CN105506064B - Method and kit for detecting susceptibility of ankylosing spondylitis - Google Patents

Abstract

The invention discloses a method for detecting susceptibility of ankylosing spondylitis and a kit thereof, wherein the method for detecting susceptibility of ankylosing spondylitis comprises the following steps: detecting the PSMB7 gene, transcript and/or protein of the individual, comparing the detected gene, transcript and/or protein with the normal PSMB7 gene, transcript and/or protein, and indicating that the possibility of the individual suffering from ankylosing spondylitis is higher than that of the normal population if the difference exists; the kit comprises: a primer for specifically amplifying PSMB7 gene or transcript, wherein the primer amplifies an amplification product with the length of 100-2000bp and containing the 306 th site in SEQ ID NO. 1. The invention can be used for early auxiliary diagnosis of ankylosing spondylitis, and can ensure that some carriers can take reasonable preventive measures before onset of disease, thereby improving the life cycle and the life quality of the carriers and having great application value and social benefit.

Description

Method and kit for detecting susceptibility of ankylosing spondylitis

Technical Field

The invention relates to a method and a kit for detecting susceptibility of ankylosing spondylitis, in particular to a method and a kit for detecting susceptibility of ankylosing spondylitis by using genotypes.

Background

Ankylosing spondylitis, also known as von bechterev's disease or maritstrumepell's disease, is a chronic, progressive, chronic inflammatory disease in which the medial-axial joints are involved. Mainly affecting the sacroiliac joint, the spinal joints and the paraspinal tissues of the pelvis. The main symptoms are low back pain, spinal stiffness and limited range of motion, and the bilateral sacroiliitis (sacrolitis) is shown by X-ray. Since the disease usually attacks the sacroiliac joint first and then affects the spine repeatedly, and finally causes the bony ankylosis of the spine, it is currently called Ankylosing Spondylitis (AS) at home and abroad.

Ankylosing spondylitis has a significant ethnicity, and the incidence rates vary greatly among different ethnic groups. The incidence rate of Indians is the highest, and the incidence rate of North American Indians is 2.7-6.3%. The second is caucasian, and the incidence rate of the caucasian is 0.1 to 1.4 percent. Yellow people are lower than white people, and China is about 0.3 percent. The incidence of black people is the lowest, about 1/4 for white people, and is only 0.2% in africa.

Ankylosing spondylitis is well developed in adults aged 20 to 40 years, especially young males aged 20 to 30 years.

Ankylosing spondylitis is autosomal dominant inheritance, has obvious family inheritance tendency, and the genetic factor is more than 90%. According to investigation, the incidence of relatives of ankylosing spondylitis patients is about twice AS high AS that of common people, AS patients with the sibling recurrence risk ratio of more than 82.90% have HLA-B27 positive, and the positive rate in normal people is 8%; the positivity of HLA-B27 accounts for 50% for children, and the positivity of HLA-B27 accounts for 25% for generating ankylosing spondylitis. It has been essentially concluded that HLA-B27 plays a role in the pathogenesis of AS AS an independent causative gene. Since only 1% to 5% of HLA-B27-positive individuals developed AS, it was suggested that other genes were involved in the onset of AS.

The product encoded by the PSMB7 gene belongs to the proteasome B type family, and is a 20S core beta subunit in ubiquitin-protease complex. The proteasome is a complex of proteolytic enzymes present in the cytoplasm and nucleus, responsible for degrading most proteins in the cell, and plays a key role in the expression of MHC class I molecules, including HLA-B27 molecules, and associated antigen presentation. The antigens presented by mhc class i molecules are all derived from degradation products of the proteasome. The function of proteasome is regulated by its structural composition, and its core part is a tetracyclic cylindrical structure composed of alpha and beta subunits, which becomes immunoproteasome after binding with PA 28. PSMB7 is an important structural subunit in the β -loop. Changes in the beta loop affect the rate of degradation of the protein and the cleavage site, as well as degradation of endogenous proteins and presentation of antigen. Polymorphism of PSMB7 gene affects the combination of immunoproteasome and target protein to be degraded, and results in the change of the amount and property of antigen peptide produced by degrading endogenous protein, including specific antigen peptide capable of inducing AS generation. Therefore, the PSMB7 gene has further research value on the relationship between the PSMB7 gene and ankylosing spondylitis.

Disclosure of Invention

The invention aims to solve the technical problem of providing a method for detecting susceptibility of ankylosing spondylitis and a kit thereof. The invention provides a basis for the auxiliary diagnosis (especially the early diagnosis) of the ankylosing spondylitis and the treatment of the ankylosing spondylitis by detecting the susceptibility to the ankylosing spondylitis by using the genotype.

In a first aspect of the present invention, there is provided a method for detecting susceptibility to ankylosing spondylitis in an individual, comprising the steps of:

detecting the PSMB7 gene, transcript and/or protein of the individual, comparing with the normal PSMB7 gene, transcript and/or protein, and indicating that the possibility of the individual suffering from ankylosing spondylitis is higher than that of the normal population.

In another preferred embodiment, the method detects the gene or transcript of PSMB7 and compares the differences with the normal PSMB7 nucleotide sequence.

In another preferred embodiment, the difference is the following Single Nucleotide Polymorphism (SNP):

c → G at position 306; wherein the nucleotide position numbering is based on SEQ ID NO. 1.

In a second aspect of the present invention, there is provided a method for detecting the presence of a single nucleotide polymorphism of PSMB7 gene in a sample, comprising the steps of:

1) amplifying the PSMB7 gene of the sample by using PSMB7 gene specific primers to obtain an amplification product;

2) detecting the presence or absence of the following single nucleotide polymorphisms in the amplification product:

c → G at position 306; wherein the nucleotide position numbering is based on SEQ ID NO. 1.

In another preferred embodiment, the PSMB7 gene specific primer has the sequence of SEQ ID No.2 and 3.

In another preferred embodiment, the amplification product has a length of 100-2000bp and contains position 306 in SEQ ID NO. 1.

In a third aspect of the present invention, there is provided a kit for ankylosing spondylitis susceptibility detection (using genotype for ankylosing spondylitis) comprising: the primer specifically amplifies PSMB7 gene or transcript, preferably, the primer amplifies the amplification product with the length of 100-2000bp and the 306 th position in SEQ ID NO. 1.

In another preferred embodiment, the kit further comprises a reagent selected from the group consisting of:

1) a probe that binds to a mutation at position 306 in SEQ ID No. 1;

2) a mutant restriction enzyme that recognizes position 306 in SEQ ID NO. 1.

In another preferred embodiment, the mutation is selected from the group consisting of a single nucleotide polymorphism:

c → G at position 306; wherein the nucleotide position numbering is based on SEQ ID NO. 1.

The sequence of the primer can be shown as SEQ ID NO.2 and 3.

The amplification product has a length of 100-2000bp and contains the 306 th position in SEQ ID NO. 1.

The kit further comprises: taq enzyme, dNTPs, magnesium ions and conventional PCR reaction buffer solution.

The present invention has been made through intensive and extensive studies to determine and analyze SNPs of a large number of candidate genes. The genomic sequence of PSMB7 is found and proved to be closely related to ankylosing spondylitis, wherein the correlation research result shows that the distribution of SNP (C → G at position 306) (marked as rs2236386) at position 306 in SEQ ID NO.1 of PSMB7 gene in a control group and a case group has significant difference (P < 0.05), so that the SNP can be used as a specific SNP for auxiliary detection of ankylosing spondylitis (or susceptibility thereof). The present invention has been completed based on this finding.

The PSMB7 gene is sequenced in almost the whole area, a plurality of SNPs are discovered, most of the SNPs are not related to the susceptibility of ankylosing spondylitis, however, association research shows that C → G at position 306 in SEQ ID NO.1 is a SNP with very high association with the susceptibility of ankylosing spondylitis.

Specifically, the invention discloses a Single Nucleotide Polymorphism (SNP) of the PSMB7 gene and the correlation of the polymorphism and ankylosing spondylitis. The SNP of the invention is G/C polymorphism at position 306 of the sequence shown in SEQ ID NO.1, and the frequency of genotype CC in the population with ankylosing spondylitis is higher than that in the population with normal controls.

Based on the new discovery of the invention, the PSMB7 protein or polypeptide has multiple novel uses. These uses include: can be used for the adjuvant diagnosis of ankylosing spondylitis.

In another aspect, the invention also includes polyclonal and monoclonal antibodies, particularly monoclonal antibodies, specific for the polypeptide encoded by human PSMB7 gene DNA or fragments thereof. As used herein, "specific" means that the antibody binds to the human PSMB7 gene product or fragment. Preferably, these antibodies bind to the human PSMB7 gene product or fragment, but do not recognize and bind to other unrelated antigenic molecules. The antibodies of the present invention include those molecules that bind to and inhibit human PSMB7 protein, as well as those antibodies that do not affect the function of human PSMB7 protein.

The present invention includes not only intact monoclonal or polyclonal antibodies, but also immunologically active antibody fragments, such as Fab' or (Fab)2 fragments; an antibody heavy chain; an antibody light chain; a genetically engineered single chain Fv molecule; or a chimeric antibody.

The antibodies of the invention can be prepared by a variety of techniques known to those skilled in the art. For example, the purified human PSMB7 gene product, or antigenic fragment thereof, can be administered to an animal to induce the production of polyclonal antibodies. Similarly, cells expressing human PSMB7 protein or an antigenic fragment thereof can be used to immunize animals to produce antibodies. Various adjuvants may be used to enhance the immune response, including Freund's adjuvant, and the like.

The antibody of the present invention may also be a monoclonal antibody. Such monoclonal antibodies can be prepared using hybridoma technology. The antibody of the invention comprises an antibody capable of blocking the function of the human PSMB7 protein and an antibody which does not influence the function of the human PSMB7 protein. The antibodies of the invention can be obtained by conventional immunization techniques using fragments or functional regions of the human PSMB7 gene product. These fragments or functional regions can be prepared by recombinant methods or synthesized using a polypeptide synthesizer. Antibodies that bind to an unmodified form of the human PSMB7 gene product can be produced by immunizing an animal with a gene product produced in a prokaryotic cell (e.g., e.coli); antibodies that bind to post-translationally modified forms (e.g., glycosylated or phosphorylated proteins or polypeptides) can be obtained by immunizing an animal with a gene product produced in a eukaryotic cell (e.g., a yeast or insect cell).

The antibody against human PSMB7 protein can be used in immunohistochemical method to detect the amount and/or mutation of human PSMB7 protein in biopsy specimen. One preferred anti-PSMB 7 antibody is an antibody that does not recognize normal PSMB7 but recognizes mutant PSMB7, or an antibody that recognizes normal PSMB7 but does not recognize mutant PSMB 7. By using the antibodies, protein level ankylosing spondylitis susceptibility detection can be conveniently carried out.

By utilizing the PSMB7 protein, substances which interact with the PSMB7 protein, such as inhibitors, agonists or antagonists and the like, can be screened out by various conventional screening methods.

The invention also relates to diagnostic assays for quantitative and in situ detection of human PSMB7 protein levels. Such assays are well known in the art and include ELISA and the like.

One method for detecting the presence of the PSMB7 protein in a test sample is to use an antibody specific for the PSMB7 protein, which comprises: contacting the sample with an antibody specific for PSMB7 protein; observing whether an antibody complex is formed, the formation of an antibody complex indicates the presence of PSMB7 protein in the sample.

The PSMB7 protein polynucleotide can be used for auxiliary diagnosis of PSMB7 protein related diseases. In terms of diagnosis, the polynucleotide of the PSMB7 protein can be used for detecting whether the PSMB7 protein is expressed or not or whether the PSMB7 protein is abnormally expressed in a disease state. For example, the PSMB7 gene DNA sequence can be used for hybridization of biopsy specimens to judge the abnormal expression of PSMB7 protein. The hybridization techniques include Southern blotting, Northern blotting, in situ blotting, etc. The technical methods are all published mature technologies, and related kits are all available from commercial sources. A part or all of the polynucleotide of the present invention can be used as a probe to be fixed on a microarray or a DNA chip (also called a "gene chip") for analyzing differential expression analysis of genes in tissues and gene diagnosis. The PSMB7 protein transcript can also be detected by RNA-polymerase chain reaction (RT-PCR) in vitro amplification using PSMB7 protein specific primers.

The detection can be performed on cDNA as well as on genomic DNA. The mutated form of the PSMB7 protein comprises point mutation, translocation, deletion, recombination and any other abnormality compared with the DNA sequence of the normal wild-type PSMB7 gene. The mutation can be detected by known techniques such as Southern blotting, DNA sequencing, PCR and in situ hybridization. In addition, since mutation may affect the expression of protein, the presence or absence of mutation in a gene can be indirectly determined by Northern blotting or Western blotting.

The most convenient method for detecting the SNP of the invention is to amplify PSMB7 gene of a sample by PSMB7 gene specific primer to obtain an amplification product; then detecting whether the following single nucleotide polymorphisms exist in the amplification product: c → G at position 306, wherein the numbering of nucleotide positions is based on SEQ ID NO. 1.

It is understood that, after the correlation between the SNP of PSMB7 gene and ankylosing spondylitis is revealed, one skilled in the art can easily design an amplification product that can specifically amplify the position containing the SNP, and then determine whether C → G at position 306 exists by sequencing or the like. In general, the length of the primer is 15-50bp, preferably 20-30 bp. Although complete complementarity of the primer to the template sequence is preferred, it is known to those skilled in the art that specific amplification (i.e., amplification of only the desired fragment) is also possible in the presence of a primer that is not necessarily complementary to the template, particularly at the 5' end of the primer.

Kits containing these primers and methods of using these primers are within the scope of the invention, provided that the primers amplify an amplification product containing the corresponding position of the SNP of the invention. A preferred primer pair has the sequences of SEQ ID NO.2 and 3.

Although the length of the amplification product is not particularly limited, the length of the amplification product is generally 100-2000bp, preferably 150-1500bp, more preferably 200-1000 bp. These amplification products should contain position 306 of SEQ ID NO. 1.

The SNP of the invention has very high relevance with the ankylosing spondylitis, so the SNP can be used for early auxiliary diagnosis of the ankylosing spondylitis, and can ensure that some carriers can take reasonable preventive measures before the onset of disease without being muzzled, thereby improving the life cycle and the life quality of the carriers, and having great application value and social benefit.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: the conditions described in the Laboratory Manual (New York: Cold spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations.

Example 1

First, research object

Case samples were collected entirely from outpatients, and the patients were diagnosed by clinically experienced rheumatologists in the physician's physician in the Ji Hospital according to revised New York standards set forth in 1984, and confirmed by multiple follow-up visits. Control samples were selected from southern central panel of age, gender matched, individuals without a history of arthritis.

Peripheral blood samples were randomly collected from 189 AS patients and 177 healthy normal control subjects based on informed consent.

Second, Experimental methods and results

1. DNA extraction

DNA was extracted from human peripheral blood samples by the conventional phenol chloroform method (DNA was also extracted using a commercial kit) and corrected to 20 ng/. mu.l for conventional PCR amplification.

2. Primer design for use in PCR and sequencing

Based on the genomic sequence of PSMB7 in GenBank, the following primers were designed and synthesized:

sense primer P1: 5'-accgcagaaccaaattaacg-3' (shown in SEQ ID NO. 2)

Antisense primer P2: 5'-aatgaatcctcccaccatca-3' (shown in SEQ ID NO. 3).

3. PCR amplification of PSMB7 Gene

PCR amplification was performed using Taq enzyme on a GeneAmp 9700PCR instrument using the Touchdown program using the extracted DNA as a template. The reaction conditions are as follows: pre-denaturation at 94 ℃ for 2 min, denaturation at 94 ℃ for 30 sec, annealing at 63 ℃ for 40 sec, and extension at 72 ℃ for 40 sec for 10 cycles, wherein the annealing temperature decreases by 0.5 ℃ per cycle; 30 cycles of subsequent denaturation at 94 ℃ for 30 seconds, annealing at 58 ℃ for 40 seconds, and extension at 72 ℃ for 40 seconds; final extension at 72 ℃ for 7 min.

The PCR amplification product was verified by agarose gel electrophoresis. As a result, an amplification product of PSMB7 gene was obtained.

4. SNP discovery and detection

PCR products were purified with Resin, sequenced with ABI-3730 DNA sequencer (applied biosystems, ABI), and sequence-calling, genotyping, and SNP-confirmation were performed with Polyphred software (http:// drog. mbt. washington. edu/Polyphred. html, university of Washington, USA).

As a result, several SNPs were found to exist, including the following:

c → G (rs2236386) at position 306 in SEQ ID NO. 1.

Wherein, rs 2236386:

ggctgtgcctgggacttgggtctgtctgtctctgaagcaccgcagaaccaaattaacggtagaaacaagtccagtctgagcggcaggagacctggtcgctggtgcgttcaccttggacaagtcctttcccggttccgggtgtcactttctcctccgtgccacttggggcgatatacttcaaacatctctggcctctctaggaaccagaatctctaagatcaatcgttctagtgcggcgtttgccccgccccttcagcccccgttgccagcgacgccggaagtgctcggctctttttgtttgcaccCgcctccgacccggaactgctttcttgggaagatggcggctgtgtcggtgtatgctccaccagttggaggcttctcttttgataactgccgcaggtgccgccttgcttggggcttggttcagggagggagaagagtgcggtgctcgggtgggagctgggccaaggtgtgatacgagaattctgagggaagaattcatggcggggcctgtggcccgcggctagtgtgactcaggggaatcagggcctgggcgtgggtttctcctgaagccgggtctgctctcccaccttcccagcgcactcggccaccgtttatctccgtctcctaaggcacgtcacggcccctttgtaagactgtcttcgttggaaaacgggagaacagtgctgatggtgggaggattcattaaa (shown in SEQ ID NO. 1). Wherein, in the sequence "C"is the SNP site.

5. SNP genotyping and association analysis

SNP genotyping was performed by direct one-way sequencing. I.e., typing and correlation analysis in ankylosing spondylitis patients and normal control groups.

And carrying out descriptive statistical analysis on the genotyping result, and carrying out chi-square test on the tabulation. Whether the genotypes were different between the patients and the controls was observed, and the analysis results were as follows:

according to the research method of case-control correlation analysis, chi-square calculation is carried out on the genotyping result, and the result shows that the genotype GG of the polymorphic site rs2236386 has obvious statistical difference. Wherein, in case samples, the distribution of rs2236386 is shown as (24 cases of genotype GG, 81 cases of genotype GC and 84 cases of genotype CC); in contrast, the control samples (genotype GG 12, genotype GC 95, and genotype CC 80) showed significant differences.

Genotype GG is more abundant in cases than in controls, with chi-square test P ═ 0.039. Individuals of genotype GG are at a 2.12-fold higher risk of developing ankylosing spondylitis than individuals of the other two genotypes (CC + GC) (95% CI: 1.03-4.38).

The above results show that: c → G changes, which increases the susceptibility of ankylosing spondylitis, and the genotype GG of PSMB7 gene polymorphism site rs2236386 is obviously related to the pathogenesis of ankylosing spondylitis. Namely: the SNP site at position 306 in SEQ ID NO.1 has a correlation with the occurrence of ankylosing spondylitis.

Example 2 ankylosing spondylitis susceptibility detection kit

As described in example 1, the C → G mutation at position 306 in SEQ ID NO.1 is closely related to ankylosing spondylitis. Therefore, PSMB7 gene specific primers can be designed based on the mutation and then detected by amplification using the DNA of the patient as a template.

A kit (100 human trials) was prepared containing:

1) sense primer P1 (shown in SEQ ID NO. 2) at a concentration of 100 pmol;

2) antisense primer P2 (shown in SEQ ID NO. 3) at a concentration of 100 pmol; and

3) PCR reaction solution containing Taq enzyme, dNTP, magnesium ions and PCR reaction buffer solution.

Randomly selecting a test group of 100 individuals, wherein the test group comprises: the method is used for detecting the ankylosing spondylitis of the patient, and comprises the following steps of (1) not knowing whether the patient is suffering from the ankylosing spondylitis, a patient known to suffer from the ankylosing spondylitis and a normal person without the ankylosing spondylitis.

3ml of peripheral blood of the subject to be detected in the test group was extracted, and DNA was extracted from the blood using a conventional method (or using a specific commercial kit). The PCR primer in the ankylosing spondylitis detection kit is diluted to 10 mu mol/l, and the extracted DNA is used as a template to perform PCR reaction with the provided primer. After purification of the PCR product, sequencing was performed using ABI 3730 DNA sequencer, and sequence interpretation and SNP confirmation were performed using Polyphred software.

Alternatively, the amplification product and the normal control are chromatographed by Denaturing High Performance Liquid Chromatography (DHPLC), and C → G at position 306 in SEQ ID NO.1 can be detected.

And (3) detection results: for subjects with C → G at position 306 in the PSMB7 gene, further examination was performed by conventional methods to confirm whether or not the subjects had ankylosing spondylitis. The test result shows that the susceptibility ratio of ankylosing spondylitis of the test object containing C → G at position 306 is obviously higher than that of the normal population without G (higher by at least 200%).

Therefore, it was revealed that the detection of C → G at position 306 of PSMB7 gene allows the adjuvant detection of ankylosing spondylitis.

Example 3 adjuvant detection of susceptibility to ankylosing spondylitis

The test of example 2 was repeated except that 70 persons (who did not know whether there was any ankylosing spondylitis symptom before the test) were randomly selected for the test.

A kit (100 human trials) was prepared containing:

1) sense primer P1 (shown in SEQ ID NO. 2) at a concentration of 100 pmol;

2) antisense primer P2 (shown in SEQ ID NO. 3) at a concentration of 100 pmol; and

3) PCR reaction solution containing Taq enzyme, dNTP, magnesium ions and PCR reaction buffer solution.

3ml of peripheral blood of the subject to be examined was extracted, and DNA was extracted from the blood using a conventional method (or using a specific commercial kit). The PCR primer in the ankylosing spondylitis detection kit is diluted to 10 mu mol/l, and the extracted DNA is used as a template to perform PCR reaction with the provided primer. After purification of the PCR product, sequencing was performed using ABI 3730 DNA sequencer, and sequence interpretation and SNP confirmation were performed using Polyphred software.

The results also confirmed that the proportion of ankylosing spondylitis was significantly higher in the test subject containing C → G at position 306 than in the test subject having GG at this position (at least 200% higher).

Claims (3)

1. The application of a genotype detection reagent in preparing a ankylosing spondylitis detection kit is characterized in that the genotype detection reagent comprises: the primer specifically amplifies PSMB7 gene or transcript, detects C → G mononucleotide polymorphism at position 306 in SEQ ID NO.1, and the length of the amplified product is 100-2000 bp.

2. Use according to claim 1, characterized in that: the sequences of the primers are shown as SEQ ID NO.2 and 3.

3. The use of claim 1, wherein the kit further comprises: taq enzyme, dNTPs, magnesium ions and conventional PCR reaction buffer solution.