CN107298697A - Human PD-L1 protein Y123Site phosphorylation antibody and preparation method and application thereof - Google Patents
Human PD-L1 protein Y123Site phosphorylation antibody and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a human PD-L1 protein Y123A site phosphorylation antibody, a preparation method and application thereof, belonging to the technical field of biomedicine for preparing the antibody, human PD-L1 protein Y123The preparation method of the site phosphorylation antibody comprises the following steps: coupling hapten synthetic peptide with carrier protein through cysteine C to obtain antigen; immunizing an animal by using the antigen obtained in the step 1), and collecting antiserum; purifying and selecting the antiserum obtained in the step 2), namely the PD-L1 protein Y123A site-phosphorylated antibody; the amino acid sequence of the hapten synthetic peptide is CSYGGADYKRITVK, and a phosphate group is added to the tyrosine Y site of the hapten synthetic peptide. Human PD-L1 protein Y of the invention123The site phosphorylation antibody can be applied to diagnosis, treatment and prognosis judgment of malignant tumors.
Description
Technical field
The invention belongs to the field of biomedicine technology of Antibody preparation, more particularly to a kind of human PD-L 1 protein Y123Site phosphorus
It is acidified antibody and its preparation method and application.
Background technology
According to global cancer statistics in 2016, global whole year there are about 1.5 thousand ten thousand cancer new cases, and 8,200,000 patients die from cancer
Disease；Wherein, 57% cancer patient and 65% cancer mortality patient come from developing country.Generation, the development of tumour are individual
Sufficiently complex process, T cell plays vital effect in the antineoplastic immune system of body.Exempt to escape body
The monitoring of epidemic disease system, tumour cell raises apoptosis ligand 1 in tumor microenvironment by various mechanism
The expression of (programmed cell death lagand 1, PD-L1, also referred to as B7-H1), PD-L1 is negative with T cell surface
Property immunologic test point-apoptosis acceptor 1 (PD-1, also referred to as CD279) combine, T cell function is suppressed, it is impossible to
Immune system sends the signal of attack tumour.PD-1/PD-L1 inhibitor can block PD-1 and PD-L1 combination, block negative sense
Adjustment signal, makes T cell activity recovery, so as to strengthen immune response.
PD-L1 is optionally high to be expressed in tumor tissues.PD-L1 is a transmembrane glycopeptide being made up of 290 amino acid
In vain, B7-CD28 superfamily members are belonged to, its amino acid sequence such as SEQ ID NO：Shown in 1.Current studies have shown that PD-L1's
MRNA is transcribed in Normal Human Tissue and organ extensively, wherein, high transcription, spleen, lymph in placenta, heart, lungs and liver
Do not transcribed in low transcription in knot, thymus gland, central nervous system.However, corresponding PD-L1 albumen is not translated in normal but extensively
In tissue, only translate on a small quantity in tonsillotome, lungs, the macrophage of liver and immune special permission region, PD-L1 albumen
Translated in eyes and placenta.Translations of the PD-L1 in the transcription and epicyte protein level in mRNA level in-site has differences, this
Imply a kind of expression of important mechanism regulating PD-L1 albumen.In recent years, hand is detected by immunohistochemical method (IHC)
Art or biopsy specimen, it is found that PD-L1 albumen is a variety of swollen in melanoma, breast cancer, non-small cell lung cancer (NSCLC), kidney etc.
High expression in knurl, and low expression or do not express in the normal tissue.PD-L1 albumen low expressions in normal structure and inductivity
It is that it is different from other co-suppression signal path most outstanding features that height, which is expressed in tumor tissues, which imply that PD-L1 albumen
Selectively it is expressed in tumor tissues, it is closely related with tumor microenvironment.These are found to be immunotherapy of tumors and provided
Important target spot, to obtain more preferable curative effect and lower toxic side effect.
With the proposition of accurate medical concept, the research about immunization therapy is got growing concern for.PD-1/PD-
L1 immunotherapies are the class anticancer immunotherapies currently got most of the attention, it is intended to resist swollen using the immune system of human body itself
Knurl：It is by blocking PD-1/PD-L1 signal paths to make death of neoplastic cells, it is adaptable to polytype tumour.Each research institution by
Step carries out the effect of corresponding Clinical Project, the single medication of investigation and conjoint therapy treating cancer, to excavate such medicine
Clinical value.At present, opdivo is applied to melanoma in Japan, and melanoma and non-small cell lung are applied in the U.S.
Cancer, in the granted melanoma idicatio of European Union, while opdivo has obtained European Union CHMP support for the application of lung cancer therapy.
MSD Corp. keytruda is expected in the recent period by non-small cell lung cancer idicatio in the U.S.'s granted melanoma idicatio
Approval, while the application that keytruda is used for melanoma treatment has obtained European Union CHMP supports.However, Astrazeneca AB
MEDI4736 and the RG7446 of Roche Holding Ag not yet harvest any idicatio at present.The well-known medical market survey institute in the whole world
Evaluatepharma is predicted：Opdivo is by as one kind in most successful immunotherapeutic agents preparation.
Concentration is compared in research currently for PD-1/PD-L1, but is the absence of the exploitation of novel targets, " immunization therapy " treatment
Pattern specification not enough.Although PD-1 inhibitor has basic difference, resistance problems with targeting medicine (being directed to cancer cell)
Still exist, it is necessary to further research and develop novel targets.
Existing research finds that the generation development of tumour is frequently accompanied by PD-L1 protein phosphorylations modification exception.PD-L1
Sulphation modification plays key effect in the physiology and pathologic process that PD-1/PD-L1 signal paths are mediated after protein translation,
The phosphorylation level of PD-L1 albumen has very important significance for the control accurate of PD-1/PD-L1 signal paths.
Therefore, it is necessary to develop a kind of human PD-L 1 protein phosphorylation antibody, the antibody protein is applied to malignant tumour
During diagnosis, treatment and prognosis judge, while developing the novel targets of cancer.
The content of the invention
Present invention aims to overcome that the deficiency that prior art is present, and a kind of human PD-L 1 albumen for cancer is provided
Y123Site phosphorylation antibody and its preparation method and application.To achieve the above object, the technical scheme taken of the present invention is：One
Plant human PD-L 1 protein Y123The hapten synthesis peptide of site phosphorylation antibody, the amino acid sequence of the hapten synthesis peptide is such as
SEQ ID NO：Shown in 2, the tyrosine Y-site of the hapten synthesis peptide is added with phosphate group.
As the improvement of above-mentioned technical proposal, the N-terminal of the hapten synthesis peptide is connected with a cysteine C.
In addition, the present invention also provides the synthetic method of the hapten synthesis peptide, comprise the following steps successively：Using polypeptide
Synthetic technology synthesis polypeptide, the amino acid sequence of the polypeptide is SYGGADYKRITVK, and the tyrosine Y-site of the polypeptide adds
Phosphorate acid groups, and the N-terminal of the polypeptide connects a cysteine C.The hapten synthesis peptide is：(NH2-)CSYGGAD
(pY)KRITVK(-COOH)。
In addition, the present invention also provides a kind of human PD-L 1 protein Y123The preparation method of site phosphorylation antibody, including it is following
Step：
1) the hapten synthesis peptide described in claim 1 is passed through into cysteine C and carrier protein (carrier hemocyanin
KLH or bovine serum albumin BSA) it is coupled, produce antigen；
2) step 1 is utilized) animal is immunized the antigen, and collect antiserum；
3) by step 2) antiserum purified and selected, as PD-L1 protein Ys123Site phosphorylation antibody.
As the improvement of above-mentioned technical proposal, the step 2) immunization wayses are specially：1 time trigger injection, 3~4 times plus
Injection is penetrated and 1 direct injection；The initiation injection and booster shots are：Antigen synthetic peptide is moved with adjuvant combined immunization
Thing, is subcutaneously injected, gluteus distinguishes intramuscular injection with huckle both sides in the relatively thin and loose position both sides of neck, skin of back,
The intracutaneous injection of waist both sides, footpad injection；The direct injection is：Antigenic solution intramuscular injection.
It is used as the improvement of above-mentioned technical proposal, the step 3) in, it is purified by following steps implementation：Determine before purification
Antiserum is directed to the potency of antigen, it is determined that antiserum before purification can recognize antigen；The antiserum of collection is purified, it is determined that
Antiserum energy specific recognition phosphorylated human PD-L1 albumen after purification.
Preferably, whether the antiserum of collection is tested with Elisa determines antiserum for the potency of antigen and its can know
Other antigen；Purified using affine separation-affinity purification circulating technology antagonistic Serum, with Dot blot and Western blot
Experiment determines whether antiserum after purification being capable of specific recognition phosphorylated human PD-L1 albumen.
In addition, the present invention also provides the human PD-L 1 protein Y that the preparation method is prepared123Site phosphorylation antibody.
In addition, the present invention also provides a kind of pharmaceutical preparation, the pharmaceutical preparation includes the human PD-L 1 protein Y123Site
Phospho-AB.
In addition, the present invention also provides the human PD-L 1 protein Y123Site phosphorylation antibody is examined in preparation for malignant tumour
The application for the pharmaceutical preparation that disconnected, treatment and prognosis judge.
In addition, the present invention also provides the application that the pharmaceutical preparation judges in diagnosis of malignant tumor, treatment and prognosis.
The beneficial effects of the present invention are：The present invention provides a kind of human PD-L 1 protein Y123Site phosphorylation antibody and its system
PD-L1 protein Ys are listed in Preparation Method and application, technical scheme123Site phosphorylation antibody, its hapten synthesis peptide
And both preparation methods, human PD-L 1 protein Y123Site phosphorylation antibody can detect human PD-L 1 protein phosphorylation level
With termination PD-1/PD-L1 cell-signaling pathways, research PD-L1 protein phosphorylations are so contributed to believe in PD-1/PD-L1 cells
The effect of number path and occurrence mechanism on cancer, diagnosis or treatment for clinical tumor disease, which are provided, searches out potential effect target
Point；Simultaneously can also be by human PD-L 1 protein Y123Site phosphorylation antibody applies to diagnosis of malignant tumor, treatment and prognosis and judged
In.
Brief description of the drawings
Fig. 1 is the antigen protein electrophoretogram in the embodiment of the present invention 3；
Fig. 2 is the antigen Western blot figures by primary antibody of negative serum in the embodiment of the present invention 3；
Fig. 3 is antiserum titre Elisa analysis charts in the embodiment of the present invention 3；
Fig. 4 is using antiserum before purification as the antigen of primary antibody to close Western blot figures in the embodiment of the present invention 4；
Fig. 5 is using antiserum after purification as the antigen of primary antibody to close Western blot figures in the embodiment of the present invention 4；
Fig. 6 is the immunohistochemistry figure of phosphorylation PDL-1 albumen in people's lung squamous cell carcinoma cancers in the embodiment of the present invention 4；
Fig. 7 is the phosphorylation PDL-1 eggs of people's normal esophageal scaly epithelium and esophageal squamous cell carcinoma in the embodiment of the present invention 4
White immunohistochemical staining figure.
Embodiment
For the object, technical solutions and advantages of the present invention are better described, below in conjunction with specific embodiment, subordinate list and attached
The invention will be further described for figure.
Embodiment 1 determines human PD-L 1 protein phosphorylation site
Being screened first by bioinformatics software NetPhorest 2.0 and NetPhos 2.0 may in human PD-L 1 albumen
Occurs the site of phosphorylation, then looking into by mass spectrum document and protein phosphorylation site database PhosohoSitePlus
Look for, determine Y123Site is a phosphorylation site in human PD-L 1 amino acid sequence；Software CLC Protein are utilized simultaneously
Workbench 5 calculates the antigenicity and hydrophily of human PD-L 1 amino acid sequence, final to determine human PD-L 1 protein-specific phosphorus
Polyadenylation sites are 123 amino acid-tyrosine Y123。
There is multiple phosphorylation sites (such as Y in human PD-L 1 albumen28Site), because follow-up antigenicity and hydrophily are discontented
Sufficient test requirements document, is not suitable as phosphorylation site.
Embodiment is 2-in-1 into hapten synthesis peptide
According to the design of hapten synthesis peptide, in Y123A phosphate group is added on site, HPLC (efficient liquid is carried out
Phase chromatography) separate, purify, the purity for obtaining hapten synthesis peptide is more than 95%.Correspondingly, one section of Y is synthesized123Site
The synthetic peptide of non-phosphorylating, and cysteine by N-terminal is coupled with bovine serum albumin and is used as the affine filler for separating chromatography, warp
Purity after HPLC is isolated and purified is more than 90%.
Embodiment 3 prepares antiserum
3.1 prepare negative serum
3mL blood is taken in heparin tube from the ear vein of the new zealand white rabbit (2~3kg, female is healthy and strong) for injection
In, use cotton balls hemostasis by compression.Blood is put into room temperature 1h or so, treats that blood clotting forms placement 2h at clot, 4 DEG C and analyses serum
Go out, 2500g centrifugation 10min, draw supernatant, labeled as negative control sera, dispense and be stored in -20 DEG C it is to be measured.
3.2 immune preceding screening-Western blot (protein blot)：
BCA (Bicinchoninic acid) protein concentration detection kit determines solubilized phosphoantigen (pY123-BSA)
Concentration and non-phosphorylating antigen (Y123- BSA) concentration, respectively 0.845mg/mL and 1.1mg/mL (coefficient R=
0.991).By pY123- BSA and Y123- BSA carries out protein electrophoresis, as a result as shown in Figure 1：Swimming lane 1 is Y123- BSA, swimming lane 2 is
pY123-BSA.Because synthetic peptide is artificial synthesized, therefore protein band is one section of thick wide band；Because of phosphorylation modification so that
pY123- BSA molecular weight is larger and band is slightly above Y123- BSA band.
Take the purifying antigen (pY after dissolving123-BSA、Y123- BSA) 5 μ g, add appropriate 5 × SDS sample-loading buffers, boiling
10min is boiled in water-bath makes protein denaturation, 10000 × g centrifugations 10min；SDS-PAGE electrophoretic separation glue volumetric concentration be
10%, the volumetric concentration of concentration glue is 5%.Sample loading gun loading is used in order, in no sample well plus isometric 5 ×
Sds gel sample-loading buffer；100V 70min are changed to after 60V 20min electrophoresis, until bromophenol blue reaches the bottom of separation gel, are closed
Close power supply.Transferring film condition：Constant current 180mA, the time is 180min.5% skimmed milk power is closed, 37 DEG C of 2h of shaking table.Transfer film is put
Enter with TBST buffer solutions by 1：5000 prepare immune preceding antiserum dilution, and level slowly shakes up, and 4 DEG C overnight.37 DEG C of works of next day
With 20min, primary antibody solution is abandoned, 1 × TBST washes film 10min, is repeated 4 times.Film is placed in 1 × TBST by 1：5000 dilutions
In the secondary antibody dilution of the goat anti-rabbit igg of HRP marks, 37 DEG C of shaking table, 50min.Two corresponding anti-solution is abandoned, 1 × TBST washes film 10min,
It is repeated 3 times.Developed according to producer's explanation using ECL kits, photographed to record.As a result as shown in Fig. 2 not occurring purpose band,
Do not occur the antibody for purpose tissue or cell extract, be gedanken experiment animal；Wherein swimming lane 1,2,3 is respectively Y123-
BSA、pY123-BSA、BSA。
3.3 animal immunes (duration is about 73d)
3.31 draw antigenic solution, another syringe draws equal amounts Freund's complete adjuvant with an asepsis injector
(CFA), connected therebetween, aspirated back and forth with plastic tube, until forming the emulsion emulsified completely, dripped and do not expand in water
Dissipate.
3.32 first immunisations are injected through relatively thin, the loose position both sides subcutaneous (s.c) of neck, skin of back respectively, gluteus
Injected with huckle both sides difference muscle (i.m), waist both sides carry out intracutaneous (i.d) and injected, and rabbit palmula injection location etc. is more
Position multi-point injection antigen emulsion.The antigen total amount about 0.61mg of first immunisation.
First time booster immunization is carried out after 3.33 first immunisation 20d, CFA conducts are replaced with incomplete Freund's adjuvant (IFA)
Immunologic adjuvant prepares antigen emulsion and injected by first immunisation mode, and this time the antigen total amount of booster immunization is each about
0.9mg。
Carried out after 3.34 immune 12d plus strong second immune, CFA is replaced with IFA as immunologic adjuvant and prepares antigen emulsus
Liquid is simultaneously injected by first immunisation mode, and this time the antigen total amount of booster immunization is each about 0.9mg.
After 3.35 immune 10d, from ear vein blood sampling 5mL, blood is put into room temperature 1h or so, treats that blood clotting forms clot, 4
2h is placed at DEG C separates out serum, 3500g centrifugation 10min, draws supernatant, labeled as positive antiserum, dispenses and be stored in -20
It is DEG C to be measured.
3.36 positive antiserums in proportion 1：1000、1：5000、1：10000 dilutions, carry out Elisa detection antibody titers,
OD values to be measured are more than 1.0, the P/N values difference 7.0,7.4 and 7.3 of the positive antiserum of each dilution factor, then now serum resists
Body potency is at least 1：5000.Antibody titer reaches expected level (Elisa potency ＞ 1：1000), carried out most before a large amount of blood samplings
A booster immunization afterwards：Antigenic solution (pY123- KLH) direct intramuscular injection rabbit, about 0.6mg.
After 3.37 last time booster immunization 3d, rabbit carries out the big blood sampling of abdominal aorta, largely collects antiserum.It will collect
Room temperature is stood overnight after the beaker for having blood is closed, and makes clot contraction.The serum of precipitation is sub-packed in 50mL by next day sterile working
In centrifuge tube, 4000g centrifugation 10min take supernatant, often pipe packing 1mL, labeled as immune rear antiserum (about 51mL altogether), storage
In -20 DEG C.
3.38 immune rear sero-fast titrations：Antigen is diluted with antigen coat liquid, it is 2 μ g/mL to make its concentration, by table
In 1 concentration gradient dilution antiserum (serum origin and the new zealand white rabbit being immunized, number of animals be respectively RB7771 and
RB7772), and according to the explanation of Elisa kits operated, testing result is as shown in table 1 and Fig. 3.In table 1 and Fig. 3,
SA represents the antibody after Streptavidin-biotin-antigen system affinity purification；(-) is represented without the system affinity purification
Antibody, i.e. negative control；Blank is blank control；P-Ab refers to hapten synthesis peptide phosphorylation in antigen, and NP-Ab is anti-
The non-phosphorylation of hapten synthesis peptide in original.
Table 1 is immune rear sero-fast titration result
The specificity identification human PD-L 1 protein Y of embodiment 4123Site phosphorylation antibody
4.1Western blot detect the specificity of purified antibodies：
BCA protein concentrations detection kit determines solubilized pY123- BSA and Y123- BSA concentration, concentration is respectively
0.692mg/mL and 0.647mg/mL (coefficient R=0.9952).The μ g of purifying antigen 5 after dissolving are respectively taken, appropriate 5 are added
× SDS sample-loading buffers, boiling water bath, which boils 10min, makes protein denaturation, 10000 × g centrifugations 10min.On Western blot
Sample is analyzed, and wherein primary antibody solution is 1：500 purified antibodies dilution and 1：10000 antiserum dilution before purification.Knot
As shown in Figure 4 and Figure 5, Fig. 4 primary antibody is antiserum dilution before purification to fruit, and BSA swimming lanes are without there is specific band, Y123-
BSA swimming lane and pY123- BSA swimming lane has specific band；Fig. 5 primary antibody is antiserum dilution after purification, BSA swimming
Road and Y123- BSA swimming lane is without there is specific band, pY123- BSA swimming lane has specific band.Therefore, after purification
(i.e. the antigen of phosphorylation, can also recognize people's phosphorylation PD-L1 to the hapten synthesis peptide-BSA of antibody energy specific recognition phosphorylation
Albumen).
4.2 immune group chemical identification antibody specificities
First by people's lung phosphorus cancerous tissue, after specimens paraffin embedding slices, microscopy after immunohistochemical staining is done, its result such as Fig. 6
Shown, phosphorylation PDL-1 albumen navigates to cell membrane, and cytoplasm is navigated on a small quantity, can be used as positive control.Then respectively with just
Often tissue and cancer of the esophagus tumor tissues do paraffin section, the microscopy after immune group chemical staining, and its result is as shown in fig. 7,400x
Epithelial cell is coloured without specificity in visible normal esophageal squamous epithelial tissue under times, and tumour cell is special in esophageal squamous cell carcinoma tissue
Opposite sex coloring positive rate accounts for 40%., phosphorylation PDL-1 albumen is primarily targeted for cell membrane, cytoplasm navigated on a small quantity.With sun
Property results of comparison is consistent.Illustrate the expression of phosphorylation PDL-1 albumen in the detectable human esophageal carcinoma of the present invention.
The preservation of 4.3 antibody
The positive combined hybrid liquid of IgG is added into final concentration of 2.5%BSA (wt/vol), 0.01%Tween-20 (vol/
Vol) and 25% glycerine mixed liquor (vol/vol), often pipe be packed as 1mL, it is -80 DEG C long-term to preserve.
The human PD-L 1 protein Y of embodiment 5123Application of the site phosphorylation antibody in tumour
5.1 present invention prepare the human PD-L 1 protein Y of high specific123Site phosphorylation antibody can use Western
The differential expression of tumour cell after the detectable normal cell of blot experiments, tumour cell and medication.
The antibody that 5.2 present invention are provided can be applied to detect in the immunological testings such as ICC, Elisa in actual applications
Phosphorylation modification situation after the transcription of human PD-L 1 albumen, so as to inquire into the modification of human PD-L 1 protein phosphorylation in tumor-related illness
In meaning.
The activation of 5.3 human PD-L 1 albumen depends on phosphorylation modification, prevents its phosphorylation from preventing withering for lymphocyte
Die, therefore antibody is easy to study human PD-L 1 protein Y in actual applications123Site phosphorylation is modified for particular biological event
Such as immunoregulatory influence, so as to inquire into its effect during the disease developments such as tumour.
The antibody that 5.4 present invention are provided contributes to the phosphorylation modification for inquiring into PD-L1 albumen to develop in tumor disease
During mechanism of action, be more conducive to suppress research of the medicine to tumor inhibition effect in clinical practice.
5.5 the present invention be directed to the specific Y of human PD-L 1 albumen123Phosphorylation polyclonal antibody prepared by site, helps to grind
Studying carefully mediation, it occurs the zymogenesis of phosphorylation, inquires into human PD-L 1 albumen various biological function in T cell signal path and egg
In vain/nucleic acid interaction network, so as to find the latent effect target spot of diagnosis and the treatment of clinical tumor disease.
Finally, it should be noted that above example is to illustrate technical scheme rather than to present invention protection
The limitation of scope, although being explained in detail with reference to preferred embodiment to the present invention, one of ordinary skill in the art should manage
Solution, technical scheme can be modified or replaced on an equal basis, without departing from technical solution of the present invention essence and
Scope.
Sequence table
<110>Zhang Hao
<120>A kind of site phosphorylation antibody of human PD-L 1 protein Y 123 and its preparation method and application
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 290
<212> PRT
<213> Homo sapiens
<400> 1
Met Arg Ile Phe Ala Val Phe Ile Phe Met Thr Tyr Trp His Leu Leu
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Asn Ala Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr
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Gly Ser Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu
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Asp Leu Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile
50 55 60
Ile Gln Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser
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Tyr Arg Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn
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Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr
100 105 110
Arg Cys Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val
115 120 125
Lys Val Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val
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Asp Pro Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr
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Gly Lys Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn
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Cys Thr Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu
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Val Ile Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Thr His
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Leu Val Ile Leu Gly Ala Ile Leu Leu Cys Leu Gly Val Ala Leu Thr
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Phe Ile Phe Arg Leu Arg Lys Gly Arg Met Met Asp Val Lys Lys Cys
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Gly Ile Gln Asp Thr Asn Ser Lys Lys Gln Ser Asp Thr His Leu Glu
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<211> 14
<212> PRT
<213>Artificial sequence
<400> 2
Cys Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys
1 5 10
Claims (10)
1. a kind of human PD-L 1 protein Y123The hapten synthesis peptide of site phosphorylation antibody, it is characterised in that：The haptens is closed
Into the amino acid sequence such as SEQ ID NO of peptide：Shown in 2, the tyrosine Y-site of the hapten synthesis peptide is added with phosphate
Group.
2. hapten synthesis peptide as claimed in claim 1, its feature is just：The N-terminal connection of the hapten synthesis peptide
There is a cysteine C.
3. the synthetic method of hapten synthesis peptide as claimed in claim 2, it is characterised in that：Comprise the following steps successively：Using
Peptide synthesis technology synthesis polypeptide, the amino acid sequence of the polypeptide is SYGGADYKRITVK, Y, the tyrosine of the polypeptide
Point addition phosphate group, the N-terminal of the polypeptide connects a cysteine C.
4. a kind of human PD-L 1 protein Y123The preparation method of site phosphorylation antibody, it is characterised in that：Comprise the following steps：
1) the hapten synthesis peptide described in claim 1 is coupled by cysteine C with carrier protein, produces antigen；
2) step 1 is utilized) animal is immunized the antigen, and collect antiserum；
3) by step 2) antiserum purified and selected, as PD-L1 protein Ys123Site phosphorylation antibody.
5. human PD-L 1 protein Y as claimed in claim 4123The preparation method of site phosphorylation antibody, it is characterised in that：The step
Rapid 2) immunization wayses are specially：1 initiation injection, 3~4 booster shots and 1 direct injection；It is described to trigger injection and strengthen
Injection is：By antigen synthetic peptide and adjuvant combined immunization animal, it is subcutaneously injected in neck, back both sides, gluteus and huckle
Distinguish intramuscular injection, the intracutaneous injection of waist both sides, footpad injection in both sides；The direct injection is：Antigenic solution intramuscular injection.
6. human PD-L 1 protein Y as claimed in claim 4123The preparation method of site phosphorylation antibody, it is characterised in that：The step
It is rapid 3) in, be purified by following steps implementation：The antiserum determined before purification is directed to the potency of antigen, it is determined that anti-blood before purification
Antigen can be recognized clearly；The antiserum of collection is purified, it is determined that antiserum energy specific recognition phosphorylated human PD- after purification
L1 albumen.
7. the human PD-L 1 protein Y that the preparation method as any one of claim 4~6 is prepared123Site phosphorylation resists
Body.
8. a kind of pharmaceutical preparation, it is characterised in that：Including the human PD-L 1 protein Y described in claim 7123Site phosphorylation resists
Body.
9. human PD-L 1 protein Y as claimed in claim 7123Site phosphorylation antibody is being prepared for diagnosis of malignant tumor, treatment
And the application of the pharmaceutical preparation of prognosis judgement.
10. the application that pharmaceutical preparation as claimed in claim 8 judges in diagnosis of malignant tumor, treatment and prognosis.
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Cited By (3)
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| CN109694412A (en) * | 2018-12-12 | 2019-04-30 | 深圳市雅臣智能生物工程有限公司 | Block the IgY and small molecule Fab antibody, preparation and application of PD-1/PD-L1 access |
| CN111363031A (en) * | 2020-03-03 | 2020-07-03 | 中国人民解放军军事科学院军事医学研究院 | pSer131 polyclonal antibody of BNIP3, preparation method and application thereof |
| CN112480249A (en) * | 2020-11-26 | 2021-03-12 | 北京大学第三医院（北京大学第三临床医学院） | Preparation method and application of phosphorylated antibody product of AKT new substrate HIP-55 |
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| CN105585636A (en) * | 2015-11-24 | 2016-05-18 | 南方医科大学 | Human NOTCH1 NICD protein Ser2162 locus phosphorylation antibody and preparation method and application thereof |
| CN106939047A (en) * | 2016-01-04 | 2017-07-11 | 钜川生物医药 | A kind of PD-L1 antibody and preparation method thereof |
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| CN105585636A (en) * | 2015-11-24 | 2016-05-18 | 南方医科大学 | Human NOTCH1 NICD protein Ser2162 locus phosphorylation antibody and preparation method and application thereof |
| CN106939047A (en) * | 2016-01-04 | 2017-07-11 | 钜川生物医药 | A kind of PD-L1 antibody and preparation method thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109694412A (en) * | 2018-12-12 | 2019-04-30 | 深圳市雅臣智能生物工程有限公司 | Block the IgY and small molecule Fab antibody, preparation and application of PD-1/PD-L1 access |
| CN111363031A (en) * | 2020-03-03 | 2020-07-03 | 中国人民解放军军事科学院军事医学研究院 | pSer131 polyclonal antibody of BNIP3, preparation method and application thereof |
| CN112480249A (en) * | 2020-11-26 | 2021-03-12 | 北京大学第三医院（北京大学第三临床医学院） | Preparation method and application of phosphorylated antibody product of AKT new substrate HIP-55 |
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|---|---|
| CN107298697B (en) | 2019-05-24 |
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