CN106975072A - Applications of the heat shock protein gp96 in treatment autoimmune hemolytic anemia - Google Patents

Abstract

The invention provides applications of the heat shock protein gp96 in treatment autoimmune hemolytic anemia, belong to protein engineering and biological medicine applied technical field.The heat shock protein gp96 of present invention amino acid sequence is as shown in SEQ ID NO.2 or its specific fragment, present invention discover that heat shock protein gp96 can induce the generation of regulatory T cells, reduce anti-Autoerythrocyte antibody titer, improve erythrocyte number and content of hemoglobin, available for the medicine for preparing treatment autoimmune hemolytic anemia, have broad application prospects.

Description

Applications of the heat shock protein gp96 in treatment autoimmune hemolytic anemia

Technical field

The present invention relates to protein engineering and biological medicine applied technical field, specifically, it is related to heat shock protein Applications of the gp96 in treatment autoimmune hemolytic anemia.

Background technology

Autoimmune hemolytic anemia (Autoimmune hemolytic anemia, AIHA) is vivo immunization function Regulation is disorderly, produces autoantibody and (or) complement adsorption in erythrocyte surface, accelerates red blood cell to break by antigen-antibody reaction A kind of hemolytic anemia caused by bad.Autoimmune hemolytic anemia can be divided into according to needed for antibody acts on erythrocyte membrane Optimum temperature, warm antibody and cold anitibody type can be divided into.The common medicine for the treatment of mankind itself's hemolytic anemia is sugared skin at present Matter hormone, it suppresses T cell and produces IL-2 and play a role by suppressing phagocytosis and processing of the macrophage to antigen.But Its adverse reaction is more, and is easily recurred after drug withdrawal.

Heat shock protein (Heat shock protein, HSP) is that a class is 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, Multiple subfamilies 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 content most abundant heat shock protein on endocytoplasmic reticulum.Research Show that gp96 has important effect in immunological regulation.The external source gp96 injection mouse of low dosage can significantly activate exempting from for mouse The immune system of mouse is significantly inhibited after epidemic disease system, the external source gp96 injection mouse of high dose.Gp96 regulation immunologic functions may It is related to regulatory T cells (Treg).Do not have been reported that heat shock protein gp96 in treatment autoimmune hemolytic anemia at present In application.

The content of the invention

It is an object of the invention to provide new opplications of the heat shock protein gp96 in treatment autoimmune hemolytic anemia.

Present invention firstly provides heat shock protein gp96, 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 the 783rd 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 above-mentioned heat shock protein gp96 in treatment autoimmune hemolytic anemia medicine is prepared Using.

The invention provides encoding heat shock proteins gp96 gene, 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) nucleotide sequence under strict conditions with sequence hybridization shown in SEQ ID NO.1；

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

Treatment autoimmune hemolytic anemia medicine is being prepared the invention provides encoding heat shock proteins GP96 gene Application in thing.

The invention provides the biomaterial of the gene containing encoding heat shock proteins GP96, the biomaterial is load Body, host cell, transgenic cell line, engineering bacteria, insect or yeast.

Further, the invention provides above-mentioned biomaterial in treatment autoimmune hemolytic anemia medicine is prepared Application.

The invention provides above-mentioned heat shock protein gp96 answering in the medicine that induction regulatory T cells are produced is prepared With.

The regulatory T cells are CD4⁺CD25⁺FOXP3⁺Regulatory T cells.

The invention provides above-mentioned heat shock protein gp96 in the medicine for preparing the anti-spontaneous erythrocyte antibody (EA) titre of reduction Application.

The medicine of raising erythrocyte number and content of hemoglobin is being prepared the invention provides above-mentioned heat shock protein gp96 Application in thing.

The invention provides the medicine containing above-mentioned heat shock protein gp96.Described medicine has following at least one work( Energy：

(1) induction regulatory T cells are produced；

(2) anti-Autoerythrocyte antibody titer is reduced；

(3) erythrocyte number and content of hemoglobin are improved.

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

(1) extraction purification is obtained from cell or tissue；

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

Described " importing the nucleic acid molecules for encoding the heat shock protein gp96 in acceptor " can be by importing into 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 plasmid pFastBac1 multiple cloning sites insetion sequence table shown in SEQ ID NO.1 is obtained The recombinant plasmid arrived.

The recombinant plasmid pFastBac1-gp96 concretely recognizes plasmid pFastBac1 EcoR I and Xba I (plasmid pFastBac1 is cut into a large fragment and one to fragment between sequence by restriction endonuclease EcoR I and Xba I Small fragment, 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.

Heat shock protein gp96 dosage can be to inject heat shock protein gp96 described in the μ g of 100 μ g~500 to mammal (heat shock protein gp96 as described in the heat shock protein gp96 or 500 μ g as described in 100 μ g).It is demonstrated experimentally that heat of the present invention is stopped Gram albumen gp96 has the generation of induction regulatory T cells, the function of the anti-Autoerythrocyte antibody titer of reduction.Therefore, heat is stopped Gram albumen gp96 has important application value to the symptom for treating autoimmune hemolytic anemia.

Brief description of the drawings

In SDS-PAGE the and western blot qualification results for the gp96 extracts that Fig. 1 is prepared for human placenta, figure, 1：Molecular weight standard；2：The SDS-PAGE of the gp96 extracts prepared with human placenta；3：Western blot reflect Fixed figure.

Fig. 2 is SDS-PAGE the and western blot qualification results that gp96 albumen is recombinated using expressed by Hansenula yeast.Figure In, 1：Molecular weight standard；2：The zymotic fluid of step 2；3：Eluent after affinity chromatography；4：Elution after ion-exchange chromatography Liquid；5：Western blot qualification figures.

Fig. 3 is the qualification result for the SDS-PAGE and Western Blot that gp96 is recombinated using insect cell expression.

Embodiment

The present invention is further described in detail with reference to embodiment, the embodiment provided is only for explaining The bright present invention, the scope being not intended to be limiting of the invention.Experimental method in embodiment, is routine unless otherwise specified Method.Material, reagent used etc., unless otherwise specified, are commercially obtained in embodiment.

Quantitative test in following examples, is respectively provided with three repetition experiments, results averaged.

BALB/C mice, female, 18-20g, purchased from Nanjing model animal research institute；BALB/C mice hereinafter referred to as Mouse.SD rats, male, 6 week old, body weight 120g, purchased from Nanjing model animal research institute.Obtain mouse spleen lymphocyte Specific method refers to following document：Zhen Liu, Xinghui Li, Lipeng Qiu, et al.Treg suppress CTL Responses upon immunization with HSP gp96.Eur.J.mmunol, 2009,39 (11)：3110–3120.

HepG2 cells (human liver cancer cell) are ATCC Products, and catalog number is HB-8065^TM.Sf9 cells are Invitrogen Products, catalog number is 11496-015.Cellfectin II reagent are Life Technologies Products, catalog number is 10362-100.Plasmid pFastBac^TM1 is that Invitrogen companies produce Product, catalog number is 10359-016.Gp96 monoclonal antibodies are Santa Cruz Products, and catalog number is sc- 56399.The monoclonal antibody of the goat anti-rat of horseradish peroxidase-labeled is the limited public affairs of Beijing Zhong Shan Golden Bridge biotechnology Product is taken charge of, catalog number is ZB-2307.HiTrap-Q Sepharose ion exchange columns are GE Products, product Catalog number (Cat.No.) is 17-5053-01.The 10/300GL molecular sieve chromatographies of Superdex 200 are GE Products, and catalog number is 17517501.Escherichia coli DH10Bac competent cells are Beijing Yuanpinghao Biological Technology Co., Ltd.'s product, catalog number For CL108-01.FITC-anti-CD3, PE-anti-CD4, Percp-Cy5.5-anti-CD25, fixation/rupture of membranes agent and APC- Anti-Foxp3 is eBioscience Products, and catalog number is respectively 11-0031-63,12-0041-81,45- 0251-80,00-5521-00 and 17-5773-80.Insect-XPRESS^TM Protein-free Insect Cells Medium with L-Glutamine are LONZA Products, and catalog number is 12-730Q.

The heat shock protein gp96 of embodiment 1 acquisition

(1) gp96 albumen is purified in human placenta

1st, tissue homogenate

Homogenate buffer is formulated：In pH 7.0 sodium bicarbonate buffer liquid plus PMSF (phenylmethylsulfonyl fluoride, molecular formula is C₇H₇FO₂S) (it is placed in beaker on ice and prepares to final concentration 1mM：PMSF a few hours interior decomposition in aqueous, the solution is not It can stay overnight).

Beaker is taken on ice, human placenta is cut into rapidly to the fragment of about 1-2 millimeters of diameter in beaker, then added Organize the homogenate buffer of 8 times of weight.Tissue pieces are stirred evenly and pour into glass homogenizer, after homogenate to bottom tissue block disappearance again It is homogenized more than 15 times up and down.Homogenate pours into centrifuge tube, 50,000g centrifugation 60min, take supernatant add 1/10 volume 10 × PBS (pH7.5,200mM NaCl), the ConA-Sepharose for step 2 is chromatographed.

2nd, ConA-Sepharose affinity chromatographys

Carrier is ConA-Sepharose (being purchased from GE companies, production number is 17-0440-01), by " 1ml carriers/4g groups Knit " calculate usage amount.The internal diameter and column length of chromatographic column are 1.6 × 2.5cm.

The supernatant that step 1 is obtained peristaltic pump loading, 0.25ml/min.Add in PBS (pH7.5,200mM NaCl) PMSF to final concentration 1mM, as eluent first, chromatographic column (0.5ml/ is crossed with peristaltic pump by the eluent first of 10 times of column volumes Min), to remove uncombined albumen.Add α-D- glucopyranoses in PBS (pH7.5,200mM NaCl) to final concentration 10% (10mg/ml), plus PMSF to final concentration 1mM, as eluent second, post layer is crossed with peristaltic pump by the eluent second of 3 times of column volumes Analyse (0.25ml/min), collect eluent.

3rd, HiTrap-Q Sepharose ion-exchange chromatographies

Carrier is that (chromatographic column is HiTrap Q HP to HiTrap-Q Sepharose, purchased from GE companies, and production number is 17- 1153-01).The internal diameter and column length of chromatographic column are 0.7 × 2.5cm.

Carrier is filled after post, first (1ml/min) is cleaned with 5ml PBS (pH7.5,200mM NaCl).Step 2 is obtained Eluent loading (1ml/min), then crosses chromatographic column (1ml/min) by 5ml PBS (pH7.5,200mM NaCl), to remove not Associated proteins.Then 10ml PBS (pH7.5,300mM NaCl) are crossed into chromatographic column (1ml/min), to remove uncombined albumen. Then 3ml PBS (pH7.5,600mM NaCl) are crossed into chromatographic column (1ml/min), collects eluent, as gp96 extracts.

Gp96 extracts are subjected to 10%SDS-PAGE, then coomassie brilliant blue staining.

Gp96 extracts are subjected to western blot, primary antibody is that the anti-gp96 antibody of rat (is purchased from santa cruz, product Number be sc-56339).

In addition to Human plactnta, it is also possible to following cell or tissue preparation gp96 extracts：Melanoma (B16) cell, Human hepatoma cell line HepG2 culture cell, clinical operation excision human tumour tissue (breast cancer, glioma, kidney, intestinal cancer, Liver cancer, stomach cancer), extraction of the extracting method with reference to Human plactnta.As a result Fig. 1 is seen.

(2) gp96 albumen is recombinated using expressed by Hansenula yeast

1st, recombinant plasmid pHFMDZ-R1L2GAmy-gp96 structure

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

2. the cDNA using step 1. enters performing PCR as template and expanded, and obtains pcr amplification product.

PCR primer is as follows：

Fp(5'-CCGgaattcATGGACGATGAAGTTGATG-3')

Rp(5'-CCGctcgagCTATTAGAATTCATCTTTTTCAGCTGTAG-3')

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

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

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

6. LEU2 fragments, LEU2 nucleotides sequence are inserted in recombinant plasmid pHFMDZ-R1-gp96 BglII restriction enzyme sites Row are as shown in SEQ ID No.3.BamHI restriction enzyme sites insert alpha-amylase reporting system as shown in SEQ ID No.4, obtain weight Group plasmid pHFMDZ-R1L2GAmy-gp96.

2nd, the expression of gp96 albumen

1. the recombinant plasmid pHFMDZ-R1L2GAmy-gp96 obtained step 1 using electrotransformation imports Hansenula yeast (being purchased from ATCC, production number is MYA-335) cell, obtains recombinant bacterium.

2. 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 culture 48h, be then transferred to 100mL SYN6 culture mediums (be purchased from Shanghai Jie Mei genes pharmaceuticals, Production number is GMS12116.1), 30 DEG C of culture 48h obtain seed culture fluid.

3. 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, glycerine is added according to the concentration of glycerine in zymotic fluid, Final glycerol concentration is controlled 0.5% or so, while controlling dissolved oxygen more than 20%.Detect that thalline is wet according to the generation situation of thalline Weight, waits thalline weight in wet base to reach and stops adding glycerine when 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.Cell is washed with PBS 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 subjected to affinity chromatography, 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) pillar is rinsed to OD values<0.01,1.5h is eluted with PBS (imidazoles containing 200mM), eluent is collected.All operations are at 4 DEG C Lower to carry out, flow velocity is 0.5mL/min.

The zymotic fluid that every liter of step 2 is obtained can obtain the gp96 albumen of 50 milligrams of purity more than 90% after purification.If Contain heterologous foreign protein in protein eluate, the eluent of affinity chromatography can be subjected to ion-exchange chromatography again, mainly comprised the following steps： 200mM NaCl PBS liquid balance pillar, loading, 300mM NaCl PBS liquid washes impurity, 800mM NaCl PBS liquid elution 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 subjected to 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 is shown, in the eluent after ion-exchange chromatography Gp96 purity of protein it is very high.

(3) gp96 albumen is recombinated using insect cell expression

1st, recombinant plasmid pFastBac^TM1-gp96 structure

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

2. 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).

1. and 2. 3. after completing step, the cDNA 1. obtained using step is template, and the F1 and R1 1. synthesized using step is draws Thing enters performing PCR amplification, obtains pcr amplification product.

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

5. the restriction enzyme EcoR I and digested plasmid pFastBac of Xba I is used^TM1, reclaim about 4700bp carrier bone Frame.

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

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

According to sequencing result, structure is carried out to recombinant plasmid pFastBac1-gp96 and is described as follows：By plasmid pFastBac1 EcoR I and the recognition sequences of Xba I between fragment (plasmid pFastBac1 is by restriction endonuclease EcoR I and Xba I A large fragment and a small fragment are cut into, the DNA is the small fragment) replace with pair shown in SEQ ID NO.1 in sequence table Ssdna molecule.Recombinant plasmid pFastBac 1-gp96 expression restructuring heat shock proteins gp96, gp96 amino acid sequence such as sequence In list shown in SEQ ID NO.2.

2nd, gp96 expression

1. the recombinant plasmid pFastBac1-gp96 cotransfection Sf9 cells (every 1 × 10 step 1 built⁶Individual 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.

2. Sf9 cell suspending liquids 1 (are contained 1 × 10⁸Individual Sf9 cells) 27 DEG C of 8~10h of culture, obtain cultivating cell；Then The addition P1 generations virus (dosage is 0.05~0.1MOI) into the culture cell, 27 DEG C of incubations 72h, 4000rpm centrifuge 5min, Supernatant is P2 generation viruses.

3. (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 is P3 generation viruses.

Using gp96 monoclonal antibodies as primary antibody, the monoclonal antibody conduct of the goat anti-rat of HRPO mark Secondary antibody, western hybridization is carried out to P3 generation viruses, 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 that gp96 is expressed in Sf9 cells.

3rd, gp96 purifying

1. (4.5 × 10 are contained to 300ml Sf9 cell suspending liquids 3⁸Individual Sf9 cells) in add P3 generations virus (dosage be 5MOI), 27 DEG C, 100~120rpm culture 72h, obtain suspension.

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

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

4. 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 5mL pH7.5,200mM PBS；10mL pH7.5,300mM is used again PBS rinse (flow velocity is 1mL/min)；Finally rinse that (flow velocity is with 3mL pH7.5,600mM PBS 1mL/min), collect after post solution and used molecular cut off to be concentrated by ultrafiltration for 50KD super filter tube, and obtain 1mL or so Concentrate.The concentrate is containing gp96.

5. the concentrate that 4. step obtains being splined on into 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 pH7.5,150mM PBS, is collected as 9~12mL Place penetrates liquid, further uses molecular cut off to be concentrated by ultrafiltration for 50KD super filter tube, obtains gp96 solution.Using BCA methods determine the protein concentration in gp96 solution, finally dispense, are stored in -80 DEG C.

The gp96 that 5. step is obtained solution carries out SDS-PAGE electrophoretic analysis, and experimental result is shown in Fig. 3, and (swimming lane is from left-hand The right side is respectively HMW standard protein and gp96).The solution for the restructuring heat shock protein gp96 that 5. step 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.As a result show, gp96 solution shows unimodal molecular weight band, corresponding molecular weight With being expected unanimously.

Determinations of the people source heat shock protein gp96 of embodiment 2 in activating regulatory T cell middle dosage

First, mice group is immune is randomly divided into placenta gp96 treatment groups 1, tire by 100 body weight for 18~22g mouse Disk gp96 treatment groups 2, placenta gp96 treatment groups 3, yeast gp96 treatment groups 1, gp96 treatment groups 2, yeast gp96 treatment groups 3, elder brother Worm gp96 treatment groups 1, insect gp96 treatment groups 2, insect gp96 treatment groups 3 and control group (every group 10), are carried out as follows respectively Processing：

(1) placenta gp96 treatment groups 1：Test the 1st day, the gp96 that intraperitoneal injection embodiment 1 is extracted using human placenta Solution；Test the 8th day, the solution for the gp96 that embodiment 1 is extracted using human placenta is injected intraperitoneally again；Test the 22nd My god, the solution for the gp96 that embodiment 1 is extracted using human placenta is injected intraperitoneally again.Every injection gp96 dosage is every time 100μg。

Placenta gp96 treatment groups 2：Carried out according to the operating procedure of placenta gp96 treatment groups 1, only inject gp96 agent by every Measure 100 μ g and replace with 200 μ g.

Placenta gp96 treatment groups 3：Carried out according to the operating procedure of placenta gp96 treatment groups 1, only inject gp96 agent by every Measure 100 μ g and replace with 400 μ g.

(2) yeast gp96 treatment groups 1：Test the 1st day, intraperitoneal injection embodiment 1 is prepared using expressed by Hansenula yeast Gp96 solution；Test the 8th day, the solution for the gp96 that embodiment 1 is extracted using human placenta is injected intraperitoneally again；Experiment the 22 days, the solution for the gp96 that embodiment 1 is extracted using human placenta is injected intraperitoneally again.Every injection gp96 dosage is equal every time For 100 μ g.

Yeast gp96 treatment groups 2：Carried out according to the operating procedure of yeast gp96 treatment groups 1, only inject gp96 agent by every Measure 100 μ g and replace with 200 μ g.

Yeast gp96 treatment groups 3：Carried out according to the operating procedure of yeast gp96 treatment groups 1, only inject gp96 agent by every Measure 100 μ g and replace with 400 μ g.

(3) insect gp96 treatment groups 1：Test the 1st day, intraperitoneal injection embodiment 1 is prepared using insect cell expression Gp96 solution；Test the 8th day, the solution for the gp96 that embodiment 1 is prepared using insect cell expression is injected intraperitoneally again；Experiment 22nd day, the solution for the gp96 that embodiment 1 is prepared using insect cell expression is injected intraperitoneally again.Every injection gp96 agent every time Amount is 100 μ g.

Insect gp96 treatment groups 2：Carried out according to the operating procedure of insect gp96 treatment groups 1, only inject gp96 agent by every Measure 100 μ g and replace with 200 μ g.

Insect gp96 treatment groups 3：Carried out according to the operating procedure of insect gp96 treatment groups 1, only inject gp96 agent by every Measure 100 μ g and replace with 400 μ g.

Control group：The 1st day intraperitoneal injection pH7.4,0.01mol/L PBS is tested, is tested the 8th day, abdominal cavity again Inject pH7.4,0.01mol/L PBS；Test the 22nd day, pH7.4,0.01mol/L PBS bufferings are injected intraperitoneally again Liquid.Per injection dosage is 100 μ L/.

2nd, mouse lymphocyte separation takes into the mouse of the 8th day after step one, obtains mouse spleen lymphocyte (every Mouse takes 3 × 10⁶Individual spleen lymphocyte).

3rd, determinations of the gp96 in activating regulatory T cell middle dosage

1st, the mouse spleen lymphocyte for taking step 2 to obtain (is less than 1 × 10⁶It is individual), 1000rpm centrifugation 10min are collected Mouse spleen lymphocyte.

2nd, complete after step 1, take the mouse spleen lymphocyte, add 100 μ L and contain 5% (mass volume ratio) BSA's PH7.4,0.01mol/L PBS are resuspended, 4 DEG C of closing 20min；Then 1000rpm centrifuges 10min, with 100 μ L of supernatant liquid Precipitation is resuspended, mixed liquor first is obtained.

3rd, complete after step 2, take the mixed liquor first, add FITC-anti-CD3, PE-anti-CD4 and Percp- Cy5.5-anti-CD25,4 DEG C of lucifuges are incubated 20min.

4th, complete after step 3, take the mixed liquor first, add 1mL pH7.4,0.01mol/L PBSs, concussion is mixed Even, 1000rpm centrifugation 10min collect mouse spleen lymphocyte, then buffered with 400 μ L pH7.4,0.01mol/L PBS Liquid is resuspended, and obtains mixed liquor second.

5th, complete after step 4, take the mixed liquor second, add 600 μ L and fix/rupture of membranes agent, 4 DEG C of rupture of membranes 30min (actual behaviour 1h is no more than during work)；Then 1000rpm centrifuges 10min, collects precipitation, obtains precipitation 1.

6th, complete after step 5, take the precipitation 1, add 1 × cleaning solutions of 1mL and be resuspended, 1000rpm centrifugation 10min are obtained Precipitation 2 and supernatant third.Take 100 μ L of supernatant liquid third that precipitation 2 is resuspended, obtain mixed liquor third；APC- is added into mixed liquor third Anti-Foxp3,4 DEG C of lucifuges are incubated 30min.

7th, complete after step 6, take the mixed liquor third, add 1 × cleaning solutions of 1mL and be resuspended, 1000rpm centrifugation 10min, Precipitation is collected, precipitation 3 is obtained.PH7.4,0.01mol/L PBSs of the 400 μ L containing 4% (mass volume ratio) paraformaldehyde is taken to buffer Precipitation 3 is resuspended in liquid, then with CD4 in flow cytometry analysis mouse spleen lymphocyte⁺CD25⁺FOXP3⁺Regulatory T cells Frequency.Experimental result is shown in Table 1,

The influence (average value ± sd values) of the gp96 immunization on rat regulatory T cells of table 1

Note：Compared with control group,^*P<0.01.

Experimental result is shown in Table 1, as a result shows, placenta, yeast and insect in totally 9 groups of gp96 treatment groups mouse spleen lymph CD4 in cell⁺CD25⁺FOXP3⁺The frequency of regulatory T cells is significantly above control group.Therefore, the inferior ferment of human placenta, the Chinese The gp96 albumen that mother's recombination expression and insect cell are recombinantly expressed can significantly induce CD4⁺CD25⁺FOXP3⁺Regulatory T cells Generation.And the gp96 of 200 μ g gp96 and 400 μ g human placentas, Hansenula yeast recombination expression and insect cell recombination expression Albumen can significantly induce higher CD4⁺CD25⁺FOXP3⁺The generation of regulatory T cells.

Applications of the gp96 of embodiment 3 in treatment autoimmune hemolytic anemia

First, anti-Autoerythrocyte antibody titer analysis

1st, the healthy BALB/C mice that the body weight that 120 six week old are immunized in mice group is 18~22g, is randomly divided into 4 groups (every group 30), are handled as follows respectively：

Gp96 treatment groups 1：Test the 1st day, intraperitoneal injection embodiment 1 prepares the gp96 of human placenta extraction solution； Test the 8th day, the solution that embodiment 1 prepares the gp96 of human placenta extraction is injected intraperitoneally again；Test the 22nd day, again abdomen Human placenta prepared by chamber injection embodiment 1 extracts gp96 solution.Per injection dosage is 200 μ g gp96/.

Gp96 treatment groups 2：Carried out according to the operating procedure of gp96 treatment groups 1, injection gp96 albumen is only changed to yeast Bacterium recombinantly expresses gp96 albumen.

Gp96 treatment groups 3：Carried out according to the operating procedure of gp96 treatment groups 1, injection gp96 albumen is only changed to insect Cell recombinantly expresses gp96 albumen.

Control group：The 1st day intraperitoneal injection pH7.4,0.01mol/L PBS is tested, is tested the 8th day, abdominal cavity again Inject pH7.4,0.01mol/L PBS；Test the 22nd day, pH7.4,0.01mol/L PBS bufferings are injected intraperitoneally again Liquid.Per injection dosage is 100 μ L/.

2nd, the foundation of mouse oneself soup-dissolving courage and uprightness anemia model takes SD rat painstaking effort, is washed with pH7.2PBS three times, and pressure product is red Cell adds a certain amount of PBS, is made into 1 × 10⁹Individual/ml red cell suspensions.Respectively at 0,7,14,21 days with the 1 of fresh configuration × 10⁹Mouse 0.2ml/ is immunized only (ip) in individual/ml Rat Erythrocytes suspension.Mouse blood 3 was taken to drip from retroorbital venous clump in 28 days, point Dissipate in PBS, wash 3 times, 2.5% red cell suspension row Coombs experiments are made into PBS, detect anti-Autoerythrocyte antibody drop Degree.

3rd, Coombs is tested in the orifice plate of " V " shape 96, and 25 μ l 0.1%PBSG is added per hole, and the first round is added Coombs serum 25ml, with multi-head dilution device successively two-fold dilution.Last round gives over to cell controls.2.5% is added per hole Mouse red blood cell suspension 25ml.2h is stored at room temperature after mixing, result is read, antibody titer takes natural logrithm.

The gp96 of table 2 is immune to produce antibody Autoerythrocyte antibody titer influence (average value ± sd values) to mouse

Note：Compared with control group,^*P<0.01.

The testing result that mouse produces anti-Autoerythrocyte antibody is shown in Table 2.As a result show, compared with normal group mouse, warp The immune anti-Autoerythrocyte antibody titer of mouse of SD Rat Erythrocytes is significantly raised.The mouse of 3 groups of gp96 treatment groups resists itself Erythrocyte antibody (EA) titre is close to normal group mouse level.The anti-Autoerythrocyte antibody titer of mouse of gp96 treatment groups 3 is slightly below Other 2 groups of gp96 treatment groups, but without significant difference.

4th, peripheral blood cell counts blood routine detection uses conventional method, takes 1 times/day of mouse docking blood sampling, the first day adopts before medication Blood is used as blank control.Red blood cell and leucocyte are counted using improvement Neubauer tallies, and Hayem is used during red blood cell count(RBC) Dilution, the measure of content of hemoglobin uses HiCN conversion fluids, and extinction is determined with TU-1800PC ultraviolet-uisible spectrophotometers Degree；The measure of granulophilocyte ratio is dyestuff with 10g/L brilliant cresyl blue ethanol solutions, is made after blood film, under low power lens Select the uniform portion of red cell distribution to be located at the granulophilocyte counted under oil mirror in 1000 red blood cells, and be scaled percentage Than.

The gp96 of table 3 is immune on mouse blood routine influence (average value ± sd values)

Note：Compared with control group,^*P<0.01.

The blood routine testing result of mouse is shown in Table 3.As a result show, compared with control group, the mouse handled through gp96 is red thin Born of the same parents' quantity, hemoglobin is significantly raised, the reduction of granulophilocyte ratio.Significantly improve the disease of autoimmune hemolytic anemia Shape.The above results show, mouse is immunized with gp96, the disease of autoimmune hemolytic anemia can be effectively treated or mitigate Shape.

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>Applications of the heat shock protein gp96 in treatment autoimmune hemolytic anemia

<130> KHP171110209.2

<160> 6

<170> PatentIn version 3.3

<210> 1

<211> 2352

<212> DNA

<213>Artificial sequence

<400> 1

atggacgatg aagttgatgt ggatggtaca gtagaagagg atctgggtaa aagtagagaa 60

ggatcaagga cggatgatga agtagtacag agagaggaag aagctattca gttggatgga 120

ttaaatgcat cacaaataag agaacttaga gagaagtcgg aaaagtttgc cttccaagcc 180

gaagttaaca gaatgatgaa acttatcatc aattcattgt ataaaaataa agagattttc 240

ctgagagaac tgatttcaaa tgcttctgat gctttagata agataaggct aatatcactg 300

actgatgaaa atgctctttc tggaaatgag gaactaacag tcaaaattaa gtgtgataag 360

gagaagaacc tgctgcatgt cacagacacc ggtgtaggaa tgaccagaga agagttggtt 420

aaaaaccttg gtaccatagc caaatctggg acaagcgagt ttttaaacaa aatgactgaa 480

gcacaggaag atggccagtc gtcttctgaa ttgattggcc agtttggtgt cggtttctat 540

tccgccttcc ttgtagcaga taaggttatt gtcacttcaa aacacaacaa cgatacccag 600

cacatctggg agtctgactc caatgaattt tctgtaattg ctgacccaag aggaaacact 660

ctaggacggg gaacgacaat tacccttgtc ttaaaagaag aagcatctga ttaccttgaa 720

ttggatacaa ttaaaaatct cgtcaaaaaa tattcacagt tcataaactt tcctatttat 780

gtatggagca gcaagactga aactgttgag gagcccatgg aggaagaaga agcagccaaa 840

gaagagaaag aagaatctga tgatgaagct gcagtagagg aagaagaaga agaaaagaaa 900

ccaaagacta aaaaagttga aaaaactgtc tgggactggg aacttatgaa tgatatcaaa 960

ccaatatggc agagaccatc aaaagaagta gaagaagatg aatacaaagc tttctacaaa 1020

tcattttcaa aggaaagtga tgaccccatg gcttatattc actttactgc tgaaggggaa 1080

gttaccttca aatcaatttt atttgtaccc acatctgctc cacgtggtct gtttgacgaa 1140

tatggatcta aaaagagcga ttacattaag ctctatgtgc gccgtgtatt catcccagac 1200

gacttccatg atatgatgcc taaatacctc aattttgtca agggtgtggt ggactcagat 1260

gatctcccct tgaatgtttc ccgcgagact cttcagcaac ataaactgct taaggtgatt 1320

aggaagaagc ttgttcgtaa aacgctggac atgatcaaga agattgctga tgataaatac 1380

aatgatactt tttggaaaga atttggtacc aacatcaagc ttggtgtgat tgaagaccac 1440

tcgaatcgaa cacgtcttgc taaacttctt aggttccagt cttctcatca tccaactgac 1500

attactagcc tagaccagta tgtggaaaga atgaaggaaa aacaagacaa aatctacttc 1560

atggctgggt ccagcagaaa agaggctgaa tcttctccat ttgttgagcg acttctgaaa 1620

aagggctatg aagttattta cctcacagaa cctgtggatg aatactgtat tcaggccctt 1680

cccgaatttg atgggaagag gttccagaat gttgccaagg aaggagtgaa gttcgatgaa 1740

agtgagaaaa ctaaggagag tcgtgaagca gttgagaaag aatttgagcc tctgctgaat 1800

tggatgaaag ataaagccct taaggacaag attgaaaagg ctgtggtgtc tcagcgcctg 1860

acagaatctc cgtgtgcttt ggtggccagc cagtacggat ggtctggcaa catggagaga 1920

atcatgaaag cacaagcgta ccaaacgggc aaggacatct ctacaaatta ctatgcgagt 1980

cagaagaaaa catttgaaat taatcccaga cacccgctga tcagagacat gcttcgacga 2040

attaaggaag atgaagatga taaaacagtt ttggatcttg ctgtggtttt gtttgaaaca 2100

gcaacgcttc ggtcagggta tcttttacca gacactaaag catatggaga tagaatagaa 2160

agaatgcttc gcctcagttt gaacattgac cctgatgcaa aggtggaaga agagcccgaa 2220

gaagaacctg aggagacagc agaagacaca acagaagaca cagagcaaga cgaagatgaa 2280

gaaatggatg tgggaacaga tgaagaagaa gaaacagcaa aggaatctac agctgaaaaa 2340

gatgaattct aa 2352

<210> 2

<211> 783

<212> PRT

<213>Artificial sequence

<400> 2

Met Asp Asp Glu Val Asp Val Asp Gly Thr Val Glu Glu Asp Leu Gly

1 5 10 15

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

20 25 30

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

35 40 45

Leu Arg Glu Lys Ser Glu Lys Phe Ala Phe Gln Ala Glu Val Asn Arg

50 55 60

Met Met Lys Leu Ile Ile Asn Ser Leu Tyr Lys Asn Lys Glu Ile Phe

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Asp Thr Gly Val Gly Met Thr Arg Glu Glu Leu Val Lys Asn Leu Gly

130 135 140

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

145 150 155 160

Ala Gln Glu Asp Gly Gln Ser Ser Ser Glu Leu Ile Gly Gln Phe Gly

165 170 175

Val Gly Phe Tyr Ser Ala Phe Leu Val Ala Asp Lys Val Ile Val Thr

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Leu Asp Thr Ile Lys Asn Leu Val Lys Lys Tyr Ser Gln Phe Ile Asn

245 250 255

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

260 265 270

Met Glu Glu Glu Glu Ala Ala Lys Glu Glu Lys Glu Glu Ser Asp Asp

275 280 285

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

290 295 300

Lys Val Glu Lys Thr Val Trp Asp Trp Glu Leu Met Asn Asp Ile Lys

305 310 315 320

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

325 330 335

Ala Phe Tyr Lys Ser Phe Ser Lys Glu Ser Asp Asp Pro Met Ala Tyr

340 345 350

Ile His Phe Thr Ala Glu Gly Glu Val Thr Phe Lys Ser Ile Leu Phe

355 360 365

Val Pro Thr Ser Ala Pro Arg Gly Leu Phe Asp Glu Tyr Gly Ser Lys

370 375 380

Lys Ser Asp Tyr Ile Lys Leu Tyr Val Arg Arg Val Phe Ile Pro Asp

385 390 395 400

Asp Phe His Asp Met Met Pro Lys Tyr Leu Asn Phe Val Lys Gly Val

405 410 415

Val Asp Ser Asp Asp Leu Pro Leu Asn Val Ser Arg Glu Thr Leu Gln

420 425 430

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

435 440 445

Leu Asp Met Ile Lys Lys Ile Ala Asp Asp Lys Tyr Asn Asp Thr Phe

450 455 460

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

465 470 475 480

Ser Asn Arg Thr Arg Leu Ala Lys Leu Leu Arg Phe Gln Ser Ser His

485 490 495

His Pro Thr Asp Ile Thr Ser Leu Asp Gln Tyr Val Glu Arg Met Lys

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

Cys Ala Leu Val Ala Ser Gln Tyr Gly Trp Ser Gly Asn Met Glu Arg

625 630 635 640

Ile Met Lys Ala Gln Ala Tyr Gln Thr Gly Lys Asp Ile Ser Thr Asn

645 650 655

Tyr Tyr Ala Ser Gln Lys Lys Thr Phe Glu Ile Asn Pro Arg His Pro

660 665 670

Leu Ile Arg Asp Met Leu Arg Arg Ile Lys Glu Asp Glu Asp Asp Lys

675 680 685

Thr Val Leu Asp Leu Ala Val Val Leu Phe Glu Thr Ala Thr Leu Arg

690 695 700

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

705 710 715 720

Arg Met Leu Arg Leu Ser Leu Asn Ile Asp Pro Asp Ala Lys Val Glu

725 730 735

Glu Glu Pro Glu Glu Glu Pro Glu Glu Thr Ala Glu Asp Thr Thr Glu

740 745 750

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

755 760 765

Glu Glu Glu Thr Ala Lys Glu Ser Thr Ala Glu Lys Asp Glu Phe

770 775 780

<210> 3

<211> 28

<212> DNA

<213>Artificial sequence

<400> 3

ccggaattca tggacgatga agttgatg 28

<210> 4

<211> 38

<212> DNA

<213>Artificial sequence

<400> 4

ccgctcgagc tattagaatt catctttttc agctgtag 38

<210> 5

<211> 25

<212> DNA

<213>Artificial sequence

<400> 5

ggaattcatg gacgatgaag ttgat 25

<210> 6

<211> 29

<212> DNA

<213>Artificial sequence

<400> 6

gctctagact attagaattc atctttttc 29

Claims (10)

1. applications of the heat shock protein gp96 in treatment autoimmune hemolytic anemia medicine is prepared, the heat shock protein Its amino acid sequence of gp96 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 the 783rd amino acids sequence；Or

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

2. application of the encoding heat shock proteins gp96 gene in treatment autoimmune hemolytic anemia medicine is prepared, described The nucleotide sequence of encoding heat shock proteins gp96 gene 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 increased 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) nucleotide sequence under strict conditions with sequence hybridization shown in SEQ ID NO.1；

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

3. the biomaterial of the gene containing encoding heat shock proteins gp96 answering in prevention and/or treating cancer medicine is prepared With the biomaterial is carrier, host cell, transgenic cell line, engineering bacteria, insect or yeast；

The nucleotide sequence of the gene of the encoding heat shock proteins gp96 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 increased 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) nucleotide sequence under strict conditions with sequence hybridization shown in SEQ ID NO.1；

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

4. applications of the heat shock protein gp96 in the medicine that induction regulatory T cells are produced is prepared, the heat shock protein Its amino acid sequence of gp96 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 the 783rd amino acids sequence；Or

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

5. application as claimed in claim 4, it is characterised in that the regulatory T cells are CD4⁺CD25⁺FOXP3⁺Regulatory T Cell.

6. applications of the heat shock protein gp96 in preparing reduction to resist the medicine of spontaneous erythrocyte antibody (EA) titre, the heat shock protein Its amino acid sequence of white gp96 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 the 783rd amino acids sequence；Or

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

7. applications of the heat shock protein gp96 in the medicine for improving erythrocyte number and content of hemoglobin is prepared, the heat is stopped Its amino acid sequence of gram albumen gp96 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 the 783rd amino acids sequence；Or

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

8. the medicine with induction regulatory T cells generation function containing heat shock protein gp96, the heat shock protein Its amino acid sequence of gp96 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 the 783rd amino acids sequence；Or

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

9. there is the medicine for reducing anti-Autoerythrocyte antibody titer function, the heat shock protein containing heat shock protein gp96 Its amino acid sequence of white gp96 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 the 783rd amino acids sequence；Or

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

10. there is the medicine for improving erythrocyte number and content of hemoglobin function, the heat containing heat shock protein gp96 Its amino acid sequence of shock protein gp96 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 the 783rd amino acids sequence；Or

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