WO2020211229A1 - 一种免疫力水平评估的方法及装置 - Google Patents

一种免疫力水平评估的方法及装置 Download PDF

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WO2020211229A1
WO2020211229A1 PCT/CN2019/101357 CN2019101357W WO2020211229A1 WO 2020211229 A1 WO2020211229 A1 WO 2020211229A1 CN 2019101357 W CN2019101357 W CN 2019101357W WO 2020211229 A1 WO2020211229 A1 WO 2020211229A1
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seq
bcr
immunity
variable region
tcr
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PCT/CN2019/101357
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French (fr)
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张志新
卓越
侯百东
华兆琳
叶树剑
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成都益安博生物技术有限公司
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    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B15/00—ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment
    • G16B15/20—Protein or domain folding
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • G16B20/20—Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B30/00—ICT specially adapted for sequence analysis involving nucleotides or amino acids
    • G16B30/10—Sequence alignment; Homology search
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B35/00—ICT specially adapted for in silico combinatorial libraries of nucleic acids, proteins or peptides
    • G16B35/10—Design of libraries

Definitions

  • the invention relates to the field of immunity detection, in particular to a method for assessing the level of immunity through immune atlas analysis.
  • the main methods currently used to analyze immune function on the market are: 1) five items of immunity, which detect the content of immunoglobulin and complement in the blood; 2) blood routine, use the method of cell counting to analyze the number of white blood cells and the number of white blood cells in the peripheral blood The increase in cytokine indicates that there is an inflammatory response in the body; 3) Lymphocyte subgroup analysis, using flow cytometry and PCR technology to analyze the number and relative proportion of each subgroup of white blood cells in the peripheral blood.
  • Immunoglobulin G immunoglobulin G
  • immunoglobulin A immunoglobulin G
  • immunoglobulin A immunoglobulin A
  • immunoglobulin M immunoglobulin M
  • complement C3 and C4 content immunoglobulin and complement are the main effect components of humoral immunity.
  • diseases such as infections, autoimmune diseases, immunodeficiency diseases, etc.
  • the concentration of these indicators will increase or decrease relative to the reference value, which is useful for evaluation.
  • Immunity the clinical value of diagnosing diseases.
  • the five tests of immunity are aimed at humoral immunity and cannot assess cellular immunity well.
  • humoral immunity only the overall levels of IgG, IgA, IgM and complement C3 and C4 can be detected, and in-depth analysis at the molecular sequence level cannot be performed.
  • Immune cells are white blood cells, which are mainly divided into lymphocytes and macrophages, which are the basic components of the immune system. Routine blood testing is to classify and count the white blood cells in the peripheral blood through microscope observation.
  • the total number of white blood cells above the upper limit of the reference value is called leukopenia, and the lower limit of the reference value is called leukopenia. Its increase and decrease are mainly affected by the number of neutrophils, and changes in the number of lymphocytes can also cause changes in the total number of white blood cells. From physiological changes to malignant tumors, the total number of white blood cells may be abnormal. Doctors can make clinical diagnosis based on the results of routine blood tests. Routine blood tests can only roughly judge the overall level of cellular immunity, and cannot distinguish immunity against specific diseases, nor can it judge the classification and diversity of immune cells at the genetic level.
  • lymphocyte subpopulation analysis has good detection results for some specific diseases. For example, the results of epidemiological statistics show that people with positive HLA-B27 genotype have several inherited immune diseases, which are much higher than ordinary people. Spondylitis, for example, is about 87 times.
  • the CD3/HLA-B27 double-labeled antibody is used in the test to delineate the T lymphocyte subsets, and then analyze whether the cell surface shows HLA-B27 tissue antigen, which can effectively help clinical differential diagnosis of ankylosing spondylitis and other seronegative bone and joint diseases.
  • HLA-B27 tissue antigen can effectively help clinical differential diagnosis of ankylosing spondylitis and other seronegative bone and joint diseases.
  • lymphocyte subsets there are many types. If a comprehensive analysis is performed, the amount of peripheral blood that needs to be collected, the cost, and the time are unacceptable. It is difficult to obtain a comprehensive immune system status if only a few lymphocyte subsets are analyzed.
  • the existing immunoassay methods generally only target a certain aspect of the immune system, and can only perform a relatively rough assessment, and lack comprehensiveness, sensitivity and accuracy.
  • B and T lymphocytes are two important types of cells in the adaptive immune system. Among them, B cells express cell surface antigen receptor BCR/antibody in the early development process, and express as antibodies after stimulation by antigen and cofactors to secrete out of the cell; T cells express cell surface antigen receptor TCR. In the early development of B or T lymphocytes, the diversity of BCR/antibody and TCR produced by genetic recombination is the basis for establishing normal immune function.
  • the molecular structure of BCR/antibody and TCR is divided into constant region and variable region, and its diversity comes from the change of variable region sequence.
  • the diversity of BCR/antibody and TCR variable region sequences comes from the V(D)J gene recombination that occurs during the formation of immune cells: the genome contains multiple variable(V), diversity(D) and joining(J) genes, These genes are separated from each other on the chromosomes of hematopoietic stem cells. When hematopoietic stem cells differentiate into immune cells, randomly selected V, D, and J genes are linked together to form the variable regions of BCR/antibody and TCR.
  • CDR complementarity determining region
  • each B or T cell expresses only one BCR/antibody or TCR.
  • the diversity of BCR/antibody and TCR may be destroyed in the case of aging, deterioration of physical condition, or disease.
  • next-generation sequencing technology it is possible to detect genetic changes at the molecular level. There is an urgent need for a method to detect and evaluate the immune system at the molecular genetic level.
  • the present invention is based on high-throughput sequencing technology and target region-specific amplification technology, and realizes the use of a small amount of peripheral blood samples, at the genetic molecular level, the overall level of the immune system Accurate and quantitative assessment provides a method and device for assessing the level of the immune system.
  • the method provided by the present invention uses the separation of lymphocytes from the venous blood of the subject, the extraction of mRNA and reverse transcription to generate cDNA, the specific amplification of TCR and/or BCR variable regions for library construction and sequencing, and the test is obtained from the sequencing data
  • the immunity level of the person overcomes the characteristics that the prior art can only target a certain aspect of the immune system and can only perform relatively rough evaluations, and lacks comprehensiveness, sensitivity and accuracy.
  • the present invention provides the following technical solutions:
  • the present invention provides a method for assessing the immunity level of a subject, including: separating lymphocytes from a blood sample of the subject, obtaining lymphocyte RNA and performing reverse transcription; using specific primers BCR amplification and / or TCR variable region of a polynucleotide, for sequencing of the variable region polynucleotide sequence information obtained; D 50 value calculation BCR and / or TCR variable region sequence, determined in accordance with the subject D 50 The level of immunity.
  • the method described above may further include the following technical features:
  • the method is used for non-diagnostic purposes.
  • the above methods for assessing the level of immunity can be used in scientific research, such as scientific research on animal or human trials of immune cell therapy. It is necessary to track the type, number, and diversity of immune cells in the body.
  • the specific primers for amplifying the variable region of BCR are SEQ ID NO: 1-SEQ ID NO: 31, including BCR-specific reverse transcription primers and BCR-specific amplification primers, wherein BCR The specific reverse transcription primers are SEQ ID NO:1-SEQ ID NO:3; the BCR specific amplification primers are SEQ ID NO:4-SEQ ID NO:21.
  • the specific primers for amplifying the TCR variable region are SEQ ID NO: 32-SEQ ID NO: 81, including TCR-specific reverse transcription primers and TCR-specific amplification primers, wherein TCR
  • the specific reverse transcription primer is SEQ ID NO: 32-SEQ ID NO: 35; the TCR specific amplification primer is SEQ ID NO: 32-SEQ ID NO: 81.
  • variable region is defined by BCR and/or TCR complementarity determining region 3 (CDR3), wherein the variable region sequence is divided into functional variable regions and non-functional variable regions.
  • CDR3 TCR complementarity determining region 3
  • the D 50 is determined according to the level of immunity of a subject, the D 50 is determined by the following steps:
  • the numbers are N1, N2...NC, and are sorted from most to least copy number (N1 ⁇ N2 ⁇ ...NC-1 ⁇ NC), the number of clone types with the most copies and occupying 50% of the total variable region sequence is recorded as
  • the ratio of H, H to C is D 50 . among them,
  • the present invention provides a device for evaluating the immunity level of a subject, a sequencing unit, and the sequencing unit is used to analyze the BCR and/or TCR variable region polynucleotides of the lymphocytes of the subject.
  • a judging unit which is connected to the D 50 determining unit, and judges the subject's immunity level according to the D 50 value.
  • FIG. 1 Electrophoresis of lymphocyte RNA; Figure 2. Electrophoresis of BCR amplification products; Figure 3. Electrophoresis of TCR amplification products
  • the present invention is based on high-throughput sequencing technology and target region-specific amplification technology. Lymphocytes are separated from the peripheral blood of the subject, RNA is extracted, and cDNA is obtained by reverse transcription, and the BCR and/or TCR variable regions are amplified by specific primers. Obtain its sequence information and evaluate the subject's immunity level. Realize the use of a small amount of peripheral blood samples, at the genetic molecular level, a comprehensive, accurate and quantitative assessment of the overall level of the immune system.
  • a method for assessing the immunity level of a subject including the following steps:
  • Step 1 Lymphocyte separation, BCR and/or TCR variable region sequence acquisition
  • Lymphocytes were separated from the subject's venous blood using conventional methods, and the lymphocyte mRNA was extracted and reverse transcription performed. Using specific primers to amplify the variable regions of BCR and/or TCR, build a library for sequencing.
  • Step 2 Assess the subject's immunity level
  • D 50 is calculated and used as an index for evaluating the diversity of the BCR/TCR sequence.
  • the total number of functional variable region CDR3 sequences is denoted as N
  • the number of different sequence types of functional variable region CDR3 sequences is C
  • the number of copies of each sequence is N1, N2...NC
  • the number of copies is Sort from most to least (N1 ⁇ N2 ⁇ «NC-1 ⁇ NC)
  • the number of clone types with the most copies and occupying 50% of the total functional variable region CDR3 sequences is recorded as H
  • the ratio of H to C is D 50 .
  • D 50 ⁇ 0.05, weakened immunity, may have suffered from serious diseases or have a high risk of disease
  • Trizol reagent (Ambion, article number 15596026). Generally, 5 ⁇ 10 6 to 1 ⁇ 10 7 cells are taken. The number of cells must be strictly counted. If there are too many cells, it will lead to incomplete RNase inhibition and lead to RNA degradation. The specific steps are as follows:
  • the detection result is that the total amount of RNA>5 ⁇ g, the volume>20 ⁇ l, the OD260/280:1.8, and the 2% agarose electrophoresis detection, there are three bands, 28sRNA, 18sRNA, 5sRNA, of which 28S is 1 ⁇ 2 of the brightness of the 18S band Times, the 5S band is the shallowest (see Figure 1).
  • the name and sequence of the BRTmix primers are shown in the following table.
  • the primer concentration is 1:1:1, and the total concentration is 20uM.
  • a specific amplification reaction is performed on the BCR gene of cDNA.
  • the PCR enzyme used is Taq enzyme from Tongke Company (Cat. No. TK01015).
  • the reaction system and conditions are shown in the following table:
  • VHmix primers are 5'-end multiplex PCR primers.
  • the names and sequences of the primers are shown in the table below. They are mixed 1:1, and the total concentration is 20uM.
  • VHmix Sequence 5’-3’ Serial number 5VH1a CAGGTGCAGCTGGTGCAGTCTGGGG SEQ ID NO: 4 5VH1b CAGGTCCAGCTTGTGCAGTCTGGGG SEQ ID NO: 5 5VH1C CAGGTTCAGCTGGTGCAGTCTGGAG SEQ ID NO: 6 5VH2 CAGRTCACCTTGARGGAGTCTGGTCCT SEQ ID NO: 7 5VH3a GAGGTGCAGCTGGTGGAGTCTGGGG SEQ ID NO: 8 5VH3b CAGGTGCAGCTGGTGGAGTCTGGGG SEQ ID NO: 9 VH3-23 GAGGTGCAGCTGTTGGAGTCTGGGG SEQ ID NO: 10 VH3-74 GAGGTGCAGCTGGTGGAGTCCGGGG SEQ ID NO: 11 5VH4 CAGGTGCAGCTGCAGGAGTCGGGC SEQ ID NO: 12 5VH4-34 CAGGTGCAGCTACAGCAGTGGGGC SEQ ID NO: 13 5VH4-39 CA
  • JHmix primers are 3’-end multiplex PCR primers. The names and sequences of the primers are shown in the table below. They are mixed at 1:1.
  • the concentration is 10uM, and the lowercase letter part is matched with the protection sequence of the second round 3’ end primer barcode
  • the second round of PCR reaction was carried out with Taq enzyme from Tongke Company (Cat. No. TK01015), the reaction system and reaction conditions were as follows:
  • the first round of PCR products 2ul VHmix primer (20um) 1ul JHxBCX(20uM) 1ul 10 ⁇ Buffer 2ul Mgcl 2 1.6ul dNTP(10um) 0.8ul Enzyme 1.2ul ddH 2 O 10.4ul total 20ul
  • the VHmix primer is the 5'-end multiplex PCR primer, that is, the VHmix in the first round of PCR.
  • JHxBCX is a 3'-end primer.
  • the primer name and sequence are shown in the following table. It contains primer sequences containing 10 different barcodes.
  • the primer sequence includes barcode sequence (underlined part), barcode protection recognition sequence (lowercase letter part) and specific amplification Sequence, one sample chooses one JHxBCX primer corresponding to a unique barcode, so as to distinguish different samples according to the barcode sequence.
  • the 3'end primers of the 6 samples are selected from the primers SEQ ID NO: 22-27 in the following table.
  • JHxBCX Sequence 5’-3’ Serial number JHxBC1 gag ATCACGTT tctcacCTGAGGAGACGGTGACC SEQ ID NO: 22 JHxBC2 gag CGATGTTT tctcacCTGAGGAGACGGTGACC SEQ ID NO: 23 JHxBC3 gag TTAGGCAT tctcacCTGAGGAGACGGTGACC SEQ ID NO: 24 JHxBC4 gag TGACCACTt ctcacCTGAGGAGACGGTGACC SEQ ID NO: 25 JHxBC5 gag ACAGTGGT tctcacCTGAGGAGACGGTGACC SEQ ID NO: 26 JHxBC6 gag GCCAATGT tctcacCTGAGGAGACGGTGACC SEQ ID NO: 27 JHxBC7 gag CAGATCTG tctcacCTGAGGAGACGGTGACC SEQ ID NO: 28 JHxBC8 gag ACTTGATG tctcacCTGAGGAGACGGTGACC SEQ ID NO
  • the target band is at more than 400 bp, as shown in Figure 2.
  • Magnetic bead purification one sample can be purified after the second round of PCR translation in 1 to 2 tubes. Add equal volume to PCR product
  • the DNA fragments were sorted and purified by magnetic beads (BMSX), and eluted with 30ul water. Use micro spectroscopy
  • the concentration of the purified product is measured by a meter.
  • TRTmix is a reverse transcription primer and also serves as a 3'primer for PCR reaction.
  • the name and sequence are shown in the following table. They are mixed at 1:1:1 and the total concentration is 20uM.
  • TRTmix Sequence 5’-3’ Numbering hTCRb-RT1 tgggagat SEQ ID NO: 32 hTCRb-RT2 cttttggg SEQ ID NO: 33 hTCRb-RT3 ccagtgtg SEQ ID NO: 34 hTCRb-RT4 ctctgcttctgatggctcaaacacagc SEQ ID NO: 35
  • V ⁇ mix primers are 5'-end multiplex PCR primers.
  • the primer names and sequences are shown in the following table. They are mixed 1:1 and the total concentration is 20uM.
  • TRBV26 gatgctgtagttacacacaattcccaagacacag SEQ ID NO: 39 TRBV25 gaagctgacatctaccagaccccaagatacc SEQ ID NO: 40 TRBV24 gatgctgatgttacccagaccccaaggaata SEQ ID NO: 41 TRBV27 gaagcccaagtgacccagaacccaagatac SEQ ID NO: 42 TRBV28 gatgtgaaagtaacccagagctcgagatatctagtc SEQ ID NO: 43 TRBV19 gatggtggaatcactcagtccccaaagtacc SEQ ID NO: 44 TRBV10 gatgctgaaatcacccagagcccaag SEQ ID NO: 45 TRBV6-4 attgctgggatcacccaggcaccaa SEQ ID NO: 46 TRBV6-19 aatgctggtgtcactcagaccccaa SEQ ID NO: 47 TR
  • TRTmix is a reverse transcription primer and also serves as a 3'primer for PCR reaction.
  • the name and sequence are shown in the following table. They are mixed at 1:1:1 and the total concentration is 20uM.
  • the TCR region was specifically amplified using Taq enzyme (product number TK01015) from Tongke Company.
  • the reaction system and conditions are shown in the following table:
  • hTCRCbBCX primer (20uM) 0.5ul 10 ⁇ Buffer 2ul Mgcl 2 1.6ul dNTP(10um) 0.8ul Enzyme 1.2ul ddH 2 O 9.4ul total 20ul
  • the multiple primers at the 5'end of V ⁇ mix are the same as the V ⁇ mix in the same reaction of RT-PCR, and the concentration is also 20uM.
  • the 3'end hTCRCbBCX primer (20uM) is selected from one of the primers in the following table.
  • the primers in the following table contain different barcocde sequences.
  • One sample is selected for one hTCRCbBCX primer.
  • One sample corresponds to only one barcode (capital letters).
  • the 3'end primers of the 6 samples are SEQ ID NO: 72-SEQ ID NO: 77.
  • the amplification effect was detected by electrophoresis, and the target band was about 350bp, as shown in Figure 3.
  • One sample can be purified after the second round of PCR with 1-2 tubes. Add 1.4 times the volume of DNA fragment separation and purification magnetic beads (Bemeg Biotech product number BMSX) to the PCR product, and eluted with 30ul water. A micro spectrophotometer was used to detect the concentration of the purified product.
  • DNA fragment separation and purification magnetic beads Bemeg Biotech product number BMSX
  • kits including Ion Plus Fragment Library Kit (Cat. No. 4471252), Ion Xpress Barcode Adaptors 1-16 Kit (Cat. No. 4471250), Agencourt AMPure XP (Cat. No. A63881), and the instruments include PCR, Qubit quantifier and nucleic acid quantification Reagents.
  • Ion chef-automated template preparation instrument after installing reagents and consumables, the experiment can be performed automatically, and the library can be added with ISP magnetic beads, enrichment, and loading chips.
  • the reagents used include Ion 520/530 ExT- chefs-4rxns&4 Init NEW-For 600bp (Cat. No. A30670), Ion 530 Chip Kit (Cat. No. A27764).
  • the reverse-processed FQ file is formatted to obtain a Fasta file.
  • Alignment file Capture the alignment data of the functional sequence, save it separately, and the file name is productive.
  • the productive_CDR3_count file performs D 50 calculation.
  • the specific calculation method is as follows:
  • the sequence information of the BCR/TCR variable region obtained by sequencing.
  • the total number of CDR3 sequences in the variable region of all functional genes is recorded as N
  • the number of CDR3 types of different sequences (clones) is recorded as C
  • the copy number of each clone is recorded It is N1, N2...NC and sorted from most to least copy number (N1 ⁇ N2 ⁇ ...NC-1 ⁇ NC)
  • the number of clone types with the most copy number and occupying 50% of the total functional CDR3 sequences is recorded as H
  • the ratio of H to C is defined as D 50 , which is used as an index to evaluate the diversity of BCR/TCR sequences.
  • TCR sequencing data is processed, and the QC results are shown in the following table:
  • the immunity level and health status of the subjects are evaluated.
  • D 50 ⁇ 0.05, low immunity, may have suffered from serious diseases or have a high risk of disease
  • Example 2 The method of the present invention evaluates the immune level of a large number of subjects
  • Example 2 According to the method described in Example 1, 124 subjects were tested and analyzed by the immune profile.
  • the D 50 values corresponding to all subjects are as follows:
  • EXAB24 0.158 EXAB25 0.065 EXAB26 0.072 EXAB67 0.0799 EXAB27 0.054 EXAB28 0.040 EXAB29 0.004 EXAB30 0.033 EXAB31 0.104 EXAB32 0.115 EXAB33 0.034 EXAB34 0.118 EXAB35 0.152 EXAB36 0.115 EXAB37 0.000 EXAB38 0.129 EXAB39 0.067 EXAB40 0.045 EXAB41 0.065 EXAB42 0.170 EXAB43 0.042 EXAB44 0.118 EXAB45 0.110 EXAB46 0.133 EXAB47 0.070 EXAB48 0.083 EXAB49 0.069 EXAB50 0.022 EXAB51 0.037 EXAB52 0.149 EXAB53 0.124 EXAB54 0.137 EXAB55 0.127 EXAB56 0.084 EXAB57 0.129 EXAB58 0.171 EXAB59 0.087 EXAB60 0.053 EXAB61
  • the subjects are classified according to the value of D 50 , and the health status of the subjects is analyzed.
  • D 50 >0.05 95% of the subjects are healthy, and when D 50 ⁇ 0.05, 60% of the subjects are Tumor patients. It is recommended that healthy subjects with D 50 ⁇ 0.05 undergo an in-depth physical examination, and 2 of them are found to have early cancer, which is expected to develop into leukemia within 2-3 years. Therefore, the subject's immune diversity (D 50 ) can be used to judge their health status.
  • D 50 ⁇ 0.05, low immunity, may have suffered from serious diseases or have a high risk of disease
  • the D 50 of the first subject is >0.15, and the immunity is excellent, and the D 50 of the second and third subjects is between 0.10-0.15 ,
  • the immunity is normal, the D 50 of subjects 4, 5, and 6 is ⁇ 0.05, the immunity is low, and they may have suffered from serious diseases or have a high risk of disease.
  • the results of the immune atlas analysis were compared with the health status of the subjects. Subjects No. 4, 5, and 6 were all tumor patients, which conformed to the drawn rules.
  • the index's 50-D symbol CDR3 diversity as an indicator to assess the level of immunizing the subject.
  • first and second are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. Therefore, the features defined with “first” and “second” may explicitly or implicitly include at least one of the features.
  • a plurality of means at least two, such as two, three, etc., unless otherwise specifically defined.
  • the terms “installed”, “connected”, “connected”, “fixed” and other terms should be understood in a broad sense, for example, it can be a fixed connection or a detachable connection. , Or integrated; it can be mechanically connected, or it can be electrically connected or can communicate with each other; it can be directly connected or indirectly connected through an intermediate medium, it can be the internal communication of two components or the interaction relationship between two components, Unless otherwise clearly defined.
  • the specific meaning of the above-mentioned terms in the present invention can be understood according to specific circumstances.

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Abstract

一种评估受试者免疫力水平的方法和装置，包括从受试者样本中分离淋巴细胞，提取淋巴细胞的RNA并进行逆转录，利用特异性引物获得BCR和/或TCR可变区多核苷酸，对所述多核苷酸进行测序获得序列信息，计算BCR和/或TCR可变区序列的D50值，从而判断受试者的免疫力水平。

Description

一种免疫力水平评估的方法及装置 技术领域
本发明涉及免疫力检测领域，具体涉及一种通过免疫图谱分析评估免疫力水平的方法。
背景技术
目前市场上用来分析免疫功能的主要方法有：1)免疫五项，检测血液中免疫球蛋白和补体的含量；2)血常规，利用细胞计数的方法分析外周血中白细胞的数量，白细胞数目的增高表明体内存在炎症反应；3)淋巴细胞亚群分析，利用流式细胞分析以及PCR技术分析外周血中白细胞各个亚群的数目和相对比例。
免疫五项检测
通过单向免疫扩散试验、酶联免疫吸附试验(ELISA)、放射免疫试验(RIA)、免疫固定电泳、免疫比浊法等方法，检测血液中免疫球蛋白G(IgG)、免疫球蛋白A(IgA)、免疫球蛋白M(IgM)、补体C3和C4的含量。免疫球蛋白和补体是体液免疫的主要效应成分，在某些疾病(如感染、自身免疫疾病、免疫缺陷病等)情况下，这些指标的浓度相对参考值将出现升高或降低，从而具有评估免疫力、诊断疾病的临床价值。然而免疫五项检测针对体液免疫，不能很好评估细胞免疫。在评估体液免疫时，只能检测IgG、IgA、IgM和补体C3、C4的总体水平，不能在分子序列层次上进行深度分析。
血常规检测
免疫细胞即白细胞，主要分为淋巴细胞和巨噬细胞，是免疫系统的基本组成单位。血常规检测是通过显微镜观测对外周血中的白细胞进行分类和计数。白细胞总数高于参考值上限称白细胞增多，低于参考值下限为白细胞减少。其增多和减少主要受中性粒细胞数量的影响，淋巴细胞等数量的改变也会引起白细胞总数的变化。从生理性变化到恶性肿瘤都有可能引起白细胞总数异常，医生可结合血常规检测侧结果进行临床诊断。血常规检测只能大致判断细胞免疫整体水平的状况，无法分辨针对具体疾病的免疫，也无法在基因水平判断免疫细胞的分类和多样性。
淋巴细胞亚群分析
通过流式细胞分析或PCR技术，对外周血中免疫细胞的相对计数、绝对计数及其变化进行监控，分析疾病状态下的免疫状况(如肿瘤、感染性疾病、免疫性疾病等)，以此辅助诊断、追踪病情发展及决定用药时机。最常检测的亚群包括T细胞(CD3)、B细胞(CD19)、NK细胞(CD16+56)、辅助性T细胞(CD3+CD4+)和抑制性T细胞(CD3+CD8+)等。淋巴细胞亚群分析针对一些特定疾病具有较好检测效果，例如流行病学统计结果显示，HLA-B27基因型阳性 的人患数个遗传性免疫疾病的机率，比一般人要高很多，以强直性脊柱炎为例，约为87倍。因此检测时选用CD3/HLA-B27双标记抗体，圈定T淋巴细胞亚群，再分析其细胞表面是否表现HLA-B27组织抗原，可以有效帮助临床鉴别诊断强直性脊柱炎等血清阴性骨关节疾病。然而淋巴细胞亚群种类繁多，如进行全面分析，则需要采集的外周血量、费用及时间均难以接受。只进行少数几种淋巴细胞亚群分析，则难以获取全面的免疫系统状况。
目前，现有的免疫检测方法，普遍存在只能针对免疫系统的某一方面，且只能进行较粗略的评估，缺乏全面性、灵敏度和精准性。
无论体液免疫还是细胞免疫，根本上免疫能力来自于相关的免疫细胞。B和T淋巴细胞是获得性免疫系统中重要的两类细胞。其中，B细胞在早期发育过程中表达细胞表面抗原受体BCR/抗体，在抗原和辅助因子刺激后表达成抗体分泌到细胞外；T细胞表达细胞表面抗原受体TCR。在B或T淋巴细胞早期发育过程中，由基因重组产生的BCR/抗体和TCR的多样性是建立正常免疫功能的基础。
BCR/抗体和TCR的分子结构分为恒定区和可变区，其多样性来自可变区序列的变化。BCR/抗体和TCR可变区序列的多样性来自于免疫细胞形成过程中发生的V(D)J基因重组：基因组中含有多个variable(V)、diversity(D)和joining(J)基因，这些基因在造血干细胞的染色体上是彼此分割的。当造血干细胞分化为免疫细胞的过程中，随机选取的V、D、J基因被连接在一起，形成了BCR/抗体和TCR的可变区。BCR/抗体和TCR的可变区序列中，又以互补性决定区(complementarity determining region，CDR)最为关键，因为它决定了BCR/抗体和TCR所能结合的抗原类型，其中第三个互补决定区(CDR3)是序列变化最显著，也是在抗原决定过程中权重最高的区域，因此CDR3的多样性就代表了BCR/TCR的多样性。
正常情况下，每个B或者T细胞只表达一种BCR/抗体或者TCR。但是，在年龄增长、身体状况恶化或罹患疾病等情况下，BCR/抗体和TCR的多样性可能被破坏。随着二代测序技术的发展，为基于分子水平检测基因变化提供了可能。目前急需一种在分子基因水平检测评估免疫系统的方法。
发明内容
本发明针对现有免疫检测方法的不足，基于高通量测序技术和目标区域特异性扩增技术，实现了使用很少量外周血样本，在基因分子水平，对免疫系统整体的水平进行全面、精确且量化的评估，提供一种免疫系统水平评估的方法和装置。
本发明所提供的方法，使用从受试者静脉血中分离淋巴细胞，提取mRNA逆转录生成cDNA，特异性扩增TCR和/或BCR可变区进行建库测序，从测序数据中获得受试者的免疫力水平。克服了现有技术只能针对免疫系统的某一方面，且只能进行比较粗略的评估的特点，缺乏全面性、灵敏度和精准性的问题。
具体而言，本发明提供了如下技术方案：
根据本发明第一方面，本发明提供了一种评估受试者免疫力水平的方法，包括：从受试者血液样本中分离淋巴细胞，获得淋巴细胞的RNA并进行逆转录；利用特异性引物扩增BCR和/或TCR可变区多核苷酸，对可变区多核苷酸进行测序获得序列信息；计算BCR和/或TCR可变区序列的D 50值，根据D 50确定受试者的免疫力水平。
根据本发明的实施例，以上所述的方法可以进一步包括如下技术特征：
在本发明的一些实施例中，所述方法用于非诊断目的。以上评估免疫力水平的方法可以用于科研，例如科学研究免疫细胞治疗的动物或人体试验时，需要追踪体内免疫细胞的种类、数量、多样性等。
在本发明的一些实施例中，扩增BCR可变区的特异性引物为SEQ ID NO:1-SEQ ID NO:31，包括BCR特异性逆转录引物和BCR特异性扩增引物，其中，BCR特异性逆转录引物为SEQ ID NO:1-SEQ ID NO:3；BCR特异性扩增引物为SEQ ID NO:4-SEQ ID NO:21。
在本发明的一些实施例中，扩增TCR可变区的特异性引物为SEQ ID NO:32-SEQ ID NO:81，包括TCR特异性逆转录引物和TCR特异性扩增引物，其中，TCR特异性逆转录引物为SEQ ID NO:32-SEQ ID NO:35；TCR特异性扩增引物为SEQ ID NO:32-SEQ ID NO:81。
在本发明的一些实施例中，所述可变区界定采用BCR和/或TCR互补决定区3(CDR3)，其中可变区序列有分为功能性可变区和非功能性可变区。
在本发明的一些实施例中，根据D 50确定受试者的免疫力水平，所述D 50是通过下列步骤确定的：
测序所得BCR和/或TCR可变区多核苷酸序列信息，比对确定功能性基因可变区序列总数N，比对到功能性基因的不同序列的类型数为C，每一种序列的拷贝数为N1、N2……NC，并按拷贝数从多到少排序(N1≥N2≥……NC-1≥NC)，拷贝数最多且占据可变区序列总数50％的克隆类型数记为H，H与C的比值为D 50。其中，
当D 50<0.05，免疫力低下，可能已经罹患严重疾病或具有很高患病风险；
当0.05≤D 50<0.10，免疫力较差，处于亚健康状态；
当0.10≤D 50<0.15，免疫力正常；
当0.15≤D 50，免疫力优秀。
根据本发明的第二方面，本发明提供一种评估受试者免疫力水平的装置，测序单元，所述测序单元用于对受试者淋巴细胞的BCR和/或TCR可变区多核苷酸进行序列测定，以便获得测序数据；D 50确定单元，所述测序单元与D 50计算单元连接，基于所述测序数据，确定所述D 50。判定单元，所述判定单元与D 50确定单元连接，根据D 50值判定受试者的免疫力水平。
附图说明
图1、淋巴细胞RNA电泳图；图2、BCR扩增产物电泳图；图3、TCR扩增产物电泳图
图4、受试者健康状况直接影响TCR多样性(D 50)图
具体实施方式
下面详细描述本发明的实施例，所述实施例的示例在附图中示出，其中自始至终相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附图描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。
本发明基于高通量测序技术和目标区域特异性扩增技术，从受试者外周血分离淋巴细胞，提取RNA，逆转录获得cDNA，利用特异性引物扩增BCR和/或TCR可变区域，获得其序列信息，评估受试者免疫力水平。实现了使用很少量外周血样本，在基因分子水平，对免疫系统整体的水平进行全面、精确且量化的评估。
在本发明的至少一些实施方式，提供了一种评估受试者免疫力水平的方法，包括以下步骤：
步骤一：淋巴细胞分离、BCR和/或TCR可变区域序列的获得
利用常规方法从受试者静脉血分离淋巴细胞，提取淋巴细胞的mRNA并进行逆转录。采用特异性引物扩增BCR和/或TCR可变区域，建库测序。
步骤二：评估受试者免疫力水平
根据测序所得的BCR和/或TCR可变区序列信息，计算D 50，用作评估BCR/TCR序列多样性的指标。
其中，功能性可变区CDR3序列的总数记为N，功能性可变区CDR3序列的不同序列的类型数为C，每一种序列的拷贝数为N1、N2……NC，并按拷贝数从多到少排序(N1≥N2≥……NC-1≥NC)，拷贝数最多且占据功能性可变区CDR3序列总数50％的克隆类型数记为H，H与C的比值为D 50。
免疫力水平的判断标准如下：
(1)D 50<0.05，免疫力低下，可能已经罹患严重疾病或具有很高患病风险；
(2)0.05≤D 50<0.10，免疫力较差，处于亚健康状态；
(3)0.10≤D 50<0.15，免疫力正常；
(4)0.15≤D 50，免疫力优秀。
以下结合对具体样本依据本发明的方法进行目标区域单体型的确定、基因型的确定、单体型或基因型确定后的用途进行详细的描述及结果展示。下面示例，仅用于解释本发明，而不能理解为对本发明的限制。在本发明中所使用的“第一”、“第二”、“第三”等仅用于方便描述目的，而不能理解为指示或暗示相对重要性，也不能理解为之间有先后顺序关系。 本发明的描述中，除非另有说明，“多个”的含义是两个或两个以上。
实施例1免疫图谱分析及免疫水平评估方法的建立
一、淋巴细胞分离和mRNA的提取
1、淋巴细胞分离
取6个受试者静脉血10ml，置于紫盖EDTA抗凝管中，用人淋巴细胞分离液(天津灏阳，货号LTS1077)分离淋巴细胞，具体操作步骤如下：
(1)将采集的新鲜血液10ml加入50ml离心管中，加入生理盐水定容到25ml，轻轻吹打混匀。
(2)在新的50ml离心管中，加入25ml的淋巴细胞分离液(等量)，然后用移液管吸取血样轻轻的沿壁加于分离液液面之上，500g，20℃，离心30min，升速为2，降速为1(Thermol公司低速离心机)。
(3)离心后，用10ml移液管小心吸掉大部分上层分离液，再吸取第二层环状乳白色淋巴细胞层到新的50ml离心管中，加入生理盐水定容到50ml，轻轻上下颠倒混匀，300g，20℃离心8min，弃上清。
(4)再次清洗细胞，先将管底细胞弹散，之后再加入生理盐水定容到20ml，轻轻上下颠倒混匀，400g，20℃离心5min，弃上清。
(5)向50ml离心管中加入2ml PBS轻轻吹打均匀混悬细胞，获得分离的淋巴细胞。
(6)细胞计数：取100μl混悬液，加900μl PBS稀释10倍，混匀后进行细胞计数(可根据细胞量自行调整稀释倍数)，细胞计数公式为：细胞数密度＝4大格细胞总数×稀释倍数×10 4/4＝细胞数/ml
2、淋巴细胞基因组mRNA的提取及检测
按照Trizol试剂(Ambion，货号15596026)说明书操作，一般取5×10 6～1×10 7个细胞，细胞量一定要严格计数，细胞不宜过多，会导致RNA酶抑制不彻底，导致RNA降解。具体操作步骤如下：
(1)取分离的5×10 6个淋巴细胞的混悬液，400g，4℃，离心5min，弃上清。
(2)轻弹离心管底部，松散细胞沉淀物。向淋巴细胞中加入1mL Trizol试剂，用枪吹打混匀，看不到沉淀，室温静止5min，使细胞充分裂解。
(3)加入200μl氯仿，手动剧烈摇晃15s后，室温静置5min。
(4)4℃，12000g离心15min，吸取上层水相，转移至另一离心管，约450μl。
(5)加入500μl异丙醇混匀，室温静置10min。
(6)4℃，12000g离心10min，弃上清，RNA沉于管底。
(7)加入1ml 75％乙醇，温和震荡EP管，使RNA沉淀整片悬浮。
(8)4℃，7500g离心5min，弃上清，室温放置5-8min，挥发酒精，不能过于干燥。
(9)用30μl DEPC/无酶水溶解RNA沉淀，少量分装后，做好标记，-80℃冰箱保存。
(10)取2μl RNA，用微量紫外分光光度计检测浓度和OD值，取2μl RNA电泳检测条带，使用2％的凝胶，DL2000Marker，同时立刻进行反转录实验。
检测结果为RNA总量＞5μg，体积＞20μl，OD260/280:1.8以上，同时2％琼脂糖电泳检测，出现三条带，为28sRNA、18sRNA、5sRNA，其中28S为18S条带亮度的1～2倍，5S条带最浅(见图1)。
3、逆转录及PCR扩增
3.1 BCR基因特异性扩增
1)逆转录反应
使用同科公司试剂盒(TonkBio TM First chain cDNA synthesis kit)进行逆转录反应，反应体系和反应条件如下：
Figure PCTCN2019101357-appb-000001
其中BRTmix引物的名称和序列如下表所示，引物浓度1：1:1混合，总浓度为20uM。
BRTmix 序列5’-3’ 序号
hIgM-RT AGGAAGTCCTGTGCGAGGCA SEQ ID NO:1
hIgG-RT CGGGGAAGTAGTCCTTGACC SEQ ID NO:2
hIgA-RT CGCTCCAGGTCACACTGAGT SEQ ID NO:3
2)第一轮PCR：
对cDNA的BCR基因进行特异性扩增反应，其中所用的PCR酶为同科公司Taq酶(货号TK01015)，反应体系及条件如下表所示：
逆转录产物 2ul
VHmix引物(20um) 0.5ul
JHmix(10uM) 1ul
10×Buffer 2ul
Mgcl 2 1.6ul
dNTP(10um) 0.8ul
Taq酶 1.2ul
ddH 2O 10.9ul
total 20ul
PCR程序：
Figure PCTCN2019101357-appb-000002
其中，VHmix引物为5’端多重PCR引物，引物名称和序列见下表，按1：1混合，总浓度为20uM。
VHmix 序列5’-3’ 序号
5VH1a CAGGTGCAGCTGGTGCAGTCTGGGG SEQ ID NO:4
5VH1b CAGGTCCAGCTTGTGCAGTCTGGGG SEQ ID NO:5
5VH1C CAGGTTCAGCTGGTGCAGTCTGGAG SEQ ID NO:6
5VH2 CAGRTCACCTTGARGGAGTCTGGTCCT SEQ ID NO:7
5VH3a GAGGTGCAGCTGGTGGAGTCTGGGG SEQ ID NO:8
5VH3b CAGGTGCAGCTGGTGGAGTCTGGGG SEQ ID NO:9
VH3-23 GAGGTGCAGCTGTTGGAGTCTGGGG SEQ ID NO:10
VH3-74 GAGGTGCAGCTGGTGGAGTCCGGGG SEQ ID NO:11
5VH4 CAGGTGCAGCTGCAGGAGTCGGGC SEQ ID NO:12
5VH4-34 CAGGTGCAGCTACAGCAGTGGGGC SEQ ID NO:13
5VH4-39 CAGCTGCAGCTGCAGGAGTCGGGC SEQ ID NO:14
5VH5 GARGTGCAGCTGGTGCAGTCTGGAG SEQ ID NO:15
5VH6 CAGGTACAGCTGCAGCAGTCAGGTCC SEQ ID NO:16
5VH7 CAGGTGCAGCTGGTGCAATCTGGGTC SEQ ID NO:17
JHmix引物为3’端多重PCR引物，引物名称和序列见下表所示，按1：1混合，总
浓度为10uM，其中小写字母部分是与第二轮3’端引物barcode的保护序列配对
JHmix 序列5’-3’ 编号
JH145 tctcacCTGAGGAGACGGTGACCAGGGT SEQ ID NO:18
JH2 tctcacCTGAGGAGACAGTGACCAGGGT SEQ ID NO:19
JH3 tctcacCTGAAGAGACGGTGACCATTGTCCCTTG SEQ ID NO:20
JH6 tctcacCTGAGGAGACGGTGACCGTGGTC SEQ ID NO:21
不进行电泳检测，接着进行第二轮扩增。
3)第二轮PCR:
使用同科公司Taq酶(货号TK01015)进行第二轮PCR反应，反应体系和反应条件如下：
第一轮PCR产物 2ul
VHmix引物(20um) 1ul
JHxBCX(20uM) 1ul
10×Buffer 2ul
Mgcl 2 1.6ul
dNTP(10um) 0.8ul
酶 1.2ul
ddH 2O 10.4ul
total 20ul
PCR程序：
Figure PCTCN2019101357-appb-000003
其中VHmix引物为5’端多重PCR引物，即第一轮PCR中的VHmix。
JHxBCX为3’端引物，引物名称和序列如下表所示，包含含有10个不同barcode的引物序列，引物序列包括barcode序列(下划线部分)、barcode保护识别序列(小写字母部分)和特异性扩增序列，一个样品选用一种JHxBCX引物，对应唯一一个barcode，以此根据barcode序列区分不同的样本,6个样本的3’端引物分别选自下表中引物SEQ ID NO:22-27，。
JHxBCX 序列5’-3’ 序号
JHxBC1 gag ATCACGTTtctcacCTGAGGAGACGGTGACC SEQ ID NO:22
JHxBC2 gag CGATGTTTtctcacCTGAGGAGACGGTGACC SEQ ID NO:23
JHxBC3 gag TTAGGCATtctcacCTGAGGAGACGGTGACC SEQ ID NO:24
JHxBC4 gag TGACCACTtctcacCTGAGGAGACGGTGACC SEQ ID NO:25
JHxBC5 gag ACAGTGGTtctcacCTGAGGAGACGGTGACC SEQ ID NO:26
JHxBC6 gag GCCAATGTtctcacCTGAGGAGACGGTGACC SEQ ID NO:27
JHxBC7 gag CAGATCTGtctcacCTGAGGAGACGGTGACC SEQ ID NO:28
JHxBC8 gag ACTTGATGtctcacCTGAGGAGACGGTGACC SEQ ID NO:29
JHxBC9 gag GATCAGCGtctcacCTGAGGAGACGGTGACC SEQ ID NO:30
JHxBC10 gag TAGCTTGTtctcacCTGAGGAGACGGTGACC SEQ ID NO:31
4)电泳检测扩增效果，目的条带在400多bp处，见图2。
5)磁珠纯化：一个样本做1～2管第二轮PCR翻译后即可纯化。在PCR产物中加入等体积
的DNA片段分选纯化磁珠(百迈格生物货号BMSX)，使用30ul水洗脱。使用微量分光光
度计检测纯化后产物浓度。
6)等浓度混合6个不同barcode样本，建库测序及后续分析。
3.2 TCR扩增
1)RT-PCR反应(逆转录及第一轮PCR)
使用QIAGEN onestep RT-PCR试剂盒(货号210212)对提取的RNA立刻进行反转录，之后在同一体系内连续进行第一轮扩增。
RNA 1ug
TRTmix引物(20uM) 0.625ul
Vβmix引物(20um) 0.625ul
5×onestep RT-PCR Buffer 5ul
RNase inhibitor 0.25ul
dNTP(10um) 1ul
酶 1ul
Add RNase-free H 2O to 25ul
RT-PCR程序：
Figure PCTCN2019101357-appb-000004
TRTmix为逆转录引物，也作为PCR反应3’端引物，名称和序列如下表所示，按1：1：1混合，总浓度为20uM。
TRTmix 序列5’-3’ 编号
hTCRb-RT1 tgggagat SEQ ID NO:32
hTCRb-RT2 cttttggg SEQ ID NO:33
hTCRb-RT3 ccagtgtg SEQ ID NO:34
hTCRb-RT4 ctctgcttctgatggctcaaacacagc SEQ ID NO:35
其中Vβmix引物为5’端多重PCR引物，引物名称和序列如下表所示，按1：1混合，总浓度为20uM。
Vβmix 序列5’-3’ 编号
TRBV20 ggtgctgtcgtctctcaacatccgag SEQ ID NO:36
TRBV29 agtgctgtcatctctcaaaagccaagcag SEQ ID NO:37
TRBV30 tctcagactattcatcaatggccagcgacc SEQ ID NO:38
TRBV26 gatgctgtagttacacaattcccaagacacag SEQ ID NO:39
TRBV25 gaagctgacatctaccagaccccaagatacc SEQ ID NO:40
TRBV24 gatgctgatgttacccagaccccaaggaata SEQ ID NO:41
TRBV27 gaagcccaagtgacccagaacccaagatac SEQ ID NO:42
TRBV28 gatgtgaaagtaacccagagctcgagatatctagtc SEQ ID NO:43
TRBV19 gatggtggaatcactcagtccccaaagtacc SEQ ID NO:44
TRBV10 gatgctgaaatcacccagagcccaag SEQ ID NO:45
TRBV6-4 attgctgggatcacccaggcaccaa SEQ ID NO:46
TRBV6-19 aatgctggtgtcactcagaccccaa SEQ ID NO:47
TRBV23 catgccaaagtcacacagactccagga SEQ ID NO:48
TRBV15 gatgccatggtcatccagaacccaagatac SEQ ID NO:49
TRBV3 gacacagctgtttcccagactccaaaatac SEQ ID NO:50
TRBV1 cccagacaccaaaatacctggtcacagc SEQ ID NO:51
TRBV4 cacctggtcatgggaatgacaaataagaagtc SEQ ID NO:52
TRBV13 gctgctggagtcatccagtcccc SEQ ID NO:53
TRBV9 gattctggagtcacacaaaccccaaagcac SEQ ID NO:54
TRBV5-1 agatatctgatcaaaacgagaggacagca SEQ ID NO:55
TRBV5-3 acacacctgatcaaaacgagaggacagca SEQ ID NO:56
TRBV18 aatgccggcgtcatgcagaacccaaga SEQ ID NO:57
TRBV16 ggtgaagaagtcgcccagactccaaaac SEQ ID NO:58
TRBV21 gacaccaaggtcacccagagacctag SEQ ID NO:59
TRBV17 gagcctggagtcagccagaccc SEQ ID NO:60
TRBV12-1 cccaggcacaaagtgacagagatgggacaa SEQ ID NO:61
TRBV12-4 ccccggcacgaggtgacagagatgggacaa SEQ ID NO:62
TRBV14 gaagctggagttactcagttccccagc SEQ ID NO:63
TRBV2 gaacctgaagtcacccagactcccagc SEQ ID NO:64
TRBV11-3 gaagctggagtggttcagtctcccag SEQ ID NO:65
TRBV11-1 gaagctgaagttgcccagtcccccag SEQ ID NO:66
TRBV7-9 ccccagacacaagatcacaaagaggggac SEQ ID NO:67
TRBV7-6 ctcccaggtacaaagtcacaaagaggggac SEQ ID NO:68
TRBV7-8 cccctaggtacaaagtcgcaaagagaggac SEQ ID NO:69
TRBV7-1 ccctgagacacaaggtagcaaagaagggaaa SEQ ID NO:70
TRBV7-2 cccccagtaacaaggtcacagagaagggaaa SEQ ID NO:71
TRTmix为逆转录引物，也作为PCR反应3’端引物，名称和序列如下表所示，按1：1：1混合，总浓度为20uM。
2)第二轮PCR：
使用同科公司Taq酶(货号TK01015)对TCR区域进行特异性扩增，反应体系和条件如下表所示：
RT-PCR产物 4ul
Vβmix引物(20um) 0.5ul
hTCRCbBCX引物(20uM) 0.5ul
10×Buffer 2ul
Mgcl 2 1.6ul
dNTP(10um) 0.8ul
酶 1.2ul
ddH 2O 9.4ul
total 20ul
PCR程序：
Figure PCTCN2019101357-appb-000005
其中Vβmix5’端多重引物，与RT-PCR相同反应的Vβmix相同，浓度也为20uM。
3’端hTCRCbBCX引物(20uM)选自下表中的一种引物，下表中的引物含有不同的barcocde序列，一个样品选用一种hTCRCbBCX引物，一个样对应唯一一个barcode(大写字母)，在本实施例中6个样本的3’端引物分别为SEQ ID NO:72-SEQ ID NO:77。
hTCRCbBCX 序列5’-3’ 编号
hTCRCbBC1 gagTTACTCGCgcacagcgacctcgggtgggaac SEQ ID NO:72
hTCRCbBC2 gagTCGTTAGCgcacagcgacctcgggtgggaac SEQ ID NO:73
hTCRCbBC3 gagTACCGAGCgcacagcgacctcgggtgggaac SEQ ID NO:74
hTCRCbBC4 gagTGTTCTCCgcacagcgacctcgggtgggaac SEQ ID NO:75
hTCRCbBC5 gagTTCGCACCgcacagcgacctcgggtgggaac SEQ ID NO:76
hTCRCbBC6 gagTTGCGTACgcacagcgacctcgggtgggaac SEQ ID NO:77
hTCRCbBC7 gagTCTACGACgcacagcgacctcgggtgggaac SEQ ID NO:78
hTCRCbBC8 gagTGACAGACgcacagcgacctcgggtgggaac SEQ ID NO:79
hTCRCbBC9 gagTAGAACACgcacagcgacctcgggtgggaac SEQ ID NO:80
hTCRCbBC10 gagTCATCCTAgcacagcgacctcgggtgggaac SEQ ID NO:81
3)电泳检测
电泳检测扩增效果，目的条带在350bp左右，见图3。
4)磁珠纯化：
一个样本做1～2管第二轮PCR后即可纯化。在PCR产物中加入1.4倍体积DNA片段分选纯化磁珠(百迈格生物货号BMSX)，使用30ul水洗脱。使用微量分光光度计检测纯化后产物浓度。
5)等浓度混合6个不同barcode样本(最多10个，对应10种hTCRCbBCX引物)，建库测序及后续分析。
4、建库、测序及数据分析获得免疫图谱
4.1建库及高通量测序
1)建库：
严格按照操作手册操作，加上测序接头及定量工作。
根据试剂盒说明书进行建库，包括Ion Plus Fragment Library Kit(货号4471252)、Ion Xpress Barcode Adaptors 1-16Kit(货号4471250)、Agencourt AMPure XP(货号A63881)，仪器包括PCR仪、Qubit定量仪及核酸定量试剂。
2)模板制备及上机测序
Ion chef-自动化模板制备仪，安装好试剂及耗材即可自动进行实验，完成文库加上ISP磁珠、富集、loading芯片工作。
Ion S5-测序仪，完成测序工作及初步数据处理。
使用试剂包括Ion 520/530 ExT-Chef-4rxns&4 Init NEW-For 600bp(货号A30670)、Ion 530 Chip Kit(货号A27764)。
4.2数据分析获得免疫图谱
高通量测序完成后，使用FQ文件进行数据分析，序列进行Igblast，从中抓取出需用于D 50计算的功能性的不同种类的CDR3氨基酸序列，同时统计对应的数量，数量按从高到低排序，保存文件名为受试者编号_productive_CDR3_count.csv。具体如下：
(1)使用网上免费平台分析，链接https://usegalaxy.org/。原数据FQ文件进行reverse反向处理。
(2)合并正反向数据后分barcode，得到单个样本FQ文件。
(3)单个样本FQ文件进行FastQC质检，下载结果。
(4)对单个样本FQ文件再次进行reverse反向处理。
(5)反向处理的FQ文件进行格式转换，得到Fasta文件。
(6)单个样本Fasta文件进行VDJ数据比对(推荐使用免费平台Igblast比对，链接https://www.ncbi.nlm.nih.gov/igblast/)，得到比对文件。
(7)比对文件抓取功能性序列的比对数据，单独保存，文件名productive。
(8)再从功能性序列的比对数据中进一步抓取不同种类的CDR3氨基酸序列，同时统计对应的数量，数量按从高到低排序，单独保存，文件名为productive_CDR3_count
(9)productive_CDR3_count文件进行D 50计算，具体计算方式如下：
5、评估受试者免疫力水平
测序所得的BCR/TCR可变区序列信息，其中所有功能性基因可变区CDR3序列的总数记为N，不同序列(克隆)的CDR3类型数记为C，每一种克隆的拷贝数被记为N1、N2……NC并按 拷贝数从多到少排序(N1≥N2≥……NC-1≥NC)，拷贝数最多且占据功能性CDR3序列总数50％的克隆类型数记为H，H与C的比值被定义为D 50，用作评估BCR/TCR序列多样性的指标。
6个受试者的数据分析及D 50计算结果如下：
TCR测序数据进行处理，QC结果见下表：
Figure PCTCN2019101357-appb-000006
D 50相关数据统计见下表：
Figure PCTCN2019101357-appb-000007
3、根据D 50数据，对受试者的免疫水平、健康状况进行评估，我们定义通过免疫图谱分析评估受试者免疫力水平的标准为：
1)D 50<0.05，免疫力低下，可能已经罹患严重疾病或具有很高患病风险；
2)0.05≤D 50<0.10，免疫力较差，处于亚健康状态；
3)0.10≤D 50<0.15，免疫力正常；
4)0.15≤D 50，免疫力优秀。
实施例2本发明方法对大量受试者免疫水平评估
根据实施例1中描述的方法对124个受试者进行免疫图谱检测分析，所有的受试者对应的D 50值如下表：
编号 D 50 身体状况
4GS 0.111 正常
71-1 0.109 正常
65-1 0.011 乳腺癌
66号 0.065 白血病早期
97号 0.063 正常
75号 0.113 肝炎
76号 0.152 T淋巴母细胞瘤白血病(2016.7.4确诊)
77号 0.004 淋巴瘤
78号 0.037 正常
92号 0.064 正常
93号 0.038 正常
94号 0.003 白血病M4(骨髓移植)
95号 0.01 急性髓系白血病
96号 0.004 急性前B淋巴细胞白血病
97号 0.063 正常
98号 0.079 淋巴瘤
16号 0.12 正常
88号 0.075 正常
89号 0.057 正常
73-2 0.113 正常
71-2 0.081 正常
80号 0.048 NKT淋巴瘤
86号 0.034 骨髓移植
85号 0.018 淋巴瘤
87号 0.091 糖尿病
11ny 0.086 正常
11-2ny 0.129 回输NK
12-1ny 0.004 肠癌(没有回输CIK)
12-2ny 0.158 回输Cik一周
12-3ny 0.072 第二次回输CIK前
13-1ny 0.093 胃癌
13-2ny 0.155 回输CIK
14ny 0.133 正常
15ny 0.11 正常
16ny 0.032 正常
17ny 0.023 正常
18ny 0.034 正常
8-2-1ny 0.029 正常
8-2-2ny 0.011 正常
19ny 0.13 正常
20ny 0.027 正常
21ny 0.109 丙肝
22ny 0.053 糖尿病
23-1ny 0.094 正常
23-2ny 0.101 正常
24ny 0.04 肝病
100号 0.052 正常
11-3gs 0.111 正常
66-2 0.076 正常
65-4 0.001 正常
1ny 0.049 肺癌
2ny 0.021 肺癌
3ny 0.0196 肺癌
4ny 0.006 肺癌
5ny 0.021 肺癌
6ny 0.033 胆囊切除
7ny 0.001 肺癌
8ny 0.037 正常
9ny 0.042 肺癌
10ny 0.08 正常
EXAB1 0.038 正常
EXAB2 0.048 正常
EXAB3 0.063 正常
EXAB4 0.073 正常
EXAB5 0.077 正常
EXAB6 0.04 正常
EXAB7 0.185 正常
EXAB8 0.105 正常
EXAB9 0.068 正常
EXAB10 0.105 正常
EXAB64 0.05 正常
EXAB12 0.169 正常
EXAB13 0.064 正常
EXAB14 0.0435 正常
EXAB15 0.124 正常
EXAB16 0.019 正常
EXAB17 0.058 正常
EXAB18 0.18 正常
EXAB66 0.066 正常
EXAB11 0.15 正常
EXAB19 0.171 正常
EXAB20 0.155 正常
EXAB21 0.078 正常
EXAB22 0.2 正常
EXAB23 0.116 正常
EXAB24 0.158 正常
EXAB25 0.065 正常
EXAB26 0.072 正常
EXAB67 0.0799 正常
EXAB27 0.054 正常
EXAB28 0.04 正常
EXAB29 0.0035 正常
EXAB30 0.033 正常
EXAB31 0.104 正常
EXAB32 0.115 正常
EXAB33 0.034 正常
EXAB34 0.118 正常
EXAB35 0.152 正常
EXAB36 0.115 正常
EXAB37 0.000 正常
EXAB38 0.129 正常
EXAB39 0.067 正常
EXAB40 0.045 正常
EXAB41 0.065 正常
EXAB42 0.170 正常
EXAB43 0.042 正常
EXAB44 0.118 正常
EXAB45 0.11 正常
EXAB46 0.133 正常
EXAB47 0.07 正常
EXAB48 0.083 正常
EXAB49 0.069 正常
EXAB50 0.022 正常
EXAB51 0.037 正常
EXAB52 0.149 正常
EXAB53 0.124 正常
EXAB54 0.137 正常
EXAB55 0.127 正常
EXAB56 0.084 正常
EXAB57 0.129 正常
EXAB58 0.171 正常
EXAB59 0.087 正常
EXAB60 0.053 正常
EXAB61 0.043 正常
其中健康人：
编号 D 50
4GS 0.111
71-1 0.109
97号 0.063
78号 0.037
92号 0.064
93号 0.038
97号 0.063
16号 0.120
88号 0.075
89号 0.057
73-2 0.113
71-2 0.081
11ny 0.086
11-2ny 0.129
13-2ny 0.155
14ny 0.133
15ny 0.110
16ny 0.032
17ny 0.023
18ny 0.034
8-2-1ny 0.029
10ny 0.08
EXAB1 0.038
EXAB64 0.05
EXAB3 0.063
EXAB4 0.073
EXAB5 0.077
EXAB6 0.04
EXAB7 0.185
EXAB8 0.105
EXAB9 0.068
EXAB10 0.105
8ny 0.037
100号 0.052
11-3gs 0.111
EXAB12 0.169
EXAB13 0.064
EXAB14 0.0435
EXAB15 0.124
EXAB16 0.019
EXAB17 0.058
EXAB18 0.18
EXAB66 0.066
EXAB11' 0.15
EXAB19 0.171
EXAB20 0.155
EXAB21 0.078
EXAB22 0.2
EXAB23 0.116
EXAB24 0.158
EXAB25 0.065
EXAB26 0.072
EXAB67 0.0799
EXAB27 0.054
EXAB28 0.040
EXAB29 0.004
EXAB30 0.033
EXAB31 0.104
EXAB32 0.115
EXAB33 0.034
EXAB34 0.118
EXAB35 0.152
EXAB36 0.115
EXAB37 0.000
EXAB38 0.129
EXAB39 0.067
EXAB40 0.045
EXAB41 0.065
EXAB42 0.170
EXAB43 0.042
EXAB44 0.118
EXAB45 0.110
EXAB46 0.133
EXAB47 0.070
EXAB48 0.083
EXAB49 0.069
EXAB50 0.022
EXAB51 0.037
EXAB52 0.149
EXAB53 0.124
EXAB54 0.137
EXAB55 0.127
EXAB56 0.084
EXAB57 0.129
EXAB58 0.171
EXAB59 0.087
EXAB60 0.053
EXAB61 0.043
白血病人：
编号 D 50 病例
66号 0.065 白血病早期
76号 0.152 T淋巴母细胞瘤白血病(骨髓移植)
77号 0.004 淋巴瘤
94号 0.003 白血病M4(骨髓移植)
95号 0.01 急性髓系白血病
96号 0.004 急性前B淋巴细胞白血病
98号 0.079 淋巴瘤
80号 0.048 NKT淋巴瘤
86号 0.034 骨髓移植
85号 0.018 骨髓移植
91号 0.012 淋巴瘤
癌症病人：
编号 D 50 病例
24ny 0.04 肝癌
1ny 0.049 肺癌
2ny 0.021 肺癌
3ny 0.0196 肺癌
4ny 0.006 肺癌
5ny 0.021 肺癌
7ny 0.001 肺癌
9ny 0.042 肺癌
12-1ny 0.004 肠癌
13-1ny 0.093 胃癌
65-1 0.011 乳腺癌
理想情况下，每一种CDR3克隆只有一个功能性拷贝，D 50＝0.5；当受试者免疫力下降，或罹患肿瘤、免疫缺陷等疾病时，D 50数值会急剧下降。来自75位健康受试者及20位肿瘤患者的免疫图谱分析表明，大多数健康受试者的D 50>0.05，而绝大多数罹患肿瘤受试者的D 50<0.05。健康人群与白血病患者的D 50存在显著差异(p＝0.0014),健康人群与癌症患者的D 50存在显著差异(p＝0.00007),而白血病患者与癌症患者的D 50没有显著差异(p＝0.498)。因此，受试者健康状况与免疫多样性(D 50)具有显著正相关性。
根据D 50数值大小对受试者进行分类，再对照受试者健康状况进行分析，D 50>0.05时，95％受试者是健康人，而D 50<0.05时，60％受试者是肿瘤患者。建议D 50<0.05的健康受试者进行深入体检后，有2人查出癌变早期，预期在2-3年内可能发展为白血病。因此，受试者免疫多样性(D 50)可以用于判断其健康状况。
因此，我们定义通过免疫图谱分析评估受试者免疫力水平的标准为：
1)D 50<0.05，免疫力低下，可能已经罹患严重疾病或具有很高患病风险；
2)0.05≤D 50<0.10，免疫力较差，处于亚健康状态；
3)0.10≤D 50<0.15，免疫力正常；
4)0.15≤D 50，免疫力优秀。
据此标准，2.2.5中所述6位受试者，可以看出1号受试者的D 50>0.15，免疫力优秀，2、3号受试者的D 50在0.10-0.15之间，免疫力正常，4、5、6号受试者D 50<0.05，免疫力低下，可能已经罹患严重疾病或具有很高患病风险。免疫图谱分析结果与受试者健康状况对照，4、5、6号受试者都是肿瘤患者，符合得出的规律。
综上所述，可以确定了标志CDR3多样性的指标D 50作为评估受试者免疫水平的指标。
在本发明的描述中，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括至少一个该特征。在本发明的描述中，“多个”的含义是至少两个，例如两个，三个等，除非另有明确具体的限定。
在本发明中，除非另有明确的规定和限定，术语“安装”、“相连”、“连接”、“固定”等术语应做广义理解，例如，可以是固定连接，也可以是可拆卸连接，或成一体；可以是机械连接，也可以是电连接或彼此可通讯；可以是直接相连，也可以通过中间媒介间接相连，可以是两个元件内部的连通或两个元件的相互作用关系，除非另有明确的限定。对于本领域的普通技术人员而言，可以根据具体情况理解上述术语在本发明中的具体含义。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (12)

  1. 一种评估受试者免疫力水平的方法，其特征在于，所述方法为非诊断目的，包括以下步骤：
    (1)从受试者血液样本中分离淋巴细胞，获得淋巴细胞的RNA并进行逆转录；
    (2)利用特异性引物获得BCR和/或TCR可变区多核苷酸，对所述多核苷酸进行测序获得序列信息；
    (3)基于所述序列信息计算BCR和/或TCR可变区序列的D 50值；
    (4)根据D 50确定受试者的免疫力水平。
  2. 根据权利要求1所述的方法，其特征在于，所述扩增BCR可变区的特异性引物为NO:1-SEQ ID NO:31，包括BCR特异性逆转录引物和BCR特异性扩增引物，其中，BCR特异性逆转录引物为SEQ ID NO:1-SEQ ID NO:3；BCR特异性扩增引物为SEQ ID NO:4-SEQ ID NO:31。
  3. 根据权利要求1所述的方法，其特征在于，所述扩增TCR可变区的特异性引物为NO:32-SEQ ID NO:81，包括TCR特异性逆转录引物和TCR特异性扩增引物，其中，TCR特异性逆转录引物为SEQ ID NO:32-SEQ ID NO:35；TCR特异性扩增引物为SEQ ID NO:32-SEQ ID NO:81。
  4. 根据权利要求1-3任一项所述的方法，其特征在于，所述的BCR和/或TCR可变区序列D 50值的计算方法如下：测序所得BCR和/或TCR可变区多核苷酸序列信息，比对确定功能性基因可变区序列总数为N，比对到功能性基因的不同序列的类型数为C，每一种序列的拷贝数为N1、N2……NC，并按拷贝数从多到少排序，拷贝数最多且占据功能性可变区序列总数50％的克隆类型数记为H，H与C的比值为D 50。
  5. 根据权利要求4所述的方法，其特征在于，所述的可变区界定采用BCR和/或TCR互补决定区3(CDR3)。
  6. 根据权利要求1-5任一项所述的方法，所述样本为外周血样。
  7. 根据权利要求1-6任一项所述的方法，其特征在于：
    (1)D 50<0.05，免疫力低下，可能已经罹患严重疾病或具有很高患病风险；
    (2)0.05≤D 50<0.10，免疫力较差，处于亚健康状态；
    (3)0.10≤D 50<0.15，免疫力正常；
    (4)0.15≤D 50，免疫力优秀。
  8. 一种评估受试者免疫力水平的装置，其特征在于，包括以下：
    测序单元，所述测序单元用于对受试者淋巴细胞的BCR和/或TCR可变区多核 苷酸进行序列测定，以便获得测序数据；
    D 50确定单元，所述测序单元与D 50计算单元连接，基于所述测序数据，确定BCR和/或TCR可变区序列的D 50值；
    判定单元，所述判定单元与D 50确定单元连接，根据D 50值判定受试者的免疫力水平。
  9. 根据权利要求8所述的装置，其特征在于，所述的D 50的计算方法如下：测序所得BCR和/或TCR可变区多核苷酸序列信息，比对确定功能性基因可变区序列总数为N，比对到功能性基因的不同序列的类型数为C，每一种序列的拷贝数为N1、N2……NC，并按拷贝数从多到少排序，拷贝数最多且占据功能性可变区序列总数50％的克隆类型数记为H，H与C的比值为D 50。
  10. 根据权利要求9所述的方法，其特征在于，所述的可变区界定采用BCR和/或TCR互补决定区3(CDR3)。
  11. 根据权利要求8-10任一项所述的装置，其特征在于：
    (1)D 50<0.05，免疫力低下，可能已经罹患严重疾病或具有很高患病风险；
    (2)0.05≤D 50<0.10，免疫力较差，处于亚健康状态；
    (3)0.10≤D 50<0.15，免疫力正常；
    (4)0.15≤D 50，免疫力优秀。
  12. 根据权利要求8所述装置，所述受试者淋巴细胞从外周血样中获得。
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