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CN1511958A - System of T cell surface antigen peptide screening human tissue compatibility antigen (HLA) and its application in producing MHC-antigen peptide polymer - Google Patents

System of T cell surface antigen peptide screening human tissue compatibility antigen (HLA) and its application in producing MHC-antigen peptide polymer Download PDF

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Publication number
CN1511958A
CN1511958A CNA021599742A CN02159974A CN1511958A CN 1511958 A CN1511958 A CN 1511958A CN A021599742 A CNA021599742 A CN A021599742A CN 02159974 A CN02159974 A CN 02159974A CN 1511958 A CN1511958 A CN 1511958A
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peptide
antigen
cell
hla
phage
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CNA021599742A
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徐小宁
王小宁
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Abstract

The present invention belongs to the field of biomedicine preparation, is T-cell surface antigen peptide for screening human histocompatibility antigen and its application in preparing MHC-antigen peptide polymer. The MHC-peptide tetramer prepared with the histoantigen peptide may be used in monitoring homoplastic transplantation rejection reaction and directing reasonable use of immunological inhibitor clinically.

Description

The system of the t cell surface antigen peptide of a kind of screening human histocompatibility antigen (HLA), and the application in preparation MHC-antigen peptide polymer
Technical field:
The present invention relates to the method for the t cell surface antigen peptide of a kind of screening human histocompatibility antigen (HLA), and the application in preparation MHC-antigen peptide polymer, field of biological pharmacy belonged to.
Background technology:
Homologue, organ transplantation are a kind of important treatment technologies of medical field, are widely used.A large amount of patients' life has successfully been saved in the homotransplantation of kidney, heart, liver, marrow.More complicated and practical homotransplantation (many internal organs, pancreatic islets transplantation etc.) is also in research is used.The major cause of homotransplantation failure is because host T cell is attacked transplanting to produce, and causes graft to be ostracised.The major antigen of knowing the T cell recognition already is the HLA antigen on the graft, comprises I class and II class antigen.Suitable mutually tissue matching reduces the valid approach of transplant rejection between graft and host.Yet because it is very low to find HLA to join the chance that type is consistent fully, the HLA that most organ transplantation is adopted joins the half-matched of type or the organ that is not harmonious.The host then is suppressed by giving effective immunosuppressor for the rejection of graft.Thereby the organ transplantation patient will use immunosuppressor all the life, and because the danger that the non-specific immunity that immunosuppressor produces infringement produces complication to the patient.Reduce patients ' life quality, also cause the very big economical load of patient simultaneously.More complicated is: when clinical adverse appears in the patient, be difficult to judge that sometimes this reaction is because host's immunological rejection causes, or the murder by poisoning of immunosuppressor causes.And the clinical solution of these two kinds of harm just in time is conflicting: promptly will strengthen the immunosuppressor consumption when rejection occurring, and the consumption of immunosuppressor occur will reducing when immunosuppressor is damaged.Thereby it is very important and practical inventing first aid, the individual medication guide that a kind of technology that can accurately differentiate above-mentioned 2 kinds of clinical adverse causes of disease produces the patient who transplants for histoorgan.Can be used as the target cell of this detection for the specific T cell of graft: when this cell rising, rejection may appear in prompting.And the clinical toxicity that occurs under the situation of this vanished cell reaction is likely that immunosuppressor is excessive and causes.By detecting this class T cell, can carry out differential diagnosis effectively, instruct clinical use reasonable plan, also can instruct simultaneously its long-term reasonably medication according to each patient's individual difference, thereby can under the prerequisite that guarantees the graft long-term surviving, improve patients ' life quality, lower economical load.
Can use MHC-peptide multimer technology for detection specific T-cells at present, and can be with its sorting, activation, amplification.Not only can be the clinical foundation that diagnosis is provided, and can utilize these T cells to carry out biotherapy (strengthening or the inhibition immune response).The preparation of MHC-peptide multimer, key points of application are to adopt corresponding T cell recognition epitope peptide, and the size of these peptides is generally at 9-15 amino-acid residue.Yet, though know that up to the present the HLA antigen on the graft is the main identifying object of T cell.Have only that seldom HLA epi-position is resolved comes out, and be used for detection specificity T cell.Because HLA has the polymorphism of height, it is very difficult screening corresponding epitope peptide according to existing t cell epitope triage techniques again.The present invention is that another efficient screening t cell epitope peptide is used for MHC-peptide multimer screening system invention basis at us, adopt T2 like cell-display technique of bacteriophage screening-the HLA antigen peptide of group host T cell recognition.This group peptide can be used for preparing the MHC-peptide multimer.A kind of efficiently, special thereby develop, the method for detection, sorting, activation, the anti-homotransplant T of amplification host specificity cell is used for the detection of homograft rejecting reaction and immunosuppressor individualized treatment.
Summary of the invention:
The experimental procedure of existing screening human histocompatibility antigen peptide is complicated, and will synthesize a large amount of human histocompatibility antigen's peptide sections, and cost is very high.
The purpose of this invention is to provide a kind of method of screening human histocompatibility antigen's peptide.
Another object of the present invention provides the application that the human histocompatibility antigen's peptide with the screening of this system prepares the MHC-peptide multimer.
That utilizes that method of the present invention can be quick and a large amount of screens purpose peptide section.
A kind of efficient screening T cell antigen epitope peptide of the present invention prepares the method for MHC-peptide multimer (MHC tetramer), and its step is as follows:
(1) T2 like cell system different HLA antigens (comprising I class and II class antigen), that the endogenous antigen peptide offers to express defective is expressed in the preparation of iRNA technology;
(2) the preparation people mainly reaches the peptide chain gene pool of minor histocompatibility antigen;
(3) gene pool that utilizes step (2) to prepare, preparation phage display peptide display system;
(4) the T2 like cell system of step (1) preparation is mixed with the phage of step (3) preparation;
(5) uncombined phage is removed in washing;
(6) utilize phage-resistance capsid protein antibody test phage bonded situation and condition;
(7) carry out gradient elution with elutriant and remove the not high phage of affinity;
(8) adding phage-sensitive host e. coli makes the phage-infect intestinal bacteria on the T2 like cell, and is increased;
(9) by further separating the phage plasmid in the intestinal bacteria, and to wherein cDNA cloning and sequencing, filter out and MHC molecule bonded peptide section sequence;
(10) synthetic peptide section of being screened is bonded to the T2 like cell with this cytositimulation t cell responses, confirms peptide section t cell epitope activity.
Specific embodiment:
Embodiment 1: preparation endogenous antibody peptide is expressed the T2 like cell system of defective
The TAP gene of preparation T2 cell, the RNAi test kit of use Benitec company according to the step of product description of test, is prepared the T2 like cell of TAP genetic flaw.
The fragmentation of above-mentioned T2 like cell, extract the gene of T2 like cell according to the method for " molecular cloning ", increase with TAP promotor and terminator with round pcr again, carry out gel electrophoresis again, discovery does not have the TAP genonema.
Embodiment 2: preparation human histocompatibility antigen peptide chain gene pool
The T cell is bred in the DMEM that contains 4.5g/L glucose and 15%FBS, uses the ordinary method isolation of RNA, and with test kit the RNA reverse transcription become cDNA (Indianapolis, Ind).The cDNA that the first step prepares increases with PCR method again.
The DNA for preparing is carried out enzyme simultaneously with enzyme XhoI and SpeI cut, and gene is inserted into (production of Dyax company) in the phage display system.Obtain containing the phage system of various peptide sections.
Embodiment 3: the high phage of screening affinity
Use the PBS washed cell, there not being bonded phage flush away, increase the ionic strength of washings again, continue washing, in conjunction with untight phage flush away, constantly increase the ionic strength of washings, constantly wash removal, filter out at last and the high phage of T2 like cell avidity in conjunction with untight phage.
Embodiment 4: the amplification screen with the high phage of T2 like cell avidity
Adding phage-sensitive host e. coli makes the phage-infect intestinal bacteria on the T2 like cell, add the LB substratum, cultivated 24-48 hour down for 37 ℃, collect intestinal bacteria, by further separating the phage plasmid in the intestinal bacteria, and to wherein cDNA cloning and sequencing, filter out and MHC molecule bonded peptide section sequence, be YIGEVLVSV, SPSVDKARAEL, SPAVDKAQAEL.
Embodiment 5: with the synthetic MHC-peptide antibody polymer of human histocompatibility antigen's peptide section
Water-bath or freezing incubator precooling 1L fold damping fluid (being loaded in the beaker of 1L of stirrer), add reduced form paddy Guang acid anhydride peptide to 5mM at folding damping fluid, and oxidized form paddy Guang acid anhydride peptide is to 0.5mM, PMSF to 0.2mM.Calculate the necessary amounts that adds 1uM heavy chain, 2uM light chain and 30mg/L peptide, heavy chain and light chain are added in the 5ml injection damping fluid, in room temperature suction 5ml syringe.And in 500ul distilled water or DMSO, dilute human histocompatibility antigen's polypeptide according to human histocompatibility antigen's amino acid sequence of polypeptide (hydrophobicity).The resuspended damping fluid of 1L is put in high-speed stirring on the agitator (avoiding reaction solution to bubble), dropwise adds human histocompatibility antigen's polypeptide in the reaction solution that stirs with pipettor.Fiercely heavy chain and light chain are injected in the reaction solution of stirring in the position near stirrer as far as possible with No. 26 syringe needles.After adding light chain, it is slightly opaque that folding damping fluid should become.Deposit in 10 ℃, spend the night.With renaturation reactant ultrafiltration and concentration to 7ml.With Superdex75 gel-filtration purifying MHC-human histocompatibility antigen peptide list aggressiveness.Detect with SDS-PAGE, and identify its conformation with the ELISA method, one anti-be BB7.2.
The fluorescently-labeled yolk antibody of single aggressiveness of MHC-I and preparation is mixed in proportion, and 37 ℃ of temperature are bathed 1h, with Superdex200 gel-filtration purifying.FPLC detects.
The fluorescently-labeled monoclonal antibody of single aggressiveness of MHC-I and preparation is mixed in proportion, and 37 ℃ of temperature are bathed 1h, with Superdex200 gel-filtration purifying.FPLC detects.
The application clinically of embodiment 6:MHC-HLA-A2.1 peptide multimer
With above-mentioned MHC-HLA-A2.1 peptide tetramer or the polymer of preparing, and on the tetramer or polymer in conjunction with one the colour developing factor, this tetramer or polymer are mixed with tissue, organ transplantation patient's serum, separate and washing, flush away does not have bonded MHC-human histocompatibility antigen's peptide tetramer or polymer, with the quantity of cell counter detection T cell, learn that the concentration of T cell uprises, rejection may appear in prompting.Thereby can instruct clinical rational to use immunosuppressor.
The application clinically of embodiment 7:MHC-HLA-B7 peptide multimer
With above-mentioned MHC-HLA-B7 peptide tetramer or the polymer of preparing, and on the tetramer or polymer in conjunction with one the colour developing factor, this tetramer or polymer are mixed with tissue, organ transplantation patient's serum, separate and washing, flush away does not have bonded MHC-human histocompatibility antigen's peptide tetramer or polymer, with the quantity of cell counter detection T cell, learn that the concentration of T cell uprises, rejection may appear in prompting.Thereby can instruct clinical rational to use immunosuppressor.

Claims (7)

1. the method for t cell surface antigen peptide of a screening human histocompatibility antigen (HLA), it may further comprise the steps:
(1) expresses T2 like cell system different HLA antigens (comprising I class and II class antigen), that the endogenous antigen peptide offers to express defective with the preparation of iRNA technology;
(2) the preparation people mainly reaches the peptide chain gene pool of minor histocompatibility antigen;
(3) gene pool that utilizes step (2) to prepare, preparation phage display peptide display system;
(4) the T2 like cell system of step (1) preparation is mixed with the phage of step (3) preparation;
(5) uncombined phage is removed in washing;
(6) utilize phage-resistance capsid protein antibody test phage bonded situation and condition;
(7) carry out gradient elution with elutriant and remove the not high phage of affinity;
(8) adding phage-sensitive host e. coli makes the phage-infect intestinal bacteria on the T2 like cell, and is increased;
(9) by further separating the phage plasmid in the intestinal bacteria, and to wherein cDNA cloning and sequencing, filter out and MHC molecule bonded peptide section sequence;
(10) synthetic peptide section of being screened is bonded to the T2 like cell with this cytositimulation t cell responses, confirms peptide section t cell epitope activity.
2. the described a kind of efficient screening T cell antigen epitope peptide of claim 1 prepares the method for MHC-peptide multimer, and the described elutriant of its step (7) is the positively charged ion eluent.
3. the method for the t cell surface antigen peptide of claim 1 described a kind of screening human histocompatibility antigen (HLA), the peptide chain that obtains exists on people HLA I class and the I quasi-molecule, can be by the T cell antigen epitope peptide of TXi Baoshouti (TCR) identification.
4. the method for the t cell surface antigen peptide of a kind of screening human histocompatibility antigen as claimed in claim 3 (HLA), described can be HLA-A2.1 by the T cell antigen epitope peptide of TXi Baoshouti (TCR) identification, sequence is YIGEVLVSV.
5. the method for the t cell surface antigen peptide of a kind of screening human histocompatibility antigen as claimed in claim 3 (HLA), described can be HLA-B7 by the T cell antigen epitope peptide of TXi Baoshouti (TCR) identification, sequence is SPSVDKARAEL.
6. the method for the t cell surface antigen peptide of a kind of screening human histocompatibility antigen as claimed in claim 3 (HLA), described can be SPAVDKAQAEL by the T cell antigen epitope peptide sequence of TXi Baoshouti (TCR) identification.
7. the T cell antigen epitope peptide on the HLA antigen in the claim 4,5 or 6 is used to prepare the MHC tetramer or polymer, and be used for that tool repels the T cell of transplanted tissue of institute, organ dysfunction in the specific detection homologue, organ transplantation patient body, thereby can be used for detecting homograft rejection, and guide clinical rational to use immunosuppressor.
CNA021599742A 2002-12-31 2002-12-31 System of T cell surface antigen peptide screening human tissue compatibility antigen (HLA) and its application in producing MHC-antigen peptide polymer Pending CN1511958A (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101696974B (en) * 2009-09-29 2013-03-06 才新 HLA antibody specificity detecting method, cell dish and reagent kit
CN105358984A (en) * 2013-03-15 2016-02-24 普罗格诺西斯生物科学公司 Methods for detecting peptide/MHC/TCR binding
CN112110995A (en) * 2019-06-19 2020-12-22 上海交通大学医学院 Tumor neoantigen polypeptide and application thereof
CN112279911A (en) * 2007-01-30 2021-01-29 埃皮瓦克斯公司 Regulatory T cell epitopes, compositions and uses thereof

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112279911A (en) * 2007-01-30 2021-01-29 埃皮瓦克斯公司 Regulatory T cell epitopes, compositions and uses thereof
CN101696974B (en) * 2009-09-29 2013-03-06 才新 HLA antibody specificity detecting method, cell dish and reagent kit
CN105358984A (en) * 2013-03-15 2016-02-24 普罗格诺西斯生物科学公司 Methods for detecting peptide/MHC/TCR binding
CN112110995A (en) * 2019-06-19 2020-12-22 上海交通大学医学院 Tumor neoantigen polypeptide and application thereof

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