CN110499324A - A method of for identifying the bacterial expression vector and screening and identification tumour neoantigen of tumour neoantigen - Google Patents

Abstract

The method of the bacterial expression vector and screening and identification tumour neoantigen that the invention discloses a kind of for identifying tumour neoantigen, is related to lesion detection technical field.Expression vector can stablize expression tumour neoantigen, and including multiple insertion genetic fragments with specific function, being inserted into genetic fragment includes the first genetic fragment that can effectively improve tumour antigen submission efficiency and the second genetic fragment for effectively improving cloning efficiency.Additionally provide a kind of method that flux carries out the verifying of tumour neoantigen immunologic function and then quickly and effectively screens tumour neoantigen.

Description

A kind of bacterial expression vector and screening and identification tumour for identifying tumour neoantigen is new The method of antigen

Technical field

The present invention relates to lesion detection technical fields, in particular to a kind of for identifying the bacterium of tumour neoantigen The method of expression vector and screening and identification tumour neoantigen.

Background technique

Currently, cancer has been to be only second to the second largest cause of the death of the mankind of cardiovascular and cerebrovascular disease, morbidity and mortality by It is cumulative to add, serious burden is brought for people's health benefit and socio-economic development.The pathogenesis of all cancers is all come From the genomic dna sequence of cancer cell change caused by gene mutation or chromosomal variation (Stratton, M.R., 2009), so there are certain difficulty in treating cancer.The means for the treatment of cancer related generally to operation, radiotherapy, chemotherapy, Endocrine therapy and biological therapy etc., the wherein high recurrence rate of surgical resection therapy, and some cancers such as leukaemia are cannot Using operative treatment；And chemotherapy there are serious side effect and generates drug resistance.

Ready-made immunotherapy is a kind of attractive and prospect cancer treatment method, and there are mainly three types of types: immune Checkpoint inhibitor (immune checkpoint blockade), adoptive cell therapy (adoptive cell Therapy), tumor vaccine (cancer vaccine).

Immunologic test point inhibitor is exactly to use in human monoclonal antibody and immunologic test point repressor, such as cytotoxicity T lymphocyte GAP-associated protein GAP 4 (CTLA4), apoptosis albumen 1 (PD-1) and its ligand PDL1, mechanism inhibit The immune response of the T cell adjusting to tumour can be improved in these albumen but only small part patient can have this therapy There is response, and immune related adverse events can be generated.

Clinically especially blood class cancer achieves adoptive cell therapy such as Chimeric antigen receptor T cell (CAR-T) Significant clinical effectiveness, so U.S.'s food and drug security bureau examine the B cell by being directed to acute lymphoblastic leukemia The CAR-T cell therapy of anti-CD19, although this method is in solid tumor due to lacking surface antigen (Klebanoff, 2016), it is difficult to It invades solid tumor and inhibits the tumor microenvironment of immune response still in the exploratory stage；The antigen used high table in tumour cell It reaches, and more or less has low expression with specific organization's organ in normal cell, this will lead to serious targeting, non-tumour poison Property, but the attention rate of the therapy increasingly rises and will become a kind of promising tumor therapy of tool.

Tumor vaccine (cancer vaccine) is immunoreacted using tumour specific antigen stimulation tumour cell.In tumour There are two kinds of tumour antigens in cell: tumor associated antigen (tumor associated antigens, TAAs) and tomour specific Property antigen (tumor specific antigens).Tumor associated antigen refers to the albumen over-expressed in tumour cell, and Also there are expression, such as epithelical cell growth factor HER2, the reverse transcriptase of telomere TERT of tumour high-expression in normal cell Deng still, these antigens are there are a degree of central tolerance and lack specificity completely, it is equally possible to generate targeting, non-swollen The danger of tumor toxicity.

Tumour specific antigen refer to only it is special in tumour cell, in the albuminoid that normal cell is not expressed, wherein Including oncogenic virus antigen (Oncogenic viral antigens) and neoantigen (neoantigens).Oncogenic virus antigen It is the tumour for having viral DNA insertion genome and generating.Neoantigen is the expression product that mutation is generated by body cell DNA, is had The special sum of high tumor is easy to cause to be immunoreacted, and will not generate targeting, non-tumor toxicity.In general, if these Abnormal Eggss White (tumour cell or APCs) in the cell is degraded to the small peptide section with epitope, small peptide section and MHC-I class or MHC- II class high affinity molecule combines, and is presented to cell surface with composite form, is identified by T cell, causes T cell activation, And then tumour cell is attacked and is removed by effector T cell.It is still to be solved for how identifying and verify the validity of tumour neoantigen Problem.

In consideration of it, the present invention is specifically proposed.

Summary of the invention

The purpose of the present invention is to provide a kind of for identifying the bacterial expression vector of tumour neoantigen.The bacterial expression carries Body can stablize expression tumour neoantigen, promote cloning efficiency, improve tumour neoantigen submission efficiency.

Another object of the present invention is to provide a kind of methods of screening and identification tumour neoantigen.This method passes through building energy It is enough to stablize expression candidate tumor neoantigen, and promote the carrier of antigen presenting cell present antigen efficiency, it realizes quick in vitro Effective identification has the tumour neoantigen of immunologic function.

The present invention is implemented as follows:

A kind of includes multiple insertion genetic fragments on carrier for identifying the bacterial expression vector of tumour neoantigen, multiple Being inserted into gene includes the first gene hly segment and the second gene ccdB segment；The protein sequence of first gene hly fragment coding is such as Shown in SEQ ID NO.1, the sequence of the albumen of the second gene ccdB fragment coding is as shown in SEQ ID NO.2；

Preferably, the nucleic acid sequence of the first gene hly segment is as shown in SEQ ID NO.3；

Preferably, the nucleic acid sequence of the second gene ccdB segment is as shown in SEQ ID NO.4.

Further, the first gene is hly gene, and hly gene is cloned from Liszt's strain, or is closed using gene At method directly synthesize acquisition, MHC I type tumor antigen presentation efficiency can be dramatically increased.Principle is: hly gene can be with Express Listeria haemolysis fibroin (LLO), antigen presenting cell phagocytosis expression hly gene bacterium after, haemolysis fibroin into Enter in antigen presenting cell, destroy the lysosome membrane of antigen presenting cell, helps the intracorporal Antigenic Peptide escape of lyase, leave lyase The Antigenic Peptide of body is spread in cytoplasm, and then enters MHC I type antigen presentation system, realizes antigen submission.

Second gene is ccdB gene, and ccdB gene encoding bacterial toxic protein CcdB, CcdB albumen can make DNA gyrase Complex poisoning, and inhibit the activity of gyrase, to inhibit DNA replication dna, make cytotoxic.The present invention is gene constructed by ccdB Selected marker when converting on to expression vector as plasmid adds ccdB gene between two recombination site sequences of carrier, The expression product of ccdB gene can inhibit the growth of Bacillus coli communis, not have the carrier of incision or recirculation in clone It cannot grow in post-conversion, and the carrier being inserted into after gene (recombinant fragment) recombination can be grown in post-conversion, to greatly mention Cloning efficiency is risen.

In the present invention using in preferred embodiment, above-mentioned carrier is ZS91 carrier.ZS91 carrier is to be with T7 promoter System, the system can make almost all of cellular resources all be used for express express target protein, furthermore can be by controlling inductive condition The amount for controlling t7 rna polymerase, to control the expression quantity of product.

The nucleic acid sequence of carrier is as shown in SEQ ID NO.5.The carrier includes the first gene hly segment and the second gene CcdB segment.

A kind of method of screening and identification tumour neoantigen comprising two restriction enzyme sites of Nde1-Kpn1 are designed on carrier, The DNA sequence dna of candidate tumor neoantigen to be identified is interposed therebetween.

Further, hly genetic fragment is cloned from Liszt's strain, or direct using the method for gene chemical synthesis Synthesis obtains.The method of clone's hly genetic fragment includes that PCR method obtains hly segment, then the hly gene cloned is connected It is connected in ZS91 carrier to intermediate vector, then by digestion.

In the present invention using in preferred embodiment, above-mentioned building bacterial expression vector further includes in bacterial expression vector It is inserted into OVA antigen (ovalbumin) fragment sequence between two restriction enzyme sites of Nde1-Kpn1 and after being located at the second gene, will be inserted into Carrier after OVA segment converts bacterial cell, and inducing expression will drench after the bacterium conversion murine antigen presenting cell of induction with T Bar cell co-cultures, and detects the bacterium submission efficiency of OVA；

Preferably, OVA antigen sequence is OVA21-388 or 3 × OVA8；The DNA sequence dna of OVA21-388 such as SEQ ID Shown in NO.6, the DNA sequence dna of 3 × OVA8 is as shown in SEQ ID NO.7；The amino acid sequence of OVA21-388 such as SEQ ID NO.8 Shown, the amino acid sequence of 3 × OVA8 is as shown in SEQ ID NO.9；

Preferably, murine antigen presenting cell is mouse hybridoma cell DC2.4, and T lymphocyte is that Mouse Hybridoma Cells are thin Born of the same parents' B3Z cell；

Preferably, thin using Elispot detection B3Z using the secretion of cellular elements in ELISA method detection B3Z cell The secretion of extracellular molecule；

Preferably, using the secretion of IL-2 in ELISA method detection B3Z cell, using in Elispot detection B3Z cell The secretion of IFN-γ.

Further, lymphocyte can be from genital tract (for example, vagina tissue) or relevant lymphoid tissue), peritonaeum Chamber, spleen, thymus gland, lung, liver, kidney, nerve fiber, internal film tissue, cavum peritoneale, marrow or the separation of its hetero-organization obtain, and can be logical It crosses in the tissue of flushing or other modes removal and separates cell, be also possible to the methods of Beads enrichment or flow cytometry and carry out carefully The separation of born of the same parents.

The detection that the antigen presentation effect of bacterial expression vector may be implemented by being inserted into OVA antigen fragment, thus effectively Whether the bacterial expression vector for prejudging above-mentioned building can efficiently present neoantigen.It avoids the submission low efficiency of carrier itself and leads The problem of cause can not carry out the detection and identification of neoantigen, prevent carrier has an impact the timeliness of subsequent experimental.

In the present invention using in preferred embodiment, above-mentioned building bacterial expression vector further includes by bacterial expression vector With 32 antigen sequences for deriving from CEF are inserted into after Nde1-Kpn1 digestion, the carrier after the antigen sequence of CEF will be inserted into and be transferred to In bacterium and the DC cell of the bacterium conversion people of induction is detected and is inserted into the thin of the carrier after the antigen sequence of CEF by inducing expression Bacterium submission efficiency；Finally by the antigen presenting cell DC cell of the bacterium conversion people of induction, and co-cultured with T lymphocyte.

Preferably, using the secretion of IL-2 in ELISA method detection T cell, using IFN- in Elispot detection T cell The secretion of γ.

Preferably, the CEF is CMV, EBV and Influenza.

By being inserted into 32 antigen sequences for deriving from CEF on bacterial expression vector, it is ensured that the expression vector energy Immune response is generated to HLA- I, the antigen sequence of CEF is used as positive control in the present invention, can examine the thin of above-mentioned building Presentation effect of the bacterium expression vector in human body cell, avoid cannot achieve due to carrier itself neoantigen detection and Identification.

In the present invention using in preferred embodiment, the above method further includes the DNA sequence dna for synthesizing candidate tumor neoantigen, And be inserted between the Nde1-Kpn1 restriction enzyme site on bacterial expression vector with the candidate tumor neoantigen DNA sequence dna after synthesis, thin Inducing expression candidate tumor neoantigen in bacterium；

Preferably, the bacterium of inducing expression candidate tumor neoantigen is BL21.

It is existing after obtaining candidate tumor neoantigen DNA sequence dna, it is also necessary to the clone such as conventional, BP or TA, step are complicated Cumbersome, design time is long, at high cost, and experimental period is long.The present invention can by the DNA sequence dna of the candidate tumor neoantigen of synthesis and Expression vector carries out genetic recombination, realizes the expression of target gene, greatly shortens the test period.

In addition, the candidate tumor neoantigen DNA sequencing fragment that the prior art needs to synthesize is longer, the DNA sequence of about 300bp Column, it is subsequent identification candidate tumor neoantigen segment immunity, need simultaneously compared with not mutated WT, due to segment compared with It is long, higher cost.Segment is longer to be also unfavorable for constructing mini-gene.Since segment is longer, specifically which section can not be determined DNA sequence dna in the effect for playing antigen, and it is unpredictable for the mutation generated in the segment whether can be to it as candidate The function of tumour neoantigen has an impact.And the present invention passes through the core fragment of predicting candidate tumour neoantigen, realizes short antigen The synthesis of Peptide D NA sequence, and then transformation efficiency is improved, it reduces costs, shortens the test period.

In addition, prior art needs are double to turn cLLO and candidate tumor neoantigen expression of peptides, efficiency is lower.Specifically, existing Plasmid containing cLLO and candidate tumor neoantigen expression of peptides is transformed into bacterium by technology needs respectively, and it is thin then to infect DC Born of the same parents, there may be incompatible appearances between different plasmids, and are finally got the upper hand with a kind of plasmid for final result, and in competence It after cell receives heat shock, is not easy to predict and control into plasmid situation therein, and plasmid itself can compete nutrition with bacterium Substance, when wherein plasmid is excessive, bacteria growth is slow, is unfavorable for the expression of target gene.

Expressing cLLO and the method for candidate tumor neoantigen peptide simultaneously in the present invention is directly by LLO plasmid construction to ZS91 On carrier, so that only single plasmid is transformed into bacterium, efficiency is improved, and bacterium burden is reduced；Or preparation expression cLLO BL21 competent bacteria, by the plasmid conversion containing LLO as gone out stable impression according to resistance screening in BL21 competent bacteria Target gene, is then transformed into stable cell line by state cell.It in this way can stable expression cLLO and candidate tumor be new simultaneously Antigenic Peptide increases transformation efficiency.

It is applied in preferred embodiment in the present invention, it is above-mentioned also to be wrapped before the DNA sequence dna of synthesis candidate tumor neoantigen Include the DNA sequence dna prediction for carrying out candidate tumor neoantigen, the DNA sequence dna of predicting candidate tumour neoantigen include to tumor sample or Tissue carries out full exon sequencing and RNA sequencing, identifies the site of mutation and the expression of mutated gene, utilizes Bioinformatic methods The peptide fragment of mutation and the strongest peptide fragment of HLA molecule binding ability of patient are filtered out as candidate tumor neoantigen；

Preferably, Bioinformatic methods include NetMHC, NetCTLpan and IEDB.

Candidate tumor can be accurately synthesized by the DNA sequence dna of Bioinformatic methods predicting candidate tumour neoantigen The DNA sequence dna of neoantigen.In addition, in other embodiments, tune can also be inserted near the DNA sequence dna of candidate tumor neoantigen Gene is saved, to promote the transcription of candidate tumor neoantigen segment, is such as inserted into the gene of ciphering activation albumen, activator protein can be tied The neighbouring DNA sequence dna of promoter is closed, the binding ability of RNA polymerase and promoter sequence is improved, to enhance RNA polymerase Transcriptional activity；The adjusting molecule in translation process can be added, such as adjusts initiation codon to the distance of purine segment to change Translation efficiency；Epigenetics modification can be carried out on this basis, such as to repairing after the translation of the effective histone of DNA replication dna Decorations, methylation modification.By NetMHC, NetCTLpan and IEDB Bioinformatic methods combine the accuracy rate that prediction can be improved, Reduce the error and inaccuracy of single method prediction bring candidate tumor neoantigen quantity.

In the present invention using in preferred embodiment, the above method further includes candidate tumor neoantigen in antigen presenting cell In immunogenicity detection, the immunity detection in antigen presenting cell includes that measurement antigen presenting cell and inducing expression are candidate Activation of the detection antigen presenting cell to T lymphocyte after the bacterium of tumour neoantigen co-cultures；

Preferably, antigen presenting cell is that the antigen presenting cell DC2.4 or human antigen's presenting cells DC of animal origin are thin Born of the same parents；

Preferably, detect T lymphocyte Activation include detect T lymphocyte proliferation, the phosphorylation of receptor or Non-phosphorylating, the secretory volume variation of one or more immune mediators, the expression quantity variation of one or more immune mediators, Yi Zhonghuo The variation of various kinds of cell surface marker expression quantity harvests supernatant and measures the secretion of one or more polypeptides relevant to activation The variation of amount and expression quantity；Preferably, immune mediator is cell factor, any in soluble mediators and cell surface marker A combination of one or more；

Preferably, cell factor be selected from TRAIL, IFN-γ, IL-12p70, IL-2, TNF-α, MIP1- α, MIP1- β, CXCL9, CXCL10, MCP1, RANTES, IL-1 β, IL-4, IL-6, IL-8, IL-9, IL-10, IL-13, IL-15, CXCL11, IL-3, IL-5, IL-17, IL-18, IL-21, IL-22, IL-23A, IL-24, IL-27, IL-31, IL-32, TGF-β, CSF, GM Any one or more in CSF and TRANC；

Preferably, soluble mediators are selected from granzyme A, granzyme B, sFas, sFasL, in perforin and particle cytolysin Any one or more；

Preferably, cell surface marker is selected from CD107a, CD107b, CD25, CD69, CD45RA, CD45RO, CD137 (4-1BB), CD44, CD62L, CD27, CCR7, CD154 (CD40L), KLRG-1, CD71, HLA-DR, CD122 (IL-2RB), CD28, IL7Ra (CD127), CD38, CD26, CD134 (OX-40), CTLA-4 (CD152), LAG-3, TIM-3 (CD366), CD39, PD1 (CD279), FoxP3, TIGIT, CD160, BTLA, any one or more in 2B4 (CD244) and KLRG1；

Preferably, cell factor is IFN-γ and TNF-α；The horizontal method of cell factor is measured as Elispot analysis；

Preferably, the bacterium of inducing expression candidate tumor neoantigen includes with antigen presenting cell post activation T lymphocyte Following two co-cultivations step: the bacterium of DC cell and inducing expression candidate tumor neoantigen co-cultures, and DC cell and T lymph are thin Born of the same parents co-culture.

Further, it is possible to the cytokine secretion in culture supernatants be detected, such as by ELISA or Elispot to T The cell factor of lymphocyte release is detected.The feelings of T lymphocyte proliferation are determined by the methods of detection ' H thymidine incorporation Condition.Antigen presenting cell and T lymphocyte can come from identical individual, can be from different individuals.

Further, antigen presenting cell can selected from mucosal tissue (such as: nose, mouth, bronchial tissue, tracheal tissue, stomach Enteron aisle, genital tract (for example, vagina tissue) or associated lymphoid tissue), cavum peritoneale, lymph node, spleen, marrow, thymus gland, lung, liver, Kidney, nerve fiber, endocrine tissue or its hetero-organization, can be the chorista taken out or flushed out in surgical procedure, It can be antigen presenting cell (such as endothelial cell, fibroblast, epithelium and interstitial cell, acidophilia from non-full-time Granulocyte, neuron/Deiter's cells).

Further, the ratio of antigen presenting cell and bacterium can be 1:1, and bacterium and antigen presenting cell are at 0-40 DEG C It is mixed -5 hours 45 minutes, after mixed culture, needs to be collected by centrifugation cell, and wash not by antigen presenting cell The bacterium of internalization.Using antibiotic or not antibiotic culture medium resuspension cell is had, it can contacted with lymphocyte Preceding fixation and kill antigen presenting cell.

Preferably, selection handles cell with paraformaldehyde.

Further, the antigen presenting cell of T lymphocyte and Antigen co-cultures under the conditions of 37 DEG C, and the time is 72 small When, the ratio of T lymphocyte and antigen presenting cell can be 1:1, cultivate in 96 holes, the number of antigen presenting cell in every hole Amount can be 1 × 10⁴-1×10⁶, the quantity of T lymphocyte can be 1 × 10⁴-1×10⁶。

In the present invention using in preferred embodiment, the above method further includes being waited using the epitope peptide verifying of tumour neoantigen The function of tumour neoantigen is selected, the method for the epitope peptide functional verification of tumour neoantigen includes external synthesis candidate tumor neoantigen Then small peptide, Antigen presenting cell are co-cultured with T lymphocyte, the cell factors such as detection T lymphocyte secretion of gamma-IFN Level.

In the present invention using in preferred embodiment, above-mentioned verifying candidate tumor neoantigen function further includes MHC class I HLA Tetramer verifying, the method for MHC class I HLA Tetramer verifying include synthesis candidate tumor neoantigen table Position peptide synthesizes MHC/peptide complex with kit, co-cultures with T lymphocyte, with flow cytomery MHC/ The binding ability of peptide complex and T lymphocyte, to identify the function of candidate tumor neoantigen.

The invention has the following advantages:

The present invention is a kind of for identifying the bacterial expression vector of tumour neoantigen by providing.The bacterial expression vector passes through Be inserted into the first genetic fragment can in subsequent expression Listeria hemolysin LLO albumen, hemolysin can in Escherichia coli with Antigen coexpression, is then swallowed by antigen presenting cell, increases antigen presentation efficiency.It can be with table by the second genetic fragment of insertion Up to ccdB albumen, ccdB albumen can inhibit the growth of Bacillus coli communis, not have the carrier for cutting ccdB converting in clone After cannot grow, and the carrier being inserted into after gene (recombinant fragment) recombination can be grown in post-conversion, thus significant increase gram Grand efficiency.

The present invention also provides a kind of methods of screening and identification tumour neoantigen.This method is by building for stablizing expression The bacterial expression vector of tumour neoantigen may be implemented quickly and effectively to identify tumour neoantigen.

Detailed description of the invention

In order to illustrate the technical solution of the embodiments of the present invention more clearly, below will be to needed in the embodiment attached Figure is briefly described, it should be understood that the following drawings illustrates only certain embodiments of the present invention, therefore is not construed as pair The restriction of range for those of ordinary skill in the art without creative efforts, can also be according to this A little attached drawings obtain other relevant attached drawings.

Fig. 1 is the ZS91-hly-ccdB carrier figure that embodiment 1 constructs；

Fig. 2 is the carrier figure of ZS91-hly-3OVA in embodiment 2；

Fig. 3 is the result figure for the secretion that ELISA detects B3Z cell IL-2 in embodiment 2；

Fig. 4 is submission effect picture of the OVA small fragment of embodiment 3 in bacterial system；

Fig. 5 is the expression LLO carrier figure in experimental example 1；

Fig. 6 converts the figure of bacterial growth after bacterium for the ZS91-hly carrier and ZS91-hly-ccdB carrier of experimental example 1；

Fig. 7 is locating effect figure of the ZS91-hly-mCherry in bacterial system；

Fig. 8 is that bacterial invasion mammal cell line A549, the GFP albumen of ZS91-hly-GFP conversion is determined in cell Position effect picture.

Specific embodiment

It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, according to normal conditions or manufacturer builds The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase Product.

Feature and performance of the invention are described in further detail with reference to embodiments.

Some researches show that Tumor mutations load (tumor mutation burden, TMB), i.e. Oncogenome remove embryo Somatic mutation quantity (somatic mutation) after system's mutation (germline mutation), with generation neoantigen Possibility correlation (Schumacher, 2015).And under effective antitumour immune response there are the special T cell of neoantigen and To present mutation small peptide tumour cell have cytotoxicity (Rajasagi, 2014；DuPage, 2012).The present invention utilizes Somatic mutation quantity after Oncogenome removal germline mutation predicts the type of neoantigen.By exempting to antitumor The special T cell progress immune detection of the lower neoantigen of epidemic disease reaction, can screen and identify neoantigen.

Embodiment 1

Present embodiments provide a kind of construction method of bacterial expression vector.The method of carrier construction specifically includes following step It is rapid:

(1) hly genetic fragment is cloned from Liszt's strain, and hly gene fragment clone is expressed among ZS91 load On body；

(2) it is inserted into ccdB genetic fragment on the ZS91 medial expression vector of step (1), increases clone's neoantigen DNA sequence The efficiency in ZS91 is arranged, the ZS91-hly-ccdB bacterial expression vector figure after building is shown referring to Fig.1, overall length 8242bp.

Embodiment 2

A kind of method for present embodiments providing bacterial expression vector examined and constructed in embodiment 1.It specifically includes as follows Step:

(1) 3 × OVA is synthesized₈(EQLESIINFEKLTEWQLESIINFEKLTEWQLESIINFEKL) and CEF sequence TEW Column.The amino acid sequence of CEF is as shown in SEQ ID NO.11, and the DNA sequence dna of CEF is referring to shown in SEQ ID NO.10.

(2) 3 × OVA8 is detected in the secretion of B3Z cell IL-2 using ELISA.

Firstly, carrying out inducing expression to 3 × OVA8.It will carry on clone ZS91-hly-3 × OVA8 of 3 × OVA8, ZS91 Empty carrier is as positive control, and the carrier figure of the ZS91-hly-3OVA8 after building is referring to shown in Fig. 2, overall length 5942bp.

The ZS91-hly-3OVA8 carrier built is transformed into competent bacteria BL21 again, monoclonal is chosen and shakes bacterium, bacterium Liquid is diluted in 100mL LB (Amp) with 1:100, final concentration 1mM IPTG is added, in 30 DEG C of inducing expressions；It is fixed with 1%PFA Bacterium 30min；It is cleaned with PBS, is resuspended in the PBS containing 30% glycerol, freezes spare.

Then the bacterium submission of OVA is carried out.DC2.4 single cell suspension kind plate is put into incubator culture in 96 orifice plates；It is added Ready bacterial antigens or OVA peptide, are fixed with or without PFA, 37 DEG C of 5%CO₂Incubator culture 20 hours.

Finally, the secretion of ELISA detection B3Z cell IL-2.Testing result is referring to shown in Fig. 3, from the figure 3, it may be seen that of the invention The carrier of the ZS91-hly-3OVA8 of offer can be carried out efficient tumour neoantigen submission and cause the significant work of T lymphocyte Change, achievees the effect that Antigen epitope peptide post activation T lymphocyte is the same directly on DC cell.

Embodiment 3

A kind of method for present embodiments providing screening and identification tumour neoantigen.Specifically comprise the following steps:

(1) discovery and prediction of clinical tumor sample neoantigen: tumor tissues are sequenced for full exon and RNA, identification The site of mutation and the expression of mutated gene predict neoantigen using NetMHC, NetCTLpan and IEDB, according to prediction result The DNA sequence dna of external synthesis candidate tumor neoantigen is simultaneously cloned into the ZS91 bacterial expression vector of the building of embodiment 1, is passed through The DNA sequence dna of the candidate tumor neoantigen of synthesis is introduced ZS91 bacterial expression vector by Nde I and Kpn I restriction enzyme.

(2) the inducing expression candidate tumor neoantigen in bacterium BL21 bacterial strain.

(3) secretion of Elispot detection T lymphocyte INF- γ is utilized.

3 × OVA8 building is transformed into bacterium competence BL21, with the expression of IPTG induction small peptide.Take DC2.4 cell Suspension is mixed with the bacterial suspension induced, 4 DEG C of placement 1h；Elispot plate is added after mixing after being cleaned with PBS with B3Z cell Middle co-cultivation 48h；4 DEG C of refrigerators are placed 10 minutes, hypotonic lysis cell；Throw away liquid in hole, every hole be added 260ul 1 × Washing buffer, discards liquid in hole after one minute, buckle on blotting paper dry；The biology that 100ul has diluted is added in every hole The antibody working solution (secondary antibody) of element label, 37 DEG C of deposited casees are interior to be incubated for 1 hour；Throw away liquid in hole, every hole be added 260ul 1 × Washing buffer, discards liquid in hole after one minute, buckle on blotting paper dry；The enzyme mark that 100ul has diluted is added in every hole Avidin working solution, 37 DEG C of deposited casees are interior to be incubated for 1 hour；Portal interior liquid, every hole addition 1 × washing of 260ul buffer, and 1 Liquid in hole is discarded after minute, is buckled on blotting paper dry；The AEC developing solution that 100ul now matches is added in every hole, and room temperature, which is protected from light, stands 5- 30 minutes, the color development stopping time is selected according to spot situation；Liquid in pouring aperture opens board bottom seat, by film front and back sides and pedestal Use ddH₂O is washed 3-5 times, color development stopping.Plate is placed in room temperature shady place naturally dry, after close pedestal；Record spot.

As a result referring to shown in Fig. 4, with the secretion situation of Elispot detection INF- γ, the results showed that 3 × OVA8 has tumour Neoantigen function.

(4) candidate tumor neoantigen and CEF are detected in PBMC cellular immunity.

Secure good health the blood of people, passes through Ficoll-Paque (Amersham Pharmacia, Uppsala, Sweden) It is unicellular that gradient centrifugation procedureization obtains peripheral blood.

The T lymphocyte for being separated CD3+ from peripheral blood using the human CD3Mcirobead of Miltenyi company, is pressed It is carried out according to kit specification.

By culture, the BL21 bacterium that inducing expression neoantigen is added co-cultures DCs, then presses DCs:T cell=1:60 T cell is added to be co-cultured in 96 holes or 384 hole PVDF ELISPOT plates, after 18-24h, measures IFN-γ and TNF-α.

Elispot analysis: 96 hole PVDF ELISPOT plates are coated with (8 μ g/ at 4 DEG C with antibody (IFN γ or TNF α) overnight ML), then plate is washed 4 times with PBS, with 37 DEG C of closing 2h of 1%BSA or skim milk.The DCs and T of bacterium will be loaded Cell is in CO₂37 DEG C of incubator co-culture in 18-24h in ELISPOT plate.Cell and culture medium in pouring aperture simultaneously use PBST Then 37 DEG C of incubations of biotin labeling detection antibody (such as anti-IFN- γ biotinylated mAb 7-B6-1) are added in washing 1-2h is washed with PBST, adds enzyme mark Avidin (such as streptavidin-HRP, PBS+0.5%BSA), 37 DEG C 1 hour. AEC developing solution is eventually adding in color development at room temperature 15-45min, ELISPOT plate is placed in the automatic plate reader of Biosys Bioreader Interior record spot number.

(5) accuracy for the candidate tumor neoantigen identified using small peptide verifying in bacterial system.It is used in the present embodiment The following two kinds method validation is conducive to the accuracy for promoting the candidate tumor neoantigen of identification, in other embodiments in this way It can be used alone MHC class I HLA Tetramer verifying.

One: MHC class I HLA Tetramer of method verifying: synthesis candidate tumor neoantigen small peptide first uses reagent Box (T-Select MHC Tetramer, MBL) synthesizes MHC/peptide complex, and identification candidate tumor neoantigen drenches T The binding ability of bar cell.

Experimental example 1

Go out hly genetic fragment from PCR in Listeria DNA in embodiment 1 to be inserted on ZS91 carrier, building expression LLO Carrier.Express LLO carrier referring to Figure 5.It is inserted into GATEWAY recombination site in T7 promoter downstream, and includes cloning site I-Kpn I of Nde, it may be achieved recombinant clone or conventional enzyme cutting clone；CcdB gene will be cloned into load in such a way that digestion connects On body, carrier figure is shown referring to Fig.1.

The ZS91-hly carrier of building and ZS91-hly-ccdB carrier are converted into bacterium respectively, the bacterial growth after conversion Figure is referring to shown in Fig. 6, and bacterium can not grow after being inserted into ccdB as shown in Figure 6, and not be inserted into the bacterium normal growth of ccdB, with reason It is consistent by result, further demonstrate the accuracy for the carrier that the present invention constructs.

Experimental example 2

The secretion effect of the IL-2 of each carrier in embodiment 2 is as shown in Figure 4.ZS91-hly-3 is constructed on bacteria carrier × OVA8, testing result show that 3 × OVA8 is co-cultured after loading DC2.4 with B3Z cell, can be normally carried out the secretion of IL-2, I.e. carrier system works normally, being capable of high efficient expression OVA antigen and to carry out DC cell delivery be in that DC cell is subsequent can be with effective stimulus The proliferation of T cell, to realize purpose of the system for the external functional verification of candidate tumor antigens.

Experimental example 3

Inducing expression ZS91-hly-mCherry in the carrier, mCherry by normal expression and can be published on bacterium In cytoplasm (referring to shown in Fig. 7).

The further locating effect of the bacterial invasion mammalian cell line cell A549 of research ZS91-hly-GFP conversion, As a result referring to Fig. 8, hly can help GFP albumen to leave lysosome as shown in Figure 8, be distributed in entire cell.

The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any to repair Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.

SEQUENCE LISTING

<110>raw Kang Yuan biotechnology (Beijing) Co., Ltd in

<120>a kind of method of bacterial expression vector and screening and identification tumour neoantigen for identifying tumour neoantigen

<160> 11

<170> PatentIn version 3.5

<210> 1

<211> 506

<212> PRT

<213>artificial sequence

<400> 1

Met Lys Asp Ala Ser Ala Phe Asn Lys Glu Asn Ser Ile Ser Ser Met

1 5 10 15

Ala Pro Pro Ala Ser Pro Pro Ala Ser Pro Lys Thr Pro Ile Glu Lys

20 25 30

Lys His Ala Asp Glu Ile Asp Lys Tyr Ile Gln Gly Leu Asp Tyr Asn

35 40 45

Lys Asn Asn Val Leu Val Tyr His Gly Asp Ala Val Thr Asn Val Pro

50 55 60

Pro Arg Lys Gly Tyr Lys Asp Gly Asn Glu Tyr Ile Val Val Glu Lys

65 70 75 80

Lys Lys Lys Ser Ile Asn Gln Asn Asn Ala Asp Ile Gln Val Val Asn

85 90 95

Ala Ile Ser Ser Leu Thr Tyr Pro Gly Ala Leu Val Lys Ala Asn Ser

100 105 110

Glu Leu Val Glu Asn Gln Pro Asp Val Leu Pro Val Lys Arg Asp Ser

115 120 125

Leu Thr Leu Ser Ile Asp Leu Pro Gly Met Thr Asn Gln Asp Asn Lys

130 135 140

Ile Val Val Lys Asn Ala Thr Lys Ser Asn Val Asn Asn Ala Val Asn

145 150 155 160

Thr Leu Val Glu Arg Trp Asn Glu Lys Tyr Ala Gln Ala Tyr Pro Asn

165 170 175

Val Ser Ala Lys Ile Asp Tyr Asp Asp Glu Met Ala Tyr Ser Glu Ser

180 185 190

Gln Leu Ile Ala Lys Phe Gly Thr Ala Phe Lys Ala Val Asn Asn Ser

195 200 205

Leu Asn Val Asn Phe Gly Ala Ile Ser Glu Gly Lys Met Gln Glu Glu

210 215 220

Val Ile Ser Phe Lys Gln Ile Tyr Tyr Asn Val Asn Val Asn Glu Pro

225 230 235 240

Thr Arg Pro Ser Arg Phe Phe Gly Lys Ala Val Thr Lys Glu Gln Leu

245 250 255

Gln Ala Leu Gly Val Asn Ala Glu Asn Pro Pro Ala Tyr Ile Ser Ser

260 265 270

Val Ala Tyr Gly Arg Gln Val Tyr Leu Lys Leu Ser Thr Asn Ser His

275 280 285

Ser Thr Lys Val Lys Ala Ala Phe Asp Ala Ala Val Ser Gly Lys Ser

290 295 300

Val Ser Gly Asp Val Glu Leu Thr Asn Ile Ile Lys Asn Ser Ser Phe

305 310 315 320

Lys Ala Val Ile Tyr Gly Gly Ser Ala Lys Asp Glu Val Gln Ile Ile

325 330 335

Asp Gly Asn Leu Gly Asp Leu Arg Asp Ile Leu Lys Lys Gly Ala Thr

340 345 350

Phe Asn Arg Glu Thr Pro Gly Val Pro Ile Ala Tyr Thr Thr Asn Phe

355 360 365

Leu Lys Asp Asn Glu Leu Ala Val Ile Lys Asn Asn Ser Glu Tyr Ile

370 375 380

Glu Thr Thr Ser Lys Ala Tyr Thr Asp Gly Lys Ile Asn Ile Asp His

385 390 395 400

Ser Gly Gly Tyr Val Ala Gln Phe Asn Ile Ser Trp Asp Glu Val Asn

405 410 415

Tyr Asp Pro Glu Gly Asn Glu Ile Val Gln His Lys Asn Trp Ser Glu

420 425 430

Asn Asn Lys Ser Lys Leu Ala His Phe Thr Ser Ser Ile Tyr Leu Pro

435 440 445

Gly Asn Ala Arg Asn Ile Asn Val Tyr Ala Lys Glu Cys Thr Gly Leu

450 455 460

Ala Trp Glu Trp Trp Arg Thr Val Ile Asp Asp Arg Asn Leu Pro Leu

465 470 475 480

Val Lys Asn Arg Asn Ile Ser Ile Trp Gly Thr Thr Leu Tyr Pro Lys

485 490 495

Tyr Ser Asn Lys Val Asp Asn Pro Ile Glu

500 505

<210> 2

<211> 101

<212> PRT

<213>artificial sequence

<400> 2

Met Gln Phe Lys Val Tyr Thr Tyr Lys Arg Glu Ser Arg Tyr Arg Leu

1 5 10 15

Phe Val Asp Val Gln Ser Asp Ile Ile Asp Thr Pro Gly Arg Arg Met

20 25 30

Val Ile Pro Leu Ala Ser Ala Arg Leu Leu Ser Asp Lys Val Ser Arg

35 40 45

Glu Leu Tyr Pro Val Val His Ile Gly Asp Glu Ser Trp Arg Met Met

50 55 60

Thr Thr Asp Met Ala Ser Val Pro Val Ser Val Ile Gly Glu Glu Val

65 70 75 80

Ala Asp Leu Ser His Arg Glu Asn Asp Ile Lys Asn Ala Ile Asn Leu

85 90 95

Met Phe Trp Gly Ile

100

<210> 3

<211> 1521

<212> DNA

<213>artificial sequence

<400> 3

atgaaggatg catctgcatt caataaagaa aattcaattt catccatggc accaccagca 60

tctccgcctg caagtcctaa gacgccaatc gaaaagaaac acgcggatga aatcgataag 120

tatatacaag gattggatta caataaaaac aatgtattag tataccacgg agatgcagtg 180

acaaatgtgc cgccaagaaa aggttacaaa gatggaaatg aatatattgt tgtggagaaa 240

aagaagaaat ccatcaatca aaataatgca gacattcaag ttgtgaatgc aatttcgagc 300

ctaacctatc caggtgctct cgtaaaagcg aattcggaat tagtagaaaa tcaaccagat 360

gttctccctg taaaacgtga ttcattaaca ctcagcattg atttgccagg tatgactaat 420

caagacaata aaatcgttgt aaaaaatgcc actaaatcaa acgttaacaa cgcagtaaat 480

acattagtgg aaagatggaa tgaaaaatat gctcaagctt atccaaatgt aagtgcaaaa 540

attgattatg atgacgaaat ggcttacagt gaatcacaat taattgcgaa atttggtaca 600

gcatttaaag ctgtaaataa tagcttgaat gtaaacttcg gcgcaatcag tgaagggaaa 660

atgcaagaag aagtcattag ttttaaacaa atttactata acgtgaatgt taatgaacct 720

acaagacctt ccagattttt cggcaaagct gttactaaag agcagttgca agcgcttgga 780

gtgaatgcag aaaatcctcc tgcatatatc tcaagtgtgg cgtatggccg tcaagtttat 840

ttgaaattat caactaattc ccatagtact aaagtaaaag ctgcttttga tgctgccgta 900

agcggaaaat ctgtctcagg tgatgtagaa ctaacaaata tcatcaaaaa ttcttccttc 960

aaagccgtaa tttacggagg ttccgcaaaa gatgaagttc aaatcatcga cggcaacctc 1020

ggagacttac gcgatatttt gaaaaaaggc gctactttta atcgagaaac accaggagtt 1080

cccattgctt atacaacaaa cttcctaaaa gacaatgaat tagctgttat taaaaacaac 1140

tcagaatata ttgaaacaac ttcaaaagct tatacagatg gaaaaattaa catcgatcac 1200

tctggaggat acgttgctca attcaacatt tcttgggatg aagtaaatta tgatcctgaa 1260

ggtaacgaaa ttgttcaaca taaaaactgg agcgaaaaca ataaaagcaa gctagctcat 1320

ttcacatcgt ccatctattt gccaggtaac gcgagaaata ttaatgttta cgctaaagaa 1380

tgcactggtt tagcttggga atggtggaga acggtaattg atgaccggaa cttaccactt 1440

gtgaaaaata gaaatatctc catctggggc accacgcttt atccgaaata tagtaataaa 1500

gtagataatc caatcgaata a 1521

<210> 4

<211> 306

<212> DNA

<213>artificial sequence

<400> 4

atgcagttta aggtttacac ctataaaaga gagagccgtt atcgtctgtt tgtggatgta 60

cagagtgata ttattgacac gcccgggcga cggatggtga tccccctggc cagtgcacgt 120

ctgctgtcag ataaagtctc ccgtgaactt tacccggtgg tgcatatcgg ggatgaaagc 180

tggcgcatga tgaccaccga tatggccagt gtgccggtct ccgttatcgg ggaagaagtg 240

gctgatctca gccaccgcga aaatgacatc aaaaacgcca ttaacctgat gttctgggga 300

atatag 306

<210> 5

<211> 8242

<212> DNA

<213>artificial sequence

<400> 5

tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60

cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120

ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180

gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240

acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300

ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360

ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420

acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480

tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540

tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat 600

tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 660

actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc 720

gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga 780

aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc 840

agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac 900

cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 960

aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat 1020

tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag 1080

tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca 1140

taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac 1200

ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg 1260

tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca 1320

tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac 1380

cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa 1440

cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 1500

gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1560

gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 1620

agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1680

aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 1740

agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 1800

cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 1860

accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 1920

aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 1980

ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 2040

cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 2100

gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttatt aatcagataa 2160

aatatttcta gatttcagtg caatttatct cttcaaatgt agcacctgaa gtcagcccca 2220

tacgatataa gttgtaattc tcatgtttga cagcttatca tcgataagct ttaatgcggt 2280

agtttatcac agttaaattg ctaacgcagt caggcaccgt gtatgaaatc taacaatgcg 2340

ctcatcgtca tcctcggcac cgtcaccctg gatgctgtag gcataggctt ggttatgccg 2400

gtactgccgg gcctcttgcg ggatatcaag gagatatacc atgaaggatg catctgcatt 2460

caataaagaa aattcaattt catccatggc accaccagca tctccgcctg caagtcctaa 2520

gacgccaatc gaaaagaaac acgcggatga aatcgataag tatatacaag gattggatta 2580

caataaaaac aatgtattag tataccacgg agatgcagtg acaaatgtgc cgccaagaaa 2640

aggttacaaa gatggaaatg aatatattgt tgtggagaaa aagaagaaat ccatcaatca 2700

aaatagtgca gacattcaag ttgtgaatgc aatttcgagc ctaacctatc caggtgctct 2760

cgtaaaagcg aattcggaat tagtagaaaa tcaaccagat gttctccctg taaaacgtga 2820

ttcattaaca ctcagcattg atttgccagg tatgactaat caagacaata aaatagttgt 2880

aaaaaatgcc actaaatcaa acgttaacaa cgcagtaaat acattagtgg aaagatggaa 2940

tgaaaaatat gctcaagctt atccaaatgt aagtgcaaaa attgattatg atgacaaaat 3000

ggcttacagt gaatcacaat taattgcgaa atttggtaca gcatttaaag ctgtaaataa 3060

tagcttgaat gtaaacttcg gcgcaatcag tgaagggaaa atgcaagaag aagtcattag 3120

ttttaaacaa atttactata acgtgaatgt taatgaacct acaagacctt ccagattttt 3180

cggcaaagct gttactaaag agcagttgca agcgcttgga gtgaatgcag aaaatcctcc 3240

tgcatatatc tcaagtgtgg cgtatggccg tcaagtttat ttgaaattat caactaattc 3300

ccatagtact aaagtaaaag ctgcttttga tgctgccgta agcggaaaat ctgtctcagg 3360

tgatgtagaa ctaacaaata tcatcaaaaa ttcttccttc aaagccgtaa tttacggagg 3420

ttccgcaaaa gatgaagttc aaatcatcga cggcaacctc ggagacttac gcgatatttt 3480

gaaaaaaggc gctactttta atcgagaaac accaggagtt cccattgctt atacaacaaa 3540

cttcctaaaa gacaatgaat tagctgttat taaaaacaac tcagaatata ttgaaacaac 3600

ttcaaaagct tatacagatg gaaaaattaa catcgatcac tctggaggat acgttgctca 3660

attcaacatt tcttgggatg aagtaaatta tgatcctgaa ggtaacgaaa ttgttcaaca 3720

taaaaactgg agcgaaaaca ataaaagcaa gctagctcat ttcacatcgt ccatctattt 3780

gccaggtaac gcgagaaata ttaatgttta cgctaaagaa tgcactggtt tagcttggga 3840

atggtggaga acggtaattg atgaccggaa cttaccactt gtgaaaaata gaaatatctc 3900

catctggggc accacgcttt atccgaaata tagtaataaa gtagataatc caatcgaata 3960

ataagacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca 4020

agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc 4080

gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc 4140

aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt 4200

gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg 4260

tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata 4320

taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc 4380

agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc 4440

atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg 4500

gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta 4560

tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg 4620

atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg 4680

gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca 4740

ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc 4800

agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg 4860

cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc 4920

gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc 4980

aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc 5040

atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg 5100

cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt 5160

ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag 5220

gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat 5280

taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc 5340

atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc 5400

gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc 5460

gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc 5520

gtagaggatc gagatctcga tcccgcgaaa ttaatacgac tcactatagg ggaattgtga 5580

gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga gatataccca 5640

tatgctcggg ccccaaataa tgattttatt ttgactgata gtgacctgtt cgttgcaaca 5700

aattgatgag caatgctttt ttataatgcc aactttgtac aaaaaagctg aacgagaaac 5760

gtaaaatgat ataaatatca atatattaaa ttagattttg cataaaaaac agactacata 5820

atactgtaaa acacaacata tccagtcact atgaatcaac tacttagatg gtattagtga 5880

cctgtagtcg accgacagcc ttccaaatgt tcttcgggtg atgctgccaa cttagtcgac 5940

cgacagcctt ccaaatgttc ttctcaaacg gaatcgtcgt atccagccta ctcgctattg 6000

tcctcaatgc cgtattaaat cataaaaaga aataagaaaa agaggtgcga gcctcttttt 6060

tgtgtgacaa aataaaaaca tctacctatt catatacgct agtgtcatag tcctgaaaat 6120

catctgcatc aagaacaatt tcacaactct tatacttttc tcttacaagt cgttcggctt 6180

catctggatt ttcagcctct atacttacta aacgtgataa agtttctgta atttctactg 6240

tatcgacctg cagactggct gtgtataagg gagcctgaca tttatattcc ccagaacatc 6300

aggttaatgg cgtttttgat gtcattttcg cggtggctga gatcagccac ttcttccccg 6360

ataacggaga ccggcacact ggccatatcg gtggtcatca tgcgccagct ttcatccccg 6420

atatgcacca ccgggtaaag ttcacgggag actttatctg acagcagacg tgcactggcc 6480

agggggatca ccatccgtcg cccgggcgtg tcaataatat cactctgtac atccacaaac 6540

agacgataac ggctctctct tttataggtg taaaccttaa actgcatttc accagtccct 6600

gttctcgtca gcaaaagagc cgttcatttc aataaaccgg gcgacctcag ccatcccttc 6660

ctgattttcc gctttccagc gttcggcacg cagacgacgg gcttcattct gcatggttgt 6720

gcttaccaga ccggagatat tgacatcata tatgccttga gcaactgata gctgtcgctg 6780

tcaactgtca ctgtaatacg ctgcttcata gcacacctct ttttgacata cttcgggtat 6840

acatatcagt atatattctt ataccgcaaa aatcagcgcg caaatacgca tactgttatc 6900

tggcttttag taagccggat ccacgcgatt acgccccgcc ctgccactca tcgcagtact 6960

gttgtaattc attaagcatt ctgccgacat ggaagccatc acagacggca tgatgaacct 7020

gaatcgccag cggcatcagc accttgtcgc cttgcgtata atatttgccc atggtgaaaa 7080

cgggggcgaa gaagttgtcc atattggcca cgtttaaatc aaaactggtg aaactcaccc 7140

agggattggc tgagacgaaa aacatattct caataaaccc tttagggaaa taggccaggt 7200

tttcaccgta acacgccaca tcttgcgaat atatgtgtag aaactgccgg aaatcgtcgt 7260

ggtattcact ccagagcgat gaaaacgttt cagtttgctc atggaaaacg gtgtaacaag 7320

ggtgaacact atcccatatc accagctcac cgtctttcat tgccatacgg aattccggat 7380

gagcattcat caggcgggca agaatgtgaa taaaggccgg ataaaacttg tgcttatttt 7440

tctttacggt ctttaaaaag gccgtaatat ccagctgaac ggtctggtta taggtacatt 7500

gagcaactga ctgaaatgcc tcaaaatgtt ctttacgatg ccattgggat atatcaacgg 7560

tggtatatcc agtgattttt ttctccattt tagcttcctt agctcctgaa aatctcgata 7620

actcaaaaaa tacgcccggt agtgatctta tttcattatg gtgaaagttg gaacctctta 7680

cgtgccgatc aacgtctcat tttcgccaaa agttggccca gggcttcccg gtatcaacag 7740

ggacaccagg atttatttat tctgcgaagt gatcttccgt cacaggtatt tattcggcgc 7800

aaagtgcgtc gggtgatgct gccaacttag tcgactacag gtcactaata ccatctaagt 7860

agttgattca tagtgactgg atatgttgtg ttttacagta ttatgtagtc tgttttttat 7920

gcaaaatcta atttaatata ttgatattta tatcatttta cgtttctcgt tcagctttct 7980

tgtacaaagt tggcattata agaaagcatt gcttatcaat ttgttgcaac gaacaggtca 8040

ctatcagtca aaataaaatc attatttgcc atcggtaccc tcgagcacca ccaccaccac 8100

cactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct 8160

gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg 8220

aaaggaggaa ctatatccgg at 8242

<210> 6

<211> 1104

<212> DNA

<213>artificial sequence

<400> 6

atggaccacc atgccaatga gaacatcttc tactgcccca ttgccatcat gtcagctcta 60

gccatggtat acctgggtgc aaaagacagc accaggacac agataaataa ggttgttcgc 120

tttgataaac ttccaggatt cggagacagt attgaagctc agtgtggcac atctgtaaac 180

gttcactctt cacttagaga catcctcaac caaatcacca aaccaaatga tgtttattcg 240

ttcagccttg ccagtagact ttatgctgaa gagagatacc caatcctgcc agaatacttg 300

cagtgtgtga aggaactgta tagaggaggc ttggaaccta tcaactttca aacagctgca 360

gatcaagcca gagagctcat caattcctgg gtagaaagtc agacaaatgg aattatcaga 420

aatgtccttc agccaagctc cgtggattct caaactgcaa tggttctggt taatgccatt 480

gtcttcaaag gactgtggga gaaaacattt aaggatgaag acacacaagc aatgcctttc 540

agagtgactg agcaagaaag caaacctgtg cagatgatgt accagattgg tttatttaga 600

gtggcatcaa tggcttctga gaaaatgaag atcctggagc ttccatttgc cagtgggaca 660

atgagcatgt tggtgctgtt gcctgatgaa gtctcaggcc ttgagcagct tgagagtata 720

atcaactttg aaaaactgac tgaatggacc agttctaatg ttatggaaga gaggaagatc 780

aaagtgtact tacctcgcat gaagatggag gaaaaataca acctcacatc tgtcttaatg 840

gctatgggca ttactgacgt gtttagctct tcagccaatc tgtctggcat ctcctcagca 900

gagagcctga agatatctca agctgtccat gcagcacatg cagaaatcaa tgaagcaggc 960

agagaggtgg tagggtcagc agaggctgga gtggatgctg caagcgtctc tgaagaattt 1020

agggctgacc atccattcct cttctgtatc aagcacatcg caaccaacgc cgttctcttc 1080

tttggcagat gtgtttcccc ttaa 1104

<210> 7

<211> 132

<212> DNA

<213>artificial sequence

<400> 7

atggagcagc ttgagagtat aatcaacttt gaaaaactga ctgaatggca gcttgagagt 60

ataatcaact ttgaaaaact gactgaatgg cagcttgaga gtataatcaa ctttgaaaaa 120

ctgactgaat gg 132

<210> 8

<211> 367

<212> PRT

<213>artificial sequence

<400> 8

Met Asp His His Ala Asn Glu Asn Ile Phe Tyr Cys Pro Ile Ala Ile

1 5 10 15

Met Ser Ala Leu Ala Met Val Tyr Leu Gly Ala Lys Asp Ser Thr Arg

20 25 30

Thr Gln Ile Asn Lys Val Val Arg Phe Asp Lys Leu Pro Gly Phe Gly

35 40 45

Asp Ser Ile Glu Ala Gln Cys Gly Thr Ser Val Asn Val His Ser Ser

50 55 60

Leu Arg Asp Ile Leu Asn Gln Ile Thr Lys Pro Asn Asp Val Tyr Ser

65 70 75 80

Phe Ser Leu Ala Ser Arg Leu Tyr Ala Glu Glu Arg Tyr Pro Ile Leu

85 90 95

Pro Glu Tyr Leu Gln Cys Val Lys Glu Leu Tyr Arg Gly Gly Leu Glu

100 105 110

Pro Ile Asn Phe Gln Thr Ala Ala Asp Gln Ala Arg Glu Leu Ile Asn

115 120 125

Ser Trp Val Glu Ser Gln Thr Asn Gly Ile Ile Arg Asn Val Leu Gln

130 135 140

Pro Ser Ser Val Asp Ser Gln Thr Ala Met Val Leu Val Asn Ala Ile

145 150 155 160

Val Phe Lys Gly Leu Trp Glu Lys Thr Phe Lys Asp Glu Asp Thr Gln

165 170 175

Ala Met Pro Phe Arg Val Thr Glu Gln Glu Ser Lys Pro Val Gln Met

180 185 190

Met Tyr Gln Ile Gly Leu Phe Arg Val Ala Ser Met Ala Ser Glu Lys

195 200 205

Met Lys Ile Leu Glu Leu Pro Phe Ala Ser Gly Thr Met Ser Met Leu

210 215 220

Val Leu Leu Pro Asp Glu Val Ser Gly Leu Glu Gln Leu Glu Ser Ile

225 230 235 240

Ile Asn Phe Glu Lys Leu Thr Glu Trp Thr Ser Ser Asn Val Met Glu

245 250 255

Glu Arg Lys Ile Lys Val Tyr Leu Pro Arg Met Lys Met Glu Glu Lys

260 265 270

Tyr Asn Leu Thr Ser Val Leu Met Ala Met Gly Ile Thr Asp Val Phe

275 280 285

Ser Ser Ser Ala Asn Leu Ser Gly Ile Ser Ser Ala Glu Ser Leu Lys

290 295 300

Ile Ser Gln Ala Val His Ala Ala His Ala Glu Ile Asn Glu Ala Gly

305 310 315 320

Arg Glu Val Val Gly Ser Ala Glu Ala Gly Val Asp Ala Ala Ser Val

325 330 335

Ser Glu Glu Phe Arg Ala Asp His Pro Phe Leu Phe Cys Ile Lys His

340 345 350

Ile Ala Thr Asn Ala Val Leu Phe Phe Gly Arg Cys Val Ser Pro

355 360 365

<210> 9

<211> 43

<212> PRT

<213>artificial sequence

<400> 9

Glu Gln Leu Glu Ser Ile Ile Asn Phe Glu Lys Leu Thr Glu Trp Gln

1 5 10 15

Leu Glu Ser Ile Ile Asn Phe Glu Lys Leu Thr Glu Trp Gln Leu Glu

20 25 30

Ser Ile Ile Asn Phe Glu Lys Leu Thr Glu Trp

35 40

<210> 10

<211> 1449

<212> DNA

<213>artificial sequence

<400> 10

ctgactaagg ggattttagg atttgtgttc acgctcaccg tgcccgaagt atgcttcatg 60

tattcagatt tccacttcat caatgagcgg tatgttctct ctatcgtccc gtcaggcccc 120

ctcaaagccg agatagaaga tctaagagta ttgagcttca tcaaagggac gaaggtggtc 180

ccagacccta agaaaacagg aggacccata tatagaagaa tagacggaca gagaaatctc 240

ccttttgaca gaacaaccgt tatggcagca ttcaataccc tggaactaag aagcagatac 300

tgggccataa ggaccaggga actaagaagc agatactggg ccataaggac caggagtgga 360

ggaggtgcac ttgccagttg tatgggcctc atatacaaca ggatggtttt tatgtgcctc 420

ggtggcctgc tcacaatggt agccggcgct cagaacgcgg ggctctgcac cctggtggcc 480

atgctagaag agaccggcag gcccgcggtg tttgaccgaa agtcagatgc aaaatcaacc 540

aaactggtct ctgactactg caacgtcctc aacaaggaat ttacagccag aaaatgccgg 600

gccaagttta agcaactgct gcagcactac aatgccggct ttctccgggg tcgtgcgtat 660

gggctagact tgcttcgtag agatcaggcc aaatggagac tgcaaaccct ggccgccgga 720

aagcgatacc gaaaaatcta tgatttgata gaactgtgtg gctctggaat ggcataccca 780

ttacataaac aacacggcat ggccccgtgt ctggcccgca acctggtgcc aatggtggct 840

acggttcagg gtcaggagcg taagacgccc cgcgtcaccg gcggcggcgc aatggcgagc 900

gccgccgagg agtcagatga ggaagaggct attgtagcct acactttgat gctgaacatc 960

cccagcatca acgtgcacca ctacccgtcg gcgggactgc tggtctccga cggaggccca 1020

aatttataca acattagaat ccaaatgtgc actgaactca aactcagtga ttatgatgga 1080

cgatctgcac tcatattgag agggtcggtt gctcacaagt cctgcctgaa acacagtcgg 1140

gtgcgggcct acacctacag caaggtgctg ggccgtctgg ctcgtttgcg tgctgaggca 1200

caggtcaaac aggctagttg cagggccata gtaactgact ttagtgtaat caatgccatt 1260

gaaatgattc ctgccaccat aggcaccgct atgtacaagc tcctaaaaaa ggtcaaacga 1320

ccccctatat ttataagacg tctgcacagg ttgctccaga ccgaggagaa ccttttagat 1380

tttgtgcgtt tcatgggtgt caagtaccag gagttcttct gggacgccaa cgacatctac 1440

cgcatcttc 1449

<210> 11

<211> 483

<212> PRT

<213>artificial sequence

<400> 11

Leu Thr Lys Gly Ile Leu Gly Phe Val Phe Thr Leu Thr Val Pro Glu

1 5 10 15

Val Cys Phe Met Tyr Ser Asp Phe His Phe Ile Asn Glu Arg Tyr Val

20 25 30

Leu Ser Ile Val Pro Ser Gly Pro Leu Lys Ala Glu Ile Glu Asp Leu

35 40 45

Arg Val Leu Ser Phe Ile Lys Gly Thr Lys Val Val Pro Asp Pro Lys

50 55 60

Lys Thr Gly Gly Pro Ile Tyr Arg Arg Ile Asp Gly Gln Arg Asn Leu

65 70 75 80

Pro Phe Asp Arg Thr Thr Val Met Ala Ala Phe Asn Thr Leu Glu Leu

85 90 95

Arg Ser Arg Tyr Trp Ala Ile Arg Thr Arg Glu Leu Arg Ser Arg Tyr

100 105 110

Trp Ala Ile Arg Thr Arg Ser Gly Gly Gly Ala Leu Ala Ser Cys Met

115 120 125

Gly Leu Ile Tyr Asn Arg Met Val Phe Met Cys Leu Gly Gly Leu Leu

130 135 140

Thr Met Val Ala Gly Ala Gln Asn Ala Gly Leu Cys Thr Leu Val Ala

145 150 155 160

Met Leu Glu Glu Thr Gly Arg Pro Ala Val Phe Asp Arg Lys Ser Asp

165 170 175

Ala Lys Ser Thr Lys Leu Val Ser Asp Tyr Cys Asn Val Leu Asn Lys

180 185 190

Glu Phe Thr Ala Arg Lys Cys Arg Ala Lys Phe Lys Gln Leu Leu Gln

195 200 205

His Tyr Asn Ala Gly Phe Leu Arg Gly Arg Ala Tyr Gly Leu Asp Leu

210 215 220

Leu Arg Arg Asp Gln Ala Lys Trp Arg Leu Gln Thr Leu Ala Ala Gly

225 230 235 240

Lys Arg Tyr Arg Lys Ile Tyr Asp Leu Ile Glu Leu Cys Gly Ser Gly

245 250 255

Met Ala Tyr Pro Leu His Lys Gln His Gly Met Ala Pro Cys Leu Ala

260 265 270

Arg Asn Leu Val Pro Met Val Ala Thr Val Gln Gly Gln Glu Arg Lys

275 280 285

Thr Pro Arg Val Thr Gly Gly Gly Ala Met Ala Ser Ala Ala Glu Glu

290 295 300

Ser Asp Glu Glu Glu Ala Ile Val Ala Tyr Thr Leu Met Leu Asn Ile

305 310 315 320

Pro Ser Ile Asn Val His His Tyr Pro Ser Ala Gly Leu Leu Val Ser

325 330 335

Asp Gly Gly Pro Asn Leu Tyr Asn Ile Arg Ile Gln Met Cys Thr Glu

340 345 350

Leu Lys Leu Ser Asp Tyr Asp Gly Arg Ser Ala Leu Ile Leu Arg Gly

355 360 365

Ser Val Ala His Lys Ser Cys Leu Lys His Ser Arg Val Arg Ala Tyr

370 375 380

Thr Tyr Ser Lys Val Leu Gly Arg Leu Ala Arg Leu Arg Ala Glu Ala

385 390 395 400

Gln Val Lys Gln Ala Ser Cys Arg Ala Ile Val Thr Asp Phe Ser Val

405 410 415

Ile Asn Ala Ile Glu Met Ile Pro Ala Thr Ile Gly Thr Ala Met Tyr

420 425 430

Lys Leu Leu Lys Lys Val Lys Arg Pro Pro Ile Phe Ile Arg Arg Leu

435 440 445

His Arg Leu Leu Gln Thr Glu Glu Asn Leu Leu Asp Phe Val Arg Phe

450 455 460

Met Gly Val Lys Tyr Gln Glu Phe Phe Trp Asp Ala Asn Asp Ile Tyr

465 470 475 480

Arg Ile Phe

Claims (10)

1. a kind of for identifying the bacterial expression vector of tumour neoantigen, which is characterized in that include multiple insertions on the carrier Genetic fragment, multiple insertion genes include the first gene hly segment and the second gene ccdB segment；First gene The protein sequence of hly fragment coding is as shown in SEQ ID NO.1, and the sequence of the albumen of the second gene ccdB fragment coding is such as Shown in SEQ ID NO.2；

Preferably, the nucleic acid sequence of the first gene hly segment is as shown in SEQ ID NO.3；

Preferably, the nucleic acid sequence of the second gene ccdB segment is as shown in SEQ ID NO.4.

2. bacterial expression vector according to claim 1, which is characterized in that the nucleic acid sequence of the carrier such as SEQ ID Shown in NO.5.

3. a kind of method of screening and identification tumour neoantigen, which is characterized in that it includes setting on carrier as claimed in claim 2 Two restriction enzyme sites of Nde1-Kpn1 are counted, the DNA sequence dna of candidate tumor neoantigen to be identified is inserted into two enzymes of Nde1-Kpn1 Between enzyme site.

4. the method for screening and identification tumour neoantigen according to claim 3, which is characterized in that building bacterial expression vector Further include the insertion OVA antigen sequence between two restriction enzyme sites of Nde1-Kpn1 of bacterial expression vector, OVA antigen sequence will be inserted into Carrier after column converts bacterial cell, inducing expression, by the bacterium conversion murine antigen presenting cell and and T lymphocyte of induction It co-cultures, detects the submission efficiency of OVA；

Preferably, the OVA antigen sequence is OVA21-388 or 3 × OVA8；The DNA sequence dna of the OVA21-388 such as SEQ Shown in ID NO.6, the DNA sequence dna of the 3 × OVA8 is as shown in SEQ ID NO.7；The amino acid sequence of the OVA21-388 is such as Shown in SEQ ID NO.8, the amino acid sequence of the 3 × OVA8 is as shown in SEQ ID NO.9；

Preferably, the murine antigen presenting cell is mouse hybridoma cell system DC2.4, and mouse T lymphocyte is that mouse is miscellaneous Hand over oncocyte system B3Z；

Preferably, using the secretion of IL-2 in ELISA method detection B3Z cell, using IFN- in Elispot detection B3Z cell The secretion of γ.

5. the method for screening and identification tumour neoantigen according to claim 3, which is characterized in that building bacterial expression vector Further include being inserted into 32 antigen sequences for deriving from CEF after by bacterial expression vector Nde1-Kpn1 digestion, CEF will be inserted into Carrier after antigen sequence is transferred in bacterium and inducing expression, will drench after the antigen presenting cell of the bacterium conversion people of induction with T Bar cell co-cultures, and detects antigen submission efficiency；

Preferably, using the secretion of IL-2 in ELISA method detection T lymphocyte, using in Elispot detection T lymphocyte The secretion of IFN-γ；

Preferably, the CEF is CMV, EBV and Influenza.

6. the method for screening and identification tumour neoantigen according to claim 3, which is characterized in that the method also includes people Work synthesizes the DNA sequence dna of candidate tumor neoantigen, is inserted between the Nde1-Kpn1 restriction enzyme site on bacterial expression vector, in bacterium Middle inducing expression candidate tumor neoantigen；

Preferably, the bacterium of inducing expression candidate tumor neoantigen is BL21.

7. the method for screening and identification tumour neoantigen according to claim 6, which is characterized in that described candidate swollen synthesizing It further include the DNA sequence dna prediction for carrying out candidate tumor neoantigen, predicting candidate tumour neoantigen before the DNA sequence dna of tumor neoantigen DNA sequence dna include that full exon sequencing and RNA sequencing are carried out to tumor sample or tissue, identify site and the mutation base of mutation The expression of cause filters out the strongest a variety of peptides of HLA molecule binding ability of the peptide fragment and patient of mutation using Bioinformatic methods Duan Zuowei candidate tumor neoantigen；

Preferably, the Bioinformatic methods include NetMHC, NetCTLpan and IEDB.

8. the method for screening and identification tumour neoantigen according to claim 6, which is characterized in that the method also includes waiting The external Function detection for selecting tumour neoantigen, bacterium and antigen presenting cell including inducing expression candidate tumor neoantigen are trained altogether Measurement of the detection antigen presenting cell to T lymphocyte activation situation after supporting；

Preferably, the antigen presenting cell is the DC cell of mouse hybridoma cell system DC2.4 or people；

It preferably, include the proliferation of detection T lymphocyte, the phosphorylation of receptor or non-to the measurement of T lymphocyte activation situation Phosphorylation, the secretory volume variation of one or more immune mediators, the expression quantity variation of one or more immune mediators are a kind of or more Kind cell surface marker object expression quantity variation, harvests supernatant and measures the secretory volume of one or more polypeptides relevant to activation With the variation of expression quantity；Preferably, the immune mediator is cell factor, appointing in soluble mediators and cell surface marker Meaning a combination of one or more；

Preferably, the cell factor be selected from TRAIL, IFN-γ, IL-12p70, IL-2, TNF-α, MIP1- α, MIP1- β, CXCL9, CXCL10, MCP1, RANTES, IL-1 β, IL-4, IL-6, IL-8, IL-9, IL-10, IL-13, IL-15, CXCL11, IL-3, IL-5, IL-17, IL-18, IL-21, IL-22, IL-23A, IL-24, IL-27, IL-31, IL-32, TGF-β, CSF, GM Any one or more in CSF and TRANC；

Preferably, the soluble mediators are selected from granzyme A, granzyme B, sFas, sFasL, in perforin and particle cytolysin Any one or more；

Preferably, the cell surface marker is selected from CD107a, CD107b, CD25, CD69, CD45RA, CD45RO, CD137, CD44, CD62L, CD27, CCR7, CD154, KLRG-1, CD71, HLA-DR, CD122, CD28, IL7Ra, CD38, CD26, It is any one in CD134, CTLA-4, LAG-3, TIM-3, CD39, PD1, FoxP3, TIGIT, CD160, BTLA, 2B4 and KLRG1 Kind is a variety of；

Preferably, the cell factor is IFN-γ and TNF-α；The horizontal method of cell factor is measured as Elispot analysis；

Preferably, the cell culture mode that the detection antigen submission effect detection is related to is the combination that following two co-cultures: The co-cultivation of the bacterium of antigen presenting cell and inducing expression candidate tumor neoantigen, antigen presenting cell and T lymphocyte are trained altogether It supports.

9. the method for screening and identification tumour neoantigen according to claim 8, which is characterized in that the method also includes benefits The function of candidate tumor neoantigen is verified with the epitope peptide of tumour neoantigen, the epitope peptide functional verification of the tumour neoantigen Method includes external synthesis candidate tumor neoantigen small peptide, and then Antigen presenting cell is co-cultured with T lymphocyte, inspection Survey the level of T lymphocyte secretion of gamma-IFN cell factor.

10. the method for screening and identification tumour neoantigen according to claim 9, which is characterized in that the verifying is candidate swollen The accuracy of tumor neoantigen immune response further includes MHC class I HLA Tetramer verifying, the MHC class I The method of HLA Tetramer verifying includes external synthesis candidate tumor neoantigen epitope peptide, synthesizes MHC/ with kit Peptide complex is co-cultured with T lymphocyte, with flow cytomery MHC/peptide complex and T lymph The binding ability of cell.