CN112226506A

CN112226506A - Idiopathic pulmonary fibrosis diagnosis marker CCT6A and application thereof in preparation of diagnosis or prognosis tool

Info

Publication number: CN112226506A
Application number: CN202011218095.6A
Authority: CN
Inventors: 王兰; 闫培硕; 余国营
Original assignee: Henan Normal University
Current assignee: Henan Normal University
Priority date: 2020-11-04
Filing date: 2020-11-04
Publication date: 2021-01-15

Abstract

The invention discloses a diagnostic marker CCT6A for idiopathic pulmonary fibrosis diseases and application thereof in preparation of a diagnostic or prognostic tool, belonging to the technical field of medical diagnostic markers and tools. According to the invention, clinical diagnosis is realized by detecting the expression of the CCT6A gene and the expression product thereof, the research discovers CCT6A with higher level in the serum of an IPF patient and discovers high-expression CCT6A in lung fibroblasts of the IPF patient, which shows that the CCT6A can regulate the activity of the fibroblasts in the pulmonary fibrosis process and further regulate the pulmonary fibrosis process. The research result of the invention provides a non-invasive method for clinically carrying out early diagnosis of idiopathic pulmonary fibrosis, and is suitable for further clinical popularization and application.

Description

Idiopathic pulmonary fibrosis diagnosis marker CCT6A and application thereof in preparation of diagnosis or prognosis tool

Technical Field

The invention belongs to the technical field of medical diagnosis markers and tools, and particularly relates to a diagnostic marker CCT6A for idiopathic pulmonary fibrosis diseases and application thereof in preparation of a diagnosis or prognosis tool.

Background

Pulmonary fibrosis is a chronic progressive fatal disease, the pathological features of which are destruction of lung parenchyma, significant changes in alveolar epithelial cell phenotype, deposition of extracellular matrix, and abnormal proliferation and accumulation of fibroblasts. It is now known that more than 200 causes of pulmonary fibrosis, such as genetic diseases, autoimmune diseases, exposure to toxic substances in the environment, drugs and radiation, etc. There is a growing body of evidence that not only inter-heterogeneity and intra-heterogeneity of tissue cells in an individual but also dynamic changes of the individual at various levels, such as cells and molecules, are widespread during the development of pulmonary fibrosis. By utilizing the technology of the multiomics, the mechanism of complex diseases can be better and comprehensively understood, disease markers can be screened, drug action targets can be researched, and special functional proteins can be screened. According to statistics, about 500 million new pulmonary fibrosis patients are added in the world every year, the prognosis is poor, and the median survival time after diagnosis is 2-3 years. The etiology of the pulmonary fibrosis is unclear, early diagnosis is easy to be confused, and the like, and means for diagnosing the pulmonary fibrosis in clinic often lacks specificity, so that more efficient and stable means are urgently needed for early warning or judging the pulmonary fibrosis. Therefore, the development of a method for accurately diagnosing idiopathic pulmonary fibrosis is a problem to be solved.

In eukaryotes, the protein encoded by CCT6A is a molecular chaperone protein, a member of chaperone proteins comprising the TCP1 complex (CCT) (also known as the TCP1 loop complex (try), comprising two identical stacked loops, each loop containing eight different homologous proteins (1-8 or α - θ), which folds a variety of proteins in an ATP-dependent manner, including actin and tubulin, studies have shown that 5% -10% of newly synthesized cytoplasmic proteins are folded by CCT, indicating its key regulatory role in cytoskeletal organization and cell cycle, studies have shown that CCT6A is significantly elevated in expression levels in a variety of human tumor cell lines, can regulate a variety of cell survival and apoptosis-related genes, such as transducin and transcriptional activator 3(STAT3), LOX-1, VHL, and p53, among others, CCT6A has been identified as a specific component of the endogenous extracellular signal-regulated kinase (ERK)1/2 signal complex, the complex has a phosphorylation site for mitogen-activated protein kinase (MAPK)/ERK.

Disclosure of Invention

The invention aims to provide a diagnostic marker CCT6A for idiopathic pulmonary fibrosis and application thereof in preparing a diagnostic or prognostic tool, and the invention discovers that the content of CCT6A in blood of patients with idiopathic pulmonary fibrosis is higher than that of normal people through experimental research, and the immunohistochemical result of lung slices of the patients with idiopathic pulmonary fibrosis shows that the expression level of CCT6A in fibroblasts is increased. The fibroblast plays an important role in synthesis, abnormal deposition and remodeling of ECM, is directly or indirectly involved in the formation of fibrosis, and the expression of CCT6A in patients with idiopathic pulmonary fibrosis is increased, which indicates that the CCT6A possibly plays an important regulatory role in the formation process of the pulmonary fibrosis, so that a tool for diagnosing idiopathic pulmonary fibrosis diseases can be developed.

The invention adopts the following technical scheme for realizing the aim, and the diagnostic marker CCT6A for the idiopathic pulmonary fibrosis is characterized in that the diagnostic marker is a CCT6A gene expression product, and a coding sequence of a CCT6A gene comprises any one of the following DNA molecules:

(1) a DNA sequence shown as SEQ ID NO.1 in the sequence table;

(2) a DNA sequence which hybridizes with the DNA sequence defined in (1) under strict conditions and codes the same functional protein;

(3) and (3) a DNA sequence which has more than 70% of homology with the DNA sequence defined in (1) or (2) and encodes the same functional protein.

Preferably, the CCT6A gene expression product comprises a CCT6A protein, a functional equivalent of the CCT6A protein, and a partial peptide of the CCT6A protein, wherein the partial peptide of the CCT6A protein contains a functional domain associated with idiopathic pulmonary fibrosis; functional equivalents of the CCT6A protein include proteins encoded by a CCT6A protein conservative variant protein, or an active fragment thereof, or an active derivative, an allelic variant, a natural mutant, an induced mutant, a DNA that hybridizes to the DNA of CCT6A under high or low stringency conditions.

Preferably, the CCT6A protein is a protein having the following amino acid sequence:

(1) a protein consisting of an amino acid sequence shown by SEQ ID NO.2 in a sequence table;

(2) a protein which is derived from the amino acid sequence shown in SEQ ID NO.2 and has the same function with the amino acid sequence shown in SEQ ID NO.2 after the substitution and/or deletion and/or addition of one or more amino acid residues on the amino acid sequence shown in SEQ ID NO.2, wherein the number of the substituted, deleted or added amino acids is usually 1-50;

(3) and polypeptides consisting of amino acid sequences with at least 80 percent of homology with the amino acid sequence shown in SEQ ID NO.2, also called polypeptides with sequence identity.

Preferably, the sources of the CCT6A gene and its expression product include, but are not limited to, blood, interstitial fluid, urine, saliva, lung tissue, bronchoalveolar lavage samples or exhaled breath condensate or spinal fluid.

The application of the idiopathic pulmonary fibrosis diagnosis marker CCT6A in preparing a diagnosis or prognosis tool is characterized in that: the diagnosis or prognosis tool comprises a product for diagnosing idiopathic pulmonary fibrosis by detecting the expression level of CCT6A gene through RT-PCR, real-time quantitative PCR, immunohistochemistry, in-situ hybridization, a chip, a high-throughput sequencing platform or LC-MS/MS mass spectrum, wherein CCT6A with higher level is found in the serum of an IPF patient, and CCT6A with high expression is found in lung fibroblasts of the IPF patient, which shows that the CCT6A can regulate the activity of the fibroblasts in the process of pulmonary fibrosis, and further regulates the process of pulmonary fibrosis.

Preferably, the product for diagnosing idiopathic pulmonary fibrosis by RT-PCR at least comprises a pair of primers for specifically amplifying CCT6A gene; the product for diagnosing the idiopathic pulmonary fibrosis by real-time quantitative PCR at least comprises a pair of primers for specifically amplifying CCT6A gene; the product for diagnosing idiopathic pulmonary fibrosis by immunodetection comprises an antibody specifically binding to an expression product of CCT6A gene; the product for diagnosing idiopathic pulmonary fibrosis by in situ hybridization comprises a probe hybridized with a nucleic acid sequence of the CCT6A gene; the product for diagnosing idiopathic pulmonary fibrosis by the chip comprises a protein chip and a gene chip, wherein the protein chip comprises an antibody specifically bound with CCT6A protein, and the gene chip comprises a probe hybridized with a nucleic acid sequence of CCT6A gene.

Preferably, the product for diagnosing idiopathic pulmonary fibrosis through real-time quantitative PCR at least comprises a pair of primers for specifically amplifying CCT6A gene, wherein the forward primer sequence in the primer sequences is shown as SEQ ID NO. 3-SEQ ID NO.7, and the corresponding reverse primer sequence is shown as SEQ ID NO. 9-SEQ ID NO. 13.

Preferably, the tool is used for diagnosing idiopathic pulmonary fibrosis by detecting the expression of CCT6A gene in a sample, and the tool comprises a chip, a kit, a test paper or a high-throughput sequencing platform.

Preferably, the chip comprises a gene chip or a protein chip, the gene chip comprises a solid phase carrier and oligonucleotide probes fixed on the solid phase carrier, the oligonucleotide probes comprise oligonucleotide probes for detecting the transcription level of the CCT6A gene and aiming at the CCT6A gene, and the gene chip is used for detecting the expression levels of a plurality of genes including the CCT6A gene; the protein chip comprises a solid phase carrier and an antibody specific to the CCT6A protein fixed on the solid phase carrier, and can be used for detecting the expression level of a plurality of proteins including the CCT6A protein; the kit comprises a gene detection kit, an enzyme-linked immunosorbent assay kit or a protein immunodetection kit, wherein the gene detection kit comprises a reagent for detecting the transcription level of CCT6A gene, the enzyme-linked immunosorbent assay kit comprises a binding antibody and a reagent for detecting the antibody, and the protein immunodetection kit comprises a specific antibody of CCT6A protein; the test paper comprises reagents required in the process of detecting the expression level of the CCT6A gene by using RT-PCR, real-time quantitative PCR, immunodetection, in-situ hybridization or a chip method; the high-throughput sequencing platform comprises test paper for detecting the transcription level of the CCT6A gene, the test paper comprises a test paper carrier and oligonucleotide fixed on the test paper carrier, the oligonucleotide can detect the transcription level of the CCT6A gene, a probe hybridized with a nucleic acid sequence of the CCT6A gene is DNA, RNA, a DNA-RNA chimera, PNA or other derivatives, and the probe can complete specific hybridization and be specifically combined with a target nucleotide sequence.

Preferably, the antibody specific to the CCT6A protein comprises a monoclonal antibody or a polyclonal antibody, and the antibody specific to the CCT6A protein comprises a complete antibody molecule, any fragment of an antibody or a modification.

Compared with the prior art, the invention has the following beneficial effects: the invention finds that the protein coded by CCT6A is molecular chaperone protein and has key action in key regulation and control of cytoskeleton tissues and cell cycles, and the invention realizes clinical diagnosis by detecting the expression of CCT6A gene and expression products thereof. In the research, CCT6A with a higher level is found in serum of an IPF patient, and CCT6A with high expression is found in lung fibroblasts of the IPF patient, which shows that CCT6A can regulate the activity of the fibroblasts in the process of pulmonary fibrosis and further regulate the process of the pulmonary fibrosis. The research result of the invention provides a non-invasive method for clinically carrying out early diagnosis of idiopathic pulmonary fibrosis, and is suitable for further clinical popularization and application.

Drawings

FIG. 1 is a graph of the differential expression of CCT6A protein compared to normal human using LC-MS/MS mass spectrometry to detect idiopathic pulmonary fibrosis;

FIG. 2 is a graph showing the differential expression of the CCT6A protein in comparison to normal humans in detection of idiopathic pulmonary fibrosis using ELISA;

FIG. 3 is a graph of the differential expression of the CCT6A gene in comparison to normal humans using QPCR in the detection of idiopathic pulmonary fibrosis;

fig. 4 is a graph of the differential expression of the CCT6A protein in detecting idiopathic pulmonary fibrosis compared to normal humans using immunohistochemistry.

Detailed Description

The present invention is described in further detail below with reference to examples, but it should not be construed that the scope of the above subject matter of the present invention is limited to the following examples, and that all the technologies realized based on the above subject matter of the present invention belong to the scope of the present invention.

The experimental research of the invention finds that the content of CCT6A in the blood of patients with idiopathic pulmonary fibrosis is higher than that of normal people, and the immunohistochemical result of lung sections of the patients with idiopathic pulmonary fibrosis shows that the expression level of CCT6A in fibroblasts is increased. The fibroblast plays an important role in synthesis, abnormal deposition and remodeling of ECM, is directly or indirectly involved in the formation of fibrosis, and the expression of CCT6A in patients with idiopathic pulmonary fibrosis is increased, which indicates that the CCT6A possibly plays an important regulatory role in the formation process of the pulmonary fibrosis, so that a tool for diagnosing idiopathic pulmonary fibrosis diseases can be developed.

According to one aspect of the invention, the invention provides application of a product for detecting CCT6A gene expression in preparing a tool for diagnosing idiopathic pulmonary fibrosis.

Further, the above-mentioned test products include: the expression level of the CCT6A protein is detected by RT-PCR, real-time quantitative PCR, immunohistochemistry, in situ hybridization, chip, ELISA or high-throughput sequencing platform, LC-MS/MS mass spectrum to diagnose the product of idiopathic pulmonary fibrosis.

Further, the product for diagnosing idiopathic pulmonary fibrosis by using RT-PCR at least comprises a pair of primers for specifically amplifying CCT6A gene; the product for diagnosing idiopathic pulmonary fibrosis by using real-time quantitative PCR at least comprises a pair of primers for specifically amplifying CCT6A gene; the product for the immunohistochemical diagnosis of idiopathic pulmonary fibrosis comprises: an antibody that specifically binds to a CCT6A protein; the product for diagnosing idiopathic pulmonary fibrosis by in situ hybridization comprises: a probe that hybridizes to a nucleic acid sequence of the CCT6A gene; the product for diagnosing idiopathic pulmonary fibrosis by using the chip comprises: protein chips and gene chips; wherein, the protein chip comprises an antibody which is specifically combined with the CCT6A protein, and the gene chip comprises a probe which is hybridized with the nucleic acid sequence of the CCT6A gene.

In a specific embodiment of the invention, the product for diagnosing idiopathic pulmonary fibrosis by using real-time quantitative PCR at least comprises a pair of primers for specifically amplifying CCT6A gene, wherein the sequences of the primers are shown as SEQ ID NO. 3-SEQ ID NO.7 and SEQ ID NO. 9-SEQ ID NO. 13.

The tool of the invention comprises a chip, a kit, test paper or a high-throughput sequencing platform. The high-throughput sequencing platform is a special diagnostic tool, and a product for detecting CCT6A gene expression can be applied to the platform to realize the detection of the expression condition of the CCT6A gene. With the development of high-throughput sequencing technology, the construction of a gene expression profile of a person becomes very convenient work. By comparing the gene expression profiles of patients with diseases and normal people, the abnormality of which gene is related to the disease can be easily analyzed. Therefore, the knowledge that the abnormality of the CCT6A gene is related to idiopathic pulmonary fibrosis in high-throughput sequencing also belongs to the use of the CCT6A gene, and is also within the protection scope of the present invention.

According to another aspect of the present invention, the present invention also provides a tool for diagnosing idiopathic pulmonary fibrosis, wherein the product comprises a chip, a kit, a test strip, or a high throughput sequencing/mass spectrometry platform.

Wherein, the chip comprises a gene chip and a protein chip; the gene chip comprises a solid phase carrier and oligonucleotide probes fixed on the solid phase carrier, wherein the oligonucleotide probes comprise oligonucleotide probes for detecting the transcription level of a CCT6A gene and aiming at a CCT6A gene; the protein chip comprises a solid phase carrier and a specific antibody of CCT6A protein fixed on the solid phase carrier; the gene chip can be used for detecting the expression levels of a plurality of genes including the CCT6A gene (for example, a plurality of genes related to idiopathic pulmonary fibrosis). The protein chip can be used for detecting the expression level of a plurality of proteins including CCT6A protein (such as a plurality of proteins related to idiopathic pulmonary fibrosis). By simultaneously detecting a plurality of markers with the idiopathic pulmonary fibrosis, the accuracy of diagnosing the idiopathic pulmonary fibrosis can be greatly improved.

Wherein, the kit comprises a gene detection kit, an enzyme-linked immunosorbent assay kit and a protein immunodetection kit; the gene detection kit comprises a reagent for detecting the transcription level of the CCT6A gene; the enzyme-linked immunosorbent assay kit comprises a binding antibody and a reagent for detecting the antibody; the protein immunoassay kit comprises a specific antibody of the CCT6A protein. Further, the reagents include reagents required in the process of detecting the expression level of the CCT6A gene using RT-PCR, real-time quantitative PCR, immunodetection, in situ hybridization or a chip method. Preferably, the reagents comprise primers and/or probes for the CCT6A gene. Primers and probes which can be used for detecting the expression level of the CCT6A gene are easily designed according to the nucleotide sequence information of the CCT6A gene.

Further, the high-throughput sequencing platform comprises a reagent for detecting the expression level of the CCT6A gene. The test paper comprises a test paper carrier and oligonucleotides fixed on the test paper carrier, wherein the oligonucleotides can detect the transcription level of the CCT6A gene. The probe that hybridizes to the nucleic acid sequence of the CCT6A gene may be DNA, RNA, a DNA-RNA chimera, PNA, or other derivatives. The length of the probe is not limited, and any length may be used as long as specific hybridization and specific binding to the target nucleotide sequence are achieved. The length of the probe may be as short as 25, 20, 15, 13 or 10 bases in length. Also, the length of the probe can be as long as 60, 80, 100, 150, 300 base pairs or more, even for the entire gene. Since different probe lengths have different effects on hybridization efficiency and signal specificity, the length of the probe is usually at least 14 base pairs, and at most, usually not more than 30 base pairs, and the length complementary to the nucleotide sequence of interest is optimally 15 to 25 base pairs. The probe self-complementary sequence is preferably less than 4 base pairs so as not to affect hybridization efficiency.

Further, the specific antibody of the CCT6A protein comprises a monoclonal antibody and a polyclonal antibody. The specific antibody of the CCT6A protein comprises a complete antibody molecule, any fragment or modification of an antibody. So long as the fragment can retain the binding ability to the CCT6A protein. The preparation of antibodies for use at the protein level is well known to those skilled in the art and any method may be used in the present invention to prepare such antibodies.

In a specific embodiment of the invention, the primer sequence aiming at the CCT6A gene is shown as SEQ ID NO. 3-SEQ ID NO. 14.

Sources of the CCT6A gene and its expression product for diagnosing idiopathic pulmonary fibrosis include, but are not limited to, blood, interstitial fluid, urine, saliva, lung tissue, bronchoalveolar lavage samples or exhaled breath condensate, spinal fluid and other body fluids. In a specific embodiment of the present invention, the source of the CCT6A gene and its expression product for diagnosing idiopathic pulmonary fibrosis is blood. In a particular embodiment of the invention, the blood is peripheral blood taken from patients with idiopathic pulmonary fibrosis and from normal persons.

The specific sequence of the CCT6A gene (NM-001009186.2) of the invention can be queried in the International public nucleic acid sequence database GeneBank.

The coding sequence of the CCT6A gene comprises any one of the following DNA molecules:

(1) a DNA sequence shown as SEQ ID NO.1 in the sequence table;

(2) a DNA sequence which hybridizes with the DNA sequence defined in 1) under strict conditions and codes the same functional protein;

(3) and (3) a DNA molecule which has 70%, preferably 90% or more homology with the DNA sequence defined in (1) or (2) and encodes a protein having the same function.

In a specific embodiment of the invention, the coding sequence of the CCT6A gene is a DNA sequence shown in SEQ ID NO. 1.

In the context of the present invention, the CCT6A gene expression product includes a CCT6A protein and a partial peptide of the CCT6A protein. The partial peptide of the CCT6A protein contains functional domains related to idiopathic pulmonary fibrosis.

The CCT6A protein includes CCT6A protein and any functional equivalent of CCT6A protein. The functional equivalents comprise a CCT6A protein conservative variation protein, or an active fragment or an active derivative thereof, an allelic variant, a natural mutant, an induced mutant, and a protein coded by DNA capable of hybridizing with DNA of CCT6A under high or low stringency conditions.

The CCT6A protein is a protein with the following amino acid sequence:

(1) a protein consisting of an amino acid sequence shown by SEQ ID NO.2 in a sequence table.

(2) A protein which is derived from the amino acid sequence shown in SEQ ID NO.2, is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.2, has the same function with the amino acid sequence shown in SEQ ID NO. 2. The number of amino acids to be substituted, deleted or added is usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10.

(3) A polypeptide consisting of an amino acid sequence having at least 80% homology (also referred to as sequence identity) with the amino acid sequence shown in SEQ ID NO.2, more preferably at least about 90% to 95% homology, often 96%, 97%, 98%, 99% homology with the amino acid sequence shown in SEQ ID NO. 2.

In a specific embodiment of the invention, the CCT6A protein is a protein having an amino acid sequence shown in SEQ ID No. 2.

In general, it is known that modification of one or more amino acids in a protein does not affect the function of the protein.

One skilled in the art will recognize that individual amino acid changes or small percentage amino acids or individual additions, deletions, insertions, substitutions to an amino acid sequence are conservative modifications, wherein a change in a protein results in a protein with a similar function. Conservative substitution tables providing functionally similar amino acids are well known in the art.

An example of a protein modified by the addition of an amino acid or amino acid residues is a fusion protein of the CCT6A protein. There is no limitation on the peptide or protein fused with the CCT6A protein as long as the resulting fusion protein retains the biological activity of the CCT6A protein.

The CCT6A protein also comprises non-conservative modification of an amino acid sequence shown in SEQ ID NO.2, as long as the modified protein can still retain the biological activity of the CCT6A protein. The number of amino acids mutated in such modified proteins is typically 10 or less, such as 6 or less, such as 3 or less.

In the context of the present invention, diagnosing idiopathic pulmonary fibrosis includes both determining whether a subject has suffered from idiopathic pulmonary fibrosis and determining whether a subject is at risk of suffering from idiopathic pulmonary fibrosis, and predicting the prognosis of a subject with idiopathic pulmonary fibrosis.

Experimental methods without specific conditions noted in the examples, molecular cloning is generally performed according to conventional conditions: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations.

Example 1

Proteomic analysis using mass spectrometry for screening of differentially expressed genes in idiopathic pulmonary fibrosis patients and normal humans

1. Clinical study subjects:

30 patients with idiopathic pulmonary fibrosis are selected, wherein 17 men and 13 women have age in the range of 40-83 years, and the diagnosis standard meets the diagnosis and treatment standard of idiopathic pulmonary fibrosis revised in ATS year.

Exclusion criteria: (1) patients with other lung diseases such as bronchial asthma, pulmonary interstitial fibrosis, and lung cancer; (2) those with other site infections; (3) patients with serious cardiovascular and cerebrovascular diseases, diabetes, blood system diseases, malignant tumor, organ failure, and hepatitis; (4) those suffering from immune system diseases or who have recently used immunosuppressive agents.

Normal control: the method comprises selecting healthy people 30 for physical examination, wherein the healthy people are 18 men and 12 women, and have the age range of 52-76.

And (3) inclusion standard: no history of chronic cough, expectoration, asthma, etc.; there is no history of upper respiratory tract infection and lung infection recently; those without other systemic infections; those without other lung diseases; those who have not recently used immunosuppressive agents or who have no systemic immune system; patients without organ failure or severe cardiovascular and cerebrovascular diseases and tumors; those without allergic diseases. The selected subjects were excluded from the lung function test and compared with the idiopathic pulmonary fibrosis group, and the differences in gender and age were not statistically significant and comparable.

All subjects signed informed consent.

2. Sample collection

In the early morning on an empty stomach, 10mL of peripheral venous blood was collected and anticoagulated with EDTA.

Study subjects: 30 patients with idiopathic pulmonary fibrosis, 30 normal patients were selected according to the method of example 1.

3. Extracting blood total peptide, detecting mass spectrum, analyzing bioinformatics, and screening out differential protein. Based on the results of previous high throughput sequencing, we selected the CCT6A gene (whose expression is up-regulated in patients with idiopathic pulmonary fibrosis) according to the size of P value.

Example 2

Verification of differences in expression of gene mRNA in idiopathic pulmonary fibrosis patients and normal persons using QPCR

1. Study subjects:

as in example 1.

2. Blood Total RNA extraction

Extraction of total RNA from blood was performed using QIAGEN blood RNA extraction kit:

(1) mu.L (or 0.25g) of whole blood was applied to RNase-Free filter column, centrifuged at 13000rpm for 2 minutes, the bottom solution was collected, and 0.75mL of lysis buffer RLS was added.

(2) The homogenate was vigorously shaken and mixed, and incubated at 15-30 ℃ for 5 minutes to completely decompose the nucleoprotein body.

(3) Optional steps are as follows: centrifugation was carried out at 12000rpm for 10 minutes at 4 ℃ and the supernatant was carefully removed and transferred to a new RNase-free centrifuge tube.

(4) 0.2mL of chloroform was added per 1mL of RLS. The sample tube cap was closed, shaken vigorously for 15 seconds and incubated for 3 minutes at room temperature.

(5) After centrifugation at 12000rpm for 10 minutes at 4 ℃ the sample will separate into three layers: the lower organic phase, the middle layer and the upper colorless aqueous phase, RNA is present in the aqueous phase. The volume of the aqueous layer was about 60% of the volume of the added RLS, and the aqueous layer was transferred to a fresh tube for further processing.

(6) 70% ethanol is added in 1 volume, the mixture is inverted and mixed (precipitation may occur), and the resulting solution is transferred to an adsorption column RA (which is sleeved in a collection tube) together with the possible precipitation.

(7) Centrifuging at 10000rpm for 45 s, discarding the waste liquid, and sleeving the adsorption column back to the collection tube again.

(8) Add 500. mu.L deproteinized solution RE, centrifuge at 12000rpm for 45 seconds, and discard the waste liquid.

(9) 700. mu.L of the rinsing solution RW was added, and centrifuged at 12000rpm for 60 seconds, and the waste liquid was discarded.

(10) 500. mu.L of the rinsing solution RW was added, and centrifuged at 12000rpm for 60 seconds, and the waste liquid was discarded.

(11) The adsorption column RA was returned to the empty collection tube and centrifuged at 12000rpm for 2 minutes to remove the rinse as much as possible so as to prevent the residual ethanol in the rinse from inhibiting the downstream reaction.

(12) Taking out the adsorption column RA, placing into a centrifugal tube without RNase, adding 50-80 μ L of RNase-free water at the middle part of the adsorption membrane according to the expected RNA yield, standing at room temperature for 2 min, centrifuging at 12000rpm for 1 min, and collecting the eluate.

3. Determination of concentration and purity of RNA samples

The concentration and purity of the RNA were determined by Nanodrop2000 and RNA integrity was checked by agarose gel electrophoresis.

4. Reverse transcription to synthesize mRNA cDNA

cDNA was synthesized by reverse transcription of 1. mu.g of total RNA using reverse transcription buffer. A25-mu-L reaction system is adopted, 1 mu g of total RNA is taken as template RNA for each sample, and the following components are respectively added into a PCR tube: DEPC water, 5 Xreverse transcription buffer, 10mmol/L dNTP, 0.1mmol/L DTT, 30. mu. mmol/L Oligo dT, 200U/. mu. L M-MLV, template RNA. Incubate at 42 ℃ for 1h, 72 ℃ for 10min, and centrifuge briefly.

5、qPCR

(1) Primer design

The qPCR amplification primer is designed according to the coding sequences of CCT6A gene and beta-actin gene in Genbank and synthesized by Shanghai biological engineering technical service company. The specific primer sequences are as follows:

CCT6A gene:

the forward primer is 5'-GTGGCGATTCAGATAAAGG-3';

the reverse primer was 5'-AATGTGACAGAACGAGGGT-3'.

Beta-actin gene:

the forward primer is 5'-GTCACCAACTGGGACGACAT-3';

the reverse primer was 5'-TAGCAACGTACATGGCTGGG-3'.

(2) Amplification of

Reaction system: 25 μ L reaction, 3 parallel channels per sample. The following reaction system was prepared: 12.5 mu L of SYBR Green polymerase chain reaction system, 1 mu L of forward primer (5 mu M), 1 mu L of reverse primer (5 mu M), 2.0 mu L of template cDNA and 8.5 mu L of enzyme-free water; all operations were performed on ice.

And (3) amplification procedure: 95 ℃ 5min, (95 ℃ 5s, 60 ℃ 60s) 40 cycles. SYBR Green is used as a fluorescent marker, PCR reaction is carried out on a Light Cycler fluorescent real-time quantitative PCR instrument, a target band is determined through melting curve analysis and electrophoresis, and relative quantification is carried out through a delta CT method.

6. Statistical method

The experiments were performed in 3 replicates, the results were expressed as mean ± sd, statistically analyzed using SPSS13.0 statistical software, and the differences between the different groups were determined by t-test to be statistically significant when P < 0.05.

Example 3

ELISA verification of CCT6A expression in idiopathic pulmonary fibrosis patients

1. Study subjects:

as in example 1.

2. Coating the capture antibody:

the antibody was diluted to 1-10ug/mL with buffer. 0.1mL was added to the reaction well overnight at 4 ℃. The next day, the well solutions were discarded and washed 3 times with wash buffer.

3. Loading and washing:

adding 50uL of a diluted sample to be detected into the coated reaction hole, and incubating for 1 hour at 37 ℃. Then washed (blank wells, negative control wells and positive control wells are done simultaneously).

4. Adding an enzyme-labeled antibody:

to each reaction well, 50uL of a freshly diluted enzyme-labeled antibody (dilution after titration) was added. Incubating for 0.5-1 hour at 37 ℃, and washing.

5. Adding a substrate solution for color development:

0.1mL of a TMB substrate solution prepared temporarily is added into each reaction hole, and the reaction is carried out at 37 ℃ for 10-30 minutes.

6. And (3) terminating the reaction:

0.05mL of 2M sulfuric acid was added to each reaction well.

7. OD value measurement:

the OD value of each well was measured after zeroing with blank control wells at 450nm on a multifunctional microplate reader to compare the expression level of CCT6A in each sample.

Example 4

Immunohistochemical analysis of CCT6A expression in pulmonary tissue of patients with idiopathic pulmonary fibrosis

(1) The slices were placed in an oven at 60 ℃ and baked for 1 hour.

(2) Dewaxing and hydrating: xylene i, 10 minutes → xylene ii, 10 minutes → absolute ethanol i, 5 minutes → absolute ethanol ii, 5 minutes → 95% alcohol by volume fraction, 5 minutes → 90% alcohol by volume fraction, 5 minutes → 80% alcohol by volume fraction, 5 minutes → 70% alcohol by volume fraction, 5 minutes → distilled water, 5 minutes.

(3) Incubate with 3 wt% Triton-100 for 5min at room temperature and wash 3 times with PBS for 2 min each.

(4) Endogenous peroxidase blocking solution was added dropwise, incubated at room temperature for 10 minutes, and PBS was washed 3 times, 2 minutes each.

(5) The sections were immersed in citrate buffer, then placed in a microwave oven and heated to 95 ℃ and held at temperature for 10 minutes for antigen retrieval, the sections were removed at room temperature and washed 3 times with PBS for 5 minutes each.

(6) The immunostaining blocking solution was added dropwise and incubated at 37 ℃ for 20 minutes.

(7) Add appropriate concentration of primary antibody working solution dropwise and incubate at 37 ℃ for 2 hours or 4 ℃ overnight.

(8) PBS was washed three times for 3 minutes each.

(9) Add secondary antibody working solution and incubate at 37 ℃ for 30 min.

(10) PBS was washed three times for 3 minutes each.

(11) Horseradish peroxidase-labeled working solution was added and incubated at 37 ℃ for 20 minutes.

(12) PBS was washed three times for 3 minutes each.

(13) Adding DAB developing solution, developing at 37 deg.C for 5min or at room temperature for 10min, and stopping developing with distilled water.

(14) Hematoxylin counterstaining, tap water stop staining.

(15) And (3) dehydrating and transparency: and (3) sealing the chip by using neutral resin in the reverse order of the step (2).

(16) And observing the dyeing condition under a microscope, and selecting a visual field for photographing and analyzing.

The results as shown in fig. 1, the expression level of CCT6A protein in blood of patients with idiopathic pulmonary fibrosis is higher than that of normal persons (fig. 1) compared to normal persons; the ELISA results of fig. 2 further show that the expression level of CCT6A gene in blood of idiopathic pulmonary fibrosis patients is significantly increased, the difference is statistically significant (P <0.05), and the results are similar to proteomics experiments; figure 3 shows a significant increase in the mRNA level of the CCT6A gene in lung tissue of patients with idiopathic pulmonary fibrosis; immunohistochemistry results indicated a significant increase in lung fibroblasts in patients with idiopathic pulmonary fibrosis (figure 4).

The foregoing embodiments illustrate the principles, principal features and advantages of the invention, and it will be understood by those skilled in the art that the invention is not limited to the foregoing embodiments, which are merely illustrative of the principles of the invention, and that various changes and modifications may be made therein without departing from the scope of the principles of the invention.

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<400> 1

agaagacccg gatagttcct cccggccacg ccgcgccggc tctgggcact cagcatcgtt 60

tccttttcct ccgctggagc agctatggcg gcggtgaaga ccctgaaccc caaggccgag 120

gtggcccgag cgcaggcggc gctggcggtc aacatcagcg cagcgcgggg tctgcaggac 180

gtgctaagga ccaacctggg gcccaagggc accatgaaga tgctcgtttc tggcgctgga 240

gacatcaaac ttactaaaga cggcaatgtg ctgcttcacg aaatgggcct tcatcctaga 300

ataatcactg aaggatttga agctgcaaag gaaaaggccc ttcagttttt ggaagaagtc 360

aaagtaagca gagagatgga cagggaaaca cttatagatg tggccagaac atctcttcgt 420

actaaagttc atgctgaact tgcagatgtc ttaacagagg ctgtagtgga ctccattttg 480

gccattaaaa agcaagatga acctattgat ctcttcatga ttgagatcat ggagatgaaa 540

cataaatctg aaactgatac aagcttaatc agagggcttg ttttggacca cggagcacgg 600

catcctgata tgaagaaaag ggtggaggat gcatacatcc tcacttgtaa cgtgtcatta 660

gagtatgaga aaacagaagt gaattctggc tttttttaca agagtgcaga agagagagaa 720

aaactcgtga aagctgaaag aaaattcatt gaagataggg ttaaaaaaat aatagaactg 780

aaaaggaaag tctgtggcga ttcagataaa ggatttgttg ttattaatca aaagggaatt 840

gacccctttt ccttagatgc tctttcaaaa gaaggcatag ttgctctgcg cagagctaaa 900

aggagaaata tggagaggct gactcttgct tgtggtgggg tagccctgaa ttcttttgac 960

gacctaagtc ctgactgctt gggacatgca ggacttgtat atgagtatac attgggagaa 1020

gagaagttta cctttattga gaaatgtaac aaccctcgtt ctgtcacatt attgatcaaa 1080

ggaccaaata agcacacact cactcagatc aaagatgcag tgagggacgg cttgagggct 1140

gtcaaaaatg ctattgatga tggctgtgtg gttccaggtg ctggtgccgt ggaagtggca 1200

atggcagaag ccctgattaa acataagccc agtgtaaagg gcagggcaca gcttggagtc 1260

caagcatttg ctgatgcatt gctcattatt cccaaggttc ttgctcagaa ctctggtttt 1320

gaccttcagg aaacattagt taaaattcaa gcagaacatt cagaatcagg tcagcttgtg 1380

ggtgtggacc tgaacacagg tgagccaatg gtggcagcag aagtaggcgt atgggataac 1440

tattgtgtaa agaaacagct tcttcactcc tgcactgtga ttgccaccaa cattctcttg 1500

gttgatgaga tcatgcgagc tggaatgtct tctctgaaag gttgaattga agcttcctct 1560

gtatctgaat cttgaagact gcaaagtgat cctgaggatt acagctgtgg aatttttgtc 1620

caagcttcaa ataattttga aagaaatttt cccatatgaa aaaaggagag aacactggca 1680

tctgttgaaa tttggaagtt ctgaaattat agtattttta aaaattgcac tgaagtgtat 1740

acacataaag caggtctttt atccagtgaa caggatgttt tgctttagca gcagtgacat 1800

aaaattccat gttagataag catatgttac ttaccttgtt attaaatatt tcttgaaaag 1860

caaattttaa tggtttaatt ttatgtggac gtatgttaaa ttatccaact accctattgt 1920

taagcatttg gttttaaaat ttttatgcta atataaatgc tcaagtaatt taaaatattg 1980

aaagcatccc tgttggtata aatttctgag taaatgcatt ggatcagttg gactttgaac 2040

gcctttgaaa tggctttgct aaaatgctcc cgccacaaag ttgtaggaaa tgggaagagg 2100

agtcaactag aggcaaggga gttgagagag ctgcaactgt aaagggcaag aacaggcaga 2160

ggtaaaaaga tgatggaagg tgtggtgact aagggccacg gttattgggt gaaatttgag 2220

attgtaggcc aactgtattt tcaagcttct gaacttaggc aaaatattca tcgcaaagtc 2280

tctagcgtca tatttttctc acccaaatta cgtttccacg agattattta tatatagttg 2340

gtctatctct gcagtccttg aaggtgaagt tgtgtgttac taggctgtgt tttgggatgt 2400

cagcagtggc ctgaagtgag ttgtgcaata aatgttaagt tgaaacctc 2449

<210> 2

<211> 486

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 2

Met Ala Ala Val Lys Thr Leu Asn Pro Lys Ala Glu Val Ala Arg Ala

1 5 10 15

Gln Ala Ala Leu Ala Val Asn Ile Ser Ala Ala Arg Gly Leu Gln Asp

20 25 30

Val Leu Arg Thr Asn Leu Gly Pro Lys Gly Thr Met Lys Met Leu Val

35 40 45

Ser Gly Ala Gly Asp Ile Lys Leu Thr Lys Asp Gly Asn Val Leu Leu

50 55 60

His Glu Met Gly Leu His Pro Arg Ile Ile Thr Glu Gly Phe Glu Ala

65 70 75 80

Ala Lys Glu Lys Ala Leu Gln Phe Leu Glu Glu Val Lys Val Ser Arg

85 90 95

Glu Met Asp Arg Glu Thr Leu Ile Asp Val Ala Arg Thr Ser Leu Arg

100 105 110

Thr Lys Val His Ala Glu Leu Ala Asp Val Leu Thr Glu Ala Val Val

115 120 125

Asp Ser Ile Leu Ala Ile Lys Lys Gln Asp Glu Pro Ile Asp Leu Phe

130 135 140

Met Ile Glu Ile Met Glu Met Lys His Lys Ser Glu Thr Asp Thr Ser

145 150 155 160

Leu Ile Arg Gly Leu Val Leu Asp His Gly Ala Arg His Pro Asp Met

165 170 175

Lys Lys Arg Val Glu Asp Ala Tyr Ile Leu Thr Cys Asn Val Ser Leu

180 185 190

Glu Tyr Glu Lys Thr Glu Val Asn Ser Gly Phe Phe Tyr Lys Ser Ala

195 200 205

Glu Glu Arg Glu Lys Leu Val Lys Ala Glu Arg Lys Phe Ile Glu Asp

210 215 220

Arg Val Lys Lys Ile Ile Glu Leu Lys Arg Lys Val Cys Gly Asp Ser

225 230 235 240

Asp Lys Gly Phe Val Val Ile Asn Gln Lys Gly Ile Asp Pro Phe Ser

245 250 255

Leu Asp Ala Leu Ser Lys Glu Gly Ile Val Ala Leu Arg Arg Ala Lys

260 265 270

Arg Arg Asn Met Glu Arg Leu Thr Leu Ala Cys Gly Gly Val Ala Leu

275 280 285

Asn Ser Phe Asp Asp Leu Ser Pro Asp Cys Leu Gly His Ala Gly Leu

290 295 300

Val Tyr Glu Tyr Thr Leu Gly Glu Glu Lys Phe Thr Phe Ile Glu Lys

305 310 315 320

Cys Asn Asn Pro Arg Ser Val Thr Leu Leu Ile Lys Gly Pro Asn Lys

325 330 335

His Thr Leu Thr Gln Ile Lys Asp Ala Val Arg Asp Gly Leu Arg Ala

340 345 350

Val Lys Asn Ala Ile Asp Asp Gly Cys Val Val Pro Gly Ala Gly Ala

355 360 365

Val Glu Val Ala Met Ala Glu Ala Leu Ile Lys His Lys Pro Ser Val

370 375 380

Lys Gly Arg Ala Gln Leu Gly Val Gln Ala Phe Ala Asp Ala Leu Leu

385 390 395 400

Ile Ile Pro Lys Val Leu Ala Gln Asn Ser Gly Phe Asp Leu Gln Glu

405 410 415

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

420 425 430

Gly Val Asp Leu Asn Thr Gly Glu Pro Met Val Ala Ala Glu Val Gly

435 440 445

Val Trp Asp Asn Tyr Cys Val Lys Lys Gln Leu Leu His Ser Cys Thr

450 455 460

Val Ile Ala Thr Asn Ile Leu Leu Val Asp Glu Ile Met Arg Ala Gly

465 470 475 480

Met Ser Ser Leu Lys Gly

485

<210> 3

<211> 19

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 3

gtggcgattc agataaagg 19

<210> 4

<211> 19

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 4

caaccctcgt tctgtcaca 19

<210> 5

<211> 18

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 5

cctgactgct tgggacat 18

<210> 6

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 6

ctgtggcgat tcagataaag 20

<210> 7

<211> 18

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 7

agatgctcgt ttctggcg 18

<210> 8

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 8

gtcaccaact gggacgacat 20

<210> 9

<211> 19

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 9

aatgtgacag aacgagggt 19

<210> 10

<211> 21

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 10

gttatcccat acgcctactt c 21

<210> 11

<211> 17

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 11

ccaccattgg ctcacct 17

<210> 12

<211> 21

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 12

ataatgtgac agaacgaggg t 21

<210> 13

<211> 21

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 13

cgataagtgt ttccctgtcc a 21

<210> 14

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 14

tagcaacgta catggctggg 20

Claims

1. The diagnostic marker CCT6A for idiopathic pulmonary fibrosis is CCT6A gene expression product, and the coding sequence of CCT6A gene includes any one of the following DNA molecules:

(1) a DNA sequence shown as SEQ ID NO.1 in the sequence table;

2. The idiopathic pulmonary fibrosis disease diagnostic marker CCT6A of claim 1, characterized in that: the CCT6A gene expression product comprises a CCT6A protein, a functional equivalent of the CCT6A protein and a partial peptide of the CCT6A protein, wherein the partial peptide of the CCT6A protein contains a functional domain related to idiopathic pulmonary fibrosis; functional equivalents of the CCT6A protein include proteins encoded by a CCT6A protein conservative variant protein, or an active fragment thereof, or an active derivative, an allelic variant, a natural mutant, an induced mutant, a DNA that hybridizes to the DNA of CCT6A under high or low stringency conditions.

3. The diagnostic marker of idiopathic pulmonary fibrosis CCT6A of claim 2, characterized in that the CCT6A protein is a protein having the amino acid sequence:

4. The idiopathic pulmonary fibrosis disease diagnostic marker CCT6A of claim 1, characterized in that: sources of the CCT6A gene and its expression product include, but are not limited to, blood, interstitial fluid, urine, saliva, lung tissue, bronchoalveolar lavage samples, or exhaled breath condensate or spinal fluid.

5. The use of the idiopathic pulmonary fibrosis disease diagnostic marker CCT6A of any one of claims 1-4 in the preparation of a diagnostic or prognostic tool, wherein: the diagnosis or prognosis tool comprises a product for diagnosing idiopathic pulmonary fibrosis by detecting the expression level of CCT6A gene through RT-PCR, real-time quantitative PCR, immunohistochemistry, in-situ hybridization, a chip, a high-throughput sequencing platform or LC-MS/MS mass spectrum, wherein CCT6A with higher level is found in the serum of an IPF patient, and CCT6A with high expression is found in lung fibroblasts of the IPF patient, which shows that the CCT6A can regulate the activity of the fibroblasts in the process of pulmonary fibrosis, and further regulates the process of pulmonary fibrosis.

6. Use according to claim 5, characterized in that: the product for diagnosing idiopathic pulmonary fibrosis by RT-PCR at least comprises a pair of primers for specifically amplifying CCT6A gene; the product for diagnosing the idiopathic pulmonary fibrosis by real-time quantitative PCR at least comprises a pair of primers for specifically amplifying CCT6A gene; the product for diagnosing idiopathic pulmonary fibrosis by immunodetection comprises an antibody specifically binding to an expression product of CCT6A gene; the product for diagnosing idiopathic pulmonary fibrosis by in situ hybridization comprises a probe hybridized with a nucleic acid sequence of the CCT6A gene; the product for diagnosing idiopathic pulmonary fibrosis by the chip comprises a protein chip and a gene chip, wherein the protein chip comprises an antibody specifically bound with CCT6A protein, and the gene chip comprises a probe hybridized with a nucleic acid sequence of CCT6A gene.

7. Use according to claim 5, characterized in that: the product for diagnosing idiopathic pulmonary fibrosis through real-time quantitative PCR at least comprises a pair of primers for specifically amplifying CCT6A genes, wherein the forward primer sequence in the primer sequences is shown as SEQ ID NO. 3-SEQ ID NO.7, and the corresponding reverse primer sequence is shown as SEQ ID NO. 9-SEQ ID NO. 13.

8. Use according to claim 5, characterized in that: the tool is used for diagnosing idiopathic pulmonary fibrosis by detecting the expression of CCT6A gene in a sample, and comprises a chip, a kit, test paper or a high-throughput sequencing platform.

9. Use according to claim 8, characterized in that: the chip comprises a gene chip or a protein chip, the gene chip comprises a solid phase carrier and an oligonucleotide probe fixed on the solid phase carrier, the oligonucleotide probe comprises an oligonucleotide probe aiming at a CCT6A gene and used for detecting the transcription level of the CCT6A gene, and the gene chip is used for detecting the expression level of a plurality of genes including the CCT6A gene; the protein chip comprises a solid phase carrier and an antibody specific to the CCT6A protein fixed on the solid phase carrier, and can be used for detecting the expression level of a plurality of proteins including the CCT6A protein; the kit comprises a gene detection kit, an enzyme-linked immunosorbent assay kit or a protein immunodetection kit, wherein the gene detection kit comprises a reagent for detecting the transcription level of CCT6A gene, the enzyme-linked immunosorbent assay kit comprises a binding antibody and a reagent for detecting the antibody, and the protein immunodetection kit comprises a specific antibody of CCT6A protein; the test paper comprises reagents required in the process of detecting the expression level of the CCT6A gene by using RT-PCR, real-time quantitative PCR, immunodetection, in-situ hybridization or a chip method; the high-throughput sequencing platform comprises test paper for detecting the transcription level of the CCT6A gene, the test paper comprises a test paper carrier and oligonucleotide fixed on the test paper carrier, the oligonucleotide can detect the transcription level of the CCT6A gene, a probe hybridized with a nucleic acid sequence of the CCT6A gene is DNA, RNA, a DNA-RNA chimera, PNA or other derivatives, and the probe can complete specific hybridization and be specifically combined with a target nucleotide sequence.

10. The idiopathic pulmonary fibrosis disease diagnostic marker CCT6A of claim 2, characterized in that: the specific antibody of the CCT6A protein comprises a monoclonal antibody or a polyclonal antibody, and the specific antibody of the CCT6A protein comprises a complete antibody molecule, any fragment or modification of an antibody.