CN107298697B - 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 PDF

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CN107298697B
CN107298697B CN201710740882.9A CN201710740882A CN107298697B CN 107298697 B CN107298697 B CN 107298697B CN 201710740882 A CN201710740882 A CN 201710740882A CN 107298697 B CN107298697 B CN 107298697B
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张灏
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Guangzhou Huake Biotechnology Co ltd
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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 animals with the antigens of step 1), and collecting the antiseraClearing; 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

A kind of human PD-L 1 protein Y123Site phosphorylation antibody and its preparation method and application
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 Ys123Site phosphorus It is acidified antibody and its preparation method and application.
Background technique
According to global cancer statistics in 2016, whole world whole year, 8,200,000 patients died of cancer there are about 1.5 thousand ten thousand cancer new cases Disease；Wherein, 57% cancer patient and 65% cancer mortality patient are from developing country.Generation, the development of tumour are Sufficiently complex process, T cell play a crucial role in the antineoplastic immune system of body.Exempt to escape body The monitoring of epidemic disease system, tumour cell raise apoptosis ligand 1 by various mechanism in tumor microenvironment The expression of (programmed cell death lagand 1, PD-L1, also referred to as B7-H1), PD-L1 and T cell surface are born Property immunologic test point-apoptosis receptor 1 (PD-1, also referred to as CD279) combine, T cell function is suppressed, Bu Nengxiang Immune system issues the signal of attack tumour.PD-1/PD-L1 inhibitor can block the combination of PD-1 and PD-L1, block negative sense Adjustment signal makes T cell activity recovery, to enhance immune response.
PD-L1 is selectively high to be expressed in tumor tissues.PD-L1 is the transmembrane glycopeptide being made of 290 amino acid It is white, belong to B7-CD28 superfamily member, amino acid sequence is as shown in SEQ ID NO:1.Studies have shown that PD-L1 at present MRNA is transcribed extensively in Normal Human Tissue and organ, wherein high transcription, spleen, lymph in placenta, heart, lungs and liver It ties, low transcription in thymus gland, is not transcribed in central nervous system.However, corresponding PD-L1 albumen is not translated extensively in normal but In tissue, only a small amount of translation is in tonsillotome, lungs, the macrophage of liver and immune special permission region, PD-L1 albumen It is translated in eyes and placenta.Translation 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, discovery PD-L1 albumen are a variety of swollen in melanoma, breast cancer, non-small cell lung cancer (NSCLC), kidney etc. High expression in tumor, and low expression or do not express in the normal tissue.PD-L1 albumen low expression in normal tissue and inductivity It is that it is different from other most significant features of co-suppression signal path 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 provide Important target spot, to obtain better curative effect and lower toxic side effect.
With the proposition of accurate medical concept, the research in relation to immunization therapy is got growing concern for.PD-1/ PD- L1 immunotherapy is a kind of anticancer immunotherapy currently to attract attention, it is intended to be resisted using the immune system of human body itself swollen Tumor: it is suitable for multiple types tumour by blocking PD-1/PD-L1 signal path to make death of neoplastic cells.Each research institution by Step carries out corresponding Clinical Project, the effect of single medication and conjoint therapy treating cancer is investigated, to excavate such drug Clinical value.Currently, opdivo has been suitable for melanoma in Japan, it is suitable for melanoma and non-small cell lung in the U.S. Cancer, in the granted melanoma idicatio of European Union, while opdivo has obtained the support of European Union CHMP for the application of lung cancer therapy. MSD Corp. keytruda is expected in the recent period in the U.S.'s granted melanoma idicatio through non-small cell lung cancer adaptation Card approval, while keytruda has obtained European Union CHMP support for the application of melanoma treatment.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 prediction: opdivo will become one of most successful immunotherapeutic agents preparation.
Compare concentration for the research of PD-1/PD-L1 at present, but is the absence of the exploitation of novel targets, " immunization therapy " treatment Mode standardizes not enough.Although PD-1 inhibitor and targeting medicine (being directed to cancer cell) have basic difference, resistance problems It still remains, it is necessary to further research and develop novel targets.
It is existing the study found that the occurrence and development of tumour to be frequently accompanied by the modification of PD-L1 protein phosphorylation abnormal. PD-L1 Sulphation modification plays key effect in the physiology and pathologic process that PD-1/PD-L1 signal path mediates after protein translation, The phosphorylation level of PD-L1 albumen has very important significance for the control accurate of PD-1/PD-L1 signal path.
Therefore, it is necessary to develop a kind of human PD-L 1 protein phosphorylation antibody, which is applied to malignant tumour During diagnosis, treatment and prognosis determine, while developing the novel targets of cancer.
Summary of the invention
Present invention aims to overcome that the shortcomings of the prior art, 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 adopted by the invention is as follows: one Kind 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 Shown in SEQ ID NO:2, the tyrosine Y-site of the hapten synthesis peptide is added with phosphate group.
As an improvement of the above technical solution, the N-terminal of the hapten synthesis peptide is connected with a cysteine C.
In addition, the present invention also provides the synthetic methods of the hapten synthesis peptide, successively the following steps are included: using polypeptide The amino acid sequence of synthetic technology synthesis polypeptide, the polypeptide is SYGGADYKRITVK, and the tyrosine Y-site of the polypeptide adds Add phosphate group, the N-terminal of the polypeptide connects a cysteine C.The hapten synthesis peptide are as follows: (NH2-) CSYGGAD (pY)KRITVK(-COOH)。
In addition, the present invention also provides a kind of human PD-L 1 protein Ys123The preparation method of site phosphorylation antibody, including it is following Step:
1) 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 to get antigen；
2) animal is immunized using antigen described in step 1), and collects antiserum；
3) the step 2) antiserum is purified and is selected, as PD-L1 protein Y123Site phosphorylation antibody.
As an improvement of the above technical solution, the step 2) immunization ways specifically: 1 initiation injection, 3~4 times plus Injection is penetrated and 1 direct injection；The initiation injection and booster shots are equal are as follows: move antigen synthetic peptide and adjuvant combined immunization Object, in the relatively thin and loose position two sides subcutaneous injection of neck, skin of back, intramuscular injection is distinguished in gluteus and huckle two sides, The intracutaneous injection of waist two sides, footpad injection；The direct injection are as follows: antigenic solution intramuscular injection.
As an improvement of the above technical solution, it in the step 3), is purified by following steps implementation: measuring before purification Antiserum is directed to the potency of antigen, determines that antiserum before purification can identify antigen；The antiserum of collection is purified, is determined Antiserum energy specific recognition phosphorylated human PD-L1 albumen after purification.
Preferably, whether the antiserum of collection is tested measurement antiserum for the potency of antigen with Elisa and its can be known Other antigen；It is purified using affine separation-affinity purification circulating technology antagonistic Serum, with Dot blot and Western Blot test 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 Ys that the preparation method is prepared123Site phosphorylation antibody.
In addition, the pharmaceutical preparation includes the human PD-L 1 protein Y the present invention also provides a kind of pharmaceutical preparation123Site Phospho-AB.
In addition, the present invention also provides the human PD-L 1 protein Ys123Site phosphorylation antibody is examined in preparation for malignant tumour The application for the pharmaceutical preparation that disconnected, treatment and prognosis determine.
In addition, the present invention also provides the applications that the pharmaceutical preparation determines 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 Preparation Method and application list PD-L1 protein Y in technical solution of the present invention123Site phosphorylation antibody, its hapten synthesis peptide And the preparation method of the two, human PD-L 1 protein Y123Site phosphorylation antibody can detect that human PD-L 1 protein phosphorylation is horizontal With suspension PD-1/PD-L1 cell-signaling pathways, facilitate to study PD-L1 protein phosphorylation in this way in PD-1/PD-L1 cell letter The effect of number access and occurrence mechanism on cancer provide for the diagnosis or treatment of clinical tumor disease and search out potential effect target Point；It simultaneously can also be by human PD-L 1 protein Y123Site phosphorylation antibody applies to diagnosis of malignant tumor, treatment and prognosis and determines In.
Detailed description of the invention
Fig. 1 is the antigen protein electrophoretogram in the embodiment of the present invention 3；
Fig. 2 is to scheme in the embodiment of the present invention 3 by the antigen Western blot of primary antibody of negative serum；
Fig. 3 is antiserum titre Elisa analysis chart in the embodiment of the present invention 3；
Fig. 4 is to close Western blot figure by the antigen of primary antibody of antiserum before purification in the embodiment of the present invention 4；
Fig. 5 is to close Western blot figure by the antigen of primary antibody of antiserum after purification 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 of people's normal esophageal scaly epithelium and esophageal squamous cell carcinoma in the embodiment of the present invention 4 Immunoreaction scorings chemical staining figure.
Specific embodiment
Purposes, technical schemes and advantages in order to better illustrate the present invention, 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 The site of phosphorylation occurs, then looking by mass spectrum document and protein phosphorylation site database PhosohoSitePlus It looks for, determines Y123Site is a phosphorylation site in human PD-L 1 amino acid sequence；Software CLC Protein is 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 are multiple phosphorylation site (such as Y for human PD-L 1 albumen28Site), since subsequent antigenicity and hydrophily are discontented Sufficient test requirements document, is not suitable as phosphorylation site.
Embodiment 2 synthesizes hapten synthesis peptide
According to the design of hapten synthesis peptide, in Y123A phosphate group is added on site, carries out HPLC (efficient liquid Phase chromatography) separation, purifying, the purity for obtaining hapten synthesis peptide is 95% or more.Correspondingly, one section of Y is synthesized123Site The synthetic peptide of non-phosphorylating, and the affine filler for separating chromatography, warp are used as with bovine serum albumin coupling by the cysteine of N-terminal Purity after HPLC is isolated and purified is 90% or more.
Embodiment 3 prepares antiserum
3.1 prepare negative serum
Take 3mL blood in heparin tube from the ear vein of the new zealand white rabbit (2~3kg, female are healthy and strong) for injection In, with cotton balls hemostasis by compression.Blood is set into room temperature 1h or so, waits for blood to solidify to form clot, 2h is placed at 4 DEG C analyses serum Out, 2500g be centrifuged 10min, draw supernatant, be 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 measures solubilized phosphoantigen (pY123- BSA antigen (the Y of concentration and non-phosphorylating)123- BSA) concentration, respectively 0.845mg/mL and 1.1mg/mL (phase relation Number R=0.991).By pY123- BSA and Y123- BSA carries out protein electrophoresis, and as a result swimming lane 1 is Y as shown in Figure 1:123- BSA, swimming lane 2 be pY123-BSA.Due to synthetic peptide be it is artificial synthesized, protein band is one section of thick wide band；Because phosphorylation modification makes pY123The molecular weight of-BSA is larger and band is slightly above Y123The band of-BSA.
Take dissolved purifying antigen (pY123-BSA、Y123- BSA) 5 μ g, 5 × SDS sample-loading buffer appropriate, boiling is added 10min is boiled in water-bath makes protein denaturation, and 10000 × g is centrifuged 10min；The volumetric concentration of SDS-PAGE electrophoretic separation glue is 10%, the volumetric concentration that glue is concentrated is 5%.Sample loading gun loading is used in order, in unused sample well plus isometric 5 × Sds gel sample-loading buffer；It is changed to 100V 70min after 60V 20min electrophoresis, until bromophenol blue reaches the bottom of separation gel, is closed Close power supply.Transferring film condition: constant current 180 mA, time 180min.The closing of 5% skimmed milk power, 37 DEG C of 2h of shaking table.Transfer film is put Enter and prepare immune preceding antiserum dilution by 1:5000 with TBST buffer, level slowly shakes up, and 4 DEG C overnight.37 DEG C of next day works With 20min, primary antibody solution is abandoned, 1 × TBST washes film 10min, is repeated 4 times.Film is placed in diluted by 1:5000 with 1 × TBST In the secondary antibody diluent of the goat anti-rabbit igg of HRP label, 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 kit, is 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, is 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), it is connected with plastic tube therebetween, is aspirated back and forth, until forming the emulsion emulsified completely, drop Yu Shuizhong does not expand It dissipates.
3.32 first immunisations are respectively through neck, position two sides subcutaneous (s.c) injection that skin of back is relatively thin, loose, gluteus With huckle two sides difference muscle (i.m) injection, waist two sides carry out intradermal (i.d) injection, and rabbit palmula position injection etc. is more Position multi-point injection antigen emulsion.About 0.61 mg of antigen total amount of first immunisation.
After 3.33 first immunisation 20d carry out first time booster immunization, use incomplete Freund's adjuvant (IFA) replace CFA as Immunologic adjuvant prepares antigen emulsion and is injected by first immunisation mode, and this time the antigen total amount of booster immunization is each about 0.9mg。
It is carried out after 3.34 immune 12d plus second strong immune, uses IFA to replace CFA as immunologic adjuvant and prepare 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 set into room temperature 1h or so, waits for blood to solidify to form clot, 4 2h is placed at DEG C is precipitated serum, and 3500g is centrifuged 10min, draws supernatant, is labeled as positive antiserum, dispenses and be stored in -20 It is DEG C to be measured.
1:1000,1:5000,1:10000 dilute 3.36 positive antiserums in proportion, carry out Elisa and detect antibody titer, OD value to be measured is 1.0 or more, and the P/N value difference 7.0,7.4 and 7.3 of the positive antiserum of each dilution, then serum is anti-at this time Body potency is at least 1:5000.Antibody titer reaches expected horizontal (the potency > 1:1000 of Elisa), carries 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 having the beaker of blood to close, and makes clot contraction.The serum of precipitation is sub-packed in 50mL by next day sterile working In centrifuge tube, 4000g is centrifuged 10min, takes supernatant, and every pipe dispenses 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, makes 2 μ g/mL of its concentration, presses (serum origin and the new zealand white rabbit being immunized, number of animals is respectively RB7771 to concentration gradient dilution antiserum in table 1 And RB7772), and illustrate to be operated according to Elisa kit, testing result is as shown in table 1 and Fig. 3.In table 1 and Fig. 3 In, SA indicates the antibody after Streptavidin-biotin-antigen system affinity purification；(-) indicates affine pure without the system The antibody of change, i.e. negative control；Blank is blank control；P-Ab refers to that hapten synthesis peptide phosphorylation, NP-Ab are in antigen The non-phosphorylation of hapten synthesis peptide in antigen.
Table 1 is immune rear sero-fast titration result
4 specificity identification human PD-L 1 protein Y of embodiment123Site phosphorylation antibody
The specificity of 4.1Western blot detection purified antibodies:
BCA protein concentration detection kit measures solubilized pY123- BSA and Y123The concentration of-BSA, concentration are respectively 0.692mg/mL and 0.647mg/mL (coefficient R=0.9952).Dissolved 5 μ g of purifying antigen is respectively taken, is added appropriate 5 × SDS sample-loading buffer, boiling water bath, which boils 10min, makes protein denaturation, and 10000 × g is centrifuged 10min.On Western blot Sample analysis, wherein primary antibody solution is the purified antibodies dilution of 1:500 and the antiserum dilution before purification of 1:10000.Knot Fruit is as shown in Figure 4 and Figure 5, and the primary antibody of Fig. 4 is antiserum dilution before purification, and BSA swimming lane is without there is specific band, Y123- The swimming lane and pY of BSA123The swimming lane of-BSA has specific band；The primary antibody of Fig. 5 is antiserum dilution after purification, BSA Swimming lane and Y123The swimming lane of-BSA is without there is specific band, pY123The swimming lane of-BSA has specific band.Therefore, after purification The hapten synthesis peptide-BSA of antibody energy specific recognition phosphorylation (i.e. the antigen of phosphorylation can also identify people's phosphorylation PD- L1 albumen).
4.2 immune group chemical identification antibody specificities
User's lung phosphorus cancerous tissue first after specimens paraffin embedding slices, is microscopy after immunohistochemical staining, result such as Fig. 6 Shown, phosphorylation PDL-1 albumen navigates to cell membrane, navigates to cytoplasm 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, and the microscopy after immune group chemical staining, 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 about 40%., phosphorylation PDL-1 albumen is primarily targeted for cell membrane, navigates to cytoplasm 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
Final concentration of 2.5%BSA (wt/vol), 0.01%Tween-20 (vol/ is added in IgG positive combined hybrid liquid Vol) and the mixed liquor of 25% glycerol (vol/vol), every pipe are packed as 1mL, -80 DEG C of long-term preservations.
5 human PD-L 1 protein Y of embodiment123Application of the site phosphorylation antibody in tumour
The human PD-L 1 protein Y of high specific is prepared in 5.1 present invention123Site phosphorylation antibody can use Western The differential expression of tumour cell after the detectable normal cell of blot experiment, tumour cell and medication.
5.2 antibody provided by the invention can be applied to detect in the immunological testings such as ICC, Elisa in practical applications Phosphorylation modification situation after the transcription of human PD-L 1 albumen, 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 that from can preventing withering for lymphocyte It dies, therefore antibody is convenient for studying human PD-L 1 protein Y in practical applications123Site phosphorylation is modified for particular biological event Such as immunoregulatory influence, to inquire into its effect during the disease developments such as tumour.
5.4 antibody provided by the invention help to inquire into the phosphorylation modification of PD-L1 albumen in tumor disease occurrence and development Mechanism of action in the process is more conducive to the research for inhibiting drug to tumor inhibition effect in clinical application.
5.5 the present invention be directed to the specific Y of human PD-L 1 albumen123The phosphorylation polyclonal antibody of site preparation, helps to grind Study carefully the zymogenesis for mediating it that phosphorylation occurs, inquire into human PD-L 1 albumen multiple biological function in T cell-signaling pathways and Albumen/nucleic acid interaction network, to find the latent effect target spot of the diagnosing and treating of clinical tumor disease.
Finally, it should be noted that above embodiments protect the present invention to illustrate technical solution of the present invention The limitation of range, although the invention is described in detail with reference to the preferred embodiments, those skilled in the art should be managed Solution, can modify to technical solution of the present invention or replace on an equal basis, without departing from technical solution of the present invention essence and Range.
Sequence table
<110>Zhang Hao
<120>a kind of 123 site phosphorylation antibody of human PD-L 1 protein Y 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
1 5 10 15
Asn Ala Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr
20 25 30
Gly Ser Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu
35 40 45
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
65 70 75 80
Tyr Arg Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn
85 90 95
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
130 135 140
Asp Pro Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr
145 150 155 160
Pro Lys Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser
165 170 175
Gly Lys Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn
180 185 190
Val Thr Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr
195 200 205
Cys Thr Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu
210 215 220
Val Ile Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Thr His
225 230 235 240
Leu Val Ile Leu Gly Ala Ile Leu Leu Cys Leu Gly Val Ala Leu Thr
245 250 255
Phe Ile Phe Arg Leu Arg Lys Gly Arg Met Met Asp Val Lys Lys Cys
260 265 270
Gly Ile Gln Asp Thr Asn Ser Lys Lys Gln Ser Asp Thr His Leu Glu
275 280 285
Glu Thr
290
<210> 2
<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 (5)

1. a kind of human PD-L 1 protein Y123The hapten synthesis peptide of site phosphorylation antibody, it is characterised in that: the haptens closes At the amino acid sequence of peptide as shown in SEQ ID NO:2, the hapten synthesis peptide is added with close to the tyrosine Y-site of C-terminal Phosphate group.
2. the synthetic method of hapten synthesis peptide as described in claim 1, it is characterised in that: successively the following steps are included: using Peptide synthesis technology synthesis polypeptide, the amino acid sequence of the polypeptide are SYGGADYKRITVK, junket of the polypeptide close to C-terminal Propylhomoserin Y-site adds phosphate group, and the N-terminal of the polypeptide connects a cysteine C.
3. a kind of human PD-L 1 protein Y123The preparation method of site phosphorylation antibody, it is characterised in that: the following steps are included:
1) hapten synthesis peptide described in claim 1 is coupled by cysteine C and carrier protein to get antigen；
2) animal is immunized using antigen described in step 1), and collects antiserum；
3) the step 2) antiserum is purified and is selected, as PD-L1 protein Y123Site phosphorylation antibody.
4. human PD-L 1 protein Y as claimed in claim 3123The preparation method of site phosphorylation antibody, it is characterised in that: the step Rapid 2) immunization ways specifically: 1 initiation injection, 3~4 booster shots and 1 direct injection；The initiation injection and reinforcement Injection is equal are as follows: by antigen synthetic peptide and adjuvant combined immunization animal, is subcutaneously injected in neck, back two sides, gluteus and huckle Distinguish intramuscular injection, the intracutaneous injection of waist two sides, footpad injection in two sides；The direct injection are as follows: antigenic solution intramuscular injection.
5. human PD-L 1 protein Y as claimed in claim 3123The preparation method of site phosphorylation antibody, it is characterised in that: the step It is rapid 3) in, be purified by following steps implementation: measuring the potency that antiserum before purification is directed to antigen, determine anti-blood before purification Antigen can be identified clearly；The antiserum of collection is purified, determines antiserum energy specific recognition phosphorylated human PD- after purification L1 albumen.
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