阅读说明：本技术 利用尾静脉注射进行基因免疫开发小鼠单克隆抗体的方法 (Method for developing mouse monoclonal antibody by gene immunization through tail vein injection ) 是由 雷坤 代腾飞 秦伏波 万定一 张永霞 于 2019-10-18 设计创作，主要内容包括：本发明公开了利用尾静脉注射进行基因免疫开发小鼠单克隆抗体的方法,本发明具体包括以下步骤：S1、表达载体的构建和佐剂质粒的扩增和纯化,S2、小鼠尾静脉注射免疫,S3、在第四次免疫之后进行血清免疫效果检测,S4、亚克隆及筛选,S5、抗体生产及抗体应用验证,本发明涉及抗体开发技术领域。该利用尾静脉注射进行基因免疫开发小鼠单克隆抗体的方法,不需要制备大量的重组蛋白,节约了实验材料,相对于基因枪和电穿孔技术进行基因免疫,不需要复杂的仪器设备,实验材料容易获取,只需要少量的重组蛋白进行检测,节约抗原,能够缩短免疫周期,节省时间,免疫时间缩短为1个月,针对难以重组表达的多次跨膜蛋白。(The invention discloses a method for developing a mouse monoclonal antibody by gene immunization through tail vein injection, which specifically comprises the following steps: s1, construction of an expression vector, amplification and purification of an adjuvant plasmid, S2, tail vein injection immunization of a mouse, S3, detection of serum immune effect after fourth immunization, S4, subcloning and screening, S5, antibody production and antibody application verification, and the invention relates to the technical field of antibody development. According to the method for developing the mouse monoclonal antibody by utilizing the tail vein injection for gene immunization, a large amount of recombinant protein does not need to be prepared, experimental materials are saved, and compared with gene immunization by a gene gun and an electroporation technology, the method does not need complex instruments and equipment, the experimental materials are easy to obtain, only a small amount of recombinant protein is needed for detection, the antigen is saved, the immunization period can be shortened, the time is saved, the immunization time is shortened to 1 month, and the method is specific to multiple transmembrane proteins which are difficult to recombine and express.)

1. The method for developing the mouse monoclonal antibody by utilizing tail vein injection to carry out gene immunization is characterized by comprising the following steps: the method specifically comprises the following steps:

s1, construction of an expression vector and amplification and purification of an adjuvant plasmid: firstly, constructing an expression vector by adopting a promoter and an antigen gene sequence, and then constructing a plasmid containing a GM-CSF (granulocyte-macrophage colony-stimulating factor) gene sequence to complete the preparation of an immune adjuvant, wherein any one of the plasmids is used as the immune adjuvant to improve the immune efficiency;

s2, mouse tail vein injection immunization: adding the expression plasmid and the immunologic adjuvant in the step S1 into lactated ringer' S buffer solution, wherein the final concentrations are respectively 500 mug/ml and 2.5-5 mug/ml, and then carrying out tail vein injection on the mouse, wherein the injection volume is 1.5-2ml each time, and the immunization is carried out once a week;

s3, detecting the immune effect of the serum after the fourth immunization: selecting the mice with the best effect in the step S2 for immune shock, killing the mice on the third day after the shock, performing spleen fusion, and performing cell fusion with mouse myeloma cells SP2/0 after the spleen is taken;

s4, subcloning and screening: detecting clone supernatant of the fused cells obtained in the step S3 by a specific screening method, subcloning the screened positive clones by a limiting dilution method, and establishing strains after subcloning for three times;

s5, antibody production and antibody application verification: transferring the cell strain established in the step S4 to a serum-free culture medium special for hybridoma for culture, centrifuging to collect supernatant after the cell growth density reaches 80%, purifying the supernatant by a protein A column, and carrying out endogenous verification on the purified antibody.

2. The method for developing a mouse monoclonal antibody by gene immunization using tail vein injection according to claim 1, wherein: the expression vector in the step S1 is any one of PTT3, pATX3 or pATX 4.

3. The method for developing a mouse monoclonal antibody by gene immunization using tail vein injection according to claim 1, wherein: in the step S1, the promoter is any one of CMV, CAG or human ubiquitin C.

4. The method for developing a mouse monoclonal antibody by gene immunization using tail vein injection according to claim 1, wherein: the antigen gene sequence in the step S1 is the full-length or fragment gene sequence of the antigen.

5. The method for developing a mouse monoclonal antibody by gene immunization using tail vein injection according to claim 1, wherein: in the step S2, blood sampling is carried out on the eye vein of the mouse before immunization, the blood sampling is used as a blank control, 6 mice are immunized in total, wherein 5 mice are injected with 1.5-2ml of plasmid in the tail vein, the 6 th mouse is injected with lactated ringer' S of the same volume as the negative control, immunization is carried out once every other week, and immunization shock is carried out 3 days before fusion.

6. The method for developing a mouse monoclonal antibody by gene immunization using tail vein injection according to claim 1, wherein: the immune shock in step S3 can be performed by using gene plasmid shock, purified recombinant protein or extract containing natural protein.

7. The method for developing a mouse monoclonal antibody by gene immunization using tail vein injection according to claim 1, wherein: the screening method in the step S4 is any one of indirect elisa, cellular immunofluorescence or flow cytometry.

8. The method for developing a mouse monoclonal antibody by gene immunization using tail vein injection according to claim 1, wherein: the culture conditions for culturing the established cell line in the step S5 are as follows: the cultivation temperature was 37 ℃ and 5% CO2 incubator.

Technical Field

The invention relates to the technical field of antibody development, in particular to a method for developing a mouse monoclonal antibody by utilizing tail vein injection for gene immunization.

Background

In the last 90 th century, people have begun to develop novel vaccines by using DNA immunization technology, the basic principle of DNA vaccines: the artificial synthetic plasmid vector containing virus DNA is injected into muscle, the plasmid carries out gene expression in vivo and synthesizes corresponding protein antigen, thereby causing the immune response of the organism, and the research and improvement of people on DNA vaccine are mainly in the following 2 aspects: (1) by adopting a more efficient immunization approach and introducing DNA plasmids into cells of an organism by using a gene gun and an electroporation technology, the immunization effect is obviously improved; (2) the mixed immune impact, firstly using DNA plasmid to make immunity, then using recombinant protein, inactivated virus or attenuated live vaccine to impact, in general, the effect of making gene immunity in small animal is superior to that of large animal, and this characteristic also indicates that said method can be used to develop high-quality mouse monoclonal antibody.

The classical antibody development process using mouse myeloma cells usually requires recombinant protein expression, and is very difficult for full-length expression of certain proteins, particularly multiple transmembrane GPCR proteins and ion channel proteins, and the integrity of protein sequences and structures is very critical for developing functional antibodies, and no matter natural protein extraction and purification or recombinant protein expression and purification, the spatial structure of the protein is very likely to change during the operation process, and the probability that antibodies generated through gene immunization can recognize conformational epitopes of the protein is very high, mainly because foreign genes can be directly expressed and synthesized in vivo into full-length proteins with natural conformations, thereby causing corresponding immune responses.

The invention adopts the tail vein injection method to carry out gene immunization, optimizes the injection volume, the immunization dose, the plasmid vector and the expression promoter, does not need to prepare a large amount of recombinant protein compared with the method for developing antibodies by recombinant protein immunized mice, saves experimental materials, shortens the immunization time to 1 month, shortens the experimental period, and does not need expensive instruments and complex operation compared with gene guns and electroporation technology to carry out gene immunization, thereby leading the technology to have wide application prospect.

Disclosure of Invention

Technical problem to be solved

Aiming at the defects of the prior art mentioned in the background technology, the invention provides a method for developing a mouse monoclonal antibody by utilizing tail vein injection to carry out gene immunization, the injection volume, the immunization dose, the plasmid vector and the expression promoter are optimized, compared with the method for developing the antibody by utilizing recombinant protein to immunize the mouse, a large amount of recombinant protein does not need to be prepared, the experimental material is saved, meanwhile, the immunization time is shortened to 1 month, the experimental period is shortened, compared with the gene immunization by a gene gun and an electroporation technology, expensive instruments and complex operation are not needed, and therefore, the technology has wide application prospect.

(II) technical scheme

In order to achieve the purpose, the invention is realized by the following technical scheme: the method for developing the mouse monoclonal antibody by utilizing tail vein injection to carry out gene immunization specifically comprises the following steps:

s1, construction of an expression vector and amplification and purification of an adjuvant plasmid: firstly, constructing an expression vector by adopting a promoter and an antigen gene sequence, and then constructing a plasmid containing a GM-CSF (plasmid-macrophage binding-stimulating factor) gene sequence to complete the preparation of an immune adjuvant, wherein any one of the plasmids is used as the immune adjuvant to improve the immune efficiency;

s2, mouse tail vein injection immunization: adding the expression plasmid and the immunologic adjuvant in the step S1 into lactated ringer' S buffer solution, wherein the final concentrations are respectively 500 mug/ml and 2.5-5 mug/ml, and then carrying out tail vein injection on the mouse, wherein the injection volume is 1.5-2ml each time, and the immunization is carried out once a week;

s3, detecting the immune effect of the serum after the fourth immunization: selecting the mice with the best effect in the step S2 for immune shock, killing the mice on the third day after the shock, performing spleen fusion, and performing cell fusion with mouse myeloma cells SP2/0 after the spleen is taken;

s4, subcloning and screening: detecting clone supernatant of the fused cells obtained in the step S3 by a specific screening method, subcloning the screened positive clones by a limiting dilution method, and establishing strains after subcloning for three times;

s5, antibody production and antibody application verification: transferring the cell strain established in the step S4 to a serum-free culture medium special for hybridoma for culture, centrifuging to collect supernatant after the cell growth density reaches 80%, purifying the supernatant by a proteinA column, and carrying out endogenous verification on the purified antibody.

Preferably, the expression vector in step S1 is any one of PTT3, pATX3, or pATX 4.

Preferably, the promoter in step S1 is any one of CMV, CAG, and human ubiquitin C.

Preferably, the antigen gene sequence in step S1 is the full-length or fragment gene sequence of the antigen.

Preferably, in the step S2, the blood is collected from the veins of eyes of the mice before immunization, and used as a blank control, 6 mice are immunized in total, wherein 5 mice are injected with 1.5-2ml of plasmid in the tail vein, the 6 th mouse is injected with the same volume of lactated ringer' S disease as a negative control, the immunization is performed once every other week, and the immunization shock is performed 3 days before the fusion.

Preferably, the immune shock in step S3 can be performed by using gene plasmid shock, purified recombinant protein or extract containing natural protein.

Preferably, the screening method in step S4 is any one of indirect elisa, cellular immunofluorescence or flow cytometry.

Preferably, the culture conditions for culturing the established cell line in step S5 are as follows: the cultivation temperature was 37 ℃ and 5% CO2 incubator.

(III) advantageous effects

The invention provides a method for developing a mouse monoclonal antibody by gene immunization by tail vein injection. Compared with the prior art, the method has the following beneficial effects:

(1) the method for developing the mouse monoclonal antibody by utilizing tail vein injection for gene immunization specifically comprises the following steps: s1, construction of an expression vector and amplification and purification of an adjuvant plasmid: firstly, constructing an expression vector by adopting a promoter and an antigen gene sequence, and then constructing a plasmid containing a GM-CSF gene sequence to complete the preparation of an immune adjuvant, wherein any one of the plasmids is used as the immune adjuvant to improve the immune efficiency, and S2, mouse tail vein injection immunization: adding the expression plasmid and the immunologic adjuvant in the step S1 into lactated ringer' S buffer solution to final concentrations of 100-: selecting the mice with the best titer in the step S2 for immune shock, killing the mice on the third day after the shock, performing spleen fusion, performing cell fusion with mouse myeloma cells SP2/0 after the spleen is taken, S4, subcloning and screening: detecting clone supernatant of the fused cells obtained in the step S3 by a specific screening method, subcloning the screened positive clones by a limiting dilution method, establishing strains after three times of subcloning, S5, and verifying antibody production and antibody application: transferring the cell strain established in the step S4 to a serum-free culture medium special for hybridoma for culture, centrifuging and collecting the supernatant when the cell growth density reaches 80%, purifying the supernatant by a protein A column, performing endogenous verification on the purified antibody, realizing gene immunization by a tail vein injection method, optimizing the injection volume, the immunization dose, a plasmid vector and an expression promoter, and compared with a method for developing the antibody by a recombinant protein immune mouse, the method does not need to prepare a large amount of recombinant protein, saves experimental materials, performs gene immunization by a gene gun and an electroporation technology, does not need complicated instruments and equipment, is easy to obtain the experimental materials, only needs a small amount of recombinant protein for detection, and saves antigens.

(2) The method for developing the mouse monoclonal antibody by utilizing tail vein injection to carry out gene immunization can shorten the immunization period, save time, shorten the immunization time to 1 month, and play an immunization effect on multiple transmembrane proteins which are difficult to be recombined and expressed, so that the technology has wide application prospect.

Drawings

FIG. 1 is a flow chart of the present invention;

FIG. 2 is a graph showing the exposure result of the mouse immune serum western-blot detection of the present invention;

FIG. 3 is a graph showing the exposure results of 21H11 subjected to an endogenous western blot assay according to the present invention;

FIG. 4 is a graph showing the result of exposure of IHC-nonnegative-cellling cancer in pathological tissue examination of 21H11 according to the present invention;

FIG. 5 is a graph showing the results of exposure of IHC-squamous cell carcinogena of the present invention for pathological tissue examination of 21H 11;

FIG. 6 is a data statistics plot of flow cytometry assays of 2C2 and 18H5 according to the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Referring to fig. 1-6, an embodiment of the present invention provides a technical solution: the method for developing the mouse monoclonal antibody by utilizing tail vein injection to carry out gene immunization specifically comprises the following steps:

preparing S1, human PD-L1 carrier plasmid and mouse GM-CSF adjuvant plasmid;

s2, injecting the tail vein of the mouse for immunization;

s3, cell fusion and screening;

s4, subcloning;

s5, and producing and verifying the antibody.

First step, preparation of human PD-L1 vector plasmid and mouse GM-CSF adjuvant plasmid

PDL1 gene sequence:

>PDL1cDNA with Kozak and RS-764bp

GCCGCCACCATGAGGATCTTCGCCGTGTTTATCTTTATGACCTACTGGCACCTGCTGAATGCCTTCACCGTGACAGTGCCCAAGGACCTGTACGTGGTGGAGTACGGCTCCAATATGACCATCGAGTGTAAGTTCCCTGTGGAGAAGCAGCTGGACCTGGCCGCCCTGATCGTGTACTGGGAGATGGAGGATAAGAATATCATCCAGTTTGTGCACGGCGAGGAGGACCTGAAGGTGCAGCACAGCAGCTACAGACAGAGAGCCAGGCTGCTGAAGGACCAGCTGTCCCTGGGCAATGCCGCCCTGCAGATCACCGACGTGAAGCTGCAGGACGCCGGCGTGTACAGATGCATGATCTCCTACGGCGGCGCCGATTACAAGAGGATCACCGTGAAGGTGAATGCCCCTTACAACAAGATCAACCAGAGGATCCTGGTGGTGGACCCCGTGACCAGCGAGCACGAGCTGACATGTCAGGCCGAGGGCTACCCCAAGGCCGAGGTCATTTGGACAAGCTCCGATCACCAGGTGCTGAGCGGCAAGACAACCACAACAAACTCCAAGAGAGAGGAGAAGCTGTTCAATGTGACAAGCACACTGAGGATCAATACCACAACAAATGAGATCTTTTACTGCACCTTCAGAAGACTGGACCCCGAGGAGAACCACACCGCCGAGCTGGTCATTCCTGAGCTGCCCCTGGCCCACCCTCCCAATGAGAGGGGCAGCCACCACCACCACCATCACTGA

Cloning vector:pcDNA3.4

Cloning strategy:EcoRI/NotI

mGM-CSF gene sequence:

>GM-CSF

gctagcgccgccaccATGTGGCTGCAGAATTTACTTTTCCTGGGCATTGTGGTCTACAGCCTCTCAGCACCCACCCGCTCACCCATCACTGTCACCCGGCCTTGGAAGCATGTAGAGGCCATCAAAGAAGCCCTGAACCTCCTGGATGACATGCCTGTCACgTTGAATGAAGAGGTAGAAGTCGTCTCTAACGAGTTCTCCTTCAAGAAGCTAACATGTGTGCAGACCCGCCTGAAGATATTCGAGCAGGGTCTACGGGGCAATTTCACCAAACTCAAGGGCGCCTTGAACATGACAGCCAGCTACTACCAGACATACTGCCCCCCAACTCCGGAAACGGACTGTGAAACACAAGTTACCACCTATGCGGATTTCATAGACAGCCTTAAAACCTTTCTGACTGATATCCCCTTTGAATGCAAAAAACCAAGCCAAAAATGAtctaga

Cloning vector:pcDNA3.1(+)

Cloning strategy:NheI/XbaI.

step two, mouse immunization:

preliminary experiment (1): optimizing injection volume

Adding an expression plasmid and an immunologic adjuvant into a lactated ringer's buffer solution, wherein the concentrations of the expression plasmid and the immunologic adjuvant are respectively 500 mug/ml and 2.5-5 mug/ml, carrying out tail vein injection on the mice, and optimizing the tail vein injection volume of each mouse, wherein the injection volumes are respectively as follows: 0.5ml, 1ml and 2ml, immunized once a week, and after the fourth immunization, mouse sera were collected to test titers.

Step three, detecting the ELISA titer of the immune serum of the mouse:

after the third immunization, the mouse serum titers were measured indirectly by Elisa.

The indirect elisa test for detecting the serum titer of the immune mice comprises the following steps:

3.1 coating

The diluted PD-L1 protein was added to a well of an enzyme-labeled plate at a concentration of 2ug/ml in a carbonate buffer solution having a pH of 9.6 in an amount of 100. mu.L per well, and the plate was incubated at 37 ℃ in an incubator for 2 hours with a sealing membrane attached thereto.

3.2 sealing

Taking out the coated enzyme label plate, patting the plate dry on a piece of straw paper, adding 300 mul of skimmed milk sealing solution into each hole, covering the plate, and placing the plate in a 37 ℃ incubator for 1.5-2 hours.

3.3 sample application

Immune serum was diluted with phosphate buffer at pH 7.4 in a gradient: 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000 and 1:64000 at a final volume of 100. mu.L per well, and then covered with a lid and incubated in a 37 ℃ incubator for one hour.

3.4 washing

The ELISA plate was removed, the primary antibody was spun off, washed 3 times with PBST, and blotted dry on a piece of paper.

3.5 Dilute of Secondary antibody

Goat Anti-Mouse (H + L) -HRP was diluted to an appropriate concentration, and the diluted secondary antibody was added to wells of an enzyme-labeled plate in an amount of 100. mu.L per well in sequence, incubated at 37 ℃ in an incubator for 30 min.

3.6 washing

The ELISA plate was removed, the secondary antibody was spun off, washed 3 times with PBST, and blotted dry on a piece of paper.

3.7 color development

Pouring the prepared TMB color development liquid into a clean sample adding groove sleeved with a PE glove, sequentially adding 100 mu L of TMB color development liquid into the holes of the enzyme-labeled plate by using a line gun, covering the holes with a cover, and placing the holes in an incubator for 5-10 min.

3.8 termination

And opening the microplate reader, preheating for 1 minute, taking the microplate out of the incubator, sequentially adding 2M sulfuric acid into the wells of the microplate in an amount of 50 mu L per well, and immediately reading.

3.9 reading

Setting 450-type 620nm wavelength, wiping the bottom of the ELISA plate with a water absorption towel, then placing the cleaned ELISA plate into a clamping groove of an ELISA reader, clicking to read, and storing detection data.

Table 1: serum detection titer data after third immunization of mice

Table 2: blank serum detection titer data before mouse immunization

Fourthly, mouse immune serum western-blot detection

4.1 SDS-PAGE gel electrophoresis

Putting a PD-L1 recombinant protein sample into a gel sample adding buffer solution, heating for 5 minutes at 98 ℃ to denature the protein, adding 0.5 mu g of the recombinant protein into each electrophoresis channel, connecting an electrophoresis device with a power supply, adjusting the voltage to 100V, allowing current to flow to an anode, and increasing the voltage to 200V when bromophenol blue migrates through the concentrated gel; and turning off the power supply when the bromophenol blue migrates to the position of 0.5cm at the bottom of the separation gel.

4.2 transfer film

Removing the gel glass plate from the electrophoresis device, washing the gel glass plate clean with deionized water, preparing for an immunoblotting operation, placing the gel glass plate in a container containing an electrophoretic transfer buffer solution, soaking for 15-20min, wearing gloves, cutting filter paper and an NC membrane, avoiding contaminating the filter paper and the membrane as much as possible, soaking the cut filter paper and the membrane in the electrophoretic transfer buffer solution, removing bubbles left on the membrane, opening the transfer box and placing the transfer box in a shallow tray, completely soaking a sponge pad with the transfer buffer solution, placing the sponge pad on the wall of the transfer box, placing a piece of soaked filter paper on the sponge, carefully placing the gel on the filter paper, avoiding bubbles (wetting the gel surface with the transfer buffer solution and wearing gloves, carefully placing the NC membrane on the gel surface, gently lowering the NC membrane from one side of the gel to avoid bubbles, taking care that the gloves or tweezers are certainly contacted with the membrane), washing the buffer tank with deionized water, placing a stirrer in the buffer tank, soaking another piece of sponge with transfer buffer, placing on the gel-membrane sandwich, closing the transfer box and inserting into the transfer tank, placing the ice box into the buffer tank, and filling with 4 deg.C pre-cooled transfer buffer. The whole device is placed in an ice-water bath, a transfer electrode is connected, and the constant current 200mA is transferred for 1 h.

4.3 sealing

After the electrotransformation is finished, the NC membrane is placed in 5% skimmed milk powder (PBS preparation) for sealing, and the serum of the third immunized mouse is subjected to western-blot detection on the recombinant protein at 37 ℃ for 1 hour or 4 ℃ overnight.

4.4 immunoassay

Rinsing the closed membrane with PBST for 2-3 times, washing the sample adding tank, rinsing with distilled water, air drying, covering the membrane with disposable gloves, cutting membrane strips (generally about 3mm wide) according to the mark and experimental design, sequentially placing the membrane strips in the sample adding tank, respectively taking 3 groups of mouse serum with highest immune serum titer, diluting with skimmed milk at a ratio of 1:1000 times, adding into the sample tank, ensuring that all parts of the membrane are contacted with the solution, incubating on a shaking table for 1H at room temperature, discarding primary antibody, placing the membrane strips in the sample adding tank, adding 5-10ml PBST in each tank, washing on the shaking table for 3min, changing the solution, repeating for 5 times, selecting appropriate enzyme labeled secondary antibody and dilution concentration according to the experimental needs and design, adding goat anti-mouse IgG (H + L) -HRP secondary antibody in each sample adding tank for about 3ml, incubating on the shaking table for 45min at room temperature, and ensuring that all parts of the membrane are contacted with the solution, discarding the secondary antibody, placing the membrane strip in the sample adding groove, adding 5-10ml PBST in each groove, washing for 3min on a shaking table, changing the solution, and repeating for 5 times.

4.5 Exposure

Diluting and mixing A, B luminous liquid in proportion, slightly rinsing an NC film with deionized water, drying filter paper corner joints by suction, covering A, B mixed liquid drops by a reverse pasting method, turning off a lamp until a light green fluorescent strip is visible (about 5 min), drying the filter paper corner joints by suction, placing the filter paper corner joints in a preservative film, fixing the preservative film in a film box, quickly covering the film box, closing the film box, and exposing according to the intensity of the visible fluorescence. Taking out the film, completely immersing the film in the developing solution for 1-2min immediately, rinsing with clear water, placing the film in the fixing solution until the film is completely fixed, washing with clear water, drying in the air, calibrating a Marker, analyzing and scanning, wherein the exposure result is shown in figure 2, and a lane 1: 2ml- #2 mice, skim milk 1:1000 fold dilution, lane 2: 1ml- - #3 mice, skim milk 1:1000 fold dilution, lane 3: 0.5ml- #3 mice, skim milk 1:1000 fold dilution, conclusion: when the immunization volume (dosage) is more than 1ml, the tail vein injection can achieve the ideal effect.

The fifth step: cell fusion

5.1 animal treatment

Balb/c mice which had been boosted were fixed and the eyeballs removed to obtain blood. The mice were then sacrificed by cervical dislocation and placed in 75% alcohol for at least 30 seconds.

5.2 immune serum Collection and preservation

The whole mouse blood was allowed to stand at room temperature for 1 hour and then stored overnight at 4 ℃. Centrifuging the whole blood of the mouse at 3000rpm for 15min the next day, carefully sucking the upper serum as a hybridoma screening positive control, and subpackaging and storing at-20 ℃ in a refrigerator.

5.3 mouse spleen treatment

The mouse limbs were held with pins with the face up, the epidermis cut open with a first set of forceps and scissors, the abdominal wall muscle layer cut open with a second set of forceps and scissors, and the spleen was separated and removed with a third set of forceps and scissors.

5.4 splenocyte harvesting

3 petri dishes with a diameter of 10cm were each added with 10ml of 1640 minimal medium. Rinsing the spleen once in a first petri dish; in a second dish, remove residual connective tissue from the spleen surface with forceps (taking care not to tear the spleen capsule); in the third culture dish, two glass slides are used for frosting and lightly milling, and spleen capsule is broken, so that spleen cells can be obtained.

The well-milled spleen cell suspension was pipetted with a 10ml pipette, filtered through a cell sieve, and transferred to a 50ml sterile centrifuge tube.

5.5 splenocyte wash

And (3) absorbing 10ml of 1640 basic culture medium again, repeatedly washing the culture dish for 2-3 times, filtering by using a cell sieve, transferring to the 50ml sterile centrifuge tube, centrifuging for 6min at 1500rpm, discarding the supernatant, repeatedly beating 10ml of 1640 basic culture medium for 10-15 times, fully suspending spleen cell sediment, adding 30ml of 1640 basic culture medium, repeatedly beating for 5 times, uniformly mixing, and centrifuging for 6min at 1500 rpm. Repeating the above steps once, discarding the supernatant, repeatedly beating with 5ml 1640 basic culture medium for 10-15 times, re-suspending the splenocytes, taking out about 0.2ml of cell suspension, counting the splenocytes after 20-40 times of dilution, and standing at room temperature before fusion.

5.6 mouse myeloma cell treatment

Collecting sp2/0 cells in a 50ml sterile centrifuge tube, centrifuging for 5min at 1000rpm, discarding the supernatant, repeatedly beating 10-15 times with 10ml1640 basic culture medium, suspending myeloma cell sediment, adding 30ml 1640 basic culture medium, repeatedly beating 5 times, mixing uniformly, centrifuging for 5min at 1000rpm, repeating the steps once, discarding the supernatant, and repeatedly beating 10-15 times with 5ml 1640 basic culture medium to resuspend myeloma cell sediment. About 0.2ml of the cell suspension was taken out, diluted 10-20 times and then counted, and left at room temperature before fusion.

5.7 spleen cell-myeloma cell fusion

Before the beginning of fusion, a constant-temperature water bath is opened, the temperature is adjusted to 37 ℃, PEG and 1640 basic culture medium are put in the water bath for preheating, and according to the cell counting result, the required spleen cells and myeloma cells are respectively mixed uniformly according to the ratio of 5: 1 proportion is mixed in a 50ml centrifuge tube, centrifugation is carried out for 5min at 1000rpm, supernatant is discarded, the wall of the centrifuge tube is flicked, cell precipitation is loosened, the centrifuge tube is placed in a water bath at 37 ℃, preheated PEG (1 ml is added in 1 min) is added into the cell precipitation at a constant speed, in the process of adding the PEG, the tip of a gun head is used for stirring gently while rotating the centrifuge tube, the mixture is kept still for 90s, and preheated 1640 basic culture medium (the first time) is added at a constant speed: adding 1ml of the mixture within 1min, and stirring gently while adding; pre-heated 1640 minimal medium (second time) was added at constant rate: adding 2ml of the mixture within 1min while stirring gently; pre-heated 1640 minimal medium (third time) was added at constant rate: adding 9ml of the mixture within 3min, and stirring gently while adding; adding preheated 1640 basic culture medium at constant speed, stirring gently while adding until the volume is 40ml, placing the centrifugal tube in a water bath at 37 ℃ for standing for 3min, centrifuging the fused cell suspension at 800rpm for 5min, removing supernatant, and loosening cell precipitate.

5.8 termination of fusion

Adding 5ml HAT culture medium, gently beating suspension cell sediment for 10 times, adding appropriate amount of HAT culture medium according to the number of splenocytes, beating, mixing, and inoculating to 96-well cell culture plate.

And a sixth step: clonal supernatant screening

6.1 cell suspension preparation

And (3) preparing cell suspension from the positive clone obtained by indirect elisa screening, and accurately counting the number of cells of the cell suspension according to a cell counting method, wherein the number of the cells is about 10 per milliliter generally.

6.2 limiting dilution

Putting a new 24-hole culture plate on a super-clean workbench, respectively adding 900ul of 15% HT selection culture medium into A1, A2 and A3 holes, uniformly mixing hybridoma cells in the 24-hole culture plate subjected to limited dilution, taking 100 microliter of cell suspension, adding the cell suspension into the A1 hole of the new 24-hole culture plate, repeatedly blowing and beating the cells for about 10 times by using a 1ml pipette, then taking 100 microliter of the cell suspension from the A1 hole single channel pipette (20-100ul) to the A2 hole, repeating the operation until the cell suspension is diluted to the A3 hole, taking 120 cells from the A3 hole and putting the cells into a V-shaped groove, sucking 15% HT selection culture medium twice by using a 10ml pipette, enabling the total volume of the culture medium in the V-shaped groove to be 16ml, and repeatedly blowing and beating the cells for about 8 times. When a 96-well culture plate is inoculated, 1-6 six rows are added to 200 mu l/well to form 1.5 cells per well, 6.4ml of the rest cell suspension is supplemented with 6.4ml of 15% HT selection medium, the cell suspension is repeatedly blown and beaten for about 8 times, and 7-12 six rows are added to 200 mu l/well to form 0.75 cell per well.

6.3 cell clone culture

Placing the cell in an 8% CO2 incubator at 37 ℃, culturing for 5 days, and then, on the seventh day, observing small cell clones on an inverted microscope, marking a plate cover with a mark '1' for a single cell clone cluster, marking a plate cover with a mark 'check' for two or more cell clone clusters, and recording and counting the results.

6.4 about days 8-9 culture supernatants can be harvested for antibody detection

The one with good and strong positive growth of the monoclonal growth hole is selected, transferred to a 24-pore plate and then subjected to cloning culture or expanded culture.

The seventh step: purified antibodies and validation of antibody applications

We finally obtained 10 antibodies by the above method, and performed subtype and titer identification on the 10 antibodies, and the identification results are shown in table 3 below.

TABLE 310 subtype and potency identification data sheet for antibodies

Wherein 21H11 is subjected to endogenous western blot detection, the detection result is shown in figure 3, the first lane: non-small cell lung cancer tissue lysate, loading 30 μ g, second lane: breast cancer tissue lysate, loading 30 μ g, third lane: human cervical cancer cell lysate (HeLa celline), loading 30 μ g, fourth lane: retinal epithelial cells (ARPE-19celline), loading 30. mu.g.

In which pathological tissue detection was performed on 21H11, and the detection images are shown in fig. 4 and 5.

The flow cytometry detection was performed on 2C2 and 18H5, and the detection results are shown in fig. 6.

The list of the above 10 monoclonal tests is shown in Table 4 below:

TABLE 410 monoclonal test List of strains

pcdna3.1(+) vector sequence:

GACGGATCGGGAGATCTCCCGATCCCCTATGGTCGACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTGGTACCGAGCTCGGATCCACTAGTCCAGTGTGGTGGAATTCTGCAGATATCCAGCACAGTGGCGGCCGCTCGAGTCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTATCCCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGCATCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGGGGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATTTTCGGATCTGATCAGCACGTGTTGACAATTAATCATCGGCATAGTATATCGGCATAGTATAATACGACAAGGTGAGGAACTAAACCATGGCCAAGTTGACCAGTGCCGTTCCGGTGCTCACCGCGCGCGACGTCGCCGGAGCGGTCGAGTTCTGGACCGACCGGCTCGGGTTCTCCCGGGACTTCGTGGAGGACGACTTCGCCGGTGTGGTCCGGGACGACGTGACCCTGTTCATCAGCGCGGTCCAGGACCAGGTGGTGCCGGACAACACCCTGGCCTGGGTGTGGGTGCGCGGCCTGGACGAGCTGTACGCCGAGTGGTCGGAGGTCGTGTCCACGAACTTCCGGGACGCCTCCGGGCCGGCCATGACCGAGATCGGCGAGCAGCCGTGGGGGCGGGAGTTCGCCCTGCGCGACCCGGCCGGCAACTGCGTGCACTTCGTGGCCGAGGAGCAGGACTGACACGTGCTACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACCGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTC。

to sum up the above

The invention can realize gene immunization by adopting a tail vein injection method, optimizes the injection volume, the immunization dose, the plasmid vector and the expression promoter, does not need to prepare a large amount of recombinant protein compared with a method for developing an antibody by a recombinant protein immunized mouse, saves experimental materials, performs gene immunization compared with a gene gun and an electroporation technology, does not need complicated instruments, is easy to obtain experimental materials, only needs a small amount of recombinant protein for detection, saves antigen, can shorten the immunization period and save time, shortens the immunization time to 1 month, and can play an immunization effect aiming at multiple transmembrane proteins which are difficult to recombine and express, thereby having wide application prospect.

It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.