CN102462841A

CN102462841A - Method for promoting immunological activity of gp96 protein and application thereof

Info

Publication number: CN102462841A
Application number: CN2010105399522A
Authority: CN
Inventors: 孟颂东; 李星辉; 刘振; 闫晓丽; 王赛锋; 李扬; 邱立鹏; 陈立钊; 胡坤
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2010-11-09
Filing date: 2010-11-09
Publication date: 2012-05-23

Abstract

The invention discloses a method for promoting immunological activity of gp96 protein and application thereof. The invention provides application of immunomodulators in the improvement of the immunological activities of gp96 protein-related maters; the immunomodulators are at least one of cyclophosphane, anti-CD25 antibody and an interfering RNA (Ribonucleic Acid) of Foxp3; the gp96 protein-related maters are the gp96 protein shown by a sequence 1 of a sequence table, N355 fragments or gp96-protein-contained extracts; and the interfering RNA of the Foxp3 is shown by a sequence 5 of the sequence table. The invention further provides various vaccines which comprise the immunomodulators and the shown gp96 protein-related maters. The invention has a great value to the treatment of various cancers, such as hepatitis B, melanoma, and the like.

Description

Proteic immunocompetent method of a kind of promotion gp96 and application thereof

Technical field

The present invention relates to proteic immunocompetent method of a kind of promotion gp96 and application thereof.

Background technology

Heat shock protein (heat shock proteins HSPs) has unique immunologic function, and its amynologic mechanism comprises two aspects: the one, and HSPs participates in the T cellular antigens and presents; The 2nd, HSPs effectively excites the natural immunity.

Heat shock protein is present in cytoplasm and the endoplasmic reticulum, by a plurality of member compositions, and like HSP60, HSP70, HSP90 and gp96 etc.Heat shock protein plays a significant role in protein folding and transportation as molecular chaperones on the one hand; The antigen polypeptide that can combine 5-25mer in the cell on the other hand gets into cell and bonded epitope is through antigen-presenting cell (APC) surface C D91 molecule and passs MHC I class and II quasi-molecule, thus startup specific T-cells immunne response.

Heat shock protein itself is unique natural adjuvant that derives from mammalian cell of finding up to now; It can act on other immunocytes such as APC and T cell; Promote DC ripe; Irritation cell produces immune factor IL-6, IL-12, TNF etc., thus the enhancing body nonspecific immune reaction.There are some researches show gp96 for Tol l appearance receptor (Toll-likereceptors among the APC; TLRs) the performance immunologic function plays crucial effects; The TLRs forfeiture causes autarcetic function in the DC cell of gp96 gene delection, and the ability that causes the transgenic mice opposing to be infected obviously reduces.

The heat-shock protein-polypeptide compound therapeutic vaccine has been used for clinical trial.Because tumor cell is a kind of cell of variation, therefore give non-specific or specific immune factor or cell exogenously, strengthen or enlarge the immunity of body, can reach the purpose of removing interior tumor cell.Based on this principle, multiple nonspecific immunity Therapeutic Method like the methods such as infusion that give exogenous interleukin-22, interferon, LAK cell all in clinical use, but curative effect and unsatisfactory, one of the main reasons is to exist tumour immunity to escape.Tumor cell is usually lost the expression of tumor antigen or is changed its antigen presentation spectrum, makes the tumor vaccine that designs to known cancer antigen can not effectively excite the immunoreation to tumor cell.Research shows that tumor has the individual specificity, and the specificity of tumor not only shows between the tumor type, also shows between the Different Individual, and promptly in the same kind tumor patient, also there are marked difference in the oncobiology of Different Individual and immunological characteristic.Therefore various antigens in the natural combination tumor cell of HSP are developed to the individuation knubble vaccine with heat shock protein, can fully take into account the difference between individual patients, to reach optimum therapeuticing effect.Carrying out individualized treatment also is one of inevitable direction of current medical science and materia medica development.

Over nearly 6 years; HSP gp96-polypeptide complex is used for clinical trial to carry out in the hospital or the Cancer center of states such as the U.S., Britain, Germany, Italy and Russia in succession; Be mainly used in gastric cancer, carcinoma of prostate, renal carcinoma, the treatment of tumor such as melanoma and infectious diseases such as HIV, genital herpes.The clinical III phase has been accomplished in the treatment of renal carcinoma and malignant melanoma at present, and rectal cancer and lymphoma have been accomplished the clinical II phase.This Therapeutic Method has been ratified listing in Russia.See that from existing clinical data the gp96-antigenic compound is unquestionable as autovaccine treatment tumor efficiency, it is clinical that renal cell carcinoma and melanoma have been accomplished the III phase; Patient's quantity of receiving treatment is big; And have statistical significance, and M1a and M1b phase patient accept the immunity more than 10 times, and average survival period prolongs 18.4 months (31.2vs 12.8 months than the treatment that the doctor selects; P=0.03, dangerous than HR=0.45).The III phase clinical data that does not shift the patients with renal cell carcinoma treatment is found that medium risk patient (n=362) (comprises the I/II phase; The T1/2/3 of III phase) accepts the treatment of gp96 autovaccine; Recurrence time prolongs 45% (P＜0.01; Dangerous than HR=0.55), to compare recurrence time with contrast and prolong about 1.8 years, patient's survival period of receiving treatment simultaneously prolongs.

Regulatory T cells (Treg) has suppressed activation, increment, differentiation and the effector function of a lot of cell types, comprises CD4 ⁺, CD8 ⁺T cell, B cell, NK cell, NKT cell and DC cell.Treg is one type of CD4 ⁺CD25 ⁺The T cell.Treg is divided into two main monoids: thymus-derived natural Treg (nTreg) and the inductive Treg of periphery (iTreg).They exercise the inhibition regulatory function through different approach, and nTreg mainly works through the direct contact of cell-cell, and iTreg is through the TGF β 1 or the IL-10/TGF β 1 performance inhibit feature of high concentration.Remove the Treg cell and cause the spontaneous generation autoimmune disease of mice.But also evidence suggests, remove and perhaps reduce of the immunoreation of Treg cell ability enhancing body to diseases such as virus, tumors.

Summary of the invention

The purpose of this invention is to provide proteic immunocompetent method of a kind of promotion gp96 and application thereof.

The invention provides the application of immunomodulator in the immunocompetence that improves gp96 albumen related substances; The sequence 1 of the gp96 albumen shown in the sequence 1 that said gp96 albumen related substances is a sequence table, sequence table is from the N355 fragment shown in amino terminal the 1st to the 355 amino acids residue or contain the proteic extract of gp96.

The present invention also protects a kind of vaccine adjuvant, for (a) as follows or (b) or (c): the vaccine adjuvant of (a) being made up of immunomodulator and said N355 fragment; (b) vaccine adjuvant of forming by immunomodulator and said gp96 albumen; (c) vaccine adjuvant of forming by immunomodulator and the proteic extract of the said gp96 of containing.

The present invention also protects a kind of Hepatitis B virus vaccine and/or suppresses the vaccine of hepatitis B virus, is made up of said gp96 albumen related substances, hepatitis B surface antigen (HBsAg), hepatitis B core albumen (HBcAg) and immunomodulator.

The present invention also protects a kind of Hepatitis B virus vaccine and/or suppresses the vaccine of hepatitis B virus, is made up of gp96 albumen related substances, hepatitis B surface antigen epi-position 362-371, hepatitis B core albumen epi-position 87-95, hepatitis B polymerase epi-position 140-148 and immunomodulator; Said hepatitis B surface antigen epi-position 362-371 is shown in the sequence 6 of sequence table; Said hepatitis B core albumen epi-position 87-95 is shown in the sequence 7 of sequence table; Said hepatitis B polymerase epi-position 140-148 is shown in the sequence 8 of sequence table.

The present invention also protects a kind of melanoma vaccines, is made up of said gp96 albumen related substances and immunomodulator.

More than at least a in the RNA interfering of arbitrary said immunomodulator antibody that is cyclophosphamide, anti-CD25 and Foxp3.The RNA interfering of said Foxp3 is shown in the sequence 5 of sequence table.

More than the proteic extract of arbitrary said gp96 of containing be that homogenate with exsomatize animal tissue or tumor tissue in vitro or stripped tumor cell carries out the ConA-Sepharose chromatography successively and HiTrap-Q Sepharose ion-exchange chromatography obtains; Said stripped animal tissue is isolated mouse liver, isolated pig liver or stripped people's Placenta Hominis; Said tumor tissues is breast carcinoma, cerebral glioma, renal carcinoma, intestinal cancer, hepatocarcinoma or gastric cancer; Said tumor cell is human liver cancer cell HepG2 or MC B16; Said ConA-Sepharose chromatography comprises the steps: that (1) is splined on chromatographic column with said homogenate, and flow velocity is 0.25ml/min; (2) with the chromatographic column of the eluent first washing step (1) of 10 times of chromatographic column column volumes, flow velocity is 0.5ml/min; The method for preparing of said eluent first is: in NaCl concentration is the PBS of 200mM pH7.5, adding Phenylmethanesulfonyl fluoride (PMSF) to final concentration is 1mM; (3) with the chromatographic column of the eluent second washing step (2) of 3 times of chromatographic column column volumes, flow velocity is 0.25ml/min, collects eluent, is eluent third; The method for preparing of said eluent second is: in NaCl concentration is the PBS of 200mMpH7.5, adding α-D-Glucopyranose. to final concentration is 10g/mL, and adding Phenylmethanesulfonyl fluoride to final concentration is 1mM; Said HiTrap-Q Sepharose ion-exchange chromatography comprises the steps: that (a) is splined on chromatographic column with said eluent third, and flow velocity is 1ml/min; (b) use the chromatographic column of 5ml NaCl concentration as the PBS washing step (a) of 200mM pH7.5, flow velocity is 1ml/min; (c) use the chromatographic column of 10ml NaCl concentration as the PBS washing step (b) of 300mM pH7.5, flow velocity is 1ml/min; (d) use the chromatographic column of 3ml NaCl concentration as the PBS washing step (c) of 600mM pH7.5, flow velocity is 1ml/min, and the eluent that obtains is and contains the proteic extract of gp96.

More than arbitrary said vaccine each component all separately the packing.

But gp96 albumen is natural extract both, escherichia coli also capable of using or yeast expression.

Gp96 albumen, N355 fragment and contain the proteic extract of gp96 and hepatitis B antigen epi-position immune mouse can obviously improve the quantity of Treg; The function of Treg also obviously strengthens simultaneously; Comprise the inhibition of CD4+ and CD8+T cell growth and the inhibit feature of secretion of gamma-IFN are obviously strengthened; Treg and CD8+T, hepatitis B antigen specific T cell, secretion of gamma-IFN cell are negative correlation, explain that Treg has inhibitory action to the T cell.Gp96 albumen activates CTL through antigen presentation on the one hand, can activate Treg again simultaneously, and Treg suppresses the CTL activity conversely again, so gp96 has the two-ways regulation function to hepatitis B T cellular immunization.In above-mentioned immunity, add immunomodulator; Can remove the Treg that gp96 induces generation; The T cell quantity is significantly improved; Comprise that CD8+T, hepatitis B antigen specific T cell, secretion of gamma-IFN cell quantity have obvious increase (P＜0.01), explain that immunomodulator can significantly strengthen the hepatitis B specific T-cells immunne response that gp96 causes.Mouse test shows that Hepatitis B virus vaccine provided by the invention can effectively activate the HBV specific T-cells, and the HBV S antigen reduction in the serum is more than 50% after 8 weeks for mouse immune, and virus sweep is more than 90% in the liver cell.More than research shows that vaccine has good anti-HBV active.Suppress Treg for antibody or the downward modulation Foxp3 etc. of the cyclophosphamide of injected in mice low dosage, anti-CD25, then vaccine can significantly improve (P＜0.01) to the inhibition of hepatitis B virus duplication.With gp96 and mouse melanin tumor antigen assembled in vitro, preparation gp96 tumor vaccine.Through the mouse immuning test of various dose, what the gp96 vaccine of discovery high dose caused is lower than low dose group on the contrary to melanomatous immunological rejection.It is machine-processed that the gp96 immunoregulation has been illustrated in research, activates the CTL killing tumor cell through antigen presentation on the one hand, can activate Treg simultaneously again and suppress the CTL activity, reduces the immunologic tumor rejection reaction.In gp96 tumor vaccine immunization experiment, inject the cyclophosphamide of low dosage simultaneously, remove gp96 and induce the Treg of generation, then significantly improve the activity of gp96 tumor vaccine.In the above-mentioned experiment, use the antibody of anti-CD25 and the RNA interfering of Foxp3 (siRNA) can reduce the quantity of Treg equally, reach the active purpose of enhancing gp96 vaccine immunity.

Description of drawings

Fig. 1 is the segmental evaluation collection of illustrative plates of N355.

Fig. 2 is the proteic evaluation collection of illustrative plates of rgp96; 1: molecular weight standard; 2: the fermentation liquid of step (two); 3: the eluent behind the affinity chromatograph; 4: the eluent behind the ion-exchange chromatography; The proteic western blot of 5:rgp96.

Fig. 3 is the evaluation collection of illustrative plates of gp96 extract.

Fig. 4 is the immunocompetence relatively (fluidic cell testing result) of rgp96 albumen and gp96 extract among the embodiment 2; A is HBc87-95, and B is HBc87-95+gp96, and C is HBc87-95+rgp96, and D is HBc87-95+IFA.

Fig. 5 is the result that the T cytoactive detects and Tetramer detects among the embodiment 4; A is flow cytometer testing result (the hepatitis B virus specific CTL reaction that immunity causes); B accounts for the ratio (immunity cause t cell responses) of splenocyte for the CD8+T cell, and C accounts for the ratio (the hepatitis B specific T-cells reaction that immunity causes) of CD8+T cell for the Tetramer positive cell; 1 is first group, and 2 is second group, and 3 is the 3rd group, and 4 is the 4th group, and 5 is the 5th group, and 6 is the 6th group.

Fig. 6 is the ELISPOT testing result of complex immunity.

Fig. 7 is a Treg testing result among the embodiment 4; A is flow cytometer testing result (the hepatitis B virus specific CTL reaction that immunity causes), and B accounts for the ratio of CD4+T cell for the Treg positive cell; 1 is first group, and 2 is second group, and 3 is the 3rd group, and 4 is the 4th group, and 5 is the 5th group, and 6 is the 6th group.

Fig. 8 is that immunity is to the active promotion of mice Treg; A:FACS detects Treg to CD4 and cd8 t cell inhibition of proliferation; B: first group, second group and the 5th group of immunity are to CD4 and the effect of cd8 t cell inhibition of proliferation; C: the 3rd group, the 4th group and the 6th group of immunity are to CD4 and the effect of cd8 t cell inhibition of proliferation; D: first group, second group and the 5th group of immunity are to the cell inhibiting effect of secretion of gamma-IFN; E: the 3rd group, the 4th group and the 6th group of immunity are to the cell inhibiting effect of secretion of gamma-IFN; 1 is first group, and 2 is second group, and 3 is the 3rd group, and 4 is the 4th group, and 5 is the 5th group, and 6 is the 6th group.

Fig. 9 is the correlation analysis of Treg and T cell in the immune mouse; A:Treg and total CD8+ cell; B:Treg and hepatitis B specific T-cells; The T cell of C:Treg and secretion of gamma-IFN.

Figure 10 is 1 result of the step 1 of embodiment 5.

Figure 11 is 2 result of the step 1 of embodiment 5.

Figure 12 is the result of the step 2 of embodiment 5.

Figure 13 is the result of the step 3 of embodiment 5.

The specific embodiment

Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment like no specified otherwise, is conventional method.Used test material among the following embodiment like no specified otherwise, is to buy from routine biochemistry reagent shop and obtains.BALB/c mouse: available from Beijing dimension tonneau China company; HBV transgenic mouse: available from the 458th hospital of Guangzhou PLA.Quantitative test in following examples all is provided with repeated experiments three times, results averaged.All adopt subcutaneous injection to carry out immunity (in the practical application, also can select other immunization ways at random for use) in following examples like intradermal injection or intramuscular injection.Hepatitis B surface antigen (HBsAg): available from Beijing Tiantan Bio-pharmaceuticals joint-stock company.Hepatitis B core albumen (HBcAg): available from abcam company, production code member ab49013.The antibody of anti-CD4: available from BioLegend, catalog number 100407.The antibody of anti-CD8: available from BioLegend, catalog number 100705.The antibody of anti-CD25: available from BioLegend, production code member 101906.The antibody of anti-FoxP3 is available from eBioscience, catalog number 77-5775.HBc87-95: be hepatitis B core albumen epi-position 87-95, sequence is SYVNTNMGL, and is synthetic by Shanghai gill biochemical corp.HBsAg362-371: be hepatitis B surface antigen epi-position 362-371, sequence is WYWGPSLYSI, and is synthetic by Shanghai gill biochemical corp.Hepatitis B polymerase epi-position 140-148, sequence is HYFQTRHYL, and is synthetic by Shanghai gill biochemical corp.Transcription factor FoxP3 is the molecular marker of special Treg cell, to CD4 ⁺CD25 ⁺Treg cells whose development and functionating have important effect; The RNA interfering of FoxP3: 5 '-GGACACUCAAUGAAAUCUA-3 ', synthetic by the sharp rich company in Guangzhou.Gp96 albumen (HUMAN HEAT SHOCK PROTEINS Hhsp HSP70; GENBANK ACCESSION NO.AY040226) aminoacid sequence is shown in the sequence 1 of sequence table, and its coded sequence is shown in the sequence 2 of sequence table.Gp96 protein fragments (N355 fragment) is the polypeptide that the sequence 1 of sequence table is formed from amino terminal the 1st to 355 amino acids residue, its coded sequence like the sequence 2 of sequence table from shown in 5 ' terminal the 1st to 1065 nucleotide.

The preparation of embodiment 1, N355 fragment, gp96 albumen and the proteic extract of gp96

One, the segmental preparation of N355

Utilize escherichia coli expression N355 fragment, concrete grammar is following:

1, the following N355 primer of design is right, by the handsome company in Shanghai synthetic primer:

Forward primer: 5 '-CGCGGATCCGACGATGAAGTTGATGTGGAT-3 ';

Downstream primer: 5 '-CCGCTCGAGTTAAGTAAAGTGAATATAAGCCATG-3 '.

2, extract the mRNA of human liver cancer cell HepG2 (available from ATCC, production number HB-8065), cDNA is synthesized in reverse transcription.

3, the cDNA with step 2 is a template, to carrying out pcr amplification, obtains pcr amplification product (containing the segmental coded sequence of N355) with the N355 primer.

4,, reclaim the enzyme action product with restricted enzyme BarnHI and Xho I double digestion pcr amplification product.

5,, reclaim carrier framework with restricted enzyme BarnHI and Xho I double digestion pGEX-6P-1 plasmid (available from GE company, production code member 27-4597-01).

6, the enzyme action product of step 4 and the carrier framework of step 5 are connected, obtain connecting product.

7, will connect product transformed into escherichia coli DH5 α (available from sky root biochemical technology company, production number CB101-03) competence, picking list bacterium colony carries out enzyme action to be identified, enzyme action identifies that male bacterium colony extracts the plasmid evaluation of checking order.Sequencing result shows, has obtained recombiant plasmid pGEX-N355 (skeleton carrier is pGEX-6P-1, between BarnHI and Xho I restriction enzyme site, has inserted the segmental coded sequence of N355).

8, with recombiant plasmid pGEX-N355 transformed into escherichia coli BL21 (DE3) (available from sky root biochemical technology company; Production number CB105-02) competent cell; Picking list bacterium colony inserts 2 * YT culture medium (the tryptone 16g that contains the 100mg/mL ampicillin from the flat board; Yeast extract 10g, sodium chloride 5g adds water and is settled to 1000mL) activation; Get activating solution (bacterium liquid) and insert 2 * YT culture medium, 37 ℃ are cultured to OD ₆₀₀Value adds IPTG (making its final concentration is 1mmol/L) then for 0.6-1.0, induces 4h for 37 ℃, collects thalline.

9, thalline is with PBS buffer (140mmol/L NaCl, 2.7mmol/L KCl, 10mmol/L Na ₂HPO ₄1.8mmol/LKH ₂PO ₄PH 7.3) resuspended, ultrasonication under the condition of ice bath (200W, broken 4s stops 6s, 99 3 circulations), 4 ℃ of 12000 centrifugal 20min of commentaries on classics/min collects supernatant then.

10, supernatant is carried out affinity chromatograph for 4 ℃; Carrier is that Glutathione-Sepharose 4B is (available from GE company; Production code member 17-5132-01), adopt reduced glutathion elution buffer (10mmol/L reduced glutathione, 50mmol/L Tris-HCl; PH8.0) eluting is collected eluent.

11, eluent is replaced with enzyme action buffer system (50mmol/L Tri s-HCl, pH8.0 with the ultrafiltration and concentration method; 150mmol/LNaCl; 1mmol/L DTT; 1mmol/L EDTA, pH 8.0), add excessive PreScission Protease enzyme (PSP albumen; Available from GE company, production code member 27-0843-01), 4 ℃ of enzyme action 16h.

12, the enzyme action system behind the enzyme action is replaced with the PBS buffer with the ultrafiltration and concentration method, carries out affinity chromatograph then, carrier is Glutathione-Sepharose 4B; Adopt the PBS buffer to carry out eluting; GST and PSP are attached on the chromatographic column, collect eluent, are the N355 fragment.

13, will contain the segmental solution of N355 and carry out 10%SDS-PAGE, and examine then and dye, see the swimming lane 1 of Fig. 1.The segmental purity of N355 reaches more than 95%.The N355 fragment is carried out western blot, and one anti-is rat anti gp96 antibody (available from santacruz, production number sc-56399), sees the swimming lane 2 of Fig. 1.

Two, the preparation of reorganization gp96 albumen (rgp96 albumen)

(1) structure of recombiant plasmid pHFMDZ-R1L2GAmy-gp96

1, extract the mRNA of human liver cancer cell HepG2, cDNA is synthesized in reverse transcription.

2, the cDNA with step 1 is that template is carried out pcr amplification, obtains pcr amplification product.

Forward primer: 5 '-CCGgaattcATGGACGATGAAGTTGATG-3 ';

Downstream primer: 5 '-CCGctcgagCTATTAGAATTCATCTTTTTCAGCTGTAG-3 '.

3,, reclaim the enzyme action product with restricted enzyme EcoRI and XhoI double digestion pcr amplification product.

4,, reclaim carrier framework with restricted enzyme EcoRI and XhoI double digestion plasmid pHFMDZ-R1A (available from Invitrogen company, production number V20520).

5, the enzyme action product of step 3 and the carrier framework of step 4 are connected, obtain recombiant plasmid pHFMDZ-R1-gp96.

Sequencing result shows that the skeleton carrier of recombiant plasmid pHFMDZ-R1-gp96 is pHFMDZ-R1A, between EcoRI and XhoI restriction enzyme site, has inserted the proteic coded sequence of gp96).

6, insert LEU2 fragment (DNA shown in the sequence 3 of sequence table) at the BglII of recombiant plasmid pHFMDZ-R1-gp96 restriction enzyme site; The BamHI restriction enzyme site inserts AMS reporting system (DNA shown in the sequence 4 of sequence table), obtains recombiant plasmid pHFMDZ-R1L2GAmy-gp96.

(2) the proteic expression of rgp96

1, adopts electrotransformation that recombiant plasmid pHFMDZ-R1L2GAmy-gp96 is imported Hansenula yeast (available from ATCC, production number MYA-335) cell, obtain the bacterium of recombinating.

2, the bacterium of will recombinating is inoculated in 5mL SD fluid medium (available from the outstanding U.S. gene in Shanghai pharmaceuticals; Production number GMS12117.7); Cultivate 48h for 37 ℃, be transferred to 100mL SYN6 culture medium (available from the outstanding U.S. gene in Shanghai pharmaceuticals, production number GMS12116.1) then; Cultivate 48h, obtain seed culture fluid for 30 ℃.

3, two bottles of seed culture fluids are inoculated in the 5L fermentation tank that contains 2L SYN6 culture medium 30 ℃ of cultivations; Use ammonia control pH to maintain 5.5; Glycerol content in 1 fermentation liquid of every 4h detection is added glycerol according to the concentration of glycerol in the fermentation liquid, and control glycerol final concentration is controlled dissolved oxygen more than 20% simultaneously about 0.5%; Generation situation according to thalline detects the thalline weight in wet base; Stop to add glycerol when reaching 180-200g/L Deng the thalline weight in wet base; Begin to induce reorganization rgp96 albumen to produce (add methanol, methanol concentration is maintained about 0.5-0.8%), induce beginning to stop fermentation after 72 hours.

(3) the proteic separation and purification of rgp96

With the centrifugal collection thalline of the fermentation liquid of step (two), with PBS buffer washed cell 2 times, in ball mill (DYNO-MILLmodel KDL), the workbook that provides according to producer uses the method smudge cells of bead ball milling; The centrifugal 20min of cell 12000 commentaries on classics/min after the fragmentation collects supernatant; Supernatant filters with 0.45 μ m filter membrane, obtains filtrating, and filtrating is concentrated, and obtains concentrated solution.

Filtrating is carried out affinity chromatograph, and concrete steps are following: adopt the Ni-NTAPurification System of handsome company (Invitrogen Corporation) to carry out affinity chromatograph, key step is: earlier with PBS balance pillar 2h; Use PBS (containing the 20mM imidazoles) balance pillar 2h then; Concentrated solution is gone up appearance with PBS (containing the 20mM imidazoles) dilution back; Wash pillar to OD value＜0.01 with PBS (containing the 20mM imidazoles); With PBS (containing the 200mM imidazoles) eluting 1.5h, collect eluent (being rgp96 albumen); All operations carries out under 4C, and flow velocity is 0.5mL/min.

Can obtain the rgp96 albumen of 50 milligrams of purity more than 90% behind the fermentation liquid purification of every liter of step (two).If contain the allos foreign protein in the albumen eluent, can the eluent of affinity chromatograph be carried out ion-exchange chromatography (Q post), the PBS liquid balance of key step: 200mMNaCl; Last appearance; The PBS liquid of 300mM NaCl is washed impurity; The PBS liquid of 800mM NaCl is washed destination protein.

The fermentation liquid of step (two), eluent (rgp96 albumen) and the eluent behind the ion-exchange chromatography behind the affinity chromatograph are carried out 10%SDS-PAGE, examine then and dye, see Fig. 2.The proteic purity of rgp96 reaches more than 90%.Rgp96 albumen is carried out western blot, and one is anti-for rat anti gp96 antibody (available from santa cruz, production number sc-56399), sees Fig. 2.

Three, the preparation of gp96 extract

Prepare the gp96 extract with mouse liver, method is following:

1, tissue homogenate

The homogenate buffer prescription: (Phenylmethanesulfonyl fluoride, molecular formula are C in the sodium bicarbonate buffer liquid of pH7.0, to add PMSF ₇H ₇FO ₂S) (place beaker on ice to dispose to final concentration 1mM; PMSF decomposes in several hours in aqueous solution, and this solution can not spend the night).

Get beaker and place on ice, the BALB/c mouse hepatic tissue is cut into rapidly the fragment of the about 1-2 millimeter of diameter with shears in beaker, add the homogenate buffer of organizing 8 times of weight then; To organize fragment to stir and pour glass homogenizer into, homogenate more than 15 times up and down again after homogenate to bottom piece of tissue disappears; Homogenate is poured centrifuge tube into, and 50, the centrifugal 60min of 000g gets 10 * PBS (pH7.5,200mM NaCl) that supernatant adds 1/10 volume, is used for the ConA-Sepharose chromatography of step 2.

2, ConA-Sepharose chromatography

Carrier is ConA-Sepharose (available from a GE company, production number 17-0440-01), calculates use amount by " 1ml carrier/4g tissue ".The internal diameter of chromatographic column and column length are 1.6 * 2.5cm.

With supernatant with on the peristaltic pump appearance, 0.25ml/min.In PBS (pH7.5,200mM NaCl), add PMSF, as the eluent first to final concentration 1mM; With peristaltic pump the eluent first of 10 times of column volumes is crossed chromatographic column (0.5ml/min), not conjugated protein to remove.In PBS (pH7.5,200mM NaCl), add α-D-Glucopyranose. to final concentration 10% (10g/mL), add PMSF to final concentration 1mM, as eluent second; With peristaltic pump the eluent second of 3 times of column volumes is crossed chromatographic column (0.25ml/min), collect eluent.

3, HiTrap-Q Sepharose ion-exchange chromatography

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

After carrier adorned post, earlier with 5ml PBS (pH7.5,200mM NaCl) cleaning (1ml/min).With appearance (1ml/min) on the eluent of step 2; Then 5ml PBS (pH7.5,200mM NaCl) is crossed chromatographic column (1ml/min), not conjugated protein to remove; Then 10ml PBS (pH7.5,300mM NaCl) is crossed chromatographic column (1ml/min), not conjugated protein to remove; Then 3ml PBS (pH7.5,600mM NaCl) is crossed chromatographic column (1ml/min), collect eluent, be the gp96 extract.

The gp96 extract is carried out 10%SDS-PAGE, examine then and dye, see the swimming lane 1 of Fig. 3.The gp96 extract is carried out westernblot, and one anti-is rat anti gp96 antibody (available from santa cruz, production number sc-56399), sees the swimming lane 2 of Fig. 3.

Except mouse liver, also available like preparation gp96 extract in the undertissue: people's tumor tissues (breast carcinoma, cerebral glioma, renal carcinoma, intestinal cancer, hepatocarcinoma, gastric cancer) and people's Placenta Hominis of murine melanoma (B16), pig liver, human hepatoma cell line HepG2's cultured cell, clinical operation excision; Method for distilling is with reference to the extraction of mouse liver.

Embodiment 2, rgp96 albumen and gp96 extract immunocompetence are relatively

BALB/c mouse is divided into four groups at random, 10 every group, carries out following immunity (immune volume equates) respectively:

First group (HBc87-95): use the HBc87-95 immune mouse, 50 micrograms/inferior;

Second group (HBc87-95+gp96): with the gp96 extract of the step 3 of embodiment 1 preparation and HBc87-95 immune mouse together, gp96 extract 20 micrograms/inferior, HBc87-9550 microgram/inferior;

The 3rd group (HBc87-95+rgp96): with the rgp96 albumen of the step 2 of embodiment 1 preparation and HBc87-95 immune mouse together, rgp96 protein 20 microgram/inferior, HBc87-9550 microgram/inferior;

The 4th group (HBc87-95+IFA): with incomplete Freund and HBc87-95 immune mouse together, incomplete Freund 20 micrograms/inferior, HBc87-9550 microgram/inferior.

In 1,2,3 each immunity of week once, in the 4th week, detect T cytoactive (antibody of the anti-CD8 of employing FITC labelling and the Pentamer of PE labelling) with flow cytometer.The flow cytometer testing result see Fig. 4 (on).The two positive cells of CD8 and Pentamer-PE are HBc87-95 peptide specific CD8+T cell, calculate the percentage ratio that it accounts for the CD8+T cell, see Fig. 4 (descending).Rgp96 albumen and the immunity of gp96 extract all can significantly improve t cell responses, not obviously difference (P＞0.01) of the CLT that rgp96 albumen and the immunity of gp96 extract cause; The hepatitis B specific CTL quantity of second group and the 3rd group is significantly higher than first group (P＜0.01), also is higher than the 4th group.

Embodiment 3, carry gp96 expression carrier and gp96 extract immunocompetence relatively

The proteic coded sequence of gp96 is inserted between the EcoR I and Xho I restriction enzyme site of expression vector pCDNA3.1 (available from Invitrogen company, production code member V790-20), obtain recombiant plasmid.

BALB/c mouse is divided into five groups at random, 10 every group, carries out following immunity (immune volume equates) respectively:

First group: use the HBc87-95 immune mouse, 50 micrograms/inferior;

Second group: with recombiant plasmid and HBc87-95 immune mouse together, recombiant plasmid 20 micrograms/inferior, HBc87-9550 microgram/inferior;

The 3rd group: with recombiant plasmid and HBc87-95 immune mouse together, recombiant plasmid 50 micrograms/inferior, HBc87-9550 microgram/inferior;

The 4th group: with recombiant plasmid and HBc87-95 immune mouse together, recombiant plasmid 500 micrograms/inferior, HBc87-9550 microgram/inferior;

The 5th group (HBc87-95+gp96): with the gp96 extract of the step 3 of embodiment 1 preparation and HBc87-95 immune mouse together, gp96 extract 20 micrograms/inferior, HBc87-9550 microgram/inferior.

In 1,2,3 each immunity of week once, in the 4th week, detect the T cytoactive with flow cytometer.The recombiant plasmid immunity can significantly improve t cell responses, and with respect to first group, hepatitis B specific CTL quantity has remarkable rising (P＜0.01).Not obviously difference (P＞0.01) of the CLT that recombiant plasmid and the immunity of gp96 extract cause.

Embodiment 4, rgp96 albumen or N355 fragment are to the two-ways regulation of immunity

6-8 BALB/c mouse in age in week is divided into five groups, 10 every group, distinguishes immunity (immune volume equates) as follows:

First group: with the rgp96 albumen and the immune together BALB/c mouse of HBc87-95 of the step 2 of embodiment 1 preparation, rgp96 protein 10 microgram/inferior, HBc87-9550 microgram/inferior;

Second group: with the rgp96 albumen and the immune together BALB/c mouse of HBc87-95 of the step 2 of embodiment 1 preparation, rgp96 protein 10 0 microgram/inferior, HBc87-9550 microgram/inferior;

The 3rd group: with the N355 fragment and the immune together BALB/c mouse of HBc87-95 of the step 1 of embodiment 1 preparation, N355 fragment 10 micrograms/inferior, HBc87-9550 microgram/inferior;

The 4th group: with the N355 fragment and the immune together BALB/c mouse of HBc87-95 of the step 1 of embodiment 1 preparation, N355 fragment 100 micrograms/inferior, HBc87-9550 microgram/inferior;

The 5th group: use the HBc87-95 immune mouse, 50 micrograms/inferior;

The 6th group: use the HBc87-95 immune mouse, 50 micrograms/inferior.

In 1,2,3 each immunity of week once,, detect T cytoactive (adopting antibody and the Tetrame of the anti-CD8 of FITC labelling), and carry out ELISPOT detection and Treg detection with flow cytometer in the 4th week.Flow cytometer detects sees that the ratio that Fig. 5 A, CD8 positive cell account for splenocyte sees Fig. 5 B, and the Tetrame positive cell accounts for the ratio of CD8 positive cell and sees Fig. 5 C.The ELISPOT testing result is seen Fig. 6.The Treg testing result is seen Fig. 7.Rgp96 albumen or the immunity of N355 fragment can significantly improve t cell responses; With respect to the 5th group and the 6th group, total T cell and hepatitis B specific CTL quantity have remarkable rising (P＜0.01); Rgp96 albumen or the immunity of N355 fragment can significantly improve hepatitis B virus Specific CTL Cells active (P＜0.01); The high dose immunity is starkly lower than low dose group to the activation of virus specific t cell and total T cell.Rgp96 obviously improves the quantity of Treg; Compare with contrast; Immunity of rgp96 low dosage or high dose immunity Treg have increased by 13.86% or 26.70% (P＜0.05 or 0.01) respectively, and immunity of N355 low dosage and high dose immunity Treg have increased by 8.77% or 12.54% respectively, (P＜0.05).Immunity to mice Treg active promotion see Fig. 8, comprise that the inhibition (P＜0.05 or 0.01) that CD4+ and CD8+T cell are grown and inhibition (P＜the 0.05 or 0.01) function of secretion of gamma-IFN obviously strengthen.Treg and T cell are the negative correlation (see figure 9) in correlation analysis discovery rgp96 albumen or the N355 fragment mice immunized, the CD8 of Treg and total CD8+T cell (r=0.84, P＜0.01), hepatitis B specific CTL (r=0.94, P＜0.01) and secretion of gamma-IFN ⁺T cell (r=0.79, P＜0.01) all is remarkable negative correlation.In sum, the segmental immunoregulation mechanism of rgp96 albumen or N355 as follows: CTL is activated through antigen presentation in (1); (2) activate Treg, Treg suppresses the CTL activity conversely again.Therefore gp96 albumen (or its fragment) has the two-ways regulation function to hepatitis B T cellular immunization, and it is significant for improving the gp96 immunocompetence how to control equilibrium point.

Embodiment 5, immunomodulator are to rgp96 albumen or the immunocompetent facilitation of N355 fragment

One, cyclophosphamide is to rgp96 albumen or the segmental immunocompetent facilitation of N355

1, high dose immunity

6-8 BALB/c mouse in age in week is divided into six groups, 10 every group, distinguishes immunity (volume equates) as follows:

First group: with the rgp96 albumen and the immune together BALB/c mouse of HBc87-95 of the step 2 of embodiment 1 preparation, rgp96 protein 10 0 microgram/inferior, HBc87-9550 microgram/inferior;

Second group: with the immune together BALB/c mouse of rgp96 albumen, HBc87-95 and cyclophosphamide of the step 2 of embodiment 1 preparation, rgp96 protein 10 0 microgram/inferior, HBc87-9550 microgram/inferior, 0.4 milligram of cyclophosphamide/inferior;

The 3rd group: with the N355 fragment and the immune together BALB/c mouse of HBc87-95 of the step 1 of embodiment 1 preparation, N355 fragment 100 micrograms/inferior, HBc87-9550 microgram/inferior;

The 4th group: with the immune together BALB/c mouse of N355 fragment, HBc87-95 and cyclophosphamide of the step 1 of embodiment 1 preparation, N355 fragment 100 micrograms/inferior, HBc87-9550 microgram/inferior, 0.4 milligram of cyclophosphamide/inferior;

The 5th group: with HBc87-95 immunity BALB/c mouse, HBc87-9550 microgram/inferior;

The 6th group: with HBc87-95 and the immune together BALB/c mouse of cyclophosphamide, HBc87-9550 microgram/inferior, 0.4 milligram of cyclophosphamide/inferior.

In 1,2,3 each immunity of week once, the 4th week was detected (antibody of the antibody of the anti-CD25 of employing PE labelling and the anti-FoxP3 of APC labelling) with flow cytometer.The result sees Figure 10; A is the flow cytometer testing result; B is the inhibitory action (CD25 and FoxP3 two positive cells account for the percentage ratio of CD4 positive cell) of cyclophosphamide to the quantity of Treg; C is the facilitation (CD8 positive cell account for the percentage ratio of splenocyte) of cyclophosphamide to the CD8+ cell quantity; D is the facilitation (Tetrame positive cell account for the percentage ratio of CD8 positive cell) of cyclophosphamide to hepatitis B specific T-cells quantity; E is the facilitation (IFN-γ positive cell account for the percentage ratio of CD8 positive cell) of cyclophosphamide to the T cell quantity of secretion of gamma-IFN; 1 is first group, and 2 is second group, and 3 is the 3rd group, and 4 is the 4th group, and 5 is the 5th group, and 6 is the 6th group.HBc87-95 can effectively be removed by cyclophosphamide and high dose rgp96 albumen (or N355 fragment) is induced the Treg of generation; The T cell quantity of T cell quantity, hepatitis B specific T-cells and secretion of gamma-IFN is significantly improved; Unite and use cyclophosphamide can make the T cell quantity of total T cell, hepatitis B specific T-cells and secretion of gamma-IFN increase by 21.72%, 52.99% and 63.88% (P＜0.05 or 0.01) respectively.

2, low dosage immunity

Second group: with the immune together BALB/c mouse of rgp96 albumen, HBc87-95 and cyclophosphamide of the step 2 of embodiment 1 preparation, rgp96 protein 10 microgram/inferior, HBc87-9550 microgram/inferior, 0.4 milligram of cyclophosphamide/inferior;

The 4th group: with the immune together BALB/c mouse of N355 fragment, HBc87-95 and cyclophosphamide of the step 1 of embodiment 1 preparation, N355 fragment 10 micrograms/inferior, HBc87-9550 microgram/inferior, 0.4 milligram of cyclophosphamide/inferior;

In 1,2,3 each immunity of week once.The 4th week was detected with flow cytometer.The result sees Figure 11; A is the facilitation (CD8 positive cell account for the percentage ratio of splenocyte) of cyclophosphamide to the CD8+ cell quantity; B is the facilitation (Tetrame positive cell account for the percentage ratio of CD8 positive cell) of cyclophosphamide to hepatitis B specific T-cells quantity; C is the facilitation (IFN-γ positive cell accounts for the percentage ratio of CD8 positive cell) of T cell quantity of the secretion of gamma-IFN of cyclophosphamide; 1 is first group, and 2 is second group, and 3 is the 3rd group, and 4 is the 4th group, and 5 is the 5th group, and 6 is the 6th group.HBc87-95 can effectively be removed by cyclophosphamide and low dosage rgp96 albumen (or N355 fragment) is induced the Treg of generation, the T cell quantity of T cell quantity, hepatitis B specific T-cells and secretion of gamma-IFN be significantly improved (P＜0.05).Rgp96 albumen (or N355 fragment) and cyclophosphamide mix use, can effectively improve the activation capacity (P＜0.05 or 0.01) of rgp96 albumen (or N355 fragment) to the T cell.

Two, the antibody of anti-CD25 is to rgp96 albumen or the segmental immunocompetent facilitation of N355

6-8 BALB/c mouse in age in week is divided into nine groups, 10 every group, distinguishes immunity (volume equates) as follows:

Second group: with the immune together BALB/c mouse of antibody of rgp96 albumen, HBc87-95 and the anti-CD25 of the preparation of the step 2 of embodiment 1, rgp96 protein 10 microgram/inferior, HBc87-9550 microgram/inferior, antibody 30 micrograms of anti-CD25/inferior;

The 4th group: with the immune together BALB/c mouse of antibody of N355 fragment, HBc87-95 and the anti-CD25 of the preparation of the step 1 of embodiment 1, N355 fragment 10 micrograms/inferior, HBc87-9550 microgram/inferior, antibody 30 micrograms of anti-CD25/inferior;

The 5th group: with the rgp96 albumen and the immune together BALB/c mouse of HBc87-95 of the step 2 of embodiment 1 preparation, rgp96 protein 10 0 microgram/inferior, HBc87-9550 microgram/inferior;

The 6th group: with the immune together BALB/c mouse of antibody of rgp96 albumen, HBc87-95 and the anti-CD25 of the preparation of the step 2 of embodiment 1, rgp96 protein 10 0 microgram/inferior, HBc87-9550 microgram/inferior, antibody 30 micrograms of anti-CD25/inferior;

The 7th group: with the N355 fragment and the immune together BALB/c mouse of HBc87-95 of the step 1 of embodiment 1 preparation, N355 fragment 100 micrograms/inferior, HBc87-9550 microgram/inferior;

The 8th group: with the immune together BALB/c mouse of antibody of N355 fragment, HBc87-95 and the anti-CD25 of the preparation of the step 1 of embodiment 1, N355 fragment 100 micrograms/inferior, HBc87-9550 microgram/inferior, antibody 30 micrograms of anti-CD25/inferior;

The 9th group: with HBc87-95 immunity BALB/c mouse, HBc87-9550 microgram/inferior.

In 1,2,3 each immunity of week once; The 4th week was detected (antibody of the antibody of the anti-CD25 of employing PE labelling and the anti-CD4 of FITC labelling) with flow cytometer.The antibody of anti-CD25 can effectively remove HBc87-95 and high dose rgp96 albumen (or N355 fragment) is induced the Treg of generation, and the T cell quantity of T cell quantity, hepatitis B specific T-cells and secretion of gamma-IFN is significantly improved; Uniting the antibody that uses anti-CD25 can make the T cell quantity of total T cell, hepatitis B specific T-cells and secretion of gamma-IFN increase by 31.52%, 63.79% and 75.75% (P＜0.05 or 0.01) respectively.Use the antibody of anti-CD25 effectively to remove HBc87-95 and low dosage rgp96 albumen (or N355 fragment) is induced the Treg of generation, the T cell quantity of T cell quantity, hepatitis B specific T-cells and secretion of gamma-IFN be significantly improved (P＜0.05); The antibody of rgp96 albumen (or N355 fragment) and anti-CD25 mixes use, can effectively improve the activation capacity (P＜0.05 or 0.01) of rgp96 albumen (or N355 fragment) to the T cell.The result of first group and second group sees Figure 12; A is the flow cytometer testing result, and B is the inhibitory action (CD25 positive cell account for the percentage ratio of CD4 positive cell) of the antibody of anti-CD25 to the quantity of Treg.The CD25 antibody treatment can obviously be removed Treg, makes Treg quantity reduce by 9.2 times, and difference is (P＜0.01) extremely significantly.

Three, the RNA interfering of FoxP3 is to the active facilitation of gp96 protein immunization

Second group: with the immune together BALB/c mouse of RNA interfering of rgp96 albumen, HBc87-95 and the FoxP3 of the preparation of the step 2 of embodiment 1, rgp96 protein 10 microgram/inferior, HBc87-95 50 micrograms/inferior, RNA interfering 500 micrograms of FoxP3/inferior;

The 4th group: with the immune together BALB/c mouse of RNA interfering of N355 fragment, HBc87-95 and the FoxP3 of the preparation of the step 1 of embodiment 1, N355 fragment 10 micrograms/inferior, HBc87-9550 microgram/inferior, RNA interfering 500 micrograms of FoxP3/inferior;

The 6th group: with the immune together BALB/c mouse of RNA interfering of rgp96 albumen, HBc87-95 and the FoxP3 of the preparation of the step 2 of embodiment 1, rgp96 protein 10 0 microgram/inferior, HBc87-9550 microgram/inferior, RNA interfering 500 micrograms of FoxP3/inferior;

The 8th group: with the immune together BALB/c mouse of RNA interfering of N355 fragment, HBc87-95 and the FoxP3 of the preparation of the step 1 of embodiment 1, N355 fragment 100 micrograms/inferior, HBc87-9550 microgram/inferior, RNA interfering 500 micrograms of FoxP3/inferior;

In 1,2,3 each immunity of week once; The 4th week was detected (antibody of the antibody of the anti-CD25 of employing PE labelling and the anti-FoxP3 of APC labelling) with flow cytometer.The RNA interfering of FoxP3 can effectively remove HBc87-95 and high dose rgp96 albumen (or N355 fragment) is induced the Treg of generation, and the T cell quantity of T cell quantity, hepatitis B specific T-cells and secretion of gamma-IFN is significantly improved; Uniting the RNA interfering that uses FoxP3 can make the T cell quantity of total T cell, hepatitis B specific T-cells and secretion of gamma-IFN increase by 11.63%, 42.67% and 55.57% (P＜0.05 or 0.01) respectively.Use the RNA interfering of FoxP3 effectively to remove HBc87-95 and low dosage rgp96 albumen (or N355 fragment) is induced the Treg of generation, the T cell quantity of T cell quantity, hepatitis B specific T-cells and secretion of gamma-IFN be significantly improved (P＜0.05); The RNA interfering of rgp96 albumen (or N355 fragment) and FoxP3 mixes use, can effectively improve the activation capacity (P＜0.05 or 0.01) of rgp96 albumen (or N355 fragment) to the T cell.The result of first group and second group sees Figure 13; A is the flow cytometer testing result, and B is the inhibitory action of the RNA interfering of FoxP3 to the quantity of Treg.The RNA interfering of FoxP3 is handled can obviously remove Treg, suppresses the quantity of Treg, makes Treg quantity reduce by 7.5 times, and difference is (P＜0.01) extremely significantly.

The preparation of embodiment 6, hepatitis B protein vaccine and effect assessment

One, the composition of hepatitis B protein vaccine first and effect assessment

1, the composition of hepatitis B protein vaccine first

Hepatitis B protein vaccine first is made up of rgp96 albumen, hepatitis B surface antigen (HBsAg), hepatitis B core albumen (HBcAg) and the cyclophosphamide of the step 2 preparation of embodiment 1, and each component is packed separately.

2, the effect assessment of hepatitis B protein vaccine first

(1) preparation of vaccine precursor

Rgp96 albumen, hepatitis B surface antigen (HBsAg) and the hepatitis B core albumen (HBcAg) of the preparation of the step 2 of embodiment 1 are carried out assembled in vitro, obtain the vaccine precursor.The solvent of assembly system is a water, and solute and concentration thereof are following: 20mM HEPES (pH7.2), 20mM NaCl, 2mM MgCl ₂, 100mM KCl, 1mM PMSF, 2ug/ul rgp96 albumen, 1ug/ul HBsAg, 1ug/ulHBcAg; Assembling condition: 45 ℃ 10 minutes (also can adopt 45 ℃ 10-30 minute, or 4 ℃ 30-120 minute).

Stability :-80 ℃ of preservations, sds gel electrophoresis is not seen obvious degradation or is formed polymer after 6 months.

(2) grouping administration

8 age in week the HBV transgenic mouse be divided into two groups, 10 every group, respectively immunity (volume equates) as follows:

First group: 10 microlitre vaccine precursors and 0.4 milligram of cyclophosphamide mix, as single immunization dosage;

Second group: each immune 10 microlitre vaccine precursors.

In 1,2,3 each immunity of week once; The expression of HBcAg in HBsAg content and the liver cell in the 8 weeks back detection mice serum.HBsAg in serum content adopts ELISA to detect (the hepatitis B virus surface antigen diagnostic kit is available from Shanghai Kehua Bio-engineering Co., Ltd).The expression ratio of HBcAg adopts SABC to detect (antibody is HBcAg rabbit multi-resistance, is ShiJi Co., Ltd available from Beijing health, production number CWP196) in the liver cell.HBsAg content in first group of mice serum (OD value 0.66 ± 0.13) is significantly less than second group of mice (1.21 ± 0.23), and the two difference reaches significant level (P＜0.05).The expression ratio (9% ± 2) of HBcAg is significantly less than second group of mice (20% ± 3) in first group of mouse liver cell, and the two difference reaches significant level (P＜0.05).Explain in the vaccine of the present invention, owing to added cyclophosphamide, the inhibitory action of hepatitis B virus duplication is significantly improved.

Two, the composition of hepatitis B protein vaccine second and effect assessment

1, the composition of hepatitis B protein vaccine second

Hepatitis B protein vaccine second is made up of the antibody of rgp96 albumen, hepatitis B surface antigen (HBsAg), hepatitis B core albumen (HBcAg) and the anti-CD25 of the step 2 preparation of embodiment 1, and each component is packed separately.

2, the effect assessment of hepatitis B protein vaccine second

(1) preparation of vaccine precursor

2 (1) with step 1.

(2) grouping administration

First group: the antibody of 10 microlitre vaccine precursors and the anti-CD25 of 30 micrograms mixes, as single immunization dosage;

Second group: each immune 10 microlitre vaccine precursors.

In 1,2,3 each immunity of week once; The expression of HBcAg in HBsAg content and the liver cell in the 8 weeks back detection mice serum.The detection method of the expression ratio of HBcAg is with 2 (2) of step 1 in HBsAg in serum content and the liver cell.HBsAg content in first group of mice serum (OD value 0.60 ± 0.11) is significantly less than second group of mice (1.30 ± 0.25), and the two difference reaches utmost point significant level (P＜0.01).The expression ratio (8% ± 3) of HBcAg is significantly less than second group of mice (18% ± 3) in first group of mouse liver cell, and the two difference reaches significant level (P＜0.05).Explain in the vaccine of the present invention, owing to added CD25 antibody, the inhibitory action of hepatitis B virus duplication is significantly improved.

Three, the composition of hepatitis B protein vaccine third and effect assessment

1, the composition of hepatitis B protein vaccine third

Hepatitis B protein vaccine third is made up of the RNA interfering of rgp96 albumen, hepatitis B surface antigen (HBsAg), hepatitis B core albumen (HBcAg) and the FoxP3 of the step 2 preparation of embodiment 1, and each component is packed separately.

2, the effect assessment of hepatitis B protein vaccine third

(1) preparation of vaccine precursor

2 (1) with step 1.

(2) grouping administration

First group: the RNA interfering of 10 microlitre vaccine precursors and 500 microgram FoxP3 mixes, as single immunization dosage;

Second group: each immune 10 microlitre vaccine precursors.

In 1,2,3 each immunity of week once; The expression of HBcAg in HBsAg content and the liver cell in the 8 weeks back detection mice serum.The detection method of the expression ratio of HBcAg is with 2 (2) of step 1 in HBsAg in serum content and the liver cell.HBsAg content in first group of mice serum (OD value 0.80 ± 0.11) is significantly less than second group of mice (1.30 ± 0.25), and the two difference reaches utmost point significant level (P＜0.01).The expression ratio (10% ± 3) of HBcAg is significantly less than second group of mice (18% ± 3) in first group of mouse liver cell, and the two difference reaches significant level (P＜0.05).Explain in the vaccine of the present invention, owing to added the RNA interfering of FoxP3, the inhibitory action of hepatitis B virus duplication is significantly improved.

Four, the composition of hepatitis B protein vaccine fourth and effect assessment

1, the composition of hepatitis B protein vaccine fourth

Hepatitis B protein vaccine fourth is made up of the antibody of rgp96 albumen, hepatitis B surface antigen (HBsAg), hepatitis B core albumen (HBcAg), cyclophosphamide and the anti-CD25 of the step 2 preparation of embodiment 1, and each component is packed separately.

2, the effect assessment of hepatitis B protein vaccine fourth

(1) preparation of vaccine precursor

2 (1) with step 1.

(2) grouping administration

First group: the antibody of 10 microlitre vaccine precursors, 0.4 milligram of cyclophosphamide and the anti-CD25 of 30 micrograms mixes, as single immunization dosage;

Second group: each immune 10 microlitre vaccine precursors.

In 1,2,3 each immunity of week once; The expression of HBcAg in HBsAg content and the liver cell in the 8 weeks back detection mice serum.The detection method of the expression ratio of HBcAg is with 2 (2) of step 1 in HBsAg in serum content and the liver cell.

HBsAg content in first group of mice serum (OD value 0.46 ± 0.14) is significantly less than second group of mice (1.25 ± 0.20), and the two difference reaches significant level (P＜0.05).The expression ratio (5% ± 2) of HBcAg is significantly less than second group of mice (20% ± 2) in first group of mouse liver cell, and the two difference reaches significant level (P＜0.05).Explain in the vaccine of the present invention, owing to added the antibody of cyclophosphamide and anti-CD25, the inhibitory action of hepatitis B virus duplication is significantly improved.Compare with the HBV transgenic mouse that not additional immunomodulator carries out administration; Above-mentioned four kinds of vaccines cause that all the HBsAg antigen in the HBV transgenic mouse serum reduces more than 50%; Virus sweep 70-80% above (HBcAg turns out cloudy) in the liver cell, difference all reaches extremely significantly (P＜0.01).More than research shows that vaccine of the present invention has good anti-HBV active.

The composition of embodiment 7, hepatitis B epiposition vaccine and effect assessment

One, the composition of hepatitis B epiposition vaccine first and effect assessment

1, the composition of hepatitis B epiposition vaccine first

Hepatitis B epiposition vaccine first is made up of rgp96 albumen, hepatitis B surface antigen epi-position 362-371, hepatitis B core albumen epi-position 87-95, hepatitis B polymerase epi-position 140-148 and the cyclophosphamide of the step 2 preparation of embodiment 1, and each component is packed separately.

2, the effect assessment of hepatitis B epiposition vaccine first

(1) preparation of vaccine precursor

Rgp96 albumen, hepatitis B surface antigen epi-position 362-371, hepatitis B core albumen epi-position 87-95 and the hepatitis B polymerase epi-position 140-148 of the preparation of the step 2 of embodiment 1 are carried out assembled in vitro, obtain hepatitis B protein vaccine precursor.The solvent of assembly system is a water, and solute and concentration thereof are following: 20mM HEPES (pH7.2), 20mM NaCl, 2mM MgCl ₂, 100mM KCl, 1mMPMSF, 2ug/ul rgp96 albumen, above-mentioned three kinds of each 3ug/ul of epi-position; Assembling condition: 45 ℃ 10 minutes (also can adopt 45 ℃ 10-30 minute, or 4 ℃ 30-120 minute).

(2) grouping administration

Second group: each immune 10 microlitre vaccine precursors.

In 1,2,3 each immunity of week once; The expression of HBcAg in HBsAg content and the liver cell in the 8 weeks back detection mice serum.The detection method of the expression ratio of HBcAg is with 2 (2) of the step 1 of embodiment 6 in HBsAg in serum content and the liver cell.HBsAg content in first group of mice serum (OD value 0.62 ± 0.11) is significantly less than second group of mice (1.11 ± 0.20), and the two difference reaches significant level (P＜0.05).The expression ratio (8% ± 2) of HBcAg is significantly less than second group of mice (19% ± 2) in first group of mouse liver cell, and the two difference reaches significant level (P＜0.05).Explain in the vaccine of the present invention, owing to added cyclophosphamide, the inhibitory action of hepatitis B virus duplication is significantly improved.

Two, the composition of hepatitis B epiposition vaccine second and effect assessment

1, the composition of hepatitis B epiposition vaccine second

Hepatitis B epiposition vaccine second is made up of the antibody of rgp96 albumen, hepatitis B surface antigen epi-position 362-371, hepatitis B core albumen epi-position 87-95, hepatitis B polymerase epi-position 140-148 and the anti-CD25 of the step 2 preparation of embodiment 1, and each component is packed separately.

2, the effect assessment of hepatitis B epiposition vaccine second

(1) preparation of vaccine precursor

2 (1) with step 1.

(2) grouping administration

Second group: each immune 10 microlitre vaccine precursors.

In 1,2,3 each immunity of week once; The expression of HBcAg in HBsAg content and the liver cell in the 8 weeks back detection mice serum.The detection method of the expression ratio of HBcAg is with 2 (2) of the step 1 of embodiment 6 in HBsAg in serum content and the liver cell.HBsAg content in first group of mice serum (OD value 0.54 ± 0.09) is significantly less than second group of mice (1.19 ± 0.18), and the two difference reaches utmost point significant level (P＜0.01).The expression ratio (7% ± 3) of HBcAg is significantly less than second group of mice (17% ± 2) in first group of mouse liver cell, and the two difference reaches significant level (P＜0.05).Explain in the vaccine of the present invention, owing to added CD25 antibody, the inhibitory action of hepatitis B virus duplication is significantly improved.

Three, the composition of hepatitis B epiposition vaccine third and effect assessment

1, the composition of hepatitis B epiposition vaccine third

Hepatitis B epiposition vaccine third is made up of the RNA interfering of rgp96 albumen, hepatitis B surface antigen epi-position 362-371, hepatitis B core albumen epi-position 87-95, hepatitis B polymerase epi-position 140-148 and the FoxP3 of the step 2 preparation of embodiment 1, and each component is packed separately.

2, the effect assessment of hepatitis B epiposition vaccine third

(1) preparation of vaccine precursor

2 (1) with step 1.

(2) grouping administration

Second group: each immune 10 microlitre vaccine precursors.

In 1,2,3 each immunity of week once; The expression of HBcAg in HBsAg content and the liver cell in the 8 weeks back detection mice serum.The detection method of the expression ratio of HBcAg is with 2 (2) of the step 1 of embodiment 6 in HBsAg in serum content and the liver cell.HBsAg content in first group of mice serum (OD value 0.90 ± 0.08) is significantly less than second group of mice (1.30 ± 0.25), and the two difference reaches utmost point significant level (P＜0.01).The expression ratio (12% ± 3) of HBcAg is significantly less than second group of mice (18% ± 3) in first group of mouse liver cell, and the two difference reaches significant level (P＜0.05).Explain in the vaccine of the present invention, owing to added the RNA interfering of FoxP3, the inhibitory action of hepatitis B virus duplication is significantly improved.

Four, the composition of hepatitis B epiposition vaccine fourth and effect assessment

1, the composition of hepatitis B epiposition vaccine fourth

Hepatitis B epiposition vaccine fourth is made up of the antibody of rgp96 albumen, hepatitis B surface antigen epi-position 362-371, hepatitis B core albumen epi-position 87-95, hepatitis B polymerase epi-position 140-148, cyclophosphamide and the anti-CD25 of the step 2 preparation of embodiment 1, and each component is packed separately.

2, the effect assessment of hepatitis B protein vaccine fourth

(1) preparation of vaccine precursor

2 (1) with step 1.

(2) grouping administration

Second group: each immune 10 microlitre vaccine precursors.

In 1,2,3 each immunity of week once; The expression of HBcAg in HBsAg content and the liver cell in the 8 weeks back detection mice serum.The detection method of the expression ratio of HBcAg is with 2 (2) of the step 1 of embodiment 6 in HBsAg in serum content and the liver cell.HBsAg content in first group of mice serum (OD value 0.41 ± 0.13) is significantly less than second group of mice (1.02 ± 0.19), and the two difference reaches significant level (P＜0.05).The expression ratio (5% ± 2) of HBcAg is significantly less than second group of mice (19% ± 3) in first group of mouse liver cell, and the two difference reaches significant level (P＜0.05).Explain in the vaccine of the present invention, owing to added the antibody of cyclophosphamide and anti-CD25, the inhibitory action of hepatitis B virus duplication is significantly improved.

Compare with the HBV transgenic mouse that not additional immunomodulator carries out administration; Above-mentioned four kinds of vaccines cause that all the HBsAg antigen in the HBV transgenic mouse serum reduces more than 60%; Virus sweep 80-90% above (HBcAg turns out cloudy) in the liver cell, difference all reaches extremely significantly (P＜0.01).More than research shows that vaccine of the present invention has good anti-HBV active.

The composition of embodiment 8, melanoma vaccines and effect assessment

One, the preparation of gp96 extract

With MC B16 (available from ATCC, production number CRL-6475) preparation gp96 extract, method is following:

1, tissue homogenate

Cultivate MC B16, collecting cell adds the homogenate buffer of organizing 8 times of weight; To organize fragment to stir and pour glass homogenizer into, homogenate more than 15 times up and down again after homogenate to bottom piece of tissue disappears; Homogenate is poured centrifuge tube into, and 50, the centrifugal 60min of 000g gets 10 * PBS (pH7.5,200mM NaCl) that supernatant adds 1/10 volume, is used for the ConA-Sepharose chromatography of step 2.

2, ConA-Sepharose chromatography

With 2 of the step 3 of embodiment 1.

3, HiTrap-Q Sepharose ion-exchange chromatography

With 3 of the step 3 of embodiment 1.

Two, the composition of melanoma vaccines

The melanoma vaccines first is made up of the gp96 extract and the cyclophosphamide of step 1 preparation, and each component is packed separately.

Melanoma vaccines second is made up of the gp96 extract of step 1 preparation and the antibody of anti-CD25, and each component is packed separately.

Melanoma vaccines third is made up of the gp96 extract of step 1 preparation and the RNA interfering of FoxP3, and each component is packed separately.

Three, grouping immunity

C57BL/6J mice (age in 6-8 week is available from Beijing dimension tonneau China company, strain C57BL/6J)) be divided into four groups, 10 every group, immunity respectively (volume equates) as follows:

First group: every subcutaneous injection 5 * 10 ⁴Individual MC B16; Begin immunity after 10 days, each two components of immune melanoma vaccines first simultaneously, gp96 extract 20 micrograms/inferior, 0.4 milligram of cyclophosphamide/inferior, every at a distance from immunity in 3 days once, immune 5 times altogether; Last immunity weighing tumor weight after 2 days.

Second group: every subcutaneous injection 5 * 10 ⁴Individual MC B16; Begin immunity after 10 days, each two components of immune melanoma vaccines second simultaneously, gp96 extract 20 micrograms/inferior, antibody 30 micrograms of anti-CD25/inferior, whenever at a distance from immunity in 3 days once, immunity is 5 times altogether; Last immunity weighing tumor weight after 2 days.

The 3rd group: every subcutaneous injection 5 * 10 ⁴Individual MC B16; Begin immunity after 10 days, each two components of immune melanoma vaccines third simultaneously, gp96 extract 20 micrograms/inferior, RNA interfering 500 micrograms of FoxP3/inferior, whenever at a distance from immunity in 3 days once, immunity is 5 times altogether; Last immunity weighing tumor weight after 2 days.

The 4th group: every subcutaneous injection 5 * 10 ⁴Individual MC B16; Begin immunity after 10 days, gp96 extract 20 micrograms of each immune step 1 preparation/inferior, whenever at a distance from immunity in 3 days once, immunity is 5 times altogether; Last immunity weighing tumor weight after 2 days.

First group of mouse tumor weight is 0.92g ± 0.17; Second group of mouse tumor weight is 0.62g ± 0.15; The 3rd group of mouse tumor weight is 1.25g ± 0.27; All be significantly less than the 4th group of mouse tumor weight (3.12g ± 0.35), three vaccine processed group and the 4th group of difference all reach utmost point significant level (P＜0.01).

Injection MC B16 picks up counting, and statistics is respectively organized the survival rate of mice after 45 days.The survival rate of first group of mice is that the survival rate of 45%, the second group of mice is that the survival rate of 45%, the three group of mice is that the survival rate of 45%, the four group of mice has only 15%, three vaccine processed group and the 4th group more all to have significant difference (P＜0.05).Explain that injection cyclophosphamide, the antibody of anti-CD25 or the RNA interfering of FoxP3 all can significantly improve the gp96 extract to the tumor suppression effect.

Claims

1. the application of immunomodulator in the immunocompetence that improves gp96 albumen related substances; Said immunomodulator is at least a in the RNA interfering of antibody and Foxp3 of cyclophosphamide, anti-CD25; The sequence 1 of the gp96 albumen shown in the sequence 1 that said gp96 albumen related substances is a sequence table, sequence table is from the N355 fragment shown in amino terminal the 1st to the 355 amino acids residue or contain the proteic extract of gp96; The RNA interfering of said Foxp3 is shown in the sequence 5 of sequence table.

2. use according to claim 1, it is characterized in that: the proteic extract of the said gp96 of containing is that the homogenate with exsomatize animal tissue or tumor tissue in vitro or stripped tumor cell carries out the ConA-Sepharose chromatography successively and HiTrap-Q Sepharose ion-exchange chromatography obtains; Said stripped animal tissue is isolated mouse liver, isolated pig liver or stripped people's Placenta Hominis; Said tumor tissues is breast carcinoma, cerebral glioma, renal carcinoma, intestinal cancer, hepatocarcinoma or gastric cancer; Said tumor cell is human liver cancer cell HepG2 or MC B16;

Said ConA-Sepharose chromatography comprises the steps:

(1) said homogenate is splined on chromatographic column, flow velocity is 0.25ml/min;

(2) with the chromatographic column of the eluent first washing step (1) of 10 times of chromatographic column column volumes, flow velocity is 0.5ml/min; The method for preparing of said eluent first is: in NaCl concentration is the PBS of 200mM pH7.5, adding Phenylmethanesulfonyl fluoride to final concentration is 1mM;

(3) with the chromatographic column of the eluent second washing step (2) of 3 times of chromatographic column column volumes, flow velocity is 0.25ml/min, collects eluent, is eluent third; The method for preparing of said eluent second is: in NaCl concentration is the PBS of 200mM pH7.5, adding α-D-Glucopyranose. to final concentration is 10g/mL, and adding Phenylmethanesulfonyl fluoride to final concentration is 1mM;

Said HiTrap-Q Sepharose ion-exchange chromatography comprises the steps:

(a) said eluent third is splined on chromatographic column, flow velocity is 1ml/min;

(b) use the chromatographic column of 5ml NaCl concentration as the PBS washing step (a) of 200mM pH7.5, flow velocity is 1ml/min;

(c) use the chromatographic column of 10ml NaCl concentration as the PBS washing step (b) of 300mM pH7.5, flow velocity is 1ml/min;

(d) use the chromatographic column of 3ml NaCl concentration as the PBS washing step (c) of 600mM pH7.5, flow velocity is 1ml/min, and the eluent that obtains is and contains the proteic extract of gp96.

3. vaccine adjuvant, for (a) as follows or (b) or (c):

(a) vaccine adjuvant of forming by immunomodulator and N355 fragment; Said immunomodulator is at least a in the RNA interfering of antibody and Foxp3 of cyclophosphamide, anti-CD25; The RNA interfering of said Foxp3 is shown in the sequence 5 of sequence table; The sequence 1 of said N355 sequence table fragment is from shown in amino terminal the 1st to the 355 amino acids residue;

(b) vaccine adjuvant of forming by immunomodulator and gp96 albumen; Said immunomodulator is at least a in the RNA interfering of antibody and Foxp3 of cyclophosphamide, anti-CD25; Said gp96 albumen is shown in the sequence 1 of sequence table; The RNA interfering of said Foxp3 is shown in the sequence 5 of sequence table;

(c) by immunomodulator with contain the vaccine adjuvant that the proteic extract of gp96 is formed; Said immunomodulator is at least a in the RNA interfering of antibody and Foxp3 of cyclophosphamide, anti-CD25; The RNA interfering of said Foxp3 is shown in the sequence 5 of sequence table.

4. a Hepatitis B virus vaccine is made up of gp96 albumen related substances, hepatitis B surface antigen, hepatitis B core albumen and immunomodulator; Said immunomodulator is at least a in the RNA interfering of antibody and Foxp3 of cyclophosphamide, anti-CD25; The sequence 1 of the gp96 albumen shown in the sequence 1 that said gp96 albumen related substances is a sequence table, sequence table is from the N355 fragment shown in amino terminal the 1st to the 355 amino acids residue or contain the proteic extract of gp96; The RNA interfering of said Foxp3 is shown in the sequence 5 of sequence table.

5. a vaccine that suppresses hepatitis B virus is made up of gp96 albumen related substances, hepatitis B surface antigen, hepatitis B core albumen and immunomodulator; Said immunomodulator is at least a in the RNA interfering of antibody and Foxp3 of cyclophosphamide, anti-CD25; The sequence 1 of the gp96 albumen shown in the sequence 1 that said gp96 albumen related substances is a sequence table, sequence table is from the N355 fragment shown in amino terminal the 1st to the 355 amino acids residue or contain the proteic extract of gp96; The RNA interfering of said Foxp3 is shown in the sequence 5 of sequence table.

6. a Hepatitis B virus vaccine is made up of gp96 albumen related substances, hepatitis B surface antigen epi-position 362-371, hepatitis B core albumen epi-position 87-95, hepatitis B polymerase epi-position 140-148 and immunomodulator; Said immunomodulator is at least a in the RNA interfering of antibody and Foxp3 of cyclophosphamide, anti-CD25; The sequence 1 of the gp96 albumen shown in the sequence 1 that said gp96 albumen related substances is a sequence table, sequence table is from the N355 fragment shown in amino terminal the 1st to the 355 amino acids residue or contain the proteic extract of gp96; The RNA interfering of said Foxp3 is shown in the sequence 5 of sequence table; Said hepatitis B surface antigen epi-position 362-371 is shown in the sequence 6 of sequence table; Said hepatitis B core albumen epi-position 87-95 is shown in the sequence 7 of sequence table; Said hepatitis B polymerase epi-position 140-148 is shown in the sequence 8 of sequence table.

7. a vaccine that suppresses hepatitis B virus is made up of gp96 albumen related substances, hepatitis B surface antigen epi-position 362-371, hepatitis B core albumen epi-position 87-95, hepatitis B polymerase epi-position 140-148 and immunomodulator; Said immunomodulator is at least a in the RNA interfering of antibody and Foxp3 of cyclophosphamide, anti-CD25; The sequence 1 of the gp96 albumen shown in the sequence 1 that said gp96 albumen related substances is a sequence table, sequence table is from the N355 fragment shown in amino terminal the 1st to the 355 amino acids residue or contain the proteic extract of gp96; The RNA interfering of said Foxp3 is shown in the sequence 5 of sequence table; Said hepatitis B surface antigen epi-position 362-371 is shown in the sequence 6 of sequence table; Said hepatitis B core albumen epi-position 87-95 is shown in the sequence 7 of sequence table; Said hepatitis B polymerase epi-position 140-148 is shown in the sequence 8 of sequence table.

8. a melanoma vaccines is made up of gp96 albumen related substances and immunomodulator; Said immunomodulator is at least a in the RNA interfering of antibody and Foxp3 of cyclophosphamide, anti-CD25; The sequence 1 of the gp96 albumen shown in the sequence 1 that said gp96 albumen related substances is a sequence table, sequence table is from the N355 fragment shown in amino terminal the 1st to the 355 amino acids residue or contain the proteic extract of gp96; The RNA interfering of said Foxp3 is shown in the sequence 5 of sequence table.

9. arbitrary described vaccine in vaccine adjuvant as claimed in claim 3 or the claim 4 to 8 is characterized in that: the proteic extract of the said gp96 of containing is that the homogenate with exsomatize animal tissue or tumor tissue in vitro or stripped tumor cell carries out the ConA-Sepharose chromatography successively and HiTrap-Q Sepharose ion-exchange chromatography obtains; Said stripped animal tissue is isolated mouse liver, isolated pig liver or stripped people's Placenta Hominis; Said tumor tissues is breast carcinoma, cerebral glioma, renal carcinoma, intestinal cancer, hepatocarcinoma or gastric cancer; Said tumor cell is human liver cancer cell HepG2 or MC B16;

Said ConA-Sepharose chromatography comprises the steps:

(2) with the chromatographic column of the eluent first washing step (1) of 10 times of chromatographic column column volumes, flow velocity is 0.5ml/min; The method for preparing of said eluent first is: in NaCl concentration is the PBS of 200mM pH7.5, adding Phenylmethanesulfonyl fluoride (PMSF) to final concentration is 1mM;

Said HiTrap-Q Sepharose ion-exchange chromatography comprises the steps:

10. vaccine as claimed in claim 9 or vaccine adjuvant is characterized in that: each component of said vaccine is all packed separately.