CN106913863B - Application of compound of polypeptide of NUP188 protein and heat shock protein gp96 in preparation of medicine for treating and preventing cancer - Google Patents

Abstract

The invention provides an NUP188 protein and application of polypeptide thereof, belonging to the technical field of protein engineering and biomedical application. The amino acid sequence of the NUP188 protein is shown as SEQ ID NO.2 or a specific fragment thereof, and the invention discovers that the NUP188 protein and the polypeptide thereof, and a compound formed by the NUP188 protein and the polypeptide and the hot shock protein gp96 through 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 NUP188 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 an application of NUP188 protein and polypeptide thereof in treating and preventing cancer.

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.

The 188kDa Nucleoporin (NUP 188homolog, NUP188) is an important component of the Nup93 sub-complex next to the largest nucleopore complex, and the sub-complex in which the NUP188 protein is located is essential for controlling transport of proteins outside the nuclear membrane to the inside of the membrane and the integrity of the nuclear membrane. In addition, the NUP188 protein is reported to play an important role in mitosis, and can directly influence the attachment of tubulin bundle on the centromere, and finally can influence the normal separation or stability of chromosome, while the marker characteristics of most tumor cells are abnormal in cell chromosome.

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 a NUP188 protein, and the amino acid sequence of the NUP188 protein comprises 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 end; 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 application of the NUP188 protein in preparation of a medicine for preventing and/or treating cancer.

The invention provides a gene for coding NUP188 protein, and the nucleotide sequence of the gene comprises the following components: i) the 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 for coding NUP188 protein in preparing a medicament for preventing and/or treating cancer.

The invention provides a biological material containing a gene for coding NUP188 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 medicament containing NUP188 protein or polypeptide thereof.

The invention provides a medicine containing a complex formed by NUP188 protein and heat shock protein gp96 or a complex formed by polypeptide containing NUP188 protein 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 the NUP188 protein or the polypeptide thereof and the heat shock protein gp96 in vitro in a natural adsorption or heat shock mode; or

(2) Introducing nucleic acid molecules encoding NUP188 protein or its polypeptide and heat shock protein gp96 into receptor, culturing, and purifying to obtain the compound.

The expression of the nucleic acid molecule encoding the NUP188 protein or the polypeptide thereof and the heat shock protein gp96 in the receptor can be realized by Hansenula.

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 application of a compound consisting of the NUP188 protein and the polypeptide thereof and a heat shock protein gp96 in preparation of 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-mannopyranoseThe glycosides are all 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, separating and purifying 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' (recognition by the restriction enzyme EcoRI is underlined)Sequence) 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.

According to the sequencing result, the structure of the recombinant plasmid pFastBac1-gp96 is described as follows: 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 a sequence 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 (dosage is 0.05-0.1 MOI) into the cultured cells, and incubating at 27 DEG CAnd centrifuging for 5min at 4000rpm for 72h, wherein the supernatant is P2 generation virus.

3. To Sf9 cell suspension 2 (containing 1.6X 10)⁸Sf9 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 NUP188 Polypeptides 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 Orbitrap Fusion high resolution mass spectrometer, analyzed for polypeptide sequence, and matched from the database, with the results shown in table 1.

TABLE 1 matching of protein polypeptides binding to mouse placental gp96

Preparation of II, NUP188 polypeptide-gp 96 complex

1. Inputting the resulting amino acid sequence of the NUP188 protein obtained in the table 1 into the website

http:// tools. immuneepitope.org/mhci/epitope prediction of MHC class I molecules, 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 NUP188 protein polypeptides

MHC I restricted type	Polypeptide sequence	Polypeptide position
			H-2Kd	IYMLLQRLTTVI	593-604
H-2Kb	IIFLYYAYF	217-225
			HLA-A2	MLLQRLTTVISPPV	595-608

2. The above polypeptides were each chemically synthesized, and the synthesized polypeptides were reconstituted with cellular DMSO (Sigma-Aldrich, 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 adopting a 50kD ultrafiltration tube to respectively prepare NUP188 polypeptide-gp 96 complexes with different restriction epitopes

EXAMPLE 5 preventive and therapeutic Effect of polypeptide-gp 96 Complex on Breast cancer

Prevention effect of polypeptide-gp 96 complex 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: the NUP188 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 result shows that the NUP188 polypeptide-gp 96 complex has obvious prevention effect on breast cancer induced by DMBA. After the NUP188 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 NUP188 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: the NUP188 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 2 was injected subcutaneously in 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. The results show that the NUP188 polypeptide-gp 96 complex has obvious therapeutic effect on an induced breast cancer model inoculated subcutaneously. After the NUP188 polypeptide-gp 96 compound is used for treating subcutaneous tumors, the growth speed of the subcutaneous tumors is obviously reduced, and the tumor volume is reduced.

Example 6 prevention and treatment of liver cancer with NUP188 polypeptide-gp 96 Complex

Prevention effect of NUP188 polypeptide-gp 96 complex 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: the NUP188 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 NUP188 polypeptide-gp 96 complex has obvious prevention effect on DEN-induced liver cancer. After the NUP188 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 NUP188 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: the NUP188 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; on day 18And (4) carrying out third immunization. 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 NUP188 polypeptide-gp 96 complex has obvious therapeutic effect on an induced liver cancer model inoculated subcutaneously. After the NUP188 polypeptide-gp 96 compound is used for treating subcutaneous tumors, the growth speed of the subcutaneous tumors is obviously reduced, and the tumor volume is reduced.

Example 7 killing Effect of specific CTL induced by NUP188 polypeptide-gp 96 Complex 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 NUP188 polypeptide (HLA-A2) -gp96 complex was added to each group 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 round of stimulation with NUP188 polypeptide-gp 96 complex were performed on the seventh and fourteenth days, respectively, and IL-2 was added the following 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, namely human breast cancer cell lines MCF-7 (positive for expression of HLA-A2), polypeptide-incubated T2 cells (positive for expression of HLA-A2) and T2 cells.

Third, detection and use of specific killing effect of breast cancer cells

Non-radioactive cytotoxicity assay (Promega, Cat. No. G1780) cytotoxic Activity assayThe main steps are as follows (see the kit instruction for details):

1. inoculating MCF-7 cells, polypeptide-incubated T2 cells and T2 cells as target cells, wherein the number of the target cells is 5 × 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. The cell killing rate (%) was calculated as [ (experimental OD value-effector cell spontaneous release group OD value-target cell spontaneous release group OD value)/(target cell maximum release group OD value-target cell spontaneous release group OD value) ] × 100% ]

The cell killing result is shown in figure 6, which shows that the NUP188 polypeptide-gp 96 complex can induce epitope-specific cytotoxic T Cells (CTL) from human peripheral blood lymphocytes (PBMC), and the CTL can kill human breast cancer cells MCF-7, and shows that the NUP188 polypeptide-gp 96 complex has the capacity of preventing and treating human breast cancer.

Example 8 killing Effect of specific CTL induced by NUP188 polypeptide-gp 96 Complex on human hepatoma cells

Preparation of human polypeptide-specific effector cells: specific CTL induced by NUP188 polypeptide-gp 96 complex was prepared in the same manner as in example 7 by adjusting the concentration of the effector cells to an appropriate concentration in RPMI-1640 medium containing 5% fetal bovine serum.

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

Detection of specific killing effect of 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 the NUP188 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 NUP188 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> NUP188 protein and heat shock protein gp96 in preparation of medicine for treating and preventing cancer

<130>KHP171110227.2

<160>11

<170>PatentIn version 3.5

<210>1

<211>5247

<212>DNA

<213> NUP88 protein

<400>1

atggcggcgg ccgccggcgg gccgtgtgtg aggagcagta gagaactgtg gactattctg 60

cttggaaggt cagctctgag agagctgagt cagattgagg cagaactgaa taaacattgg 120

cggcgattgt tagaggggct ttcttactac aaacctccca gtccaagttc agctgaaaaa 180

gtgaaagcta ataaagatgt agcttcacca ttgaaggaac tgggtttaag aatcagcaag 240

tttttgggtc ttgatgaaga acagagtgtg cagttactcc agtgttacct gcaagaggac 300

tacaggggta ctcgggactc agtaaagaca gtactgcaag atgagaggca gagccaggcc 360

ttaatcctga agattgcaga ttattattat gaagaaagaa cctgtattct tcgttgtgtc 420

ttacaccttc tcacttactt ccaagatgaa agacacccct atagggttga atatgcagac 480

tgtgttgata aattggagaa ggaactagtt tcaaaataca gacagcagtt cgaagagctt 540

tataaaactg aagcaccaac ttgggagaca catggaaatc tcatgacaga gcgccaagtg 600

tctcgctggt ttgttcagtg ccttcgggaa cagtccatgc tgctagaaat tattttcctt 660

tattatgcat actttgagat ggcacccagt gacttacttg tattaaccaa gatgtttaaa 720

gagcaaggat ttggtagtag gcagaccaat aggcacctgg tggatgagac tatggatcct 780

tttgtagatc ggattggcta cttcagtgcc ctcatcctgg tggagggcat ggatatcgag 840

tccttgcata agtgtgcttt ggatgacaga agagaactgc atcagtttgc gcaggatggg 900

cttatttgtc aggatatgga ctgtttaatg ttgacctttg gggacattcc acatcatgcc 960

ccagtgcttt tggcctgggc tctcctccgt cacactctga acccagaaga gacaagcagt 1020

gtggtccgga agataggtgg cacagccatc cagctgaatg tgtttcagta cttgacccga 1080

ttgctccagt cccttgccag tgggggaaat gattgcacca ccagcactgc atgcatgtgt 1140

gtctatggac tgctctcttt cgttctgacc tcgttggagc tgcacaccct gggcaatcag 1200

caggatataa ttgatacagc atgtgaagta ttggccgacc cttctcttcc ggaactgttc 1260

tggggaacag agccaacttc tggccttggg atcattctgg acagtgtgtg tggaatgttt 1320

ccccaccttc tctccccact cctgcaactg ctccgagccc tggtatcagg gaagtccaca 1380

gccaaaaagg tgtatagctt cttggataag atgtctttct acaatgaact ttataaacac 1440

aagcctcatg atgtgatctc ccatgaagat ggaactcttt ggcggagaca aacacccaaa 1500

ctcctttatc cccttggggg tcaaaccaac cttcgcatac ctcaaggcac tgtgggccaa 1560

gtaatgttgg atgatagggc atacctggta cgctgggaat actcctatag cagctggacc 1620

ctctttacct gcgagattga aatgttgctt catgttgttt caactgcaga tgtgattcag 1680

cactgccagc gagtcaaacc catcattgat ctcgtccata aggtcatcag tacagacctg 1740

tcgatagcag actgtctcct gcccatcaca tctcgcatct acatgctgct gcagcggtta 1800

acgacagtga tctccccacc tgtggatgtc attgcttctt gtgtcaactg cttaactgtt 1860

ttggctgccc gcaatccagc aaaggtctgg actgatcttc gtcacacagg ttttttacca 1920

tttgtggccc atcctgtctc cagcctgagt cagatgatta gtgcggaagg gatgaatgct 1980

ggagggtacg gaaacctctt gatgaacagt gaacagcctc agggcgagta tggggttact 2040

attgcctttc tgcgcttgat caccaccctt gtcaaggggc aacttggtag tacccagagc 2100

caaggacttg taccctgtgt aatgtttgtg ctgaaggaga tgcttcccag ctaccataag 2160

tggcgctaca actctcatgg agtgagggaa cagattggtt gcctgatctt ggagctgatt 2220

catgcgatac tgaacctgtg ccacgagaca gacctgcaca gcagtcatac tcccagcctg 2280

cagtttctct gcatctgcag cctggcatac acagaagcag gacagacagt tatcaatatc 2340

atgggcattg gcgtggacac cattgacatg gtgatggctg ctcagcctcg aagtgatggg 2400

gcagagggcc aggggcaggg ccagctgctg atcaagacag tgaaactggc attctccgtc 2460

accaacaatg ttattcggct gaaacctcct tctaatgtgg tgtcccccct ggaacaggct 2520

ctctcacaac atggtgctca tggaaacaac ctcattgctg ttctagccaa atacatctac 2580

cacaaacatg accctgcttt gccacgtctt gccattcagc tgctgaaacg tctggccacg 2640

gtggccccaa tgtcagtgta tgcttgtctg ggcaatgatg cggctgccat tcgtgatgcc 2700

ttcctgaccc gattgcagag caaaattgag gacatgcgca tcaaagtcat gattctagag 2760

ttcctcactg ttgcagtaga gacccagcca ggcctcatcg aactgtttct gaacctggaa 2820

gttaaggatg gcagtgatgg ctcaaaggaa ttcagccttg ggatgtggag ctgtctccat 2880

gcagtgctgg agctgattga ttcccaacag caagatcgat actggtgccc acccctgctg 2940

catcgtgccg ccattgcctt tttgcatgct ctgtggcagg atcggaggga cagtgccatg 3000

ctggtcctcc gaaccaaacc caagttttgg gaaaatttaa ccagtccgct gtttggaacc 3060

ctttctcctc cctctgaaac atcagagccc agcatcctgg aaacctgtgc cctaatcatg 3120

aagataattt gcttggagat atactatgta gtaaagggtt cattagacca gtcattaaag 3180

gatacactga agaaattttc catcgagaaa cgctttgcct actggtcagg gtatgtcaag 3240

tcattggcag ttcacgtggc cgaaacagaa ggcagcagct gcacctcctt gttagagtac 3300

cagatgctgg tgtccgcctg gaggatgctt ctcatcattg ccaccactca tgcagatata 3360

atgcacctga ctgactctgt ggtgcgtcgc cagctctttc ttgacgtgct tgatggaacc 3420

aaagcattac tcctagttcc agcctcagtg aactgccttc gccttggctc catgaagtgc 3480

actctgctgc ttatcctcct ccggcagtgg aagagagagt taggttctgt ggatgaaatc 3540

cttggaccct tgacggagat cctggaggga gtgctgcagg ccgaccagca actcatggag 3600

aagaccaagg ccaaggtgtt ctcagcattc atcacagtgt tgcaaatgaa ggagatgaaa 3660

gtaagtgaca tcccccagta ctcccagctg gtgctgaatg tctgtgagac cctccaagag 3720

gaagtgattg cactcttcga ccagacccgc cacagtctgg cattaggcag tgccacagag 3780

gacaaggaca gcatggagac tgacgactgt tctcggtccc ggcacaggga ccagcgtgat 3840

ggggtgtgtg tcctgggcct gcacctggcc aaggagctgt gtgaggtaga cgaggatggt 3900

gactcctggc tgcaggtaac ccgcaggctc cccatcctac ccaccctcct caccactcta 3960

gaggtgagcc ttcgcatgaa gcagaacctg catttcactg aggccacatt gcatctgctc 4020

ctcaccctgg ctcgcactca gcagggagcc acagcagtgg ctggagctgg catcacccag 4080

agcatttgtt tgccccttct gagtgtgtac cagctgagca ccaacggcac agcacagaca 4140

cctagtgcct ctcggaagtc cctggatgcc ccctcttggc caggagtcta ccgcctgtcc 4200

atgtccctga tggagcagct gctcaaaact ctgcgctaca acttcctgcc tgaggccctg 4260

gacttcgtgg gtgtccacca ggagcggacc ttacagtgcc tcaacgcagt gaggacagtg 4320

cagagtctgg cctgcctgga ggaggcggac cacaccgtgg gttttattct gcagctctct 4380

aacttcatga aggagtggca cttccacctg cctcagctca tgcgtgatat ccaggtcaac 4440

ctgggttact tgtgccaggc atgtacctct ctcctgcaca gtcgaaagat gctgcagcat 4500

tacttacaga acaaaaatgg ggatggcctc ccctcagctg ttgcccagcg agtccagagg 4560

ccaccgtctg ctgcttctgc tgccccctcc tcctcaaagc agcccgctgc tgacacagag 4620

gcatcagagc agcaggcctt gcacacagtc cagtatggcc ttctcaagat cctcagcaag 4680

acgctggcag ccctgcgcca cttcacccca gatgtctgcc agattctgct ggatcagtcc 4740

ctggaccttg ctgaatacaa cttcctgttt gccctgagct ttaccactcc cacctttgac 4800

tccgaagtgg ccccctcctt cgggaccctt ctggccacag tgaatgtggc cctcaacatg 4860

cttggagagc tggacaagaa aaaggagccc ctcacccagg cagtggggct cagcacacag 4920

gcagaaggga ccaggacgtt aaagtccctc ctgatgttta ccatggaaaa ctgcttctac 4980

ctgctcatct ctcaggcgat gcggtacctt agggacccgg ctgtgcaccc ccgggacaaa 5040

cagcggatga agcaggagct cagctctgag ttgagcacgc tgctgtccag cctctcgcgc 5100

tacttccgcc ggggagcccc cagctcccct gccactggtg tcctcccctc gccgcagggc 5160

aagtccacct ctctctccaa agccagccct gagagtcagg agcctctgat ccagttggtg 5220

caggcgtttg tccggcatat gcaaaga 5247

<210>2

<211>1749

<212>PRT

<213> NUP88 protein

<400>2

Met Ala Ala Ala Ala Gly Gly Pro Cys Val Arg Ser Ser Arg Glu Leu

1 5 10 15

Trp Thr Ile Leu Leu Gly Arg Ser Ala Leu Arg Glu Leu Ser Gln Ile

20 25 30

Glu Ala Glu Leu Asn Lys His Trp Arg Arg Leu Leu Glu Gly Leu Ser

35 40 45

Tyr Tyr Lys Pro Pro Ser Pro Ser Ser Ala Glu Lys Val Lys Ala Asn

50 55 60

Lys Asp Val Ala Ser Pro Leu Lys Glu Leu Gly Leu Arg Ile Ser Lys

65 70 75 80

Phe Leu Gly Leu Asp Glu Glu Gln Ser Val Gln Leu Leu Gln Cys Tyr

85 90 95

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

100 105 110

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

115 120 125

Tyr Tyr Glu Glu Arg Thr Cys Ile Leu Arg Cys Val Leu His Leu Leu

130 135 140

Thr Tyr Phe Gln Asp Glu Arg His Pro Tyr Arg Val Glu Tyr Ala Asp

145 150 155 160

Cys Val Asp Lys Leu Glu Lys Glu Leu Val Ser Lys Tyr Arg Gln Gln

165 170 175

Phe Glu Glu Leu Tyr Lys Thr Glu Ala Pro Thr Trp Glu Thr His Gly

180 185 190

Asn Leu Met Thr Glu Arg Gln Val Ser Arg Trp Phe Val Gln Cys Leu

195 200 205

Arg Glu Gln Ser Met Leu Leu Glu Ile Ile Phe Leu Tyr Tyr Ala Tyr

210 215 220

Phe Glu Met Ala Pro Ser Asp Leu Leu Val Leu Thr Lys Met Phe Lys

225 230 235 240

Glu Gln Gly Phe Gly Ser Arg Gln Thr Asn Arg His Leu Val Asp Glu

245 250 255

Thr Met Asp Pro Phe Val Asp Arg Ile Gly Tyr Phe Ser Ala Leu Ile

260 265 270

Leu Val Glu Gly Met Asp Ile Glu Ser Leu His Lys Cys Ala Leu Asp

275 280 285

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

290 295 300

Asp Met Asp Cys Leu Met Leu Thr Phe Gly Asp Ile Pro His His Ala

305 310 315 320

Pro Val Leu Leu Ala Trp Ala Leu Leu Arg His Thr Leu Asn Pro Glu

325 330 335

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

340 345 350

Asn Val Phe Gln Tyr Leu Thr Arg Leu Leu Gln Ser Leu Ala Ser Gly

355 360 365

Gly Asn Asp Cys Thr Thr Ser Thr Ala Cys Met Cys Val Tyr Gly Leu

370 375 380

Leu Ser Phe Val Leu Thr Ser Leu Glu Leu His Thr Leu Gly Asn Gln

385 390 395 400

Gln Asp Ile Ile Asp Thr Ala Cys Glu Val Leu Ala Asp Pro Ser Leu

405 410 415

Pro Glu Leu Phe Trp Gly Thr Glu Pro Thr Ser Gly Leu Gly Ile Ile

420 425 430

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

435 440 445

Gln Leu Leu Arg Ala Leu Val Ser Gly Lys Ser Thr Ala Lys Lys Val

450 455 460

Tyr Ser Phe Leu Asp Lys Met Ser Phe Tyr Asn Glu Leu Tyr Lys His

465 470 475 480

Lys Pro His Asp Val Ile Ser His Glu Asp Gly Thr Leu Trp Arg Arg

485 490 495

Gln Thr Pro Lys Leu Leu Tyr Pro Leu Gly Gly Gln Thr Asn Leu Arg

500 505 510

Ile Pro Gln Gly Thr Val Gly Gln Val Met Leu Asp Asp Arg Ala Tyr

515 520 525

Leu Val Arg Trp Glu Tyr Ser Tyr Ser Ser Trp Thr Leu Phe Thr Cys

530 535 540

Glu Ile Glu Met Leu Leu His Val Val Ser Thr Ala Asp Val Ile Gln

545 550 555 560

His Cys Gln Arg Val Lys Pro Ile Ile Asp Leu Val His Lys Val Ile

565 570 575

Ser Thr Asp Leu Ser Ile Ala Asp Cys Leu Leu Pro Ile Thr Ser Arg

580 585 590

Ile Tyr Met Leu Leu Gln Arg Leu Thr Thr Val Ile Ser Pro Pro Val

595 600 605

Asp Val Ile Ala Ser Cys Val Asn Cys Leu Thr Val Leu Ala Ala Arg

610 615 620

Asn Pro Ala Lys Val Trp Thr Asp Leu Arg His Thr Gly Phe Leu Pro

625 630 635 640

Phe Val Ala His Pro Val Ser Ser Leu Ser Gln Met Ile Ser Ala Glu

645 650 655

Gly Met Asn Ala Gly Gly Tyr Gly Asn Leu Leu Met Asn Ser Glu Gln

660 665 670

Pro Gln Gly Glu Tyr Gly Val Thr Ile Ala Phe Leu Arg Leu Ile Thr

675 680 685

Thr Leu Val Lys Gly Gln Leu Gly Ser Thr Gln Ser Gln Gly Leu Val

690 695 700

Pro Cys Val Met Phe Val Leu Lys Glu Met Leu Pro Ser Tyr His Lys

705 710 715 720

Trp Arg Tyr Asn Ser His Gly Val Arg Glu Gln Ile Gly Cys Leu Ile

725 730 735

Leu Glu Leu Ile His Ala Ile Leu Asn Leu Cys His Glu Thr Asp Leu

740 745 750

His Ser Ser His Thr Pro Ser Leu Gln Phe Leu Cys Ile Cys Ser Leu

755 760 765

Ala Tyr Thr Glu Ala Gly Gln Thr Val Ile Asn Ile Met Gly Ile Gly

770 775 780

Val Asp Thr Ile Asp Met Val Met Ala Ala Gln Pro Arg Ser Asp Gly

785 790 795 800

Ala Glu Gly Gln Gly Gln Gly Gln Leu Leu Ile Lys Thr Val Lys Leu

805 810 815

Ala Phe Ser Val Thr Asn Asn Val Ile Arg Leu Lys Pro Pro Ser Asn

820 825 830

Val Val Ser Pro Leu Glu Gln Ala Leu Ser Gln His Gly Ala His Gly

835 840 845

Asn Asn Leu Ile Ala Val Leu Ala Lys Tyr Ile Tyr His Lys His Asp

850 855 860

Pro Ala Leu Pro Arg Leu Ala Ile Gln Leu Leu Lys Arg Leu Ala Thr

865 870 875 880

Val Ala Pro Met Ser Val Tyr Ala Cys Leu Gly Asn Asp Ala Ala Ala

885 890 895

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

900 905 910

Arg Ile Lys Val Met Ile Leu Glu Phe Leu Thr Val Ala Val Glu Thr

915 920 925

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

930 935 940

Ser Asp Gly Ser Lys Glu Phe Ser Leu Gly Met Trp Ser Cys Leu His

945 950 955 960

Ala Val Leu Glu Leu Ile Asp Ser Gln Gln Gln Asp Arg Tyr Trp Cys

965 970 975

Pro Pro Leu Leu His Arg Ala Ala Ile Ala Phe Leu His Ala Leu Trp

980 985 990

Gln Asp Arg Arg Asp Ser Ala Met Leu Val Leu Arg Thr Lys Pro Lys

995 1000 1005

Phe Trp Glu Asn Leu Thr Ser Pro Leu Phe Gly Thr Leu Ser Pro

1010 1015 1020

Pro Ser Glu Thr Ser Glu Pro Ser Ile Leu Glu Thr Cys Ala Leu

1025 1030 1035

Ile Met Lys Ile Ile Cys Leu Glu Ile Tyr Tyr Val Val Lys Gly

1040 1045 1050

Ser Leu Asp Gln Ser Leu Lys Asp Thr Leu Lys Lys Phe Ser Ile

1055 1060 1065

Glu Lys Arg Phe Ala Tyr Trp Ser Gly Tyr Val Lys Ser Leu Ala

1070 1075 1080

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

1085 1090 1095

Glu Tyr Gln Met Leu Val Ser Ala Trp Arg Met Leu Leu Ile Ile

1100 1105 1110

Ala Thr Thr His Ala Asp Ile Met His Leu Thr Asp Ser Val Val

1115 1120 1125

Arg Arg Gln Leu Phe Leu Asp Val Leu Asp Gly Thr Lys Ala Leu

11301135 1140

Leu Leu Val Pro Ala Ser Val Asn Cys Leu Arg Leu Gly Ser Met

1145 1150 1155

Lys Cys Thr Leu Leu Leu Ile Leu Leu Arg Gln Trp Lys Arg Glu

1160 1165 1170

Leu Gly Ser Val Asp Glu Ile Leu Gly Pro Leu Thr Glu Ile Leu

1175 1180 1185

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

1190 1195 1200

Ala Lys Val Phe Ser Ala Phe Ile Thr Val Leu Gln Met Lys Glu

1205 1210 1215

Met Lys Val Ser Asp Ile Pro Gln Tyr Ser Gln Leu Val Leu Asn

1220 1225 1230

Val Cys Glu Thr Leu Gln Glu Glu Val Ile Ala Leu Phe Asp Gln

1235 1240 1245

Thr Arg His Ser Leu Ala Leu Gly Ser Ala Thr Glu Asp Lys Asp

1250 1255 1260

Ser Met Glu Thr Asp Asp Cys Ser Arg Ser Arg His Arg Asp Gln

1265 1270 1275

Arg Asp Gly Val Cys Val Leu Gly Leu His Leu Ala Lys Glu Leu

1280 1285 1290

Cys Glu Val Asp Glu Asp Gly Asp Ser Trp Leu Gln Val Thr Arg

1295 1300 1305

Arg Leu Pro Ile Leu Pro Thr Leu Leu Thr Thr Leu Glu Val Ser

1310 1315 1320

Leu Arg Met Lys Gln Asn Leu His Phe Thr Glu Ala Thr Leu His

1325 1330 1335

Leu Leu Leu Thr Leu Ala Arg Thr Gln Gln Gly Ala Thr Ala Val

1340 1345 1350

Ala Gly Ala Gly Ile Thr Gln Ser Ile Cys Leu Pro Leu Leu Ser

1355 1360 1365

Val Tyr Gln Leu Ser Thr Asn Gly Thr Ala Gln Thr Pro Ser Ala

1370 1375 1380

Ser Arg Lys Ser Leu Asp Ala Pro Ser Trp Pro Gly Val Tyr Arg

1385 1390 1395

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

1400 1405 1410

Asn Phe Leu Pro Glu Ala Leu Asp Phe Val Gly Val His Gln Glu

1415 1420 1425

Arg Thr Leu Gln Cys Leu Asn Ala Val Arg Thr Val Gln Ser Leu

1430 1435 1440

Ala Cys Leu Glu Glu Ala Asp His Thr Val Gly Phe Ile Leu Gln

1445 1450 1455

Leu Ser Asn Phe Met Lys Glu Trp His Phe His Leu Pro Gln Leu

1460 1465 1470

Met Arg Asp Ile Gln Val Asn Leu Gly Tyr Leu Cys Gln Ala Cys

1475 1480 1485

Thr Ser Leu Leu His Ser Arg Lys Met Leu Gln His Tyr Leu Gln

1490 1495 1500

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

1505 1510 1515

Gln Arg Pro Pro Ser Ala Ala Ser Ala Ala Pro Ser Ser Ser Lys

1520 1525 1530

Gln Pro Ala Ala Asp Thr Glu Ala Ser Glu Gln Gln Ala Leu His

1535 1540 1545

Thr Val Gln Tyr Gly Leu Leu Lys Ile Leu Ser Lys Thr Leu Ala

1550 1555 1560

Ala Leu Arg His Phe Thr Pro Asp Val Cys Gln Ile Leu Leu Asp

1565 1570 1575

Gln Ser Leu Asp Leu Ala Glu Tyr Asn Phe Leu Phe Ala Leu Ser

1580 1585 1590

Phe Thr Thr Pro Thr Phe Asp Ser Glu Val Ala Pro Ser Phe Gly

1595 1600 1605

Thr Leu Leu Ala Thr Val Asn Val Ala Leu Asn Met Leu Gly Glu

1610 1615 1620

Leu Asp Lys Lys Lys Glu Pro Leu Thr Gln Ala Val Gly Leu Ser

1625 1630 1635

Thr Gln Ala Glu Gly Thr Arg Thr Leu Lys Ser Leu Leu Met Phe

1640 1645 1650

Thr Met Glu Asn Cys Phe Tyr Leu Leu Ile Ser Gln Ala Met Arg

1655 1660 1665

Tyr Leu Arg Asp Pro Ala Val His Pro Arg Asp Lys Gln Arg Met

1670 1675 1680

Lys Gln Glu Leu Ser Ser Glu Leu Ser Thr Leu Leu Ser Ser Leu

1685 1690 1695

Ser Arg Tyr Phe Arg Arg Gly Ala Pro Ser Ser Pro Ala Thr Gly

1700 1705 1710

Val Leu Pro Ser Pro Gln Gly Lys Ser Thr Ser Leu Ser Lys Ala

1715 1720 1725

Ser Pro Glu Ser Gln Glu Pro Leu Ile Gln Leu Val Gln Ala Phe

1730 1735 1740

Val Arg His Met Gln Arg

1745

<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

115 120 125

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

Ile Tyr Met Leu Leu Gln Arg Leu Thr Thr Val Ile

1 5 10

<210>10

<211>9

<212>PRT

<213>H-2Kb

<400>10

Ile Ile Phe Leu Tyr Tyr Ala Tyr Phe

1 5

<210>11

<211>14

<212>PRT

<213>HLA-A2

<400>11

Met Leu Leu Gln Arg Leu Thr Thr Val Ile Ser Pro Pro Val

1 5 10

Claims (5)

1. A medicine comprises a complex of a polypeptide of NUP188 protein and a heat shock protein gp 96;

the amino acid sequence of the polypeptide of the NUP188 protein is as follows:

i) an amino acid sequence shown as SEQ ID NO. 9; or

ii) the amino acid sequence shown in SEQ ID NO. 10; or

iii) the amino acid sequence shown as SEQ ID NO. 11;

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

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

the polypeptide of the NUP188 protein and the heat shock protein gp96 form a complex in vitro in a natural adsorption or heat shock mode.

The application of a complex formed by combining a polypeptide of the NUP188 protein 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 the NUP188 protein is as follows:

i) an amino acid sequence shown as SEQ ID NO. 9; or

ii) the amino acid sequence shown in SEQ ID NO. 10; or

iii) the amino acid sequence shown as SEQ ID NO. 11;

the cancer is breast cancer or liver cancer.

5. The use of claim 4, wherein the medicament has the function of at least one of:

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

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

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