundamental research and the required cost of clinical treatment.

The present domestic pig amelogenin (pAm) that there is not yet the one-component of synthetic.Am is the expression product of Am gene in the enameloblast.Pig has two Am genes, is positioned respectively on X chromosome and the Y chromosome.Reverse transcription PCR confirms that main transcript comes from X chromosome, and the amelogenin gene on the Y chromosome is also transcribed, accounts for 10% of total transcript.Pig Am gene comprises 7 exons at least, has the mode that has at least 9 kinds to select shearing, and the content of nine kinds of different Am in tissue of coding is also unequal.It is modal a kind of cut modes that exon 4 is removed in shearing, the most common in the enamel of growing by the Am of its coding.The main component that studies confirm that ripe pig Am is the translation product of Am gene on the X chromosome---the Mr1.96 that formed by 175 amino-acid residues * 10 ⁴Albumen.

Summary of the invention

The object of the present invention is to provide a kind of Recombinant Swine amelogenin and preparation method thereof, and contain the recombinant prokaryotic expression vector of total length pig amelogenin gene and the coli strain that is transformed by this recombinant prokaryotic expression vector.

The invention provides a kind of preparation method of Recombinant Swine amelogenin, may further comprise the steps:

Step 1, synthetic total length pig amelogenin gene;

Step 2 makes up the recombined pronucleus expression plasmid PGEX4T1-pAm that contains said gene;

Step 3, the recombined pronucleus expression plasmid PGEX4T1-pAm that step 2 is obtained transforms bacterial strain E.coli. BL21;

Step 4, the recombinant bacterial strain expressed fusion protein GST-pAm that induces step 3 to obtain;

Step 5, the GST label protein of the fusion rotein GST-pAm that excision step 4 obtains obtains Recombinant Swine amelogenin pAm.

Wherein, the base sequence of the pig of total length described in the step 1 amelogenin gene is shown in SEQ ID NO.1.

Preferably, in step 2, make up recombined pronucleus expression plasmid PGEX4T1-pAm and adopt restriction endonuclease ECoR I and Sal I, enzyme is cut primer and is respectively:

5 '-CCGGAATTCATGCCTCTACCACCTCATCCTG-3 ' and

5’-ACGCGTCGACTTAATCCACTTCCTCCCG CTTG-3’。

Preferably, in the step 4, induce the recombinant bacterial strain expressed fusion protein GST-pAm of step 3 acquisition by isopropylthio-β-D galactoside (IPTG).

Preferably, step 5 is: the fusion rotein GST-pAm that purification step 4 obtains, excision GST label protein, the Recombinant Swine amelogenin pAm of acquisition purifying.

Wherein, the fusion rotein GST-pAm of step 4 acquisition is by the GST affinitive layer purification.

Preferably, excision GST label protein adopts zymoplasm in the step 5.

The invention provides a kind of Recombinant Swine amelogenin of aforesaid method preparation.

Described Recombinant Swine amelogenin is by total length pig amelogenin gene (pAm gene) coding, and wherein, the base sequence of described total length pig amelogenin gene is shown in SEQ ID NO.1.

The invention provides a kind of recombinant prokaryotic expression vector that contains total length pig amelogenin gene, wherein, in prokaryotic expression plasmid PGEX4T1, include SEQ ID NO.1 nucleotide sequence.

The coli strain that the present invention also provides a kind of above-mentioned recombinant prokaryotic expression vector that contains total length pig amelogenin gene to transform.

Use the expressed fusion rotein of PGEX4T series plasmid with glutathione s-transferase (GST) mark, GST itself is transferring enzyme important in the detoxification processes, utilize the solubility that it increases foreign protein because it is highly solvable, and it can great expression play a kind of effect that promotes that expression amount improves in intestinal bacteria.Between GST and multiple clone site, exist simultaneously and can with the proteolytic enzyme recognition site of GST excision, GST can be excised.E.coli. BL21 is that specialized designs is used for the Host Strains at expression in escherichia coli heterology albumen, and it can accurately control the advantage of expression level by changing the IPTG induced concentration, and has lon and ompT defective type characteristics, can increase protein stability.According to the needs of research purpose, the encoding sequence of synthetic total length pig amelogenin first, and by prokaryotic expression plasmid in intestinal bacteria successful expression fusion rotein.But the clonal expression bacterial strain prolonged preservation of this invention gained, and massive duplication at any time, a large amount of synthetic Recombinant Swine amelogenins (pAm) comparatively conveniently, also for future amelogenin mass production and commercialization provide may.

Description of drawings

Fig. 1 is the structural representation of prokaryotic expression carrier plasmid PGEX4T1.

Fig. 2 is total length pig amelogenin gene PCR amplified production agarose gel electrophoresis figure: M is dna profiling, and 1 is total length pig amelogenin gene amplification product, 2 negative contrasts.

Fig. 3 identifies that for the restructuring plasmid enzyme restriction electrophorogram: M is dna profiling; 1 is goal gene fragment pAm, 2 be plasmid PGEX4T1 through EcoR I and Sal I double digestion sample, 3 is that recombinant plasmid is through EcoR I and Sal I double digestion sample.

Fig. 4 is Recombinant Swine amelogenin SDS-PAGE gel electrophoresis figure: M is the lower molecular weight standard protein; C is the sample after the control strain that transformed blank plasmid PGEX4T1 is induced; 1 ~ 5 is respectively the sample after 1 ~ No. 5 cloning by expression is induced, and M is the lower molecular weight standard protein.

Fig. 5 is that the purification result figure of Recombinant Swine amelogenin: A is the SDS-PAGE electrophorogram, and B is Western-blot evaluation figure.Wherein Marker is the lower molecular weight standard protein, and rPAm is the Recombinant Swine amelogenin behind the purifying, and EMD is the ENAMELIN sample that extract in the laboratory.

Fig. 6 is Recombinant Swine amelogenin rPAm mass spectrometry results figure.

Embodiment

The invention will be further described below in conjunction with embodiment, to understand better the present invention.

I, clone's total length pig glaze protein gene (pAm gene)

One, main experiment material

1. main agents and solution

(1) glue reclaims test kit (day root company) in a small amount

(2) PCR reagent: Taq archaeal dna polymerase, 10 * amplification buffer, MgCL2,10mmol/L 4 * dNTP(Promeg company)

(3) DNA marker(days root companies)

(4) 6 * agarose electrophoresis sample solutions (Takara).

(5) 50 * TAE damping fluids: 242g Tris alkali, 57.1ml glacial acetic acid, 100ml 0.5mol/L EDTA PH=8.0 add distilled water and are settled to 1L.

2. laboratory apparatus

(1) desk centrifuge GB7676-87(Shanghai centrifugal machine factory)

(2) U-3000 spectrophotometer (Hitachi company)

(3) 9600-PCR instrument (PE company)

(4) constant current constant voltage electrophoresis apparatus DY-501(Pharmacia company)

Two, experimental technique

1. design of primers

(1) base sequence of pAm gene is shown in SEQ ID NO.1, and it is as follows to have characteristics:

1 ~ 59 includes 5 ' cap structure, the structures such as 5 ' non-translational region and promotor, and 60 ~ 107 coded signal peptides, 108 ~ 629 coding full-length proteins, 630 ~ 778 is 3 ' non-translational region, comprises the PolyA tail, so the amplification scope of target gene is 108 ~ 629.Choose two restriction endonucleases not having restriction enzyme site in the pAm gene order and in plasmid, have a cloning site as goal gene being cloned into restriction endonuclease in the carrier.Design is with the primer of restriction enzyme site, guarantees that the PCR product introduces restriction enzyme site when comprising complete pAm gene order.

With DNA Star software the pAm gene order is analyzed, these sequence 108 ~ 629 interdigits do not exist the restriction endonuclease of restriction enzyme site as follows:

AatII AccI AclI AfeI AflII AflIII AgeI AhdI ApaI ApaLI ApoI AscI AseI AvaII AvrII BaeI BaeI' BanII BbeI BbsI BbvCI BcgI BcgI' BciVI BclI BfaI BglI BglII BlpI BplI BplI' BpmI Bpu10I BsaAI BsaBI BsaHI BsaI BsaWI BsaXI BsaXI' BseMII BseRI BsiEI BsiHKAI BsiWI BsmBI BsmFI BsmI BspEI BspHI BsrBI BsrDI BsrFI BsrGI BssHII BssSI BstBI BstEII BstUI BstXI BstZ17I Bsu36I BtrI BtsI ClaI DdeI DraI DraIII DrdI EagI EarI EciI EcoICRI EcoNI EcoO109I EcoRI EcoRV FseI FspI HaeII HgaI HhaI HinP1I HincII HpaI Hpy99I HpyCH4III HpyCH4IV KasI MboII MfeI MluI MspA1I NaeI NarI NdeI NgoMIV NheI NotI NruI NsiI NspI PacI PciI PmeI PmlI Ppu10I PpuMI PshAI PsiI PspOMI PvuI PvuII RsrII SacI SacII SalI SanDI SapI Sau96I SbfI ScaI SexAI SfiI SfoI SgfI SgrAI SmlI SnaBI SpeI SphI SrfI SspI StuI SwaI TaqI TatI TfiI TscI Tsp509I TspRI Tth111I XbaI XcmI XhoI XmnI

(2) structural representation of vector plasmid PGEX4T1 is seen Fig. 1.

Behind the cloning site that relatively exists in non-existent restriction enzyme site and the plasmid in the pAm gene order, choose EcoR I and Sal I as goal gene being cloned into restriction endonuclease in the carrier.Require the design primer as follows according to above-mentioned design of primers: Am-EcoR I 5 ' CCG GAA TTC ATG CCT CTA CCA CCT CAT CCT G 3 '; Am-Sal I 5 ' ACGC GTC GAC TTA ATC CAC TTC CTC CCG CTT G 3 '.

2. contain synthetic and the amplification of the pAm gene of restriction enzyme site

(1) is preced with the pAm gene that biotechnology Development Co., Ltd synthetic contains restriction enzyme site by the Shanghai rising sun.

(2) pcr amplification pAm gene

Take the pAm gene of synthetic as template, amplification total length pig amelogenin gene.Replace dna profiling with sterilized water, as negative control.The product loading is carried out the agarose gel electrophoresis check.Reaction system is as follows: ddH2O 9.8 μ l; 10 * Buffer, 2 μ l; MgCl21.2 μ l; 4dNTPs 2 μ l; Template cDNA 2 μ l; Primer Am-EcoR I 1 μ l; Am-Sal I 1 μ l; Taq enzyme 1 μ l.Reaction parameter: 94 ℃, 5 minutes; (94 ℃, 30 seconds; 56 ℃, 30 seconds; 72 ℃, 45 seconds) * 30 circulations; 72 ℃, 5 minutes, reaction finished.

3. agarose gel electrophoresis detects the PCR product

(1) configuration 1.5% sepharose is put into the electrophoresis chamber that contains TAE after solidifying.

(2) get 0.5 μ l, 6 * sampling liquid on point template, add 2 μ l sample blendings.

(3) loading, 80V, 30min.

(8) observation electrophoresis result and taking pictures under the ultraviolet lamp.

4.PCR product reclaims (test kit is reclaimed in rubber tapping): pcr amplification product is reclaimed

(1) PCR product electrophoretic band is cut off the Agarose plug that contains DNA under ultraviolet lamp, make it as far as possible little.Put into the 1.5ml centrifuge tube, weighing (glue weight=gross weight-blank pipe weight).

(2) ratio that adds 300 μ l Buffer S1 in every 100mg agarose adds S1 liquid, and 50 ℃ of water-baths 10 minutes shook up once, until glue dissolves fully in per 2 minutes.

(3) add the Virahol of 1/3 Buffer S1 volume, 50 ℃ of water-baths 1 minute, mixing.

(4) dissolved gum liquid is transferred in the adsorption column 15000 rpm, centrifugal 1 minute.Outwell the liquid in the collection tube, adsorption column is put into same collection tube.Such as the glue volume greater than column capacity, then centrifugal complete after, again move liquid in same post, recentrifuge.

(5) in post, add 500 μ l BufferW1,10000 rpm, centrifugal 15 seconds.Discard centrifugate, pillar places in the same recovery tube again.

(6) in post, add 500 μ l BufferW1, left standstill centrifugal 1 minute of 10000 rpm 1 minute.Discard centrifugate, pillar places in the same recovery tube again.

(7) with pillar with 10000 rpm recentrifuge 1 minute.

(8) pillar is placed in the new 1.5ml centrifuge tube, post mould central authorities add 30 μ l sterilized waters, and 50 ℃ of water-baths or room temperature were placed 2 minutes.

(9) 15000rpm centrifugal 1 minute, collects centrifugate.

(10) get 2 μ l centrifugates, agarose gel electrophoresis detects, and electrophoresis step is the same.

5. goal gene is identified in order-checking

The PCR product that rubber tapping is reclaimed is served the order-checking of marine life engineering corporation, and with DNA Star software the standard sequence of sequencing result and Gene Bank announcement is compared.

Three, result

1. the evaluation of goal gene

The agarose gel electrophoresis detected result as shown in Figure 2, the about 540bp of sample sets specific amplification products size behind the pcr amplification and expects that the pAm mRNA coding region base length of gained is consistent.And negative control is without the specificity product.

The pig amelogenin standard sequence that the amplified production of order-checking confirmation PCR and Gene Bank announce is in full accord.

II, structure contain the recombined pronucleus expression plasmid PGEX4T1-pAm of pAm gene

One, main experiment material

1. raw material

(1) PGEX4T1 vector plasmid (GE healthcare company)

(2) bacillus coli DH 5 alpha competence bacterium (Shanghai bio-engineering corporation)

2. main agents

(1) restriction enzyme EcoR I, Sal I, 10 * K Buffer(TakaRa company, Dalian)

(2) T4 DNA Ligase(TakaRa company, Dalian)

(3) penbritin (Roche company)

(4) paraxin

(5) RnaseA that provides in the test kit is all added solution P1,2 ~ 8 ℃ of preservations behind the mixing before plasmid extraction test kit (day root company, Beijing) uses in a small amount.Add 4 times of volume dehydrated alcohols before using among the rinsing liquid PW.

(6) gel reclaims test kit (day root company, Beijing) in a small amount

(7) liquid LB substratum: microbial culture tryptone 10g, microbial culture yeast extract 5g, NaCl 10g.After the mentioned component preparation, add an amount of distilled water, fully dissolving on the magnetic force heating stirrer, 5M NaOH regulates pH to 7.0, mends distilled water to 1000ml.121 ℃ of high pressure steam sterilizations 20 minutes cool off rear 4 ℃ of Refrigerator stores.

(8) solid LB substratum: microbial culture tryptone 10g, microbial culture yeast extract 5g, NaCl 10g, agar (agarose) 15g.After the mentioned component preparation, add an amount of distilled water, fully dissolving on the magnetic force heating stirrer, 5M NaOH regulates pH to 7.0, mends distilled water to 1000ml.121 ℃ of high pressure steam sterilizations 20 minutes, adding penbritin to final concentration after taking out in Bechtop is 100 μ g/ml, casts plate, cools off rear 4 ℃ of Refrigerator stores.

3. key instrument

(1) YJ-1FB Medical purification worktable (Wujiang treating plant head factory)

(2) desk centrifuge GB7676-87(Shanghai centrifugal machine factory)

(3) U-3000 spectrophotometer (Hitachi company)

(4) 9600-PCR instrument (PE company)

(5) Molecular Imager Fx and Quantity One software (Bio-Rad company)

(6) constant-temperature table (granary, YHZ-22 Jiangsu Wang Xiu testing installation factory)

Two, experimental technique

1. the preparation of carrier: PGEX4T1 plasmid extraction

(1) the E.Coli.DH5 α bacterial strain that contains plasmid PGEX4T1 that will buy is inoculated on the LB solid medium plate that contains penbritin 100 μ g/ml with method of scoring, and 37 ℃ of incubations spend the night activated spawn.

(2) 4 single colony inoculations of the next afternoon picking are in 4 flasks that contain 5ml LB liquid nutrient medium (penbritin 100 μ g/ml), and in the shaking table 37 ℃, 200rpm spends the night.

(3) change above bacterium liquid over to 4 1.5ml centrifuge tubes, 10000rpm, centrifugal 1 minute, abandon supernatant, blot raffinate.

(4) in centrifuge tube, add 250 μ l solution P1, the vibration mixing.

(5) add 250 μ l solution P2, leniently spin upside down abundant cracking thalline 4-6 time.

(6) add 350 μ l solution P3, leniently spin upside down 6-8 time immediately, abundant mixing is seen 12000rpm after the white flocks, centrifugal 10 minutes.

(7) column equilibration: adsorption column CB3 is put into collection tube, add 500 μ l balance liquid BL, 12000rpm, centrifugal 1 minute.Outwell waste liquid in the collection tube.

(8) the careful supernatant with in the step 6 is drawn to adsorption column CB3, and room temperature was placed 1-2 minute, centrifugal 1 minute of 12000rpm.Outwell waste liquid in the collection tube.

(9) in adsorption column CB3, add 700 μ l rinsing liquid PW, centrifugal 1 minute of 12000rpm.Outwell waste liquid in the collection tube.

(10) in adsorption column CB3, add 500 μ l rinsing liquid PW, centrifugal 1 minute of 12000rpm.Outwell waste liquid in the collection tube.

(11) adsorption column CB3 is relay in the recovery collector, centrifugal 2 minutes of 12000rpm eliminates raffinate.

(12) adsorption column CB3 is put into a clean centrifuge tube, add 50 μ l elution buffer EB to adsorption film central authorities, room temperature was placed 2 minutes.Centrifugal 2 minutes of 12000rpm collects plasmid solution in the centrifuge tube.

2. the structure of recombinant plasmid

(1) double digestion of goal gene pAm and plasmid PGEX4T1

Goal gene pAm and plasmid PGEX4T1 carry out double digestion 4h with ECoR I and Sal I respectively in 37 ℃ of H type universal buffering liquid.Reaction system sees the following form 1,2.

Table 1 goal gene endonuclease reaction system (μ l)

pAm	10×H Buffer	ECoRⅠ	SalⅠ	Total
					75	10	7.5	7.5	100

Table 2 PGEX4T1 endonuclease reaction system (μ l)

PGEX4T1	10×H Buffer	ECoRⅠ	SalⅠ	Total
					35	5	5	5	100

Enzyme is cut the product recovery of tapping rubber respectively, and operation steps is the same.Get 1 μ l and reclaim product and carry out agarose gel electrophoresis, the electrophoresis step condition is the same, and the Agarose plug behind the Molecular Imager Fx scanning electrophoresis, Quantity One software determine that enzyme cuts the concentration of product.

(2) enzyme is cut the connection of product

Purpose fragment pAm is connected and spends the night at 14 ℃ (12 ℃～16 ℃) with T4DNA ligase with the double digestion product of vector plasmid PGEX4T1.Reaction system sees Table 3.

Table 3 ligation system (μ l)

PGEX4T1 double digestion product	PAm double digestion product	Ligation buffer	Ligase	Total
						3	14	2	1	20

(3) conversion of competence bacterium

With connecting fluid transformed competence colibacillus bacterium as sample sets; With plasmid PGEX4T1 transformed competence colibacillus bacterium as the blank group; Bacterium DH5 α is inoculated in to contain on the antibiotic LB substratum makes negative control.

A) in Bechtop, get two 1.5ml centrifuge tubes, numbering 1#, 2#.Add respectively 100 μ l competence bacteriums.Add 1 μ l plasmid PGEX4T1 mixing (blank) in the 1# pipe.Add 20 μ l connecting fluid mixings (sample) in the 2# pipe.

B) 0 ℃ of ice bath is 0.5～1 hour.

C) 42 ℃ of water-baths are 90 seconds.

D) rapid 0 ℃ of ice bath is 2 minutes.

E) in pipe, add 800 μ l liquid LB substratum (not containing microbiotic) in the Bechtop.

F) 37 ℃, 200rpm shaking culture 45 minutes.

G) a certain amount of bacterium liquid is coated the agar plate surface that contains 100 μ g/ml penbritins with aseptic shop bacterium device.

H) negative control: get the fresh DH5 α bacterium liquid 100 μ l that spend the night and be coated with plate.Blank: get 1# pipe bacterium liquid 100 μ l and be coated with plate.Centrifugal 3 minutes of sample: 2# pipe 10000rpm, drawing has the bacterium liquid 200 μ l of precipitation to be coated with plate.

I) keep flat 20 minutes after, be inverted to cultivate 10～14 hours for 37 ℃.

3. the check of recombinant plasmid

Single bacterium colony extracting plasmid in the picking sample plate, with the single bacterium colony in the blank plate and plasmid PGEX4T1 in contrast, use respectively double digestion to come test-target fragment pAm whether to insert plasmid PGEX4T1, the positive colony that filters out is served the order-checking of marine life engineering corporation and is identified.

(1) a small amount of extracting plasmid:

2 single bacterium colonies of difference picking change over to respectively and contain 5ml LB liquid nutrient medium (containing penbritin 100 μ g/ml) in vitro in sample plate/blank plate, interior 37 ℃ of shaking table, and 200rpm spends the night.Extracting plasmid method is the same.

(2) enzyme is cut check

With 2 kinds of clones' enzyme sample recombinant plasmid PGEX4T1-pAm is carried out enzyme and cut, and compare with empty plasmid PGEX4T1.Reaction system such as following table, enzyme are cut product loading agarose electrophoresis and are detected.

Table 4 endonuclease reaction system (μ l)

Recombinant plasmid/empty plasmid	10×H Buffer	EcoRⅠ	SalⅠ	H2O	Total
						10	2	1	1	6	20

(3) order-checking check

To serve the order-checking of marine life engineering corporation through the sample recombinant plasmid of double digestion preliminary screening, and by the total length Am gene standard sequence contrast that DNA Star software and Gene Bank announce, identify simultaneously the restriction enzyme site of Insert Fragment both sides, initial and termination codon.

Four, result

1. recombinant plasmid PGEX4T1-pAm enzyme is cut evaluation

As seen be dispersed in single colony growth in the sample plate, illustrate to transform successfully.Recombinant prokaryotic expression vector plasmid PGEX4T1-pAm shows that through the gel electrophoresis spectrum of EcoR I and Sal I double digestion size is about the goal gene pAm band of 540bp and the vector plasmid band of 4.9kbp, respectively with goal gene pAm and the electrophoretic band of plasmid PGEX4T1 behind double digestion concordant (as shown in Figure 3).

2. recombinant plasmid sequencing result

Sequencer address shows that the pAm gene order of the fragment inserted in the recombinant plasmid and bibliographical information is in full accord, and fragment two ends restriction enzyme site, initiation codon and termination codon are all accurate.

III, preparation contain the recombinant strains of recombinant plasmid PGEX4T1-pAm

One, main experiment material

1. main agents

(1) PGEX4T1 vector plasmid (GE healthcare company)

(2) e. coli bl21 competence bacterium (day root company)

(3) penbritin (Roche company)

2. key instrument

(1) YJ-1FB Medical purification worktable (Wujiang treating plant head factory)

(2) desk centrifuge GB7676-87(Shanghai centrifugal machine factory)

(3) constant-temperature table (granary, YHZ-22 Jiangsu Wang Xiu testing installation factory)

Two, experimental technique

1. the conversion of competence bacterium

Recombinant expression plasmid PGEX4T1-pAm and blank plasmid PGEX4T1 through identifying are transformed respectively expression strain E.coli. BL21 competent cell, obtain recombinant strains and blank bacterial strain.

(1) in Bechtop, gets two 1.5ml centrifuge tubes, numbering 1#, 2#.Add respectively 100 μ l competence bacteriums.Add the mixing competence bacterium (blank) that the blank plasmid PGEX4T1 of 1 μ l transforms in the 1# pipe.Add the mixing competence bacterium (sample) that 1 μ l recombinant plasmid PGEX4T1-pAm transforms in the 2# pipe.

(2) 0 ℃ of ice baths 0.5～1 hour.

(3) 42 ℃ of water-baths 90 seconds.

(4) rapid 0 ℃ of ice bath is 2 minutes.

(5) in pipe, add 800 μ l liquid LB substratum (not containing microbiotic) in the Bechtop.

(6) 37 ℃, 200rpm shaking culture 45 minutes.

(7) a certain amount of bacterium liquid is coated the agar plate surface that contains 100 μ g/ml penbritins with aseptic shop bacterium device.

(8) blank: get 1# pipe bacterium liquid 200 μ l and be coated with plate; Sample: get 2# pipe bacterium liquid 200 μ l and be coated with plate.

(9) keep flat 20 minutes after, be inverted to cultivate 10～14 hours for 37 ℃.

The abduction delivering of IV, Recombinant Swine amelogenin, separation and purification and evaluation

One, main experiment material

1. main agents

(1) isopropylthio-β-D galactoside (IPTG) (Biolab company)

(2) SDS-PAGE lower molecular weight standard protein (Shanghai biochemical research institute)

（3）Glutathione Sepharose 4B Medium column

（4）Triton X-100

(5) preparation 10 * PBS, 1.4 M NaCl, 27 mM KCl, 100 mM Na2HPO4,18 mM KH2PO4 regulate pH to 7.3 with 5M NaOH respectively, then are settled to 1L, 121 ℃ of high pressure steam sterilizations 20 minutes.

(6) Elution buffer, 50 mM Tris-HCl, 10 mM reduced glutathion reduced glutathione (pH 8.0)

(7) the required reagent of polyacrylamide gel electrophoresis:

A) 30% acrylamide soln: acrylamide (Acr) 37.5g, two acrylamide (Bic) the 1g ultrapure waters of methene dissolve rear 4 ℃ of preservations to 100ml.Return to room temperature during use and without precipitation.

B) 1.5mol/L Tris(PH8.8): Tris (MW121.14) 45.43g, transfer pH to 8.8 with concentrated hydrochloric acid after the dissolving, be settled to 250ml, room temperature preservation.

C) 1.0mol/L Tris(PH6.8): Tris (MW121.14) 15.14g, transfer pH to 6.8 with concentrated hydrochloric acid after the dissolving, be settled to 250ml, room temperature preservation.

D) 10% sodium lauryl sulphate (SDS)

E) 10% ammonium persulphate

F) TEMED(N, N, N, N-tetra methylethlene di total length Amine, N, N, N, N-Tetramethyl Ethylene Diamine)

G) 1 * Tris-glycine electrophoretic buffer: 6g Tris, 8g glycine, 1g SDS, distilled water constant volume be to 1L(25mmol/L Tris alkali, 250 mmol/L electrophoresis level glycine, PH8.30,1%SDS)

H) destainer methyl alcohol: glacial acetic acid: water=5: 1: 4

Add 0.25% coomassie brilliant blue R250 in the staining fluid destainer

I) 5 * SDS-PAGE electrophoresis sample solution (ancient cooking vessel state company)

J) transfering buffering liquid: glycine (MW75.07) 2.9g, Tris(MW121.14) 5.8g, SDS 0.37g, methyl alcohol 200ml, distilled water is settled to 1000ml.

K) 10X ponceau dye liquor: Ponceau S 2g, trichoroacetic acid(TCA) 30g, sulphosalicylic acid 30g, distilled water is settled to 100ml.

L) wash-out antibody damping fluid PBST contains 1 * PBS of 0.1%Tween-20.

2. key instrument

(1) YJ-1FB Medical purification worktable (Wujiang treating plant head factory)

(2) desk centrifuge GB7676-87(Shanghai centrifugal machine factory)

(3) constant-temperature table (granary, YHZ-22 Jiangsu Wang Xiu testing installation factory)

(4) Cole-Parmer instrument co. Ultrasonic Cell Disruptor

(5) Mettler AZ260 electronic balance

(6) Bio-RAD vertical electrophoresis apparatus

Two, experimental technique

1. the abduction delivering of Recombinant Swine amelogenin fusion rotein

(1) selects recombinant strains 5 strains and 1 strain of blank bacterial strain that obtains in the III, be inoculated into 2ml and contain in two anti-liquid LB substratum (acillin 100 μ g/ml), 37 ℃ of 200rpm shaken overnight activation.

(2) the above-mentioned bacterium that spends the night activation is inoculated into by 20% and contains 37 ℃ of 250rpm shaking culture in the antibiotic 4ml liquid LB substratum, surveyed the OD600 value every 1 hour.

(3) when the OD600 value reaches 0.6～0.8, adding isopropylthio-β-D galactoside (IPTG) to final concentration in the super clean bench is 0.3mmol/L, continues 37 ℃ of 200rpm shaking culture, induces 4 hours.

(5) get 1ml bacterium liquid 12000rpm and received bacterium in centrifugal 1 minute, abandon supernatant, add 25 μ l H2O and the resuspended thalline of 25 μ l, 2 * sample-loading buffer.

(6) heating of all samples boiling water bath after will inducing 5min carries out 12% SDS-PAGE electrophoresis detection behind centrifugal 3 min of 5000 rpm.

2.SDS-PAGE gel electrophoresis

(1) clean sheet glass: a hand fastening sheet glass, the another hand dips in a washing powder and cleans gently.All clean later with the tap water punching on the two sides, stands in the basket after clean with distilled water flushing again and dry.

(2) encapsulating: put into folder after the sheet glass alignment and tighten.Then vertical card is prepared encapsulating on the top of the shelf.Join 10% separation gel by previous methods, shake up immediately behind the adding TEMED and get final product encapsulating.Then add the fluid-tight of one deck water on the glue.When between Dang Shui and the glue refracted ray being arranged, illustrate that glue is solidifying.Upper water and water is blotted with thieving paper removes photoresist.Join 4% concentrated glue by previous methods, add to shake up immediately behind the TEMED and get final product encapsulating.Then remaining space is filled concentrated glue inserts comb in the concentrated glue.Glue is flowed down in order to avoid Bubble formation is arranged in the glue along sheet glass.To make comb maintenance level when inserting comb.By the time after concentrated gelling is solid, put it in the electrophoresis chamber, add the both sides that two hands after enough electrophoresis liquid pinch respectively comb and gently it is extracted straight up.

(3) loading: take out in the EP pipe of loading sample to 200 μ l, add 2 * SDS sample-loading buffer to final concentration and be 1 *.Sample boils 5-10min and makes protein denaturation in boiling water, centrifugal 3 minutes of 10000rpm.To well, slowly add sample with the adherent absorption sample of microsyringe.

(4) electrophoresis: constant voltage is 100V, and electrophoresis has just been run out of to bromjophenol blue can stop electrophoresis

(5) dyeing and decolouring: sheet glass prized concentrated glue is scraped off gently, carefully peel separation gel.At the about 45min of decolorization swinging table dyeing, then water rinses out the dye liquor of not catching with staining fluid.Changing destainer decolours at decolorization swinging table.

3. the affinitive layer purification of restructuring pAm fusion rotein

(1) the pAm expression strain is inoculated into 50ml and contains in two anti-liquid LB substratum (acillin 100 μ g/ml and paraxin), 37 ℃ of 200rpm shaken overnight activation.

(2) bacterium of the above-mentioned activation of spending the night is inoculated into by 4% contains 37 ℃ of 250rpm shaking culture in the two anti-liquid LB substratum of 1L, surveyed the OD600 value every 1 hour.

(3) when the OD600 value reaches 0.8～1.0, adding isopropylthio-β-D galactoside (IPTG) to final concentration is 0.2mmol/L, continues 37 ℃ of 200rpm shaking culture, induces 4 hours.

(4) collect bacterium liquid to the 500ml centrifugal bottle, 4 ℃ of 5000 rpm collected thalline in centrifugal 30 minutes.

(5) proceed as follows on ice, with the resuspended thalline of 1 * PBS, transfer in the 50ml centrifuge tube, centrifugal 30 minutes of 4 ℃ of 12000 rpm.

(6) again wash bacterial sediment on ice with 1 * PBS.

(7) electronic balance weighing bacterial precipitation weighs 3 grams.

(8) with 30 ml(10 times volume) 1 * PBS in resuspended thalline on ice, add N,O-Diacetylmuramidase 30 μ l, repeatedly blow and beat mixing.

(9) in carrying out ultrasonic bacteria breaking on ice, programming (net cycle time: 100 seconds; Pulse mode: made a call to 1 second, stopped 1 second).Repeat 2～3 times to the bacteria suspension shape that is translucent.

(10) adding 20%TritonX-100 is 1% to final concentration, repeatedly piping and druming dissolving, and room temperature is placed and was helped dissolving in 30 minutes.

Centrifugal 1 hour of (11) 4 ℃ of 10000rpm collect supernatant.

(12) supernatant is crossed Glutathione Sepharose 4B post, discarded effluent liquid.

(13) with the repeatedly rinsing of the PBS that contains TrixtonX-100 of 10 times of column volumes, thoroughly remove unconjugated foreign protein.

(14) add Thrombin enzymic digestion liquid, 22 ℃ are spent the night.

(15) with ℃ preservation of 1 * PBS eluted protein sample-80.

(16) get 25 μ l wash-outs after sample add 25 μ l, 2 * sample-loading buffer, boiling water bath heating 5min carries out the SDS-PAGE electrophoresis detection behind the centrifugal 3min of 5000 rpm.

4. the evaluation of restructuring pAm fusion rotein

(1) electrophoresis: step is with the SDS-PAGE gel electrophoresis

(2) transferring film:

A) turn a film and need prepare the filter paper of 6 7.0～8.3cm and the nitrocellulose filter of 1 7.3～8.6cm, place transfering buffering liquid to soak the nitrocellulose filter that cuts.Put into clip, two blocks of sponge pads, glass rod, the filter paper that transferring film uses and the film that soaked in the enamel tray that shifts liquid being added with.Clip is opened the black one side maintenance level that makes.Fill up a sponge pad in the above, at mat pad three metafiltration paper, on the other hand fixedly filter paper proficiency is rolled wherein bubble with glass rod.

B) carefully peel separation gel and be placed on the filter paper, with hand adjustment is whole it is alignd with filter paper, membrane cover on glue, be covered completely whole glue and bubble removing.At 3 filter paper of film loam cake and remove bubble.Cover at last another sponge pad, roll several times and just can close clip.Whole operating in the transfer liquid carried out, and constantly roll the bubble that degass.The filter paper on film both sides can not be in contact with one another, and can be short-circuited after the contact.

C) clip is put into the transfer groove groove, be made the black flour of folder to the black flour of groove, the fine flour of folder is to red of groove.Meeting heat production during electrotransfer is one side lower the temperature at the ice cube of putting of groove.General with 60V transferase 12 h or 250mA transferase 12 h.

D) film being dyed 5min(with 1 * ponceau dye liquor after having turned shakes on decolorization swinging table).Then water rinses out the dye liquor of not catching and just can see albumen on the film.Film is dried for subsequent use.

(3) immune response:

A) film is soaked from bottom to top with the PBS that contains 0.5% skim-milk after, shake sealing 1h under the room temperature on the decolorization swinging table.

B) primary antibodie (goat-anti people, total length Amelogenin, Santa Cruz) is diluted to proper concn (1:200) with the PBS that contains 0.5% skim-milk; The together preservative film of the suitable size of tearing is laid on the experiment table top, uses water-soaked so that preservative film keeps smooth for four jiaos; Antibody-solutions is added on the preservative film; From confining liquid, take out film, suck debris with filter paper after, membranin faced down to be put on the antibody liquid level, lifts four jiaos of films to drive residual bubble out of; 4 ℃ of overnight incubation are at room temperature washed three times on the decolorization swinging table with PBST, each 10min.

C) the same method is prepared two anti-diluents (anti-sheep IgG, Rock Land, 1: 5000) and is contacted with film, hatch 1～2h under the room temperature after, at room temperature wash three times each 10min on the decolorization swinging table with PBST.

(4) gel images analysis: film is scanned and analysis image with Odyssay Infrared fluorescence scanning system (Gene company limited).

Three, experimental result

1. the SDS-PAGE detected through gel electrophoresis of restructuring pAm fusion rotein

Detect as shown in Figure 4, have more an obvious newborn protein band before inducing after the control strain that has transformed blank plasmid PGEX4T1 is induced at about 46KD place, illustrate that it is the albumen that produces after IPTG induces, its protein size is big or small basically identical with affinity labelling glutathione s-transferase (GST).

And the sample of recombinant bacterial strain after inducing has an obvious newborn protein band at about 46KD, with the fusion rotein size basic identical (the GST size is 26KD, and the pAm size is about 20KD) of estimating.

Soluble analysis to expressing protein shows that fusion protein expression products exists with the inclusion body form.

2. the affinitive layer purification of Recombinant Swine amelogenin pAm

Recombinant expression plasmid PGEX4T1-pAm can pass through gsh-agarose resin affinitive layer purification through IPTG abduction delivering product fusion rotein GST-pAm, proteolytic cleavage except visible purifying behind the GST label after pAm albumen, the result is as shown in Figure 5.

3. the evaluation of recombinant protein pAm

The albumen pAm that purifying obtains through Westen-blot checking can with anti-amelogenin polyclonal antibody generation compatible reaction, confirm that the albumen that obtains by abduction delivering and purifying is the Recombinant Swine amelogenin.

Adopt mass spectrometry to determine that the pig amelogenin molecular weight of purifying is 25372.72Da, the result as shown in Figure 6.

More than specific embodiments of the invention are described in detail, but it is just as example, the present invention is not restricted to specific embodiment described above.To those skilled in the art, any equivalent modifications that the present invention is carried out and substituting also all among category of the present invention.Therefore, not breaking away from impartial conversion and the modification of doing under the spirit and scope of the present invention, all should contain within the scope of the invention.

Claims (10)

1. the preparation method of a Recombinant Swine amelogenin is characterized in that, may further comprise the steps:

Step 1, synthetic total length pig amelogenin gene;

Step 2 makes up the recombined pronucleus expression plasmid PGEX4T1-pAm that contains said gene;

Step 3, the recombined pronucleus expression plasmid PGEX4T1-pAm that step 2 is obtained transforms bacterial strain E.coli. BL21;

Step 4, the recombinant bacterial strain expressed fusion protein GST-pAm that induces step 3 to obtain;

Step 5, the GST label protein of the fusion rotein GST-pAm that excision step 4 obtains obtains the Recombinant Swine amelogenin.

2. the preparation method of Recombinant Swine amelogenin according to claim 1 is characterized in that, the base sequence of the pig of total length described in the step 1 amelogenin gene is shown in SEQ ID NO.1.

3. the preparation method of Recombinant Swine amelogenin according to claim 1, it is characterized in that, in step 2, make up recombined pronucleus expression plasmid PGEX4T1-pAm and adopt restriction endonuclease ECoR I and Sal I, enzyme is cut primer and is respectively 5 '-CCGGAATTCATGCCTCTACCACCTCATCCTG-3 ' and 5 '-ACGCG TCGACTTAATCCACTTCCTCCCGCTTG-3 '.

4. the preparation method of Recombinant Swine amelogenin according to claim 1 is characterized in that, in the step 4, induces the recombinant bacterial strain expressed fusion protein GST-pAm of step 3 acquisition by isopropylthio-β-D galactoside.

5. the preparation method of Recombinant Swine amelogenin according to claim 1 is characterized in that, step 5 is: the fusion rotein GST-pAm that purification step 4 obtains, excision GST label protein, the Recombinant Swine amelogenin pAm of acquisition purifying.

6. the preparation method of Recombinant Swine amelogenin according to claim 5 is characterized in that, the fusion rotein GST-pAm that step 4 obtains is by the GST affinitive layer purification.

7. the preparation method of Recombinant Swine amelogenin according to claim 1 or 5 is characterized in that, excision GST label protein adopts zymoplasm in the step 5.

8. Recombinant Swine amelogenin of method preparation as claimed in claim 1.

9. a recombinant prokaryotic expression vector that contains total length pig amelogenin gene is characterized in that, includes SEQ ID NO.1 nucleotide sequence in prokaryotic expression plasmid PGEX4T1.

10. coli strain that is transformed by the described recombinant prokaryotic expression vector of claim 9.

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