CN101497921A

CN101497921A - Use of PRM1 gene

Info

Publication number: CN101497921A
Application number: CNA2008100431011A
Authority: CN
Inventors: 黄健; 滕小梅; 韩泽广
Original assignee: Shanghai Human Genome Research Center
Current assignee: Shanghai Human Genome Research Center
Priority date: 2008-02-02
Filing date: 2008-02-02
Publication date: 2009-08-05

Abstract

The invention discloses the application of a PRM1 gene which is used for preparing a product for diagnosing colon cancer. The PRM1 gene can be taken as a specific marker gene used for diagnosing the colon cancer, so that the diagnosis of the colon cancer is more accurate and quicker.

Description

The application of PRM1 gene

Technical field

The present invention relates to a kind of gene, relate in particular to a kind of application of PRM1 gene.

Background technology

Big colorectal carcinoma is one of China's kinds of tumor, accounts for the 5th of the national malignant tumour cause of the death.And the sickness rate of big colorectal carcinoma is the trend that raises year by year, and age of onset is tending towards rejuvenation.China has the big colorectal carcinoma of 130,000 people's New Developments every year approximately, and the speedup of sickness rate is the twice of world average level, reaches average annual 4%, and at present big colorectal carcinoma has become sickness rate one of the fastest malignant tumour that rises in the most of area of China.Therefore seek very important (the S Zheng of effective immunotherapy target spot, SR Cai.Colorectal cancer epidemiology and prevention study inchina.The Chinese-German J Clinical Oncology, 2003,2:72-75).

Tumor-testis (cancer-testis, CT) can express in multiple tissue-derived tumour, but only limit to express in the sexual cell of testis in human healthy tissues by gene.Because sexual cell is not expressed the human leucocyte antigen molecule, and there is blood-testis barrier in the body, so the CT antigen that testis tissue is expressed can not cause organism immune response, and be expressed in the CT antigen of tumor tissues, can cause specific immune response, so this class antigen is regarded as having tumour-specific again.Utilize this characteristic, find the specificity CT gene relevant with tumour, can promote the development of antigen-specific tumor vaccine, for immunotherapy of tumors provides new opportunity (ELke J, Dirk J, Knuth Alexander.Clinicalcancer vaccine trials.Curr Opin Immunol, 2002,14 (2): 178-182).

PRM1 (Protamine 1) gene is called protamine P1 again, is positioned on the karyomit(e) 16p13.2, and total length is 426 bases, contains 2 exons, and 2 introns are encoded one and contained 50 amino acid whose nucleoprotein (Choudhary, S.K.; Wykes.A haploid expressed gene cluster exists as a singlechromatin domain in human sperm.J.Biol.Chem, 1995,270:8755-8762), i.e. protamine P1.Protamine be spermatid nuclear dna bonded major protein (Cho.C, Willis.Haploinsufficiency of protamine-1 or-2 causes infertility in mice.Nature Genet, 2001,28:82-86).Human protamine gene cluster comprises three genes of closely regulating, PRM1, PRM2 and TNP2, in the sperm forming process, the product of three genes can repack gamete (the Martins RP that paternal genome is formed with function, Krawetz SA.Nuclear organization of the protamine locus.SocReprod Fertil Suppl, 2007,64:1-12).In the formation of sperm, brought into play vital role.In research in the past, PRM1 be considered to the male sex do not give birth to very important relation (Ravel C, Chantot-Bastaraud S.Mutations in the protamine 1 gene associated with male infertility.Mol HumReprod, 2007,13 (7): 461-464).

People PRM1 gene is one of member of CT antigen family, it be unclear that about the relation of PRM1 gene and colorectal carcinoma, does not also see the bibliographical information mistake.

Summary of the invention

One of the technical problem to be solved in the present invention provides a kind of application of PRM1 gene, and this PRM1 gene can be used as the tagged molecule of colorectal carcinoma diagnosis, has improved the accuracy of colorectal carcinoma diagnosis.

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

In one aspect of the invention, provide the application of a kind of PRM1 gene in the product of preparation diagnosing colon cancer.

The product of described diagnosing colon cancer comprises: with the product of RT-PCR, real-time quantitative PCR or immunodetection diagnosing colon cancer.

In the present invention, described product with the RT-PCR diagnosing colon cancer comprises the primer of a pair of specific amplified PRM1 gene at least.

Described product with the real-time quantitative PCR diagnosing colon cancer comprises the primer of a pair of specific amplified PRM1 gene at least.

Described product with the immunodetection diagnosing colon cancer comprises: with PRM1 protein-specific bonded antibody, comprise polyclonal antibody and monoclonal antibody.

In the present invention, can use a series of methods known in the art to prepare at the special antibody of PRM1 albumen.For example, the people PRM1 gene product or its antigen fragment of purifying is injected in the animal body to produce polyclonal antibody.Equally, the cell of expressing human PRM1 albumen or its antigen fragment also can be used for animal is caused immunity and produces antibody.Antibody prepared in accordance with the present invention also can be monoclonal antibody, and these monoclonal antibodies can prepare (for example, Kohler et al., Nature 256:495,1975 with hybridoma technology; Kohler et al., Eur.J.Immunol.6:511,1976; Kohler et al., Eur.J.Immunol.6:292,1976).Antibody of the present invention comprises the antibody that can prevent the PRM1 function, also can be the antibody that does not influence people PRM1 function.Each antibody-like can produce by the fragment of people PRM1 gene product or functional domain are caused immunity, and people PRM1 gene product and fragment thereof can produce or synthesize with Peptide synthesizer with recombination method.With the PRM1 gene product bonded antibody of non-modified forms, can utilize the gene product that for example produces among the E.coli at prokaryotic cell prokaryocyte to come immune animal and obtain.With posttranslational modification form such as glycosylation or phosphorylation PRM1 albumen or polypeptide bonded antibody, can utilize the gene product that in eukaryotic cell such as yeast or insect cell, produces to come immune animal and obtain.

The present invention experimental results show that the expression of PRM1 gene in colon cancer tissue apparently higher than cancer beside organism, so the PRM1 gene can be used as the special marker gene of diagnosing colon cancer, makes the colorectal carcinoma diagnosis more accurately, fast.

Description of drawings

Fig. 1 is that the present invention detects the expression figure of PRM1 gene in human healthy tissues by RT-PCR;

Fig. 2 is that the present invention detects the differential expression figure of PRM1 gene in human colon carcinoma tissue and cancer beside organism by RT-PCR;

Fig. 3 is the present invention detects PRM1 gene differential expression in human colon carcinoma tissue and cancer beside organism by real-time fluorescence quantitative RT-PCR a case line chart.

Embodiment

The present invention is further detailed explanation below in conjunction with drawings and Examples.

Following examples only are used to the present invention is described and are not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions among the embodiment, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment 1

The RT-PCR experiment detects the expression of PRM1 gene in human healthy tissues and colon cancer tissue.

1. separate tissue (Tissue isolation)

Used colorectal carcinoma of the present invention and cancer beside organism are all from the operation patients of clinical colorectal carcinoma.The colon cancer tissue of excision is once exsomatizing, cut rapidly focus and around healthy tissues beyond 5 centimeters, and put into liquid nitrogen and preserve.The other diagnosis of cancer and cancer is final foundation with pathological diagnosis all.15 kinds of human normal tissues (brain, the heart, lung, spleen, kidney, stomach, oesophagus, small intestine, ovary, mammary gland, prostate gland, pancreas, liver, tire liver and testis) RNA is all available from Clontech company.Reversed transcriptive enzyme is M-MLV Reverse Transcriptase, Promega CO..PCR reagent Taq archaeal dna polymerase, dNTP etc. are the precious Bioisystech Co., Ltd in Dalian.The primer of PRM1 gene (GeneID:5619) is Forward:5 '-CAGAGTTCCACCTGCTCACA-3 ' (SEQ ID NO:1), Reverse:5 '-TTCTCAGGCAGGAGTTTGGT-3 ' (SEQ ID NO:2), and PCR product length is 280bp.β-actin primer as internal reference is Forward:5 '-TCACCCACACTGTGCCCATCTACGA-3 ' (SEQ ID NO:3), Reverse:5 '-CAGCGGAACCGCTCATTGCCAATGG-3 ' (SEQ ID NO:4); β-actin amplification fragment length is 400bp.

2. the extraction agent box of total RNA

Extracting RNA reagent employing TRIzol reagent (GIBCO/BRL), this reagent are based on the extraction process production of one step of acidic phenol.Being used for used vessel of extracting RNA and water all will not have the processing of RNA enzyme, to guarantee the environment of no RNA enzyme in the experiment.

3.RNA extraction steps

To grind vessel such as pestle and homogenizer and do roasting 4h, remove the RNA enzyme, cooling at 200 ℃; Add precooling in the liquid nitrogen, will organize from liquid nitrogen and take out rapidly, be crushed into powder; With curet tissue is put into the homogenizer that adds TRIzol reagent in advance, homogenate number minute; Liquid after the homogenate is changed in the centrifuge tube of no RNA enzyme, behind the adding chloroform, 4 ℃ of centrifugal layerings; The upper strata water is changed in the centrifuge tube of a no RNA enzyme, behind the adding chloroform, 4 ℃ of centrifugal layerings; The upper strata water is changed in the centrifuge tube of a no RNA enzyme, add Virahol, 4 ℃ of centrifugation RNA; Precipitate 2 times with 75% washing with alcohol; Deionized water dissolving precipitation with no RNA enzyme.Extractive RNA quality evalution: ultraviolet spectrophotometer is measured 260/280 ratio (ratio is all 1.7～2.0); And observation has or not degraded in MOPS denaturing formaldehyde glue, and the used RNA of the present invention does not all have degraded.In order to remove the pollution of genomic dna, all RNA all digests with the DNA enzyme I (U.S. Ambion company) of no RNA enzyme.

4.cDNA synthetic

Get total RNA2 μ g, random primer 1 μ l, 70 ℃ of insulation 3min, sex change 5min on ice immediately.Add 5 * buffer, DTT and 0.05gL ^-1Each 2 μ l of dNTP and the reversed transcriptive enzyme of 1 μ l, fully behind the mixing, 42 ℃ of 2h.Template uses final concentration to be generally 0.01gL ^-1

5. sxemiquantitative RT-PCR

With cDNA is template, and pcr amplification PRM1 gene is simultaneously with the contrast of β-actin as the template amount.The PCR condition is: 94 ℃ of pre-sex change 3min; 94 ℃ of sex change 30s, 57 ℃ of annealing 30s, 72 ℃ are extended 40s, totally 35 circulations (β-actin is 25 circulations); 72 ℃ are extended 5min.Observe the PCR product with 2% agarose gel electrophoresis.

6. experimental result shows, the PRM1 gene is only expressed in testis, does not all have the (see figure 1) of expression in other tissue.Utilize sxemiquantitative RT-PCR method to detect the expression of PRM1 gene in cancerous human colon tumor tissue and cancer beside organism, found that the PRM1 gene obviously raises in 33% (3/9) colorectal carcinoma, 3 routine positive findingses are seen Fig. 2.In Fig. 2, " N " refers to cancer beside organism, and " C " refers to colon cancer tissue.

7. according to above-mentioned experimental result, can pass through the RT-PCR diagnosing colon cancer: the PCR primer of design PRM1 gene, the content of PRM1 gene RNA in the detection tumor tissues, the rna content height then illustrates the possibility height of suffering from colorectal carcinoma, otherwise then low.

The experiment of embodiment 2 real-time fluorescence quantitative RT-PCRs

Use the novel real-time fluorescence quantitative PCR instrument (Thermal Cycler DiceDetection System, Japan) of TaKaRa biotech firm, detect the expression of PRM1 gene in the cancer beside organism of 12 pairs of cancerous human colon tumor tissues and correspondence thereof.Reaction system is as follows: cumulative volume 20ul, SYBR Premix EX Taq 10ul, each 0.4ul of primer (10uM), DNA 2ul, ddH ₂O 7.2ul, the mixing reagent that turns upside down is put into the PCR instrument after centrifugal and is carried out amplified reaction.Instrument uses the description operation by producer.β-Actin is used to be used as internal reference.The equal triplicate of described experiment is to guarantee result's reliability.PRM1 expression of gene level in colorectal carcinoma and the cancer beside organism is calculated by the following method: the average β-actin_Ct of PRM1 Δ Ct=mean P RM1_Ct-, PRM1 Δ Δ Ct=PRM1 Δ Ct-cancerous tissue-PRM1 Δ Ct_ cancer beside organism, the multiple relation of the PRM1 gene in cancer and the cancer beside organism is with 2 ^{-PRMl Δ Δ Ct}Calculate.Experimental data SPSS software statistics (χ ²Check), P＜0.05 has been defined as statistical significance.

The result shows that compare with cancer beside organism, the PRM1 gene has the rise (P＜0.01) that is higher than 2 times in 50% (6/12) colon cancer tissue, and therefore, the expression of PRM1 gene in colorectal carcinoma group and the other group of cancer has the notable difference (see figure 3).In Fig. 3, " * " represents p＜0.01, and horizontal line is represented the intermediate value of every group of detected value Δ Ct in the square frame, and the outer short-term up and down of square frame is represented maximum and the Schwellenwert in the detected value respectively.The positive rate of fluorescence quantitative PCR detection of the present invention is higher than the result of above-mentioned common RT-PCR, and this may be relevant with the highly sensitive of quantitative fluorescent PCR.

This experimental result shows, can pass through the real-time fluorescence quantitative PCR diagnosing colon cancer: the PCR primer of design PRM1 gene, and the content of PRM1 gene RNA in the detection colon cancer tissue, the rna content height then illustrates the possibility height of suffering from colorectal carcinoma, otherwise then low.

Embodiment 3 immunodetection

1. antigen protein obtains

(1) utilizes gene engineering expression: the cDNA sequence that can from the Genbank database, obtain people PRM1 gene, obtain encoder block by pcr amplification, insert in prokaryotic organism or the eukaryote expression vector, express PRM1 albumen, and press the purification system protein purification of gene engineering expression product.

(2) by cultivating the human body derived cell or the tissue of high expression level PRM1 gene, separation and purification PRM1 albumen again.

2. Antibody Preparation

Can adopt following several method to prepare antibody:

(1) cytogamy method: with the PRM1 protein immune animal (comprising rabbit, goat etc.) of above-mentioned preparation, obtain spleen cell, merge with the myeloma cell again, and the Monoclonal Antibody technology prepares monoclonal antibody routinely.

(2) utilize the phage display storehouse, the spleen IgG variable region of clone immune animal also is expressed as gene engineering monoclonal antibody.

(3) utilize the protein immune animal of purifying, the preparation polyvalent antibody.

3. detect

(1) with the antibody (how anti-or monoclonal antibody) of preparation, carry out the pathology detection of colorectal carcinoma with histochemical method, positive signal is a colorectal carcinoma.

(2) get the patients serum, detect with the ELISA method, positive reaction is the suspicious patient of colorectal carcinoma.

(3) with PRM1 antibody as one of probe of protein chip, be used for the kinds of tumors diagnosis.

Sequence table

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Claims

1. the application of a PRM1 gene is characterized in that, the application of described PRM1 gene in the product of preparation diagnosing colon cancer.

2. application as claimed in claim 1 is characterized in that, the product of described diagnosing colon cancer comprises: with the product of RT-PCR, real-time quantitative PCR or immunodetection diagnosing colon cancer.

3. application as claimed in claim 2 is characterized in that, described product with the RT-PCR diagnosing colon cancer comprises the primer of a pair of specific amplified PRM1 gene at least.

4. application as claimed in claim 2 is characterized in that, described product with the real-time quantitative PCR diagnosing colon cancer comprises the primer of a pair of specific amplified PRM1 gene at least.

5. application as claimed in claim 2 is characterized in that, described product with the immunodetection diagnosing colon cancer comprises: with PRM1 protein-specific bonded antibody.