WO2020259189A1 - Application of sox11 as a diagnostic marker for schizophrenia - Google Patents

Abstract

Provided is an application of SOX11 as a diagnostic marker for schizophrenia; specifically provided is the application of a SOX11 mRNA or protein quantitative detection agent in the preparation of a reagent or kit for diagnosing schizophrenia.

Description

Application of SOX11 as a diagnostic marker for schizophrenia

Cross references to related applications

This application requires a Chinese patent application with the application number 201910602071.1 and the name "SOX11 as a diagnostic marker for schizophrenia" submitted to the Chinese Patent Office on July 5, 2019, and a Chinese patent application on June 27, 2019 The bureau’s application number is 201910569911.9, the priority of the Chinese patent application titled "SOX11 as a diagnostic marker for schizophrenia", the entire content of which is incorporated into this application by reference.

Technical field

The present disclosure relates to the field of biomedical detection, in particular to the application of SOX11 as a diagnostic marker for schizophrenia.

Background technique

Schizophrenia (SC) is a chronic disabling disease with complex etiology and high phenotypic heterogeneity. There are often obstacles in perception, thinking, emotion, and behavior, and generally unconscious and intellectual obstacles. At present, it is generally believed that the lifetime prevalence of schizophrenia in the general population is 1%. It often starts in young adults and most of them have a poor prognosis. More than 50% of patients have long-term intermittent mental problems, and about 20% of patients have residuals. Symptoms and lead to mental disability. The unemployment rate of patients with schizophrenia is as high as 80%-90%, the relative risk of suicide increases by 12 times, and the life expectancy is reduced by about 10-20 years.

The concept of schizophrenia has been put forward for more than 100 years. However, the pathogenesis and pathological mechanism of schizophrenia are still unclear. This has become an urgent and difficult problem in the medical field. In the past 50 years, a large number of genetic epidemiological studies have shown that genetic factors are one of the important reasons for the occurrence and development of schizophrenia. As predicted on the basis of genetic epidemiological research results in the 1960s, schizophrenia is a highly polygenic disease. Genome-wide association studies have identified more than 100 gene loci containing common alleles with small genetic effects, and hundreds of such gene loci have overall genetic effects. This indicates that there are a series of population frequency single nucleotide polymorphisms (single nucleotide polymorphism, SNP) have the risk of disease.

At present, the clinical diagnosis of schizophrenia is mainly based on the patient’s detailed medical history and psychiatric symptoms, and then make comprehensive judgments with reference to the age of onset, disease stage, and disease course. Therefore, it is a process that depends on the subjective experience of the doctor as a whole. Diagnosis results often vary from person to person. In order to make the diagnosis of schizophrenia more objective and improve the consistency and accuracy of the diagnosis, in recent years, researchers from all over the world have been committed to finding biomarkers of schizophrenia and establishing effective detection methods. A biomarker refers to a certain characteristic biochemical index in the course of general physiology or pathology or treatment that can be objectively measured and evaluated. Through its measurement, the progress of the organism's current biological process can be known. The chemical nature of biomarkers can be DNA, RNA, protein, or metabolites, which are generally obtained from body fluids. The application of biomarkers to schizophrenia can effectively assist the uncertain human factors in the current diagnosis, reduce the rate of error/misdiagnosis, and help guide medication and rehabilitation.

Therefore, there is an urgent need in the art for biomarkers that can be used to diagnose first-episode schizophrenia, which should be reliable, and should also have good sensitivity and specificity.

Summary of the invention

The present disclosure provides an objective detection method for diagnosing schizophrenia, that is, detecting the expression level of Sox 11 in peripheral blood.

Specifically, the present disclosure relates to the application of SOX11 mRNA or protein quantitative detection agent in preparing reagents or kits for diagnosing schizophrenia.

The present disclosure also relates to a method for diagnosing schizophrenia by detecting the expression amount of SOX11 mRNA or protein in a sample of a subject.

SOX11 mRNA or protein has high specificity and sensitivity for the diagnosis of schizophrenia, and is a peripheral blood index, which can be easily obtained; single index detection method requires only one index to be measured, which is convenient for future promotion. It can also be combined with other detection methods to get more reliable results.

Description of the drawings

In order to more clearly describe the specific embodiments of the present disclosure or the technical solutions in the prior art, the following will briefly introduce the drawings that need to be used in the specific embodiments or the description of the prior art. Obviously, the appendix in the following description The drawings are some embodiments of the present disclosure. For those of ordinary skill in the art, other drawings may be obtained based on these drawings without creative work.

Figure 1 is an example of the expression of SOX11 mRNA in the serum of violent schizophrenia patients and normal people;

Figure 2 An embodiment of the present application shows the expression of SOX11 protein in blood samples of violent schizophrenia patients and normal people.

Detailed ways

In order to better illustrate the purpose, technical solutions and advantages of the present disclosure, the present disclosure will be further illustrated below in conjunction with specific embodiments. If the specific conditions are not specified in the implementation, it shall be carried out in accordance with the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used without the manufacturer's indication are all conventional products that can be purchased commercially.

Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, but methods and materials similar or equivalent to those described herein can also be used in the present disclosure.

The present disclosure relates to the application of SOX11 mRNA or protein quantitative detection agent in preparing reagents or kits for diagnosing schizophrenia.

The present disclosure also relates to a quantitative detection agent for SOX11 mRNA or protein, which is used in the diagnosis of schizophrenia.

The severity and/or susceptibility of the disorder and/or disease can also be determined based on the difference in expression levels. For example, when the marker gene is one of the genes described herein, the degree of increase in the expression level of the marker gene is related to the existence and/or severity of the condition and/or disease.

SOX11 is a nuclear transcription factor that belongs to the C subgroup of the HMG-box gene related to SRY (sex determining region on Y-chromosome). This subgroup contains three members: SOX4, SOX11 and SOX12. Human SOX11 is located on chromosome 2p25.3, and the encoded SOX11 protein is 46.7kDa and contains 441 amino acids. The expression of SOX11 is essential for embryonic neurogenesis and tissue remodeling. It is usually expressed in the nervous system during human embryonic development and is necessary for neurite growth and neuron survival.

SOX11 is clinically believed to be related to certain tumors, for example, it is overexpressed in more than 90% of mantle cell lymphomas (MCL), including the rare Cyclin D1 negative cases; in the vast majority of B and T lymphoblastic leukemias / Lymphoma and half of childhood Burkitt lymphoma also have strong expression; in some hairy cell leukemias may have weak expression. The expression of SOX11 is also speculated to be related to the survival of high-grade epithelial ovarian cancer.

Although SOX11 is closely related to the nervous system, there is no related report of SOX11 as a schizophrenia marker in the prior art. At present, there are very few reports on the expression level of SOX 11 gene or the role of related gene mutations in human mental diseases, and reports related to its mechanism regulation are even rarer. The inventors of the present disclosure unexpectedly discovered that the levels of SOX11 mRNA (such as SOX11 mRNA in peripheral blood) or protein levels in patients with mental illness and control populations have significant differences; the diagnosis can be performed alone/in combination with other molecular markers, and the results are objective and reliable. It has a good application prospect in the diagnosis of schizophrenia patients.

In the present disclosure, the term "schizophrenic disorder" described herein refers to a series of mental disorders including schizophrenia and related psychosis. The term is intended to specifically include schizophrenia (generally including first-episode schizophrenia or relapsed schizophrenia), schizophrenia-like schizophrenia, affective schizophrenia, delusional disorder, short-term mental disorder and short-term mental disorder, such as mental As defined in the Diagnostic and Statistical Manual of Disorders, 4th Edition (DSM-IV-TR).

In one or more embodiments, the schizophrenia is violent.

Symptoms of aggressive behavior that are common in violent schizophrenia Patients who have psychotic symptoms are likely to have aggressive behavior. In particular, psychiatric patients suffering from schizophrenia, paranoid psychosis and affective psychosis are the high-risk groups of violent behavior.

The following clinical symptoms are common in violent schizophrenia: auditory hallucinations, delusions, and enlarged suicide.

The term "quantitative detection agent for SOX11 mRNA" in the present disclosure should not only be understood as a detection agent for SOX11 mRNA, but should include other detection reagents known to those skilled in the art that can reflect the expression level of SOX11 mRNA. For example, the expression of SOX11 mRNA can be indirectly detected by quantitatively detecting the cDNA obtained by reverse transcription of SOX11 mRNA.

In this disclosure, if there is no special statement, "SOX11 mRNA" and "SOX11 protein" can be referred to as "markers", "marker genes" (specifically SOX11 mRNA), "biochemical markers", and "marker polypeptides" ( Specifically refers to SOX11 protein), "schizophrenia markers". It specifically refers to a molecule to be used as a target for the analysis of a patient's experimental sample.

The SOX11 mRNA used as a marker in the present disclosure is expected to include its full-length ribonucleotide sequence, or naturally-occurring variants, or full-length sequences and fragments of variants, especially fragments that can be detected and determined for specific sequences . For example, it comprises at least 7, 8, 9, 10, 11, 12, 15 or 20 consecutive ribonucleotides of the full-length ribonucleotide sequence.

The SOX11 protein used as a marker in the present disclosure is expected to include naturally-occurring variants of the protein and fragments of the protein or the variants, particularly immunologically detectable fragments. The immunologically detectable fragment contains, for example, at least 5, 6, 7, 8, 9, 10, 11, 12, 15 or 20 consecutive amino acids of the marker polypeptide. The expression "SOX11 protein" includes the complete protein sequence of SOX11 and the marker polypeptide defined above.

Those skilled in the art can recognize that ribonucleotides/proteins/polypeptides released by cells or ribonucleotides present in the extracellular matrix may be damaged (for example, during inflammation), and may be degraded or Cut into fragments like this. As the skilled artisan will appreciate, mRNA, protein or fragments thereof may also be present as part of the complex. Such a complex can also be used as a marker in the sense of the present disclosure. Additionally, in the alternative, the marker polypeptide or variants thereof may carry post-translational modifications. Non-limiting examples of post-translational modifications are glycosylation, acylation and/or phosphorylation.

"Naturally occurring variants" should be understood to mean that higher animal genes are usually accompanied by high frequency polymorphisms. There are also many molecules that produce isotypes that contain mutually different amino acid sequences during splicing. Any gene associated with a schizophrenia-related disease that has an activity similar to that of the marker gene is included in the marker gene, even if it has a nucleotide sequence difference due to polymorphism or isotype.

RNA quantitative detection reagents can be selected from reagents known to those skilled in the art, such as nucleic acids that can hybridize to the RNA and labeled with fluorescent labels; in common cases, RNA detection reagents can be selected from RT-PCR primers, and Used to amplify the product of RT-PCR-cDNA primers.

In one or more embodiments, the SOX11 mRNA quantitative detection agent includes a reagent suitable for at least one of the following methods:

Real-time fluorescent quantitative PCR, digital PCR, fluorescent dye method, resonance light scattering method, sequencing or biological mass spectrometry.

In one or more embodiments, the SOX11 mRNA quantitative detection agent is a probe or primer that can specifically bind to SOX11 mRNA or SOX11 cDNA.

It should also be understood that the marker gene may include homologues of other species except human. Therefore, unless explicitly stated, the expression "marker gene" refers to a homolog of a marker gene unique to a species or an exogenous marker gene that has been introduced into an individual. Similarly, it should be understood that "a homolog of a marker gene" can be used as a probe to hybridize to a human marker gene under stringent conditions. Such stringent conditions are known to those skilled in the art (who can select appropriate conditions to produce the same stringency through experiment or experience). A polynucleotide containing the nucleotide sequence of the marker gene or the complementary strand of the nucleotide sequence of the marker gene and having a nucleotide sequence of at least 15 nucleotides can be used as a primer or a probe. Therefore, the "complementary strand" refers to one strand of double-stranded DNA composed of A:T (U for RNA) and G:C base pairs relative to the other strand.

In addition, "complementary" means not only a sequence that is completely complementary to a region of at least 15 consecutive nucleotides, but also a sequence having at least 40% in some cases, 50% in some cases, and 60% in some cases. %, 70% in some cases, 80% in some cases, 90% in some cases, and in some cases 95% or higher nucleotide sequence homology. The degree of homology between nucleotide sequences can be determined using an algorithm such as BLAST.

Such polynucleotides can be used as probes for detecting marker genes or as primers for amplifying marker genes. When used as primers, polynucleotides generally comprise 15 bp to 100 bp, and in certain embodiments 15 bp to 35 bp of nucleotides. When used as a probe, DNA contains the entire nucleotide sequence of the marker gene (or its complementary strand), or a partial sequence thereof having at least 15 bp nucleotides. When used as a primer, the 3'region must be complementary to the marker gene, and the 5'region can be linked to a restriction endonuclease recognition sequence or tag. "Polynucleotide" can be DNA or RNA. Such polynucleotides can be synthetic or naturally occurring. Similarly, DNA used as a hybridization probe is usually labeled. Those skilled in the art can easily understand such marking methods. As used herein, the term "oligonucleotide" means a polynucleotide having a relatively low degree of polymerization. Oligonucleotides are also included in polynucleotides.

For example, Northern hybridization, dot blot hybridization, or DNA microarray technology can be used for detection of disorders and/or diseases using hybridization technology. In addition, gene amplification techniques such as the RT-PCR method can be used. By using the PCR amplification monitoring method in the gene amplification step of RT-PCR, the expression of marker genes can be analyzed more quantitatively.

In one or more embodiments, the probe or primer has a detectable label.

In the PCR gene amplification monitoring method, the detection target (reverse transcript of DNA or RNA) is hybridized with a probe labeled with a fluorescent dye and a quencher that absorbs fluorescence. When PCR is performed and Taq polymerase degrades the probe with its 5'-3' exonuclease activity, the fluorescent dye and the quencher are separated from each other, thereby detecting fluorescence. Real-time detection of fluorescence. By simultaneously measuring a standard sample in which the copy number of the target is known, the cycle number (in which PCR amplification is linear) can be used to determine the copy number of the target in the subject sample. Likewise, those skilled in the art recognize that the PCR amplification monitoring method can be performed using any appropriate method.

In one or more embodiments, the quantitative detection agent for SOX11 mRNA is a qRT-PCR primer for SOX11 mRNA, the upstream primer is agcaagaaatgcggcaagc, as shown in SEQ ID NO:1, and the downstream primer is atccagaaacacgcacttgac, as shown in SEQ ID NO: shown in 2.

In one or more embodiments, the quantitative detection agent for SOX11 protein is a reagent required to specifically measure SOX11 protein.

In one or more embodiments, the reagent (specific binding agent) required to specifically measure the SOX11 protein is, for example, a ligand or receptor (if present) of the SOX11 protein, agglutination that binds to the SOX11 protein Protein, aptamers that bind to SOX11 protein, or antibodies and antibody fragments that bind to SOX11 protein. Specific binding agent having an affinity of at least 10 ⁷ l / mol for its corresponding target molecule. Specific binding agent to its target molecule, for example, 10 ⁸ l / mol, e.g., affinity or 10 ⁹ l / mol of. The skilled person will understand that using the term "specific" means that other biomolecules present in the sample do not significantly bind to the specific binding agent of the SOX11 protein.

In one or more embodiments, the level of binding to biomolecules other than the target molecule (SOX11 protein) produces a binding affinity that is at most only 10% or less, only 5 percent of the affinity to the target molecule, respectively. % Or less, only 2% or less, or only 1% or less. In one or more embodiments, the specific binding agent will simultaneously meet the aforementioned minimum criteria for affinity and specificity.

In one or more embodiments, the detection agent capable of specifically binding to the SOX11 polypeptide is an antibody or an antibody fragment.

The term "antibody" includes polyclonal antibodies and monoclonal antibodies, and the term "antibody fragment" includes antigen compound binding fragments of these antibodies, including Fab, F(ab') ₂ , Fd, Fv, scFv, bispecific antibodies and antibodies The smallest recognition unit, and single-chain derivatives of these antibodies and fragments, such as scFv-Fc, etc. The type of antibody can choose IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD. In addition, the term "antibody" includes naturally-occurring antibodies and non-naturally-occurring antibodies, including, for example, chimeric, bifunctional, humanized, and human antibodies, and related synthetic antibodies. Isoforms. The term "antibody" can be used interchangeably with "immunoglobulin".

In one or more embodiments, the antibodies or antibody fragments can be obtained by immunizing with SOX11 protein or fragments thereof as antigens. The immunized subjects can include humans and all livestock (such as domestic animals and pets) and wild animals and poultry. Birds, which include, without limitation, cattle, horses, cows, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, geese, turkeys, and cockfighting Wait.

In one or more embodiments, the antibody or antibody fragment is labeled with an indicator that shows signal strength.

In one or more embodiments, the kit further includes one or more detection agents for schizophrenia markers.

The schizophrenia markers described herein can be combined with other risk factors for schizophrenia. In certain embodiments, such additional risk factors are selected from SNPs, microsatellites, or indel polymorphisms.

In one or more embodiments, the kit further includes reverse transcription reagents, blood extraction reagents, internal reference primers, fluorescent substances for indicating the amount of DNA synthesis, qPCR reaction buffer, dNTPs, DNA polymerase , One or more of the water.

In one or more embodiments, the internal reference primer is selected from primers of GAPDH, tubulin or actin.

In one or more embodiments, the water is nuclease-free water, such as reverse osmosis water, distilled water, deionized water, and reverse osmosis water .

In one or more embodiments, the DNA polymerase is selected from Taq, Bst, Vent, Phi29, Pfu, Tru, Tth, T11, Tac, Tne, Tma, Tih, Tf1, Pwo, Kod, Sac, Sso , Poc, Pab, Mth, Pho, ES4 DNA polymerase, Klenow fragment.

In one or more embodiments, the blood extraction reagent is selected from an anticoagulant (for example, citric acid or its salt, EDTA or its salt), lymphocyte separation solution and the like.

The present disclosure also provides a method for diagnosing schizophrenia (especially violent schizophrenia) in a human individual, the method comprising:

(a) Obtain a sample containing SOX11 mRNA or protein of the subject; (b) Measure the expression of SOX11 mRNA or protein in the sample, (c) Optionally, measure one or more other spirits in the sample The concentration of the schizophrenia marker; (d) the measurement result of step (b) and the measurement result of optional step (c) are used to diagnose schizophrenia.

In one or more embodiments, in step a), the above-mentioned reagents required for specific and quantitative measurement of SOX11 mRNA/protein are used for measurement.

The term "subject" refers to any animal, including mammals, such as but not limited to primates (such as humans), rodents (such as mice, rats, rabbits), dogs, cats, pigs, cows, Sheep, horse.

In one or more embodiments, the sample is a body fluid of the subject, such as blood, serum or plasma and cerebrospinal fluid, tissue or tissue lysate, cell culture supernatant, semen, saliva sample, Or other appropriate samples containing SOX11 mRNA/protein, such as peripheral blood;

In one or more embodiments, the sample is total RNA extracted from peripheral blood;

In one or more embodiments, the method is an in vitro method.

The technical solution provided by the present disclosure is helpful for more objective diagnosis of schizophrenia. The term "diagnosis" includes assessing the susceptibility to schizophrenia, the severity of schizophrenia, or the diagnosis of schizophrenia along with other indicators/conditions, or the prognostic assessment of schizophrenia.

The method of diagnosing a schizophrenia disorder may include an additional step including reporting the susceptibility to at least one entity selected from the group consisting of an individual, an individual’s guardian, an individual’s agent, a genetic service provider, a doctor, a medical institution And medical insurance companies. In certain embodiments, other single entities, including any of the aforementioned entities, can be targeted by such reports, and the same applies to any combination of the aforementioned entities.

Even if the subject is not diagnosed as suffering from a disorder and/or disease in routine testing (although the symptoms suggest such a disease), whether such a subject suffers from the disorder and/or disease can be determined by following the method described herein Perform testing to easily determine.

More specifically, in certain embodiments, when the marker gene is one of the genes described herein, its symptoms at least suggest an increase or decrease in the expression level of the marker in subjects who are susceptible to the disorder and/or disease It indicates that the symptoms are mainly caused by the condition and/or disease.

In addition, testing can be used to determine whether the condition and/or disease has improved in the subject. In other words, the method described herein can be used to judge the therapeutic effect of the treatment for the condition and/or disease. In addition, when the marker is one of the markers described herein (especially SOX11 mRNA/protein), an increase or decrease in the expression level of the marker gene in a subject who has been diagnosed with the disorder and/or disease means The disease has moved forward.

The present disclosure provides a method for diagnosing schizophrenia, including:

Measure the expression of SOX11 mRNA or protein in the subject’s sample, and

Analyze the measurement results of SOX11 mRNA or protein expression to diagnose schizophrenia.

In one or more embodiments, the method includes

(a) Obtain samples containing SOX11 mRNA or protein of the subject;

(b) Measure the expression level of SOX11 mRNA or protein in the sample,

(c) Measuring the concentration of one or more other markers of schizophrenia in the sample;

(d) Use the measurement result of step (b) and the measurement result of step (c) to diagnose schizophrenia.

In one or more embodiments, the method includes

(i) Measure the expression of SOX11 mRNA or protein in the subject sample

(ii) Measure SOX11 mRNA or protein expression in the control sample, and

(iii) Compare the expression level of SOX11 mRNA or protein in the subject sample and the control sample,

The expression level of SOX11 mRNA or protein in the subject sample is lower than the expression level of SOX11 mRNA or protein in the control sample is an indication of schizophrenia.

In one or more embodiments, the control sample is a sample from a healthy subject or a sample from a non-schizophrenic subject.

The present disclosure also provides a method for judging the therapeutic effect of schizophrenia, including

(1) Measure the expression of SOX11 mRNA or protein in the subject's sample before treatment;

(2) Measure the expression level of SOX11 mRNA or protein in the subject's sample after treatment; and

(3) Compare the expression level of SOX11 mRNA or protein in subject samples before and after treatment,

The expression level of SOX11 mRNA or protein in the subject sample after treatment is lower than the expression level of SOX11 mRNA or protein in the subject sample before treatment is an indication that schizophrenia continues to progress or the treatment effect is not good.

In one or more embodiments, the subject is a mammal, such as a primate, such as a human.

In one or more embodiments, the sample is at least one of blood, serum, plasma, cerebrospinal fluid, tissue, tissue lysate, cell culture supernatant, semen, or saliva sample, such as peripheral blood.

In one or more embodiments, the SOX11 mRNA measurement is performed by qRT-PCR using SOX11 mRNA primers. The upstream primer of the SOX11 mRNA primer is shown in SEQ ID NO:1, and the downstream primer is shown in SEQ ID NO: shown in 2.

In one or more embodiments, the schizophrenia is violent schizophrenia.

The embodiments of the present disclosure will be described in detail below in conjunction with examples, but those skilled in the art will understand that the following examples are only used to illustrate the present disclosure and should not be regarded as limiting the scope of the present disclosure. If specific conditions are not indicated in the examples, it shall be carried out in accordance with the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used without the manufacturer's indication are all conventional products that are commercially available.

Example 1

The schizophrenic patients came from a hospital specializing in schizophrenia (Ankang Hospital of the Public Security Department of Jilin Province) and a family member in Siping City, Jilin Province who suffered from schizophrenia. A total of 46 people were violent schizophrenia patients. Normal people come from normal members of the same family in Siping and other normal people who have nothing to do with this family, a total of 39 people. This study has been reviewed by the Medical Ethics Committee of Ankang Hospital of the Public Security Department of Jilin Province, and the clinical trial protocol is ethical. Both the case group and the control group were formally informed in writing.

Experimental procedure: The blood samples were processed with red blood cell lysate to obtain white blood cells; total RNA was extracted from the above peripheral blood white blood cell samples; SOX11 was specifically transcribed into cDNA using the total RNA as a template, and real-time quantitative PCR (qPCR) technology was used to detect the mRNA expression level of SOX11 , Compare the level of SOX11 in the blood of schizophrenia patients and normal people.

SOX11 qRCP reaction system:

Prepare PCR reaction solution according to the following components (on ice)

SOX11 qRCP reaction conditions:

Reaction conditions

Two-step PCR amplification standard procedure:

Stage 1: pre-denaturation

Reps: 1

95°C for 30 seconds

Stage 2: PCR reaction

Reps: 40

95°C for 5 seconds

60°C for 30 seconds

Dissociation stage

Result analysis:

Derive the SOX11 qPCR Ct value, use the formula △Ct=Ct (target gene)-Ct (internal reference gene) to calculate △Ct. Use the △Ct of the sample to be studied in this experiment minus the △Ct of the control sample and compare All the results take the opposite number, and the calculation is -△△Ct. Finally, the power of 2 is performed on -△△Ct, that is, 2 ^-△△Ct to get Fold Change.

The qPCR test results showed that the expression of SOX11 mRNA in the blood of violent schizophrenia patients was significantly different from that of normal people, and the expression of SOX11 mRNA in the blood of violent schizophrenia patients was significantly lower than that of normal controls. The result is shown in Figure 1.

Example 2

SOX11 protein level detection (Western blot)

The Western blot method was used to detect the protein level expression in the samples of schizophrenia patients and normal people. The specific methods are as follows:

(1) Glue: Wash the glass plate, dry it, and install the vertical electrophoresis plate. Separate gel was prepared according to the formula, 7ml of each gel was poured into a vertical electrophoresis plate, anhydrous ethanol was gently added for liquid sealing, and the gel was allowed to stand at room temperature for 30 minutes to solidify, then the anhydrous ethanol was poured out and dried with absorbent paper. Prepare concentrated glue according to the formula, pour 3ml of each piece of glue into the separating glue, insert a comb, and let it stand at room temperature for 30 minutes to solidify;

(2) Sample processing: Take the sample (containing 50μg protein) and mix it with 5*loading buffer at a ratio of 4:1, and place it on a metal bath at 97°C for 5 minutes;

(3) Sample loading: unplug the comb, add the sample and protein Marker to the loading wells in turn, the loading volume of each well should be the same, and the empty wells should be filled with 1× loading buffer.

(4) Electrophoresis: Perform electrophoresis at 80V constant voltage, when bromophenol blue runs into the separation gel, switch to 120V constant voltage electrophoresis, and wait until the bromophenol blue dye runs to the bottom of the gel;

(5) Electric transfer: Install in the electric transfer glue folder in the following order: anode → sponge layer → filter paper three layers → NC (PVDF) membrane → gel → filter paper three layers → sponge layer → cathode, remove the glue, membrane and filter paper Clamp the air bubbles between them and move them into the electroporation tank, inject electroporation buffer, and electroporate at 200mA constant current on ice for 2 hours;

(6) Take out the PVDF membrane, cut a notch in the upper right corner to indicate the front, put it in 5% skimmed milk powder, and seal it on a 70rpm shaker at room temperature for 1 hour;

(7) Primary antibody incubation (Novus Biologicals, nbp1-85823, 0.4ug/ml), overnight at 4°C;

(8) Wash the membrane with PBST, 5min×4 times; then add the fluorescent secondary antibody and incubate for 1 hour at room temperature;

(9) Wash the membrane with PBST, 5min×4 times;

(10) Use Odessey instrument to check, save the results and carry out digital analysis.

Western blot data shows that the Sox11 protein expression in the blood of schizophrenic patients (1#, 3#, 4#, 7#, 9#, 10#, 12#) is similar to that of normal people (2#, 5#, 6#, 8#). , 11#) is significantly lower (p<0.05).

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present disclosure, not to limit it; although the present disclosure has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand: It is still possible to modify the technical solutions recorded in the foregoing embodiments, or equivalently replace some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the technical solutions of the embodiments of the present disclosure Range.

Industrial applicability

SOX11 mRNA or protein has high specificity and sensitivity for the diagnosis of schizophrenia, and is a peripheral blood index, which can be easily obtained; single index detection method requires only one index to be measured, which is convenient for future promotion. It can also be combined with other detection methods to get more reliable results.

Claims (20)

Application of SOX11 mRNA or protein quantitative detection agent in preparing reagents or kits for diagnosing schizophrenia. SOX11 mRNA or protein quantitative detection agent, used in the diagnosis of schizophrenia. The application according to claim 1 or the quantitative detection agent for application according to claim 2, wherein the schizophrenia is violent. The application or the quantitative detection agent for the application according to any one of claims 1 to 3, wherein the SOX11 mRNA quantitative detection agent includes a reagent suitable for at least one of the following methods: Real-time fluorescent quantitative PCR, digital PCR, fluorescent dye method, resonance light scattering method, sequencing or biological mass spectrometry. The application or the quantitative detection agent for the application according to any one of claims 1 to 4, wherein the quantitative detection agent for SOX11 mRNA is a probe or primer capable of specifically binding to SOX11 mRNA or SOX11 cDNA. The application or the quantitative detection agent for the application according to claim 5, wherein the probe or primer has a detectable label. The application or the quantitative detection agent for the application according to any one of claims 1 to 6, wherein the quantitative detection agent for SOX11 mRNA is a qRT-PCR primer of SOX11 mRNA, and its upstream primer is as SEQ ID NO :1, the downstream primer is shown in SEQ ID NO: 2. The application or the quantitative detection agent for the application according to any one of claims 1 to 7, wherein the quantitative detection agent for SOX11 protein comprises a lectin, an aptamer antibody, or an aptamer antibody capable of specifically binding to SOX11 protein One or more of antibody fragments; Preferably, the SOX11 protein quantitative detection agent is an antibody or an antibody fragment. The use according to any one of claims 1, 3 to 8, wherein the kit further includes one or more detection agents for schizophrenia markers. The application or the quantitative detection agent for the application according to claim 9, wherein the schizophrenia marker is selected from SNP, microsatellite, or indel polymorphism. The application according to any one of claims 1, 3 to 10, wherein the kit further includes reverse transcription reagents, blood extraction reagents, internal reference primers, fluorescent substances for indicating the amount of DNA synthesis, qPCR reaction One or more of buffer, dNTPs, DNA polymerase, and water. Methods of diagnosing schizophrenia include: Measure the expression of SOX11 mRNA or protein in the subject’s sample, and Analyze the measurement results of SOX11 mRNA or protein expression to diagnose schizophrenia. The method of claim 12, wherein the method comprises (a) Obtain samples containing SOX11 mRNA or protein of the subject; (b) Measure the expression level of SOX11 mRNA or protein in the sample, (c) Measuring the concentration of one or more other schizophrenia markers in the sample; and (d) Use the measurement result of step (b) and the measurement result of step (c) to diagnose schizophrenia. The method of claim 12, wherein the method comprises (i) Measure the expression of SOX11 mRNA or protein in the subject sample (ii) Measure SOX11 mRNA or protein expression in the control sample, and (iii) Compare the expression level of SOX11 mRNA or protein in the subject sample and the control sample, The expression level of SOX11 mRNA or protein in the subject sample is lower than the expression level of SOX11 mRNA or protein in the control sample is an indication of schizophrenia. The method according to claim 14, wherein the control sample is a sample from a healthy subject or a sample from a non-schizophrenic subject. Methods of judging the treatment effect of schizophrenia, including (1) Measure the expression of SOX11 mRNA or protein in the subject's sample before treatment; (2) Measure the expression level of SOX11 mRNA or protein in the subject's sample after treatment; and (3) Compare the expression level of SOX11 mRNA or protein in the subject sample before and after the treatment. The expression level of SOX11 mRNA or protein in the subject sample after the treatment is lower than the SOX11 mRNA or protein expression in the subject sample before the treatment. Or protein expression is an indication that schizophrenia continues to progress or treatment is not effective. The method according to any one of claims 12 to 16, wherein the subject is a mammal, preferably a primate, more preferably a human. The method according to any one of claims 12 to 17, wherein the sample is at least one of blood, serum, plasma, cerebrospinal fluid, tissue, tissue lysate, cell culture supernatant, semen or saliva sample Species, preferably peripheral blood. The method according to any one of claims 12 to 18, wherein the SOX11 mRNA measurement is performed by qRT-PCR using SOX11 mRNA primers, and the upstream primer of the SOX11 mRNA primer is as SEQ ID NO:1 As shown, the downstream primer is shown in SEQ ID NO: 2. The method according to any one of claims 12 to 19, wherein the schizophrenia is violent schizophrenia.

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