CN107868126A - Polypeptide, preparation method and applications - Google Patents
Polypeptide, preparation method and applications Download PDFInfo
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Abstract
The present invention proposes polypeptide, preparation method and applications.The polypeptide has SEQ ID NO：Amino acid sequence shown in 1~4.Polypeptide according to embodiments of the present invention is compared to wild type Sox2, and in terms of inducing somatic reprogramming, efficiency improves at least 1.5 times.
Description
Technical field
The present invention relates to biological field, in particular it relates to polypeptide, preparation method, nucleic acid, construct and change
Cell fate ground method.
Background technology
Using Oct4, Sox2, Klf4, four kinds of transcription factor induction reprogrammings of c-Myc (OSKM) produce iPS cells, disclose
The possibility of transcription factor induced expression in cell fate conversion.Another method is fixed transcription factor mixture
It can realize that the orientation of different type body cell is exchanged, referred to as transdifferentiation, also referred to as pedigree reprograms.In transdifferentiation, one
Individual cells pedigree can be converted to another pedigree without versatility intermediate state.This in recent years method there has been weight
The breakthrough wanted, by skipping the intermediate steps of versatility, can produce cardiac muscle cell, endothelial cell, liver cell, pancreatic beta cell,
Neuron, NSC etc..These methods are possible to improve people to the degenerative disease such as type of diabetes 1, Parkinson's, old
The understanding of year property macular degeneration and hepatopathy, and provide generally acknowledged therapeutic scheme using gene therapy and regenerative medicine.This causes can
To establish disease model and be used for drug screening and inspection.The use of animal in experiment can be saved by such mode.
In addition, invalid " attempting and wrong " therapy and more customization therapies are avoided to reduce the pain of patient.Therefore, pedigree is advised
Draw and be likely to be used for routine clinical diagnosis, and be that few patients are customized according to reaction of the patients own cells to medicine
Therapeutic scheme.It can be transplanted in addition, cell has with degenerative disease in patient body.Including sendai virus self-replacation
RNAs and additional carrier including non-fusion technology iPS can be obtained under conditions of Good Manufacture Practice (GMP) it is thin
Born of the same parents.Similar technology is also developed in transdifferentiation.In order to further improve the efficiency of complicated donorcells, speed, and
The raising of success rate, these technologies are considered as consistent with diagnostic application.
The member of Sox transcription factor families can promote to reprogram in many transcription factor complexs.Can by with
One high flow colony box (HMG box) with 79 amino acid is combined with DNA.This phase for DNA and other albumen
Interaction is highly important.Conventional research indicates the HMG with exceptional function by Sox2 and Sox17 cell types
The importance of the protein-protein interaction of box mediations.By being processed to the amino acid sequence of HMG boxes, Ke Yi
Sox17 and Sox2 kinds integrate new function, and the mechanism that these features exchange is to change the regulation and control combined with Oct4 dependences
DNA sequence dna.
However, how on the basis of existing achievement in research, the induced efficiency of induced multi-potent stem cell is further improved, is
The target that scientists are pursued diligently.
The content of the invention
In this invention, inventor establishes an emerging method, referred to as by cell selection and sequencing orient into
Change the reprogramming factor (DERBYseq).By applying this method, confirm that mutant Sox two kinds of mutant are producing first
It is more excellent relative to the Sox2 performances of wild type during iPS.
Therefore, in the first aspect of the present invention, the present invention proposes a kind of polypeptide of separation.According to the implementation of the present invention
Example, the polypeptide have SEQ ID NO：Amino acid sequence shown in 1~4.
MYNMMETELKPPGPQQASGGGGGGGNATAAATGGNQKNSPDRVKRPMNAFMVWSRGQRRKMAQENPKMHNSEISKRL
GAEWKLLSNTEKRPFRDEARRLRALHMKEHPDYKYRPRRKTKTLMKKDKYTLPGGLLAPGGNSMASGVGVGAGLGGG
LNQRMDSYAHMNGWSNGSYSMMQEQLGYPQHPGLNAHGAAQMQPMHRYVVSALQYNSMTSSQTYMNGSPTYSMSYSQ
QGTPGMALGSMGSVVKSEASSSPPVVTSSSHSRAPCQAGDLRDMISMYLPGAEVPEPAAPSRLHMAQHYQSGPVPGT
AKYGTLPLSHM(SEQ ID NO：1).
MYNMMETELKPPGPQQASGGGGGGGNATAAATGGNQKNSPDRVKRPMNAFMVWSRGQRRKMAQENPKMHNSEISKRL
GAEWKLLSATEKRPFHDEAKRLRALHMKEHPDYKYRPRRKTKTLMKKDKYTLPGGLLAPGGNSMASGVGVGAGLGGG
LNQRMDSYAHMNGWSNGSYSMMQEQLGYPQHPGLNAHGAAQMQPMHRYVVSALQYNSMTSSQTYMNGSPTYSMSYSQ
QGTPGMALGSMGSVVKSEASSSPPVVTSSSHSRAPCQAGDLRDMISMYLPGAEVPEPAAPSRLHMAQHYQSGPVPGT
AKYGTLPLSHM(SEQ ID NO：2).
MYNMMETELKPPGPQQASGGGGGGGNATAAATGGNQKNSPDRVKRPMNAFMVWSRGQRRKMAQENPKMHNSEISKRL
GAEWKLLSDTEKRPFTDEAKRLRALHMKEHPDYKYRPRRKTKTLMKKDKYTLPGGLLAPGGNSMASGVGVGAGLGGG
LNQRMDSYAHMNGWSNGSYSMMQEQLGYPQHPGLNAHGAAQMQPMHRYVVSALQYNSMTSSQTYMNGSPTYSMSYSQ
QGTPGMALGSMGSVVKSEASSSPPVVTSSSHSRAPCQAGDLRDMISMYLPGAEVPEPAAPSRLHMAQHYQSGPVPGT
AKYGTLPLSHM(SEQ ID NO：3).
MYNMMETELKPPGPQQASGGGGGGGNATAAATGGNQKNSPDRVKRPMNAFMVWSRGQRRKMAQENPKMHNSEISKRL
GAEWKLLSFTEKRPFRDEATRLRALHMKEHPDYKYRPRRKTKTLMKKDKYTLPGGLLAPGGNSMASGVGVGAGLGGG
LNQRMDSYAHMNGWSNGSYSMMQEQLGYPQHPGLNAHGAAQMQPMHRYVVSALQYNSMTSSQTYMNGSPTYSMSYSQ
QGTPGMALGSMGSVVKSEASSSPPVVTSSSHSRAPCQAGDLRDMISMYLPGAEVPEPAAPSRLHMAQHYQSGPVPGT
AKYGTLPLSHM(SEQ ID NO：4).
Polypeptide according to embodiments of the present invention is compared to wild type Sox2, and in terms of inducing somatic reprogramming, efficiency carries
High at least 1.5 times, preferably 4~5 times of (such as SEQ ID NO:The polypeptide of amino acid sequence shown in 1 and 2).
In the second aspect of the present invention, the present invention proposes a kind of method for obtaining polypeptide noted earlier.According to the present invention
Embodiment, methods described includes：(1) using Sox2 and Sox17 as template, dashed forward at random in HMG protein-interactings surface region
Become the 46th, 53 and 56 amino acids, to build the random library of Sox mutant；(2) random library of the mutant is entered
Row transfection handles and collects virus, mutant described in the expressing viral；(3) virus is infected into body cell, and will infection
Somatic Cell Culture 12~15 days afterwards, the body cell carry the transgenosis green fluorescent protein of Oct4 promoters driving；(4) utilize
The cell that Schwann Cells art sorting step (3) obtains is flowed, is carried respectively from green fluorescence feminine gender and green fluorescent protein positive population
Take genomic DNA and carry out high-flux sequence and screening, to obtain the candidate Sox that can strengthen generation induction type multipotential stem cell
Mutant protein；Wherein, the wild type Sox of at least 1.5 times of the induced efficiency of multipotential stem cell induced efficiency is target
The index of polypeptide.Polypeptide is obtained during induction type multipotential stem cell is produced relative to wild type by the above method
Soc2, efficiency significantly improve.
According to an embodiment of the invention, the above method can further include at least one following additional technical feature：
According to an embodiment of the invention, the random library of the Sox mutant includes carrying Sox mutant and NNK codings
The construct of son, the carrier of the construct is pMX.Wherein, pMX is a kind of retroviral vector.
According to an embodiment of the invention, the transfection processing is by the way that the random library of the mutant is transfected into Plat-E
What cell was realized.Plat-E cells embed the gene that virus assembly needs, and Retroviral Transfer, transfection effect are done suitable for pMX
Rate is high, produces malicious efficiency high.
According to an embodiment of the invention, the virus is retrovirus.
According to an embodiment of the invention, the body cell is embryo fibroblast.And then reprogramming efficiency further carries
It is high.
In the third aspect of the present invention, the present invention proposes a kind of nucleic acid of separation, it is characterised in that has SEQ ID
NO：Nucleotide sequence shown in 5~8.
ATGTATAACATGATGGAGACGGAGCTGAAGCCGCCGGGCCCGCAGCAAGCTTCGGGGGGCGGCGGCGGAGGAGGCAA
CGCCACGGCGGCGGCGACCGGCGGCAACCAGAAGAACAGCCCGGACCGCGTCAAGAGGCCCATGAACGCCTTCATGG
TATGGTCCCGGGGGCAGCGGCGTAAGATGGCCCAGGAGAACCCCAAGATGCACAACTCGGAGATCAGCAAGCGCCTG
GGCGCGGAGTGGAAACTTTTGTCCAATACCGAGAAGCGGCCGTTCCGGGACGAGGCCCGGCGGCTGCGCGCTCTGCA
CATGAAGGAGCACCCGGATTATAAATACCGGCCGCGGCGGAAAACCAAGACGCTCATGAAGAAGGATAAGTACACGC
TTCCCGGAGGCTTGCTGGCCCCCGGCGGGAACAGCATGGCGAGCGGGGTTGGGGTGGGCGCCGGCCTGGGTGGCGGG
CTGAACCAGCGCATGGACAGCTACGCGCACATGAACGGCTGGAGCAACGGCAGCTACAGCATGATGCAGGAGCAGCT
GGGCTACCCGCAGCACCCGGGCCTCAACGCTCACGGCGCGGCACAGATGCAACCGATGCACCGCTACGTCGTCAGCG
CCCTGCAGTACAACTCCATGACCAGCTCGCAGACCTACATGAACGGCTCGCCCACCTACAGCATGTCCTACTCGCAG
CAGGGCACCCCCGGTATGGCGCTGGGCTCCATGGGCTCTGTG
GTCAAGTCCGAGGCCAGCTCCAGCCCCCCCGTGGTTACCTCTTCCTCCCACTCCAGGGCGCCCTGCCAGGCCGGGGA
CCTCCGGGACATGATCAGCATGTACCTCCCCGGCGCCGAGGTGCCGGAGCCCGCTGCGCCCAGTAGACTGCACATGG
CCCAGCACTACCAGAGCGGCCCGGTGCCCGGCACGGCCAAATACGGCACACTGCCCCTGTCGCACATGTGA(SEQID
NO：5).
ATGTATAACATGATGGAGACGGAGCTGAAGCCGCCGGGCCCGCAGCAAGCTTCGGGGGGCGGCGGCGGAGGAGGCAA
CGCCACGGCGGCGGCGACCGGCGGCAACCAGAAGAACAGCCCGGACCGCGTCAAGAGGCCCATGAACGCCTTCATGG
TATGGTCCCGGGGGCAGCGGCGTAAGATGGCCCAGGAGAACCCCAAGATGCACAACTCGGAGATCAGCAAGCGCCTG
GGCGCGGAGTGGAAACTTTTGTCCGCTACCGAGAAGCGGCCGTTCCATGACGAGGCCAAGCGGCTGCGCGCTCTGCA
CATGAAGGAGCACCCGGATTATAAATACCGGCCGCGGCGGAAAACCAAGACGCTCATGAAGAAGGATAAGTACACGC
TTCCCGGAGGCTTGCTGGCCCCCGGCGGGAACAGCATGGCGAGCGGGGTTGGGGTGGGCGCCGGCCTGGGTGGCGGG
CTGAACCAGCGCATGGACAGCTACGCGCACATGAACGGCTGGAGCAACGGCAGCTACAGCATGATGCAGGAGCAGCT
GGGCTACCCGCAGCACCCGGGCCTCAACGCTCACGGCGCGGCACAGATGCAACCGATGCACCGCTACGTCGTCAGCG
CCCTGCAGTACAACTCCATGACCAGCTCGCAGACCTACATGAACGGCTCGCCCACCTACAGCATGTCCTACTCGCAG
CAGGGCACCCCCGGTATGGCGCTGGGCTCCATGGGCTCTGTGGTCAAGTCCGAGGCCAGCTCCAGCCCCCCCGTGGT
TACCTCTTCCTCCCACTCCAGGGCGCCCTGCCAGGCCGGGGACCTCCGGGACATGATCAGCATGTACCTCCCCGGCG
CCGAGGTGCCGGAGCCCGCTGCGCCCAGTAGACTGCACATGGCCCAGCACTACCAGAGCGGCCCGGTGCCCGGCACG
GCCAAATACGGCACACTGCCCCTGTCGCACATGTGA(SEQID NO：6).
ATGTATAACATGATGGAGACGGAGCTGAAGCCGCCGGGCCCGCAGCAAGCTTCGGGGGGCGGCGGCGGAGGAGGCAA
CGCCACGGCGGCGGCGACCGGCGGCAACCAGAAGAACAGCCCGGACCGCGTCAAGAGGCCCATGAACGCCTTCATGG
TATGGTCCCGGGGGCAGCGGCGTAAGATGGCCCAGGAGAACCCCAAGATGCACAACTCGGAGATCAGCAAGCGCCTG
GGCGCGGAGTGGAAACTTTTGTCCGATACCGAGAAGCGGCCGTTCACTGACGAGGCCAAGCGGCTGCGCGCTCTGCA
CATGAAGGAGCACCCGGATTATAAATACCGGCCGCGGCGGAAAACCAAGACGCTCATGAAGAAGGATAAGTACACGC
TTCCCGGAGGCTTGCTGGCCCCCGGCGGGAACAGCATGGCGAGCGGGGTTGGGGTGGGCGCCGGCCTGGGTGGCGGG
CTGAACCAGCGCATGGACAGCTACGCGCACATGAACGGCTGGAGCAACGGCAGCTACAGCATGATGCAGGAGCAGCT
GGGCTACCCGCAGCACCCGGGCCTCAACGCTCACGGCGCGGCACAGATGCAACCGATGCACCGCTACGTCGTCAGCG
CCCTGCAGTACAACTCCATGACCAGCTCGCAGACCTACATGAACGGCTCGCCCACCTACAGCATGTCCTACTCGCAG
CAGGGCACCCCCGGTATGGCGCTGGGCTCCATGGGCTCTGTGGTCAAGTCCGAGGCCAGCTCCAGCCCCCCCGTGGT
TACCTCTTCCTCCCACTCCAGGGCGCCCTGCCAGGCCGGGGACCTCCGGGACATGATCAGCATGTACCTCCCCGGCG
CCGAGGTGCCGGAGCCCGCTGCGCCCAGTAGACTGCACATGGCCCAGCACTACCAGAGCGGCCCGGTGCCCGGCACG
GCCAAATACGGCACACTGCCCCTGTCGCACATGTGA(SEQID NO:7)。
ATGTATAACATGATGGAGACGGAGCTGAAGCCGCCGGGCCCGCAGCAAGCTTCGGGGGGCGGCGGCGGAGGAGGCAA
CGCCACGGCGGCGGCGACCGGCGGCAACCAGAAGAACAGCCCGGACCGCGTCAAGAGGCCCATGAACGCCTTCATGG
TATGGTCCCGGGGGCAGCGGCGTAAGATGGCCCAGGAGAACCCCAAGATGCACAACTCGGAGATCAGCAAGCGCCTG
GGCGCGGAGTGGAAACTTTTGTCCTTTACCGAGAAGCGGCCGTTCCGGGACGAGGCCACTCGGCTGCGCGCTCTGCA
CATGAAGGAGCACCCGGATTATAAATACCGGCCGCGGCGGAAAACCAAGACGCTCATGAAGAAGGATAAGTACACGC
TTCCCGGAGGCTTGCTGGCCCCCGGCGGGAACAGCATGGCGAGCGGGGTTGGGGTGGGCGCCGGCCTGGGTGGCGGG
CTGAACCAGCGCATGGACAGCTACGCGCACATGAACGGCTGGAGCAACGGCAGCTACAGCATGATGCAGGAGCAGCT
GGGCTACCCGCAGCACCCGGGCCTCAACGCTCACGGCGCGGCACAGATGCAACCGATGCACCGCTACGTCGTCAGCG
CCCTGCAGTACAACTCCATGACCAGCTCGCAGACCTACATGAACGGCTCGCCCACCTACAGCATGTCCTACTCGCAG
CAGGGCACCCCCGGTATGGCGCTGGGCTCCATGGGCTCTGTGGTCAAGTCCGAGGCCAGCTCCAGCCCCCCCGTGGT
TACCTCTTCCTCCCACTCCAGGGCGCCCTGCCAGGCCGGGGACCTCCGGGACATGATCAGCATGTACCTCCCCGGCG
CCGAGGTGCCGGAGCCCGCTGCGCCCAGTAGACTGCACATGGCCCAGCACTACCAGAGCGGCCCGGTGCCCGGCACG
GCCAAATACGGCACACTGCCCCTGTCGCACATGTGA(SEQID NO:8)
The polypeptide of nucleic acid coding according to embodiments of the present invention is compared to wild type Sox2, in inducing somatic reprogramming side
Face, efficiency improve at least 1.5 times, preferably 4~5 times.
In the fourth aspect of the present invention, the present invention proposes a kind of construct.According to an embodiment of the invention, the structure
Guide and support with foregoing nucleic acid.Body cell is imported using construct according to embodiments of the present invention, realizes polypeptide noted earlier
Overexpression, and then the efficiency of reprogramming of somatic cells can be effectively improved.
In the fifth aspect of the present invention, the present invention proposes a kind of method for changing cell fate.According to the reality of the present invention
Example is applied, methods described includes：The cell is overexpressed foregoing polypeptide.Using method according to embodiments of the present invention, carefully
Born of the same parents' reprogramming efficiency improves at least 1.5 times, preferably 4~5 times.
According to an embodiment of the invention, the above method can further include at least one following additional technical feature：
According to an embodiment of the invention, the cell is body cell.
According to an embodiment of the invention, the body cell is fibroblast.It is overexpressed in fibroblast noted earlier
Polypeptide, fibroblast reprogramming efficiency are higher.
According to an embodiment of the invention, the cell is overexpressed Oct4 and Klf4 simultaneously.And then reprogramming efficiency is further
Improve.
According to an embodiment of the invention, the cell is overexpressed Oct4, Klf4 and c-Myc simultaneously.And then reprogramming efficiency
Further improve.
Brief description of the drawings
Fig. 1 is the Multiple sequence alignments figure of the HMG domains of homologous Sox albumen according to embodiments of the present invention；
Fig. 2 is DERBYSeq schematic flow sheets according to embodiments of the present invention；
Fig. 3 is DERBYSeq data analysis schematic flow sheets according to embodiments of the present invention,
Wherein, volcano figure illustrates the differential enrichment sequence variants of DESeq2 analyses, positive negative with GFP using GFP
Between amplification subsequence, be respectively derived from the induction of four factor OSK+eSox2 libraries and the induction of three factor OK+eSox2 libraries be real
Test, color coding represents the enrichment of the read in two libraries, and X-axis represents multiple and Y-axis represents the P values (padj) corrected；
Fig. 4 is that the iPSC of the eSox2 experiment best from OKM and expression according to embodiments of the present invention is averagely cloned
Number,
Wherein, x-axis represents the date of reprogramming, and upper strata represents versus wild type Sox2 and Sox17, in the positive enrichments of GFP
In the most prominent mutant, expression contrast the wild type Sox2 and Sox17 of lower floor are the most prominent in the negative enrichments of GFP
Mutant；
Fig. 5 is being averaged for best Sox mutant (eSox) of the expression of the factors of OSK 3 participation according to embodiments of the present invention
IPSC clones number,
It represents versus wild type Sox2 and Sox17 at the middle and upper levels, the mutant the most prominent in the positive enrichments of GFP, under
Expression contrast the wild type Sox2 and Sox17 of layer, are enriched with mutant the most prominent in GFP feminine genders.
Fig. 6 is that fluorescence according to embodiments of the present invention sorts cell and complete opening scanning,
Wherein, it is shown that the best eSox2 mutant filtered out in the induction of 4 factors was given birth in the iPS of the 10th day
Into efficiency, and from efficiency of 3 factors at 12 days；
Fig. 7 is the immunocytochemistry experiment shows of iPS cells caused by eSox2NRR according to embodiments of the present invention
Endogenous pluripotency marker's gene Oct4, Sox2 and Nanog expression；
Fig. 8 is the caryogram of iPSC derived from eSox2NRR according to embodiments of the present invention；
Fig. 9 be it is according to embodiments of the present invention under the conditions of Sox2, Sox17, Sox4, Sox18, three NRR mutant
Reprogramming ability,
Wherein, repeat to test to be derived from biology three times, clone's reading is represented with log2, error bar standard deviation S D
It is (+/-) to represent；
Figure 10 is that volcano figure according to embodiments of the present invention shows that airflow classification GFP positive fragment variables differential is enriched with (GFP
+) and green fluorescent protein (GFP) negative cells transduction three factor determination+esox17 libraries experiment,
Wherein, X-axis represents multiple change, and Y-axis represents P values；
Figure 11 is clone's number that esox17 according to embodiments of the present invention and OK inductions iPSC obtains in different number of days；
Figure 12 is that flow cytometer according to embodiments of the present invention and full plate scan esox17iPSC before display programs 10 days
Formation efficiency；
Figure 13 is clone's number that esox17 according to embodiments of the present invention and OKM inductions iPSC obtained at the 8th day,
Wherein, error bar represents standard deviation (+/-) average value；
Figure 14 is the life that complete opening scanning according to embodiments of the present invention shows the 8th day eSox17 and isogeneous induction iPSC of OKM mono-
Into efficiency；And
Figure 15 is the sequence alignment figure of eSox2 according to embodiments of the present invention and eSox17 in HMG domains.
Embodiment
Embodiments of the invention are described below in detail, the example of the embodiment is shown in the drawings.Below with reference to
The embodiment of accompanying drawing description is exemplary, it is intended to for explaining the present invention, and is not considered as limiting the invention.
Inventor establishes the method for being oriented molecular evolution with transcription factor library in mammalian cell, passes through
Inducing mouse embryonic fibroblast (MEF) turns into the method that multipotent stem cells (iPSC) demonstrate inventor.Inventor's
Method include by being strategically mutated to molecular surface, library screening and, pass through cell selection and high-flux sequence knowledge
Not high performance Sox mutant.Inventor is template using Sox2 and Sox17, in HMG protein-interactings surface random mutation
3 amino acid generate 8000 different mutant to build eSox transcription factors library.Inventor containing Oct4 and Klf4,
Have c-Myc and without c-Myc in the case of, have the iPS cells of green fluorescence by the way that MEF is changed into screen library.Use
The fluorescence-assisted cell sorting separation GFP positives and GFP negative cells, genome is extracted from GFP feminine genders and GFP positive populations
DNA carries out high-flux sequence and screening.Final inventor determines the tens of kinds of eSox candidate albumens that can strengthen generation iPS,
And they are verified.Wherein eSox albumen the most prominent enhances 4- relative to the Sox2 of the wild type efficiency reprogrammed
5 times.The Sox17 of wild type can not produce iPS, many to form ability of the mutant with efficiently induction iPSC based on Sox17.Hair
A person of good sense thinks that DERBYseq screenings can be used for mankind's reprogramming, and produces the feature of regenerating medicine in vivo and in vitro
Cell.
It is discussed in detail below with reference to accompanying drawing and the method for obtaining Sox mutant is screened by DERBYseq.
The site of embodiment 1 selects and library construction
Based on the past research of inventor, the E46 in HMG regions, I53, K56 (L46, V53, E56 in Sox17) position in Sox2
Point is chosen as random mutation point.The region random mutation of three selections turns into pMX-Sox2 and utilizes NNK coding structure bags
Random library containing 8000 mutant.For the Bacteria Culture of GENEWIZ companies purchase in 100mL conical flasks, plasmid proposes greatly examination
Agent box extraction Library plasmid (Tiangen).
The reprogramming screening of the cell of embodiment 2
2.1 libraries transfect and prepared by virus
Defrosting Plat-E cells, cultivated using in PlatE culture mediums (DMEM for including 10%FBS).Transfection is in 10cm
The culture dish kind culture inoculation cells of 7-8 million are cultivated, and when reaching in 70%-80%, change liquid, configuration transfection liquid 10ug's
Polyethylenimine (the Polysciences of PMX plasmids, final concentration of 1ug/ul；#23966)、1ml Opti-MEM
(Thermo Fisher Scientific；#31985070).Transfection liquid is added in culture medium ware, after transfection 12 hours
Change fresh culture.Virus is collected in 48h and 72h and is collected by filtration with 0.45 micron of filter.
2.2 retroviral infection embryo fibroblasts
From GIBH Animal Houses, (the MEF cells from mice embryonic are collected in embryo to MEC
The reporter gene of the transgenosis green fluorescent protein of oct4 promoters driving is carried at 13.5 days, on MEF cells), each six hole
Plate is inoculated with 30000 or so cells/wells, and for 8-10h in MEF medium cultures, composition is DMEM in high glucose culture medium before transfection
(Thermo), 10% hyclone (FBS, Natocor, #SFBE), 1x glutamic acid (Thermo Fisher Scientific；#
35050061), 1x nonessential amino acid (Thermo Fisher Scientific；#11140050) and 0.5 × penicillin/chain
Mycin (Hyclone；#SV30010).Culture medium (Sigma- of the Retroviral supernatant filtering containing 8mg/ml per 1mL
Aldrich；#40804ES76).At interval of 24 hours culture mediums for adding the filtering containing four factors, add at twice.According to
Reference shows that mES culture mediums can be used for reprogramming process (14).After virus infection 48 hours, substituted with mES culture mediums
MEF culture mediums.From the culture medium start recording date is changed, it is designated as the 0th day.Reprogrammed cell maintains 37 DEG C in CO2 incubators
Middle culture (BB15Thermo Fisher Scientific), and observe cellular change situation (Axio with phase contrast microscope
Vert.A1Zeiss microscope), mES culture mediums are changed once for every 24 hours.
2.3 flow cytometries sort cell
After the reprogramming of MEF cells is 12 to 15 days of iPS cells, mES culture mediums are removed, cell two is washed with 1X DPBS
It is secondary, and with 0.25% pancreas enzyme -EDTA 1mM (Thermo Fisher Scientific；#25300054) digest, by the molten of separation
Liquid is filtered by 40 μM of BD cellular filters, and the cell concentration of each sample is in ten thousand cells of 6-7/ml or so.Cell is cultivated in FACS
It is resuspended under base, (FACS culture mediums are made up of DPBS (1X)+2mm EDTA+0.1% bovine serum albumin(BSA)s or serum).To each
Sample, manually in triplicate, and it is merged into a pipe, for cell classification twice.By using GFP laser channelings 488nm
(Beckman Coulter-MoFloTM Astrios.) classifies to cell, and each sample collects about 20000~100000
Individual cell.
Embodiment 3 two generation sequencing library prepares and sequencing
Using quick gDNA micro prep kits (Zymo research#) from the GFP positives and GFP negative cells
Extract genomic DNA.Pass through DreamTaq Green PCR Master Mix (Thermo Fisher Scientific；#
K1082 15 PCR cycles) are carried out, it is outer using the forward and backward Sox2 and Sox17 of exogeneous primer generation PMX skeletons, extraction
The eSox libraries that source is integrated.Using the PCR primer of PCR purification kits (Tiangen#DP209) purifying external source, there is mutation position
The target spot of point is expanded by 6 PCR cycles of primer with 3 random sequences, and primer includes barcode and Illumina
Sequencing compatibility joint.PCR primer with 250 base-pairs length carries out electrophoresis and pure by Tiangen gel purification kits
Change.Library is sequenced using Illumina HiSeq2500 and matches the pairing of 150bp ends.15% Phix DNA are added to
Complexity is added above.For extracting the primer sequence of foreign gene and being connect for the illumina with coding of amplification
Head lists out in table 1.
The caryogram of embodiment 4 is identified
IPS cells are cultivated in 2i conditions download 6cm plates, until 70% converges, add 20ug/ml colchicin
(Aladdin；#477305) after 1 hour, with trypsin digestion and cell, cell is collected by centrifugation in 200xg 3min, with 8 milliliters
Cell is resuspended in 0.075M KCl, is incubated 20min at 37 DEG C, adds 10 milliliters of fixer (acetic acid：Methanol 1:3), it is gently mixed,
It is incubated 10 minutes at 37 DEG C.Supernatant is centrifuged off, the fixer for adding precooling is titrated to 10 milliliters.Cell distribution is cold at one
Gai Zhong, it is incubated 3 hours at 75 DEG C.(Sigma-Aldrich after trypsin treatment and Giemsa dyeing；#48900) in microscope
(Olympus BX51) is analyzed.
The immunochemistry of embodiment 5 is analyzed
Induced multi-potent stem cell is cultivated in 2i culture mediums, and in 0.1% gelatin coating plate (Sigma-Aldrich；#
G1393 continuous passage purifies in).2i culture mediums are DMEM/F12 (Thermo Fisher Scientific；#
C11320500BT)：Neurobasal medium(Thermo Fisher Scientific；#21103049)=1:1 mixing,
Add 1x N2 (Thermo Fisher Scientific；#17502048),1x B27(Thermo Fisher
Scientific；#17504044), 1x glutamic acid (Thermo Fisher Scientific), 1x nonessential amino acid
(Thermo Fisher Scientific), 1mM Sodium Pyruvates (Thermo Fisher Scientific), 0.055mM β-mercapto
Base ethanol (MP Biomedicals), 0.5x penicillin/streptomycins, 1,000U/ml LIF, 3 μM of CHIR99021
(Selleck；#S2924-25mg),1μM PD0325901(Selleck；#S1036-25mg) is under 2i culture medium conditions, often
The cells of 1-2 million are cultivated in individual 24 orifice plate, are cultivated 48 hours in 37 DEG C of CO2 incubators.Cell is washed with 1XDPBS three times
And mix 15min at room temperature with 4% paraformaldehyde, then with 0.2% Triton X-100 (Sigma-Aldrich；#
9002-93-1) dissolve 10%BSA (MPBIO；#0218054991) and 1XDPBS, at ambient temperature by cell in mixed liquor
30min is incubated, it is then with DPBS that cell is clear twice, and with NANOG (Novus；#NB100-58842),Oct4(Santa
Cruz；#sc-5279), Sox2 (Santa Cruz；Primary antibody #sc-17320) is incubated overnight in 4 °.Then, with 1X DPBS
Wash cell three times, each 5min, be incubated at room temperature with secondary antibody lucifuge respectively in connection with primary antibody 2h.(donkey-anti-rabbit:
Thermo Fisher Scientific#A24870,rabbit-anti-mouse:Thermo Fisher Scientific#
A21063,donkey-anti-goat:Abcam#ab6949).Cell 5min then is washed with DPBS again, washing is three times.With
These cells 1X DAPI (Thermo Fisher Scientific afterwards；#R37606) is dyed, and is taken pictures in phase contrast microscope
Observe (Axio Vert.A1ZEISS).
Conclusion
1) directed differentiation platform of the transcription factor in mammalian cell is established
On HMG domain sequences the 46,53,57th three primary amino acids (on HMG domains with close to Oct4 phases
Interaction), sequence alignment such as Fig. 1, by random use NNK codon mutations build library, generate except terminator codon it
Outer one contains the library of 8000 mutant.Such as Fig. 2, by the same Oct4 of mutant library (O), Klf4 (K), c-Myc (M) or
OK, it is imported into by transfection in the MEC of Oct4-GFP reporter genes.Induced in mES culture mediums
Ips cells can efficiently strengthen the transcription factor for the mutant for generating iPSC to screen under the conditions of OK and OSK.It is thin by streaming
Born of the same parents' art is classified to cell, and extraction genomic DNA carries out the library gene of low circulation number PCR amplification insertions.In the second wheel
The joint that the sequencing of the generations of illumina bis- is added in PCR is sequenced.After obtaining high-flux sequence result, analysis process such as Fig. 3 will
Original DNA sequence dna translates into amino acid sequence, counts what different mutants occurred in GFP feminine genders and GFP positive cell groups
Number.Inventor is given a mark using DESeq2 to the mutant of differential enrichment in GFP feminine genders and GFP positive colonies,
Based on P values and enrichment times, most prominent candidate mutant is as OKM+eSox libraries and candidate's egg of OK+eSox libraries confirmation
In vain, such as Fig. 4.
2) DERBYSeq can obtain more efficient functional mutant that reprogramming of somatic cells is iPSC cells
By proving that the eSox2 mutant versus wild type Sox2 that DERBYSeq is filtered out have more Gao Chong with OK and OKM
Programming efficiency, the reprogramming of mutation induction are accelerated relative to wild type, speed, can shift to an earlier date 3-4 days and observe ips grams
It is grand, as shown in Figure 5.In addition, the 10th day and 12 days under the conditions of 3 factor eSox2+OK under the conditions of 4 factor eSox2+OKM,
It was observed that clone's number be 3-4 times of Sox2 of wild type.Facs analysis and full plate scan image show eSox2 mutant AHK
Increased really with NRR generation iPSC abilities, this coincide with data above.Main Almightiness type label Oct4,
Sox2, Nanog, which demonstrate the eSox2NRR at five generations, can derive iPSC, such as Fig. 6, and caryogram identification is just, such as Fig. 7, hair
Tested again in the case of NRR mutation are present after a person of good sense, three other paralog Sox transcription factor Sox4, Sox17,
Sox18.Sox17NRR illustrates the higher efficiency of versus wild type Sox2, but Sox4NRR and Sox18NRR produces iPSC's
Ability is poor, such as Fig. 9.
3) DERBYSeq can make Sox17 from being changed into high reprogramming energy without becoming ability again
Inventor next explore whether can use can not play reprogramming effect gene come identify new transcription because
Son.Inventor have selected Sox17 and choose L46, tri- sites of V53, E57 structure saturation mutation library, build the structure in library
It is identical with screening technique with Sox2, using OG2-MEF, retroviral vector and LIF+ Serum Systems.Inventor's
Sox17DERBYSeq screenings determine several mutant, and these mutant are enriched in eSox17 libraries and OK transcription factor body
In the FACS classification of GFP positive cells caused by system, such as Figure 10.Efficiency highest eSox17 mutant generates 15 times of iPS
Number is cloned, and speed increases, such as Figure 11,12,13, inventor is found that the eSox mutation of 12 functional property mutation
Body, they can strengthen wild type Sox reprogramming ability respectively, such as Figure 14, and the sequence alignment of mutant is as shown in figure 15.Always
For, inventor attempts to improve wild type Sox2 using DERBYSeq technologies and increases new function on wild type Sox17
It is very successful.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means specific features, structure, material or the spy for combining the embodiment or example description
Point is contained at least one embodiment or example of the present invention.In this manual, to the schematic representation of above-mentioned term not
Identical embodiment or example must be directed to.Moreover, specific features, structure, material or the feature of description can be with office
Combined in an appropriate manner in one or more embodiments or example.In addition, in the case of not conflicting, the skill of this area
Art personnel can be tied the different embodiments or example and the feature of different embodiments or example described in this specification
Close and combine.
Although embodiments of the invention have been shown and described above, it is to be understood that above-described embodiment is example
Property, it is impossible to limitation of the present invention is interpreted as, one of ordinary skill in the art within the scope of the invention can be to above-mentioned
Embodiment is changed, changed, replacing and modification.
SEQUENCE LISTING
<110>Chinese Academy of Sciences Guangzhou Institute of Biomedicine and Health
<120>Polypeptide, preparation method and applications
<130> PIDC3174992
<160> 8
<170> PatentIn version 3.3
<210> 1
<211> 319
<212> PRT
<213> Artificial
<220>
<223>The amino acid sequence of Sox mutant proteins
<400> 1
Met Tyr Asn Met Met Glu Thr Glu Leu Lys Pro Pro Gly Pro Gln Gln
1 5 10 15
Ala Ser Gly Gly Gly Gly Gly Gly Gly Asn Ala Thr Ala Ala Ala Thr
20 25 30
Gly Gly Asn Gln Lys Asn Ser Pro Asp Arg Val Lys Arg Pro Met Asn
35 40 45
Ala Phe Met Val Trp Ser Arg Gly Gln Arg Arg Lys Met Ala Gln Glu
50 55 60
Asn Pro Lys Met His Asn Ser Glu Ile Ser Lys Arg Leu Gly Ala Glu
65 70 75 80
Trp Lys Leu Leu Ser Asn Thr Glu Lys Arg Pro Phe Arg Asp Glu Ala
85 90 95
Arg Arg Leu Arg Ala Leu His Met Lys Glu His Pro Asp Tyr Lys Tyr
100 105 110
Arg Pro Arg Arg Lys Thr Lys Thr Leu Met Lys Lys Asp Lys Tyr Thr
115 120 125
Leu Pro Gly Gly Leu Leu Ala Pro Gly Gly Asn Ser Met Ala Ser Gly
130 135 140
Val Gly Val Gly Ala Gly Leu Gly Gly Gly Leu Asn Gln Arg Met Asp
145 150 155 160
Ser Tyr Ala His Met Asn Gly Trp Ser Asn Gly Ser Tyr Ser Met Met
165 170 175
Gln Glu Gln Leu Gly Tyr Pro Gln His Pro Gly Leu Asn Ala His Gly
180 185 190
Ala Ala Gln Met Gln Pro Met His Arg Tyr Val Val Ser Ala Leu Gln
195 200 205
Tyr Asn Ser Met Thr Ser Ser Gln Thr Tyr Met Asn Gly Ser Pro Thr
210 215 220
Tyr Ser Met Ser Tyr Ser Gln Gln Gly Thr Pro Gly Met Ala Leu Gly
225 230 235 240
Ser Met Gly Ser Val Val Lys Ser Glu Ala Ser Ser Ser Pro Pro Val
245 250 255
Val Thr Ser Ser Ser His Ser Arg Ala Pro Cys Gln Ala Gly Asp Leu
260 265 270
Arg Asp Met Ile Ser Met Tyr Leu Pro Gly Ala Glu Val Pro Glu Pro
275 280 285
Ala Ala Pro Ser Arg Leu His Met Ala Gln His Tyr Gln Ser Gly Pro
290 295 300
Val Pro Gly Thr Ala Lys Tyr Gly Thr Leu Pro Leu Ser His Met
305 310 315
<210> 2
<211> 0
<212> PRT
<213> Artificial
<220>
<223>The amino acid sequence of Sox mutant proteins
<400> 2
<210> 3
<211> 319
<212> PRT
<213> Artificial
<220>
<223>The amino acid sequence of Sox mutant proteins
<400> 3
Met Tyr Asn Met Met Glu Thr Glu Leu Lys Pro Pro Gly Pro Gln Gln
1 5 10 15
Ala Ser Gly Gly Gly Gly Gly Gly Gly Asn Ala Thr Ala Ala Ala Thr
20 25 30
Gly Gly Asn Gln Lys Asn Ser Pro Asp Arg Val Lys Arg Pro Met Asn
35 40 45
Ala Phe Met Val Trp Ser Arg Gly Gln Arg Arg Lys Met Ala Gln Glu
50 55 60
Asn Pro Lys Met His Asn Ser Glu Ile Ser Lys Arg Leu Gly Ala Glu
65 70 75 80
Trp Lys Leu Leu Ser Asp Thr Glu Lys Arg Pro Phe Thr Asp Glu Ala
85 90 95
Lys Arg Leu Arg Ala Leu His Met Lys Glu His Pro Asp Tyr Lys Tyr
100 105 110
Arg Pro Arg Arg Lys Thr Lys Thr Leu Met Lys Lys Asp Lys Tyr Thr
115 120 125
Leu Pro Gly Gly Leu Leu Ala Pro Gly Gly Asn Ser Met Ala Ser Gly
130 135 140
Val Gly Val Gly Ala Gly Leu Gly Gly Gly Leu Asn Gln Arg Met Asp
145 150 155 160
Ser Tyr Ala His Met Asn Gly Trp Ser Asn Gly Ser Tyr Ser Met Met
165 170 175
Gln Glu Gln Leu Gly Tyr Pro Gln His Pro Gly Leu Asn Ala His Gly
180 185 190
Ala Ala Gln Met Gln Pro Met His Arg Tyr Val Val Ser Ala Leu Gln
195 200 205
Tyr Asn Ser Met Thr Ser Ser Gln Thr Tyr Met Asn Gly Ser Pro Thr
210 215 220
Tyr Ser Met Ser Tyr Ser Gln Gln Gly Thr Pro Gly Met Ala Leu Gly
225 230 235 240
Ser Met Gly Ser Val Val Lys Ser Glu Ala Ser Ser Ser Pro Pro Val
245 250 255
Val Thr Ser Ser Ser His Ser Arg Ala Pro Cys Gln Ala Gly Asp Leu
260 265 270
Arg Asp Met Ile Ser Met Tyr Leu Pro Gly Ala Glu Val Pro Glu Pro
275 280 285
Ala Ala Pro Ser Arg Leu His Met Ala Gln His Tyr Gln Ser Gly Pro
290 295 300
Val Pro Gly Thr Ala Lys Tyr Gly Thr Leu Pro Leu Ser His Met
305 310 315
<210> 4
<211> 319
<212> PRT
<213> Artificial
<220>
<223>The amino acid sequence of Sox mutant proteins
<400> 4
Met Tyr Asn Met Met Glu Thr Glu Leu Lys Pro Pro Gly Pro Gln Gln
1 5 10 15
Ala Ser Gly Gly Gly Gly Gly Gly Gly Asn Ala Thr Ala Ala Ala Thr
20 25 30
Gly Gly Asn Gln Lys Asn Ser Pro Asp Arg Val Lys Arg Pro Met Asn
35 40 45
Ala Phe Met Val Trp Ser Arg Gly Gln Arg Arg Lys Met Ala Gln Glu
50 55 60
Asn Pro Lys Met His Asn Ser Glu Ile Ser Lys Arg Leu Gly Ala Glu
65 70 75 80
Trp Lys Leu Leu Ser Phe Thr Glu Lys Arg Pro Phe Arg Asp Glu Ala
85 90 95
Thr Arg Leu Arg Ala Leu His Met Lys Glu His Pro Asp Tyr Lys Tyr
100 105 110
Arg Pro Arg Arg Lys Thr Lys Thr Leu Met Lys Lys Asp Lys Tyr Thr
115 120 125
Leu Pro Gly Gly Leu Leu Ala Pro Gly Gly Asn Ser Met Ala Ser Gly
130 135 140
Val Gly Val Gly Ala Gly Leu Gly Gly Gly Leu Asn Gln Arg Met Asp
145 150 155 160
Ser Tyr Ala His Met Asn Gly Trp Ser Asn Gly Ser Tyr Ser Met Met
165 170 175
Gln Glu Gln Leu Gly Tyr Pro Gln His Pro Gly Leu Asn Ala His Gly
180 185 190
Ala Ala Gln Met Gln Pro Met His Arg Tyr Val Val Ser Ala Leu Gln
195 200 205
Tyr Asn Ser Met Thr Ser Ser Gln Thr Tyr Met Asn Gly Ser Pro Thr
210 215 220
Tyr Ser Met Ser Tyr Ser Gln Gln Gly Thr Pro Gly Met Ala Leu Gly
225 230 235 240
Ser Met Gly Ser Val Val Lys Ser Glu Ala Ser Ser Ser Pro Pro Val
245 250 255
Val Thr Ser Ser Ser His Ser Arg Ala Pro Cys Gln Ala Gly Asp Leu
260 265 270
Arg Asp Met Ile Ser Met Tyr Leu Pro Gly Ala Glu Val Pro Glu Pro
275 280 285
Ala Ala Pro Ser Arg Leu His Met Ala Gln His Tyr Gln Ser Gly Pro
290 295 300
Val Pro Gly Thr Ala Lys Tyr Gly Thr Leu Pro Leu Ser His Met
305 310 315
<210> 5
<211> 960
<212> DNA
<213> Artificial
<220>
<223>Encode the nucleotide sequence of the nucleic acid of Sox mutant proteins
<400> 5
atgtataaca tgatggagac ggagctgaag ccgccgggcc cgcagcaagc ttcggggggc 60
ggcggcggag gaggcaacgc cacggcggcg gcgaccggcg gcaaccagaa gaacagcccg 120
gaccgcgtca agaggcccat gaacgccttc atggtatggt cccgggggca gcggcgtaag 180
atggcccagg agaaccccaa gatgcacaac tcggagatca gcaagcgcct gggcgcggag 240
tggaaacttt tgtccaatac cgagaagcgg ccgttccggg acgaggcccg gcggctgcgc 300
gctctgcaca tgaaggagca cccggattat aaataccggc cgcggcggaa aaccaagacg 360
ctcatgaaga aggataagta cacgcttccc ggaggcttgc tggcccccgg cgggaacagc 420
atggcgagcg gggttggggt gggcgccggc ctgggtggcg ggctgaacca gcgcatggac 480
agctacgcgc acatgaacgg ctggagcaac ggcagctaca gcatgatgca ggagcagctg 540
ggctacccgc agcacccggg cctcaacgct cacggcgcgg cacagatgca accgatgcac 600
cgctacgtcg tcagcgccct gcagtacaac tccatgacca gctcgcagac ctacatgaac 660
ggctcgccca cctacagcat gtcctactcg cagcagggca cccccggtat ggcgctgggc 720
tccatgggct ctgtggtcaa gtccgaggcc agctccagcc cccccgtggt tacctcttcc 780
tcccactcca gggcgccctg ccaggccggg gacctccggg acatgatcag catgtacctc 840
cccggcgccg aggtgccgga gcccgctgcg cccagtagac tgcacatggc ccagcactac 900
cagagcggcc cggtgcccgg cacggccaaa tacggcacac tgcccctgtc gcacatgtga 960
<210> 6
<211> 960
<212> DNA
<213> Artificial
<220>
<223>Encode the nucleotide sequence of the nucleic acid of Sox mutant proteins
<400> 6
atgtataaca tgatggagac ggagctgaag ccgccgggcc cgcagcaagc ttcggggggc 60
ggcggcggag gaggcaacgc cacggcggcg gcgaccggcg gcaaccagaa gaacagcccg 120
gaccgcgtca agaggcccat gaacgccttc atggtatggt cccgggggca gcggcgtaag 180
atggcccagg agaaccccaa gatgcacaac tcggagatca gcaagcgcct gggcgcggag 240
tggaaacttt tgtccgctac cgagaagcgg ccgttccatg acgaggccaa gcggctgcgc 300
gctctgcaca tgaaggagca cccggattat aaataccggc cgcggcggaa aaccaagacg 360
ctcatgaaga aggataagta cacgcttccc ggaggcttgc tggcccccgg cgggaacagc 420
atggcgagcg gggttggggt gggcgccggc ctgggtggcg ggctgaacca gcgcatggac 480
agctacgcgc acatgaacgg ctggagcaac ggcagctaca gcatgatgca ggagcagctg 540
ggctacccgc agcacccggg cctcaacgct cacggcgcgg cacagatgca accgatgcac 600
cgctacgtcg tcagcgccct gcagtacaac tccatgacca gctcgcagac ctacatgaac 660
ggctcgccca cctacagcat gtcctactcg cagcagggca cccccggtat ggcgctgggc 720
tccatgggct ctgtggtcaa gtccgaggcc agctccagcc cccccgtggt tacctcttcc 780
tcccactcca gggcgccctg ccaggccggg gacctccggg acatgatcag catgtacctc 840
cccggcgccg aggtgccgga gcccgctgcg cccagtagac tgcacatggc ccagcactac 900
cagagcggcc cggtgcccgg cacggccaaa tacggcacac tgcccctgtc gcacatgtga 960
<210> 7
<211> 960
<212> DNA
<213> Artificial
<220>
<223>Encode the nucleotide sequence of the nucleic acid of Sox mutant proteins
<400> 7
atgtataaca tgatggagac ggagctgaag ccgccgggcc cgcagcaagc ttcggggggc 60
ggcggcggag gaggcaacgc cacggcggcg gcgaccggcg gcaaccagaa gaacagcccg 120
gaccgcgtca agaggcccat gaacgccttc atggtatggt cccgggggca gcggcgtaag 180
atggcccagg agaaccccaa gatgcacaac tcggagatca gcaagcgcct gggcgcggag 240
tggaaacttt tgtccgatac cgagaagcgg ccgttcactg acgaggccaa gcggctgcgc 300
gctctgcaca tgaaggagca cccggattat aaataccggc cgcggcggaa aaccaagacg 360
ctcatgaaga aggataagta cacgcttccc ggaggcttgc tggcccccgg cgggaacagc 420
atggcgagcg gggttggggt gggcgccggc ctgggtggcg ggctgaacca gcgcatggac 480
agctacgcgc acatgaacgg ctggagcaac ggcagctaca gcatgatgca ggagcagctg 540
ggctacccgc agcacccggg cctcaacgct cacggcgcgg cacagatgca accgatgcac 600
cgctacgtcg tcagcgccct gcagtacaac tccatgacca gctcgcagac ctacatgaac 660
ggctcgccca cctacagcat gtcctactcg cagcagggca cccccggtat ggcgctgggc 720
tccatgggct ctgtggtcaa gtccgaggcc agctccagcc cccccgtggt tacctcttcc 780
tcccactcca gggcgccctg ccaggccggg gacctccggg acatgatcag catgtacctc 840
cccggcgccg aggtgccgga gcccgctgcg cccagtagac tgcacatggc ccagcactac 900
cagagcggcc cggtgcccgg cacggccaaa tacggcacac tgcccctgtc gcacatgtga 960
<210> 8
<211> 960
<212> DNA
<213> Artificial
<220>
<223>Encode the nucleotide sequence of the nucleic acid of Sox mutant proteins
<400> 8
atgtataaca tgatggagac ggagctgaag ccgccgggcc cgcagcaagc ttcggggggc 60
ggcggcggag gaggcaacgc cacggcggcg gcgaccggcg gcaaccagaa gaacagcccg 120
gaccgcgtca agaggcccat gaacgccttc atggtatggt cccgggggca gcggcgtaag 180
atggcccagg agaaccccaa gatgcacaac tcggagatca gcaagcgcct gggcgcggag 240
tggaaacttt tgtcctttac cgagaagcgg ccgttccggg acgaggccac tcggctgcgc 300
gctctgcaca tgaaggagca cccggattat aaataccggc cgcggcggaa aaccaagacg 360
ctcatgaaga aggataagta cacgcttccc ggaggcttgc tggcccccgg cgggaacagc 420
atggcgagcg gggttggggt gggcgccggc ctgggtggcg ggctgaacca gcgcatggac 480
agctacgcgc acatgaacgg ctggagcaac ggcagctaca gcatgatgca ggagcagctg 540
ggctacccgc agcacccggg cctcaacgct cacggcgcgg cacagatgca accgatgcac 600
cgctacgtcg tcagcgccct gcagtacaac tccatgacca gctcgcagac ctacatgaac 660
ggctcgccca cctacagcat gtcctactcg cagcagggca cccccggtat ggcgctgggc 720
tccatgggct ctgtggtcaa gtccgaggcc agctccagcc cccccgtggt tacctcttcc 780
tcccactcca gggcgccctg ccaggccggg gacctccggg acatgatcag catgtacctc 840
cccggcgccg aggtgccgga gcccgctgcg cccagtagac tgcacatggc ccagcactac 900
cagagcggcc cggtgcccgg cacggccaaa tacggcacac tgcccctgtc gcacatgtga 960
Claims (10)
1. a kind of polypeptide of separation, it is characterised in that the polypeptide has SEQ ID NO：Amino acid sequence shown in 1~4.
A kind of 2. method for obtaining polypeptide described in claim 1, it is characterised in that including：
(1) using Sox2 and Sox17 as template, the of HMG protein-interacting surface region random mutation HMG domain sequences
46,53 and 56 amino acids, to build the random library of Sox mutant；
(2) random library of the mutant is carried out transfecting processing and collects virus, mutant described in the expressing viral；
(3) virus is infected into body cell, and Somatic Cell Culture 12~15 days, the body cell will carries Oct4 and open after infection
The transgenosis green fluorescent protein of mover driving；
(4) using the cell for flowing Schwann Cells art sorting step (3) acquisition, from green fluorescence feminine gender and green fluorescent protein positive group
Genomic DNA is extracted in body respectively and carries out high-flux sequence and screening, with obtain can strengthen it is competent thin more than generation induction type
Candidate's Sox mutant proteins of born of the same parents；
Wherein, wild type Sox of the induced efficiency of the multipotential stem cell more than 1.5 times induced efficiency is the finger of target polypeptides
Mark.
3. according to the method for claim 2, it is characterised in that the random library of the Sox mutant includes carrying Sox and dashed forward
The construct of variant and NNK coding, the carrier of the construct is pMX,
Optionally, the transfection, which is handled, is realized by the way that the random library of the mutant is transfected into Plat-E cells,
Optionally, the virus is retrovirus；
Optionally, the body cell is embryo fibroblast.
4. a kind of nucleic acid of separation, it is characterised in that there is SEQ ID NO：Nucleotide sequence shown in 5~8.
5. a kind of construct, it is characterised in that carry the nucleic acid described in claim 4.
A kind of 6. method for changing cell fate, it is characterised in that including：The cell is overexpressed more described in claim 1
Peptide.
7. according to the method for claim 6, it is characterised in that the cell is body cell.
8. according to the method for claim 7, it is characterised in that the body cell is fibroblast.
9. according to the method for claim 6, it is characterised in that the cell is overexpressed Oct4 and Klf4 simultaneously.
10. according to the method for claim 6, it is characterised in that the cell is overexpressed Oct4, Klf4 and c-Myc simultaneously.
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| PCT/CN2018/077401 WO2019095584A1 (en) | 2017-11-17 | 2018-02-27 | eSOX17 POLYPEPTIDES FOR EFFICIENT CELLULAR REPROGRAMMING, DERBY-SEQ METHOD OF OBTAINING THE SAME AND USE THEREOF |
| PCT/CN2018/077403 WO2019095585A1 (en) | 2017-11-17 | 2018-02-27 | eSOX2 POLYPEPTIDES FOR EFFICIENT CELLULAR REPROGRAMMING, DERBY-SEQ METHOD OF OBTAINING THE SAME AND USE THEREOF |
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| US20110014620A1 (en) * | 2009-07-08 | 2011-01-20 | Claudio Basilico | Methods for identification of bone anabolic agents |
| US20110281281A1 (en) * | 2010-05-12 | 2011-11-17 | Ipierian, Inc. | Integration-free human induced pluripotent stem cells from blood |
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| US20110020814A1 (en) * | 2009-06-05 | 2011-01-27 | Ipierian, Inc. | Methods and compositions for selection of stem cells |
| WO2011056147A1 (en) * | 2009-11-04 | 2011-05-12 | Agency For Science, Technology And Research | Mutant sox proteins and methods of inducing pluripotency |
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| US20110281281A1 (en) * | 2010-05-12 | 2011-11-17 | Ipierian, Inc. | Integration-free human induced pluripotent stem cells from blood |
| CN107286232A (en) * | 2017-05-23 | 2017-10-24 | 中国科学院广州生物医药与健康研究院 | Efficient induction of the POU domains engineered protein to multipotential stem cell |
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| CN111808171A (en) * | 2020-07-07 | 2020-10-23 | 深圳大学 | Targeting peptide of tumor stem cell marker Oct4 and application thereof |
| CN111808171B (en) * | 2020-07-07 | 2022-02-01 | 深圳大学 | Targeting peptide of tumor stem cell marker Oct4 and application thereof |
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