CN103074425B - Uses of the CD263 gene - Google Patents

Abstract

The invention provides a CD263 gene application, and relates to the preparation of products to distinguish latent tuberculosis infection and active tuberculosis. The preferred products include the products utilizing real-time quantitative PCR or gene chip detection to distinguish the latent tuberculosis infection and the active tuberculosis. According to the experimental results, the expression of the CD236 gene is obviously higher in the blood of tuberculosis patients than in healthy people or latent infection crowds, and therefore, the CD263 gene can serve as a special marking gene for the diagnosis of tuberculosis, so as to allow the tuberculosis diagnosis to be more accurate and quicker.

Description

Uses of the CD263 gene

Technical field:

The invention relates to the technical field of bioengineering, in particular to the application of CD263 gene.

Background technique:

Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis infection. Mycobacterium tuberculosis can not only cause pulmonary tuberculosis (85%), but also cause tuberculosis in multiple organs outside the lungs. Although there are currently effective anti-tuberculosis drugs, tuberculosis is still the number one killer of infectious diseases, and about 2 million people die from tuberculosis every year in the world; about one-third of the world's population is infected with Mycobacterium tuberculosis, which is called About 10% of people with latent tuberculosis infection (LTBI) will eventually develop active tuberculosis.

Due to the lack of effective tuberculosis vaccine at present, the prevention and control of tuberculosis mainly depends on early detection, treatment and isolation of active tuberculosis patients. However, the current detection technology for diagnosing active tuberculosis has serious deficiencies and cannot meet the requirements of clinical and tuberculosis prevention and control: 1) Microbiological examination of sputum Mycobacterium tuberculosis has high specificity and is currently the gold standard for diagnosing active tuberculosis, but there are sensitive Low efficiency (less than 40%), long time-consuming (1-2 months for tuberculosis culture), and high requirements for laboratory biosafety. 2) Although the detection of Mycobacterium tuberculosis gene has achieved the purpose of rapid diagnosis (1 day), the sensitivity of genetic detection directly from sputum samples has not been significantly improved, and there are problems of false negatives and false positives. 3) Antibody detection in immunological testing is considered unsuitable for the diagnosis of tuberculosis by the World Health Organization; cellular immunological testing includes tuberculin skin test (TST) and tuberculosis interferon release test (IGRA), which cannot effectively distinguish patients with active tuberculosis and latent tuberculosis infection, although the sensitivity of the latter in patients with active tuberculosis was significantly higher than that of other tests.

CD263, also called TRAIL/Apo-2L, DCR1, DCR1-TNFR. It encodes a decoy receptor, whose expression can protect normal cells from TRAIL's killing effect and regulate TRAIL-induced apoptosis. Its protective effect on cells is to block the apoptosis induced by death receptors by participating in competitive binding to TRAIL. Studies have found that CD263 plays an important role in tumorigenesis. There is no report about CD263 gene as a diagnostic marker for tuberculosis.

Invention content:

The technical problem to be solved by the present invention is to provide a use of CD263 gene, which can be used as a molecular marker for distinguishing tuberculosis latent infection and active tuberculosis.

In order to solve the problems of the technologies described above, the present invention is realized through the following technical solutions:

The invention provides an application of CD263 gene for preparing a product for distinguishing tuberculosis latent infection and active tuberculosis.

The product for distinguishing latent tuberculosis infection and active tuberculosis preferably includes: a product for distinguishing latent tuberculosis infection and active tuberculosis by real-time quantitative PCR or gene chip detection.

The product for distinguishing tuberculosis latent infection and active tuberculosis by real-time quantitative PCR preferably includes at least one pair of primers for specifically amplifying CD263 gene.

The primers for specifically amplifying the CD263 gene are preferably:

CD263-F: 5'-AAGTTCGTCGTCGTCATCGT-3',

CD263-R: 5'-TTGAAGCTGTGCCTCTGTTG-3';

The product for detecting and distinguishing tuberculosis latent infection and active tuberculosis by gene chip preferably includes: a probe hybridized with the nucleic acid sequence of CD263 gene.

The kit of the invention can detect the expression of the CD263 gene of the patient, thereby diagnosing whether the patient suffers from active tuberculosis.

In the present invention, the term "primer" refers to an oligonucleotide, which can be natural or synthetic, and it can be used as a starting point for inducing DNA synthesis under certain conditions, and can induce synthesis and nucleic acid under appropriate conditions. Primer amplification products of strand complementarity, that is, in the presence of four different deoxyribonucleoside triphosphates and a polymerization reagent (i.e., DNA polymerase or reverse transcriptase), in a suitable buffer and in a suitable temperature for the amplification reaction. Preferred primers are single-stranded oligodeoxyribonucleotides. The suitable length of the primer depends on the designed use of the primer, but generally it is between 15-25 nucleotides.

In the present invention, the probe can be DNA, RNA, DNA-RNA chimera, PNA or other derivatives. The length of the probe is not limited, as long as it completes specific hybridization and specifically binds to the target nucleotide sequence, any length is acceptable.

Experiments have proved that the expression of the CD263 gene in the blood of tuberculosis patients of the present invention is significantly higher than that of healthy people and latent infected people, so the CD263 gene can be used as a specific marker gene for diagnosing tuberculosis, making the diagnosis of tuberculosis more accurate and rapid.

Description of drawings:

The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

Fig. 1 is a graph showing quantitative RT-PCR results of differential expression of CD263 gene in Example 1 of the present invention in human peripheral blood mononuclear cells (PBMC).

Fig. 2 is a microarray result graph showing the differential expression of the CD263 gene in PBMC according to Example 2 of the present invention.

Detailed ways:

Below in conjunction with embodiment and accompanying drawing, the present invention is further described:

Example 1

In this embodiment, the population is divided into three groups: tuberculosis patients, latent infection population and healthy population (20 cases each). By detecting the change of CD263 gene mRNA in each peripheral blood mononuclear cell (PBMC), it is found that it is present in tuberculosis patients. A clear trend of up-regulated expression.

In this example, quantitative RT-PCR method was used to detect the expression changes of CD263 gene in each case. Specific steps are as follows:

Step 1: Preparation of Peripheral Blood Mononuclear Cell (PBMC) Suspension

Add 5ml of Lymphocyte Separation Solution (Fresenius Kabi NOrgeAs: LYS3773) into the centrifuge tube; take 2ml of heparin anticoagulated venous blood from the above-mentioned confirmed tuberculosis patients, patients with latent infection and healthy people, and mix with an equivalent amount of 1M phosphate buffer solution (PBS ) to mix well to obtain a mixed solution, use a pipette to slowly superimpose the mixed solution on the surface of the lymphocyte separation solution along the tube wall, keep a clear interface, and centrifuge at 2000 rpm for 20 minutes; use a straw to absorb the middle cloud layer and put it into Then add 5 times the volume of 1M PBS to another centrifuge tube, centrifuge at 1500 rpm for 10 minutes to wash the cells, discard the supernatant, wash the cells again under the same conditions, and then add RPMI1640 containing 10% calf serum by volume ( Thermoscientific: SH30807.01b) 1ml, resuspended cells, and obtained 20 cases of PBMC suspension from each of the three groups of people; each case took 20 μl of PBMC suspension on a hemocytometer, and counted the cell concentration.

Step 2: RNA extraction

RNA was extracted from 20 cases of PBMC suspensions from each of the above-mentioned three groups of people using RNeasy Mini Kit from Qiagene Company (Catalog No. 74106). The specific operation is: take the above-mentioned PBMC suspension containing 1× ¹⁰⁶ cells in a centrifuge tube free of DNase and RNase, centrifuge at 3000 rpm for 10 minutes, discard the supernatant; add 350 μl Buffer RLT to the cell pellet, Mix well to lyse; add 250 μl of absolute ethanol, mix well, transfer the liquid to the RNeasy column, centrifuge at 8,000g for 30 seconds, discard the waste liquid; add 350 μl Buffer RW1 and centrifuge at 8,000g for 30 seconds, discard the waste liquid; add 80 μl DNase Solution (10μl DNase+70μl BufferRDD), digest on column for 15min, centrifuge at 8,000g for 30 seconds, discard the waste; add 350μl BufferRW1, centrifuge at 8,000g for 30 seconds, discard the waste; add 500μl Buffer RPE, centrifuge at 8,000g for 30 seconds, discard Waste liquid; spin empty, centrifuge at 8,000g for 1 minute; transfer the column to a 1.5ml centrifuge tube to remove DNase and RNase, add 40μl RNase-free ddH ₂ O, centrifuge at 10,000g for 1 minute, and collect each of the three groups of people The RNA of 20 cases was stored at -80°C until use.

Step 3: Reverse transcription

Using TAKARA's reverse transcription kit (DRR047), take 0.5 μg of RNA obtained in step 2 for reverse transcription. Compared with traditional reverse transcription kits, this kit adds a step of removing genomic DNA, which can guarantee RNA purity and specificity of amplification. The steps are as follows:

(1) Genomic DNA removal reaction

Table 1

Reagent Usage amount 5×gDNA Eraser Buffer 2μl gDNA Eraser 1μl total RNA 0.5μg RNase Free ddH ₂ O Make up to 10μl

After preparing the reaction system according to Table 1, incubate at 42°C for 2 minutes and store at 4°C.

(2) Reverse transcription reaction

The preparation of the reaction system was carried out on ice, and the specific system was as follows:

Table 2

Reagent Usage amount 5×Prime Script Buffer 2 4μl PrimeScript RT Enzyme Mix I 1μl RT primer mix 1μl RNA reaction solution to remove genomic DNA 10μg RNase Free ddH ₂ O Make up to 20μl

After preparing the reaction system according to Table 2, incubate at 37°C for 15 minutes, place at 85°C for 5 seconds, and store at 4°C after the reaction.

Step 4: Real-time quantitative PCR reaction

Template: the above-mentioned reverse transcription product was used as a template for the fluorescent quantitative PCR reaction, and the amount of the template used was 1 μl. Using the sequences of CD263 gene (NM_145201.4) and GADPH gene (NG_032578.1), two pairs of primers were designed with Primer Premier5 software.

Primers: Synthesized by Yingwei Jieji (Shanghai) Trading Co., Ltd., designed as follows:

table 3

The system of PCR reaction:

Premix Ex TaqTMII（货号：DRR081D），此产品能够抑制非特异性反应，在宽广的范围内进行更加准确的定量。本Buffer和改良后的Hot Start法用DNA聚合酶TaKaRa Ex Taq HS组合使用，可以进行再现性好、可信度高的RealTime PCR解析。The PCR reaction adopts TAKARA company's

Premix Ex TaqTMII (Catalog No.: DRR081D), this product can suppress non-specific reactions and perform more accurate quantification in a wide range. This Buffer can be used in combination with the improved Hot Start method using DNA polymerase TaKaRa Ex Taq HS to perform RealTime PCR analysis with good reproducibility and high reliability.

Table 4

Prepare the fluorescent quantitative reaction solution according to the above table, and use the ABI7500 real-time fluorescent quantitative PCR instrument for real-time quantitative PCR reaction.

The real-time quantitative PCR reaction adopts two-step PCR, and the amplification standard procedure is: 95°C for 30 seconds; 95°C for 5 seconds, 60°C for 40 seconds, 40 cycles.

According to the results of real-time quantitative PCR, ABI7500software v2.0.6 was used to process and analyze the results, and the GADPH gene was used as an internal reference gene to calculate the expression levels of tuberculosis patients and latent infected people relative to healthy people by using the method of 2 ^-ΔΔCT . The results are shown in the figure 1, where the abscissa represents different populations, the ordinate represents the relative expression level, and the larger the ordinate, the higher the expression level. The results show that the expression level of CD263 in tuberculosis patients is significantly higher than that of latent infection and healthy people. Expression level (see Figure 1), the difference was significant (P<0.05). In Figure 1, "TB" refers to patients with active TB infection, "LTBI" refers to patients with latent TB infection, and "HC" refers to healthy controls.

According to the above experimental results, quantitative RT-PCR can be used to diagnose tuberculosis latent infection and active tuberculosis: design PCR primers for CD263 gene, and detect the expression level of CD263 gene in tuberculosis tissue. If the expression level of CD263 is significantly increased, it means that you have tuberculosis The probability is high, and vice versa, so as to better distinguish between active tuberculosis infection and latent tuberculosis infection.

Example 2

In this embodiment, the crowd is divided into three groups: 9 cases of tuberculosis patients, 6 cases of latently infected people and healthy people, and detect the change of CD263 gene mRNA in each peripheral blood mononuclear cell (PBMC) by a gene chip, and find that it is significantly different in tuberculosis patients. There was a clear trend of up-regulated expression.

In this embodiment, the difference in gene expression level of CD263 gene in TB, LTBI, and HC in tuberculosis samples is detected by gene chip, including the following four steps:

Step 1: Chip Preparation

At present, glass or silicon wafers are mainly used as carriers to prepare chips, and target genes are arranged in sequence as probes on the carrier by spotting method. Target genes can be divided into genomic DNA and cDNA (or artificially synthesized DNA).

Step 2: Sample Preparation

The extraction steps of total RNA in the sample to be tested are as follows: Separate the PBMCs of tuberculosis patients, latent infected persons and healthy people, and then use Qiagene RNA extraction kit to extract total RNA (see Example 1 for the specific method). The extracted total RNA Further used for sample cDNA probe preparation, the process includes the preparation of fluorescent Cy3 and Cy5 probes (cDNA first-strand labeling), purification and quantification, and the quantified Cy3 and Cy5-labeled probes are sucked back into 1.5ml centrifuge tubes , heated and drained, and stored at -20°C until hybridization.

The quantified Cy3 and Cy5 labeled probes can be DNA, RNA, DNA-RNA chimera, PNA or other derivatives, and the length of the probes is not limited, as long as specific hybridization is completed, specific to the target nucleotide sequence Sexual union, any length is fine.

Step 3: Chip hybridization

1) Preparation: (1) Wash the coverslip, put the coverslip in ddH ₂ O, 95% ethanol, ddH ₂ O in turn, each for 3 minutes, and finally put it into a dry 50ml centrifuge tube at 1000 rpm, centrifuge for 3 minutes , remove residual water stains, and place it for use; (2) prepare a 0.1M PBS solution; (3) balance the hybridization oven, calibrate the hybridization oven with a spirit level, and keep it level; (4) prepare the following washing solution: 20×SSC solution , 10% (M/V) SDS solution, washing solution I (2×SSC/0.5% (M/V) SDS), washing solution II (1×SSC/0.1% (M/V) SDS), washing solution III (0.1 x SSC).

2) Pre-hybridization Prepare 30 μl of pre-hybridization solution (composed of 9 μl of 20×SSPE buffer, 3 μl of 50×Denhandts buffer and 18 μl of ddH ₂ O), take out the chip, add 30 μl of pre-hybridization solution onto the chip, and cover it cover glass, and then put the chip into a hybridization box with 0.1MPBS, and place it in a 42°C hybridization oven for pre-hybridization for 1 hour. After the pre-hybridization is completed, take out the chip and soak it in ddH ₂ O for a while. Put the chip into a dry centrifuge tube and centrifuge to remove residual water stains to obtain a pre-hybridized chip.

3) Hybridization Take out 1-5 μl of quantified Cy3 and Cy5-labeled probes, fully dissolve each with 9-15 μl ddH ₂ O and mix them in a 1.5ml centrifuge tube, then continue to add Cy3/Cy5 fluorescent-labeled probes to this centrifuge tube Hybridization solution (consisting of 9 μl of 20×SSPE buffer, 3 μl of 50×Denhandts buffer and 18 μl of ddH ₂ O) to obtain a hybridization mixture, then take the hybridization mixture dropwise on the pre-hybridized chip, and cover with a cover glass Finally, put the hybridization chip into a hybridization box added with 0.1M PBS, and place it in a hybridization box at 42°C for 12 to 20 hours for hybridization.

Step 4: Signal detection

After the chip hybridization reaction is completed, the chip is washed, and then scanned by a scanner such as a laser confocal scanner. After scanning, the image is converted into a digital signal based on fluorescence intensity, and the chip data is read with GenPix pro6.0 software, GenePix pro6. 0 Through the background noise of the chip and the light intensity analysis of the hybridization point, the light intensity value of each point is calibrated, and the light intensity of each hybridization point is converted into a data value.

In order to study the changes of CD263 in three different populations, we adopted the analysis method of oneway ANOVA combined with Bonferroni correlation coefficient, and the threshold was set at p<0.05. Finally, after analysis, the expression difference of CD263 gene in tuberculosis was detected by gene chip. The result As shown in Figure 2, red represents up-regulation of gene expression, and green represents down-regulation of gene expression (mark A in Figure 2 represents red, mark B represents green; "TB" refers to active tuberculosis infection, "LTBI" refers to latent tuberculosis infection , "HC" means healthy control). The results showed that the expression of CD263 gene was significantly up-regulated in tuberculosis patients, while the expression of CD263 was down-regulated in both LTBI and HC.

Claims (5)

1. the purposes of the primer of a kind of amplification CD263 gene, it is characterized in that, be used for preparing the product of distinguishing tuberculosis latent infection and active tuberculosis. 2. the purposes of the primer of amplification CD263 gene according to claim 1, it is characterized in that, the product of described discrimination tuberculosis latent infection and active tuberculosis comprises: detect and distinguish tuberculosis latent infection and active tuberculosis with real-time quantitative PCR product. 3. The purposes of the primers for amplifying CD263 gene according to claim 2, characterized in that, the product for discriminating tuberculosis latent infection and active tuberculosis with real-time quantitative PCR at least includes a pair of primers for specific amplification of CD263 gene. 4. the purposes of the primer of amplification CD263 gene according to claim 3, it is characterized in that, the primer of described specific amplification CD263 gene is: CD263-F: 5'-AAGTTCGTCGTCGTCATCGT-3', CD263-R: 5'-TTGAAGCTGTGCCTCTGTTG-3'. 5. A use of a gene chip comprising a probe hybridized with the nucleic acid sequence of the CD263 gene, characterized in that it is used to prepare a product for distinguishing tuberculosis latent infection and active tuberculosis.

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