CN107641148A - Polypeptide and its application - Google Patents
Polypeptide and its application Download PDFInfo
- Publication number
- CN107641148A CN107641148A CN201710978419.8A CN201710978419A CN107641148A CN 107641148 A CN107641148 A CN 107641148A CN 201710978419 A CN201710978419 A CN 201710978419A CN 107641148 A CN107641148 A CN 107641148A
- Authority
- CN
- China
- Prior art keywords
- polypeptide
- cell
- lung cancer
- stem cell
- present
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses a kind of polypeptide and its application.The polypeptide includes the amino acid sequence shown in any one of SEQ ID No.3; it can specifically bind the lung cancer stem cell in non-small cell lung cancer H460 cell lines; without being combined with normal pneumonocyte, other lung carcinoma cells and normal stem cell; its preparation method is simple and easy simultaneously, suitable for scale industrial production.The present invention provides important theory and practice basis for identification, separation and targeted therapy of lung cancer stem cell etc., has broad application prospects.
Description
The present invention is Application No. 201310585699.8, and the applying date is on November 19th, 2013, entitled《Polypeptide
And its application》Divisional application.
Technical field
The present invention is more particularly directed to a kind of lung cancer stem cell specific binding polypeptide and its application, belong to biomedicine field.
Background technology
The World Health Organization in 2008 explicitly points out in its report announced, and NCD just turns into the mankind the most
Fatal " killer ", there are about 5,000,000 people to die from cancer every year.But for entirety, therapeutic effect for tumour is simultaneously paid no attention to
Think.Essentially consist in that tumor pathogenesis is unclear, such as the Origin of tumour.Tumor stem cell (Cancer Stem
Cells, CSCs) theoretical proposition let us sees new hope.It is heterogeneous that tumor stem cell theory thinks that tumor tissues have
Property, in all tumour cells, only very small part cell, which has, forms and maintains tumour growth and heterogeneous ability, this
Sub-fraction cell is referred to as tumor stem cell.And it is believed that tumor stem cell is tumour starting and growth, recurs and turn
Move, and root caused by drug resistance.Research for tumor stem cell is advantageous to us and is better understood by tumor development
Process and establish a kind of more effective therapeutic strategy.
At present, the research of tumor stem cell depends on molecular marker, but most solid tumors do not find spy
Different tumor stem cell molecular marker.Researcher it is expected to establish a kind of new strategy always, established a kind of independent of spy
Different molecular marker is identified and isolated from tumor stem cell method.The surface display technologies occurred in recent years can be in unknown cell
The screening of cell-specific Binding peptide is realized in the case of surface molecular.Surface display technologies are real using genetic engineering means
Existing allogenic polypeptide（Or protein domain）The novel gene engineering skill being shown in the form of fusion protein in antimicrobial surface
Art, the polypeptide or protein domain being demonstrated can keep relatively independent space structure and bioactivity.Bacterium surface exhibition
Show technology combination fluidic cell sorting technology using quick, high-throughout feature as display technique field in the focus applied.But
How to carry out the screening of lung cancer stem cell specific binding polypeptide using above-mentioned surface display technologies and further establish one kind to disobey
The method for being identified and isolated from tumor stem cell relied in special molecular mark is still industry urgent problem to be solved.
The content of the invention
An object of the present invention is to provide a kind of polypeptide, and it includes any in SEQ ID No.1~SEQ ID No.3
Amino acid sequence shown in.
The second object of the present invention is the derivative for providing foregoing polypeptides, and it includes：
(i) more than one amino acid in the amino acid sequence shown in any one of SEQ ID No.1~SEQ ID No.3 is residual
The substituted polypeptide formed of base,
Or more than one ammonia of (ii) in the amino acid sequence shown in any one of SEQ ID No.1~SEQ ID No.3
The polypeptide for introducing substituted radical in base acid residue and being formed,
Or polypeptide described in (iii) claim 1 merges the polypeptide to be formed with selected compound,
Or selected additional amino acid sequence is blended in shown in any one of SEQ ID No.1~SEQ ID No.3 by (iv)
Amino acid sequence and the polypeptide that is formed.
Further, the derivative of the polypeptide includes：
More than one in amino acid sequence shown in any one of SEQ ID No.1~SEQ ID No.3, preferably 1~3, more
It is preferred that 1~2, particularly preferred 1 conservative amino acid it is substituted it is being formed, can be with non-small cell lung cancer H460 cell lines
In lung cancer stem cell specific binding polypeptide.
Further, the derivative of the polypeptide includes the core shown in any one of SEQ ID No.1~SEQ ID No.3
Heart amino acid sequence, and connect the peptide fragment with X1, X2 amino acid respectively at core amino acid sequence both ends, wherein X1, X2
For any one integer in 0~7.
As one of wherein more typical case, the derivative of the polypeptide has the amino shown in SEQ ID No.4
Acid sequence.
The third object of the present invention is to provide another polypeptide, and it has the amino acid sequence shown in SEQ ID No.1,
And disulfide bond is formed between two cysteines therein, forms circular polypeptides structure.
The fourth object of the present invention is that providing foregoing polypeptides or derivatives thereof is preparing non-small cell lung cancer H460 cells
It is the application in the detection of lung cancer stem cell and/or separation agent or kit.
The fifth object of the present invention is that providing foregoing polypeptides or derivatives thereof is preparing controlling for non-small cell lung cancer H460
Treat the application in medicine.
The sixth object of the present invention is the polynucleotides or its variant for providing a kind of separation, and it can encode foregoing
Polypeptide or derivatives thereof.
The seventh object of the present invention is to provide the polynucleotides comprising coding foregoing polypeptides or derivatives thereof or its variation
The expression vector of body.
The eighth object of the present invention is to provide thin comprising foregoing polynucleotides or the host of its variant or aforementioned bearer
Born of the same parents.
The nineth object of the present invention is to provide a kind of fluorescein-labeled polypeptide, and it is included：
Foregoing polypeptides or derivatives thereof,
And to mark the fluorescein molecule of the polypeptide or derivatives thereof.
The tenth object of the present invention is to provide a kind of magnetic particle of polypeptide marker, and it includes foregoing polypeptides or its derivative
Thing and magnetic particle, wherein, the magnetic particle includes magnetic nano-particle or superparamagnetic nano-particle.
The tenth object of the present invention one be to provide a kind of non-small cell lung cancer H460 cell line lung cancer stem cell detection and/
Or separation agent or kit, it includes foregoing polypeptides or derivatives thereof.
The tenth object of the present invention two is to provide a kind of pharmaceutical composition for being used to treat non-small cell lung cancer H460, bag
Containing pharmaceutical acceptable carrier and foregoing polypeptides or derivatives thereof.
The present invention using the external display technique of bacterium random peptide library and fluidic cell sorting technology filter out with it is non-small thin
The bacterium of lung cancer stem cell specific binding in born of the same parents' lung cancer H460 cell lines, reflects through gene sequencing and cell in vitro Binding experiment
After fixed, 13 peptides with the specific binding of lung cancer stem cell are obtained, there is the sequence shown in SEQ ID No.1~SEQ ID No.3,
It can be respectively designated as " HCBP-1 peptides ", " HCBP-2 peptides " and " HCBP-3 peptides ".
Compared with prior art, advantages of the present invention at least that：HCBP-1 peptides~HCBP-3 the peptides, it is particularly therein
HCBP-1 peptides can be specifically bound with the lung cancer stem cell in non-small cell lung cancer H460 cell lines, and to normal lung into fiber finer
Born of the same parents HLF cells, a variety of lung carcinoma cells and human mesenchymal stem cell act on without specific binding, show that polypeptide is thin to lung cancer tumor
The specific selection of born of the same parents.Meanwhile the preparation method of the HCBP-1 peptides~HCBP-3 peptides is simple and easy, given birth to suitable for large-scale industrial
Production.The present invention provides important theory and practice basis for identification, separation and targeted therapy of lung cancer stem cell etc., has wide
Wealthy application prospect.
It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and have in below (eg embodiment)
It can be combined with each other between each technical characteristic of body description, so as to form new or preferable technical scheme.As space is limited, exist
This no longer tires out one by one states.
Brief description of the drawings
Figure 1A -1B show the Enrichment Amplification of non-small cell lung cancer H460 tumor stem cells, wherein, Figure 1A is shown carefully
Born of the same parents' culture monolayer adherence in the environment for have serum grows, and keeps differentiation state.Figure 1B shows a small amount of cell in serum-free
Lasting separation growth, the glomerate clone ball of shape, keeps stem cell state in cultivating system.
Fig. 2A -2B show non-small cell lung cancer H460 tumor stem cells specific binding bacterium of the present invention（Hereinafter referred to as
Specified bacterial, and cell shown in figure is the tumor stem cell as blank control）Enrichment process, wherein, Fig. 2A is shown
With the increase of sorting number, specified bacterial progressively increases to the joint efficiency of tumor stem cell, and Fig. 2 B are shown with sorting
The increase of number, specified bacterial do not increase to the joint efficiency of H460 attached cells.
Fig. 3 shows present invention screening gained specified bacterial and lung normal fibroblast system HLF cells, lung cancer cell line
A549, H1299 cell, the joint efficiency of human mesenchymal stem cell MSC cells are very low.
Fig. 4 A-4B show 3 bacterial clones screened（Peptide containing HCBP-1~HCBP-3 peptides respectively）With Tumor Stem
The combination situation of cell and adherent H460 cells, Fig. 4 A show HCBP-1 bacterium and tumor stem cell joint efficiency it is reachable
52.3 %, and there was only bacterium and the tumor stem cell that 0.6 %, Fig. 4 B show HCBP-2 with the joint efficiency of attached cell
Joint efficiency only has 0.5 % up to 38.6 % with the joint efficiency of attached cell.Fig. 4 C show HCBP-3 bacterium with swelling
The joint efficiency of knurl stem cell only has 0.9 % up to 37.2 % with the joint efficiency of attached cell.
Fig. 5 shows the result of fluorescence microscope identification tumor stem cell, wherein, it is enriched the clone ball of tumor stem cell
Surface combines the HCBP-1 polypeptides of a large amount of FITC mark, and H460 cell surfaces are only in conjunction with a small amount of HCBP-1 polypeptides,
Control polypeptides are relatively low to two kinds of cell combinations.
Fig. 6 A-6B show the result of flow cytometry tumor stem cell, and wherein Fig. 6 A, which are shown, is enriched tumour
The Percentage bound of clone's glomus cell of stem cell and the control polypeptides of FITC marks only has 1.1%, and and HCBP-1 ' polypeptides knot
Conjunction rate may be up to 60%, Fig. 6 B and show that two kinds of polypeptides are relatively low to H460 cells Percentage bound.
Embodiment
For the present inventor by depth studying extensively, it is SEQ ID No.1, SEQ ID that find has core sequence first
No.2 and SEQ ID No.3, especially SEQ ID No.1 polypeptide, can specifically with non-small cell lung cancer H460 cells
Lung cancer stem cell in system combines, and confirms, carries out derivative polypeptide in certain limit according to the core sequence polypeptide, together
Sample has binding activity.It is demonstrated experimentally that the lung that the polypeptide and its derivative polypeptide can be used in non-small cell lung cancer H460 cell lines
Cancer stem cell is identified and isolated from, and not limited to this.The present invention is completed on this basis.
Active peptides
In the present invention, term " polypeptide with core sequence " refers to SEQ ID No.1~SEQ ID No.3, is particularly
Amino acid sequence shown in SEQ ID No.1 as core sequence and done with the lung cancer in non-small cell lung cancer H460 cell lines
The albumen or polypeptide of cell-bound activity.
Wherein, a kind of preferable polypeptide with core sequence is with following structural formula：
X1-LGCFPEGEMACWW-X2
In formula, X1, X2 are the peptide fragment of 0 or 1-7 amino acid.
In the present invention, term " derivative polypeptide ", refer to the lung cancer stem cell knot in non-small cell lung cancer H460 cell lines
Close activity, the variant form of former sequence is used as using sequence shown in SEQ ID No.1- SEQ ID No.3.These variant forms
Including (but being not limited to)：Compared with former sequence, the missing of 1-3 (being usually 1-2, more preferably 1) amino acid, insertion
And/or substitution, and in C-terminal and/or N-terminal addition or missing one or several amino acid.For example, in the art, use
When similar nature or similar amino acid are substituted, it will not generally change the function of protein.Again for example, in C-terminal and/or
N-terminal, which adds or lacked one or several amino acid, will not generally also change the 26S Proteasome Structure and Function of protein.In addition, the term
Also include monomer and multimeric forms polypeptide of the present invention.The term also includes linear and nonlinear polypeptide (such as cyclic peptide).
Term " polypeptide of the present invention " refers to " core sequence ", " polypeptide with core sequence " and " derivative polypeptide "
General name
Present invention additionally comprises active fragment, derivative and the analog of shown polypeptide of the present invention.As used herein, term " piece
The lung cancer stem cell that section ", " derivative " and " analog " refer to be kept substantially in non-small cell lung cancer H460 cell lines combines
The polypeptide of activity.Polypeptide fragment, the derivative or the like of the present invention can be that (i) has one or several conservative or non-conservations
The substituted polypeptide of amino acid residue (preferably conservative amino acid), or (ii) have in one or more amino acid residues
The polypeptide of substituted group, or (iii) core sequence and another compound (for example extend the compound of polypeptide half-life period, example
Such as polyethylene glycol) the formed polypeptide of fusion, or (iv) additional amino acid sequence is blended in this peptide sequence and formed more
Peptide (merge and formed with sequence labels such as targeting sequencing, secretion sequence or 6His and then albumen).According to teaching herein, this
A little fragment, derivative and analogs belong to scope known to those skilled in the art.
A kind of preferable reactive derivative refers to compared with formula i amino acid sequence or corresponding former sequence, there is at most 3,
Preferably at most 2, more preferably at most 1 amino acid is similar or similar amino acid is replaced and forms polypeptide by property.This
A little conservative variation's polypeptides carry out amino acid substitution preferably based on table 1 and produced.
Table 1
| Initial residue | Representational substitution | Preferable substitution |
| Ala (A) | Val; Leu; Ile | Val |
| Arg (R) | Lys; Gln; Asn | Lys |
| Asn (N) | Gln; His; Lys; Arg | Gln |
| Asp (D) | Glu | Glu |
| Cys (C) | Ser | Ser |
| Gln (Q) | Asn | Asn |
| Glu (E) | Asp | Asp |
| Gly (G) | Pro; Ala | Ala |
| His (H) | Asn; Gln; Lys; Arg | Arg |
| Ile (I) | Leu; Val; Met; Ala; Phe | Leu |
| Leu (L) | Ile; Val; Met; Ala; Phe | Ile |
| Lys (K) | Arg; Gln; Asn | Arg |
| Met (M) | Leu; Phe; Ile | Leu |
| Phe (F) | Leu; Val; Ile; Ala; Tyr | Leu |
| Pro (P) | Ala | Ala |
| Ser (S) | Thr | Thr |
| Thr (T) | Ser | Ser |
| Trp (W) | Tyr; Phe | Tyr |
| Tyr (Y) | Trp; Phe; Thr; Ser | Phe |
| Val (V) | Ile; Leu; Met; Phe; Ala | Leu |
The present invention also provides the analog of polypeptide of the present invention.The difference of these analogs and natural polypeptides can be amino acid sequence
On difference or do not influence difference on the modified forms of sequence, or have both at the same time.Analog also includes having not
The analog of the residue (such as D- amino acid) of natural L-amino acids is same as, and with non-naturally occurring or synthesis amino acid
The analog of (such as β, gamma-amino acid).It should be understood that the polypeptide of the present invention is not limited to the above-mentioned representational polypeptide enumerated.
Wherein, modification (not changing primary structure generally) form includes：The chemically derived form of inner or in vitro polypeptide
Such as acetylation or carboxylated.Modification also includes glycosylation, such as those in the synthesis and processing of polypeptide or further processing step
It is middle to carry out glycosylation modified and caused polypeptide.This modification can be by the way that polypeptide (be such as fed exposed to glycosylated enzyme is carried out
The glycosylase or deglycosylating enzyme of newborn animal) and complete.Modified forms also include having phosphorylated amino acid residue (such as phosphoric acid
Tyrosine, phosphoserine, phosphothreonine) sequence.Also include be modified so as to improve its anti-proteolysis performance or
Optimize the polypeptide of solubility property.
Polypeptide of the present invention can also with as pharmaceutically or physiology it is acceptable acid or alkali derived from salt form use.These
Salt includes but is not limited to the salt formed with following acid：Hydrochloric acid, hydrobromic acid, sulfuric acid, citric acid, tartaric acid, phosphoric acid, lactic acid,
Pyruvic acid, acetic acid, butanedioic acid, oxalic acid, fumaric acid, maleic acid, oxaloacetic acid, methanesulfonic acid, ethyl sulfonic acid, benzene sulfonic acid or hydroxyl second sulphur
Acid.Other salt include：The salt formed with alkali metal or alkaline-earth metal (such as sodium, potassium, calcium or magnesium), and with ester, carbamate
Or the form of other conventional " pro-drugs ".
Coded sequence
The invention further relates to the polynucleotides for being separately encoded polypeptide of the present invention.
For the sequence of the preferable coded sequence core sequence of one of which as shown in SEQ ID No.5, it encodes SEQ ID
Core polypeptide fragment shown in No.1.
The polynucleotides of the present invention can be DNA form or rna form.DNA can be coding strand or noncoding strand.Coding
The coding region sequence of mature polypeptide can or the variant of its degeneracy identical with the sequence shown in SEQ ID No.5.Such as this
Used in text, by taking core sequence as an example, " variant of degeneracy " refers to that coding has sequence shown in SEQ ID No.1 in the present invention
Polypeptide, but with the differentiated nucleotide sequence of corresponding encoded region sequence in SEQ ID No.5.
The coding nucleotide full length sequence of the present invention or its fragment can generally use PCR TRAPs, recombination method or artificial conjunction
Into method obtain.At present, it is already possible to completely by chemical synthesis come obtain encoding polypeptide of the present invention (or its fragment, or its
Derivative) DNA sequence dna.Then the DNA sequence dna can be introduced to various existing DNA moleculars as known in the art (or as carried
Body) and cell in.
The present invention also relates to the carrier of the polynucleotides comprising the present invention, and the carrier with the present invention or polypeptide of the present invention
Coded sequence is through host cell caused by genetic engineering.
On the other hand, present invention additionally comprises the monoclonal antibody of polypeptide of the present invention and polyclonal antibody, especially monoclonal
Antibody.
The preparation method of polypeptide of the present invention
One of ordinary skill in the art can prepare polypeptide of the present invention, such as biosynthesis, full chemistry synthesis using conventional method
The methods of.
It is a kind of it is preferable that with liquid phase synthesis techniques or solid phase synthesis technique, such as Boc solid phase methods, Fmoc solid phase methods
Or two methods are used in combination.Synthesis in solid state can quickly obtain sample, can be selected suitably according to the sequence signature of purpose peptide
Resin carrier and synthesis system.For example, preferable solid phase carrier is such as connected with the Wang trees of C-terminal amino acid in peptide in Fmoc systems
Fat, arm of the Wang resin structures between polystyrene, with amino acid are 4- alkoxy benzylalcohols；With 25% hexahydropyridine/dimethyl
Formamide room temperature treatment 20 minutes, to remove Fmoc blocking groups, and according to given amino acid sequence from C-terminal one by one to N-terminal
Extension.After the completion of synthesis, the proinsulin related peptide of synthesis is cut from resin with the trifluoroacetic acid containing 4% p-methyl phenol
Get off and remove protection group, may filter that except ether precipitates isolated thick peptide after resin.After the solution of products therefrom is freezed, use
Peptide needed for gel filtration and the purifying of reverse phase HPLC method.When carrying out synthesis in solid state using Boc systems, preferred resin
To be connected with the PAM resins of C-terminal amino acid in peptide, arm of the PAM resin structures between polystyrene, with amino acid is 4- hydroxyl first
Base phenyl acetamide；In Boc synthesis systems, deprotection, in and, coupling circulation in, with TFA/ dichloromethane (DCM) remove
Blocking group Boc and with diisopropylethylamine (DIEA/ dichloromethane neutralize.After the completion of peptide chain condensation, with containing p-cresol (5-
10%) hydrogen fluoride (HF), handle 1 hour, peptide chain is cut from resin, while remove blocking group at 0 DEG C.With 50-
80% acetic acid (containing a small amount of mercaptoethanol) extracts peptide, is further divided after solution is lyophilized with molecular sieve Sephadex G10 or Tsk-40f
From purifying, then purify through high pressure liquid phase to obtain required peptide again.Known various coupling agents in chemistry of peptides field can be used
Each amino acid residue is coupled with coupling method, such as dicyclohexylcarbodiimide (DCC), hydroxyl benzotriazole can be used
(HOBt) or the urea hexafluorophosphoric acid esters (HBTU) of 1,1,3,3- tetra- are directly coupled.For the obtained small peptide of synthesis, its purity with
Structure can be confirmed with RP-HPLC and mass spectral analysis.
In a preferred embodiment, biological synthesis process has following steps：
(1) with the polynucleotides (or its variant) for encoding active peptides of the present invention or derivatives thereof, or with containing more nucleosides
The recombinant expression carrier conversion of acid or suitable host cell of transduceing；
(2) host cell cultivated in suitable culture medium；
(3) polypeptide of the present invention is separated, purified from culture medium or cell.
In the present invention, polynucleotide sequence can be plugged into recombinant expression carrier.Side well-known to those having ordinary skill in the art
Method can be used to build the expression vector of DNA sequences encoding and suitable transcription/translation control signal containing the polypeptide.Comprising upper
Appropriate DNA sequence dna and the carrier of appropriate promoter or control sequence are stated, can be used for converting appropriate host cell, so that
It can express the active peptides of the present invention.Host cell can be prokaryotic, such as bacterial cell；Or low eucaryon is thin
Born of the same parents, such as yeast cells；Or higher eucaryotic cells, such as plant cell.
It can be carried out with recombinant DNA conversion host cell with routine techniques well known to those skilled in the art.When host is original
When core biology is such as Escherichia coli, can absorb DNA competent cell can harvest after exponential phase of growth, use CaCl2Method processing, institute
With the step of it is generally well-known in the art.Another method is to use MgCl2.If desired, conversion can also use the side of electroporation
Method is carried out.When host is eucaryote, following DNA transfection methods can be selected：Calcium phosphate precipitation, conventional mechanical methods are such as
Microinjection, electroporation, liposome packaging etc..
The transformant of acquisition can use conventional method culture, express the active peptides of the present invention.It is thin according to host used
Born of the same parents, culture medium used may be selected from various conventional mediums in culture.Cultivated under conditions of suitable for host cell growth.
After host cell growth is to appropriate cell density, inducing selection (such as temperature transition or chemical induction) with suitable method
Promoter, cell is further cultured for a period of time.
Polypeptide of the present invention can be expressed or be secreted into extracellular in the cell or on cell membrane.It is if desired, available
Its physics, chemical and other characteristics are separated and purified described active peptides by various separation methods.These methods are
It is well-known to those skilled in the art.The example of these methods includes but is not limited to：The renaturation process of routine, use albumen precipitation
Agent processing (salting-out method), centrifugation, the broken bacterium of infiltration, super processing, ultracentrifugation, sieve chromatography (gel filtration), adsorption chromatography, from
The combination of sub- displacement chromatography, high performance liquid chroma- tography (HPLC) and other various liquid chromatography technologies and these methods.
Main advantages of the present invention include：
(a) polypeptide of the present invention can be specifically bound with the lung cancer stem cell in non-small cell lung cancer H460 cell lines；
(b) polypeptide of the present invention can carry out fluorescence molecule mark or be coupled to magnetic particle surface, specific recognition lung cancer stem cell,
So as to carry out being identified and isolated from for tumor stem cell；
(c) can be prepared by the method for synthesis in solid state, purity is high, and yield is big, and cost is low.
The present invention is expanded on further with reference to specific embodiment.It should be understood that these embodiments are merely to illustrate the present invention
Rather than limitation the scope of the present invention.The experimental method of unreceipted actual conditions in the following example, generally according to conventional strip
Part, such as Sambrook et al., molecular cloning：Laboratory manual (New York: Cold Spring Harbor
Laboratory Press, 1989) condition described in, or according to the condition proposed by manufacturer.Unless otherwise indicated,
Otherwise percentage and number are percentage by weight and parts by weight.
The enrichment of lung cancer stem cell in the non-small cell lung cancer H460 cell lines of embodiment 1：
By the H460 cell dissociations of adhere-wall culture into single cell suspension, with 1 × 104Individual/ml density is inoculated into serum-free
In DMEM/F12 culture mediums, 20 ng/ml EGF and bFGF and 2% B27 are added.Every other day with containing the new of growth factor
Fresh culture medium carries out half amount and changes liquid, until needing Secondary Culture.When clone ball length is to 100-200 μm, low-speed centrifugal collects clone
Ball, 1 ml pancreatin is added, 37 DEG C of 2 min of digestion, blows and beats repeatedly to single cell suspension, adds 10 ml PBS, mix centrifugation 1000
Rpm, 5 min, PBS are washed 2 times, and PBS is resuspended, cell count, with 1 × 104Individual/ml density renewed vaccination is to serum-free
In DMEM/F12 culture mediums, Secondary Culture is carried out.
Figure 1A shows that cell culture monolayer adherence in the environment for have serum grows, and keeps differentiation state.Figure 1B is shown
The lasting separation growth in serum free culture system of a small amount of cell, shape glomerate clone ball, keeps stem cell state.It is logical
Single clone's glomus cell is inoculated into 96 orifice plates by the method for crossing limiting dilution, and a cell is only contained in each hole, this
The single clone's glomus cell of kind can grow up to a larger clone ball again in the time of 10 days, and these clone balls are thin
Born of the same parents can be with continuous passage 20 more than generation, and indicating these clone's glomus cells has the spy of stem cell self-renewing and infinite multiplication
Property.Therefore a small amount of tumor stem cell be present in explanation non-small cell lung cancer H460 cell lines, and free serum culture body can be passed through
System is enriched with.
Embodiment 2 screens the polypeptide with the lung cancer stem cell specific binding in H460 cell lines：
Bacterium random peptide library used in the present invention comes from University of California at Santa Barbara department of chemistry engineering Patrick
S.Daugherty laboratories.Its detailed building process is：OmpX (outer membrane protein X) gene order is entered
Row transformation, makes the separated of s53 and s54 residues on its 2nd loop, and formation is free on extracellular COOH ends and NH2 ends, turned into
CPX（circularly permuted outer membrane protein OmpX）.Afterwards by the X2CX7CX2 of random synthesis
Polypeptid coding sequence be connected in CPX sequences, it is expressed in CPX extracellular NH2 ends, by improved CPX fragments connect
It is connected to pBAD33（Guzman, L. et al., Tight regulation, modulation, and high-level
Expression by vectors containing the arabinose PBAD promoter, J. Bacteriol.,
1995,177, p4124-4130）AlajGFP1 in plasmid（Bessette, P.H. et al. Rapid isolation of
high-affinity protein binding peptides using bacterial display. (2004)
Protein Eng. Des. Sel. 17, 731-739）In the multiple cloning sites in downstream, improved pBAD33 plasmids can be with
CPX and surface display polypeptide expression are controlled using araBAD promoters.Improved pBAD33 plasmids are transformed into large intestine bar
Bacterium MC1061 bacterial strains, it is possible to utilize arabinose（arabinose）Control the displaying of CPX and rondom polypeptide in bacterium surface
（Dane, K.Y. et al. Isolation of cell specific peptide ligands using
fluorescent bacterial display libraries (2006) J. Immunol Methods. 309, 120-
129）.
（1）Utilize the bacterium of selected by flow cytometry apoptosis expression and tumor stem cell specific binding
Bacterium random peptide library is recovered, and at least takes the bacterium amount of 10 times of storage capacity in LB culture mediums, expands growth at 37 DEG C, is used
0.02% 1 hour Induction of bacterial of (m/v) arabinose room temperatures, by bacterium：Cell is 100：1 ratio and non-small cell lung cancer
Cell line H460 cell incubations, 4 DEG C, 30 min, 1000 rpm are centrifuged, 4 DEG C, 5 min, take the same amount of clone of supernatant
Glomus cell is incubated, 4 DEG C, 30 min, is centrifuged 1000 rpm, 4 DEG C, 5 min, 3 times, is abandoned supernatant, and PBS (pH 7.4) is resuspended,
It combines situation to flow cytometry, sub-elects with reference to germy cell, the cell sorted out is placed in into Bacteria Culture
It is incubated overnight in base, is augmented with the bacterium of specific binding, repeats the in-vitro screening step 5 time or so of the above, bacterium and tumour
When the joint efficiency of stem cell reaches 40 % and no longer increased, collect bacterium solution and the bacterium solution of collection is inoculated into 5 mL LB cultures
In base, it is incubated overnight.
（2）The specificity verification of bacterium
Bacterium of last wheel sorting is cultivated, induced, and in proportion respectively with lung fibroblast HLF cells, lung cancer
Cell line A549 cells, H1299 cells, human mesenchymal stem cell MSC cell incubations, 4 DEG C, 30 min, centrifuge 1000 rpm, 4
DEG C, 5 min, 3 times, abandon supernatant, PBS (pH 7.4) is resuspended, flow cytometry its combine situation.
（3）The sequencing of specific binding polypeptide
10 μ l are taken out from the bacterium of last wheel sorting to be coated on LB solid mediums, are incubated overnight at 37 DEG C.From flat
50 monoclonal bacteriums are selected on plate at random, numbering clone1-clone50, are inoculated into respectively in 5mL LB fluid nutrient mediums,
It is incubated overnight under conditions of 37 DEG C, 200 revs/min, and the bacterium to being incubated overnight carries out the sequencing of polypeptide respectively, here,
Polypeptide is sequenced for Suzhou Jin Weizhi bio tech ltd.
Fig. 2A is shown as the increase of sorting number, bacterium progressively increase to the joint efficiency of tumor stem cell.Fig. 2 B
Show as the increase of sorting number, bacterium do not increase to the joint efficiency of H460 attached cells.
Fig. 3 shows bacterium and its lung normal fibroblast system HLF cells, other lung carcinoma cells of last wheel sorting
It is A549, H1299 cell, very low with the joint efficiency of human mesenchymal stem cell MSC cells.
Sequencing result, which is shown, shares 3 peptide sequences in 50 clones, the wherein polypeptide of LGCFPEGEMACWW sequences occurs
Frequency highest (for 66.7%), be named as " HCBP-1 peptides ", LKCDWYGINMCVR sequences occur frequency be 20.0%, name
For " HCBP-2 peptides ", the frequency that LNCGFMSLWECWY sequences occur is 13.3%, is named as " HCBP-3 peptides ".
Fig. 4 A show HCBP-1 bacterium and the joint efficiency of tumor stem cell up to 52.3 %, and with attached cell
Joint efficiency only has 0.6 %, Fig. 4 B to show HCBP-2 bacterium and the joint efficiency of tumor stem cell up to 38.6 %, and
There was only 0.5 % with the joint efficiency of attached cell.Fig. 4 C show that HCBP-3 bacterium and the joint efficiency of tumor stem cell can
Up to 37.2 %, and there was only 0.9 % with the joint efficiency of attached cell.
Therefore airflow classification is progressively enriched the bacterium combined with tumor stem cell, and has obtained 3 high joint efficiencies
Specific polypeptide.
The fluorescence labeling polypeptide of embodiment 3 carries out tumor stem cell identification：
Bio tech ltd of hypo Thailand is respectively synthesized the HCBP-1 ' polypeptides and sequence that sequence is SEQ ID No.4 on Shanghai
SEQ ID No.6 control polypeptides are classified as, and FITC fluorescein marks are carried out in N-terminal.
（1）The identification of tumor stem cell is carried out using fluorescence microscope
a）Cell climbing sheet：The cover glass of aseptic process is put into 6 orifice plates, and H460 cells are inoculated into 6 orifice plates containing cover glass,
Culture medium is the culture mediums of RPMI 1640 containing 10 % FBS, is incubated overnight, makes cell in the adherent stretching, extension in cover glass surface.Gram
Grand ball grows to 100 μm or so, and low-speed centrifugal is collected, and serum free medium carries out gravity treatment, adds 6 containing cover glass
In orifice plate, it is incubated overnight so that clone ball pastes on the cover slip.
B) cell is fixed：Culture medium in orifice plate is suctioned out, 2 ml PBS are washed 2 times, and the % poly first of 1 ml 4 is added per hole
Aldehyde, room temperature fix 10 min, and 2 ml PBS are washed 2 times.
C) close：The BSA of 1 ml 3% are added per hole, room temperature closes 10 min, and 2 ml PBS are washed 2 times.
D) it is incubated：Final concentration of 3 × 10 are added per hole3The fluorescent polypeptide of mM FITC marks, room temperature lucifuge are incubated 30
Min, 2 ml PBS are washed 2 times.
e）Redye：Adding final concentration of 2 μ g/ml Hoechst 33342 per hole, room temperature lucifuge dyes 10 min, and 2
Ml PBS are washed 2 times.
f）Mounting, fluorescence microscope are taken pictures.
Fig. 5 shows the combination situation of HCBP-1 polypeptides and control polypeptides to two kinds of cells, is enriched tumor stem cell
The HCBP-1 ' polypeptides of a large amount of FITC marks that combine of clone's ball surface, and H460 cell surfaces are only in conjunction with a small amount of
HCBP-1 ' polypeptides, control polypeptides are relatively low to two kinds of cell combinations.
（2）The identification of tumor stem cell is carried out using flow cytometer
a）Into single cell suspension, density is 1 × 10 for H460 cells and clone ball cell dissociation6Individual/ml.
b）Every group of cell adds the polypeptide of final concentration of 0.5 μM of FITC marks, and 4 DEG C, lucifuge is incubated 30min.
c）4 DEG C, 1000 rpm centrifuge 5 min, and 1 ml PBS are washed 2 times.
d）0.5 ml PBS are resuspended, flow cytomery or sorting.
Fig. 6 shows the combination situation of HCBP-1 ' polypeptides and control polypeptides to two kinds of cells, and wherein Fig. 6 A are shown
The Percentage bound for being enriched clone's glomus cell of tumor stem cell and the control polypeptides of FITC marks only has 1.1%, and and HCBP-
The Percentage bound of 1 ' polypeptide may be up to 60%.Fig. 6 B show that two kinds of polypeptides are relatively low to H460 cells Percentage bound.
Therefore, by previous experiments it can be confirmed that the HCBP-1 peptides of the present invention have the property that：
（1）It can be specifically bound with the lung cancer stem cell in non-small cell lung cancer H460 cell lines, and to normal lung into fiber finer
The joint efficiency of born of the same parents HLF cells, lung cancer cell line A549, H1299, H460 cell and human mesenchymal stem cell MSC cells is very
It is low.
（2）Tumor stem cell, which is carried out, using the polypeptide of fluorescence labeling is identified and isolated from work.
（3）The preparation method of the HCBP-1 peptides is simple to operate, easy to implement, beneficial to progress large-scale industrial production.
All it is incorporated as referring in this application in all documents that the present invention refers to, it is independent just as each document
It is incorporated as with reference to such.In addition, it is to be understood that after the above-mentioned instruction content of the present invention has been read, those skilled in the art can
To be made various changes or modifications to the present invention, these equivalent form of values equally fall within the model that the application appended claims are limited
Enclose.
Sequence table
<110>Suzhou Institute of Nano-tech. and Nano-bionics, Chinese Academy of Sciences
<120>Polypeptide and its application
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 13
<212> PRT
<213>Artificial sequence (artificial sequence)
<400> 1
Leu Gly Cys Phe Pro Glu Gly Glu Met Ala Cys Trp Trp
1 5 10
<210> 2
<211> 13
<212> PRT
<213>Artificial sequence (artificial sequence)
<400> 2
Leu Lys Cys Asp Trp Tyr Gly Ile Asn Met Cys Val Arg
1 5 10
<210> 3
<211> 13
<212> PRT
<213>Artificial sequence (artificial sequence)
<400> 3
Leu Asn Cys Gly Phe Met Ser Leu Trp Glu Cys Trp Tyr
1 5 10
<210> 4
<211> 21
<212> PRT
<213>Artificial sequence (artificial sequence)
<400> 4
Gly Gly Leu Gly Cys Phe Pro Glu Gly Glu Met Ala Cys Trp Trp Ser
1 5 10 15
Gly Gly Ser Gly Lys
20
<210> 5
<211> 39
<212> DNA
<213>Artificial sequence (artificial sequence)
<400> 5
ccaccagcac gccatctcgc cctcggggaa gcaccccaa 39
<210> 6
<211> 21
<212> PRT
<213>Artificial sequence (artificial sequence)
<400> 6
Gly Ser Ser Gly Cys Ser Ser Gly Ser Ser Gly Ser Cys Ser Gly Ser
1 5 10 15
Ser Gly Ser Ser Lys
20
Claims (10)
1. a kind of polypeptide, it is characterised in that its amino acid sequence is as shown in SEQ ID No.3.
2. polypeptide described in claim 1 is preparing what non-small cell lung cancer H460 cell line lung cancer stem cell detected and/or separated
Application in reagent or kit.
3. application of the polypeptide described in claim 1 in the medicine for preparing non-small cell lung cancer H460.
4. the polynucleotides or its variant of separation, it is characterised in that it can encode the polypeptide described in claim 1.
5. include the polynucleotides of polypeptide or the expression vector of its variant described in coding claim 1.
6. include the host cell of carrier described in polynucleotides described in claim 4 or its variant or claim 5.
7. a kind of fluorescein-labeled polypeptide, it is characterised in that it is included：
Polypeptide described in claim 1,
And to mark the fluorescein molecule of the polypeptide.
A kind of 8. magnetic particle of polypeptide marker, it is characterised in that it includes polypeptide and magnetic particle described in claim 1,
Wherein, the magnetic particle includes magnetic nano-particle or superparamagnetic nano-particle.
9. non-small cell lung cancer H460 cell line lung cancer stem cell detects and/or the reagent or kit of separation, it is characterised in that
It includes polypeptide described in claim 1.
10. the pharmaceutical composition for treating non-small cell lung cancer H460, it is characterised in that will comprising pharmaceutical acceptable carrier and right
Seek 1 polypeptide.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710978419.8A CN107641148A (en) | 2013-11-19 | 2013-11-19 | Polypeptide and its application |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310585699.8A CN104650188B (en) | 2013-11-19 | 2013-11-19 | Polypeptide and its application |
| CN201710978419.8A CN107641148A (en) | 2013-11-19 | 2013-11-19 | Polypeptide and its application |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310585699.8A Division CN104650188B (en) | 2013-11-19 | 2013-11-19 | Polypeptide and its application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107641148A true CN107641148A (en) | 2018-01-30 |
Family
ID=53241872
Family Applications (3)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710972298.6A Pending CN107602667A (en) | 2013-11-19 | 2013-11-19 | Polypeptide and its application |
| CN201310585699.8A Active CN104650188B (en) | 2013-11-19 | 2013-11-19 | Polypeptide and its application |
| CN201710978419.8A Pending CN107641148A (en) | 2013-11-19 | 2013-11-19 | Polypeptide and its application |
Family Applications Before (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710972298.6A Pending CN107602667A (en) | 2013-11-19 | 2013-11-19 | Polypeptide and its application |
| CN201310585699.8A Active CN104650188B (en) | 2013-11-19 | 2013-11-19 | Polypeptide and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (3) | CN107602667A (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002008273A2 (en) * | 2000-07-21 | 2002-01-31 | Millennium Pharmaceuticals, Inc. | 47508, a novel human histone deacetylase family member and uses thereof |
| CN102382175A (en) * | 2011-11-02 | 2012-03-21 | 中国科学院苏州纳米技术与纳米仿生研究所 | Polypeptide for specifically targeting lung cancer cell, and preparation method and application thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102775474B (en) * | 2012-07-31 | 2014-03-12 | 中国科学院苏州纳米技术与纳米仿生研究所 | Polypeptide and applications thereof |
| CN102757482B (en) * | 2012-07-31 | 2013-12-18 | 中国科学院苏州纳米技术与纳米仿生研究所 | Polypeptide and application thereof |
-
2013
- 2013-11-19 CN CN201710972298.6A patent/CN107602667A/en active Pending
- 2013-11-19 CN CN201310585699.8A patent/CN104650188B/en active Active
- 2013-11-19 CN CN201710978419.8A patent/CN107641148A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002008273A2 (en) * | 2000-07-21 | 2002-01-31 | Millennium Pharmaceuticals, Inc. | 47508, a novel human histone deacetylase family member and uses thereof |
| CN102382175A (en) * | 2011-11-02 | 2012-03-21 | 中国科学院苏州纳米技术与纳米仿生研究所 | Polypeptide for specifically targeting lung cancer cell, and preparation method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| PRAVEEN K DUBEY 等: "Liposomes Modified With Cyclic RGD Peptide for Tumor Targeting", 《J DRUG TARGET》 * |
| 温浙盛 等: "肿瘤细胞膜模型筛选抗肿瘤多肽新方法的建立", 《癌症》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104650188A (en) | 2015-05-27 |
| CN104650188B (en) | 2017-11-24 |
| CN107602667A (en) | 2018-01-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110845603B (en) | Human collagen 17-type polypeptide, production method and use thereof | |
| CN109593126B (en) | Polypeptide, its production method and purposes | |
| CN106554410B (en) | A kind of recombination human source collagen and its encoding gene and preparation method | |
| CN112552393B (en) | Recombinant human III-type collagen and pichia pastoris recombinant expression system thereof | |
| CN110194795A (en) | A kind of recombination human source collagen and its application | |
| CN112745394B (en) | Recombinant human-like collagen and preparation method and application thereof | |
| CN107245494A (en) | Solution expression with high efficiency and purification process of the A β 42 in Escherichia coli | |
| CN109966484B (en) | Immunopotentiator, preparation method, avian influenza vaccine and application | |
| CN113637068B (en) | Recombinant I-type humanized collagen C1L5T and preparation method and application thereof | |
| CN113186109A (en) | Saccharomyces cerevisiae expression long-acting recombinant fibronectin and application thereof in cosmetics | |
| WO2003106494A1 (en) | A human collagen-like protein and the method of producing it | |
| CN114853881B (en) | Recombinant humanized fusion collagen and efficient hydroxylation method and application thereof | |
| CN112745385A (en) | Recombinant humanized collagen, industrial preparation method and product application thereof | |
| CN108840938A (en) | A kind of fusion protein being made of pig albumin and Porcine interferon-gamma and preparation method thereof and a kind of Recombinant Swine long-acting interferon γ | |
| CN116874590B (en) | Recombinant III type collagen and preparation method thereof | |
| CN107540739A (en) | A kind of tumor-targeting polypeptide | |
| WO1993013132A1 (en) | Novel megakaryocyte amplifier | |
| CN104650188B (en) | Polypeptide and its application | |
| CN116554309A (en) | Recombinant human III type collagen and preparation method and application thereof | |
| CN108484749B (en) | Recombinant soluble human bone-targeted interferon gamma-1 b and preparation method thereof | |
| CN104725485B (en) | One kind restructuring active peptide and its synchronic preparation method | |
| CN102925470B (en) | A kind of method of recombinant expressed production human thymosin in yeast | |
| CN117986355B (en) | Recombinant humanized III type collagen and preparation method and application thereof | |
| CN109608535A (en) | A kind of the chicken alpha interferon peptide chain and its recombinant expression engineered strain of optimization | |
| CN110343164A (en) | A kind of pig interferon alpha-mutant and its preparation method and application of 7 site mutation of high activity |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20180130 |
|
| WD01 | Invention patent application deemed withdrawn after publication |