CN108732361A - A kind of antineoplastic polypeptide and its blood testing ELISA kit - Google Patents
A kind of antineoplastic polypeptide and its blood testing ELISA kit Download PDFInfo
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Abstract
The invention discloses a kind of antineoplastic polypeptides, for CAD and the artificial synthesized polypeptide being made of 72 amino acid, in antineoplastic polypeptide optimization process, the conventional polypeptide layout strategy of amino acid selectively replacement and additions and deletions is not used only, amino acid side chain is also changed structure strategy to be fused in design concept, obtains ideal polypeptide structure to a greater extent.Antineoplastic polypeptide peptide chain length of the present invention is short, can be obtained using conventional Fmoc method synthesis in solid state, is PD-1 inhibitor, has preferable inhibitory activity to tumour.Monoclonal antibody and ELISA kit are further obtained on the basis of antineoplastic polypeptide, the content in drug discovery process for detecting antineoplastic polypeptide in blood.
Description
Technical field
The invention belongs to polypeptide designs and synthesis in solid state, Antibody preparation and detection and pharmacology activity research field, specifically relate to
And a kind of antineoplastic polypeptide and its blood testing ELISA kit.
Technical background
Malignant tumour is a kind of disease of serious damage human health, the four of all deaths caused by China's tumour
/ mono-.According to statistics, 250 people just suffer from malignant tumour in every 100,000 people in the whole nation within 2011.Recent decades, China's malignant tumour
Incidence is all improved every year, and the ascendant trend of incidence is intended to straight line.It is estimated that will in every 100,000 people in China in 2049
There are 400 people's cancer strickens.The treatment means of tumour are still largely based on drug therapy at present.Thus, research and
Novel efficient, less toxic antitumor drug is developed to have very important significance.Wherein, the small peptide compounds tool that amino acid is oriented to
There is extraordinary antitumor activity.In addition to this, amino acid carrier has extraordinary bioaffinity and biocompatibility.?
In some antitumor drug molecules, amino acid is introduced, the selectivity and biocompatibility of drug can be improved, and it can be reduced
To the toxicity of normal cell.On the other hand, peptides are one of bioactive molecules of Almightiness type, false peptide and pseudo- peptides with
And its bioactivity of derivative is very more, for example, antibacterial, antiviral and anticancer isoreactivity.
Body T cell is immunized plays important function in the generation, evolution of control tumour.Wherein tumor by local soaks
Lubricant nature CD8+T lymphocytes have played key effect in antitumor immunity of organism response.Discovered in recent years, in tumor patient body
There are immunosuppression mechanisms, interfere removing of the body to tumour, tumour is caused to be colonized for a long time, show as T lymphocyte functions barrier
Hinder and weakens with proliferative capacity.PD-1 has been acknowledged as the surface marker of debilitating T lymphocytes.Block PD-1/PD-L1 accesses
The function of debilitating T cell can partly be restored.Known PD-1 (programmed death 1) programmed death receptor 1 is
A kind of important immunosuppression molecule.It is the immunological regulation of target spot to antitumor, anti-infective, anti-autoimmune disease using PD-1
And organ transplant survival etc. has important meaning.Its ligand PD-L1 also can be used as target spot, and corresponding antibody can also play phase
Same effect.
According to the space structure design polypeptide of albumen, to inhibit the function of albumen to study at present in three-dimensional structure
Hot spot, and also there are multiple products to list.High investment based on the research of PD-1 antibody and uncertainty, for the spy of PD-1
The research of specific polypeptide is also a new research direction.
False peptide（pseudo-peptide）With the change of difference Partial Fragment only in peptide fragment of peptide.The acquisition of false peptide and knot
Structure confirmation method is similar to the synthetic method of general peptide, is only that the change of raw material amino acid.False peptide has tolerance enzymolysis, stablizes
The advantages that property is good, bioavilability is high, false peptide research has become drug as the important means of polypeptide drug structure optimization and grinds
Very active field in hair.
Invention content
A kind of antineoplastic polypeptide of present invention offer is CAD and artificial synthesized by 72 amino acid groups
At polypeptide, the software of the CAD is the independent development of applicant team, and operation principle is：With certain length
Random small peptide make it in the surface migration of PD-1 protein moleculars, calculate each atom and the adjacent original of PD-1 protein surfaces on the small peptide
The active force of son, and energy variation that is mobile, reversing peptide fragment generation, optimize more various possible combinations, select repeatedly
Minimum energy is selected, as determining conformation.In antineoplastic polypeptide optimization process, amino acid is not used only and selectively replaces and increases
Amino acid side chain is also changed structure strategy and is fused in design concept, obtained to a greater extent by the conventional polypeptide layout strategy deleted
Ideal polypeptide structure.Antineoplastic polypeptide peptide chain length of the present invention is short, can be obtained using conventional Fmoc method synthesis in solid state, and
And there is preferable inhibitory activity to tumour.
One aspect of the present invention discloses a kind of antineoplastic polypeptide, sequence such as SEQ ID NO：Shown in 1.
Another aspect of the present invention discloses a kind of antineoplastic polypeptide, passes through sequence such as SEQ ID NO：In polypeptide chain shown in 1
A few amino acids side-chain structure improvement and design form, the 17th amino acid Tyr, the 37th amino acid Phe, in sequence
68 amino acid Tyr at least one replace with（Abbreviation Baa in this patent, CAS：
1252914-17-2）.Baa exists with the hydroxyl of front and back amino acid and amino condensation as the form of peptide bond in peptide chain.
Further, amino acid is L-type in the antineoplastic polypeptide, i.e., absolute configuration is S types.
In currently preferred technical solution, the antineoplastic polypeptide sequence is：
| SEQ ID | SEQ |
| NO：1 | Ser-Val-Phe-Trp-Lys-Gly-Gly-Arg-Glu-His-His-Asp-Lys-His-Leu-Ala-Tyr-Ile-Arg-Phe-Asp-Ile-Pro-Gly-Ser-Gly-Pro-Phe-Asn-Asn-Arg-Asp-Leu-Gln-Gly- Ser-Phe-Ile-Asn-Glu-Trp-Asn-Arg-Leu-Gly-Lys-Ile-Gly-Thr-Ala-Met-Arg-Asp-Phe-Leu-Thr-Tyr-Ala-Pro-Pro-Thr-Asp-Trp-Ile-Leu-Cys-Asn-Tyr-Ile-Gly- Thr-Ala |
| NO：2 | Ser-Val-Phe-Trp-Lys-Gly-Gly-Arg-Glu-His-His-Asp-Lys-His-Leu-Ala-Baa-Ile-Arg-Phe-Asp-Ile-Pro-Gly-Ser-Gly-Pro-Phe-Asn-Asn-Arg-Asp-Leu-Gln-Gly- Ser-Phe-Ile-Asn-Glu-Trp-Asn-Arg-Leu-Gly-Lys-Ile-Gly-Thr-Ala-Met-Arg-Asp-Phe-Leu-Thr-Tyr-Ala-Pro-Pro-Thr-Asp-Trp-Ile-Leu-Cys-Asn-Tyr-Ile-Gly- Thr-Ala |
| NO：3 | Ser-Val-Phe-Trp-Lys-Gly-Gly-Arg-Glu-His-His-Asp-Lys-His-Leu-Ala-Tyr-Ile-Arg-Phe-Asp-Ile-Pro-Gly-Ser-Gly-Pro-Phe-Asn-Asn-Arg-Asp-Leu-Gln-Gly- Ser-Baa-Ile-Asn-Glu-Trp-Asn-Arg-Leu-Gly-Lys-Ile-Gly-Thr-Ala-Met-Arg-Asp-Phe-Leu-Thr-Tyr-Ala-Pro-Pro-Thr-Asp-Trp-Ile-Leu-Cys-Asn-Tyr-Ile-Gly- Thr-Ala |
| NO：4 | Ser-Val-Phe-Trp-Lys-Gly-Gly-Arg-Glu-His-His-Asp-Lys-His-Leu-Ala-Tyr-Ile-Arg-Phe-Asp-Ile-Pro-Gly-Ser-Gly-Pro-Phe-Asn-Asn-Arg-Asp-Leu-Gln-Gly- Ser-Phe-Ile-Asn-Glu-Trp-Asn-Arg-Leu-Gly-Lys-Ile-Gly-Thr-Ala-Met-Arg-Asp-Phe-Leu-Thr-Tyr-Ala-Pro-Pro-Thr-Asp-Trp-Ile-Leu-Cys-Asn-Baa-Ile-Gly- Thr-Ala |
| NO：5 | Ser-Val-Phe-Trp-Lys-Gly-Gly-Arg-Glu-His-His-Asp-Lys-His-Leu-Ala-Baa-Ile-Arg-Phe-Asp-Ile-Pro-Gly-Ser-Gly-Pro-Phe-Asn-Asn-Arg-Asp-Leu-Gln-Gly- Ser-Baa-Ile-Asn-Glu-Trp-Asn-Arg-Leu-Gly-Lys-Ile-Gly-Thr-Ala-Met-Arg-Asp-Phe-Leu-Thr-Tyr-Ala-Pro-Pro-Thr-Asp-Trp-Ile-Leu-Cys-Asn-Tyr-Ile-Gly- Thr-Ala |
| NO：6 | Ser-Val-Phe-Trp-Lys-Gly-Gly-Arg-Glu-His-His-Asp-Lys-His-Leu-Ala-Baa-Ile-Arg-Phe-Asp-Ile-Pro-Gly-Ser-Gly-Pro-Phe-Asn-Asn-Arg-Asp-Leu-Gln-Gly- Ser-Phe-Ile-Asn-Glu-Trp-Asn-Arg-Leu-Gly-Lys-Ile-Gly-Thr-Ala-Met-Arg-Asp-Phe-Leu-Thr-Tyr-Ala-Pro-Pro-Thr-Asp-Trp-Ile-Leu-Cys-Asn-Baa-Ile-Gly- Thr-Ala |
| NO：7 | Ser-Val-Phe-Trp-Lys-Gly-Gly-Arg-Glu-His-His-Asp-Lys-His-Leu-Ala-Tyr-Ile-Arg-Phe-Asp-Ile-Pro-Gly-Ser-Gly-Pro-Phe-Asn-Asn-Arg-Asp-Leu-Gln-Gly- Ser-Baa-Ile-Asn-Glu-Trp-Asn-Arg-Leu-Gly-Lys-Ile-Gly-Thr-Ala-Met-Arg-Asp-Phe-Leu-Thr-Tyr-Ala-Pro-Pro-Thr-Asp-Trp-Ile-Leu-Cys-Asn-Baa-Ile-Gly- Thr-Ala |
| NO：8 | Ser-Val-Phe-Trp-Lys-Gly-Gly-Arg-Glu-His-His-Asp-Lys-His-Leu-Ala-Baa-Ile-Arg-Phe-Asp-Ile-Pro-Gly-Ser-Gly-Pro-Phe-Asn-Asn-Arg-Asp-Leu-Gln-Gly- Ser-Baa-Ile-Asn-Glu-Trp-Asn-Arg-Leu-Gly-Lys-Ile-Gly-Thr-Ala-Met-Arg-Asp-Phe-Leu-Thr-Tyr-Ala-Pro-Pro-Thr-Asp-Trp-Ile-Leu-Cys-Asn-Baa-Ile-Gly- Thr-Ala |
Antineoplastic polypeptide of the present invention is that artificial synthetic method is prepared, and is specifically obtained for Fmoc method synthesis in solid state.This
Field technology personnel know after known peptide sequence, and a variety of methods may be used and prepared, only with artificial synthesis and
Speech just has many variety classes.The present invention is purified using high performance liquid chromatography, it can also be used in those skilled in the art
He purifies known method, such as：Immunoabsorption.Further, since the particularity of the polypeptide of biological field, this field skill
Art personnel are it is well known that using sequencing（Edman edman degradation Edmans, tandem mass spectrum）, mass spectrum and nuclear-magnetism organically combine come the side of realization
Just, accurate structural identification.
It is PD-1 inhibitor that another aspect of the present invention, which discloses a kind of antineoplastic polypeptide, and in test example part, the present invention's is anti-swollen
Tumor polypeptide is outstanding to being showed in the affinity measurement of PD-1, prompts may have positive treatment to make PD-1 positive cancer patients
With especially lung cancer and melanoma patients.When finding the arrival of test group mouse tumor terminal in the experiment of heteroplastic transplantation tumor
Between obviously increase, further confirm the therapeutic effect of antineoplastic polypeptide of the present invention to cancer.
Find that antineoplastic polypeptide of the present invention has good drug safety by cytology toxicity test, through kunming mice
Abdominal cavity single-dose acute toxicity test finds to have no any toxic reaction when highest dosage 2000mg/kg.
Another aspect of the present invention discloses a kind of monoclonal antibody of antineoplastic polypeptide and preparation method thereof, and further public
A kind of kit of the monoclonal antibody comprising antineoplastic polypeptide is opened, this antibody and kit can be used for detecting blood moderate resistance
The content of oncopeptide.The preparation of monoclonal antibody has been at present more mature technology, can be with known to antigen
The preparation flow that monoclonal antibody is directly applied mechanically by Reagent Company obtains.
In antineoplastic polypeptide body in metabolism and clinical application, content is all important observation index, blood inspection in blood
Preparing for the quasi- kit of mark is most important, and the sample in kit is all made of unified technique and obtains, stable quality, testing result
Reliably.
Kit of the present invention is enzyme linked immunological（ELISA）Kit, including antineoplastic polypeptide monoclonal antibody, into
One step, including following reagent：
（1）Standard antineoplastic polypeptide：Antineoplastic polypeptide is dissolved in common buffer solution with determining amount and is frozen, more in order to ensure
Peptide quality can be preserved in the form of mother liquor, and multiple singles are used as possible with concentration variation in order to avoid multigelation is damaged
Use packaging.
（2）Antineoplastic polypeptide monoclonal antibody（Primary antibody）.
（3）Enzyme labelled antibody（Secondary antibody）：It can amplify the letter of primary antibody by developing the color with the antibody of an anti-binding with enzyme label
Number, realize quantitative detection.Those skilled in the art can also by other means realize that secondary antibody marks, not in the present invention one
One enumerates, the mode of enzyme label also there are many, it is anti-with horseradish peroxidase enzyme mark goat anti-mouse in application method of the present invention
For body.
Specific implementation mode
Embodiment 1：Antineoplastic polypeptide synthesis in solid state, purifying and structural identification
Antineoplastic polypeptide of the present invention is all made of the acquisition of Fmoc solid-phase synthesis, and high-efficient liquid phase chromatogram purification simultaneously passes through sequence
Analysis and mass spectrum verification.Its concrete operations is as follows：
1, the synthesis in solid state of antineoplastic polypeptide
1）Using DMF as solvent, various alpha-amidos are by a concentration of 0.25M of fmoc-protected amino acid solution, HBTU solution, HOBt
A concentration of 0.33M of solution, a concentration of 200ml/L of piperidine solution, a concentration of 174.2ml/L of DIEA solution.
2）Weigh 0.05mmol Fmoc-Ala-Wang resins（Functional group content 0.33mmol/g）It is placed in solid phase reactor
In, add 8ml DCM swellings overnight, decompression pumps solvent.The piperidine solution 8ml of a concentration of 200ml/L is added, reacts at room temperature
5min is drained；The piperidine solution 8ml for adding a concentration of 200ml/L, reacts 20min, drains at room temperature；Use 8ml's successively
DMF is washed 3 times, each 1min.The accurate alpha-amido for measuring 0.2mmol is by fmoc-protected amino acid solution, 0.2mmol
HBTU solution, 0.195mmol HOBt solution be added solid phase reactor in, react 5min after be added 0.4mmol DIEA, room temperature
Lower nitrogen bubble oscillating reactions 2h.It is washed 3 times with the DMF of 8ml successively, each 1min.The piperidines that a concentration of 200ml/L is added is molten
Liquid 8ml, reacts 5min at room temperature, drains；The piperidine solution 8ml for adding a concentration of 200ml/L, reacts 20min at room temperature, takes out
It is dry；It is washed 3 times with the DMF of 8ml successively, each 1min.This is continuously synthesized one by one from the C-terminal of peptide chain to N-terminal according to step as above
The antineoplastic polypeptide of invention.
The Fmoc Preservation tactics of amino acid of the present invention are specially：Fmoc-Ala-OH, Fmoc-Arg (Pbf)-OH,
Fmoc-Asn (Trt)-OH, Fmoc-Asp (OtBu)-OH, Fmoc-Cys (Trt)-OH, Fmoc-Gln (Trt)-OH, Fmoc-Glu
(OtBu)-OH, Fmoc-Gly-OH, Fmoc-His (Trt)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Lys (Boc)-
OH, Fmoc-Met-OH, Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Ser (tBu)-OH, Fmoc-Thr (tBu)-OH,
Fmoc-Trp (Boc)-OH, Fmoc-Tyr (tBu)-OH, Fmoc-Val-OH, Fmoc-Baa-OH.
3）After to be synthesized, the resin of polypeptide chain is connected with DMF and DCM successively cleaning respectively, after by resin as true
Drying for standby in empty drying box.
It needs to stress, amino acid R bases involved in some technical solutions of antineoplastic polypeptide of the present invention
The R group of the special amino acid classes that are modified of group, Baa and Paa are relatively stable group, without sulfydryl, hydroxyl and
Additional amino needs to protect, it is only necessary to protect amino using Fmoc, ensure the accurate of synthesis in solid state to a certain extent
Rate reduces purifying difficulty, improves combined coefficient.
2, the cutting of antineoplastic polypeptide
1）After resin drying, resin is broken up as possible with scraper, is transferred in heart bottle, magneton is added, delays under ice-water bath
The slow cutting liquid 10ml being added for removing resin（TFA in cutting liquid：Pure water：Thioanisole：Phenol：Dithioglycol ratio is
82.5:5:5:5:2.5）, 2h is reacted under ice-water bath.
2）It waits for that reaction is completed, reaction solution is transferred along together with resin into filter, is filtered with water pump, the filter that will be obtained
Liquid is placed in round-bottomed flask, and is dried up under nitrogen flowing.
3）It is sticky to wait for that the sample in round-bottomed flask is blown to, removes nitrogen tube, 4 DEG C of ice of about 20ml are poured into round-bottomed flask
Ether is fully broken up after mixing, and then in refrigerated centrifuge, 8000r/min centrifuges 15min at 4 DEG C, abandons supernatant, then at
It breaks up, centrifuges in 4 DEG C of ice ether of 20ml；It repeats operation 3 times, precipitation is dried in vacuo again to get antineoplastic polypeptide
Crude product.
3, the purifying and identification of antineoplastic polypeptide
Purification is carried out to antineoplastic polypeptide using RP-HPLC method.Using C18 semi-preparative columns, mobile phase：A phases
（Water phase）：Deionized water（Containing 0.1% TFA (m/v)）；B phases（Organic phase）：80% acetonitrile solution（Containing 0.1% TFA (m/v)）；
Flow velocity：5ml/min；Gradient：A phases with per minute 1% variation from 50% drop to 10%, B phases with per minute 1% variation from
50% is raised to 90%.According to high-efficient liquid phase chromatogram Fractional Collections eluent, the eluent revolving corresponding to main peak removes organic phase, so
Solid powder is obtained after freezing freezes afterwards, errorless through sequencing sequence, mass spectrum verification has feature corresponding with theoretical molecular weight
Peak.
Embodiment 2：The preparation of antineoplastic polypeptide monoclonal antibody
The preparation of monoclonal antibody has been at present more mature technology, can directly provide antineoplastic polypeptide by Reagent Company's system
It is standby, or be made by following methods, operations described below step is only sketched, and is not addressed place and is all made of technology hand in the prior art
Section or the conventional technical means of those skilled in the art.
（1）Immune animal obtains bone-marrow-derived lymphocyte
Polypeptide is emulsified using Freund's adjuvant, mouse is cooked and is immunized three times, and does primary reinforcement in first three day for taking its spleen and exempts from
Epidemic disease, the affinity of antibody that may make are preferable.
（2）Cell fusion
The myeloma cell with the mouse of immune mouse homology is taken, not secreting type and deficient, the present embodiment can be used to use enzyme
Deficiency lacks TK（Thymidine kinase）And HGPRT（Hypoxanthine guanine phosphoribosyltransferase）.
Cell fusion can use chemical fusion method and electro fusion method etc..The present embodiment uses chemical fusion method, chemistry used
Fusion agent is PEG（Polyethylene glycol）, molecular weight is bigger, and fusion rate is higher, but toxicity is also stronger simultaneously, therefore the present embodiment
It is≤4000 that PEG, which selects molecular weight, is carried out in the alkaline environment of pH8.0~pH8.8 or so.
（3）Cell screening
Cell screening is the selectivity culture of cell, it is therefore an objective to which the hybridoma for screening fusion is selectively cultivated using HAT
Base（Add hypoxanthine HyPoxanthine H, aminopterin Aminoopterin A and thymidine Thymidine in culture medium
T）.In HAT culture mediums, the myeloma cell that do not merge cannot utilize remedial pathway synthetic DNA due to a lack of TK and HGPRT, and
Glutamine and urine nucleotide monophosphate synthetic DNA, this approach can only be utilized to be blocked again by aminopterin, therefore inevitably
Want dead.After deactivating HAT culture mediums, with HT culture mediums.The B cell and T cell not merged all can in the case of normal culture
Naturally it withers away.In this way, only have the hybridoma of fusion due to obtaining TK and HGPRT from splenocyte, and it is thin with myeloma
The characteristic of born of the same parents' energy infinite multiplication, therefore can be proliferated.
The hybridoma grown in HAT culture mediums, only minority are the thin of the predetermined monoclonal antibody specific of secretion
Born of the same parents, it is therefore necessary to be screened.Using enzyme linked immunosorbent assay (ELISA)（ELISA）, filter out monoclonal antibody needed for capable of generating
Positive hybridoma cell.
（4）Cloning
Hybridizing clones for detecting antibody positive should carry out cloning as early as possible, and otherwise antibody secreted cell can be overstepping one's bounds by antibody
The cell secreted is inhibited.Cloning method uses limiting dilution assay or FACS（Fluorescence-activation sorter）Partition method.
（5）Mass propagation
A large amount of preparations of monoclonal antibody can use extracorporeal culture-ing and ascites method.The present invention uses ascites method.The cell of cloning
Mass propgation can be carried out in vitro, collected supernatant and obtained a large amount of single cloning antibody.But extracorporeal culture-ing
Obtained monoclonal antibody is limited, no more than specific cell concentration, and to change culture solution daily.And hybridoma is thin in vivo
Born of the same parents' breeding can overcome these limitations, take Syngenic mice, first pre-processed in its Intraperitoneal injection norphytane, 1~2 week pneumoretroperitoneum
Hybridoma is injected, has apparent ascites to generate after a week.The ascites antibody content prepared in this way is high, miscellaneous in ascites
Albumen is also less, is convenient for the purifying of antibody.
（6）Antibody purification
Before monoclonal antibody purifying, ascites is pre-processed using silica absorption method or filter centrifugation method, is then carried out single
Anti- slightly carries, and thick extracting method selects ammonium sulfate precipitation method, octanoic acid-ammonium sulfate precipitation method or the euglobulin precipitation method, using DEAE
Ion exchange column, gel filtration or affinity chromatography carry out the final purification of monoclonal antibody, titration, and packing freezes.
Embodiment 3：Blood testing enzyme linked immunological kit
In antineoplastic polypeptide body in metabolism and clinical application, content is all important observation index, blood testing mark in blood
Preparing for quasi- kit is most important, and the sample in kit is all made of unified technique and obtains, and stable quality, testing result can
It leans on.
Kit of the present invention is enzyme linked immunological kit, including following reagent：
（1）Standard antineoplastic polypeptide：Antineoplastic polypeptide is dissolved in common buffer solution with determining amount and is frozen, more in order to ensure
Peptide quality can be preserved in the form of mother liquor, and multiple singles are used as possible with concentration variation in order to avoid multigelation is damaged
Use packaging.
（2）Antineoplastic polypeptide monoclonal antibody（Primary antibody）：Preparation method is as described in Example 2.
（3）Enzyme labelled antibody（Secondary antibody）：It can amplify the letter of primary antibody by developing the color with the antibody of an anti-binding with enzyme label
Number, realize quantitative detection.Those skilled in the art can also by other means realize that secondary antibody marks, not in the present invention one
One enumerates, the mode of enzyme label also there are many, it is anti-with horseradish peroxidase enzyme mark goat anti-mouse in application method of the present invention
For body.
Embodiment 4：Enzyme linked immunological kit application method
1, standard group：Standard antineoplastic polypeptide solution is diluted to multiple concentration with PBS, and 50 μ are added per hole into 96 hole elisa Plates
l.Test group：It takes serum dilution PBS to dilute 100 times, 50 μ l is added per hole into 96 hole elisa Plates.It was incubated under room temperature
Night evaporating water completes coating process.
2, ELISA Plate is put into 60 DEG C of heating 30min in incubator.
3, it per 200 μ l confining liquids of hole, is incubated 3 hours and closes in 37 DEG C of insulating boxs.Confining liquid is concentration 3%（Quality volume is dense
Degree）Skimmed milk power PBS solution.
4, confining liquid is poured out, per 200 μ l PBST of hole, board-washing 4 times.PBST is concentration 1 ‰（Volumetric concentration）Tween 20
PBS solution.
5, antineoplastic polypeptide monoclonal antibody is added, per hole 50 μ l, 4 DEG C of incubation 4h.
6, liquid is poured out, per 200 μ l PBST of hole, board-washing 4 times.
7, horseradish peroxidase enzyme mark goat anti-mouse antibody is added, per hole 50 μ l, 4 DEG C of incubation 2h.
8, liquid is poured out, per 200 μ l PBST of hole, board-washing 4 times.
9, the substrate developing solution of Extemporaneous is added, being incubated 30min per 75 μ l of hole, in general 37 DEG C of insulating boxs will appear
Apparent color change.Substrate developing solution（The NaHPO of 2.57ml 0.2mol/L4The lemon of solution and 2.43ml 0.1mol/L
PH to 5.0 is adjusted after acid solution mixing, using the preceding hydrogen peroxide that 15 μ l 3% are added, 4mg OPD add distilled water to 10ml）.
10, terminate liquid is added（The H of 2mol/L2SO4）Reaction is terminated, per 25 μ l of hole.
11, microplate reader measures the absorption value at 492.
12, standard group draws standard curve according to absorption value, and calculates antineoplastic polypeptide of the present invention in serum
Concentration.
Test example 1：Measurement of the antineoplastic polypeptide to the affinity of PD-1
（1）Antineoplastic polypeptide is configured to the solution of a concentration of 1mg/mL with 1 × PBS, is further configured to concentration with 1 × PBS
For the solution of 100 μ g/mL, 2.5 μ L is taken to be dropped in the SPR chips of carboxylated（Purchased from Plexera companies, the type SPR standard configurations of K × 5
Substrate）On, polypeptide is fixed on SPR chips by the specific reaction of streptavidin and biotin.It then successively will be dense
Degree is the PD-1 solution of 0.1 μ g/mL, 1 μ g/mL, 10 μ g/mL, 50 μ g/mL（1 × PBS dilutes）Pass through chip list as mobile phase
Face, binding time 150s, Dissociation time 130s, live again time 200s.Using 1 × PBS as dissociation solution, using 0.5% phosphoric acid as
It lives again liquid.In the type SPR instrument of K × 5（Plexera）The combination dissociation curve of upper record polypeptide and TNF-α.
（2）With the SPR curves of polypeptide described in Langmuir formula fittings and PD-1.Be computed can obtain balance combination/
Dissociation constant.The equilibrium dissociation constant KD values of polypeptide and PD-1 see the table below：
| SEQ ID | KD values（nM） |
| NO：1 | 3.42 |
| NO：4 | 0.97 |
| NO：5 | 1.36 |
| NO：7 | 0.35 |
As seen from the above table, antineoplastic polypeptide of the invention has good affinity to PD-1, prompts to PD-1 positive cancers
Patient may have positive therapeutic effect, especially lung cancer and melanoma patients.
Test example 2：The heteroplastic transplantation tumor mouse model test of pesticide effectiveness
The female AJ mouse between 6-8 week old are grouped at random according to weight, every group 10.It will be dissolved in 200 μ l at the 0th day
The 2 × 10 of DMEM culture mediums6A SA1/N fibrosarcoma cells are subcutaneously implanted mouse in right armpit.Passed through peritonaeum at the 4th, 8 and 11 day
Inner injecting and administering, control group give PBS, and test group gives the polypeptide of 1mg/kg.Tumour growth is monitored twice a week to mouse, is held
It is about 10 weeks continuous.Three-dimensional measurement is carried out to tumour using electronic caliper（Highly × width × length）And calculate gross tumor volume.
When tumour reaches tumour terminal（1500mm3）Or mouse is put to death when weight loss of the display more than 15%.Different groups reach swollen
The average time of tumor terminal see the table below：
| SEQ ID | Tumour terminal（It） |
| Control group | 26 |
| NO：1 | 36 |
| NO：4 | 40 |
| NO：5 | 37 |
| NO：7 | 38 |
By upper table it can be found that the arrival time of test group mouse tumor terminal obviously increases, wherein SEQ ID NO：4 tumours are whole
Point has respectively reached 40 days, and during monitoring tumour growth twice a week, control group tumour continued propagation increases, and respectively tests
Group does not find the apparent growth of tumour during administration, it was demonstrated that therapeutic effect of the antineoplastic polypeptide of the present invention to cancer.
Sequence table
<110>Cui Xiangju
<120>A kind of antineoplastic polypeptide and its blood testing ELISA kit
<141> 2018-06-17
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 72
<212> PRT
<213> Artificial Sequence
<400> 1
Ser Val Phe Trp Lys Gly Gly Arg Glu His His Asp Lys His Leu Ala
1 5 10 15
Tyr Ile Arg Phe Asp Ile Pro Gly Ser Gly Pro Phe Asn Asn Arg Asp
20 25 30
Leu Gln Gly Ser Phe Ile Asn Glu Trp Asn Arg Leu Gly Lys Ile Gly
35 40 45
Thr Ala Met Arg Asp Phe Leu Thr Tyr Ala Pro Pro Thr Asp Trp Ile
50 55 60
Leu Cys Asn Tyr Ile Gly Thr Ala
65 70
Claims (7)
1. a kind of antineoplastic polypeptide, sequence such as SEQ ID NO：Shown in 1.
2. antineoplastic polypeptide as described in claim 1, characterized in that the 17th amino acid Tyr, the 37th ammonia in sequence
Base acid Phe, the 68th amino acid Tyr at least one replace with。
3. antineoplastic polypeptide as claimed in claim 2, is selected from：
Ser-Val-Phe-Trp-Lys-Gly-Gly-Arg-Glu-His-His-Asp-Lys-His-Leu-Ala-Baa-Ile-
Arg-Phe-Asp-Ile-Pro-Gly-Ser-Gly-Pro-Phe-Asn-Asn-Arg-Asp-Leu-Gln-Gly-Ser-Phe-
Ile-Asn-Glu-Trp-Asn-Arg-Leu-Gly-Lys-Ile-Gly-Thr-Ala-Met-Arg-Asp-Phe-Leu-Thr-
Tyr-Ala-Pro-Pro-Thr-Asp-Trp-Ile-Leu-Cys-Asn-Tyr-Ile-Gly-Thr-Ala；
Ser-Val-Phe-Trp-Lys-Gly-Gly-Arg-Glu-His-His-Asp-Lys-His-Leu-Ala-Tyr-Ile-
Arg-Phe-Asp-Ile-Pro-Gly-Ser-Gly-Pro-Phe-Asn-Asn-Arg-Asp-Leu-Gln-Gly-Ser-Baa-
Ile-Asn-Glu-Trp-Asn-Arg-Leu-Gly-Lys-Ile-Gly-Thr-Ala-Met-Arg-Asp-Phe-Leu-Thr-
Tyr-Ala-Pro-Pro-Thr-Asp-Trp-Ile-Leu-Cys-Asn-Tyr-Ile-Gly-Thr-Ala；
Ser-Val-Phe-Trp-Lys-Gly-Gly-Arg-Glu-His-His-Asp-Lys-His-Leu-Ala-Tyr-Ile-
Arg-Phe-Asp-Ile-Pro-Gly-Ser-Gly-Pro-Phe-Asn-Asn-Arg-Asp-Leu-Gln-Gly-Ser-Phe-
Ile-Asn-Glu-Trp-Asn-Arg-Leu-Gly-Lys-Ile-Gly-Thr-Ala-Met-Arg-Asp-Phe-Leu-Thr-
Tyr-Ala-Pro-Pro-Thr-Asp-Trp-Ile-Leu-Cys-Asn-Baa-Ile-Gly-Thr-Ala；
Ser-Val-Phe-Trp-Lys-Gly-Gly-Arg-Glu-His-His-Asp-Lys-His-Leu-Ala-Baa-Ile-
Arg-Phe-Asp-Ile-Pro-Gly-Ser-Gly-Pro-Phe-Asn-Asn-Arg-Asp-Leu-Gln-Gly-Ser-Baa-
Ile-Asn-Glu-Trp-Asn-Arg-Leu-Gly-Lys-Ile-Gly-Thr-Ala-Met-Arg-Asp-Phe-Leu-Thr-
Tyr-Ala-Pro-Pro-Thr-Asp-Trp-Ile-Leu-Cys-Asn-Tyr-Ile-Gly-Thr-Ala；
Ser-Val-Phe-Trp-Lys-Gly-Gly-Arg-Glu-His-His-Asp-Lys-His-Leu-Ala-Baa-Ile-
Arg-Phe-Asp-Ile-Pro-Gly-Ser-Gly-Pro-Phe-Asn-Asn-Arg-Asp-Leu-Gln-Gly-Ser-Phe-
Ile-Asn-Glu-Trp-Asn-Arg-Leu-Gly-Lys-Ile-Gly-Thr-Ala-Met-Arg-Asp-Phe-Leu-Thr-
Tyr-Ala-Pro-Pro-Thr-Asp-Trp-Ile-Leu-Cys-Asn-Baa-Ile-Gly-Thr-Ala；
Ser-Val-Phe-Trp-Lys-Gly-Gly-Arg-Glu-His-His-Asp-Lys-His-Leu-Ala-Tyr-Ile-
Arg-Phe-Asp-Ile-Pro-Gly-Ser-Gly-Pro-Phe-Asn-Asn-Arg-Asp-Leu-Gln-Gly-Ser-Baa-
Ile-Asn-Glu-Trp-Asn-Arg-Leu-Gly-Lys-Ile-Gly-Thr-Ala-Met-Arg-Asp-Phe-Leu-Thr-
Tyr-Ala-Pro-Pro-Thr-Asp-Trp-Ile-Leu-Cys-Asn-Baa-Ile-Gly-Thr-Ala；
Ser-Val-Phe-Trp-Lys-Gly-Gly-Arg-Glu-His-His-Asp-Lys-His-Leu-Ala-Baa-Ile-
Arg-Phe-Asp-Ile-Pro-Gly-Ser-Gly-Pro-Phe-Asn-Asn-Arg-Asp-Leu-Gln-Gly-Ser-Baa-
Ile-Asn-Glu-Trp-Asn-Arg-Leu-Gly-Lys-Ile-Gly-Thr-Ala-Met-Arg-Asp-Phe-Leu-Thr-
Tyr-Ala-Pro-Pro-Thr-Asp-Trp-Ile-Leu-Cys-Asn-Baa-Ile-Gly-Thr-Ala。
4. application of the antineoplastic polypeptide as PD-1 inhibitor as described in claim 1-3.
5. application of the antineoplastic polypeptide as described in claim 1-3 in antitumor drug.
6. including the monoclonal antibody of the antineoplastic polypeptide described in claim 1-3.
7. including the blood testing kit of monoclonal antibody described in claim 6.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102924578A (en) * | 2011-08-09 | 2013-02-13 | 哈药集团技术中心 | Anti-tumor polypeptide, preparation method and anti-tumor applications thereof |
| CN104258373A (en) * | 2014-09-17 | 2015-01-07 | 吉林大学 | Application of antineoplastic polypeptide TT-1 in preparing antineoplastic medicines |
-
2018
- 2018-06-17 CN CN201810624951.4A patent/CN108732361A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102924578A (en) * | 2011-08-09 | 2013-02-13 | 哈药集团技术中心 | Anti-tumor polypeptide, preparation method and anti-tumor applications thereof |
| CN104258373A (en) * | 2014-09-17 | 2015-01-07 | 吉林大学 | Application of antineoplastic polypeptide TT-1 in preparing antineoplastic medicines |
Non-Patent Citations (2)
| Title |
|---|
| QIAO LI等: "Discovery of peptide inhibitors targeting human programmed death 1 (PD-1) receptor", 《ONCOTARGET》 * |
| 姚金凤等: "多肽类药物代谢研究进展", 《中国药理学通报》 * |
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