CN106632676A - Mouse anti-swine PD-L1 (Programmed Death 1 Ligand 1) monoclonal antibody and application thereof - Google Patents

Abstract

The invention discloses a mouse anti-swine PD-L1 (Programmed Death 1 Ligand 1) monoclonal antibody. The monoclonal antibody is secreted by a hybridoma cell strain 3B5 of which the collection number is CCTCC No:C2016195; the hybridoma cell strain 3B5 is collected in the China Center for Type Culture Collection at Wuhan University on November 30, 2016. The monoclonal antibody is Ig G1 immunoglobulin; the potency of hybridoma cell strain 3B5 supernatant is 1:210; the potency of ascitic fluid is 1:1.024*10<5>. Test results show that the monoclonal antibody does not undergo any cross reaction with a pET32a carrier protein or nonspecific proteins; Western-blot identifies that a monoclonal antibody secreted by a positive hybridoma cell strain undergoes a specific reaction with recombinant proteins; flow cytometry assay confirms that the monoclonal antibody secreted by the hybridoma cell strain can be effectively combined with a PD-L1 protein on a PBMC (Peripheral Blood Mononuclear Cell). The MAb lays a basis for blockage of a PD-1/PD-L1 pathway, and has important significance to researches on the relationships between the expression level changes of the PD-L1 on certain tissue cells in swine and diseases.

Description

The anti-pig PD-L1 monoclonal antibodies of one plant of mouse and its application

Technical field

The present invention relates to the anti-pig PD-L1 monoclonal antibodies of one plant of mouse and its application, belong to antibody engineering technical field.

Background technology

Programmed death factor ligand -1 (Ligand 1, PD-L1 of Programmed Death 1) belong to B7 families into Member, be I type transmembrane glycoproteins, be made up of signal peptide, adventitia area, transmembrane region and the part of intracellular region four, be expressed in Various Tissues and Cell, its acceptor is the programmed death factor 1 (PD-1 molecules) of CD28 families.Research shows that PD-1 is combined with PD-L1 can be with Suppress the activation of T cell and B cell, negative regulation played to immune response and is acted on, the regulation and control be for body itself in order to avoid Damage of the excessive immune response to body tissue, is a kind of self-protective mechanism.But in the interaction of cause of disease and host, certain A little bacteriums, virus and parasite can be by the expression of PD-L1 molecules on PD-1 molecules on rise specific T-cells or target cell The immune clearance of body is escaped, persistent infection is caused.In recent years research shows that PD-1/PD-L suppresses approach in autoimmunity Important function has been played in the generation development of property disease and chronic viral infection.PD-1/PD- is intervened using blocking-up type antibody L signal can effectively improve the anti-chronic viral infection immunity of body, thus prepare the anti-pig PD-L1 monoclonal antibodies of mouse have it is important Theoretical research and actual application value.

The content of the invention

In order to solve the above problems, it is an object of the invention to provide the anti-pig PD-L1 monoclonal antibodies of one plant of mouse and its application, The monoclonal antibody can be combined with pig PD-L1 protein-specifics.

To achieve these goals, the technical solution adopted in the present invention is：

The anti-pig PD-L1 monoclonal antibodies of one plant of mouse, described monoclonal antibody is CCTCC NO by deposit number： The hybridoma cell strain 3B5 secretions of C2016195, described hybridoma cell strain 3B5 is preserved in China typical culture collection The heart, preservation address is Wuhan, China Wuhan University, and preservation date is on November 30th, 2016.

Described monoclonal antibody is lgG1 immunoglobulin like protein.

A kind of application of described anti-pig PD-L1 monoclonal antibodies of mouse in detection pig PD-L1 protein reagents are prepared.

Described monoclonal antibody is preparing pig PD-L1 protein diagnostics reagent or the application in auxiliary diagnostic.

Described diagnostic reagent includes Immunohistochemical detection reagent or sero-immunity detection reagent.

Described sero-immunity detection reagent is MBP enzyme linked immuno-adsorbent assay reagent.

A kind of pharmaceutical composition comprising described monoclonal antibody.

Beneficial effects of the present invention：

Pig PD-L1 extracellular region protein of the present invention with abduction delivering after purification excites mouse body as immunogene, effectively Specific immune response to target molecule.Hybridoma is screened with ELISA method, the supernatant for filtering out is positive Growth often has a plural hybridoma colonies in hole, some colonies may not secretory antibody, or secretion is not institute Antibody being needed, therefore, to be separated in time using clonal culture protocol.The most frequently used hybridoma clone cultivation is limiting dilution Method and soft agar cultivation.In the present invention, clone's culture is carried out to hybridoma using limiting dilution assay, it is final to obtain 1 plant The hybridoma cell strain 3B5 of the anti-pig PD-L1 monoclonal antibodies of continuous release specific murine, monoclonal antibody be Ig G1 immunoglobulin like protein, it is non-other Hypotype；The potency of cell conditioned medium is 1：2¹⁰；The potency 1 of ascites：1.024×10⁵.By ELISA test for identification, as a result it is positive Reaction, this plant of monoclonal antibody of preliminary proof is anti-PD-L1 extracellular region proteins, then proves that this plant of monoclonal antibody is carried with pET32a with cross matching Body protein and unrelated protein no cross reaction, further prove that sifted out hybridoma is special, is reflected with Western-blot Determine positive hybridoma secrete monoclonal antibody and specific reaction occurs with recombinant protein, finally confirm miscellaneous with Flow cytometry Knurl secrete monoclonal antibody is handed over effectively to be combined with PD-L1 albumen on PMBC (PBMC).

This plant of MAb that the present invention is obtained provides basis to block PD-1/PD-L1 paths, to studying some tissues in pig body The expression change of PD-L1 is significant with the relation of disease on cell.

Description of the drawings

Fig. 1 is the specific outcome of Western-blot test for identification monoclonal antibodies, in figure, M：Marker；1：PD-L1 Albumen；2：Rosetta supernatants；3：The unloaded body proteins of pET-32a (+).

Fig. 2 is the combination situation of PD-L1 albumen on Flow cytometry monoclonal antibody and Swine PBMC.

Specific embodiment

The content of the invention is further illustrated below in conjunction with specific embodiment, without in the case of specified otherwise, is adopted Operational means, verification method etc. are all the routine techniques means of those skilled in the art, are specifically referred to《Molecular Cloning: A Laboratory refers to South》And other normal experiment methods.The present invention simply highlights the innovative point of technical scheme, thus prior art here is not done into one The explanation of step.

Experiment material

Murine myeloma cell NS0 is frozen by this laboratory；6～8 week old BALB/c mouses are tested purchased from Shanghai Si Laike Company of Animals Ltd., raises in this laboratory animal room.1640 culture mediums, HAT and HT culture mediums are purchased from GIBCO companies of the U.S.； Hyclone is purchased from Hangzhou Chinese holly biology Co., Ltd；Freund's complete adjuvant, incomplete Freund's adjuvant and soluble one pack system Tmb substrate solution is purchased from Sigma Co., USA；Polyethylene glycol (PEG) 1500 is purchased from Roche companies of the U.S.；HRP marks sheep anti mouse IgG is purchased from Invitrogen companies of the U.S.；Tween-20 is purchased from Guangzhou Ding Guo Bioisystech Co., Ltd；Mouse IgG hypotypes are identified Kit is purchased from Roche companies of the U.S.；Tissue Culture Plate, cell cryopreservation tube are purchased from Shanghai Wei Ke biochemical reagents Co., Ltd；Its Its reagent is domestic pure analysis pure reagent.

GNK solution：Weigh KCl 0.4g, NaCl 8g, Na₂HPO₄·12H₂O 3.56g、NaH₂PO₄·H₂O 0.69g, phenol Red 0.01g, glucose 2g, plus distilled water (DDW) is settled to 1000mL, adjusts pH value to 7.2,115 DEG C of autoclavings, 4 DEG C of guarantors Deposit standby.

The preparation of the anti-pig PD-L1 monoclonal antibodies of the mouse of embodiment 1

1st, immunogenic preparation

According to the gene order of pig PD-L1 in GenBank and prokaryotic expression carrier polyclone enzyme enzyme site, its is designed extracellular Area's primer, the cDNA with pig peripheral blood mononuclearcell (PBMC) expands PD-L1 extracellular domain fragments as template using round pcr, And be connected with pMD18-T-simple carriers, pMD-PD-L1 recombinant plasmids are built, convert into DH5 α and screened by bacterium solution PCR Positive colony is simultaneously sequenced, using restriction enzyme EcoR I and Xho I respectively to positive recombinant plasmid pMD-PD-L1 and protokaryon table Double digestion is carried out up to carrier pET-32a (+) and reclaimed, using T₄Digestion purpose fragment and carrier that DNA ligase connection is reclaimed Fragment, builds pET-32a-PD-L1 prokaryotic expression carriers, and connection product converted into DH5 α, it is identified it is correct after convert to place Main bacterium Rosetta (DE3) carries out abduction delivering.The expression of recombinant protein, Western-blot are analyzed using SDS-PAGE The antigentic specificity of identification recombinant protein.Then restructuring destination protein is isolated and purified using nickel post, with the albumen of purifying Isopyknic complete Freund's adjuvant or incomplete Freund's adjuvant are added, is emulsified on refiner, be respectively prepared Freund's complete adjuvant Antigen and Freund's incomplete adjuvant antigen.Wherein, the concrete steps being related to have：

The abduction delivering of the 1.1 Host Strains Rosetta (DE3) containing pET-32a-PD-L1 prokaryotic expression carriers

By the bacterial classification Rosetta (DE3) containing recombinant expression plasmid pET-32a-PD-L1 in containing Amp (100 μ g/mL) LB In fluid nutrient medium, 37 DEG C of overnight Amplification Cultures.Take above-mentioned culture bacterium by volume 1:100 it is inoculated in fresh corresponding 2 × YT nutrient solutions, 37 DEG C are cultivated to OD₆₀₀It is worth for 0.5, IPTG to final concentration of 0.5mmol/L is added, to induce the table of destination protein Reach, continue to cultivate 4h in 37 DEG C, after the product 5000rpm of induction, 4 DEG C of centrifugation 10min, collects thalline, by volume 1:20 Plus the resuspended thalline of PBS, lysozyme is added to final concentration of 100 μ g/mL, 30 DEG C of water-bath 30min；Ultrasonication 15min, Precipitation is collected after 8000rpm, 4 DEG C of centrifugation 20min, with appropriate 2M urea washes liquid is resuspended 10min on ice is put, 8000rpm, 4 DEG C Centrifugation 20min, repetition wash 4-5 time, finally with 8M urea dissolve inclusion body, 8000rpm, 4 DEG C centrifugation 20min, collection supernatant put- 80 DEG C save backup.

The SDS-PAGE analyses of expression product：12% separation gel (mass fraction), about 15.0mL, each component is shown in Table 1.Will be each After component is well mixed, sol solution will be separated and added immediately in mounted glass glue flat board, to apart from glass plate top About 1.5cm, then gently covers the water layer of about 1.0cm on sol solution is separated, to prevent air in oxygen to gel polymerisation Inhibitory action.About 30min is stored at room temperature, after gel polymerisation is complete, the water layer of covering is outwelled, gel top is rinsed with water several Blot the water on gel top after secondary as far as possible with filter paper.

The preparation of the separation gel of table 1 12%

Prepare 5% and concentrate glue (mass fraction), about 6.1mL, each component is shown in Table 2.After each component is well mixed, will concentrate Sol solution is added immediately to glass plate, and liquid level is flushed with edge in glass plate, is immediately gently inserted comb between glass plate, as far as possible Avoid producing bubble.After about 30min concentration glue polymerizations completely, comb is carefully extracted.Add appropriate 1 × Tris-Gly running buffers Liquid, uses electrophoresis wash buffer comb hole, the sample being carefully added into after processing before loading.Concentration glue stage voltage is 80V, treats bromine Phenol is blue into up voltage during separation gel to 120V, until bromophenol blue frontal migration is to gel bottom.It is careful to peel off after electrophoresis is finished Gel, adds appropriate coomassie brilliant blue R_250 dyeing liquor slowly to shake dyeing 2h, takes out gel and rinses for several times in water, then With Coomassie brilliant blue destainer decolouring 3h, need to change therebetween and decolourize 3 times.After protein band is clear, observe result, with gel into As system is taken pictures and is preserved.As a result show, the relative molecular mass of expression product is 45kDa, relative with expected recombinant protein Molecular mass is consistent.

Table 2 5% concentrates the preparation of glue

The Western-blot analyses of expression product：Jing after SDS-PAGE electrophoresis, cut needs the portion of transferring film to destination protein Point.Respectively by gel, foam-rubber cushion and pvdf membrane (soaking 1～2min in methyl alcohol) in advance in electrotransfer buffer solution balance 30～ 60min.Then according to be followed successively by foam-rubber cushion, gel, pvdf membrane, the order of foam-rubber cushion from top to bottom putting well successively, particularly exist To avoid the generation of bubble when gel is put on pvdf membrane as far as possible, the bubble excluded between each layer is finally gently compressed with hand, To eliminate the generation of bubble.Put well and covered on half-dried translator, be connected to electrophoresis apparatus, both positive and negative polarity is connected successively, turn under the conditions of 25V 30～45min of film.Gel after electrotransfer is terminated is put into coomassie brilliant blue R_250 dyeing liquor and dyes, to check transfer effect Really.

Transfer removes pvdf membrane after finishing, and proceeds as follows：

Closing：Pvdf membrane is put into the appropriate glass dish of size, and carries out mark, then washed with TBST, use matter Amount fraction 5% skimmed milk power-TBST room temperatures are closed overnight；

Wash film I：Confining liquid is discarded, is shaken slowly with TBST and is washed film three times, each 5min～10min；

It is anti-plus one：By 6 × His monoclonal antibodies (the μ g/mL of working concentration 2) and (work of goat-anti human PD-L 1 monoclonal antibody The μ g/mL of concentration 0.4) respectively with adding after 1% skimmed milk power-TBST dilutions, 37 DEG C are shaken slowly 2h；

Wash film II：Discard one to resist, shaken slowly with TBST and wash film three times, each 5min～10min；

It is anti-plus two：Horseradish peroxidase-labeled sheep anti mouse (two resist) is diluted into (1 with 1% skimmed milk power-TBST:1000) After add, 37 DEG C of slow shaking 60min；

Wash film III：Discard two to resist, slowly shaken with TBST and wash film three times, each 5min～10min；

Colour developing：Nitrite ion is prepared with reference to the specification in AEC colour reagent boxes, pvdf membrane is placed in nitrite ion, treated out After existing specific reaction band, rinsed with terminating reaction, Taking Pictures recording result with distilled water immediately.

Western-blot results show that the recombinant protein can be with 6 × His monoclonal antibodies and goat-anti human PD-L 1 antibody Recognized, it was demonstrated that PD-L1 extracellular region gene fragments obtain correct expression in prokaryotic expression system, and former with good reaction Property.

The purifying of 1.2pET-32a-PD-L1 restructuring destination proteins

According to the operation manual of ProteinPure-Ni-NTA Resin affinity columns, protein purification is carried out, concrete steps are such as Under：

Balance：With the equilibration buffer chromatographic column of 5～10 times of volumes.The His label restructuring strong for binding ability Albumen improves its specific binding, and low concentration imidazoles (10mM～20mM) can be added in equilibrium liquid.

Loading：Sample buffer should be as consistent with level pad as possible.Shift to an earlier date in order to avoid blocking chromatographic column sample Filter or be centrifuged.

Washing：With the equilibration buffer solution chromatographic column of 5～10 times of column volumes, efflux is collected.

Wash-out：The imidazoles wash-out destination protein of variable concentrations can be adopted.

Then the destination protein to collecting wash-out is carried out after SDS-PAGE identifications, using purifying of the gradient dialysis to identification Albumen is concentrated and desalination, is concretely comprised the following steps：The appropriate eluent containing destination protein is added in the bag filter handled well, is used Clip is sequentially placed into the urea liquid of 8mol/L, 6mol/L, 4mol/L, 2mol/L, 1mol/L, 0.5mol/L after clipping, often 4 DEG C of dialysis 3h of individual gradient, finally carry out dialysis in PBS solution.Jing after silica gel suitably concentration, collect restructuring destination protein solution and survey The concentration of amount destination protein.

2nd, animal immune

Female BAl BIc/c the mouse of 36 week old or so are selected, immunity, 50 μ are carried out using the subcutaneous multi-point injection method of nape part G/, per the dosage of only 200 μ L.The antigen emulsified with Freund's complete adjuvant carries out first immunisation, subcutaneous multi-point injection；Then use The antigen of incomplete Freund's adjuvant emulsification carries out respectively secondary immunity and three immunity, per minor tick 14d, three immunity 10 days Afterwards, antibody titer is surveyed with indirect ELISA, when antibody titer reaches 1:Can be merged after 1600,3～5d enters before cell fusion Row booster immunization, tail vein or the μ g of lumbar injection 25 are not added with the antigen of adjuvant.

As a result show, the serum antibody titer of 3 BALB/c mouse is respectively 3.2 × 10 after first immunisation³、6.4×10³With 6.4×10³；Potency is respectively 1.28 × 10 after three immunity⁴、2.56×10⁴With 2.56 × 10⁴(being shown in Table 3).

The serum titer of the immunity BALB/c mouse of table 3

3rd, the recovery of NS0 myeloma cell and culture

In fusion the last week, recovery myeloma cell NS0 is placed in 37 DEG C, 5%CO₂Overnight incubation in incubator, next day changes Liquid.Fusion the same day, the NS0 cells taken in exponential phase are gently blown with suction pipe, collect into centrifuge tube, 1000r/min from Heart 10min, abandoning supernatant again suspends cell after washed once with GNK solution, cell count, and adjustment cell density is 10⁶Individual/mL, puts standby in incubator.

4th, the preparation of feeder cells

1d prepares feeder cells before fusion.Take Kunming mouse 1 lethal, 75% alcohol disinfecting body surface, aseptic operation scissors is beaten The skin of abdomen of mouse is opened, peritonaeum is exposed.Then the HAT culture mediums of 5mL precoolings are squeezed into mouse peritoneal with 5mL syringes, gently Light extrusion belly, suctions out the liquid of injection again, collects Turnover of Mouse Peritoneal Macrophages.40mL, 100 μ are diluted to HAT culture mediums L/ holes are added in 4 piece of 96 porocyte culture plates, put 37 DEG C, 5%CO₂Cultivate in incubator.

5th, cell fusion

GNK solution, 50% (mass fraction) PEG solution are preheated in 40 DEG C of water-baths；Mouse for fusion is taken a blood sample and divides It is then lethal to be dipped into alcohol from serum；NS0 cells are collected in 50mL centrifuge tubes, 1000r/min centrifugation 10min are abandoned Clearly, washed once with GNK solution；Mouse skin, peritonaeum are cut off successively, abdominal cavity is exposed, and are taken spleen and are positioned over grinding on nylon gauze, And washed down with GNK solution, splenocyte is collected in 50mL centrifuge tubes, 1000r/min centrifugation 10min abandon supernatant；By splenocyte It is mixed in 50mL centrifuge tubes that (spleens cell number is about 2.6 × 10 with NS0 myeloma cell⁸Individual, NS0 cells used are about 3 bottles 80% 10mL NS0 cells, splenocyte：NS0 number of cells is about 10:, plus GNK solution is to 40mL, 1000r/min centrifugations 1) 10min, abandons supernatant, breaks up cell mass；This fusion pipe is moved into 40 DEG C of water-baths, the 50%PEG that will be preheated with 1mL suction pipes (pH8.0) it is added drop-wise in fusion pipe, side edged is shaken gently for fusion pipe, adds in 1min, and continues slowly to shake in a water bath Fusion pipe 1.5min.The GNK solution 15mL of 40 DEG C of preheatings are slowly added dropwise immediately.Add step be：1mL/30s, 3mL/30s, 11mL/30s.Then GNK solution is slowly added to 40mL, 37 DEG C of water-baths stand 5min, 1000r/min centrifugation 10min, abandon Clearly.Cell mass is broken up, plus the piping and druming of 40mL HAT culture mediums is mixed, and is added in 96 porocyte culture plates containing feeder cells, per hole 100 μ L, put 37 DEG C of 5%CO₂Cultivate in incubator.

6th, the culture of fused cell

Cell after fusion is placed in CO₂Cultivate in incubator, next day checks for pollution, such as find that pollution is added dropwise immediately 1% NaOH.The visible little clone of general 3～4d；Half amount changes liquid in the hole for clone occur during 7d；10d or so can draw few Amount supernatant is used to screen, and adds the HAT culture mediums of equivalent；14d or so can half amount replacing HT cultures to the positive hole screened Base, while going to Amplification Culture in 24 well culture plates to the cell in the positive hole of screening, prepares cloning, will in screening process HAT culture mediums are gradually replaced by 1640 complete mediums.

7th, the screening of positive hybridoma cell

With 5 μ g/mL purification of Recombinant PD-L1 extracellular region protein wrapper sheets, 50 μ L/ holes, by hybridization of the indirect ELISA to merging Oncocyte culture supernatant is screened for the first time, and the strong positive clones hole to screening carries out second specificity screening, uses carrier Albumen, normal E. coli supernatant and other unrelated proteins carry out wrapper sheet detection, and its detection method is identical with first time, while Control group is set up, the stronger positive hole of the specificity to screening goes to Amplification Culture in 24 well culture plates, prepares cloning.

Concrete screening step is as follows：It is coated with the albumen of 5 μ g/mL, 50 μ L/ holes, overnight, secondary daily PBST is washed 4 DEG C of coatings Plate, then closes 2h with 5% skim milk (PBST dissolvings) in 37 DEG C, supernatant is abandoned, with confining liquid 1:1 dilution clone hole to be detected Supernatant (one resists), is advisable per the μ L of hole 50.The positive, negative control, blank being done simultaneously and being compareed without clone's hole supernatant, 37 DEG C incubate 30min is educated, supernatant is abandoned, PBST is washed 5 times, it is anti-plus two, two anti-1:1000 times of dilutions, 50 μ L/ holes, 37 DEG C of incubation 30min are abandoned Clearly, washed 5 times with PBST, be subsequently adding TMB developers, develop the color 5～10min, surveys OD₄₅₀Value.

8th, the clone of positive hybridoma cell

Limiting dilution is carried out when the cell hole length of positive colony is to 80%, limiting dilution the previous day prepares feeder cells. Take out a little cell suspension to be counted, cloning is carried out to the cell in positive hole using limiting dilution assay.Its step is as follows：

With 10 μ L trypan blues and 10 μ L cell suspensions after the mixing of PCR pipe moderate proportions, living cells is carried out under the microscope Count, formula：Cell number/mL=N/4 × 10³(N is the living cells of 4 block plaids in corner under microscope to × 10 × extension rate Sum), the viable count contained by every milliliter of cell suspension is calculated, appropriate cell suspension is then taken, diluted with 1640 culture mediums To 10mL, and will be containing 2 × 10³Individual living cells (dilution factor I), with the orifice plate of cell suspension bed board half block 96 of dilution factor I, 100 μ L/ holes, 20 cells/wells；The cell suspension plus the complete mediums of 9mL 1640 (dilution factor II) of 1mL dilution factors I are taken, is used The another orifice plate of half block 96 of cell suspension bed board of dilution factor II, 100 μ L/ holes, 2 cells/wells；The cell for taking 3mL dilution factors II hangs Liquid adds the complete mediums of 7mL 1640 (dilution factor III), and with the cell suspension of dilution factor III one piece of 96 orifice plate is spread, 100 μ L/ holes, 0.6 cells/well；Above-mentioned culture plate is placed in into CO₂Cultivate in incubator, after culture 7d, monoclonal hole is marked simultaneously partly Amount changes liquid, when the 1/10 of monoclonal length to bottom hole, carries out bioactivity and specific detection, filters out specific higher and sun The stronger cell of property carries out repeated cloning；Simultaneously every time clone's remaining cell is proceeded into 24 porocyte culture plates cultures (and in original Another batch of culture medium is added in hole, in case the two pollutes or cell death simultaneously), proceed to then and expanded in 6 orifice plates or cell Big culture, and it is frozen in time；Through multiple clone operations, until when the cell hole Positive rate of all clones is 100%, i.e., Can determine that the hybridoma cell strain for obtaining secrete monoclonal antibody；After obtaining the hybridoma cell strain of stably excreting antibody, and When be enlarged culture and it is frozen.

9th, the preparation of ascites, purifying

By 8～12 week old female BAl BIcs/c mouse peritoneal injection 0.5mL atoleine sensitization.7d pneumoretroperitoneums inject hybridoma Cell, cell infusion amount is 5 × 10⁶Individual/only, and observe mouse web portion after inoculating cell daily, treat that mouse web portion substantially expands, use Asepsis injector suctions out ascites, and 3 000r/min centrifugation 20min take supernatant packing, mark, and -20 DEG C save backup.Using IgG The monoclonal antibody-purified kit extraction purification monoclonal antibody of class, the purifying examination provided according to the rich Science and Technology Ltd. of the Aurion of Beijing hundred Agent box operation instructions step operation.

The anti-pig PD-L1 monoclonal antibody specificity identifications of the mouse of embodiment 2

1st, indirect ELISA identifies the specificity of monoclonal antibody

3 subclones of Jing, obtain the hybridoma of 1 plant of secretion specific monoclonal antibody, and hybridoma cell strain is named as 3B5. Respectively with the unloaded body protein (bag of pig PD-L1 albumen, chicken PD-L1 albumen, Rosetta (DE3) supernatants and pET-32a (+) for obtaining 5 μ g/mL are by concentration) coated elisa plate, 50 μ L/ holes.Add Mab supernatant to be checked, 50 μ L/ holes (1:200 dilutions), 37 DEG C Incubation 30min, PBST board-washings 3 times.The anti-50 μ L of mountain sheep anti-mouse igg two of the HRP marks of 1: 1000 times of dilution are added, 37 DEG C are placed in Incubation 30min, PBST are washed 3 times, add 50 μ L TMB nitrite ions, act on 5-10min, use 2mol/L H₂SO₄Terminate anti- Should, read each detection hole OD₄₅₀Numerical value, each hole OD₄₅₀Value (the results are shown in Table 4) with P/N ＞ 2.1 as Positive judgement standards.As a result table It is bright, this plant of monoclonal antibody can with Recombinant Swine PD-L1 albumino reactions and with the unloaded body proteins of pET32a (+) and unrelated protein reaction, tool There is good specificity

The monoclonal antibody specificity identification result of table 4

2nd, the specificity of Western-blot test for identification monoclonal antibody

Jing after SDS-PAGE electrophoresis, cut needs the part of transferring film to restructuring PD-L1 extracellular regions destination protein.Respectively will be solidifying Glue, foam-rubber cushion and pvdf membrane (soaking 1～2min in methyl alcohol) balance in advance 30～60min in electrotransfer buffer solution.Then press According to be followed successively by from top to bottom foam-rubber cushion, gel, pvdf membrane, foam-rubber cushion order put well successively, particularly gel is being put into To avoid the generation of bubble when on pvdf membrane as far as possible, the bubble excluded between each layer finally gently be compressed with hand, to eliminate bubble Generation.Put well on half-dried translator cover, be connected to electrophoresis apparatus, both positive and negative polarity is connected successively, under the conditions of 25V transferring film 30～ 45min.Gel after electrotransfer terminates is put into coomassie brilliant blue R_250 dyeing liquor and dyes, to check transfer effect.

Transfer removes pvdf membrane after finishing, and pvdf membrane is washed with TBST, is closed with 5% skimmed milk power-TBST room temperatures Night；Confining liquid is discarded, is shaken slowly with TBST and is washed film three times, every time 5～10min；One is added to resist：By in hybridoma 3B5 cultures Clear dilution with 1% skimmed milk power-TBST covers pvdf membrane after (1: 100), and 37 DEG C are shaken slowly 2h；Discard one to resist, PBST shakes wash slowly Film 3 times, each 5-10min；Add the mountain sheep anti-mouse igg two anti-(1: 1000 dilution) of HRP marks, 37 DEG C of slow shaking 1h；Abandon Two resist, and are slowly shaken with TBST and wash film three times, every time 5～10min；Specification according to AEC colour reagent boxes prepares nitrite ion, Pvdf membrane is placed in nitrite ion, after specific reaction band to appear, is rinsed with terminating reaction with distilled water immediately, note of taking pictures Record result.Rosetta supernatants pET-32a (+) empty carrier protein control group (result is shown in Fig. 1) is set simultaneously.As a result show, should There is specific reaction in strain monoclonal antibody and pig PD-L1 albumen, and specific band occur, but not with control group Rosetta supernatants with The albumino reaction of pET-32a (+) empty carrier, occurs without band.

The anti-pig PD-L1 monoclonal antibody Identification of Biological Characteristics of the mouse of embodiment 3

1st, the detection of antibody titer

The Hybridoma Cell Culture supernatant collected is pressed into 1:1、1:2¹、1:2²、1:2³、1:2⁴、1:2⁵、1:2⁶、1:2⁷、1:2⁸、 1:2⁹、1:2¹⁰、1:2¹¹Dilution, the ascites obtained after hybridoma injection mouse peritoneal is by 1: 100,1: 200,1: 400,1: 800th, after 1: 1600,1: 3200,1: 6400,1: 12800,1: 25600,1: 51200,1: 102400,1: 204800 dilution, by weight Group PD-L1 extracellular region protein wrapper sheets, by indirect elisa method its OD is determined₄₅₀Value, detects respectively hybridoma supernatant and abdomen Water potency.Parallel negative control experiments are done with mice serum before immunity, it is anti-to occur with restructuring PD-L1 extracellular region proteins target antigen The greatest dilution of the monoclonal antibody answered is its potency, each hole OD₄₅₀Value is with P/N >=2.1 as positive.As a result show, cell The potency of supernatant is 1：2¹⁰；The potency of ascites is 1: 1.024 × 10⁵。

2nd, monoclonal antibody hypotype identification

The Ig subclass of said monoclonal antibody, the strict by specification of operating procedure are identified using Ig subgroup identifications RNA isolation kit Carry out.As a result show 3B5 secrete monoclonal antibody be IgG1 immunoglobulin like protein, non-other hypotypes.

3rd, hybridoma cell strain secretes the repeated pruning of monoclonal antibody

The hybridoma cell strain supernatant of serial passages in vitro is collected, with indirect elisa method its potency is determined, freeze-stored cell, Interval is recovered frozen cell after 1 month, detects the antibody titer in its culture supernatant, continuous detection 3 months, is evaluated with this The stability of the secretory antibody.As a result show, the hybridoma of clone purification is recovered after 3 months in liquid nitrogen cryopreservation, many times of Jing Pass on and still be able to stably excreting high-titer antibody, antibody titer is up to 1: 2¹¹。

4th, the Flow cytometry of monoclonal antibody

Swine peripheral blood lymphocytes are separated with reference to the specification of PBLC separating liquid, is comprised the following steps that：

(1) from pig vena cava anterior collection anticoagulation 5mL, (anti-coagulants is 10% sodium citrate, and whole blood and anti-coagulants ratio are 10:1), anticoagulation is gently mixed with equivalent PBS；

(2) 4mL lymphocyte separation mediums are taken in 12ml glass centrifuge tubes, above-mentioned mixing blood 6mL is slowly added along tube wall Enter (note in centrifuge tube：Action is light, it is impossible to mix), horizontal centrifugal, 2000rpm, room temperature centrifugation 20min；

(3) the white cell thin between plasma layer and separating liquid is drawn with capillary, moves into new 12mL glass centrifugation Guan Zhong, adds 2～5 times of volumes PBS, gently suspension cell, 1000rpm, room temperature centrifugation 10min, abandons supernatant, adds 10mL PBS, gently suspension cell, repeated centrifugation once, abandons supernatant；

(4) red cell volume in leucocyte is observed, is suitably added 1～2mL erythrocyte cracked liquids, on ice splitting erythrocyte 5min, adds 10mL PBS, 1000rpm, room temperature centrifugation 10min, abandons supernatant, adds 0.5mL PBS suspension cells so as to disperse It is standby after cell count for individual cells suspension.

Take 1 × 10⁶Individual PBMCs is added in the monoclonal antibody of the anti-PD-L1 of final concentration of 10 μ g/mL, and reaction system is 100 μ L, mouse IgG1 are isotype antibody controls, and 37 DEG C of effect 1h are washed 3 times with PBS, add the sheep anti-mouse igg (1 of FITC marks: 500 times of dilutions), 37 DEG C of effect 1h are washed 3 times with PBS, and with 1mL PBS suspension cells, (result is shown in figure to Flow cytometry 2).Fig. 2 results show that the fluorescence intensity of the fluorescence intensity ratio isotype antibody controls of monoclonal antibody is remarkably reinforced, and illustrate to prepare Monoclonal antibody can be very good combine PBMC on PD-L1 albumen.

Claims (7)

1. anti-pig PD-L1 monoclonal antibodies of one plant of mouse, it is characterised in that described monoclonal antibody is CCTCC by deposit number NO：The hybridoma cell strain 3B5 secretions of C2016195, described hybridoma cell strain 3B5 is preserved in Chinese Typical Representative culture guarantor Tibetan center, preservation address is Wuhan, China Wuhan University, and preservation date is on November 30th, 2016.

2. anti-pig PD-L1 monoclonal antibodies of mouse according to claim 1, it is characterised in that described monoclonal antibody is LgG1 immunoglobulin like protein.

3. a kind of anti-pig PD-L1 monoclonal antibodies of mouse as described in any one of claim 1-2 are preparing detection pig PD-L1 albumen Application in reagent.

4. application according to claim 3, it is characterised in that described monoclonal antibody is examined in preparation pig PD-L1 albumen Application in disconnected reagent or auxiliary diagnostic.

5. application according to claim 4, it is characterised in that described diagnostic reagent is tried including Immunohistochemical detection Agent or sero-immunity detection reagent.

6. application according to claim 5, it is characterised in that described sero-immunity detection reagent is Enzyme-linked Immunosorbent Assay Detection reagent.

7. a kind of pharmaceutical composition of the monoclonal antibody comprising described in claim 1.