CN106822869A

CN106822869A - The application of DEF8 albumen and its polypeptide in treatment with pre- anti-cancer

Info

Publication number: CN106822869A
Application number: CN201710157562.0A
Authority: CN
Inventors: 孟颂东; 郑华国; 李杨
Original assignee: Beijing Thermal Biological Technology Co Ltd
Current assignee: Foshan Rexiu Biotechnology Co ltd
Priority date: 2017-03-16
Filing date: 2017-03-16
Publication date: 2017-06-13
Anticipated expiration: 2037-03-16
Also published as: CN106822869B

Abstract

The invention provides a kind of DEF8 albumen and its purposes of polypeptide, belong to protein engineering and biological medicine applied technical field.The amino acid sequence of DEF8 albumen of the invention is as shown in SEQ ID NO.2 or its specific fragment, present invention discover that DEF8 albumen and its polypeptide, and the compound that they are formed thermal shock or naturally with heat not albumen gp96 by way of absorption can be prevented and treated including the ND including breast cancer, liver cancer, can be used to prepare the medicine for preventing and treating cancer, have broad application prospects.

Description

The application of DEF8 albumen and its polypeptide in treatment with pre- anti-cancer

Technical field

The present invention relates to protein engineering and biological medicine applied technical field, specifically, be related to DEF8 albumen and its Application of the polypeptide in treatment with pre- anti-cancer.

Background technology

Breast cancer is one of most common malignant tumour of women.According to statistics, breast cancer incidence accounts for the various evils of whole body Property tumour 7-10%, the cancer of the uterus is alreadyd exceed in many areas, as having a strong impact on the physically and mentally healthy even threat to life of women One of most common tumour.The morbidity of breast cancer and the gene of patient, habits and customs, Foods, bearing status etc. It is closely related, different ethnic groups, the breast cancer incidence of region have obvious difference.The hotspot of breast cancer, it is main to concentrate In North America, Northern Europe and Oceania, especially Caucasian female is in the majority.The middle hair area of breast cancer, concentrates on South America, southern Europe and with color Row.Asia is then the low hair area of breast cancer.For example, Caucasian female all one's life breast cancer incidence in the U.S.'s is 13.1%, i.e., Averagely just having 1 people per 8-9 people may suffer from breast cancer, and the breast cancer incidence of Asia women is 4-7%.WHO is counted, entirely The annual new breast cancer of ball up to 1,300,000 people or so, dead 500,000 people or so.In the metropolitan breast such as China Beijing, Shanghai, Tianjin Gland cancer morbidity has leapt to the various carcinomas first places of women, and has the trend of obvious rising.

Liver cancer refers to the malignant tumour for betiding liver, including two kinds of primary carcinoma of liver and metastatic hepatic carcinoma.Primary Hepatic Cancer is clinically one of most common malignant tumour.Worldwide in male cancer patient, liver cancer ratio ranked sixth, dead Rate comes second；In female cancer patients, liver cancer ratio ranked seventh, and the death rate ranked sixth.2008, the whole world had 748, 300 newly-increased liver cancer cases, there is 695,900 liver cancer patient death.And have one in these newly-increased liver cancer cases and death Half in China.Liver cancer incidence highest region is main in East Asia, Southeast Asia, middle non-sum the west African state.Parts of Asia and Why liver cancer incidence is higher for African areas to the south of the Sahara, it may be possible to because these regional HBV are prevailing, because these are regional 8% resident chronic infection HBV, HBV was infected in the liver cancer patient of developing country 60%.

Marrow founder subculture cell differentially expressed protein 8 (Differentially expressed in FDCP 8) finds earliest Its variant expression in hemopoietic system.DEF8 albumen has extensive distribution, especially in PBL Expression is high, but its expression is nearly no detectable in thymus gland and tire liver, and DEF8 albumen macrophage and granulocyte point Expressed during change and significantly lowered.But the function on DEF8 albumen is up to the present still unclear.

Heat shock protein (Heat shock protein, HSP) is a class highly conserved in biological evolution and deposit extensively It is the protein in protokaryon and eucaryote.HSP according to degree of homology and molecular size range can be divided into HSP110, HSP90, The multiple subfamily such as HSP70, HSP60, HSP40, small molecule HSP and ubiquitin.Heat shock protein (Heat shock protein, HSP) gp96 belongs to the member of HSP90 subfamilies, and the albumen is the most abundant heat shock protein of content on endocytoplasmic reticulum.Heat is stopped Gram albumen gp96 albumen has polypeptide binding characteristic, and it can receive the polypeptide fragment from TAP complexs in endoplasmic reticulum, assists MHC I quasi-molecules are assembled to, submission is on cell membrane.The heat shock protein gp96 of different tissue sources can carry it The polypeptide fragment of source tissue's internal specific expression.

The content of the invention

It is an object of the invention to provide a kind of energy prevention and the target proteinses and its application process of polypeptide for the treatment of cancer.

Present invention firstly provides a kind of DEF8 albumen, the amino acid sequence of the DEF8 albumen contains as follows：

I) amino acid sequence shown in SEQ ID NO.2；Or

Ii) amino acid sequence shown in SEQ ID NO.2 from N-terminal the 2nd to last amino acids sequence；Or

Iii) amino acid sequence shown in SEQ ID NO.2 be substituted, lack and/or increase one or more amino acid and Amino acid sequence with identical function.

The invention provides application of the above-mentioned DEF8 albumen in prevention and/or treating cancer medicine is prepared.

The invention provides the gene of encoding D EF8 albumen, the nucleotide sequence of the gene contains as follows：

I) nucleotide sequence shown in SEQ ID NO.1；Or

Ii) nucleotide sequence shown in SEQ ID NO.1 be substituted, lack and/or increase one or more nucleotides and Express the nucleotide sequence of identical function protein；Or

Iii) there is the nucleotide sequence of 75% and above homology with the nucleotides shown in SEQ ID NO.1；Or

Iv) under strict conditions with the nucleotide sequence of sequence hybridization shown in SEQ ID NO.1；

The stringent condition be in 0.1 × SSPE containing 0.1%SDS or the 0.1 × SSC solution containing 0.1%SDS, Hybridize at 65 DEG C, and film is washed with the solution.

The invention provides encoding D EF8 albumen gene prepare prevention and/or treating cancer medicine in application.

The invention provides the biomaterial of the gene containing encoding D EF8 albumen, the biomaterial is carrier, host Cell, transgenic cell line, engineering bacteria, insect or yeast.

Further, the application the invention provides above-mentioned biomaterial in prevention and/or treating cancer medicine is prepared.

The medicine is to treat or prevent breast cancer or the medicine of liver cancer, with following at least one function：

(1) incidence of the breast cancer of chemicals induction is reduced；

(2) the liver cancer incidence of chemicals induction is reduced；

(3) it is slowed or shut off the growth of breast cancer tumour stove set up；

(4) it is slowed or shut off the growth of hepatic carcinoma stove set up；

(5) reduce or stop the transfer of the breast cancer tumour stove set up；

(6) reduce or stop the transfer of the hepatic carcinoma stove set up；

(7) induction produces the CTL cells of Breast Cancer-Specific and breast cancer cell is killed；

(8) induction produces the CTL cells of liver cancer-specific and HCC is killed.

The invention provides the medicine containing DEF8 albumen or its polypeptide.

The invention provides containing DEF8 albumen and heat shock protein gp96 composition compound or contain DEF8 albumen The medicine of the compound that polypeptide is constituted with heat shock protein gp96.

The amino acid sequence of the heat shock protein gp96 contains the sequence or SEQ ID NO.4 described in SEQ ID NO.4 Shown amino acid sequence is substituted, lacks and/or increases one or more amino acid and the amino acid sequence with identical function Row.

The acquisition pattern of the heat shock protein gp96 is following any：

(1) extracted from the placenta tissue of isolated mammalian；

(2) nucleic acid molecules that will encode the heat shock protein gp96 are imported in acceptor, and culture using yeast, is fed Newborn animal cell expression diagram of system is reached, and purifying is obtained.

The isolated mammalian can originate for people, mouse.The mouse includes but is not limited to C57BL/6 and BALB/c mouse.

The compound is to obtain in the following manner：

(1) DEF8 albumen or its polypeptide are formed multiple with absorption or thermal shock mode naturally in vitro with heat shock protein gp96 Compound；Or

(2) encoding D EF8 albumen or its polypeptide are imported in acceptor with the nucleic acid molecules of encoding heat shock proteins gp96, training Support, purifying obtains compound.

Described " encoding D EF8 albumen or its polypeptide are imported in acceptor with the nucleic acid molecules of encoding heat shock proteins gp96 " Can be by expressed by Hansenula yeast.

" nucleic acid molecules that will encode the heat shock protein gp96 are imported in acceptor " can import by acceptor Recombinant vector is realized；The recombinant vector can be that the encoding gene insertion of the heat shock protein gp96 is set out into what plasmid was obtained Recombinant plasmid.The recombinant vector concretely recombinant plasmid pFastBac1-gp96.The recombinant plasmid pFastBac1- DNA moleculars of the gp96 concretely in the MCS insetion sequence table of plasmid pFastBac1 shown in SEQ ID NO.3 is obtained The recombinant plasmid for arriving.

The EcoR I and Xba I of plasmid pFastBac1 are concretely recognized sequence by the recombinant plasmid pFastBac1-gp96 (plasmid pFastBac1 is cut into a large fragment and a small pieces to fragment between row by restriction endonuclease EcoR I and Xba I Section, the DNA is the small fragment) replace with the recombinant plasmid that the DNA molecular shown in SEQ ID NO.1 is obtained.

The acceptor can be Sf9 cells.

The compound constituted with heat shock protein gp96 the invention provides above-mentioned DEF8 albumen and its polypeptide is controlled in preparation Treat and/or prevent the application in cancer drug.

The medicine is to treat or prevent breast cancer or the medicine of liver cancer, with following at least one function：(1) reduce The incidence of the breast cancer of chemicals induction；

(2) the liver cancer incidence of chemicals induction is reduced；

(3) it is slowed or shut off the growth of breast cancer tumour stove set up；

(4) it is slowed or shut off the growth of hepatic carcinoma stove set up；

(5) reduce or stop the transfer of the breast cancer tumour stove set up；

(6) reduce or stop the transfer of the hepatic carcinoma stove set up；

Using said medicine of the invention, for immunization, immunizing dose is no less than altogether to be no less than 10 μ g every time 3 times, hypodermic injection, intracutaneous injection or intraperitoneal injection mode is taken to be immunized.After medicine (vaccine) of the invention immune several weeks, The cell of the Immunel response of body, removing in-vivo tumour stem cell and/or Tumor dormancy cell, and canceration can be triggered, So as to reach the purpose of prevention and treatment cancer.The present invention can be anti-for autologous or allogeneic tumor as tumor prevention vaccine Control, reduce the incidence of cancer in healthy population, also can be used as tumor therapeutic vaccine, patient is postoperative to be treated and can effectively prevent immediately Anti- recurrence is shifted, or as chemotherapeutic auxiliary treatment.The immune protection that can be used for allogeneic tumor of the invention.

Brief description of the drawings

Fig. 1 is SDS-PAGE the and Western blot qualification results of placenta gp96.

Fig. 2 is SDS-PAGE the and Western Blot qualification results of Yeast expression gp96, in figure, 1：Molecular weight standard； 2：The zymotic fluid of step 2；3：Eluent after affinity chromatography；4：Eluent after ion-exchange chromatography；5：western blot Qualification figure.

Fig. 3 is the qualification result of the SDS-PAGE and Western Blot of insect expression gp96.

Fig. 4 is therapeutic action (gross tumor volume) of the polypeptide-gp96 compounds to breast cancer.

Fig. 5 is therapeutic action (gross tumor volume) of the polypeptide-gp96 compounds to liver cancer.

Fig. 6 is the specific CTL of polypeptide-gp96 compounds induction for human breast cancer cell lethal effect.

Fig. 7 specific CTL of polypeptide-gp96 compounds induction is for human milk HCC lethal effect.

Specific embodiment

Experimental technique in following embodiments, unless otherwise specified, is conventional method.Material used in embodiment, Reagent etc., unless otherwise specified, commercially obtains.Quantitative test in following examples, is respectively provided with three repetitions Experiment, results averaged.

Female C57BL/6 is Beijing experimental animal Co., Ltd of dimension tonneau China product with female BAl BIc/C mice； Hereinafter abbreviation mouse.Polypeptide is synthesized by Shanghai gill biochemistry Co., Ltd.HepG2 cells (human liver cancer cell) are ATCC companies Product, catalog number is HB-8065^TM.MCF-7 cells (human breast cancer cell) are ATCC Products, and catalog number is HTB-22^TM.H22 and HHCC cells are purchased from Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences's preclinical medicine cell centre, resource number Respectively 3111C0001CCC000309,3111C0002000000069.Sf9 cells are Invitrogen Products, product Catalog number (Cat.No.) is 11496-015.Cellfectin II reagent are Life technologies Products, catalog number It is 10362-100.Plasmid pFastBac^TM1 is Invitrogen Products, and catalog number is 10359-016.Gp96 Dan Ke Grand antibody is Santa Cruz Products, and catalog number is sc-56399.The goat of horseradish peroxidase-labeled is anti-big The monoclonal antibody of mouse is Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge product, and catalog number is ZB-2307.HiTrap-Q Sepharose ion exchange columns are GE Products, and catalog number is 17-5053-01.Superdex 200 10/ 300GL molecular sieve chromatographies are GE Products, and catalog number is 17517501.Escherichia coli DH10Bac competent cells It is Beijing Yuanpinghao Biological Technology Co., Ltd.'s product, catalog number is CL108-01.Insect-XPRESS^TM Protein- Free Insect Cells medium with L-Glutamine are LONZA Products, and catalog number is 12-730Q. BSA、PMSF、NaHCO₃、MnCl₂、CaCl₂、NaCl₂, that Tris, methyl α-D- mannopyranose glycosides are Sigma-Aldrich is public Department product, catalog number be respectively V900933, P7626,792519, V900197,793639,746398, T1378, M6882。

Solution A：Solute and its concentration are PMSF 1mM and NaHCO₃30mM；Solvent is distilled water；PH value is 7.4.

Solution B：Solute and its concentration are 2mM MnCl₂、2mM CaCl₂, 500mM NaCl and PMSF 1mM；Solvent is PH7.4,20mM Tris-HCl buffer solutions.

Solution C：Solute and its concentration be 10% (mass volume ratio) methyl α-D- mannopyranoses glycosides, 500mM NaCl and 1mM PMSF；Solvent is pH7.4,20mM Tris-HCl buffer solutions.

Cleaning fluid：Solution B is diluted to 10 times of volumes with distilled water.

ConA agarose Gel columns are GE Products, and catalog number is 17-0440-01, the specification of the post for 1.6 × 2.5cm, filled media is Con A-Sepharose 4B.Hitrap Q anion-exchange columns are GE Products, catalogue Number it is 17-1153-01, the specification of the post is 0.7 × 2.5cm.The IgG antibody of HRP marks is SEROTEC Products, product Catalog number (Cat.No.) is STAR117P.1 × cleaning solution is pH7.4,0.01mol/L containing 0.1% (percent by volume) Triton-X100 PBS.50kD and 3kD super filter tubes are Merck Millipore Products, catalog number be respectively UFC905096, UFC500324。

The extraction of gp96 in the placenta of embodiment 1

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

(1) placenta tissue of prepartal mouse is separated, the in vitro placenta tissue of mouse is obtained.Take the in vitro placenta tissue of mouse Or the in vitro placenta tissue of people, shred, by mass volume ratio 1g：4mL adds solution A, is then ground with glass homogenizer.

(2) after completing step (1), 16500g centrifugation 1h obtain supernatant first.

(3) after completing step (2), supernatant first is taken, 16500g centrifugation 50min obtain supernatant second.

(4) after completing step (3), supernatant second is taken, by volume 9：1 adds solution B, and sample solution is obtained after mixing.

(5) after completing step (4), by sample solution loading to ConA agarose Gel columns.

(6) after completing step (5), the ConA agarose Gel columns, real-time monitoring in elution process are eluted with cleaning fluid Ultraviolet absorption value, Detection wavelength is 280nm, until the ultraviolet absorption value of eluted product is less than 0.01.

(7) after completing step (6), the ConA agarose Gel columns are eluted with solution C, after discarding the post excessively for flowing out first 0.5 column volume of solution, what is flowed out after then collecting crosses 1 column volume of solution after post；By the ConA agarose Gel columns After being incubated 50min, 1.5 column volumes of solution after post were regathered.Solution merges after the post excessively that will be collected twice, and as ConA is washed De- liquid.

(8) after completing step (7), by ConA eluents loading to Hitrap Q anion-exchange columns.

(9) after completing step (8), linear gradient elution, flow velocity are carried out with the PBS of pH7.4,12mM containing NaCl It is 1mL/min.Gradient elution program：NaCl contents are made at the uniform velocity to be increased to by 300mM in the PBS of pH7.4 12mM 800mM, 20 column volumes of linear gradient elution.Collect and combine the eluent that NaCl contents are 400~450mM, as eluent First.

(10) after completing step (9), eluent first is taken, is concentrated by ultrafiltration with super filter tube, obtain the solution of pgp96. In the solution of pgp96, pgp96 concentration is 5mg/mL.

By the solution of pgp96 carry out SDS-PAGE electrophoretic analysis and Western blot (using gp96 monoclonal antibodies as Primary antibody, the IgG antibody of HRP marks is used as secondary antibody), experimental result is shown in Fig. 1 (arrow is signified for pgp96).Result shows, pgp96 Solution display unimodal molecular weight band, corresponding molecular weight be 96kDa.

Embodiment 2 is prepared using the restructuring gp96 albumen of expressed by Hansenula yeast

First, the structure of recombinant plasmid pHFMDZ-R1L2GAmy-gp96

1st, the mRNA of human liver cancer cell HepG2, reverse transcription synthesis cDNA are extracted.

2nd, enter performing PCR amplification by template of the cDNA of step 1, obtain amplified production.

PCR primer is as follows：

Fp(5'-CCGgaattcATGGACGATGAAGTTGATG-3')

Rp(5'-CCGctcgagCTATTAGAATTCATCTTTTTCAGCTGTAG-3')

3rd, with restriction enzyme EcoRI and XhoI double digestion pcr amplification product, digestion products are reclaimed.

4th, (Invitrogen companies are purchased from, are produced with restriction enzyme EcoRI and XhoI double digestion plasmid pHFMDZ-R1A Article Number is V20520), reclaim carrier framework.

5th, the carrier framework of the digestion products of step 3 and step 4 is connected, obtains recombinant plasmid pHFMDZ-R1-gp96 simultaneously Sequencing.Sequencing result shows that the skeleton carrier of recombinant plasmid pHFMDZ-R1-gp96 is pHFMDZ-R1A, in EcoRI and XhoI The coded sequence of gp96 albumen is inserted between restriction enzyme site.

2nd, the expression of gp96 albumen

1st, the recombinant plasmid pHFMDZ-R1-gp96 for being obtained step 1 using electrotransformation is imported Hansenula yeast and (is purchased from ATCC, production number is MYA-335) cell, obtain recombinant bacterium.

2nd, recombinant bacterium is inoculated in 5mL SD fluid nutrient mediums and (is purchased from Shanghai Jie Mei genes pharmaceuticals, production number is GMS12117.7), 37 DEG C of culture 48h, be then transferred to 100mL SYN6 culture mediums (purchased from Shanghai Jie Mei genes pharmaceuticals, Production number is GMS12116.1), 30 DEG C of culture 48h obtain seed culture fluid.

3rd, two bottles of seed culture fluids are inoculated in the 5L fermentation tanks containing 2L SYN6 culture mediums, 30 DEG C of cultures.Use ammonia Water management pH maintains 5.5, and glycerol content in 1 zymotic fluid is detected per 4h, and the concentration according to glycerine in zymotic fluid adds glycerine, Control final glycerol concentration is 0.5% or so, while control dissolved oxygen is more than 20%.Generation situation detection thalline according to thalline is wet Weight, stops adding glycerine when waiting thalline weight in wet base to reach 180-200g/L, starts induction restructuring gp96 albumen generations and (adds first Alcohol, makes methanol concentration maintain 0.5-0.8% or so), induction stops fermentation after starting 72 hours, obtains zymotic fluid.

3rd, gp96 albumen is isolated and purified

The zymotic fluid centrifugation that step 2 is obtained, collects thalline.With PBS washed cell 2 times, in ball mill In (DYNO-MILL model KDL), the operation manual provided according to producer uses the method smudge cells of bead ball milling, breaks Cell 12000rpm/min centrifugation 20min after broken, collect supernatant.Supernatant is filtered with 0.45 μm of filter membrane, is filtered Liquid.Filtrate is concentrated, concentrate is obtained.

Filtrate is carried out into affinity chromatography, is comprised the following steps that：Using handsome company (Invitrogen Corporation) Ni-NTA Purification System carry out affinity chromatography, mainly comprise the following steps：First pillar 2h, Ran Houyong are balanced with PBS PBS (imidazoles containing 20mM) balance pillars 2h.Loading after concentrate is diluted with PBS (imidazoles containing 20mM), with PBS (miaows containing 20mM Azoles) rinse pillar to OD values<0.01,1.5h is eluted with PBS (imidazoles containing 200mM), collect eluent.All operations are at 4 DEG C Under carry out, flow velocity is 0.5mL/min.

The zymotic fluid that every liter of step 2 is obtained can obtain 50 milligrams of gp96 albumen of purity more than 90% after purification.If Contain heterologous foreign protein in protein eluate, the eluent of affinity chromatography can again be carried out ion-exchange chromatography, mainly comprise the following steps： The PBS liquid balance pillar of 200mM NaCl, loading, the PBS liquid of 300mM NaCl washes impurity, the PBS liquid wash-out of 800mM NaCl Destination protein.

Eluent after eluent and ion-exchange chromatography after the zymotic fluid of step 2, affinity chromatography is carried out 10% SDS-PAGE, then coomassie brilliant blue staining.Gp96 albumen is carried out into western blot, primary antibody is the anti-gp96 antibody of rat (being purchased from santa cruz, production number is sc-56399), as a result sees Fig. 2.Fig. 2 shows, in the eluent after ion-exchange chromatography Gp96 purity of protein it is very high.

The preparation of the restructuring heat shock protein gp96 of the insect cell expression of embodiment 3

First, recombinant plasmid pFastBac^TMThe structure of 1-gp96

1st, the RNA of HepG2 cells is extracted using Trizol methods, reverse transcription is then carried out and is obtained cDNA.

2nd, according to the sequence of people gp96 genes (No. GenBank is AY040226.1), artificial synthesized primers F 1：5’- GGAATTCATGGACGATGAAGTTGAT-3 ' (underscore is the recognition sequences of restriction enzyme EcoR I) and R1：5’- GCTCTAGACTATTAGAATTCATCTTTTTC-3 ' (underscore is the recognition sequences of restriction enzyme Xba I).

3rd, be template with the cDNA that step 1 is obtained after completing step 1 and 2, with step it is 2-in-1 into F1 and R1 enter as primer Performing PCR is expanded, and obtains pcr amplification product.

4th, with restriction enzyme EcoR I and the double digestion pcr amplification products of Xba I, digestion products are reclaimed.

5th, with the restriction enzyme EcoR I and digested plasmid pFastBac of Xba I^TM1, reclaim the carrier framework of about 4700bp.

6th, digestion products are connected with carrier framework, obtain connection product.

7th, the connection product conversion Escherichia coli DH10Bac competent cells for obtaining step 6, obtain recombinating large intestine bar Bacterium, then extracts the plasmid of the recombination bacillus coli, obtains recombinant plasmid pFastBac1-gp96.

Structure is carried out according to sequencing result to recombinant plasmid pFastBac1-gp96 to be described as follows：By plasmid pFastBac1 EcoR I and the recognition sequences of Xba I between fragment (plasmid pFastBac1 is cut into by restriction endonuclease EcoR I and Xba I One large fragment and a small fragment, the DNA are the small fragment) replace with the double-stranded DNA shown in SEQ ID NO.3 in sequence table Molecule.Recombinant plasmid pFastBac 1-gp96 expression restructuring heat shock protein gp96 (hereinafter referred to as rgp96), the amino of rgp96 Acid sequence is as shown in SEQ ID NO.4 in sequence table.

2nd, the expression of rgp96

1st, the recombinant plasmid pFastBac1-gp96 cotransfection Sf9 cells (every 1 × 10 for building step one⁶Individual Sf9 cells About transfect 4 μ g recombinant plasmids pFastBac1-gp96；During cotransfection, transfection reagent is Cellfectin II reagent, Culture medium is Insect-XPRESS^TMProtein-free Insect Cells medium with L-Glutamine, 27 DEG C 72h, centrifugation are incubated, supernatant is P1 generation viruses.

2nd, Sf9 cell suspending liquids 1 (are contained 1 × 10⁸Individual Sf9 cells) 27 DEG C of 8~10h of culture, obtain cultured cells；Then To P1 generations virus (dosage is 0.05~0.1MOI) is added in the cultured cells, 27 DEG C are incubated 72h, 4000rpm centrifugation 5min, Supernatant is P2 generation viruses.

3rd, (1.6 × 10 are contained to Sf9 cell suspending liquids 2⁸Individual Sf9 cells) in add P2 generation virus (dosage be 0.05~ 0.1MOI), 27 DEG C, 100~120rpm culture 72h, 4000rpm centrifugation 5min, supernatant are P3 generation viruses.

The monoclonal antibody conduct of the goat anti-rat marked as primary antibody, HRPO using gp96 monoclonal antibodies Secondary antibody, western hybridization is carried out to P3 for virus, and western hybridization specific steps refer to following document：Zhang Yueming, Duan Yueqiang, Expression and its mirror of Luo Deyan, Yao Huijuan, Wang Xiliang, Li Zhi the Kui mouse soluble IL-5 α acceptors in Bac-to-Bac systems Fixed [J] Products in China magazines, 2013,26：5.Western hybrid experiment results show, rgp96 tables in Sf9 cells Reach.

3rd, the purifying of rgp96

1st, (4.5 × 10 are contained to the Sf9 cell suspending liquids 3 of 300ml⁸Individual Sf9 cells) in add P3 generations virus (dosage be 5MOI), 27 DEG C, 100~120rpm culture 72h, obtain suspension.

2nd, the suspension is taken, 7000rpm centrifugation 20min obtain supernatant 1.

3rd, the supernatant 1 is taken, through 0.22mm membrane filtrations, sample solution is obtained.

4th, the sample solution is splined on HiTrap-Q Sepharose ion exchange columns (flow velocity is 1mL/min), Then first (flow velocity is 1mL/min) is rinsed with the PBS of pH7.5,200mM of 5mL；Again with pH7.5,300mM of 10mL PBS rinse (flow velocity is 1mL/min)；Finally rinse that (flow velocity is with the PBS of pH7.5,600mM of 3mL 1mL/min), collect after post solution and used molecular cut off to be concentrated by ultrafiltration for the super filter tube of 50KD, obtain 1mL or so Concentrate.The concentrate is to contain rgp96.

5th, the concentrate that step 4 is obtained is splined on the 10/300GL molecular sieve chromatographies of Superdex 200 (flow velocity is 0.25mL/min), (flow velocity is 0.25mL/min) is then washed with the PBS of pH7.5,150mM, is collected as 9~12mL Place penetrates liquid, further uses molecular cut off to be concentrated by ultrafiltration for the super filter tube of 50KD, obtains the solution of rgp96.Adopt The protein concentration in the solution of rgp96 is determined with BCA methods, is finally dispensed, be stored in -80 DEG C.

The solution of the rgp96 that step 5 is obtained carries out SDS-PAGE electrophoretic analysis, experimental result see Fig. 3 (swimming lane by a left side to The right side is respectively HMW standard protein and rgp96).The solution of the restructuring heat shock protein gp96 that step 5 is obtained is carried out (using gp96 monoclonal antibodies as primary antibody, the monoclonal of the goat anti-rat of HRPO mark resists Western blot Body is used as secondary antibody), experimental result is shown in Fig. 3.Result shows that the solution of rgp96 shows unimodal molecular weight band, corresponding molecular weight It is consistent with expection.

The compound that the DEF8 polypeptides of embodiment 4 are combined with placenta gp96 is prepared and identification

First, the peptide identification that mouse placenta gp96 is combined

1st, polypeptide wash-out：The concentration for taking the extraction of 5mg embodiments 1 is 10mg/mL mouse placenta pgp96, adds 5 μ L to contain The aqueous solution of 20% (volume ratio) trifluoroacetic acid, 1h is incubated in 4 DEG C.

2nd, by the Incubating Solution addition 3kD super filter tubes in step 1,12,000rpm centrifugation 30min take and penetrate liquid.

3rd, the liquid injection high pressure liquid chromatographs of Agilent 1200 that penetrate in step 2 are carried out into desalting and purifying (chromatographic column Model SB-C182.1mm × 250mm), program is as follows：A phases：5% (volume ratio) acetonitrile solution, containing 0.1% trifluoro Acetic acid；B phases：Acetonitrile, containing 0.1% trifluoroacetic acid；0-30min 100%A, 30-70min 100%B.

4th, the eluent of 30-70 minutes in collection step 3, freezes.

5th, it is the freeze-dried powder of step 4 is molten with the aqueous formic acid weight of 0.1% (volume ratio), inject Orbitrap Fusion high resolution mass spectrometers, analyze polypeptide sequence, and matched from database.Matching result is shown in Table 1.

The polypeptide matching result that table 1 is combined with mouse placenta gp96

2nd, prepared by DEF8 polypeptides-gp96 compounds

1st, the DEF8 albumen result amino acid sequences for obtaining table 1 are input into network address http:// Tools.immuneepitope.org/mhci/ carries out the Antigen Epitope Prediction of MHC I quasi-molecules, and optimal H-2K is chosen respectively^d、H- 2K^bAnd HLA-A2 restricted epitopes and chemical synthesis, during selected amino acid sequence should be the mankind during prediction HLA-A2 epitopes Homologous protein.Epitope the selection result is shown in Table 2.

The epitope the selection result of the DEF8 polypeptides of table 2

MHC I restricted types	Polypeptide sequence	Polypeptide position
				RYLALMVSRPVL	273-284
	CSMRYLAL	270-277
			HLA-A2	LLFNYVEELVEI	292-303

2nd, difference chemical synthesis above polypeptide (Shanghai gill biochemistry Co., Ltd), the polypeptide cell grade DMSO that will synthesize It is 20mg/mL concentration that (Sigma-Aldrich companies, catalog number (Cat.No.) D2650) redissolves, in taking 1mg polypeptides and 1mg embodiments 3 respectively The restructuring rgp96 of preparation, 4mL volumes are diluted to pH7.4 0.01mol/L PBSs.55 DEG C of thermal shocks 10 minutes, in room Temperature cooling 30 minutes.Then uncombined polypeptide is washed away using 50kD super filter tubes, different restricted epitopes is prepared respectively DEF8 polypeptide-gp96 compounds.

Prevention and treatment of the DEF8 polypeptide-gp96 compounds of embodiment 5 to breast cancer

First, prevention effect of the DEF8 polypeptides-gp96 compounds to primary breast cancer

Take 8 week old C57BL/6 mouse 20 and be only divided into two groups, every group 10, be handled as follows respectively：

First group：DEF8 polypeptides (H-2K prepared by abdominal part hypodermic embodiment 4^b)-gp96 compounds, it is immunized three times (each 0.2ml), single immunization dosage is 20ug/；

Second group：Rgp96 prepared by abdominal part hypodermic embodiment 3, is immunized three times (each 0.2ml), single immunization agent Measure as 20ug/ only；

In two groups of the above：Test carries out being immunized for the first time on the 1st day；Second is carried out within 8th day to be immunized；Carry out the 3rd within 22nd day It is secondary immune.

1 week after the completion of immune starts the DMBA that every mouse presses the edible olive oil of 50mg/kg dosage oral administration gavage (Sigma-Aldrich companies, catalog number (Cat.No.) D3254) solution, 1 times a week, continuous 5 weeks, induced breast cancer morbidity.

Own mouse produces breast cancer tumour to start (about 14 weeks or so after modeling) and detects tumour growth weekly, and counts swollen Knurl incidence and calculating gross tumor volume.Gross tumor volume computing formula V=ab²/ 2 (V-volume, a-tumour major diameter, b-tumour are short Footpath).Always it was observed that 34 weeks, calculating Tumor incidence, tumour number and average tumor size.Statistics is shown in Table 3.From result As can be seen that DEF8 polypeptide-gp96 compounds have obvious prevention effect to the breast cancer that DMBA is induced.Immune DEF8 polypeptides- The incidence of disease of breast cancer is substantially reduced after gp96 compounds, and disease time is postponed, and tumour number and gross tumor volume reduce.

The mouse breast cancer tumour statistics of the DMBA of table 3 inductions

2nd, therapeutic action of the DEF8 polypeptides-gp96 compounds to induced breast cancer model

Take 20 female Balb/c mouse of 6-8 weeks, every subcutaneous inoculation 6 × 10 respectively⁵TUBO cells, the 2nd day will be small Mouse is divided into two groups, every group 10, is handled as follows respectively：

First group：DEF8 polypeptides (H-2K prepared by abdominal part hypodermic embodiment 4^d)-gp96 compounds, it is immunized three times (each 0.2ml), single immunization dosage is 20ug/；

Second group：Gp96 prepared by abdominal part hypodermic embodiment 3, is immunized three times (each 0.2ml), single immunization dosage For 20ug/ only；

In two groups of the above：Carry out within the 2nd day after inoculated tumour cell immune for the first time；Second is carried out within 5th day to be immunized；8th It is immune that it carries out third time.Since first day immune, observation tumour growth situation, records tumor size, by following public affairs daily Formula calculates gross tumor volume：V=ab²/ 2 (V-volume, a-tumour major diameter, b-tumour minor axis).Tumor volume change is shown in Fig. 4.From Result can be seen that DEF8 polypeptide-gp96 compounds has obvious therapeutic action to the induction type breast cancer model of subcutaneous vaccination. The speed of growth of hypodermic tumour substantially slows down after DEF8 polypeptide-gp96 complex therapies, and gross tumor volume diminishes.

Prevention and treatment of the DEF8 polypeptide-gp96 compounds of embodiment 6 to liver cancer

First, prevention effect of the DEF8 polypeptides-gp96 compounds to primary carcinoma of liver

20 6 week old female C57BL/6 mouse are taken, two groups are equally divided into, every group 10, is handled as follows respectively：

1 week after the immune end of last time, two groups of mouse are changed to containing 30 μ g/mL DEN (Sigma- per daily drink Aldrich, catalog number (Cat.No.) 73861) sterile distilled water, it is continuous to raise 40 weeks, mouse, taking-up liver are put to death during to the 40th week It is compared com-parison and analysis.Comparative result is shown in Table 4.From the results, it was seen that what DEF8 polypeptide-gp96 compounds were induced DEN Liver cancer has obvious prevention effect.The incidence of disease of liver cancer is substantially reduced after immune DEF8 polypeptide-gp96 compounds, and disease time is pushed away Late, tumour number and gross tumor volume reduce.

The rat liver cancer statistics of the DEN of table 4 inductions

2nd, therapeutic action of the DEF8 polypeptides-gp96 compounds to induction liver cancer model

By the rat liver cancer H22 cells of liquid nitrogen cryopreservation in 37 DEG C of water-baths quick-thawing, cell density is adjusted to 1 × 10⁷/ ML, 2 BALB/c mouses of intraperitoneal inoculation, every 0.2mL.After mouse web portion swells, mouse cervical dislocation is put to death, abdomen of sterilizing Portion, extracts ascites and merges, and cell density to 1 × 10 is adjusted with PBS⁷/ mL, subcutaneous vaccination 20 BALB/c mouses, every 0.2mL.Mouse is divided into two groups after 12 days, every group 10, is handled as follows respectively：

In two groups of the above：Carry out within the 12nd day after inoculated tumour cell immune for the first time；Second is carried out within 15th day to be immunized；The Carry out within 18 days third time immune.Since first day immune, observation tumour growth situation, records tumor size, by following daily Formula calculates gross tumor volume：V=ab²/ 2 (V-volume, a-tumour major diameter, b-tumour minor axis).Tumor volume change is shown in Fig. 5. From the results, it was seen that DEF8 polypeptide-gp96 compounds have obvious therapeutic action to the induction type liver cancer model of subcutaneous vaccination. The speed of growth of hypodermic tumour substantially slows down after DEF8 polypeptide-gp96 complex therapies, and gross tumor volume diminishes.

The specific CTL of the DEF8 polypeptide-gp96 compounds of embodiment 7 induction is for human breast cancer cell lethal effect

First, the preparation of people source polypeptid specificity effector cell

1st, the anti-freezing of user's lymphocyte separation medium (Cellgro, 25-072-CI) separation HLA-A2 positive volunteers is new Fresh whole blood, obtains PMNC (PBMC), with containing 10% hyclone (Gibco, 10099-141-FBS) RPMI-1640 complete mediums (Gibco, 12633012) adjustment cell concentration is 1.0X10⁶/ ml, is inoculated in 24 orifice plates, per hole 1ml。

2nd, every group of next day be separately added into DEF8 polypeptides (HLA-A2)-gp96 compounds to final concentration of 10 μ g/ml.

3rd, IL-2 (PeproTech companies, catalog number (Cat.No.) 212-12) to final concentration of 50U/ml is added per hole within the 3rd day, per 2- 3 days half amounts are changed liquid and supplement IL-2 to final concentration of 50U/ml.

4th, carrying out the second wheel, third round DEF8 polypeptide-gp96 compounds respectively at the 7th day, fortnight stimulates, next day Add IL-2 to final concentration of 50U/ml.

5th, 3 days after third round stimulates, people source polypeptide effector cell CTL is obtained.

2nd, target cell：Human breast carcinoma cell lines MCF-7 (HLA-A2 expression is positive), the T2 cells (HLA-A2 that polypeptide is incubated Expression is positive), T2 cells.

3rd, the detection of mammary tumor cells specific lethal effect：UseNon-radioactive cell toxicity is examined Surveying (Promega, catalog number (Cat.No.) G1780) carries out cytotoxicity assay, and key step is following (referring to kit operation instructions)：

1st, T2 cells, the cells of Τ 2 being incubated using MCF-7 cells, polypeptide as target cell, inoculation target cell population is 5 × 10³/ hole, 10 are compared according to effect target:1 ratio adds above-mentioned effector cell, effector cell to be inoculated in 96 well culture plates with the holes of 50 μ 1/ In, the μ 1 of final volume 100；

In addition the spontaneous LDH releases group of another laying effect cell, for calibrating the spontaneous LDH (each groups for discharging of effector cell Effector cell adds 96 orifice plates, RPMI-1640 culture mediums of the μ 1 containing 5% hyclone of supplement 50 to final volume 100 with the holes of 50 μ 1/ μ1；).The spontaneous LDH releases group of target cell, for correcting the spontaneous LDH for discharging of target cell, (each group target cell is with the holes of 50 μ 1/ Add 96 orifice plates, RPMI-1640 culture mediums of the μ 1 containing 5% hyclone of supplement 50 to the μ 1 of final concentration 100；).Target cell is maximum LDH release groups, during for calculating as determine 100% LDH discharge reference (cell loading is with the spontaneous release of target cell Group；).Volume correction control group, for correcting because the Volume Changes for adding lysate to cause (are added containing 5% hyclone The μ 1 of RPMI-1640 culture mediums 100；).Culture medium ground control group, for correct by culture medium serum produce LDH activity with And the phenol red background absorption for causing (adds the μ 1 of RPMI-1640 culture mediums 100 containing 5% hyclone；).

2nd, after cell inoculation, 4min is centrifuged using 250g, is then incubated 4h in 37 DEG C of incubators；Before supernatant is harvested 45min, to addition lysate (10 ×), the holes of 10 μ 1/ in target cell maximum LDH release groups；250g centrifugation 4min are then used by, are received Obtain supernatant；

3rd, in supernatant to the ELISA Plates of 0 μ of transferase 45 1, with detection buffer substrate, the holes of 50 μ of substrate 1/ for preparing are added to In ELISA Plate, flat board is covered, room temperature lucifuge reaction 30min is detected to every empty addition in the terminate liquids of 50 μ 1,1h in ELIASA 490nm light absorption values OD.

4th, cell killing rate is calculated

Killing rate (%)=[(the spontaneous spontaneous release group OD of release group OD values-target cell of experimental group OD values-effector cell Value)/(the target cell maximum release group OD values-spontaneous release group OD values of target cell)] × 100%

Cell killing result is shown in Fig. 6.Result shows that DEF8 polypeptide-gp96 compounds can be from the PBLC of people (PBMC) cytotoxic T cell (CTL) of epitope specificity is induced in, the cell can be killed to human breast cancer cell line Bcap-37, Show the ability that DEF8 polypeptide-gp96 compounds have prevention and treatment human breast carcinoma.

The specific CTL of the polypeptide-gp96 compounds of 8 DEF of embodiment 8 induction is for human liver cancer cell lethal effect

First, the preparation of people source polypeptid specificity effector cell：It is special that DEF8 polypeptides (HLA-A2)-gp96 compounds are induced Property CTL preparation methods are adjusted to debita spissitudo, as effect with embodiment 7 with the RPMI-1640 culture mediums containing 5% hyclone Answer cell.

2nd, target cell：Human liver cancer cell HHCC (HLA-A2 expression is positive), (HLA-A2 is expressed the T2 cells that polypeptide is incubated It is positive), T2 cells

3rd, the detection of liver cancer cell specificity lethal effect：Target cell is changed to human liver cancer by detection method with embodiment 7 Cell HHCC, experimental group compares 10 by effect target:1 ratio adds effector cell and target cell, while setting up, effector cell is spontaneous to release Put the spontaneous release group of group, target cell, target cell maximum release group, ground control group, volume correction control group.Incubated under the conditions of 37 DEG C Cell pyrolysis liquid is added after educating 4h, supernatant is harvested and is done LDH detections.Cell killing result is shown in Fig. 7.Result show DEF8 polypeptides- Gp96 compounds can induce the cytotoxic T cell of epitope specificity from the PBLC (PBMC) of people (CTL), the cell can be killed to human liver cancer cell HHCC, show that DEF8 polypeptide-gp96 compounds have prevention and treatment human liver cancer Ability.

Although above the present invention is described in detail with a general description of the specific embodiments, On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.

SEQUENCE LISTING

<110>Beijing Re Xiu Bioisystech Co., Ltd

<120>The application of DEF8 albumen and its polypeptide in treatment with pre- anti-cancer

<130> KHP171110225.0

<160> 11

<170> PatentIn version 3.5

<210> 1

<211> 1536

<212> DNA

<213>DEF8 albumen

<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 albumen

<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 Pro Arg 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

115 120 125

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

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

Application of the 1.DEF8 albumen in prevention and/or treating cancer medicine is prepared, described its amino acid sequence of DEF8 albumen contains Have as follows：

I) amino acid sequence shown in SEQ ID NO.2；Or

Ii) amino acid sequence shown in SEQ ID NO.2 from N-terminal the 2nd to last amino acids sequence；Or

Iii) amino acid sequence shown in SEQ ID NO.2 is substituted, lacks and/or increases one or more amino acid and has The amino acid sequence of identical function.
2. application of the gene of encoding D EF8 albumen in prevention and/or treating cancer medicine is prepared, the encoding D EF8 albumen Gene nucleotide sequence contain it is as follows：

I) nucleotide sequence shown in SEQ ID NO.1；Or

Ii) nucleotide sequence shown in SEQ ID NO.1 is substituted, lacks and/or increases one or more nucleotides and expression The nucleotide sequence of identical function protein；Or

Iii) there is the nucleotide sequence of 75% and above homology with the nucleotides shown in SEQ ID NO.1；Or

Iv) under strict conditions with the nucleotide sequence of sequence hybridization shown in SEQ ID NO.1；

The stringent condition is in 0.1 × SSPE containing 0.1%SDS or the 0.1 × SSC solution containing 0.1%SDS, at 65 DEG C Lower hybridization, and wash film with the solution.
3. application of the biomaterial of the gene containing encoding D EF8 albumen in prevention and/or treating cancer medicine is prepared, institute Biomaterial is stated for carrier, host cell, transgenic cell line, engineering bacteria, insect or yeast；

The nucleotide sequence of the gene of the encoding D EF8 albumen contains as follows：

I) nucleotide sequence shown in SEQ ID NO.1；Or

Ii) nucleotide sequence shown in SEQ ID NO.1 is substituted, lacks and/or increases one or more nucleotides and expression The nucleotide sequence of identical function protein；Or

Iii) there is the nucleotide sequence of 75% and above homology with the nucleotides shown in SEQ ID NO.1；Or

Iv) under strict conditions with the nucleotide sequence of sequence hybridization shown in Seq ID NO.1；

The stringent condition is in 0.1 × SSPE containing 0.1%SDS or the 0.1 × SSC solution containing 0.1%SDS, at 65 DEG C Lower hybridization, and wash film with the solution.
4. the application as described in claim 1-3 is any, it is characterised in that the medicine has following at least one function：

(1) incidence of the breast cancer of chemicals induction is reduced；

(2) the liver cancer incidence of chemicals induction is reduced；

(3) it is slowed or shut off the growth of breast cancer tumour stove set up；

(4) it is slowed or shut off the growth of hepatic carcinoma stove set up；

(5) reduce or stop the transfer of the breast cancer tumour stove set up；

(6) reduce or stop the transfer of the hepatic carcinoma stove set up；

(7) induction produces the CTL cells of Breast Cancer-Specific and breast cancer cell is killed；

(8) induction produces the CTL cells of liver cancer-specific and HCC is killed.
5. the medicine containing DEF8 albumen or its polypeptide, the DEF8 albumen its amino acid sequence contains as follows：

I) amino acid sequence shown in SEQ ID NO.2；Or

Ii) amino acid sequence shown in SEQ ID NO.2 from N-terminal the 2nd to last amino acids sequence；Or

Iii) amino acid sequence shown in SEQ ID NO.2 is substituted, lacks and/or increases one or more amino acid and has The amino acid sequence of identical function.
6. a kind of medicine, it is characterised in that the compound containing DEF8 albumen and heat shock protein gp96 compositions contains DEF8 The compound that the polypeptide of albumen is constituted with heat shock protein gp96；

Described its amino acid sequence of DEF8 albumen contains as follows：

I) amino acid sequence shown in SEQ ID NO.2；Or

Ii) amino acid sequence shown in SEQ ID NO.2 from N-terminal the 2nd to last amino acids sequence；Or

Iii) amino acid sequence shown in SEQ ID NO.2 is substituted, lacks and/or increases one or more amino acid and has The amino acid sequence of identical function；

The amino acid sequence of the heat shock protein gp96 contains shown in sequence or SEQ ID NO.4 described in SEQ ID NO.4 Amino acid sequence be substituted, lack and/or increase one or more amino acid and the amino acid sequence with identical function.
7. medicine as claimed in claim 6, it is characterised in that the acquisition pattern of the heat shock protein gp96 is following One：

(1) extracted from the placenta tissue of isolated mammalian；

(2) nucleic acid molecules that will encode the heat shock protein gp96 are imported in acceptor, and culture is moved using yeast, lactation Thing cell expression system is expressed, and purifying is obtained.
8. medicine as claimed in claims 6 or 7, it is characterised in that the compound is to obtain in the following manner：

(1) DEF8 albumen or its polypeptide are formed compound with absorption or thermal shock mode naturally in vitro with heat shock protein gp96 Thing；Or

(2) encoding D EF8 albumen or its polypeptide are imported in acceptor with the nucleic acid molecules of encoding heat shock proteins gp96, it is culture, pure Change and obtain compound.
9.DEF8 albumen and its polypeptide are preparing treatment and/or prevention cancer drug with the compound of heat shock protein gp96 compositions In application.
10. application as claimed in claim 9, it is characterised in that the medicine has following at least one function：

(1) incidence of the breast cancer of chemicals induction is reduced；

(2) the liver cancer incidence of chemicals induction is reduced；

(3) it is slowed or shut off the growth of breast cancer tumour stove set up；

(4) it is slowed or shut off the growth of hepatic carcinoma stove set up；

(5) reduce or stop the transfer of the breast cancer tumour stove set up；

(6) reduce or stop the transfer of the hepatic carcinoma stove set up；

(7) induction produces the CTL cells of Breast Cancer-Specific and breast cancer cell is killed；

(8) induction produces the CTL cells of liver cancer-specific and HCC is killed.