CN103372209B - Application of antibody of gp96 protein in preparation of cancer cell inhibitor - Google Patents

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

Application of gp96 protein antibody in preparation of cancer cell inhibitor

technical field

The invention relates to the application of the gp96 protein antibody in the preparation of cancer cell inhibitors.

Background technique

Heat shock protein (gp96 protein) exists in the endoplasmic reticulum membrane of almost all cells of humans and animals, and also exists on the plasma membrane of some cells. It is a member of the heat shock protein 90 family (HSP90).

The full-length human gp96 protein consists of 803 amino acids, and the 1st to 21st amino acid residues from the N-terminal constitute the signal peptide, so the mature gp96 protein consists of 782 amino acids. The gp96 protein is a glycoprotein, and the molecule has 6 potential glycosylation sites.

The crystal structure of canine gp96 (highly homologous to human gp96, with a homology of 98.5) and ATP has been resolved, and a single molecule is divided into an N-terminal domain, a middle (M) domain and a C-terminal domain. The gp96 protein exists in the form of a homodimer, its N-terminus binds to ATP, and its C-terminus forms a dimerization. The entire dimer twists left-handed around the molecular axis from the C-terminus to the N-terminus to form a twisted V-shape.

The binding of gp96 protein to ATP and the function of hydrolyzing ATP are related to its conformational change. The conformational change involves the 90° rotation of the N-terminal domain and the M-segment domain. The conformational change of gp96 protein triggers the binding or release of other proteins.

Contents of the invention

The purpose of the present invention is to provide the application of the gp96 protein antibody in the preparation of cancer cell inhibitors.

Application of the antibody to the gp96 protein shown in Sequence 1 of the Sequence Listing in the preparation of products; the product has the functions shown in any of the following (1) to (6); (1) inhibiting cancer cell proliferation; (2) promoting Cancer cell apoptosis; (3) inhibiting tumor growth; (4) inhibiting tumor invasion; (5) inhibiting tumor transplantation; (6) treating and/or preventing tumors.

The antibody to the gp96 protein can be an antibody obtained by using the gp96 protein and/or the surface antigen of the gp96 protein as an immunogen. The surface antigen of the gp96 protein can specifically be a polypeptide consisting of amino acid residues 1 to 355 at the N-terminal of Sequence 1 in the sequence listing (abbreviated as N355 fragment). The gp96 protein antibody can specifically be a monoclonal antibody obtained by using the gp96 protein and the N355 fragment as immunogens.

The antibody to the gp96 protein can specifically be a monoclonal antibody secreted by the mouse monoclonal hybridoma cell A-HSP96-6.

The mouse monoclonal hybridoma cell A-HSP96-6 has been preserved in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures (CGMCC for short) on April 18, 2012. The address is: Courtyard 3, No. 1 Beichen West Road, Chaoyang District, Beijing No.), the deposit number is CGMCC No.6007.

In the above (1) and the above (2), the cancer cells are breast cancer cells, liver cancer cells, lung cancer cells, prostate cancer cells or gastric cancer cells. In the above (3) to the above (6), the tumor is a tumor caused by breast cancer cells, a tumor caused by liver cancer cells, a tumor caused by lung cancer cells, a tumor caused by prostate cancer cells or a tumor caused by gastric cancer cells.

The invention also protects the mouse monoclonal hybridoma cell A-HSP96-6.

The invention also protects the monoclonal antibody secreted by the mouse monoclonal hybridoma cell A-HSP96-6.

The present invention also protects a product whose active ingredient is an antibody to the gp96 protein shown in Sequence 1 of the Sequence Listing; the product has the functions shown in any of the following (1) to (6); (1) inhibits cancer Cell proliferation; (2) promote cancer cell apoptosis; (3) inhibit tumor growth; (4) inhibit tumor invasion; (5) inhibit tumor transplantation; (6) treat and/or prevent tumors.

The antibody to the gp96 protein can specifically be a monoclonal antibody secreted by the mouse monoclonal hybridoma cell A-HSP96-6.

In the above (1) and the above (2), the cancer cells are breast cancer cells, liver cancer cells, lung cancer cells, prostate cancer cells or gastric cancer cells. In the above (3) to the above (6), the tumor is a tumor caused by breast cancer cells, a tumor caused by liver cancer cells, a tumor caused by lung cancer cells, a tumor caused by prostate cancer cells or a tumor caused by gastric cancer cells.

The present invention also protects the application of the gp96 protein shown in Sequence 1 of the sequence listing as a target in the development of products; the product has the functions shown in any of the following (1) to (6); (1) inhibits the proliferation of cancer cells (2) promote cancer cell apoptosis; (3) inhibit tumor growth; (4) inhibit tumor invasion; (5) inhibit tumor transplantation; (6) treat and/or prevent tumors.

In (1) and (2), the cancer cells are breast cancer cells, liver cancer cells, lung cancer cells, prostate cancer cells or gastric cancer cells; in the (3) to (6), the The tumor is a tumor derived from breast cancer cells, a tumor derived from liver cancer cells, a tumor derived from lung cancer cells, a tumor derived from prostate cancer cells, or a tumor derived from stomach cancer cells.

Any one of the breast cancer cells shown above can specifically be SKBr3 cells or MDA-MB-231 cells. Any one of the above liver cancer cells can specifically be SK-Hep-1 cells or Bel-7402 cells. Any one of the lung cancer cells mentioned above can specifically be NCI-H460 cells or A549 cells. Any one of the above prostate cancer cells can specifically be PC-3 cells or DU-145 cells. Any of the above gastric cancer cells can specifically be BGC-823 cells or MNK-45 cells.

The present invention finds that the gp96 protein antibody has the functions of inhibiting the proliferation of cancer cells, promoting the apoptosis of cancer cells, inhibiting tumors and the like. The invention has great value for the treatment of cancer.

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. Quantitative experiments in the following examples were all set up to repeat the experiments three times, and the results were averaged. ATCC is the abbreviation of American type culture collection, website http://www.atcc.org/ . .JCRB is the Japanese cell bank, the website is http://cellbank.nibio.go.jp , located in TOKYO.

PBS buffer: 8g NaCl, 0.2g KCl, 3.625g Na ₂ HPO ₄ ·12H ₂ O, 0.24g KH ₂ PO ₄ , add water to 1L, adjust pH to 7.3.

SKBr3 cells (also known as SK-BR-3 cells, belonging to breast cancer cells): ATCC number is HTB-30.

MDA-MB-231 cells (belonging to breast cancer cells): ATCC code is HTB-26;

SK-Hep-1 cells (also known as SK-HEP-1 cells, belonging to liver cancer cells): ATCC code is HTB-52;

Bel-7402 cells (belonging to liver cancer cells): China Center for Type Culture Collection, CCTCC No. GDC0035 ( http://www.cctcc.org/biodb/cell/search_pure.php page query content input bel-7402);

NCI-H460 cells (belonging to lung cancer cells): ATCC code is HTB-177;

A549 cells (belonging to lung cancer cells): ATCC code is CCL-185;

PC-3 cells (belonging to prostate cancer cells): ATCC number is CRL-1435;

DU-145 cells (also known as DU 145, belonging to prostate cancer cells): ATCC number is HTB-81.

BGC-823 cells (belonging to gastric cancer cells): Shanghai Institute of Cell Biology, Chinese Academy of Sciences.

MNK-45 cells (also known as MNK45 cells, belonging to gastric cancer cells): JCRB: JCRB number is JCRB0254.

The gp96 protein is shown in sequence 1 of the sequence listing, and its coding sequence is shown in sequence 2 of the sequence listing. The gN355 fragment is a polypeptide consisting of amino acid residues 1 to 355 from the amino terminal of Sequence 1 in the sequence listing, and its coding sequence is shown in nucleotides 1 to 1065 from the 5' end of Sequence 2 in the sequence listing.

Embodiment 1, prepare the monoclonal antibody of heat shock protein (gp96 protein)

1. Purification of gp96 protein

1. Soak 70g-80g of isolated human placenta tissue, remove the capsule-like substance and connective tissue, shred it, add it to 200mL NaHCO ₃ buffer solution (containing 2mL PMSF) at pH 7.4, and grind the tissue.

2. Centrifuge the ground tissue fluid at 4°C and 13,000 rpm for 35 minutes, and take the supernatant.

3. Add the supernatant of step 2 to PMSF to make it 1% by volume, and then carry out ammonium sulfate precipitation with 50% saturation (that is, add 29.1g ammonium sulfate to 100mL supernatant), and stand at 4°C for 4 hours , and then centrifuged at 4° C. and 13500 rpm for 35 minutes, and the supernatant was taken.

4. Add the supernatant of step 3 to PMSF to make it 1% by volume, and then carry out ammonium sulfate precipitation with 70% saturation (that is, add 12.5g ammonium sulfate to 100mL supernatant), and stand overnight at 4°C , and then centrifuged at 4°C and 16500rpm for 90 minutes to collect the precipitate.

5. Dissolve the precipitate with 100mL affinity chromatography buffer (add PMSF 2mL when used), and then carry out Con-A Sepharose column affinity chromatography.

The relevant parameters of Con-A Sepharose column affinity chromatography are as follows: the column length is 20cm, and the inner diameter is 10mm; Con-A Sepharose column material (GE Healthcare, USA) is used to pack the chromatography column 8-10mL;

The formula of affinity chromatography buffer: PBS buffer containing 200mM NaCl and 1mM PMSF.

The formula of affinity chromatography eluent: PBS buffer containing 200mM NaCl, 1mM PMSF and 10g/mLα-D-glucopyranose.

The column passing process of Con-A Sepharose column affinity chromatography is as follows: First, equilibrate the Con-A Sepharose column with affinity chromatography buffer for at least 3 column volumes at 4°C; then load the sample at a flow rate of 1 mL/min, The sample was loaded cyclically for 12 hours; after the sample was loaded, 25 mL of affinity chromatography eluent (adding PMSF 1 mM when used) was used for circular elution overnight, and the next day, 3 column volumes were eluted with affinity chromatography eluent. All the eluate after passing through the column was collected and filtered with a 0.22 μm filter.

6. The eluate collected in step 5 was subjected to Hitrap Q anion exchange column affinity chromatography.

Hitrap Q anion exchange column affinity chromatography adopts Hitrap Q anion exchange column (chromatographic column is HiTrapQ HP; purchased from GE Company, product number 17-1153-01). The inner diameter and column length of the chromatography column are 0.7×2.5 cm.

Ion exchange liquid A is PBS buffer.

Ion exchange solution B was PBS buffer containing 1M NaCl.

The process of passing through the affinity chromatography of Hitrap Q anion exchange column is as follows: Equilibrate the HitrapQ anion exchange column with 5-10 column volumes with ion exchange A solution and load the sample; Eluting and eluting impurity proteins (i.e. the volume ratio of ion exchange A liquid and ion exchange B liquid is 8: 2), then within 30 minutes the volume fraction of ion exchange B liquid in the eluent was changed from 20% Increase linearly to 100%, the flow rate is 1mL/min; collect the post-column eluate with a retention time of 10min to 15min.

7. Concentrate the eluate collected in step 6 to 500 μL, and then perform Superdex-200 molecular sieve gel chromatography.

Molecular sieve buffer is PBS buffer.

Equilibrate the Hiload 10/60 Superdex 200pg gel chromatography column with molecular sieve buffer, then inject the concentrated protein sample into the ATKA FPLC through the loop, and collect the protein peak with a molecular weight of about 94kDa, which is the purified gp96 protein.

2. Preparation of N355 Fragment

Using Escherichia coli to express the N355 fragment, the specific method is as follows:

1. Design the following N355 primer pair, and the primers will be synthesized by Shanghai Yingjun Company:

Upstream primer: 5'-CGCGGATCCGACGATGAAGTTGATGTGGAT-3';

Downstream primer: 5'-CCGCTCGAGTTAAGTAAAGTGAATATAAGCCATG-3'.

2. Extract the mRNA of human liver cancer cell HepG2 (purchased from ATCC, product number HB-8065), and synthesize cDNA by reverse transcription.

3. Using the cDNA in step 2 as a template, perform PCR amplification with the N355 primer pair to obtain a PCR amplification product (coding sequence containing the N355 fragment).

4. Digest the PCR amplification product with restriction endonucleases BarnHI and Xho I, and recover the digested product.

5. Digest the pGEX-6P-1 plasmid (purchased from GE, product number 27-4597-01) with restriction endonucleases BarnHI and Xho I, and reclaim the vector backbone.

6. Ligate the digestion product of step 4 with the vector backbone of step 5 to obtain a ligation product.

7. Transform the ligation product into competent Escherichia coli DH5α (purchased from Tiangen Biochemical Technology Company, product number CB101-03), pick a single colony for enzyme digestion identification, and extract plasmids from colonies that are positive for enzyme digestion identification for sequencing identification. Sequencing results showed that the recombinant plasmid pGEX-N355 was obtained (the backbone vector was pGEX-6P-1, and the coding sequence of the N355 fragment was inserted between the BarnHI and Xho I restriction sites).

8. Transform the recombinant plasmid pGEX-N355 into Escherichia coli BL21(DE3) (purchased from Tiangen Biochemical Technology Co., Ltd., product number CB105-02) competent cells, pick a single colony from the plate and insert it into a medium containing 100 mg/mL ampicillin. 2×YT medium (tryptone 16g, yeast extract 10g, sodium chloride 5g, add water to make up to 1000mL) activation; take the activation solution (bacteria liquid) into 2×YT medium, culture at 37°C to OD ₆₀₀ value 0.6-1.0, then add IPTG (to make the final concentration 1mmol/L), induce for 4 hours at 37°C, and collect the bacteria.

9. The cells were resuspended in PBS buffer (140mmol/L NaCl, 2.7mmol/L KCl, 10mmol/L Na ₂ HPO ₄ , 1.8mmol/L KH ₂ PO ₄ ; pH7.3), and ultrasonically disrupted in an ice bath (200W, break for 4s and stop for 6s, 99 times for 3 cycles), then centrifuge at 12000 rpm at 4°C for 20min, and collect the supernatant.

10. The supernatant was subjected to affinity chromatography at 4°C. The carrier was Glutathione-Sepharose 4B (purchased from GE, product number 17-5132-01), and reduced glutathione elution buffer (10mmol/L reducedglutathione , 50mmol/L Tris-HCl, pH8.0) elution, collect eluate.

11. Replace the eluate with an enzyme digestion buffer system (50mmol/L Tris-HCl, pH8.0; 150mmol/L NaCl; 1mmol/L DTT; 1mmol/L EDTA, pH8.0) by ultrafiltration and concentration, and add Excessive PreScission Protease enzyme (PSP protein; purchased from GE, product number 27-0843-01), digested at 4°C for 16h.

12. Replace the digestion system with PBS buffer by ultrafiltration and concentration, and then perform affinity chromatography. The carrier is Glutathione-Sepharose 4B, which is eluted with PBS buffer, and GST and PSP are bound to the chromatography On the column, collect the eluate, which is the N355 fragment.

3. Obtaining hybridoma cells

On the first day, 100 μg gp96 protein (100 μL, volume adjusted with sterile PBS buffer) was evenly mixed with an equal volume of Freund’s complete adjuvant, and then immunized BALB/C mice by subcutaneous multipoint injection (purchased from Beijing Weitongli Co., Ltd. Hua Laboratory Animal Technology Co., Ltd.). On the 14th day, 50 μg gp96 protein was mixed with an equal volume of Freund's incomplete adjuvant to boost the immunization once, and on the 28th day, the same method was used to boost the immunization again. One week after the last booster immunization, test the serum antibody titer by ELISA, because the antibody titer obtained at this time is low. Seven booster immunizations were carried out to the mice, followed by four booster immunizations of gp96 protein (50 μg each time) and three booster immunizations of N355 fragments (50 μg each time). On the third day after the last booster immunization, the serum titer was measured again by ELISA.

A total of 6 mice were subjected to the above experiments. The mouse splenocytes with the highest serum antibody titer were fused with myeloma cells SP2/0 (catalog number TCM18, Shanghai Cell Bank, Chinese Academy of Sciences) at a ratio of 5:1, selected for culture by HAT screening medium, and limited dilution Hybridoma cells were obtained by the method, and the screening was repeated 3-5 times until the ELISA results were all positive, and a cell line capable of stably secreting specific monoclonal antibodies was obtained so far.

The specific immunization process is shown in Table 1.

Table 1 specific process

Four strains of hybridoma cells were obtained, one of which was named mouse monoclonal hybridoma cell A-HSP96-6, and the other three strains were named mouse monoclonal hybridoma cell I, mouse monoclonal hybridoma cell II and Mouse monoclonal hybridoma III. The mouse monoclonal hybridoma cell A-HSP96-6 has been preserved in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures (CGMCC for short) on April 18, 2012. The address is: Courtyard 3, No. 1 Beichen West Road, Chaoyang District, Beijing No.), the deposit number is CGMCC No.6007.

4. Preparation and purification of monoclonal antibodies

1. Incremental cultivation method

The preparation method of cell culture medium (7.4): add calf serum and sodium bicarbonate to RPMI-1640 medium, the final concentration of calf serum is 10% (mass percentage composition), the final concentration of sodium bicarbonate is 0.2 % (mass percentage).

The four hybridoma cells obtained in Step 3 were respectively placed in cell culture medium, cultured at 37°C for 2 days, and the obtained culture solution was purified by octanoic acid-saturated ammonium sulfate method to obtain monoclonal antibodies (stored at -20°C).

Protein concentration (mg/ml) in monoclonal antibody = 1.45×OD ₂₈₀ −0.74×OD ₂₆₀ .

Calculate the protein concentration in the mAb using the formula above. The protein concentration in the monoclonal antibody obtained from the mouse monoclonal hybridoma cell A-HSP96-6 was 1 mg/ml. The protein concentration in the monoclonal antibody obtained from the mouse monoclonal hybridoma I was 0.6 mg/ml. The protein concentration in the monoclonal antibody obtained from mouse monoclonal hybridoma II was 0.7 mg/ml. The protein concentration in the monoclonal antibody obtained from mouse monoclonal hybridoma III was 0.5 mg/ml.

Example 2. Use of gp96 antibody to significantly reduce tumor cell proliferation

1. The use of gp96 antibody significantly reduces the proliferation of breast cancer cell SKBr3.

Detect the effect of various monoclonal antibodies prepared in Step 4 of Example 1 on various cancer cells (SKBr3 cells, SK-Hep-1 cells) by CCK-8 kit (purchased from Japan Tongjin Chemical Research Institute, article number CK04-05). , NCI-H460 cells, PC-3 cells or BGC-823 cells) proliferation inhibition. The specific operation steps are as follows:

1. Add cell solution (50,000 cells/ml) into a 96-well plate, set up 24 replicate wells for each type of cell, and add 0.1ml of cell solution to each well.

2. Group processing

After the cells adhere to the wall, add monoclonal antibody to 12 wells, add 0.1ml monoclonal antibody to each well, the final concentration of monoclonal antibody is 50ug/ml, as the experimental group;

After the cells adhere to the wall, add PBS buffer solution to 12 wells, and add 0.1ml PBS buffer solution to each well as a control group;

Both the experimental group and the control group were cultured normally at 37°C, and the time was counted from the grouping treatment, and samples were taken at different time detection points (0, 3, 6, 12 hours), and samples from 3 wells were taken each time.

3. Add 10ul of CCK-8 detection reagent (a component of the CCK-8 kit) to each sample well, incubate at 37°C for 2 hours, and then measure the OD value at 490nm (OD _490nm value).

Cell growth inhibition rate=(average value of OD _490nm value of control group-average value of OD _490nm value of experimental group)/average value of OD _490nm value of control group×100%.

The average value of the OD _490nm value of the control group is the average value of the OD _490nm value of each sample hole at each time detection point of the control group, which is 1.36.

The average value of the OD _490nm value of the experimental group is the average value of the OD _490nm value of each sample hole at each time detection point of the experimental group.

When using the monoclonal antibody obtained by the mouse monoclonal hybridoma cell A-HSP96-6: the cell growth inhibition rate of the SKBr3 cell experimental group was 41.5%, and the cell growth inhibition rate of the SK-Hep-1 cell experimental group was 40.5%. The cell growth inhibition rate of the NCI-H460 cell experiment group was 43%, the cell growth inhibition rate of the PC-3 cell experiment group was 42%, and the cell growth inhibition rate of the BGC-823 cell experiment group was 40%. The effect of the monoclonal antibody obtained by the other three mouse monoclonal hybridoma cells is slightly lower than that of the monoclonal antibody obtained by the mouse monoclonal hybridoma cell A-HSP96-6, but the growth inhibition rate of each cell is also within 25%. % to 35%. The results showed that the monoclonal antibody prepared in Example 1 could significantly inhibit the proliferation (growth) of SKBr3 cells, SK-Hep-1 cells, NCI-H460 cells, PC3 cells and BGC-823 cells.

Example 3. Using gp96 antibody to induce tumor cell apoptosis

The promotion effect of various monoclonal antibodies prepared in step 4 of Example 1 on the apoptosis of cancer cells (SKBr3 cells, SK-Hep-1 cells, NCI-H460 cells, PC-3 cells or BGC-823 cells) was detected respectively. Specific steps are as follows:

1. Add the cell solution and inoculate in a 6-well cell culture plate, 0.1ml per well, 200,000 cells/well.

2. Group processing:

Experimental group (set up three replicate wells for each cell): after the cells adhere to the wall, add 0.1ml of monoclonal antibody to each well, the final concentration of monoclonal antibody is 50ug/ml, and normal culture at 37°C for 24 hours;

Negative control group (three replicate wells for each cell): after the cells adhere to the wall, add 0.1ml of PBS buffer to each well, and culture at 37°C for 24 hours.

3. Use the kit produced by Invitrogen After the Apoptosis Assay kit stains the cells, the results are analyzed by flow cytometry. The specific operation steps are as follows:

(1) Routinely digest cells with trypsin and wash twice with PBS buffer.

(2) Suspend the cells gently with 20ul 1×Annexin V Buffer, add 1ul FITC annexin V, mix gently, and stain at room temperature for 15 minutes in the dark.

(3) Add 1× Annexin V Buffer to the reaction tube to make the final volume 200ul.

(4) Add PI at a concentration of 100ug/ml to make the final concentration 1ug/ml, and stain for about 3 minutes at room temperature in the dark to detect on the machine (the apoptosis rate is directly measured by flow cytometry).

The calculation formula for the increased apoptosis rate of the experimental group compared with the negative control group is: the apoptosis rate of the experimental group-the apoptosis rate of the negative control group.

The results are the average value of three replicate wells.

When using the monoclonal antibody obtained from the mouse monoclonal hybridoma cell A-HSP96-6: for SKBr3 cells, the apoptosis rate of the negative control group was 6.8%, and the apoptosis rate of the experimental group was 34.6% higher than that of the negative control group %; for SK-Hep-1 cells, the apoptosis rate of the negative control group was 6.8%, and the apoptosis rate of the experimental group was 33.7% higher than that of the negative control group; for NCI-H460 cells, the apoptosis rate of the negative control group was 33.7%. The apoptosis rate of the experimental group was 6.8%, and the apoptosis rate of the experimental group was 35.2% higher than that of the negative control group; for PC-3 cells, the apoptosis rate of the negative control group was 6.8%, and the apoptosis rate of the experimental group was higher than that of the negative control group. The apoptosis rate was 34.3%; for BGC-823 cells, the apoptosis rate in the negative control group was 6.8%, and the apoptosis rate in the experimental group was 33.8% higher than that in the negative control group. The effect of the monoclonal antibody obtained by the other three mouse monoclonal hybridoma cells is slightly lower than that obtained by the mouse monoclonal hybridoma cell A-HSP96-6, but for each cell, the experimental group is better than the negative control The increased apoptosis rates of the groups also all ranged from 21% to 28%. The results show that the monoclonal antibody prepared in Example 1 can obviously promote the apoptosis of each cancer cell.

Example 4. The use of gp96 antibody significantly inhibited the growth of mammary gland tumors in nude mice.

The inhibitory effects of various monoclonal antibodies prepared in Step 4 of Example 1 on the growth of transplanted tumors of breast cancer cells (SKBR3 cells or MDA-MB-231 cells) were tested respectively. Specific steps are as follows:

1. BALB/c nude mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were subcutaneously inoculated with breast cancer cells cultured to logarithmic growth phase, and each mouse was inoculated with 2 million cells.

2. After 7 days, tumors were seen to form in the mice, and the mice with tumors were randomly divided into two groups, and the following treatments were performed respectively:

gp96 antibody group (5 mice): carry out intraperitoneal injection treatment with monoclonal antibody solution (monoclonal antibody diluted with PBS buffer solution), each injection of 0.5ml (containing 100ug monoclonal antibody protein), weekly treatment 3 times (on the first, third and sixth days of the week);

Negative control group (5 mice): carry out intraperitoneal injection treatment with PBS buffer solution, inject 0.5ml each time, treat 3 times a week (respectively on the first day, the third day and the sixth day of the week) .

3. The nude mice were sacrificed after 5 weeks of continuous treatment, the tumor weight was weighed and the tumor inhibition rate was calculated (the average value of the group was calculated).

The formula for calculating the tumor inhibition rate is as follows: (tumor weight of mice in the negative control group-tumor weight of mice in the gp96 antibody group)/tumor weight of mice in the negative control group×100%.

When using the monoclonal antibody obtained from the mouse monoclonal hybridoma cell A-HSP96-6: for the nude mice inoculated with SKBr3 cells, the tumor weight of the mice in the negative control group was 1.12g (p<0.01), and the tumor weight of the gp96 antibody group The inhibition rate was 33.8%; for the nude mice inoculated with MDA-MB-231 cells, the tumor weight of the mice in the negative control group was 1.30 g (p<0.01), and the tumor inhibition rate in the gp96 antibody group was 35.6%. The effect of the monoclonal antibody obtained from the other three mouse monoclonal hybridoma cells was slightly lower than that of the monoclonal antibody obtained from the mouse monoclonal hybridoma cell A-HSP96-6, but the tumor inhibition rate on each breast cancer cell was also the same. Between 23% and 26%. The results show that the monoclonal antibody prepared in Example 1 can effectively inhibit the growth of breast cancer tumors.

Example 5. The use of gp96 antibody significantly inhibited the growth of lung cancer tumors in nude mice.

The inhibitory effects of various monoclonal antibodies prepared in Step 4 of Example 1 on the growth of transplanted tumors of lung cancer cells (NCI-H460 cells or A549 cells) were tested respectively. Specific steps are as follows:

1. BALB/c nude mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were subcutaneously inoculated with lung cancer cells cultured to logarithmic growth phase, and each mouse was inoculated with 2 million cells.

2. After 7 days, tumors were seen to form in the mice, and the mice with tumors were randomly divided into two groups, and the following treatments were performed respectively:

gp96 antibody group (5 mice): carry out intraperitoneal injection treatment with monoclonal antibody solution (monoclonal antibody diluted with PBS buffer solution), each injection of 0.5ml (containing 100ug monoclonal antibody protein), weekly treatment 3 times (on the first, third and sixth days of the week);

Negative control group (5 mice): carry out intraperitoneal injection treatment with PBS buffer solution, inject 0.5ml each time, treat 3 times a week (respectively on the first day, the third day and the sixth day of the week) .

3. The nude mice were sacrificed after 5 weeks of continuous treatment, the tumor weight was weighed and the tumor inhibition rate was calculated (the average value of the group was calculated).

The formula for calculating the tumor inhibition rate is as follows: (tumor weight of mice in the negative control group-tumor weight of mice in the gp96 antibody group)/tumor weight of mice in the negative control group×100%.

When using the monoclonal antibody obtained from the mouse monoclonal hybridoma cell A-HSP96-6: for the nude mice inoculated with NCI-H460 cells, the tumor weight of the mice in the negative control group was 1.25 g (p<0.01), and the weight of the tumor in the gp96 antibody group was 1.25 g (p<0.01). The tumor inhibition rate was 33.3%; for the nude mice inoculated with A549 cells, the tumor weight of the mice in the negative control group was 1.08g (p<0.01), and the tumor inhibition rate in the gp96 antibody group was 34.6%. The effect of the monoclonal antibody obtained from the other three mouse monoclonal hybridoma cells was slightly lower than that of the monoclonal antibody obtained from the mouse monoclonal hybridoma cell A-HSP96-6, but the tumor inhibition rate on each breast cancer cell was also the same. Between 22% and 24%. The results show that the monoclonal antibody prepared in Example 1 can effectively inhibit the growth of lung cancer tumors.

Example 6, gp96 antibody inhibits prostate cancer tumor growth

The inhibitory effects of various monoclonal antibodies prepared in Step 4 of Example 1 on the growth of transplanted tumors of prostate cancer cells (PC-3 cells or DU-145 cells) were tested respectively. Specific steps are as follows:

1. BALB/c nude mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were subcutaneously inoculated with prostate cancer cells cultured to logarithmic growth phase, and each mouse was inoculated with 2 million cells.

2. After 7 days, tumors were seen to form in the mice, and the mice with tumors were randomly divided into two groups, and the following treatments were performed respectively:

gp96 antibody group (5 mice): carry out intraperitoneal injection treatment with monoclonal antibody solution (monoclonal antibody diluted with PBS buffer solution), each injection of 0.5ml (containing 100ug monoclonal antibody protein), weekly treatment 3 times (on the first, third and sixth days of the week);

Negative control group (5 mice): carry out intraperitoneal injection treatment with PBS buffer solution, inject 0.5ml each time, treat 3 times a week (respectively on the first day, the third day and the sixth day of the week) .

3. The nude mice were sacrificed after 5 weeks of continuous treatment, the tumor weight was weighed and the tumor inhibition rate was calculated (the average value of the group was calculated).

The formula for calculating the tumor inhibition rate is as follows: (tumor weight of mice in the negative control group-tumor weight of mice in the gp96 antibody group)/tumor weight of mice in the negative control group×100%.

When using the monoclonal antibody obtained from the mouse monoclonal hybridoma cell A-HSP96-6: for the nude mice inoculated with PC-3 cells, the tumor weight of the mice in the negative control group was 1.52 g (p<0.01), and the weight of the tumor in the gp96 antibody group was 1.52 g (p<0.01). The tumor inhibition rate was 37.2%; for the nude mice inoculated with DU-145 cells, the tumor weight of the mice in the negative control group was 1.40 g (p<0.01), and the tumor inhibition rate in the gp96 antibody group was 30.6%. The effect of the monoclonal antibody obtained from the other three mouse monoclonal hybridoma cells was slightly lower than that of the monoclonal antibody obtained from the mouse monoclonal hybridoma cell A-HSP96-6, but the tumor inhibition rate on each breast cancer cell was also the same. Between 26% and 29%. The results show that the monoclonal antibody prepared in Example 1 can effectively inhibit the growth of prostate cancer tumors.

Example 7, gp96 antibody inhibits gastric cancer tumor growth

The inhibitory effects of various monoclonal antibodies prepared in Step 4 of Example 1 on the growth of transplanted tumors of gastric cancer cells (BGC-823 cells or MNK-45 cells) were tested respectively. Specific steps are as follows:

1. BALB/c nude mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were subcutaneously inoculated with gastric cancer cells cultured to logarithmic growth phase, and each mouse was inoculated with 2 million cells.

2. After 7 days, tumors were seen to form in the mice, and the mice with tumors were randomly divided into two groups, and the following treatments were performed respectively:

gp96 antibody group (5 mice): carry out intraperitoneal injection treatment with monoclonal antibody solution (monoclonal antibody diluted with PBS buffer solution), each injection of 0.5ml (containing 100ug monoclonal antibody protein), weekly treatment 3 times (on the first, third and sixth days of the week);

Negative control group (5 mice): carry out intraperitoneal injection treatment with PBS buffer solution, inject 0.5ml each time, treat 3 times a week (respectively on the first day, the third day and the sixth day of the week) .

3. The nude mice were sacrificed after 5 weeks of continuous treatment, the tumor weight was weighed and the tumor inhibition rate was calculated (the average value of the group was calculated).

The formula for calculating the tumor inhibition rate is as follows: (tumor weight of mice in the negative control group-tumor weight of mice in the gp96 antibody group)/tumor weight of mice in the negative control group×100%.

When using the monoclonal antibody obtained from the mouse monoclonal hybridoma cell A-HSP96-6: for the nude mice inoculated with MNK-45 cells, the tumor weight of the mice in the negative control group was 1.28g (p<0.01), and the tumor weight in the gp96 antibody group was 1.28g (p<0.01). The tumor inhibition rate was 32.4%; for the nude mice inoculated with BGC-823 cells, the tumor weight of the mice in the negative control group was 1.16g (p<0.01), and the tumor inhibition rate in the gp96 antibody group was 35.6%. The effect of the monoclonal antibody obtained from the other three mouse monoclonal hybridoma cells was slightly lower than that of the monoclonal antibody obtained from the mouse monoclonal hybridoma cell A-HSP96-6, but the tumor inhibition rate on each breast cancer cell was also the same. Between 24% and 27%. The results show that the monoclonal antibody prepared in Example 1 can effectively inhibit the growth of gastric cancer tumors.

Example 8, gp96 antibody inhibits the growth of liver cancer

The inhibitory effects of various monoclonal antibodies prepared in Step 4 of Example 1 on the growth of transplanted tumors of liver cancer cells (SK-Hep-1 cells or Bel-7402 cells) were tested respectively. Specific steps are as follows:

1. BALB/c nude mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were subcutaneously inoculated with liver cancer cells cultured to logarithmic growth phase, and each mouse was inoculated with 2 million cells.

2. After 7 days, tumors were seen to form in the mice, and the mice with tumors were randomly divided into two groups, and the following treatments were performed respectively:

gp96 antibody group (5 mice): carry out intraperitoneal injection treatment with monoclonal antibody solution (monoclonal antibody diluted with PBS buffer solution), each injection of 0.5ml (containing 100ug monoclonal antibody protein), weekly treatment 3 times (on the first, third and sixth days of the week);

Negative control group (5 mice): carry out intraperitoneal injection treatment with PBS buffer solution, inject 0.5ml each time, treat 3 times a week (respectively on the first day, the third day and the sixth day of the week) .

3. The nude mice were sacrificed after 5 weeks of continuous treatment, the tumor weight was weighed and the tumor inhibition rate was calculated (the average value of the group was calculated).

The formula for calculating the tumor inhibition rate is as follows: (tumor weight of mice in the negative control group-tumor weight of mice in the gp96 antibody group)/tumor weight of mice in the negative control group×100%.

When using the monoclonal antibody obtained from the mouse monoclonal hybridoma cell A-HSP96-6: for the nude mice inoculated with SK-Hep-1 cells, the tumor weight of the negative control group mice was 1.45g (p<0.01), gp96 The tumor inhibition rate of the antibody group was 30.5%. For the nude mice inoculated with Bel-7402 cells, the tumor weight of the mice in the negative control group was 1.26g (p<0.01), and the tumor inhibition rate of the gp96 antibody group was 34.1%. The effect of the monoclonal antibody obtained from the other three mouse monoclonal hybridoma cells was slightly lower than that of the monoclonal antibody obtained from the mouse monoclonal hybridoma cell A-HSP96-6, but the tumor inhibition rate on each breast cancer cell was also the same. Between 23% and 27%. The results show that the monoclonal antibody prepared in Example 1 can effectively inhibit the growth of liver cancer tumors.

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.