CN1314805C - A novel APO and antibody thereof and preparation and use - Google Patents
A novel APO and antibody thereof and preparation and use Download PDFInfo
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- CN1314805C CN1314805C CNB200410029588XA CN200410029588A CN1314805C CN 1314805 C CN1314805 C CN 1314805C CN B200410029588X A CNB200410029588X A CN B200410029588XA CN 200410029588 A CN200410029588 A CN 200410029588A CN 1314805 C CN1314805 C CN 1314805C
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Abstract
The present invention provides a novel cDNA sequence of an apoptosis promoting ligand (APO), a coded amino acid sequence thereof, a function thereof for promoting apoptosis and an application thereof, and provides a preparation method and an application for an antibody thereof. The cDNA sequence is formed from 381 basic groups, and the length of the amino acid sequence is the length of 126 amino acids. The protein of the cDNA sequence, which is expressed in colibacillus, has obvious inhibiting effects on bacillus growth, and the protein of the cDNA sequence, which is expressed in the mammalian cell, can cause cell apoptosis. Thus, a polypeptide molecule encoded by the cDNA sequence can be used as an inducting agent for promoting tumor cell apoptosis clinically to treat tumors, and performs effects on differentiation induction and treatment for tumors. The present invention determines epitope by analyzing the amino acid sequence to synthesize a polypeptide for preparing a monoclonal antibody, and a protein stamp technique indicates that the monoclonal antibody can specifically recognize the novel apoptosis promoting ligand (APO). Thus, the monoclonal antibody can be used for preventing normal cells from apoptosis so as to protect the normal tissue and avoid wrong apoptosis.
Description
Technical field the invention belongs to biomedical professional domain, relate to a kind of novel Apoptoietin (Apoptoietin, abbreviate APO as) cDNA sequence and aminoacid sequence, and the function information of this gene, also relate to this novel Apoptoietin (APO) thus antibody preparation and inducing apoptosis of tumour cell treat tumour and prevent apoptosis wild phase related disorders such as the neural system degeneration aspect purposes.
The background technology apoptosis is procedural, normal necrocytosis, is the important means that body is removed unnecessary cell.Apoptosis and cell proliferation, differentiation and aging play complementary and equilibrated effect, and be most important in metazoan growth, morphogenesis and maintenance process.A kind of basic biological phenomena as cell, apoptosis result out of control will be serious: when apoptosis is not enough, canceration, virus disease and autoimmune disease easily take place: excessive acquired immune deficiency syndrome (AIDS), hepatitis gravis and degeneration nervous system disorders such as senile dementia, the Parkinson's disease etc. of then may producing of apoptosis.
The purpose of this invention is to provide a kind of novel Apoptoietin (APO).This invention can be used as brand-new clinically apoptosis of tumor cells promotor, for the cancer patients provides new treatment approach.
The present invention is a bait with nervous process chemotropic factor β-Netrin, people's tire brain cDNA library in the screening 4-6 month in pregnant age, and it is highly consistent with the cDNA of neuronatin to screen 10 clones altogether.The cDNA sequence of representative clone JKY2290 wherein is 381 bases.Its length amino acid sequence is 126 amino acid.Analyze by BLAST, the omnidistance homology of the cDNA sequence of 381 bases and people's neuronatin, but 126 aminoacid sequences of its coding are not found homologous sequence in the GenBank database.Known have two kinds according to neuronatin cDNA sequence prediction product, is respectively neuronatin-α and neuronatin-β, and these three kinds of product comparison backs are found that the aminoacid sequence that we were sieved to is a kind of brand-new neuronatin coded product.
The present invention is template with JKY2290, with the APO subclone to prokaryotic expression carrier pGEX4T-2, in intestinal bacteria, can pass through sec.-propyl-β-D-galactoside (isopropylthio-β-D-galactoside, IPTG) abduction delivering, adding IPTG is different with not adding the influence of IPTG to rate of bacterial growth, inducible protein is expressed after adding IPTG, can produce the obvious suppression effect to bacterial growth.
The present invention is template with JKY2290, with the APO subclone to carrier for expression of eukaryon pEGFP-N1, transfection eucaryon mammalian cell (COS7), increase along with the transfection time, considerable change takes place in the location of green fluorescence signal in cell of reflection APO Subcellular Localization, in whole cytoplasm, distribute at first, after insert to nucleus, and nuclear in the gathering.Simultaneously, tangible phenomena of apoptosis (karyomit(e) is assembled, and nuclear membrane advances to split) appears.
The present invention thinks to the functional study of this gene that from above result APO can cause the apoptosis of cell.This invention might search out the inductor of new tumour cell, and oncotherapy aspect clinically plays a role.
The epitope of the present invention by this aminoacid sequence of GOLDKEY software analysis selected the synthetic polypeptide of CVPSPEEHLARSVRGQ encoded peptide section, and by after carbodiimide method and the BSA coupling as immunity antigen.Get BSA coupling polypeptide 300 μ g as antigen immune Balb/c mouse, cell fusion method prepares monoclonal antibody routinely.Prepared antibody can the novel Apoptoietin of specific recognition (APO) proteins encoded.
Antibody provided by the present invention can neutralize with intracellular novel Apoptoietin (APO), be used to prevent the generation of apoptosis, thereby protective tissue is avoided the apoptosis that should not take place.Therefore, can be used for the control and the treatment of apoptosis wild phase related disorders such as neural system degeneration.
Summary of the invention
1. novel Apoptoietin provided by the present invention (APO) has following cDNA sequence:
CGGACTCCGAGACCAGCGGATCTCGGCAAACCCTCTTTCTCGACCACCCACCTA
CCATTCTTGGAACCATGGCGGCAGTGGCGGCGGCCTCGGCTGAACTGCTCATCA
TCGGCTGGTACATCTTCCGCGTGCTGCTGCAGGTGTTCAGGTACTCCCTGCAGA
AGCTGGCATACACGGTGTCGCGGACCGGGCGGCAGGTGTTGGGGGAGCGCAGGC
AGCGAGCCCCCAACTGAGGCCCCAGCTCCCAGCCCTGGGCGGCCGTATCATCAG
GTGCTCCTGTGCATCTCGGCCAGCACGGGAGCCAGTGCCGCGCAGGAATGTGGG
GTCCCCTGTGTTCCCTCGCCAGAGGAGCACTTGGCAAGGTCAGTGAGGGGCCAG
TAG
2. novel Apoptoietin provided by the present invention (APO) has following aminoacid sequence:
RTPRPADLGKPSFSTTHLPFLEPWRQWRRPRLNCSSSAGTSSACCCRCSGTPCR
SWHTRCRGPGGRCWGSAGSEPPTEAPAPSPGRPYHQVLLCISASTGASAAQECG
VPCVPS?PEEHLARSVRGQ
3. novel Apoptoietin provided by the present invention (APO) has following biological characteristics:
At the albumen of expression in escherichia coli the growth of bacterium is had the obvious suppression effect, expressed proteins can cause the apoptosis of cell in mammalian cell.
4. the preparation method of novel Apoptoietin provided by the present invention (APO) antibody
1) antigenic preparation: synthetic peptide C VPSPEEHLARSVRGQ, with carbodiimide method and BSA coupling, coupled product is as antigen.
2) the antigen immune Balb/c mouse of preparation prepares monoclonal antibody by traditional cell fusion method the preparation of antibody: with 1).
3) purifying antibody: with 2) monoclonal antibody of gained through centrifugal, precipitation, desalination, slightly carry antibody, then through affinity purification, promptly get antibody purification.
4) specificity, sensitivity, the stability of evaluation antibody.
5. the purposes of novel Apoptoietin provided by the present invention (APO) in short apoptosis of tumor cells and oncotherapy.
6. novel Apoptoietin provided by the present invention (APO) antibody is used to prevent the normal cell apoptosis, thereby can be used for the treatment of apoptosis-associated diseases such as nerve retrograde affection.
Description of drawings
Fig. 1: the cDNA sequence of novel Apoptoietin (APO)
Fig. 2: the aminoacid sequence of novel Apoptoietin (APO)
Fig. 3: novel Apoptoietin (APO) cDNA sequence and GenBank nucleic acid database complete sequence similarity comparative result
Fig. 4: the sequence alignment result of 10 clones coding products.The blue amino-acid residue of representing unanimity, the sequence of fore portion unanimity is the vector encoded sequence
Fig. 5: the growth curve after IPTG induces.Novel Apoptoietin (APO) abduction delivering in intestinal bacteria, the growth of pair cell has the obvious suppression effect.Specifically, the growth velocity comparative result is 0mM＞0.1mM＞0.5mM IPTG.
Fig. 6: different time green fluorescence location changes.Specifically, behind pEGFP-N1 empty carrier and pEGFP-N1/APO carrier difference transfection eucaryon mammalian cell, different time is observed the green fluorescence signal and is changed in intracellular location.After the empty carrier transfection, increase the location no change in time, fluorescence is uniform distribution in full cell.Along with the increase of transfection time, considerable change takes place in the location of the green fluorescence of fusion rotein in cell, in whole cytoplasm, distribute at first, after insert to nucleus, and gathering in nuclear.Simultaneously, tangible phenomena of apoptosis appears.
The karyomorphism of transfected cell is determined in Fig. 7: DAPI dyeing.Specifically, the cell that can see green fluorescence transfection pEGFP-N1/APO carrier.Simultaneously, by 4 ', 6-diamino-2-phenylindone (4 ', 6-diamidino-2-phenylindole, DAPI) dyeing can be observed the nucleus aggegation, and nuclear membrane advances to split.And do not have the cell of fluorescence still to keep nuclear integrity.
Fig. 8: Western blot result.By the specificity of Western blot detection antibody, used antigen is prokaryotic expression protein, and molecular weight is 37KD.Ascites (the 2B of different hybridoma cell strain preparations
2, 2F
8) detect same specific band.
Fig. 9: mouse brain paraffin section immunohistochemical methods result.With prepared monoclonal anti physical efficiency and intracellular albumen specific recognition, (diaminobenzidine, DAB) colour developing can be seen positive cell dyeing to diaminobenzidine.
Embodiment
1. the homologous recombination in prokaryotic cell prokaryocyte (E.coli) with adenovirus carrier and novel Apoptoietin (APO) obtains the gene recombination medicine that adenovirus carrier and novel Apoptoietin (APO) make up.Use the gene recombination medicine, can be prepared into clinical grade gene therapy goods, the tumor cell induction agent as new clinically is used for treatment for cancer.
2. antibody provided by the present invention can be prepared into antibody drug, can neutralize with intracellular novel Apoptoietin (APO), is used to prevent the generation of apoptosis, thereby protective tissue is avoided wrong apoptosis.Can be used for treating apoptosis wild phase related disorders such as degeneration nervous system disorders (senile dementia, Parkinson's disease etc.) clinically.
Sequence table
＜110〉Institute of Radiation Medicine, Academy of Military Medical Sciences, PLA
＜120〉a kind of novel Apoptoietin (APO) and antibody thereof: preparation and purposes
<130>
<160>1
<170>PatentIn?version?3.1
<210>1
<211>381
<212>DNA
<213>
<400>1
cggactccga?gaccagcgga?tctcggcaaa?ccctctttct?cgaccaccca?cctaccattc 60
ttggaaccat?ggcggcagtg?gcggcggcct?cggctgaact?gctcatcatc?ggctggtaca 120
tcttccgcgt?gctgctgcag?gtgttcaggt?actccctgca?gaagctggca?tacacggtgt 180
cgcggaccgg?gcggcaggtg?ttgggggagc?gcaggcagcg?agcccccaac?tgaggcccca 240
gctcccagcc?ctgggcggcc?gtatcatcag?gtgctcctgt?gcatctcggc?cagcacggga 300
gccagtgccg?cgcaggaatg?tggggtcccc?tgtgttccct?cgccagagga?gcacttggca 360
aggtcagtga?ggggccagta?g 381
Claims (3)
1. a novel Apoptoietin Apoptoietin (APO), the cDNA sequence of the described novel Apoptoietin that it is characterized in that encoding is:
CGGACTCCGAGACCAGCGGATCTCGGCAAACCCTCTTTCTCGACCACCCACCTAC
CATTCTTGGAACCATGGCGGCAGTGGCGGCGGCCTCGGCTGAACTGCTCATCATC
GGCTGGTACATCTTCCGCGTGCTGCTGCAGGTGTTCAGGTACTCCCTGCAGAAGC
TGGCATACACGGTGTCGCGGACCGGGCGGCAGGTGTTGGGGGAGCGCAGGCAGCG
AGCCCCCAACTGAGGCCCCAGCTCCCAGCCCTGGGCGGCCGTATCATCAGGTGCT
CCTGTGCATCTCGGCCAGCACGGGAGCCAGTGCCGCGCAGGAATGTGGGGTCCCC
TGTGTTCCCTCGCCAGAGGAGCACTTGGCAAGGTCAGTGAGGGGCCAGTAG。
2. novel Apoptoietin APO according to claim 1 is characterized in that the aminoacid sequence of this novel Apoptoietin is:
RTPRPADLGKPSFSTTHLPFLEPWRQWRRPRLNCSSSAGTSSACCCRCSGTPCRS
WHTRCRGPGGRCWGSAGSEPPTEAPAPS?PGRPYHQVLLCISASTGASAAQECGVP
CVPSPEEHLARSVRGQ。
3. the described novel Apoptoietin APO of claim 1 is in the purposes of preparation in the antitumor drug.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB200410029588XA CN1314805C (en) | 2004-03-26 | 2004-03-26 | A novel APO and antibody thereof and preparation and use |
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| CNB200410029588XA CN1314805C (en) | 2004-03-26 | 2004-03-26 | A novel APO and antibody thereof and preparation and use |
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| CN1673369A CN1673369A (en) | 2005-09-28 |
| CN1314805C true CN1314805C (en) | 2007-05-09 |
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Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE10303974A1 (en) | 2003-01-31 | 2004-08-05 | Abbott Gmbh & Co. Kg | Amyloid β (1-42) oligomers, process for their preparation and their use |
| JP5486808B2 (en) | 2005-11-30 | 2014-05-07 | アッヴィ・インコーポレイテッド | Monoclonal antibody against amyloid beta protein and use thereof |
| PL1954718T3 (en) | 2005-11-30 | 2015-04-30 | Abbvie Inc | Anti-a globulomer antibodies, antigen-binding moieties thereof, corresponding hybridomas, nucleic acids, vectors, host cells, methods of producing said antibodies, compositions comprising said antibodies, uses of said antibodies and methods of using said antibodies |
| US8455626B2 (en) | 2006-11-30 | 2013-06-04 | Abbott Laboratories | Aβ conformer selective anti-aβ globulomer monoclonal antibodies |
| US20100311767A1 (en) | 2007-02-27 | 2010-12-09 | Abbott Gmbh & Co. Kg | Method for the treatment of amyloidoses |
| CN104744591B (en) | 2010-04-15 | 2022-09-27 | Abbvie德国有限责任两合公司 | Amyloid beta binding proteins |
| CN105348387B (en) | 2010-08-14 | 2020-08-25 | Abbvie 公司 | Amyloid beta binding proteins |
| CN105924502A (en) * | 2014-06-22 | 2016-09-07 | 马恒标 | Tissue protecting active peptide SL11 and application thereof |
| CN114181322A (en) * | 2021-12-13 | 2022-03-15 | 厦门医学院 | Polypeptide for inhibiting lung cancer cell proliferation |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002098441A2 (en) * | 2001-06-07 | 2002-12-12 | Azign Bioscience A/S | Method of treating metabolic disorders using neuronatin polypeptides |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2002098441A2 (en) * | 2001-06-07 | 2002-12-12 | Azign Bioscience A/S | Method of treating metabolic disorders using neuronatin polypeptides |
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