CN109021062A - A kind of screening technique of tumour neoantigen - Google Patents
A kind of screening technique of tumour neoantigen Download PDFInfo
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Abstract
The present invention provides a kind of screening techniques of tumour neoantigen, comprising: the corresponding polypeptide sequence encoded of A, the mutated gene for obtaining tumor tissue cell；B, the polypeptide sequence is subjected to affinity prediction with major histocompatibility complex MHCI respectively, and the affinity filtered out is more than the polypeptide sequence of specified threshold；C, the polypeptide sequence of coding corresponding to the mutated gene is carried out to the prediction of polypeptide restriction enzyme site, and retain restriction enzyme site not the affinity be more than specified threshold polypeptide sequence among, can not be digested can be independently at the polypeptide sequence of peptide fragment, as the candidate polypeptide sequence of tumour neoantigen.By upper, by the screening technique of the tumour neoantigen of the application, screening to tumour neoantigen, facilitate it is subsequent further targetedly tested accordingly, can greatly reduce the number of experiment, realize time saving, laborsaving and reduction of expenditure.
Description
Technical field
The present invention relates to antigen selection field more particularly to a kind of screening techniques of tumour neoantigen.
Background technique
Tumor vaccine (tumor vaccine) is one of hot spot of research in recent years, and principle is by tumour antigen with a variety of shapes
Formula is such as: tumour cell, tumor correlated albumen or polypeptide, the gene for expressing tumour antigen import patient's body, tumour are overcome to draw
The immunosuppressive condition risen enhances immunogenicity, activates the immune system of patient itself, and Cellular Immunity and body fluid is induced to exempt from
Epidemic disease response, to achieve the purpose that control or remove tumour.In April, 2010, U.S. Food and Drug Administration (FDA) approval
Provenge/sipuleucel-T for treating advanced prostate cancer, become first self active immunotherapy medicine and
First real therapeutic cancer vaccine paves the way (1,2) for the research and development of other similar products.
Have within 2017 2 technical teams obtained in the personalized tumor vaccine clinical test based on NGS it is gratifying at
Fruit, the clinical trial results of team of the U.S.: in 6 melanoma patients of vaccine inoculation, 4 human tumours are completely disappeared, and 32
Without recurrence in month, in addition 2 human tumours are still had, and tumour also completely disappears after receiving adjuvant treatment；The clinic of German team
Test result: in the patient of 13 vaccine inoculations, 8 human tumours are completely disappeared and in 23 months without recurrence, remaining 5 patient due to
Tumour has been spread when vaccine inoculation, has 2 people tumor regression occur, wherein 1 people receives completed tumor regression 1 after adjuvant treatment,
2.This technology or treatment method adjust using individuation knubble neoantigen or activating immune system kill tumour, from principle
Tumour is possible to become chronic disease in conjunction with other tumor therapeuticing methods, if larger scale clinical is proved to be successful, future market is latent
Power is huge (3,4).
But be all at present the screening carried out by the means of experiment one by one for the development of tumor vaccine, which takes
When, it is laborious, spend larger, and be not easy to find suitable tumor vaccine (tumour antigen), therefore, it is new to need a kind of pair of tumour at present
The method of the screening of antigen obtains suitable tumor vaccine by the screening to tumour neoantigen, subsequent further to facilitate
It is targetedly tested accordingly, greatly to reduce the number of experiment, realizes time saving, laborsaving and reduction of expenditure.
Summary of the invention
In view of this, the main purpose of the present invention is to provide a kind of screening techniques of tumour neoantigen, by tumour
The screening of neoantigen, with facilitate it is subsequent further targetedly tested accordingly, can greatly reduce the number of experiment,
Realize time saving, laborsaving and reduction of expenditure.
A kind of screening technique of tumour neoantigen provided by the present application, comprising steps of
A, the corresponding polypeptide sequence encoded of mutated gene of tumor tissue cell is obtained；
B, the polypeptide sequence is subjected to affinity prediction with major histocompatibility complex MHCI respectively, and filtered out
Affinity is more than the polypeptide sequence of specified threshold；
C, the polypeptide sequence of coding corresponding to the mutated gene is carried out to the prediction of polypeptide restriction enzyme site, and retains digestion
Site not the affinity be more than specified threshold polypeptide sequence among, can not be digested can be independently at the more of peptide fragment
Peptide sequence, as the candidate polypeptide sequence of tumour neoantigen.
By upper, the screening technique of tumour neoantigen provided by the present application passes through the screening to tumour neoantigen: will be described more
Peptide sequence carries out affinity prediction with major histocompatibility complex MHCI respectively and is screened (the affinity of polypeptide and MHCI
It ensure that polypeptide MHCI compound can successfully be identified by T cell surface receptor TCR, to activate T cell, cause relevant thin
Born of the same parents immune response) and the prediction of polypeptide restriction enzyme site screened (whether the prediction of polypeptide restriction enzyme site can reflect polypeptide
It is easy to be cut and be presented on MHC, whether the prediction of polypeptide restriction enzyme site can assist prediction core polypeptide can be by just definite
Cut), selective affinity it is strong and can not be digested can be independently at the polypeptide sequence of peptide fragment, as the time of tumour neoantigen
Select polypeptide sequence, facilitate it is subsequent further targetedly tested accordingly, can greatly reduce the number of experiment, realize
Time saving, laborsaving and reduction of expenditure.
Preferably, tumour neoantigen described in step C further include: the tumor tissues, the tumor tissues GAP-associated protein GAP, institute
State the mutant DNA sequences or mutant rna sequence of tumor tissue cell.
Preferably, after the step C further include:
D, core polypeptide sequence transporter related to antigen processing is subjected to affinity prediction, and prediction result is shown
Show core polypeptide sequence of the affinity within specified range as tumour neoantigen.
By upper, the affinity of polypeptide transporter related to antigen processing reflect polypeptide in presentation can smoothly into
Row, therefore preferable affinity can guarantee that polypeptide can be by successfully submission to MHCI molecule to a certain extent, therefore it is pre-
It can also be predicted as supplementary means when polypeptide is surveyed with MHCI affinity size.
Preferably, after the step C or D further include:
E, the core polypeptide is subjected to the test of docking in structure with major histocompatibility complex MHCI, and will produced
The structure of the docking conformation of raw compound is scored by the experience scoring functions of free energy, and it is highest right to retain scoring
Connect conformation.
By upper, structure-based polypeptide and MHCI molecular docking are mutual between research polypeptide ligand and receptor biological macromolecular
Action rule predicts a kind of effective means of its binding pattern and affinity.Here the purpose docked is reliable, conjunction in order to obtain
The epitope polypeptide of reason conformation in conjunction with the molecule of MHCI.It is scored again with the experience scoring functions of Conjugated free energy, quantitative estimation table
The size of position polypeptide and MHCI molecule affinity.
Preferably, after the step E, further includes:
F, pass through the highest docking conformation of scoring and major histocompatibility complex MHCI described in molecular dynamics simulation
Between interaction and motion change；And analysis obtains core polypeptide sequence and major histocompatibility complex MHCI accordingly
Junction sequence composition.
By upper, molecular dynamics simulation is the interaction that macromolecular and polypeptide are simulated according to the basic principle of Newtonian mechanics
With motion change, carry out the rule for the biological phenomena behind that inquiry experiment means can't resolve.We pass through molecular dynamics simulation
Means inquire into the action rule and motion change between MHCI and polypeptide complex, can intuitively embody polypeptide under stable state
Interaction and affinity between MHCI, can accurately predict whether polypeptide can steadily be combined with MHCI.
Preferably, after the step F, further includes:
G, when judge the junction sequence form in there are mutating acids, and judge the mutating acid and
When major histocompatibility complex MHCI is tightly combined；By the sequence of candidate polypeptide sequence tumour neoantigen as a filter
Column.
By upper, illustrate why polypeptide can be combined steadily with MHCI, just because of the generation of the mutating acid, because
This can be by the sequence of candidate polypeptide sequence tumour neoantigen as a filter.
Preferably, the step A, comprising:
A, the DNA of tumor tissue cell is extracted, and DNA sequencing is carried out to it；
B, the DNA sequence dna after sequencing is compared with the normal histiocytic DNA sequence dna, obtains the DNA of mutation
Sequence；
C, its polypeptide sequence for corresponding to coding is obtained by biological software according to the DNA sequence dna of the mutation.
By upper, the corresponding polypeptide sequence encoded of the mutated gene of available tumor tissue cell through the above steps.
Preferably, the step A, the polypeptide sequence are as follows: the polypeptide sequence containing 8-30 amino acid residue.
By upper, the affinity of the polypeptide sequence of the length containing 8-30 amino acid residue is preferable, influences if too long more
The affinity of peptide sequence, it is too short, influence the effect of the polypeptide.
In conclusion the screening technique of tumour neoantigen provided by the present application, passes through the screening to tumour neoantigen: prediction
Polypeptide and MHCI affinity, at the same be aided with proteasomal cleavage sites prediction, TAP transhipment prediction and structure-based MHC with
Polypeptide docking test, and scored by molecular dynamics simulation and carry out molecular dynamics between highest docking conformation and MHCI
Simulation；Tumour neoantigen after being screened, facilitate it is subsequent further targetedly tested accordingly, can greatly subtract
The number tested less realizes time saving, laborsaving and reduction of expenditure.
Detailed description of the invention
Fig. 1 is a kind of flow chart of the screening technique of tumour neoantigen provided by the embodiments of the present application；
Fig. 2 is the schematic diagram of the several docking conformations generated after the polypeptide of the embodiment of the present application is docked with MHCI；
Fig. 3 is core polypeptide VLAKKLKFV and MHCI (specially HLA-A*0201) flexible docking of the embodiment of the present application
The schematic diagram of the docking conformation of highest scoring afterwards；
Fig. 4 is that the core polypeptide VLAKKLKFV and MHCI (specially HLA-A*0201) of the embodiment of the present application meet object point
The schematic diagram of conformation after subdynamics simulation；
Fig. 5 is Mutated residues Lys4 and MHCI (specially HLA- in the core polypeptide VLAKKLKFV of the embodiment of the present application
A*0201) engagement groove combination image schematic diagram；
Fig. 6 is the molecular docking marking result of 4 polypeptides designed in the embodiment of the present application；
Fig. 7 is the molecular docking conformation of 4 polypeptides and HLA-A*0201 designed in the embodiment of the present application；
Fig. 8 is 4 polypeptides designed in the application the present embodiment parent with the T2 cell of HLA-A*0201 in an experiment
With power experimental result.
Specific embodiment
The application is illustrated below in conjunction with the attached drawing in the embodiment of the present application.
Embodiment one
As described in Figure 1, the present embodiment provides a kind of screening techniques of tumour neoantigen, comprising steps of
S101 obtains the corresponding polypeptide sequence encoded of mutated gene of the tumor tissue cell of patient.Specifically, packet
It includes:
A, the DNA of tumor tissue cell is extracted by SDS method, and DNA sequencing is carried out to it；
B, the DNA sequence dna after sequencing is compared with the histiocytic DNA sequence dna of normal wild type, obtain with
The DNA sequence dna of the different mutation of the histiocytic DNA sequence dna of normal wild type.Wherein, normally this is histiocytic
DNA sequence dna acquisition modes can be by obtaining in existing database.Wherein, the database may is that COSMIC,
NCBI, UCSC, Ensembl, TCGA etc..
C, the polypeptide sequence of the corresponding coding of the DNA sequence dna for the mutation being obtained by biological software.Wherein, the biology
Learning software can be DNA-man, be also possible to other softwares that DNA sequence dna can be translated into amino acid sequence.Wherein, described
Polypeptide sequence are as follows: the polypeptide sequence at least containing 8-30 amino acid residue.
The present embodiment of the application is by taking two peptide fragments difference that the following two kinds tumor tissue cell obtains as an example:
>s1_ACPP_E34K
DRSVLAKKLKFVTLVFRHGDRSPID
>s2_MECOM_Q216K
EDSDKLFESKAELADHQKF
The polypeptide sequence is carried out affinity prediction with major histocompatibility complex MHCI respectively, and sieved by S102
Select the polypeptide sequence that affinity is more than specified threshold.
Specifically, the affinity of polypeptide and MHCI ensure that polypeptide MHCI compound can be smooth by T cell surface receptor TCR
Ground identification, to activate T cell, causes relevant cell immune response.Therefore prediction polypeptide and MHCI affinity are particularly significant,
Whether the affine degree of the polypeptide and MHCI are for that successfully can be used as tumour neoantigen vaccine very crucial the polypeptide.
The source MHCI of the application is HLA-A2402, HLA-A0201, HLA-B1501, tetra- heterozygosis MHCI of HLA-B4402
Type.It is carried out with two polypeptides in step S101 based on artificial neural network (wherein, the affinity with to it respectively
Prediction technique is including but not limited to: artificial neural network, deep learning, support vector machines, specific score matrix, 3D-QSAR
The prediction technique of (ComaFA, CoMSIA), Hidden Markov Model and other knowledge based libraries) affinity prediction:
As a result as follows:
Table one: sequence D RSVLAKKLKFVTLVFRHGDRSPID and HLA-A2402 affinity prediction result.
Table two: sequence D RSVLAKKLKFVTLVFRHGDRSPID and HLA-A0201 affinity prediction result.
Table three: sequence D RSVLAKKLKFVTLVFRHGDRSPID and HLA-B1501 affinity prediction result.
Table four: sequence D RSVLAKKLKFVTLVFRHGDRSPID and HLA-B4402 affinity prediction result.
Table five: sequence EDSDKLFESKAELADHQKF and HLA-A2402 affinity prediction result.
Table six: sequence EDSDKLFESKAELADHQKF and HLA-A0201 affinity prediction result.
Table seven: sequence EDSDKLFESKAELADHQKF and HLA-B1501 affinity prediction result.
Table eight: sequence EDSDKLFESKAELADHQKF and HLA-B4402 affinity prediction result.
By finding that the affinity of VLAKKLKFV_ACPP and HLA-A0201 is 59.32Nm to interpretation of result as above；
The affinity of KLFESKAEL_MECOM and HLA-A0201 is 19.05Nm；KLKFVTLVF_ACPP is with HLA-A1501 affinity
13.34Nm；AELADHQKF_MECOM and HLA-B4402 affinity are 15.25Nm.The above polypeptide is affine with related MHC I type
Power is excellent.In the case study on implementation we use≤affinity of 500Nm to be subjected to predicted value, 0~150Nm is strong affinity.
HLA-A0201 parting is distributed extremely wide in asian population, therefore our selective affinities are more than the polypeptide sequence of specified threshold
9 peptide of core that VLAKKLKFV_ACPP is DRSVLAKKLKFVTLVFRHGDRSPID is arranged, KLFESKAEL_MECOM is
9 peptide of core of EDSDKLFESKAELADHQKF.
Polypeptide sequence in S101, is carried out the prediction of polypeptide restriction enzyme site by S103, and retains restriction enzyme site not in step
The affinity obtained in S102 be more than it is among the polypeptide sequence of specified threshold, can independent polypeptide sequence, as swollen
The candidate polypeptide sequence of tumor neoantigen.
Specifically, the prediction of polypeptide restriction enzyme site can reflect whether polypeptide is easy to be cut and be presented on MHC, polypeptide
The prediction of restriction enzyme site can assist whether prediction core polypeptide can correctly be cut.It is pre- with prediction multienzyme shear ability
Method of determining and calculating is mutually mixed, can shearing site of the Accurate Prediction polypeptide in albumen, thus assist prediction neoantigen polypeptide at antigen
Potentiality.> s1_ACPP_E34K is predicted respectively by the modes such as artificial neural network or correlation regression analysis:
DRSVLAKKLKFVTLVFRHGDRSPID and > s2_MECOM_Q216K:EDSDKLFESKAELADHQKF are in albumen multienzyme
Cleavage site.
As a result as follows:
Table nine: sequence D RSVLAKKLKFVTLVFRHGDRSPID multienzyme cleavage site prediction.
By predicting to find to sequence D RSVLAKKLKFVTLVFRHGDRSPID multienzyme cleavage site, core polypeptide
The both ends of VLAKKLKFV can accurately be cut in multienzyme.
Table ten: sequence EDSDKLFESKAELADHQKF multienzyme cleavage site prediction.By right
The prediction discovery of EDSDKLFESKAELADHQKF multienzyme cleavage site, discovery is for EDSDKLFESKAELADHQKF at the 9th
On have a strong restriction enzyme site, be difficult core polypeptide as KLFESKAEL occur in body state, therefore the polypeptide is filtered
Fall.And VLAKKLKFV can be retained independently at peptide fragment, the candidate polypeptide sequence as tumour neoantigen.
The candidate polypeptide sequence (core polypeptide VLAKKLKFV) transporter related to antigen processing is carried out parent by S104
It is predicted with power, and shows core polypeptide sequence of the affinity within specified range as tumour neoantigen prediction result.
Specifically, the affinity of polypeptide transporter related to antigen processing reflect polypeptide can be smooth in presentation
Carry out, thus preferable affinity can guarantee to a certain extent polypeptide can by successfully submission to MHCI molecule, therefore its
It can also be predicted as supplementary means when polypeptide is predicted with MHCI affinity size.Based on Cascade SVM
Algorithm made of method training, for predicting the affinity of polypeptide transporter related to antigen processing, with " jack-knife " method
Verifying has obtained 88% correlation.
11 core polypeptide VLAKKLKFV of table transporter affinity prediction related to antigen processing
It is found with polypeptide affinity analysis, two polypeptides show medium affinity with TAP, in the SOP in our settings
It can be used as potential candidate polypeptide Deng the polypeptide with high affinity.Have by prediction discovery core polypeptide VLAKKLKFV higher
Antigen process related transporter affinity.
S105, by the candidate polypeptide sequence (core polypeptide VLAKKLKFV) and major histocompatibility complex MHCI
The docking test in structure is carried out, and the structure of the docking conformation of the compound of generation is passed through to the experience scoring functions of free energy
It scores, and retains the highest docking conformation of scoring.
Specifically, structure-based polypeptide is phase between research polypeptide ligand and receptor biological macromolecular with MHCI molecular docking
Interaction rule, predicts a kind of effective means of its binding pattern and affinity.Here the purpose docked be in order to obtain it is reliable,
Reasonable epitope polypeptide conformation in conjunction with MHCI (referring specifically to HLA-A*0201 herein) molecule.Wherein, structure-based MHCI with
Polypeptide joint mode, method is including but not limited to the distribution of peptide-binding groove electrostatic, energy match, spatial match, grid computing, segment
Growth, simulated annealing, genetic algorithm etc.；Joint mode classification includes rigidity docking, docking semi-flexible and flexible docking.To taking over
Cheng Zhong, according to geometry, complementary, energy complement and the principle of chemical environment complementation come Real-Time Evaluation epitope polypeptide molecule and MHCI
The quality of intermolecular interaction, and find the best combination mode of the two.It is commented again with the experience scoring functions of Conjugated free energy
Point, the size of quantitative estimation epitope polypeptide and MHCI molecule affinity.
We carry out flexible docking by Rosetta docking procedure, to polypeptide and MHCI, generate 200 docking conformations.Such as
It is the several schematic diagrames for docking conformation generated after the polypeptide of the embodiment of the present application carries out flexible docking with MHCI shown in Fig. 2.
And the structure of the docking conformation of the compound of generation is scored by the experience scoring functions of free energy, it is right
It takes and point is as follows:
| total_score | rm sBB | descrip tion |
| -574.774 | 13.556 | top_1.pdb |
| -574.001 | 9.972 | top_2.pdb |
| -573.248 | 2.625 | top_3.pdb |
| -572.879 | 2.534 | top_4.pdb |
| -572.448 | 1.461 | top_5.pdb |
| -572.329 | 4.19 | top_6.pdb |
| -572.216 | 1.742 | top_7.pdb |
| -572.139 | 1.804 | top_8.pdb |
| -572.052 | 6.753 | top_9.pdb |
| -571.849 | 2.073 | top_10.pdb |
12 core polypeptide VLAKKLKFV of table transporter affinity prediction related to antigen processing
As shown in figure 3, we select the highest docking conformation that scores, as subsequent analysis conformation.
S106 passes through the highest docking conformation of scoring and major histocompatibility complex described in molecular dynamics simulation
Interaction and motion change between MHCI；And analysis obtains the candidate polypeptide sequence (core polypeptide accordingly
VLAKKLKFV it) is formed with the sequence of the junction of major histocompatibility complex MHCI.
Specifically, molecular dynamics simulation is the phase interaction for simulating macromolecular and polypeptide according to the basic principle of Newtonian mechanics
With with motion change, carry out the rule for the biological phenomena behind that inquiry experiment means can't resolve.We pass through molecular dynamics mould
Quasi- means inquire into the action rule and motion change between MHCI and polypeptide complex, can intuitively embody more under stable state
Interaction and affinity between peptide and MHCI, can accurately predict whether polypeptide can steadily be combined with MHCI.Wherein,
The molecular dynamics simulation of MHCI and polypeptide complex, method is including but not limited to NAMD, Amber, Gromacs etc..We into
The molecular dynamics simulation of row 10ps, as a result as follows:
Table 13: molecular dynamics simulation result
As shown in figure 4, after molecular dynamics simulation between core polypeptide VLAKKLKFV and MHCI (HLA-A*0201)
Conformation.Found by the further analysis to molecular dynamics result: the anchor residues that two sections of core polypeptide respectively with
Glu63, Lys66, Tyr171, Trp123 and Lys146 of HLA-A*0201 etc. are combined closely with hydrogen bond etc..
S107, when judging that the candidate polypeptide sequence (core polypeptide VLAKKLKFV) and the ajor histocompatibility are multiple
The amino acid of the fit junction MHCI is mutating acid, and when judge that the mutating acid and MHCI are tightly combined, by institute
State the sequence of candidate polypeptide sequence tumour neoantigen as a filter.
Specifically, polypeptide VLAKKLKFV wild-type sequence is VLAEKLKFV, it is Glu4Lys mutation, and our molecule
Close combination has occurred in the engagement groove of dynamics simulation Lys4 and HLA-A*0201 as the result is shown.It finds based on the above results
VLAKKLKFV can have extremely strong affinity with HLA-A*0201.
In order to be better described the application screening technique acquisition polypeptide sequence affinity effect, the application also into
Go following test:
One, docking for polypeptide set and HLA-A*0201 molecule is carried out using molecular docking programs ZDOCK, to polypeptide and HLA
Affinity carries out qualitative analysis:
It is as follows to design peptide molecule:
| Serial number | Polypeptide title | Polypeptide sequence | netMHC4.0(nM) |
| 1 | positive ctrl | NH2-YLLPAIVHI-CONH2 | 4.32 |
| 2 | negative ctrl | NH2-LGKRGSKPK-CONH2 | 43819.37 |
| 3 | core-pep1 | NH2-VLAKKLKFV-CONH2 | 59.32 |
| 4 | core-pep2 | NH2-KLFESKAEL-CONH2 | 119.05 |
Table 14: the polypeptide sequence of design
Peptide molecule small data set is constructed according to above-mentioned polypeptide sequence, carries out receptor HLA-A*0201 modeling and combination
The determination in region, and carry out docking for receptor HLA-A*0201 and peptide molecule small data set, finally, relatively and analyzing respectively
The docking score of a polypeptide sequence.
As shown in fig. 6, for the molecular docking marking result of 4 polypeptides designed in the embodiment of the present application；It gives a mark higher,
The affinity for representing polypeptide and corresponding MHCI is higher.It is found by analysis, what the screening technique above-mentioned of the application screened
The docking score of the polypeptide sequence of serial number 3 be it is highest, followed by serial number 1 control polypeptide sequence (sequence be it is existing
The good polypeptide sequence of known affinity), be the polypeptide sequence of serial number 4 again, 1 is minimum.And the result be based on
The software netMHC4.0 software prediction of knowledge base it is almost the same (NetMHC4.0 software prediction be polypeptide and HLA molecule parent
With the numerical value of power, unit Nm, numerical value is lower, and affinity is stronger)
Two, polypeptide affinity detects
1, T2 cell culture: T2 cell is purchased from ATCC, with 20%FBS IMDM (Gibco) complete medium culture；
2, the polypeptide sequence predicted uses synthesis in solid state, and Purity >=95% freezes after being dissolved with DMSO and protects in -80 DEG C
It deposits；
3, following raw material: T2 cell, 1X10^6cells/well is added in 24 orifice plates；Natural human β-2microglobulin
(Prospec), final concentration of 0.5 μM；Final concentration gradient is arranged in every polypeptide are as follows: 2.5 μM, 5 μM, 10 μM, 20 μM, 40 μM, 80 μ
M is separately added into 24 orifice plates, is incubated for 16h altogether in 37 DEG C of 5%CO2 incubators.Experimental setup blank group and control group (are not added
Polypeptide)；
4, cell is transferred in 1.5ml centrifuge tube, is cleaned 2 times with 1ml 1XPBS, abandon supernatant；
5, FITC Mouse Anti-Human HLA-A2 (BD Biosciences, Oxford, U.K.) is added, 4 DEG C are kept away
Light is incubated for 1h；
6, it is cleaned 2 times with 1ml 1XPBS, abandons supernatant；
7, cell is resuspended with 500 μ L 1XPBS, and is transferred in flow cytometer showed pipe；
8, (BD Biosciences) is detected with flow type analyzer；
9, testing result is analyzed using Flow-Jo and GraphPad Prism；
10, testing result is indicated with fluorescence index (FI) value, FI=MFI sample/MFI background.
As a result as shown in figure 8, as seen from Figure 8, the highest of result 3 and 1 of affinity, followed by 4 and 2, the result with
Molecular docking fractional result in Fig. 6 is almost the same.
In conclusion the screening technique of tumour neoantigen provided by the present application, passes through the screening to tumour neoantigen: prediction
Polypeptide and MHCI affinity, at the same be aided with proteasomal cleavage sites prediction, TAP transhipment prediction and structure-based MHC with
Polypeptide docking test, and scored by molecular dynamics simulation and carry out molecular dynamics between highest docking conformation and MHCI
Simulation；Tumour neoantigen after being screened, is further targetedly tested accordingly, can greatly reduce experiment
Number realizes time saving, laborsaving and reduction of expenditure.
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all in spirit of the invention and
Within principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (8)
1. a kind of screening technique of tumour neoantigen, which is characterized in that comprising steps of
A, the corresponding polypeptide sequence encoded of mutated gene of tumor tissue cell is obtained；
B, the polypeptide sequence is subjected to affinity prediction with major histocompatibility complex MHCI respectively, and filtered out affine
Power is more than the polypeptide sequence of specified threshold；
C, the polypeptide sequence of coding corresponding to the mutated gene is carried out to the prediction of polypeptide restriction enzyme site, and retains restriction enzyme site
Not the affinity be more than specified threshold polypeptide sequence among, can not be digested can be independently at the polypeptide sequence of peptide fragment
Column, as the candidate polypeptide sequence of tumour neoantigen.
2. the method according to claim 1, wherein tumour neoantigen described in step C further include: the tumor group
It knits, the tumor tissues GAP-associated protein GAP, the mutant DNA sequences of the tumor tissue cell or mutant rna sequence.
3. the method according to claim 1, wherein after the step C further include:
D, candidate polypeptide sequence transporter related to antigen processing is subjected to affinity prediction, and prediction result is shown into parent
With candidate polypeptide sequence of the power within specified range as tumour neoantigen.
4. according to the method described in claim 3, it is characterized in that, after the step C or D further include:
E, the candidate polypeptide sequence is subjected to the test of docking in structure with major histocompatibility complex MHCI, and will produced
The structure of the docking conformation of raw compound is scored by the experience scoring functions of free energy, and it is highest right to retain scoring
Connect conformation.
5. according to the method described in claim 4, it is characterized in that, after the step E, further includes:
F, by between the highest docking conformation of scoring and major histocompatibility complex MHCI described in molecular dynamics simulation
Interaction and motion change；And analysis obtains the knot of candidate polypeptide sequence and major histocompatibility complex MHCI accordingly
Sequence composition at conjunction.
6. according to the method described in claim 5, it is characterized in that, after the step F, further includes:
G, when judge the junction sequence form in there are mutating acids, and judge the mutating acid with mainly
When histocompatibility complex MHCI is tightly combined；By the sequence of candidate polypeptide sequence tumour neoantigen as a filter.
7. the method according to claim 1, wherein the step A, comprising:
A1, the DNA for extracting tumor tissue cell, and DNA sequencing is carried out to it；
A2, the DNA sequence dna after sequencing is compared with the histiocytic DNA sequence dna of normal wild type, obtains mutation
DNA sequence dna；
The polypeptide sequence of the corresponding coding of A3, the DNA sequence dna that the mutation is obtained by biological software.
8. the method according to claim 1, wherein polypeptide sequence described in step A are as follows: contain 8-30 amino acid
The polypeptide sequence of residue.
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109706065A (en) * | 2018-12-29 | 2019-05-03 | 深圳裕策生物科技有限公司 | Tumor neogenetic antigen load detection device and storage medium |
| CN110257478A (en) * | 2019-06-20 | 2019-09-20 | 杭州师范大学 | A kind of rapid screening method of effective neoantigen peptide of tumour individuation vaccine |
| CN110433285A (en) * | 2019-06-20 | 2019-11-12 | 杭州师范大学 | A kind of individuation knubble antigen peptide vaccine and preparation method thereof |
| CN110499324A (en) * | 2019-09-02 | 2019-11-26 | 中生康元生物科技(北京)有限公司 | A method of for identifying the bacterial expression vector and screening and identification tumour neoantigen of tumour neoantigen |
| CN110675913A (en) * | 2019-01-16 | 2020-01-10 | 倍而达药业（苏州）有限公司 | Screening method of tumor neoantigen based on HLA typing and structure |
| CN111105843A (en) * | 2019-12-31 | 2020-05-05 | 杭州纽安津生物科技有限公司 | HLA type I molecule and polypeptide affinity prediction method |
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| WO2020187143A1 (en) * | 2019-03-15 | 2020-09-24 | 痕准生物科技有限公司 | Method for identifying neoantigens |
| CN111798919A (en) * | 2020-06-24 | 2020-10-20 | 上海交通大学 | Tumor neoantigen prediction method, prediction device and storage medium |
| CN113292642A (en) * | 2020-04-13 | 2021-08-24 | 倍而达药业（苏州）有限公司 | Third-generation epidermal growth factor receptor inhibitor drug-resistant mutation neoantigen and application thereof |
| CN113980899A (en) * | 2021-11-29 | 2022-01-28 | 杭州艾沐蒽生物科技有限公司 | Method for high-throughput screening of antigen-specific TCR |
| CN114446383A (en) * | 2022-01-24 | 2022-05-06 | 电子科技大学 | Quantum computation-based ligand-protein interaction prediction method |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103180730A (en) * | 2010-05-14 | 2013-06-26 | 综合医院公司 | Compositions and methods of identifying tumor specific neoantigens |
| CN106397574A (en) * | 2016-10-21 | 2017-02-15 | 上海交通大学医学院附属新华医院 | Antigen epitope peptide and application thereof |
| WO2017139694A1 (en) * | 2016-02-12 | 2017-08-17 | Nantomics, Llc | High-throughput identification of patient-specific neoepitopes as therapeutic targets for cancer immunotherapies |
| WO2017194170A1 (en) * | 2016-05-13 | 2017-11-16 | Biontech Rna Pharmaceuticals Gmbh | Methods for predicting the usefulness of proteins or protein fragments for immunotherapy |
| CN107704727A (en) * | 2017-11-03 | 2018-02-16 | 杭州风起智能科技有限公司 | Neoantigen Activity Prediction and sort method based on tumour neoantigen characteristic value |
-
2018
- 2018-08-06 CN CN201810885573.5A patent/CN109021062B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103180730A (en) * | 2010-05-14 | 2013-06-26 | 综合医院公司 | Compositions and methods of identifying tumor specific neoantigens |
| WO2017139694A1 (en) * | 2016-02-12 | 2017-08-17 | Nantomics, Llc | High-throughput identification of patient-specific neoepitopes as therapeutic targets for cancer immunotherapies |
| WO2017194170A1 (en) * | 2016-05-13 | 2017-11-16 | Biontech Rna Pharmaceuticals Gmbh | Methods for predicting the usefulness of proteins or protein fragments for immunotherapy |
| CN106397574A (en) * | 2016-10-21 | 2017-02-15 | 上海交通大学医学院附属新华医院 | Antigen epitope peptide and application thereof |
| CN107704727A (en) * | 2017-11-03 | 2018-02-16 | 杭州风起智能科技有限公司 | Neoantigen Activity Prediction and sort method based on tumour neoantigen characteristic value |
Non-Patent Citations (4)
| Title |
|---|
| MUHAMMAD USMAN MIRZA等: "Towards peptide vaccines against Zika virus: Immunoinformatics combined with molecular dynamics simulations to predict antigenic epitopes of Zika viral proteins", 《SCIENTIFIC REPORTS》 * |
| 尚伟主编: "《临床肿瘤生物免疫治疗》", 31 January 2006, 天津科学技术出版社 * |
| 林昊等主编: "《简明生物信息学》", 30 November 2014, 电子科技大学出版社 * |
| 樊代明主编: "《肿瘤研究前沿 第2卷》", 31 December 2002, 西安交通大学出版社 * |
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| CN110675913A (en) * | 2019-01-16 | 2020-01-10 | 倍而达药业（苏州）有限公司 | Screening method of tumor neoantigen based on HLA typing and structure |
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| CN110499324A (en) * | 2019-09-02 | 2019-11-26 | 中生康元生物科技(北京)有限公司 | A method of for identifying the bacterial expression vector and screening and identification tumour neoantigen of tumour neoantigen |
| CN111105843B (en) * | 2019-12-31 | 2023-07-21 | 杭州纽安津生物科技有限公司 | HLAI type molecule and polypeptide affinity prediction method |
| CN111105843A (en) * | 2019-12-31 | 2020-05-05 | 杭州纽安津生物科技有限公司 | HLA type I molecule and polypeptide affinity prediction method |
| CN113292642B (en) * | 2020-04-13 | 2023-02-28 | 倍而达药业（苏州）有限公司 | Third-generation epidermal growth factor receptor inhibitor drug-resistant mutation neoantigen and application thereof |
| CN113292642A (en) * | 2020-04-13 | 2021-08-24 | 倍而达药业（苏州）有限公司 | Third-generation epidermal growth factor receptor inhibitor drug-resistant mutation neoantigen and application thereof |
| CN111798919A (en) * | 2020-06-24 | 2020-10-20 | 上海交通大学 | Tumor neoantigen prediction method, prediction device and storage medium |
| CN113980899A (en) * | 2021-11-29 | 2022-01-28 | 杭州艾沐蒽生物科技有限公司 | Method for high-throughput screening of antigen-specific TCR |
| CN114446383A (en) * | 2022-01-24 | 2022-05-06 | 电子科技大学 | Quantum computation-based ligand-protein interaction prediction method |
| CN114446383B (en) * | 2022-01-24 | 2023-04-21 | 电子科技大学 | Quantum calculation-based ligand-protein interaction prediction method |
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