CN101988096A - In-situ hybridization detection kit of MAGEA3 (Melanoma-Associated Antigen Antiger 3) as well as detection method and application thereof - Google Patents

Abstract

The invention relates to an in-situ hybridization detection kit of MAGEA3 (Melanoma-Associated Antigen Antiger 3), comprising a hybridization probe and a marker, wherein the hybridization probe sequence is shown in SEQ ID No.1. The invention also provides an in-situ hybridization detection method of the MAGEA3. In addition, the invention also provides application of the kit to the preparation of medicaments for detecting liver cancer diseases. The kit provided by the invention has the advantages of high sensitivity and strong specificity; and the detection method of the invention is convenient and simple for operation and can be widely used and popularized in municipal or higher-grade hospitals.

Description

A kind of hybridization in situ detection kit of MAGEA3 gene and detection method thereof and application

[technical field]

The present invention relates to a kind of test kit, specifically about a kind of hybridization in situ detection kit and detection method and application of MAGEA3 gene

[background technology]

Liver cancer is one of China's common cancer, and the mortality ratio height is only second to stomach, esophagus and occupies the 3rd in the dead cis-position of malignant tumour; Partly then accounting for second in the geographic rural area, be only second to cancer of the stomach.China dies from about 110,000 people of liver cancer every year, accounts for 45% of whole world PLC mortality number.Onset is often hidden, and how to chance on liver cancer in hepatopathy is followed up a case by regular visits to or in the health check-up generaI investigation.This moment, patient was both asymptomatic, and physical examination also lacks the sign of tumour itself, and this phase is referred to as the subclinical phase.In a single day symptom appears in liver cancer, and its course of disease of the person of prescription on individual diagnosis has entered middle and advanced stage mostly.

Liver cancer changes to the formation cancer before cancer, need several years, how to accomplish early detection, diagnosis, prognosis detection and the recurrence after the diagnosis and treatment, shift that to detect in early days be the key that reduces M ﹠ M.

(melanoma-associated antiger MAGE) is a class tumour specific antigen to melanoma associated antigen, by the MAGE gene family coding that is positioned at X chromosome.In liver cancer tissue, MAGE-1 and MAGE-3 gene are specificity overexpression, and in liver cirrhosis and normal hepatocytes biopsy, do not express, have only indivedual hepatocellular carcinoma cancer beside organism sample can express MAGE-3mRNA, point out this mRNA of two kinds to be used to detect the liver cancer cell hematogenous spread as the specific tumour marker.Hepatocellular carcinoma patient's postoperative peripheral blood MAGE and (or) MAGE-3mRNA lasting masculin or transfer positive person's poor prognosis to by feminine gender, monitoring follow up a case by regular visits to peripheral blood MAGE and (or) but MAGE-3mRNA early prediction transfer and relapse.But bibliographical information is also arranged, in hepatocellular carcinoma patient's liver cancer tissue the positive rate of MAGE-4 genetic expression and hepatocellular carcinoma patient TNM by stages, diameter of tumor, have or not complete coating, have or not TTPV, lifetime etc. is all irrelevant.

Along with molecular biotechnology is perfect day by day, functional genomics, the deep expansion of research such as cancer genomics, so far, we might do more accurate early diagnosis on gene level, just can accomplish the early prediction diagnosis in canceration early stage or cancer cells formation (during mono-clonal).

Hybridization in situ technique (in situ hybridization) combines molecular biology and cytochemistry technology, is probe with the nucleic acid molecule of mark, in the technology of histocyte in situ detection specific nucleic acid molecule.Its principle is to make the nucleic acid strand (being probe) that contains distinguished sequence, process mark, under optimum conditions with histocyte in the complementary nucleic acid strand be that target nucleic acid is hybridized, with radioautograph or immunocytochemistry label probe is surveyed again, thereby shown special DNA or RNA molecule at cell in-situ.

[summary of the invention]

The objective of the invention is provides a kind of purposes of hybridization in situ detection kit of MAGEA3 gene at deficiency of the prior art.

One purpose more of the present invention is that a kind of hybridization in situ detection kit of MAGEA3 gene is provided.

Another purpose of the present invention is that a kind of in situ hybridization detection method of MAGEA3 gene is provided.

For achieving the above object, the technical scheme taked of the present invention is:

The application of a kind of hybridization in situ detection kit of MAGEA3 gene in preparation detection liver cancer diseases medicine, described test kit comprises hybridization probe, marker, described hybridization probe sequence is shown in SEQ IDNO.1.MAGEA3 gene order number: NM_0053621753bp, mRNA, chromosome x q28 ", cds:210...1154bp.

The RNA sequence of described hybridization probe sequence shown in SEQ ID NO.1.

Described marker is selected from radionuclide or non-radioactive marker.

Described radionuclide is selected from ³H, ³⁵S, ¹²⁵I or ³²A kind of among the P.

Described non-radioactive marker is selected from a kind of in vitamin H, digoxin, alkaline phosphatase, horseradish peroxidase or the fluorescein.

Described non-radioactive marker is preferably from digoxin.

Described test kit also comprises synergistic agent, and described synergistic agent is the alkaline phosphatase enzyme antibody.

For realizing above-mentioned second purpose, the technical scheme that the present invention takes is:

A kind of hybridization in situ detection kit of MAGEA3 gene comprises hybridization probe, marker, and wherein said hybridization probe sequence is shown in SEQ ID NO.1, and described marker is selected from radionuclide or non-radioactive marker.

For realizing above-mentioned the 3rd purpose, the technical scheme that the present invention takes is:

A kind of in situ hybridization detection method of MAGEA3 gene, this method may further comprise the steps:

A, the hybridization probe in the test kit is contacted with RNA to be measured in the substrate, form hybridization complex;

The hybridization complex that b, detection a step obtain.

The condition that forms hybridization complex in the described a step is: the temperature of nucleic acid hybridization is 42 ℃; The time of nucleic acid hybridization is 16-24 hour, and described substrate is selected blood cell sample or histocyte sample for use.

The component of diagnostic kit of the present invention is by hybridization probe, hybridization solution, developer, compositions such as synergistic agent.The nucleic acid hybridization principle of this test kit is that the molecular biology insider all knows, and the concrete operations step is to carry out quantitative analysis, result's report under sample disposal, prehybridization, hybridization, immunohistochemical staining, the mirror, and wherein Za Jiao concrete steps comprise:

Instrumentation:

1). sample to be measured is put into reactive tank;

2). instrument discards liquid automatically, adds Digestive system automatically;

3). instrument discards liquid automatically, and the back is fixing automatically;

4). instrument discards liquid automatically, automatically prehybridization (42 ℃);

5). instrument discards liquid automatically, cleans automatically;

6). instrument discards liquid automatically, automatically hybridization (42 ℃);

7). instrument discards liquid automatically, cleans automatically;

8). instrument discards liquid automatically, and automatic and DIG antibody is cultivated (room temperature);

9). instrument discards liquid automatically, cleans colour developing automatically;

10). take out the mounting microscopy.

The invention has the advantages that:

1, test kit provided by the invention has characteristics highly sensitive, high specificity.

2, detection method of the present invention is convenient and simple for operation, can be widely used and promoted in Municipal Hospitals.

3, clinical meaning of the present invention is that more early stage tracking detects liver cancer generation dynamic process, and the detection and the screening implement that are used for prevention of hcc medical science.Diagnostic kit of the present invention is with other detects the oncofetal protein mark clinically, and the medical imaging inspection has remarkable difference.The present invention can detect the MAGEA3 unconventionality expression on gene level, before medical imaging inspection and other inspection is not found the occupancy liver lesion, before the cancer biochemical indicator does not produce unusually, also do not form before the lump, can accomplish the information acquisition of above abnormal gene expression early, give real prognosis early diagnosis of clinical liver cancer sufferer.So just might implement early diagnosis, early prevention, the early treatment of liver cancer, might from the source, thoroughly effect a radical cure the liver cancer foul disease.

[description of drawings]

Fig. 1 is a hepatocarcinoma patient MAGEA3 genetic expression picture in the embodiment of the invention.

Fig. 2 is a patient with liver cirrhosis MAGEA3 genetic expression picture in the embodiment of the invention.

Fig. 3 is a normal people MAGEA3 genetic expression picture in the embodiment of the invention.

[embodiment]

Below in conjunction with accompanying drawing the specific embodiment of the invention is elaborated.

Embodiment 1

A kind of hybridization in situ detection kit of MAGEA3 gene comprises hybridization probe, marker, synergistic agent, and wherein, described hybridization probe sequence is shown in SEQ ID NO.1.The hybridization probe digoxigenin labeled.Other liquid and sample in the test kit are composed as follows:

Digestive system 100 μ l/ manage 1 pipe/box colourless transparent liquid

Protection liquid 100 μ l/ pipe 1 pipe/box colourless transparent liquid

Prehybridization solution 1300 μ l/ manage 2 pipe/box colourless transparent liquids

Justice hybridization solution 10 μ l/ pipe 1 pipe/box colourless transparent liquid

Antisense hybridization solution 10 μ l/ manage 1 pipe/box colourless transparent liquid

Confining liquid 1000 μ l/ manage 1 pipe/box colourless transparent liquid

Alkaline phosphatase enzyme antibody 1 μ l/ manages 1 pipe/box colourless transparent liquid

Developer A 175 μ l/ manage 1 pipe/box yellow liquid

Developer B 320 μ l/ manage 1 pipe/box colourless transparent liquid

Light yellow or the colourless transparent liquid of damping fluid I 10x 90ml/ bottle 1 bottle/box

Light yellow or the colourless transparent liquid of damping fluid II 10x 80ml/ bottle 1 bottle/box

Light yellow or the colourless transparent liquid of damping fluid III 10x 20m/ bottle 3 bottle/boxes

Light yellow or the colourless transparent liquid of damping fluid IV 10x 90ml/ bottle 1 bottle/box

Stationary liquid 90ml/ bottle 1 bottle/box colourless transparent liquid

6/box of positive control sample

Mentioned reagent composition explanation: (all reagent are available from SIGMA)

1, Digestive system: the 20mg/ml Proteinase K, the 100mg Proteinase K adds DEPC-H ₂O 5ml;

2, the glycine of protection liquid: 0.2g adds 1 * damping fluid I of 1ml;

3, prehybridization solution: 1 * damping fluid II 7.5ml

50×D?3ml

10mg/ml?yest?t-RNA?750ul

11mg/ml?SALMON?TESTES?DNA?682ul

0.04M?EDTA?3ml

50％?formamide?15ml

4, the bloking of confining liquid: 0.03g (buying from Roche Holding Ag) adds 1ml 1 * damping fluid III;

5,10x damping fluid I:(PH7.1-7.4)

NaCl?80g

Na ₂HPO ₄.12H ₂O?360g

KCl 2g

KH ₂PO ₄?2g

Add tri-distilled water to 1l, and autoclaving;

6,10x damping fluid II:(PH7.0)

NaCl 175.3g

Trisodium Citrate 88.2g

Several of HCl

Add tri-distilled water to 1l, and autoclaving;

7, damping fluid III:(PH7.9)

Tris 121.1g

NaCl 87.66g

About HCl 60ml

Add tri-distilled water to 1l, and autoclaving;

8, damping fluid IV:

1M Tris-HCl (PH9.5): Tirs 121.1g adds about HCl 3ml, adds water 900ml, transfers PH to 9.5, adds water to 1l, and autoclaving;

1M NaCl:NaCl 58.44 adds water to 1l, and autoclaving;

0.5M MgCl ₂: 101.65g MgCl ₂.6H ₂O adds water to 1l, and autoclaving;

9, stationary liquid: Paraformaldehyde 96 40g adds 1 * damping fluid I to 1l, and heat (about 50-60 degree) is stirred to dissolving a little;

10, developer A:NBT 1g adds 70%DMF11.44ml;

11, developer B:BCIP 1g adds 100%DMF30ml.

Test kit of the present invention can many person-portions use or person-portion use.

Embodiment 2

A kind of MAGEA3 gene hybridization in situ detection method and test kit thereof are used

One, sample disposal

1, with the centrifuge tube of 10ml, dress 4.5ml lymphocyte separation medium, again the 3ml anticoagulation is slowly added contain lymphocyte separation medium (blood: in centrifuge tube lymphocyte separation medium=1: 1.5), the centrifugal 10min of 2000r/min;

2, draw the middle layer white corpuscle to another centrifuge tube, in this pipe, add 1 * damping fluid I of about twice again, mixing, the centrifugal 10min of 1500g/min;

3, abandon supernatant. precipitation adds 1 * damping fluid I of about twice, mixing, the centrifugal 10min of 1500g/min;

4, abandon supernatant, and test tube mouth excess liquid is gone with the tissue suction.Again precipitation is made suspension, drop in push jack on the slide, seasoning.(hospital with good conditionsi can use the pelleter film-making.) 3ml blood, can do 4 slice, thin pieces;

5, with 40ml 4% stationary liquid, in glass jar, fixedly 30min uses 1 * damping fluid I to wash 5min again.Every cylinder can be put 16;

6, sample can be kept at-20 ℃, or continues to do experiment.

Two, reagent in the test kit is mixed with working concentration

1, with 10 * damping fluid I with tri-distilled water by being diluted to 1 * damping fluid I at 1: 10;

2, with 20 * damping fluid II with tri-distilled water by being diluted to 2 * damping fluid II at 1: 10;

By being diluted to 0.2 * damping fluid II at 1: 100; By being diluted to 0.1 * damping fluid II at 1: 200;

3, with 10 * damping fluid III with tri-distilled water by being diluted to 1 * damping fluid III at 1: 10;

4,10 * damping fluid IV with tri-distilled water by be diluted at 1: 10 * damping fluid IV (get 1#, 2#, each 10ml of 3#, add water to 100ml both can).

Three, experimental procedure:

1, gets two of every person's samples to be checked, two of (other two give over to check with) and positive control samples (test at every turn and do a pair of positive control);

2, in glass jar, add Digestive system (Digestive system 100ul adds 1 * damping fluid 199.9ml, is working concentration) 20ml.37 ℃ of water-bath preheatings 10 minutes.Put 16 slides into, handle 12min, use 1 * damping fluid I to wash 5min again for 37 ℃;

3, wash 10min with 0.2% protection liquid (protection liquid 1ml adds 1 * damping fluid I99ml and is working concentration), tri-distilled water is washed 5min, and above process is all carried out at glass jar.The slide seasoning;

4, slide is put into the box of preserving moisture, add prehybridization solution 20ul/ sheet. covered, the lid box of tightly preserving moisture is placed in 42 ℃ of constant water bath box more than the 3h;

5, take out slide, discard cover glass, slide is put into glass jar, with 70%, 90%, 95% ethanol is respectively washed 2min, seasoning;

6, slide is put into the box of preserving moisture, two of every patient specimens, one adds just hybridization solution 20ul/ sheet, and another adds antisense hybridization solution 20ul/ sheet, covered, the lid box of tightly preserving moisture is placed on 16-24h in 42 ℃ of constant water bath box;

7, take out slide, discard cover glass, slide is put into glass jar

In 42 ℃ of constant water bath box, wash twice, each 15min with 2 * damping fluid II

In 42 ℃ of constant water bath box, wash once each 15min with 0.2 * damping fluid II

In 42 ℃ of constant water bath box, wash twice, each 15min with 0.1 * damping fluid II;

8, wash 30s with 1 * damping fluid III, take out slide, seasoning;

9, slide is put into the box of preserving moisture, add 0.5%l confining liquid (the 1ml confining liquid adds 5ml 1 * damping fluid III) 100ul/ sheet, cover the box of tightly preserving moisture, at room temperature act on 30min;

10, take out slide, III washes 30s with 1 * damping fluid, seasoning;

11, slide is put into the box of preserving moisture, add alkaline phosphatase enzyme antibody (adding 1.8ml 1 * damping fluid III) 100ul/ sheet, cover the box of tightly preserving moisture and at room temperature act on 30min;

12, take out slide, wash 3 times, each 15min with 1 * damping fluid III;

13,1 * damping fluid IV washes 2min, adds developer (developer A73.3ul, developer B157.5ul are added among 30ml 1 * damping fluid IV, mixing), more than the room temperature lucifuge 12h;

14, wash 5min with tri-distilled water, seasoning, (add with glycerine 10% 1 * damping fluid I mixing) mounting microscopy.

Four, the result judges

100-300 cell of counting calculates the per-cent of catching the purple cell under light microscopic.

What the positive control sample added the antisense hybridization solution should catch purple more than 80%.

All add the negative internal reference of just hybridization solution should be colourless.

The cDNA of digoxigenin labeled, RNA and oligonucleotide probe, not only probe has a biotin labeling advantage, also having overcome biotin labeled probe is organized the endogenous vitamin H to do shortcomings such as sorrow in the crossover process in position), this hybridization probe and the leukocytic RNA nucleic acid to be measured of blood of human body are hybridized, method with immunohistochemical methods develops the color again, under light microscopic, observe existence and the location of mRNA,, judge the expression amount of goal gene according to painted cell count.

The inventive method is a nucleic acid hybridization in situ technology commonly used at present, this method is by detecting the MAGEA3 gene expression amount in the substrate cell, be used for determining whether liver cancer takes place, because the MAGEA3 gene is low the expression in the normal people, if MAGEA3 genetic expression height, illustrate that cancer takes place, thereby obtain the diagnostic message of cancer.

The embodiment of the invention is sampled as: 10 of hepatocarcinoma patients, 10 of patient with liver cirrhosis, 10 of normal control groups.Take out all people's to be checked peripheral blood 3-5 milliliter (separation white corpuscle) and do in situ hybridization.The result represents that all liver cancer patient MAGEA3 genes have overexpression, cell dyeing; Patient with liver cirrhosis MAGEA3 gene is not expressed; Normal control group MAGEA3 gene is not expressed the cell dye-free.Concrete outcome is asked for an interview the MAGEA3 genetic expression figure that Fig. 1 is a hepatocarcinoma patient; Fig. 2 is patient with liver cirrhosis MAGEA3 expression figure; Fig. 3 is a normal people MAGEA3 genetic expression picture.

Embodiment 3

Detect liver cancer diseases and detect parallel laboratory test between the liver cancer diseases with the MAGEA3 kit gene with the IL10 kit gene.

Specificity, susceptibility, accuracy for each comfortable liver cancer diseases of scientific evaluation said gene.We use the method for parallel test, detect the mRNA of said gene simultaneously, detection technique adopts the nucleic acid hybridization in situ technology, with same routine liver cancer diseases peripheral blood of patients, detect MAGEA3 gene and IL10 (interleukin-11 0) (IL10 gene order NM_0005721629bp simultaneously, mRNA.1q31-q32 ", cds:60 ... the 596bp) mRNA of gene (carrying out same procedure and step and reagent that the hybridization in situ technique of embodiment 1 and embodiment 2 is all adopted in nucleic acid hybridization in situ, immunohistochemical staining, mirror numeration down, report as a result etc.).Find the MAGEA3 gene in the liver cancer diseases patient expression amount than the expression amount height of IL10 gene same disease patient.The result shows that specificity, susceptibility, the accuracy of the diagnosis of MAGEA3 gene pairs liver cancer diseases are better than IL10 gene, and in situ hybridization genetic expression figure shows that MAGEA3 expression of gene amount is 70%, and IL10 expression of gene amount is 50%.The index that test kit of the present invention is done in the liver cancer diseases diagnosis has very important clinical meaning.

The above only is a preferred implementation of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the inventive method; can also make some improvement and replenish, these improvement and replenish and also should be considered as protection scope of the present invention.

SEQUENCE?LISTING

＜110〉Rui bends biotechnology (Shanghai) Co., Ltd.

＜120〉a kind of hybridization in situ detection kit of MAGEA3 gene and detection method thereof and application

<130>/

<160>1

<170>PatentIn?version?3.3

<210>1

<211>1753

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>1

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Claims (10)

1. the application of the hybridization in situ detection kit of a MAGEA3 gene in preparation detection liver cancer diseases medicine, described test kit comprises hybridization probe, marker, described hybridization probe sequence is shown in SEQ ID NO.1.

2. application according to claim 1 is characterized in that: the RNA sequence of described hybridization probe sequence shown in SEQ ID NO.1.

3. application according to claim 1 and 2 is characterized in that: described marker is selected from radionuclide or non-radioactive marker.

4. application according to claim 3 is characterized in that: described radionuclide is selected from ³H, ³⁵S, ¹²⁵I or ³²A kind of among the P.

5. application according to claim 3 is characterized in that: described non-radioactive marker is selected from a kind of in vitamin H, digoxin, alkaline phosphatase, horseradish peroxidase or the fluorescein.

6. application according to claim 5 is characterized in that: described non-radioactive marker is preferably from digoxin.

7. application according to claim 1 and 2 is characterized in that: described test kit also comprises synergistic agent, and described synergistic agent is the alkaline phosphatase enzyme antibody.

8. the hybridization in situ detection kit of a MAGEA3 gene comprises hybridization probe, marker, it is characterized in that, described hybridization probe sequence is shown in SEQ ID NO.1, and described marker is selected from radionuclide or non-radioactive marker.

9. the in situ hybridization detection method of a MAGEA3 gene is characterized in that, this method may further comprise the steps:

A, the hybridization probe in the described test kit of claim 8 is contacted with RNA to be measured in the substrate, form hybridization complex;

The hybridization complex that b, detection a step obtain.

10. detection method according to claim 9 is characterized in that: the condition that forms hybridization complex in a step is: the temperature of nucleic acid hybridization is 42 ℃; The time of nucleic acid hybridization is 16-24 hour, and described substrate is selected blood cell sample or histocyte sample for use.

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