CN107630085B

CN107630085B - Application of molecular marker in male osteoporosis

Info

Publication number: CN107630085B
Application number: CN201710946152.4A
Authority: CN
Inventors: 王丽; 马青; 王玉功
Original assignee: Individual
Current assignee: Individual
Priority date: 2017-10-12
Filing date: 2017-10-12
Publication date: 2020-09-29
Anticipated expiration: 2037-10-12
Also published as: CN107630085A

Abstract

The invention discloses application of a molecular marker in male osteoporosis, wherein the molecular marker is BTNL 3. Experiments prove that the BTNL3 gene and the protein coded by the gene are up-regulated in patients with osteoporosis, and the detection of the expression level of BTNL3 indicates that the gene can be used for early diagnosis of osteoporosis.

Description

Application of molecular marker in male osteoporosis

Technical Field

The invention belongs to the field of biological medicines, and relates to application of a molecular marker in male osteoporosis, wherein the molecular marker is BTNL 3.

Background

Osteoporosis (osteoporotis) is a systemic bone disease in which bone fracture is easily caused by a decrease in bone density and bone mass due to various causes, a destruction of bone microarchitecture, and an increase in bone fragility. Osteoporosis is divided into primary and secondary categories. The occurrence and development of the primary osteoporosis are closely related to endocrine disturbance, the whole body nutrition state, the immune function and genetic genes.

With the rapid development of economy and the acceleration of industrialization, the non-infectious disease threat syndrome facing human health is increasing in weight, osteoporosis, osteoporotic fracture and the like are even more inexorable killers harmful to health, and are widely considered as one of the major health problems affecting human health.

In the elderly, sex hormone levels gradually decline with age. In men, serum testosterone levels are closely related to age, initially increasing with age, peaking at 7-10 years of age, and then decreasing with age. Osteoporosis is frequently associated with the elderly in postmenopausal women and men, and is related to age.

Although the current research considers that the onset of osteoporosis is related to age and sex, and is closely related to genetic factors, environmental factors and the like, the pathogenesis of osteoporosis is still unclear, and the early diagnosis, the monitoring of the treatment effect and the prognosis evaluation of osteoporosis play an important role in preventing and treating osteoporosis. In recent years, with the development of biotechnology, genetic factor research has become a popular topic in the field of osteoporosis diagnosis and treatment, and studies have shown that osteoporosis is related to in vivo gene changes, and for example, patent nos. 201610272604.0 and 201610271798.2 report that differential expression of genes is related to the development of osteoporosis, and it is seen that early diagnosis of osteoporosis using genes is a trend of future development.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a molecular marker for early diagnosis of male osteoporosis, so as to realize specific diagnosis and prevention of osteoporosis.

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

the invention provides application of BTNL3 gene in preparing a product for diagnosing osteoporosis.

Further, the product comprises a reagent for detecting the expression level of BTNL3 in the sample.

Further, BTNL3 is up-regulated in osteoporosis.

Further, the reagent detects the expression level of BTNL3 by RT-PCR, real-time quantitative PCR, immunoassay, in situ hybridization or a chip.

Further, the reagent for detecting the expression level of BTNL3 by RT-PCR at least comprises a pair of primers for specifically amplifying the BTNL3 gene; the reagent for detecting the expression level of the BTNL3 by real-time quantitative PCR at least comprises a pair of primers for specifically amplifying the BTNL3 gene; the reagent for detecting the expression level of BTNL3 through immunity comprises an antibody and/or a ligand which is specifically bound with BTNL3 protein; the reagent for detecting the expression level of BTNL3 by in situ hybridization comprises a probe that hybridizes to a nucleic acid sequence of BTNL3 gene; the reagent for detecting the expression level of BTNL3 by the chip comprises a probe hybridized with a nucleic acid sequence of BTNL3 gene, or an antibody and/or a ligand specifically bound with BTNL3 protein.

In the present invention, a "sample" is a sample containing cells or cellular material from which nucleic acids, polypeptides, or other analytes can be obtained. Examples of biological samples include, but are not limited to: urine, blood, serum, plasma, cerebrospinal fluid, pleural fluid, bronchial lavage, sputum, peritoneal fluid, bladder wash, secretions (e.g., breast secretions), oral wash, swabs (e.g., oral swabs), isolated cells, tissue samples, touch preps, and fine needle punctures. Preferably, the sample is human peripheral blood.

The invention provides a product for diagnosing osteoporosis, which comprises a preparation, a chip or a kit, wherein the preparation, the chip or the kit comprises a reagent for detecting the expression level of BTNL 3.

Further, the reagents for detecting the expression level of BTNL3 in the chip include a probe that specifically recognizes the BTNL3 gene, or an antibody or ligand that specifically binds to a protein encoded by BTNL 3.

Further, the reagents for detecting the expression level of BTNL3 in the kit include primers that specifically amplify the BTNL3 gene; or a probe specifically recognizing the BTNL3 gene; or an antibody or ligand that specifically binds to a protein encoded by BTNL 3.

Further, the primer sequence of the specific amplification BTNL3 gene is shown as SEQ ID NO.3 and SEQ ID NO. 4.

Further, the kit comprises a SYBR Green polymerase chain reaction system and a primer pair for amplifying housekeeping genes; the SYBR Green polymerase chain reaction system comprises: PCR buffer, dNTPs, SYBR Green fluorescent dye.

Further, the detection kit also comprises an instruction manual and a label.

The gene chip or the gene detection kit of the invention can be used for detecting the expression levels of a plurality of genes (for example, a plurality of genes related to osteoporosis) including the BTNL3 gene. The protein chip or protein immunoassay kit can be used for detecting the expression level of a plurality of proteins (such as a plurality of proteins related to osteoporosis) including the BTNL3 protein. The multiple markers of osteoporosis are detected simultaneously, so that the accuracy of osteoporosis diagnosis can be greatly improved.

The invention has the advantages and beneficial effects that:

the invention discovers that the molecular marker BTNL3 is related to the occurrence and development of male osteoporosis for the first time, and diagnoses whether patients have osteoporosis and risk of osteoporosis by detecting the change of a subject BTNL3, thereby realizing early-finding and early-treating of patients with osteoporosis and improving the life quality of the patients.

Drawings

FIG. 1 is a graph of the detection of the expression of the BTNL3 gene in osteoporosis patients using QPCR;

fig. 2 is a graph of detecting differential expression of the BTNL3 gene at the protein level using immunoblotting.

Detailed Description

Through extensive and intensive research and a large number of screens, the invention discovers that BTNL3 presents specific high expression in male osteoporosis patients for the first time, and a better way and a better method are searched for the early detection of osteoporosis.

Biomarkers

A "biomarker," also referred to as a "molecular marker," is any gene or protein whose expression level in a tissue or cell is altered compared to the expression level of a normal or healthy cell or tissue.

Any method available in the art for detecting expression of a molecular marker is encompassed herein. Expression of the molecular markers of the invention can be detected at the nucleic acid level (e.g., RNA transcript) or at the protein level. By "detecting expression" is intended the determination of the amount or presence of an expression product of an RNA transcript or its molecular marker gene. Thus, "detecting expression" includes instances where a molecular marker is determined to be not expressed, not to be detected, expressed at a low level, expressed at a normal level, or overexpressed. To determine overexpression, the body sample being tested can be compared with a corresponding body sample from a healthy person. That is, the "normal" level of expression is the level of expression of the molecular marker. This sample may be presented in a normalized form. In some embodiments, the determination of the overexpression of the molecular marker is performed by comparing a body sample with a corresponding body sample from a healthy person.

One skilled in the art will recognize that the utility of the present invention is not limited to quantifying gene expression of any particular variant of the marker genes of the present invention. As a non-limiting example, the coding sequence or amino acid sequence of one representative human BTNL3 is shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. BTNL3 includes polypeptides comprising SEQ ID No.2 and other BTNL3 native sequence polypeptides, such as naturally occurring variants and native sequence polypeptides, which are encoded by the nucleotide sequence shown in SEQ ID No. 1.

The polynucleotide of the present invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand.

The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, a synthetic polypeptide, preferably a recombinant polypeptide. The polypeptides of the invention may or may not also include an initial methionine residue.

Polynucleotides encoding the mature polypeptide of BTNL3 include: a coding sequence encoding only the mature polypeptide; the coding sequence for the mature polypeptide and various additional coding sequences; the coding sequence (and optionally additional coding sequences) as well as non-coding sequences for the mature polypeptide. The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, and may also include additional coding and/or non-coding sequences.

The present invention also relates to variants of the above polynucleotides which encode polypeptides having the same amino acid sequence as the present invention or fragments, analogs and derivatives of the polypeptides. The variant of the polynucleotide may be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include substitution variants, deletion variants and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the encoded polypeptide.

The invention also relates to nucleic acid fragments, including sense and antisense nucleic acid fragments, which hybridize to the sequences described above. As used herein, a "nucleic acid fragment" is at least 15 nucleotides, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides in length. The nucleic acid fragments can be used in amplification techniques of nucleic acids (e.g., PCR) to determine and/or isolate a polynucleotide encoding a BTNL3 protein.

The full-length nucleotide sequence of human BTNL3 or a fragment thereof of the present invention can be obtained by PCR amplification, recombination, or artificial synthesis. For the PCR amplification method, primers can be designed based on the disclosed nucleotide sequences, particularly open reading frame sequences, and the sequences can be amplified using a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art as a template. When the sequence is long, two or more PCR amplifications are often required, and then the amplified fragments are spliced together in the correct order. Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.

Detection method

The present invention can be detected using a variety of nucleic acid and protein techniques known to those of ordinary skill in the art, including but not limited to: nucleic acid sequencing, nucleic acid hybridization, nucleic acid amplification technology and protein immunization technology.

The nucleic acid amplification technique of the invention is selected from the group consisting of Polymerase Chain Reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), Transcription Mediated Amplification (TMA), Ligase Chain Reaction (LCR), Strand Displacement Amplification (SDA) and Nucleic Acid Sequence Based Amplification (NASBA). Among them, PCR requires reverse transcription of RNA into DNA before amplification (RT-PCR), TMA and NASBA to directly amplify RNA.

Generally, PCR uses multiple cycles of denaturation, annealing of primer pairs to opposite strands, and primer extension to exponentially increase the copy number of a target nucleic acid sequence; RT-PCR Reverse Transcriptase (RT) is used to prepare complementary DNA (cDNA) from mRNA, and the cDNA is then amplified by PCR to produce multiple copies of the DNA; TMA autocatalytically synthesizes multiple copies of a target nucleic acid sequence under substantially constant conditions of temperature, ionic strength and pH, wherein multiple RNA copies of the target sequence autocatalytically generate additional copies, TMA optionally including the use of blocking, partial, terminating and other modifying moieties to improve the sensitivity and accuracy of the TMA process; LCR with target nucleic acid adjacent region hybridization of two sets of complementary DNA oligonucleotides. The DNA oligonucleotides are covalently linked by DNA ligase in repeated cycles of heat denaturation, hybridization, and ligation to produce a detectable double-stranded ligated oligonucleotide product; the SDA uses multiple cycles of the following steps: primer sequence pairs anneal to opposite strands of the target sequence, primer extension in the presence of dNTP α S to produce double-stranded hemiphosphorothioated (phosphorothioated) primer extension products, endonuclease-mediated nicking of the hemimodified restriction enzyme recognition site, and polymerase-mediated extension from the 3' end of the nick to displace the existing strand and produce a strand for the next round of primer annealing, nicking and strand displacement, thereby causing geometric amplification of the products.

Non-amplified or amplified nucleic acids of the invention can be detected by any conventional means.

Nucleic acid hybridization techniques of the invention include, but are not limited to, In Situ Hybridization (ISH), microarrays, and Southern or Northern blots. In Situ Hybridization (ISH) is a hybridization of specific DNA or RNA sequences in a tissue section or section using a labeled complementary DNA or RNA strand as a probe (in situ) or in the entire tissue if the tissue is small enough (whole tissue embedded ISH). DNA ISH can be used to determine the structure of chromosomes. RNA ISH is used to measure and locate mRNA and other transcripts (e.g., ncRNA) within tissue sections or whole tissue embedding. Sample cells and tissues are typically treated to fix the target transcript in situ and to increase probe access. The probe is hybridized to the target sequence at high temperature, and then excess probe is washed away. The localization and quantification of base-labeled probes in tissues labeled with radiation, fluorescence or antigens is performed using autoradiography, fluorescence microscopy or immunohistochemistry, respectively. ISH can also use two or more probes labeled with radioactive or other non-radioactive labels to detect two or more transcripts simultaneously.

Southern and Northern blots were used to detect specific DNA or RNA sequences, respectively. DNA or RNA extracted from the sample is fragmented, separated by electrophoresis on a matrix gel, and then transferred to a membrane filter. The filter-bound DNA or RNA is hybridized to a labeled probe complementary to the sequence of interest. Detecting the hybridization probes bound to the filter. A variation of this procedure is a reverse Northern blot, in which the substrate nucleic acid immobilized to the membrane is a collection of isolated DNA fragments and the probe is RNA extracted from the tissue and labeled.

Protein immunization techniques include sandwich immunoassays, such as sandwich ELISA, in which detection of a biomarker is performed using two antibodies that recognize different epitopes on the biomarker; radioimmunoassay (RIA), direct, indirect or contrast enzyme-linked immunosorbent assay (ELISA), Enzyme Immunoassay (EIA), Fluorescence Immunoassay (FIA), western blot, immunoprecipitation, and any particle-based immunoassay (e.g., using gold, silver or latex particles, magnetic particles, or quantum dots). The immunization can be carried out, for example, in the form of microtiter plates or strips.

The immunization method according to the present invention may be based on, for example, any of the following methods.

Immunoprecipitation is the simplest immunoassay method; this method measures the amount of precipitate that is formed after the reagent antibody has been incubated with the sample and reacted with the target antigen present therein to form insoluble aggregates. The immunoprecipitation can be either qualitative or quantitative.

In a particle immunoassay, multiple antibodies are attached to the particle and the particle is capable of binding many antigenic molecules simultaneously. This greatly accelerates the speed of the visible reaction. This allows for a fast and sensitive detection of the biomarker.

In immunoturbidimetry (immunonephelometry), the interaction of an antibody and a target antigen on a biomarker causes the formation of an immune complex that is too small to precipitate. However, these complexes will scatter incident light, which can be measured using a turbidimeter. The concentration of the antigen (i.e. biomarker) can be determined within a few minutes of the reaction.

Radioimmunoassay (RIA) methods use radioisotopes such as I125 to label antigens or antibodies. The isotope used emits gamma rays, which are usually measured after removal of unbound (free) radiolabel. The main advantages of RIA compared to other immunoassays are higher sensitivity, easy signal detection and confirmation, fast assay. The main disadvantages are the health and safety risks posed by the use of radiation and the time and expense associated with maintaining the licensed radiation safety and disposal procedures. For this reason, RIA has been largely replaced by enzyme immunoassays in routine clinical laboratory practice.

Enzyme Immunoassays (EIAs) have evolved as alternatives to Radioimmunoassays (RIA). These methods use enzymes to label the antibody or target antigen. The sensitivity of EIA is close to that of RIA and there is no risk caused by radioisotopes. One of the most widely used EIA methods for detection is enzyme-linked immunosorbent assay (ELISA). The ELISA method may use two antibodies, one specific for the target antigen and the other coupled to an enzyme, the addition of an enzyme substrate causing the generation of a chemiluminescent or fluorescent signal.

Fluorescence Immunoassay (FIA) refers to an immunoassay that uses a fluorescent label or an enzyme label that acts on a substrate to form a fluorescent product. Fluorescence measurements are inherently more sensitive than chromatic (spectrophotometric) measurements. Thus, the FIA method has higher analytical sensitivity than the EIA method using absorption (optical density) measurement.

Chemiluminescent immunoassays use a chemiluminescent label that produces light when excited by chemical energy; the emission is measured using a photodetector.

Thus, the immunization method according to the present invention can be carried out using well-known methods. Any direct (e.g., using a sensor chip) or indirect method may be used in the detection of the biomarkers of the invention.

Probe needle

"Probe" refers to a molecule that can be used to measure the expression of a particular gene. Exemplary probes include PCR primers and gene-specific DNA oligonucleotide probes, such as microarray probes immobilized on a microarray substrate, quantitative nuclease protection test probes, probes attached to molecular barcodes, and probes immobilized on beads.

The term "probe" as used herein refers to a molecule that binds to a specific sequence or subsequence or other portion of another molecule. Unless otherwise indicated, "probe" generally refers to a polynucleotide probe that is capable of binding to another polynucleotide (often referred to as a "target polynucleotide") by complementary base pairing. Depending on the stringency of the hybridization conditions, a probe can bind to a target polynucleotide that lacks complete sequence complementarity to the probe. The probe may be directly or indirectly labeled, and includes within its scope a primer. Hybridization modalities, including, but not limited to: solution phase, solid phase, mixed phase or in situ hybridization assays.

As the probe, a labeled probe in which a polynucleotide for cancer detection is labeled, such as a fluorescent label, a radioactive label, or a biotin label, can be used. Methods for labeling polynucleotides are known per se. The presence or absence of the test nucleic acid in the sample can be checked by: immobilizing the test nucleic acid or an amplification product thereof, hybridizing with the labeled probe, washing, and then measuring the label bound to the solid phase. Alternatively, the polynucleotide for cancer detection may be immobilized, a nucleic acid to be tested may be hybridized therewith, and the nucleic acid to be tested bound to the solid phase may be detected using a labeled probe or the like. In this case, the polynucleotide for cancer detection bound to the solid phase is also referred to as a probe. Methods for assaying test nucleic acids using polynucleotide probes are also well known in the art. The process can be carried out as follows: the polynucleotide probe is contacted with the test nucleic acid at or near Tm (preferably within ± 4 ℃) in a buffer for hybridization, washed, and the hybridized labeled probe or template nucleic acid bound to the solid phase probe is then measured.

The size of the polynucleotide used as a probe is preferably 18 or more nucleotides, more preferably 20 or more nucleotides, and the entire length of the coding region or less. When used as a primer, the polynucleotide is preferably 18 or more nucleotides in size, and 50 or less nucleotides in size. These probes have a base sequence complementary to a specific base sequence of a target gene. Here, the term "complementary" may or may not be completely complementary as long as it is a hybrid. These polynucleotides usually have a homology of 80% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 100% with respect to the specific nucleotide sequence. These probes may be DNA or RNA, or they may be polynucleotides in which part or all of the nucleotides are substituted with artificial nucleic acids such as PN, LNA, ENA, GNA, TNA, etc.

Chip and kit

The term "chip", also referred to as an "array", refers to a solid support comprising attached nucleic acid or peptide probes. Arrays typically comprise a plurality of different nucleic acid or peptide probes attached to the surface of a substrate at different known locations. These arrays, also known as "microarrays," can generally be produced using either mechanosynthesis methods or light-guided synthesis methods that incorporate a combination of photolithography and solid-phase synthesis methods. The array may comprise a flat surface, or may be nucleic acids or peptides on beads, gels, polymer surfaces, fibers such as optical fibers, glass, or any other suitable substrate. The array may be packaged in a manner that allows for diagnostic or other manipulation of the fully functional device.

A "microarray" is an ordered array of hybridization array elements, such as polynucleotide probes (e.g., oligonucleotides) or binding agents (e.g., antibodies), on a substrate. The matrix may be a solid matrix, for example, a glass or silica slide, beads, a fiber optic binder, or a semi-solid matrix, for example, a nitrocellulose membrane. The nucleotide sequence may be DNA, RNA or any permutation thereof.

Various probe arrays have been described in the literature and can be used in the context of the present invention to detect markers that may be associated with the phenotypes described herein. For example, a DNA probe array chip or a larger DNA probe array wafer (otherwise, individual chips may be obtained by breaking the wafer) is used in one embodiment of the present invention. The DNA probe array wafer generally comprises a glass wafer on which a high-density array of DNA probes (short DNA fragments) is placed. Each of these wafers may hold, for example, about 6000 million DNA probes for identifying longer sample DNA sequences (e.g., from an individual or population, e.g., containing a marker of interest). The identification of sample DNA by the DNA probe set on the glass wafer was performed by DNA hybridization. When a DNA sample is hybridized to an array of DNA probes, the sample binds to those probes whose sample DNA sequences are complementary. By assessing that the individual sample DNA hybridizes more strongly to those probes, it is possible to determine whether a known nucleic acid sequence is present in the sample, and thus whether a marker found in the nucleic acid is present. This approach can also be used to perform ASH by controlling hybridization conditions to allow discrimination of single nucleotides, e.g., for SNP identification and sample genotyping of one or more SNPs. Arrays provide a convenient embodiment for the simultaneous (or tandem) detection of multiple polymorphic markers.

In the present invention, the kit can be used for detecting the expression level of a BTNL3 gene or protein, comprising a ligand, and/or a chip of the invention for BTNL3 detection and/or quantification. Optionally together with instructions for the kit.

Kits include one or more sterile containers, which may be in the form of a box, ampoule, bottle, vial, tube, bag, pouch, blister pack, or other suitable container known in the art. Such containers may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for containing medicaments.

In the present invention, the term "including" is used to mean, and is used interchangeably with, the phrase "including but not limited to".

Antibodies

In the present invention, the term "antibody" refers to a natural or synthetic antibody that selectively binds to an antigen of interest. The term includes polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, fragments or polymers of those immunoglobulin molecules, as well as human or humanized forms of immunoglobulin molecules that selectively bind an antigen of interest, are also included within the scope of the term "antibody" so long as they exhibit the desired biological activity. "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, with such variants generally being present in minor amounts, except for possible variants that may arise during the course of production of the monoclonal antibody. Such monoclonal antibodies typically include an antibody comprising a polypeptide sequence that binds to a target, wherein the target-binding polypeptide sequence is obtained by a process that includes selecting a single target-binding polypeptide sequence from a plurality of polypeptide sequences.

Monoclonal antibodies also include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.

"humanized" forms of non-human (e.g., murine) antibodies refer to chimeric immunoglobulins, immunoglobulin chains or fragments thereof such as Fv, Fab ', F (ab')²Or other antigen binding subsequences of antibodies.

An "antibody fragment" comprises a portion of a full-length antibody, typically the antigen-binding or variable region thereof. Examples of antibody fragments include Fab, Fab ', F (ab')²And Fv fragments; a diabody; a linear antibody; a single chain antibody molecule; and multispecific antibodies formed from antibody fragments.

"Fv" is the smallest antibody fragment that contains the entire antigen recognition and binding site. The fragment consists of a dimer of one heavy chain variable domain and one light chain variable domain in tight, non-covalent association. Six hypervariable loops (3 loops each for the heavy and light chains) are shed from the folded structure of these two domains, contributing to the amino acid residues that bind antigen and conferring antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) can have the ability to recognize and bind antigen, with only a lower affinity than the entire binding site.

"functional fragments" of an antibody of the invention refer to those fragments that retain the binding of the polypeptide with substantially the same affinity as the intact full chain molecule from which they are derived and that are active in at least one assay (e.g., inhibiting the TH 2-induced asthma pathway, such as in a mouse model, or inhibiting the biological activity of the antigen to which the antibody fragment binds in vitro).

Polyclonal antibodies include antibodies derived by immunizing an animal (e.g., a mouse) producing a human antibody with BTNL3 protein. When a chimeric antibody or a humanized antibody is prepared, amino acids in the variable region (e.g., FR) and/or constant region may be replaced with other amino acids, or the like.

Statistical method

In the present invention, the experiment is repeated at least 3 times, the result data are expressed in the form of mean value ± standard deviation, statistical analysis is performed by using SPSS18.0 statistical software, and the difference between the two is determined by t test, and the statistical significance is considered when P is less than 0.05.

The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. Experimental procedures without specific conditions noted in the examples, generally following conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press,1989), or according to the manufacturer's recommendations.

Example 1 screening of Gene markers associated with osteoporosis

1. Sample collection

Blood was collected from 6 male osteoporosis patients and healthy persons each, and none of the above samples had a history of smoking, drinking, maternal family, coffee or carbonated beverage, lack of physical activity, no disease affecting bone metabolism, or use of related drugs. The samples were obtained with informed consent, and all samples were obtained with consent from the tissue ethics committee.

2. Preparation and Mass analysis of RNA samples

2.1 preparation of RNA samples

Total RNA was extracted using an RNA extraction kit from Promega corporation. The method comprises the following specific steps:

1) taking 1ml of whole blood collected in a test tube treated by heparin or EDTA, and putting the whole blood into a sterile centrifuge tube;

2) centrifuging at 3000rpm (400g) for 5min, carefully sucking off the supernatant from the top of the sample;

3) adding 1ml of blood cell lysate, carefully sucking and placing for 4-5 times, re-suspending the precipitate, and centrifuging at 3000rpm for 5 min;

4) repeat step 3 twice (three times total);

5) avoid cell pellet, carefully aspirate almost all supernatant, retain only 100 μ l supernatant; confirming that BME has been added to the RNA lysate, then adding 175. mu.l of RNA lysate to the cells, pipetting, resuspending and lysing the cells;

6) adding 350 μ l of RNA diluent, mixing by inversion for 3-4 times, centrifuging at 13000g for 10min at room temperature, and transferring clear lysate to a sterile centrifuge tube;

7) adding 200 μ l 95% ethanol into the clarified lysate, and mixing by pipetting for 3-4 times; transferring the mixture into a centrifugal column assembly, and centrifuging for 1min at 13000 g;

8) taking down the centrifugal column from the centrifugal column assembly body, discarding the liquid in the collecting pipe, putting the centrifugal column back on the collecting pipe, adding 600 mu l of RNA washing liquid into the centrifugal column assembly body, and centrifuging for 1min at 13000 g;

9) discarding the liquid from the collection tube, loading the column back onto the collection tube, and adding 50 μ l of freshly prepared DNase incubation mixture directly onto the membrane inside the column;

10) incubating at room temperature for 15min, adding 200 μ l DNase termination buffer (ethanol is confirmed to be added) into the centrifugal column, and centrifuging at 13000g for 1 min;

11) adding 600 μ l RNA washing solution (ethanol is added), 13000g, centrifuging for 1 min;

12) emptying the collection tube, adding 250 μ l of RNA washing solution (added with ethanol) into the centrifugal column, and centrifuging at high speed for 2 min;

13) transferring the centrifugal column from the collecting tube to an elution tube, adding 100. mu.l of nuclease-free water to the membrane, placing the assembly of the centrifugal column into a centrifuge with the cap of the elution tube facing outwards, centrifuging at 13000g for 1min, discarding the centrifugal column, capping the elution tube with RNA, and storing at-70 ℃.

2.2 Mass analysis of RNA samples

The concentration and purity of the extracted RNA were determined using Nanodrop2000, RNA integrity was determined by agarose gel electrophoresis, and RIN was determined by Agilent 2100. The concentration is more than or equal to 200 ng/mul, and the OD260/280 is between 1.8 and 2.2.

3. Removal of rRNA

Ribosomal RNA was removed from total RNA using Ribo-Zero kit.

4. Construction of cDNA library

The construction of cDNA library was carried out using the Truseq RNA sample Prep Kit from Illumina, the detailed procedures were as described in the specification.

5. Sequencing on machine

The cDNA library was sequenced using the Hiseq4000 sequencing platform, the specific procedures were as described in the specification.

6. High throughput transcriptome sequencing data analysis

And (3) performing bioinformatics analysis on a sequencing result, performing RNA-seq reading positioning by using TopHat v1.3.1, standardizing the number of RNA-seq fragments by using Cufflinks v1.0.3 to calculate the relative abundance of the transcript, detecting differential expression by using cuffdiff, and considering that mRNA is significantly differentially expressed when P is less than 0.05.

7. Results

The RNA-seq results show that the expression level of the BTNL3 gene in osteoporosis patients is significantly higher than that of healthy people.

Example 2 QPCR validation of differential expression of the BTNL3 Gene

1. Large sample QPCR validation was performed on BTNL3 gene differential expression. 80 cases of each of the blood of the osteoporosis patients and the healthy persons were selected in accordance with the sample collection method in example 1.

2. The RNA extraction procedure was as described in example 1.

3. Reverse transcription

Reverse transcription of mRNA Using FastQuant cDNA first Strand Synthesis kit (cat # KR106)

4、QPCR

Amplification was carried out using SuperReal PreMix Plus (SYBR Green) (cat # FP205) and the experimental procedures were performed according to the product instructions.

1) Primer design

QPCR amplification primers were designed based on the coding sequences of the BTNL3 gene and GAPDH gene in Genebank and were synthesized by Bomeide Biopsies. The specific primer sequences are as follows:

BTNL3 gene:

the forward primer is 5'-AGCCTGTATGATGTGGAGAT-3' (SEQ ID NO. 3);

the reverse primer was 5'-AGCAAGGTGGATGGAACA-3' (SEQ ID NO. 4).

The primer sequence of housekeeping gene GAPDH is as follows:

a forward primer: 5'-GGAGCGAGATCCCTCCAAAAT-3' (SEQ ID NO.5)

Reverse primer: 5'-GGCTGTTGTCATACTTCTCATGG-3' (SEQ ID NO.6)

2) Prepare 20 μ l PCR reaction system according to Table 1:

TABLE 1 PCR reaction System

3) And (3) PCR reaction conditions: : 95 ℃ for 15min, (95 ℃ for 10s, 55 ℃ for 30s, 72 ℃ for 32s) x 40 cycles, 95 ℃ for 15s, 60 ℃ for 60s, 95 ℃ for 15 s. SYBR Green is used as a fluorescent marker, PCR reaction is carried out on a Light Cycler fluorescent quantitative PCR instrument, a target band is determined through melting curve analysis and electrophoresis, relative quantification is carried out by a delta-CT method, and each sample is subjected to 3 times of repeated experiments.

5. Results

As shown in fig. 1, BTNL3 gene expression was significantly upregulated in osteoporotic patients compared to the control group, with the difference being statistically significant (P <0.05), consistent with the RNA-sep results.

Example 3 differential expression of BTNL3 as verified at the protein level

1. Each histone was extracted according to the RIPA protein lysate kit instructions, and the protein concentration in the sample was detected using the BCA protein concentration assay kit.

2. The conventional Western-blot method is used for detecting the change of the BTNL3 protein, each group of experiments are repeated for 3 times, beta-actin is used as an internal reference, the absorbance of a BTNL3 protein band is quantitatively analyzed, and the expression quantity is represented by the ratio of the absorbance of the BTNL3 protein to the absorbance of the beta-actin.

3. Results

The results are shown in figure 2, with a statistically significant difference (P <0.05) in protein levels of BTNL3 in male osteoporosis patients compared to healthy humans.

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.

Sequence listing

<110> Wanglima king yugong

Application of <120> molecular marker in male osteoporosis

<160>6

<170>SIPOSequenceListing 1.0

<210>1

<211>1401

<212>DNA

<213>Homo sapiens

<400>1

atggcttttg tgctcatttt ggttctcagt ttctacgagc tggtgtcagg acagtggcaa 60

gtcactggac cgggcaagtt tgtccaggcc ttggtggggg aggacgccgt gttctcctgc 120

tccctctttc ctgagaccag tgcagaggct atggaagtgc ggttcttcag gaatcagttc 180

catgctgtgg tccacctcta cagagatggg gaagactggg aatctaagca gatgccacag 240

tatcgaggga gaactgagtt tgtgaaggac tccattgcag gggggcgtgt ctctctaagg 300

ctaaaaaaca tcactccctc ggacatcggc ctgtatgggt gctggttcag ttcccagatt 360

tacgatgagg aggccacctg ggagctgcgg gtggcagcac tgggctcact tcctctcatt 420

tccatcgtgg gatatgttga cggaggtatc cagttactct gcctgtcctc aggctggttc 480

ccccagccca cagccaagtg gaaaggtcca caaggacagg atttgtcttc agactccaga 540

gcaaatgcag atgggtacag cctgtatgat gtggagatct ccattatagt ccaggaaaat 600

gctgggagca tattgtgttc catccacctt gctgagcaga gtcatgaggt ggaatccaag 660

gtattgatag gagagacgtt tttccagccc tcaccttggc gcctggcttc tattttactc 720

gggttactct gtggtgccct gtgtggtgtt gtcatgggga tgataattgt tttcttcaaa 780

tccaaaggga aaatccaggc ggaactggac tggagaagaa agcacggaca ggcagaattg 840

agagacgccc ggaaacacgc agtggaggtg actctggatc cagagacggc tcacccgaag 900

ctctgcgttt ctgatctgaa aactgtaacc catagaaaag ctccccagga ggtgcctcac 960

tctgagaaga gatttacaag gaagagtgtg gtggcttctc agggtttcca agcagggaaa 1020

cattactggg aggtggacgt gggacaaaat gtagggtggt atgtgggagt gtgtcgggat 1080

gacgtagaca gggggaagaa caatgtgact ttgtctccca acaatgggta ttgggtcctc 1140

agactgacaa cagaacattt gtatttcaca ttcaatcccc attttatcag cctccccccc 1200

agcacccctc ctacacgagt aggggtcttc ctggactatg agggtgggac catctccttc 1260

ttcaatacaa atgaccagtc ccttatttat accctgctga catgtcagtt tgaaggcttg 1320

ttgagaccct atatccagca tgcgatgtat gacgaggaaa aggggactcc catattcata 1380

tgtccagtgt cctggggatg a 1401

<210>2

<211>466

<212>PRT

<213>Homo sapiens

<400>2

Met Ala Phe Val Leu Ile Leu Val Leu Ser Phe Tyr Glu Leu Val Ser

1 5 10 15

Gly Gln Trp Gln Val Thr Gly Pro Gly Lys Phe Val Gln Ala Leu Val

20 25 30

Gly Glu Asp Ala Val Phe Ser Cys Ser Leu Phe Pro Glu Thr Ser Ala

35 40 45

Glu Ala Met Glu Val Arg Phe Phe Arg Asn Gln Phe His Ala Val Val

50 55 60

His Leu Tyr Arg Asp Gly Glu Asp Trp Glu Ser Lys Gln Met Pro Gln

65 70 75 80

Tyr Arg Gly Arg Thr Glu Phe Val Lys Asp Ser Ile Ala Gly Gly Arg

85 90 95

Val Ser Leu Arg Leu Lys Asn Ile Thr Pro Ser Asp Ile Gly Leu Tyr

100 105 110

Gly Cys Trp Phe Ser Ser Gln Ile Tyr Asp Glu Glu Ala Thr Trp Glu

115 120 125

Leu Arg Val Ala Ala Leu Gly Ser Leu Pro Leu Ile Ser Ile Val Gly

130 135 140

Tyr Val Asp Gly Gly Ile Gln Leu Leu Cys Leu Ser Ser Gly Trp Phe

145 150 155 160

Pro Gln Pro Thr Ala Lys Trp Lys Gly Pro Gln Gly Gln Asp Leu Ser

165 170 175

Ser Asp Ser Arg Ala Asn Ala Asp Gly Tyr Ser Leu Tyr Asp Val Glu

180 185 190

Ile Ser Ile Ile Val Gln Glu Asn Ala Gly Ser Ile Leu Cys Ser Ile

195 200 205

His Leu Ala Glu Gln Ser His Glu Val Glu Ser Lys Val Leu Ile Gly

210 215 220

Glu Thr Phe Phe Gln Pro Ser Pro Trp Arg Leu Ala Ser Ile Leu Leu

225 230 235 240

Gly Leu Leu Cys Gly Ala Leu Cys Gly Val Val Met Gly Met Ile Ile

245 250 255

Val Phe Phe Lys Ser Lys Gly Lys Ile Gln Ala Glu Leu Asp Trp Arg

260 265 270

Arg Lys His Gly Gln Ala Glu Leu Arg Asp Ala Arg Lys His Ala Val

275 280 285

Glu Val Thr Leu Asp Pro Glu Thr Ala His Pro Lys Leu Cys Val Ser

290 295 300

Asp Leu Lys Thr Val Thr His Arg Lys Ala Pro Gln Glu Val Pro His

305 310 315 320

Ser Glu Lys Arg Phe Thr Arg Lys Ser Val Val Ala Ser Gln Gly Phe

325 330 335

Gln Ala Gly Lys His Tyr Trp Glu Val Asp Val Gly Gln Asn Val Gly

340 345 350

Trp Tyr Val Gly Val Cys Arg Asp Asp Val Asp Arg Gly Lys Asn Asn

355 360 365

Val Thr Leu Ser Pro Asn Asn Gly Tyr Trp Val Leu Arg Leu Thr Thr

370 375 380

Glu His Leu Tyr Phe Thr Phe Asn Pro His Phe Ile Ser Leu Pro Pro

385 390 395 400

Ser Thr Pro Pro Thr Arg Val Gly Val Phe Leu Asp Tyr Glu Gly Gly

405 410 415

Thr Ile Ser Phe Phe Asn Thr Asn Asp Gln Ser Leu Ile Tyr Thr Leu

420 425 430

Leu Thr Cys Gln Phe Glu Gly Leu Leu Arg Pro Tyr Ile Gln His Ala

435 440 445

Met Tyr Asp Glu Glu Lys Gly Thr Pro Ile Phe Ile Cys Pro Val Ser

450 455 460

Trp Gly

465

<210>3

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

agcctgtatg atgtggagat 20

<210>4

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

agcaaggtgg atggaaca 18

<210>5

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

ggagcgagat ccctccaaaa t 21

<210>6

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>6

ggctgttgtc atacttctca tgg 23

Claims

1. Use of a reagent for the detection of the expression level of BTNL3 in a blood sample for the preparation of a product for the diagnosis of osteoporosis in a male, characterized in that BTNL3 is up-regulated in the blood sample of a male patient with osteoporosis compared to a healthy male.

2. The use of claim 1, wherein the reagents comprise reagents for detecting the expression level of BTNL3 by RT-PCR, real-time quantitative PCR, immunodetection, in situ hybridization, or a chip.

3. The use of claim 2, wherein the reagents for detecting the expression level of BTNL3 by RT-PCR comprise at least one pair of primers that specifically amplify the BTNL3 gene; the reagent for detecting the expression level of the BTNL3 by real-time quantitative PCR at least comprises a pair of primers for specifically amplifying the BTNL3 gene; the reagent for immunologically detecting the expression level of BTNL3 comprises an antibody that specifically binds to BTNL3 protein; the reagent for detecting the expression level of BTNL3 by in situ hybridization comprises a probe that hybridizes to a nucleic acid sequence of BTNL3 gene; the reagent for detecting the expression level of BTNL3 by the chip comprises a probe hybridized with a nucleic acid sequence of BTNL3 gene or an antibody specifically bound with BTNL3 protein.

4. The use of claim 1, wherein the product comprises a formulation, chip or kit.

5. The use of claim 4, characterized in that the reagents in the chip for detecting the expression level of BTNL3 comprise a probe specifically recognizing the BTNL3 gene or an antibody specifically binding to a protein encoded by BTNL 3.

6. The use of claim 4, wherein the reagents in the kit for detecting the expression level of BTNL3 comprise a primer specifically amplifying the BTNL3 gene; or a probe specifically recognizing the BTNL3 gene; or an antibody that specifically binds to a protein encoded by BTNL 3.

7. The use according to claim 6, wherein the primer sequence for the specific amplification of the BTNL3 gene is as shown in SEQ ID No.3 and SEQ ID No. 4.

8. The use of claim 4, wherein the kit comprises a SYBR Green polymerase chain reaction system, a primer pair for amplifying a housekeeping gene; the SYBR Green polymerase chain reaction system comprises: PCR buffer, dNTPs, SYBR Green fluorescent dye.