CN106243215B - A kind of human TNF related apoptosis-inducing ligand I receptor excitability antigen epitope polypeptide and its antibody and application - Google Patents
A kind of human TNF related apoptosis-inducing ligand I receptor excitability antigen epitope polypeptide and its antibody and application Download PDFInfo
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- CN106243215B CN106243215B CN201610680242.9A CN201610680242A CN106243215B CN 106243215 B CN106243215 B CN 106243215B CN 201610680242 A CN201610680242 A CN 201610680242A CN 106243215 B CN106243215 B CN 106243215B
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- antibody
- antigen epitope
- trail
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
- C07K16/065—Purification, fragmentation
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
Abstract
The invention discloses a kind of human TNF related apoptosis-inducing ligand I receptor excitability antigen epitope polypeptide and its antibody and applications.The present invention passes through molecular biology and immunological technique, it was found that a new TRAIL I receptor excitability epitope, amino acid sequence is as shown in SEQ ID NO.1.Studies have shown that the antigen epitope polypeptide has immunogenicity, the Balb/c mouse stimulated through the Antigenic Peptide can produce corresponding antibody, which can improve the effect of TRAIL killing tumor cell in vitro.Therefore, the invention also provides the antibody assists purposes and the antigen epitope polypeptide in TRAIL anti-tumor drug preparing the purposes in broad-spectrum anti-tumor vaccine in preparation.
Description
Technical field
The present invention relates to a kind of antigen epitope polypeptide, further relate to for antibody of the antigen epitope polypeptide and application thereof, it is special
Other, the present invention relates to a kind of newfound apoptosis induction ligand related to human tumor necrosis factor (TRAIL) I receptor excitabilities
Antigen epitope polypeptide further relates to stimulate tumor necrosin relative death caused by Balb/c mouse to lure as the antigen epitope polypeptide
Ligand i receptor specific antibody is led, further relates to the antibody in the killing for improving tumor necrosin relative death inducing ligand
The active purposes of tumour cell.The invention belongs to molecular biology and immunological technique.
Background technique
Malignant tumour has become seriously threatens human health and the lethal principal disease died now.In recent years, knubble biological is controlled
Treatment achieves greater advance, especially monoclonal antibody therapeutics for tumor and is successfully applied to clinic, obtains remarkable result.TRAIL
It is tumor necrosis factor superfamily newcomer.Other superfamily members are different from, as TNF-α and FasL are toxic to normal cell
Effect is limited applies in clinic as anti-tumor drug, and TRAIL shows as selective induction expression TRAIL specific receptors
Apoptosis and unsystematic cytotoxicity occur for tumour cell, make oncotherapy research hotspot.A large number of studies show that
TRAIL has very strong killing tumour ability, is most to have one of candidate drug of the antineoplaston of application prospect, has entered
The research of II clinical trial phase.But due to the presence of the tumor type low to TRAIL tolerance or sensibility, thus blocks TRAIL and make
It is anti-tumor drug in clinical application.
TRAIL can be tied with its five receptors (death receptor DR4 or DR5 and Decoy receptor DcR1, DcR2 and OPG)
It closes.In some tumour cells, Decoy receptor DcR1, DcR2 or OPG may cause to swell with death receptor competitive binding TRAIL
Drug resistance of the oncocyte to TRAIL.Therefore, researcher develops for selective death receptor DR4 or DR5 specific monoclonal antibody, it is intended to
Tumor effect is killed instead of TRAIL performance.Monoclonal antibody is only in conjunction with its corresponding unique receptor, the specificity with height.Death receptor
Monoclonal antibody great potential is shown to specific killing tumour cell effect.Preclinical test shows that monoclonal antibody is combined with other drugs and answers
With with synergistic effect, the effect of oncotherapy can be improved.
Currently, a variety of antibody for TRAIL excitability receptor have been in clinical trial, what they had can be direct
Instead of TRAIL in conjunction with excitability receptor killing tumor cell, what is had itself is unable to killing tumor cell, but can be improved
TRAIL killing tumor cell, such as AMG 655.2012, mouse was immunized in Marie P.Piechocki application DR5 DNA vaccination,
The antibody of anti-DR5 is isolated in success from immunized mice serum, these antibody can in vivo, Cytotoxicity in vitro tumour cell,
But killing activity possessed by the antibody of the different mouse generation after same vaccine is immune is not quite similar, this is particularly likely that
Because the DNA vaccination for DR5 produces Multiple Antibodies, to affect its activity.Therefore screening excitability DR4 or DR5 is anti-
Former epitope is to develop vaccine to provide strong support to replace or improve TRAIL killing activity.
Summary of the invention
The purpose of the present invention is to provide a kind of newfound human TNF related apoptosis-inducing ligand I receptor excitability antigen epitope polypeptides, should
Antigen epitope polypeptide has immunogenicity, and the tumor necrosis factor as caused by antigen epitope polypeptide stimulation Balb/c mouse is related
Apoptosis induction ligand I receptor specific antibody, which has, improves tumor necrosin relative death inducing ligand killing tumor cell
The purposes of effect.
In order to achieve the above object, present invention employs following technological means:
A kind of human TNF related apoptosis-inducing ligand I receptor excitability antigen epitope polypeptide of the invention, it is characterised in that the antigen table
The amino acid sequence of position polypeptide is as shown in SEQ ID NO.1.
The nucleotide sequence of the coding human TNF related apoptosis-inducing ligand I receptor excitability antigen epitope polypeptide is also in guarantor of the invention
Within the scope of shield.
Further, the invention also provides resist human TNF related apoptosis-inducing ligand I receptor excitability epitope of the present invention more
The antibody of peptide.
In the present invention, it is preferred to, the antibody is to be prepared by the following method to obtain: will be through described in claim 1
The immune Balb/c mouse of human TNF related apoptosis-inducing ligand I receptor excitability antigen epitope polypeptide peripheral blood with 8000rpm be centrifuged 5min with
The impurity including red blood cell is removed, is then purified using ProteinA/G Sepharose, human TNF related apoptosis-inducing ligand I type is obtained
The antibody of receptor agonism antigen epitope polypeptide.
Antibody activity detection experiments have shown that, being individually added into antibody of the present invention cannot effectively induce HeLa cell to wither
Die, and antibody+TRAIL can induction HeLa Apoptosis effectively, and work to be better than and TRAIL be used alone, thus say
Antibody caused by bright P7 antigen can effectively improve the ability of TRAIL killing HeLa cell
Therefore, further, the invention also provides the antibody in preparation auxiliary TRAIL anti-tumor drug
Purposes.And the antigen epitope polypeptide is preparing the purposes in broad-spectrum anti-tumor vaccine.
Detailed description of the invention
Fig. 1 is antibody specificity testing result；
Fig. 2 is antibody activity testing result.
Specific embodiment
The invention will now be further described with reference to specific embodiments, the advantages and features of the present invention will be with description and
It is apparent.But examples are merely exemplary for these, and it is not intended to limit the scope of the present invention in any way.Those skilled in the art
Member it should be understood that without departing from the spirit and scope of the invention can details to technical solution of the present invention and form into
Row modifications or substitutions, but these modifications and replacement are fallen within the protection scope of the present invention.
Embodiment one, human TNF related apoptosis-inducing ligand I receptor extracellular region structural domain divide and synthesis
1, the division of the extracellular peptide fragment of human TNF related apoptosis-inducing ligand I receptor
Human TNF related apoptosis-inducing ligand I receptor extracellular region is divided into 11 groups, shown in table 1, for one group with 20 amino acid to carry out polypeptide
Screening.
The division of table 1:TRAILI receptor extracellular region peptide fragment
| Peptide name section | Amino acid sequence |
| P1 | ASGTEAAAATPSKVWGSSAG |
| P2 | RIEPRGGGRGALPTSMGQHG |
| P3 | PSARARAGRAPGPRPAREAS |
| P4 | PRLRVHKTFKFVVVGVLLQV |
| P5 | VPSSAATIKLHDQSIGTQQW |
| P6 | EHSPLGELCPPGSHRSEHPG |
| P7 | ACNRCTEGVGYTNASNNLFA |
| P8 | CLPCTACKSDEEERSPCTTT |
| P9 | RNTACQCKPGTFRNDNSAEM |
| P10 | CRKCSRGCPRGMVKVKDCTP |
| P11 | WSDIECVHKESGNGHN |
2, the synthesis and coupling of Antigenic Peptide
11 groups of TRAIL I receptor Antigenic Peptides are synthesized by Qiang Yao Biotechnology Co., Ltd, and respectively with KLH (blood
Azurin) and BSA (bovine serum albumin(BSA)) be coupled.
The screening of embodiment two, anti-human TRAIL I receptor agon-ist epitopes
1, animal immune: the polypeptide being coupled with KLH synthesized using embodiment one is immunogene (KLH is negative control),
Be immunized Balb/c mouse after Freund's complete adjuvant emulsification, the polypeptides of 2 Zhou Houyong embodiments one and incomplete Freund's adjuvant carry out the
Secondary immunity after being immunized three times, takes mice serum to carry out bioactivity, and potency reaches 16000 or more mouse, direct abdominal cavity note
Aforementioned polypeptides booster immunization is penetrated, puts to death mouse after 3 days, separates peripheral blood.The mouse of P7 group Antigenic Peptide stimulation produces as the result is shown
Specific antibody is given birth to.
2, the purifying of antibody: P7 Antigenic Peptide (shown in ACNRCTEGVGYTNASNNLFA, SEQ ID NO.1) is post-stimulatory
Mouse peripheral blood, to remove the impurity such as red blood cell, then uses ProteinA/G Sepharose pure with 8 000rpm centrifugation 5min
Change antibody.
3, the detection of antibody specificity: 1 × 105HeLa cell is digested through 0.25% pancreatin, and PBS washing is added diluted
Immune serum, and the mice serum being immunized using KLH is incubated for 30min as negative control on ice, and after PBS washing, FITC label is added
Sheep anti-mouse igg, PBS washing after, flow cytometer detection situation in conjunction with cell surface receptor.It is produced the result shows that being stimulated through P7 Antigenic Peptide
Raw antibody can in conjunction with the TRAIL receptor I of HeLa cell surface, and with positive control antibodies no significant difference, as a result such as
Fig. 1.
The detection of 4 antibody activities: by HeLa cell with 5 × 103It is a/hole be inoculated with 96 orifice plates, be respectively set negative control group,
TRAIL group, antibody group, antibody+TRAIL group, TRAIL concentration are 1 μ g/ml, and antibody concentration is 1:10 dilution, and 24 as a child
Mtt assay detects Apoptosis.HeLa Apoptosis cannot effectively be induced by being individually added into antibody group as the result is shown, and antibody+
TRAIL group can induction HeLa Apoptosis effectively, and work and be better than TRAIL group, thus illustrate produced by P7 antigen
Antibody can effectively improve the ability of TRAIL killing Hela cell, as a result such as Fig. 2.
Claims (3)
1. a kind of human TNF related apoptosis-inducing ligand I receptor excitability antigen epitope polypeptide, it is characterised in that the ammonia of the antigen epitope polypeptide
Base acid sequence is as shown in SEQ ID NO.1.
2. encoding the nucleotide sequence of human TNF related apoptosis-inducing ligand I receptor excitability antigen epitope polypeptide described in claim 1.
3. purposes of the antigen epitope polypeptide described in claim 1 in preparation auxiliary TRAIL induction hela Apoptosis antibody.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998032856A1 (en) * | 1997-01-28 | 1998-07-30 | Human Genome Sciences, Inc. | Death domain containing receptor 4 (dr4: death receptor 4), member of the tnf-receptor superfamily and binding to trail (ap02-l) |
| CN101479296A (en) * | 2006-04-30 | 2009-07-08 | 北京同为时代生物技术有限公司 | TRAIL receptor-binding agents and uses of the same |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8329179B2 (en) * | 1997-01-28 | 2012-12-11 | Human Genome Sciences, Inc. | Death domain containing receptor 4 antibodies and methods |
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- 2016-08-17 CN CN201610680242.9A patent/CN106243215B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998032856A1 (en) * | 1997-01-28 | 1998-07-30 | Human Genome Sciences, Inc. | Death domain containing receptor 4 (dr4: death receptor 4), member of the tnf-receptor superfamily and binding to trail (ap02-l) |
| CN101479296A (en) * | 2006-04-30 | 2009-07-08 | 北京同为时代生物技术有限公司 | TRAIL receptor-binding agents and uses of the same |
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