CN1978463A

CN1978463A - Mesenchymal stem cell affinity peptide screening and use

Info

Publication number: CN1978463A
Application number: CN 200510127973
Authority: CN
Inventors: 裴雪涛; 赵敬湘; 杨丽平; 王韫芳; 闫舫; 师伟; 秦立蓬; 管利东
Original assignee: Institute of Field Blood Transfusion Chinese Academy of Military Medical Sciences
Current assignee: Institute of Field Blood Transfusion Chinese Academy of Military Medical Sciences
Priority date: 2005-12-09
Filing date: 2005-12-09
Publication date: 2007-06-13
Anticipated expiration: 2025-12-09
Also published as: CN100475836C

Abstract

This invention relates to biotechnology field of cell and polypeptide. The aim of this invention is screening a small peptide that can strongly combine with MSC, and using it to separate and purify MSC and using it as carrying agent to prepare targeted therapeutic agent. The stated binding peptide sequence of MSC is ringed seven peptide sequence that abundantly contains Ser, Thr, Asn, Lys. Finally this invention uses phage display technique to screen small peptide that can strongly combine with MSC in random libraries of ringed seven peptides by affinity screening with MSC. MSC can link to solid phase sustentaculum through small peptide screened by using this invention. It can be used for screening, separating and purifying MSC. It can be used to prepare tracer agent of MSC after linking with fluorescent tumor marker. It can be used as carrying agent of MSC and medicine to prepare praeparatum about targeted therapy.

Description

The screening of mesenchymal stem cell affinity peptide and purposes

Technical field

The invention belongs to the biological medicine technology field, relate to and adopt the application of phage random peptide library the screening of mescenchymal stem cell surface affinity peptide and the affinity peptide that filters out.

Background technology

(mesenchymal stem cells is to derive from mesoblastic adult stem cell with height self ability and multidirectional differentiation potential MSC) to mescenchymal stem cell.Extensively be present between whole body reticular tissue and organ in the matter, the abundantest with content in the myeloid tissue.In recent years report from prematurity of fetus Cord blood, umbilical vein subendothelial layer, periosteum, tendon, blood vessel, fatty tissue etc. and also be separated to MSC.MSC has high differentiation potential, and wherein mesenchymal stem cells MSCs can be divided into sophisticated mesenchymal cell under different inductive conditions, as scleroblast, chondroblast, adipocyte, Tenocyte cell, skein cell, vascular endothelial cell etc.Present research also demonstrates the whole art differentiation of MSC might cross over the germinal layer boundary, surmount the traditional mesenchymal cell that is divided into the mesoderm source, and transform to parenchyma, as be divided into myocardial cell, neurocyte etc., this achievement in research not only has the important in theory meaning, its multidirectional differentiation potential and do not exist advantages such as tissue matching and immunological rejection to make its seed cell as organizational project when transplanting provides the good curing approach at aspects such as the reparation of bone and cartilaginous tissue damage, muscle tissue degenerative disease, coronary heart disease.MSC is easy to the importing and the expression of foreign gene, in the process of external long-term cultivation, remain the potential of multidirectional differentiation, the genetic background quite stable is therefore quite wide in the application prospect of aspects such as organizational project trauma repair, cell replacement treatment, hematopoiesis support, gene therapy.Another characteristic of mescenchymal stem cell is to have migration chemotactic ability, can be to the direction migration of tumor cell invasion, it is that carrier carries therapeutic gene and realized targeted therapy (Nedime Serakinci to tumour with MSC that many researchs reports are abroad arranged at present, Oncogene, 2004,23:5095-5098; Nakamural K, Gene Therapy, 2004,11:1155-1164), these results of study are for being that the targeted therapy of cell carrier provides experimental basis with MSC.

The method of separation MSC commonly used has full marrow method and centrifuging, and full marrow method is regularly changed liquid and removed not attached cell promptly according to the adherent characteristic of stem cell, is cell etc. as hematopoiesis, reaches the purpose of separation and purification MSC.Centrifuging is extracted monocyte and is carried out adherent culture promptly according to the difference of cellular constituent proportion, but it is longer to obtain time of high purity MSC.In order to obtain more highly purified MSC, the someone utilizes flow cytometer method, immunomagnetic beads method etc. that MSC is carried out separation and purification, but required plant and instrument costliness.

Phage display rondom polypeptide storehouse technology is based on display technique of bacteriophage (phage display).1985 by Smith (Smith GP., Science, 1985,228:1315-1317) by genetic engineering means the less important coat protein pIII of exogenous protein and phage is formed fusion rotein first and be showed in the phage particle surface, can keep relatively independent space conformation and biological activity, and not influence the infection ability of recombinant phage the host bacterium.Compare with other gene expression systems, the maximum characteristics of display technique of bacteriophage are exactly that it will be demonstrated polypeptide or protein and gene coupling effectively, constitute an entity, thereby have also just obtained its gene when obtaining certain polypeptide structure.

The screening of phage display random peptide library is biological elutriation (biopanning) process of an affinity purification (affinity purification).Its detailed process is: earlier phage peptide library and target molecule are interacted for some time, then non-specific combination phage is removed in washing, and specificity is eluted amplification in conjunction with phage, and then drops into the elutriation of next round.Continue to repeat elutriation, wash-out, amplification, final elutriation goes out the recombinant phage clone of specific combination.By analyzing the structure and the sequence of the peptide that is screened, for the interaction mechanism of (as antigen and antibody, acceptor and part, enzyme-to-substrate) between the protein molecule provides theoretical foundation, the bioactive peptide that screens function simultaneously can enter Clinical Application and exploitation.

Adopted the interaction of phage random peptide library technical study acceptor and part to screen the peptide storehouse with the acceptor molecule of purifying mostly in the past, thereby obtain and acceptor molecule bonded peptide section, and when understanding few to the structural information of a certain acceptor molecule, and under surface of cell membrane acceptor condition of unknown, the natural receptor molecule that obtains purifying will be very difficult, and the acceptor molecule of purifying also may lose its native conformation and function, at this moment, the intact cell of adopt expressing this receptor is as a kind of easy selection of can yet be regarded as of screening target.At present, the cell that is used to screen the phage random peptide library technology comprises the tumour cell etc. of cell, thrombocyte, peripheral blood neutrophil and the vitro culture of transfection acceptor gene.

The object of the present invention is to provide a kind of technology of utilizing phage random peptide library in-vitro screening cell surface affinity peptide, and obtained can with mescenchymal stem cell bonded ring seven peptide aminoacid sequence, the little peptide that utilizes the present invention to screen connects screening, separation and the purifying that can be used for MSC on solid support, with the tracer agent that can be used for preparing MSC after fluorescent marker connects, can be used as the preparation that carrier between MSC and medicine can be used for preparing targeted therapy.The present invention will be in preparation neoplasm targeted therapy medicine, drug screening, cell paste has splendid application prospect in spike and the cell sorting, and will have a tremendous social and economic benefits.

Summary of the invention

For achieving the above object, technological approaches of the present invention is a separation and Culture mescenchymal stem cell at first, in the time of at the bottom of cell is in logarithmic phase and covers with bottle, carry out affine screening with the ring seven peptide storehouse, by strengthening proof strength, through the three-wheel screening, ELISA identifies, obtain the stronger clone of affinity, check order and carry out sequential analysis.With the Liu Shi method synthetic peptide being carried out affinity identifies.Concrete technical scheme is as follows:

(1) separation and Culture of mescenchymal stem cell

1) mescenchymal stem cell can derive from the marrow of mouse, people's marrow, peripheral blood, embryo, bleeding of the umbilicus etc.

2) separation of human marrow mesenchymal stem cell: under the aseptic condition, puncture through the bilateral posterior superior iliac spine, gather marrow, through the Percoll (U.S., Sigma company product, relative density 1.073g/ml) collector's BMNC after the density gradient centrifugation, adherent culture 72h removes the mesenchymal stem cells MSCs that is of adherent growth behind the suspension cell.The cellular form that cultivation is gone down to posterity as shown in Figure 1.

3) separation of mouse mescenchymal stem cell: aseptic condition separates femur and tibia down, go out marrow, blow and beat into cell suspension, centrifugal, abandon supernatant, the PBS re-suspended cell presets equal-volume lymphocyte separation medium (U.S., Sigma company product with adherent the joining gently of cell suspension, relative density 1.083g/ml) in the centrifuge tube, make to be suspended from the upper strata, centrifugal, the mononuclearcell of collecting cloud tunica albuginea layer is in another centrifuge tube, after the PBS washing, adherent culture 72h removes the mesenchymal stem cells MSCs that is of adherent growth behind the suspension cell, obtains progressively purifying after cultivating through going down to posterity.

(2) evaluation of mescenchymal stem cell surface marker and purity

Get the 3rd and be commissioned to train and support the mescenchymal stem cell of amplification, the trysinization collecting cell, centrifugal.Behind PBS washed cell 2～3 times, with 1ml PBS re-suspended cell, counting.Adjusting cell concn with PBS is 1 * 106/ml, by 5 * 105 cell/pipes with the cell average mark to a plurality of EP pipes, a plurality of antibody with PE or FITC mark make up respectively, add in each pipe, hatch 20～30min under the room temperature, wash 2 times with PBS then, add 0.5ml PBS re-suspended cell after, the surface marker of cultivating amplifying cells detected and identify with flow cytometer.

(3) phage random peptide library screening

1) phage random peptide library can be commercial dodecapeptide storehouse (Ph.D.-12TM Phage Display PeptideLibrary Kit), seven peptide storehouses (Ph.D.-7TM Phage Display Peptide Library Kit) and ring seven peptide storehouse (the peptide storehouse of the different lengths of (Ph.D.-C7CTM Phage Display Peptide Library Kit) and oneself structure thereof.

2) the polypeptide both sides that suppress the ring seven peptide storehouse (Ph.D.-C7C) of disulfide linkage to be showed have respectively added a halfcystine.Through peroxidation, form the polypeptide of cyclisation during the phage assembling.The storage capacity in general peptide storehouse is subjected to the restriction of electric commentaries on classics condition, reaches as high as 109 storage capacity, is enough to cover the combined probability of 7 peptides, but combined probability that can only cover part 12 peptides.In addition, 7 cycle peptide library make it easier and the acceptor mortise because the cyclisation of peptide has strengthened its space conformation.Therefore select that 7 cycle peptide library are easier to screen the peptide affine with MSC for use, this is a special preferred peptide storehouse of the present invention.

3) screening method: at first in flat board or culturing bottle, cultivate adherent mesenchyme in cell, to mescenchymal stem cell logarithmic phase and density be 90% o'clock, remove nutrient solution, the PBS washing; Add the serum-free medium that contains phage peptide library, hatch 15min～1h for 37 ℃; Absorption contains the serum-free medium of phage peptide library, with the PBS washed cell 3～6 times that contains 0.1%～0.5% polysorbas20 (Tween 20); Glycine buffer with pH2.2 washes and the strong bonded phage of cell surface at last, and use rapidly in Tris (pH9.8) damping fluid and after, stay 10 μ l to carry out titer determination, residue changes in the fresh bacterium liquid (ER2738), hatch 15min～30mm for 37 ℃, 37 ℃ of violent jolting 4～5h amplification phages then, the PEG method is purified, and enters the next round screening again.Phage number after counting each screening back and the amplification.For improving the affinity of phage display peptide and cell, in screening process subsequently, gradually reduce the binding time of phage and cell, and strengthen washing intensity.

(4) phage titre is measured

Each screening of taking turns all need be measured the titre of input and output phage, and calculated yield.Phage to be measured by gradient (1: 10～1: 108) dilution, is added among the LB that contains host bacterium E.coliER2738 (OD=0.2～0.3), and mixing is laid in the bottom platform of 37 ℃ of preheatings in the top-layer agar that adding is dissolved.In 37 ℃ of overnight incubation, calculate its clone's number and phage titre (TU).TU/L==locus coeruleus number * extension rate * 10 ⁵

(5) evaluation of the amplification of plaque and phage positive colony

Last phage of taking turns wash-out is paved plate, tens of random chooses are separated good blue phage spot and are added 37 ℃ of violent jolting 4～5h amplification phages in the resistance substratum that contains host bacterium (as ER2738) on the IPTG/X-gal agar plate, the centrifuging and taking supernatant, multiple once centrifugal, get 80% supernatant, preserve or add glycerine to final concentration 50% ,-20 ℃ of preservations for 4 ℃.The mono-clonal supernatant carries out EL ISA evaluation after measuring titre.

The MSCs cell inoculation of separation and purification is cultivated in 96 well culture plates, treated the cell well-grown at the bottom of monolayer adherence is paved with plate the time, with PBS flushing 2 times, stationary liquid is fixed (glutaraldehyde as 0.25%, 10% formaldehyde, acetone etc.); With PBS flushing 2 times, 3% hydrogen peroxide is handled the activity of cell endogenous peroxydase; PBS flushing rear enclosed liquid (spends the night or 37 ℃ of sealing 1h～3h as 2～5% skimmed milks, 0.1%～0.5%BSA) 4 ℃ of sealing; Every hole adds the phage of purifying, and (1 * 108～1 * 1010pfu) hatches 1h～3h, and is contrast with wild-type M13 phage and PBS; With the PBS washed cell that contains 0.05%～0.3% polysorbas20 (Tween20) 3～6 times; Every hole adds anti-M13 phage mAb (dilution in 1: 2000～1: 5000) the 50 μ L of horseradish peroxidase (HRP) mark, hatches 1h～3h; With the PBS washed cell that contains 0.05%～0.3% polysorbas20 (Tween 20) 3～6 times, developer (is surveyed A450nm. as colour developing backs such as tetramino p-diaminodiphenyl (TMB), O-Phenylene Diamines (OPD).Computation of mean values ± standard deviation is selected positive colony from the phage clone that above-mentioned screening obtains.

(6) positive phage clones determined dna sequence and analysis

Extract ssDNA with reference to phage single-chain DNA extraction agent box, precipitation is dissolved in the 30 μ l ultrapure waters.Get 5 μ lssDNA and carry out electrophoretic analysis, residue phage ssDNA carries out sequencing, amino acid occurs in the comparative sequences ratio and rule.

(7) design of polypeptide, synthetic

Select the template that the frequency of occurrences is the highest and peptide is synthesized in the positive strong sequence conduct of ELISA in the sequencing result.Synthetic peptide adopts mass spectroscopy to determine accuracy and purity.Can carry out mark (as FITC, Cy5.5 etc.) to synthetic peptide as required.

(8) affinity of synthetic peptide and MSC detects

Can there be several different methods to detect the affinity of synthetic peptide and MSC

1) competition with wild type phage M13 combines test

Carry out aforesaid screening process with joining in the long culture plate that mescenchymal stem cell arranged with 1: 1 mixed with wild-type M13 phage respectively after the positive phage clones amplification.Because wild-type M13 does not contain the LacZ gene, so on the IPTG/X-gal agar plate, can not show blue spot, therefore count blue, hickie number respectively, and calculate it relatively and binding ability (the Zhang JB of wild-type M13 phage according to following method, Cancer Lett, 2001,171 (2): 153-164.)

The peptide storehouse phage of the M13 * input of the peptide storehouse phage/output of the M13 * output of relative bonding force=input

2) Liu Shi detects

With the mescenchymal stem cell trysinization of cultivating, PBS washing back is hatched 20min～30min with the synthetic peptide of fluorescent mark (as FITC, Cy5.5 etc.) at 37 ℃, and negative control adopts the MSC that does not add synthetic peptide.After the PBS washing three times, Liu Shi detects fluorescently-labeled ratio and intensity.

Description of drawings

The form of Fig. 1 human marrow mesenchymal stem cell (* 100)

The form (* 100) of Fig. 2 rat mescenchymal stem cell

Fig. 3 rat bone marrow mesenchymal stem cells Liu Shi identifies

(1) FITC-CD90 positive expression

(2) FITC-CD45 positive expression

(3) PE-CD31 positive expression

(4) FITC-CD44 positive expression

The DNA electrophoretic analysis of Fig. 4 positive colony phage clone

Synthetic peptide of Fig. 5 FITC mark and rat MSC bonded Fluirescence observation

Synthetic peptide of Fig. 6 FITC mark and rat MSC bonded Liu Shi analyze

(1) rat brain glioma 9L cell negative control

(2) rat brain glioma 9L cell synthesizes combining of peptide with the FITC mark

(3) rat bone marrow mesenchymal stem cells negative control

(4) rat bone marrow mesenchymal stem cells synthesizes combining of peptide with the FITC mark

The enrichment of table 1 positive bacteriophage

The EL ISA method of table 2 positive phage clones is identified

The aminoacid sequence of table 38 positive phage clones

Specific embodiments

Accompanying drawings specific implementation method of the present invention is as follows:

Embodiment 1, with the rat separation and Culture of example explanation MSC

The experimental procedure of present embodiment is as follows:

Select healthy wistar rat in 6 ages in week (about 150 grams, male and female are not limit) for use, aseptic condition separates femur, shin bone down, goes out marrow, blows and beats into cell suspension, 1200rpm, centrifugal 5min.Abandon supernatant, the PBS re-suspended cell joins in the centrifuge tube that presets equal-volume lymphocyte separation medium (proportion 1.083) gently with cell suspension is adherent, makes to be suspended from the upper strata 1800rpm, centrifugal 25min.The mononuclearcell of collecting cloud tunica albuginea layer is in another centrifuge tube, and the PBS washing is (the centrifugal 5min of 1200rpm) once, abandons supernatant, and 10% newborn calf serum L-DMEM is resuspended with containing.With 1 * 10 ⁵/ ml inoculating cell is incubated at 5%CO under temperature of saturation, 37 ℃, pH7.2 condition in culturing bottle ₂Constant incubator.Change liquid during 3d first, change liquid twice later on weekly, when treating that cell merges near 90%, tryptic digestion, 1 * 10 ⁴/ ml goes down to posterity, be labeled as the first-generation, inverted microscope is observed day by day, treat cell be paved with bottle at the bottom of the time, repeat aforesaid operations, the amplification of going down to posterity repeatedly, go down to posterity with 0.25% tryptic digestion, 2～3min at every turn, the amount and the digestion time of strict control enzyme with MSC and possible miscellaneous lymphocyte, unicellular separating, thereby obtain the MSC (see figure 2) of purifying.

The evaluation of embodiment 2, rat marrow MSC surface marker and purity

Getting the 4th is commissioned to train and supports the rat bone marrow mesenchymal stem cells of amplification, 0.25% trysinization collecting cell, the centrifugal 5min of 1200rpm.Behind PBS washed cell 2 times, with 1ml PBS re-suspended cell, counting.Adjusting cell concn with PBS is 1 * 10 ⁶Individual/ml, by 5 * 10 ⁵Individual cell/pipe with the cell average mark to a plurality of EP pipes, each 5 μ l of a plurality of antibody CD90, CD45, CD44 and CD31 (Peprotech company product) with PE or FITC mark add in each pipe respectively, hatch 30min under the room temperature, wash 2 times with PBS then, after adding 0.5ml PBS re-suspended cell, the surface marker of cultivating amplifying cells is detected and identify with flow cytometer.The result shows that rat bone marrow mesenchymal stem cells CD90, the CD45, CD44 and the CD31 that cultivate amplification express positive (see figure 3).

The screening of embodiment 3, MSC affinity peptide

The experimental procedure of present embodiment is as follows:

The MSC in the 2nd generation of separation and purification or the 3rd generation is seeded in to be added with in L-DMEM substratum 6 well culture plates of (containing 10% foetal calf serum) cultivates 2d, treat that the cell well-grown is at the bottom of monolayer adherence is paved with plate the time, draw nutrient solution, with L-DMEM substratum flushing 2 times, add 1ml and contain the stoste of 10 μ L Ph.D.-C7C phage peptide libraries (titre is 6.5 * 10 ¹²Pfu/mL) L-DMEM cultivates and hatches 1h based on 37 ℃.With the PBS liquid washing that contains 0.1%Tween20 4 times, each 1min.The phage of removal and the non-specific combination of MSC, every then hole add the glycine buffer of 1mL pH2.2 and place 8min on ice, and shake frequently so that wash-out goes out to be attached to the phage of cell surface.The phage that sucking-off elutes is used the neutralization of Tris (pH9.8) damping fluid rapidly.Stay 10 μ l to carry out titer determination, residue changes in LB (tetracyclin resistance) nutrient solution that 20ml contains the fresh bacterium liquid of 4ml (ER2738, OD=0.2～0.4), hatch 15min for 37 ℃, 37 ℃ of violent jolting 4～5h amplification phages then, the PEG method is purified, and enters the next round screening again.Phage number after counting each screening back and the amplification.Every enrichment condition of taking turns positive bacteriophage sees Table 1.For improving specificity, the 2nd, 3,4 take turns the binding time (45min that gradually reduces phage and cell, 30min, 15min), strengthen phage (0.1%PBST, the 0.2%PBST of the weak avidity of wash-out, 0.3%PBST) through 4 take turns the screening process of wash-out-amplification-wash-out after, the 4th phage of taking turns wash-out is paved plate, and 30 of random chooses are separated purifications of increasing in a large number of good blue phage spots on the IPTG/X-gal agar plate, are used for the evaluation of EILSA positive colony.

Table 1

Round	Input(pfu)	Output(pfu)	Recovery(％)
Round	Input(pfu)	Output(pfu)	Recovery(％)	1 2 3 4	1.0×10 ¹¹1.0×10 ¹¹1.0×10 ¹¹1.0×10 ¹¹	6.6×10 ⁶ 8.7×10 ⁶ 1.1×10 ⁹ 1.3×10 ⁸	6.6×10 ^-5 8.7×10 ^-5 1.1×10 ^-2 1.3×10 ^-3

The evaluation of the amplification of embodiment 4, plaque and phage positive colony

Picking is expanded to OD=0.4～0.5 at the ER2738 mono-clonal of LB (Tet resistance) growth in LB (Tet resistance) substratum, with the dilution in 1: 10 of LB (Tet resistance) nutrient solution, add 2ml in each test tube.Take turns screening back from 4 respectively and measure on the flat board of titre at random that 50 locus coeruleus of picking increase, 37 ℃, 250rpm, jolting 5h; 4 ℃ of nutrient solutions, 10000rpm, centrifugal 5min gets the supernatant repeated centrifugation once, gets 80% supernatant, preserves or add glycerine to final concentration 50% ,-20 ℃ of preservations for 4 ℃.The mono-clonal supernatant carries out EL ISA evaluation after measuring titre.

Phage E LISA: the MSC in the 2nd generation of separation and purification or the 3rd generation is seeded in to be added with in L-DMEM substratum 96 well culture plates of (containing 10% foetal calf serum) cultivates, treat that the cell well-grown is at the bottom of monolayer adherence is paved with plate the time, draw nutrient solution, with PBS flushing 2 times, 4 ℃ of 0.25% glutaraldehyde are 30min fixedly, with PBS flushing 2 times, add hydrogen peroxide (10 μ LH ₂O ₂/ 10ml PBS) normal temperature effect 30min removes endogenous peroxydase.PBS flushing 2 times adds 2% skimmed milk, and 4 ℃ of sealings are spent the night.Every hole adds the phage 1 * 10 of purifying ⁹, incubated at room 2h.0.1%PBST washes 4 times, and every hole adds the anti-M13 phage of HRP mark mAb (New England Biolabs) the 50 μ L of dilution in 1: 2000, incubated at room 2h.0.1%PBST washes 4 times, and A450nm is surveyed in TMB colour developing back.Each clone all establishes 6 parallel holes, by the negative contrast of PBS, is contrast with M13 simultaneously with bag, computation of mean values ± standard deviation.From the phage clone that above-mentioned screening obtains, select positive colony, obtain 8 positive colonies (table 2) altogether than control value＞2.

Table 2

	Positive colony (A450)	Contrast M13 (A450)	Negative control PBS (A450)
	Positive colony (A450)	Contrast M13 (A450)	Negative control PBS (A450)	1 2 3 4 5 6 7 8	0.233 0.230 0.280 0.258 0.240 0.248 0.358 0.263	0.118 0.118 0.118 0.118 0.118 0.118 0.118 0.118	0.104 0.104 0 104 0.104 0.104 0.104 0.104 0.104

Embodiment 5, single stranded DNA extract and identify

Positive phage clones is increased, promptly get 50 μ L mono-clonal supernatants and join 5ml and contain in the resistance substratum of ER2738 37 ℃, 250rpm cultivates 5h; 4 ℃ of nutrient solutions, 10000rpm, centrifugal 5min gets the supernatant repeated centrifugation once, gets 80% supernatant.Get 1ml phage supernatant, add 400 μ LPEG, 4 ℃ of 10min precipitation phages; 4 ℃, the centrifugal 10min of 12000rpm obtains the phage precipitation; Add 200 μ L Loddie Buffr (10mMTris-HCl, pH8.0,1mM EDTA, 4M NaCl, the preservation of normal temperature black out) and heavily revolve phage; Add 500 μ L ethanol, the centrifugal 10min of room temperature 12000rpm behind the mixing room temperature placement 10min abandons supernatant, with 70% washing with alcohol precipitation, drying at room temperature 5min adds 30 μ L DNA Free Warter dissolving, get 5 μ L and be used for electrophoretic analysis, all the other are used for sequencing.As seen the ssDNADNA band that extracts 8 positive colonies from electrophorogram (Fig. 4) is 3.4kb, and that confirm extraction is the ssDNA of phage.

Embodiment 6, positive colony dna sequencing and polypeptid acid sequence analysis

SsDNA to 8 positive colonies carries out the dna sequencing analysis by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd, and its encoding amino acid sequence sees Table 3.Amino acid to 8 positive colonies is analyzed, and the probability that discovery Asn, Lys, Thr, Ser occur in the positive colony of screening is bigger, therefore selects for use Ser-Thr-Asn-Pro-Lys-Val-Leu to carry out chemosynthesis.For the ease of detecting, this peptide is added FITC as fluorescent mark, be FITC-Ahx-Cys-Ser-Thr-Asn-Pro-Lys-Val-Leu-Cys by BeiJing ZhongKe Yaguang Biology Science Co., Ltd's synthetic sequence.

Table 3

Numbering	Aminoacid sequence
Numbering	Aminoacid sequence							1 2 3 4 5 6 7 8	Asp Ser Ser Ser Asn Thr Ser Ser	Ser Leu Thr Asp Thr Leu Pro Asp	Pro Glu Asn Pro Ile Thr Phe Asp	Asn Lys Pro Leu Met Asn Asn Gln	Lys Asn Lys Arg Arg Tyr Thr Ile	Thr Thr Val Gln Ser Ser Lys Ile	Ser Ser Leu Leu Asp Ser Ala Trp

Embodiment 7, synthetic peptide are to the detection of MSC affinity

1 * 10 ⁴MSC be inoculated in 96 orifice plates, grow to 90% density after, remove nutrient solution, contain with 100 μ L in the serum-free medium of 100 μ MFITC mark peptides, hatch 30min for 37 ℃, fluorescent microscope detects visible 100% cell and presents green fluorescence, sees Fig. 5.

Detect the affinity of synthetic peptide to MSC with stream Schwann Cells instrument, step is as follows: separation and purification Wistar rat MSC, 2 * 10 ⁵The digestion back is washed one time with PBS, adding 200 μ L concentration is the synthetic peptide of 100 μ M, hatch 30min in 37 ℃ of constant incubators, with PBS flushing 2 times, carrying out Liu Shi after 1% Paraformaldehyde 96 is fixing detects, set up negative control except that own control, also with the negative contrast of incoherent glioma cell 9L at random.The Liu Shi detected result shows almost green fluorescence on the 100%MSC mark, and fluorescence intensity is 29.7 times of self negative control, and 3.8 times of negative contrast illustrate that synthetic peptide has binding specificity to MSC.See Fig. 6.

Sequence table

<210>1

<211>21

<212>DNA

<400>1

ctatcaggct tattctgatc a 21

<210>2

<211>21

<212>DNA

<400>2

agcgacctct tcttatgctc a 21

<210>3

<211>21

<212>DNA

<400>3

agctgcttag gcttccaaga c 21

<210>4

<211>21

<212>DNA

<400>4

tcactaggcg acgccgtcga c 21

<210>5

<211>21

<212>DNA

<400>5

ttatgctaat catcctcact a 21

<210>6

<211>21

<212>DNA

<400>6

tgagactgat taataagcag c 21

<210>7

<211>21

<212>DNA

<400>7

agcggaaaat tatgcttccg c 21

<210>8

<211>21

<212>DNA

<400>8

agcctactag tctcctccaa c 21

Claims

1, one group can with the little peptide of mescenchymal stem cell (MSC) high-affinity bonded, its aminoacid sequence from the N-terminal to the C-terminal is:

Asp Ser Pro Asn Lys Thr Ser

Ser Leu Glu Lys Asn Thr Ser

Ser Thr Asn Pro Lys Val Leu

Ser Asp Pro Leu Arg Gln Leu

Asn Thr Ile Met Arg Ser Asp

Thr Leu Thr Asn Tyr Ser Ser

Ser Pro Phe Asn Thr Lys Ala

Ser Asp Asp Gln Ile Ile Trp。

2, according to the reverse sequence of the described little peptide of claim 1, its aminoacid sequence from the N-terminal to the C-terminal is:

Ser Thr Lys Asn Pro Ser Asp

Ser Thr Asn Lys Glu Leu Ser

Leu Val Lys Pro Asn Thr Ser

Leu Gln Arg Leu Pro Asp Ser

Asp Ser Arg Met Ile Thr Asn

Ser Ser Tyr Asn Thr Leu Thr

Ala Lys Thr Asn Phe Pro Ser

Trp Ile Ile Gln Asp Asp Ser。

3, according to the described little peptide of claim 1, the gene that it is characterized in that described little peptide is one of following nucleotide sequences:

1) dna sequence dna of sequence 1～8 in the sequence table;

2) with sequence table in the dna sequence dna that limits of sequence 1～8 have 90% above homology, and the identical function protein DNA sequence of encoding.

4, according to the fusion rotein of the described little peptide of claim 1.

5, have the purposes of the little peptide of high-affinity with MSC, it is characterized in that by with the high-affinity of MSC, this peptide is connected in screening, separation and the purifying that can be used for MSC on the solid support.

6, have the purposes of the little peptide of high-affinity with MSC, it is characterized in that can be used for preparing the preparation of targeted therapy as the carrier between MSC and medicine.

7, have the purposes of the little peptide of high-affinity with MSC, it is characterized in that and the tracer agent that can be used for preparing MSC after fluorescent marker connects.