CN106822869B - Application of compound of polypeptide of DEF8 protein and heat shock protein gp96 in preparation of medicine for treating and preventing cancer - Google Patents

Abstract

The invention provides DEF8 protein and application of polypeptide thereof, belonging to the technical field of protein engineering and biomedical application. The amino acid sequence of the DEF8 protein is shown as SEQ ID NO.2 or a specific fragment thereof, and the DEF8 protein, the polypeptide thereof and a compound formed by the protein and the hot protein gp96 in a hot shock or natural adsorption mode can prevent and treat neoplastic diseases including breast cancer and liver cancer, can be used for preparing a medicament for preventing and treating the cancer and has wide application prospect.

Description

Application of compound of polypeptide of DEF8 protein and heat shock protein gp96 in preparation of medicine for treating and preventing cancer

Technical Field

The invention relates to the technical field of protein engineering and biomedical application, in particular to application of DEF8 protein and polypeptide thereof in treating and preventing cancers.

Background

Breast cancer is one of the most common malignancies in women. According to data statistics, the incidence rate of breast cancer accounts for 7-10% of various malignant tumors of the whole body, and in many areas, the breast cancer exceeds uterine cancer, so that the breast cancer becomes one of the most common tumors which seriously affect the physical and mental health of women and even endanger life. The incidence of breast cancer is closely related to the genetic genes, living habits, common food, fertility conditions and the like of patients, and the incidence of breast cancer of different races and regions has obvious difference. The high incidence of breast cancer is mainly concentrated in north america, northern europe and oceania, and especially in white women. The middle-onset of breast cancer is concentrated in south america, south europe and israel. Asia is a low-incidence area of breast cancer. For example, the lifetime breast cancer incidence rate of caucasian women in the United states is 13.1%, i.e., on average, 1 person per 8-9 people may have breast cancer, while the breast cancer incidence rate of Asian women is 4-7%. According to WHO statistics, the number of new breast cancer is about 130 thousands of people every year and about 50 thousands of people die worldwide. The breast cancer in the large cities of Beijing, Shanghai, Tianjin and the like in China jumps over the first place of various cancers of women and has a trend of obvious rising.

Liver cancer refers to malignant tumor occurring in liver, including primary liver cancer and metastatic liver cancer. Primary liver cancer is one of the most common malignant tumors in clinic. Among male cancer patients worldwide, liver cancer ranks sixth in proportion and mortality ranks second; among female cancer patients, liver cancer ranks seventh in proportion and mortality ranks sixth. In 2008, there were 748,300 new cases of liver cancer worldwide, and 695,900 patients with liver cancer died. Half of these new cases of liver cancer and death are in China. The highest incidence of liver cancer is mainly found in east asia, southeast asia, middle africa and west africa. The higher incidence of liver cancer in parts of Asia and sub-Saharan areas of Africa may be due to the prevalence of HBV in these areas, since 8% of the residents in these areas are chronically infected with HBV and 60% of liver cancer patients in developing countries are infected with HBV.

Bone marrow progenitor cells Differentially expressed protein 8 (differentiated expressed in FDCP 8) was first found to be Differentially expressed in the hematopoietic system. DEF8 protein is widely distributed, and is highly expressed particularly in peripheral blood leukocytes, but its expression is hardly detectable in thymus and fetal liver, and is significantly down-regulated upon DEF8 protein macrophage and granulocyte differentiation. However, the function of DEF8 protein has not been known so far.

Heat Shock Proteins (HSPs) are a class of proteins that are highly conserved in biological evolution and widely found in prokaryotes and eukaryotes. HSPs can be classified into multiple subfamilies, such as HSP110, HSP90, HSP70, HSP60, HSP40, small HSP and ubiquitin, according to the degree of homology and molecular weight. Heat Shock Protein (HSP) gp96 belongs to a member of the HSP90 subfamily, and is the most abundant Heat shock protein on the endoplasmic reticulum. The heat shock protein gp96 has polypeptide binding properties, and can accept polypeptide fragments from the TAP complex in the endoplasmic reticulum to facilitate their assembly into MHC class I molecules for presentation on cell membranes. The heat shock protein gp96 derived from different tissues can carry polypeptide fragments specifically expressed in the tissues from which it is derived.

Disclosure of Invention

The invention aims to provide a target protein capable of preventing and treating cancer and an application method of a polypeptide thereof.

The invention firstly provides DEF8 protein, and the amino acid sequence of the DEF8 protein contains the following components:

i) an amino acid sequence shown as SEQ ID NO. 2; or

ii) the amino acid sequence shown in SEQ ID NO.2 from the 2 nd to the last amino acid sequence from the N terminal; or

iii) the amino acid sequence shown in SEQ ID NO.2 is substituted, deleted and/or added with one or more amino acids and has the same function.

The invention provides an application of the DEF8 protein in preparing a medicament for preventing and/or treating cancer.

The invention provides a gene for coding DEF8 protein, and the nucleotide sequence of the gene comprises the following components:

i) a nucleotide sequence shown as SEQ ID NO. 1; or

ii) a nucleotide sequence which is shown in SEQ ID NO.1 and expresses the same functional protein by replacing, deleting and/or adding one or more nucleotides; or

iii) a nucleotide sequence having 75% or more homology with the nucleotide shown in SEQ ID NO. 1; or

iv) a nucleotide sequence which hybridizes under stringent conditions to the sequence shown in SEQ ID NO. 1;

the stringent conditions are hybridization at 65 ℃ in a 0.1 XSSPE containing 0.1% SDS or a 0.1 XSSC solution containing 0.1% SDS, and washing the membrane with the solution.

The invention provides an application of a gene coding DEF8 protein in preparing a medicament for preventing and/or treating cancer.

The invention provides a biological material containing a gene coding DEF8 protein, wherein the biological material is a vector, a host cell, a transgenic cell line, an engineering bacterium, an insect or a yeast.

Further, the invention provides an application of the biological material in preparing a medicament for preventing and/or treating cancer.

The medicine is used for treating or preventing breast cancer or liver cancer, and has at least one of the following functions:

(1) reducing the incidence of chemical-induced breast cancer;

(2) reducing the incidence of liver cancer induced by chemicals;

(3) slowing or stopping the growth of established breast cancer foci;

(4) slowing or stopping the growth of established liver cancer tumor foci;

(5) reducing or stopping metastasis of established breast cancer foci;

(6) reducing or stopping metastasis of established liver cancer foci;

(7) inducing and generating breast cancer specific CTL cells and killing the breast cancer cells;

(8) inducing to generate specific CTL cell of liver cancer and killing the liver cancer cell.

The invention provides a drug containing DEF8 protein or polypeptide thereof.

The invention provides a medicine containing a compound consisting of DEF8 protein and heat shock protein gp96 or a compound consisting of DEF8 protein polypeptide and heat shock protein gp 96.

The amino acid sequence of the heat shock protein gp96 contains the amino acid sequence which is shown in SEQ ID NO.4 or the amino acid sequence which is shown in SEQ ID NO.4 and has the same function after one or more amino acids are substituted, deleted and/or added.

The heat shock protein gp96 is obtained by any one of the following methods:

(1) extracting from isolated mammalian placental tissue;

(2) introducing the nucleic acid molecule coding the heat shock protein gp96 into a receptor, culturing, expressing by using a yeast and mammalian cell expression system, and purifying to obtain the protein.

The isolated mammal may be of human or murine origin. The mice include but are not limited to C57BL/6 and BALB/C mice.

The complex is obtained by:

(1) forming a complex of DEF8 protein or a polypeptide thereof and heat shock protein gp96 in vitro by natural adsorption or heat shock; or

(2) The nucleic acid molecule coding DEF8 protein or its polypeptide and heat shock protein gp96 are introduced into receptor, cultured and purified to obtain the compound.

The "introduction of a nucleic acid molecule encoding DEF8 protein or a polypeptide thereof and encoding heat shock protein gp96 into a recipient" can be expressed by hansenula polymorpha.

Said "introducing a nucleic acid molecule encoding said heat shock protein gp96 into a receptor" can be effected by introducing a recombinant vector into the receptor; the recombinant vector can be a recombinant plasmid obtained by inserting the coding gene of the heat shock protein gp96 into a starting plasmid. The recombinant vector can be specifically a recombinant plasmid pFastBac1-gp 96. The recombinant plasmid pFastBac1-gp96 can be specifically a recombinant plasmid obtained by inserting a DNA molecule shown by SEQ ID NO.3 in a sequence table into the polyclonal site of a plasmid pFastBac 1.

The recombinant plasmid pFastBac1-gp96 can be specifically a recombinant plasmid obtained by replacing a fragment between EcoRI and XbaI recognition sequences of the plasmid pFastBac1 (the plasmid pFastBac1 is cut into a large fragment and a small fragment by restriction endonucleases EcoRI and XbaI, and the DNA is the small fragment) with a DNA molecule shown in SEQ ID NO. 1.

The receptor may be Sf9 cells.

The invention provides an application of a compound consisting of the DEF8 protein and the polypeptide thereof and a heat shock protein gp96 in preparing a medicament for treating and/or preventing cancer.

The medicine is used for treating or preventing breast cancer or liver cancer, and has at least one of the following functions: (1) reducing the incidence of chemical-induced breast cancer;

(2) reducing the incidence of liver cancer induced by chemicals;

(3) slowing or stopping the growth of established breast cancer foci;

(4) slowing or stopping the growth of established liver cancer tumor foci;

(5) reducing or stopping metastasis of established breast cancer foci;

(6) reducing or stopping metastasis of established liver cancer foci;

(7) inducing and generating breast cancer specific CTL cells and killing the breast cancer cells;

(8) inducing to generate specific CTL cell of liver cancer and killing the liver cancer cell.

The medicine of the invention is applied to immune objects, the immune dose is not less than 10 mug each time, and is not less than 3 times in total, and the immunization is carried out by adopting a mode of subcutaneous injection, intradermal injection or intraperitoneal injection. The medicine (vaccine) of the present invention can initiate specific immunoreaction of body after being immunized for several weeks, eliminate in vivo tumor stem cell and/or tumor dormancy cell and cancerated cell, so as to reach the aim of preventing and treating cancer. The invention can be used as a tumor preventive vaccine for preventing and treating autologous or allogeneic tumors and reducing the incidence rate of cancers in healthy people, and can also be used as a tumor therapeutic vaccine for preventing metastasis and recurrence of patients by immediate postoperative treatment or as adjuvant treatment of chemotherapy. The invention can also be used for the immunological prevention and treatment of allogeneic tumors.

Drawings

FIG. 1 shows the SDS-PAGE and Western blot identification of placental gp 96.

FIG. 2 shows the identification of yeast expressing gp96 by SDS-PAGE and Western Blot, in which 1: a molecular weight standard; 2: the fermentation liquor of step 2; 3: eluent after affinity chromatography; 4: eluent after ion exchange chromatography; 5: western blot identification.

FIG. 3 shows the identification of insect gp96 expression by SDS-PAGE and Western Blot.

FIG. 4 is a graph of the therapeutic effect (tumor volume) of polypeptide-gp 96 complex on breast cancer.

FIG. 5 shows the therapeutic effect of polypeptide-gp 96 complex on liver cancer (tumor volume).

FIG. 6 shows the killing effect of specific CTL induced by polypeptide-gp 96 complex on human breast cancer cells.

FIG. 7 shows that specific CTL induced by the polypeptide-gp 96 complex has killing effect on human breast cancer cells.

Detailed Description

The experimental procedures in the following examples are conventional unless otherwise specified. The materials, reagents and the like used in the examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

The female C57BL/6 and female BALB/C mice are products of the GJINGWEITONGHUA laboratory animals LLC company; hereinafter referred to as mouse. The polypeptide was synthesized by Shanghai Jier Biochemical Co., Ltd. HepG2 cell (human hepatoma cell) was a product of ATCC company under the catalog number HB-8065^TM. MCF-7 cells (human breast cancer cells) are available from ATCC under the catalog number HTB-22^TM. H22 and HHCC cells were purchased from the basic medicine cell center of the institute of basic medicine of Chinese academy of medical sciences with resource numbers 3111C0001CCC000309 and 3111C0002000000069, respectively. Sf9 cells were Invitrogen, catalog number 11496-015. Cellffectin II reagent is a product of Life technologies, Inc., catalog number 10362-. Plasmid pFastBac^TM1 is Invitrogen corporation, catalog number 10359-. The gp96 monoclonal antibody is available from Santa Cruz, Inc., under the sc-56399 catalog. The goat anti-rat monoclonal antibody marked by horseradish peroxidase is a product of Beijing Zhonghua Jinqiao biotechnology limited company, and the catalog number of the product is ZB-2307. HiTrap-Q Sepharose ion exchange column is product of GE company, and has catalog number of 17-5053-01. Superdex 20010/300 GL molecular sieve chromatographic column is a product of GE company, and the catalog number is 17517501. The escherichia coli DH10Bac competent cell is a product of Beijing original Hao Biotechnology Limited, and the catalog number of the product is CL 108-01. Insect-XPRESS^TMProtein-free Instrument with L-Glutamine is a product of LONZA, and the product catalog number is 12-730Q. BSA, PMSF, NaHCO₃、MnCl₂、CaCl₂、NaCl₂Tris, methyl alpha-D-mannopyranoside are products of Sigma-Aldrich company, and the catalog numbers are V900933, P7626, 792519, V900197, 793639, 746398, T1378 and M6882 respectively.

Solution A: the solute and the concentration thereof are PMSF 1mM and NaHCO ₃30 mM; the solvent is distilled water; the pH was 7.4.

Solution B: solute and concentration thereof are 2mM MnCl₂、2mM CaCl₂500mM NaCl and PMSF 1 mM; the solvent is Tris-HCl buffer (20 mM) with pH 7.4.

Solution C: solute and the concentration thereof are 10 percent (mass volume ratio) of methyl alpha-D-mannopyranoside, 500mM NaCl and 1mM PMSF; the solvent is Tris-HCl buffer (20 mM) with pH 7.4.

Cleaning solution: solution B was diluted to 10 volumes with distilled water.

The ConA Sepharose column is a product of GE corporation, catalog number is 17-0440-01, the specification of the column is 1.6X 2.5cm, and the packing medium is Con A-Sepharose 4B. The Hitrap Q anion exchange column is available from GE under catalog number 17-1153-01, and the column size is 0.7X 2.5 cm. The HRP labeled IgG antibody is available from SEROTEC corporation under catalog number STAR 117P. 1 × washing solution is 0.01mol/LPBS buffer containing 0.1% (volume percent) Triton-X100 and pH 7.4. 50kD and 3kD ultrafiltration tubes are made by Merck Millipore company, and the catalog numbers are UFC905096 and UFC500324 respectively.

Example 1 extraction of gp96 from placenta

The extraction process of heat shock protein gp96 (hereinafter referred to as pgp96) in tissue is as follows:

(1) separating the placenta tissue of the mouse before delivery to obtain the isolated placenta tissue of the mouse. Taking a mouse in-vitro placenta tissue or a human in-vitro placenta tissue, shearing into pieces, and mixing the cut pieces according to the mass-volume ratio of 1 g: 4mL of solution A was added and then ground with a glass homogenizer.

(2) After completion of step (1), 16500g was centrifuged for 1h to give supernatant A.

(3) And (3) after the step (2) is finished, taking the supernatant A, and centrifuging 16500g for 50min to obtain the supernatant B.

(4) After the step (3) is finished, taking the supernatant B, and mixing the supernatant B with the supernatant B according to the volume ratio of 9: 1, adding the solution B, and uniformly mixing to obtain a sample solution.

(5) After completion of step (4), the sample was applied to a ConA sepharose column.

(6) And (5) eluting the ConA sepharose column by using a cleaning solution, and monitoring the ultraviolet absorption value in real time in the elution process until the ultraviolet absorption value of the eluted product is lower than 0.01, wherein the detection wavelength is 280 nm.

(7) After the step (6) is finished, eluting the ConA sepharose column by using the solution C, discarding 0.5 column volumes of the post-column solution which flows out firstly, and then collecting 1 column volume of the post-column solution which flows out later; after incubating the ConA sepharose column for 50min, the post-column solution was collected for 1.5 column volumes. And combining the two collected solutions after passing through the column to obtain the ConA eluent.

(8) After completion of step (7), the ConA eluate was applied to a Hitrap Q anion exchange column.

(9) After completion of step (8), linear gradient elution was carried out with NaCl-containing PBS buffer (pH 7.4, 12 mM) at a flow rate of 1 mL/min. Gradient elution procedure: the NaCl content was increased from 300mM to 800mM with a constant gradient in PBS buffer pH 7.412mM, and the column was eluted with a linear gradient over 20 column volumes. Collecting and combining the eluent with the NaCl content of 400-450 mM to obtain the eluent A.

(10) After step (9) is completed, the eluent A is taken and subjected to ultrafiltration concentration by an ultrafiltration tube to obtain a solution of pgp 96. In the solution of pgp96, the concentration of pgp96 was 5 mg/mL.

The solution of pgp96 was subjected to SDS-PAGE analysis and Western blot (gp 96 monoclonal antibody as primary antibody and HRP-labeled IgG antibody as secondary antibody) and the results of the experiment are shown in FIG. 1 (indicated by the arrow as pgp 96). The results show that the solution of pgp96 shows a single molecular weight band, corresponding to a molecular weight of 96 kDa.

Example 2 preparation of recombinant gp96 protein expressed by Hansenula polymorpha

Construction of recombinant plasmid pHFMDZ-R1L2GAmy-gp96

1. Extracting mRNA of human liver cancer cell HepG2, and synthesizing cDNA through reverse transcription.

2. And (3) carrying out PCR amplification by taking the cDNA in the step (1) as a template to obtain an amplification product.

The PCR primers were as follows:

Fp(5'-CCGgaattcATGGACGATGAAGTTGATG-3')

Rp(5'-CCGctcgagCTATTAGAATTCATCTTTTTCAGCTGTAG-3')

3. and (3) double-digesting the PCR amplification product by using restriction enzymes EcoRI and XhoI, and recovering the digested product.

4. The plasmid pHFMDZ-R1A (purchased from Invitrogen, product No. V20520) was double-digested with restriction enzymes EcoRI and XhoI, and the vector backbone was recovered.

5. And (4) connecting the enzyme digestion product in the step (3) with the vector skeleton in the step (4) to obtain a recombinant plasmid pHFMDZ-R1-gp96 and sequencing the recombinant plasmid. The sequencing result shows that the skeleton vector of the recombinant plasmid pHFMDZ-R1-gp96 is pHFMDZ-R1A, and the coding sequence of gp96 protein is inserted between EcoRI and XhoI enzyme cutting sites.

Expression of gp96 protein

1. The recombinant plasmid pHFMDZ-R1-gp96 obtained in step 1 was introduced into Hansenula polymorpha (purchased from ATCC and having a product number of MYA-335) cells by electrotransformation to obtain recombinant bacteria.

2. The recombinant strain was inoculated into 5mL of SD liquid medium (purchased from Shanghai Jimei Gene medicine, product No. GMS12117.7), cultured at 37 ℃ for 48 hours, then transferred to 100mL of SYN6 medium (purchased from Shanghai Jimei gene medicine, product No. GMS12116.1), and cultured at 30 ℃ for 48 hours, to obtain a seed culture solution.

3. Two flasks of seed culture were inoculated into a 5L fermentor containing 2L of SYN6 medium and cultured at 30 ℃. Controlling the pH value to be 5.5 by using ammonia water, detecting the content of glycerol in the fermentation liquor for 1 time every 4h, supplementing the glycerol according to the concentration of the glycerol in the fermentation liquor, controlling the final concentration of the glycerol to be about 0.5%, and simultaneously controlling the dissolved oxygen to be more than 20%. Detecting the wet weight of the thallus according to the generation condition of the thallus, stopping glycerol supplementation when the wet weight of the thallus reaches 180-200g/L, starting to induce the recombinant gp96 protein to generate (methanol is supplemented to maintain the methanol concentration at about 0.5-0.8%), and stopping fermentation 72 hours after the induction is started to obtain fermentation liquor.

Thirdly, separation and purification of gp96 protein

And (3) centrifuging the fermentation liquor obtained in the step (2) and collecting thalli. The cells were washed 2 times with PBS buffer, disrupted by glass bead milling in a ball MILL (DYNO-MILL model KDL) according to the manufacturer's manual, centrifuged at 12000rpm/min for 20min, and the supernatant was collected. The supernatant was filtered through a 0.45 μm filter to obtain a filtrate. And concentrating the filtrate to obtain a concentrated solution.

Carrying out affinity chromatography on the filtrate, and specifically comprising the following steps: affinity chromatography was performed using Ni-NTA Purification System from Invitrogen Corporation, and the main steps were: the column was equilibrated first with PBS for 2h and then with PBS (containing 20mM imidazole) for 2 h. The concentrate was diluted with PBS (containing 20mM imidazole) and loaded, the column was washed with PBS (containing 20mM imidazole) to OD <0.01, eluted with PBS (containing 200mM imidazole) for 1.5h, and the eluate was collected. All operations were carried out at 4 ℃ and the flow rate was 0.5 mL/min.

After purification of fermentation liquor obtained in the step 2, 50mg of gp96 protein with purity of more than 90% can be obtained. If the protein eluent contains heterologous hybrid protein, the eluent of affinity chromatography can be further subjected to ion exchange chromatography, and the main steps are as follows: the column was equilibrated with 200mM NaCl in PBS, loaded, and washed with 300mM NaCl in PBS to elute impurities and 800mM NaCl in PBS to elute the target protein.

And (3) performing 10% SDS-PAGE on the fermentation liquor obtained in the step (2), the eluent obtained after affinity chromatography and the eluent obtained after ion exchange chromatography, and then staining with Coomassie brilliant blue. The gp96 protein was subjected to a western blot, and the primary antibody was a rat anti-gp 96 antibody (purchased from santa cruz, product No. sc-56399), and the results are shown in FIG. 2. FIG. 2 shows that the gp96 protein was highly pure in the eluate after ion exchange chromatography.

EXAMPLE 3 preparation of recombinant Heat shock protein gp96 expressed by insect cells

Recombinant plasmid pFastBac^TMConstruction of 1-gp96

1. The RNA of HepG2 cells was extracted by Trizol method and then reverse transcribed to obtain cDNA.

2. According to the sequence of human gp96 gene (GenBank number is AY040226.1), primer F1 is artificially synthesized: 5' -GGAATTCATGGACGATGAAGTTGAT-3' (restriction enzyme EcoRI recognition sequence underlined) and R1: 5' -GCTCTAGACTATTAGAATTCATCTTTTTC-3' (recognition sequence for the restriction enzyme Xba I is underlined).

3. After steps 1 and 2 are completed, PCR amplification is carried out by taking the cDNA obtained in step 1 as a template and taking F1 and R1 synthesized in step 2 as primers to obtain a PCR amplification product.

4. The PCR amplification product is double digested with restriction enzymes EcoRI and XbaI, and the digested product is recovered.

5. The plasmid pFastBac was digested with the restriction enzymes EcoRI and Xba I^TM1, recovering a vector backbone of about 4700 bp.

6. And connecting the enzyme digestion product with a carrier skeleton to obtain a connecting product.

7. And (3) transforming the connecting product obtained in the step (6) into an escherichia coli DH10Bac competent cell to obtain a recombinant escherichia coli, and then extracting the plasmid of the recombinant escherichia coli to obtain a recombinant plasmid pFastBac1-gp 96.

The recombinant plasmid pFastBac1-gp96 was structurally described as follows based on the sequencing results: the fragment between the EcoRI and XbaI recognition sequences of plasmid pFastBac1 (plasmid pFastBac1 was cut into one large and one small fragment by the restriction endonucleases EcoRI and XbaI, the DNA being the small fragment) was replaced by a double stranded DNA molecule as shown in SEQ ID No.3 of the sequence Listing. The recombinant plasmid pFastBac1-gp96 expresses recombinant heat shock protein gp96 (hereinafter abbreviated as rgp96), and the amino acid sequence of the rgp96 is shown as SEQ ID NO.4 in a sequence table.

Expression of di, rgp96

1. The recombinant plasmid pFastBac1-gp96 constructed in the step one was co-transfected into Sf9 cells (every 1X 10)⁶Sf9 cells were transfected with approximately 4. mu.g of recombinant plasmid pFastBac1-gp 96; in the process of co-transfection, the transfection reagent is Cellffectin IIreagent, and the culture medium is Insect-XPRESS^TMProtein-free Instrument with L-Glutamine, incubation at 27 deg.C for 72h, and centrifuging to obtain supernatant as P1 generation virus.

2. Sf9 cell suspension 1 (containing 1X 10)⁸Sf9 cells) are cultured for 8-10 h at 27 ℃ to obtain cultured cells; then adding P1 generation virus (the dose is 0.05-0.1 MOI) into the cultured cells, incubating for 72h at 27 ℃, centrifuging for 5min at 4000rpm, and obtaining the supernatant which is the P2 generation virus.

3. To Sf9 cell suspension 2 (containing 1.6:)10⁸Sf9 cells) are added with P2 generation virus (the dosage is 0.05-0.1 MOI), cultured for 72h at the temperature of 27 ℃ and at the rpm of 100-120, and centrifuged for 5min at the rpm of 4000 for obtaining the supernatant which is the P3 generation virus.

The gp96 monoclonal antibody is used as a primary antibody, and the goat anti-rat monoclonal antibody marked by horseradish peroxidase is used as a secondary antibody, the P3 generation virus is subjected to western hybridization, and the specific steps of the western hybridization refer to the following documents: yangming, chuanqiang, rodelitis, yaohuaazan, jonquil, lissengquine. expression of mouse soluble IL-5 α receptor in Bac-to-Bac system and its identification [ J ]. journal of chinese biologies, 2013, 26: 5. results of western hybridization experiments showed that rgp96 was expressed in Sf9 cells.

Purification of tri, rgp96

1. To 300ml of Sf9 cell suspension 3 (containing 4.5X 10)⁸Sf9 cells) is added with P3 virus (the dose is 5MOI), and the mixture is cultured for 72 hours at the temperature of 27 ℃ and the rpm of 100-120 to obtain suspension.

2. The suspension was centrifuged at 7000rpm for 20min to obtain supernatant 1.

3. And (3) filtering the supernatant fluid 1 through a filter membrane of 0.22mm to obtain a supernatant fluid.

4. Loading the sample solution on a HiTrap-Q Sepharose ion exchange chromatography column (flow rate is 1mL/min), and then washing with 5mL of PBS buffer solution (flow rate is 1mL/min) with pH7.5 and 200 mM; then washed with 10mL of 300mM PBS buffer (flow rate 1mL/min) at pH 7.5; and finally, washing with 3mL of PBS buffer solution (the flow rate is 1mL/min) with the pH value of 7.5 and the concentration of the solution after passing through the column, and performing ultrafiltration concentration by using an ultrafiltration tube with the molecular weight cutoff of 50KD to obtain about 1mL of concentrated solution. The concentrate contained rgp 96.

5. And (3) loading the concentrated solution obtained in the step (4) onto a Superdex 20010/300 GL molecular sieve chromatographic column (flow rate is 0.25mL/min), washing with PBS (phosphate buffer solution) with pH7.5 and 150mM (flow rate is 0.25mL/min), collecting 9-12 mL penetration liquid, and further performing ultrafiltration concentration by adopting an ultrafiltration tube with molecular weight cutoff of 50KD to obtain a solution of rgp 96. The protein concentration in the solution of rgp96 was determined by BCA method, finally dispensed and stored at-80 ℃.

The solution of rgp96 obtained in step 5 was analyzed by SDS-PAGE and the results are shown in FIG. 3 (lanes from left to right are high molecular weight standard protein and rgp96, respectively). Western blot was performed on the solution of the recombinant heat shock protein gp96 obtained in step 5 (using gp96 monoclonal antibody as primary antibody and horseradish peroxidase-labeled goat-anti-rat monoclonal antibody as secondary antibody), and the experimental results are shown in FIG. 3. The results show that the solution of rgp96 shows a single molecular weight band, corresponding to a molecular weight consistent with the expectation.

Example 4 preparation and characterization of complexes of DEF8 polypeptide binding to placental gp96

Identification of mouse placental gp 96-binding polypeptide

1. Polypeptide elution: 5mg of mouse placenta pgp96 extracted in example 1 and having a concentration of 10mg/mL was added to 5. mu.L of an aqueous solution containing 20% (by volume) trifluoroacetic acid and incubated at 4 ℃ for 1 hour.

2. The incubation solution in step 1 was added to a 3kD ultrafiltration tube, centrifuged at 12,000rpm for 30min, and the permeate was taken.

3. Injecting the penetrating liquid in the step 2 into an Agilent 1200 high-pressure liquid chromatograph for desalting and purifying (chromatographic column model SB-C18)

2.1mm × 250mm), the procedure is as follows: phase A: 5% (volume ratio) acetonitrile water solution containing 0.1% trifluoroacetic acid; phase B: acetonitrile, containing 0.1% trifluoroacetic acid; 0-30min 100% A, 30-70min 100% B.

4. Collecting the eluate of step 3 for 30-70min, and lyophilizing.

5. The lyophilized powder of step 4 was reconstituted with 0.1% (by volume) aqueous formic acid, injected into an orbitrapfiltration high resolution mass spectrometer, analyzed for polypeptide sequence, and matched from the database. The matching results are shown in table 1.

TABLE 1 matching of protein polypeptides binding to mouse placental gp96

Preparation of Di, DEF8 polypeptide-gp 96 Complex

1. Inputting the DEF8 protein result amino acid sequence obtained in the table 1 into the website http:// tools. immuneepitope.org/mhci/performing epitope prediction of MHC class I molecules, and respectively selecting optimal H-2K^d、H-2K^bAnd HLA-A2 restrictive epitope and chemically synthesized, and the amino acid sequence selected when predicting the HLA-A2 epitope should be homologous protein in human. The epitope screening results are shown in table 2.

TABLE 2 epitope screening results for DEF8 protein Polypeptides

MHC I restricted type	Polypeptide sequence	Polypeptide position
			H-2Kd	RYLALMVSRPVL	273-284
H-2Kb	CSMRYLAL	270-277
			HLA-A2	LLFNYVEELVEI	292-303

2. The above polypeptides were each chemically synthesized (Shanghai Jier Biochemical Co., Ltd.), the synthesized polypeptides were reconstituted with cell-grade DMSO (Sigma-Aldrich Co., Cat. No. D2650) to a concentration of 20mg/mL, and 1mg of the polypeptide and 1mg of the recombinant rgp96 prepared in example 3 were each diluted to a volume of 4mL with PBS buffer pH 7.40.01mol/L. The mixture was hot-hit at 55 ℃ for 10 minutes and cooled at room temperature for 30 minutes. Then washing away the unbound polypeptide by using a 50kD ultrafiltration tube to respectively prepare DEF8 polypeptide-gp 96 complexes with different restriction epitopes.

Example 5 prevention and treatment of Breast cancer with DEF8 polypeptide-gp 96 Complex

First, DEF8 polypeptide-gp 96 compound has preventive effect on primary breast cancer

20 8-week-old C57BL/6 mice were divided into two groups of 10 mice each, and treated as follows:

a first group: DEF8 polypeptide (H-2K) prepared in example 4 was injected subcutaneously into the abdomen^b) Gp96 complex, immunized three times (0.2 ml each) with a single immunization dose of 20 ug/mouse;

second group: the rgp96 prepared in example 3 was injected subcutaneously into the abdomen three times (0.2 ml each) with a single immunization dose of 20 ug/mouse;

in the above two groups: experiment day 1 first immunization; second immunization on day 8; the third immunization was performed on day 22.

After 1 week of immunization, a solution of DMBA (Sigma-Aldrich, Cat. No. D3254) in 50mg/kg of intragastric edible olive oil was administered orally to each mouse 1 time per week for 5 consecutive weeks to induce the onset of breast cancer.

Tumor growth was measured weekly from the initiation of breast cancer tumor development in mice (about 14 weeks after molding), and tumor incidence and tumor volume were counted. Tumor volume calculation formula V ═ ab²V-volume, a-tumor major diameter, b-tumor minor diameter. Tumor incidence, number of tumors and mean tumor size were calculated for 34 weeks of observation. The statistical results are shown in Table 3. The results show that the DEF8 polypeptide-gp 96 complex has obvious prevention effect on breast cancer induced by DMBA. After the DEF8 polypeptide-gp 96 compound is immunized, the incidence rate of breast cancer is obviously reduced, the incidence time is delayed, and the number and the volume of tumors are reduced.

TABLE 3 statistical results of DMBA-induced breast cancer tumors in mice

Therapeutic effect of DEF8 polypeptide-gp 96 complex on induced breast cancer model

20 female Balb/c mice of 6-8 weeks were inoculated subcutaneously with 6X 10 mice each⁵TUBO cells, mice divided into two groups of 10 on day 2, were treated as follows:

a first group: DEF8 polypeptide (H-2K) prepared in example 4 was injected subcutaneously into the abdomen^d) Gp96 complex, immunized three times (0.2 ml each) with a single immunization dose of 20 ug/mouse;

second group: gp96 prepared in example 3 was injected subcutaneously into the abdomen three times (0.2 ml each time) and the single immunization dose was 20 ug/mouse;

in the above two groups: the first immunization was performed on day 2 after tumor cell inoculation; second immunization on day 5; the third immunization was performed on day 8. Starting from the first day of immunization, tumor growth was observed daily, tumor size was recorded, and tumor volume was calculated according to the following formula: ab ═ V²V-volume, a-tumor major diameter, b-tumor minor diameter. The change in tumor volume is shown in FIG. 4. From the results, it can be seen that DEF8 polypeptide-gp 96 complex has significant therapeutic effect on an induced breast cancer model inoculated subcutaneously. After the DEF8 polypeptide-gp 96 compound is used for treating subcutaneous tumors, the growth rate of the subcutaneous tumors is obviously reduced, and the tumor volume is reduced.

Example 6 prevention and treatment of liver cancer by DEF8 polypeptide-gp 96 Complex

First, DEF8 polypeptide-gp 96 compound has prevention effect on primary liver cancer

20 female C57BL/6 mice, 6 weeks old, were divided equally into two groups of 10 mice each, and treated as follows:

a first group: DEF8 polypeptide (H-2K) prepared in example 4 was injected subcutaneously into the abdomen^b) Gp96 complex, immunized three times (0.2 ml each) with a single immunization dose of 20 ug/mouse;

second group: the rgp96 prepared in example 3 was injected subcutaneously into the abdomen three times (0.2 ml each) with a single immunization dose of 20 ug/mouse;

in the above two groups: experiment day 1 first immunization; second immunization on day 8; the third immunization was performed on day 22.

1 week after the end of the last immunization, both groups of mice were fed continuously for 40 weeks with daily drinking water changed to sterile distilled water containing 30. mu.g/mL DEN (Sigma-Aldrich, Cat. No. 73861), and by 40 weeks, the mice were sacrificed and livers were removed for comparative analysis. The results of the comparison are shown in Table 4. The results show that the DEF8 polypeptide-gp 96 complex has obvious prevention effect on DEN-induced liver cancer. After the DEF8 polypeptide-gp 96 compound is immunized, the incidence rate of liver cancer is obviously reduced, the incidence time is delayed, and the number and the volume of tumors are reduced.

TABLE 4 DEN-induced statistical results of mouse liver cancer

Therapeutic effect of DEF8 polypeptide-gp 96 complex on induction of liver cancer model

Rapidly thawing mouse liver cancer H22 cells frozen in liquid nitrogen in water bath at 37 deg.C, and adjusting cell density to 1 × 10⁷Per mL, 2 BALB/c mice were intraperitoneally inoculated, 0.2mL each. After the abdominal distension of the mouse, the mouse is dislocated and killed, the abdominal part is disinfected, the ascites is extracted and combined, the cell density is adjusted to 1 multiplied by 10 by PBS⁷Each mouse was inoculated subcutaneously with 20 BALB/c mice, 0.2 mL. After 12 days, the mice were divided into two groups of 10 mice each, and treated as follows:

a first group: DEF8 polypeptide (H-2K) prepared in example 4 was injected subcutaneously into the abdomen^d) Gp96 complex, immunized three times (0.2 ml each) with a single immunization dose of 20 ug/mouse;

second group: the rgp96 prepared in example 3 was injected subcutaneously into the abdomen three times (0.2 ml each) with a single immunization dose of 20 ug/mouse;

in the above two groups: the first immunization was performed on day 12 after tumor cell inoculation; a second immunization on day 15; the third immunization was performed on day 18. Starting from the first day of immunization, tumor growth was observed daily, tumor size was recorded, and tumor volume was calculated according to the following formula: ab ═ V²V-volume, a-tumor major diameter, b-tumor minor diameter. Changes in tumor volume are shown in figure 5. The results show that the DEF8 polypeptide-gp 96 complex has obvious therapeutic effect on an induced liver cancer model inoculated subcutaneously. After the DEF8 polypeptide-gp 96 compound is used for treating subcutaneous tumors, the growth rate of the subcutaneous tumors is obviously reduced, and the tumor volume is reduced.

Example 7 DEF8 polypeptide-gp 96 Complex-induced specific CTL killing Effect on human Breast cancer cells

Preparation of human polypeptide specific effector cell

1. Anticoagulated fresh whole blood of HLA-A2 positive volunteers was separated using human lymphocyte separation medium (Cellgro,25-072-CI) to obtain Peripheral Blood Mononuclear Cells (PBMC), and the cell concentration was adjusted to 1.0X10 with RPMI-1640 complete medium (Gibco, 12633012) containing 10% fetal bovine serum (Gibco, 10099-141-FBS)⁶Perml, 1ml per well, seeded in 24-well plates.

2. The following day each group was added DEF8 polypeptide (HLA-A2) -gp96 complex to a final concentration of 10. mu.g/ml.

3. On the third day IL-2(PeproTech, Cat. 212-12) was added to a final concentration of 50U/ml per well, and half the fluid changes and IL-2 supplementation were performed to a final concentration of 50U/ml every 2-3 days.

4. A second and a third rounds of DEF8 polypeptide-gp 96 stimulation were performed on the seventh and fourteenth days, respectively, and IL-2 was added the next day to a final concentration of 50U/ml.

5. 3 days after the third round of stimulation, the human polypeptide effector cells CTL are obtained.

Secondly, target cells: human breast cancer cell line MCF-7 (positive for HLA-A2), T2 cell (positive for HLA-A2) and T2 cell incubated by polypeptide.

And thirdly, detecting the specific killing effect of the breast cancer cells: use of

The cytotoxic activity was measured by non-radioactive cytotoxicity assay (Promega, catalog No. G1780) using the following main procedure (see kit instructions for details):

1. t2 cell, T2 cell incubated with MCF-7 cell, polypeptideCells as target cells, the number of target cells inoculated was 5X 10³The effector cells are added into each well according to the ratio of the effective target to the effective target of 10:1, and the effector cells are inoculated into a 96-well culture plate at the rate of 50 mu 1 per well, and the final volume is 100 mu 1;

in addition, an effector cell spontaneous LDH release group was set to calibrate the spontaneous LDH release from the effector cells (each group of effector cells was added to a 96-well plate at 50. mu.1/well, supplemented with 50. mu.1 RPMI-1640 medium containing 5% fetal bovine serum to a final volume of 100. mu.1;). Target cells spontaneous LDH release groups, used to correct target cells spontaneous LDH release (each group of target cells at 50u 1/hole into 96-well plates, supplemented with 50u 1 containing 5% fetal bovine serum RPMI-1640 medium to a final concentration of 100 u 1;). The maximal LDH release group of target cells was used as a reference for determining 100% LDH release when calculated (cells loaded with the same target cells spontaneous release group;). Volume correction control group to correct volume change due to addition of lysate (addition of RPMI-1640 medium containing 5% fetal bovine serum 100. mu.1;). A control group of media background was used to correct the LDH activity produced by serum in the media and the background absorption caused by phenol red (100. mu.1 of RPMI-1640 medium containing 5% fetal bovine serum; addition).

2. After cell inoculation, centrifugation was carried out for 4min using 250g followed by incubation for 4h in an incubator at 37 ℃; lysate (10 ×), 10 μ 1/well, was added to the maximal LDH-releasing group of target cells 45min before harvesting the supernatant; then centrifuging for 4min by using 250g, and harvesting a supernatant;

3. transferring 50 mu 1 of supernatant into an ELISA plate, preparing a substrate by using a detection buffer solution, adding 50 mu 1/hole of the prepared substrate into the ELISA plate, covering the plate, reacting for 30min at room temperature in a dark place, adding 50 mu 1 of stop solution into each hole, and detecting the light absorption value OD at 490nm in an ELISA instrument within 1 h.

4. Calculation of cell killing Rate

The killing rate (%) < x 100%

The results of cell killing are shown in FIG. 6. The results show that DEF8 polypeptide-gp 96 complex can induce epitope-specific cytotoxic T Cells (CTL) from human peripheral blood lymphocytes (PBMC), the CTL can kill human breast cancer cells MCF-7, and the DEF8 polypeptide-gp 96 complex has the capacity of preventing and treating human breast cancer.

Example 8 DEF8 polypeptide-gp 96 Complex-induced specific CTL killing Effect on human hepatoma cells

Firstly, preparing human polypeptide specific effector cells: DEF8 polypeptide (HLA-A2) -gp96 complex-induced specific CTL was prepared in the same manner as in example 7, using RPMI-1640 medium containing 5% fetal bovine serum to adjust the concentration to an appropriate level, and used as effector cells.

Secondly, target cells: HHCC (HLA-A2 positive) of human liver cancer cell, T2 cell (HLA-A2 positive) incubated by polypeptide and T2 cell

Thirdly, detecting the specific killing effect of the liver cancer cells: the detection method is the same as that in example 7, target cells are replaced by human liver cancer cells HHCC, effector cells and target cells are added into an experimental group according to the ratio of the effective target to the target of 10:1, and an effector cell spontaneous release group, a target cell maximum release group, a background control group and a volume correction control group are simultaneously established. And incubating for 4h at 37 ℃, adding cell lysate, and harvesting supernatant for LDH detection. The results of cell killing are shown in FIG. 7. The result shows that DEF8 polypeptide-gp 96 complex can induce epitope specific cytotoxic T Cells (CTL) from human peripheral blood lymphocytes (PBMC), the cells can kill human liver cancer cells HHCC, and the DEF8 polypeptide-gp 96 complex has the capacity of preventing and treating human liver cancer.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

SEQUENCE LISTING

<110> Beijing thermo-shock Biotechnology Ltd

Application of complex of polypeptide of <120> DEF8 protein and heat shock protein gp96 in preparation of medicine for treating and preventing cancer

<130>KHP171110225.0

<160>11

<170>PatentIn version 3.5

<210>1

<211>1536

<212>DNA

<213> DEF8 protein

<400>1

atggccatcc tgtccctgcg agcccctggg ccctggcagg cgatgcaggt atgggcagac 60

aggacgctgt tgactccgca caccggggtg acttctcagg ttctcggggt ggcagctgca 120

gtgatgacac cgcttcctgg tggtcacgcc gcgggcagga cgcgggaggc caggtgggat 180

gctatggaat atgatgagaa gctggcccgt ttccggcagg cccacctcaa ccccttcaac 240

aagcagtctg ggccgagaca gcatgagcag ggccctgggg aggaggtccc ggacgtcact 300

cctgaagagg ccctgcctga gctgccccct ggggagccgg aattccgctg ccctgaacgc 360

gtgatggatc tcggcctgtc tgaggaccac ttctcccgcc ctgtgggtct gttcctggcc 420

tctgacgtcc agcagctgcg gcaggcgatc gaggagtgca agcaggtgat tctggagctg 480

cccgagcagt cggagaagca gaaggatgcc gtggtgcgac tcatccacct ccggctgaag 540

ctccaggagc tgaaggaccc caatgaggat gagccaaaca tccgagtgct ccttgagcac 600

cgcttttaca aggagaagag caagagcgtc aagcagacct gtgacaagtg taacaccatc 660

atctgggggc tcattcagac ctggtacacc tgcacagggt gttattaccg ctgtcacagt 720

aagtgcttga acctcatctc caagccctgt gtgagctcca aagtcagcca ccaagctgaa 780

tacgaactga acatctgccc tgagacaggg ctggacagcc aggattaccg ctgtgccgag 840

tgccgggcgc ccatctctct gcggggtgtg cccagtgagg ccaggcagtg cgactacacc 900

ggccagtact actgcagcca ctgccactgg aacgacctgg ctgtgatccc tgcacgcgtt 960

gtacacaact gggactttga gcctcgaaag gtttctcgct gcagcatgcg ctacctggcg 1020

ctgatggtgt ctcggcccgt actcaggctc cgggagatca accctctgct gttcagctac 1080

gtggaggagc tggtggagat tcgcaagctg cgccaggaca tcctgctcat gaagccgtac 1140

ttcatcacct gcagggaggc catggaggct cgtctgctgc tgcagctcca ggatcggcag 1200

cattttgtgg agaacgacga gatgtactct gtccaggacc tcctggacgt gcatgccggc 1260

cgcctgggct gctcgctcac cgagatccac acgctcttcg ccaagcacat caagctggac 1320

tgcgagcggt gccaggccaa gggcttcgtg tgtgagctct gcagagaggg cgacgtgctg 1380

ttcccgttcg acagccacac gtctgtgtgc gccgactgct ccgcggtctt ccacagggac 1440

tgctactacg acaactccac cacttgtccc aagtgtgccc ggctcagcct gaggaagcag 1500

tcgctcttcc aggagccagg tcccgatgtg gaggcc 1536

<210>2

<211>512

<212>PRT

<213> DEF8 protein

<400>2

Met Ala Ile Leu Ser Leu Arg Ala Pro Gly Pro Trp Gln Ala Met Gln

1 5 10 15

Val Trp Ala Asp Arg Thr Leu Leu Thr Pro His Thr Gly Val Thr Ser

20 25 30

Gln Val Leu Gly Val Ala Ala Ala Val Met Thr Pro Leu Pro Gly Gly

35 40 45

His Ala Ala Gly Arg Thr Arg Glu Ala Arg Trp Asp Ala Met Glu Tyr

50 55 60

Asp Glu Lys Leu Ala Arg Phe Arg Gln Ala His Leu Asn Pro Phe Asn

65 70 75 80

Lys Gln Ser Gly ProArg Gln His Glu Gln Gly Pro Gly Glu Glu Val

85 90 95

Pro Asp Val Thr Pro Glu Glu Ala Leu Pro Glu Leu Pro Pro Gly Glu

100 105 110

Pro Glu Phe Arg Cys Pro Glu Arg Val Met Asp Leu Gly Leu Ser Glu

115 120 125

Asp His Phe Ser Arg Pro Val Gly Leu Phe Leu Ala Ser Asp Val Gln

130 135 140

Gln Leu Arg Gln Ala Ile Glu Glu Cys Lys Gln Val Ile Leu Glu Leu

145 150 155 160

Pro Glu Gln Ser Glu Lys Gln Lys Asp Ala Val Val Arg Leu Ile His

165 170 175

Leu Arg Leu Lys Leu Gln Glu Leu Lys Asp Pro Asn Glu Asp Glu Pro

180 185 190

Asn Ile Arg Val Leu Leu Glu His Arg Phe Tyr Lys Glu Lys Ser Lys

195 200 205

Ser Val Lys Gln Thr Cys Asp Lys Cys Asn Thr Ile Ile Trp Gly Leu

210 215 220

Ile Gln Thr Trp Tyr Thr Cys Thr Gly Cys Tyr Tyr Arg Cys His Ser

225 230 235 240

Lys Cys Leu Asn Leu Ile Ser Lys Pro Cys Val Ser Ser Lys Val Ser

245 250 255

His Gln Ala Glu Tyr Glu Leu Asn Ile Cys Pro Glu Thr Gly Leu Asp

260 265 270

Ser Gln Asp Tyr Arg Cys Ala Glu Cys Arg Ala Pro Ile Ser Leu Arg

275 280 285

Gly Val Pro Ser Glu Ala Arg Gln Cys Asp Tyr Thr Gly Gln Tyr Tyr

290 295 300

Cys Ser His Cys His Trp Asn Asp Leu Ala Val Ile Pro Ala Arg Val

305 310 315 320

Val His Asn Trp Asp Phe Glu Pro Arg Lys Val Ser Arg Cys Ser Met

325 330 335

Arg Tyr Leu Ala Leu Met Val Ser Arg Pro Val Leu Arg Leu Arg Glu

340 345 350

Ile Asn Pro Leu Leu Phe Ser Tyr Val Glu Glu Leu Val Glu Ile Arg

355 360 365

Lys Leu Arg Gln Asp Ile Leu Leu Met Lys Pro Tyr Phe Ile Thr Cys

370 375 380

Arg Glu Ala Met Glu Ala Arg Leu Leu Leu Gln Leu Gln Asp Arg Gln

385 390 395 400

His Phe Val Glu Asn Asp Glu Met Tyr Ser Val Gln Asp Leu Leu Asp

405 410 415

Val His Ala Gly Arg Leu Gly Cys Ser Leu Thr Glu Ile His Thr Leu

420 425 430

Phe Ala Lys His Ile Lys Leu Asp Cys Glu Arg Cys Gln Ala Lys Gly

435 440 445

Phe Val Cys Glu Leu Cys Arg Glu Gly Asp Val Leu Phe Pro Phe Asp

450 455 460

Ser His Thr Ser Val Cys Ala Asp Cys Ser Ala Val Phe His Arg Asp

465 470 475 480

Cys Tyr Tyr Asp Asn Ser Thr Thr Cys Pro Lys Cys Ala Arg Leu Ser

485 490 495

Leu Arg Lys Gln Ser Leu Phe Gln Glu Pro Gly Pro Asp Val Glu Ala

500 505 510

<210>3

<211>2409

<212>DNA

<213> Heat shock protein gp96

<400>3

atgagggccc tgtgggtgct gggcctctgc tgcgtcctgc tgaccttcgg gtcggtcaga 60

gctgacgatg aagttgatgt ggatggtaca gtagaagagg atctgggtaa aagtagagaa 120

ggatcaagga cggatgatga agtagtacag agagaggaag aagctattca gttggatgga 180

ttaaatgcat cacaaataag agaacttaga gagaagtcgg aaaagtttgc cttccaagcc 240

gaagttaaca gaatgatgaa acttatcatc aattcattgt ataaaaataa agagattttc 300

ctgagagaac tgatttcaaa tgcttctgat gctttagata agataaggct aatatcactg 360

actgatgaaa atgctctttc tggaaatgag gaactaacag tcaaaattaa gtgtgataag 420

gagaagaacc tgctgcatgt cacagacacc ggtgtaggaa tgaccagaga agagttggtt 480

aaaaaccttg gtaccatagc caaatctggg acaagcgagt ttttaaacaa aatgactgaa 540

gcacaggaag atggccagtc aacttctgaa ttgattggcc agtttggtgt cggtttctat 600

tccgccttcc ttgtagcaga taaggttatt gtcacttcaa aacacaacaa cgatacccag 660

cacatctggg agtctgactc caatgaattt tctgtaattg ctgacccaag aggaaacact 720

ctaggacggg gaacgacaat tacccttgtc ttaaaagaag aagcatctga ttaccttgaa 780

ttggatacaa ttaaaaatct cgtcaaaaaa tattcacagt tcataaactt tcctatttat 840

gtatggagca gcaagactga aactgttgag gagcccatgg aggaagaaga agcagccaaa 900

gaagagaaag aagaatctga tgatgaagct gcagtagagg aagaagaaga agaaaagaaa 960

ccaaagacta aaaaagttga aaaaactgtc tgggactggg aacttatgaa tgatatcaaa 1020

ccaatatggc agagaccatc aaaagaagta gaagaagatg aatacaaagc tttctacaaa 1080

tcattttcaa aggaaagtga tgaccccatg gcttatattc actttactgc tgaaggggaa 1140

gttaccttca aatcaatttt atttgtaccc acatctgctc cacgtggtct gtttgacgaa 1200

tatggatcta aaaagagcga ttacattaag ctctatgtgc gccgtgtatt catcacagac 1260

gacttccatg atatgatgcc taaatacctc aattttgtca agggtgtggt ggactcagat 1320

gatctcccct tgaatgtttc ccgcgagact cttcagcaac ataaactgct taaggtgatt 1380

aggaagaagc ttgttcgtaa aacgctggac atgatcaaga agattgctga tgataaatac 1440

aatgatactt tttggaaaga atttggtacc aacatcaagc ttggtgtgat tgaagaccac 1500

tcgaatcgaa cacgtcttgc taaacttctt aggttccagt cttctcatca tccaactgac 1560

attactagcc tagaccagta tgtggaaaga atgaaggaaa aacaagacaa aatctacttc 1620

atggctgggt ccagcagaaa agaggctgaa tcttctccat ttgttgagcg acttctgaaa 1680

aagggctatg aagttattta cctcacagaa cctgtggatg aatactgtat tcaggccctt 1740

cccgaatttg atgggaagag gttccagaat gttgccaagg aaggagtgaa gttcgatgaa 1800

agtgagaaaa ctaaggagag tcgtgaagca gttgagaaag aatttgagcc tctgctgaat 1860

tggatgaaag ataaagccct taaggacaag attgaaaagg ctgtggtgtc tcagcgcctg 1920

acagaatctc cgtgtgcttt ggtggccagc cagtacggat ggtctggcaa catggagaga 1980

atcatgaaag cacaagcgta ccaaacgggc aaggacatct ctacaaatta ctatgcgagt 2040

cagaagaaaa catttgaaat taatcccaga cacccgctga tcagagacat gcttcgacga 2100

attaaggaag atgaagatga taaaacagtt ttggatcttg ctgtggtttt gtttgaaaca 2160

gcaacgcttc ggtcagggta tcttttacca gacactaaag catatggaga tagaatagaa 2220

agaatgcttc gcctcagttt gaacattgac cctgatgcaa aggtggaaga agagcccgaa 2280

gaagaacctg aagagacagc agaagacaca acagaagaca cagagcaaga cgaagatgaa 2340

gaaatggatg tgggaacaga tgaagaagaa gaaacagcaa aggaatctac agctgaaaaa 2400

gatgaattg 2409

<210>4

<211>803

<212>PRT

<213> Heat shock protein gp96

<400>4

Met Arg Ala Leu Trp Val Leu Gly Leu Cys Cys Val Leu Leu Thr Phe

1 5 10 15

Gly Ser Val Arg Ala Asp Asp Glu Val Asp Val Asp Gly Thr Val Glu

20 25 30

Glu Asp Leu Gly Lys Ser Arg Glu Gly Ser Arg Thr Asp Asp Glu Val

35 40 45

Val Gln Arg Glu Glu Glu Ala Ile Gln Leu Asp Gly Leu Asn Ala Ser

50 55 60

Gln Ile Arg Glu Leu Arg Glu Lys Ser Glu Lys Phe Ala Phe Gln Ala

65 70 75 80

Glu Val Asn Arg Met Met Lys Leu Ile Ile Asn Ser Leu Tyr Lys Asn

85 90 95

Lys Glu Ile Phe Leu Arg Glu Leu Ile Ser Asn Ala Ser Asp Ala Leu

100 105 110

Asp Lys Ile Arg Leu Ile Ser Leu Thr Asp Glu Asn Ala Leu Ser Gly

115 120125

Asn Glu Glu Leu Thr Val Lys Ile Lys Cys Asp Lys Glu Lys Asn Leu

130 135 140

Leu His Val Thr Asp Thr Gly Val Gly Met Thr Arg Glu Glu Leu Val

145 150 155 160

Lys Asn Leu Gly Thr Ile Ala Lys Ser Gly Thr Ser Glu Phe Leu Asn

165 170 175

Lys Met Thr Glu Ala Gln Glu Asp Gly Gln Ser Thr Ser Glu Leu Ile

180 185 190

Gly Gln Phe Gly Val Gly Phe Tyr Ser Ala Phe Leu Val Ala Asp Lys

195 200 205

Val Ile Val Thr Ser Lys His Asn Asn Asp Thr Gln His Ile Trp Glu

210 215 220

Ser Asp Ser Asn Glu Phe Ser Val Ile Ala Asp Pro Arg Gly Asn Thr

225 230 235 240

Leu Gly Arg Gly Thr Thr Ile Thr Leu Val Leu Lys Glu Glu Ala Ser

245 250 255

Asp Tyr Leu Glu Leu Asp Thr Ile Lys Asn Leu Val Lys Lys Tyr Ser

260 265 270

Gln Phe Ile Asn Phe Pro Ile Tyr Val Trp Ser Ser Lys Thr Glu Thr

275 280 285

Val Glu Glu Pro Met Glu Glu Glu Glu Ala Ala Lys Glu Glu Lys Glu

290 295 300

Glu Ser Asp Asp Glu Ala Ala Val Glu Glu Glu Glu Glu Glu Lys Lys

305 310 315 320

Pro Lys Thr Lys Lys Val Glu Lys Thr Val Trp Asp Trp Glu Leu Met

325 330 335

Asn Asp Ile Lys Pro Ile Trp Gln Arg Pro Ser Lys Glu Val Glu Glu

340 345 350

Asp Glu Tyr Lys Ala Phe Tyr Lys Ser Phe Ser Lys Glu Ser Asp Asp

355 360 365

Pro Met Ala Tyr Ile His Phe Thr Ala Glu Gly Glu Val Thr Phe Lys

370 375 380

Ser Ile Leu Phe Val Pro Thr Ser Ala Pro Arg Gly Leu Phe Asp Glu

385 390 395 400

Tyr Gly Ser Lys Lys Ser Asp Tyr Ile Lys Leu Tyr Val Arg Arg Val

405 410 415

Phe Ile Thr Asp Asp Phe His Asp Met Met Pro Lys Tyr Leu Asn Phe

420 425 430

Val Lys Gly Val Val Asp Ser Asp Asp Leu Pro Leu Asn Val Ser Arg

435 440 445

Glu Thr Leu Gln Gln His Lys Leu Leu Lys Val Ile Arg Lys Lys Leu

450 455 460

Val Arg Lys Thr Leu Asp Met Ile Lys Lys Ile Ala Asp Asp Lys Tyr

465 470 475 480

Asn Asp Thr Phe Trp Lys Glu Phe Gly Thr Asn Ile Lys Leu Gly Val

485 490 495

Ile Glu Asp His Ser Asn Arg Thr Arg Leu Ala Lys Leu Leu Arg Phe

500 505 510

Gln Ser Ser His His Pro Thr Asp Ile Thr Ser Leu Asp Gln Tyr Val

515 520 525

Glu Arg Met Lys Glu Lys Gln Asp Lys Ile Tyr Phe Met Ala Gly Ser

530 535 540

Ser Arg Lys Glu Ala Glu Ser Ser Pro Phe Val Glu Arg Leu Leu Lys

545 550 555 560

Lys Gly Tyr Glu Val Ile Tyr Leu Thr Glu Pro Val Asp Glu Tyr Cys

565 570 575

Ile Gln Ala Leu Pro Glu Phe Asp Gly Lys Arg Phe Gln Asn Val Ala

580 585 590

Lys Glu Gly Val Lys Phe Asp Glu Ser Glu Lys Thr Lys Glu Ser Arg

595 600 605

Glu Ala Val Glu Lys Glu Phe Glu Pro Leu Leu Asn Trp Met Lys Asp

610 615 620

Lys Ala Leu Lys Asp Lys Ile Glu Lys Ala Val Val Ser Gln Arg Leu

625 630 635 640

Thr Glu Ser Pro Cys Ala Leu Val Ala Ser Gln Tyr Gly Trp Ser Gly

645 650 655

Asn Met Glu Arg Ile Met Lys Ala Gln Ala Tyr Gln Thr Gly Lys Asp

660 665 670

Ile Ser Thr Asn Tyr Tyr Ala Ser Gln Lys Lys Thr Phe Glu Ile Asn

675 680 685

Pro Arg His Pro Leu Ile Arg Asp Met Leu Arg Arg Ile Lys Glu Asp

690 695 700

Glu Asp Asp Lys Thr Val Leu Asp Leu Ala Val Val Leu Phe Glu Thr

705 710 715 720

Ala Thr Leu Arg Ser Gly Tyr Leu Leu Pro Asp Thr Lys Ala Tyr Gly

725 730 735

Asp Arg Ile Glu Arg Met Leu Arg Leu Ser Leu Asn Ile Asp Pro Asp

740 745 750

Ala Lys Val Glu Glu Glu Pro Glu Glu Glu Pro Glu Glu Thr Ala Glu

755 760 765

Asp Thr Thr Glu Asp Thr Glu Gln Asp Glu Asp Glu Glu Met Asp Val

770 775 780

Gly Thr Asp Glu Glu Glu Glu Thr Ala Lys Glu Ser Thr Ala Glu Lys

785 790 795 800

Asp Glu Leu

<210>5

<211>28

<212>DNA

<213> Artificial sequence

<400>5

ccggaattca tggacgatga agttgatg 28

<210>6

<211>38

<212>DNA

<213> Artificial sequence

<400>6

ccgctcgagc tattagaatt catctttttc agctgtag 38

<210>7

<211>25

<212>DNA

<213> Artificial sequence

<400>7

ggaattcatg gacgatgaag ttgat 25

<210>8

<211>29

<212>DNA

<213> Artificial sequence

<400>8

gctctagact attagaattc atctttttc 29

<210>9

<211>12

<212>PRT

<213>H-2Kd

<400>9

Arg Tyr Leu Ala Leu Met Val Ser Arg Pro Val Leu

1 5 10

<210>10

<211>8

<212>PRT

<213>H-2Kb

<400>10

Cys Ser Met Arg Tyr Leu Ala Leu

1 5

<210>11

<211>12

<212>PRT

<213>HLA-A2

<400>11

Leu Leu Phe Asn Tyr Val Glu Glu Leu Val Glu Ile

1 5 10

Claims (5)

1. A medicament, comprising a complex of a DEF8 protein-containing polypeptide and a heat shock protein gp 96;

the amino acid sequence of the polypeptide of DEF8 protein is shown in SEQ ID NO.9 or SEQ ID NO. 10;

the amino acid sequence of the heat shock protein gp96 is shown in SEQ ID NO. 4.

2. The medicament of claim 1, wherein the gp96 is obtained by introducing a nucleic acid molecule encoding gp96 into a recipient, culturing, expressing in a yeast or mammalian cell expression system, and purifying.

3. The medicament according to claim 1 or 2, wherein the complex is obtained by:

the polypeptide of DEF8 protein and the heat shock protein gp96 form a complex in vitro by natural adsorption or heat shock.

The application of a complex consisting of a polypeptide of DEF8 and a heat shock protein gp96 in preparing a medicament for treating and/or preventing cancer, wherein the amino acid sequence of the polypeptide of DEF8 is shown as SEQ ID No.9 or SEQ ID No. 10; the cancer is breast cancer or liver cancer.

5. The use of claim 4, wherein when the amino acid sequence of the polypeptide of DEF8 is as set forth in SEQ ID No.9 or SEQ ID No.10, the medicament has the function of at least one of:

(1)H-2K^bthe restricted polypeptide sequence CSMRYLAL can reduce the incidence of chemical-induced breast cancer;

(2)H-2K^bthe restricted polypeptide sequence CSMRYLAL can reduce the incidence of liver cancer induced by chemicals;

(3)H-2K^dthe restricted polypeptide sequence RYLALMVSRPVL slows or stops the growth of an established breast cancer tumor focus;

(4)H-2K^dthe restricted polypeptide sequence RYLALMVSRPVL slows down or stops the growth of an established tumor focus of liver cancer.

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