CN103372209B - Application of antibody of gp96 protein in preparation of cancer cell inhibitor - Google Patents
Application of antibody of gp96 protein in preparation of cancer cell inhibitor Download PDFInfo
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Abstract
The invention discloses an application of an antibody of gp96 protein in preparation of a cancer cell inhibitor. The invention discloses an application of the antibody of the gp96 protein showed in sequence 1 in a sequence table in preparation of a product. The product comprises the any one of the functions (1)-(6): (1) inhibiting amplification of cancer cells; (2) promoting apoptosis of the cancer cells; (3) inhibiting growth of cancer; (4) inhibiting tumor invasion; (5) inhibiting tumor transplantation; and (6) treating and/or preventing tumor. The application disclosed by the invention has a great value for treating cancer.
Description
Technical field
The application of the antibody that the present invention relates to gp96 albumen in preparing inhibition of cancer cell agent.
Background technology
Heat shock protein(HSP) (gp96 albumen) is present in the endoplasmic reticulum of the nearly all cell of humans and animals, is also present on the plasma membrane of some cell the member in Ta Shi heat shock protein 90 family (HSP90).
People's total length gp96 albumen is comprised of 803 amino acid, from the 1st to 21 amino-acid residues of N-terminal, forms signal peptide, and therefore ripe gp96 albumen is comprised of 782 amino acid.Gp96 albumen is glycoprotein, and this molecule has 6 potential glycosylation sites.
The gp96 of dog class is (with people's gp96 height homology, having 98.5 homology) crystalline structure of being combined with ATP resolves, individual molecule is divided into N end structure territory, centre (M) structural domain and C end structure territory, gp96 albumen exists with the form of homodimer, its N end is in conjunction with ATP, C end forms dimerization, and whole dimer holds N end around the left-handed distortion of molecule axis, to form the V font of distortion from C.
Gp96 albumen is relevant with its conformational change to the combination of ATP and the function of performance hydrolysising ATP, and its conformational change relates to the rotation of 90 ° in N end structure territory and M segment structure territory, and gp96 protein conformation changes combination or the release causing with other albumen.
Summary of the invention
The application of the antibody that the object of this invention is to provide gp96 albumen in preparing inhibition of cancer cell agent.
The application of the antibody of gp96 albumen shown in the sequence 1 of sequence table in preparing product; Described product has the function shown in arbitrary in (1) to (6) as follows; (1) anticancer propagation; (2) promote cancer cell-apoptosis; (3) suppress tumor growth; (4) suppress tumor invasion; (5) suppress tumour transplatation; (6) treat and/or prevent tumour.
The antibody of described gp96 albumen can be take the antibody that the surface antigen of gp96 albumen and/or gp96 albumen obtains as immunogen.The surface antigen of described gp96 albumen specifically can be the sequence 1 of sequence table from the polypeptide (being called for short N355 fragment) of N-terminal the 1st to 355 amino acids residues compositions.The antibody specific of described gp96 albumen can be take the monoclonal antibody that described gp96 albumen and described N355 fragment obtain as immunogen.
The antibody specific of described gp96 albumen can be the monoclonal antibody of mouse monoclonal hybridoma A-HSP96-6 secretion.
Mouse monoclonal hybridoma A-HSP96-6 has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center and (has been called for short CGMCC on 04 18th, 2012, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), preserving number is CGMCC No.6007.
In described (1) and described (2), described cancer cells is breast cancer cell, liver cancer cell, lung carcinoma cell, prostate cancer cell or stomach cancer cell.Described (3) in described (6), the tumour that the tumour that described tumour is the tumour that causes of breast cancer cell, liver cancer cell causes, the tumour that lung carcinoma cell causes, tumour that prostate cancer cell causes or stomach cancer cell cause.
The present invention also protects described mouse monoclonal hybridoma A-HSP96-6.
The present invention also protects the monoclonal antibody of described mouse monoclonal hybridoma A-HSP96-6 secretion.
The present invention also protects a kind of product, the antibody of gp96 albumen shown in the sequence 1 that its activeconstituents is sequence table; Described product has the function shown in arbitrary in (1) to (6) as follows; (1) anticancer propagation; (2) promote cancer cell-apoptosis; (3) suppress tumor growth; (4) suppress tumor invasion; (5) suppress tumour transplatation; (6) treat and/or prevent tumour.
The antibody specific of described gp96 albumen can be the monoclonal antibody of described mouse monoclonal hybridoma A-HSP96-6 secretion.
In described (1) and described (2), described cancer cells is breast cancer cell, liver cancer cell, lung carcinoma cell, prostate cancer cell or stomach cancer cell.Described (3) in described (6), the tumour that the tumour that described tumour is the tumour that causes of breast cancer cell, liver cancer cell causes, the tumour that lung carcinoma cell causes, tumour that prostate cancer cell causes or stomach cancer cell cause.
The present invention also protects gp96 albumen application in development as target spot shown in the sequence 1 of sequence table; Described product has the function shown in arbitrary in (1) to (6) as follows; (1) anticancer propagation; (2) promote cancer cell-apoptosis; (3) suppress tumor growth; (4) suppress tumor invasion; (5) suppress tumour transplatation; (6) treat and/or prevent tumour.
In described (1) and described (2), described cancer cells is breast cancer cell, liver cancer cell, lung carcinoma cell, prostate cancer cell or stomach cancer cell; Described (3) in described (6), the tumour that the tumour that described tumour is the tumour that causes of breast cancer cell, liver cancer cell causes, the tumour that lung carcinoma cell causes, tumour that prostate cancer cell causes or stomach cancer cell cause.
Shown in arbitrary above, breast cancer cell specifically can be SKBr3 cell or MDA-MB-231 cell.Arbitrary described liver cancer cell specifically can be SK-Hep-1 cell or Bel-7402 cell above.Arbitrary described lung carcinoma cell specifically can be NCI-H460 cell or A549 cell above.Arbitrary described prostate cancer cell specifically can be PC-3 cell or DU-145 cell above.Arbitrary described stomach cancer cell specifically can be BGC-823 cell or MNK-45 cell above.
The present invention's discovery, the antibody of gp96 albumen has anticancer propagation, promotes cancer cell-apoptosis, suppresses the effects such as tumour.The present invention has great value for the treatment of cancer.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples, all arranges and repeats experiment, results averaged for three times.ATCC is writing a Chinese character in simplified form of US mode culture collection warehousing (American type culture collection), network address
http:// www.atcc.org/..JCRB be Japanese cell bank, network address is
http:// cellbank.nibio.go.jp, be positioned at TOKYO.
PBS damping fluid: 8g NaCl, 0.2g KCl, 3.625g Na
2hPO
412H
2o, 0.24g KH
2pO
4, add water to 1L, adjust pH7.3.
SKBr3 cell (claim again SK-BR-3 cell, belong to breast cancer cell): ATCC is numbered HTB-30.
MDA-MB-231 cell (belonging to breast cancer cell): ATCC is numbered HTB-26;
SK-Hep-1 cell (claim again SK-HEP-1 cell, belong to liver cancer cell): ATCC is numbered HTB-52;
Bel-7402 cell (belonging to liver cancer cell): Chinese Typical Representative culture collection center, CCTCC be numbered GDC0035 (
http:// www.cctcc.org/biodb/cell/search_pure.phppage query contents input bel-7402);
NCI-H460 cell (belonging to lung carcinoma cell): ATCC is numbered HTB-177;
A549 cell (belonging to lung carcinoma cell): ATCC is numbered CCL-185;
PC-3 cell (belonging to prostate cancer cell): ATCC is numbered CRL-1435;
DU-145 cell (claim again DU 145, belong to prostate cancer cell): ATCC is numbered HTB-81.
BGC-823 cell (belonging to stomach cancer cell): Shanghai Inst. of Cytobiology, Chinese Academy of Sciences.
MNK-45 cell (claim again MNK45 cell, belong to stomach cancer cell): JCRB:JCRB is numbered JCRB0254.
Gp96 albumen is as shown in the sequence 1 of sequence table, and its encoding sequence is as shown in the sequence 2 of sequence table.GN355 fragment is the polypeptide that the sequence 1 of sequence table forms from N-terminal the 1st to 355 amino acids residues, and its encoding sequence is if the sequence 2 of sequence table is from as shown in the 1st to 1065 Nucleotide of 5 ' end.
Embodiment 1, prepare the monoclonal antibody of heat shock protein(HSP) (gp96 albumen)
One, the purifying of gp96 albumen
1, the in vitro human placenta of 70g-80g is soaked, remove coating shape material and reticular tissue, after shredding, add the NaHCO of 200mL pH7.4
3in buffered soln (containing 2mL PMSF) and organize grinding.
2, by centrifugal 35 minutes of 4 ℃, tissue juice, 13000rpm after grinding, get supernatant.
3, the supernatant of step 2 is added to PMSF, making its volumn concentration is 1%, then carries out the ammonium sulfate precipitation (being to add 29.1g ammonium sulfate in 100mL supernatant) of 50% saturation ratio, and 4 ℃ standing 4 hours, then centrifugal 35 minutes of 4 ℃, 13500rpm, get supernatant.
4, the supernatant of step 3 is added to PMSF, making its volumn concentration is 1%, then carries out the ammonium sulfate precipitation (being to add again 12.5g ammonium sulfate in 100mL supernatant) of 70% saturation ratio, 4 ℃ of standing over night, then centrifugal 90 minutes of 4 ℃, 16500rpm, get precipitation.
5, with 100mL affinity chromatography damping fluid (used time adds PMSF 2mL) dissolution precipitation, then carry out Con-ASepharose post affinity chromatography.
The correlation parameter of Con-A Sepharose post affinity chromatography is as follows: column length is 20cm, and internal diameter is 10mm; Adopt Con-A Sepharose post material (GE Healthcare, the U.S.) dress chromatography column 8-10mL;
The formula of affinity chromatography damping fluid: containing the PBS damping fluid of 200mM NaCl and 1mM PMSF.
The formula of affinity chromatography elutriant: containing the PBS damping fluid of 200mM NaCl, 1mM PMSF and 10g/mL α-D-Glucopyranose.
Con-A Sepharose post affinity chromatography to cross post flow process as follows: first, at 4 ℃ by least 3 column volumes of affinity chromatography damping fluid balance for Con-A Sepharose post; Then loading, flow velocity is 1mL/ minute, makes sample circulation loading 12 hours; After having gone up sample, with 25mL affinity chromatography elutriant (used time adds PMSF 1mM) recycling elution, spend the night, use again 3 column volumes of affinity chromatography elutriant wash-out next day, all elutriants of crossing after post are collected, with 0.22 μ m filter, filter.
6, the elutriant of step 5 being collected carries out Hitrap Q anion-exchange column affinity chromatography.
Hitrap Q anion-exchange column affinity chromatography adopts Hitrap Q anion-exchange column, and (chromatography column is HiTrapQ HP; Purchased from GE company, production number 17-1153-01).The internal diameter of chromatography column and column length are 0.7 * 2.5cm.
Ion-exchange A liquid is PBS damping fluid.
Ion-exchange B liquid is the PBS damping fluid containing 1M NaCl.
The post flow process excessively of Hitrap Q anion-exchange column affinity chromatography is as follows: by loading after 5-10 column volume of ion-exchange A liquid balance HitrapQ anion-exchange column; After loading, first use the elutriant wash-out foreign protein (volume ratio that is ion-exchange A liquid and ion-exchange B liquid is 8: 2) containing 20% ion-exchange B liquid, then in 30 minutes, ion-exchange B liquid shared volume parts in elutriant is risen to 100% by 20% linearity, flow velocity is 1mL/ minute; Elutriant after the post excessively that collection retention time is 10min to 15min.
7, after the post excessively of step 6 being collected, elutriant is concentrated into 500 μ L, then carries out Superdex-200 molecular sieve gel chromatography.
Molecular sieve damping fluid is PBS damping fluid.
With molecular sieve damping fluid balance Hiload 10/60 Superdex 200pg gel chromatography column, then the protein sample having concentrated is encircled and injected ATKA FPLC by loop, collect the protein peak of the about 94kDa of molecular weight, be the gp96 albumen of purifying.
Two, the preparation of N355 fragment
Utilize escherichia coli expression N355 fragment, concrete grammar is as follows:
1, design following N355 primer pair, by the handsome company in Shanghai synthetic primer:
Upstream primer: 5 '-CGCGGATCCGACGATGAAGTTGATGTGGAT-3 ';
Downstream primer: 5 '-CCGCTCGAGTTAAGTAAAGTGAATATAAGCCATG-3 '.
2, the mRNA that extracts human liver cancer cell HepG2 (purchased from ATCC, production number HB-8065), cDNA is synthesized in reverse transcription.
3, take the cDNA of step 2 is template, with N355 primer pair, carries out pcr amplification, obtains pcr amplification product (containing the encoding sequence of N355 fragment).
4,, with restriction enzyme BarnHI and Xho I double digestion pcr amplification product, reclaim enzyme and cut product.
5, with restriction enzyme BarnHI and Xho I double digestion pGEX-6P-1 plasmid (purchased from GE company, production code member 27-4597-01), reclaim carrier framework.
6, the carrier framework of the enzyme of step 4 being cut to product and step 5 is connected, and obtains connecting product.
7, will connect product and transform bacillus coli DH 5 alpha (purchased from Tian Gen biochemical technology company, production number CB101-03) competence, picking list bacterium colony carries out enzyme and cuts evaluation, and enzyme is cut and identified that positive bacterium colony extracts the plasmid evaluation of checking order.Sequencing result shows, has obtained recombinant plasmid pGEX-N355 (skeleton carrier is pGEX-6P-1, has inserted the encoding sequence of N355 fragment between BarnHI and Xho I restriction enzyme site).
8, recombinant plasmid pGEX-N355 is transformed to e. coli bl21 (DE3) (purchased from Tian Gen biochemical technology company, production number CB105-02) competent cell, from flat board, the access of picking list bacterium colony is containing 2 * YT substratum (Tryptones 16g of 100mg/mL penbritin, yeast extract 10g, sodium-chlor 5g, adds water and is settled to 1000mL) activation; Get activation solution (bacterium liquid) access 2 * YT substratum, 37 ℃ are cultured to OD
600value, for 0.6-1.0, then adds IPTG (making its final concentration is 1mmol/L), and 37 ℃ of induction 4h, collect thalline.
9, PBS damping fluid (140mmol/L NaCl, 2.7mmol/L KCl, 10mmol/L Na for thalline
2hPO
4, 1.8mmol/L KH
2pO
4; PH7.3) resuspended, ultrasonication under condition of ice bath (200W, broken 4s stops 6s, 99 3 circulations), then 4 ℃ of 12000 centrifugal 20min of turn/min, collects supernatant.
10,4 ℃ of supernatants are carried out to affinity chromatography, carrier is that Glutathione-Sepharose 4B is (purchased from GE company, production code member 17-5132-01), adopt reduced glutathion elution buffer (10mmol/L reduced glutathione, 50mmol/L Tris-HCl, pH8.0) wash-out, collects elutriant.
11, elutriant is replaced with to enzyme by ultrafiltration and concentration method and cuts buffer system (50mmol/L Tris-HCl, pH8.0; 150mmol/L NaCl; 1mmol/L DTT; 1mmol/L EDTA, pH8.0), add excessive PreScission Protease enzyme (PSP albumen; Purchased from GE company, production code member 27-0843-01), 4 ℃ of enzymes are cut 16h.
12, the enzyme system of cutting after enzyme is cut is replaced with PBS damping fluid by ultrafiltration and concentration method, then carries out affinity chromatography, and carrier is Glutathione-Sepharose 4B, adopt PBS damping fluid to carry out wash-out, GST and PSP are attached on chromatography column, collect elutriant, are N355 fragment.
Three, the acquisition of hybridoma
The 1st day, 100 μ g gp96 albumen (100 μ L adjust volume with aseptic PBS damping fluid) are evenly mixed by subcutaneous multi-point injection immune balb/c mice (purchased from Beijing Vital River Experimental Animals Technology Co., Ltd.) with isopyknic not formula Freund's complete adjuvant.In the time of the 14th day, with 50 μ g gp96 albumen, mix with isopyknic Freund's incomplete adjuvant, once, in the time of the 28th day, in kind booster immunization is once again for booster immunization.After one week after last booster immunization, with ELISA, detect serum antibody titer, because the antibody titer now obtaining is lower.Again mouse is carried out to booster immunization 7 times, be followed successively by 4 gp96 albumen booster immunizations (each 50 μ g) and 3 N355 fragment booster immunizations (each 50 μ g).After booster immunization the 3rd day, surveys serum titer with ELISA again the last time.
Altogether 6 mouse are carried out to above-mentioned experiment.Get mouse boosting cell and myeloma cell SP2/0 (catalog number TCM18 that serum antibody titer is the highest, Chinese Academy of Sciences's Shanghai cell bank) in the ratios of 5: 1, merge, by HAT screening culture medium, select to cultivate, with limiting dilution assay, obtain hybridoma, through 3-5 time, repeat screening, until ELISA result is all positive, so far obtain the cell strain of energy stably excreting monoclonal antibody specific.
Concrete immunologic process is in Table 1.
Table 1 detailed process
Obtain four strain of hybridoma, incite somebody to action a wherein strain called after mouse monoclonal hybridoma A-HSP96-6, other three strains are called after mouse monoclonal hybridoma I, mouse monoclonal hybridoma II and mouse monoclonal hybridoma III respectively.Mouse monoclonal hybridoma A-HSP96-6 has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center and (has been called for short CGMCC on 04 18th, 2012, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), preserving number is CGMCC No.6007.
Four, the preparation and purification of monoclonal antibody
1, increment culture method
The preparation method of cell culture medium (7.4): add calf serum and sodium bicarbonate in RPMI-1640 substratum, the final concentration of calf serum is 10% (quality percentage composition), and the final concentration of sodium bicarbonate is 0.2% (quality percentage composition).
Four strain of hybridoma that step 3 is obtained are placed in respectively cell culture medium, cultivate 2 days for 37 ℃, by sad-saturated ammonium sulphate method, the nutrient solution obtaining are carried out to purifying, obtain monoclonal antibody (20 ℃ of preservations).
Protein concn in monoclonal antibody (mg/ml)=1.45 * OD
280-0.74 * OD
260.
Adopt above formula to calculate the protein concn in monoclonal antibody.Protein concn in the monoclonal antibody that mouse monoclonal hybridoma A-HSP96-6 obtains is 1mg/ml.Protein concn in the monoclonal antibody that mouse monoclonal hybridoma I obtains is 0.6mg/ml.Protein concn in the monoclonal antibody that mouse monoclonal hybridoma II obtains is 0.7mg/ml.Protein concn in the monoclonal antibody that mouse monoclonal hybridoma III obtains is 0.5mg/ml.
Embodiment 2, use gp96 antibody significantly reduce tumor cell proliferation
One, use gp96 antibody significantly to reduce the propagation of breast cancer cell SKBr3.
By CCK-8 test kit (purchased from Japanese colleague's chemistry institute, article No. CK04-05), detect respectively various monoclonal antibodies prepared by the step 4 of embodiment 1 restraining effect to various cancer cells (SKBr3 cell, SK-Hep-1 cell, NCI-H460 cell, PC-3 cell or BGC-823 cell) propagation.Concrete operation step is as follows:
1, enchylema (concentration is 50000 cell/ml) is added in 96 orifice plates, every kind of cell arranges 24 multiple holes, and every hole adds 0.1ml enchylema.
2, packet transaction
After cell attachment, in 12 holes, add monoclonal antibody, every hole adds 0.1ml monoclonal antibody, and the final concentration of monoclonal antibody is 50ug/ml, as experimental group;
After cell attachment, in 12 holes, add PBS damping fluid, every hole adds 0.1mlPBS damping fluid, as a control group;
Equal 37 ℃ of normal cultivations of experimental group and control group, start timing from packet transaction, in different time check point (0,3,6,12 hours) sampling, get the sample in 3 holes respectively at every turn.
3, every hole sample adds CCK-8 detection reagent (component of CCK-8 test kit) 10ul, hatches the OD value (OD that measures 490nm after 2 hours for 37 ℃
490nmvalue).
Inhibitory rate of cell growth=(control group OD
490nmmean value-experimental group the OD of value
490nmthe mean value of value)/control group OD
490nmmean value * 100% of value.
Control group OD
490nmthe mean value of value is each time detecting of control group and puts each sample aperture OD
490nmthe mean value of value is 1.36.
Experimental group OD
490nmthe mean value of value is each time detecting of experimental group and puts each sample aperture OD
490nmthe mean value of value.
While adopting the monoclonal antibody that mouse monoclonal hybridoma A-HSP96-6 obtains: the inhibitory rate of cell growth of SKBr3 cell experiment group is 41.5%, the inhibitory rate of cell growth of SK-Hep-1 cell experiment group is 40.5%, the inhibitory rate of cell growth of NCI-H460 cell experiment group is 43%, the inhibitory rate of cell growth of PC-3 cell experiment group is that the inhibitory rate of cell growth of 42%, BGC-823 cell experiment group is 40%.The monoclonal antibody that the effect of the monoclonal antibody that other three kinds of mouse monoclonal hybridomas obtain obtains a little less than mouse monoclonal hybridoma A-HSP96-6, but to the growth inhibition ratio of each cell also all between 25% to 35%.Result shows, the monoclonal anti physical efficiency of embodiment 1 preparation significantly suppresses the propagation (growth) of SKBr3 cell, SK-Hep-1 cell, NCI-H460 cell, PC3 cell and BGC-823 cell.
Embodiment 3, use gp96 antibody induction apoptosis of tumor cells
Detect respectively various monoclonal antibodies prepared by the step 4 of embodiment 1 promoter action to cancer cells (SKBr3 cell, SK-Hep-1 cell, NCI-H460 cell, PC-3 cell or BGC-823 cell) apoptosis.Concrete steps are as follows:
1, enchylema is added and be inoculated in 6 porocyte culture plates, every hole 0.1ml, 200,000 cells/well.
2, packet transaction:
Experimental group (every kind of cell arranges three multiple holes): after cell attachment, every hole adds 0.1ml monoclonal antibody, and the final concentration of monoclonal antibody is 50ug/ml, 37 ℃ of normal cultivations 24 hours;
Negative control group (every kind of cell arranges three multiple holes): after cell attachment, every hole adds 0.1mlPBS damping fluid, 37 ℃ of normal cultivations 24 hours.
3, the test kit that uses Invitrogen company to produce
apoptosis Assay kit to cell dyeing after by flow cytometry analysis result, concrete operation steps is as follows:
(1) with the conventional peptic cell of pancreatin, with PBS damping fluid, wash twice.
(2) with 20ul 1 * Annexin V Buffer, cell is hanged gently, mix gently room temperature lucifuge dyeing 15 minutes after adding the FITC annexinV of 1ul.
(3) to adding 1 * Annexin V Buffer to make final volume in reaction tubes, be 200ul.
(4) adding concentration is the PI of 100ug/ml, and making its final concentration is 1ug/ml, and the dyeing of room temperature lucifuge can be gone up machine testing (apoptosis rate is directly measured by flow cytometer) in about 3 minutes.
The calculation formula of the apoptosis rate that experimental group increases than negative control group is: experimental group apoptosis rate-negative control group apoptosis rate.
Result is all got the mean value in three multiple holes.
While adopting the monoclonal antibody that mouse monoclonal hybridoma A-HSP96-6 obtains: for SKBr3 cell, the apoptosis rate of negative control group is 6.8%, and the apoptosis rate that experimental group increases than negative control group is 34.6%; For SK-Hep-1 cell, the apoptosis rate of negative control group is 6.8%, and the apoptosis rate that experimental group increases than negative control group is 33.7%; For NCI-H460 cell, the apoptosis rate of negative control group is 6.8%, and the apoptosis rate that experimental group increases than negative control group is 35.2%; For PC-3 cell, the apoptosis rate of negative control group is 6.8%, and the apoptosis rate that experimental group increases than negative control group is 34.3%; For BGC-823 cell, the apoptosis rate of negative control group is 6.8%, and the apoptosis rate that experimental group increases than negative control group is 33.8%.The monoclonal antibody that the effect of the monoclonal antibody that other three kinds of mouse monoclonal hybridomas obtain obtains a little less than mouse monoclonal hybridoma A-HSP96-6, but concerning each cell, the apoptosis rate that experimental group increases than negative control group is also all between 21% to 28%.Result shows, the monoclonal anti physical efficiency of embodiment 1 preparation obviously promotes the apoptosis of each cancer cells.
Embodiment 4, use gp96 antibody significantly suppress breast tumor growth in nude mouse.
Detect respectively various monoclonal antibodies prepared by the step 4 of embodiment 1 restraining effect to the growth of xenografted of breast cancer cell (SKBr3 cell or MDA-MB-231 cell).Concrete steps are as follows:
1, the breast cancer cell subcutaneous vaccination BALB/c nude mice (purchased from Beijing Vital River Experimental Animals Technology Co., Ltd.) of logarithmic phase will be cultured to, every inoculation 2,000,000 cells.
2, after 7 days, visible mouse forms tumour, and the mouse that forms tumour is divided into two groups at random, carries out respectively following treatment and processes:
Gp96 antibody group (5 mouse): carry out abdominal injection treatment with monoclonal antibody solution (by PBS damping fluid dilution monoclonal antibody), each every injection 0.5ml (containing 100ug monoclonal antibody protein), weekly treatment 3 times (respectively at the first day in this week, the 3rd day and the 6th day);
Negative control group (5 mouse): carry out abdominal injection treatment with PBS damping fluid, each every injection 0.5ml, weekly treatment 3 times (respectively at the first day in this week, the 3rd day and the 6th day).
3, treat continuously after 5 weeks and put to death nude mice, take tumor weight and calculate tumor control rate (all calculating the mean value of this group).
The calculation formula of tumor control rate is as follows: tumor weight * 100% of (weight of tumor weight-gp96 antibody group mouse tumor of negative control group mouse)/negative control group mouse.
While adopting the monoclonal antibody that mouse monoclonal hybridoma A-HSP96-6 obtains: for the nude mice of inoculation SKBr3 cell, the tumor weight of negative control group mouse is 1.12g (p < 0.01), and the tumor control rate of gp96 antibody group is 33.8%; For the nude mice of inoculation MDA-MB-231 cell, the tumor weight of negative control group mouse is 1.30g (p < 0.01), and the tumor control rate of gp96 antibody group is 35.6%.The monoclonal antibody that the effect of the monoclonal antibody that other three kinds of mouse monoclonal hybridomas obtain obtains a little less than mouse monoclonal hybridoma A-HSP96-6, but to the tumor control rate of each breast cancer cell also all between 23% to 26%.Result shows, the monoclonal anti physical efficiency of embodiment 1 preparation effectively suppresses breast cancer tumour growth.
Embodiment 5, use gp96 antibody significantly suppress lung cancer tumor growth in nude mouse.
Detect respectively various monoclonal antibodies prepared by the step 4 of embodiment 1 restraining effect to the growth of xenografted of lung carcinoma cell (NCI-H460 cell or A549 cell).Concrete steps are as follows:
1, the lung carcinoma cell subcutaneous vaccination BALB/c nude mice (purchased from Beijing Vital River Experimental Animals Technology Co., Ltd.) of logarithmic phase will be cultured to, every inoculation 2,000,000 cells.
2, after 7 days, visible mouse forms tumour, and the mouse that forms tumour is divided into two groups at random, carries out respectively following treatment and processes:
Gp96 antibody group (5 mouse): carry out abdominal injection treatment with monoclonal antibody solution (by PBS damping fluid dilution monoclonal antibody), each every injection 0.5ml (containing 100ug monoclonal antibody protein), weekly treatment 3 times (respectively at the first day in this week, the 3rd day and the 6th day);
Negative control group (5 mouse): carry out abdominal injection treatment with PBS damping fluid, each every injection 0.5ml, weekly treatment 3 times (respectively at the first day in this week, the 3rd day and the 6th day).
3, treat continuously after 5 weeks and put to death nude mice, take tumor weight and calculate tumor control rate (all calculating the mean value of this group).
The calculation formula of tumor control rate is as follows: tumor weight * 100% of (weight of tumor weight-gp96 antibody group mouse tumor of negative control group mouse)/negative control group mouse.
While adopting the monoclonal antibody that mouse monoclonal hybridoma A-HSP96-6 obtains: for the nude mice of inoculation NCI-H460 cell, the tumor weight of negative control group mouse is 1.25g (p < 0.01), and the tumor control rate of gp96 antibody group is 33.3%; For the nude mice of inoculation A549 cell, the tumor weight of negative control group mouse is 1.08g (p < 0.01), and the tumor control rate of gp96 antibody group is 34.6%.The monoclonal antibody that the effect of the monoclonal antibody that other three kinds of mouse monoclonal hybridomas obtain obtains a little less than mouse monoclonal hybridoma A-HSP96-6, but to the tumor control rate of each breast cancer cell also all between 22% to 24%.Result shows, the monoclonal anti physical efficiency of embodiment 1 preparation effectively suppresses lung cancer tumor growth.
Embodiment 6, gp96 antibody suppression prostate cancer tumor growth
Detect respectively various monoclonal antibodies prepared by the step 4 of embodiment 1 restraining effect to the growth of xenografted of prostate cancer cell (PC-3 cell or DU-145 cell).Concrete steps are as follows:
1, the prostate cancer cell subcutaneous vaccination BALB/c nude mice (purchased from Beijing Vital River Experimental Animals Technology Co., Ltd.) of logarithmic phase will be cultured to, every inoculation 2,000,000 cells.
2, after 7 days, visible mouse forms tumour, and the mouse that forms tumour is divided into two groups at random, carries out respectively following treatment and processes:
Gp96 antibody group (5 mouse): carry out abdominal injection treatment with monoclonal antibody solution (by PBS damping fluid dilution monoclonal antibody), each every injection 0.5ml (containing 100ug monoclonal antibody protein), weekly treatment 3 times (respectively at the first day in this week, the 3rd day and the 6th day);
Negative control group (5 mouse): carry out abdominal injection treatment with PBS damping fluid, each every injection 0.5ml, weekly treatment 3 times (respectively at the first day in this week, the 3rd day and the 6th day).
3, treat continuously after 5 weeks and put to death nude mice, take tumor weight and calculate tumor control rate (all calculating the mean value of this group).
The calculation formula of tumor control rate is as follows: tumor weight * 100% of (weight of tumor weight-gp96 antibody group mouse tumor of negative control group mouse)/negative control group mouse.
While adopting the monoclonal antibody that mouse monoclonal hybridoma A-HSP96-6 obtains: for the nude mice of inoculation PC-3 cell, the tumor weight of negative control group mouse is 1.52g (p < 0.01), and the tumor control rate of gp96 antibody group is 37.2%; For the nude mice of inoculation DU-145 cell, the tumor weight of negative control group mouse is 1.40g (p < 0.01), and the tumor control rate of gp96 antibody group is 30.6%.The monoclonal antibody that the effect of the monoclonal antibody that other three kinds of mouse monoclonal hybridomas obtain obtains a little less than mouse monoclonal hybridoma A-HSP96-6, but to the tumor control rate of each breast cancer cell also all between 26% to 29%.Result shows, the monoclonal anti physical efficiency of embodiment 1 preparation effectively suppresses prostate cancer tumor growth.
Embodiment 7, gp96 antibody suppression cancer of the stomach tumor growth
Detect respectively various monoclonal antibodies prepared by the step 4 of embodiment 1 restraining effect to the growth of xenografted of stomach cancer cell (BGC-823 cell or MNK-45 cell).Concrete steps are as follows:
1, the stomach cancer cell subcutaneous vaccination BALB/c nude mice (purchased from Beijing Vital River Experimental Animals Technology Co., Ltd.) of logarithmic phase will be cultured to, every inoculation 2,000,000 cells.
2, after 7 days, visible mouse forms tumour, and the mouse that forms tumour is divided into two groups at random, carries out respectively following treatment and processes:
Gp96 antibody group (5 mouse): carry out abdominal injection treatment with monoclonal antibody solution (by PBS damping fluid dilution monoclonal antibody), each every injection 0.5ml (containing 100ug monoclonal antibody protein), weekly treatment 3 times (respectively at the first day in this week, the 3rd day and the 6th day);
Negative control group (5 mouse): carry out abdominal injection treatment with PBS damping fluid, each every injection 0.5ml, weekly treatment 3 times (respectively at the first day in this week, the 3rd day and the 6th day).
3, treat continuously after 5 weeks and put to death nude mice, take tumor weight and calculate tumor control rate (all calculating the mean value of this group).
The calculation formula of tumor control rate is as follows: tumor weight * 100% of (weight of tumor weight-gp96 antibody group mouse tumor of negative control group mouse)/negative control group mouse.
While adopting the monoclonal antibody that mouse monoclonal hybridoma A-HSP96-6 obtains: for the nude mice of inoculation MNK-45 cell, the tumor weight of negative control group mouse is 1.28g (p < 0.01), and the tumor control rate of gp96 antibody group is 32.4%; For the nude mice of inoculation BGC-823 cell, the tumor weight of negative control group mouse is 1.16g (p < 0.01), and the tumor control rate of gp96 antibody group is 35.6%.The monoclonal antibody that the effect of the monoclonal antibody that other three kinds of mouse monoclonal hybridomas obtain obtains a little less than mouse monoclonal hybridoma A-HSP96-6, but to the tumor control rate of each breast cancer cell also all between 24% to 27%.Result shows, the monoclonal anti physical efficiency of embodiment 1 preparation effectively suppresses cancer of the stomach tumor growth.
Embodiment 8, gp96 antibody suppression liver cancer growth
Detect respectively various monoclonal antibodies prepared by the step 4 of embodiment 1 restraining effect to the growth of xenografted of liver cancer cell (SK-Hep-1 cell or Bel-7402 cell).Concrete steps are as follows:
1, the liver cancer cell subcutaneous vaccination BALB/c nude mice (purchased from Beijing Vital River Experimental Animals Technology Co., Ltd.) of logarithmic phase will be cultured to, every inoculation 2,000,000 cells.
2, after 7 days, visible mouse forms tumour, and the mouse that forms tumour is divided into two groups at random, carries out respectively following treatment and processes:
Gp96 antibody group (5 mouse): carry out abdominal injection treatment with monoclonal antibody solution (by PBS damping fluid dilution monoclonal antibody), each every injection 0.5ml (containing 100ug monoclonal antibody protein), weekly treatment 3 times (respectively at the first day in this week, the 3rd day and the 6th day);
Negative control group (5 mouse): carry out abdominal injection treatment with PBS damping fluid, each every injection 0.5ml, weekly treatment 3 times (respectively at the first day in this week, the 3rd day and the 6th day).
3, treat continuously after 5 weeks and put to death nude mice, take tumor weight and calculate tumor control rate (all calculating the mean value of this group).
The calculation formula of tumor control rate is as follows: tumor weight * 100% of (weight of tumor weight-gp96 antibody group mouse tumor of negative control group mouse)/negative control group mouse.
While adopting the monoclonal antibody that mouse monoclonal hybridoma A-HSP96-6 obtains: for the nude mice of inoculation SK-Hep-1 cell, the tumor weight of negative control group mouse is 1.45g (p < 0.01), and the tumor control rate of gp96 antibody group is 30.5%; For the nude mice of inoculation Bel-7402 cell, the tumor weight of negative control group mouse is 1.26g (p < 0.01), and the tumor control rate of gp96 antibody group is 34.1%.The monoclonal antibody that the effect of the monoclonal antibody that other three kinds of mouse monoclonal hybridomas obtain obtains a little less than mouse monoclonal hybridoma A-HSP96-6, but to the tumor control rate of each breast cancer cell also all between 23% to 27%.Result shows, the monoclonal anti physical efficiency of embodiment 1 preparation effectively suppresses liver cancer tumor growth.
Claims (7)
1. the application of the antibody of gp96 albumen shown in the sequence 1 of sequence table in preparing product; Described product has the function shown in arbitrary in (1) to (4) as follows: (1) anticancer propagation; (2) promote cancer cell-apoptosis; (3) suppress tumor growth; (4) treatment tumour;
In described (1) and described (2), described cancer cells is breast cancer cell, liver cancer cell, lung carcinoma cell, prostate cancer cell or stomach cancer cell; Described (3) in described (4), the tumour that the tumour that described tumour is the tumour that causes of breast cancer cell, liver cancer cell causes, the tumour that lung carcinoma cell causes, tumour that prostate cancer cell causes or stomach cancer cell cause.
2. application as claimed in claim 1, is characterized in that: the antibody of described gp96 albumen is the monoclonal antibody of mouse monoclonal hybridoma A-HSP96-6 secretion; The preserving number of described mouse monoclonal hybridoma A-HSP96-6 is CGMCC No.6007.
3. mouse monoclonal hybridoma A-HSP96-6, its preserving number is CGMCC No.6007.
4. the monoclonal antibody that described in claim 3, mouse monoclonal hybridoma A-HSP96-6 secretes.
5. a product, the antibody of gp96 albumen shown in the sequence 1 that its activeconstituents is sequence table; Described product has the function shown in arbitrary in (1) to (4) as follows: (1) anticancer propagation; (2) promote cancer cell-apoptosis; (3) suppress tumor growth; (4) treatment tumour;
In described (1) and described (2), described cancer cells is breast cancer cell, liver cancer cell, lung carcinoma cell, prostate cancer cell or stomach cancer cell; Described (3) in described (4), the tumour that the tumour that described tumour is the tumour that causes of breast cancer cell, liver cancer cell causes, the tumour that lung carcinoma cell causes, tumour that prostate cancer cell causes or stomach cancer cell cause.
6. product as claimed in claim 5, is characterized in that: the antibody of described gp96 albumen is monoclonal antibody claimed in claim 4.
7. the application in Dispersal risk as target spot of gp96 albumen shown in the sequence 1 of sequence table;
Described antibody has the function shown in arbitrary in (1) to (4) as follows: (1) anticancer propagation; (2) promote cancer cell-apoptosis; (3) suppress tumor growth; (4) treatment tumour;
In described (1) and described (2), described cancer cells is breast cancer cell, liver cancer cell, lung carcinoma cell, prostate cancer cell or stomach cancer cell; Described (3) in described (4), the tumour that the tumour that described tumour is the tumour that causes of breast cancer cell, liver cancer cell causes, the tumour that lung carcinoma cell causes, tumour that prostate cancer cell causes or stomach cancer cell cause.
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