CN100445296C - Polypeptide organic compounds and application in xenogeneic transplantation - Google Patents
Polypeptide organic compounds and application in xenogeneic transplantation Download PDFInfo
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- CN100445296C CN100445296C CNB2006100501640A CN200610050164A CN100445296C CN 100445296 C CN100445296 C CN 100445296C CN B2006100501640 A CNB2006100501640 A CN B2006100501640A CN 200610050164 A CN200610050164 A CN 200610050164A CN 100445296 C CN100445296 C CN 100445296C
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- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The present invention provides a polypeptide organic compound which is selected from one of a, b, c, d, e, f, g, h respectively with the molecular weight of 915.96, 963.06, 763.86, 963.15, 902.07, 1031.18, 987.13 and 951.14. The present invention can be specifically combined with a B-type blood resistant monoclonal antibody to be dissolved in water, and amino acid is connected by peptide bonds. The polypeptide organic compound of the present invention can be specifically combined with the pig resistant antigenic antibody prestored in a human body, and accordingly, a function of the polypeptide organic compound is sealed. The hyperacute rejection reaction of xenogeneic organ transplantation can be specifically blocked, a combining site with a natural alpha-Gal resistant antibody prestored in the human body is specific, and a function on sealing the antibody is just performed but the integral immune reaction of the human body can not be influenced. A side effect caused by a general immunosuppressant can be prevented.
Description
Technical field
The invention belongs to organic compound, relate generally to polypeptide class organic compound, especially about the polypeptide organic compound of synthetic and the application in xenotransplantation thereof.
Technical background
The progress of medical technology makes human homogeneous organ transplantation become possibility.Since the source deficiency of human organ, the organ that a large amount of patients can not get being badly in need of, and utilizing the organ replacement human organ of pig is the focus of studying at present.But because there is heterologous antigen epi-position Gal-α-1 in the cell surface of pig, 3-Gal can combine with the natural antibody that prestores in the human body and produce hyperacute rejection, thereby causes the failure of xenotransplant.People can seal or remove the natural anti-α of people-Gal antibody striving to find a kind of method, for approach is opened up in xenotransplant.Display technique of bacteriophage is a kind of strong instrument that can screen with target molecule bonded specific polypeptide, successfully applies to epitope, the research of specific receptors agonist or inhibitor and vaccine.We utilize display technique of bacteriophage in this experiment, and to two kinds of peptide storehouses, affine screening has been carried out in linear 7 peptide storehouses and C7C library.The polypeptide of showing in the linear 7 peptide storehouses does not have structural limitations, has the structure of relative flexibility.The polypeptide that C7C peptide storehouse is showed is owing to the restriction that a pair of disulfide linkage is arranged forms ring-type, and conformation is relatively stable.Adopt two kinds of peptide storehouses to screen simultaneously, purpose is that comparison is owing to the different differences of the polypeptide that filters out on aminoacid sequence that cause of structure.Because anti-Type B blood monoclonal antibody has the characteristic in conjunction with α-Gal epitope, therefore we are target molecule with anti-Type B blood monoclonal antibody, filter out the polypeptide that to simulate α-Gal epitope, natural anti-α-Gal antibody can be blocked specifically, the xenotransplant hyperacute rejection can be used for.
Clinical immunosuppressor commonly used has ciclosporin A, glucocorticosteroid, azathioprine, endoxan etc. at present, can reduce whole immune response, but it is invalid to the xenotransplant hyperacute rejection, and multiple side effect is arranged, bone marrow depression, liver renal toxicity, upper gastrointestinal hemorrhage, alopecia or the like just arranged as common.The polypeptide compound that we screen can be blocked the xenotransplant hyperacute rejection specifically, but does not influence the whole immune response of human body, the side effect that can avoid immunosuppressor commonly used to cause.
Summary of the invention
The objective of the invention is to overcome the rejection that occurs in the present xenotransplantation clinically, design and provide a kind of polypeptide organic compound.
Polypeptide organic compound provided by the invention is selected from a kind of among eight kinds of a, b, c, d, e, f, g, the h, can combine with anti-Type B blood monoclonal antibody specificity, and is water-soluble, links to each other by peptide bond between each amino acid.
The aminoacid sequence of polypeptide organic compound a provided by the invention is:
Phe-His-Glu-Asn-Trp-Pro-Ser, molecular weight are 915.96; The aminoacid sequence of polypeptide organic compound b is: Phe-His-Glu-Phe-Trp-Pro-Thr, molecular weight are 963.06; The aminoacid sequence of polypeptide organic compound c is: Ser-Met-Leu-Asp-Thr-Pro-Thr, molecular weight are 763.86; The aminoacid sequence of polypeptide organic compound d is: Cys-Leu-Pro-Thr-Ile-Thr-Asn-Thr-Cys, (wherein forming disulfide linkage between the 1st Cys and the 9th 's the Cys), molecular weight is 963.15; The aminoacid sequence of polypeptide organic compound e is: Cys-His-Ile-Leu-Gly-Ser-Thr-Ala-Cys, (wherein forming disulfide linkage between the 1st Cys and the 9th 's the Cys), molecular weight is 902.07; The aminoacid sequence of polypeptide organic compound f is: Cys-His-Pro-Thr-Trp-Ser-Ser-Leu-Cys, (wherein forming disulfide linkage between the 1st Cys and the 9th 's the Cys), molecular weight is 1031.18; The aminoacid sequence of polypeptide organic compound g is: Cys-His-Gln-Thr-Pro-Leu-Ser-Thr-Cys, (wherein forming disulfide linkage between the 1st Cys and the 9th 's the Cys), molecular weight is 987.13; The aminoacid sequence of polypeptide organic compound h is: Cys-Lys-Pro-Thr-Ser-Thr-Leu-Thr-Cys, (wherein forming disulfide linkage between the 1st Cys and the 9th 's the Cys), molecular weight is 951.14.
Second purpose of the present invention provides polypeptide organic compound a, b, c, d, e, f, g, the h application in the medicine of hyperacute rejection in the control xenotransplantation in preparation treatment transplant operation.
Advantage of the present invention has:
1. polypeptide organic compound of the present invention, because molecular composition has only 7～9 amino acid, thus can be synthetic in a large number with artificial chemical synthesis process, can obtain a large amount of purer little peptides.
2. can combine with the antigenic antibodies specific of anti-pig that prestores in the human body, thus the effect of sealing it, the hyperacute rejection that takes place in the time of can suppressing the pig organ transplantation to human body.
The binding site of the natural anti-α-Gal antibody that prestores in polypeptide organic compound 3. of the present invention and the human body is specific, just in time plays the blocking antibody effect, thereby has avoided owing to the not strong side effect that brings of action site specificity.
4. because the composition amino acid quantity of polypeptide organic compound of the present invention is less, utilized and onset by body easily after being developed to medicine.
Description of drawings
Fig. 1 is the competitive and anti-B monoclonal antibody bonded ELISA inhibiting rate graphic representation of three kinds of linear polypeptides and stachyose.
Fig. 2 is the competitive and anti-B monoclonal antibody bonded ELISA inhibiting rate graphic representation of five kinds of C7C cyclic peptide and stachyose.
The agglutination reaction that Fig. 3 suppresses the swine erythrocyte that human matural antibodies causes for polypeptide compound is figure as a result.
Embodiment
The present invention reaches accompanying drawing with the following Examples and is described further.
Embodiment 1: as target protein, obtain positive phage clones with the display technique of bacteriophage screening with anti-Type B blood monoclonal antibody.
Display technique of bacteriophage be utilize Protocols in Molecular Biology with the random sequence oligonucleotides fragment cloning of one group of certain-length of synthetic in the particular expression carrier, make its expression product be presented on phage surface with the form of fusion rotein.Because comprised all possible sequence of amino acid of the little peptide of this length in the peptide storehouse, each phage surface albumen presents a kind of peptide section wherein, be convenient to screening; The phage that screens can be increased by bacterium.Use the phage peptide library ehec infection, the oligonucleotide fragment at random that is recombined into phage is duplicated in intestinal bacteria, and in the coat protein of phage, express.Then, target protein is coated on the enzyme plate.With after target protein mixes, wash plate with this phage peptide library.If the coat protein on the phage can combine with target protein, just can not washed off.With acid or affinity elution liquid phage is eluted at last.Sieve 3-4 takes turns so continuously, just can sift out the phage stronger with the target protein bonding force.The dna sequence dna of surveying this phage obtain the recombinating sequence of oligonucleotide has also been known the sequence of corresponding polypeptide.Can obtain polypeptide by the method for artificial chemosynthesis then.
Polypeptide organic compound provided by the invention comprises eight kinds of a, b, c, d, e, f, g, h, can combine with anti-Type B blood monoclonal antibody specificity, and is water-soluble, links to each other by peptide bond between each amino acid.
The aminoacid sequence of polypeptide organic compound a provided by the invention is: Phe-His-Glu-Asn-Trp-Pro-Ser, molecular weight are 915.96; The aminoacid sequence of polypeptide organic compound b is: Phe-His-Glu-Phe-Trp-Pro-Thr, molecular weight are 963.06; The aminoacid sequence of polypeptide organic compound c is: Ser-Met-Leu-Asp-Thr-Pro-Thr, molecular weight are 763.86; The aminoacid sequence of polypeptide organic compound d is: Cys-Leu-Pro-Thr-Ile-Thr-Asn-Thr-Cys, (wherein forming disulfide linkage between the 1st Cys and the 9th 's the Cys), molecular weight is 963.15; The aminoacid sequence of polypeptide organic compound e is: Cys-His-Ile-Leu-Gly-Ser-Thr-Ala-Cys, (wherein forming disulfide linkage between the 1st Cys and the 9th 's the Cys), molecular weight is 902.07; The aminoacid sequence of polypeptide organic compound f is: Cys-His-Pro-Thr-Trp-Ser-Ser-Leu-Cys, (wherein forming disulfide linkage between the 1st Cys and the 9th 's the Cys), molecular weight is 1031.18; The aminoacid sequence of polypeptide organic compound g is: Cys-His-Gln-Thr-Pro-Leu-Ser-Thr-Cys, (wherein forming disulfide linkage between the 1st Cys and the 9th 's the Cys), molecular weight is 987.13; The aminoacid sequence of polypeptide organic compound h is: Cys-Lys-Pro-Thr-Ser-Thr-Leu-Thr-Cys, (wherein forming disulfide linkage between the 1st Cys and the 9th 's the Cys), molecular weight is 951.14.
Embodiment 2: the experiment of stachyose competitive ELISA
Test method: in 96 hole enzyme plates, wrap and spent the night for 4 ℃ by the anti-Type B blood of 100 μ l monoclonal antibody, with 37 ℃ of sealings of 5%BSA 2 hours, wash plate 3 times with TBS then, add that 100 μ l positive bacteriophages and final concentration are respectively 100,20,4,0.8,0.16, the stachyose mixed solution of 0.032mM reaction 1 hour, other establish do not add stachyose positive bacteriophage liquid as blank.Wash plate 6 times with the TBS that contains 0.1% Tween 20, add the anti-M13 antibody of 100 μ l horseradish peroxidase-labeled, in conjunction with 1 hour, wash plate 6 times with the TBS that contains 0.1%Tween 20, TMB develops the color, and measures absorbancy with microplate reader.Calculate inhibiting rate according to formula: [A
450(-s)-A
450(+s)]/A
450(-s) * 100%, A
450(-s) be not for adding the absorbancy of stachyose, A
450(+s) is for adding the absorbancy of stachyose.
Test-results: stachyose can effectively suppress combining of polypeptide and antibody, illustrates that stachyose and polypeptide are incorporated into the same position of antibody, i.e. α-Gal epitope binding site.This has illustrated that positive colony just is combined in stachyose and anti-Type B blood monoclonal antibody bonded site probably.The result is referring to Fig. 1, and 2.Experiment finds that on the whole, stachyose than linear polypeptide inhibiting rate height, illustrates that the linear polypeptide of screening is stronger than the avidity of ring type polypeptide antagonist to cyclic peptide.
Above example has illustrated that the binding site (α-Gal binding site) of polypeptide organic compound and anti-B monoclonal antibody is specific, just in time plays the anti-B monoclonal antibody action of sealing, thereby has avoided owing to the not strong side effect that brings of action site specificity.
Embodiment 3: polypeptide suppresses the agglutination reaction of human serum and swine erythrocyte
Test method: earlier be suspended in fresh 1% swine erythrocyte for preparing in the phosphate buffered saline buffer of pH 7.4 standby.In the hole of U type blood-coagulation-board, successively by behind the doubling dilution of hole, every hole adds the A type human serum of the suitable dilution of 40 μ l and the swine erythrocyte of 40 μ l 1% respectively to 40 μ l polypeptide with phosphoric acid buffer.Then this mixed solution was at room temperature shaken 1 minute with oscillator earlier, leave standstill again and observe the aggegation result after 1 hour.If erythroprecipitin is the slick round dot in an edge to the bottom in hole, then this is not for there being aggegation.If red corpuscle forms a network, be the slick round dot in an edge at the bottom of not sinking to the hole, this is for aggegation occurring.Blank, the positive and negative control are established in test.
Test-results: this polypeptide in advance can with the anti-α-Gal antibodies in the A type human serum, make its inactivation.Like this this antibody just can not be again with swine erythrocyte on Gal-α-1, the combination of 3-Gal antigen, thereby the blocking-up people swine erythrocyte agglutination reaction that anti-α-Gal antibody causes.The result is referring to Fig. 3.In Fig. 3, A1～A2, H1～H2,01～02 all is blanks, and with leaving standstill 1 hour behind the swine erythrocyte of 40 μ l 1% and the 80 μ l phosphate buffered saline buffer mixings, red corpuscle is sunken to the agglutination plate bottom, smooth round dot occurs, and this result shows and aggegation do not occur.A3～A4, H3～H4,03～04 positive contrast with the swine erythrocyte of 40 μ l 1%, was left standstill 1 hour behind 40 μ l1% human serums and the 40 μ l phosphate buffered saline buffer mixings, and red corpuscle does not sink to presenting smooth round dot, but presents network-like structure, aggegation occurs.B, two of B ' are stachyose and suppress swine erythrocyte aggegation result of experiment, (concentration is followed successively by 200mM with the stachyose of the different concns of phosphate buffered saline buffer dilution to have added 40 μ l in 1～4 hole successively, 40mM, 8mM, 1.6mM), add swine erythrocyte and 40 μ l, 1% human serum of 40 μ l 1% then, left standstill behind the mixing 1 hour.Aggegation does not appear in B1～B2, B ' 1～B ' 2, and B3, B ' 3 begin to have network-like structure to occur, and obvious agglutinative phenomenon is arranged, and illustrate that stachyose can suppress the swine erythrocyte aggegation that natural anti-α-Gal antibody brings out by the people.C1-4 and C ' 1-4 are that phage display peptide a suppresses swine erythrocyte aggegation result of experiment, (concentration is followed successively by 0.1mM to have added phage with the different concns of phosphate buffered saline buffer dilution in 1～4 hole successively, 0.02mM, 0.004mM, 0.0008mM) 40 μ l, add swine erythrocyte and 40 μ l, 1% human serum of 40 μ l 1% again, left standstill behind the mixing 1 hour.C1, aggegation does not appear in C ' 1, and C2, C ' 2 begin to have network-like structure to occur, and obvious agglutinative phenomenon is arranged.E1-4 and E ' 1-4, I1-4 and I ' 1-4, J1-4 and J ' 1-4, K1-4 and K ' 1-4, L1-4 and L ' 1-4, M1-4 and M ' 1-4, P1-4 and P ' 1-4, Q1-4 and Q ' 1-4 are respectively phage display peptide b, h, e, k, d, g, c, the experimental result of f, experimental technique and phage concentration such as preceding.(its aminoacid sequence is: Ser-Ser-Thr-Ile-Ala-Asn-Thr) except phage display peptide k, each phage clone all can be under 0.1mM concentration anticoagulant effectively, phage display peptide k may antagonist the binding ability deficiency, fail obvious anticoagulant.G1-4 and N1-4 are without the wild type phage (negative control) of crossing screening, from linear 7 peptide storehouses and C7C peptide storehouse, aggegation all occurs respectively.D1-4 and D ' 1-4, F1-4 and F ' 1-4, R1-4 and R ' 1-4 are respectively the polypeptide a of chemosynthesis, b, the experimental result of f, experimental technique such as preceding, peptide concentration all are followed successively by 20mM, 4mM, 0.8mM, 0.16mM, but visible polypeptide a and b anticoagulant when concentration is 4mM, and the concentration that polypeptide f needs is 20mM.The negative contrast of S1-4, the peptide sequence of adding are Cys-Val-Gln-Pro-Ser-His-Ser-Ser-Cys, aggegation all occurs; The above results repeats 3 times, presents good consistence.
Above example with image directly mode illustrated that the polypeptide organic compound is except can be with anti-B monoclonal antibody combines, can also combine with the antigenic antibody of anti-pig (anti-α-Gal antibody) specificity that prestores in the human body, thereby seal its effect, can suppress the generation of the xenotransplantation hyperacute rejection of human matural antibodies (anti-α-Gal antibody) triggering.
Need not further to elaborate, believe and adopt the disclosed content in front, those skilled in the art can use the present invention to greatest extent.Therefore, the preferred specific embodiments of front is interpreted as only illustrating, but not limits the scope of the invention by any way.
Claims (1)
1. the application in the medicine of polypeptide organic compound hyperacute rejection in preparation treatment xenotransplantation, the aminoacid sequence of this polypeptide organic compound is: Phe-His-Glu-Phe-Trp-Pro-Thr, molecular weight are 963.06.
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| CNA2008100013148A Division CN101230095A (en) | 2006-03-31 | 2006-03-31 | Polypeptide organic compound and use thereof in hetero transplantation |
| CNA2008100013222A Division CN101225107A (en) | 2006-03-31 | 2006-03-31 | Polypeptide organic compound and use thereof in hetero transplantation |
| CNA200810001310XA Division CN101230092A (en) | 2006-03-31 | 2006-03-31 | Polypeptide organic compound and use thereof in hetero transplantation |
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| CN1054772A (en) * | 1990-03-15 | 1991-09-25 | 普罗托斯分子设计有限公司 | Synthetic polypeptide |
| DE19749277A1 (en) * | 1997-11-07 | 1999-05-20 | Univ Leipzig | Removing albumin from liquid by affinity chromatography, for purification of fluids or albulmin recovery |
| CN1412197A (en) * | 2002-11-15 | 2003-04-23 | 浙江大学 | 17 peptide organic compound and its application |
| WO2005098435A2 (en) * | 2004-04-05 | 2005-10-20 | Universite Bordeaux 2 | Peptides and peptidomimetics binding to cd23 |
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| CN1054772A (en) * | 1990-03-15 | 1991-09-25 | 普罗托斯分子设计有限公司 | Synthetic polypeptide |
| DE19749277A1 (en) * | 1997-11-07 | 1999-05-20 | Univ Leipzig | Removing albumin from liquid by affinity chromatography, for purification of fluids or albulmin recovery |
| CN1412197A (en) * | 2002-11-15 | 2003-04-23 | 浙江大学 | 17 peptide organic compound and its application |
| WO2005098435A2 (en) * | 2004-04-05 | 2005-10-20 | Universite Bordeaux 2 | Peptides and peptidomimetics binding to cd23 |
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