CN103372209A - 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 present invention relates to the application of antibody in the agent of preparation inhibition of cancer cell of gp96 albumen.
Background technology
Heat shock protein (gp96 albumen) is present in the endoplasmic reticulum of the nearly all cell of humans and animals, also is present on the plasma membrane of some cell, and it is the member in the heat shock protein 90 family (HSP90).
People's total length gp96 albumen is comprised of 803 aminoacid, forms signal peptide from the 1st to 21 amino acid residue of N-terminal, and therefore ripe gp96 albumen is comprised of 782 aminoacid.Gp96 albumen is glycoprotein, and this molecule has 6 potential glycosylation sites.
The gp96 of dog class is (with people's gp96 height homology, 98.5 homology is arranged) crystal structure of being combined with ATP resolves, individual molecule is divided into N end structure territory, centre (M) domain and C end structure territory, gp96 albumen exists with the form of homodimer, its N end is in conjunction with ATP, the C end forms dimerization, and whole dimer holds the N end to form the V font of distortion around the left-handed distortion of molecule axis from C.
The function of the combination of gp96 albumen and ATP and performance hydrolysising ATP is relevant with its conformation change, and its conformation change relates to 90 ° the rotation in N end structure territory and M segment structure territory, and the gp96 protein conformation changes combination or the release of initiation and other albumen.
Summary of the invention
The purpose of this invention is to provide the application of antibody in the agent of preparation inhibition of cancer cell of gp96 albumen.
The application of antibody in preparing product of gp96 albumen shown in the sequence 1 of sequence table; Described product has the function shown in arbitrary in following (1) to (6); (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 tumor.
The antibody of described gp96 albumen can be the antibody that obtains as immunogen take the surface antigen of gp96 albumen and/or gp96 albumen.The surface antigen of described gp96 albumen specifically can be the sequence 1 of sequence table from the polypeptide (being called for short the N355 fragment) of N-terminal the 1st to 355 amino acids residue composition.The antibody specific of described gp96 albumen can be the monoclonal antibody that obtains as immunogen take described gp96 albumen and described N355 fragment.
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 common micro-organisms center and (has been called for short CGMCC on 04 18th, 2012, the 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 cancerous cell is breast cancer cell, hepatoma carcinoma cell, lung carcinoma cell, prostate gland cancer cell or stomach cancer cell.Described (3) in described (6), the tumor that the tumor that described tumor is the tumor that causes of breast cancer cell, hepatoma carcinoma cell causes, the tumor that lung carcinoma cell causes, tumor that prostate gland 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, and its active component is the antibody of gp96 albumen shown in the sequence 1 of sequence table; Described product has the function shown in arbitrary in following (1) to (6); (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 tumor.
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 cancerous cell is breast cancer cell, hepatoma carcinoma cell, lung carcinoma cell, prostate gland cancer cell or stomach cancer cell.Described (3) in described (6), the tumor that the tumor that described tumor is the tumor that causes of breast cancer cell, hepatoma carcinoma cell causes, the tumor that lung carcinoma cell causes, tumor that prostate gland cancer cell causes or stomach cancer cell cause.
The present invention also protects gp96 albumen shown in the sequence 1 of sequence table as the application of target spot in development; Described product has the function shown in arbitrary in following (1) to (6); (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 tumor.
In described (1) and described (2), described cancerous cell is breast cancer cell, hepatoma carcinoma cell, lung carcinoma cell, prostate gland cancer cell or stomach cancer cell; Described (3) in described (6), the tumor that the tumor that described tumor is the tumor that causes of breast cancer cell, hepatoma carcinoma cell causes, the tumor that lung carcinoma cell causes, tumor that prostate gland cancer cell causes or stomach cancer cell cause.
More than arbitrary shown in breast cancer cell specifically can be SKBr3 cell or MDA-MB-231 cell.More than arbitrary described hepatoma carcinoma cell specifically can be SK-Hep-1 cell or Bel-7402 cell.More than arbitrary described lung carcinoma cell specifically can be NCI-H460 cell or A549 cell.More than arbitrary described prostate gland cancer cell specifically can be PC-3 cell or DU-145 cell.More than arbitrary described stomach cancer cell specifically can be BGC-823 cell or MNK-45 cell.
The present invention finds that the antibody of gp96 albumen has the effects such as anticancer propagation, promotion cancer cell-apoptosis, inhibition tumor.The present invention has great value for the treatment of cancer.
The specific embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment if no special instructions, is conventional method.Used test material among the following embodiment if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples all arranges repeated experiments three times, results averaged.ATCC be US mode culture collection warehousing (American type culture collection) write a Chinese character in simplified form network address
Http:// www.atcc.org/.JCRB be Japanese cell bank, network address is
Http:// cellbank.nibio.go.jp, be positioned at TOKYO.
PBS buffer: 8g NaCl, 0.2g KCl, 3.625g Na
2HPO
412H
2O, 0.24g KH
2PO
4, add water to 1L, transfer pH7.3.
SKBr3 cell (claim again the 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 the SK-HEP-1 cell, belong to hepatoma carcinoma cell): ATCC is numbered HTB-52;
Bel-7402 cell (belonging to hepatoma carcinoma 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 gland cancer cell): ATCC is numbered CRL-1435;
DU-145 cell (claim again DU 145, belong to prostate gland 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 the MNK45 cell, belong to stomach cancer cell): JCRB:JCRB is numbered JCRB0254.
Gp96 albumen is shown in the sequence 1 of sequence table, and its coded sequence is shown in the sequence 2 of sequence table.The gN355 fragment is the polypeptide that the sequence 1 of sequence table forms from amino terminal the 1st to 355 amino acids residue, its coded sequence such as the sequence 2 of sequence table from shown in 5 ' terminal the 1st to 1065 nucleotide.
The monoclonal antibody of embodiment 1, preparation heat shock protein (gp96 albumen)
One, the purification of gp96 albumen
1, the human placenta that 70g-80g is exsomatized soaks, and removes peplos shape material and connective tissue, shreds the NaHCO of rear adding 200mL pH7.4
3In the buffer solution (containing 2mL PMSF) and organize grinding.
2,4 ℃ of tissue fluids, the 13000rpm after will grinding is centrifugal 35 minutes, gets supernatant.
3, the supernatant with step 2 adds PMSF, making its volumn concentration is 1%, then carries out the ammonium sulfate precipitation (being to add 29.1g ammonium sulfate in the 100mL supernatant) of 50% saturation, and 4 ℃ left standstill 4 hours, then centrifugal 35 minutes of 4 ℃, 13500rpm are got supernatant.
4, the supernatant with step 3 adds PMSF, making its volumn concentration is 1%, then carries out the ammonium sulfate precipitation (being to add again 12.5g ammonium sulfate in the 100mL supernatant) of 70% saturation, 4 ℃ of hold over night, then centrifugal 90 minutes of 4 ℃, 16500rpm are got precipitation.
5, with 100mL affinity chromatograph buffer (time spent adds PMSF 2mL) dissolution precipitation, then carry out Con-ASepharose post affinity chromatograph.
The relevant parameter of Con-A Sepharose post affinity chromatograph is as follows: column length is 20cm, and internal diameter is 10mm; Adopt Con-A Sepharose post material (GE Healthcare, the U.S.) dress chromatographic column 8-10mL;
The prescription of affinity chromatograph buffer: the PBS buffer that contains 200mM NaCl and 1mM PMSF.
The prescription of affinity chromatograph eluent: the PBS buffer that contains 200mM NaCl, 1mM PMSF and 10g/mL α-D-Glucopyranose..
Con-A Sepharose post affinity chromatograph to cross the post flow process as follows: at first, 4 ℃ with Con-A Sepharose post with at least 3 column volumes of affinity chromatograph buffer balance; Then loading, flow velocity is 1mL/ minute, makes sample circulation loading 12 hours; Gone up behind the sample and spent the night with 25mL affinity chromatograph eluent (time spent adds PMSF 1mM) recycling elution, used again 3 column volumes of affinity chromatograph eluent eluting next day, all eluents of crossing behind the post have been collected, filtered with 0.22 μ m filter.
6, the eluent of step 5 being collected carries out Hitrap Q anion-exchange column affinity chromatograph.
Hitrap Q anion-exchange column affinity chromatograph adopts Hitrap Q anion-exchange column, and (chromatographic column is HiTrapQ HP; Available from GE company, production number 17-1153-01).The internal diameter of chromatographic column and column length are 0.7 * 2.5cm.
Ion exchange A liquid is the PBS buffer.
Ion exchange B liquid is the PBS buffer that contains 1M NaCl.
The excessively post flow process of Hitrap Q anion-exchange column affinity chromatograph is as follows: with loading behind 5-10 column volume of ion exchange A liquid balance HitrapQ anion-exchange column; Loading is complete rear first with the eluent eluting foreign protein (volume ratio that is ion exchange A liquid and ion exchange B liquid is 8: 2) that contains 20% ion exchange B liquid, then in 30 minutes ion exchange B liquid shared volume parts in eluent is risen to 100% by 20% linearity, flow velocity is 1mL/ minute; Collect retention time and be the eluent behind the post of crossing of 10min to 15min.
7, eluent is concentrated into 500 μ L behind the excessively post of step 6 being collected, and then carries out Superdex-200 molecular sieve gel chromatography.
The molecular sieve buffer is the PBS buffer.
With molecular sieve buffer balance Hiload 10/60 Superdex 200pg gel chromatography column, the protein sample that then will concentrate injects ATKA FPLC by the loop ring, collects the protein peak of the about 94kDa of molecular weight, is the gp96 albumen of purification.
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:
Forward primer: 5 '-CGCGGATCCGACGATGAAGTTGATGTGGAT-3 ';
Downstream primer: 5 '-CCGCTCGAGTTAAGTAAAGTGAATATAAGCCATG-3 '.
2, extract the mRNA of human liver cancer cell HepG2 (available from ATCC, production number HB-8065), cDNA is synthesized in reverse transcription.
3, take the cDNA of step 2 as template, to carrying out pcr amplification, obtain pcr amplification product (coded sequence that contains the N355 fragment) with the N355 primer.
4, with restricted enzyme BarnHI and Xho I double digestion pcr amplification product, reclaim the enzyme action product.
5, with restricted enzyme BarnHI and Xho I double digestion pGEX-6P-1 plasmid (available from GE company, production code member 27-4597-01), reclaim carrier framework.
The carrier framework of 6, the enzyme action product of step 4 being connected with step connects, and obtains connecting product.
7, will connect product and transform bacillus coli DH 5 alpha (available from sky root biochemical technology company, production number CB101-03) competence, picking list bacterium colony carries out enzyme action to be identified, enzyme action identifies that positive bacterium colony extracts the plasmid evaluation of checking order.Sequencing result shows, has obtained recombiant plasmid pGEX-N355 (skeleton carrier is pGEX-6P-1, has inserted the coded sequence of N355 fragment between BarnHI and Xho I restriction enzyme site).
8, recombiant plasmid pGEX-N355 is transformed e. coli bl21 (DE3) (available from sky root biochemical technology company, production number CB105-02) competent cell, the access of picking list bacterium colony contains 2 * YT culture medium (tryptone 16g of 100mg/mL ampicillin from the flat board, yeast extract 10g, sodium chloride 5g adds water and is settled to 1000mL) activation; Get activating solution (bacterium liquid) access 2 * YT culture medium, 37 ℃ are cultured to OD
600Then value adds IPTG (making its final concentration is 1mmol/L) for 0.6-1.0, induces 4h for 37 ℃, collects thalline.
9, thalline PBS buffer (140mmol/L NaCl, 2.7mmol/L KCl, 10mmol/L Na
2HPO
4, 1.8mmol/L KH
2PO
4PH7.3) resuspended, ultrasonication under the condition of ice bath (200W, broken 4s stops 6s, 99 3 circulations) then turns for 4 ℃ 12000/the centrifugal 20min of min, collects supernatant.
10,4 ℃ of supernatants are carried out affinity chromatograph, carrier is that Glutathione-Sepharose 4B is (available from GE company, production code member 17-5132-01), adopt reduced glutathion elution buffer (10mmol/L reduced glutathione, 50mmol/L Tris-HCl, pH8.0) eluting is collected eluent.
11, eluent is replaced with enzyme action buffer system (50mmol/L Tris-HCl, pH8.0 with the ultrafiltration and concentration method; 150mmol/L NaCl; 1mmol/L DTT; 1mmol/L EDTA, pH8.0), add excessive PreScission Protease enzyme (PSP albumen; Available from GE company, production code member 27-0843-01), 4 ℃ of enzyme action 16h.
12, the enzyme action system behind the enzyme action is replaced with the PBS buffer with the ultrafiltration and concentration method, then carries out affinity chromatograph, carrier is Glutathione-Sepharose 4B, adopt the PBS buffer to carry out eluting, GST and PSP are attached on the chromatographic column, collect eluent, are the N355 fragment.
Three, the acquisition of hybridoma
The 1st day, 100 μ g gp96 albumen (100 μ L adjust volume with aseptic PBS buffer) are evenly mixed by subcutaneous multi-point injection immune balb/c mice (available from Beijing Vital River Experimental Animals Technology Co., Ltd.) with isopyknic not formula Freund's complete adjuvant.Mix with isopyknic incomplete Freunds adjuvant with 50 μ g gp96 albumen in the time of the 14th day, booster immunization once, in kind booster immunization is once again in the time of the 28th day.After the week behind the last booster immunization, detect serum antibody titer with ELISA, because the antibody titer that obtain this moment is lower.Again mice is carried out 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).Serum titer is surveyed with ELISA again in behind the booster immunization the 3rd day the last time.
Altogether 6 mices are carried out above-mentioned experiment.Get the highest mouse boosting cell of serum antibody titer and myeloma cell SP2/0 (catalog number TCM18, Chinese Academy of Sciences's Shanghai cell bank) merges in 5: 1 ratios, select to cultivate by the HAT screening culture medium, obtain hybridoma with limiting dilution assay, repeat screening through 3-5 time, until ELISA result is all positive, so far obtain the cell strain of energy stably excreting monoclonal antibody specific.
Concrete immunologic process sees 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 common micro-organisms center and (has been called for short CGMCC on 04 18th, 2012, the 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): in the RPMI-1640 culture medium, add calf serum and sodium bicarbonate, 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 place respectively cell culture medium, cultivate 2 days for 37 ℃, with sad-saturated ammonium sulfate method the culture fluid that obtains are carried out purification, obtain monoclonal antibody (20 ℃ of preservations).
Protein concentration in the monoclonal antibody (mg/ml)=1.45 * OD
280-0.74 * OD
260
Adopt the protein concentration in the above formula calculating monoclonal antibody.Protein concentration in the monoclonal antibody that mouse monoclonal hybridoma A-HSP96-6 obtains is 1mg/ml.Protein concentration in the monoclonal antibody that mouse monoclonal hybridoma I obtains is 0.6mg/ml.Protein concentration in the monoclonal antibody that mouse monoclonal hybridoma II obtains is 0.7mg/ml.Protein concentration 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.
Detect respectively the various monoclonal antibodies of step 4 preparation of embodiment 1 by CCK-8 test kit (available from Japanese colleague's chemistry institute, article No. CK04-05) to the inhibitory action of various cancerous cell (SKBr3 cell, SK-Hep-1 cell, NCI-H460 cell, PC-3 cell or BGC-823 cell) propagation.Concrete operation step is as follows:
1, (concentration is that 50000 cells/ml) add in 96 orifice plates, and every kind of cell arranges 24 multiple holes, and every hole adds the 0.1ml Cell sap with Cell sap.
2, packet transaction
Behind cell attachment, in 12 holes, add monoclonal antibody, every hole adds the 0.1ml monoclonal antibody, and the final concentration of monoclonal antibody is 50ug/ml, as experimental group;
Behind cell attachment, in 12 holes, add the PBS buffer, every hole adds the 0.1mlPBS buffer, in contrast group;
Equal 37 ℃ of normal cultivations of experimental group and matched group begin timing from packet transaction, in different time test point (0,3,6,12 hours) sampling, get the sample in 3 holes respectively at every turn.
3, every hole sample adding CCK-8 detectable (component of CCK-8 test kit) 10ul is hatched the OD value (OD that measure 490nm after 2 hours for 37 ℃
490nmValue).
Inhibitory rate of cell growth=(matched group OD
490nmThe meansigma methods of value-experimental group OD
490nmThe meansigma methods of value)/matched group OD
490nmThe meansigma methods of value * 100%.
Matched group OD
490nmThe meansigma methods of value is each time detecting of matched group and puts each sample aperture OD
490nmThe meansigma methods of value is 1.36.
Experimental group OD
490nmThe meansigma methods of value is each time detecting of experimental group and puts each sample aperture OD
490nmThe meansigma methods of value.
When 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 hybridomies 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%.The result shows that the monoclonal anti physical ability 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 the various monoclonal antibodies of step 4 preparation of embodiment 1 to the facilitation of cancerous cell (SKBr3 cell, SK-Hep-1 cell, NCI-H460 cell, PC-3 cell or BGC-823 cell) apoptosis.Concrete steps are as follows:
1, the Cell sap adding is 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): behind cell attachment, every hole adds the 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): behind cell attachment, every hole adds the 0.1mlPBS buffer, 37 ℃ of normal cultivations 24 hours.
3, the test kit that uses Invitrogen company to produce
Apoptosis Assay kit to cell dyeing after by the flow cytometry analysis result, concrete operating procedure is as follows:
(1) with the conventional peptic cell of pancreatin, washes twice with the PBS buffer.
(2) with 20ul 1 * Annexin V Buffer cell is hanged gently, add behind the FITC annexinV of 1ul gently mixing, room temperature lucifuge dyeing 15 minutes.
(3) adding 1 * Annexin V Buffer in the reaction tube, to make final volume 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.
Experimental group than the computing formula of the apoptosis rate that negative control group increases is: experimental group apoptosis rate-negative control group apoptosis rate.
The result all gets the meansigma methods in three multiple holes.
When adopting the monoclonal antibody that mouse monoclonal hybridoma A-HSP96-6 obtains: for the SKBr3 cell, the apoptosis rate of negative control group is 6.8%, and experimental group is 34.6% than the apoptosis rate that negative control group increases; For the SK-Hep-1 cell, the apoptosis rate of negative control group is 6.8%, and experimental group is 33.7% than the apoptosis rate that negative control group increases; For the NCI-H460 cell, the apoptosis rate of negative control group is 6.8%, and experimental group is 35.2% than the apoptosis rate that negative control group increases; For the PC-3 cell, the apoptosis rate of negative control group is 6.8%, and experimental group is 34.3% than the apoptosis rate that negative control group increases; For the BGC-823 cell, the apoptosis rate of negative control group is 6.8%, and experimental group is 33.8% than the apoptosis rate that negative control group increases.The monoclonal antibody that the effect of the monoclonal antibody that other three kinds of mouse monoclonal hybridomies 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%.The result shows that the monoclonal anti physical ability of embodiment 1 preparation obviously promotes the apoptosis of each cancerous cell.
Embodiment 4, use gp96 antibody significantly suppress breast tumor growth in the nude mouse.
Detect respectively the various monoclonal antibodies of step 4 preparation of embodiment 1 to the inhibitory action of the growth of xenografted of breast cancer cell (SKBr3 cell or MDA-MB-231 cell).Concrete steps are as follows:
1, will be cultured to the breast cancer cell subcutaneous vaccination BALB/c nude mice (available from Beijing Vital River Experimental Animals Technology Co., Ltd.) of exponential phase, every inoculation 2,000,000 cells.
2, after 7 days, visible mice forms tumor, and the mice that forms tumor is divided into two groups at random, carries out respectively following treatment and processes:
Gp96 antibody group (5 mices): carry out the lumbar injection treatment with monoclonal antibody solution (with PBS buffer dilution monoclonal antibody), each every injection 0.5ml (containing the 100ug monoclonal antibody protein), weekly treatment 3 times (respectively at this all first day, the 3rd day and the 6th day);
Negative control group (5 mices): carry out lumbar injection treatment with the PBS buffer, each every injection 0.5ml, weekly treatment 3 times (respectively at this all first day, the 3rd day and the 6th day).
3, treat continuously rear execution of 5 weeks nude mice, take by weighing tumor weight and calculate tumor control rate (all calculating the meansigma methods of this group).
The computing formula of tumor control rate is as follows: the tumor weight of (weight of the tumor weight of negative control group mice-gp96 antibody group mouse tumor)/negative control group mice * 100%.
When 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 mice 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 mice 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 hybridomies 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%.The result shows, the monoclonal anti physical ability establishment breast cancer tumour growth of embodiment 1 preparation.
Embodiment 5, use gp96 antibody significantly suppress pulmonary carcinoma tumor growth in the nude mouse.
Detect respectively the various monoclonal antibodies of step 4 preparation of embodiment 1 to the inhibitory action of the growth of xenografted of lung carcinoma cell (NCI-H460 cell or A549 cell).Concrete steps are as follows:
1, will be cultured to the lung carcinoma cell subcutaneous vaccination BALB/c nude mice (available from Beijing Vital River Experimental Animals Technology Co., Ltd.) of exponential phase, every inoculation 2,000,000 cells.
2, after 7 days, visible mice forms tumor, and the mice that forms tumor is divided into two groups at random, carries out respectively following treatment and processes:
Gp96 antibody group (5 mices): carry out the lumbar injection treatment with monoclonal antibody solution (with PBS buffer dilution monoclonal antibody), each every injection 0.5ml (containing the 100ug monoclonal antibody protein), weekly treatment 3 times (respectively at this all first day, the 3rd day and the 6th day);
Negative control group (5 mices): carry out lumbar injection treatment with the PBS buffer, each every injection 0.5ml, weekly treatment 3 times (respectively at this all first day, the 3rd day and the 6th day).
3, treat continuously rear execution of 5 weeks nude mice, take by weighing tumor weight and calculate tumor control rate (all calculating the meansigma methods of this group).
The computing formula of tumor control rate is as follows: the tumor weight of (weight of the tumor weight of negative control group mice-gp96 antibody group mouse tumor)/negative control group mice * 100%.
When 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 mice 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 mice 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 hybridomies 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%.The result shows, the monoclonal anti physical ability establishment pulmonary carcinoma tumor growth of embodiment 1 preparation.
Embodiment 6, gp96 antibody suppression carcinoma of prostate tumor growth
Detect respectively the various monoclonal antibodies of step 4 preparation of embodiment 1 to the inhibitory action of the growth of xenografted of prostate gland cancer cell (PC-3 cell or DU-145 cell).Concrete steps are as follows:
1, will be cultured to the prostate gland cancer cell subcutaneous vaccination BALB/c nude mice (available from Beijing Vital River Experimental Animals Technology Co., Ltd.) of exponential phase, every inoculation 2,000,000 cells.
2, after 7 days, visible mice forms tumor, and the mice that forms tumor is divided into two groups at random, carries out respectively following treatment and processes:
Gp96 antibody group (5 mices): carry out the lumbar injection treatment with monoclonal antibody solution (with PBS buffer dilution monoclonal antibody), each every injection 0.5ml (containing the 100ug monoclonal antibody protein), weekly treatment 3 times (respectively at this all first day, the 3rd day and the 6th day);
Negative control group (5 mices): carry out lumbar injection treatment with the PBS buffer, each every injection 0.5ml, weekly treatment 3 times (respectively at this all first day, the 3rd day and the 6th day).
3, treat continuously rear execution of 5 weeks nude mice, take by weighing tumor weight and calculate tumor control rate (all calculating the meansigma methods of this group).
The computing formula of tumor control rate is as follows: the tumor weight of (weight of the tumor weight of negative control group mice-gp96 antibody group mouse tumor)/negative control group mice * 100%.
When 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 mice 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 mice 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 hybridomies 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%.The result shows, the monoclonal anti physical ability establishment carcinoma of prostate tumor growth of embodiment 1 preparation.
Embodiment 7, gp96 antibody suppression gastric cancer tumor growth
Detect respectively the various monoclonal antibodies of step 4 preparation of embodiment 1 to the inhibitory action of the growth of xenografted of stomach cancer cell (BGC-823 cell or MNK-45 cell).Concrete steps are as follows:
1, will be cultured to the stomach cancer cell subcutaneous vaccination BALB/c nude mice (available from Beijing Vital River Experimental Animals Technology Co., Ltd.) of exponential phase, every inoculation 2,000,000 cells.
2, after 7 days, visible mice forms tumor, and the mice that forms tumor is divided into two groups at random, carries out respectively following treatment and processes:
Gp96 antibody group (5 mices): carry out the lumbar injection treatment with monoclonal antibody solution (with PBS buffer dilution monoclonal antibody), each every injection 0.5ml (containing the 100ug monoclonal antibody protein), weekly treatment 3 times (respectively at this all first day, the 3rd day and the 6th day);
Negative control group (5 mices): carry out lumbar injection treatment with the PBS buffer, each every injection 0.5ml, weekly treatment 3 times (respectively at this all first day, the 3rd day and the 6th day).
3, treat continuously rear execution of 5 weeks nude mice, take by weighing tumor weight and calculate tumor control rate (all calculating the meansigma methods of this group).
The computing formula of tumor control rate is as follows: the tumor weight of (weight of the tumor weight of negative control group mice-gp96 antibody group mouse tumor)/negative control group mice * 100%.
When 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 mice 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 mice 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 hybridomies 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%.The result shows, the monoclonal anti physical ability establishment gastric cancer tumor growth of embodiment 1 preparation.
Embodiment 8, gp96 antibody suppression liver cancer growth
Detect respectively the various monoclonal antibodies of step 4 preparation of embodiment 1 to the inhibitory action of the growth of xenografted of hepatoma carcinoma cell (SK-Hep-1 cell or Bel-7402 cell).Concrete steps are as follows:
1, will be cultured to the hepatoma carcinoma cell subcutaneous vaccination BALB/c nude mice (available from Beijing Vital River Experimental Animals Technology Co., Ltd.) of exponential phase, every inoculation 2,000,000 cells.
2, after 7 days, visible mice forms tumor, and the mice that forms tumor is divided into two groups at random, carries out respectively following treatment and processes:
Gp96 antibody group (5 mices): carry out the lumbar injection treatment with monoclonal antibody solution (with PBS buffer dilution monoclonal antibody), each every injection 0.5ml (containing the 100ug monoclonal antibody protein), weekly treatment 3 times (respectively at this all first day, the 3rd day and the 6th day);
Negative control group (5 mices): carry out lumbar injection treatment with the PBS buffer, each every injection 0.5ml, weekly treatment 3 times (respectively at this all first day, the 3rd day and the 6th day).
3, treat continuously rear execution of 5 weeks nude mice, take by weighing tumor weight and calculate tumor control rate (all calculating the meansigma methods of this group).
The computing formula of tumor control rate is as follows: the tumor weight of (weight of the tumor weight of negative control group mice-gp96 antibody group mouse tumor)/negative control group mice * 100%.
When 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 mice 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 mice 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 hybridomies 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%.The result shows, the monoclonal anti physical ability establishment hepatocarcinoma tumor growth of embodiment 1 preparation.
Claims (10)
1. the application of antibody in preparing product of gp96 albumen shown in the sequence 1 of sequence table; Described product has the function shown in arbitrary in following (1) to (6); (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 tumor.
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. application as claimed in claim 1 or 2 is characterized in that: in described (1) and described (2), described cancerous cell is breast cancer cell, hepatoma carcinoma cell, lung carcinoma cell, prostate gland cancer cell or stomach cancer cell; Described (3) in described (6), the tumor that the tumor that described tumor is the tumor that causes of breast cancer cell, hepatoma carcinoma cell causes, the tumor that lung carcinoma cell causes, tumor that prostate gland cancer cell causes or stomach cancer cell cause.
4. mouse monoclonal hybridoma A-HSP96-6, its preserving number is CGMCC No.6007.
5. the monoclonal antibody of the described mouse monoclonal hybridoma of claim 4 A-HSP96-6 secretion.
6. product, its active component are the antibody of gp96 albumen shown in the sequence 1 of sequence table; Described product has the function shown in arbitrary in following (1) to (6); (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 tumor.
7. product as claimed in claim 6, it is characterized in that: the antibody of described gp96 albumen is monoclonal antibody claimed in claim 5.
8. such as claim 6 or 7 described products, it is characterized in that: in described (1) and described (2), described cancerous cell is breast cancer cell, hepatoma carcinoma cell, lung carcinoma cell, prostate gland cancer cell or stomach cancer cell; Described (3) in described (6), the tumor that the tumor that described tumor is the tumor that causes of breast cancer cell, hepatoma carcinoma cell causes, the tumor that lung carcinoma cell causes, tumor that prostate gland cancer cell causes or stomach cancer cell cause.
9. gp96 albumen shown in the sequence 1 of sequence table is as the application of target spot in development; Described product has the function shown in arbitrary in following (1) to (6); (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 tumor.
10. application as claimed in claim 9 is characterized in that: in described (1) and described (2), described cancerous cell is breast cancer cell, hepatoma carcinoma cell, lung carcinoma cell, prostate gland cancer cell or stomach cancer cell; Described (3) in described (6), the tumor that the tumor that described tumor is the tumor that causes of breast cancer cell, hepatoma carcinoma cell causes, the tumor that lung carcinoma cell causes, tumor that prostate gland cancer cell causes or stomach cancer cell cause.
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| CN104356233B (en) * | 2014-10-11 | 2017-09-29 | 中国科学院微生物研究所 | A kind of human heat shock protein gp96 scFv antibody and preparation method and application |
| CN118126201A (en) * | 2024-05-06 | 2024-06-04 | 云南大学 | Cell membrane targeting GRP94 chimeric antigen receptor, engineering cell and application |
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| CN104356233B (en) * | 2014-10-11 | 2017-09-29 | 中国科学院微生物研究所 | A kind of human heat shock protein gp96 scFv antibody and preparation method and application |
| CN118126201A (en) * | 2024-05-06 | 2024-06-04 | 云南大学 | Cell membrane targeting GRP94 chimeric antigen receptor, engineering cell and application |
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