CN114181895A - Method for screening CTL epitope by self-constructed SLA-2-HB01-pCDH/sT2 cell line - Google Patents

Abstract

The invention belongs to the field of biological medicines, and particularly relates to a CTL epitope screening method of an autonomously constructed SLA-2-HB01-pCDH/sT2 cell line. The invention transfects exogenous SLA-2-HB01 gene to sT2 cell, and establishes stable SLA-2 gene expression model. By loading positive epitope peptides such as EB155 and the like on the cell surface and detecting an SLA-2-peptides-beta 2m complex expressed on the cell surface through FACs, the SLA-2-HB01-pCDH/sT2 is judged to have the function of presenting an exogenous polypeptide epitope. To promote the formation of cell surface complexes, the addition of β 2m contributes to an increase in the efficiency of antigen polypeptide presentation. In the results of the invention, the As63, EB155 and Hu62 peptides can be presented by SLA-2-HB01- -pCDH/sT2 cell line, wherein As63 is presented in SLA-2-HB01-pCDH/sT2, SLA-2-HB01-Flag-pCDH/sT2 and SLA-2-HB01-3 Xflag-pCDH/sT 2 cell line with basically consistent presentation efficiency, thus the sT2 expressing SLA-2-HB01 gene in series constructed by the invention has the function of presenting antigen, thereby being used for screening polypeptide epitope of porcine virus at cellular level and further proving that the addition of Flag tag at the C-terminal of SLA-2 heavy chain molecule does not affect the antigen presentation function of cell line.

Description

Method for screening CTL epitope by self-constructed SLA-2-HB01-pCDH/sT2 cell line

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to a CTL epitope screening method of an autonomously constructed SLA-2-HB01-pCDH/sT2 cell line.

Background

The traditional vaccine is difficult to control swine viruses such as foot and mouth disease viruses and is easy to cause the phenomenon of zoonosis, so that the development of a novel safe vaccine is one of the key points of the current research. With the continuous fusion development of immunology and bioinformatics, the virus antigen is used for predicting SLA-2 restrictive CD8⁺CTL epitope and further construction of polypeptide epitope vaccine are hot spots of research.It is therefore of great importance how to select suitable viral epitopes.

Epitopes (epitope), also known as antigenic epitopes, antigenic determinants, chemical groups in antigenic molecules that determine the specificity of the antigen, can bind specifically to TCR or BCR, and finally provoke the immune response of the body to form an immune response against pathogenic microorganisms. Epitopes generally include B cell epitopes, Th epitopes and CTL epitopes. Among them, CTL epitopes are directly related to immune response of infected viruses in host cells, and the immune prevention effect of stimulated killer T cells is the most remarkable, so that the CTL epitopes become hot spots for the research of the current viral epitopes.

CTL epitope is formed by binding antigen protein and MHC class I molecule and being specifically recognized by TCR to induce CD8⁺CTLs produce short peptides of the immune response. T cell epitope is presented by MHC class I molecule on cell surface, and is recognized by TCR to form three molecule complex, which activates corresponding CD8⁺T cells are converted into sensitized CTL which plays a cytotoxic role to kill target cells.

At present, there are many techniques for screening CTL epitopes, and the commonly used methods are:

MHC tetramer (MHC tetramer) technology: altman et al propose by means of the biotin-avidin cascade amplification principle. According to the specific binding relationship between Biotin and avidin 4:1, Biotin-protein ligase (Biotin-protein ligand A, BirA) is utilized to identify a specific sequence of a protein, 1 BirA enzyme substrate peptide sequence for identifying Biotin is added at the N end of an alpha chain of an MHC molecule, the BirA enzyme can catalyze Biotin to be bound to pMHC, 4 purified pMHC monomers and 1 streptavidin marked by avidin form a tetramer complex, so that the affinity of the pMHC and TCR is improved, and antigen specific T lymphocytes are rapidly detected and separated through FACs with higher sensitivity.

Phage Display Technology (PDT): the technology for screening and modifying antigen polypeptide is a gene engineering technology which fuses exogenous protein or antibody variable regions with specific protein on the surface of a bacteriophage, constructs a protein library and screens specific protein with higher affinity. The technology has large library capacity, but has low specificity and sensitivity, and can not realize the expression of whole protein molecules. The method can only carry out the screening of B cell epitope and Th epitope, but can not carry out the screening of CTL epitope.

Construction of heavy and light chain complexes in vitro screening: in vitro binding of the restricted polypeptide epitope to the MHC class I molecule can also be achieved by mimicking the in vitro binding of the polypeptide epitope to the MHC class I molecule alpha chain molecule. This combination is in two ways: one is polypeptide epitope combined with single expression MHC class I molecule alpha chain and beta 2m chain; the second is the binding of polypeptide epitopes to MHC class I molecule complexes.

The study of polypeptide epitopes binding to the alpha and beta 2m chains of single-expression MHC class I molecules began with the study of HLA-A2. In 1991, Sliver et al used a denaturant to separate a beta 0 chain and a beta 12m chain of HLA-A2, and then interacted the separated beta 2 chain, beta 32m chain and polypeptide under renaturation conditions, and found that the three were recombined to form a complex, thereby realizing the in vitro reconstruction of the alpha chain, the polypeptide and the beta 2m chain of the MHC class I molecule for the first time; then, Carboczi et al express a large amount of alpha chains and beta 2m chains of HLA-A2 in E.coli to obtain a large amount of inclusion bodies, dissolve the inclusion bodies by using 8mol/L urea, respectively finish the in vitro reconstruction of influenza virus antigen peptide, nonapeptide of type 1 human immunodeficiency virus envelope protein gp120, HLA-A2 alpha chains and beta 2m chains by adopting an in vitro dilution renaturation method, separate and purify an HLA-A2 complex by using a molecular sieve, detect and find that the renaturation efficiency is as high as 10-15%, and simultaneously find that heavy chain alpha and light chain beta 2m cannot form an HLA-A2 complex under the condition of lacking HLA-A2 restrictive polypeptide; the crystallography research of the obtained compound shows that the obtained crystal data are consistent with the crystallography data of HLA-A2 separated from human lymphocytes, and the crystal resolution reaches the aim of

The method is proved to play a role in identifying and screening the antigen peptide.

The study of binding of polypeptide epitopes to MHC class I complexes began first with murine and human MHC class I binding studies to polypeptides. In 1992, Godeau spliced the mouse H-2Kd extracellular region to β 2m using a glycine-serine rich 15 amino acid linker and then transferred to a baculovirus vector and expressed in insect cells, which showed that the expressed protein could bind to the H-2Kd restriction polypeptide fragment in vitro, indicating that the reconstituted complex protein had activity for binding to the polypeptide. Sylvester-Hvid et al performed in e.coli expression and folding of mouse heavy chain, β 2m and polypeptide in vitro, and biochemical and serological experimental identification showed that expressed MHC-polypeptide complex could be partially correctly folded in vitro and could bind antigen polypeptide. In 2002, Oleksiewicz and the like covalently connect a section of CSFV (CSFV) epitope peptide with a pig SLA-1 heavy chain gene and a pig beta 2m light chain gene, and express by using an expression vector pET-22b, and ELISA detection shows that the SLA-1-peptides-beta 2m recombinant protein complex expressed by pronucleus can be partially and correctly folded in vitro. In 2006, the subject group reported a study of in vitro construction of porcine SLA class I molecular complexes with open antigen polypeptide binding grooves to bind and screen foot-and-mouth disease virus 9 peptides, indicating that the recombinant SLA-2- (G4S) 3-. beta.2m protein complex can be used to identify nonapeptides, i.e., candidate T cell epitopes.

Although the method can play a role in screening polypeptide epitopes by simulating and constructing a complex in vitro, the required experimental conditions are harsh, the required protein amount is very large, and the requirements can be met only by performing prokaryotic expression generally, however, the protein expressed by a prokaryotic expression system has low activity, difficult folding, instability, easy depolymerization and the like, and the most important problem is that only a single epitope can be analyzed in each reaction. Therefore, the epitope screening method using the conventional CTL is inefficient. Therefore, selection of rapid and effective CTL epitopes is urgent.

Disclosure of Invention

In order to overcome the defects of the prior art, the FMDV target epitope verified by an in vitro crystallization technology is selected and added into an autonomously constructed SLA-2-HB01-pCDH/sT2 cell line, so that the cell line has the function of presenting the epitope.

The above purpose of the invention is realized by the following technical scheme:

a method for screening CTL epitope by using self-constructed SLA-2-HB01-pCDH/sT2 cell line comprises the following steps:

step S1: polypeptide epitope synthesis

The polypeptide of Hu62, AS63, EB155 and the like which can be renatured with SLA-2-HB01 protein molecules in vitro are synthesized by combining experimental data of a crystal crystallization technology at the early stage of a laboratory, and are VP1 structural protein sequences of common epidemic disease pathogen A type and Asia I (Asia 1) type FMDV virus strains HQ116378-FMDV-A, Asia1/Jiangsu/Wuxi and Ebola virus Ebola AY9 of China, the sequences are synthesized by Beijing Asian Biotech limited company in Beijing, see Table 1, wherein the polypeptide of Hu62 and AS63 is also subjected to C-terminal biotin labeling.

Table 1 synthetic epitopes

NetMHC 2.8Server applies weight matrix to predict the binding score of the polypeptide and SLA-1-HB (the rank threshold of the high-binding-force polypeptide is 0.1; the rank threshold of the weak-binding-force polypeptide is 1.0)

Polypeptide IC50 values predicted using artificial neural networks in NetMHC 2.8Server (Strong binding/SB lower than 50 nmol/L; Weak binding/WB lower than 500nmol/L)

Step S2: affinity experiments for PK15, sT2, and SLA-2-HB01-pCDH/sT2 cell lines

(1) Selecting PK15, sT2 and SLA-2-HB01-pCDH/sT2 cells with good states, respectively counting 3 cells which are 4 multiplied by 10⁶Cell, 5% CO at 37 ℃₂After culturing in an incubator for 12h, EB155 peptide is loaded in the cells respectively, the concentration is 50ug/mL, and the cells are cultured for 16 h. Simultaneously setting a comparison group;

(2) after incubating the cells, taking out the cells from the incubator, washing the cells for 2-3 times by precooling 1 XPBS, taking care when washing to avoid losing proteins on the cell surface, digesting and collecting the cells, counting and taking 1 XP 10⁶(ii) individual cells;

(3) 4% paraformaldehyde, fixed at room temperature for 20min, and washed 2 times with 1 × PBS after precooling. Blocking with 3% BSA filtered through a 0.22 μm microfilm for 10min at room temperature, washing with 1 × PBS for 2 times;

(4) adding 200 μ L of 1:500 times 1 × PBS diluted monoclonal antibody of β 2m into 3 cells, incubating at 4 deg.C for 1h, washing with cold 1 × PBS for 2-3 times, and removing unbound specific antibody;

(5) adding 200 mu L of PE Rat anti-Mouse IgG solution diluted by 1:500 times of 1 XPBS, incubating for 1h at room temperature in the dark, washing for 2 times by precooling 1 XPBS, adding 500 mu L of 1 XPBS for resuspension, filtering, and detecting by a flow cytometer.

Step S3: screening peptide conditions

(1) Selection of exogenous peptide addition conditions

Selecting sT2 and SLA-2-HB01-pCDH/sT2 cell lines with good state, counting and then connecting 4 x10⁶One plate of sT2 cells and four plates of SLA-2-HB01-pCDH/sT2 cells at 37 ℃ in 5% CO₂After 12h of culture in an incubator, different treatments were performed: sT2 cells were untreated; SLA-2-HB01-pCDH/sT2 was untreated; SLA-2-HB01-pCDH/sT2 was added at 3. mu.g/mL. beta.2m; SLA-2-HB01-pCDH/sT2 was added at 50. mu.g/mL AS 63; SLA-2-HB01-pCDH/sT2 was charged with 3. mu.g/mL. beta.2m and 50. mu.g/mL AS63 at 37 ℃ with 5% CO₂Culturing for 16h in an incubator;

② after incubating the cells, taking out the cells from the incubator, precooling 1 × PBS and washing for 2-3 times, taking care when washing to avoid losing protein on the cell surface, digesting and collecting the cells, counting and taking 1 × 10⁶(ii) individual cells;

③ 4% paraformaldehyde, fixing at room temperature for 20min, and washing with precooling 1 × PBS for 2 times. Blocking with 0.22 microfilm filtered 3% BSA at room temperature for 10min, washing with 1 × PBS for 2 times;

adding 200 mu L of beta 2m monoclonal antibody diluted by 1:500 times of 1 XPBS into the cells, incubating for 1h at 4 ℃, washing for 2-3 times by cold 1 XPBS, and removing the unbound special antibody;

adding 200 mu L of 1:500 times of 1 XPBS diluted PE Rat anti-Mouse IgG solution, incubating for 1h in the dark at room temperature, washing for 2 times by precooled 1 XPBS, adding 500 mu L of 1 XPBS for resuspension, filtering, and detecting by a flow cytometer.

(2) Screening peptide timing

On the basis of earlier experiments, cell lines of sT2 and SLA-2-HB01-pCDH/sT2 with better states are selected, counted and then connected with 4 multiplied by 10⁶Individual cells, sT2 cell plate, SLA-2-HB01-pCDH/sT2 cell 6 plate, at 37 deg.C、5％CO₂After 12h of incubation in an incubator, 5 plates of SLA-2-HB01-pCDH/sT2 cells were loaded with 50. mu.g/mL AS63 and 3. mu.g/mL. beta.2 m, respectively, followed by 5% CO at 37 ℃%₂Culturing in an incubator for 3h, 6h, 12h, 16h and 18 h; after the cell incubation, the flow assay was performed in the same manner as in step S2 (1).

(3) Peptide selection in transfected sT2 cell line

On the basis of earlier stage experiments, selecting sT2, SLA-2-HB01-pCDH/sT2, SLA-2-HB01-Flag-pCDH/sT2 and SLA-2-HB01-3 Xflag-pCDH/sT 2 cell lines with good states, and respectively inoculating 4X 10 cells⁶After culturing the cells, 50. mu.g/mL AS63 and 3. mu.g/mL. beta.2m were added, followed by 5% CO at 37 ℃₂After culturing for 18h in an incubator and incubating the cells, the flow assay was performed in the same manner as in step S2 (1).

Step S4: CTL epitope screening

Selecting SLA-2-HB01-pCDH/sT2 cell line with better state, counting and then connecting 4X 10⁶The cells were treated with epitope peptide at a ratio of 50. mu.g/mL and beta.2m at a ratio of 3. mu.g/mL, and then incubated at 37 ℃ with 5% CO₂After incubation for 18h in the incubator, different cell counts were collected and after counting 1X 10 cells were aspirated from the prepared cell suspension⁶Adding 500-800 μ L PBS into each cell in a new centrifuge tube, washing the cells once, centrifuging at 1500rpm for 5min, discarding the supernatant, adding 4% paraformaldehyde, fixing at room temperature for 20min, and washing with 1 × PBS after precooling for 2 times. Then 3% BSA 100. mu.L room temperature blocking 10 min. After the blocking, 500. mu.L of PBS was added and gently mixed by a gun at 1500rpm for 5min, and the supernatant was discarded. 100 mu L of the pre-diluted 500 x beta 2m monoclonal antibody is added, the staining is carried out for 1h at 4 ℃, and the centrifugal tube is flicked by fingers in the middle and mixed for 2 times. After the primary anti-staining was completed, 500. mu.L of 1 XPBS was added to the tube, and the mixture was mixed by a gun at 1500rpm for 5min, and the supernatant was discarded. Staining secondary antibody, adding 100 μ L APC Rat anti-Mouse IgG diluted 500 Xin advance into the centrifuge tube, staining at 4 deg.C or on ice in dark place for 30min, and flicking and mixing for 2 times. After the staining was completed, 500. mu.L of PBS was added, and after mixing, the supernatant was discarded at 1500rpm for 5 min. 300-600. mu.L of 1 XPBS was added depending on the number of cells after centrifugation. Avoiding light, filtering the cells toAnd (4) carrying out on-machine detection in a marked new centrifugal tube by using a flow cytometer.

Step S5: laser confocal detection

The cell lines of SLA-2-HB01-Flag-pCDH/sT2 and SLA-2-HB01-3 Xflag-pCDH/sT 2 are selected and respectively treated with and without peptides.

(1) Cell surface direct immunofluorescent staining peptides

Cell slide: laser confocal special dish (hole diameter 20nm, thickness 0.13-0.17mm), and mixing cells (density 2 × 10)⁴mL) 200. mu.L of the suspension is transferred to a special laser confocal culture dish, after inoculation, the cells are ensured to be single cells, and the cells are uniformly paved in the dish by a cross shaking method (more edges and less middle parts are avoided); after 12-24h of inoculation, adopting biotin-labeled peptides (peptide 100 mu g/mL; beta 2m 3 mu g/mL) to incubate for 18h at 37 ℃, wherein the cell growth is slow;

secondly, taking out the cells from the incubator, and washing the cells for 2 times by precooling 1 multiplied by PBS (carefully washing the cells to avoid losing proteins on the surfaces of the cells);

③ 4% paraformaldehyde, fixed at room temperature for 20min, and washed with 1 × PBS for 2 times. Mixing 5% BSA, 5% skimmed milk powder and 2% FBS as blocking solution, blocking at room temperature for 10min, and washing with 1 × PBS for 2 times;

adding 1:2000 for dilution

(for detecting whether the biotin-labeled peptides can be presented on the cell surface by SLA-2) and sealed for 1h at room temperature in the absence of light. Wash 2 times with 1 XPBS for 3min each time. Removing excess unbound antibody components;

fifthly, adding 1mL of 1 XPBS into the cell culture dish, and putting the cell culture dish into a wet box to be protected from light. And (5) observing under laser confocal condition, and taking a picture.

(2) Cell surface indirect immunofluorescence staining-beta 2m

Cell slide: mixing cells (density 2X 10)⁴mL) 200. mu.L of the suspension is transferred to a special laser confocal culture dish, cells are ensured to be single cells after inoculation, and the cells are uniformly paved in the dish by a cross shaking method (more edges and less middle parts are avoided); inoculation 12-2After 4h, the cells were incubated for 18h at 37 ℃ with biotin-labeled peptides (peptide 100. mu.g/mL,. beta.2m.sup.3. mu.g/mL), during which time the cells grew more slowly;

secondly, taking out the cells from the incubator, and washing the cells for 2 times by precooling 1 multiplied by PBS (carefully washing the cells to avoid losing proteins on the surfaces of the cells);

③ 4 percent paraformaldehyde, fixing for 20min at room temperature, and washing for 2 times by 1 × PBS;

mixing 5% BSA, 5% skimmed milk powder and 2% FBS as a blocking solution, blocking for 10min at room temperature, and washing with 1 × PBS for 1 time;

adding primary antibody (beta 2m) (detecting whether the peptide marked by biotin can be presented on the cell surface by SLA-2), and incubating for 2h at 4 deg.C or on ice, or staying overnight. Washing with 1 × PBS for 2 times to remove unbound specific antibody;

sixthly, 1 × PBS, 1:1000 dilution

Donkey anti-mouse IgG, protected from light for 1h at room temperature; washing with 1 × PBS for 2-3 times;

seventhly, 1mL of 1 XPBS is added into the cell culture dish and put into a wet box to be protected from light. And (5) observing and photographing under laser confocal condition.

(3) Intracellular direct immunofluorescence staining-SLA-2-HB 01-Flag, SLA-2-HB01-3 Xflag

Cell slide: mixing cells (density 2X 10)⁴mL) 200. mu.L of the suspension is transferred to a special laser confocal culture dish, after inoculation, the cells are ensured to be single cells, and the cells are uniformly paved in the dish by a cross shaking method (more edges and less middle parts are avoided); after 12-24h of inoculation, adopting biotin-labeled peptides (peptide 100 mu g/mL; beta 2m 3 mu g/mL) to incubate for 18h at 37 ℃, wherein the cell growth is slow;

secondly, taking out the cells from the incubator, and washing the cells for 2 times by precooling 1 multiplied by PBS (carefully washing the cells to avoid losing proteins on the surfaces of the cells);

③ 4 percent paraformaldehyde, fixing for 20min at room temperature, and washing for 2 times by 1 × PBS;

0.1 percent Triton-X100, 10min (permeable membrane) at room temperature, and washing for 2 times by 1 XPBS;

5% BSA, 5% skimmed milk powder and 2% FBS are mixed as a blocking solution and blocked for 10min at room temperature, and the mixture is washed 1 times by PBS;

sixthly, adding the mixture to 1:50 for dilution

anti-Flag or DYLight405 (dilution times according to experiment requirements) was incubated for 1h at room temperature in the dark. Pre-cooled 1 × PBS wash 2 times for 3min each. Removing excess unbound antibody components;

seventhly, 1mL of cold 1 × PBS is added into the cell culture dish and put into a wet box to be protected from light. And (5) observing under laser confocal condition, and taking a picture.

(4) Three antibodies were simultaneously immunofluorescent stained

Cell slide: mixing cells (density 2X 10)⁴mL) 200. mu.L of the suspension is transferred to a special laser confocal culture dish, after inoculation, the cells are ensured to be single cells, and the cells are uniformly paved in the dish by a cross shaking method (the number of the cells on the sides is avoided, and the number of the cells in the middle is small); after 12-24h of inoculation, adopting biotin-labeled peptides (peptide 100 mu g/mL; beta 2m 3 mu g/mL) to incubate for 18h at 37 ℃, wherein the cell growth is slow;

② the cells were taken out from the incubator and washed 2 times with precooled 1 XPBS. 4% paraformaldehyde, and fixing at room temperature for 20 min. Then washed 2 times with 1 × PBS. Mixing 5% BSA, 5% skimmed milk powder and 2% FBS as blocking solution, blocking at room temperature for 10min, and washing with 1 × PBS for 1 time;

③ adding beta 2m primary antibody, dyeing for 1h at 4 ℃, washing for 2 times by precooled 1 XPBS, and removing the unbound antibody;

fourthly, diluting with 1 XPBS at a ratio of 1:1000

The donkey anti-mouse IgG is protected from light for 1h at room temperature; washing with 1 × PBS for 2-3 times;

adding 1:10000 for dilution

Washing with 1 × PBS for 3-5 times (3 min each time) at 4 deg.C in a dark place for 1h, and removing excessive unbound antibody component;

sixthly, adding 0.1 percent of Triton-X100 into each dish, keeping out of the sun for 10min, washing for 2 times by using precooled 1 XPBS, sucking the surface, adding 5 percent of BSA, 5 percent of skim milk powder and 2 percent of FBS into each dish, mixing as a sealing solution, and sealing for 10min at room temperature in the absence of the sun;

seventhly, adding 1:10000 diluted DYLight405 into each dish, keeping out of the sun for 1h at room temperature, and washing with precooled 1 × PBS for 5 times for 3min each time;

add 1mL of 1 XPBS, put into a wet box and keep out of the sun. And (5) observing under laser confocal condition, and taking a picture.

Compared with the prior art, the invention has the beneficial effects that:

the invention transfects exogenous SLA-2-HB01 gene to sT2 cell, and establishes stable SLA-2 gene expression model. Positive epitope peptides such as EB155 and the like are loaded on the surface of the cell, SLA-2-peptides-beta 2m complex expressed on the surface of the cell is detected through FACs, and the function that sT2 has the exogenous polypeptide epitope presenting function is judged. To promote the formation of cell surface complexes, the addition of β 2m contributes to an increase in the efficiency of antigen polypeptide presentation. In the results of the invention, the As63, EB155 and Hu62 peptides can be presented by SLA-2-HB01-pCDH/sT2 cell lines, wherein As63 is presented in SLA-2-HB01-pCDH/sT2, SLA-2-HB01-Flag-pCDH/sT2 and SLA-2-HB01-3 Xflag-pCDH/sT 2 cell lines with basically consistent presentation efficiency, and further proves that the addition of a Flag tag at the C-terminal of the SLA-2 heavy chain molecule does not influence the antigen presentation function of the cell lines.

As the result of Western blotting experiments shows that the expression quantity of beta 2m protein in different cells is the same, and the beta 2m gene of the pig is monomorphic, exogenous SLA-2 heavy chain can be only considered to be transfected during design, and the beta 2m of the light chain can be expressed by using the cell line. For a variety of tests, porcine β 2m monoclonal antibodies were also used to test the expression of cell surface SLA-I-peptides, and polypeptides bound by cell line SLA-2 and exogenous SLA-2 and presented by the cells were analyzed. Because the fluorescent antibody used in the early-stage flow detection is the PE-labeled secondary antibody, in order to prevent the non-specific binding of the secondary antibody and other proteins on the cell surface, a control group only staining the secondary antibody is arranged in the FACs experiment, and the peak values of the two are completely coincided after the comparison with blank cells, which indicates that the cell surface is not non-specifically bound with PE, and the experimental group data is real and reliable.

The eukaryon expression vector expressing SLA-2-HB01 gene in the autonomously constructed sT2 cell line of the invention has eGFP green fluorescence which is particularly bright, and in FACs detection experiments, eGFP and FITC excitation light areas almost completely coincide to cause background influence on PE detection of FL2 channel, so that in FACs detection, sT2 cell is used for regulating voltage of flow cell, and then PE fluorescence compensation is carried out by using the detection result of SLA-2-HB01-pCDH/sT2 cell. A large number of experimental results show that when the APC-Rat anti-Mouse Ig fluorescence labeled secondary antibody is used for FACs experiments, eGFP has no background influence on FL4 channel detection, so that when CTL epitope screening and other function detection is carried out on the constructed sT2 cell line, APC Rat anti-Mouse IgG is selected as the fluorescence labeled secondary antibody, flow regulation voltage and fluorescence compensation are not needed each time, the purposes of saving materials and time are achieved, and errors in experimental results are eliminated.

In the LSCM experimental result, the Flag tag and the 3 Xflag tag exist at the C-terminal of the SLA-2-HB01 gene, when the SLA-2-HB01 molecule presents exogenous peptide to express on the cell surface, the C-terminal of the SLA-2-HB01 gene is in the cytoplasmic region of the cell membrane, therefore, when the Flag tag monoclonal antibody is stained, the cell should be subjected to membrane penetration treatment, so that the Flag tag and the Flag monoclonal antibody can be specifically combined without being influenced by the cell membrane. LSCM assay results showed that no specific binding of Alexa594-anti Flag antibody (1: 50 dilution) to Flag tag was seen in SLA-2-HB01-Flag-pCDH/sT 2-loaded Biotin-AS63 peptide cells; under the same conditions, binding of the Alexa594-anti Flag antibody (1: 50 dilution) to the corresponding cell membrane surface 3 Xflag tag protein was observed in the SLA-2-HB01-3 Xflag-pCDH/sT 2 cell line. When examined with DYLight405-anti Flag antibody, the dilution of staining with DYLight405-anti Flag antibody concentration required in SLA-2-HB01-Flag-pCDH/sT2 cells under the same treatment and observation conditions was 1:2000, whereas the concentration required in SLA-2-HB01-3 Xflag-pCDH/sT 2 cells was lower, the minimum dilution of staining was 1: 10000. these results indicate that both Flag and 3 xflag tag proteins bind specifically to DYLight405-anti Flag antibodies, but both are relatively easy to detect with 3 xflag and require lower concentrations of antibodies, less non-specific. The reason is probably that the Flag tag only has 8 AA, the short part of the sequence can not be efficiently and specifically combined with the Flag monoclonal antibody in the cell membrane, and the 3 Xflag tag is the superposition of 3 Flag tags, has long sequence and is easily recognized and specifically combined by the Flag monoclonal antibody. Since the excitation lights of Alexa594 and Alexa647 are relatively close and not easily distinguished, which may cause the influence of related background, the detection of Flag tag in the later LSCM experiment was performed with DYLight405-anti Flag (DYKDDDDK Ab same epitope as Sigmas FLAG-DL405) antibody.

Drawings

FIG. 1 shows affinity detection of PK15, sT2 and SLA-2-HB01-pCDH/sT2 cells, wherein A is the affinity detection result of EB155 peptide loaded cells of PK15, sT2 and SLA-2-HB01-pCDH/sT2 cells; b is the average fluorescence intensity result of flow cytometry detection;

FIG. 2 shows peptide screening conditions, in which the A, SLA-2-HB01-pCDH/sT2 cell line is loaded with peptide conditions for flow cytometry detection; b, flow-averaged fluorescence intensity results for peptide-loaded conditions of the SLA-2-HB01-pCDH/sT2 cell line; c, flow cytometry detection results of different time of peptide loading of SLA-2-HB01-pCDH/sT2 cell line; d, flow-average fluorescence intensity results of SLA-2-HB01-pCDH/sT2 cell line loading peptides at different times;

FIG. 3 is a graph showing the results of peptide-presenting cell lines of SLA-2-HB01-pCDH/sT2, SLA-2-HB01-Flag-pCDH/sT2 and SLA-2-HB01-3 Xflag-pCDH/sT 2, wherein A is SLA-2-HB01-pCDH/sT2, SLA-2-HB01-Flag-pCDH/sT2 and SLA-2-HB01-3 Xflag-pCDH/sT 2; b is peptide flow type average fluorescence intensity result presented under the same conditions of SLA-2-HB01-pCDH/sT2, SLA-2-HB01-Flag-pCDH/sT2 and SLA-2-HB01-3 Xflag-pCDH/sT 2;

FIG. 4 shows functional verification of CTL epitope peptide screening of cell line, wherein AS63 and Hu156 peptides are loaded in A, C, SLA-2-HB01-pCDH/sT2 cells respectively; b, loading the Hu62 and EB155 peptides in SLA-2-HB01-pCDH/sT2 cells; D-F, SLA-2-HB01-pCDH/sT2 cells were loaded with AHu62, AS127, AHu63 and Ahu154 peptides, respectively;

fig. 5 is a confocal laser-induced dyeing structure, wherein a: SLA-2-HB01-Flag-pCDH/sT2 cell line load biotin labeling peptide, each structure staining label; b: SLA-2-HB01-3 XFlag-pCDH/sT 2 cell line load biotin labeling peptide, each structure staining label;

FIG. 6 shows Flag staining in SLA-2-HB01-Flag-pCDH/sT2, SLA-2-HB01-3 Xflag-pCDH/sT 2, in which the A, Alexa594-anti Flag antibody detects the SLA-2-HB01-Flag-pCDH/sT2 cell line in bright field (20X); b, Alexa594-anti Flag antibody detects SLA-2-HB01-Flag-pCDH/sT2 cell line fluorescent field (20X); c, Alexa594-anti Flag antibody detection of SLA-2-HB01-3 Xflag-pCDH/sT 2 cell line brightfield (20X); d, detection of SLA-2-HB01-3 XFlag-pCDH/sT 2 cell line fluorescent field by Alexa594-anti Flag antibody (20X);

FIG. 7 is a diagram showing the confocal laser detection of SLA-2-HB01-Flag and SLA-2-HB01-3 Xflag, wherein A1-A3 are respectively the bright field, the fluorescence field and the combination of the two fields of the DYLight405-anti Flag (1: 2000) detection Flag in the SLA-2-HB01-Flag-pCDH/sT2 cell line; B1-B3, namely a bright field, a fluorescent field and a combined picture of the two fields of the DYLight405-anti Flag (1: 2000) detection Flag after the SLA-2-HB01-Flag-pCDH/sT2 cell line is loaded with the peptide; C1-C3, namely a bright field, a fluorescent field and a combined picture of the two fields of the DYLight405-anti Flag (1: 10000) detection Flag after the SLA-2-HB01-Flag-pCDH/sT2 cell line is loaded with the peptide; D1-D3, which are respectively a bright field, a fluorescent field and a combined picture of the DYLight405-anti Flag (1: 10000) detection Flag in an SLA-2-HB01-3 Xflag-pCDH/sT 2 cell line; E1-E3, which are respectively a bright field, a fluorescent field and a combined picture of the DYLight405-anti Flag (1: 10000) detection Flag after peptide loading in an SLA-2-HB01-3 Xflag-pCDH/sT 2 cell line;

FIG. 8 shows the staining of SLA-2-peptide-. beta.2m molecules, in which, A-E, a control SLA-2-HB01-3 XFlag-pCDH/sT 2 cell line; a, bright field (40 ×); b, detecting the expression of the cell membrane surface Biotin-AS63 peptide (40 x) by a fluorescence visual field; c, detecting the expression of beta 2m on the surface of the cell membrane by a fluorescence visual field (40 ×); d, fluorescence visual field detection of cell membrane surface SLA-2-HB01-3 XFlag expression (40X); e, SLA-2-HB01-3 XFlag-pCDH/sT 2 cells SLA-2-HB01-3 XFlag-peptide-. beta.2m Tri-molecule co-staining (40X). F-J, panel SLA-2-HB01-3 XFlag-pCDH/sT 2 cell line loaded with Biotin-AS 63; f, bright field (40 ×); g, detecting the expression of the cell membrane surface Biotin-AS63 peptide (40 x) by a fluorescence visual field; h, fluorescent visual field detection of cell membrane surface β 2m expression (40 ×); i, detecting the expression of SLA-2-HB01-3 XFlag on the surface of the cell membrane by a fluorescence visual field (40X); three molecules of SLA-2-HB01-3 XFlag-peptide-. beta.2m co-staining in J, SLA-2-HB01-3 XFlag-pCDH/sT 2-loaded peptide cells (40X).

Detailed Description

The invention is described in more detail below with reference to specific examples, without limiting the scope of the invention. Unless otherwise specified, the experimental methods adopted by the invention are all conventional methods, and experimental equipment, materials, reagents and the like used in the experimental method can be obtained from commercial sources.

The sources of the cells and related reagents adopted by the invention are as follows:

sT2 cells, pCDH/sT2, SLA-2-HB01-pCDH/sT2, SLA-2-HB01-Flag-pCDH/sT2 and SLA-2-HB01-3 Xflag-pCDH/sT 2 were derived from this laboratory and were all cultured in the conventional conditions of DMEM containing 10% FBS.

Polypeptide: all peptide fragments were synthesized by Beijing Zhongke Sudoku Biotech Co., Ltd, with a purity of > 98% by HPLC detection, and were stored in 0.5 mg/tube at-80 ℃ separately.

Antibody: the SLA-2-HB01 monoclonal antibody and the pig beta 2m monoclonal antibody are synthesized and stored in the laboratory; natural Streptavidin protein

ab134348 was purchased from abcam; DYKDDDDK Absame epitope as Sigmas FLAG-DL405(DYLight 405) was purchased from Sigma;

donkey anti-mouse IgG from Invitrogen; APC Rat anti-Mouse IgG (product No. 560098) and PE Rat anti-Mouse IgG were purchased from BD Co.

Example 1 affinity experiments for PK15, sT2 and SLA-2-HB01-pCDH/sT2 cell lines

EB155 peptide is used as PK15, sT2 and SLA-2-HB01-pCDH/sT2 cell line as positive control peptide, and is loaded in three cell lines respectively, because the expression quantity of beta 2m in the cell lines is basically consistent in the detection of the previous Western blotting experiment, the pig beta 2m monoclonal antibody is selected as the detection of cell surface peptide. Since SLA-I-peptides are presented to the cell membrane surface as a whole, detection of β 2m expression at the cell membrane surface indicates that epitopic peptides are presented to the cell surface by SLA class I molecules. The experimental group also set PE-labeled IgG as a background control group, which was not different from the PK15 and sT2 cells alone, indicating that the staining result is the cell flow detection result of specific binding of PE and the beta 2m monoclonal antibody. In the PK15 and sT2 cell lines, it can be seen that the sT2 cell can present exogenous peptides, and the transfected sT2 cell line is also confirmed to have the function of more effectively presenting exogenous peptides, as shown in fig. 1.

Example 2 peptide conditional screening

SLA-2-HB01-pCDH/sT2 cell line can continuously and stably express SLA-2-HB01 protein in a large quantity, and under different experimental conditions, the cell line is added with 50ug/mL AS63 exogenous peptide and incubated for 18h with 3 ug/mL beta 2m, the presentation efficiency is best, and the experimental results are shown in figure 2. This condition also further illustrates that when β 2m is loaded in a cell line, there is no effect on the expression of β 2m protein on the cell line surface. In three cell lines of SLA-2-HB01-pCDH/sT2, SLA-2-HB01-Flag-pCDH/sT2 and SLA-2-HB01-3 Xflag-pCDH/sT 2 which are transfected with SLA-2-HB01 gene, the function of presenting peptides is not influenced by the addition of a label at the C-terminal of the SLA I class according to the flow results, as shown in FIG. 3.

Example 3 CTL epitope screening functional validation

After peptide screening conditions were determined, whether peptides could bind to SLA-2-HB01 and whether affinity was enhanced was examined by using a binding assay in which peptides were loaded onto SLA-2-HB01-pCDH/sT2 cells, 50. mu.g/mL exogenous peptides and 3. mu.g/mL. beta.2 m were added to the cell line, incubated at 37 ℃ for 18h, and then cell membrane surface. beta.2 m expression was measured by a. beta.2 m monoclonal antibody to determine SLA-2-peptide expression, and CTL epitope peptides were selected, as shown in FIG. 4. The Fluorescence Index (FI) was calculated by determining the mean fluorescence intensity (. beta.2m) on the cell line by FACs analysis (MFI). According to the following formula: FI (MFI [ SLA-2-HB01-pCDHsT2 cells + peptide ]/MFI [ SLA-2-HB01-pCDHsT2 cells with a peptide ]) -1 demonstrates the binding ability of SLA-2 to peptides. As a result, AS63, Hu62, and EB155 were found to bind to SLA-2-HB01, while none of the other control peptides were bound and presented. Table 2 is presented for the ability of the predicted epitope to bind to SLA-2-HB 01.

TABLE 2 CTL epitope peptide binding ability to SLA-2-HB01

Example 4 laser confocal experiments to verify cell line function

After 100 mu g/mL Biotin-AS63 peptide and 3 mu g/mL beta 2m are added into a laser confocal plate of SLA-2-HB01-Flag-pCDH/sT2 cells and incubated for 18h at 37 ℃, the SLA I-peptide-beta 2m complex is subjected to trimolecular cell staining, and the specific staining structure is shown in figure 5. LSCM assay results showed that no fluorescence was observed for binding of Alexa594-anti Flag antibody (1: 50 dilution) to Flag tag on the surface after loading Biotin-AS63 peptide in SLA-2-HB01-Flag-pCDH/sT2 cells in the wavelength range of 590-617nm AS shown in FIG. 6B, but that for binding of Alexa594-anti Flag antibody (1: 50 dilution) to the corresponding cell membrane surface 3 Xflag was observed in SLA-2-HB01-3 Xflag-pCDH/sT 2 cell line under the same conditions AS shown in FIG. 6D.

In the wavelength range of 400-420nm, cell membrane surface anti-Flag fluorescence was detected in both SLA-2-HB01-Flag-pCDH/sT2 and SLA-2-HB01-3 Xflag-pCDH/sT 2 cell lines incubated with 100. mu.g/mL Biotin-AS63 peptide and 3. mu.g/mL. beta.2 m 37 ℃ for 18h, AS shown in FIG. 7, but the desired DYLight405-anti Flag antibody concentration in SLA-2-HB01-3 Xflag-pCDH/sT 2 cells was lower, with a minimum dilution of staining of 1:10000 (see fig. 7D and 7E), but at a DYLight405-anti Flag antibody dilution concentration of 1: at 10000, there was no fluorescence reaction in SLA-2-HB01-Flag-pCDH/sT2 cells, as shown in FIG. 7C. While the dilution of DYLight405-anti Flag antibody concentration staining required in SLA-2-HB01-Flag-pCDH/sT2 cells was 1:2000, see fig. 7A and 7B.

100 mu g/mL Biotin-AS63 and 3 mu g/mL beta 2m are loaded in an SLA-2-HB01-3 XFlag-pCDH/sT 2 cell line and incubated together at 37 ℃ for 18h, an SLA-2-HB01-3 XFlag-pCDH/sT 2 cell line without peptides and beta 2m is selected AS a control, and the experimental group and the control group are subjected to DYLight-550-anti Biotin (1: 2000), Alexa647 (1: 1000) and DYLight405-anti Flag (1: 10000) co-staining after 18h, so that in the cell line loaded with the Biotin-AS63 peptides, the SLA-2-3 XFlag molecules, the beta 2m molecules and the Biotin-AS63 molecules can be detected on the surface of a cell membrane, and the SLA-2-3 XFlag-peptide-beta 2m molecular structure can be simultaneously expressed on the surface of the cell membrane, as shown in fig. 8, it was further demonstrated that the construction of the transfected sT2 cell line can function to present exogenous peptides to the cell surface.

The embodiments described above are merely preferred embodiments of the invention, rather than all possible embodiments of the invention. Any obvious modifications to the above would be obvious to those of ordinary skill in the art, but would not bring the invention so modified beyond the spirit and scope of the present invention.

Claims (9)

1. A method for screening CTL epitope by using self-constructed SLA-2-HB01-pCDH/sT2 cell line is characterized by comprising the following steps:

step S1: synthesizing polypeptide epitope; wherein the polypeptide sequence is as follows:

step S2: affinity experiments for PK15, sT2, and SLA-2-HB01-pCDH/sT2 cell lines;

step S3: screening peptide conditions;

step S4: screening CTL epitopes;

step S5: and (3) laser confocal detection.

2. The method for screening CTL epitopes by using an autonomously constructed SLA-2-HB01-pCDH/sT2 cell line as claimed in claim 1, wherein the step S2 comprises the following steps:

(1) selecting PK15, sT2 and SLA-2-HB01-pCDH/sT2 cells with good states, respectively counting 3 cells which are 4 multiplied by 10⁶Cell, 5% CO at 37 ℃₂After culturing in an incubator for 12h, respectively loading EB155 peptides with the concentration of 50ug/mL on the cells, culturing for 16h, and setting a control group;

(2) after incubating the cells, taking out the cells from the incubator, precooling the cells, washing the cells for 2-3 times by 1 × PBS, digesting and collecting the cells, counting the cells, and taking out the cells with the size of 1 × 10⁶(ii) individual cells;

(3) fixing 4% paraformaldehyde at room temperature for 20min, washing with precooled 1 × PBS for 2 times, blocking with 3% BSA filtered by 0.22 μm microfilm at room temperature for 10min, and washing with 1 × PBS for 2 times;

(4) adding 200 μ L of 1:500 times 1 × PBS diluted monoclonal antibody of β 2m into 3 cells, incubating at 4 deg.C for 1h, washing with cold 1 × PBS for 2-3 times, and removing unbound specific antibody;

(5) adding 200 mu L of PE Rat anti-Mouse IgG solution diluted by 1:500 times of 1 XPBS, incubating for 1h at room temperature in the dark, washing for 2 times by precooling 1 XPBS, adding 500 mu L of 1 XPBS for resuspension, filtering, and detecting by a flow cytometer.

3. The method for screening CTL epitopes by using an autonomously constructed SLA-2-HB01-pCDH/sT2 cell line as claimed in claim 1, wherein the step S3 comprises the following steps:

(1) selection of exogenous peptide addition conditions

Selecting sT2 and SLA-2-HB01-pCDH/sT2 cell lines with good state, counting and then connecting 4 x10⁶One plate of sT2 cells and four plates of SLA-2-HB01-pCDH/sT2 cells at 37 ℃ in 5% CO₂After 12h of culture in an incubator, different treatments were performed: sT2 cells were untreated; SLA-2-HB01-pCDH/sT2 was untreated; SLA-2-HB01-pCDH/sT2 was added at 3. mu.g/mL. beta.2m; SLA-2-HB01-pCDH/sT2 was added at 50. mu.g/mL AS 63; SLA-2-HB01-pCDH/sT2 was charged with 3. mu.g/mL. beta.2m and 50. mu.g/mL AS63 at 37 ℃ with 5% CO₂Culturing for 16h in an incubator;

② after incubating the cells, taking out the cells from the incubator, precooling 1 × PBS to wash for 2-3 times, digesting and collecting the cells, counting and taking 1 × 10⁶(ii) individual cells;

③ 4 percent paraformaldehyde, fixing for 20min at room temperature, precooling 1 multiplied by PBS and washing for 2 times, then sealing for 10min at room temperature by 3 percent BSA filtered by a 0.22 micro-membrane, and washing for 2 times by 1 multiplied by PBS;

adding 200 mu L of beta 2m monoclonal antibody diluted by 1:500 times of 1 XPBS into the cells, incubating for 1h at 4 ℃, washing for 2-3 times by cold 1 XPBS, and removing the unbound special antibody;

adding 200 mu L of 1:500 times of 1 XPBS diluted PE Rat anti-Mouse IgG solution, incubating for 1h in the dark at room temperature, washing for 2 times by precooling 1 XPBS, adding 500 mu L of 1 XPBS for resuspension, filtering, and detecting by a flow cytometer;

(2) screening peptide timing

On the basis of earlier experiments, cell lines of sT2 and SLA-2-HB01-pCDH/sT2 with better states are selected, counted and then connected with 4 multiplied by 10⁶Cells, sT2 cell plate, SLA-2-HB01-pCDH/sT2 cell 6 plate, 5% CO at 37 ℃%₂After 12h of incubation in an incubator, 5 plates of SLA-2-HB01-pCDH/sT2 cells were loaded with 50. mu.g/mL AS63 and 3. mu.g/mL. beta.2 m, respectively, followed by 5% CO at 37 ℃%₂Culturing in an incubator for 3h, 6h, 12h, 16h and 18 h; after the cell incubation, the flow detection was performed in the same manner as in step S2 (1);

(3) peptide selection in transfected sT2 cell line

On the basis of earlier stage experiments, selecting sT2, SLA-2-HB01-pCDH/sT2, SLA-2-HB01-Flag-pCDH/sT2 and SLA-2-HB01-3 Xflag-pCDH/sT 2 cell lines with good states, and respectively inoculating 4X 10 cells⁶After culturing the cells, 50. mu.g/mL AS63 and 3. mu.g/mL. beta.2m were added, followed by 5% CO at 37 ℃₂After culturing for 18h in an incubator and incubating the cells, the flow assay was performed in the same manner as in step S2 (1).

4. The method for screening CTL epitopes by using an autonomously constructed SLA-2-HB01-pCDH/sT2 cell line as claimed in claim 1, wherein the step S4 comprises the following steps:

selecting SLA-2-HB01-pCDH/sT2 cell line with better state, counting and then connecting 4X 10⁶The cells were treated with epitope peptide at a ratio of 50. mu.g/mL and beta.2m at a ratio of 3. mu.g/mL, and then incubated at 37 ℃ with 5% CO₂After incubation for 18h in the incubator, collecting different cell counts, and sucking out the cell suspension from the prepared cell suspension after counting1×10⁶Adding 500-800 μ L PBS into each cell in a new centrifuge tube, washing the cells once, centrifuging at 1500rpm for 5min, discarding the supernatant, adding 4% paraformaldehyde, fixing at room temperature for 20min, pre-cooling for 2 times with 1 × PBS, and sealing with 100 μ L of 3% BSA at room temperature for 10 min; after the sealing is finished, adding 500 mu L PBS and lightly mixing the mixture by a gun, carrying out 5min at 1500rpm, discarding the supernatant, adding 100 mu L of 500 x beta 2m monoclonal antibody which is diluted in advance, dyeing the mixture for 1h at 4 ℃, and lightly flicking the centrifugal tube by fingers in the middle to mix the mixture for 2 times; after the first-antibody staining is finished, adding 500 mu L of 1 XPBS into a centrifuge tube, uniformly mixing by using a gun, rotating at 1500rpm for 5min, and discarding the supernatant; staining secondary antibody, adding 100 μ L of APC Rat anti-Mouse IgG diluted by 500 Xin advance into a centrifuge tube, staining at 4 deg.C or on ice in dark place for 30min, and flicking and mixing for 2 times; after dyeing, adding 500 mu L PBS, mixing uniformly, and discarding the supernatant at 1500rpm for 5 min; adding 300-600 mu L of 1 multiplied by PBS for resuspension according to the amount of the centrifuged cells; and (4) keeping out of the light, filtering the cells into a new marked centrifugal tube by using a filter screen, and performing on-machine detection by using a flow cytometer.

5. The method for screening CTL epitopes by using an autonomously constructed SLA-2-HB01-pCDH/sT2 cell line as defined in claim 1, wherein the step S5 comprises the following steps: the selected cell lines are SLA-2-HB01-Flag-pCDH/sT2 and SLA-2-HB01-3 Xflag-pCDH/sT 2 cell lines, and peptide adding and peptide non-adding treatments are respectively carried out, and the operation is as follows:

(1) performing peptide direct immunofluorescence staining on the cell surface;

(2) carrying out beta 2m indirect immunofluorescence staining on the cell surface;

(3) SLA-2-HB01-Flag, SLA-2-HB01-3 Xflag direct immunofluorescence staining are carried out in the cells;

(4) three antibodies were simultaneously immunofluorescent stained.

6. The method for screening CTL epitopes according to claim 5, wherein the method for screening CTL epitopes by using an autonomously constructed SLA-2-HB01-pCDH/sT2 cell line is as follows (1):

cell slide: the special laser confocal dish with the diameter of 20nm and the thickness of 0.13-0.17mm is adopted,taking 200 μ L of density of 2 × 10⁴Transferring the/mL cells to a special laser confocal culture dish, wherein the inoculated cells are single cells and the cells are uniformly paved in the dish by a cross shaking method; after 12-24h of inoculation, adopting peptides marked by biotin, wherein the peptides are 100 mu g/mL; beta 2m is 3 mu g/mL, and the mixture is incubated for 18h at 37 ℃;

taking out the cells from the incubator, precooling the cells, and washing the cells for 2 times by using 1 multiplied by PBS;

③ fixing 4 percent paraformaldehyde at room temperature for 20min, washing with 1 multiplied by PBS for 2 times, mixing 5 percent BSA, 5 percent skimmed milk powder and 2 percent FBS as a sealing solution, sealing at room temperature for 10min, and washing with 1 multiplied by PBS for 2 times;

adding 1:2000 for dilution

550-streptavidin, sealing for 1h at room temperature in dark, washing with 1 × PBS for 2 times, 3min each time, and removing the un-bound excessive antibody components;

fifthly, adding 1mL of 1 XPBS into the cell culture dish, placing the dish in a wet box to avoid light, observing the dish under laser confocal conditions, and taking a picture.

7. The method for screening CTL epitopes by using an autonomously constructed SLA-2-HB01-pCDH/sT2 cell line as claimed in claim 5, wherein the step (2) comprises the following steps:

cell slide: taking 200 μ L of density of 2 × 10⁴The cells of/mL are transferred to a special laser confocal culture dish, the inoculated cells are single cells, and the cells are uniformly spread in the dish by a cross shaking method; after 12-24h of inoculation, adopting biotin-labeled peptides, wherein the peptides are 100 mu g/mL, the beta 2m is 3 mu g/mL, and incubating for 18h at 37 ℃;

taking out the cells from the incubator, precooling the cells, and washing the cells for 2 times by using 1 multiplied by PBS;

③ 4 percent paraformaldehyde, fixing for 20min at room temperature, and washing for 2 times by 1 × PBS;

mixing 5% BSA, 5% skimmed milk powder and 2% FBS as a blocking solution, blocking for 10min at room temperature, and washing with 1 × PBS for 1 time;

adding primary anti-beta 2m, incubating at 4 deg.C or on ice for 2h, or overnight, washing with 1 × PBS for 2 times, and removing unbound special antibody;

sixthly, 1 × PBS, 1:1000 dilution

647Donkey anti-mouse IgG, keeping away from light for 1h at room temperature; washing with 1 × PBS for 2-3 times;

seventhly, 1mL of 1 XPBS is added into the cell culture dish, the dish is placed into a wet box to be protected from light, and the dish is observed and photographed under the laser confocal condition.

8. The method for screening CTL epitopes by using an autonomously constructed SLA-2-HB01-pCDH/sT2 cell line as claimed in claim 5, wherein the step (3) comprises the following steps:

cell slide: taking 200 μ L of density of 2 × 10⁴The cells per mL are transferred to a laser confocal special culture dish, the inoculated cells are single cells, and the cells are uniformly spread in the dish by a cross shaking method; after 12-24h of inoculation, adopting biotin-labeled peptides, wherein the peptides are 100 mu g/mL, the beta 2m is 3 mu g/mL, and incubating for 18h at 37 ℃;

taking out the cells from the incubator, precooling the cells, and washing the cells for 2 times by using 1 multiplied by PBS;

③ 4 percent paraformaldehyde, fixing for 20min at room temperature, and washing for 2 times by 1 × PBS;

0.1 percent Triton-X100, penetrating the membrane for 10min at room temperature, and washing for 2 times by 1 times of PBS;

5% BSA, 5% skimmed milk powder and 2% FBS are mixed as a blocking solution and blocked for 10min at room temperature, and the mixture is washed 1 times by PBS;

sixthly, adding the mixture to 1:50 for dilution

594anti-Flag or DYLight405, incubating for 1h at room temperature in the dark, washing for 2 times with precooled 1 × PBS, 3min each time,

removing the surplus antibody component which is not combined;

adding 1mL of cold 1 XPBS into a cell culture dish, placing the dish in a wet box to avoid light, observing the dish under laser confocal conditions, and taking a picture.

9. The method for screening CTL epitopes by using an autonomously constructed SLA-2-HB01-pCDH/sT2 cell line as claimed in claim 5, wherein the step (4) comprises the following steps:

cell slide: taking 200 μ L of density of 2 × 10⁴The cells per mL are transferred to a laser confocal special culture dish, the inoculated cells are single cells, and the cells are uniformly spread in the dish by a cross shaking method; after 12-24h of inoculation, adopting biotin-labeled peptides, wherein the peptides are 100 mu g/mL, the beta 2m is 3 mu g/mL, and incubating for 18h at 37 ℃;

taking out the cells from the incubator, precooling the cells for washing for 2 times by 1 XPBS, fixing the cells for 20min at room temperature by 4% paraformaldehyde, then washing for 2 times by 1 XPBS, mixing 5% BSA, 5% skimmed milk powder and 2% FBS as a sealing solution, sealing for 10min at room temperature, and washing for 1 time by 1 XPBS;

③ adding beta 2m primary antibody, dyeing for 1h at 4 ℃, washing for 2 times by precooled 1 XPBS, and removing the unbound antibody;

fourthly, diluting with 1 XPBS at a ratio of 1:1000

647Donkey anti-mouse IgG protected from light for 1h at room temperature; washing with 1 × PBS for 2-3 times;

adding 1:10000 for dilution

550-streptavidin, keeping away from light at 4 ℃ for 1h, washing with 1 × PBS for 3-5 times, 3min each time, and removing the excess antibody components which are not combined;

sixthly, adding 0.1 percent of Triton-X100 into each dish, keeping out of the sun for 10min, washing for 2 times by using precooled 1 XPBS, sucking the surface, adding 5 percent of BSA, 5 percent of skim milk powder and 2 percent of FBS into each dish, mixing as a sealing solution, and sealing for 10min at room temperature in the absence of the sun;

seventhly, adding 1:10000 diluted DYLight405 into each dish, keeping out of the sun for 1h at room temperature, and washing with precooled 1 × PBS for 5 times for 3min each time;

adding 1mL of 1 XPBS, placing the mixture into a wet box to avoid light, observing the mixture under laser confocal conditions, and taking a picture.

Images