CN106243215A - A kind of human TNF related apoptosis-inducing ligand I receptor excitability antigen epitope polypeptide and antibody thereof and application - Google Patents
A kind of human TNF related apoptosis-inducing ligand I receptor excitability antigen epitope polypeptide and antibody thereof and application Download PDFInfo
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- CN106243215A CN106243215A CN201610680242.9A CN201610680242A CN106243215A CN 106243215 A CN106243215 A CN 106243215A CN 201610680242 A CN201610680242 A CN 201610680242A CN 106243215 A CN106243215 A CN 106243215A
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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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- C07K16/065—Purification, fragmentation
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- 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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Abstract
The invention discloses a kind of human TNF related apoptosis-inducing ligand I receptor excitability antigen epitope polypeptide and antibody thereof and application.The present invention passes through molecular biology and immunological technique, it was found that a new TRAIL I receptor excitability epitope, its aminoacid sequence is as shown in SEQ ID NO.1.Research shows, described antigen epitope polypeptide has immunogenicity, and the Balb/c Mus stimulated through this antigenic peptides can produce corresponding antibody, and this antibody can improve the effect of TRAIL killing tumor cell in vitro.Therefore, the invention allows for described antibody purposes in preparation auxiliary TRAIL anti-tumor drug, and the purposes that described antigen epitope polypeptide is in preparing broad-spectrum anti-tumor vaccine.
Description
Technical field
The present invention relates to a kind of antigen epitope polypeptide, further relate to antibody for this antigen epitope polypeptide and application thereof, special
Other, the present invention relates to a kind of newfound apoptosis induction ligand related to human tumor necrosis factor (TRAIL) I receptor excitability
Antigen epitope polypeptide, further relates to be stimulated tumor necrosin relative death produced by Balb/c Mus to lure by this antigen epitope polypeptide
Lead ligand i receptor specific antibody, further relate to this antibody in the killing improving tumor necrosin relative death inducing ligand
The active purposes of tumor cell.The invention belongs to molecular biology and immunological technique.
Background technology
Malignant tumor has become current serious threat human health the lethal principal disease died.In recent years, knubble biological is controlled
Treatment achieves greater advance, and especially monoclonal antibody therapeutics for tumor is successfully applied to clinic, obtains remarkable result.TRAIL
It is tumor necrosis factor superfamily newcomer.It is different from other superfamily members, as TNF-α and FasL are toxic to normal cell
Effect limits applies in clinic as antitumor drug, and TRAIL shows as selective induction and expresses TRAIL specific receptors
Tumor cell generation apoptosis and unsystematic cytotoxicity, make oncotherapy study hotspot.Numerous studies show
TRAIL has the strongest killing tumor ability, is one of candidate medicine of antineoplaston having application prospect most, has entered
II clinical trial phase research.But due to the existence of the tumor type low to TRAIL tolerance or sensitivity, thus retardance TRAIL makees
For antitumor drug at clinical application.
TRAIL can tie with its five receptor (death receptor DR4 or DR5 and Decoy receptor DcR1, DcR2 and OPG)
Close.In some tumor cells, Decoy receptor DcR1, DcR2 or OPG may cause swelling with death receptor competition binding TRAIL
The oncocyte drug resistance to TRAIL.Therefore, researcher development is for selectivity death receptor DR4 or DR5 specific monoclonal antibody, it is intended to
Replace TRAIL to play and kill tumor effect.Monoclonal antibody, only in conjunction with its corresponding unique receptor, has the specificity of height.Death receptor
Monoclonal antibody specific killing tumor cell effect is demonstrated great potential.Preclinical test shows that monoclonal antibody is combined with other drug and answers
With having cooperative effect, the effect of oncotherapy can be improved.
At present, the multiple antibody for TRAIL excitability receptor has been in clinical trial, and what they had can be direct
Replace TRAIL be combined killing tumor cell with excitability receptor, have self can not killing tumor cell, but can improve
TRAIL killing tumor cell, such as AMG 655.2012, Marie P.Piechocki application DR5 DNA vaccination immune mouse,
The antibody of anti-DR5 is isolated in success from immunized mice serum, these antibody can in vivo, Cytotoxicity in vitro tumor cell,
But the killing activity that the antibody that the different mices after same vaccine immunity produce is had is not quite similar, and this is particularly likely that
Because the DNA vaccination for DR5 creates Multiple Antibodies, thus have impact on its activity.Therefore screening excitability DR4 or DR5 resist
Former epi-position provides powerful support for replacement or raising TRAIL killing activity for the offer that develops vaccine.
Summary of the invention
It is an object of the invention to provide a kind of newfound human TNF related apoptosis-inducing ligand I receptor excitability antigen epitope polypeptide, should
Antigen epitope polypeptide has immunogenicity, this antigen epitope polypeptide stimulate tumor necrosis factor produced by Balb/c Mus to be correlated with
Apoptosis induction ligand I receptor specific antibody has raising tumor necrosin relative death inducing ligand killing tumor cell
The purposes of effect.
In order to achieve the above object, present invention employs techniques below means:
A kind of human TNF related apoptosis-inducing ligand I receptor excitability antigen epitope polypeptide of the present invention, it is characterised in that described antigen table
The aminoacid sequence of position polypeptide is as shown in SEQ ID NO.1.
The nucleotide sequence of the human TNF related apoptosis-inducing ligand I receptor excitability antigen epitope polypeptide described in coding is also the guarantor of the present invention
Within the scope of protecting.
Further, the invention allows for anti-human TNF related apoptosis-inducing ligand I receptor excitability epitope of the present invention many
The antibody of peptide.
In the present invention, it is preferred to, described antibody is to be prepared by the following method to obtain: will be through described in claim 1
Human TNF related apoptosis-inducing ligand I receptor excitability antigen epitope polypeptide immunity Balb/c Mus peripheral blood with 8000rpm be centrifuged 5min with
Remove the impurity including erythrocyte, then use ProteinA/G Sepharose to be purified, it is thus achieved that human TNF related apoptosis-inducing ligand I type
The antibody of receptor agonism antigen epitope polypeptide.
Antibody activity detection test shows, is individually added into antibody of the present invention and can not effectively induce HeLa cell to wither
Die, and antibody+TRAIL can induction HeLa apoptosis effectively, and work to be better than and be used alone TRAIL, thus say
Antibody produced by bright P7 antigen can effectively improve TRAIL and kill the ability of HeLa cell
Therefore, further, the invention allows for described antibody in preparation auxiliary TRAIL anti-tumor drug
Purposes.And the purposes that described antigen epitope polypeptide is in preparing broad-spectrum anti-tumor vaccine.
Accompanying drawing explanation
Fig. 1 is antibody specificity testing result；
Fig. 2 is antibody activity testing result.
Detailed description of the invention
Below in conjunction with specific embodiment further describe the present invention, advantages of the present invention and feature will be with describe and
Apparent.But these embodiments are only exemplary, the scope of the present invention is not constituted any restriction.People in the art
Member it should be understood that to enter the details of technical solution of the present invention and form lower without departing from the spirit and scope of the present invention
Row amendment or replacement, but these amendments and replacement each fall within 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 outer peptide fragment of human TNF related apoptosis-inducing ligand I receptor born of the same parents
It is one group by human TNF related apoptosis-inducing ligand I receptor extracellular region with 20 aminoacid, is divided into 11 groups, shown in table 1, to carry out polypeptide
Screening.
The division of table 1:TRAILI receptor extracellular region peptide fragment
| Peptide name section | Aminoacid 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 of antigenic peptides and coupling
11 groups of TRAIL I receptor antigenic peptides are synthesized by Qiang Yao bio tech ltd, and respectively with KLH (blood
Azurin) and BSA (bovine serum albumin) carry out coupling.
Embodiment two, the screening of anti-human TRAIL I receptor agon-ist epitopes
1, animal immune: the polypeptide with KLH coupling using embodiment one synthesis is immunogen (KLH is negative control),
With immunity Balb/c Mus after Freund's complete adjuvant emulsifying, after 2 weeks, carry out the with the polypeptide of embodiment one and incomplete Freund's adjuvant
Secondary immunity, after three immunity, takes mice serum and carries out bioactivity, and titer reaches the mice of more than 16000, and direct abdominal cavity is noted
Penetrate aforementioned polypeptides booster immunization, by sacrifice after 3 days, separate peripheral blood.The mice that result display P7 group antigenic peptides stimulates produces
Give birth to specific antibody.
2, the purification of antibody: by post-stimulatory for P7 antigenic peptides (shown in ACNRCTEGVGYTNASNNLFA, SEQ ID NO.1)
Mouse peripheral blood is centrifuged 5min to remove the impurity such as erythrocyte with 8 000rpm, then uses ProteinA/G Sepharose pure
Change antibody.
3, the detection of antibody specificity: 1 × 105HeLa cell washs through 0.25% trypsinization, PBS, adds dilution
Immune serum, and with mice serum immune for KLH as negative control, after hatching 30min, PBS washing on ice, add FITC labelling
Sheep anti-mouse igg, PBS washing after, flow cytometer detection is combined situation with cell surface receptor.Result shows to stimulate through P7 antigenic peptides to produce
Raw antibody can be combined with the TRAIL receptor I of HeLa cell surface, and with positive control antibodies no significant difference, result is such as
Fig. 1.
The detection of 4 antibody activities: by HeLa cell with 5 × 10396 orifice plates are inoculated in individual/hole, be respectively provided with negative control group,
TRAIL group, antibody group, antibody+TRAIL group, TRAIL concentration is 1 μ g/ml, and antibody concentration is 1:10 dilution factor, and 24 as a child
Mtt assay detection apoptosis.Result shows that being individually added into antibody group can not effectively induce HeLa apoptosis, and antibody+
TRAIL group can induction HeLa apoptosis effectively, and work and be better than TRAIL group, thus explanation P7 antigen is produced
Antibody can effectively improve TRAIL kill Hela cell ability, result such as Fig. 2.
Claims (6)
1. a human TNF related apoptosis-inducing ligand I receptor excitability antigen epitope polypeptide, it is characterised in that the ammonia of described antigen epitope polypeptide
Base acid sequence is as shown in SEQ ID NO.1.
2. the nucleotide sequence of coding human TNF related apoptosis-inducing ligand I receptor excitability antigen epitope polypeptide described in claim 1.
The antibody of the human TNF related apoptosis-inducing ligand I receptor excitability antigen epitope polypeptide described in the most anti-claim 1.
4. antibody as claimed in claim 3, it is characterised in that be to be prepared by the following method to obtain:
Periphery by the Balb/c Mus through the human TNF related apoptosis-inducing ligand I receptor excitability antigen epitope polypeptide immunity described in claim 1
Blood is centrifuged 5min to remove impurity including erythrocyte with 8000rpm, then uses ProteinA/G Sepharose to enter
Row purification, it is thus achieved that the antibody of human TNF related apoptosis-inducing ligand I receptor excitability antigen epitope polypeptide.
5. the purposes in preparation auxiliary TRAIL anti-tumor drug of the antibody described in claim 3 or 4.
6. the purposes in preparing broad-spectrum anti-tumor vaccine of the antigen epitope polypeptide described in claim 1.
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Citations (3)
| 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) |
| US20080241155A1 (en) * | 1997-01-28 | 2008-10-02 | Human Genome Sciences, Inc. | Death domain containing receptor 4 |
| 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 |
-
2016
- 2016-08-17 CN CN201610680242.9A patent/CN106243215B/en active Active
Patent Citations (3)
| 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) |
| US20080241155A1 (en) * | 1997-01-28 | 2008-10-02 | Human Genome Sciences, Inc. | Death domain containing receptor 4 |
| 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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