T cell receptor and B cell receptor detection system based on spatial omics

Abstract

The invention relates to a TCR and / or BCR detection system based on spatial omics, and aims to solve the problems of high detection cost, low spatial resolution, incapability of accurately positioning cells and the like in the existing spatial VDJ technology. The invention provides a T cell receptor and / or B cell receptor detection system based on a spatial omics method, the system comprises: a module (1), which constructs a coding primer pool according to a VJ region sequence of TCR and / or BCR to be detected, the VJ region is formed by rearranging a V region and a J region, the coding primer pool is used for each V region and J region to be detected, and the coding primer pool is used for coding the TCR and / or BCR to be detected. Or specific coding sequences are distributed to the V region and the CDR3 region respectively; the module (2) is used for amplifying a to-be-detected VJ region of a to-be-detected tissue sample through the coding primer pool and fixing an amplification product; and the module (3) is used for positioning the amplification product by using the signal probe group and reading the coding sequence, so that the position information of the VJ region is obtained, and the sequence information of the VJ region is obtained according to the coding sequence.

Description

Technical Field

[0001] The present invention relates to the technical field of spatial omics, and in particular to a T cell receptor and B cell receptor detection system based on spatial omics. Background Art

[0002] Autoimmune diseases associated with gene polymorphisms such as systemic lupus erythematosus have complex mechanisms and are difficult to cure. Their pathogenesis is related to immunoglobulins and T cell receptors. The risk of gene base editing in lupus patients is increased, which is associated with the T cell receptor pathway. Determining the spatial expression location information and sequence information of related TCR and / or BCR is of great significance for studying the pathogenesis of autoimmune diseases such as lupus.

[0003] Engineered T cell therapy involves injecting T cells with modified specific T cell receptors into the patient's body to target and attack pathological cells that express specific signal-presenting molecules to achieve disease treatment. Although this therapy has an excellent prognosis, it has fatal risks of self-targeting or off-targeting, which causes the engineered T cell receptors to recognize and attack normal cells. Therefore, it requires high testing and customization costs, and is currently difficult to promote on a large scale.

[0004] The rearrangement and combination of the variable region V(D)J in the T cell receptor and B cell receptor at the gene level determines the type of molecule that the former specifically recognizes. V(D)J detection is a common technology for studying T cell-B cell receptors, also known as V(D)J sequencing. This technology uses sequencing or imaging to detect the gene rearrangement of the variable region of the T cell receptor or B cell receptor at the RNA level, and can detect the rearrangement combination of the V(D)J region, thereby inferring the amino acid sequence and recognition characteristics of the T cell receptor or B cell receptor. It is often used to study the signal presentation mechanism of complex diseases such as autoimmune diseases. The existing commercial spatial V(D)J technology uses a 100-micron diameter microsphere array to capture RNA, and then performs downstream amplification and high-throughput sequencing to achieve T cell-B cell receptor determination. Compared with traditional V(D)J sequencing, spatial V(D)J technology can accurately detect the spatial expression sites of each T cell-B cell receptor and display cell signal transduction pathways. However, the spatial resolution of this type of microsphere array technology is usually 100 microns, which cannot achieve single-cell level imaging detection below 10 microns, and cannot accurately locate T cell-B cell receptor information at the single-cell level.

[0005] In summary, the existing technology has problems such as high cost of T cell receptor detection in T cell therapy, low resolution of spatial V(D)J technology, and inability to accurately locate cells. Summary of the invention

[0006] The object of the present invention is to provide a spatial omics system, method and detection kit for detecting TCR and / or BCR variable region V(D)J. In the present invention, V(D)J refers to the variable region of TCR or BCR, wherein the D region may be present or absent, and therefore, V(D)J represents the VJ region or the VDJ region, wherein the VJ region is formed by rearrangement of the V region and the J region, and the VDJ region is formed by rearrangement of the V region, the D region and the J region.

[0007] In order to solve the above technical problems, the present invention provides the following technical solutions: 1. Detection system and detection method

[0008] The present invention provides a T cell receptor and / or B cell receptor detection system based on a spatial omics method, the system comprising:

[0009] Module (1) constructs a coding primer pool according to the VJ region sequence and / or VDJ region of the TCR and / or BCR to be tested, wherein the VJ region is formed by rearrangement of the V region and the J region, and the VDJ region is formed by rearrangement of the V region, the D region and the J region, and the coding primer pool assigns a specific coding sequence to each region to be tested in the VJ region or the VDJ region, for example, assigning coding sequences to the regions to be tested in groups 1)-3): 1) V region and J region detection; 2) V region and CDR3 region detection; 3) V region, D region and J region detection;

[0010] Module (2), which uses the coding primer pool to amplify the VJ region and / or VDJ region to be tested of the tissue sample to be tested, and fixes the amplified product;

[0011] Module (3) uses a signal probe set to perform multiple rounds of imaging, locate the amplified product and read the coding sequence, thereby obtaining the position information of the VJ region and / or VDJ region, and obtaining its sequence information based on the coding sequence.

[0012] The present invention also provides a method for detecting T cell receptors and / or B cell receptors based on a spatial omics method, the method comprising:

[0013] Step (1), constructing a coding primer pool according to the VJ region sequence and / or VDJ region of the TCR and / or BCR to be tested, wherein the VJ region is formed by rearrangement of the V region and the J region, and the VDJ region is formed by rearrangement of the V region, the D region and the J region, and the coding primer pool assigns a specific coding sequence to each region to be tested in the VJ or VDJ region, for example, assigning coding sequences to the regions to be tested in groups 1)-3): 1) V region and J region; 2) V region and CDR3 region; 3) V region, D region and J region; Step (1) is an optional step for the method;

[0014] Step (2), amplifying the VJ region and / or VDJ region to be tested of the tissue sample to be tested by using the coding primer pool, and fixing the amplified product;

[0015] Step (3) uses the signal probe set to perform multiple rounds of imaging, locate the amplified product and read the coding sequence, thereby obtaining the position information of the VJ or VDJ region, and obtaining its sequence information based on the coding sequence.

[0016] In some specific embodiments, the TCR and / or BCR to be tested are TCR and / or BCR sequences associated with a disease, or TCR sequences associated with TCR-T cell therapy, or TCR and / or BCR sequences that need to be detected in other biotechnology scenarios.

[0017] The system and method of the present invention are used to detect the V(D)J sequence of disease-associated TCR and / or BCR, or the V(D)J sequence of TCR for TCR-T therapy, or the V(D)J sequence of TCR and / or BCR that needs to be detected in other biotechnology scenarios. Preferably, the disease is an autoimmune disease, for example, systemic lupus erythematosus.

[0018] In some specific embodiments, the disease-associated TCR and / or BCR V(D)J sequences are obtained by obtaining TCR and / or BCR V(D)J sequences through public databases, and using computer codes (e.g., RNAsee, Stitchr, and / or biopython) to screen TCR and / or BCR V region and J region sequences, or V region and CDR3 region sequences, or V region, D region and J region sequences, or V region, D region and CDR3 region sequences associated with the disease. Preferably, the disease is an autoimmune disease (e.g., systemic lupus erythematosus). Taking systemic lupus erythematosus as an example, the screening index is a subset of V region sequences and J region sequences that are significantly different from normal controls in the disease data. If there is no prior knowledge, the top 16 highly expressed sequences are selected from the V region sequence library and the J region sequence library. 1) Detect the VJ region of TCR or BCR by detecting the V region and J region

[0019] In some specific embodiments, the VJ region of a TCR or BCR is detected by detecting the V region and the J region; the coding primer pool in the detection system or the detection method comprises primer set 1 and primer set 2, wherein the primer set 1 comprises one or more V region primers and one or more J region primers;

[0020] The primer set 1 includes one or more V region primers and one or more J region primers;

[0021] The V region primer includes a 5' end sequence that is reverse complementary to the V region sequence of the TCR and / or BCR to be tested and a 3' end artificial sequence that includes the V region coding sequence;

[0022] The J region primer includes a 5' end sequence that is reverse complementary to the J region sequence of the TCR and / or BCR to be tested and a 3' end artificial sequence that includes the J region coding sequence;

[0023] The primer set 2 includes one or more VJ primers and one or more JV primers, and the 5' ends of the VJ primers and the JV primers are modified with a phosphate group;

[0024] The VJ primer includes a 5' end sequence that is reverse complementary to a portion of the artificial sequence of the V region primer, a spacer sequence, and a 3' end sequence that is reverse complementary to a portion of the artificial sequence of the J region primer, and the 5' end and 3' end of the VJ primer include sequences that are reverse complementary to a portion of the V region coding sequence and the J region coding sequence, respectively;

[0025] The JV primer includes a 5' end sequence that is reverse complementary to a portion of the artificial sequence of the J region primer, a spacer sequence, and a 3' end sequence that is reverse complementary to a portion of the artificial sequence of the V region primer, and the 5' end and 3' end of the JV primer include sequences that are reverse complementary to a portion of the J region coding sequence and the V region coding sequence, respectively;

[0026] The 3' end of the VJ primer and the 5' end of the JV primer are connected by DNA ligase to form a complete sequence that is reverse complementary to the J region coding sequence;

[0027] The 3' end of the JV primer and the 5' end of the VJ primer are connected by DNA ligase to form a complete sequence that is reverse complementary to the V region coding sequence;

[0028] In some specific embodiments, the total length of each primer in the primer set 1 is 40 to 60 nt, and the length of the coding sequence of each primer is 2 to 4 nt;

[0029] In some specific embodiments, the primer set 1 sequence includes the nucleic acid sequence shown in SEQ ID NO:1-16, wherein the J region primer includes the nucleic acid sequence shown in SEQ ID NO:1-8, and the V region primer includes the nucleic acid sequence shown in SEQ ID NO:9-16; the primer set 2 includes the nucleic acid sequence shown in SEQ ID NO:17-32.

[0030] In an embodiment where the VJ region of a TCR or BCR is detected by detecting the V region and the J region, the signal probe set of the system or method comprises: a V region nucleic acid probe set and a J region nucleic acid probe set;

[0031] The V region nucleic acid probe set is used to read the V region coding sequence, wherein the reading position is referred to as a reading position, and for each reading position, the V region nucleic acid probe set includes 4 nucleic acid probes labeled by different fluorescent groups, wherein the 4 probes include a sequence that is reverse complementary to the artificial sequence of the V region primer and a sequence corresponding to the coding sequence; wherein, in the sequence corresponding to the coding sequence, the nucleotides corresponding to the coding positions of the 4 probes are A, T, G and C, respectively, and the corresponding sequences outside the coding positions are degenerate sequences; optionally, the sequence corresponding to the coding sequence is located at the 5' end, 3' end or in the middle of the nucleic acid probe;

[0032] The J region nucleic acid probe set is used to read the J region coding sequence, wherein the reading position is referred to as a reading position, and for each reading position, the J region nucleic acid probe set includes 4 nucleic acid probes labeled by different fluorescent groups, wherein the 4 probes include a sequence that is reverse complementary to the artificial sequence of the J region primer and a sequence corresponding to the coding sequence; wherein, in the sequence corresponding to the coding sequence, the nucleotides corresponding to the coding positions of the 4 probes are A, T, G and C, respectively, and the corresponding sequences outside the coding positions are degenerate sequences; optionally, the sequence corresponding to the coding sequence is located at the 5' end, 3' end or in the middle of the nucleic acid probe;

[0033] Preferably, the fluorescent group labeled with the signal probe set is selected from red, orange, green and blue fluorescent groups; more preferably, the fluorescent group is selected from Cy5, Cy3, AF488 and AMCA fluorescent groups;

[0034] In some specific embodiments, for the detection of less than 16 J region sequences and / or the detection of less than 16 V region sequences, the number of coding bits of the J region coding sequence and / or the V region coding sequence is 2 bits (i.e., composed of 2 nucleotides). In some specific embodiments, in the sequence reading of the 2-bit coded V region probe and the 2-bit coded J region probe, the nucleic acid probes of the V region nucleic acid probe group and the J region nucleic acid probe group include the nucleic acid sequences shown in SEQ ID NOs:33-48. In some specific embodiments, the 5' end of the nucleic acid sequence shown in SEQ ID NOs:33, 37, 41, 45 is modified with a Cy5 fluorescent group; the 5' end of the nucleic acid sequence shown in SEQ ID NOs:34, 38, 42, 46 is modified with a Cy3 fluorescent group; the 5' end of the nucleic acid sequence shown in SEQ ID NOs:35, 39, 43, 47 is modified with an AF488 group; the 5' end of the nucleic acid sequence shown in SEQ ID NOs:36, 40, 44, 48 is modified with an AMCA group. 2) Detection of the VJ region of TCR or BCR by detecting the V region and CDR3 region

[0035] In an embodiment of detecting the VJ region of a TCR or BCR by detecting the V region and the J region, based on the V(D)J structural features of the TCR and / or BCR, the J region primer can be replaced with a CDR3 region primer when all conditions other than the J region primer remain unchanged, and the V-CDR3 region of the TCR and / or BCR is detected by the V region and the CDR3 region; the coding primer pool in the detection system or the detection method comprises primer set 1 and primer set 2, wherein the primer set 1 comprises one or more V region primers and one or more CDR3 region primers;

[0036] The CDR3 region primer includes a 5' end sequence that is reverse complementary to the CDR3 region sequence of the TCR and / or BCR to be tested and a 3' end artificial sequence including a J region coding sequence;

[0037] The V region primer and primer set 2 are the same as those in the embodiment of detecting the VJ region of TCR or BCR by detecting the V region and CDR3 region;

[0038] In an embodiment of detecting the V-CDR3 region of TCR and / or BCR by targeting the V region and CDR3 region, the primer set 1 sequence includes the nucleic acid sequences shown in SEQ ID NOs: 9-16 and SEQ IDs: 49-56, wherein the CDR3 region primers include the nucleic acid sequences shown in SEQ ID NOs: 49-56, and the V region primers include the nucleic acid sequences shown in SEQ ID NOs: 9-16;

[0039] In the embodiment where the V-CDR3 region of TCR and / or BCR is detected by detecting the V region and CDR3 region, the signal probe set of the system or method is the same as the signal probe set of the system or method where the VJ region of TCR or BCR is detected by detecting the V region and J region. 3) Detect the VDJ region of TCR or BCR by detecting the V region, D region and J region

[0040] In some specific embodiments, the VDJ region of TCR or BCR is detected by detecting the V region, D region and J region, and the coding primer pool in the detection system or method includes primer set 1 and primer set 2, wherein the primer set 1 includes one or more V region primers, one or more D region primers and one or more J region primers;

[0041] The V region primer includes a 5' end sequence that is reverse complementary to the V region sequence of the TCR and / or BCR to be tested and a 3' end artificial sequence that includes the V region coding sequence;

[0042] The D region primer includes a 5' end sequence that is reverse complementary to the D region sequence of the TCR and / or BCR to be tested and a 3' end artificial sequence that includes the D region coding sequence;

[0043] The J region includes a 5' end sequence that is reverse complementary to the J region sequence of the TCR and / or BCR to be tested and a 3' end artificial sequence that includes a J region specific coding sequence;

[0044] The primer set 2 includes one or more VD primers, one or more DJ primers and one or more JV primers, and the 5' ends of the VD primers, DJ primers and JV primers are modified with phosphate groups;

[0045] The VD primer includes a 5' end sequence that is reverse complementary to a portion of the artificial sequence of the V region primer, a spacer sequence, and a 3' end sequence that is reverse complementary to a portion of the artificial sequence of the D region primer; the 5' end and the 3' end of the VD primer include sequences that are reverse complementary to a portion of the V region coding sequence and the D region coding sequence, respectively;

[0046] The DJ primer includes a 5' end sequence that is reverse complementary to a portion of the artificial sequence of the D region primer, a spacer sequence, and a 3' end sequence that is reverse complementary to a portion of the artificial sequence of the J region primer; the 5' end and the 3' end of the DJ primer include sequences that are reverse complementary to a portion of the D region coding sequence and the J region coding sequence, respectively;

[0047] The JV primer includes a 5' end sequence that is reverse complementary to a portion of the artificial sequence of the J region primer, a spacer sequence, and a 3' end sequence that is reverse complementary to a portion of the artificial sequence of the V region primer, and the 5' end and 3' end of the JV primer include sequences that are reverse complementary to a portion of the J region coding sequence and the V region coding sequence, respectively;

[0048] The 3' end of the VD primer and the 5' end of the DJ primer are connected by DNA ligase to form a complete sequence that is reverse complementary to the D region coding sequence;

[0049] The 3' end of the DJ primer and the 5' end of the JV primer are connected by DNA ligase to form a complete sequence that is reverse complementary to the J region coding sequence;

[0050] The 3' end of the JV primer and the 5' end of the VD primer are connected by DNA ligase to form a complete sequence that is reverse complementary to the V region coding sequence.

[0051] In an embodiment in which the VDJ region of a TCR and / or BCR is detected by detecting the V region, the D region and the J region, the signal probe set of the system or method comprises: a V region nucleic acid probe set, a D region nucleic acid probe set and a J region nucleic acid probe set;

[0052] The V region nucleic acid probe set is used to read the V region coding sequence, wherein the reading position is referred to as a reading position, and for each reading position, the V region nucleic acid probe set includes 4 nucleic acid probes labeled by different fluorescent groups, wherein the 4 probes include a sequence that is reverse complementary to the artificial sequence of the V region primer and a sequence corresponding to the coding sequence; wherein, in the sequence corresponding to the coding sequence, the nucleotides corresponding to the coding positions of the 4 probes are A, T, G and C, respectively, and the corresponding sequences outside the coding positions are degenerate sequences; optionally, the sequence corresponding to the coding sequence is located at the 5' end, 3' end or in the middle of the nucleic acid probe;

[0053] The D region nucleic acid probe set is used to read the D region coding sequence, wherein the reading position is referred to as a reading position, and for each reading position, the D region nucleic acid probe set includes 4 nucleic acid probes labeled by different fluorescent groups, wherein the 4 probes include a sequence that is reverse complementary to the artificial sequence of the D region primer and a sequence corresponding to the coding sequence; wherein, in the sequence corresponding to the coding sequence, the nucleotides corresponding to the coding positions of the 4 probes are A, T, G and C, respectively, and the corresponding sequences outside the coding positions are degenerate sequences; optionally, the sequence corresponding to the coding sequence is located at the 5' end, 3' end or in the middle of the nucleic acid probe;

[0054] The J region nucleic acid probe set is used to read the J region coding sequence, wherein the reading position is referred to as a reading position, and for each reading position, the J region nucleic acid probe set includes 4 nucleic acid probes labeled by different fluorescent groups, wherein the 4 probes include a sequence that is reverse complementary to the artificial sequence of the J region primer and a sequence corresponding to the coding sequence; wherein, in the sequence corresponding to the coding sequence, the nucleotides corresponding to the coding positions of the 4 probes are A, T, G and C, respectively, and the corresponding sequences outside the coding positions are degenerate sequences; optionally, the sequence corresponding to the coding sequence is located at the 5' end, 3' end or in the middle of the nucleic acid probe;

[0055] Preferably, the fluorescent group labeled with the V region nucleic acid probe set, the D region nucleic acid probe set and / or the J region nucleic acid probe set is selected from red, orange, green and blue fluorescent groups; more preferably, the fluorescent group is selected from Cy5, Cy3, AF488 and AMCA fluorescent groups.

[0056] In some specific embodiments, the tissue sample is a section or a smear.

[0057] In some specific embodiments, the tissue sample to be tested is from a lupus animal model (preferably B6.MRL-Fas lpr / J mouse model) or lupus patients or subjects of TCR-T cell therapy or T cell donors.

[0058] In some specific embodiments, the module (2) or the step (2) comprises using the primer pool, DNA ligase and DNA polymerase to form a circular template and performing rolling circle amplification on the circularized template, and hydrogel fixation of the rolling circle amplification product; preferably, the DNA ligase is T4 ligase, and the DNA polymerase is Phi29 enzyme.

[0059] In some specific embodiments, the module (2) or the step (2) comprises: adding a primer pool to a tissue sample, incubating and eluting at room temperature, connecting the phosphate sticky ends using T4 ligase to obtain a circularized nucleic acid template, adding Phi29 enzyme for rolling circle amplification to obtain a signal amplification nucleic acid chain, eluting residual enzymes, adding polyacrylamide to solidify into a hydrogel network, and fixing the amplification product in situ.

[0060] In some specific embodiments, the module (3) or the step (3) uses a signal probe to locate the amplified product and read the coding sequence, including multiple rounds of imaging and decoding.

[0061] In some specific embodiments, each round of multiple rounds of imaging in the module (3) or step (3) includes: A) dropping the corresponding signal probe of the reading round on the surface of the tissue sample for incubation; B) eluting the unbound signal probe; C) acquiring a signal image using a laser confocal microscope; D) eluting all the signal probes.

[0062] In some specific embodiments, the multi-round decoding includes using a computer to align and decode image signals obtained from multiple rounds of imaging. Preferably, the images obtained from multiple rounds of imaging are aligned, and each round of signals of each signal point is read round by round (preferably using python code to read), thereby obtaining the coding sequence of the signal point, and decoding the V(D)J sequence label of each signal point according to the one-to-one correspondence of the coding sequence.

[0063] In some specific embodiments, the number of rounds of imaging is determined by the length of the coding sequence. For example, for a 2-bit coded V region probe and a 2-bit coded J region probe, a total of 4 rounds of imaging are performed; for a 3-bit coded V region probe and a 3-bit coded J region probe, a total of 6 rounds of imaging are performed; for a 4-bit coded V region probe and a 4-bit coded J region probe, a total of 8 rounds of imaging are performed. For example, for a 2-bit coded V region probe, a 2-bit coded D region probe, and a 2-bit coded J region probe, a total of 6 rounds of imaging are performed; for a 3-bit coded V region probe, a 3-bit coded D region probe, and a 3-bit coded J region probe, a total of 9 rounds of imaging are performed; for a 4-bit coded V region probe, a 4-bit coded D region probe, and a 4-bit coded J region probe, a total of 12 rounds of imaging are performed. 2. Test kit

[0064] The present invention also provides a kit for detecting the VJ region or VDJ region of BCR and / or TCR, the kit comprising primer set 1, primer set 2 and signal probe set in the aforementioned detection system or detection method; preferably, the kit also comprises DNA ligase and DNA polymerase, the DNA ligase is preferably T4 ligase, and the DNA polymerase is preferably Phi29 enzyme.

[0065] In some specific embodiments, the primer set 1 includes the nucleic acid sequence shown in SEQ ID NO: 1-16 or includes the nucleic acid sequence shown in SEQ ID NO: 8-16 and SEQ ID NO: 49-56, the primer set 2 includes the nucleic acid sequence shown in SEQ ID NO: 17-32 and the phosphate group is modified at the 5' end, and the signal probe set includes the nucleic acid sequence shown in SEQ ID NO: 33-48. In some preferred technical schemes, the 5' end of the nucleic acid sequence shown in SEQ ID NO: 33, 37, 41, 45 is modified with a Cy5 fluorescent group; the 5' end of the nucleic acid sequence shown in SEQ ID NO: 34, 38, 42, 46 is modified with a Cy3 fluorescent group; the 5' end of the nucleic acid sequence shown in SEQ ID NO: 35, 39, 43, 47 is modified with an AF488 group; the 5' end of the nucleic acid sequence shown in SEQ ID NO: 36, 40, 44, 48 is modified with an AMCA group. Beneficial Effects

[0066] Compared with the prior art, the present invention has at least the following beneficial effects:

[0067] 1. The spatial omics-based T cell receptor and B cell receptor detection method and system disclosed in the present invention can innovatively amplify V(D)J variable regions in situ in tissues and read the V region and / or D region and / or J region and / or CDR3 region variable region signals in segments. Compared with existing T cell receptor and B cell receptor sequencing technologies, it reduces the detection cost, avoids the cDNA reverse transcription step, and improves the detection efficiency;

[0068] 2. The spatial omics-based T cell receptor and B cell receptor detection method and system disclosed in the present invention uses multiple rounds of imaging and decoding of coding sequences using fluorescent reading probes to improve detection accuracy. Compared with the prior art, the spatial resolution is increased from 100 microns to 200 nanometers;

[0069] 3. The spatial omics-based T cell receptor and B cell receptor detection method and system disclosed in the present invention can realize single-cell level detection in situ in tissues, and can be applied to assist in the diagnosis and treatment of engineered T cells, and reduce the testing cost and fatal off-target risk of engineered T cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0071] Figure 1 This is a schematic diagram of the T cell receptor and B cell receptor detection system module based on spatial omics of the present invention;

[0072] Figure 2 This is a simplified flow chart of the T cell receptor and B cell receptor detection process based on spatial omics of the present invention;

[0073] Figure 3 Module (2) and step (2) of the present invention are schematic diagrams of a process for amplifying signals of one or more specific V(D)J regions of a sample / cell to be tested using a coding primer pool;

[0074] Figure 4 The present invention is a schematic diagram of the combination of primer set 1 and primer set 2 in the coding primer pool in the VJ region or V-CDR3 region in module (2) and step (2); wherein 1 represents the RNA target sequence of the sample tissue (VJ rearrangement, the nucleic acid structure is V region-CDR3 region-J region); 2 represents the V region primer; 3 represents the J region primer or the CDR3 region primer; 4 represents the JV primer; 5 represents the VJ primer; 6 represents the J region coding sequence or the CDR3 region coding sequence; 7 represents the V region coding sequence;

[0075] Figure 5 The present invention is a schematic diagram of the combination of primer set 1 and primer set 2 in the coding primer pool in the VDJ region in module (2) and step (2); wherein 2 represents a J region primer or a CDR3 region primer, 3 represents a V region primer, 8 represents a target sequence of a sample tissue (VDJ rearrangement, a nucleic acid structure of V region-D region-J region), and 9 represents a D region primer;

[0076] Figure 6 This is a schematic diagram of multiple rounds of imaging in module (3) and step (3) of the present invention, wherein 10 represents the V(D)J sequence encoding amplification chain; 11 represents the amplification chain encoding sequence; 12 represents the first round signal probe (correctly bound to the coding sequence site); 13 represents the first round signal probe (unbound). DETAILED DESCRIPTION

[0077] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Generally, the components of the embodiments of the present invention described and shown in the drawings here can be arranged and designed in various different configurations.

[0078] Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. In the absence of conflict, the following embodiments and features in the embodiments can be combined with each other. Example 1

[0079] like Figure 1 and Figure 2 As shown, the embodiments of the present invention provide a T cell receptor and B cell receptor V(D)J detection system based on spatial omics technology and a corresponding detection method and kit, which are suitable for detecting V(D)J variable regions in T cell receptors and B cell receptors. The detection reagents and detection methods involved in the system are specifically introduced in combination with specific application scenarios (taking V(D)J detection of lupus-associated TCR as an example).

[0080] The main reagents used are: [reagent 1. 10× incubation buffer, components: sodium citrate buffer SSC; formamide; yeast tRNA; vanadyl ribonucleoside complex RVC; RNase inhibitor], [reagent 2. Primer set 1 premix pool, components: nucleic acid sequences shown in SEQ ID No: 1-16 dissolved in 1× incubation buffer at a concentration of 10 μM], [reagent 3. Primer set 2 premix pool, components: SEQ ID No: 17-32 nucleic acid sequence is dissolved in 1× incubation buffer at a concentration of 1 μM], [reagent 4. T4 ligase, concentration 5 U / μL], [reagent 5. dNTP nucleic acid, concentration 2.5 mM], [reagent 6. Phi29 enzyme, concentration 10 U / μL], [reagent 7. Gel monomer mixture, components: acrylamide; methylene bisacrylamide; sodium citrate buffer SSC], [reagent 8. Gel monomer condensate, components: ammonium persulfate; tetramethylethylenediamine], [reagent 9. First round (J region coding position 1) fluorescently labeled signal probe premix, components: SEQ ID No: 33-36 nucleic acid sequence is dissolved in ultrapure water at a concentration of 10 μM], [reagent 10. Second round (J region coding position 2) fluorescently labeled signal probe premix, components: SEQ ID The nucleic acid sequence shown in No:37-40 is dissolved in ultrapure water at a concentration of 10 μM], [Reagent 11. The third round (V region coding position 1) fluorescently labeled signal probe premix, components: the nucleic acid sequence shown in SEQ ID No:41-No.44 is dissolved in ultrapure water at a concentration of 10 μM], [Reagent 12. The fourth round (V region coding position 2) fluorescently labeled signal probe premix, components: the nucleic acid sequence shown in SEQ ID No:45-48 is dissolved in ultrapure water at a concentration of 10 μM].

[0081] The above reagents can be commercially available reagents with suitable concentrations. A combination of one or more of the above reagents can be made into a kit.

[0082] Specific detection system and detection method:

[0083] Module (1) or step S1, primer design specifically includes:

[0084] According to the disease characteristics or target molecules to be detected, the required V(D)J sequences are preliminarily screened and a V(D)J primer pool is constructed. Specifically, the V and J region sequences, or V, D, J region sequences of T cell receptors and B cell receptors, are obtained using public databases such as VDJbase and IMGT-GENE-DB, and the V and J region sequences to be detected, or the V, D, J region sequences to be detected, are screened according to the disease and molecular detection requirements. For each V and J region sequence, a primer set 1 for reading the V and J region, or the V, D, J region, and a primer set 2 for rolling circle amplification are designed. The corresponding sequence of the J region can be replaced by the corresponding region sequence of CDR3.

[0085] Taking the detection of lupus-associated TCR as an example, module (1) or step S1 is described in detail. The target sequence focuses on the TCRβ-V region sequence and the TCRβ-J region sequence. Therefore, the TCRβV and J region sequences to be tested are screened, specifically including: based on the IMGT-GENE-DB database, 24 / 14 types of T cell receptor V / J are obtained, and according to the risk of lupus disease base editing and the corresponding expression abundance of each V / J sequence, "RNAsee", "Stitchr" and "biopython" codes are used to filter and screen the high-risk sites for base editing, and 8 / 8 types of T cell receptor V / J sequences to be tested are obtained;

[0086] Among them, primer group 1 is composed of a mixture of primers of group V and primers of group J. Figure 3 As shown, the 3' end of each primer is reverse complementary to a V region or J region sequence fragment to be detected, and the 5' end is an artificial sequence, which contains a unique coding sequence assigned to each specific V region or J region sequence, with a total length of 40 to 60 nt, and its specific sequence is shown in SEQ ID No: 1-No.16.

[0087] Primer set 2 (for rolling circle amplification) specifically includes: VJ primer and JV primer, whose nucleic acid sequences are shown in SEQ ID NO: 17-32; the 5' end of each primer is modified with a phosphate group for connection with T4 DNA ligase. Figure 3-4 As shown, primer set 2 is connected end to end with primer set 1, and a circularized nucleic acid template with reverse complementary sequences to the V region and J region coding sequences is formed by T4 DNA ligase.

[0088] Furthermore, if Figure 3 As shown, module (2) or step S2 specifically includes:

[0089] Sample preparation, incubation at room temperature with primer pool, T4 enzyme ligation, rolling circle amplification, and gel fixation to obtain the V(D)J sequence encoding amplification chain in the tissue.

[0090] Sample preparation specifically includes: for tissues such as kidney, spleen, lung, lymph node, thymus, brain, etc., immerse them in liquid nitrogen for quick freezing, use a freezing slicer to prepare 10-micron thick frozen sections, and transfer them to the surface of poly-lysine-treated adhesive slides; for blood tissue, use a red blood cell lysis reagent to lyse red blood cells, and then add them dropwise to the surface of poly-lysine adhesive slides without RNase.

[0091] The combination of primer pool specifically includes: adding the primer pool designed and constructed in step S1, that is, mixing [reagent 2. Primer group 1 premix pool] and [reagent 3. Primer group 2 premix pool] in a ratio of 1:1 on the slice or smear, and incubating at a constant temperature of 37 degrees Celsius for 48 hours; adding 100 μL 2×SSC, and eluting 3 times, each time for 15 minutes.

[0092] The T4 enzyme ligation specifically includes: after combining with the primer pool, adding 50 μL of 0.1U / μl T4 ligase, incubating at room temperature for 2 hours, and eluting 3 times using 2×SSC.

[0093] The rolling circle amplification specifically includes: after T4 enzyme connection, 50 μL of 0.2U / μl Phi29 enzyme is added, incubated at 30 degrees Celsius for 2 hours, and eluted 3 times using 2×SSC; the V(D)J sequence encoding amplification chain is obtained.

[0094] Gel fixation specifically includes: adding 100uL of [reagent 7. Gel monomer mixture] (4% acrylamide; 0.2% methylene bisacrylamide; 2×SSC; dissolved in ultrapure water) on the slice or smear after rolling circle amplification, and incubating at room temperature for 1 hour; using a pipette to suck out the gel monomer mixture on the tissue, and directly adding 50 microliters of [reagent 8. Gel monomer coagulation solution] (0.2% ammonium persulfate; 0.2% tetramethylethylenediamine) and [reagent 7. Gel monomer mixture] 1:100 mixed solution, covering the tissue solution with an acrylic coverslip, and incubating at room temperature for 1 hour in a nitrogen environment; obtaining a hydrogel-fixed V(D)J sequence encoding amplification chain.

[0095] Furthermore, if Figure 6 As shown, module (3) or step S3 specifically includes:

[0096] The tissues after V(D)J coding amplification chain amplification and fixation were subjected to multiple rounds of imaging, and the multiple rounds of image signals were aligned and decoded using a computer to obtain the V(D)J signal spatial map.

[0097] Depending on the design of the coding sequence of the circular reading primer designed in step S1, the number of rounds of imaging can be 4-6. Taking the lupus model detection as an example, 4 rounds are performed. The first round operation is: drop 100 μL Dilute DAPI dye at 1:1000 and incubate at 37 degrees Celsius for 1 hour; add signal probe group [reagent 9. First round (J region coding position 1) fluorescently labeled signal probe premix] on the slice or smear and incubate at room temperature for 2 hours; add 100 μL, 2×SSC, elute 3 times, 15 minutes each time; use a laser confocal microscope, under 40X magnification, 8×8 stitching to shoot 4-channel images, the 4-channel excitation-emission wavelengths are red (637-685nm) / orange (561-594nm) / green (488-521nm) / blue (405-450nm) channels to shoot the first round; add 100 μL, 60% concentration formamide, elute 3 times, 5 minutes each time, to elute the bound fluorescent probe; add 100 μL, 2×SSC, elute 2 times, 5 minutes each time, and then proceed to the next round of operations. The signal probe group added in the second round of operation is [reagent 10. Second round (J region coding position 2) fluorescently labeled signal probe premix], and the rest of the operation is the same as the first round. The signal probe group added in the third round of operation is [reagent 11. Third round (V region coding position 1) fluorescently labeled signal probe premix], and the rest of the operation is the same as the first round. The signal probe group added in the fourth round of operation is [reagent 12. Fourth round (V region coding position 2) fluorescently labeled signal probe premix], and the rest of the operation is the same as the first round.

[0098] Computer alignment and decoding of multiple rounds of image signals specifically include: aligning the images obtained from multiple rounds of imaging, obtaining the coding sequence based on the fluorescence signal of each signal point in each round, and then obtaining the corresponding V, J or V, D, J region sequence based on the coding sequence, thereby obtaining V(D)J information. Taking the lupus model detection as an example, 4 rounds of images are obtained, and the 4 rounds of fluorescence signals of each signal point are read one by one using python code. According to the designed coding sequence list, a cell-gene list is generated to decode the VJ rearrangement information of each signal point.

[0099] The V(D)J signal spatial map is obtained by the system or detection method of the present invention, and multi-omics data can be integrated to jointly analyze the signal pathways and molecular targets of T cell and B cell receptors in target diseases or tissues. It can be used to discover spatially specific molecular interactions and cell interactions in tissues and molecular targets at the subcellular level, and can be used as a screening method and diagnosis and treatment aid for immune-related diseases such as systemic lupus erythematosus, tumors, and rheumatism. It can also be used for the detection of T cell surface antigens and can be used as a quality inspection plan in the process of functionalized T cell therapy.

[0100] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A T cell receptor and / or B cell receptor detection system based on spatial omics method, characterized in that: The system comprises: Module (1), which constructs a coding primer pool based on the VJ region sequence of the TCR and / or BCR to be tested, wherein the VJ region is formed by rearrangement of the V region and the J region, and the coding primer pool allocates specific coding sequences to each V region and J region, or V region and CDR3 region to be tested; Module (2), which amplifies the VJ region to be tested of the tissue sample to be tested by using the coding primer pool, and fixes the amplified product; Module (3) uses a signal probe set to locate the amplified product and read the coding sequence, thereby obtaining the position information of the VJ region and obtaining its sequence information based on the coding sequence.

2. The system according to claim 1, characterized in that The encoding primer pool includes primer set 1 and primer set 2, wherein: The primer set 1 includes one or more V region primers and one or more J region primers; The V region primer includes a 5' end sequence that is reverse complementary to the V region sequence of the TCR and / or BCR to be tested and a 3' end artificial sequence that includes the V region coding sequence; The J region includes a 5' end sequence that is reverse complementary to the J region sequence of the TCR and / or BCR to be tested and a 3' end artificial sequence that includes a J region coding sequence; The primer set 2 includes one or more VJ primers and one or more JV primers, and the 5' ends of the VJ primers and the JV primers are modified with a phosphate group; The VJ primer includes a 5' end sequence that is reverse complementary to a portion of the artificial sequence of the V region primer, a spacer sequence, and a 3' end sequence that is reverse complementary to a portion of the artificial sequence of the J region primer, and the 5' end and 3' end of the VJ primer include sequences that are reverse complementary to a portion of the V region coding sequence and the J region coding sequence, respectively; The JV primer includes a 5' end sequence that is reverse complementary to a portion of the artificial sequence of the J region primer, a spacer sequence, and a 3' end sequence that is reverse complementary to a portion of the artificial sequence of the V region primer, and the 5' end and 3' end of the JV primer include sequences that are reverse complementary to a portion of the J region coding sequence and the V region coding sequence, respectively; The 3' end of the VJ primer and the 5' end of the JV primer are connected by DNA ligase to form a complete sequence that is reverse complementary to the J region coding sequence; The 3' end of the JV primer and the 5' end of the VJ primer are connected by DNA ligase to form a complete sequence that is reverse complementary to the V region coding sequence; Preferably, the primer set 1 includes the nucleic acid sequences shown in SEQ ID NOs: 1-16, and the primer set 2 includes the nucleic acid sequences shown in SEQ ID NOs: 17-32.

3. The system according to claim 2, characterized in that The encoding primer pool includes primer set 1 and primer set 2, wherein: The primer set 1 includes one or more V region primers and one or more CDR3 region primers; The V region primer includes a 5' end sequence that is reverse complementary to the V region sequence of the TCR and / or BCR to be tested and a 3' end artificial sequence that includes the V region coding sequence; The CDR3 region includes a 5' end sequence that is reverse complementary to the CDR3 region sequence of the TCR and / or BCR to be tested and a 3' end artificial sequence that includes a CDR3 region coding sequence; The primer set 2 includes one or more V-CDR3 primers and one or more CDR3-V primers, and the 5' ends of the V-CDR3 primers and the CDR3-V primers are modified with a phosphate group; The V-CDR3 primer includes a 5' end sequence that is reverse complementary to a portion of the artificial sequence of the V region primer, a spacer sequence, and a 3' end sequence that is reverse complementary to a portion of the artificial sequence of the CDR3 region primer, and the 5' end and 3' end of the V-CDR3 primer include sequences that are reverse complementary to a portion of the V region coding sequence and the CDR3 region coding sequence, respectively; The CDR3-V primer includes a 5' end sequence that is reverse complementary to a portion of the artificial sequence of the CDR3 region primer, a spacer sequence, and a 3' end sequence that is reverse complementary to a portion of the artificial sequence of the V region primer, and the 5' end and 3' end of the CDR3-V primer include sequences that are reverse complementary to a portion of the CDR3 region coding sequence and the V region coding sequence, respectively; The 3' end of the V-CDR3 primer and the 5' end of the CDR3-V primer are connected by DNA ligase to form a complete sequence that is reverse complementary to the CDR3 region coding sequence; The 3' end of the CDR3-V primer and the 5' end of the V-CDR3 primer are connected by DNA ligase to form a complete sequence that is reverse complementary to the V region coding sequence; Preferably, the primer set 1 includes the nucleic acid sequences shown in SEQ ID NOs: 9-16 and SEQ ID NOs: 49-56, and the primer set 2 includes the nucleic acid sequences shown in SEQ ID NOs: 17-32.

4. The system according to any one of claims 2 to 3, characterized in that: The signal probe group includes: 1) a V region nucleic acid probe group; and 2) a J region nucleic acid probe group or a CDR3 region nucleic acid probe group; The V region nucleic acid probe set is used to read the V region coding sequence, wherein the reading position is referred to as a reading position, and for each reading position, the V region nucleic acid probe set includes 4 nucleic acid probes labeled by different fluorescent groups, wherein the 4 probes include a sequence that is reverse complementary to the artificial sequence of the V region primer and a sequence corresponding to the coding sequence; wherein, in the sequence corresponding to the coding sequence, the nucleotides corresponding to the coding positions of the 4 probes are A, T, G and C, respectively, and the corresponding sequences outside the coding positions are degenerate sequences; optionally, the sequence corresponding to the coding sequence is located at the 5' end, 3' end or in the middle of the nucleic acid probe; The J region nucleic acid probe set is used to read the J region coding sequence, wherein the reading position is referred to as a reading position, and for each reading position, the J region nucleic acid probe set includes 4 nucleic acid probes labeled by different fluorescent groups, wherein the 4 probes include a sequence that is reverse complementary to the artificial sequence of the J region primer and a sequence corresponding to the coding sequence; wherein, in the sequence corresponding to the coding sequence, the nucleotides corresponding to the coding positions of the 4 probes are A, T, G and C, respectively, and the corresponding sequences outside the coding positions are degenerate sequences; optionally, the sequence corresponding to the coding sequence is located at the 5' end, 3' end or in the middle of the nucleic acid probe; The CDR3 region nucleic acid probe set is used to read the CDR3 region coding sequence, wherein the reading position is referred to as a reading position, and for each reading position, the CDR3 region nucleic acid probe set includes 4 nucleic acid probes labeled by different fluorescent groups, wherein the 4 probes include a sequence that is reverse complementary to the artificial sequence of the CDR3 region primer and a sequence corresponding to the coding sequence; wherein, in the sequence corresponding to the coding sequence, the nucleotides corresponding to the coding positions of the 4 probes are A, T, G and C, respectively, and the corresponding sequences outside the coding positions are degenerate sequences; optionally, the sequence corresponding to the coding sequence is located at the 5' end, 3' end or in the middle of the nucleic acid probe; Preferably, the fluorescent group labeled with the signal probe set is selected from red, orange, green and blue fluorescent groups; more preferably, the fluorescent group is selected from Cy5, Cy3, AF488 and AMCA fluorescent groups.

5. The system according to claim 4, characterized in that The V region nucleic acid probe set includes the nucleic acid sequences shown in SEQ ID NOs: 41-48, and the J region nucleic acid probe set or the CDR3 region nucleic acid probe set includes the nucleic acid sequences shown in SEQ ID NOs: 33-40.

6. The system according to any one of claims 2 to 5, characterized in that: The module (2) comprises using the coding primer pool, DNA ligase and DNA polymerase to form a circular template and perform rolling circle amplification on the circularized template, and hydrogel fixation of the rolling circle amplification product; the DNA ligase is preferably T4 ligase, and the DNA polymerase is preferably Phi29 enzyme.

7. The system according to any one of claims 1 to 5, characterized in that: The module (3) obtains the position information and sequence information of the VJ region to be tested through multiple rounds of imaging and multiple rounds of decoding.

8. The system according to any one of claims 1 to 7, characterized in that: The tissue sample to be tested comes from a lupus animal model or a lupus patient or a subject of TCR-T cell therapy or a T cell donor.

9. A kit for detecting BCR and / or TCR, characterized in that: The kit comprises primer set 1, primer set 2 and signal probe set in the system according to any one of claims 1 to 8; preferably, the kit further comprises DNA ligase and DNA polymerase, the DNA ligase is preferably T4 ligase, and the DNA polymerase is preferably Phi29 enzyme.

10. The kit according to claim 9, characterized in that The primer set 1 includes 1) the nucleic acid sequence shown in SEQ ID NO:1-16 or 2) the nucleic acid sequence shown in SEQ ID NO:9-16 and SEQ ID NO:49-56, the primer set 2 includes the nucleic acid sequence shown in SEQ ID NO:17-32, and the signal probe set includes the nucleic acid sequence shown in SEQ ID NO:33-48. Preferably, the 5' end of the nucleic acid sequence shown in SEQ ID NO:33, 37, 41, 45 is modified with a Cy5 fluorescent group; the 5' end of the nucleic acid sequence shown in SEQ ID NO:34, 38, 42, 46 is modified with a Cy3 fluorescent group; the 5' end of the nucleic acid sequence shown in SEQ ID NO:35, 39, 43, 47 is modified with an AF488 group; the 5' end of the nucleic acid sequence shown in SEQ ID NO:36, 40, 44, 48 is modified with an AMCA group.

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