RSS-based classification method in VDJ rearrangement and application thereof

Abstract

The invention provides an antibody heavy chain classification method, which comprises the following steps: dividing a base AG contained in an RSS sequence of an antibody heavy chain into two categories, namely IGHV127 and IGHV346; the invention also provides an application thereof. Compared with the prior art, the RSS-based classification method in VDJ rearrangement and the application thereof have the advantages that the RSS classification mode is redefined, classification division is performed according to the number of AG bases in the RSS sequence, the law of each type of RSS in evolution is effectively revealed, the preference of each type of RSS on antigens can be determined accordingly, and the accuracy of the RSS classification method in VDJ rearrangement and the application of the RSS-based classification method in VDJ rearrangement are improved. Various types of RSS indicate the functional difference of B cells, such as antibody types, linkage relationship between B cells and T cells, specific antigen binding and the like; most importantly, the RSS subjected to the classification mode can play a decisive role in specific recognition or antigen (or target DNA) combination, and a basis of an immune system genome is formed.

Description

Technical Field

[0001] This application relates to the field of biopharmaceutical technology, and specifically relates to a RSS-based classification method in VDJ rearrangement and its applications. Background Art

[0002] VDJ rearrangement is an important gene recombination process in the immune system. It occurs in B cells and T cells and is used to generate highly diverse immune receptors. In the human immune system, there are a large number of gene segments encoding the variable regions (V), diversity regions (D), and joining regions (J) of different antibodies or T cell receptors. VDJ rearrangement refers to the recombination of a V region, a D region, and a J region to form the variable region of the immune receptor, thereby achieving effective recognition of various pathogens.

[0003] VDJ rearrangement is completed through a series of complex steps. First, the RAG enzyme (recombination activating gene) exists in early lymphoid cells, which recognizes and binds to the RSS (Recombination Signal Sequence) site. RSS consists of a highly conserved heptamer sequence (heptamer: CACAGTG), a relatively conserved nonamer sequence (nonamer: ACAAAAACC), and a currently considered non-conserved spacer sequence, serving as the boundary of the cleavage site. Next, the RAG enzyme cleaves both sides of the locus, separating the V, D, and J regions. Then, the cleaved DNA fragments undergo random DNA ligation through the non-homologous end joining (NHEJ) or alternative end joining (AWEJ) mechanism to recombine the V, D, and J regions. This process can result in a large number of gene recombination combinations, thus generating a high degree of diversity of immune receptors. After recombination, the newly formed VDJ gene fragments will be transcribed and translated into antibodies or T cell receptors with specific immune functions. The expression of these recombined gene fragments will also undergo further selection and regulation to ensure the production of immune receptors with good functions and autoimmunity. Among them, the RSS of VDJ rearrangement is one of the most important signal sequences determining gene segment recombination, determining the DNA sequence of antibodies or T cell receptors, and thus determining their protein sequences and functions.

[0004] In antibodies, the heavy chain is mainly responsible for providing structural stability and diversity. The heavy chain plays an important role in the formation of the antigen-binding site of the antibody and determines the binding of the antibody to a specific antigen. In previous studies, researchers classified all human antibody heavy chains into IGHV1-7 according to the similarity of their encoded protein sequences (i.e., the DNA sequence of the coding region), and these 7 classes of heavy chains did not show other obvious differences except for sequence similarity. Summary of the Invention

[0005] In view of the above-mentioned technical limitations, the present application proposes a RSS-based classification method for VDJ rearrangement and its application, which overcomes the deficiencies and defects mentioned in the background art.

[0006] To achieve the above object, the present application adopts the following technical solutions:

[0007] The inventive point of the present application is to provide a classification method for antibody heavy chains. By 23 bases in the RSS sequence of antibody heavy chains, they are divided into two categories according to the bases AG they contain, namely: sequences with the number of bases AG less than or equal to 2 in the RSS spacer sequence of antibody heavy chains are divided into one category, denoted as category IGHV127; sequences with the number of bases AG greater than or equal to 3 in the RSS spacer sequence of antibody heavy chains are divided into one category, denoted as category IGHV346.

[0008] Optionally, in the above-mentioned classification method for antibody heavy chains, all human antibody heavy chains are first divided into 7 categories of IGHV1-IGHV7 according to the similarity of their encoded protein sequences, and then artificial intelligence is used to analyze and calculate the splicing events, and they are divided into 2 major categories according to the number of bases AG contained therein, namely category IGHV127 and category IGHV346.

[0009] "Artificial intelligence analyzes splicing events" is an existing research result. Specifically, through various artificial intelligence methods, it is found that the proportion of the last two bases AG at the end of human exons reaches about 50% or more, and the proportion of the last base G in the exon is about 80%, which is significantly higher than that of the remaining positions (25%). Based on this, a hypothesis that AG has a large connection with splicing events is proposed, and then a classification method classified by the number of AG is obtained through statistics in antibody heavy chains.

[0010] The second inventive point of the present application is to provide the application of the above classification method in evaluating the possibility and law of the evolution process of adaptive immunity in vertebrates.

[0011] Optionally, for the above application, the judgment criteria are as follows:

[0012] a. All or more of the antibody heavy chains of category IGHV127 appear in vertebrates with a low degree of evolution, and antibody heavy chains of category IGHV346 do not appear or appear very rarely. Vertebrates with a low degree of evolution include relatively lower chondrichthyes, osteichthyes, archosaurs, amphibians, etc.;

[0013] b. Both category IGHV127 and category IGHV346 antibody heavy chains appear in primates, laurasiatheria, rodents, etc., and the proportion of IGHV346 in all antibody heavy chains gradually increases and exceeds the proportion of IGHV127;

[0014] c. During the evolution from non-mammals to mammals, certain transitional animals showed both IGHV127-class antibody heavy chains and IGHV346-class antibody heavy chains that were similar but not identical to those of humans.

[0015] The third inventive point of this application is to provide the application of the above classification method in determining the antibody usage preference of the human immune response.

[0016] Optionally, for the above application, the determination method is as follows:

[0017] A. When the inducer of the human immune response is an exogenous protein, the antibodies produced are mainly IGHV127-class antibodies;

[0018] B. When the inducer of the human immune response is an endogenous protein, the antibodies produced are mainly IGHV346-class antibodies.

[0019] The fourth inventive point of this application is to provide the application of the above classification method in determining the functional differences of B cells.

[0020] Optionally, for the above application, the determination method for the functional differences of B cells includes the following steps:

[0021] S1. Further divide the two types of antibody heavy chains, IGHV127 and IGHV3467, in the classification method into three categories, namely IGHV346-1: single-cell populations with more than 90%-95% being IGHV346; IGHV346-2: IGHV346 in the remaining single-cell populations; IGHV127: IGHV127 in the remaining single-cell populations;

[0022] S2. Perform characteristic gene set and Gene Ontology (GO) AUCell scoring on the three types of B cells obtained in step S1, and the specific differences are as follows:

[0023] 1) In terms of antibody types, IGHV346-1 is more expressed as IgM and less or not expressed as IgG, IgA, IgE; IGHV346-2 is less expressed as IgM and more expressed as IgG, IgA, IgE; IGHV127 is rarely or almost not expressed as IgA and is at an intermediate level in the expression of IgM, IgE, and IgG;

[0024] 2) In the linkage relationship between B cells and T cells, IGHV346-1 shows a relatively high level of activation of the MHC II-related pathway, IGHV346-2 and IGHV127 show relatively low levels of activation of the MHC II-related pathway, and IGHV346-2 is the lowest; IGHV346-1 shows a relatively low level of activation of the MHC I-related pathway, IGHV346-2 and IGHV127 show relatively high levels of activation of the MHC I-related pathway, and IGHV346-2 is the highest;

[0025] 3) In terms of specific antigen binding, IGHV346-1 binds more to antigens in the form of RNA or DNA, IGHV346-2 mainly binds to protein-related antigens, and IGHV127 binds more to polysaccharide-related antigens.

[0026] In the above step 2) of S2, the linkage relationship between B cells and T cells is similar to that of the heavy chain IGHV of B cells. The TRBV present in T cells also has RSS in the structure of heptamer-spacer-nonamer. According to the amino acid similarity of its functional regions, it is divided into TRBV1 to TRBV30. Using the same classification method as the RSS spacer sequence of the B cell heavy chain (the judgment standard changes from the number of AG to the number of CT in the reverse complementary strand of AG), TRBV1 to TRBV30 can be divided into TRBV-A class with the number of CT in the RSS spacer sequence less than or equal to 2 and TRBV-B class with the number of CT in the RSS spacer sequence greater than 2 (i.e., greater than or equal to 3).

[0027] T cells are mainly divided into CD8+ T cells and CD4+ T cells. Among them, CD8+ T cells usually directly kill antigens, and CD4+ T cells can be further divided into Th1 cells, Th2 cells, Th17 cells, Tfh cells, etc. Among them, Th2 cells bind to and activate B cells to produce antibodies. In single-cell data analysis, using the above classification method (judging the number of CT), CD4+ Th1 / 2 cells, other CD4+ T cells, and CD8+ T cells can be better distinguished, that is, the number of CT in the 23nt of the TRBV RSS spacer sequence of Th1 / 2 cells is TRBV-A class with less than or equal to 2, and other CD4+ T cells and CD8+ T cells contain both TRBV-A class with the number of CT less than or equal to 2 and TRBV-B class with the number of CT greater than 2 (i.e., greater than or equal to 3).

[0028] Specifically as Figure 4 shown.

[0029] The fifth inventive point of the present application is to provide the application of the above classification method in identifying key sequences of endogenous DNA in the genome of the immune system.

[0030] Optionally, for the above application, the method for identifying the endogenous DNA is specifically:

[0031] (a) Based on the PAM site sequence NAG / NGG, a key element for the Cas protein to recognize and cleave events in the bacterial CRISPR / Cas system, which is similar to the AG element in the spacer sequence of RSS in the V(D)J region of higher animals, the possibility that the V(D)J region in higher animals evolved from the prokaryotic CRISPR / Cas system is determined;

[0032] (b) Based on the Cas (CRISPR-associated) protein, as a nuclease in the bacterial CRISPR / Cas system, being able to recognize the target DNA sequence guided by the Guide RNA (gRNA) and the PAM site and cleave the target DNA, which is similar to the cleavage process of the important protein elements RAG1 / RAG2 and their complex in the V(D)J rearrangement process of vertebrates, the possibility that the V(D)J rearrangement process in vertebrates evolved from the prokaryotic Cas cleavage process is determined;

[0033] (c) Based on the fact that the Guide RNA (gRNA) is an RNA sequence nucleic acid molecule in the CRISPR / Cas system, which can direct the position where the Cas protease cleaves the target DNA sequence and forms a complex with the Cas protease, the possibility that there is one or more RNAs assisting or guiding the cleavage process of RAG1 / RAG2 and their complex in the V(D)J rearrangement process of vertebrates is determined.

[0034] Compared with the prior art, the present application has the following advantages:

[0035] The RSS-based classification method and its application in VDJ rearrangement provided by the present application redefine the classification method of RSS. The RSS sequences under this classification form can be judged by the number of AG bases therein, which can effectively reveal the laws of various types of RSS in evolution, and can also determine the antigen preferences of various types of RSS. Various types of RSS indicate the differences in B cell functions, such as antibody types, the linkage relationship between B cells and T cells, specific antigen binding, etc.; most importantly, the RSS after this classification method plays a decisive role in specifically recognizing or binding to antigens (or target DNA), forming the basis of the immune system genome. Brief Description of the Drawings

[0036] Figure 1 Shows the distribution of the number of AG in the spacer sequences of the heavy chain antibodies of 481 vertebrates.

[0037] Figure 2 Shows the phylogenetic tree of the spacer sequences of the heavy chain antibodies of 481 vertebrate species and the distribution of the number of AG in the spacer sequences of the species.

[0038] Figure 3 Classification standard diagram showing human antibody spacer sequences; it is drawn based on the frequencies of bases at each position in all sequences. Among them, those with the number of AG bases less than or equal to 2 in the human heavy-chain antibody spacer sequences are IGHV127, and those with the number of AG bases greater than 3 in the human antibody spacer sequences are IGHV346.

[0039] Figure 4 The determination results after classifying T-cell TRBV RSS spacer sequences using a similar classification method are shown; among them, the classification method of T-cell TRBV RSS spacer sequences is extremely similar to that of B-cell heavy-chain IGHV RSS spacer sequences, that is, the classification criteria are reverse complementary CT (T cells) / AG (B cells) respectively, to determine the possibility of a common origin of T cells and B cells in the human immune system. Detailed implementation mode

[0040] To make the objectives, technical solutions, and advantages of this application clearer, the following further details this application. However, it should be understood that the description herein is only used to explain this application and not to limit the scope of this application.

[0041] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which this application belongs. The terms used in the description of this application herein are only for the purpose of describing specific embodiments and are not intended to limit this application. The reagents and instruments used herein are all commercially available, and the characterization means involved can refer to the relevant descriptions in the prior art and will not be elaborated herein.

[0042] To further understand this application, the following further details this application in combination with the best embodiments.

[0043] Example 1

[0044] A classification method for antibody heavy chains, which divides 23 bases in the antibody heavy-chain RSS sequence into two categories according to the bases AG they contain, namely: sequences with the number of AG bases less than or equal to 2 in the antibody heavy-chain RSS spacer sequence are divided into one category, denoted as category IGHV127; sequences with the number of AG bases greater than or equal to 3 in the antibody heavy-chain RSS spacer sequence are divided into one category, denoted as category IGHV346.

[0045] First, all human antibody heavy chains are divided into 7 categories of IGHV1 - IGHV7 according to the similarity of the protein sequences they encode, and then artificial intelligence is used to analyze and calculate the splicing events, which are divided into 2 major categories according to the number of bases AG contained therein, namely category IGHV127 and category IGHV346.

[0046] "Artificial intelligence analysis of splicing events" is an existing research result. Specifically, through various artificial intelligence methods, it is found that the proportion of the two bases AG at the end of human exons reaches about 50%, and the proportion of the last base G in the exon is about 80%, which is significantly higher than that of the other positions (25%). Based on this, a hypothesis that AG has a large connection with splicing events is proposed. Subsequently, a classification method classified by the number of AG is obtained through statistics in the heavy chain of antibodies.

[0047] This application also provides the application of the above classification method in evaluating the possibility and law of the evolution process of adaptive immunity in vertebrates.

[0048] The judgment criteria for the application are as follows:

[0049] a. All or most of the vertebrates with a low degree of evolution have IGHV127-class antibody heavy chains, and do not have or rarely have IGHV346-class antibody heavy chains. Vertebrates with a low degree of evolution include relatively low-level cartilaginous fish, bony fish, archosaurs, amphibians, etc.

[0050] b. Primates, Laurasiatheria, rodents, etc. simultaneously have both IGHV127 and IGHV346 classes of antibody heavy chains, and the proportion of IGHV346 in all antibody heavy chains gradually increases and exceeds the proportion of IGHV127.

[0051] c. During the evolution from non-mammals to mammals, certain transitional period animals show both IGHV127-class antibody heavy chains and IGHV346-class antibody heavy chains that are similar but not exactly the same as those of humans.

[0052] This application also provides the application of the above classification method in determining the antibody usage preference of the human immune response.

[0053] The judgment method for the application is as follows:

[0054] A. When the inducer of the human immune response is an exogenous protein, the antibodies produced are mainly IGHV127-class antibodies;

[0055] B. When the inducer of the human immune response is an endogenous protein, the antibodies produced are mainly IGHV346-class antibodies.

[0056] This application also provides the application of the above classification method in determining the functional differences of B cells.

[0057] In the application, the judgment method for the functional differences of B cells includes the following steps:

[0058] S1. Further divide the two types of antibody heavy chains, IGHV127 and IGHV3467, in the classification method into three categories, namely IGHV346-1: more than 90%-95% of the single-cell clusters are IGHV346; IGHV346-2: IGHV346 in the remaining single-cell clusters; IGHV127: IGHV127 in the remaining single-cell clusters;

[0059] S2. Perform feature gene set and Gene Ontology (GO) AUCell scoring on the three types of B cells obtained in step S1, and the specific differences are as follows:

[0060] 1) In terms of antibody types, IGHV346-1 is more expressed as IgM and less or not expressed as IgG, IgA, IgE; IGHV346-2 is less expressed as IgM and more expressed as IgG, IgA, IgE; IGHV127 is rarely or hardly expressed as IgA and is at an intermediate level in the expression of IgM, IgE, and IgG;

[0061] 2) In the linkage relationship between B cells and T cells, IGHV346-1 shows a higher level of activation of the MHC II-related pathway, IGHV346-2 and IGHV127 show a lower level of activation of the MHC II-related pathway, and IGHV346-2 is the lowest; IGHV346-1 shows a lower level of activation of the MHC I-related pathway, IGHV346-2 and IGHV127 show a higher level of activation of the MHC I-related pathway, and IGHV346-2 is the highest;

[0062] 3) In terms of specific antigen binding, IGHV346-1 binds more to antigens in the form of RNA or DNA, IGHV346-2 mainly binds to protein-related antigens, and IGHV127 binds more to polysaccharide-related antigens.

[0063] In the second item of step S2 above, the linkage relationship between B cells and T cells is similar to that of the IGHV of the B cell heavy chain. The TRBV existing in T cells also has RSS in the structure of heptamer-spacer-nonamer. According to the amino acid similarity of its functional regions, it is divided into TRBV1 to TRBV30. Using the same classification method as the RSS spacer sequence of the B cell heavy chain (the judgment standard changes from the number of AGs to the number of CTs in the reverse complementary strand of AG), TRBV1 to TRBV30 can be divided into TRBV-A class with the number of CTs in the RSS spacer sequence less than or equal to 2 and TRBV-B class with the number of CTs in the RSS spacer sequence greater than 2 (i.e., greater than or equal to 3).

[0064] T cells are mainly divided into CD8+ T cells and CD4+ T cells. Among them, CD8+ T cells usually directly kill antigens, and CD4+ T cells can be further divided into Th1 cells, Th2 cells, Th17 cells, Tfh cells, etc. Among them, Th2 cells bind to and activate B cells to produce antibodies. In single-cell data analysis, using the above classification method (determining the number of CTs), CD4+ Th1 / 2 cells, other CD4+ T cells, and CD8+ T cells can be better distinguished. That is, the number of CTs in the 23nt TRBV RSS spacer sequence of Th1 / 2 cells is all TRBV-A types with a CT number less than or equal to 2, while other CD4+ T cells and CD8+ T cells contain both TRBV-A types with a CT number less than or equal to 2 and TRBV-B types with a CT number greater than 2 (i.e., greater than or equal to 3).

[0065] Specifically, as Figure 4 shown.

[0066] This application also provides the application of the above classification method in identifying key sequences of endogenous DNA in the immune system genome.

[0067] In the application, the method for identifying the endogenous DNA is specifically as follows:

[0068] (a) Based on the fact that the key element PAM site sequence NAG / NGG for Cas protein to recognize and cleave events in the bacterial CRISPR / Cas system is similar to the AG element in the spacer sequence of V(D)J regions in higher animals, determine the possibility that the V(D)J regions in higher animals evolved from the prokaryotic CRISPR / Cas system;

[0069] (b) Based on the fact that Cas (CRISPR-associated) protein, as a nuclease in the bacterial CRISPR / Cas system, can recognize the target DNA sequence guided by Guide RNA (gRNA) and PAM site and cleave the target DNA, which is similar to the cleavage process of the important protein elements RAG1 / RAG2 and their complex in the V(D)J rearrangement process of vertebrates, determine the possibility that the V(D)J rearrangement process in vertebrates evolved from the prokaryotic Cas cleavage process;

[0070] (c) Based on the fact that Guide RNA (gRNA) is an RNA sequence nucleic acid molecule in the CRISPR / Cas system, which can direct the position where Cas protease cleaves the target DNA sequence and forms a complex with Cas protease, determine the possibility that there is one or more RNAs assisting or guiding the cleavage process of RAG1 / RAG2 and their complex in the V(D)J rearrangement process of vertebrates.

[0071] Example 2

[0072] RSS categories in human VDJ rearrangement:

[0073] In antibodies, the heavy chain is mainly responsible for providing structural stability and diversity. The heavy chain plays an important role in the formation of the antigen-binding site of the antibody and determines the binding of the antibody to a specific antigen. In the applicant's research, it was found that the spacer sequences of the antibody heavy chain RSS are not random or non-conservative, but are divided into two major categories according to a certain rule. In previous studies, researchers classified all human antibody heavy chains into IGHV1-7 according to the similarity of their encoded protein sequences (i.e., the DNA sequences of the coding regions), and these 7 classes of heavy chains did not show any other obvious differences except sequence similarity. Here, through the study of using AI to analyze and calculate splicing events, the applicant found that the spacer sequences of the antibody heavy chain RSS can be divided into 2 classes by the number of AG bases, that is, the RSS spacer sequences of IGHV1, IGHV2, and IGHV7 contain only 3 or fewer AG bases, denoted as IGHV127; the RSS spacer sequences of IGHV3, IGHV4, and IGHV6 contain 4 or more AG bases, denoted as IGHV346. Since the antibody heavy chain RSS contains 23 bases, that is, the human antibody heavy chain can be divided into 2 classes only by the number of AG bases in these 23 bases.

[0074] Example 3

[0075] The law of RSS categories in evolution:

[0076] Adaptive immunity is a highly specific and memory immune response that first appeared in vertebrates. During the evolutionary process of vertebrates, as the complexity of their living environment increased and the diversity of infectious pathogens, the body gradually developed a more flexible and efficient immune system. The evolution of adaptive immunity is a gradual and progressive process. Continuous evolution has made the immune system more complex and efficient, increasing the survival chance and quality of life of vertebrates.

[0077] Therefore, the applicant counted the RSS of 481 vertebrate antibodies, covering from lower fish (bony fish, cartilaginous fish) to higher mammals (even-toed ungulates, primates, etc.), and obtained the following results:

[0078] 1. In lower cartilaginous fish, bony fish, archosaurs, amphibians, etc., all or most of them have IGHV127-class antibody heavy chains, and IGHV346-class antibody heavy chains do not appear or appear very rarely;

[0079] 2. Primates, Laurasiatheria, rodents, etc. including humans have both IGHV127 and IGHV346 classes of antibody heavy chains, and the proportion of IGHV346 in all antibody heavy chains gradually increases and exceeds the proportion of IGHV127;

[0080] 3. During the evolution from non-mammals to mammals, certain transitional animals (such as the platypus) showed both IGHV127-class antibody heavy chains and IGHV346-class antibody heavy chains that were similar but not identical to those of humans. The above results explained the possible evolutionary process and rules of adaptive immunity in vertebrates from the perspective of sequences.

[0081] The specific number of AG in the RSS spacer sequence of the heavy-chain antibodies of 481 vertebrates is shown in Table 1.

[0082] Table 1

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095] Example 4

[0096] Antigen preference of human RSS classes:

[0097] These two types of antibodies, IGHV127 and IGHV346, showed different preferences for antigens from different sources in the human body. When the human body encounters an immune response caused by exogenous proteins, the antibodies produced by the human body are mainly IGHV127-class antibodies; when the human body encounters an immune response caused by endogenous proteins, the antibodies produced by the human body are mainly IGHV346-class antibodies.

[0098] According to literature retrieval and database analysis, about 90% of the antibodies produced by autoimmune diseases are IGHV346 antibodies, while for the antibodies produced by other bacteria, viruses and other antigens, the ratio of IGHV127 to IGHV346 is about 6:4. Among them, for the new coronavirus (COVID-19) that has been widely spread in recent years, due to the large number of vaccines, the applicant has counted the differences in the antibodies produced by patients to different vaccines. For mRNA vaccines, about 90% of the recipients produce IGHV346 antibodies, and very few patients produce IGHV127 antibodies; for inactivated virus vaccine recipients and acutely infected patients, both IGHV127 and IGHV346 antibodies exist, and the proportions of the two types of antibodies are basically the same.

[0099] In the above results, the mechanism of action of mRNA vaccines in producing antigens is similar to that of antigens in autoimmune diseases, that is, they are all produced by the body's own RNA translation, and the proportion of IGHV346 antibodies is basically the same; while the antigens in inactivated virus vaccines or acute infections are all exogenously provided antigens, so the proportions of the two types of antibodies are also very similar. These results all suggest that there are certain differences in the preference of IGHV127 and IGHV346 antibodies for antigens.

[0100] Example 5

[0101] Functional differences between human RSS-class B cells:

[0102] By analyzing the single-cell immune repertoire sequencing data in multiple data sets, the above two types of antibody heavy chains can be further divided into three categories, namely,

[0103] IGHV346-1: more than 90% to 95% of the single-cell populations are IGHV346;

[0104] IGHV346-2: IGHV346 in the remaining single-cell clusters;

[0105] IGHV127: IGHV127 in the remaining single cell populations.

[0106] These three types of B cells (antibody heavy chains) cover all B cells, but have great differences in function, especially in antibody types, the linkage between B cells and T cells, and specific antigen binding.

[0107] The specific differences obtained by comparing the characteristic gene sets and Gene Ontology (GO) AUCell scores of these three types of B cells are as follows:

[0108] a) In terms of antibody types, IGHV346-1 is more expressed as IgM and less or not expressed as IgG, IgA, IgE; IGHV346-2 is less expressed as IgM and more expressed as IgG, IgA, IgE; IGHV127 is rarely or hardly expressed as IgA and is at an intermediate level in the expression of IgM, IgE, and IgG.

[0109] b) In the linkage relationship between B cells and T cells, IGHV346-1 shows a higher level of activation of the MHC II-related pathway, while IGHV346-2 and IGHV127 show a lower level of activation of the MHC II-related pathway, with IGHV346-2 being the lowest. MHC II mainly induces an immune response through CD4+ T cells (Th cells), which is consistent with the relatively high binding level of IGHV346-1 to the CD4 receptor; on the contrary, for the activation of the MHC I-related pathway, IGHV346-1 shows a lower level, while IGHV346-2 and IGHV127 show a higher level, with IGHV346-2 being the highest. MHC I mainly initiates an immune attack by binding to CD8+ T cells (cytotoxic T cells), which is consistent with the relatively low binding level of IGHV346-1 to the CD8 receptor and the relatively high binding levels of IGHV346-2 and IGHV127 to the CD8 receptor.

[0110] c) In terms of specific antigen binding, IGHV346-1 binds more to antigens in the form of RNA or DNA, IGHV346-2 mainly binds to protein-related antigens, and IGHV127 binds more to polysaccharide-related antigens.

[0111] These differences suggest that B cells can exhibit some unique functional characteristics at the genomic level and are not randomly matched in acquired adaptive immunity.

[0112] Example 6

[0113] RSS is the basis of the immune system genome:

[0114] The CRISPR / Cas system is an acquired immune system in bacteria that is used to combat foreign DNA, plasmids, and phages that invade bacteria. Different from the immune systems of ordinary prokaryotes, the CRISPR / Cas system is an acquired immune system, which means that this immune system has "memory". It can remember the foreign DNA and phages that have invaded, and cut their genomes when they invade again. The cut genomes will become linear, unable to replicate and express, and will be degraded by enzymes in the bacteria. Among them, the PAM (Protospacer Adjacent Motif) site is the key sequence for the CRISPR / Cas system to recognize foreign DNA. The PAM site is located after the DNA target and is the key signal for the CRISPR / Cas system to recognize and cut foreign DNA. The CRISPR / Cas system judges whether it pairs with the target DNA sequence according to the PAM sequence. The existence of the PAM sequence enables the system to distinguish foreign DNA from the DNA of its own genome. In the CRISPR / Cas9 system, the PAM site is usually a sequence of 3 base pairs. Generally, the PAM sequence is NGG (N represents any nucleotide, and G represents guanosine), and the most common one is AGG. This sequence form has a high sequence similarity and functional similarity with the highly repetitive AG / AGG sequences in the V region RSS spacer sequence during VDJ rearrangement of the IGHV gene in higher vertebrates. Subsequently, the guide RNA (gRNA) guides the Cas protein to cut on the target DNA. The gRNA usually consists of two parts: crRNA and tracrRNA. The crRNA (CRISPR RNA) is generated by extracting the spacer sequence of the CRISPR region and contains a sequence complementary to the target DNA. The tracrRNA (trans-activating CRISPR RNA) is an RNA molecule that forms a gRNA complex after binding to the crRNA. The biological function produced by the combined action of the gRNA and the Cas protein is extremely similar to the biological function of the RAG protein catalyzing VDJ rearrangement during VDJ rearrangement of the IGHV gene in higher vertebrates.

[0115] The CRISPR / Cas defense system of bacteria or archaea against foreign DNA (such as viral genomes) has great similarities with the adaptive immunity of vertebrates in terms of sequence structure, the role of biological components, and overall biological functions. Whether it is the PAM site in CRISPR / Cas or the RSS in the VDJ rearrangement of the vertebrate immune system, both play a decisive role in specifically recognizing or binding antigens (or target DNA) and are the basis of the immune system genome.

[0116] Example 7

[0117] To verify the specific application of this classification method, in the experiment, by obtaining the Cas protein sequences and Cas protein domain information of various bacteria and archaea, and aligning them with the RAG1 / RAG2 proteins of various vertebrates with evolutionary relationships to construct a phylogenetic tree, and then analyzing their possible evolutionary directions and positions.

[0118] Example 8

[0119] To verify the specific application of this classification method, in the experiment, by obtaining the RAG1 / RAG2 proteins of various vertebrates with evolutionary relationships and their protein domain information, and then conducting a comparative search in the NCBI bacterial non-redundant protein sequence database (NR), and then specifically analyzing the sequence associations or evolutionary relationships between the search results and Cas and related proteins.

[0120] The above are only the preferred embodiments of the present application and are not intended to limit the present application. Any modifications, equivalent replacements, or improvements made within the spirit and principle of the present application shall be included within the protection scope of the present application.

Claims

1. A classification method for antibody heavy chains, characterized in that, The 23 bases in the RSS sequence of the antibody heavy chain are classified into two categories according to the bases AG they contain. They are: the sequences with the number of bases AG less than or equal to 2 in the RSS spacer sequence of the antibody heavy chain are classified into one category, denoted as category IGHV127; the sequences with the number of bases AG greater than or equal to 3 in the RSS spacer sequence of the antibody heavy chain are classified into one category, denoted as category IGHV346.

2. The classification method of an antibody heavy chain according to claim 1, wherein The classification method is to first classify all human antibody heavy chains into 7 categories of IGHV1 - IGHV7 according to the similarity of the encoded protein sequences, and then use artificial intelligence to analyze and calculate the splicing events, which are classified into 2 major categories according to the number of bases AG contained therein, namely category IGHV127 and category IGHV346.

3. Application of the classification method according to claim 1 or 2 in evaluating the possibility and law of the evolution process of adaptive immunity in vertebrates.

4. The application according to claim 3, wherein The determination criteria are as follows: a. Vertebrates with a low degree of evolution all or mostly have antibody heavy chains of category IGHV127, and do not have or rarely have antibody heavy chains of category IGHV346. Vertebrates with a low degree of evolution include relatively lower chondrichthyes, osteichthyes, archosaurs, amphibians, etc.; b. Primates, Laurasiatheria, rodents, etc. simultaneously have antibody heavy chains of both category IGHV127 and category IGHV346, and the proportion of IGHV346 in all antibody heavy chains gradually increases and exceeds the proportion of IGHV127; c. During the evolution process from non - mammals to mammals, certain transitional period animals show both antibody heavy chains of category IGHV127 and IGHV346 - like but not exactly the same as those of humans.

5. Application of the classification method according to claim 1 or 2 in determining the antibody usage preference of the human immune response.

6. The application according to claim 5, characterized in that, The determination method is as follows: A. When the inducer of the human immune response is an exogenous protein, the antibodies produced are mainly antibodies of category IGHV127; B. When the inducer of the human immune response is an endogenous protein, the antibodies produced are mainly antibodies of category IGHV346.

7. Application of the classification method according to claim 1 or 2 in determining the functional differences of B cells.

8. The application according to claim 7, characterized in that The determination method for the functional differences of B cells includes the following steps: S1. Further classify the two types of antibody heavy chains of IGHV127 and IGHV346 in the classification method into three categories, namely IGHV346 - 1: single - cell populations with more than 90% - 95% being IGHV346; IGHV346 - 2: IGHV346 in the remaining single - cell populations; IGHV127: IGHV127 in the remaining single - cell populations; S2. Perform characteristic gene set and Gene Ontology (GO) AUCell scoring on the three types of B cells obtained in step S1, and the specific differences are as follows: 1) In terms of antibody types, IGHV346-1 is more expressed as IgM and less or not expressed as IgG, IgA, IgE; IGHV346-2 is less expressed as IgM and more expressed as IgG, IgA, IgE; IGHV127 is rarely or almost not expressed as IgA and is at an intermediate level in the expression of IgM, IgE, and IgG. 2) In terms of the linkage relationship between B cells and T cells, IGHV346-1 shows a relatively high level of activation of the MHC II-related pathway, IGHV346-2 and IGHV127 show a relatively low level of activation of the MHC II-related pathway, and IGHV346-2 is the lowest; IGHV346-1 shows a relatively low level of activation of the MHC I-related pathway, IGHV346-2 and IGHV127 show a relatively high level of activation of the MHC I-related pathway, and IGHV346-2 is the highest. 3) In terms of specific antigen binding, IGHV346-1 binds more to antigens in the form of RNA or DNA, IGHV346-2 mainly binds to protein-related antigens, and IGHV127 binds more to polysaccharide-related antigens.

9. Use of the classification method according to claim 1 or 2 for identifying key sequences of endogenous DNA in the immune system genome.

10. The application according to claim 9, wherein The specific method for identifying the endogenous DNA is as follows: (a) Based on the fact that the PAM site sequence NAG\NGG, the key element for Cas protein to recognize and cleave events in the bacterial CRISPR / Cas system, is similar to the AG element in the spacer sequence of RSS in the V(D)J region of higher animals, determine the possibility that the V(D)J region in higher animals evolved from the prokaryotic CRISPR\Cas system. (b) Based on the fact that Cas (CRISPR-associated) protein, as a nuclease in the bacterial CRISPR / Cas system, can recognize the target DNA sequence guided by Guide RNA (gRNA) and PAM site and cleave the target DNA, which is similar to the cleavage process of the important protein elements RAG1 / RAG2 and their complex in the vertebrate V(D)J rearrangement process, determine the possibility that the vertebrate V(D)J rearrangement process evolved from the prokaryotic Cas cleavage process. (c) Based on the fact that Guide RNA (gRNA) is an RNA sequence nucleic acid molecule in the CRISPR / Cas system, which can direct the position where Cas protease cleaves the target DNA sequence and forms a complex with Cas protease, determine the possibility that there is one or more RNAs assisting or guiding the cleavage process of RAG1 / RAG2 and their complex in the vertebrate V(D)J rearrangement process.

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