CN102796172A

CN102796172A - Hepatitis B virus (HBV) specific human leukocyte antigen-A33 (HLA-A33) restrictive epitope peptides and application thereof

Info

Publication number: CN102796172A
Application number: CN2011101357188A
Authority: CN
Inventors: 田波; 高福; 潘煦文
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2011-05-23
Filing date: 2011-05-23
Publication date: 2012-11-28
Anticipated expiration: 2031-05-23
Also published as: CN102796172B

Abstract

The invention relates to hepatitis B virus (HBV) specific human leukocyte antigen-A33 (HLA-A33) restrictive epitope peptides and application thereof. An HLA-A33 restrictive cell level epitope peptide screening platform is constructed; and 5 important HBV specific HLA-A33 restrictive epitope peptides (namely SEQ ID No: 5, 7, 9, 10 and 19) are determined by combining computer simulation analysis, detecting on the cell level epitope peptide screening platform and using three detection technologies of trimerical protein level renaturation, tetrameric patient's blood sample detection and Elispot detection. The invention also discloses the application of the screened HBV specific HLA-A33 restrictive epitope peptides in preparation of a medicament for treating hepatitis B in examinees, and specifically, the hepatitis B of the examinees is of HLA-A33 type. The invention also provides a medicament for treating the HLA-A33 hepatitis B in the examinees, and the medicament contains one or more of the 5 HBV specific HLA-A33 restrictive epitope peptides (namely SEQ ID No: 5, 7, 9, 10 and 19) and a medicinal carrier.

Description

The HLA_A33 restricted epitope peptide and the application thereof of one group of hepatitis B virus Idiotype

Technical field

The invention belongs to field of immunology, be specifically related to the HLA_A33 restricted epitope peptide and the application thereof of hepatitis B virus (HBV) Idiotype.

Background technology

One: the meaning of finding HLA_A33 restricted type HBV specific epitopes

Hepatitis B virus (Hepatitis B virus, HBV) infecting still is at present one of important health problem in the whole world, the nearly 300,000,000 5 thousand ten thousand chronic infection populations to 2009 whole world in the end of the year, directly or indirectly reach 100 ten thousand because of HBV infects dead number every year.China HBV infection population ratio is bigger, estimates near 10% of total population.HBV infects various clinical symptoms can occur, comprises acute, chronic, the hepatitis gravis that are divided into by disease incidence speed, and the liver cirrhosis, the liver cancer that occur by development process are also arranged, finally often dead because of liver failure.Pathogenesis to above-mentioned complicacy has been done many researchs, and what clear and definite conclusion was wherein arranged is the factors such as formation of the age bracket that infects, immunoreactive difference between individuals, immunological tolerance mechanism.But in difference between individuals; Receiving antigen-specific CTL (cytotoxic lymphocytes) the identification infected liver cell of the strict restriction of MHC-I (Major Histocompatibility Complex-I) quasi-molecule is one of important mechanisms of immune clearance; Simultaneously; At acute infection period also is the major cause that causes liver injury, and in chronic infection, then is to cause one of reason that this disease carries for a long time owing to CTL mechanism is suppressed the immunological tolerance that occurs.Therefore, no matter inducing antigen-specific CTL perhaps controls the special CTL of antigenicity, in the treatment of HBV, all has vital role.And the t cell epitope peptide of identifying antigen-specific is the basis in the basis of this theoretical investigation and practical application.

Three kinds of HLA_A that are usually used in immune Research, B, DRB1 (Human leukocyte antigen_A, B; DRB1) during gene polymorphic type distributes, specifically more concentrated with the distribution of HLA_A, the wherein distribution in HLA_A site; Mainly with HLA_A02, A1, A24; A33, A30 are main, add up to account for about 86%.Therefore, the HBV specificity epitope of several important types in research HLA_A site is an important inlet analyzing difference between individuals.By the end of the year 2004, to HLA_A02,11 have had more deep research, find to have a plurality of epi-positions, and the discovery of HLA_A33 type epi-position are almost nil.And HLA_33 type crowd's genotype accounts for total crowd's 9%, adds the very akin HLA_A31 with HLA_A33, and both totals account for total crowd's about 12%, therefore, finds the HBV specificity epitope of HLA_A33, has great significance.

Two: epitope peptide is in the effect of immunological tolerance disease therapeutic

Immunotolerance virus is meant the virus that can cause the body immune system response capacity low.The mechanism of the initiation immunological tolerance of various viruses is different, but the ability drop or the disappearance that finally all can cause the B cell to produce antibody, T cell and cellular immunization, both one of or both all have, and make the lasting existence of virus.

The immunotolerance virus immunity of organism of can effectively escaping causes immunological tolerance, makes the immunologic mechanism of human body no longer resist former generation immunne response, makes virus long-term existence in vivo.HBV is typical immunotolerance virus, and susceptible mother produces 90% newborn infant becomes the virus carrier, and the HBV patient that partly is grown up also can form the chronic infection of hepatitis B.The variation of the persistent infection of HBV and antigenic overexpression and virus often causes the Th1 class cell relevant with immunomodulatory viral special in virus replication lasting in patient's body, the thymus gland to be cloned ctl response ability drop relevant with the cells infected cracking with virus sweep in rejecting, peripheral blood and the liver, disappear or clone rejecting.

Recent a series of research shows that antigen of hepatitis B virus specific cytotoxic T lymphocyte responses is significant in hepatitis B infected.The HBV specific CTL is removed the HBV effect through non-damages of performance such as a large amount of IFN-γ of secretion; Before this effect occurs in hepatocellular injury, the follow-up hepatocellular injury that causes maybe with cause that the non-specific immunity active cells soaks into after specificity is replied in liver, apoptosis activation etc. is relevant.Further research discloses, and it is closely related that the difference of HBV specificity answering and hepatitis B patient different clinical lapses to.In self limiting acute hepatitis B patient; The specific CTL effect of replying is strong; Possibly removed rapidly with virus and hepatocellular injury is gently relevant; And in chronic hepatitis B patient, a little less than the specific CTL responsibility, possibly cause inflammatory cell infiltration relevant with removing the virus capable deficiency with chronicity.Recently study and cognition arrives, and possibly be a kind of effective way that promotes chronic viral hepatitis B patient's virus sweep and body recovery through stimulating specific CTL to reply.

Summary of the invention

The invention belongs to field of immunology, be specifically related to the HLA_A33 restricted epitope peptide and the application thereof of hepatitis B virus (HBV) Idiotype.Particularly, the present invention has made up the cell levels epitope peptide screening platform of HLA_A33 restricted type, and through combining computer simulation analysis; On this cell levels epitope peptide screening platform, detect, unite the horizontal renaturation of utilization trimer protein, tetramer patient blood examination, Elispot and detect three kinds of detection techniques, the HLA_A33 restricted epitope peptide of having confirmed 5 important hepatitis B virus Idiotypes (promptly; SEQ ID No:5; 7,9,10 and 19).The present invention also provides the application in the medicine that the HLA_A33 restricted epitope peptide of the hepatitis B virus Idiotype that is screened is used for preparing the hepatitis B of treating the experimenter, and more specifically, said experimenter's hepatitis B is the HLA_A33 type.The present invention also provides the medicine of HLA_A33 type hepatitis B among a kind of experimenter of treatment, and said medicine comprises one or more in the HLA_A33 restricted epitope peptide (that is, SEQ ID No:5,7,9,10 and 19) of above-mentioned 5 kinds of HBV Idiotypes, and pharmaceutical carrier.

Particularly, the invention is characterized in the known technology of utilization, made up the cell levels epitope peptide screening platform of HLA_A33 restricted type; And, 16 HBVB have been sought, candidate's peptide of C hypotype through computer simulation analysis; And after further screening the detection of platform through above-mentioned cell levels, unite and use the horizontal renaturation of trimer protein, tetramer patient blood examination, Elispot to detect three kinds of detection techniques, confirmed 5 important epitope peptides (promptly; Its amino sequence is SEQ ID No:5; 7,9,10 and 19).

In one aspect of the invention; The invention provides a kind of screening platform of the HLA_A33 of being used for restricted type cell levels epitope peptide; This platform is to utilize VSVG pseudovirus system (see below term explanation) stable cell lines constructing technology, changes the HLA_A33 type gene after optimizing over to RMA-S cell (available from the multiple auspicious bio tech ltd in Shanghai), and this cell is the cell of a kind of TAP enzyme (antigen presentation be correlated with translocator) defective type; When cultivating for 37 ℃, do not present epi-position to cytolemma.Thereby can't detect the epi-position of this albumen on film, and after adding exogenous peptide,, and form stabilized complex with the β 2m albumen of prior adding if this albumen can combine with peptide, can on epicyte, detect this albumen existence.Can judge thus whether peptide combines with special HLA_A33 molecule.

In another aspect of the present invention, the horizontal renaturation of integrated use trimer protein of the present invention, tetramer patient blood examination, Elispot detect three kinds of detection techniques, have found HLA_A33 restricted epitope peptide and candidate's epitope peptide of 5 HBV Idiotypes.Particularly, the aminoacid sequence of the HLA_A33 restricted epitope peptide of these 5 HBV Idiotypes is respectively:

GYRWMCLRR (peptide 1, SEQ ID No:5);

YLWEWASVR (peptide 3, SEQ ID No:7);

LVSFGVWIR (peptide 5, SEQ ID No:9);

HISCLTFGR (peptide 6, SEQ ID No:10);

VVDFSQFSR (peptide 17, SEQ ID No:19).

In another aspect of the present invention; The present invention through the Elispot experimental verification secreting function that in the hepatitis B patient body, can excite IFN-γ of HLA_A33 restricted epitope peptide of above-mentioned 5 HBV Idiotypes, confirmed that through tetramer technology above-mentioned 5 peptides can combine with CD8+ cell-specific in the specific peripheral blood in the hepatitis B patient body.Particularly, the HLA_A33 restricted epitope peptide of HBV Idiotype of the present invention has that the CD8+ cell recognition has infected the cell of HBV and brought out immunoreactive ability simultaneously in the primosome.

In a preferred embodiment of the invention, the HLA_A33 restricted epitope peptide that the invention provides the hepatitis B virus Idiotype that is screened is used for preparing the application in the medicine of the hepatitis B of treating the experimenter.More specifically, said experimenter's hepatitis B is the HLA_A33 type.Said experimenter is a Mammals, comprises people and non-human mammal, for example, changes the hepatitis B model mice of people HLA_A33, perhaps is fit to the hepatitis B patient of immunotherapy, or the like.

In a preferred embodiment of the invention, the present invention provides the medicine of HLA_A33 type hepatitis B among a kind of experimenter of treatment, and the HLA_A33 restricted epitope peptide that said medicine comprises above-mentioned 5 kinds of HBV Idiotypes (promptly; SEQ ID No:5,7,9; 10 and 19) one or more in, and pharmaceutical carrier.More specifically, said experimenter is the people.

In addition, when being used for this paper, being defined as of following term:

HLA_A33: human leucocyte antigen (HLA) (Human Leucocyte Antigen) A serotype 33 hypotypes.

GR-9: in the t cell epitope of agreement was called for short, first of epitope peptide commonly used and last amino acids residue added that the length of peptide representes, can be used for representative peptide GYRWMCLRR like GR-9.Since among this paper identical AR-9 etc. have a plurality of, so this paper when narrating with peptide 1, the mode of peptide 2 is main.

VSVG pseudovirus system: VSVG is herpes stomatitis virus gp G, and it can be brought the gene in the pseudovirus of packing in the multiple eukaryotic cell into through receptor mediated endocytosis.What use in this experiment is can integrated slow virus pUC pUC.

CTL (cytotoxic lymphocytes): behind the HBV infected liver cell; Virus antigen is cracked into micromolecular oligopeptides by the proteasome of antigen presenting cell (APC); And with liver cell in the HLA-I quasi-molecule be combined into mixture, be expressed in cell surface, become t cell epitope.CTL produces adhesion through the CD8+ molecule and the HLA-I quasi-molecule on surface, and discerns above-mentioned oligopeptides epi-position by TXi Baoshouti, and this type of CTL is HBV antigen-specific CTL (HBV antigens specific cytotoxic lymphocytes, HBV sCTL).

HBV: (Hepatitis B virus): hepatitis B virus.

PBMC: PMNC

ELISPOT: enzyme linked immunological spotting method (enzyme linked immunospot assay)

Tetramer-PE: with the specific tetramer of PE mark, each monomer in the tetramer is by heavy chain, the β 2m of HLA_A33, and peptide is formed.The difference of nine kinds of different Tetramer-PE is wherein contained peptide difference among this paper.

Description of drawings

From the detailed description below in conjunction with accompanying drawing, above-mentioned feature and advantage of the present invention will be more obvious, wherein:

Fig. 1 show peptide combines result of experiment; The line of mark "+" is the peak (peptide concentration be 50mM) of known positive peptide FFVDGAANR when the cell surface that has HLA_A33 forms tripolymer; Result when the known positive peptide FDVDGAANR of mark "-" combines; Other peak is for only adding peptide and β 2m, but clone is the result of untransfected gene.The MRS-A cell of the transfection HLA_A33 that presentation of results is constructed can be used to screen can with the peptide section of HLA_A33 specific combination.

Fig. 2 shows the cell screening result of peptide 1-5 of the present invention and 7-8.S2-3 is meant the control group that does not add peptide, and L1-8 is the experimental group that adds peptide; Antibody staining is the W6/32 with the FITC mark, and cell counting is 500,000.As can be seen from the figure,

peptide

1,3,5 can obviously strengthen the trimerical expression of the special A3303 of cell surface.And the skew of the combination peak of 2,4,7,8 figure is not obvious, explain at cell levels, and in these several peptides,

peptide

1,3,5 can form tripolymer with HLA_A33 and β 2m.

The sieve chromatography figure that Fig. 3 display body is folding outward.Wherein transverse axis is the volume of wash-out, and the longitudinal axis is the absorbance value of 280nm.Fig. 3 A: peptide 1, Fig. 3 B: peptide 3, Fig. 3 C: peptide 5, Fig. 3 D: peptide 6; Fig. 3 E: peptide 17, demonstration be that (that is, peptide 1 for five peptides in 9 folding male peptides; Peptide 3, peptide 5, peptide 6; Peptide 17) the molecular sieve figure behind folding, Fig. 3 F is known positive control, Fig. 3 G is the folding result of known negative control.This presentation of results, the peak of HC+ β 2m be at peptide 1, peptide 3, peptide 5, peptide 6, peptide 17 can with the HLA_A33 heavy chain external its when folding, can form good tripolymer result.

Fig. 4: shown the result of two routine HLA_A33 type hepatitis B patient blood sample Elispot, peptide 1, peptide 3, peptide 5, peptide 6, peptide 17 all can stimulate generation greater than the spot more than 6, explain that these 5 peptides all can bring out immunoreation.

Fig. 5: shown HLA_A33 type hepatitis B patient blood sample with the painted result of the tetramer, explain in patient's body existence can with the cell of these five the formed HLA_A33 tetramer of peptide bonded, CD8+ stained positive.

Embodiment

Below come further to illustrate the present invention through embodiment.But should be appreciated that said embodiment is illustrational purpose, and be not intended to limit scope of the present invention and spirit.

The structure of the restrictive epitope peptide screening of embodiment 1:HLA_A33 platform

The structure of 1:HLA_A3303 stable cell lines:

In the present embodiment, use VSVG pseudovirus system respectively (available from Biovector Science Lab, Inc) form; (hereinafter to be referred as people HLA_A3303, GenBank:U09740.1) two ends add NheI, behind the XhoI site with people's the full gene of HLA_A3303; (nucleotide sequence is SEQ ID No:1 to complete synthesis this sequence of rising sun hat biotech firm in Shanghai; Its expression can produce the represented albumen like SEQ ID No:2), utilize NheI again, it (is that above-mentioned VSVG pseudovirus system is (available from Biovector Science Lab that two sites of XhoI are implemented in carrier plentilox 3.7-RRP; Inc) plasmid in); Form recombinant plasmid plentilox-HLA_A33, carry this recombinant plasmid 15 μ g greatly, again with 5ug VSVG plasmid; 5 μ g RSV/REV plasmids, the common transfection of 5 μ g pMDLG plasmids (above-mentioned three kinds of plasmids are the plasmid in the above-mentioned VSVG pseudovirus system equally) prepared that cell is good, cell degree of converging is the 10cm of 70-80% in 24 hours in advance ²293T cell in the Tissue Culture Dish (this cell is available from NIH), transfection is carried out with conventional liposome method.At 37 ℃, 5%CO ₂Following cultivation, changed liquid in 4 hours after, similarity condition is cultivated sucking-off cells and supernatant after 48 hours again, 2000～3000rpm, under 18 ℃ of temperature centrifugal 10 minutes.Collect supernatant.In-80 ℃ of preservations.This is the pseudovirus of the packaged HLA_A3303 of having gene.

Infect RMA-S cell (available from the multiple auspicious bio tech ltd in Shanghai) with above-mentioned pseudovirus; With the clone of tetracycline (available from AMRISO) the screening stable transfection of the concentration of 2～4 μ g/mL, detect and the painted method of cell fluorescence confirms that the clone of stable transfection is expressed in HLA_A3303 on the epicyte through Western Blot.

2: peptide synthetic

The aminoacid sequence of the positive peptide that this experiment is used is FDVDGAANR as the aminoacid sequence of FFVDGAANR, negative peptide, every peptide Synthetic 2 0mg, and 95% purity, synthetic by Shanghai gill biochemical corp.

3: peptide combines experiment

The RMS-A-A3303 cell was cultivated 14～18 hours at 26 ℃, and with the PBS re-suspended cell that contains 20% foetal calf serum (available from GIBICO), adjustment concentration is every milliliter of 2*10 ⁵Individual cell, 50 μ L are got in each experiment, and adding positive peptide and negative peptide respectively, to make ultimate density be gradient 0.1 μ M; 1 μ M, 10 μ M, 100 μ M; 1mM adds β 2m microglobulin (available from Sigma) (final concentration is 10 μ g/mL), and each experimental group repeats 3 multiple holes; Cultivate after 1 hour, place 37 ℃ to cultivate again 3 hours for 26 ℃.After cell washed with the PBS that contains 20% foetal calf serum, 4 ℃, 500g was centrifugal; RV is 50 μ L, puts on ice, adds the HLA_A antibody W6/32 (available from eBioscience) of FITC mark; Hatched under the room temperature 10 minutes, after PBS washs 3 times, the flow cytometer fluorescence intensity.

Fig. 1 show peptide combines result of experiment; The line of mark "+" is the peak (peptide concentration be 50mM) of known positive peptide FFVDGAANR when the cell surface that has HLA_A33 forms tripolymer; Result when the known positive peptide FDVDGAANR of mark "-" combines; Other peak is for only adding peptide and β 2m, but clone is the result of untransfected gene.The RMS-A cell of the transfection HLA_A33 that presentation of results is constructed can be used to screen can with the peptide section of HLA_A33 specific combination.

The screening of embodiment 2:HBV Idiotype HLA_A33 specificity epitope

With the following resource of having announced on the network, to the B of HBV, possible candidate's polypeptide of C hypotype is predicted respectively:

The 3D-QSAR program is MHCPred

Marking matrix program is SYFPEITHI

The TAP program is TAPpred

Result by the three has selected 16 peptides as candidate's polypeptide.The long and sees the following form 1.

Combine the method for experiment by above-mentioned peptide, it is that other method is constant the 50 μ M that the peptide concentration during except screening is fixed as final concentration, in candidate's peptide of screening prediction can with HLA_A3303 bonded epitope peptide.

Fig. 2 shows the cell screening result of peptide 1～5 of the present invention and 7～8.S2～3 are meant the control group that does not add peptide, and L1～8th adds the experimental group of peptide; Antibody staining is the W6/32 with the FITC mark, and cell counting is 500,000.As can be seen from the figure,

peptide

1,3,4 can form tripolymer with HLA_A33 and β 2m.

Embodiment 3: the tripolymer renaturation is to the evaluation of candidate's peptide

The 1:HLA_A3303 inclusion bodies of colibacillus is expressed and purifying:

After the codon sequence of HLA_A3303 and mRNA sequence be optimized (nucleic acid sequence encoding after the optimization is SEQ ID No:3); With the expression cassette of optimizing, to be cloned among the carrier pET30A (available from Novagen) with enzyme NdeI-XhoI, the clone of formation is called pET30-HLA_A33HC-LND; Its expression can produce the represented albumen like SEQ ID No:4; This albumen contains the α 1 of HLA_A quasi-molecule, and α 2, α 3 structural domains and one section biotinylation site sequence LND.

By the phraseology of routine, when intestinal bacteria (E.coli) bacterium liquid OD value reached 0.7, it was 0.5mMol/L that adding IPTG makes final concentration, induces after 4 hours for 37 ℃ and receives bacterium; With N,O-Diacetylmuramidase (sigma) 5mg/ (every liter of original substratum) room temperature treatment 1 hour, use-80 ℃ of freeze thawing treatment again after, with inclusion body washings (1mM DTT, 0.5%Triton X-100,50mM Tris-HCl, 100mM NaCl; PH=8.0) washing inclusion body, 13000g, adds above-mentioned washings again by 4 ℃ after centrifugal 10 minutes; It is ultrasonic that (1cm ultrasonic head, 300W under the condition of ice bath, surpass 4 seconds; Stopped 6 seconds, and surpassed altogether 200 times), washing, centrifugal, ultrasonic again; After repeating three circulations, dissolve with 8M urea.This is the HLA_A33HC-LND albumen of purifying.

2: trimerical renaturation

HLA_A33HC-LND inclusion body protein, the β 2m of 13.2mg, the synthetic candidate's peptide of 12mg of the purifying of 18.6mg are added in the (gsh of 400mM arginine hydrochloride, 2mMNaEDTA, 0.5mM Sleep-promoting factor B, 5mM reductibility: pH=8.0) in the 250mL l-arginine renaturation buffer; 4～8 ℃ of renaturation are after 12 hours; Concentrate cup (available from Millipore) with 10KD volume is reduced to 10mL; Again with the dialysis of 4 ℃ of distilled waters after 24 hours,, and damping fluid is replaced as 20mM Tris with the ultrafiltration pipe processing of the 50mL 10KD of Millipore; 50mM NaCl, pH=8.0.TV less than 4mL after, detect the efficient of renaturation altogether with Superdex 200HR molecular sieve column, form giving instructions in reply property of the schedule of proportion efficient that trimerical ratio accounts for total protein with renaturation.Renaturation result sees the following form 2.

Table 2: the comparison sheet of main positive peptide folding efficiency

The positive peptide sequence of CK+ be FFVDGAANR,

The negative peptide sequence of CK-is FDVDGAANR

From table, can find out,

peptide

1,3,5,6,11,14,17 can form the good in vitro renaturation with HLA_A3303, belong to strong binding peptide, and

peptide

12,13 is weak relatively binding peptide.

The partial results that renaturation is relevant is presented among Fig. 3.

Embodiment 4:Elispot technology is to the evaluation of candidate's peptide

Under the prerequisite that hepatitis B patient is known the inside story, get hepatitis B patient blood sample, whether by HLA_A site somatotype reagent (available from Beijing Sheng Taihua Bioisystech Co., Ltd) operation instruction of standard, detecting blood sample is the HLA_A33 type.The old XX of hepatitis B patient wherein, woods XX is the HLA_A33 type with the blood sample of waiting XX, is used for follow-up experiment and detects.

With A33 type blood sample 6mL, by the PBMC (human lymphocyte parting liquid Ficoll paque plus is available from GE healthcare) of standard program separation peripheral blood, lymphocytes culture medium (reaching section available from Shenzhen is Bioisystech Co., Ltd) adjustment concentration is 2.5*10 ⁶Individual cell/mL;

By the explanation of people IFN-gama Elispot detection kit (reaching section available from Shenzhen is Bioisystech Co., Ltd), behind lymphocytes culture medium (reaching section available from Shenzhen is Bioisystech Co., Ltd) activating reagent bar, add 100 μ L and contain 2.5*10 ⁶The above-mentioned isolating patient P BMC of individual cell/L.Adding final concentration in the experimental group is the peptide stimulation of 10 μ g/mL, and it is that 4ug/mL concanavalin A albumen (Con A) stimulates that positive control adds final concentration, and negative control does not add any stimulant, and each sample is established 3 multiple holes.In cell culture incubator, after 14 hours, remove substratum, in every hole, add the deionized water of 100 μ L precoolings, come lysing cell, abandon supernatant after 10 minutes, with washings washing precipitation 3 times in 4 degree held 37 ℃ of cultivations.

In every hole, add biotin labeled resisting-IFN-γ monoclonal antibody (test kit carries), hatched 1 hour for 37 ℃, after the cleaning; Add again and detect antibody (test kit carries); Hatch after 1 hour repetitive scrubbing once more for 37 ℃, add supporting developer, 37 ℃ after 10～20 minutes; Obviously use the tap water stopped reaction in the back Deng spot, read spot with the spot plate reading machine behind the airing and form number (SFC).

Table 3:Elispot result

Conclusion:

peptide

1,3,5,6,17 is five main peptides,

peptide

11,12 is two accessory peptides

Fig. 4: shown the result of two routine HLA A33 type hepatitis B patient blood sample Elispot, peptide 1, peptide 3, peptide 5, peptide 6, peptide 17 all can stimulate generation greater than the spot more than 6, explain that these 5 peptides all can bring out immunoreation.

Embodiment 5: tetramer technology is to the evaluation of candidate's peptide

1: tetrameric preparation

Get the 1L HLA_A33HC-LND of the tripolymerization of renaturation altogether as stated above, with the 10kD of Millipore concentrate glass be concentrated to 700 μ L volumes after, with biotinylation test kit (available from Guangzhou?FulenGen?Co., Ltd.); With this Biotinization (700 μ L trimerizing albumen, 100 μ L solution A, 100 μ L solution B; 100 μ L d-vitamin Hs, 10 μ L BirA enzymes, 0.5 μ L Pepstatin (storage liquid concentration: 2mg/mL; In DMSO), the bright peptide (leupeptin) (storage liquid concentration: 2mg/mL is in dH2O) that presses down of 0.5 μ L; 30 ℃ of reactions are after 12 hours, with molecular sieve Superdex 200 HR 10/30 collection tripolymer peak wherein.Concentrate less than behind the 1mL with the 10KD evaporating pipe, the damping fluid of substitutional solution is PBS again, and with the quantitative albumen of protein quantification test kit of Bio-Rad; Adjustment protein concentration 5mg/mL, in molar ratio, promptly; The streptavidin (available from eBioscience) of 1 part of PE mark is joined 5 parts of molar ratios in this biotinylated protein; The streptavidin that progressively adds the PE mark behind the polymerized at room temperature, is removed unconverted monomer with the evaporating pipe of 100kD again.The tetramer that keeps in Dark Place and prepare.

2: the collection of blood sample and tetramer dyeing

Agree through patient; After signing blood sample joint study agreement, get the hepatitis B patient blood sample, extract test kit with Promega blood sample genome and extract the DNA sample from China-Japan Friendship Hospital and You An hospital; Use HLA_A site somatotype reagent (available from Beijing Sheng Taihua Bioisystech Co., Ltd) to detect patient's HLA_A somatotype again; For the somatotype result is the sample of HLA_A33, further uses the PBMC in lymphocyte separation medium (Ficoll paque plus is available from the GE healthcare) sample separation; Use the RMPI 1640 substratum re-suspended cells that contain 10% foetal calf serum, and cell concn is adjusted into 2*10 ⁶/ mL joins 24 well culture plates and cultivates, and every hole adds the corresponding epitope peptide of 10 μ g/mL respectively.The human IL-2 (available from eBioscience) who adds the 30U/mL reorganization in the time of the 3rd day.Amount was changed liquid in per 3 days half later on, and added the IL-2 of 30U/mL.The 10th day collecting cell also used the tetramer and the CD8 fluorescent antibody staining for preparing, with the quantity of flow cytometer detection specificity CTL.The result is presented among Fig. 5.In the presentation of results patient body of Fig. 5 existence can with the cell of the formed HLA_A33 tetramer bonded of these five peptides (peptide 1, peptide 3, peptide 5, peptide 6, peptide 17), CD8+ stained positive.Two positive cells accounted for the ratio of total CD8+ cell before 0.417% to 1.836%, contrasted negative peptide (peptide 16) and had only 0.012%, and wherein the highest is No. 5 peptides, this and Elispot data consistent.These five and be the restrictive HBV type specificity of HLA_A33 epi-position have also been confirmed.

Should be appreciated that; Although with reference to its exemplary embodiment; The present invention is shown particularly and describe, but will be understood by those skilled in the art that, under the condition that does not deviate from by the defined the spirit and scope of the present invention of accompanying Claim; The variation of various forms and details can be carried out therein, the arbitrary combination of various embodiments can be carried out.

The sequence explanation:

SEQ ID No:1, the people HLA_A3303 gene order of synthetic, two ends have NheI, the XhoI restriction enzyme site;

SEQ ID No:2, the HLA_A3303 protein sequence of SEQ ID No:1 coding;

SEQ ID No:3 carries out the encoding sequence of codon optimized HLA_A3303 to intestinal bacteria;

SEQ ID No:4, the HLA_A3303 protein sequence of SEQ ID No:3 coding;

The B of the HBV of SEQ ID No:5-20:16 bar prediction, candidate's amino acid sequence of polypeptide of C hypotype.

Need to prove that primary HLA_A33 gene has 6 functional zone, is respectively from front to back: signal peptide, α 1, and α 2, and α 3, stride film district and intracellular region totally six parts, and the sequence of SEQ ID No:2 is consistent with this sequence; Because HLA_A33 combines with peptide, and combining back and TCR bonded functional zone with peptide, α 2 decisions by α 1; The decision of α 3 districts combines with β 2m; Thereby when external renaturation and the tetramer prepare, all be to keep these three districts, and after carboxyl terminal adds top connection (gsgsg); Increase a biotinylated label (lndifeaqkiewhe) again, the aminoacid sequence after the change is represented by SEQ IDNo:4.On behalf of complete HLA_A33 (SEQ ID No:2), this sequence can fully combine with peptide in experiment in vitro; Combine with β 2m; With after peptide, β 2m combine again with these three kinds of functions of TCR bonded.

Claims

1. the HLA_A33 restricted epitope peptide of one group of hepatitis B virus Idiotype, it is made up of following aminoacid sequence: GYRWMCLRR, YLWEWASVR, LVSFGVWIR, HISCLTFGR,, or VVDFSQFSR.

2. the HLA_A33 restricted epitope peptide of the described hepatitis B virus Idiotype of claim 1 is used for preparing the application in the medicine of the hepatitis B of treating the experimenter, and wherein said experimenter's hepatitis B is the HLA_A33 type.

3. the described application of claim 2, wherein said experimenter is a Mammals.

4. the described application of claim 3, wherein said experimenter is the people.

5. medicine of treating HLA_A33 type hepatitis B among the experimenter, it comprises in the HLA_A33 restricted epitope peptide of hepatitis B virus Idiotype of claim 1 one or more, and pharmaceutical carrier.

6. the described medicine of claim 5, wherein said experimenter is the people.