CN103074425B

CN103074425B - CD263 gene application

Info

Publication number: CN103074425B
Application number: CN201210589472.6A
Authority: CN
Inventors: 陈心春; 周伯平; 蔡毅; 杨倩婷; 张明霞; 翟景南
Original assignee: Third Peoples Hospital of Shenzhen
Current assignee: Third Peoples Hospital of Shenzhen
Priority date: 2012-12-29
Filing date: 2012-12-29
Publication date: 2014-01-01
Anticipated expiration: 2032-12-29
Also published as: CN103074425A

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

The purposes of CD263 gene

Technical field:

The present invention relates to technical field of bioengineering, particularly the purposes of CD263 gene.

Background technology:

Tuberculosis (Tuberculosis, TB) is the chronic infection disease caused by m tuberculosis infection, and mycobacterium tuberculosis not only can cause pulmonary tuberculosis (85%), and can cause the tuberculosis of the outer multiple organ of lung.Although have at present effective antitubercular agent, tuberculosis remains the No.1 killer of current infectious diseases, and annual approximately 2,000,000 people in the whole world die from tuberculosis; Mycobacterium tuberculosis is infected in the whole world approximately 1/3rd population, at these, is called as in the crowd of tubercule bacillus latent infection (LTBI), have an appointment 10% the most at last progress be active tuberculosis.

Owing to lacking at present effective tuberculosis vaccine, prevention and control lungy mainly depend on early discovery, treatment, isolation active tuberculosis patient.Yet, there is wretched insufficiency in diagnostic activities detection technique lungy now, can not meet requirement clinical and that tuberculosis prophylaxis is controlled: 1) phlegm mycobacterium tuberculosis microorganism checking specificity is high, it is the phthisical gold standard of current diagnostic activities, but there is susceptibility low (less than 40%), length consuming time (tubercule bacillus is cultivated 1-2 consuming time month), the laboratory Biosafety requires high shortcoming.2) mycobacterium tuberculosis gene test, although realized the purpose (1 day) of quick diagnosis, the gene test of directly carrying out from sputum specimen does not significantly improve aspect susceptibility, and has false negative, false-positive problem.3) in immunology detection, antibody test is assert and is not suitable for diagnosis lungy by the World Health Organization; Cellular immunology detects and comprises tuberculin skin test (TST) and tubercule bacillus Interferon, rabbit release test (IGRA), can not effectively distinguish active tuberculosis patient and tubercule bacillus latent infection person, although the susceptibility that the latter detects the active tuberculosis patient is significantly higher than other detections.

CD263, also be TRAIL/Apo-2L, DCR1, DCR1-TNFR.Its Decoy receptor of encoding, its expression can be protected the lethal effect of normal cell escape TRAIL, and the effect that regulated and controled of the apoptosis that TRAIL is induced.Its provide protection to cell be by participating in the competition property in conjunction with TRAIL, thereby the apoptosis that death receptor is induced is obstructed.The research discovery, CD263 plays an important role in the generation of tumour.Still there is no about the CD263 gene at present the report as the Diagnosis of Tuberculosis mark.

Summary of the invention:

The technical problem to be solved in the present invention is to provide a kind of purposes of CD263 gene, and the CD263 gene can be used as the molecular marker of differentiating tuberculosis latent infection and active tuberculosis.

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

The invention provides a kind of purposes of CD263 gene, for the preparation of the product of differentiating tuberculosis latent infection and active tuberculosis.

The product of described differentiation tuberculosis latent infection and active tuberculosis preferably includes: the product of differentiating tuberculosis latent infection and active tuberculosis with real-time quantitative PCR or genechip detection.

The described product by real-time quantitative PCR differentiation tuberculosis latent infection and active tuberculosis preferably at least comprises the primer of a pair of specific amplified CD263 gene.

The primer of described specific amplified CD263 gene is preferably:

CD263-F：5’-AAGTTCGTCGTCGTCATCGT-3’，

CD263-R：5’-TTGAAGCTGTGCCTCTGTTG-3’；

The described product of differentiating tuberculosis latent infection and active tuberculosis with genechip detection preferably includes: with the probe of the nucleic acid array hybridizing of CD263 gene.

Utilize test kit of the present invention, can detect the expression of patient CD263 gene, thereby whether the diagnosis patient suffers from active tuberculosis.

In the present invention, term " primer " refers to a kind of oligonucleotide, can be natural also can synthesizing, it can be used as the synthetic starting point of induce dna under certain condition, bring out primer extension product synthetic and the nucleic acid chains complementation under conditions suitable, under four kinds of different triphosphoric acid dezyribonucleosides and a kind of polymerization agent (being archaeal dna polymerase or reversed transcriptive enzyme) exist, in a kind of suitable damping fluid and carry out amplified reaction at suitable temperature.Preferred primer is the sub-thread oligodeoxyribonucleotide.The appropriate length of primer depends on the designed use of this primer, but generally between 15～25 Nucleotide.

In the present invention, described probe can be DNA, RNA, DNA-RNA mosaic, PNA or other derivative.The length of described probe is restriction not, if complete specific hybrid, with purpose nucleotide sequence specific binding, any length can.

The experiment proved that, the expression of CD263 gene of the present invention in tuberculosis patient blood be apparently higher than healthy population and latent infection crowd, so the CD263 gene can be used as the special marker gene of diagnosis of tuberculosis, makes Diagnosis of Tuberculosis more accurately, fast.

The accompanying drawing explanation:

Below in conjunction with the drawings and specific embodiments, the present invention is further detailed explanation.

Fig. 1 is the quantitative RT-PCR figure as a result of CD263 gene differential expression in human peripheral blood single nucleus cell (PBMC) of the embodiment of the present invention 1.

Fig. 2 is the chip results figure of CD263 gene differential expression in PBMC of the embodiment of the present invention 2.

Embodiment:

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

Embodiment 1

The present embodiment is divided into three groups by the crowd: tuberculosis patient, latent infection crowd and healthy population (each 20 examples), change by detecting CD263 gene mRNA in every Patients with Peripheral blood mononuclear cell (PBMC), find that it is obvious up-regulated expression trend in tuberculosis patient.

The expression that the present embodiment detects every routine CD263 gene with quantitative RT-PCR method changes.Concrete steps are as follows:

Step 1: the preparation of peripheral blood mononuclear cell (PBMC) suspension

Add lymphocyte separation medium (Fresenius Kabi NOrge As:LYS3773) 5ml in centrifuge tube; Get the above-mentioned tuberculosis patient of making a definite diagnosis, each 2ml of anticoagulant heparin venous blood of latent infection patient and healthy population fully mixes and obtains mixed solution with the phosphoric acid buffer (PBS) of equivalent 1M respectively, with pipettor, mixed solution slowly is superimposed on the lymphocyte separation medium liquid level along tube wall, keep interface clearly, 2000 rev/mins centrifugal 20 minutes; In the middle of drawing with suction pipe, the cloud and mist stratification enters after another centrifuge tube then to add the 1M PBS of 5 times of volumes, 1500 rev/mins of centrifugal 10 minutes washed cells, abandon supernatant, the same terms repeated washing cell once, the RPMI1640(Thermo scientific:SH30807.01b that then to add containing the calf serum volume percent be 10%) 1ml, re-suspended cell, obtain each 20 routine PBMC suspensions of three groups of crowds; Every example is got the PBMC suspension of 20 μ l on blood counting chamber, counting cells concentration.

Step 2: RNA extracting

The RNeasy Mini Kit(article No. 74106 of employing Qiagene company) each 20 routine PBMC suspensions of three groups of crowds obtained above carried out to the RNA extracting.Concrete operations are: get and above-mentionedly contain 1 * 10 ⁶the PBMC suspension of individual cell in the centrifuge tube that removes DNA enzyme and RNA enzyme, 3000 rev/mins centrifugal 10 minutes, abandon supernatant; Add 350 μ l Buffer RLT in cell precipitation, fully mix cracking; Add 250 μ l dehydrated alcohols, mix, liquid rotating is moved on in the RNeasy pillar, centrifugal 30 seconds of 8,000g, abandon waste liquid; Add 350 μ l Buffer RW1 with 8,000g centrifugal 30 seconds, abandon waste liquid; Add 80 μ l DNase solution (10 μ l DNase+70 μ l Buffer RDD), digest 15min on post, centrifugal 30 seconds of 8,000g, abandon waste liquid; Add 350 μ l Buffer RW1, centrifugal 30 seconds of 8,000g, abandon waste liquid; Add 500 μ l Buffer RPE, centrifugal 30 seconds of 8,000g, abandon waste liquid; Sky gets rid of, centrifugal 1 minute of 8,000g; Posts transfer, to a 1.5ml centrifuge tube that removes DNA enzyme and RNA enzyme, is added to the ddH of 40 μ l without RNase ₂o, centrifugal 1 minute of 10,000g, collect three groups of crowds each 20 examples RNA in-80 ° of C preserve, stand-by.

Step 3: reverse transcription

Adopt the reverse transcription test kit (DRR047) of TAKARA company, get the RNA0.5 μ g that step 2 obtains and carry out reverse transcription, this test kit has increased the step of removing genomic dna than classical inverse transcript reagent box, can guarantee to the full extent the purity of RNA and the specificity of amplification.Substep is as follows:

(1) removal of genomic dna reaction

Table 1

Reagent	Usage quantity
		5 * gDNA Eraser damping fluid	2μl
gDNA?Eraser	1μl
		Total RNA	0.5μg
Rnase?Free?ddH ₂O	Benefit to 10 μ l

After preparing reaction system according to table 1,42 ℃ of temperature, bathe 2min, preserve under 4 ℃.

(2) reverse transcription reaction

The reaction system preparation is all carried out on ice, and concrete system is as follows:

Table 2

Reagent	Usage quantity
		5 * Prime Script damping fluid 2	4μl
PrimeScript RT enzyme mixture I	1μl
		The RT primer mixture	1μl
Remove the RNA reaction solution of genomic dna	10μg
		Rnase?Free?ddH ₂O	Benefit to 20 μ l

After preparing reaction system according to table 2,37 ℃ of temperature, bathe 15min, place 5 seconds, reacted and put 4 ℃ of preservations for 85 ℃.

Step 4: quantitative fluorescent PCR reaction

Template: above-mentioned reverse transcription product is as the template of quantitative fluorescent PCR reaction, and the template consumption is 1 μ l.Utilize CD263 gene (NM_145201.4) and GADPH gene (NG_032578.1) sequence, with two pairs of primers of Primer Premier5 software design.

Primer: by the English Weihe River, prompt base (Shanghai) trade Co., Ltd is synthetic, designs as follows:

Table 3

The system of PCR reaction:

The PCR reaction adopts TAKARA company

premix Ex TaqTMII(article No.: DRR081D), this product can suppress nonspecific reaction, in broad scope, carries out quantitative more accurately.Hot Start method after this Buffer and improvement is used in combination with archaeal dna polymerase TaKaRa Ex Taq HS, can carry out the Real Time PCR that reproducibility is good, with a high credibility and resolve.

Table 4

According to the reaction solution of upper table preparation fluorescent quantitation, and use instrument to carry out the real-time quantitative PCR reaction for ABI7500 real-time fluorescence quantitative PCR instrument.

Real-time quantitative PCR reaction adopts two-step approach PCR, the amplification standard program: 95 ℃ 30 seconds; 95 ℃ 5 seconds, 60 ℃ 40 seconds, 40 circulations.

According to the result of real-time quantitative PCR, with ABI7500software v2.0.6, result is carried out to Treatment Analysis, take the GADPH gene as reference gene, utilize 2 ^{-Δ Δ CT}method calculate tubercular and the latent infection crowd expression amount with respect to healthy population, result as shown in Figure 1, wherein X-coordinate means different crowds, ordinate zou means relative expression quantity, ordinate zou shows that more greatly its expression level is higher, result shows, the expression level of CD263 in tuberculosis patient be apparently higher than the expression level (see figure 1) of latent infection and healthy population, significant difference (P<0.05).In Fig. 1, " TB " refers to active tuberculosis the infected, and " LTBI " refers to latent tuberculosis the infected, and " HC " refers to normal healthy controls.

According to above-mentioned experimental result, can pass through quantitative RT-PCR diagnosis of tuberculosis latent infection and active tuberculosis: the PCR primer of design CD263 gene, detect the expression amount of CD263 gene in the tuberculosis tissue, if the expression amount of CD263 significantly raises, illustrate that tuberculate possibility is high, otherwise low, better to distinguish active tuberculosis the infected and latent tuberculosis the infected.

Embodiment 2

The present embodiment is divided into three groups by the crowd: tuberculosis patient 9 examples, equal 6 examples of latent infection crowd and healthy population, change by CD263 gene mRNA in the every Patients with Peripheral blood mononuclear cell of genechip detection (PBMC), find that it is obvious up-regulated expression trend in tuberculosis patient.

The difference of CD263 gene gene expression dose in TB, LTBI and HC in genechip detection tuberculosis sample for the present embodiment comprises following four steps:

Step 1: chip preparation

Preparing at present chip, mainly to take sheet glass or silicon chip be carrier, by the point sample method, using target gene as probe, is arranged in order on carrier, and target gene can be divided into genomic dna, cDNA(or artificial-synthetic DNA).

Step 2: sample preparation

In testing sample, the extraction steps of total RNA is as follows: the PBMC that separates respectively tuberculosis patient, latent infection person and healthy population, extract test kit with Qiagene RNA again and extract its concrete grammar of total RNA(with reference to embodiment 1), the total RNA extracted is further used for the preparation of sample cDNA probe, its process comprises preparation (cDNA the first chain mark), the purifying and quantitative of fluorescence Cy3 and Cy5 probe, Cy3 quantitatively and the probe of Cy5 mark suck back to the 1.5ml centrifuge tube, heating is drained, be stored in-20 ℃, wait to hybridize.

Described Cy3 after quantitatively and the probe of Cy5 mark can be DNA, RNA, DNA-RNA mosaic, PNA or other derivative, and the length of probe is restriction not, if complete specific hybrid, with purpose nucleotide sequence specific binding, any length can.

Step 3: chip hybridization

1) prepare: (1) washboard slide, cover glass is put into to ddH successively ₂o, 95% ethanol, ddH ₂o, each 3min, finally put into 1000 rev/mins of dry 50ml centrifuge tubes, and centrifugal 3min removes residual water stainly, places stand-by; (2) the PBS solution of preparation 0.1M; (3) the balance hybrid heater, calibrate hybrid heater with water level gauge, the maintenance level; (4) be formulated as follows the liquid of developing a film: 20 * SSC solution, 10%(M/V) SDS solution, washing lotion I(2 * SSC/0.5%(M/V) SDS), washing lotion II(1 * SSC/0.1%(M/V) SDS), the solution III of developing a film (0.1 * SSC).

2) prehybridization preparation prehybridization solution 30 μ l(are by 20 * SSPE damping fluid of 9 μ l, 50 * Denhandts damping fluid and the 18 μ l ddH of 3 μ l ₂o forms), take out chip, 30 μ l prehybridization solutions are dripped on chip, covered, then put into chip the hybridizing box that is added with 0.1MPBS, is placed in 42 ℃ of hybrid heater prehybridizations 1 hour, after prehybridization completes, takes out chip ddH ₂o embathes a moment, chip is put into to dry centrifuge tube after cover glass comes off centrifugal, removes residual water stainly, obtains the chip of prehybridization.

3) probe 1～5 μ l through quantitative Cy3 and Cy5 mark is taken out in hybridization, respectively with 9～15 μ l ddH ₂o fully dissolves and is mixed in the 1.5ml centrifuge tube, then at this centrifuge tube, continues to add the hybridization solution of Cy3/Cy5 fluorescence labeling probe (by 20 * SSPE damping fluid of 9 μ l, 50 * Denhandts damping fluid and the 18 μ l ddH of 3 μ l ₂o forms) obtain hybrid mixed liquid, then get on the chip that the hybrid mixed drop is added in prehybridization, and covered obtains hybridization hybrid chip, finally this hybridization hybrid chip is put into to the hybridizing box that is added with 0.1M PBS, be placed in 42 ℃ of hybridization casees and hybridize 12～20 hours.

Step 4: signal detection

After the chip hybridization reaction finishes, carry out the chip washing, by scanner, as the laser confocal scanning instrument, scanned again, after scanning, image is converted into to the numerary signal based on fluorescence intensity, read chip data with GenPix pro6.0 software, GenePix pro6.0, by the light intensity analysis of background noise and the hybridization point of chip, to the light intensity value calibration of each point, and is converted into data value by the light intensity of each hybridization point.

In order to study the changing conditions of CD263 in three different crowds, we have taked the analytical procedure of one way ANOVA in conjunction with the Bonferroni relation conefficient, threshold value setting is p<0.05, finally by analysis, the differential expression of CD263 gene in genechip detection tuberculosis, as shown in Figure 2, redness represents the genetic expression rise to result, green represents that (in Fig. 2, sign A represents redness to down regulation of gene expression, and sign B represents green; " TB " refers to active tuberculosis the infected, and " LTBI " refers to latent tuberculosis the infected, and " HC " refers to normal healthy controls).Result is presented at CD263 genetic expression in tuberculosis patient and significantly raises, and, in LTBI and HC, the expression amount of CD263 is all lowered.

Claims

1. the purposes of the primer of the CD263 gene that increases, is characterized in that, for the preparation of the product of differentiating tuberculosis latent infection and active tuberculosis.

2. the purposes of the primer of amplification CD263 gene according to claim 1, is characterized in that, the product of described differentiation tuberculosis latent infection and active tuberculosis comprises: detect the product of differentiating tuberculosis latent infection and active tuberculosis with real-time quantitative PCR.

3. the purposes of the primer of amplification CD263 gene according to claim 2, is characterized in that, the described product by real-time quantitative PCR differentiation tuberculosis latent infection and active tuberculosis at least comprises the primer of a pair of specific amplified CD263 gene.

4. the purposes of the primer of amplification CD263 gene according to claim 3, is characterized in that, the primer of described specific amplified CD263 gene, for:

CD263-F：5’-AAGTTCGTCGTCGTCATCGT-3’，

CD263-R：5’-TTGAAGCTGTGCCTCTGTTG-3’。

5. one kind comprises and the purposes of the gene chip of the probe of the nucleic acid array hybridizing of CD263 gene, it is characterized in that, for the preparation of the product of differentiating tuberculosis latent infection and active tuberculosis.