CN112501269B

CN112501269B - A method for rapid identification of high-affinity TCR antigen cross-reactivity

Info

Publication number: CN112501269B
Application number: CN202011473454.2A
Authority: CN
Inventors: 蓝勋; 王莉惠
Original assignee: Tsinghua University
Current assignee: Tsinghua University
Priority date: 2020-12-15
Filing date: 2020-12-15
Publication date: 2022-02-18
Anticipated expiration: 2040-12-15
Also published as: CN112501269A

Abstract

The invention relates to a method for rapidly identifying the cross-reactivity of high-affinity TCR antigens. The TCR and pMHC sequences are adjusted to a constitutive promoter for inducible expression, and the element is integrated and expressed into the yeast genome, comprising the following steps: Protein display on the surface of different mating types of yeast; yeast mating experiments using different mating types of yeast integrating TCR and pMHC; diploid screening and next-generation sequencing. The present invention can provide a method for rapidly identifying the cross-reactivity of high-affinity TCR antigens, which requires no pure protein, but only needs to synthesize DNA sequences. By using the Golden-gate cloning method, a more complete antigen mutation library can be obtained within one week, which is convenient and quick; The yeast mating system breaks through the limitation of screening at the cellular level, the operation steps are simplified, the antigen sequences that interact with specific TCRs can be obtained in a short time, and the experimental results are reliable.

Description

Method for rapidly identifying high-affinity TCR antigen cross-reactivity

Technical bell area

The invention relates to the field of biotechnology, in particular to a method for rapidly identifying high-affinity TCR antigen cross-reactivity.

Background

TCRs are molecules that specifically recognize antigens and mediate immune responses on the surface of T cells. In the adaptive immune system, the TCR specifically recognizes an antigenic molecule polypeptide (pMHC) presented by a histocompatibility complex molecule, thereby activating T cells and producing an immune effect. Tumor cell immunotherapy-TCR-T therapy is a therapeutic approach that utilizes T cells that specifically bind to a target tumor antigen to kill malignant cells. The high affinity TCR evolved in vitro can improve the anti-tumor capability of the modified T cell, but can also recognize non-target antigens due to the problem of antigen cross-reactivity, thereby causing serious side effects to patients.

Currently, one of the methods for studying TCR antigen cross-reactivity is to use purified MHC-antigen multimers for the study. The method can detect the T cell immune response of 15 antigens simultaneously by relying on the UV-induction to rapidly realize the antigen peptide replacement technology and the multicolor fluorescence coding technology. The Bentzen group identified the recognition of over 1000 different pmhcs by 16 different TCRs using the developed DNAbarcode labeled MHC multimer technology. However, such methods require purification of MHC tetrameric proteins, are time-consuming and labor-intensive, and it is difficult for an inexperienced subject group to rapidly construct the corresponding tetrameric proteins. The commercial purchase of tetramers of one epitope of an antigen takes more than 1 million, which makes the antigen screening throughput greatly limited.

The second approach is to use cells presenting the antigen of interest for the study. Screening was performed using APCs expressing neoantigens as developed by the Rosenberg group, and antigen expression methods could be achieved by loading neoantigen peptides or synthesizing tandem genes. In recent years, researchers have developed T-Scan and Cytosine technologies to improve the throughput of T-cell antigen recognition screening based on MHC technology presented by cells. Although this approach reflects the reality of TCR-antigen interactions in vivo very well, cell culture takes a lot of time and money.

The third approach is that some researchers, such as Garcia topic group and zhao hui min topic group, have succeeded in covalently linking peptide fragments with MHC class I or II to construct single-chain forms for display on the yeast surface, and without protein purification, a large number of pmhcs presenting different antigens can be obtained for studying the cross-reactivity of the TCR of interest. However, this method requires the preparation of TCR tetramers by in vitro purification of the TCR of interest, and the core technology is currently mastered by only a few subject groups, and there is no commercial product.

Klavins group reported a high throughput method for detecting protein-protein interactions by engineering the yeast mating system. During the mating process of yeasts, yeasts of different mating types grow in polarity under the action of semaphores, approach to each other to generate a 'shmoo' shape, and then are combined with each other through the agglutinin on cell walls at contact positions of the yeasts of different mating types to mediate downstream mating signals, so that cells are fused. The authors knocked out sag1 in the partner lectin that mediates cell fusion, mediated cell fusion by a foreign protein displayed outside the yeast cell wall, linked the strength of protein interaction to yeast mating, and characterized the interaction between proteins in high-throughput and quantitative manner without the need for purification of the proteins. The article realizes that in a one-time mating experiment, a protein interaction network comprising thousands of pairs of interactions is identified. However, the affinity of TCR-pMHC is low, the screening difficulty is higher, and the noise is higher. This approach has not been used for TCR-pMHC interactions.

In addition, in the aspects of TCR and pMHC display, researchers such as Kranz have succeeded in displaying the V region of a single-chain TCR on the surface of yeast by Linker linkage and obtaining 10 by random in vitro⁸The library of TCR variants of (a) rapidly screen for high affinity TCRs that bind to the target antigen. China researchers such as professor of Huangjun professor of Guangdong institute of medicine and others have also successfully achieved yeast display of single-chain TCR. The artificial bank building mode breaks through the screening limitation of a natural T cell bank, and increases the screening efficiency while maintaining the antigen specificity of the TCR. pMHC display also different laboratories are using this technology. Over 10 TCRs and pMHCs have been successfully displayed outside the yeast cell wall. However, the technology is that TCR or pMHC is used for yeast display, pMHC or TCR in the other direction exists in the form of tetramer protein for interaction screening, and no report is related to research of protein interaction in combination with yeast mating technology.

Based on this, it is important for those skilled in the art to design a method for rapidly identifying high affinity TCR antigen cross-reactivity that can break through the limitations of cell screening levels without relying on protein purification or cell culture.

Disclosure of Invention

The present invention aims to provide a method for rapidly identifying the cross-reactivity of the high-affinity TCR antigen against the deficiencies of the prior art, which does not need pure protein, breaks through the limitation of screening on the cellular level, and is more likely to obtain the antigen sequence interacting with the specific TCR in a short time. And a Golden Gate cloning method can be utilized to artificially construct a mutation library of pMHC, so that the test of the TCR antigen cross reaction activity is faster and more comprehensive.

In order to achieve the purpose, the invention provides the following technical scheme:

a method for rapidly identifying high affinity TCR antigen cross-reactivity, comprising the steps of:

the S1, TCR and pMHC sequences are adjusted to be constitutive promoters to induce expression, the elements are integrated and expressed into a yeast genome, and TCR and pMHC proteins are displayed on the surfaces of different mating yeast;

s2, carrying out yeast mating experiments by using yeast with different mating types integrating TCR and pMHC;

s3, diploid screening and next generation sequencing.

Preferably, the step of S1 includes:

firstly, constructing a TCR sequence to be researched into a scTCR form to obtain a peptide fragment mutation library of pMHC;

reconstructing a pMHC library to construct a required receiving vector ysynalpha _ DEST to obtain a carrier of the ysyna-TCR and the ysynalpha-pMHC;

integrating TCR into MATa yeast with knockout of Sag1 gene to obtain a yeast strain expressing TCR sequence;

integrating pMHC into a MATalpha yeast strain to obtain a display library of pMHC;

after integrated expression, the yeast can be induced to display the target TCR and pMHC by directly culturing in a complete culture medium YPD;

after overnight incubation, the cells were transferred, sampled at different time intervals for flow analysis, the TCR and pMHC display levels on the yeast surface were determined, and the highest display level time point was selected for subsequent mating experiments.

Preferably, the MATa yeast integrates and expresses red fluorescent protein at the same time, and red fluorescence can be detected in a subsequent flow experiment; the MATalpha yeast integrates and expresses a blue fluorescent protein at the same time, and blue fluorescence can be detected in a subsequent flow-type experiment.

Preferably, the method by which the TCR sequences are constructed into the scTCR format is a yeast-directed method; assembling in a V beta-linker (GSADDAKKDAAKKDGKS) -V alpha form;

the peptide fragment of the pMHC is formed by connecting a peptide fragment-MHC heavy chain light chain through a Linker sequence and performing fusion expression with the N end of AGA 2; the peptide segment is constructed by a PCR annealing extension mode after a long primer is synthesized;

the modification method of the required receiving vector ysynalpha _ DEST for constructing the pMHC library comprises the following steps: removing 5 BSAI sites on the vector backbone by synonymous mutation and introducing 2 BSAI sites at the position of peptide fragment integration on MHC;

the preparation method of the carrier of the ysyna-TCR and the ysynalpha-pMHC comprises the following steps: the synthetic TCR sequence and the ysyna _ DEST plasmid were separately digested with NheI and XhoI restriction enzymes; carrying out double enzyme digestion on a pMHC sequence and a modified ysynalpha _ DEST plasmid, carrying out enzyme digestion in a water bath, and then cutting a target fragment under an ultraviolet lamp by using DNA gel, and obtaining a corresponding fragment; connecting at room temperature; transformed into E.coli DH5 alpha.

Preferably, the specific procedures for integrating the TCR and pMHC sequences into the yeast genome are as follows:

after a carrier of the ysyna-TCR is obtained, enzyme digestion is carried out by PmeI, and fragments comprising ARS314+ TCR + TRP + fluorescent protein are cut off by enzyme; recovering the gel to obtain fragments; the carrier of the ysynalpha-pMHC peptide segment mutation library is cut by PmeI in the same way; cutting and recycling the rubber;

converting the TCR sequence into an MATa strain by using a conventional lithium acetate conversion method, and converting the pMHC sequence into an MATalpha strain;

screening single clones through SC-TRP plates;

the correct strain construction for integrating the TCR is to select a single clone to directly streak and culture the single clone on a YPD plate, and separate the single clone for subsequent experiments;

the antigen mutation library of pMHC, scrape all yeast colonies with the scraper, centrifugate, resuspend in SC-TRP culture medium; and (5) freezing and storing.

Preferably, the step of S2 includes:

overnight culturing the constructed strains expressing TCR and pMHC;

inoculating the strain cultured overnight, and performing yeast mating experiment after culturing;

and (3) controlling the OD conditions of Mate, including initial concentration, the alpha yeast input amount for displaying the TCR and the culture medium volume, performing a yeast mating experiment in a deep-well plate, and performing mixed culture to obtain a diploid signal formed by mediation of the TCR-pMHC.

Preferably, in the screening of the diploid in S3, irrelevant TCR is used as a negative control, the experimental group and the control group are operated simultaneously, and after the subsequent second generation sequencing, the difference between the experimental group and the control group is used as a threshold value, and the sequencing data is selected to be corrected.

Preferably, the process of screening for diploids comprises the steps of:

after incubation, directly taking 300ul-600ul bacterial liquid after mating experiments and washing for 2 times by using a culture medium of SC-Lys-Leu;

then adding into the culture medium of SC-Lys-Leu for culture.

Preferably, the culture medium added to SC-Lys-Leu is replaced by sorting diploids by flow, and the specific steps comprise: the cells were sorted and cultured in YPD medium.

Preferably, the specific steps of the second generation sequencing comprise:

centrifuging the obtained positive diploid to obtain a strain, and extracting a genome by a bead milling method;

specifically amplifying a target genome by using a forward primer and a reverse primer to introduce a sequencing joint sequence of the illumina;

after being recovered by utilizing zymoclean kit glue, the zymoclean kit glue is used for resuspension and is used as a template for the second round of PCR;

performing a second round of PCR, building libraries at two ends, and introducing a sequenced barcode sequence;

detecting the concentration of double-stranded DNA by utilizing the Qubit, and performing mixed sequencing on the fragments in equal quantity;

after obtaining the second generation sequencing data, the number of mutant sequences tested was compared to all sequences to obtain a ratio value, and all ratios were corrected by dividing them by TAX 8.

Compared with the prior art, the invention has the following beneficial technical effects:

1. according to the invention, TCR and pMHC sequences are regulated from an inducible promoter to a constitutive promoter for inducible expression, and the element is integrated and expressed into a yeast genome, so that the problems that the displaying ratio of TCR and pMHC on the yeast surface is reduced and the subsequent experimental results are influenced due to the fact that plasmids are easy to lose after multiple subcultures in a yeast complete culture medium YPD are solved. The display kurtosis of the improved TCR and pMHC on the yeast surface is obviously improved, and the detection has correct function, can be directly used for subsequent yeast mating experiments, and simplifies the subsequent operation process;

2. the invention modifies the pMHC antigen library receiving vector, and utilizes the Golden-gate cloning method to construct the antigen mutation library within a week, and the construction of the antigen mutation library does not need to purify protein, only needs to synthesize a DNA sequence, and is convenient and fast;

3. the invention realizes the identification of the interaction between the TCR and the pMHC by controlling the OD condition during the matching, and solves the problems that the interaction between the TCR and the pMHC is weak and a diploid signal cannot be detected;

4. according to the method, irrelevant TCR is directly designed to be used as negative control, the experimental group and the control group are operated simultaneously, the difference between the experimental group and the control group is used as a threshold value, and sequencing data is selected to be corrected, so that the false positive caused by low TCR-pMHC interaction is avoided.

Drawings

The invention is further illustrated with reference to the following figures and examples.

FIG. 1 is a schematic diagram of a screening system using yeast display and yeast mating techniques;

FIG. 2 is a constitutive promoter-induced, integrated TCR and pMHC element design;

FIG. 3 is a graph of the surface display of plasmid-and integrant-expressed protein yeasts;

FIG. 4 is a functional identification of a TCR displayed on the surface of yeast;

FIG. 5 is a graph showing the results of repeating the test of mating conditions of yeast in Klavins, flow cytometry and detection of diploid yeast formation;

FIG. 6 is a graph of the ratio of protein displayed on the surface of plasmid-inducible and constitutively expressed yeasts at different time points;

FIG. 7 is a graph showing the results of diploid signal formation mediated by TCR-pMHC interaction as detected by the yeast mating system after modification;

FIG. 8 is a schematic diagram illustrating peptide design using the SL9 peptide as an example;

FIG. 9 is a BSAI design sequence map of the positions of the peptide fragments of the recipient vector of the pMHC antigen library.

Detailed Description

Technical solutions of the present invention will be described in detail below by way of embodiments with reference to the accompanying drawings. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto.

Example 1

This example generally describes a method for rapidly identifying cross-reactivity of high affinity TCR antigens.

As shown in fig. 1, a method for rapidly identifying high affinity TCR antigen cross-reactivity comprises the steps of:

s1, adjusting the TCR and pMHC sequences into constitutive promoters to induce expression, integrating and expressing the elements into a yeast genome, and displaying the TCR and pMHC proteins on the surfaces of different mating yeast;

s3, diploid screening and next generation sequencing.

Further, the step of S1 includes:

Further, the MATa type yeast integrates and expresses red fluorescent protein at the same time, and red fluorescence can be detected in a subsequent flow type experiment; the MATalpha yeast integrates and expresses a blue fluorescent protein at the same time, and blue fluorescence can be detected in a subsequent flow-type experiment.

Furthermore, the method for constructing the scTCR form by the TCR sequence is a yeast targeting method; assembling in a V beta-linker (GSADDAKKDAAKKDGKS) -V alpha form;

as shown in the attached figure 9, the modification method of the receiving vector ysynalpha _ DEST required by the pMHC library construction comprises the following steps: removing 5 BSAI sites on the vector backbone by synonymous mutation and introducing 2 BSAI sites at the position of peptide fragment integration on MHC;

Further, the specific procedures for integrating the TCR and pMHC sequences into the yeast genome are as follows:

screening single clones through SC-TRP plates;

the correct strain construction of the integrated TCR is to select a single clone to directly streak and culture the single clone on a YPD plate for separating the single clone for subsequent experiments;

Further, the step of S2 includes:

overnight culturing the constructed strains expressing TCR and pMHC;

and (3) controlling the OD conditions of Mate, including initial concentration, the input amount of a-type yeast for displaying TCR, the input amount of alpha yeast for displaying pMHC and the volume of a culture medium, performing a yeast mating experiment in a deep-well plate, and performing mixed culture to obtain a diploid signal formed by mediation of TCR-pMHC.

Further, in the screening of the diploid in S3, irrelevant TCR is used as a negative control, the experimental group and the control group are operated simultaneously, and after the subsequent second-generation sequencing, the difference between the experimental group and the control group is used as a threshold value, and the sequencing data is selected to be corrected.

Further, the diploid screening process comprises the following steps:

then adding into the culture medium of SC-Lys-Leu for culture.

Further, the culture medium added into SC-Lys-Leu is replaced by flow sorting diploid, and the specific steps comprise: the cells were sorted and cultured in YPD medium.

Further, the second generation sequencing comprises the following specific steps:

Example 2

This embodiment is performed based on embodiment 1, and the same points as embodiment 1 are not repeated.

This example mainly describes a specific implementation method of a method for rapidly identifying cross-reactivity of high affinity TCR antigens, comprising the steps of:

s3, diploid screening and next generation sequencing.

Further, the step of S1 includes:

after overnight culture, the cells are transferred according to 0.1OD/ml, the cells are determined to be 0h, and flow analysis can be carried out for 4h, 24h and 48h in sequence, the display levels of TCR and pMHC on the surface of yeast are detected, and the time point (4h) with the highest display level is selected for subsequent mating experiments.

the modification method of the required receiving vector ysynalpha _ DEST for constructing the pMHC library comprises the following steps: the 5 BSAI sites on the vector backbone were removed by synonymous mutations and 2 BSAI sites were introduced at the position of peptide integration on the MHC.

The preparation method of the carrier of the ysyna-TCR and the ysynalpha-pMHC comprises the following steps: the synthetic TCR sequence and the ysyna _ DEST plasmid were separately digested with NheI and XhoI restriction enzymes; carrying out double enzyme digestion on a pMHC sequence and a modified ysynalpha _ DEST plasmid, carrying out enzyme digestion in a water bath at 37 ℃ for 3h, then using 1.5% DNA gel and 120V for 40min, cutting a target fragment under an ultraviolet lamp, and recovering a corresponding fragment by using a recovery kit; connecting at room temperature for 20 min; transformed into E.coli DH5 alpha;

after a carrier of the ysyna-TCR is obtained, enzyme digestion is carried out for 4h at 37 ℃ by PmeI, fragments comprising ARS314+ TCR + TRP + fluorescent protein are cut off by enzyme, and the fragments are obtained by glue recovery; the carrier of the ysynalpha-pMHC peptide fragment mutation library is cut by PmeI for 8 h; cutting and recycling the rubber;

screening single clones through SC-TRP plates;

the correct strain construction for integration of the TCR was to pick single clones and directly streak them onto YPD plates and isolate single clones for subsequent experiments.

pMHC antigen mutation library, with scraper scraping all yeast colony, 2000g centrifugation for 5 minutes, heavy suspension in SC-TRP medium;

freezing and storing 3OD/tube according to 100X of theoretical storage capacity; the final concentration of 15% glycerol was frozen to-80 ℃.

Further, the step of S2 includes:

overnight culturing the constructed strains expressing TCR and pMHC;

inoculating the strain cultured overnight from 0.1OD, culturing for 4h, and performing yeast mating experiment;

controlling OD conditions of Mate, taking 0.05OD/ML as an initial concentration, wherein the input amount of a type yeast displaying TCR is 0.0125OD, the input amount of alpha yeast displaying pMHC is 0.0375OD, the total culture medium is 1ML, carrying out yeast mating experiments in a 96-hole sharp-bottom deep-hole plate in a shaker set to 30 ℃ and 220RPM, and carrying out mixed culture for 22h to obtain a diploid signal formed by TCR-pMHC mediation.

Further, the diploid screening process comprises the following steps:

after 22h, 300ul-600ul of bacterial liquid after the direct mating experiment is washed for 2 times by using a culture medium of SC-Lys-Leu;

then adding into 3-20ml SC-Lys-Leu culture medium to culture for 36 h.

Further, the culture medium added into 3-20ml of SC-Lys-Leu for 36h is replaced by flow sorting diploid, and the specific steps comprise: 5000 cells were sorted and cultured in 1ml of YPD medium.

centrifuging the obtained positive diploid at 4000rpm for 10 minutes to obtain a strain, and extracting a genome by a bead milling method;

specifically amplifying a target genome by using Primer 1-F (SEQ ID NO.7) and a reverse Primer 1-R (SEQ ID NO.8) to introduce a sequencing joint sequence of the illumina;

after recovering the zymoclean kit gel, resuspending the gel at 30ul, and taking the gel as a template of the second round of PCR;

putting 300ng for second round PCR, building libraries at two ends and introducing a sequenced barcode sequence;

after second generation sequencing data are obtained, comparing the number of the detected mutant sequences with all the sequences to obtain a ratio value, and dividing all the ratios by TAX 8 to carry out correction;

the experiment was repeated 3-4 technical replicates each time and was independently repeated 2 times, each independent experiment analyzed results separately, with the fold of experimental/control >1 relative to control and P value <0.5 in at least one independent experiment as the screening threshold.

Example 3

This example mainly describes the design of devices that integrate the expression of TCR and pMHC.

In current studies, yeast surface display of TCR and pMHC utilize an a-lectin-gene of interest surface display system. The a-lectin is composed of a core subunit Aga1 and a binding subunit Aga2, wherein the core subunit Aga1 and the binding subunit Aga2 are connected through a disulfide bond, and the C end of the core subunit Aga1 is covalently bound with cell wall glucan. The TCR and pMHC are fused and expressed with the C end or N end of the Aga2 protein in the yeast a-lectin, the expression of the fusion protein is induced by GAL1 promoter, which is a galactose induction system, when yeast grows in a glucose medium, the transcription of GAL1 promoter is completely inhibited, cells are transferred into a galactose-containing medium to induce the production of an Aga2p fusion gene product, and the fusion gene product is transferred to the cell surface through a relevant secretion path. The element is expressed on pCton2 carrier, and is expressed in EBY100 strain in the form of plasmid. However, if a yeast mating experiment is subsequently required, a better mating effect can be achieved in the yeast complete medium YPD, thereby finally assisting the screening. However, if the plasmid is easily lost after several subcultures in yeast complete medium YPD, the TCR and pMHC display ratio on the yeast surface is reduced, and the subsequent experimental results are affected.

Therefore, unlike inducible promoter induction in previous studies, the TCR and pMHC sequences were expressed in plasmid form, in this example, the TCR and pMHC sequences were modified to constitutive promoters to induce expression, and the elements were expressed integrated into the yeast genome.

FIG. 2 shows the design of elements for integrating and expressing TCR and pMHC, and the left and right ARS314 in the figure represent 500bp sequences at both ends of the ARS314 site of the selected yeast genome as the homology arms for subsequent homologous recombination and integration of the elements into the yeast genome. pGPD is a constitutive promoter, and induces and expresses Aga2-scTCR C-terminal fusion protein and pMHC-Aga 2N-terminal fusion protein. TRP1 represents a reporter gene that pGPD drives expression for subsequent auxotrophic screening. mCherry represented the red fluorescent protein pTEF1 driven for expression. mTurquoise represents the blue fluorescent protein that pTEF1 drives expression.

In the embodiment, the TCR and pMHC sequences are adjusted from inducible promoter induction to constitutive promoter induction expression, and the element is integrated and expressed into the yeast genome, so that the problem that the subsequent experimental result is influenced due to the reduction of the TCR and pMHC display ratio on the yeast surface because plasmids are easily lost after multiple subcultures in a yeast complete culture medium YPD is solved.

Example 4

This embodiment is based on embodiment 1, and the same points as embodiment 1 will not be described again.

This example mainly describes the surface display condition and functional identification condition of plasmid-type and integrative expression protein yeast;

since AGA2 gene is followed by an HA tag, it is shown that the protein is displayed outside the wall if it can be stained with HA-FITC antibody. The 0.1OD bacterial liquid is centrifuged at 3000rpm for 5min, washed with PBS + 0.1% BSA for 1 time, stained with HA-FITC (1: 1000) in a 100ul system at room temperature for 30min, and analyzed for FITC fluorescence intensity and ratio by LSRFortessa SORP flow analyzer. The displayed kurtosis refers to the ratio of yeast expressing FITC fluorescence to the total yeast. In the same manner, yeast expressing 868TCR was stained with a tetramer of SL 9-HLA-A0201-PE (1: 200) and confirmed to function correctly by flow analysis.

FIG. 3 shows yeast transformed with 868, A6, SL9, tax sequences on the horizontal axis and HA tag expressing yeast in the ratio detected by flow on the vertical axis. The dots represent the yeast display ratio measured 22h after induction with the GAL1 promoter, plasmid-type expression and induction. The squares represent constitutive promoter induction, integrated form expression, yeast display rate cultured for 4 h; both time points were the highest peaked protein expression in the different forms of induction.

As shown in FIG. 3, by measuring the peak degree of TCR and pMHC displayed on the yeast surface, the optimized TCR and pMHC displayed on the yeast surface with efficiency as high as more than 90% compared with the plasmid expression is found.

At the same time, we used the tetramer of SL9-HLA-a 0201 to test whether the TCR displayed on the yeast surface functions correctly, and the results are shown in fig. 4.

The horizontal axis represents the staining intensity of HLA-A0201 SL9 tetramer coupled with PE fluorescent molecules on the surface of yeast cells expressing 868TCR by plasmid or integration, and fluorescence can be detected on the PE detection channel of the flow analyzer. The vertical axis represents the expression intensity of mCherry red fluorescent protein, and fluorescence can be detected by a PE-TexRed detection channel of a flow analyzer. The sampling time points were again plasmid-inducible 22h, YPD 4 h.

Consistent with the plasmid type, all TCRs that could be displayed on the surface could be stained with tetramers, indicating that the improved TCR expressed still had correct function.

Therefore, the display kurtosis of the improved TCR and pMHC on the surface is obviously improved, and the detection has correct functions and can be directly used for subsequent yeast mating experiments.

In the embodiment, the expression forms of TCR and pMHC are modified, the display kurtosis of the improved TCR and pMHC on the surface of yeast is obviously improved, the detection has correct function, and the improved TCR and pMHC can be directly used for subsequent yeast mating experiments, so that the subsequent operation process is simplified.

Example 5

This example mainly describes the comparison of the results of the prior art yeast mating test and the modified yeast mating test.

The identification of the interaction between proteins such as BCL2 and ligands has only been accomplished in previous reports, and this relatively weak interaction has not been tested for TCR and pMHC.

The procedure reported in the literature was first repeated, overnight cultured yeast strains displaying TCR and pMHC were cultured according to 1: 3 was added to 3ml of YPD, and the mixture was subjected to Mate experiment without controlling the OD number, and mixed-cultured at 220rpm and 30 ℃.

As shown in FIG. 5, the horizontal axis represents the expression intensity of mTurquoise blue fluorescent protein expressed in MATalpha strain detected by flow. The vertical axis represents the expression intensity of mCherry red fluorescent protein expressed in MATa strain detected by flow. The boxed area in the figure represents the population expressing bifluorescence, i.e., diploid. The values in the figure are the ratio of the diploid population in the population. Wherein bfl1-noxa is a known protein interaction reported in literature, and the mating conditions of the previous system are repeated, under which the 868TCR-SL9 HLA-A0201 positive interaction group does not form two-fold. Therefore, the current yeast mating experiments cannot detect diploid signals and cannot be used for the relatively weak interaction of TCR and pMHC.

In order to optimize the system for the detection of TCR-pMHC interaction, we first try to ensure that the peak expression levels of TCR and pMHC on the yeast surface are the highest. We examined protein display kurtosis at different time points for yeast strains expressing TCR and pMHC, and the results are shown in figure 6.

FIG. 6 shows the efficiency of protein display on the yeast surface, which is the ratio of HA-tag expressing yeast detected by flow. The horizontal axis represents yeast strains transformed with 868, A6, SL9, tax, the left panel represents plasmid-type expressing yeast, the circles represent samples taken at 22h induction, flow analysis was performed after staining, the squares represent samples taken at 46h induction, flow analysis was performed after staining, the triangles represent samples taken at 72h induction, and flow analysis was performed after staining. The right panel represents constitutively expressing yeast. The circles represent samples taken after 4h of incubation, flow analysis after staining, the squares represent samples taken after 46h of incubation, flow analysis after staining, the triangles represent samples taken after 72h of incubation, and flow analysis after staining.

It can be seen that the two proteins show the highest kurtosis when the strain is cultured to the exponential phase.

The overnight cultured strain was transferred from 0.1OD, and cultured for 4 hours before yeast mating test. In addition, the OD conditions at the time of mating were controlled to 0.05OD/ML as the starting concentration, wherein the input amount of TCR-displaying yeast type a was 0.0125OD, pMHC-displaying yeast alpha was 0.0375OD, and the total medium was 1ML, and the yeast mating test was carried out in a 96-well conical-bottomed deep-well plate. The results of the detection are shown in FIG. 7.

FIG. 7 left panel vertical axis percentage of diploid population in population after yeast mating experiment by flow analysis. Statistical analysis was performed after independent repetition of 7 Mate experiments. It was found that for the SL9 peptide fragment, significant enrichment was observed in the 868TCR interaction group. The right panel of FIG. 7 is a flow chart, which shows that distinct diploids (bifluorescent populations) can be generated.

It can be seen that the optimized yeast mating system detects the diploid signal of TCR-pMHC-mediated formation.

In the embodiment, the identification of the interaction between the TCR and the pMHC is realized by controlling the OD condition during the matching, and the problem that the interaction between the TCR and the pMHC is weak and a diploid signal cannot be detected is solved.

Example 6

This embodiment is performed on the basis of embodiment 1, and the same points as embodiment 1 are not repeated.

This example mainly describes the construction of a A6TCR (SEQ ID NO.2) and 868TCR (SEQ ID NO.1) as examples to demonstrate TCR sequences.

The initial source of A6TCR (SEQ ID NO.2) used in this example was a single-chain A6TCR with an affinity KD of 53nM, obtained by yeast evolution. The specific sequence is that the variable regions V beta 13 and V alpha 2 of A6 are connected through a linker sequence GSADDAKKDAAKKDGKS, and the fusion is expressed to the C terminal of AGA2 gene. Wherein the sequence is preceded by an in-frame HA tag sequence after AGA2 for subsequent detection of correct display in yeast.

The 868TCR (SEQ ID NO.1) used in this example was derived from a yeast-derived single-chain 868TCR with an affinity KD of 450pM, and similarly the variable regions V.beta.5 and V.alpha.2 of 868 were linked by a linker sequence of GSADDAKKDAAKKDGKS to fuse to the C-terminus of the AGA2 gene. Wherein the sequence is preceded by an in-frame HA tag sequence after AGA2 for subsequent detection of correct display in yeast.

Example 7

This example mainly describes the construction of peptides/HLA-a 0201 and SL9 peptides as examples to display pMHC sequences.

The peptide fragment-Linker (ggggggsgggggggs) - β 2M-Linker (ggggsggggsggggsggggs) -HLA-a × 0201 heavy chain (α 1 α 2 α 3, wherein HLA-a × 02: 01 heavy chain is modified by Y84A mutation, which allows Linker to be linked to β 2M at both ends of the peptide through the ends of the peptide binding groove, thereby producing a single chain trimer the heavy chain is truncated at S302) and HA tag-Linker (kllqasggggsggggggsggggs) -AGA 2.

The peptide segment is constructed by PCR annealing extension after long primer is synthesized. As shown in FIG. 8, BSAI restriction sites and 6 protection bases are introduced at both ends of the peptide fragment sequence of the target. Wherein the BSAI recognition site GGTCTC is outside the peptide segment and has the same BSAI cohesive end as the subsequent vector.

Taking SL9 Peptide as an example, as shown in FIG. 8, a forward primer F (SEQ ID NO.9) and a reverse primer Peptide-R (SEQ ID NO.10) were designed. The sl93 mutation library created in the present invention was also designed by synthesizing random mutation primers for NNK in the manner described above. The forward primer was Radom 3-F: (SEQ ID NO.11), the reverse primers were identical.

Example 8

This example expresses TCR and pMHC using the integrative recipient vector used by the Klavins topic group (young et al, 2017).

The vector introduces the homology arm of ARS314, TRP1 and fluorescent protein besides AGA2 gene. The integrated recipient vector for TCR expression comprises a series of elements: upstream and downstream 500bp homology arms of yeast ARS314 for genomic integration of subsequent elements; pGPD constitutive promoter, inducing the expression of AGA 2; introducing an HA tag after the Aga2 gene for detecting the expression of the AGA2 fusion protein; receiving a target gene sequence between NheI and XhoI, wherein a screening gene ccdB (suicide gene) of the escherichia coli is expressed at the position on a receiving vector, and the target gene can only be correctly integrated and replace the correct clone of the suicide gene to survive in the subsequent escherichia coli transformation screening; the pGPD constitutive promoter TPR1 gene is used for screening subsequent transgenic yeast; pTEF1 constitutively promoted expression of the red fluorescent protein.

Two PMEI restriction sites are designed at both ends of the sequence of all the elements for linearization and integration of the sequence, and all the vectors contain an ampicillin resistance gene for subsequent Escherichia coli transformation and screening.

Similarly, the integrated receptor vector for pMHC expression, expressed blue fluorescent protein, was distinguished from the red fluorescent protein on the TCR expression vector. In addition, to speed up the construction of the antigen mutation library, the pMHC library-receiving vector was engineered to remove 5 BSAI sites on the vector backbone by synonymous mutations and to introduce 2 BSAI sites at the site of peptide stretch integration on the MHC, as shown in fig. 9.

For specific TCRs and pmhcs, such as 868TCR (SEQ ID No.1), a6TCR (SEQ ID No.2) and SL9-HLA-a 0201(SEQ ID No.3), TAX-HLA-a 0201(SEQ ID No.4), first, the synthesized TCR sequence and the ysyna _ DEST plasmid (SEQ ID No.5) were separately digested with NheI and XhoI restriction enzymes; the pMHC sequence and the ysynalpha _ DEST plasmid (SEQ ID NO.6) are subjected to double enzyme digestion, after the enzyme digestion is carried out in a water bath at 37 ℃ for 3h, 1.5 percent DNA gel is used, the solution runs for 40min at 120V, a target fragment is cut under an ultraviolet lamp, and the corresponding fragment is recovered by using a Shanghai Bocai recovery kit. The ligation was performed at room temperature for 20min under the following ligation conditions. Transformed into Escherichia coli DH5 alpha to finally obtain the carrier of ysyna-TCR and ysynalpha-pMHC. The correct vector was determined by receiving sequencing primers at both ends of the vector.

The conditions for the cleavage are as follows:

10XCutsmat	1.5
		Plasmid	1ug
NheI	0.4
		XhoI	0.4
ddH2O	Up to 15ul

the conditions for attachment are as follows:

the library amplification conditions for constructing an antigen mutation library using the Golden Gate cloning method are as follows:

2xmix	50
		Primer F(Random3-F)	10
Primer R	10
		H2O	30

after obtaining PCR, the column recovery was performed, followed by Golden Gate ligation, under the following conditions:

10xT4 ligase buffer	5
		10xBSA(1mg/ml)	5
PCR	total (262.8ng)
		Plasmid(40ng/ul)	1
BSAI	1.25
		T4 ligase	0.5
H2O	25.25

PCR and connection systems are accumulated as much as possible, at least P is one 50ul system, and 1 50ul system is connected. After the fragments are ligated, the ligation is performed by ethanol precipitation, the 3XNaAc ethanol precipitation step is referred to for DNA ethanol precipitation, and finally 8ul is used for resuspending the DNA product. Then, after the plasmid is transformed into DH5 alpha by a conventional method, the plasmid is greatly extracted to obtain a final mutation library, and the final library capacity is about 7.8x10⁵. With the increase of the theoretical storage capacity of the designed antigen mutation library, the system can be expanded, enough mutant strains are accumulated after multiple experiments, and the theory can be quickly taken to 10⁸The library of antigen mutations of (a).

Example 9

To further confirm that this method can achieve screening at the antigen mutant pool level, several single-base mutants at amino acid position 3 of SL9 (3F/3L/3W/3H/3I/3D/3S/3G) and single-base mutant at amino acid position 8 of TAX (TAX 8H/8V/8K) were constructed in this example

First, we constructed SL 9-related different mutants, SL9, 3F, 3L, 3H, 3W, 3I, 3D, 3G, 3D, TAX 8H, TAX 8V, TAX 8K, TAX 8x 14 strains, mixed in equal amounts, and performed yeast mating experiments, and screened for diploids and then performed next generation sequencing. The experiment is repeated for 3-4 technical wells each time, and is independently repeated for 2 times, and the results are respectively analyzed in each independent experiment. First, based on the second generation sequencing result, the ratio of each peptide fragment in each group is calculated, i.e. the number of sequences/total sequence measured for each peptide fragment is calculated. The resulting ratio/Tax 8 was then used for data correction, considering that the strong affinity peptide competitively affected the binding of other peptides to TCRs, affecting the screening results, whereas Tax 8 was not expected to interact with either the 868TCR or the a6 TCR. Relative to the control group, we can obtain significantly enriched peptide fragments with the fold of experimental group/control group >1.1 and P value <0.5 in at least one independent experiment as the screening threshold. Wherein the positive control SL9/TAX can be enriched. Other 3F/3L/3W/3H/3I were also verified in subsequent experiments to be recognized by the corresponding TCR.

The enriched peptide fragments that can be screened in the mini-pool screening are shown in the following table:

in the table, 8681/2 represents the independent experiment of two mutation pools and 868TCR screening, and A61/2 represents the independent experiment of two mutation pools and A6TCR screening. First, the sequence/total sequence number measured for each peptide fragment was calculated in 868TCR experimental group and a control group of a6TCR, the ratio in the figure is the ratio of the numbers of rises in experimental group and control group in respective experiments, Pvalue is the statistical analysis of two-tailed T-Test performed on rise, Pvalue <0.05 represents significant difference.

Then, in order to determine that the method is suitable for screening random mutation libraries of peptide fragments, a SL9 single-base random mutation library is established by utilizing the technologies of Golden Gate cloning and the like, 868TCR is used as a positive group, A6TCR is used as a control group, and a second-generation sequencing detection result is obtained after diploid screening is screened. The results show that we can find that the positive control SL9 is enriched with a fold >1.1 of experimental/control group, and 3 as negative control, indeed, cannot be screened out. In addition, 3F/SL9/3L/3N/3Q/3V/3H/3WSL9/3L/3Q can be screened, and SL9/3L/3Q can be obviously enriched if the screening threshold is further set as the P value <0.5 in at least one independent experiment.

Enriched peptide fragments that could be screened in the randomized pool screening are shown in the following table:

in the table, 8681/2 represents the independent experiment of two mutation pools and 868TCR screening, and A61/2 represents the independent experiment of two mutation pools and A6TCR screening. First, the sequence/total sequence number measured for each peptide fragment was calculated in 868TCR experimental group and a control group of a6TCR, the ratio in the figure is the ratio of the numbers of rises in experimental group and control group in respective experiment, the figure shows mutants with the ratio larger than that in two experiments, Pvalue is a two-tailed T-Test statistical analysis of rise, Pvalue <0.05 represents a significant difference.

Previous studies found that the peptide fragment with relatively high affinity was SL9/3W/3F/3H/3L, with the lowest affinity for 3T. The selection of peptides enriched better is the peptide with higher affinity, and the ratio of weak relative affinity to the control group is also high, while 3D/3S not enriched is almost without affinity. The coincidence degree of the peptide fragment and the peptide fragment screened by the existing literature is high, and the accuracy can reach 100% after verification.

In conclusion, the method provided by the invention has theoretical and practical operation feasibility.

Example 10

This example describes the steps of diploid screening by way of example using SL 9.

The mutation pool of SL9 at base 3 was removed from-80 ℃ and added to 1ml of YPD, centrifuged at 2.8g for 3min, washed once with 1ml of YPD, resuspended in 50ml of YPD, and cultured in a conical flask for 25 hours to make it larger than 8 OD. 5OD bacteria are taken out to 5ML of YPD culture medium, and are transferred from 0.1OD/ML to 5ML of culture medium after being cultured overnight, and OD is controlled to be 0.4-0.5 as much as possible for subsequent experiments. Then at least 600ul of the bacterial liquid is firstly washed for 2 times by using the culture medium of SC-Lys-Leu, and then added into 20ml of the culture medium of SC-Lys-Leu for culturing for 36h for diploid screening.

Example 11

This example mainly describes the method of DNA extraction.

And centrifuging the obtained positive diploid at 4000rpm for 10 minutes to obtain a strain, and extracting a genome by a bead milling method. 100ul of yeast lysate, 2% Triton X-100, 1% SDS, 100mM sodium chloride, 10mM Tris.CL pH 8.0, 1mM EDTA, pH 8.0 was added, after vortexing, approximately 100ul of disrupted beads were added, 200ul of phenol chloroform isopropanol (25: 24: 1) then the mixture is shaken for 30 minutes on a shaking instrument, 100ul of sterilized deionized water is added, and after shaking and mixing, the mixture is centrifuged at 13000rpm for 10 minutes. Then 180ul of the intermediate layer and a new EP tube are sucked, and 500ul of frozen absolute ethyl alcohol is added to the intermediate layer and the new EP tube for sedimentation for 10min at the temperature of minus 20 ℃. Then centrifuging at 12000rpm for 5 minutes, sucking the supernatant, adding 500ul 70% absolute ethyl alcohol, reversing and mixing evenly, centrifuging, sucking the supernatant, drying at room temperature for 10 minutes, and adding 100ul sterile deionized water for resuspension. And (4) constructing subsequent second generation sequencing. With the increase of bacterial liquid, the system for extracting the genome can be amplified to 200ul of lysate for cracking in an equal ratio. Others are all increasing proportionally.

The present invention has been illustrated by the above embodiments, but it should be understood that the above embodiments are for illustrative and descriptive purposes only and are not intended to limit the invention to the scope of the described embodiments. It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention, including combinations and subcombinations of features. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> Qinghua university

<120> a method for rapidly identifying high affinity TCR antigen cross reactivity

<160> 11

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1161

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atgcagttac ttcgctgttt ttcaatattt tctgttattg cttcagtttt agcacaggaa 60

ctgacaacta tatgcgagca aatcccctca ccaactttag aatcgacgcc gtactctttg 120

tcaacgacta ctattttggc caacgggaag gcaatgcaag gagtttttga atattacaaa 180

tcagtaacgt ttgtcagtaa ttgcggttct cacccctcaa caactagcaa aggcagcccc 240

ataaacacac agtatgtttt taaggacaat agctcgacga ttgaaggtag atacccatac 300

gacgttccag actacgctct gcaggctagt ggtggaggag gctctggtgg aggcggtagc 360

ggaggcggag ggtcggctag cgaagctggt gttactcaat ctccaactca tttgattaaa 420

actagaggtc aacaagttac tttgagatgt tctccaaaat ctggtcatga tactgtttct 480

tggtatcaac aagctttggg tcaaggtcca caattcatct ttcaatatgt tagaggtgaa 540

gaaagacaac gtggtaactt cccagataga ttttctggtc atcaataccc aaactattct 600

tctgaattga acattaacgc tttgttgttg ggtgactctg ctttgtattt gtgtgcttct 660

tctgatactg tttcttatga acaatatttt ggtccaggta ttagattgac tgttactgaa 720

gatttgaaaa atggttctgc tgatgatgct aaaaaagatg ctgctaagaa agatggtaaa 780

tctcaaaaag aagttgaaca aaattctggt ccattgtctg ttccagaagg tgctattgct 840

tctttgaact gtacttactc tgatagaggt tctcaatctt tcttttggta cagacaatac 900

tctggtaaat ctccagaatt gatcatgtcc atctactcta atggtgacaa agaagatggt 960

agattcactg ctcaattgaa caaggcttct caatacatct ctttgttgat cagagattct 1020

aagttgtctg attctgctac ttatttgtgt gctgttagag gtgctcatga ttatgctttg 1080

aattttggta aaggtacttc tttgttggtt actccacata ttggatccga acaaaagctt 1140

atttctgaag aggacttgta a 1161

<210> 2

<211> 1167

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atgcagttac ttcgctgttt ttcaatattt tctgttattg cttcagtttt agcacaggaa 60

ctgacaacta tatgcgagca aatcccctca ccaactttag aatcgacgcc gtactctttg 120

tcaacgacta ctattttggc caacgggaag gcaatgcaag gagtttttga atattacaaa 180

tcagtaacgt ttgtcagtaa ttgcggttct cacccctcaa caactagcaa aggcagcccc 240

ataaacacac agtatgtttt taaggacaat agctcgacga ttgaaggtag atacccatac 300

gacgttccag actacgctct gcaggctagt ggtggaggag gctctggtgg aggcggtagc 360

ggaggcggag ggtcggctag caatgcggga gtaactcaaa cacctaaatt ccaagttttg 420

aagacaggtc aaagtatgac gttacaatgt gcccaagata tgaaccatga atacatggca 480

tggtatagac aagatccagg tatgggttta agattgattc attactctgt cggtgtaggt 540

ataacagatc aaggagatgt cccagacgga tataaagtta gtagatcgac tacagaggat 600

ttcccattaa gactattatc cgctgctccc tcacaaacta gcgtttactt ttgtgcatcc 660

agaccagggt tgatgagtgc ccaaccagaa ctatattttg gtccaggcac ccggttaact 720

gttacagaag atttgataaa tggaagcgca gatgatgcaa agaaagatgc agctaagaaa 780

gatggtaaat ctcaaaagga ggtagaacaa aattctggac cactatcagt tccagaaggt 840

gcgattgctt ctttgaactg tacttactca gatagaggct cccaatcatt tttctggtat 900

agacagtatt caggcaaaag cccagaattg atcatgtcta tctatagcaa cggtgataag 960

gaggacggtc ggtttactgc tcaattgaat aaggcttctc aatatgtgtc cttattgatc 1020

agagacagtc aaccgtctga ttcagccaca tatctatgtg cagtgactac cgattcatgg 1080

ggtaaactac aatttggagc aggtacgcag gtggtagtga ctccagatgg atccgaacaa 1140

aagcttattt ctgaagagga cttgtaa 1167

<210> 3

<211> 1623

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atgcagttac ttcgctgttt ttcaatattt tctgttattg ctagcgtttt agcatctttg 60

tataatactg ttgctacttt gggtggtggt ggttctggtg gtggtggttc tggtggtggt 120

ggttctattc agaggactcc aaaaattcag gtctactcta gacatccagc tgagaatggt 180

aagtctaact tcttgaactg ctacgtatct ggtttccatc catctgatat tgaagtcgac 240

ttgttgaaaa acggtgagag gatcgaaaaa gttgagcact ctgacttgtc tttttctaag 300

gactggtcct tctacttgtt gtactacacc gagttcactc caactgaaaa ggatgaatac 360

gcttgcaggg ttaatcatgt cactttgtcc cagccaaaaa ttgtcaagtg ggacagagat 420

atgggtggtg gtggttctgg tggtggtggt tctggtggtg gtggttctgg tggtggtggt 480

tctggttctc attctatgag gtacttcttt acttctgtct ctagaccagg tagaggtgaa 540

ccaagattta ttgctgtcgg ttacgtcgat gatactcagt tcgtcaggtt tgattctgat 600

gctgcttctc aaagaatgga accaagagct ccatggattg aacaagaagg tccagaatat 660

tgggatggtg aaaccaggaa agttaaagct cactcccaaa ctcatagggt tgatttgggt 720

actttgagag gtgcttacaa ccaatctgaa gctggttctc atactgttca aaggatgtac 780

ggttgtgatg ttggttctga ttggagattc ttgaggggtt accaccaata tgcttatgac 840

ggtaaggatt acattgcttt gaaggaagac ttgaggtctt ggactgctgc tgatatggct 900

gctcaaacca ctaaacataa gtgggaagct gctcatgttg ctgaacaatt gagagcttac 960

ttggaaggta cttgcgttga atggttgaga aggtacttgg aaaacggtaa ggagactttg 1020

caaaggactg atgctccaaa aactcacatg actcatcatg ctgtctctga tcatgaagct 1080

actttgagat gttgggcttt gtctttttat cccgctgaga ttactttgac ttggcagaga 1140

gatggtgaag atcagactca agatactgaa ttggtcgaaa ctagaccagc tggtgatggt 1200

acttttcaaa aatgggctgc tgttgttgtt ccatctggtc aggaacaaag atacacttgt 1260

cacgtccaac atgaaggttt gccaaaacca ttgactttga gatgggaacc atcttctgga 1320

tcctacccat acgacgttcc agactacgct aagcttctgc aggctagtgg tggaggaggc 1380

tctggtggag gcggtagcgg aggcggaggg tcgcaggaac tgacaactat atgcgagcaa 1440

atcccctcac caactttaga atcgacgccg tactctttgt caacgactac tattttggcc 1500

aacgggaagg caatgcaagg agtttttgaa tattacaaat cagtaacgtt tgtcagtaat 1560

tgcggttctc acccctcaac aactagcaaa ggcagcccca taaacacaca gtatgttttt 1620

taa 1623

<210> 4

<211> 1623

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atgcagttac ttcgctgttt ttcaatattt tctgttattg ctagcgtttt agcattgttg 60

tttggttatc cagtttatgt tggtggtggt ggttctggtg gtggtggttc tggtggtggt 120

ggttctattc agaggactcc aaaaattcag gtctactcta gacatccagc tgagaatggt 180

aagtctaact tcttgaactg ctacgtatct ggtttccatc catctgatat tgaagtcgac 240

ttgttgaaaa acggtgagag gatcgaaaaa gttgagcact ctgacttgtc tttttctaag 300

gactggtcct tctacttgtt gtactacacc gagttcactc caactgaaaa ggatgaatac 360

gcttgcaggg ttaatcatgt cactttgtcc cagccaaaaa ttgtcaagtg ggacagagat 420

atgggtggtg gtggttctgg tggtggtggt tctggtggtg gtggttctgg tggtggtggt 480

tctggttctc attctatgag gtacttcttt acttctgtct ctagaccagg tagaggtgaa 540

ccaagattta ttgctgtcgg ttacgtcgat gatactcagt tcgtcaggtt tgattctgat 600

gctgcttctc aaagaatgga accaagagct ccatggattg aacaagaagg tccagaatat 660

tgggatggtg aaaccaggaa agttaaagct cactcccaaa ctcatagggt tgatttgggt 720

actttgagag gtgcttacaa ccaatctgaa gctggttctc atactgttca aaggatgtac 780

ggttgtgatg ttggttctga ttggagattc ttgaggggtt accaccaata tgcttatgac 840

ggtaaggatt acattgcttt gaaggaagac ttgaggtctt ggactgctgc tgatatggct 900

gctcaaacca ctaaacataa gtgggaagct gctcatgttg ctgaacaatt gagagcttac 960

ttggaaggta cttgcgttga atggttgaga aggtacttgg aaaacggtaa ggagactttg 1020

caaaggactg atgctccaaa aactcacatg actcatcatg ctgtctctga tcatgaagct 1080

actttgagat gttgggcttt gtctttttat cccgctgaga ttactttgac ttggcagaga 1140

gatggtgaag atcagactca agatactgaa ttggtcgaaa ctagaccagc tggtgatggt 1200

acttttcaaa aatgggctgc tgttgttgtt ccatctggtc aggaacaaag atacacttgt 1260

cacgtccaac atgaaggttt gccaaaacca ttgactttga gatgggaacc atcttctgga 1320

tcctacccat acgacgttcc agactacgct aagcttctgc aggctagtgg tggaggaggc 1380

tctggtggag gcggtagcgg aggcggaggg tcgcaggaac tgacaactat atgcgagcaa 1440

atcccctcac caactttaga atcgacgccg tactctttgt caacgactac tattttggcc 1500

aacgggaagg caatgcaagg agtttttgaa tattacaaat cagtaacgtt tgtcagtaat 1560

tgcggttctc acccctcaac aactagcaaa ggcagcccca taaacacaca gtatgttttt 1620

taa 1623

<210> 5

<211> 9230

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ttcttttcga tgaagatggt tctcctgcag ctatgttgct ttctatatca gcgattggac 60

ctggagtctg agagtcagcg tcatcgttta agtcaccggt tggcgtttct tgtttcaatt 120

catctgtctg taatgtgtag atcaaatttt tcaattcgtt ataatctagg taatgattct 180

gccattcagg gacagcgttg tatttgagaa agtgtgagaa tctcatttcg aggcacaata 240

atagccgcct ttaccgtagt tttgctgcac ctttatctga gagctgactg cttttttggt 300

gtgaaacact gttttctggt aataattttt caatgcatcg gattactttt cccacgtgcg 360

aaatcatcaa ttaattagat tgaaaaaagg gtaagggaaa ataagaaaga ggcggtagcc 420

taaagatacg gtaattgaaa cgtttcctat gcacaatctt aaaccttttt aggtaattga 480

ttaagttgac tgtaatatct gccgatacga aggttttctc cagcgagttt atcattatca 540

atactcgcca tttcaaagaa tacgtaaata attaatagta gtgattttcc taactttatt 600

tagtcaaaaa attagccttt taattctgct gtaacccgta catgcccaaa atagggggcg 660

ggttacacag aatatataac atcgtaggtg tctgggtgaa cagtttattc ctggcatcca 720

ctaaatataa tggagcccgc tttttaagct ggcatccaga aaaaaaaaga atcccagcac 780

caaaatattg ttttcttcac caaccatcag ttcataggtc cattctctta gcgcaactac 840

agagaacagg ggcacaaaca ggcaaaaaac gggcacaacc tcaatggagt gatgcaacct 900

gcctggagta aatgatgaca caaggcaatt gacccacgca tgtatctatc tcattttctt 960

acaccttcta ttaccttctg ctctctctga tttggaaaaa gctgaaaaaa aaggttgaaa 1020

ccagttccct gaaattattc ccctacttga ctaataagta tataaagacg gtaggtattg 1080

attgtaattc tgtaaatcta tttcttaaac ttcttaaatt ctacttttat agttagtctt 1140

ttttttagtt ttaaaacacc agaacttagt ttcgacggat tctagaacta gtggatctac 1200

aaaatgcagt tacttcgctg tttttcaata ttttctgtta ttgcttcagt tttagcacag 1260

gaactgacaa ctatatgcga gcaaatcccc tcaccaactt tagaatcgac gccgtactct 1320

ttgtcaacga ctactatttt ggccaacggg aaggcaatgc aaggagtttt tgaatattac 1380

aaatcagtaa cgtttgtcag taattgcggt tctcacccct caacaactag caaaggcagc 1440

cccataaaca cacagtatgt ttttaaggac aatagctcga cgattgaagg tagataccca 1500

tacgacgttc cagactacgc tctgcaggct agtggtggag gaggctctgg tggaggcggt 1560

agcggaggcg gagggtcggc tagccatatg caagtttgta caaaaaagct gaacgagaaa 1620

cgtaaaatga tataaatatc aatatattaa attagatttt gcataaaaaa cagactacat 1680

aatactgtaa aacacaacat atccagtcac tatggcggcc gcattaggca ccccaggctt 1740

tacactttat gcttccggct cgtataatgt gtggattttg agttaggatc cgtcgagatt 1800

ttcaggagct aaggaagcta aaatggagaa aaaaatcact ggatatacca ccgttgatat 1860

atcccaatgg catcgtaaag aacattttga ggcatttcag tcagttgctc aatgtaccta 1920

taaccagacc gttcagctgg atattacggc ctttttaaag accgtaaaga aaaataagca 1980

caagttttat ccggccttta ttcacattct tgcccgcctg atgaatgctc atccggaatt 2040

ccgtatggca atgaaagacg gtgagctggt gatatgggat agtgttcacc cttgttacac 2100

cgttttccat gagcaaactg aaacgttttc atcgctctgg agtgaatacc acgacgattt 2160

ccggcagttt ctacacatat attcgcaaga tgtggcgtgt tacggtgaaa acctggccta 2220

tttccctaaa gggtttattg agaatatgtt tttcgtctca gccaatccct gggtgagttt 2280

caccagtttt gatttaaacg tggccaatat ggacaacttc ttcgcccccg ttttcaccat 2340

gggcaaatat tatacgcaag gcgacaaggt gctgatgccg ctggcgattc aggttcatca 2400

tgccgtctgt gatggcttcc atgtcggcag aatgcttaat gaattacaac agtactgcga 2460

tgagtggcag ggcggggcgt aaacgccgcg tggatccggc ttactaaaag ccagataaca 2520

gtatgcgtat ttgcgcgctg atttttgcgg tataagaata tatactgata tgtatacccg 2580

aagtatgtca aaaagaggta tgctatgaag cagcgtatta cagtgacagt tgacagcgac 2640

agctatcagt tgctcaaggc atatatgatg tcaatatctc cggtctggta agcacaacca 2700

tgcagaatga agcccgtcgt ctgcgtgccg aacgctggaa agcggaaaat caggaaggga 2760

tggctgaggt cgcccggttt attgaaatga acggctcttt tgctgacgag aacaggggct 2820

ggtgaaatgc agtttaaggt ttacacctat aaaagagaga gccgttatcg tctgtttgtg 2880

gatgtacaga gtgatattat tgacacgccc gggcgacgga tggtgatccc cctggccagt 2940

gcacgtctgc tgtcagataa agtctcccgt gaactttacc cggtggtgca tatcggggat 3000

gaaagctggc gcatgatgac caccgatatg gccagtgtgc cggtctccgt tatcggggaa 3060

gaagtggctg atctcagcca ccgcgaaaat gacatcaaaa acgccattaa cctgatgttc 3120

tggggaatat aaatgtcagg ctcccttata cacagccagt ctgcaggtcg accatagtga 3180

ctggatatgt tgtgttttac agtattatgt agtctgtttt ttatgcaaaa tctaatttaa 3240

tatattgata tttatatcat tttacgtttc tcgttcagct ttcttgtaca aagtgggtct 3300

cgaggggggc ggatccgaac aaaagcttat ttctgaagag gacttgtaat agtgagcagg 3360

catcgagtga agtcaactat ataactgtct agaaataaat tttttcaaac cttaaccaga 3420

ttcgaaaagc ggcnnnnnnn nnnnnnnngc ttcaataaag gagcgagcac ccgataactt 3480

cgtatagcat acattatacg aacggtacat ttaatagaac agcatcgtaa tatatgtgta 3540

ctttgcagtt atgacgccag atggcagtag tggaagatat tctttattga aaaatagctt 3600

gtcaccttac gtacaatctt gatccggagc ttttcttttt ttgccgatta agaattaatt 3660

cggtcgaaaa aagaaaagga gagggccaag agggagggca ttggtgacta ttgagcacgt 3720

gagtatacgt gattaagcac acaaaggcag cttggagtat gtccgttatt aacttcacgg 3780

gttccagcgg gccattagtt aaagtttgtg gccttcaatc aacggaagca gctgaatgcg 3840

cactagacag tgatgctgac cttttaggta ttatttgtgt tcccaacagg aagcgtacta 3900

tagaccccgt aattgctaga aaaatctcct ctttggttaa ggcttacaag aactcatccg 3960

gtactcctaa gtatttggtt ggtgttttta ggaaccagcc aaaagaagat gtattagctt 4020

tggttaatga ctatggtatt gacattgttc aattgcatgg cgacgaaagt tggcaagagt 4080

accaggaatt cttgggcttg cctgttataa agcgtttagt ttttccaaag gattgtaaca 4140

tattattatc cgcagcctca caaaaaccgc atagcttcat cccgctgttt gattcagaag 4200

ccggtggtac cggcgaatta ttagattgga actccattag cgattgggtg ggtcgtcagg 4260

aaagccccga aagtcttcac tttatgttag ccggcgggtt aacaccagaa aatgttggtg 4320

atgctttacg tttgaatggt gttattggtg tggacgtgtc cgggggtgtt gagaccaatg 4380

gggtcaaaga ctctaataag attgctaact tcgtgaaaaa tgccaaaaag tgagcgaatt 4440

tcttatgatt tatgattttt attattaaat aagttataaa aaaaataagt gtatacaaat 4500

tttaaagtga ctcttaggtt ttaaaacgaa aattcttatt cttgagtaac tctttcctgt 4560

aggtcaggtt gctttctcag gtatagcatg aggtcgctct tattgaccac acctccagaa 4620

gcgaggcgaa taaaggtggc caaattaaag ccttcgagcg tcccaaaacc ttctcaagca 4680

aggttttcag tataatgtta catgcgtaca cgcgtctgta cagaaaaaaa agaaaaattt 4740

gaaatataaa taacgttctt aatactaaca taactataaa aaaataaata gggacctaga 4800

cttcaggttg tctaactcct tccttttcgg ttagagcgga tgtgggggga gggcgtgaat 4860

gtaagcgtga cataactaat tgaccgaggt cgacggtatc atcattgatg tccaccacct 4920

ggtccaccct tgtacagctc gtccatgccg ccggtggagt ggcggccctc ggcgcgttcg 4980

tactgttcca cgatggtgta gtcctcgttg tgggaggtga tgtccaactt gatgttgacg 5040

ttgtaggcgc cgggcagctg cacgggcttc ttggccttgt aggtggtctt gacctcagcg 5100

tcgtagtggc cgccgtcctt cagcttcagc ctctgcttga tctcgccctt cagggcgccg 5160

tcctcggggt acatccgctc ggaggaggcc tcccagccca tggtcttctt ctgcattacg 5220

gggccgtcgg aggggaagtt ggtgccgcgc agcttcacct tgtagatgaa ctcgccgtcc 5280

tgcagggagg agtcctgggt cacggtcacc acgccgccgt cctcgaagtt catcacgcgc 5340

tcccacttga agccctcggg gaaggacagc ttcaagtagt cggggatgtc ggcggggtgc 5400

ttcacgtagg ccttggagcc gtacatgaac tgaggggaca ggatgtccca ggcgaagggc 5460

agggggccac ccttggtcac cttcagcttg gcggtctggg tgccctcgta ggggcggccc 5520

tcgccctcgc cctcgatctc gaactcgtgg ccgttcacgg agccctccat gtgcaccttg 5580

aagcgcatga actccttgat gatggccatg ttatcctcct cgcccttgct caccattttg 5640

tagatccact agttctagaa tccgtttgta attaaaactt agattagatt gctatgcttt 5700

ctttctaatg agcaagaagt aaaaaaagtt gtaatagaac aagaaaaatg aaactgaaac 5760

ttgagaaatt gaagaccgtt tattaactta aatatcaatg ggaggtcatc gaaagagaaa 5820

aaaatcaaaa aaaaaaattt tcaagaaaaa gaaacgtgat aaaaattttt attgcctttt 5880

tcgacgaaga aaaagaaacg aggcggtctc ttttttcttt tccaaacctt tagtacgggt 5940

aattaacgac accctagagg aagaaagagg ggaaatttag tatgctgtgc ttgggtgttt 6000

tgaagtggta cggcgatgcg cggagtccga gaaaatctgg aagagtaaaa aaggagtaga 6060

aacattttga agctatggtg tgtgggggat cacttgtggg ggattgggtg tgatgtaagg 6120

attcgcggtc ctcgaaaatt aaaagtccaa cgcgcctgtt gcttcctatg tgatatgtat 6180

tatatgtaat atgcataaat atatctactg cattgtattt tgaacgtaca aagtatgcat 6240

tgtgtccctg aaaaccactg agttgcccct ttctttgaac agttctgttt ttaggtaagc 6300

cttggaacct tagctatact gacttgatac gccctgtctt tgttgtcgtt atataaagtg 6360

tgcaccttta tcatagcccc taataagctt cttgcagtag tatttgaaca gtgtatatct 6420

tcatattgct tttgttgtaa atattgcgca aaagcgacta tctttgtgat ttggttgaac 6480

ttttgcatat tcacgttatt accatcttta gtgaatgtat cgtttccgtc tcttatgaag 6540

gtaatgtccc tgataagcag tgatgtaaaa ggtacacaag gaagctgact gtggaaaata 6600

tgtttaattg ttcttctata aacgttgtag ttgttgtttg gatgtacgac gacttttagg 6660

cgctgaaaaa ggtggtctga tttaacatct atcgggagag atagtctttc tattgaatga 6720

ttttgcaatg atgtaatgat tgaagcaagt gagtttaagt ttcgtaaggt ttaaactggc 6780

tgctacccac gcccatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt 6840

ccacacaaca taggagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagg 6900

taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc 6960

cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct 7020

tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca 7080

gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac 7140

atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt 7200

ttccataggc tcggcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg 7260

cgaaacccga caggactata aagataccag gcgttccccc ctggaagctc cctcgtgcgc 7320

tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc 7380

gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc 7440

aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac 7500

tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt 7560

aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct 7620

aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc 7680

ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt 7740

ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg 7800

atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc 7860

atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa 7920

tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag 7980

gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact gcccgtcgtg 8040

tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga 8100

gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag 8160

cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa 8220

gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc 8280

atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca 8340

aggcgagtta catgatcccc catgttgtga aaaaaagcgg ttagctcctt cggtcctccg 8400

atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat 8460

aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc 8520

aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg 8580

gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg 8640

gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt 8700

gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca 8760

ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata 8820

ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac 8880

atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa 8940

gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt 9000

atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg 9060

cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt 9120

cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag 9180

cagattgtac tgagagtgca ccatagtcgg cgggaccagg gagtttaaac 9230

<210> 6

<211> 8917

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ttcttttcga tgaagatggt tctcctgcag ctatgttgct ttctatatca gcgattggac 60

ctggagtctg agagtcagcg tcatcgttta agtcaccggt tggcgtttct tgtttcaatt 120

catctgtctg taatgtgtag atcaaatttt tcaattcgtt ataatctagg taatgattct 180

gccattcagg gacagcgttg tatttgagaa agtgtgagaa tctcatttcg aggcacaata 240

atagccgcct ttaccgtagt tttgctgcac ctttatctga gagctgactg cttttttggt 300

gtgaaacact gttttctggt aataattttt caatgcatcg gattactttt cccacgtgcg 360

aaatcatcaa ttaattagat tgaaaaaagg gtaagggaaa ataagaaaga ggcggtagcc 420

taaagatacg gtaattgaaa cgtttcctat gcacaatctt aaaccttttt aggtaattga 480

ttaagttgac tgtaatatct ggtaccgact gcctcatcag taagacccgt tgaaaagaac 540

ttacctgaaa aaaacgaata tatactagcg ttgaatgtta gcgtcaacaa caagaagttt 600

aatgacgcgg aggccaaggc aaaaagattc cttgattacg taagggagtt agaatcattt 660

tgaataaaaa acacgctttt tcagttcgag tttatcatta tcaatactgc catttcaaag 720

aatacgtaaa taattaatag tagtgatttt cctaacttta tttagtcaaa aaattagcct 780

tttaattctg ctgtaacccg tacatgccca aaataggggg cgggttacac agaatatata 840

acatcgtagg tgtctgggtg aacagtttat tcctggcatc cactaaatat aatggagccc 900

gctttttaag ctggcatcca gaaaaaaaaa gaatcccagc accaaaatat tgttttcttc 960

accaaccatc agttcatagg tccattctct tagcgcaact acagagaaca ggggcacaaa 1020

caggcaaaaa acgggcacaa cctcaatgga gtgatgcaac ctgcctggag taaatgatga 1080

cacaaggcaa ttgacccacg catgtatcta tctcattttc ttacaccttc tattaccttc 1140

tgctctctct gatttggaaa aagctgaaaa aaaaggttga aaccagttcc ctgaaattat 1200

tcccctactt gactaataag tatataaaga cggtaggtat tgattgtaat tctgtaaatc 1260

tatttcttaa acttcttaaa ttctactttt atagttagtc ttttttttag ttttaaaaca 1320

ccaagaactt agtttcgaat aaacacacat aaacaaacaa agaattccct acttcataca 1380

ttttcaatta agcgtctcta tgcagttact tcgctgtttt tcaatatttt ctgttattgc 1440

tagcgtttta gcaagagacc agcgtgcaag acactgcggt ctctggtggt ggtggttctg 1500

gtggtggtgg ttctggtggt ggtggttcta ttcagaggac tccaaaaatt caggtctact 1560

ctagacatcc agctgagaat ggtaagtcta acttcttgaa ctgctacgta tctggtttcc 1620

atccatctga tattgaagtc gacttgttga aaaacggtga gaggatcgaa aaagttgagc 1680

actctgactt gtctttttct aaggactggt ccttctactt gttgtactac accgagttca 1740

ctccaactga aaaggatgaa tacgcttgca gggttaatca tgtcactttg tcccagccaa 1800

aaattgtcaa gtgggacaga gatatgggtg gtggtggttc tggtggtggt ggttctggtg 1860

gtggtggttc tggtggtggt ggttctggtt ctcattctat gaggtacttc tttacttctg 1920

tctctagacc aggtagaggt gaaccaagat ttattgctgt cggttacgtc gatgatactc 1980

agttcgtcag gtttgattct gatgctgctt ctcaaagaat ggaaccaaga gctccatgga 2040

ttgaacaaga aggtccagaa tattgggatg gtgaaaccag gaaagttaaa gctcactccc 2100

aaactcatag ggttgatttg ggtactttga gaggtgctta caaccaatct gaagctggtt 2160

ctcatactgt tcaaaggatg tacggttgtg atgttggttc tgattggaga ttcttgaggg 2220

gttaccacca atatgcttat gacggtaagg attacattgc tttgaaggaa gacttgaggt 2280

cttggactgc tgctgatatg gctgctcaaa ccactaaaca taagtgggaa gctgctcatg 2340

ttgctgaaca attgagagct tacttggaag gtacttgcgt tgaatggttg agaaggtact 2400

tggaaaacgg taaggagact ttgcaaagga ctgatgctcc aaaaactcac atgactcatc 2460

atgctgtctc tgatcatgaa gctactttga gatgttgggc tttgtctttt tatcccgctg 2520

agattacttt gacttggcag agagatggtg aagatcagac tcaagatact gaattggtcg 2580

aaactagacc agctggtgat ggtacttttc aaaaatgggc tgctgttgtt gttccatctg 2640

gtcaggaaca aagatacact tgtcacgtcc aacatgaagg tttgccaaaa ccattgactt 2700

tgagatggga accatcttct ggatcctacc catacgacgt tccagactac gctaagcttc 2760

tgcaggctag tggtggagga ggctctggtg gaggcggtag cggaggcgga gggtcgcagg 2820

aactgacaac tatatgcgag caaatcccct caccaacttt agaatcgacg ccgtactctt 2880

tgtcaacgac tactattttg gccaacggga aggcaatgca aggagttttt gaatattaca 2940

aatcagtaac gtttgtcagt aattgcggtt ctcacccctc aacaactagc aaaggcagcc 3000

ccataaacac acagtatgtt ttttaaagag acgctcgagc tgaagaggac ttgtaatagt 3060

gagagagccc acaaccaggt atcgnnnnnn nnnnnnnnnt atataactgt ctagaaataa 3120

attttttcaa agccaccttt attcgcctcg cttctgataa cttcgtataa tgtatgctat 3180

acgaacggta gaggtgtggt caataagagc gacctcatgc tatacctgag aaagcaacct 3240

gacctacagg aaagagttac tcaagaataa gaattttcgt tttaaaacct aagagtcact 3300

ttaaaatttg tatacactta ttttttttat aacttattta ataataaaaa tcataaatca 3360

taagaaattc gctcactttt tggcattttt cacgaagtta gcaatcttat tagagtcttt 3420

gaccccattg gtttcaacac ccccggacac gtccacacca ataacaccat tcaaacgtaa 3480

agcatcacca acattttctg gtgttaaccc gccggctaac ataaagtgaa gactttcggg 3540

gctttcctga cgacccaccc aatcgctaat ggagttccaa tctaataatt cgccggtacc 3600

accggcttct gaatcaaaca gcgggatgaa gctatgcggt ttttgtgagg ctgcggataa 3660

taatatgtta caatcctttg gaaaaactaa acgctttata acaggcaagc ccaagaattc 3720

ctggtactct tgccaacttt cgtcgccatg caattgaaca atgtcaatac catagtcatt 3780

aaccaaagct aatacatctt cttttggctg gttcctaaaa acaccaacca aatacttagg 3840

agtaccggat gagttcttgt aagccttaac caaagaggag atttttctag caattacggg 3900

gtctatagta cgcttcctgt tgggaacaca aataatacct aaaaggtcag catcactgtc 3960

tagtgcgcat tcagctgctt ccgttgattg aaggccacaa actttaacta atggcccgct 4020

ggaacccgtg aagttaataa cggacatact ccaagctgcc tttgtgtgct taatcacgta 4080

tactcacgtg ctcaatagtc accaatgccc tccctcttgg ccctctcctt ttcttttttc 4140

gaccgaatta attcttaatc ggcaaaaaaa gaaaagctcc ggatcaagat tgtacgtaag 4200

gtgacaagct atttttcaat aaagaatatc ttccactact gccatctggc gtcataactg 4260

caaagtacac atatattacg atgctgttct attaaatgcg ggtgctcgct cctttattga 4320

agccaaatta aagccttcga gcgtcccaaa accttctcaa gcaaggtttt cagtataatg 4380

ttacatgcgt acacgcgtct gtacagaaaa aaaagaaaaa tttgaaatat aaataacgtt 4440

cttaatacta acataactat aaaaaaataa atagggacct agacttcagg ttgtctaact 4500

ccttcctttt cggttagagc ggatgtgggg ggagggcgtg aatgtaagcg tgacataact 4560

aattgaccga ggtcgacggt atcatcaggg cccggcaccc gctccagcgc ctgcaccagc 4620

tcccttgtac aattcgtcca tacccaaggt gataccagca gcagtaacga attccaacaa 4680

aaccatgtga tctctcttct cgttagggtc cttggacaac ttagattggg tggacaagta 4740

gtggttgtct ggcaacaaaa ctggaccatc accgattggg gtattttgtt ggtagtggtc 4800

ggctaattgg acaccaccat cttcgatgtt gtgtctaatt ttaaagttgg ccttgatacc 4860

gttcttttgt ttgtcggcgg taatgtagac gttgtcagag aaatagttgt attccaactt 4920

gtgacccaag atgttaccgt cttccttgaa atcgataccc tttaattcga ttctgttgac 4980

caaagtatca ccttcaaact taacctcggc acgggtcttg taattaccat catccttgaa 5040

aaaaatggtt ctttcttgga cgtaaccttc tggcatagcg gatttaaaga aatcgtgttg 5100

tttcatgtgg tctgggtaac gagcgaaaca ttgaacaccc caagataaag tggtaaccaa 5160

agttggccat gggactggca acttaccagt ggtacaaatg aatttcaagg tcaacttacc 5220

gtaagtagcg tcaccttcac cttcaccaga aacagaaaac ttgtgaccgt taacgtcacc 5280

gtccaattca accaagattg gaacaacacc agtaaataat tcttcacctt tagataccat 5340

tttgtagatc cactagttct agaatccgtt tgtaattaaa acttagatta gattgctatg 5400

ctttctttct aatgagcaag aagtaaaaaa agttgtaata gaacaagaaa aatgaaactg 5460

aaacttgaga aattgaagac cgtttattaa cttaaatatc aatgggaggt catcgaaaga 5520

gaaaaaaatc aaaaaaaaaa attttcaaga aaaagaaacg tgataaaaat ttttattgcc 5580

tttttcgacg aagaaaaaga aacgaggcgc tctctttttt cttttccaaa cctttagtac 5640

gggtaattaa cgacacccta gaggaagaaa gaggggaaat ttagtatgct gtgcttgggt 5700

gttttgaagt ggtacggcga tgcgcggagt ccgagaaaat ctggaagagt aaaaaaggag 5760

tagaaacatt ttgaagctat ggtgtgtggg ggatcacttg tgggggattg ggtgtgatgt 5820

aaggattcgc ggtcctcgaa aattaaaagt ccaacgcgcc tgttgcttcc tatgtgatat 5880

gtattatatg taatatgcat aaatatatct actgcattgt attttgaacg tacaaagtat 5940

gcattgtgcg gccgcctttc tttgaacagt tctgttttta ggtaagcctt ggaaccttag 6000

ctatactgac ttgatacgcc ctgtctttgt tgtcgttata taaagtgtgc acctttatca 6060

tagcccctaa taagcttctt gcagtagtat ttgaacagtg tatatcttca tattgctttt 6120

gttgtaaata ttgcgcaaaa gcgactatct ttgtgatttg gttgaacttt tgcatattca 6180

cgttattacc atctttagtg aatgtatcgt ttccgtctct tatgaaggta atgtccctga 6240

taagcagtga tgtaaaaggt acacaaggaa gctgactgtg gaaaatatgt ttaattgttc 6300

ttctataaac gttgtagttg ttgtttggat gtacgacgac ttttaggcgc tgaaaaaggt 6360

ggtctgattt aacatctatc gggagagata gtctttctat tgaatgattt tgcaatgatg 6420

taatgattga agcaagtgag tttaagtttc gtaaggttta aactggctgc tacccacgcc 6480

catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatag 6540

gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgaggtaa ctcacattaa 6600

ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat 6660

gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc 6720

tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg 6780

cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag 6840

gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcg 6900

gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag 6960

gactataaag ataccaggcg ttcccccctg gaagctccct cgtgcgctct cctgttccga 7020

ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc 7080

aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg 7140

tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt 7200

ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca 7260

gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca 7320

ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag 7380

ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca 7440

agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg 7500

ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa 7560

aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta 7620

tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag 7680

cgatctgtct atttcgttca tccatagttg cctgactgcc cgtcgtgtag ataactacga 7740

tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgtgac ccacgctcac 7800

cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc 7860

ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta 7920

gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac 7980

gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat 8040

gatcccccat gttgtgaaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa 8100

gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg 8160

tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag 8220

aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc 8280

cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct 8340

caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat 8400

cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg 8460

ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc 8520

aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta 8580

tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg 8640

tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct 8700

ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga 8760

cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag 8820

cgggtgttgg cgggtgtcgg ggctggctta actatgcggc atcagagcag attgtactga 8880

gagtgcacca tagtcggcgg gaccagggag tttaaac 8917

<210> 7

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

tcgtcggcag cgtcagatgt gtataagaga cagcaaacaa agaattccct acttcatac 59

<210> 8

<211> 60

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gtctcgtggg ctcggagatg tgtataagag acagcgatcc tctcaccgtt tttcaacaag 60

<210> 9

<211> 61

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

agcgtgggtc tctagcatct ttgtataata ctgttgctac tttgggtgag agacccagca 60

c 61

<210> 10

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

gtgctgggtc tctcaccc 18

<210> 11

<211> 61

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

agcgtgggtc tctagcatct ttgnnkaata ctgttgctac tttgggtgag agacccagca 60

c 61

Claims

1. A method for rapidly identifying high affinity TCR antigen cross-reactivity, comprising the steps of:

s1, adjusting the TCR and pMHC sequences into constitutive promoter elements to induce expression, integrating and expressing the elements into a yeast genome, and displaying the TCR and pMHC proteins on the surfaces of different mating yeast;

the step of S1 includes:

the preparation method of the carrier of the ysyna-TCR and the ysynalpha-pMHC comprises the following steps: carrying out double enzyme digestion on the synthesized TCR sequence, the ysyna _ DEST plasmid, the pMHC sequence and the modified ysynalpha _ DEST plasmid by using NheI and XhoI restriction endonucleases, carrying out enzyme digestion in a water bath, cutting a target fragment by using DNA gel under an ultraviolet lamp, and recovering a corresponding fragment; connecting at room temperature; transformed into E.coli DH5 alpha;

after a carrier of the ysyna-TCR is obtained, enzyme digestion is carried out by PmeI, and fragments comprising ARS314, TCR, TRP and fluorescent protein are cut off by enzyme; recovering the gel to obtain fragments; the carrier of the ysynalpha-pMHC peptide segment mutation library is cut by PmeI in the same way; cutting and recycling the rubber;

after overnight culture, switching, sampling at different time intervals for flow analysis, detecting the display levels of TCR and pMHC on the surface of yeast, and selecting the time point with the highest display level for subsequent mating experiments;

the step of S2 includes:

overnight culturing the constructed TCR expression and pMHC display strains;

controlling OD conditions of mating, including initial concentration, input amount of a-type yeast displaying TCR, input amount of alpha yeast displaying pMHC and volume of culture medium, performing yeast mating experiment in a deep-well plate, and performing mixed culture to obtain diploid signals formed by TCR-pMHC mediation;

s3, diploid screening and next generation sequencing.

2. The method of claim 1, wherein the MATa yeast simultaneously integrates and expresses red fluorescent protein, and red fluorescence can be detected in a subsequent flow assay; the MATalpha yeast integrates and expresses a blue fluorescent protein at the same time, and blue fluorescence can be detected in a subsequent flow-type experiment.

3. The method of claim 1, wherein the cross-reactivity of the high affinity TCR antigen is rapidly determined,

the method for constructing the scTCR form by the TCR sequence is a yeast targeting method; the assembly is carried out by adopting a V beta-linker (GSADDAKKDAAKKDGKS) -V alpha form.

4. The method of claim 1, wherein the specific steps of integrating the TCR into the MATa yeast with the Sag1 gene knocked out and integrating the pMHC into the MATapha yeast strain are as follows:

screening single clones through SC-TRP plates;

the correct strain construction of integrating TCR into MATa yeast with Sag1 gene knock-out is to select single clone and directly streak and culture on YPD plate, separate single clone for subsequent experiment; scraping all MATalpha yeast colonies integrated with pMHC by using a scraper, centrifuging, and suspending in an SC-TRP culture medium to obtain a PMHC peptide fragment mutation library; and (5) freezing and storing.

5. The method of claim 1, wherein the screening of diploid in S3 comprises using unrelated TCR as negative control, performing both experimental and control groups, and after subsequent second generation sequencing, using the difference between experimental and control groups as a threshold, and optionally correcting the sequencing data.

6. The method of claim 3, wherein the diploid screening process comprises the steps of:

after incubation, directly taking 300ul-600ul bacterial liquid after mating experiment and washing for 2 times by using a culture medium of SC-Lys-Leu;

then adding into the culture medium of SC-Lys-Leu for culture.

7. The method of claim 3, wherein the diploid screening process comprises the steps of: after incubation, directly taking 300ul-600ul bacterial liquid after mating experiment and washing for 2 times by using a culture medium of SC-Lys-Leu; diploid is sorted by flow, and the specific steps comprise: the cells were sorted and cultured in YPD medium.