CN103145852A - Recombining human pleiotrophin fusion protein and method for preparing same - Google Patents
Recombining human pleiotrophin fusion protein and method for preparing same Download PDFInfo
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Abstract
The invention discloses a recombining human pleiotrophin fusion protein and a method for preparing the same. The pleiotrophin fusion protein is obtained by constructing a prokaryotic expression vector pET44a(+)-pleiotrophin, further connecting a purification tag sequence, thrombin and an enterokinase enzyme site in turn at an N terminal of the human pleiotrophin, transforming the Nus sequence into escherichia coli to obtain a transformant, and inductively expressing, separating and purifying the escherichia coli transformant. The biological activity verifies and indicates that the method is capable of promoting the cell growth of the pleiotrophin fusion protein and can be applied to the preparation of nerve nutrition drugs.
Description
Technical field
The invention belongs to biological technical field, relate to the expression of recombination fusion protein, particularly multi-functional somatomedin Pleiotrophin fusion rotein of a kind of recombinant human and preparation method thereof.
Background technology
Multi-functional somatomedin (pleiotrophin) is a kind of somatomedin obtained by University of Washington's medical center purifying in 1989, thought at that time the albumen that this factor is 17kDa, there is high affinity with heparin, and be named as HBGF-8 first (heparin.binding growth factor-8).The Ptn gene is positioned at 7q33-q34, and length is greater than 100kb, 168 amino acid of encoding, and albumen size 15.3kD, show as 18kD at the sds polyacrylamide electrophoresis.The PTN normal expression, embryonic stage, is seldom expressed in adult health tissues, and in brain, expression level is the highest, in addition, also can be expressed in uterus, Leydig cells etc.The PTN Main Function has: (1) plays an important role to generation, growth and transfer, the tumor-blood-vessel growth of tumour; (2) in central nervous system, axon growth, cynapse generation, Neuromuscular connection etc. are played an important role; (3) other are as effects such as promoting mitosis, promotion bone developments; (4) promote hematopoietic stem cell expansion and and regeneration.Therefore pleiotrophin can be used as nutritional support dopaminergic neuron medicine, alleviates Parkinsonian symptom, for Parkinsonian treatment provides new approaches simultaneously.
In view of the important biological function of Pleiotrophin, obtain the activated Pleiotrophin albumen of high purity, can be used as the genomic medicine of nutritional support dopaminergic neuron, there is the prospect that is applied to treatment of Parkinson disease.And, due to the mRNA secondary structure complexity of Pleiotrophin gene, be unfavorable for the heterogenous expression of eukaryotic system; And single expression Pleiotrophin albumen, because expression amount is less, also can affect the efficiency of late gene medicine.
Summary of the invention
The problem that the present invention solves is to provide multi-functional somatomedin Pleiotrophin fusion rotein of a kind of recombinant human and preparation method thereof, the Pleiotrophin gene has been carried out to expression and purifying, and verified that this fusion rotein has the activity that promotes growth to the classical cell model PC12 of nerve cell.
The present invention realizes by following technical solution:
The multi-functional somatomedin Pleiotrophin fusion rotein of a kind of recombinant human, also be connected with purifying flag sequence, zymoplasm and enteropeptidase restriction enzyme site in turn at the N of the multi-functional somatomedin Pleiotrophin structural domain of people end, and the Nus sequence.
The aminoacid sequence of the multi-functional somatomedin Pleiotrophin of described people is as shown in SEQ.ID.NO.1.
The flag sequence that described purifying flag sequence is 6 * His.
The aminoacid sequence of the multi-functional somatomedin Pleiotrophin fusion rotein of described recombinant human is as shown in SEQ.ID.NO.2.
A kind of prokaryotic expression carrier of expressing the multi-functional somatomedin Pleiotrophin fusion rotein of recombinant human, this prokaryotic expression carrier is pET44a
(+)-pleiotrophin, be by EcoR I/XHo I restriction enzyme site by the multi-functional somatomedin Pleiotrophin gene fragment of the people as shown in SEQ.ID.NO.3, be cloned into pET44a
(+)expression vector.
The preparation method of the multi-functional somatomedin Pleiotrophin fusion rotein of a kind of recombinant human comprises the following steps:
1) take the cDNA that comprises whole person Pleiotrophin genes encoding frame is template, usings primer pair P as primer, pcr amplification people pleiotrophin gene fragment, and described primer pair P is:
Upstream primer P1:aggaattcatgcaggctcaacagtacc;
Downstream primer P2:agctcgagtaaatccagcatcttctcc;
2) the people pleiotrophin gene fragment of pcr amplification is obtained to the fragment of two sticky ends with EcoR I/XHo I double digestion; And by the pET44a of this fragment and EcoR I/XHo I double digestion
(+)the carrier connection obtains expression vector pET44a
(+)-pleiotrophin;
3) by expression vector pET44a
(+)-pleiotrophin is transfected into competent intestinal bacteria, and screening obtains the positive transformant of destination gene expression;
4) by positive transformant picking neat in edge, bacterium colony that growth conditions is good after 37 ℃ of thermostat containers are cultivated 12h, be inoculated in the LB nutrient solution containing penbritin 100mg/L 37 ℃ of overnight incubation of shaking table; Next day, the volume ratio with 1: 100 was inoculated in fresh containing in the LB nutrient solution of penbritin 100mg/L, and 37 ℃ are continued to be cultured to bacterial density and reach OD
600=0.4～0.6, add the IPTG inducing culture 4h of final concentration 0.2mM concentration, make expression vector pET44a
(+)-pleiotrophin is expressed, then centrifugal collection bacterium;
5) ratio in the 10mL/g bacterial precipitation adds lysate, and resuspended bacterium ultrasonicly under ice bath is split bacterium, then centrifugation, and collect supernatant;
The TrisCl that described lysate is pH8.5, concentration 50mmol/L, the mixing solutions of the beta-mercaptoethanol of 5mmol/L and the PMSF of 1mmol/L;
6) supernatant of collection is splined on to the metal chelate affinity chromatography post, by the buffer A of 10 times of column volumes, washes the flow velocity wash-out of post with 0.5mL/min, then by the buffer B of 2 times of column volumes, wash post, finally use damping fluid C elution of bound albumen, collect elution peak;
The TrisCl that described buffer A is pH8.5, concentration 20mmol/L, the KCl of 100mmol/L, the mixing solutions of the beta-mercaptoethanol of 5mmol/L and the imidazoles of 20mmol/L; The TrisCl that described buffer B is pH8.5, concentration 20mmol/L, the mixing solutions of the KCl of 1mol/L and the beta-mercaptoethanol of 5mmol/L; The TrisCl that described damping fluid C is pH8.5, concentration 20mmol/L, the KCl of 100mmol/L, the mixing solutions of the beta-mercaptoethanol of 5mmol/L and the imidazoles of 100mmol/L;
The elution peak of collection is splined on to ion exchange chromatography, with the albumen of elutriant D elution of bound, collects elution peak, obtain the multi-functional somatomedin Pleiotrophin fusion rotein of recombinant human of purifying;
The TrisCl that described elutriant D is pH7.4, concentration 50mmol/L and the mixing solutions of 1mmol/L NaCl.
Compared with prior art, the present invention has following useful technique effect:
1, because Pleiotrophin is basic protein, its theoretical pI value is 9.66, thereby if has determined to want the more difficult expression of this albumen of single expression.And the present invention is by building prokaryotic expression carrier pET44a
(+)-pleiotrophin, and conversion intestinal bacteria, carry out separation and purification after abduction delivering and obtain having the fusion rotein of the multi-functional somatomedin Pleiotrophin of recombinant human of Pleiotrophin fusion rotein, this fusion rotein has the nutritional support neurone, promote the biological activity of neurocyte proliferation, the retardation that tumour cell is had to the cell cycle, can promote for the preparation of nutrition the genomic medicine of neurocyte proliferation.
2, the prokaryotic expression carrier pET44a that the present invention builds
(+)-pleiotrophin is on the basis of human cloning pleiotrophin gene, by EcoR I/XHo I double enzyme site, is cloned into expression vector pET44a
(+), connection can be by the sequence of zymoplasm and enteropeptidase identification, and the pleiotrophin fusion rotein built also comprises the purifying flag sequence of 6 * His, is convenient to like this purifying of fusion rotein; And can remove flag sequence by the restriction enzyme digestion reaction of zymoplasm and enteropeptidase when being necessary, thereby can access the pleiotrophin polypeptide.
3, this fusion rotein utilizes protokaryon intestinal bacteria system to be expressed, and has economy, is easy to the advantage of enlarged culturing, and fusion rotein impels expression amount greatly to increase, and purification tag is easy to purifying, and output is high, and the purity height is all the advantage of this fusion rotein.
The accompanying drawing explanation
Fig. 1 is the electrophoresis detection figure that pcr amplification obtains the pleiotrophin gene fragment;
Fig. 2 is pET44a
(+)the plasmid map of carrier;
Fig. 3 is pET44a
(+)the EcoR I of-pleiotrophin expression vector and XHo I double digestion are identified electrophorogram;
Fig. 4 is the figure as a result that SDS-PAGE detects the pleiotrophin expressing fusion protein;
Fig. 5 is the figure as a result that Western Blot detects pleiotrophin fusion rotein solubility expression;
Fig. 6 is the figure as a result after SDS-PAGE detects the pleiotrophin fusion protein purification;
Fig. 7 is that MTT detects the bioactive histogram of pleiotrophin fusion rotein.
Embodiment
The present invention realizes the preparation of recombinant human Pleiotrophin by biotechnology, by building prokaryotic expression carrier pET44a (+)-Pleiotrophin, and be transformed into intestinal bacteria and obtain transformant, again the intestinal bacteria transformant is carried out to abduction delivering, obtain the Pleiotrophin fusion rotein after separation and purification.The Pleiotrophin fusion rotein is carried out to SDS-PAGE and Western Blot and analyzes, its molecular weight with predict the outcome consistent; And the biological activity checking shows, the Pleiotrophin fusion rotein has the effect that promotes the PC12 Growth of Cells.Below the present invention is described in detail, the explanation of the invention is not limited.
1. the amplification of people pleiotrophin gene (NM_002825.5)
Search people Pleiotrophin gene (NM_002825.5) in GenBank.According to its mRNA nucleotide sequence, design synthetic Auele Specific Primer P, by the two ends of EcoR I and XHo I introducing pleiotrophin encoding sequence; Primer P is specially:
Upstream primer P1:aggaattcatgcaggctcaacagtacc27;
Downstream primer P2:agctcgagtaaatccagcatcttctcc27;
People's embryo liver cDNA of take is template, take primer P as primer, according to following program, is amplified containing complete ORF(encoder block) pleiotrophin cDNA sequence: 95 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 60s, 30 circulations, 72 ℃ of 10min.Glue reclaims the PCR product that amplification obtains, and carries out the agarose gel electrophoresis evaluation, and as shown in Figure 1, swimming lane M is DNA Marker to result, and the two ends that swimming lane 1 is 507bp are with the pleiotrophin gene of cloning site.
2. expression vector pET44a (+)-Pleiotrophin-
mHDdesign & formulation:
For the ease of the smooth expression of people pleiotrophin gene and the purifies and separates of pleiotrophin fusion rotein, the present invention has selected pET44a
(+)prokaryotic expression carrier, people pleiotrophin gene clone is entered to the downstream of T7 promotor, this carrier has high deliquescent e. coli protein Nus.Tag fusion partner and label while having a kind of overexpression obtained by a large amount of screening systems, can further improve the effective expression of recombination fusion protein; The sequence of amino acid sites is identified in downstream at Nus.Tag with coding zymoplasm and enteropeptidase, can carry out to fusion rotein pleiotrophin the cutting of the label in later stage like this; And the purifying His label contained makes fusion rotein be expressed and can obtain by purifying by chromatographic separation afterwards.
Specifically by following steps, build:
A. the enzyme of vector plasmid and people Pleiotrophin gene is cut
The Pleiotrophin gene that reclaims, through EcoR I/XHo I double digestion, and is reclaimed to the people pleiotrophin gene fragment with sticky end with test kit; Procaryotic cell expression plasmid pET44a
(+)be purchased from Novagen company, as shown in Figure 2, wherein, T7 is T7 promotor/primer sites to its plasmid map, 2426-2442bp; Nus.Tag is: Nus label protein, 834-2318bp; His.Tag is: histidine-tagged protein, 2325-2342bp, 801-818bp and 512-529bp; The polyclone restriction enzyme site is: 530-724bp; LacI is: lac Z allelotrope, 2833-3915bp; Ap is: penbritin drug resistance gene, 5870-6730bp; F1ori is: f1 initiation site, 6852-7299bp.By prokaryotic expression carrier pET44a
(+)with EcoR I/XHol double digestion, test kit reclaims the linearization plasmid large fragment.
The people pleiotrophin sequence of b. enzyme being cut and the pET44a of recovery
(+)linear fragment connects with the T4DNA ligase enzyme, to connect product transformed competence colibacillus DH5 α cell, after 37 ℃ of thermostat containers are cultivated 12h, picking mono-clonal colony inoculation is in containing the LB nutrient solution of acillin (Amp) 100mg/L, 37 ℃ of shaking tables are cultivated after 12h and are received bacterium and extract plasmid and carry out enzyme and cut evaluation, EcoR I/XHo I double digestion is identified electrophoresis result as shown in Figure 3, swimming lane 1, recombinant plasmid shown in 2 is cut the Insert Fragment of the approximately 507bp size that obtains expection through enzyme, through sequencing, illustrate that people pleiotrophin sequence correctly is inserted in the middle of pET44a (+) carrier, expression vector pET44a (+)-pleiotrophin successfully constructs.
3. expression vector pET44a (+)-Pleiotrophin-
mHDconversion and abduction delivering
By recombinant expression vector pET44a (+)-Pleiotrophin-
mHDbe transformed in expressive host bacterium e. coli bl21 (DE3) competent cell, after 37 ℃ of thermostat containers are cultivated 12h, picking neat in edge, the bacterium colony that growth conditions is good, be inoculated in the LB nutrient solution containing penbritin 100mg/L, and 37 ℃ of shaking culture of shaking table are spent the night.Next day, be inoculated in freshly in the LB nutrient solution of penbritin 100mg/L with the volume ratio of 1:100,37 ℃ are continued shaking culture to bacterial densities and reach OD
600=0.4～0.6, adding IPTG is 0.2mmol/L to final concentration, and inducing culture 4h makes expression vector pET44a (+)-pleiotrophin be expressed, then centrifugal collection bacterium.
4.pleiotrophin the evaluation of fusion rotein
A.10%SDS-PAGE(polyacrylamide gel electrophoresis) detect the expression of pleiotrophin fusion rotein
With the pET44a (+) that induced-pleiotrophin/BL21 reconstitution cell in contrast, IPTG induces centrifugal collection after 4h, uses the lysate dissolution of bacteria, adds 50 μ l5 * sample-loading buffers to boil 10min.Get total protein and carry out 10% SDS-PAGE electrophoresis.Newborn band after obviously inducing as seen after coomassie brilliant blue staining, the about 82.5kDa of molecular weight; Concrete outcome is as shown in Figure 4: in figure, swimming lane M is protein Marker, is respectively 97.2,66.4,44.3,29.0,20.1,14.3kDa; Swimming lane 1 is the pET44a (+) that do not induce-pleiotrophin/BL21 reconstitution cell, and swimming lane 2 is 0.2mM IPTG abduction delivering 4h; With other swimming lanes, compare, swimming lane 2 is carrying out after IPTG induces, and produces the pleiotrophin fusion rotein of the about 82.5kDa of molecular weight.
The Western Blot of b.pleiotrophin fusion rotein detects
PET44a (+) after collection IPTG induces-pleiotrophin/BL21 (DE3) recombinant bacterium, extract respectively cleer and peaceful inclusion body on the bacterium kytoplasm (collect bacterium and split centrifugal upper cleer and peaceful precipitation after bacterium), Western Blot analyzes the expression of merging pleiotrophin albumen.After the SDS-PAGE electrophoresis detected at expressing fusion protein finishes, pull down gel, according to the Bio-Rad description of product, by close negative electrode one side of gel, cellulose nitrate (NC) film is near anode one side, in the transfering buffering liquid (25mM Tris, 192mM Glycine, 20% methyl alcohol) of precooling, 100V constant voltage electrophoresis is 60 minutes, by protein delivery to the NC film; After electrophoresis finishes, take out the NC film, immerse room temperature sealing 1h in confining liquid (containing the TBST of 2%BSA) after washings TBST (20mM Tris-HCl, pH7.5,150mM NaCl, 0.05%Tween-20) cleans, the TBST room temperature is washed 3 times, each 5min; Then add mouse source His tag antibody, incubated at room 1h, the TBST room temperature is washed 3 times, each 5min; Add again two of luciferase mark to resist, incubated at room 1h, the TBST room temperature is washed 3 times, each 5min, infrared albumen is swept the film instrument and is swept film and be presented at the rear pleiotrophin fusion rotein band of inducing all be labeled as seen in cleer and peaceful inclusion body on kytoplasm, molecular weight is consistent with the SDS-PAGE electrophoresis detection, illustrates that the pleiotrophin fusion rotein is solubility expression.As shown in Figure 5, wherein, swimming lane M is protein Marker to concrete outcome, is respectively 97.2,66.4,44.3,29.0,20.1,14.3kDa; Swimming lane 1 is the pET44a (+) that do not induce-pleiotrophin/BL21 reconstitution cell; Swimming lane 2 is the supernatant after pET44a (+)-pleiotrophin/BL21 induces with 0.2mM IPTG; Swimming lane 3 for pET44a (+)-pleiotrophin/BL21 after inducing with 0.2mM IPTG inclusion body; Contrast swimming lane 1,2,3 is visible: the pleiotrophin fusion rotein has expression in the inclusion body precipitation.
And according to the expression of pET44a (+) carrier, the pleiotrophin fusion rotein is from aminoterminal, contain successively Nus sequence, zymoplasm and enteropeptidase restriction enzyme site, His label, containing 744 amino acid, the aminoacid sequence of concrete Pleiotrophin is as shown in SEQ.ID.NO.1, and the aminoacid sequence of pleiotrophin fusion rotein is (or as shown in SEQ.ID.NO.2):
MGSSHHHHHHSSMNKEILAVVEAVSNEKALPREKIFEALESALATATKKK?YEQEIDVRVQIDRKSGDFDTFRRWLVVDEVTQPTKEITLEAARYEDESLNL?GDYVEDQIESVTFDRITTQTAKQVIVQKVREAERAMVVDQFREHEGEIITG?VVKKVNRDNISLDLGNNAEAVILREDMLPRENFRPGDRVRGVLYSVRPEA?RGAQLFVTRSKPEMLIELFRIEVPEIGEEVIEIKAAARDPGSRAKIAVKTND?KRIDPVGACVGMRGARVQAVSTELGGERIDIVLWDDNPAQFVINAMAPAD?VASIVVDEDKHTMDIAVEAGNLAQAIGRNGQNVRLASQLSGWELNVMTV?DDLQAKHQAEAHAAIDTFTKYLDIDEDFATVLVEEGFSTLEELAYVPMKE?LLEIEGLDEPTVEALRERAKNALATIAQAQEESLGDNKPADDLLNLEGVD?RDLAFKLAARGVCTLEDLAEQGIDDLADIEGLTDEKAGALIMAARNICWF?GDEATSGSGHHHHHHSAGKETAAAKFERQHMDSPPPTGLVPRGSAGSGTI?DDDDKSPGARGSEFMQAQQYQQQRRKFAAAFLAFIFILAAVDTAEAGKK?EKPEKKVKKSDCGEWQWSVCVPTSGDCGLGTREGTRTGAECKQTMKTQ?RCKIPCNWKKQFGAECKYQFQAWGECDLNTALKTRTGSLKRALHNAECQ?KTVTISKPCGKLTKPKPQAESKKKKKEGKKQEKMLDLLEHHHHHH
The theoretical molecular of pleiotrophin fusion rotein is about 82.5kDa, with SDS-PAGE and Western Blot, detects consistent.
The purifying of c.pleiotrophin fusion rotein
Enlarged culturing pET44a (+)-pleiotrophin/BL21 recombinant bacterium, IPTG induces rear centrifugal collection bacterium, by the 10mL/g bacterial precipitation, adds lysate (pH8.5,50mmol/L TrisCl, 5mmol/L beta-mercaptoethanol, 1mmol/L PMSF), resuspended bacterium, ultrasonic bacterium (each 1min, 5～10 times), 12 split under ice bath, the centrifugal 10min of 000r/min, in separation, cleer and peaceful precipitation, abandon supernatant, receives heavy 20 ℃ of the shallow lake – of collection frozen.
Metal chelate affinity chromatography: regeneration NI-NTA post, buffer A (pH8.5,20mmol/LTrisCl, 100mmol/L KCl, 5mmol/L beta-mercaptoethanol, 20mmol/L imidazoles) balance NI-NTA chromatography column, by the supernatant upper prop, keep the flow velocity of 0.5mL/min, the buffer A of 10 times of column volumes is washed post, to wash away foreign protein; The buffer B of 2 times of column volumes (pH8.5,20mmol/L TrisCl, 1mol/L KCl, 5mmol/L beta-mercaptoethanol) is washed post, washes away nucleic acid; Use damping fluid C (pH8.5,20mmol/L TrisCl, 100mmol/L, 5mmol/L beta-mercaptoethanol, 100mmol/L imidazoles) elution of bound albumen again, collect elution peak, polyacrylamide gel electrophoresis shows that eluted protein comprises target protein.
Ion exchange chromatography is further purified: ion exchange chromatography damping fluid (pH7.4, 50mmol/L Trisci) balance Q Sapharose HP chromatography column, to be added in through the pleiotrophin fusion rotein of Ni-NTA chromatography column preliminary purification on Q Sapharose HP gel media, wash chromatography column with damping fluid, with elution buffer (pH7.4, 50mmol/L TrisCl, 1mol/L NaCl) albumen of elution of bound, collect elution peak, polyacrylamide gel electrophoresis is identified eluted protein, result is as shown in Figure 6: wherein, swimming lane M is protein Marker, be respectively 97.2, 66.4, 44.3, 29.0, 20.1, 14.3kDa, swimming lane 1 is the pET44a (+) that do not induce-pleiotrophin/BL21 reconstitution cell, swimming lane 2 is 0.2mMIPTG abduction delivering 4h, swimming lane 3 is the pleiotrophin fusion rotein after purifying.Freeze-drying after the recombinant human pleiotrophin fusion rotein packing of purifying is standby.
The promotion of d.pleiotrophin fusion rotein cell growth
Ordinary method is cultured to the PC12 cell of logarithmic phase, makes cell suspension, is divided into 6 groups, and every group of 5 every porocyte numbers in hole are 1 * 10
3individual volume is 150 μ L.The 1st group adds 25 μ L buffer A contrasts, the 2nd group adds pleiotrophin fusion rotein liquid 5 μ L buffer A 20 μ L, the 3rd group adds pleiotrophin fusion rotein liquid 10 μ L buffer A 15 μ L, the 4th group adds pleiotrophin fusion rotein liquid 15 μ L buffer A 10 μ L, the 5th group adds pleiotrophin fusion rotein liquid 20 μ L buffer A 5 μ L, the 6th group adds pleiotrophin fusion rotein liquid 25 μ L, act on after 72 hours and add MTT solution (5mg/ml joins with PBS) 20 μ l to continue to hatch 4 hours, the careful suction abandoned culture supernatant in hole, every hole adds 150 μ l DMSO, vibrate 10 minutes, crystallisate is fully melted.Select the 490nm wavelength, measure each hole absorbance value on microplate reader.The detected result of six groups of samples (transverse axis is the albumen irritating concentration, and the longitudinal axis is light absorption value) as shown in Figure 7, along with the also increase thereupon of raising light absorption value of protein liquid concentration, illustrates that the PC12 cell is stimulated rear rate of growth to accelerate by the pleiotrophin fusion rotein.
Claims (8)
1. the multi-functional somatomedin Pleiotrophin fusion rotein of recombinant human, it is characterized in that, comprise the multi-functional somatomedin Pleiotrophin of people, also be connected with purifying flag sequence, zymoplasm and enteropeptidase restriction enzyme site in turn at the N of the multi-functional somatomedin Pleiotrophin of people end, and the Nus sequence.
2. the multi-functional somatomedin Pleiotrophin fusion rotein of recombinant human as claimed in claim 1, is characterized in that, the aminoacid sequence of the multi-functional somatomedin Pleiotrophin of described people is as shown in SEQ.ID.NO.1.
3. the multi-functional somatomedin Pleiotrophin fusion rotein of recombinant human as claimed in claim 1, is characterized in that, the flag sequence that described purifying flag sequence is 6 * His.
4. the multi-functional somatomedin Pleiotrophin fusion rotein of recombinant human as claimed in claim 1, is characterized in that, the aminoacid sequence of the multi-functional somatomedin Pleiotrophin fusion rotein of described recombinant human is as shown in SEQ.ID.NO.2.
5. a prokaryotic expression carrier of expressing the multi-functional somatomedin Pleiotrophin fusion rotein of recombinant human, is characterized in that, this prokaryotic expression carrier is pET44a
(+)-Pleiotrophin, be by EcoR I/XHo I restriction enzyme site by the multi-functional somatomedin Pleiotrophin gene fragment of the people as shown in SEQ.ID.NO.3, be cloned into pET44a
(+)expression vector.
6. the preparation method of the multi-functional somatomedin Pleiotrophin fusion rotein of recombinant human, is characterized in that, comprises the following steps:
1) take the cDNA that comprises whole person Pleiotrophin genes encoding frame is template, pcr amplification people Pleiotrophin gene fragment;
2) after being cut, the people Pleiotrophin gene fragment enzyme of pcr amplification obtains the fragment with sticky end; And by this fragment and same two pET44a
(+)carrier connects, and obtains expression vector pET44a
(+)-Pleiotrophin;
3) by expression vector pET44a
(+)-Pleiotrophin is transfected into competent intestinal bacteria, and screening obtains the positive transformant of destination gene expression;
4) by positive transformant picking neat in edge, bacterium colony that growth conditions is good after 37 ℃ of thermostat containers are cultivated 12h, be inoculated in the LB nutrient solution containing penbritin 100mg/L 37 ℃ of overnight incubation of shaking table; Be inoculated in fresh containing in the LB nutrient solution of penbritin 100mg/L next day, 37 ℃ are continued to be cultured to bacterial density and reach OD
600=0.4～0.6, add the IPTG inducing culture 2～4h of 0.2mM concentration, make expression vector pET44a
(+)-pleiotrophin is expressed, then centrifugal collection bacterium;
5) ratio in 5～10mL/g bacterial precipitation adds lysate, and resuspended bacterium ultrasonicly under ice bath is split bacterium, then centrifugation, and collect supernatant;
6) supernatant of collection is splined on to the metal chelate affinity chromatography post, by the buffer A of 10 times of column volumes, washes the flow velocity wash-out of post with 0.5mL/min, then by the buffer B of 2 times of column volumes, wash post, finally use damping fluid C elution of bound albumen, collect elution peak;
The TrisCl that described buffer A is pH8.5, concentration 20mmol/L, the KCl of 100mmol/L, the mixing solutions of the beta-mercaptoethanol of 5mmol/L and the imidazoles of 20mmol/L; The TrisCl that described buffer B is pH8.5, concentration 20mmol/L, the mixing solutions of the KCl of 1mol/L and the beta-mercaptoethanol of 5mmol/L; The TrisCl that described damping fluid C is pH8.5, concentration 20mmol/L, the KCl of 100mmol/L, the mixing solutions of the beta-mercaptoethanol of 5mmol/L and the imidazoles of 100mmol/L;
The elution peak of collection is splined on to ion exchange chromatography, with the albumen of elutriant D elution of bound, collects elution peak, obtain the multi-functional somatomedin Pleiotrophin fusion rotein of recombinant human of purifying;
The TrisCl that described elutriant D is pH7.4, concentration 50mmol/L and the mixing solutions of 1mmol/L NaCl.
7. the preparation method of the multi-functional somatomedin Pleiotrophin fusion rotein of recombinant human as claimed in claim 6, is characterized in that, described pcr amplification people Pleiotrophin gene fragment, be to using primer pair P as primer, and described primer pair P is:
Upstream primer P1:aggaattcat gcaggctcaa cagtacc;
Downstream primer P2:agctcgagta aatccagcat cttctcc.
8. the preparation method of the multi-functional somatomedin Pleiotrophin fusion rotein of recombinant human as claimed in claim 6, it is characterized in that, the TrisCl that described lysate is pH8.5, concentration 50mmol/L, the mixing solutions of the beta-mercaptoethanol of 5mmol/L and the PMSF of 1mmol/L.
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| CN106749605A (en) * | 2016-12-15 | 2017-05-31 | 武汉海特生物制药股份有限公司 | A kind of growth factor of human nerve analog and preparation method thereof |
| CN114369170A (en) * | 2021-12-09 | 2022-04-19 | 陕西师范大学 | Therapeutic fusion protein and vaccine for glioblastoma based on targeted pleiotropic growth factor, and preparation method and application thereof |
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| CN101775404A (en) * | 2009-01-08 | 2010-07-14 | 华东理工大学 | Method for highly expressing basic protein with prokaryotic expression system |
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| CN106749605A (en) * | 2016-12-15 | 2017-05-31 | 武汉海特生物制药股份有限公司 | A kind of growth factor of human nerve analog and preparation method thereof |
| CN114369170A (en) * | 2021-12-09 | 2022-04-19 | 陕西师范大学 | Therapeutic fusion protein and vaccine for glioblastoma based on targeted pleiotropic growth factor, and preparation method and application thereof |
| CN114369170B (en) * | 2021-12-09 | 2022-09-23 | 陕西师范大学 | Therapeutic fusion protein and vaccine for glioblastoma based on targeted pleiotropic growth factor, and preparation method and application thereof |
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