CN106319066B - Primer and kit for detecting Systemic Lupus Erythematosus (SLE) B cell receptor library - Google Patents

Abstract

The invention provides a primer and a detection kit for detecting a Systemic Lupus Erythematosus (SLE) B cell receptor bank, wherein specific primer sequences in the kit are respectively shown as Seq ID No: 1-25. The kit product of the invention has the advantages of high flux, multi-index parallel detection, rapid and accurate detection and the like. Experiments show that the kit can identify and quantify the amplified B cells and plasma cell clones by screening the BCR library, and provides a basis for the establishment of the clinical characteristics, subtype analysis and individualized diagnosis and treatment of the systemic lupus erythematosus.

Description

Primer and kit for detecting Systemic Lupus Erythematosus (SLE) B cell receptor library

Technical Field

The invention relates to the field of molecular biological detection, in particular to a primer and a kit for detecting a systemic lupus erythematosus B cell receptor library.

Background

Systemic Lupus Erythematosus (SLE) is one of the most common autoimmune diseases, with high disability and mortality rates, and the appearance of multiple autoantibodies in serum and multiple systemic involvement are its major clinical features. From the data of the Chinese Lupus cooperative group, there are currently more than one million SLE patients in China, and the prevalence rate is the second in the world. The Meta analysis shows that the 5-year survival rate of the SLE patient is 94 percent, and the 10-year survival rate is 89 percent. As a multifactorial chronic rheumatic disease, SLE has a strong genetic background, and there are a variety of autoimmune responses, as well as a variety of autoantibodies. Although many molecular pathways are aberrant, multiple cell phenotypes are deregulated in lupus, B cells are a central role because autoantibodies are critical in diagnosing SLE and have been discovered before additional evidence of immune dysregulation has emerged. B cells not only secrete autoantibodies, but also play a key role in antigen presentation and cytokine secretion. B cells are important promoters and effectors of the normal immune response, in which they perform the same function but are directed against self-antigens.

The humoral immune response of the body is mainly mediated by B cells, and the humoral immune response responds to antigens by producing specific antibodies, thereby playing an important role in protecting the body. Autoantibody producing B cells are the major cell population mediating the humoral immune response of the body, the specificity of which is determined by the B Cell Receptor (BCR), the diversity of which is due to VDJ gene rearrangement. B cell recognition, binding to antigen, relies primarily on Complementarity Determining Regions (CDRs), which determine the specificity of the antibody. B cell recognition of antigens is primarily dependent on BCR, a tetramer of two heavy (H) and two light (κ or λ) chains whose variable and constant regions are encoded by multiple discrete gene fragment rearrangements on different chromosomes. The human kappa and lambda chains are formed by the rearrangement of the "Vn-Jn-C" gene, the H chain is formed by the rearrangement of the "Vn-Dn-Jn-Cn" gene, and the variable region comprises three hypervariable regions: CDR1, CDR2, CDR3, CDR3 is more diverse than CDR1, CDR2, and the key site determining antigen peptide recognition by B cells is the most diverse heavy chain CDR3 region, which consists of "IGHV (51 functional gene segments) terminal-IGHD (23 functional gene segments) -IGHJ front end (6 functional gene segments)" gene rearrangement and "V-D" and "D-J" joining intermediate inserted or spliced nucleotide sequences, both type conversion and somatic high-frequency mutation contribute to the formation of their diversity.

Due to the striking diversity of BCRs, the normal body responds to almost all foreign bodies that invade the body. Each clone B cell BCR has a specific CDR3 sequence composition, which when the antigen enters the body, causes its specific B cell clone to expand and produce a corresponding antibody response to the antigen. Since the BCR CDR3 region of B cell is the main part of B cell to recognize antigen, its length and diversity will influence its recognition and affinity to antigen, so the specificity of B cell can be considered as the result of gene recombination and high frequency mutation of somatic cell to form specific CDR3, and the characteristic of variable CDR3 sequence provides a diverse B cell receptor library for antigen selection.

By analyzing the diversity, the overlapping rate, the gene/amino acid composition characteristics of the CDR3 region, the biased access of each subfamily and the like of the B cell BCR CDR3 receptor library of the organism, the related conditions of the organism immunity in physiological and pathological states can be observed, and a new thought, a new method and a means are provided for monitoring and analyzing the immune condition of the organism in physiological and pathological states and the generation and development mechanisms of B cells.

SLE is an autoimmune disease that shows a high degree of clinical heterogeneity, characterized by lymphocyte hyperreactivity and the formation of pathogenic autoantibodies. The nature of the immune alterations that cause this disease remains unclear. Significant B cell activation and development of autoreactivity suggest that one or more physiological checkpoints governing the recognition of BCR by autoantigens, as well as B cell triggering and maturation processes may be affected.

Under the influence of T helper cells and antigens from secondary lymphoid organ follicles or ectopic follicles of autoimmune patients, mature naive B cells undergo germinal center reactions leading to somatic mutations such as clonal expansion, Ig gene rearrangement, and Ig heavy chain class switch recombination. These molecular processes are unique to B cells and can ensure the specific generation of high affinity binding to BCR and determine its ultimate effector function. Antigen-activated B cells are susceptible to tolerance induction, however clonal deletion is not sufficient to account for all tolerance induction, as many autoreactive B cells are not cleared, suggesting that other mechanisms are working to prevent further cell development into plasma or memory B cells, including induction of anergy and specific alteration of secondary v (d) J rearrangements by BCR.

The high-throughput sequencing technology can identify and quantify the amplified B cell and plasma cell clones by screening a BCR library, has the advantages of rapid and quantitative longitudinal and cross-sectional comparison, and can realize the linkage of clonal amplification and inflammation.

B cell BCR CDR3 receptor library correlation research methods are numerous, such as polymerase chain reaction-single chain conformation polymorphism analysis; with the development of nucleic acid end labeling technology, CDR3 sequences were studied using "immunochromatography analysis techniques" or "immunoscan (fingerprinting) techniques"; based on various labeling (isotope, fluorescein, etc.) of the corresponding probe, a nucleic acid hybridization technique is used to analyze the expression frequency of the CDR3 sequence composed of the corresponding V or J gene family. The methods can only roughly analyze the sequence diversity degree and the polymorphism distribution or partial sequence composition of the length of the hypervariable region, the obtained data are limited and easy to deviate, the CDR3 spectral patterns are deeply and dynamically monitored, the composition and the characteristics of individual B cell BCR CDR3 receptor library sequences and the overlapping rate among individuals are rapidly and comprehensively observed, and the association of the individual B cell BCR CDR3 receptor library sequences with diseases or disease activity conditions is researched; comparatively analyzing the B cell BCR CDR3 receptor library of the same disease patient; comparing the difference between the BCR CDR3 receptor library of B cells of an individual with a certain disease and a normal individual, and the like, the further intensive research on the B cell receptor library at the gene level is to be carried out. The progress of DNA sequencing technology has transformed the life research from single, local gene or gene fragment to whole genome, and the application of high-throughput sequencing technology developed in recent years is required for detecting the change of sequence composition accurately in large quantities.

The high-throughput sequencing technology, also known as the next generation sequencing technology or the deep sequencing technology or the second generation sequencing technology, is a revolutionary change of the traditional sequencing technology, and can perform sequence determination on hundreds of thousands to millions of DNA molecules at a time, so that the complete and detailed analysis of transcriptome and genome of one species becomes possible. The method is an efficient, accurate, high-sensitivity and automatic gene sequencing method, and mainly comprises the steps of amplifying a target sequence by using a specific primer, carrying out electrophoresis on an amplification product, and sequencing a target region with a band. At present, high-throughput sequencing is a rapid and efficient means for deeply researching an immune receptor library, and is applied to research on tumors, autoimmune diseases, inflammatory diseases, vaccines and the like. B cell receptor genes are analyzed in a large range, and the association of certain gene loci with diseases can be found; the detection of the BCRCDR3 receptor library of the body B cell can find that the sequence has single and few cloning phenomena, and provides important information for the prevention and early diagnosis of diseases.

Animal experiments have shown that large scale clonal expansion is developed against self-antigens and autoreactive clones are often considered to be expanded. Several features of the encoding BCR sequence may point to autoreactivity. For example, the Ig-rich heavy chain gene V4-34(IGHV4-34) is responsive to self epitopes and appears to be tightly regulated to prevent autoimmune responses. In SLE, compared with healthy individuals and RA patients, this IGHV4-34 positive and autoantibody producing plasma cells are widely present in peripheral blood. Another specific manifestation (response to nuclear antigens) is the increased number of amino acids in the heavy chain CDR3 region, the most prominent antigen binding region of BCR. These are usually counter-selections in the development process of B cells, but occur frequently in autoimmune diseases.

Disclosure of Invention

The invention aims to provide a primer for detecting a systemic lupus erythematosus B cell receptor bank.

Another object of the present invention is to provide a detection kit comprising the above primer.

In order to achieve the purpose of the invention, the primer for detecting the systemic lupus erythematosus B cell receptor bank comprises the following primer combinations:

1) IgH-specific PCR amplification primers with the following sequences (Seq ID No: 1-10):

2) IgK specific PCR amplification primers with the following sequences (Seq ID No: 11-17):

3) IgL-specific PCR amplification primers with the following sequences (Seq ID No: 18-25):

wherein S is G or C, and Y is T or C.

The invention also provides a kit containing the primer and used for detecting the systemic lupus erythematosus B cell receptor bank.

Preferably, the kit also comprises a reverse transcription primer oligo (dT)₂₀At least one of reverse transcriptase, dNTP mixture, DEPC treated water, DTT, reaction buffer and the like.

More preferably, the kit also comprises a systemic lupus erythematosus B cell receptor bank standard product. The systemic lupus erythematosus B cell receptor bank standard is TA cloning plasmid from BCR amplification products of healthy control people.

The kit comprises a first chain of cDNA synthesized by reverse transcription and a PCR reaction process;

(1) reverse transcription to synthesize the first strand of cDNA

① preparation of System I with 5. mu.g total RNA and 50. mu.M Oligo (dT)₂₀1 μ L of 10mM dNTP, 1 μ L of DEPC treated water to 10 μ L of the total system;

incubating at 65 deg.C for 5min, and standing on ice for 1 min;

② System II was prepared with 2. mu.L of 10 XTT reaction buffer, 25mM MgCl₂4 muL, 0.1M DTT 2 muL, 40U/muL RNase 1 muL, 200U/muL reverse transcriptase 1 muL;

incubating at 50 deg.C for 50min, terminating reaction at 85 deg.C for 5min, and cooling on ice;

(2) PCR reaction

The following system is preparedIII: 10 XPCR reaction buffer 5. mu.L, 10mM dNTP 1.5. mu.L, 50mM MgSO₄0.5. mu.L of cDNA, 2. mu.L of the first strand of cDNA, 1U of DNA polymerase, 1.5. mu.L of each of 10. mu.M forward and reverse primers, and 50. mu.L of DEPC-treated water as a whole;

PCR reaction conditions of IgH amplification primers: 94 ℃ for 2 min; 30s at 94 ℃, 30s at 60 ℃ and 1min at 68 ℃, and 50 cycles; 5min at 68 ℃; storing at 4 deg.C;

PCR reaction conditions of the IgK and IgL amplification primers are as follows: 94 ℃ for 2 min; 30s at 94 ℃, 45s at 59 ℃, 1min at 68 ℃ and 50 cycles; 5min at 68 ℃; storing at 4 ℃.

The invention further provides application of the primer or the kit in detecting a systemic lupus erythematosus B cell receptor bank.

The application comprises the following steps:

1) extracting total RNA of the sample;

2) performing reverse transcription by taking the RNA extracted in the step 1) as a template to obtain a first cDNA chain;

3) respectively amplifying IgH, IgK and IgL by PCR by taking the cDNA in the step 2) as a template;

4) analyzing the PCR amplification product.

The invention develops primers and a detection kit for detecting a Systemic Lupus Erythematosus (SLE) B cell receptor library through a large number of experiments based on a high-throughput sequencing technology. The kit product of the invention has the advantages of high flux, multi-index parallel detection, rapid and accurate detection and the like. Experiments show that the kit can identify and quantify the amplified B cells and plasma cell clones by screening the BCR library, and provides a basis for the establishment of the clinical characteristics, subtype analysis and individualized diagnosis and treatment of the systemic lupus erythematosus. The detection kit for the systemic lupus erythematosus B cell receptor bank has the advantages of no side effect, high flux, multi-index parallel detection, high sensitivity, high detection speed, good repeatability and the like.

Drawings

FIG. 1 shows the first strand cDNA synthesized in example 2 of the present invention and the result of PCR amplification; wherein 1 represents a general RT-PCR and 2 represents a specific RT-PCR.

FIG. 2 shows the result of PCR amplification of the SLE B cell receptor library standard of example 1.

FIG. 3 shows the result of the analysis of SLE patient BCR IgH V-J pairing in example 2 of the present invention. Establishing a PCR specific amplification system of BCR IgH/IgK/IgL, carrying out deep sequencing on the amplified IgH/IgK/IgL through a Miseq second-generation sequencing platform, and determining BCR clone specifically amplified in the SLE group through bioinformatics analysis.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular cloning: a laboratory manual,2001), or the conditions suggested by the manufacturer's instructions.

The following examples used systemic lupus erythematosus B cell receptor library standards were derived from the healthy control population BCR amplification product TA cloning plasmid.

EXAMPLE 1 preparation of detection kit for B-cell receptor library of systemic lupus erythematosus

1. Kit composition

(1) Specific PCR primer for amplifying IgH, IgK and IgL

1) IgH specific PCR amplification primers have the following sequences:

2) the sequence of the IgK specific PCR amplification primer is as follows:

3) the sequence of the specific PCR amplification primer of the IgL is as follows:

wherein S is G or C, and Y is T or C.

(2) Standard substance of systemic lupus erythematosus B cell receptor bank

(3)SuperScript^TMIII First-Strand Synthesis System for RT-PCR Cat.No:18080-051)

(4)

Pfx DNA polymerase (Cat. No.11708-013)

2. Application of kit

RT-PCR procedure:

reagent (I): SuperScript^TMIII First-Strand Synthesis System forRT-PCR(Cat.No:18080-051)

(II) RT-PCR process: all reagents are mixed evenly by shaking before use, and the reverse transcription system is 20 ul. (the reverse transcription RNA content is self-adjusted according to the extracted cell RNA content, the reverse transcription RNA content of each group is the same, and the RT-PCR process refers to SuperScript^TMIII First-Strand Synthesis System for RT-PCR reagent instruction, the specific flow is as follows:

(1) mix and briefly centrifuge each sample prior to use.

(2) Mix the following ingredients in a 0.2ml or 0.5ml tube:

(3) the tubes were incubated at 65 ℃ for 5min and placed on ice for at least 1 min.

(4) The following cDNA Synthesis Mix was prepared, with the addition of each component in the order given:

(5) add 10. mu.L of cDNA Synthesis Mix to each RNA/primer mixture, Mix gently, and enrich by brief centrifugation. The incubation conditions were as follows: oligo (dT)₂₀:50℃50min。

(6) The reaction was terminated at 85 ℃ for 5min and cooled on ice.

(7) The cDNA synthesis reaction product can be stored at-20 ℃ or used directly in PCR reaction.

PCR process:

reagent (I):

Pfx DNA Polymerase(Cat.No.11708-013)

(II) PCR process: all reagents were mixed by shaking before use, and the PCR system was 50. mu.L. All primer concentrations were 10. mu.M (i.e., 10. mu. mol/L).

The following VH/VK/VL mixtures were prepared:

note: when template DNA was reversed with >100ng RNA, 1. mu.l of 50mM MgSO4 was present in the PCR system.

0.5. mu.l of 50mM MgSO4 in PCR system when template DNA was reversed with <100ng RNA.

And C, carrying out amplification on the PCR reaction system by using a VH/VK/VL single primer or a primer mixture according to the system.

The mixture was shaken well and centrifuged slightly to allow the reagent components to settle to the bottom of the tube.

And (3) PCR reaction conditions:

VH reaction conditions: 94 ℃ for 2 min; 50 cycles (94 ℃, 30 s; 60 ℃, 30 s; 68 ℃, 1 min); at 68 ℃ for 5 min; infinity at 4 ℃

VK + VL reaction conditions: 94 ℃ for 2 min; 50 cycles (94 ℃, 30 s; 59 ℃, 45 s; 68 ℃, 1 min); at 68 ℃ for 5 min; infinity at 4 ℃

And (4) carrying out electrophoresis detection on the PCR product, storing the PCR product at-20 ℃, and sequencing.

Example 2 detection of BCR library in patients with systemic lupus erythematosus

1. Purpose of experiment

And carrying out amplification and high-throughput sequencing detection on the BCR library of the systemic lupus erythematosus patient.

2. Test object

1) And selecting the grouping case and recording the personal basic information on the premise of informed consent and meeting the inclusion condition.

2) The BCR α, β chain receptor pool of peripheral blood CD8+ T cells was sequenced in 5 healthy volunteers and 5 systemic lupus erythematosus.

3. Reagent preparation

The kit prepared in example 1 was used.

Standard control (systemic lupus erythematosus-specific T cell receptor bank reference).

4. The experimental process comprises the following steps:

RT-PCR procedure:

reagent (I): SuperScript^TMIII First-Strand Synthesis System for RT-PCR(Cat.No:18080-051)

(II) RT-PCR process: all reagents are mixed evenly by shaking before use, and the reverse transcription system is 20 ul. (the reverse transcription RNA content is self-adjusted according to the extracted cell RNA content, the reverse transcription RNA content of each group is the same, and the RT-PCR process refers to SuperScript^TMIII First-Strand Synthesis System for RT-PCR reagent instruction, the specific flow is as follows:

(1) mix and briefly centrifuge each sample prior to use.

(2) Mix the following ingredients in 0.2 or 0.5ml tubes:

Component	Amount
		up to 5μg total RNA	nμL(80ng RNA)
Primer:50μM oligo(dT)20	1μL
		10mM dNTP mix	1μL
DEPC-treated water	to 10μL

(3) the tubes were incubated at 65 ℃ for 5min and placed on ice for at least 1 min.

(4) The following cDNA Synthesis Mix was prepared, with the addition of each component in the order given:

(5) add 10. mu.L of cDNA Synthesis Mix to each RNA/primer mixture, Mix gently, and enrich by brief centrifugation. The incubation conditions were as follows: oligo (dT)₂₀:50℃50min。

(6) The reaction was terminated at 85 ℃ for 5min and cooled on ice.

(7) The cDNA synthesis reaction product can be stored at-20 ℃ or used directly in PCR reaction.

PCR process:

reagent (I):

Pfx DNA Polymerase(Cat.No.11708-013)

(II) PCR process: all reagents were mixed by shaking before use, and the PCR system was 50. mu.L. All primer concentrations were 10. mu.M (i.e., 10. mu. mol/L).

The following VH/VK/VL mixtures were prepared:

note: when template DNA was reversed with >100ng RNA, 1. mu.l of 50mM MgSO4 was present in the PCR system.

0.5. mu.l of 50mM MgSO4 in PCR system when template DNA was reversed with <100ng RNA.

And C, carrying out amplification on the PCR reaction system by using a VH/VK/VL single primer or a primer mixture according to the system.

The mixture was shaken well and centrifuged slightly to allow the reagent components to settle to the bottom of the tube.

And (3) PCR reaction conditions:

VH reaction conditions: 94 ℃ for 2 min; 50 cycles (94 ℃, 30 s; 60 ℃, 30 s; 68 ℃, 1 min); at 68 ℃ for 5 min; infinity at 4 ℃

VK + VL reaction conditions: 94 ℃ for 2 min; 50 cycles (94 ℃, 30 s; 59 ℃, 45 s; 68 ℃, 1 min); at 68 ℃ for 5 min; infinity at 4 ℃

And (4) carrying out electrophoresis detection on the PCR product, storing the PCR product at-20 ℃, and sequencing. The first strand of the synthesized cDNA and the PCR amplification result are shown in FIG. 1. The PCR amplification results of the SLE B cell receptor library standard are shown in FIG. 2.

Quantification was done with Nanodrop to calculate the starting amount when the library was built next. The amount of purified DNA was generally about 45 ng/. mu.l.

Purified DNA library construction and gel recovery.

Quantit quantification library.

Real-Time PCR pool quantification.

Hiseq2500P250 sequencing.

Through biological information analysis, research results show that: 1. the BCR receptor bank level of the systemic lupus erythematosus patient is greatly different from that of a healthy volunteer; 2. there was a very small fraction of characteristic clonotype B cell expansion in systemic lupus erythematosus patients analyzed by V-J paring compared to healthy volunteers. (FIG. 3)

Experimental results show that the kit can realize the regular detection of B cell response of SLE patients with systemic lupus erythematosus and the molecular characteristic judgment of B cell response of specific SLE patients.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Reference to the literature

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Claims (4)

1. The primer for detecting the systemic lupus erythematosus B cell receptor bank is characterized by comprising the following primer combinations:

1) IgH specific PCR amplification primers have the following sequences:

forward primer 5 '-3':

CAGGTGCAGCTGGTGCAGTCTGG

CAGGTCACCTTGAAGGAGTCTGG

GAGGTGCAGCTGGTGGAGTCTGG

GAGGTGCAGCTGTTGGAGTCTGG

CAGGTGCAGCTGCAGGAGTCGGG

CAGGTGCAGCTACAGCAGTGGGG

GAGGTGCAGCTGGTGCAGTCTGG

CAGGTACAGCTGCAGCAGTCAGG

CAGGTGCAGCTGGTGCAATCTGG

reverse primer 5 '-3':

GACSGATGGGCCCTTGGTGGA

2) the sequence of the IgK specific PCR amplification primer is as follows:

forward primer 5 '-3':

GACATCCAGATGACCCAGTCTCC

GATATTGTGATGACCCAGWCTCC

GAAATTGTGTTGACGCAGTCTCC

GACATCGTGATGACCCAGTCTCC

GAAACGACACTCACGCAGTCTCC

GAAATTGTGCTGACTCAGTCTCC

reverse primer 5 '-3':

CGTTGGTGCAGCCACAGT

3) the sequence of the specific PCR amplification primer of the IgL is as follows:

forward primer 5 '-3':

GGTCCTGGGCCCAGTCTGTGCTG

GGTCCTGGGCCCAGTCTGCCCTG

GCTCTGTGACCTCCTATGAGCTG

GGTCTCTCTCSCAGCYTGTGCTG

GTTCTTGGGCCAATTTTATGCTG

GGTCCAATTCYCAGGCTGTGGTG

GAGTGGATTCTCAGACTGTGGTG

reverse primer 5 '-3':

AGAGGASGGYGGGAACAGAGTGAC

wherein S is G or C, and Y is T or C.

2. A kit for detecting the B cell receptor bank of systemic lupus erythematosus, which comprises the primer of claim 1.

3. The kit according to claim 2, wherein the kit further comprises a reverse transcription primer oligo (dT)₂₀At least one of reverse transcriptase, dNTP mixture, DEPC treated water, DTT and reaction buffer.

4. The kit of claim 2 or 3, wherein the kit further comprises a standard of the B cell receptor library of systemic lupus erythematosus.

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