CN1714871A - Use of human ING4 gene as chemotherapy medicine sensitivity promoter - Google Patents

Abstract

The present invention discloses the use of human inhibitor of growth 4 (ING4) gene as tumor treating chemotherapy medicine sensitizer. The present invention also discloses one method of raising the sensitivity of tumor cell on chemotherapy medicine, and the method includes introducing ING4 gene into tumor cell before chemotherapy. Therefore, ING4 gene may be applied in the tumor treating chemotherapeutic process to promote the sensitivity of tumor cell on chemotherapy medicine, so as to reduce the dosage of chemotherapy medicine and reduce the toxic side effect of chemotherapy medicine on health cell of body.

Description

Human ING4 gene is as the purposes of chemotherapeutics sensitizer

Technical field

The present invention relates to genomics, molecular biology, cytobiology, genetic engineering and clinical medicine such as fields such as disease treatment and diagnosis.Particularly, the present invention relates to utilize new gene ING4 to improve the sensitivity of tumor chemotherapeutic drug, to be applied to clinical cancer therapy.The invention still further relates to the ING4 gene is imported the method for tumor cell and improves the means of ING4 gene expression in the tumor cell.

Background technology

The complement mark of human genome working draft the Human Genome Project (HGP) and has been obtained breakthrough, is an important milestone of human knowledge self process.It is for we understand human heredity and variation, generation, development, prevention and the treatment of disease laid a good foundation.According to ten thousand genes of the nearly 3-4 of genomic dna sequence predict human, the function of wherein a lot of genes is unknown, and disclosing the function of these unknown genes and they and the generation of human diseases and the relation of human normal physiological activity will be the important research direction of post genome project.

Large-scale cDNA order-checking can help us to recognize a lot of new genes, the finishing and also provide the important channel of discerning new gene for us to the analysis of the bioinformatics of whole genome sequence of the meticulous order-checking of genome simultaneously.The function of being familiar with these new genes is a challenge, has only the function that has fullyed understand these genes, understands their positions in the cell signal network regulation, and these gene informations could be human health service better.

Growth inhibited albumen (Inhibitor of Growth, ING) be one group of discovered in recent years can cell growth inhibiting and relevant albumen takes place with tumor.This histone has stronger conservative, and at yeast, nematicide all has its homogenic existence in the unicellular lower eukaryotes such as fruit bat.First growth inhibited protein gene ING1 is that Garkavtsev etc. utilized subtractive hybridization and hereditary straining element (Genetic suppressor element, a gene that GSE) screens in 1996 from galactophore epithelial cell.Expressing excessively of ING1 can suppress the growth of cell, and causes that cell was detained in the G1 phase of cell cycle.And the formation of the expression of ING1 antisense strand energy inducing cell colony and the growth on soft agar show the growth of ING1 energy negative regulation cell, so this new gene is named as growth suppressor gene (Inhibit of Growth).In research thereafter, discovery ING1 such as Garkavtesv only take place under the situation that wild type p53 exists the inhibitory action of growth, and there are synergism in prompting ING1 and p53 on function; Co-immunoprecipitation experiment simultaneously shows in the p53 albumen composition that is settled out with p53 antibody, can detect ING1 albumen, thereby confirms that ING1 can take place directly to interact with p53.These results show that ING1 is an important component part on the p53 signal path, play the negative regulation effect with the common cell growth of p53.After the research of ING1 gene structure in find ING1 exist 3 different shearings this, express p47ING1a, p33ING1b and p24ING1c respectively, wherein take place to interact with p53 and can be cytostatic be p33ING1b.

On the other hand, in head and neck phosphorus cell carcinoma and esophagus phosphorus cell carcinoma (ESCC), find in the genetics research to have high-frequency 13q33-34 zone heterozygosity disappearance (LOH), and the site of ING1 gene on chromosome just, this zone.Garkavtsev etc. find that in neuroblastoma the ING1 gene exists rearrangement or disappearance; Gunduz etc. find in the 6 routine ING1 exon 2s sudden change is arranged in 23 routine head and neck phosphorus cell carcinomas, these amino acid whose changes are positioned at a PHD domain or a possible motif that appraises and decides, thereby have influence on the proteic function of ING1.In breast carcinoma, find in the gastric cancer that the expression of ING1 is very low in addition, immunohistochemical staining ING1 albumen feminine gender among the ESCC, these evidence explanations ING1 may be a candidate tumor suppressor gene.

Development along with the Human Genome Project, American National biotechnology (the NCBI of information centre, National Center ofBiotechnology information) homologous other 4 growth suppressor gene (ING2 have successively been discharged with ING1, ING3, ING4, ING5), their albumen such as p33ING2, p47ING3, p29ING4 and ING4ING5 of encoding respectively, these proteic common ground are that their C ends all have conservative PHD (plant homeodomain) group.Wherein the homology of ING2 and ING3 and ING1 reaches 64% and 56% respectively; direct interaction does not all take place with p53 in studies show that ING2, the ING3 of Nagashima etc.; but can both play the negative regulation effect by the growth of p53 pair cell; wherein the similar cell that makes to ING1 of ING2 is arrested in the G0/G1 phase; and ING3 reduces S phase cell; ING2 can increase the acetylation of 383 lysines of p53 simultaneously, and ING3 does not cause this variation of p53.Recently discoverys ING2 such as Gozani is that (Phosphoinositides abridges: intranuclear receptor PtdINsPs), and the binding site of ING2 and PtdINsps is at the PHD group for important signal conduction micromolecule phosphatidylinositols.

The ING4 gene is the new growth suppressor gene family gene that Nanfang Research Centre, State Human Gene Group is cloned in the EST order-checking process to hypothalamus-hypophysis-hypothalamic pituitary adrenal axis express spectra research, ING4 and ING1 have 36% homology, similar with other ING family members, also contain a conservative PHD group at the C of ING1 end, there is a clear and definite nuclear localization signal (Nuclear Location Signal in the bioinformatic analysis discovery at the middle part of ING1, NLS), these information show that all ING4 may have the function similar to ING1.

At present, three of oncotherapy main means are: operative treatment, radiotherapy and chemotherapy.Chemotherapy is called for short chemotherapy, promptly refers to the tumor that heals with medicine.The main effect of chemotherapy in oncotherapy mainly contains aspect three.The one, as the radical treatment means, adopt chemotherapy can obtain better curative effect to hemopoietic system malignant tumor and some solid tumors, as leukemia, malignant lymphoma, choriocarcinoma, spermocytoma etc.; The 2nd, as the postoperative adjuvant chemotherapy, because of the operation and radiotherapy all belong to topical therapeutic, some are relatively limited to, sending out the very little tumor of trend can cure, but can not prevent or reduce metastasis, and chemotherapy belongs to systemic treatment, can kill remaining cancerous cell or micro metastasis in the body, make the tumor of sending out easily have considerable part to be cured, chemotherapy is occupied more and more consequence as a kind of whole body therapeutic in Comprehensive Treatment, along with the progress of the exploitation and the basic research of each kind new medicine, chemotherapy will become will study one of most active fields from now on, and range of application also can be more and more extensive.

Chemotherapy all has therapeutical effect to primary tumor, metastasis and subclinical metastasis, but the shortcoming of chemotherapy also is fairly obvious: 1, chemotherapeutics there is no specificity to killing tumor cell, it kill normally carefully be killed cause that human body occur to be felt sick, vomiting, alopecia, leukopenia ... Deng toxicity, side effect, usually make patient be difficult to tolerance; Powerful toxic and side effects makes chemotherapy be difficult to carry out, and excessively chemotherapy may shorten patient's survival period; 2, the part tumor types is insensitive to chemotherapy, adopts chemotherapy not have clinical treatment and is worth; The tumor of chemotherapy sensitivity through repeatedly after the chemotherapy tumor progressively being descended to chemosensitivity, is developed immunity to drugs, so that last the inefficacy.Therefore, the raising tumor cell is the important topic in oncotherapy field to the sensitivity of chemotherapeutics.

Growth inhibited protein family gene ING1 once was proved to be with ING2 can improve the sensitivity of different tumors to different chemotherapeutics, and we find that in the process of research ING4 gene function the high expressed of ING4 can obviously promote the drug susceptibility of different tumor cells to chemotherapeutics.

Summary of the invention

The object of the present invention is to provide the purposes of a kind of human ING4 gene as the sensitizer of tumor chemotherapeutic drug;

Another object of the present invention is to provide a kind of method that strengthens tumor cell to chemotherapy drug susceptibility.

Above-mentioned purpose of the present invention is achieved by the following technical solution:

(Inhibitor of Growth 4, ING4) gene is as the purposes of the sensitizer of tumor chemotherapeutic drug to the invention discloses human ING4.

Preferably, described tumor chemotherapeutic drug is amycin (doxorubicin) or etoposide (etoposide).

The present invention also provides human ING4 gene preparing as the purposes in the sensitizer of tumor chemotherapeutic drug.

Preferably, described tumor chemotherapeutic drug is amycin (doxorubicin) or etoposide (etoposide).

The present invention also provides a kind of method that strengthens tumor cell to chemotherapy drug susceptibility, and this method imports tumor cell with the ING4 gene before being included in chemotherapy, makes it to express.

Preferably, described method with ING4 gene importing tumor cell comprises with plasmid transfection or adenovirus mediated.

And preferred, described tumor chemotherapeutic drug is amycin (doxorubicin) and etoposide (etoposide).

The present invention also provides a kind of pharmaceutical composition that is used for the chemotherapy tumor, comprises known chemotherapeutics, and/or pharmaceutical carrier or excipient, and also comprises human ING4 gene or its encoding proteins.

Among the present invention, described " human ING4 gene " or " ING4 gene " are meant and comprise having the coded sequence shown in the SEQ ID NO:1; Perhaps with SEQ ID NO:1 in 18-767 position nucleotide sequence have the sequence of 70% homology at least; Perhaps under the moderate stringent condition with the sequence of sequence hybridization shown in the SEQ ID NO:1;

Described " encoding proteins of ING4 gene " is meant and comprises the sequence that has with aminoacid sequence at least 70% homology shown in the SEQ ID NO.2; Perhaps contain SEQ ID NO.2 polypeptide of sequence.

Compared with prior art, the present invention has following beneficial effect:

According to disclosed human ING4 gene as the purposes of the sensitizer of tumor chemotherapeutic drug and strengthen the method for tumor cell to chemotherapy drug susceptibility, the ING4 gene can be used and the chemotherapy of tumors process, thereby obviously promote the drug susceptibility of different tumor cells to chemotherapeutics, thereby can reduce the dosage of chemotherapeutics, and alleviate the toxic and side effects of chemotherapeutics to the human normal cell.

Description of drawings

Fig. 1 is prokaryotic expression GST-ING4 fusion rotein and antibody qualification result figure;

Fig. 2 is that Wester-blot identifies that ING4 crosses expression HepG2 stable cell line figure as a result;

Fig. 3 is the figure as a result that the HepG2 of cells were tested by flow cytometry chemotherapeutics processing stablizes the cell death of strain, DOX: amycin, ETO: etoposide;

Fig. 4 is DOX, and ETO handled after 48 hours, and ING4 crosses expression HepG2 stable cell line cell death increases sketch map;

Fig. 5 is the figure as a result that Bst XI enzyme action is identified recombinant adenovirus plasmid pAdING4;

Fig. 6 is the image that fluorescence microscope infects the HT1080 cell of the Ad-GFP of MOI100 and Ad-ING4;

Fig. 7 is the Western-blot image of adenovirus mediated ING4 gene high expressed in fibrosarcoma cell HT1080 cell;

Fig. 8 is that original position end-labelling (TUNEL) detects the apoptotic cell of HT1080 cell after 48 hours that amycin is handled the infection adenovirus, and the black point is an apoptotic cell;

Fig. 9 is that original position end-labelling (TUNEL) detects the apoptotic cell of HT1080 cell after 24 hours that the 30g/ml etoposide is handled the infection adenovirus, and the black point is an apoptotic cell.

The specific embodiment

Below in conjunction with drawings and Examples the present invention is further described.

Embodiment 1

The clone of people ING4 gene and pcDNA3.0-ING4 carrier for expression of eukaryon and pGEX 5x-1-ING4 construction of prokaryotic expression vector

1. design primer:

ING4-F:5 '-CCGGAATTCTGCTTCGAGATGGCTGCG-3 ' (SEQ ID NO.3) (containing EcoRI restricted enzyme point of contact)

NG4-R:5 '-CGGGGGTACCTTTCTTCTTCCGTTCTTGGGAG-3 (SEQ ID NO.4) (containing XhoI restricted enzyme point of contact)

2.PCR reaction

Composition	Volume
		Template
10 * Buffer forward primer (10pmol/ul) reverse primer (10pmol/ul) dNTP Mix (10mM of each dNTP) Pfu archaeal dna polymerase (5U/ul) dd H2O cumulative volume	0.4ul 1.0ul 0.5ul 0.5ul 0.2ul 0.1ul 7.3ul 10ul

PCR obtains total length open reading frame (ORF) fragment of ING4 from homemade human brain cDNA storehouse.

PCR condition: 94 ℃ of 4min; 94 ℃ of 40s, 60 ℃ of 45s, 72 ℃ of 1min, 35 circulations; 72 ℃ of 7min.

3.Xho1﹠amp; Ecor1 double digestion PCR product, plasmid

Composition	Volume
		Purified pcr product (1ug)/plasmid 10 * Buffer 2 Xho1 (10U/ul) Ecor1 (10U/ul) dd H2O cumulative volume	5ul 1ul 0.5ul 0.5ul 3ul 10ul

37 ℃ of enzyme action 4hr, 70 ℃ of heat inactivation 15min

4. connect

Composition	Volume
		Fragment double digestion product (50ng/ul) carrier double digestion product (400ng/ul) 10 * ligase Buffer T4DNAligase (6U/ul) dd H 2The O cumulative volume	3ul 0.3ul 0.5ul 0.5ul 0.7ul 5ul

16 ℃ connect 16hr.

5. electricity transforms: with parameter voltage 1.5kv, electric capacity 25 μ F, resistance 200 Ω carry out electricity and transform, and 1 μ l connection product is converted in the competent cell, and 37 ℃, 250rpm shaken cultivation 1 hour in the LB culture fluid is coated with flat board

6. bacterium colony PCR identifies: a plurality of bacterium colonies of picking are made PCR and are identified that the PCR reaction system is identical with the system of above-mentioned PCR respectively on long LB (Amp) flat board that bacterium colony arranged.

7. plasmid extracts: will identify positive colony plasmid pcDNA3.0-ING4 and pGEX 5x-1-ING4 through PCR, and adopt Plasmid DNA Miniprep System (Mini-M) the test kit extracting of VIOGENE company.It is correct that the gained plasmid inserts fragment through the nucleic acid sequencing checking.

Embodiment 2

Protokaryon and eukaryotic expression ING4 and anti-ING4 Polyclonal Antibody Preparation and evaluation

1.GST-ING4 the preparation of fusion rotein

The GST-p28 plasmid that 1) will build transforms the BL-21 bacterial strain, chooses the monoclonal colony inoculation in 5ml LB (Amp) culture medium, 37 ℃ of shaking table 300rpm overnight incubation.Bacterium liquid is inoculated among 1000ml 2 * YT (Amp), and 27 ℃ of 300rpm are cultured to OD ₆₀₀=0.6-0.8.Add 200 μ l 0.1M IPTG, continue overnight incubation.The centrifugal 10min of 8000rpm removes supernatant, adds 40ml PBS, the suspendible cell.Following operation is all carried out in 4 ℃.Add 1M DTT to ultimate density be 5mM, add PMSF (10mg/ml) to ultimate density be 100 μ g/ml.The ice-bath ultrasonic cell lysis becomes homogenate.Add 20%Triton X-100 to final concentration 1%, ice bath 30min.

The centrifugal 10min of 12000rpm gets supernatant, adds 1ml 50% agarose glutathion (Glutathione Sepharose4B), 4 ℃ of joltings 1 hour.

2) 4 ℃ of centrifugal 5min of 500g remove supernatant, add 4 ℃ of jolting 5min of 10ml PBS.

3) 4 ℃ of centrifugal 5min of 500g go to add 10ml PBS again behind the supernatant, transfer to behind the mixing in the pillar that purification uses, and open upper and lower cover, allow PBS flow out pillar automatically, replenish PBS simultaneously.

4) measure the OD of effluent with ultraviolet spectrophotometer ₂₈₀Value is until the OD of effluent and PBS ₂₈₀When value relatively is more or less the same, allow PBS flow to end, cover lower cover, add 1ml eluent (Glutathione Elution Buffer), cover loam cake, room temperature vibration mixing 30 minutes.

5) open upper and lower cover, allow eluent drip in the collecting pipe of 1.5ml.

6) when eluent just drips off, cover lower cover, add the 1ml eluent again, repeating step 4,5, and, when the protein content of collecting liquid is very low, stop eluting with the protein content of ultraviolet spectrophotometer measurement collection liquid.

7) will collect liquid and transfer in the bag filter of handling well, dialyse 24 hours at 4 ℃ of refrigerators with the PBS dialysis solution.Dialysis solution slowly stirs with magnetic stirring apparatus, changes dialysis solution therebetween 2-3 time.

8) the collection liquid after the dialysis is the good GST-ING4 fusion rotein of purification, with ultraviolet spectrophotometer measurement fusion proteic content, and with the purity of 10%SDS-PAGE electrophoresis detection fusion rotein.

2. animal immune

With the good GST-ING4 fusion rotein of purification immune rabbit according to a conventional method.After the immunity 2 months, get the titre of serum, and detect the specificity of antibody with Western blot with ELISA method monitoring IgG antibody.

3. antibody purification

After sensitivity and specificity reach requirement, put to death rabbit, collect blood and put room temperature and solidified l hour, put 4 ℃ of refrigerator overnight then, make blood clot retraction.

To pour in the 50ml centrifuge tube with the isolating serum of clot in second day, in 4 ℃ centrifugal 30 minutes (2500g), supernatant is incorporated in the serum of above 50ml centrifuge tube with clot, again with serum in 4 ℃ centrifugal 30 minutes (1500g).The supernatant packing is stored in-20 ℃ or by following method antibody purification.

Get 1ml 50% agarose Protein G (protein G Sepharose 4B) in 4 ℃ centrifugal 30 minutes (500g), remove supernatant, add 10ml binding buffer liquid (Wash/Binding Buffer, the 20mM sodium phosphate, 150mM NaCl, Ph7.4), mixing, 500g is centrifugal 5 minutes in 4 ℃, remove supernatant, add 0.8ml binding buffer liquid mixing again, transfer to purification with in the pillar, allow liquid flow out naturally, add the binding buffer liquid balance Protein G affinity column of 10 times of bed volumes then.

Get 1ml serum and dilute with isopyknic binding buffer liquid, join on the pillar that above balance crosses, effluent is upper prop 2 times again again.Be washed till the OD of effluent then with the binding buffer liquid of bed volume more than 10 times ₂₈₀Near background level.(100mM glycine-HCl, pH3.0) eluting antibody are collected effluent to add the 1ml eluent.Every pipe adds the neutralization buffer of 10 times of dilutions of 20 μ l (1M Tris-HCl, pH9.0), every pipe is collected about 300 μ l in the collecting pipe.Add 1ml eluent eluting again after eluting is intact, until the protein content of effluent near background level.Measure the content of antibody with ultraviolet spectrophotometer, the purity of 12%SDS-PAGE electrophoresis detection antibody, and with the specificity of Western blot detection antibody.In order to reduce the influence of GST antibody to experiment, we mix with 0.2ml 50% glutathione agarose granule with 0.5ml GST supernatant earlier.The glutathione agarose granule in conjunction with GST albumen after, with PBS flush away unconjugated albumen, repeat to wash once.Add 4 ℃ of 0.5ml ING4 antibody in conjunction with 1 hour, the supernatant after centrifugal is the anti-ING4 antibody of purification.

4. identify

Protein electrophoresis and commentaries on classics film: conventional protein electrophoresis sample separation; 10% separation gel, 60V 30min, 160V 1 hour 20 minutes.Pvdf membrane soaked 10 minutes with methanol, and deionized water rinsing was transferred in the transfering buffering liquid balance 20 minutes again; Running gel was put in the transfering buffering liquid balance 20 minutes; The ground paper backing plate also places the transfering buffering liquid balance.Placing in the following order changes the film instrument: negative pole-backing plate-running gel-pvdf membrane-backing plate-positive pole.Change film half an hour, 15 volts of voltages.

The sealing of film: 3% skim milk and 2%BSA/PBST 50ml, horizontal shaking table, room temperature sealing 4 hours; Also can first room temperature seal the some time, put into 4 ℃ of refrigerators again, spend the night;

One anti-(the anti-people ING4 of rabbit): anti-with confining liquid dilution one, the anti-people ING4 of rabbit extension rate: 1000 times; With film fully not in an anti-diluent.Horizontal shaking table, room temperature 2 hours also can be spent the night.

Washing: wash momently 2 times with PBST earlier; With＞4ml/cm ²The PBST of (membrane area), room temperature was washed 10 minutes; 3*10 minute, room temperature washing;

Two anti-(the anti-rabbit iggs of HRP-donkey): anti-with confining liquid dilution two, 10000 times of dilutions; Horizontal shaking table, room temperature 2 hours;

Washing: wash momently 2 times with PBST earlier; With＞4ml/cm ²The PBST of (membrane area), room temperature was washed 10 minutes; 3*10 minute, room temperature washing;

Detect: detectable (ECL plus) from 4 ℃ of taking-ups, is being opened forward horizontal stand to room temperature; With the A liquid of detectable and the B liquid mixed with 40: 1, the consumption of detectable is 0.1ml/cm ²Film places in the absorbent paper dry, protein powder up, detectable is added on the film, covers whole film surface; Room temperature 2 minutes; Pick up pvdf membrane with tweezers, the following corner of film is ridden on the napkin, blot unnecessary detectable; Film seals with preservative film, darkroom exposure, time: 10 seconds; Earlier with several of 10 second time multiple pressures, time expand, pressed one or several again, and time expand is washed the slice, thin piece of pressure at first tabletting the time.

The qualification result of prokaryotic expression GST-ING4 fusion rotein and antibody is seen Fig. 1.

Embodiment 3

Set up the stable cell line of expressing ING4

The HepG2 cell culture is in MEM culture medium (containing 10% hyclone, 1 * non essential amino acid), and other cell (COS-7, Hela, 293,3T3 etc.) is incubated at DMEM culture medium (containing 10% hyclone), at CO ₂37 ℃ of incubators, 5%CO2 cultivates.

1. transient transfection

A. liposome transfection method (lipofectamin, GIBCO BRL)

1) shop cell

Will cells transfected be inoculated in the culture dish by certain density, COS-7 cell inoculation density is 1～1.5 * 10 ⁵/ 35mm culture dish; The Hela cell is 2～4 * 10 ⁵/ 35mm culture dish;

2) 37 ℃, 5%CO ₂Cultivated 5-24 hour, and made cell attachment;

3) A pipe: plasmid consumption 1～2 μ g ,+100 μ l DMEM (serum-free, antibiotic-free), mixing;

B pipe: lipofectamine (lipofectamin) 3-5ul ,+100 μ l DMEM (serum-free, antibiotic-free), mixing;

A+B, mixing, room temperature leaves standstill 15-45min, general 30min;

4) cultured cell removes supernatant, washes once with DMEM (serum-free, antibiotic-free), adds 800ul DMEM (serum-free, antibiotic-free), splashes into the transfection mixed liquor, shakes up; Bad or do not tolerate serum-free medium, available complete medium as cell attachment;

5) 37 ℃, 5%CO ₂Cultivated 5-24 hour, changing liquid is DMEM (10%FCS), can the collecting cell analysis after transfection 24-72 hour.

B. calcium phosphate transfection method (ProFection ^Mammalian Transfection System, Promega)

1) in the previous day of transfectional cell cell is inoculated in the culture dish by certain density.

2) liquid was changed in transfection in preceding 3 hours, reagent is taken out from-20 ℃ of refrigerators melt, and balance is to room temperature.

3) reagent is shaken up, in A, B pipe, adds following ingredients (35mm culture dish):

A: plasmid x μ l (2-6 μ g)

2M CaCl ₂ 18.5μl

H ₂O 131.5-xμl

150μl

B：2xHBS 150μl

4) pipettor froths in the B pipe, with another pipettor with in the one after another drop of at leisure adding of the liquid in the A pipe B pipe.Adding the back room temperature left standstill 30 minutes.

5) mixed liquor after mixing leaves standstill once more, one after another drop of being added in the culture dish up and down, the culture dish that rocks from side to side, is evenly distributed on the cell calcium phosphate-DNA precipitation.

6) 37 ℃, 5%CO ₂Cultivate and change liquid after 4-16 hour.Can the collecting cell analysis after transfection 24-72 hour.

2. stable transfection

1) transfectional cell 6418 screening concentration determines

Seed cells in 6 orifice plates 0.5～1 * 10 ⁵Cells/well.The G418 that added 0,100,200,400,800,1600 μ g/ml culture fluid in second day respectively continues to cultivate to each hole.Change liquid once in per 4 days, and add the G418 of respective amount.Every day observation of cell death condition, during to the 14th day cell all the Cmin of dead used G418 be the G418 concentration of screening transfectional cell.

2) foundation of stable transfected cells strain

With the method transfectional cell of transient transfection, in 1: 10 ratio branch dish, and the G418 that adds respective amount screened cell transfecting after 48 hours.Changed liquid (containing G418) once in per 4 days.About about one month time (different cell screening asynchronism(-nization)), the cell of untransfected plasmid is all dead, and the cell of transfection band G418 resistant gene plasmid forms monoclonal.Sop up culture fluid, will be placed on the cell clone with the wetted filter paper of pancreatin (Clontech) and place 3-5 minute, filter paper is transferred in 24 orifice plates then and (added culture fluid/hole that 0.5ml contains G418 in advance), every Kong Fangyi cell clone.After cell clone has been selected 24 orifice plates are put 37 ℃ of cultivations, changed liquid (containing G418) once, and covered with in per 4 days up to cell.The cell that covers with is transferred to amplification culture in 6 orifice plates or the 35mm culture dish.

After the cell clone amplification culture, carry out RT-PCR with the sequencing primer on the plasmid and identify whether exogenous gene expresses, and Western blot identifies the height that ING4 expresses, and qualification result is seen Fig. 2 in monoclonal cell.Positive cell clone is frozen standby.

Embodiment 4

Crossing the HepG2 hepatoma cell strain of expressing ING4 increases the drug susceptibility of amycin and etoposide

HepG2 stable cell line and the control cells strain of the mistake of 1X104 being expressed ING4 are seeded in 24 orifice plates, cultivate after 24 hours for 37 ℃, the chemotherapeutics amycin (doxorubicin) and the etoposide (etoposide) that add various dose respectively, continue to cultivate 48 hours, the trypsinization collecting cell, the PBS washed cell is fixed with 70% ethanol, utilize the cells were tested by flow cytometry cell cycle, the hypodiploid cell is dead cell (Fig. 3).The result shows that the high expressed of ING4 can obviously increase the chemotherapy drug susceptibility (Fig. 4) of HepG2 hepatoma carcinoma cell.

Embodiment 5

The structure of adenovirus expression carrier, packing and amplification

1. the structure of shuttle plasmid pAdTrack-CMV-ING4,

1.1Xho1﹠amp; KpnI double digestion pcDNA3.0-ING4, pAdTrack-CMV plasmid

Composition	Volume
		1ug plasmid
10 * Buffer 2 Xho1 (10U/ul) Ecor1 (10U/ul) dd H2O cumulative volume	5ul 1ul 0.5ul 0.5ul 3ul 10ul

37 ℃ of enzyme action 4hr, 70 ℃ of heat inactivation 15min

1.2 glue reclaims the pcDNA3.0-ING4 enzyme action small fragment that contains the ING4 gene

1.3 connect

Composition	Volume
		Fragment double digestion product (50ng/ul) carrier double digestion product (400ng/ul) 10 * ligase Buffer T4DNAl igase (6U/ul) dd H 2The O cumulative volume	3ul 0.3ul 0.5ul 0.5ul 0.7ul 5ul

16 ℃ connect 16hr.

1.4 electricity transforms: with parameter voltage 1.5kv, electric capacity 25 μ F, resistance 200 Ω carry out electricity and transform, and 1 μ l is connected product be converted in the competent cell, and 37 ℃, 250rpm is shaken cultivation 1hr in the LB culture fluid, is coated with flat board.

1.5 bacterium colony PCR identifies: a plurality of bacterium colonies of picking are made PCR and are identified respectively on long LB (Amp) flat board that bacterium colony arranged.

1.6 plasmid extracts: will identify that positive colony plasmid pAdTrack-CMV-ING4 adopts Plasmid DNA Miniprep System (Mini-M) the test kit extracting of VIOGENE company through PCR.It is correct that the gained plasmid inserts fragment through the nucleic acid sequencing checking.

2.BJ5183 homologous recombination adenoviral plasmid pAdING4 in the antibacterial

2.1 preparation electroporation competence antibacterial

Place 37 ℃ to grow to OD escherichia coli BJ5183 and DH5 α ₅₅₀Be about at 0.8 o'clock, collect antibacterial, wash 2 times with 10% cold glycerol respectively, 500 times concentrate the back with the packing of 20 μ l/ pipe, put-80 ℃ of refrigerators and preserve.

2.2 homologous recombination produces adenoviral plasmid pAduPA in the antibacterial

The shuttle plasmid pAdTrack-CMV-ING that order-checking is correct QIAGEN-tip100 test kit (Qiagen) extracting, purification, get 1 μ g and successively use restricted enzyme Pme I (NEB) linearization for enzyme restriction, reclaim with QIAquickPCR Purification test kit (Qiagen) behind CIP (NEB) dephosphorylation, get 0.4 μ g and 0.1 μ g superhelix pAdEasy-1 plasmid 2,500V, 200ohms, electroporation cotransformation BJ5183 competence antibacterial under the 25 μ F conditions, 37 ℃ of shaking tables shake half an hour, kanamycin LB culture medium flat plate coated plate, select 16 minimum clones after 24 hours, QIAGEN-mini test kit (Qiagen) is taken out plasmid for a short time and is made Bst XI enzyme action and identify, qualification result is seen Fig. 5, select required recombinant adenovirus plasmid pAdING4, electroporation transforms DH5 α and increases in a large number.

3.293 cell packing amplification adenovirus AdING4 and AdGFP

With 293 cells with 2 * 10 ⁶/ hole is inoculated in 60mm culture dish (Nunc), cell density grows to 60%-80% behind the 24h, after the recovery of 8 μ g pAdING4 usefulness restricted enzyme Pac I (NEB) enzyme action, get in form (ratio is 1: 10) adding 293 cells of the linearizing pAduPA of 4 μ g with liposome-DNA complex, change the fresh training liquid that contains 10%FCS (HighClone) behind the 24h, observe GFP by fluorescence microscope (Olympus) after 3 days and express.Collect 293 cells multigelation 4 times in liquid nitrogen and 37 ℃ of water-baths after 7 days, infect 293 cells once more with 3m1/ ware virus supernatant and increase.Collecting cell after 3 days, centrifugal 7 minutes of 1500rpm carefully abandons supernatant, and is resuspended with 2ml PBS/ ware, multigelation 4 times; Repeated infection, collection step, the final viral supernatant of collecting is used for next step research.

4. measure AdING4 and AdGFP titre

With 293 cells with 5 * 10 ⁵/ hole is inoculated in six orifice plates (Nunc).After treating that next day cell density grows to 90%, will infect each porocyte respectively behind the viral supernatant doubling dilution, after 18 hours under the fluorescence microscope calculation expression GFP positive cell number measure AduPA and the AdGFP titre (expresses forming units per ml, efu/ml).

Embodiment 6

Adenovirus mediated ING4 gene is high expressed in fibrosarcoma cell HT1080 cell

With the HT1080 cell with 1 * 10 ⁵/ hole is inoculated in six orifice plates, cultivates after 24 hours, and virus is infected the HT1080 cell with MOI 25-500 titre, observes GFP after 2 days and expresses, and the results are shown in Figure 6; And cell lysis is got cell conditioned medium, the centrifugal 8min of 13000rpm.Collect centrifugal supernatant, get the expression that 40mg total protein western blot detects ING4, testing result is seen Fig. 7.

Embodiment 7

The ING4 high expressed increases the sensitivity of fibrosarcoma cell HT1080 cell to chemotherapeutics

With the HT1080 cell with 1 * 10 ⁵/ hole is inoculated in six orifice plates, cultivate after 24 hours, virus is infected the HT1080 cell with MOI 100 titres, after 6 hours, adding chemotherapeutics amycin (doxorubicin) and etoposide (etoposide) cultivated 24-48 hour, use original position end-labelling (TUNEL) and detect apoptotic cell (Fig. 8,9).The result shows that the high expressed of ING4 can obviously increase the chemotherapy drug susceptibility of HT1080 cell.

Sequence table

＜110〉Research Center of Shanghai Human Genome

＜120〉human ING4 gene is as the purposes of chemotherapeutics sensitizer

<130>NP-1305

<160>4

<170>PatentIn version 3.2

<210>1

<211>1377

<212>DNA

<213>Homo sapiens

<220>

<221>CDS

<222>(18)..(767)

<400>1

ctttgttttg cttcgag atg gct gcg ggg atg tat ttg gaa cat tat ctg 50

Met Ala Ala Gly Met Tyr Leu Glu His Tyr Leu

1 5 10

gac agt att gaa aac ctt ccc ttt gaa tta cag aga aac ttt cag ctc 98

Asp Ser Ile Glu Asn Leu Pro Phe Glu Leu Gln Arg Asn Phe Gln Leu

15 20 25

atg agg gac cta gac caa aga aca gag gac ctg aag gct gaa att gac 146

Met Arg Asp Leu Asp Gln Arg Thr Glu Asp Leu Lys Ala Glu Ile Asp

30 35 40

aag ttg gcc act gag tat atg agt agt gcc cgc agc ctg agc tcc gag 194

Lys Leu Ala Thr Glu Tyr Met Ser Ser Ala Arg Ser Leu Ser Ser Glu

45 50 55

gaa aaa ttg gcc ctt ctc aaa cag atc cag gaa gcc tat ggc aag tgc 242

Glu Lys Leu Ala Leu Leu Lys Gln Ile Gln Glu Ala Tyr Gly Lys Cys

60 65 70 75

aag gaa ttt ggt gac gac aag gtg cag ctt gcc atg cag acc tat gag 290

Lys Glu Phe Gly Asp Asp Lys Val Gln Leu Ala Met Gln Thr Tyr Glu

80 85 90

atg gtg gac aaa cac att cgg cgg ctg gac aca gac ctg gcc cgt ttt 338

Met Val Asp Lys His Ile Arg Arg Leu Asp Thr Asp Leu Ala Arg Phe

95 100 105

gag gct gat ctc aag gag aaa cag att gag tca agt gac tat gac agc 386

Glu Ala Asp Leu Lys Glu Lys Gln Ile Glu Ser Ser Asp Tyr Asp Ser

110 115 120

tct tcc agc aaa ggc aaa aag aaa ggc cgg act caa aag gag aag aaa 434

Ser Ser Ser Lys Gly Lys Lys Lys Gly Arg Thr Gln Lys Glu Lys Lys

125 130 135

gct gct cgt gct cgt tcc aaa ggg aaa aac tcg gat gaa gaa gcc ccc 482

Ala Ala Arg Ala Arg Ser Lys Gly Lys Asn Ser Asp Glu Glu Ala Pro

140 145 150 155

aag act gcc cag aag aag tta aag ctc gtg cgc aca agt cct gag tat 530

Lys Thr Ala Gln Lys Lys Leu Lys Leu Val Arg Thr Ser Pro Glu Tyr

160 165 170

ggg atg ccc tca gtg acc ttt ggc agt gtc cac ccc tct gat gtg ttg 578

Gly Met Pro Ser Val Thr Phe Gly Ser Val His Pro Ser Asp Val Leu

175 180 185

gat atg cct gtg gat ccc aac gaa ccc acc tat tgc ctt tgt cac cag 626

Asp Met Pro Val Asp Pro Asn Glu Pro Thr Tyr Cys Leu Cys His Gln

190 195 200

gtc tcc tat gga gag atg att ggc tgt gac aac cct gat tgt tcc att 674

Val Ser Tyr Gly Glu Met Ile Gly Cys Asp Asn Pro Asp Cys Ser Ile

205 210 215

gag tgg ttc cat ttt gcc tgt gtg ggg ctg aca acc aag cct cgg ggg 722

Glu Trp Phe His Phe Ala Cys Val Gly Leu Thr Thr Lys Pro Arg Gly

220 225 230 235

aaa tgg ttt tgc cca cgc tgc tcc caa gaa cgg aag aag aaa tag 767

Lys Trp Phe Cys Pro Arg Cys Ser Gln Glu Arg Lys Lys Lys

240 245

ataagggcct tggattccaa cacagtttct tccacatccc ctgacttggg ctagtgggca 827

gaggaatgcc tgtgctgggg ccaggggttc agggaggagt ggatggcaca gtgctgtcat 887

cccttctcct cccctctccc cactcccggt gctgaggctg catcagaccc tggtagggag 947

gggtgccgca gccactaacg gtatgtgctc tccttcagcc ctctccttcg gagggacgtg 1007

gtcttgccca ctgtcctttt gcctccatgc tgaggtcggt gctgtatttc agagggaggg 1067

tccttttcat tctccttgct ttgtatttaa ggactggggc atagcatggg ggcagtcccc 1127

cagacctctt cattccccct cctgtggtga gggctaggtg tgatcaacac ttttcttctc 1187

cattcccttc ctgctttttt catggtgggg atccaccagg tcatctagct ctggccctag 1247

ttgaagggca ccccttcctc tgtgccaaga ggattcatcc tgggagaggg ggcaaggtgg 1307

aatgcagata actcacatgt aaaaggaact tgggtaggta aataaaagct atacatgttg 1367

aaaaaaaaaa 1377

<210>2

<211>249

<212>PRT

<213>Homo sapiens

<400>2

Met Ala Ala Gly Met Tyr Leu Glu His Tyr Leu Asp Ser Ile Glu Asn

1 5 10 15

Leu Pro Phe Glu Leu Gln Arg Asn Phe Gln Leu Met Arg Asp Leu Asp

20 25 30

Gln Arg Thr Glu Asp Leu Lys Ala Glu Ile Asp Lys Leu Ala Thr Glu

35 40 45

Tyr Met Ser Ser Ala Arg Ser Leu Ser Ser Glu Glu Lys Leu Ala Leu

50 55 60

Leu Lys Gln Ile Gln Glu Ala Tyr Gly Lys Cys Lys Glu Phe Gly Asp

65 70 75 80

Asp Lys Val Gln Leu Ala Met Gln Thr Tyr Glu Met Val Asp Lys His

85 90 95

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

100 105 110

Glu Lys Gln Ile Glu Ser Ser Asp Tyr Asp Ser Ser Ser Ser Lys Gly

115 120 125

Lys Lys Lys Gly Arg Thr Gln Lys Glu Lys Lys Ala Ala Arg Ala Arg

130 135 140

Ser Lys Gly Lys Asn Ser Asp Glu Glu Ala Pro Lys Thr Ala Gln Lys

145 150 155 160

Lys Leu Lys Leu Val Arg Thr Ser Pro Glu Tyr Gly Met Pro Ser Val

165 170 175

Thr Phe Gly Ser Val His Pro Ser Asp Val Leu Asp Met Pro Val Asp

180 185 190

Pro Asn Glu Pro Thr Tyr Cys Leu Cys His Gln Val Ser Tyr Gly Glu

195 200 205

Met Ile Gly Cys Asp Asn Pro Asp Cys Ser Ile Glu Trp Phe His Phe

210 215 220

Ala Cys Val Gly Leu Thr Thr Lys Pro Arg Gly Lys Trp Phe Cys Pro

225 230 235 240

Arg Cys Ser Gln Glu Arg Lys Lys Lys

245

<210>3

<211>27

<212>DNA

＜213〉artificial sequence

<220>

<221>misc feature

＜223〉primer

<400>3

ccggaattct gcttcgagat ggctgcg 27

<210>4

<211>32

<212>DNA

＜213〉artificial sequence

<220>

<221>misc feature

＜223〉primer

<400>4

cgggggtacc tttcttcttc cgttcttggg ag 32

Claims (8)

1. (Inhibitor of Growth 4, ING4) gene is as the purposes of the sensitizer of tumor chemotherapeutic drug for human ING4.

2. human ING4 gene according to claim 1 is characterized in that as the purposes of the sensitizer of tumor chemotherapeutic drug described tumor chemotherapeutic drug is amycin (doxorubicin) and etoposide (etoposide).

3. human ING4 gene is preparing as the purposes in the sensitizer of tumor chemotherapeutic drug.

4. human ING4 gene according to claim 3 as the purposes in the sensitizer of tumor chemotherapeutic drug, is characterized in that described tumor chemotherapeutic drug is amycin (doxorubicin) or etoposide (etoposide) in preparation.

5. a method that strengthens tumor cell to chemotherapy drug susceptibility is characterized in that, described method imports tumor cell with the ING4 gene before being included in chemotherapy, makes it to express.

6. a kind of method that strengthens tumor cell to chemotherapy drug susceptibility according to claim 5 is characterized in that, described method with ING4 gene importing tumor cell comprises with plasmid transfection or adenovirus mediated.

7. according to claim 5 or 6 described a kind of methods that strengthen tumor cell to chemotherapy drug susceptibility, it is characterized in that described tumor chemotherapeutic drug is amycin (doxorubicin) or etoposide (etoposide).

8. the pharmaceutical composition that is used for the chemotherapy tumor comprises known chemotherapeutics, and/or pharmaceutical carrier or excipient, it is characterized in that, also comprises human ING4 gene or its encoding proteins.

Images