CN106983855A

CN106983855A - Applications of the heat shock protein gp96 in treatment myasthenia gravis

Info

Publication number: CN106983855A
Application number: CN201710157300.4A
Authority: CN
Inventors: 孟颂东; 李鑫; 鞠莹
Original assignee: Beijing Thermal Biological Technology Co Ltd
Current assignee: Foshan repast Biotechnology Co., Ltd.
Priority date: 2017-03-16
Filing date: 2017-03-16
Publication date: 2017-07-28

Abstract

The present invention relates to biomedicine field, applications of the heat shock protein gp96 in treatment myasthenia gravis is specifically disclosed.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, reduces acetylcholinergic receptor AchR antibody titers；Reduce myasthenia gravis clinical score.Available for the medicine for preparing treatment myasthenia gravis, have broad application prospects.

Description

Applications of the heat shock protein gp96 in treatment myasthenia gravis

Technical field

The present invention relates to biomedicine field, specifically, it is related to heat shock protein gp96 in treatment myasthenia gravis In application.

Background technology

Myasthenia gravis (myasthenia gravis, MG) is that one kind is mainly involved on neuromuscular junction postsynaptic membrane The autoimmune disease of acetylcholinergic receptor (acetylcholine receptor, AchR).Clinic is mainly shown as part Or whole body skeletal muscle weakness and fatiguability, exacerbation of symptoms after activity, through rest and anticholinesterase (cholinesterase Inhibitors, ChEI) treatment after symptom mitigation.Illness rate is 77~15,0/1,000,000, and annual morbidity is 4~1,1/1,000,000.Female Property illness rate be more than male, about 3:2, each age group has a morbidity, and children 1~5 years old are in the majority.The pathogenic factor of myasthenia gravis point Two major classes a, class is inborn genetic, and Ji Shaojian is unrelated with autoimmunity；Equations of The Second Kind is autoimmune disease, most common. Pathogenic factor is still not clear, generally believe with infecting, medicine, environmental factor it is relevant.Have 65% in Patients With Myasthenia Gravis simultaneously ~80% has a thymic hyperplasia, and 10%~20% occurs together thymoma.

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.At present Applications of the heat shock protein gp96 in treatment myasthenia gravis is not had been reported that.

The content of the invention

In order to solve problems of the prior art, it is an object of the invention to provide heat shock protein gp96 in treatment weight New opplication in disease gravis.

In order to realize the object of the invention, technical scheme is as follows：

Present invention firstly provides applications of the heat shock protein gp96 in the product for preparing treatment myasthenia gravis, institute Heat shock protein gp96 is stated for a1) or a2) or a3)：

A1) protein of the amino acid sequence as shown in SEQ ID No.2；

A2) in a1) N-terminal or/and the obtained fused protein of C-terminal connection label；

A3) by a1) amino acid sequence pass through one or several amino acid residues substitution and/or missing and/or addition The obtained protein with identical function.

Further, 1) or 2) or 3) acquisition pattern of the heat shock protein gp96 is：

1) cell and tissue in it is native purified；

2) yeast expressed recombinant protein is utilized；

3) insect cell expression recombinant protein is utilized.

Further, the function of the product is following A1), A2) and at least one of A3)：

A1 the generation of regulatory T cells) is induced；

A2 acetylcholinergic receptor AchR antibody titers) are reduced；

A3 myasthenia gravis clinical score) is reduced.

Wherein, the regulatory T cells are CD4⁺CD25⁺FOXP3⁺Regulatory T cells.

Preferably, the product is injection.

Present invention also offers heat shock protein gp96 encoding gene in the product for preparing treatment myasthenia gravis Application, the encoding gene be d1) d2) or d3)：

D1) nucleotide sequence is the DNA molecular shown in SEQ ID No.1；

D2 the nucleotide sequence) and d1) limited has 75% or more than 75% homogeneity, and encodes the heat shock protein Gp96 DNA molecular；

D3) under strict conditions with d1) nucleotide sequence hybridization that limits, and the coding heat shock protein gp96 DNA molecular.

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.

Present invention also offers the biomaterial containing heat shock protein GP96 encoding genes, the biomaterial is load Body, host cell, transgenic cell line, engineering bacteria, insect or yeast.

Further, application of the above-mentioned biomaterial in treatment autoimmune hemolytic anemia medicine is prepared is fallen within Protection scope of the present invention.

The present invention further provides a kind of product for treating myasthenia gravis, its active component is heat shock protein Gp96, the heat shock protein gp96 be a1) a2) or a3)：

A1) protein of the amino acid sequence as shown in SEQ ID No.2；

The function of the product is following A1), A2) and at least one of A3)：

A1 the generation of regulatory T cells) is induced；

A2 acetylcholinergic receptor AchR antibody titers) are reduced；

A3) myasthenia gravis clinical score is reduced, and alleviates the symptom of myasthenia gravis, treats myasthenia gravis.

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

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, cultivated, purifying.Using ferment Mother, mammalian cell, 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.

Plasmid pFastBac1 EcoR I and Xba I 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 injection dosage of the product is that mouse immune single dose is 100-400 μ g/18-22g body weight, people's immunizing dose For 100-1500 μ g/ person-time.

The mammal can be mouse or people.The mouse concretely Kunming mouse.

The present invention relates to raw material or reagent be ordinary commercial products, the operation being related to is unless otherwise specified This area routine operation.

On the basis of common sense in the field is met, above-mentioned each optimum condition can be mutually combined, obtain specific embodiment party Formula.

The beneficial effects of the present invention are：

Present invention finds heat shock protein gp96 have the generations of induction regulatory T cells, reduction AchR antibody titers, Reduce the function of myasthenia gravis clinical score.Therefore, heat shock protein gp96 has to the symptom for treating myasthenia gravis Important application value.

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 preferred embodiment of the present invention is described in detail below in conjunction with embodiment.It will be appreciated that following real Providing merely to play the purpose of explanation for example is applied, is not used to limit the scope of the present invention.The skill of this area Art personnel can carry out various modifications and replacement in the case of without departing substantially from spirit of the invention and spirit to the present invention.

Experimental method in following embodiments, is conventional method unless otherwise specified.

Material, reagent used etc., unless otherwise specified, are commercially obtained in following embodiments.

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

Kunming mouse, female, 22~25g of body weight, purchased from Nanjing model animal research institute.Obtain mouse spleen lymph thin The specific method of born of the same parents 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 acquisition of embodiment 1, heat shock protein gp96

(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).

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

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 5mLSD 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 shows that the gp96 purity of protein in the eluent after ion-exchange chromatography 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 2. 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 framework.

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 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 chain DNA molecule shown in sequence 1 in sequence table. Recombinant plasmid pFastBac 1-gp96 expression restructuring heat shock protein gp96 (hereinafter referred to as gp96) gp96 amino acid sequence is such as In sequence table shown in sequence 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 solution for the gp96 that 5. step is obtained carries out SDS-PAGE electrophoretic analysis, and experimental result is shown in left figure (swimming lane in Fig. 3 1 is HMW standard protein, and swimming lane 2 is gp96, and arrow is gp96).The restructuring heat shock protein that 5. step is obtained Gp96 solution carries out Western blot, and (using gp96 monoclonal antibodies as primary antibody, the goat of HRPO mark resists The monoclonal antibody of rat is used as secondary antibody), experimental result is shown in right figure in Fig. 3.As a result show, gp96 solution shows single molecule Band is measured, corresponding molecular weight is with being expected unanimously.

The determination of embodiment 2, gp96 in activating regulatory T cell middle dosage

First, mice group is immunized

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

Placenta gp96 treatment groups 1：Test the 1st day, the gp96's that intraperitoneal injection embodiment 1 is extracted using human placenta is molten Liquid；Test the 8th day, the solution for the gp96 that embodiment 1 is extracted using human placenta is injected intraperitoneally again；Test the 22nd day, then The solution for the gp96 that secondary intraperitoneal injection embodiment 1 is extracted using human placenta.Per injection dosage is 100 μ ggp96/.

Placenta gp96 treatment groups 2：Carried out according to the operating procedure of placenta gp96 treatment groups 1, only by the μ g of injection dosage 100 Gp96/ only replaces with 200 μ g gp96/ only.

Placenta gp96 treatment groups 3：Carried out according to the operating procedure of placenta gp96 treatment groups 1, only by the μ g of injection dosage 100 Gp96/ only replaces with 400 μ g gp96/ only.

Yeast gp96 treatment groups 1：Test the 1st day, the gp96's that intraperitoneal injection embodiment 1 is prepared using expressed by Hansenula yeast 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 day, Intraperitoneal injection embodiment 1 utilizes the gp96 of human placenta extraction solution again.Per injection dosage is 100 μ ggp96/ Only.

Yeast gp96 treatment groups 2：Carried out according to the operating procedure of yeast gp96 treatment groups 1, only by the μ g of injection dosage 100 Gp96/ only replaces with 200 μ g gp96/ only.

Yeast gp96 treatment groups 3：Carried out according to the operating procedure of yeast gp96 treatment groups 1, only by the μ g of injection dosage 100 Gp96/ only replaces with 400 μ g gp96/ only.

Insect gp96 treatment groups 1：Test the 1st day, the gp96's that intraperitoneal injection embodiment 1 is prepared using insect cell expression Solution；Test the 8th day, the solution for the gp96 that embodiment 1 is prepared using insect cell expression is injected intraperitoneally again；Test the 22nd My god, the solution for the gp96 that embodiment 1 is prepared using insect cell expression is injected intraperitoneally again.Per injection dosage is 100 μ Ggp96/ is only.

Insect gp96 treatment groups 2：Carried out according to the operating procedure of insect gp96 treatment groups 1, only by the μ g of injection dosage 100 Gp96/ only replaces with 200 μ g gp96/ only.

Insect gp96 treatment groups 3：Carried out according to the operating procedure of insect gp96 treatment groups 1, only by the μ g of injection dosage 100 Gp96/ only replaces with 400 μ g gp96/ only.

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 is separated

The mouse of the 8th day after step one is taken into, obtaining 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.

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.

The application of embodiment 3, gp96 in treatment myasthenia gravis

1st, the foundation of Mouse Model of Experimental Autoimmune Myasthenia Gravis

EAMG models AChR and freund adjuvant 1:Mouse is immunized after 2 mixings, first sensitization takes the μ g of AChR 15 that 200 μ are made L antigen emulsions, in back, limbs, many places intradermal vaccination of tail base portion.The μ L of antigen emulsion 100 containing 15 μ g AChR are used after 4 weeks again Secondary inoculation.Vehicle control group is immune with equivalent freund adjuvant.7~14d is judged model after final immunization.

Criterion：

(1) clinical manifestation：There are the different journeys such as withered hair, the grand back of the body, appetite decline, activity reduction and cyllopodia in EAMG mouse The myasthenia performance of degree；

(2) swimming test：EAMG mouse swimming times are significantly shorter than vehicle control group.

2nd, the clinical efficacy of gp96 protein for treatment experimental myasthenia gravis mouse

The myasthenia gravis Kunming mouse that 120 body weight are 18~22g, is randomly divided into 4 groups (every group 30), enters respectively The following processing of row：

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.

1) body weight is detected：Daily mouse of weighing from the gp96 protein immunizations treatment same day.

2) clinical score：Carry out the clinical assessment of EAMG seriousness to mouse by two observers using blind.0 point:Flesh Power is normal；1 point:Movable slight reduction, grasps powerless or croons, fatiguability；2 points:Activity is significantly reduced, and body weight substantially mitigates, In the back of a bow, a low volt during rest, preceding Clinodactyly, shake all over the body；3 points:The serious whole body is powerless, and without crooning, no grasp motion is in Moribund condition or death.Symptom occupy intermediate score respectively as 0.5,1.5,2.5 points.Carried out respectively at the 4th week after treatment clinical Scoring.

Gp96 is immune to be shown in Table 2 to the testing result that mouse weight influences.As a result show, compared with control group mice, warp The body weight that mouse is immunized in gp96 is significantly raised, and 3 groups of gp96 treatment groups are without significant difference.

The immune influences (average value ± sd values) to mouse weight of the gp96 of table 2

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

Gp96 is immune to be shown in Table 3 to the influence testing result that mice clinical scores.As a result show, compared with control group mice, Significantly reduced through the gp96 clinical scores that mouse is immunized, 3 groups of gp96 treatment groups are without significant difference.

The immune influences (average value ± sd values) to Mice Mice clinical score of the gp96 of table 3

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

The above results show, mouse is immunized with gp96, the symptom of myasthenia gravis can be effectively treated or mitigate.

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 table

<110>Beijing Re Xiu Bioisystech Co., Ltd

<120>Applications of the heat shock protein gp96 in treatment myasthenia gravis

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<170> PatentIn version 3.3

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tccgccttcc ttgtagcaga taaggttatt gtcacttcaa aacacaacaa cgatacccag 600

cacatctggg agtctgactc caatgaattt tctgtaattg ctgacccaag aggaaacact 660

ctaggacggg gaacgacaat tacccttgtc ttaaaagaag aagcatctga ttaccttgaa 720

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gaagagaaag aagaatctga tgatgaagct gcagtagagg aagaagaaga agaaaagaaa 900

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attactagcc tagaccagta tgtggaaaga atgaaggaaa aacaagacaa aatctacttc 1560

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gaagaacctg aggagacagc agaagacaca acagaagaca cagagcaaga cgaagatgaa 2280

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

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

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

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

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

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

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

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

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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

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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

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

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

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

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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

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

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

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

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

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

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

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

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

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

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

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

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

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ccgctcgagc tattagaatt catctttttc agctgtag 38

Claims

1. applications of the heat shock protein gp96 in the product for preparing treatment myasthenia gravis, it is characterised in that the heat is stopped Gram albumen gp96 is a1) or a2) or a3)：

A1) protein of the amino acid sequence as shown in SEQ ID No.2；

A3) by a1) amino acid sequence by one or several amino acid residues substitution and/or missing and/or addition obtain The protein with identical function.

2. application as claimed in claim 1, it is characterised in that the acquisition pattern of the heat shock protein gp96 1) or 2) for or 3)：

1) cell and tissue in it is native purified；

2) yeast expressed recombinant protein is utilized；

3) insect cell expression recombinant protein is utilized.

3. application as claimed in claim 1, it is characterised in that the function of the product is following A1), A2) and A3) in extremely Few one kind：

A1 the generation of regulatory T cells) is induced；

A2 acetylcholinergic receptor AchR antibody titers) are reduced；

A3 myasthenia gravis clinical score) is reduced.

4. application as claimed in claim 3, it is characterised in that the regulatory T cells are CD4⁺CD25⁺FOXP3⁺Regulatory T Cell.

5. the application as described in any one of Claims 1 to 4, it is characterised in that the product is injection.

6. application of the heat shock protein gp96 encoding gene in the product for preparing treatment myasthenia gravis, its feature exists In, the encoding gene be d1) d2) or d3)：

D1) nucleotide sequence is the DNA molecular shown in SEQ ID No.1；

D3) under strict conditions with d1) nucleotide sequence hybridization that limits, and DNA points of the coding heat shock protein gp96 Son.

7. a kind of product for treating myasthenia gravis, it is characterised in that its active component is heat shock protein gp96, the heat Shock protein gp96 be a1) a2) or a3)：

A1) protein of the amino acid sequence as shown in SEQ ID No.2；

8. product as claimed in claim 7, it is characterised in that the function of the product is following A1), A2) and A3) in extremely Few one kind：

A1 the generation of regulatory T cells) is induced；

A2 acetylcholinergic receptor AchR antibody titers) are reduced；

9. product as claimed in claim 8, it is characterised in that the regulatory T cells are CD4⁺CD25⁺FOXP3⁺Regulatory T Cell.

10. the product as described in any one of claim 7~9, it is characterised in that the acquisition pattern of the heat shock protein gp96 For：

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

Or (2) import the nucleic acid molecules for encoding the heat shock protein gp96 in acceptor, cultivate, purifying.