CN104844691B - Sox2 protein peptides aptamer and its identification - Google Patents

Abstract

The present invention discloses peptide aptamer of the targeting with reference to Sox2 albumen.Peptide aptamer compatibility that the present invention obtains is high, high specificity, therefore, the great application value of the present invention.Beneficial effects of the present invention（1）By bimolecular fluorescence complementary technology, screened from high flux library and obtain candidate peptide aptamer of the targeting with reference to Sox2 albumen；（2）By way of vector construction, the Sox2 protein expression vectors with Flag or Myc amalgamation and expressions are obtained respectively；（3）By way of vector construction, the peptide aptamer expression vector with Flag or Myc amalgamation and expressions is obtained respectively；（4）Utilize Immunoprecipitation, it is determined that the special peptide aptamer with reference to Sox2 albumen can be targetted.

Description

Sox2 protein peptides aptamer and its identification

Technical field

The invention belongs to biomedicine technical field, and in particular to screening and determine targeting with reference to the special of Sox2 albumen Peptide aptamer.

Background technology

Bimolecular fluorescence complementary technology is that one kind is directly perceived, quickly judges that target protein is positioned and interacted in living cells Technology, the technology dexterously by two complementary fragments of fluorescent protein molecule respectively with target protein amalgamation and expression.It is if glimmering Photoprotein activation recovering, then show that two target proteins are interacted.The technology has been used for different Protein-protein interactions Strong and weak comparison and the research work of protein ubiquitination etc..

Aptamer technology refers to position target using the aptamer of screening, detects and the skill of functional biological research Art.Aptamer has long half-life period, characteristic nontoxic and that acquisition can be screened by the target molecule of non-immunogenicity, and they It can also be easy to produce by chemical synthesis or expression system.Peptide aptamer refers to screen pin from random protein expression libraries To the short peptide molecules of protein target, these screened in library from high power capacity acquisition short peptide molecules can specifically with target knot Close, so as to influence the function of target, can be applied to target and its intervention study of compound function.Classical peptide aptamer screening Mainly screened and obtained from random peptide expression library by yeast-two hybrid technique.In order to which stabilization is derived from random expression library The conformation of peptide aptamer, a series of variable reading frame of encoded peptide aptamers is inserted into a scaffolding protein framework by us （The typically thioredoxin of bacterium）To form fusion protein, whereby, the peptide aptamer of fusion can form specific albumen structure As in favor of be combineding with each other between peptide aptamer and target molecule.

Sox2 albumen is a powerful transcription factor, and the evil with self-renewing, multiple tumours is maintained in stem cell dryness Property process（Such as tumour generation, tumor cell proliferation, migration, invasion and attack transfer, tumor stem cell characteristic maintains, chemotherapeutic drug resistance Deng）Many aspects there is facilitation.Therefore, the small-molecule drug that screening acquisition can target the albumen can be not only Sox2 Protein-function assays provide an effective tool, and by modification, may be derived from having a constant current modulation to specific tumors The peptide aptamer medicament of effect.

The content of the invention

It is an object of the invention to provide one to utilize bimolecular fluorescence complementary technology quickly from the random expression of peptides of high power capacity It is adapted to the peptide adaptation submethod that special target combination Sox2 albumen is screened in sublibrary.

The authentication method of special peptide aptamer is carried out using Immunoprecipitation it is an object of the invention to provide one.

Sox2 protein peptides aptamers, sequence are：FSTLFFPLFFL.Utilize the peptide aptamer based on bimolecular fluorescence complementary Library（It is prepared by the construction method in patent 201410339641.X）, using pBiFc-VN173- Sox2 as target, screening obtains The candidate peptide aptamer with reference to Sox2 must be targetted；By expression vector establishment and Immunoprecipitation, from candidate peptide aptamer The middle special high affine combination Sox2 of identification energy peptide aptamer.

Described targeting is referred to as TrxA-peptide-TrxA, peptide with reference to the candidate peptide aptamer of Sox2 albumen Refer to following 7 small peptides for including 11 amino acid, particular sequence is TrxA-TSCVFFSFLYL-TrxA, TrxA- FSWIVFRLTFL-TrxA, TrxA-LFYIYFSFWFL-TrxA, TrxA- FSTLFFPLFFL-TrxA, TrxA- TPVFLPPFSPM-TrxA, TrxA-VLLFFYVFLFL-TrxA, TrxA-HCTIIWLEFTL-TrxA, by TrxA Cysteine form disulfide bond, to form fixed conformation, special small peptide is located in TrxA expression cassettes.

Sox2 protein peptides aptamer authentication methods, comprise the following steps：

1）Using design primer, using bimolecular fluorescence complementary obtain candidate peptide aptamer TrxA-peptide-TrxA as Template, high-fidelity PCR amplification is carried out to TrxA-peptide-TrxA；

3）Candidate peptide aptamer TrxA-peptide-TrxA is transferred to pCDNA3.1-myc- using digestion connection method His-C expression vectors, obtain pCDNA3.1-myc-His-C-TrxA-peptide-TrxA expression vectors；

4) Sox2 is transferred to pCMV-Tag2B expression vectors using digestion connection method, obtains pCMV-Tag2B-Sox2 Expression vector；

5) corotation dyeing method is utilized, in blocks respectively by pCDNA3.1-myc-His-C-TrxA-peptide- TrxA and pCMV-Tag2B-Sox2 is transfected into HEK293T cells, carries out co-immunoprecipitation using Flag antibody and combines utilization Myc Identification of the antibodies；

6) candidate peptide aptamer is transferred to pCMV-Tag2B expression vectors using digestion connection method, structure obtains PCMV-Tag2B- TrxA-peptide-TrxA expression vectors；

7) Sox2 is transferred to pCDNA3.1-myc-His-C expression vectors using digestion connection method, structure obtains PCDNA3.1-myc-His-C-Sox2 expression vectors；

8) utilize corotation dyeing method, in blocks respectively by pCMV-Tag2B-TrxA-peptide-TrxA and PCDNA3.1-myc-His-C-Sox2 is transfected into HEK293T cells, carries out co-immunoprecipitation using Flag antibody and combines profit With Myc Identification of the antibodies.

Using clone technology and vector construction technology, there is specific stickiness end by what is obtained by High fidelity PCR and digestion The Sox2 ORFs orientation at end is inserted into pBiFc-VN173, obtains pBiFc-VN173- Sox2 expression vectors.

According to bimolecular fluorescence complementary technology, it is adapted to screening in sublibrary from peptide and obtains 7 candidate peptide aptamers, these Peptide aptamer is inserted in TrxA supports, and is expressed with the segment composition of VENUS PROTEIN Cs 155.

Using clone technology and vector construction technology, there is specific stickiness end by what is obtained by High fidelity PCR and digestion The TrxA-peptide-TrxA candidate peptide aptamers nucleotide fragments orientation at end is inserted into pCDNA3.1-myc-His-C expression and carried Body, obtain pCDNA3.1-myc-His-C-TrxA-peptide-TrxA expression vectors.

Using clone technology and vector construction technology, there is specific stickiness end by what is obtained by High fidelity PCR and digestion The Sox2 ORFs orientation at end is inserted into pCMV-Tag2B expression vectors, obtains pCMV-Tag2B-Sox2 expression vectors.

Using clone technology and vector construction technology, there is specific stickiness end by what is obtained by High fidelity PCR and digestion The Sox2 ORFs orientation at end is inserted into pCDNA3.1-myc-His-C expression vectors, obtains pCDNA3.1-myc-His- C-Sox2 expression vectors.

Using clone technology and vector construction technology, there is specific stickiness end by what is obtained by High fidelity PCR and digestion The TrxA-peptide-TrxA candidate peptide aptamers nucleotide fragments orientation at end is inserted into pCMV-Tag2B expression vectors, obtains PCMV-Tag2B-TrxA-peptide-TrxA expression vectors.

After all purposes plasmid extraction sequencing company will be sent to be sequenced and confirm the correctness and uniformity of sequence.

All expression vectors are distributed into two combinations and carry out cotransfection HEK293T cells, combination one is：pCDNA3.1- Myc-His-C-TrxA-peptide-TrxA and pCMV-Tag2B-Sox2；Combining two is：pCDNA3.1-myc-His-C-Sox2 And pCMV-Tag2B-TrxA-peptide-TrxA.After 48 hours, carry out co-immunoprecipitation using Flag antibody and combine utilization Myc Identification of the antibodies.

The beneficial effects of the present invention are：

（1）PBiFc-VN173-Sox2 expression vectors are constructed, the 1-172 amino acid of Sox2 albumen and VENUS albumen is residual Base carries out amalgamation and expression；

（2）Using bimolecular fluorescence complementary technology, screened from the peptide aptamer expression library of high power capacity and obtain 7 targets To the candidate peptide aptamer for combining Sox2 albumen；

（3）PCDNA3.1-myc-His-C-TrxA-peptide-TrxA, pCMV-Tag2B-Sox2 are constructed, and PCDNA3.1-myc-His-C-Sox2 and pCMV-Tag2B-TrxA-peptide-TrxA expression vectors, make target protein with Flag or Myc label proteins carry out amalgamation and expression；

（4）On the basis of sequencing result is correct, using removing endotoxin plasmid extraction kit by corresponding expression vector It is stripped, by them, cotransfection HEK293T cells, co-immunoprecipitation result show in the way of combination, peptide aptamer P42 energy Special with Sox2 albumen and high affine combination, the peptide adaptor sequence are：FSTLFFPLFFL.

Brief description of the drawings

Fig. 1 is the expression vector schematic diagram for bimolecular fluorescence complementary and co-immunoprecipitation.A is Sox2 albumen, with VENUS albumen 1-172 amino acid residue amalgamation and expressions；B is TrxA-peptide-TrxA, is merged with the C155 of VENUS albumen Expression;C is pCMV-Tag2B-Sox2 expression vectors；D expresses for pcDNA3.1/myc-His-C-TrxA-peptide-TrxA Carrier；E is pCMV-Tag2B-TrxA- peptide-TrxA expression vectors；F expresses for pcDNA3.1/myc-His-C-Sox2 Carrier.

Fig. 2 is the screening of bimolecular fluorescence complementary and the peptide aptamer schematic diagram of Sox2 albumen specific bonds.Wherein 1 is Sox2 + peptide aptamer P15；2 be Sox2+ peptide aptamers P18；3 be Sox2+ peptide aptamers P32；4 be Sox2+ peptide aptamers P42；5 are Sox2+ peptide aptamers p46；6 be Sox2+ peptide aptamers P55；7 be Sox2+ peptide aptamers P64.

Fig. 3 is co-immunoprecipitation identification and the peptide aptamer schematic diagram of Sox2 albumen specific bonds.Swimming lane 1 is untransfected The HEK293T lysates control of expression vector；Swimming lane 2 is a HEK293T cell for transfection pCMV-Tag2B-Sox2 expression vectors Lysate is co-precipitated result with Flag antibody mediated immunities；Swimming lane 3 is transfected expression vector pCMV-Tag2B-Sox2 and pcDNA3.1/ Myc-His-C-TrxA-P42-TrxA HEK293T cell pyrolysis liquids；Swimming lane 4 is transfected expression vector pCMV-Tag2B-Sox2 It is co-precipitated and is tied with Flag antibody mediated immunities with pcDNA3.1/myc-His-C-TrxA-P42-TrxA HEK293T cell pyrolysis liquids Fruit；Swimming lane 5 is transfected expression vector pcDNA3.1/myc-His-C-Sox2 HEK293T cell pyrolysis liquids；Swimming lane 6 is transfection Expression vector pCMV-Tag2B-TrxA-P42-TrxA and pcDNA3.1/myc-His-C-Sox2 HEK293T cell pyrolysis liquids； Swimming lane 7 is transfected expression vector pCMV-Tag2B-TrxA-P42-TrxA and pcDNA3.1/myc-His-C-Sox2 HEK293T Cell pyrolysis liquid is co-precipitated result with Flag antibody mediated immunities.

Embodiment

Embodiment 1

Based on bimolecular fluorescence complementary technology Sox2 fusion expression vectors structure and special peptide aptamer screening include with Lower step

Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments Method, it is conventional method unless otherwise specified.Experiment material in following embodiments, it is conventional life unless otherwise specified Change reagent store to obtain.Quantitative experiment in following examples, independent repeatedly experiment three times is disposed as, is as a result averaged Value.

Step 1）PBiFc-VN173-Sox2 expression vector establishments：Inverted with Human esophageal squamous cell cancer cell line KYSE450 RNA The cDNA of record is template, and design primer pair Sox2 genes carry out high-fidelity PCR amplification, and using restriction enzyme EcoRI and After XbaI carries out digestion to amplified production, pBiFcVN173 carriers are inserted into, are carried with forming pBiFcVN173-Sox2 expression Body.Amplimer sequence is as follows：Forward primer：5’-CGGAATTCAATGTACAACATGATGGAGAC-3 ' (end EcoRI Restriction enzyme site）；Reverse primer:5’-TGCTCTAGACATGTGTGAGAGGGGCAG-3’ （End is XbaI enzyme cutting site）.Through Digestion identification and sequencing result show：Obtain expected expression Sox2 targets and available for the carrier of double fluorescence complementaries（Fig. 1）.

PCR reaction conditions are as follows：

Step 2）Using go the extracting of endotoxin plasmid extraction kit be sequenced correct pBiFcVN173-Sox2 and PDsRED2-C1 carriers, random 10 peptide aptamer Library plasmids of picking, will using the transfection reagents of Lipofectamine 2000 The pBiFc-VC155-TrxA-peptide-TrxA Library plasmids of the random pickings of 200ng, 200ng pBiFcVN173-Sox2 With 50ng pDsRED2-C1（The red fluorescence of pDsRed2-C1 transfections is as internal reference）Carrier forms 10 various combinations and is total to respectively Transfect in the HEK293T cells of 12 orifice plates.

Step 3）The HEK293T cells of transfection are positioned over after being cultivated 24 hours in cell culture incubator, in fluorescence microscope Down it was observed that the appearance of fluorescence.PRELIMINARY RESULTS show wherein 7 peptide aptamers can specifically with Sox2 albumen specific bonds, so as to fast Fast preliminary screening obtains the peptide aptamer of site-specific combination Sox2 albumen（Fig. 2）, candidate peptide aptamer TrxA-peptide- The corresponding amino acid sequences of peptide are shown in Table 1 in TrxA, and sequence does not include the TrxA sequences of flank in list.

Table 1 is the sequencing result and amino acid sequence with the candidate peptide aptamer of Sox2 albumen specific bonds.

Embodiment 2

Targeting comprises the following steps with reference to the identification of the peptide aptamer of Sox2 albumen

1）For expressing the construction of eukaryotic expression vector of candidate peptide aptamer【TrxA-peptide-TrxA-Myc, Peptide refers to 11 AA small peptide】

Designing special primer and high-fidelity PCR amplification is carried out to TrxA-peptide-TrxA, peptide refers to P15 respectively, P18, P32, P42, P46, P55, P64.After carrying out digestion to amplified production using restriction enzyme EcoRI and BamHI, by this The endonuclease bamhi of 7 candidate peptide aptamers inserts in pcDNA3.1- myc-hisC carriers respectively, obtains pcDNA3.1-myc- HisC-TrxA-peptide-TrxA carriers, these carriers can express the peptide aptamer with myc amalgamation and expressions, be respectively PcDNA3.1-myc-hisC-TrxA- P15-TrxA, pcDNA3.1-myc-hisC-TrxA- P18-TrxA, pcDNA3.1- Myc-hisC- TrxA- P32-TrxA, pcDNA3.1-myc-hisC- TrxA- P42-TrxA, pcDNA3.1-myc-hisC - TrxA- P46-TrxA, pcDNA3.1-myc-hisC- TrxA- P55-TrxA, pcDNA3.1-myc-hisC- TrxA- P64-TrxA（Fig. 1）.

Amplimer sequence is as follows：Forward primer：5’-cgGGATCCAACCATGAGCGATAAAATTATTC AC-3’（End Hold as BamHI restriction enzyme sites）；Reverse primer： 5’-CGGAATTCCCAGGTTAGC GTCGAGGAA-3’ （End is EcoRI Restriction enzyme site）, PCR reaction conditions are as follows：

2）For expressing the construction of eukaryotic expression vector (Flag-Sox2) of Sox2 albumen

Design special primer and high-fidelity PCR amplification is carried out to Sox2, use restriction enzyme EcoRI and HindIII pairs After amplified production carries out digestion, endonuclease bamhi is inserted into pCMV-Tag2B carriers, obtains Flag and Sox2 amalgamation and expressions PCMV-Tag2B-Sox2 expression vectors（Fig. 1）, the fusion protein F lag-Sox2 of the vector expression is for subsequently immune coprecipitated Form sediment and test.Primer sequence is as follows, forward primer：5’-CGGAATTCATGTACAACATGATGGAGACG -3’；（End is EcoRI restriction enzyme sites）：Reverse primer：5’-CCCAAGCTTTCACATGTGTGAGAGG GGCA-3’ （End is HindIII enzymes Enzyme site）.PCR reaction conditions are as follows：

3）For expressing the construction of eukaryotic expression vector of candidate peptide aptamer【Flag-TrxA-peptide-TrxA, Peptide refers to the small peptide that peptide refers to 11 AA】

Design primer pair TrxA-peptide-TrxA and carry out high-fidelity PCR amplification, using restriction enzyme EcoRI and After BamHI carries out digestion to amplified production, endonuclease bamhi is inserted into pCMV-Tag2B carriers, obtains Flag and TrxA- The pCMV-Tag2B-TrxA-peptide-TrxA expression vectors of peptide-TrxA amalgamation and expressions（Respectively：pCMV-Tag2B- TrxA-P15-TrxA, pCMV-Tag2B-TrxA-P18-TrxA, pCMV-Tag2B-TrxA-P32-TrxA, pCMV-Tag2B- TrxA-P42-TrxA, pCMV-Tag2B-TrxA-P46-TrxA, pCMV-Tag2B-TrxA-P55-TrxA, pCMV-Tag2B- TrxA-P64-TrxA）（Fig. 1）, the fusion protein of these vector expressions is used for follow-up co-immunoprecipitation experiment.Primer sequence is such as Under：Forward primer：5’-cgGGATCCATGAGCGATAAAATTATTCAC-3’（End is BamHI restriction enzyme sites）；Reversely draw Thing： 5’-CGGAATTCCAGGTTAGCGTCGAGGAA-3’（End is EcoRI restriction enzyme sites）.PCR reaction conditions are as follows：

4）For expressing the construction of eukaryotic expression vector (Sox2-Myc) of Sox2 albumen

Design special primer and high-fidelity PCR amplification is carried out to Sox2, using restriction enzyme EcoRI and BamHI to expanding After increasing production thing progress digestion, endonuclease bamhi is inserted in pcDNA3.1-myc-hisC carriers and obtains pcDNA3.1-myc-hisC- Sox2 expression vectors（Fig. 1）, the carrier can express the Sox2 with myc amalgamation and expressions.Amplimer sequence is as follows：Forward direction is drawn Thing：5’-cgGGATCCAGCCACCATGTACA ACATGATGGAGAC-3’（End is BamHI restriction enzyme sites）；Reverse primer： 5’-GCGAATTCCCATGTGTGAGAGGGGCAG-3’（End is EcoRI restriction enzyme sites）.PCR reaction conditions are as follows：

5）Co-immunoprecipitation verifies the specific bond of peptide aptamer and Sox2 albumen

Fusion expression vector is divided into two groups, one group is pcDNA3.1-myc-hisC-TrxA-P15-TrxA and pCMV- Tag2B-Sox2, pcDNA3.1-myc-hisC-TrxA-P18-TrxA and pCMV-Tag2B-Sox2, pcDNA3.1-myc- HisC-TrxA-P32-TrxA and pCMV-Tag2B-Sox2, pcDNA3.1-myc-hisC-TrxA-P42-TrxA and pCMV- Tag2B-Sox2, pcDNA3.1-myc-hisC-TrxA-P46-TrxA and pCMV-Tag2B-Sox2, pcDNA3.1-myc- HisC-TrxA-P55-TrxA and pCMV-Tag2B-Sox2, pcDNA3.1-myc-hisC-TrxA-P64-TrxA and pCMV- Tag2B -Sox2。

Another group is pCMV-Tag2B-TrxA-P15-TrxA and pcDNA3.1-myc-hisC-Sox2, pCMV-Tag2B- TrxA-P18-TrxA and pcDNA3.1-myc-hisC-Sox2, pCMV-Tag2B-TrxA-P32-TrxA and pcDNA3.1-myc- HisC-Sox2, pCMV-Tag2B-TrxA-P42-TrxA and pcDNA3.1-myc-hisC-Sox2, pCMV-Tag2B-TrxA- P46-TrxA and pcDNA3.1-myc-hisC-Sox2, pCMV-Tag2B-TrxA-P55-TrxA and pcDNA3.1-myc-hisC- Sox2, pCMV-Tag2B-TrxA-P64-TrxA and pcDNA3.1-myc-hisC-Sox2.

This two groups of carrier corotation are entered into HEK293T cells using transfection reagent PEI respectively, albumen is harvested and is marked using Flag Label antibody do co-immunoprecipitation, do immunoblot experiment checking with Myc and Flag tag antibodies, from the figure 3, it may be seen that Sox2 with TrxA-P42-TrxA combines closely, and P42 is the special peptide aptamer of Sox2 albumen.

SEQUENCE LISTING

<110>Fuzhou General Hospital, Nanjing Military Area, PLA

<120>Sox2 protein peptides aptamer and its identification

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<170> PatentIn version 3.3

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Claims (1)

1.Sox2 protein peptides aptamers, it is characterised in that：The peptide adaptor sequence is：FSTLFFPLFFL.