CN102796172B - Hepatitis B virus (HBV) specific human leukocyte antigen-A33 (HLA-A33) restrictive epitope peptides and application thereof - Google Patents
Hepatitis B virus (HBV) specific human leukocyte antigen-A33 (HLA-A33) restrictive epitope peptides and application thereof Download PDFInfo
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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
Technical field
The invention belongs to field of immunology, be specifically related to HLA_A33 restricted epitope peptide and the application thereof of hepatitis B virus (HBV) Idiotype.
Background technology
One: the meaning finding HLA_A33 restricted type HBV specific epitopes
Hepatitis B virus (Hepatitis B virus, HBV) infection is still one of global important health problem at present, to the 2009 nearly 300,000,000 5 thousand ten thousand chronic infection populations in the whole world in the end of the year, the number of the direct or indirect death because of HBV infection every year reaches 100 ten thousand.China HBV infection crowd ratio is larger, estimates 10% close to total population.Can there is various clinical symptoms in HBV infection, comprise acute, chronic, the hepatitis gravis be divided into by disease incidence speed, also has the liver cirrhosis, the hepatocarcinoma that occur by development process, finally normal dead because of liver failure.Done many research to the pathogenesis of above-mentioned complexity, what wherein have clear and definite conclusion is the factor such as formation of the age bracket infected, immunoreactive individual variation, Mechanism of immunotolerance.But in individual variation, the Peptide-specific CTL (cytotoxic lymphocytes) strictly limited by MHC-I (Major Histocompatibility Complex-I) quasi-molecule identifies that infected liver cell is one of important mechanisms of immune clearance, simultaneously, it is also the main cause causing hepatic injury at acute infection period, and in chronic infection, because the immunologic tolerance of the suppressed appearance of CTL mechanism is then one of reason causing this disease to be carried for a long time.Therefore, no matter inducing antigen-specific CTL or control the special CTL of antigenicity, all has important function in the treatment of HBV.And identify that the t cell epitope peptide of antigen-specific is the basis in the basis of this theoretical research and practical application.
Be usually used in three kinds of HLA_A, B, DRB1 (Human leukocyteantigen_A of immune Research, B, DRB1) in Gene polymorphism distribution, specifically more concentrated with the distribution of HLA_A, the wherein distribution in HLA_A site, main with HLA_A02, A1, A24, A33, A30 is main, adds up to and accounts for about 86%.Therefore, the HBV specificity epitope of the several important type in research HLA_A site is the important entrance analyzing individual variation.Ended for the end of the year 2004, to HLA_A02,11 have had more deep research, find to there is multiple epi-position, and HLA_A33 type epi-position finds almost nil.And the genotype of HLA_33 type crowd accounts for total crowd's 9%, add the very akin HLA_A31 with HLA_A33, both totals account for total crowd's about 12%, therefore, find the HBV specificity epitope of HLA_A33, have great significance.
Two: epitope peptide is in the effect of immunologic tolerance disease treatment
Immunologic tolerance virus refers to the virus that body immune system respond can be caused low.The mechanism of the initiation immunologic tolerance of various virus is different, but the ability that B cell finally all can be caused to produce antibody, T cell and cellular immunization declines or disappears, both one of or both all have, and make the sustainable existence of virus.
Immunologic tolerance virus can effectively be escaped immunity of organism, causes immunologic tolerance, the immunologic mechanism of human body is no longer resisted and originates in raw immunne response, make virus long-term existence in vivo.HBV is typical immunologic tolerance virus, and susceptible mother produces 90% neonate becomes virus carrier, and part adult HBV patient also can form the chronic infection of hepatitis B.In the virus replication that the variation of the persistent infection of HBV and the overexpression of antigen and virus often causes continuing in patient body, thymus, the special Th1 class cell relevant to immunomodulating of virus is cloned the ctl response ability of being correlated with infection cell cracking with virus sweep in rejectings, peripheral blood and liver and declines, disappears or clone and reject.
Recent a series of research display, antigen of hepatitis B virus specific cytotoxic T lymphocyte responses is significant in hepatitis B infected.HBV specific CTL plays non-damaging by a large amount of IFN-γ of secretion etc. and removes HBV effect, before this effect occurs in hepatocyte injury, the follow-up hepatocyte injury caused causes after may replying with specificity that nonspecific immunity competent cell infiltrates in liver, apoptosis activation etc. is relevant.Further research discloses, and the difference of HBV specificity answering is closely related from the different Clinical Outcomes of hepatitis B patient.In self limiting acute hepatitis B patient, specific CTL response effect is strong, may be rapidly cleared in humans with virus and hepatocyte injury is gently relevant, and in chronic hepatitis B patient, specific CTL responsibility is weak, may be not enough with removing virus capable and cause inflammatory cell infiltration relevant with chronicity.Recently research is recognized, may be a kind of effective way promoting Chronic Hepatitis B virus sweep and body recovery by stimulating specific CTL response.
Summary of the invention
The invention belongs to field of immunology, be specifically related to HLA_A33 restricted epitope peptide and the application thereof of hepatitis B virus (HBV) Idiotype.Particularly, the present invention constructs the cellular level epitope peptide Screening Platform of HLA_A33 restricted type, and by conjunction with computer simulation analysis, this cellular level epitope peptide Screening Platform detects, and the horizontal renaturation of conjunctive use trimer protein, tetramer patient blood examination, Elispot detect three kinds of detection techniques, confirm the HLA_A33 restricted epitope peptide of 5 important hepatitis B virus Idiotypes (namely, SEQ ID No:5,7,9,10 and 19).The HLA_A33 restricted epitope peptide that present invention also offers screened hepatitis B virus Idiotype is for the preparation of the application in the medicine of the hepatitis B in treatment experimenter, and more specifically, the hepatitis B of described experimenter is HLA_A33 type.The present invention also provides a kind of medicine for the treatment of HLA_A33 type hepatitis B in experimenter, and described pharmaceutical pack is containing HLA_A33 restricted epitope peptide (that is, the SEQ ID No:5 of above-mentioned 5 kinds of HBV Idiotypes, 7,9,10 and 19) one or more in, and pharmaceutical carrier.
Specifically, the invention is characterized in and use known technology, construct the cellular level epitope peptide Screening Platform of HLA_A33 restricted type, and pass through computer simulation analysis, 16 HBVB are found, the candidate peptide of C hypotype, and after detection further by above-mentioned cell-based screening platform, the horizontal renaturation of conjunctive use trimer protein, tetramer patient blood examination, Elispot detect three kinds of detection techniques, confirm 5 important epitope peptides (that is, 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 for HLA_A33 restricted type cellular level epitope peptide, this platform utilizes VSVG pseudovirus system (term seen below illustrates) stable cell lines constructing technology, HLA_A33 type gene after optimizing is proceeded to RMA-S cell (purchased from Fu Xiang bio tech ltd, Shanghai), this cell is the cell of a kind of TAP enzyme (antigen presentation be correlated with transport protein) deficiency, when cultivating for 37 DEG C, do not present epi-position on cell membrane.Thus cannot detect the epi-position of this albumen on film, after adding exogenous peptide, if this albumen and peptide can be in conjunction with, and form stable complex with the β 2m albumen added in advance, can detect on epicyte that this albumen exists.Can judge whether peptide is combined with special HLA_A33 molecule thus.
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, by Tetramer technology, the secretory function that can excite IFN-γ in hepatitis B patient body of the HLA_A33 restricted epitope peptide of the present invention's above-mentioned 5 HBV Idiotypes by Elispot experimental verification, confirms that above-mentioned 5 peptides can be combined by CD8+ cell-specific in hepatitis B patient body in specific peripheral blood.Specifically, the HLA_A33 restricted epitope peptide of HBV Idiotype of the present invention has CD8+ cell recognition in primosome and has infected the cell of HBV and brought out immunoreactive ability simultaneously.
In a preferred embodiment of the invention, the invention provides the HLA_A33 restricted epitope peptide of screened hepatitis B virus Idiotype for the preparation of the application in the medicine of the hepatitis B in treatment experimenter.More specifically, the hepatitis B of described experimenter is HLA_A33 type.Described experimenter is mammal, comprises people and non-human mammal, such as, turns the hepatitis B model mice of people HLA_A33, or the hepatitis B patient of applicable immunization therapy, etc.
In a preferred embodiment of the invention, the invention provides a kind of medicine for the treatment of HLA_A33 type hepatitis B in experimenter, (namely described pharmaceutical pack contains the HLA_A33 restricted epitope peptide of above-mentioned 5 kinds of HBV Idiotypes, SEQ ID No:5,7,9,10 and 19) one or more in, and pharmaceutical carrier.More specifically, described experimenter is people.
In addition, when used herein, following term is defined as:
HLA_A33: human leucocyte antigen (HLA) (Human Leucocyte Antigen) A serotype 33 hypotype.
GR-9: in the t cell epitope of agreement is called for short, first of conventional epitope peptide and last amino acids residue add that the length of peptide represents, as GR-9 can be used for representative peptide GYRWMCLRR.Because AR-9 etc. identical herein has multiple, with peptide 1 when therefore describing herein, the mode of peptide 2 is main.
VSVG pseudovirus system: VSVG is vesicular stomatitis virus glycoprotein G, and the gene in the pseudovirus of packaging can be brought in multiple eukaryotic cell by receptor mediated endocytosis by it.In this experiment is can integrated slow virus pUC pUC.
CTL (cytotoxic lymphocytes): after HBV infection hepatocyte, virus antigen is cracked into micromolecular oligopeptide by the proteasome of antigen presenting cell (APC), and be combined into complex with HLA-I quasi-molecule in hepatocyte, be expressed in cell surface, become t cell epitope.CTL is produced by surperficial CD8+ molecule and HLA-I quasi-molecule and adheres to, and by the above-mentioned oligopeptide epi-position of φt cell receptor identification, this type of CTL is HBV Peptide-specific CTL (HBV antigens specific cytotoxiclymphocytes, HBV sCTL).
HBV:(Hepatitis B virus): hepatitis B virus.
PBMC: PERIPHERAL BLOOD MONONUCLEAR CELL
ELISPOT: enzyme-linked immunospot assay (enzyme linked immunospot assay)
Tetramer-PE: with the specific tetramer of PE labelling, each monomer in the tetramer is by heavy chain, the β 2m of HLA_A33, and peptide composition.The difference of nine kinds of different Tetramer-PE is wherein contained peptide differences herein.
Accompanying drawing explanation
Below in conjunction with in the detailed description of accompanying drawing, above-mentioned feature and advantage of the present invention will be more obvious, wherein:
Fig. 1 shows the result of peptide Binding experiment, the line of mark "+" is the peak (peptide concentration be 50mM) of known positive peptide FFVDGAANR when the cell surface with HLA_A33 forms trimer, mark "-" known positive peptide FDVDGAANR in conjunction with time result, other peak is for only to add peptide and β 2m, but cell is the result of untransfected gene.The MRS-A cell of transfection HLA_A33 constructed by result illustrates may be used for screening can with the peptide section of HLA_A33 specific bond.
Fig. 2 shows the cell screening result of peptide 1-5 and 7-8 of the present invention.S2-3 refers to the matched group not adding peptide, and L1-8 is the experimental group adding peptide; Antibody staining is the W6/32 with FITC labelling, and cell counting is 500,000.As can be seen from the figure, peptide 1,3,5 obviously can strengthen the trimerical expression of the special A3303 of cell surface.And the figure's of 2,4,7,8 is not obvious in conjunction with peak skew, illustrate at cellular level, in these several peptides, peptide 1,3,5 can form trimer with HLA_A33 and β 2m.
Fig. 3 shows the sieve chromatography figure of in vitro folding.Wherein transverse axis is the volume of eluting, 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, display be five peptides (that is, peptides 1 in 9 folding positive peptides, peptide 3, peptide 5, peptide 6, peptide 17) folding after molecular sieve figure, Fig. 3 F is known positive control, and Fig. 3 G is the folding result of known negative control.This result illustrate, the peak of HC+ β 2m at peptide 1, peptide 3, peptide 5, peptide 6, peptide 17 can with HLA_A33 heavy chain external its folding time, good trimer result can be formed.
Fig. 4: the result showing two routine HLA_A33 type hepatitis B patient blood sample Elispot, peptide 1, peptide 3, peptide 5, peptide 6, peptide 17 all can stimulate the speckle producing and be greater than more than 6, illustrates that these 5 peptides all can bring out immunoreation.
Fig. 5: the result showing HLA_A33 type hepatitis B patient blood sample tetramer staining, illustrates in patient body to there is cell that can be combined with the HLA_A33 tetramer that these five peptides are formed, CD8+ stained positive.
Detailed description of the invention
Come by the following examples to illustrate the present invention further.But should be appreciated that, described embodiment is illustrational object, is not intended to limit scope and spirit of the present invention.
The structure of the restrictive epitope peptide Screening Platform of embodiment 1:HLA_A33
The structure of 1:HLA_A3303 stable cell lines:
In the present embodiment, use VSVG pseudovirus system (purchased from Biovector Science Lab respectively, Inc) form, by the HLA_A3303 full genome of people (hereinafter referred to as people HLA_A3303, GenBank:U09740.1) two ends add NheI, behind XhoI site, in Shanghai Xu Guan biotech firm this sequence complete synthesis, (nucleotide sequence is SEQ ID No:1, its expression can produce the albumen represented by SEQ ID No:2), recycling NheI, it (is that above-mentioned VSVG pseudovirus system is (purchased from Biovector Science Lab that XhoI two sites are implemented in carrier plentilox 3.7-RRP, Inc) plasmid in) in, form recombiant plasmid plentilox-HLA_A33, carry greatly this recombiant plasmid 15 μ g, again with 5ug VSVG plasmid, 5 μ g RSV/REV plasmids, it is good that the common transfection of 5 μ g pMDLG plasmids (above-mentioned three kinds of plasmids are the plasmid in above-mentioned VSVG pseudovirus system equally) shifts to an earlier date 24 hours and prepares cell, cell confluency degree is the 10cm of 70-80%
2293T cell (this cell is purchased from NIH) in Tissue Culture Dish, transfection conventional liposome body method carries out.At 37 DEG C, 5%CO
2lower cultivation, after within 4 hours, changing liquid, similarity condition cultivates sucking-off cells and supernatant after 48 hours again, 2000 ~ 3000rpm, at 18 DEG C of temperature centrifugal 10 minutes.Collect supernatant.In-80 DEG C of preservations.This is the packaged pseudovirus with HLA_A3303 gene.
RMA-S cell (purchased from Fu Xiang bio tech ltd, Shanghai) is infected with above-mentioned pseudovirus, screen the clone of stable transfection with the puromycin (purchased from AMRISO) of the concentration of 2 ~ 4 μ g/mL, HLA_A3303 expresses on epicyte by the clone through the method determination stable transfection that Western Blot detects and cell fluorescence dyes.
2: the synthesis of peptide
The aminoacid sequence of the positive peptide of this experiment is FFVDGAANR, the aminoacid sequence of negative peptide is FDVDGAANR, every bar peptide symthesis 20mg, and 95% purity, is synthesized by gill biochemical corp, Shanghai.
3: peptide Binding experiment
Cultivated 14 ~ 18 hours at 26 DEG C by RMS-A-A3303 cell, with the PBS re-suspended cell containing 20% hyclone (purchased from GIBICO), adjustment concentration is every milliliter of 2*10
5individual cell, 50 μ L are got in each experiment, add positive peptide respectively and negative peptide makes ultimate density be gradient 0.1 μM, 1 μM, 10 μMs, 100 μMs, 1mM, adds β 2m microglobulin (purchased from Sigma) (final concentration is 10 μ g/mL), and each experimental group repeats 3 multiple holes, cultivate after 1 hour, be placed in 37 DEG C and cultivate 3 hours again for 26 DEG C.After cell washs with the PBS containing 20% hyclone, 4 DEG C, 500g is centrifugal, retention volume is 50 μ L, puts on ice, adds the HLA_A antibody W6/32 (purchased from eBioscience) of FITC labelling, incubated at room temperature 10 minutes, after PBS washs 3 times, flow cytomery fluorescence intensity.
Fig. 1 shows the result of peptide Binding experiment, the line of mark "+" is the peak (peptide concentration be 50mM) of known positive peptide FFVDGAANR when the cell surface with HLA_A33 forms trimer, mark "-" known positive peptide FDVDGAANR in conjunction with time result, other peak is for only to add peptide and β 2m, but cell is the result of untransfected gene.The RMS-A cell of transfection HLA_A33 constructed by result illustrates may be used for screening can with the peptide section of HLA_A33 specific bond.
The screening of embodiment 2:HBV Idiotype HLA_A33 specificity epitope
By the following resource that network has been announced, respectively the possible candidate polypeptide of the B of HBV, C hypotype is predicted:
3D-QSAR program is MHCPred
Marking matrix program is SYFPEITHI
TAP program is TAPpred
By the result of three, have selected 16 peptides as candidate polypeptide.The long and sees the following form 1.
By the method for above-mentioned peptide Binding experiment, be except 50 μMs except peptide concentration during screening is fixed as final concentration, other method is constant, the epitope peptide that can be combined with HLA_A3303 in the candidate peptide of screening prediction.
Fig. 2 shows the cell screening result of peptide 1 ~ 5 and 7 ~ 8 of the present invention.S2 ~ 3 refer to the matched group not adding peptide, and L1 ~ 8 are the experimental grouies adding peptide; Antibody staining is the W6/32 with FITC labelling, and cell counting is 500,000.As can be seen from the figure, peptide 1,3,5 obviously can strengthen the trimerical expression of the special A3303 of cell surface.And the figure's of 2,4,7,8 is not obvious in conjunction with peak skew, illustrate at cellular level, in these several peptides, peptide 1,3,4 can form trimer with HLA_A33 and β 2m.
Embodiment 3: trimer renaturation is to the qualification of candidate peptide
1:HLA_A3303 inclusion bodies of colibacillus expression and purification:
After the Codon sequences of HLA_A3303 and mRNA sequence are optimized (nucleic acid sequence encoding after optimization is SEQ ID No:3), by the expression cassette optimized, be cloned in carrier pET30A (purchased from Novagen) with enzyme NdeI-XhoI, the clone formed is called pET30-HLA_A33HC-LND, its expression can produce the albumen represented by SEQ ID No:4, this albumen contains the α 1 of HLA_A quasi-molecule, α 2, α 3 domain and one section of biotinylation site sequence LND.
Expression way routinely, when escherichia coli (E.coli) bacterium liquid OD value reaches 0.7, adding IPTG makes final concentration be 0.5mMol/L, 37 DEG C of inductions received bacterium after 4 hours, by lysozyme (sigma) 5mg/ (often liter of original culture medium) room temperature treatment 1 hour, after using-80 DEG C of freeze thawing treatment again, with inclusion body cleaning mixture (1mM DTT, 0.5%Triton X-100, 50mM Tris-HCl, 100mM NaCl, pH=8.0) inclusion body is washed, 13000g, 4 DEG C after centrifugal 10 minutes, add above-mentioned cleaning mixture again, ultrasonic (1cm ultrasonic head, 300W, under condition of ice bath, surpass 4 seconds, stop 6 seconds, surpass 200 times altogether), wash again, centrifugal, ultrasonic, after repeating three circulations, dissolve with 8M carbamide.This is the HLA_A33HC-LND albumen of purification.
2: trimerical renaturation
By the HLA_A33HC-LND inclusion body protein of the purification of 18.6mg, the β 2m of 13.2mg, 12mg synthesizes candidate peptide and is added in (400mM arginine hydrochloride in 250mL arginine renaturation buffer, 2mMNaEDTA, 0.5mM oxidized form of glutathione, the glutathion of 5mM reproducibility: pH=8.0), 4 ~ 8 DEG C of renaturation are after 12 hours, concentrate cup (purchased from Millipore) with 10KD and volume is reduced to 10mL, after dialysing 24 hours with distilled water 4 DEG C again, with the super filter tube process of the 50mL 10KD of Millipore, and buffer exchange is become 20mM Tris, 50mM NaCl, pH=8.0.After cumulative volume is less than 4mL, detect the efficiency of altogether renaturation with Superdex 200HR molecular sieve column, form with renaturation the schedule of proportion that trimerical ratio accounts for total protein and show annealing efficiency.Renaturation the results are shown in following table 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
As can be seen from the table, peptide 1,3,5,6,11,14,17 can form good renaturation in vitro with HLA_A3303, belong to strong binding peptide, and peptide 12,13 are relative weak binding peptide.
The partial results that renaturation is relevant shows in figure 3.
Embodiment 4:Elispot technology is to the qualification of candidate peptide
Under the prerequisite that hepatitis B patient is known the inside story, get Hepatitis B patients blood sample, by HLA_A site grouping reagents (purchased from Beijing Sheng Taihua Bioisystech Co., Ltd) operation instruction of standard, detect whether blood sample is HLA_A33 type.Wherein the blood sample of the old XX of hepatitis B patient, woods XX and time XX is HLA_A33 type, in detecting for follow-up experiment.
By A33 type blood sample 6mL, PBMC (the human lymphocyte separating medium Ficoll paque plus of peripheral blood is separated by standardization program, purchased from GE healthcare), it is 2.5*10 that lymphocytes culture medium (reaching section purchased from Shenzhen is Bioisystech Co., Ltd) adjusts concentration
6individual cell/mL;
By the explanation of people IFN-gama Elispot detection kit (reaching section purchased from Shenzhen is Bioisystech Co., Ltd), after lymphocytes culture medium (reaching section purchased from Shenzhen is Bioisystech Co., Ltd) activating reagent bar, add 100 μ L containing 2.5*10
6the patient P BMC of the above-mentioned separation of individual cell/L.Add the peptide stimulation that final concentration is 10 μ g/mL in experimental group, 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 3 multiple holes established by each sample.Cultivate 14 hours at 37 DEG C in cell culture incubator after, remove culture medium, in every hole, add the deionized water of 100 μ L pre-coolings, under 4 degree, place cell lysis, abandon supernatant after 10 minutes, with cleaning mixture washing precipitation 3 times.
Biotin labeled anti-IFN-γ monoclonal antibody (test kit carries) is added in every hole, hatch 1 hour for 37 DEG C, after cleaning, add again and detect antibody (test kit carries), to hatch after 1 hour cyclic washing again, add supporting developer for 37 DEG C, 37 DEG C after 10 ~ 20 minutes, obviously use tap water stopped reaction afterwards Deng speckle, after airing, read spot formation number (SFC) with speckle plate reading machine.
Table 3:Elispot result
Conclusion: peptide 1,3,5,6,17 is five main peptides, peptide 11, and 12 is two secondary peptides
Fig. 4: the result showing two routine HLA A33 type hepatitis B patient blood sample Elispot, peptide 1, peptide 3, peptide 5, peptide 6, peptide 17 all can stimulate the speckle producing and be greater than more than 6, illustrates that these 5 peptides all can bring out immunoreation.
Embodiment 5: Tetramer technology is to the qualification of candidate peptide
1: tetrameric preparation
Get the HLA_A33HC-LND that 1L is total to the trimer of renaturation as stated above, concentrate after cup is concentrated to 700 μ L volumes with the 10kD of Millipore, with biotinylation kit (purchased from Guangzhou FulenGen Co., Ltd.), by this protein biotinylation (700 μ L trimerizing albumen, 100 μ L solution A, 100 μ L solution B, 100 μ L D-biotins, 10 μ L BirA enzymes, 0.5 μ L Pepstatin (storage liquid concentration: 2mg/mL, in DMSO), 0.5 μ L is bright presses down peptide (leupeptin) (storage liquid concentration: 2mg/mL, in dH2O), 30 DEG C of reactions are after 12 hours, trimer peak is wherein collected with molecular sieve Superdex 200 HR 10/30.After being less than 1mL with 10KD concentration tube is concentrated again, the buffer of substitutional solution is PBS, and with the protein quantification kit albumen of Bio-Rad, adjustment protein concentration 5mg/mL, in molar ratio, namely, the streptavidin (purchased from eBioscience) of 1 part of PE labelling is joined the molar ratio in 5 parts of these biotinylated proteins, progressively add the streptavidin of PE labelling, after polymerized at room temperature, then remove unconverted monomer with the concentration tube of 100kD.Keep in Dark Place the tetramer prepared.
2: the collection of blood sample and tetramer staining
Agree to through patient, after signing blood sample joint study agreement, hepatitis B patient blood sample is got from China-Japan Friendship Hospital and You An hospital, extract test kit with Promega blood sample genome and extract DNA sample, HLA_A site grouping reagents (purchased from Beijing Sheng Taihua Bioisystech Co., Ltd) is used to detect the HLA_A typing of patient again, for the sample that genotyping result is HLA_A33, use lymphocyte separation medium (Ficoll paque plus further, purchased from GE healthcare) PBMC in sample separation, with the RMPI 1640 culture medium re-suspended cell containing 10% hyclone, and cell concentration is adjusted to 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 (purchased from eBioscience) of 30U/mL restructuring is added when the 3rd day.Within every 3 days later, half amount changes liquid, and adds the IL-2 of 30U/mL.10th day collecting cell also uses the tetramer and CD8 fluorescent antibody staining prepared, by the quantity of flow cytomery specific CTL.Result display in Figure 5.Cell that the HLA_A33 tetramer that in the result explanation patient body of Fig. 5, existence can be formed with these five peptides (peptide 1, peptide 3, peptide 5, peptide 6, peptide 17) combines, CD8+ stained positive.Two positive cell accounted for the ratio of total CD8+ cell before 0.417% to 1.836%, and contrast negative peptide (peptide 16) and only have 0.012%, wherein the highest is No. 5 peptides, this and Elispot data consistent.Also demonstrate that these five and be HLA_A33 restrictive HBV type specificity epi-position.
Should be appreciated that, although with reference to the embodiment that it is exemplary, the present invention shown particularly and describe, but will be understood by those skilled in the art that, under the condition not deviating from the spirit and scope of the present invention defined by accompanying claim, the change of various forms and details can be carried out wherein, the combination in any of various embodiment can be carried out.
Sequence illustrates:
SEQ ID No:1, the people HLA_A3303 gene order of synthetic, two ends are with NheI, XhoI restriction enzyme site;
The HLA_A3303 protein sequence that SEQ ID No:2, SEQ ID No:1 encodes;
SEQ ID No:3, carries out the coded sequence of codon optimized HLA_A3303 for escherichia coli;
The HLA_A3303 protein sequence that SEQ ID No:4, SEQ ID No:3 encodes;
The aminoacid sequence of the candidate polypeptide of B, C hypotype of the HBV of SEQ ID No:5-20:16 bar prediction.
It should be noted that, original HLA_A33 gene has 6 functional areas, from front to back respectively: signal peptide, α 1, α 2, α 3, cross-film district, and intracellular region totally six parts, the sequence of SEQ ID No:2 is consistent with this sequence; Because HLA_A33 is combined with peptide, and the functional areas be combined with TCR after being combined with peptide are by α 1, α 2 determines, α 3 district determines to be combined with β 2m, thus, when in vitro prepared by renaturation and the tetramer, be all retain these three districts, and after c-terminus adds top connection (gsgsg), increase a biotinylated label (lndifeaqkiewhe) again, the aminoacid sequence after change is represented by SEQ IDNo:4.During this sequence is tested in vitro, complete HLA_A33 (SEQ ID No:2) can be represented completely and be combined with peptide; Be combined with β 2m; With these three kinds of functions that peptide, β 2m are combined with TCR after being combined again.
Claims (3)
1. the application of HLA_A33 restricted epitope peptide in the medicine for the preparation of the hepatitis B in treatment experimenter of hepatitis B virus Idiotype, the hepatitis B of wherein said experimenter is HLA_A33 type, and the HLA_A33 restricted epitope peptide of wherein said hepatitis B virus Idiotype is LVSFGVWIR.
2. application according to claim 1, wherein said experimenter is mammal.
3. application according to claim 2, wherein said experimenter is people.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6037135A (en) * | 1992-08-07 | 2000-03-14 | Epimmune Inc. | Methods for making HLA binding peptides and their uses |
| CN1315963A (en) * | 1998-09-01 | 2001-10-03 | 基因创新有限公司 | Benzodiazepines and benzothiazepines derivatives and HBSAG peptides binding to annexins, their compositions and use |
| CN1454082A (en) * | 2000-09-08 | 2003-11-05 | 艾普免疫公司 | Inducing cellular immune responses to hepatitis B virus using peptide and nucleic acid compositions |
| WO2004058807A3 (en) * | 2002-12-24 | 2004-09-30 | Algonomics N V | Mhc class i restricted t-cell stimulating peptides from hepatitis b virus |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6037135A (en) * | 1992-08-07 | 2000-03-14 | Epimmune Inc. | Methods for making HLA binding peptides and their uses |
| CN1315963A (en) * | 1998-09-01 | 2001-10-03 | 基因创新有限公司 | Benzodiazepines and benzothiazepines derivatives and HBSAG peptides binding to annexins, their compositions and use |
| CN1454082A (en) * | 2000-09-08 | 2003-11-05 | 艾普免疫公司 | Inducing cellular immune responses to hepatitis B virus using peptide and nucleic acid compositions |
| WO2004058807A3 (en) * | 2002-12-24 | 2004-09-30 | Algonomics N V | Mhc class i restricted t-cell stimulating peptides from hepatitis b virus |
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