CN103145852B - 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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- CN103145852B CN103145852B CN201310068275.4A CN201310068275A CN103145852B CN 103145852 B CN103145852 B CN 103145852B CN 201310068275 A CN201310068275 A CN 201310068275A CN 103145852 B CN103145852 B CN 103145852B
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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 that 1989 Nian You University of Washington medical center purifying obtain, thought at that time that this factor was the albumen of 17kDa, there is high affinity with heparin, and be named as HBGF-8 first (heparin.binding growth factor-8).Ptn gene is positioned at 7q33-q34, and length is greater than 100kb, 168 amino acid of encoding, and albumen size 15.3kD, shows as 18kD at sds polyacrylamide electrophoresis.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.PTN Main Function has: (1) plays an important role to the generation of tumour, growth and transfer, tumor-blood-vessel growth; (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 because the mRNA secondary structure of Pleiotrophin gene is complicated, 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, 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 recombinant human, is also 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 Nus sequence.
The aminoacid sequence of the described multi-functional somatomedin Pleiotrophin of 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.
Express a prokaryotic expression carrier for 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.
A preparation method for the multi-functional somatomedin Pleiotrophin fusion rotein 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
(+)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) in the ratio of 10mL/g bacterial precipitation, add lysate, resuspended bacterium, ultrasonicly under ice bath splits bacterium, then centrifugation, and collect supernatant;
Described lysate is the TrisCl of 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 metal chelate affinity chromatography post, by the buffer A of 10 times of column volumes, washes post with the flow velocity wash-out of 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;
Described buffer A is the TrisCl of 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; Described buffer B is the TrisCl of pH8.5, concentration 20mmol/L, the mixing solutions of the KCl of 1mol/L and the beta-mercaptoethanol of 5mmol/L; Described damping fluid C is the TrisCl of 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;
Described elutriant D is the TrisCl of 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 determined, wants the more difficult expression of this albumen of single expression.And the present invention is by building prokaryotic expression carrier pET44a
(+)-pleiotrophin, and transform 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 nutritional support neurone, promote the biological activity of neurocyte proliferation, retardation tumour cell 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 building also comprises the purifying flag sequence of 6 * His, is convenient to like this purifying of fusion rotein; And by the restriction enzyme digestion reaction of zymoplasm and enteropeptidase, can remove flag sequence when being necessary, thereby can access pleiotrophin polypeptide.
3, this fusion rotein utilizes protokaryon intestinal bacteria system to express, 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 purity height is all the advantage of this fusion rotein.
Accompanying drawing explanation
Fig. 1 is the electrophoresis detection figure that pcr amplification obtains 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 result figure that SDS-PAGE detects pleiotrophin expressing fusion protein;
Fig. 5 is the result figure that Western Blot detects pleiotrophin fusion rotein solubility expression;
Fig. 6 is that SDS-PAGE detects the result figure after 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 intestinal bacteria transformant is carried out to abduction delivering, after separation and purification, obtain Pleiotrophin fusion rotein.Pleiotrophin fusion rotein is carried out to SDS-PAGE and Western Blot and analyzes, its molecular weight with predict the outcome consistent; And biological activity checking shows, Pleiotrophin fusion rotein has the effect that promotes 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)
In GenBank, search people Pleiotrophin gene (NM_002825.5).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, amplifies 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 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 obtaining 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 containing is expressed fusion rotein and can obtains 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 reclaiming, 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; 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 linearization plasmid large fragment.
The people pleiotrophin sequence of b. enzyme being cut and the pET44a of recovery
(+)linear fragment connects with 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 and are received bacterium after 12h 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 is correctly 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, 37 ℃ of thermostat containers are cultivated after 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, is 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 is expressed expression vector pET44a (+)-pleiotrophin, then centrifugal collection bacterium.
The evaluation of 4.pleiotrophin fusion rotein
A.10%SDS-PAGE(polyacrylamide gel electrophoresis) detect the expression of pleiotrophin fusion rotein
With pET44a (+)-pleiotrophin/BL21 reconstitution cell of inducing in contrast, centrifugal collection after IPTG induction 4h, uses 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 coomassie brilliant blue staining after visible obviously induction, 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 pET44a (+)-pleiotrophin/BL21 reconstitution cell of not inducing, and swimming lane 2 is 0.2mM IPTG abduction delivering 4h; Compare with other swimming lanes, swimming lane 2, after carrying out IPTG induction, produces the pleiotrophin fusion rotein of the about 82.5kDa of molecular weight.
The Western Blot of b.pleiotrophin fusion rotein detects
Collect pET44a (+)-pleiotrophin/BL21 (DE3) recombinant bacterium after IPTG induction, extract respectively cleer and peaceful inclusion body on 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 detecting at expressing fusion protein finishes, pull down gel, according to the Bio-Rad description of product, by gel near negative electrode one side, 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 NC film; After electrophoresis finishes, take out 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, TBST room temperature is washed 3 times, each 5min; Then add mouse source His tag antibody, incubated at room 1h, TBST room temperature is washed 3 times, each 5min; Add again two of luciferase mark to resist, incubated at room 1h, TBST room temperature is washed 3 times, each 5min, infrared albumen is swept film instrument and is swept film and be presented at pleiotrophin fusion rotein band after the induction being all labeled as seen in cleer and peaceful inclusion body on kytoplasm, molecular weight is consistent with SDS-PAGE electrophoresis detection, illustrates that 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 pET44a (+)-pleiotrophin/BL21 reconstitution cell of not inducing; Swimming lane 2 is the supernatant after 0.2mM IPTG induction for pET44a (+)-pleiotrophin/BL21; Swimming lane 3 be pET44a (+)-pleiotrophin/BL21 with after 0.2mM IPTG induction inclusion body; Contrast swimming lane 1,2,3 is visible: pleiotrophin fusion rotein has expression in inclusion body precipitation.
And according to the expression of pET44a (+) carrier, 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, detects consistent with SDS-PAGE and Western Blot.
The purifying of c.pleiotrophin fusion rotein
Enlarged culturing pET44a (+)-pleiotrophin/BL21 recombinant bacterium, centrifugal collection bacterium after IPTG induction, adds lysate (pH8.5 by 10mL/g bacterial precipitation, 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, abandons 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 supernatant upper prop, the flow velocity that keeps 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, pleiotrophin fusion rotein through Ni-NTA chromatography column preliminary purification is added on Q Sapharose HP gel media, with damping fluid, wash chromatography column, 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 pET44a (+)-pleiotrophin/BL21 reconstitution cell of not inducing, 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, acting on after 72 hours adds MTT solution (5mg/ml joins with PBS) 20 μ l to continue to hatch 4 hours, careful suction abandoned culture supernatant in hole, every hole adds 150 μ l DMSO, vibrate 10 minutes, crystallisate is fully melted.Select 490nm wavelength, in microplate reader, measure each hole absorbance value.The detected result of six groups of samples as shown in Figure 7 (transverse axis is albumen irritating concentration, and the longitudinal axis is light absorption value), along with the also increase thereupon of raising light absorption value of protein liquid concentration, illustrates that PC12 cell is stimulated rear rate of growth to accelerate by pleiotrophin fusion rotein.
Claims (3)
1. the multi-functional somatomedin Pleiotrophin fusion rotein of recombinant human, it is characterized in that, comprise the multi-functional somatomedin Pleiotrophin of people, at the N of the multi-functional somatomedin Pleiotrophin of people end, be also connected with purifying flag sequence, zymoplasm and enteropeptidase restriction enzyme site in turn, and Nus sequence;
The aminoacid sequence of the multi-functional somatomedin Pleiotrophin fusion rotein of described recombinant human is as shown in SEQ.ID.NO.2.
2. 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.
3. a preparation method for 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;
Described pcr amplification people Pleiotrophin gene fragment, is 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;
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) in the ratio of 5～10mL/g bacterial precipitation, add lysate, resuspended bacterium, ultrasonicly under ice bath splits bacterium, then centrifugation, and collect supernatant;
Described lysate is the TrisCl of 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 metal chelate affinity chromatography post, by the buffer A of 10 times of column volumes, washes post with the flow velocity wash-out of 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;
Described buffer A is the TrisCl of pH 8.5, concentration 20mmol/L, the KCl of 100mmol/L, the mixing solutions of the beta-mercaptoethanol of 5mmol/L and the imidazoles of 20mmol/L; Described buffer B is the TrisCl of pH 8.5, concentration 20mmol/L, the mixing solutions of the KCl of 1mol/L and the beta-mercaptoethanol of 5mmol/L; Described damping fluid C is the TrisCl of pH 8.5, 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; Described elutriant D is the TrisCl of pH 7.4, concentration 50mmol/L and the mixing solutions of 1mmol/L NaCl.
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