CN111647564B

CN111647564B - anti-EB virus LMP1 monoclonal antibody, cell strain and application thereof

Info

Publication number: CN111647564B
Application number: CN202010418416.0A
Authority: CN
Inventors: 李欣; 崇拓拓; 龙雨飞
Original assignee: Individual
Current assignee: Shenzhen Hospital of Southern Medical University
Priority date: 2020-05-18
Filing date: 2020-05-18
Publication date: 2023-07-04
Anticipated expiration: 2040-05-18
Also published as: CN111647564A

Abstract

The invention discloses a monoclonal antibody for resisting EB virus LMP1 and cell strain and application thereof, wherein the invention uses the sequence of SEQ ID NO.2 as antigen to obtain cell strain by adopting cell fusion, three rounds of subcloning selection are combined with indirect ELISA and FACS detection technology, hybridoma cell strain capable of stably secreting positive antibody is finally screened out, the amplification culture is carried out, and the cell supernatant is single subtype through subtype identification result; cell 7B63B10 with better FACS detection result is used for preparing ascites and purifying the antibody. The secreted antibody has high specificity and high affinity, and the established indirect ELISA method has good detection specificity and sensitivity, which indicates that the double-antibody indirect ELISA kit has good popularization and application value.

Description

anti-EB virus LMP1 monoclonal antibody, cell strain and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a monoclonal antibody for resisting EB virus LMP1, a cell strain and application thereof.

Background

EB virus (Epstein-Barr virus, EBV) is a DNA virus belonging to human type 4 herpes virus (HHV-4), the first virus to be associated directly with human tumors. It is counted that over 90% of all people worldwide are infected with this virus and can be carried throughout the life after the primary infection, thereby causing a variety of diseases: burkitt Lymphoma (BL), nasopharyngeal carcinoma (NPC), hodgkin Lymphoma (HL), post-transplant lymphoproliferative disease (PTLD), EBV-associated gastric cancer (EBVaGC), and the like.

LMP1 (Latent membrane protein, LMP1, latent membrane protein 1) is one of the key proteins responsible for B cell immortalization. The EBV encoded membrane protein has the molecular weight of 66kDa, consists of 386 amino acids (aa), and comprises a hydrophilic cytoplasmic N-terminal (1-23 aa), 6 water-conveying transmembrane regions (24-186 aa) and a C-terminal (187-386 aa) consisting of 200 amino acids; the C-terminus comprises three important C-terminal activation domains (C terminal activating regions, CTARs): CTAR1, CTAR2 and CTAR3. These regions provide anchor sites for aptamer proteins, including tumor necrosis factor receptor-related factors (TNFR-associated factors, TRAFs), tumor necrosis factor receptor-related death domain proteins (TNFR-associated death domain, TRADD), receptor-interacting protein kinases (Receptor interacting protein kinase, RIP), BS69, JAK-3 proteins, and the like, mediate cell immortalization, inhibit cell differentiation and apoptosis, promote tumor metastasis, evade host immune response, and the like, by transducing signals through NF- κ B, JNK/p38-SAPK, PI3-K/Akt, ERK-MAPK, and JAK/STAT pathways.

Therefore, finding a cell strain capable of stably secreting and specifically combining an anti-LMP 1 monoclonal antibody becomes a problem to be solved urgently, and has important significance for a deep molecular mechanism of the EB virus LMP1.

Disclosure of Invention

The primary object of the present invention is to provide a cell line secreting an anti-LMP 1 monoclonal antibody.

Another object of the present invention is to provide a monoclonal antibody secreted by the above cell line.

It is a further object of the present invention to provide the use of the cell lines and monoclonal antibodies described above.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

in a first aspect of the present invention, a hybridoma cell line 7B63B10 secreting an anti-LMP 1 monoclonal antibody is deposited with the chinese collection of typical cultures, university of martial arts, and the collection number cctccc NO: C2019160.

in a second aspect of the present invention, an anti-LMP 1 monoclonal antibody is provided, wherein the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown as SEQ ID No.5, and the amino acid sequence of the light chain variable region of the monoclonal antibody is shown as SEQ ID No. 7.

According to the embodiment of the invention, the gene sequence of the heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO.4, and the gene sequence of the light chain variable region is shown as SEQ ID NO. 6.

According to an embodiment of the invention, the monoclonal antibody has high specificity for specifically recognizing LMP1.

According to an embodiment of the invention, the Ig type of the monoclonal antibody is IgG2b.

The monoclonal antibody is secreted by the hybridoma cell strain 7B63B10.

In a third aspect of the present invention, there is provided a kit for detecting LMP1 of EB virus, comprising the above-mentioned anti-LMP 1 monoclonal antibody.

In a fourth aspect of the invention, the application of the hybridoma cell strain 7B63B10 in preparing the anti-LMP 1 monoclonal antibody is provided. According to embodiments of the invention, the monoclonal antibodies may be used to specifically recognize LMP1; the heavy chain variable region of the monoclonal antibody has an amino acid sequence shown as SEQ ID NO.5 in a sequence table; the light chain variable region has an amino acid sequence shown in SEQ ID NO.7 of the sequence Listing.

In a fifth aspect of the present invention, there is provided a kit for detecting epstein barr virus LMP1, comprising any of the monoclonal antibodies described above.

In a sixth aspect of the present invention, there is provided an epstein barr virus LMP1 detection system comprising a sampling device and a detection device, wherein the detection system adopts a flow cytometry and/or a cell fluorescence immunoassay, and the flow cytometry and/or the cell fluorescence immunoassay uses any of the monoclonal antibodies described above.

In a seventh aspect of the invention, the use of a monoclonal antibody in the preparation of a reagent for detecting LMP1 is presented.

In an eighth aspect of the present invention, there is provided a method for preparing a hybridoma cell line secreting an LMP1 monoclonal antibody, the method comprising the steps of:

according to LMP1 sequence information published by NCBI, performing escherichia coli codon optimization and performing gene synthesis;

constructing an escherichia coli expression plasmid, converting BL21 host bacteria, and carrying out a test of small expression;

amplifying the culture volume of the escherichia coli, collecting supernatant or sediment, and purifying and detecting target proteins;

immunizing a Balb/c mouse with a recombinant protein expressed by an antigen LMP1 escherichia coli;

spleen cells of immunized mice are collected and fused with Sp2/0 myeloma cells, and the fused hybridoma cells are selectively cultured by HAT culture medium;

and detecting the antibody content in the culture supernatant of the hybridoma cells by adopting an indirect ELISA method, and screening to obtain the hybridoma cell strain capable of stably secreting the monoclonal antibody.

According to an embodiment of the invention, the immunization comprises: the mice are female Balb/c mice of 6-8 weeks old, purified LMP1 recombinant protein is fully mixed and emulsified with Freund's complete adjuvant with equal volume, then Freund's incomplete adjuvant is adopted to emulsify antigen, and the mixture is fully mixed to a water-in-oil state for subcutaneous multipoint immunization, and the immunization is enhanced for 2-3 times.

According to an embodiment of the invention, the fusing comprises: taking the spleen of a mouse under the aseptic condition to prepare a cell suspension, fusing the SP2/0 cell of the myeloma of the mouse and the spleen cell under the mediation of PEG1450, and adding a DMEM culture medium to terminate after the fusion.

According to an embodiment of the invention, the number ratio of the mouse myeloma SP2/0 cells to the spleen cells is 1:5.

According to an embodiment of the invention, the detecting comprises: liquid and semi-solid fusion plates were tested.

According to an embodiment of the present invention, the liquid fusion plate detection includes: and (3) starting to detect when the liquid-exchanging cells of the fusion plate grow to more than about 1 ten thousand cells with medium size, and selecting positive Kong Zuoya clones after the quality control of the indirect ELISA is qualified.

According to an embodiment of the invention, the semi-solid melt plate detection comprises: after conventional cell fusion, cell pellet was resuspended in 20ml 2% methylcellulose concentration semisolid medium and cultured in cell culture dishes; after 7-10 days, observing the size and the number of cell clones in a culture dish, picking the cell clones to HT medium of 20% fetal bovine serum under a split microscope, culturing the cell clones for 2-3 days, and performing titer detection, and performing ELISA quality control by an indirect method to obtain subclones.

According to an embodiment of the invention, the methylcellulose semi-solid medium comprises: DMEM, MEM, 2% by mass of methylcellulose, fetal bovine serum, 50 x HAT and L-glutamine.

In a ninth aspect of the present invention, there is provided a method for producing an anti-LMP 1 monoclonal antibody, comprising the steps of injecting the hybridoma cells described above into a BALB/c mouse previously treated with Freund's incomplete adjuvant, collecting ascites from the mouse, and purifying the ascites after pretreatment.

According to an embodiment of the invention, the purification comprises: purifying by Protein G-agarose affinity chromatography column.

The beneficial effects of the invention are as follows:

the hybridoma cell strain 7B63B10 is prepared, and can stably secrete an anti-LMP 1 monoclonal antibody. The cell strain specifically recognizing the LMP1 monoclonal antibody adopts a semi-solid high-flux fusion technology, so that the subcloning period is obviously shortened, the reserve base number of positive clones is increased, and the high-affinity monoclonal antibody capable of specifically recognizing the LMP1 is obtained by combining indirect ELISA and FACS detection technologies in the screening process.

The invention takes the monoclonal antibody of the anti-LMP 1 secreted by the hybridoma cell strain 7B63B10 as a detection antibody, and the established indirect ELISA method has good detection specificity and sensitivity, which indicates that the indirect ELISA kit has good popularization and application values.

Drawings

FIG. 1 is an SDS-PAGE electrophoresis after protein purification, wherein lanes 1 to 7 are respectively, an LMP1 loading test before LMP1 purification, an LMP1 breakthrough test after LMP1 column hanging, an LMP1 test after Marker and NTA20 buffer removal, an LMP1 test after NTA60 buffer removal, an LMP1 test after NTA200 buffer removal, and an LMP1 test after NTA500 buffer removal.

FIG. 2 is an SDS-PAGE electrophoresis after purification of the antibodies, wherein lane 1 is Marker and lane 2 is LMP1 purified monoclonal antibody.

FIG. 3 is a power curve of the affinity constant of anti-LMP 1 monoclonal antibodies.

FIG. 4 is a FACS flow cytometry detection of a monoclonal antibody of the invention.

FIG. 5 is an IF cytoimmunofluorescence of a monoclonal antibody of the invention, A is MDA-MB-231 and B is Raji.

Detailed Description

The following examples are given to illustrate the technical solution of the present invention in a clear and complete manner, but are not limited thereto.

The hybridoma cell strain 7B63B10 secreting the anti-LMP 1 monoclonal antibody is preserved in China center for type culture Collection (China, university of Wuhan, and China) in 7 months and 19 days, and the preservation number is CCTCC NO: C2019160.

example 1 sequence analysis of LMP1 Gene and selection of antigen sequence

The LMP1 amino acid sequence is as follows:

MERDLERGPPGPPRPPLGPPLSSSIGLALLLLLLALLFWLYIVMSNWTGGALLVLYSFALMLIIIILIIFIFRRDLLCPLGGLGLLLLMSKYYTLCPPPPFPYASFSNALSPLSPVTLLLIALWNLHGQALYLGIVLFIFGCLLVLGLWIYFLEILWRLGATIWQLLAFILAFFLAIILLIIALYLQQNWWTLLVDLLWLLLFMAILIWMYYHGPRHTDEHHHDDSLPHPQQATDDSSHESDSNSNDGRHHLLVSGAGDGPPLCSQNLGAPGGGPDNGPQDPDNTDDNGPQDPDNTDDNGPQDPDNTDDNGPQDPDNTDDNGPQDPDNTDDNGPHDPLPHNPSDSAGNDGGPPNLTEEVENKGGDRDPPSMTDGGGGDPHLPTLLLGTSGSGGDDDDPHGPVQLSYYD(SEQ ID NO.1)

the LMP1 gene codes 408 amino acids, 6 times of transmembrane region, has hydrophilicity, and is subjected to transmembrane region analysis, signal peptide analysis, hydrophobicity analysis, disorder sequence analysis, antigenicity analysis, homology analysis and structural domain analysis. The most suitable amino acid sequence (213-408 aa) of SEQ ID NO.2 is selected and expressed as an antigen sequence in E.coli to prepare an antigen.

The antigen sequence selected was as follows:

HGPRHTDEHHHDDSLPHPQQATDDSSHESDSNSNDGRHHLLVSGAGDGPPLCSQNLGAPGGGPDNGPQDPDNTDDNGPQDPDNTDDNGPQDPDNTDDNGPQDPDNTDDNGPQDPDNTDDNGPHDPLPHNPSDSAGNDGGPPNLTEEVENKGGDRDPPSMTDGGGGDPHLPTLLLGTSGSGGDDDDPHGPVQLSYYD(SEQ ID NO.2)

nucleotide sequence corresponding to the selected antigen sequence:

CATGGTCCGCGTCATACCGATGAACATCATCACGATGATAGCCTGCCGCATCCGCAGCAGGCAACCGATGATAGTAGCCATGAAAGCGATAGCAATAGCAATGATGGTCGTCATCATCTGCTGGTTAGCGGTGCCGGTGATGGTCCGCCTCTGTGTAGCCAGAATCTGGGTGCACC TGGTGGTGGTCCGGATAATGGTCCGCAGGATCCGGATAACACAGATGATAATGGCCCTCAAGATCCTGACAATACCGACGATAACGGACCTCAGGACCCAGATAATACGGATGACAACGGTCCACAAGACCCTGATAACACTGACGACAATGGACCGCAAGACCCCGACAACACGGACGATAACGGTCCGCATGATCCTCTGCCGCATAATCCGAGCGATAGCGCAGGTAATGATGGTGGTCCTCCGAATCTGACCGAAGAGGTTGAAAATAAAGGTGGTGATCGTGATCCGCCTAGCATGACCGATGGTGGTGGCGGTGATCCGCATCTGCCGACACTGCTGCTGGGCACCAGCGGTAGCGGTGGTGATGACGATGATCCTCATGGTCCGGTTCAGCTGAGCTATTATGAT(SEQ ID NO.3)

preparation and purification of antigens

According to the coding sequence corresponding to the antigen, artificially synthesizing a gene fragment, connecting the gene fragment with a recombinant plasmid, constructing a recombinant expression plasmid, carrying out protein expression, bacteria breaking detection and purification, and preparing a monoclonal antibody as the antigen.

Results: FIG. 1 illustrates LMP1 proteins eluted with different buffers, but the present invention uses LMP1 eluted with NTA60 and NTA200 buffers to achieve the best effect, and can be used for subsequent monoclonal antibody preparation.

Preparation of LMP1 monoclonal antibodies

After the antigen prepared by the invention is immunized with the mouse, lymphocytes in the spleen of the mouse are separated and fused with SP2/0 myeloma cells, the indirect ELISA method is used for detection, and after ELISA quality control is qualified (namely negative control is <0.2 and positive control is > 1.0), monoclonal cells with high positive values are selected for limiting dilution, and repeated for the second time until finally, monoclonal cell strains capable of stably secreting positive antibodies are screened out for expansion culture.

Results: combining tables 1-4, screening 108 positive clones with OD450nm between 1.0 and 2.0 by indirect ELISA after fusion detection, selecting partial clones for FACS detection, finally selecting 14 strains with the best result for first subcloning, screening 12 strains for first subcloning, and finally screening 9 strains of fixed hybridoma cell strains; and then selecting a cell strain which has highest affinity and best FCS flow result and stably secretes positive antibody from the cell strain, and carrying out subsequent ascites preparation and antibody purification experiments.

TABLE 1 first subclone Indirect ELISA results

TABLE 2 results of first subclone supernatant flow cytometry (FACS)

TABLE 3 results of second subclone indirect ELISA

TABLE 4 results of second subclone supernatant flow cytometry (FACS)

Sequence number	Gene name	Cloning	Raji	B95-8	MDA-MB-231
						1	LMP1	2E6 1E1	41.6	19	0.93
2	LMP1	10G11 5D12	90	28.4	1.18
						3	LMP1	12H10 1E7	92.4	37.9	3.94
4	LMP1	7B63B10	96	38.4	4.29
						5	SP2/0	Negative control	1.02	1.95	1.10

Combining tables 1-4, screening 108 positive clones with OD450nm between 1.0 and 2.0 by indirect ELISA after fusion detection, selecting partial clones for FACS detection, finally selecting 14 strains with the best result for first subcloning, screening 12 strains for first subcloning, and finally screening 9 strains of fixed hybridoma cell strains; and then selecting a cell strain which has highest affinity and best FCS flow result and stably secretes positive antibody from the cell strain, and carrying out subsequent ascites preparation and antibody purification experiments.

Cell strain determination and subtype identification

And (3) amplifying and culturing the cell strain screened in the subcloning stage and stably secreting the positive antibody, collecting the supernatant for antigen detection, adopting ELISA gradient dilution and Western blot to verify the stability, collecting cells and amplifying and culturing, and collecting the supernatant again and detecting the subtype of the antibody in the supernatant.

As shown in Table 5, the hybridoma cell line with the best detection result of the present invention was 7B63B10, which showed single subtype by subtype identification, and ascites was prepared using the cell line, and the Ig type of the monoclonal antibody secreted was IgG2B.

TABLE 5 determination of stable hybridoma cell lines subtype

Plant number	2E6 1E1	12H10 1E7	7B63B10	10G11 5D12	13A1 4F3
						Subtype type	IgG1	IgG2b	IgG2b	IgG2a	IgG1
Plant number	7B10 1D10	9E9 1B12	6D9 2B2	11C12 2D6	/
						Subtype type	IgG1	IgG1	IgG1	IgG2a	/

Heavy chain variable region gene sequence of monoclonal antibody:

GAGGTGAAGCTGGTGGAATCTGGGGGAGGCTTCGTGCAGCCTGGAGGGTCCCGGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTTTGGAATGCACTGGGTTCGTCAGGCTCCAGAGAAGGGGCTGGAGTGGGTCGCATACATTAGTAGTGGCAGTAGTACCATCTACTATGCAGACACAGTGAAGGGCCGATTCACCATCTCCAGAGACAATCCCAAGAACACCCTGTTCCTGCAAATGACCAGTCTAAGGTCTGAGGACACGGCCATGTATTACTGTGCAAGAGACTGGGTTCTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA(SEQ ID NO.4)；

monoclonal antibody heavy chain variable region amino acid sequence:

EVKLVESGGGFVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEKGLEWVAYISSGSSTI YYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCARDWVLDYWGQGTTLTVSS(SEQ ID NO.5)；

monoclonal antibody light chain variable region gene sequence:

GATATTGTGATAACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCGTCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACAT TGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAATATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACACATGTTCCGTTCACGTTCGGAGGGGGGACCAAGCTGGAGCTGAAACGG(SEQ ID NO.6)；

monoclonal antibody light chain variable region amino acid sequence:

DIVITQTPLSLPVSLGDQASVSCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNR FSGVPDRFSGSGSGTDFTLNISRVEAEDLGVYFCSQSTHVPFTFGGGTKLEIKR(SEQ ID NO.7)。

the monoclonal antibodies described above were prepared in large quantities.

The monoclonal antibody of the invention is prepared by a conventional in vivo induction method, and is purified. The result shows that: as shown in FIG. 2, the purity of SDS-PAGE shows that the concentration of the monoclonal antibody LMP1 7B63B10 prepared by the invention is about 10mg/ml, and the purity is higher than 90%.

The monoclonal antibodies described above were subjected to application tests.

1. Monoclonal antibody affinity constant determination

1) Coating: coating according to the conventional coating concentration of 2ug/ml, wherein 50 mu l of each well of the 96-well plate is coated, and the mixture is incubated for 2 hours at 37 ℃ or reacted overnight at 4 ℃;

2) Closing: 200 μl/well of 2% BSA or 5% skim milk blocking solution, incubating at 37deg.C for 1 hr or reacting overnight at 4deg.C, and washing with TBST for 4 times;

3) An antibody: diluting the purified antibody according to a certain proportion, adding the diluted antibody into a 96-well plate, simultaneously adding PBS with a corresponding volume, 50 mu l/well, preventing foam from being generated as much as possible, adding uniformly, oscillating on a micro-oscillator, enabling liquid phase to be mixed more uniformly, and incubating for 1 hour at 37 ℃;

4) And (2) secondary antibody: TBST is washed for 4 times after the primary antibody is incubated, and enzyme-labeled secondary antibody is added into the primary antibody, 100 mu l/hole is incubated for 1 hour at 37 ℃; color development: after the secondary antibody is incubated, TBST is washed for 4 times, 100 mu l/hole of substrate solution is added, and the substrate solution is placed in a constant temperature box at 37 ℃ for 5 to 10 minutes;

5) Termination reaction, colorimetric: after adding 30. Mu.l/Kong Zhongzhi solution and yellowing, the absorbance at 450nm was measured using an ELISA reader.

The binding response Curve model and its equation were fitted by Curve estimate procedure in the Curve regression analysis using Beatty-established non-competitive ELISA, SPSS15.0 statistical software with mAb concentration on the X-axis and OD on the Y-axis (Table 6). Substituting the OD50 value into a curve equation, and calculating the corresponding antibody concentration.

Results: the Beatty derived formula is K= (n-1)/2 (n Ab)]’T-[Ab]T), the affinity constant is calculated. According to the Beatty derived formula and FIG. 3, the anti-LMP 1 monoclonal antibody affinity constant calculated according to the power curve model was K=9×10 ⁹ M ^-1 The present invention pertains to high affinity antibodies.

TABLE 6 OD value of anti-LMP 1 monoclonal antibody prepared by 7B63B10 cell line

Concentration of primary antibody (ug/ml)	OD value
		1	2.5774
0.5	2.622
		0.25	2.7527
0.125	2.5221
		62.5	1.5072
31.25	0.7175
		15.625	0.2089
7.813	0.0051

FACS flow cytometry Positive Rate

1) Flow cytometry experiments were performed using the EB virus positive cell line Akata-EBV to verify the purified antibody 7B63B10 of the cell line 7B63B10 of the present invention. Among them, the Akata-EBV cell line induced by goat anti-human IgG for 6 hours started to express EB virus in large amounts, so that LMP1 expression level was increased.

2) 2X 10 cells per tube ⁵ The individual was resuspended in 100ul FACS buffer (pre-chilled PBS with 2% FBS), 20 μl/tube was used Hu FcR binding Inhibitor (eBioscience 14-9161-73), blocked for 20min on ice, and the supernatant was discarded by centrifugation at 250g for 4 min.

3) An antibody: the cells were resuspended in 500ul of supernatant, centrifuged for 4min on ice for 30min and at 250g, and the supernatant was discarded; cells were resuspended using 1ml FACS Buffer, washed once and repeated once.

4) After dilution of the secondary antibody (Abcam ab 150119) 1:1000 with FACS buffer, 200 μl of cells per tube were resuspended in ice protected from light for 30min. The samples were washed twice with FACS buffer (procedure above) and resuspended with 400. Mu.l FACS buffer before detection on-machine.

Results: as shown in FIG. 4, the antibody secreted by the cell strain 7B63B10 can be applied to flow cytometry detection, and the positive rate of the flow cytometry detection is 85.4%, so that the detection requirement is met.

3. Cell immunofluorescence assay

1) The fibrio Nectin (Corning 354008) was copolymerized Jiao Min (Nest 80002), 0.05ug/ul 100ul, overnight at 4 ℃.

2) The next day, after the coating liquid was aspirated, PBS was washed three times, raji cells were added, and the cells were allowed to adhere to the wall.

3) Fixing: 4% paraformaldehyde 300 ul/dish, 15 minutes at room temperature, PBS wash three times.

4) Closing: preparing a sealing liquid: 9ml PBS, 1ml goat serum, and 10ul Tween-20; aspirate 500 ul/dish, 60 minutes at room temperature. The PBS was washed three times.

5) An antibody: antibody dilution: 10ml PBS, 0.1g BSA and 10ul Tween-20, the antibodies prepared according to the invention were diluted to 2ug/ml with a diluent, 500 ul/dish; negative controls (antibody dilution alone) were also set and washed three times with PBS after overnight at 4 ℃.

6) And (2) secondary antibody: antibody dilutions were diluted 1:1000 (secondary antibody Abcam ab 150119), washed three times with PBS at room temperature for 1 hour.

7) Nuclear staining: DAPI dye (Soy Bao C0060), 300 ul/dish was diluted with PBS at a volume ratio of 1:100 for 10 minutes at room temperature. The PBS was washed three times.

8) After addition of anti-quench coverslipping (Sigma F4680), confocal detection.

Results: cells stained with the antibodies of the invention were observed to be fluorescent, as shown in FIG. 5, EBV positive cells Raji could be bound by the antibodies, while EBV negative cells MDA-MB-231 gave negative results. It was demonstrated that the antibodies prepared according to the present invention recognize LMP1.

In summary, (1) the hybridoma cell line 7B63B10 of the invention produces an anti-LMP 1 monoclonal antibody that is highly specific for specifically recognizing LMP1 and does not have detectable cross-reactivity with LMP1 family proteins or other highly homologous proteins. Erroneous results are avoided when used in immunoassays for determining the presence or amount of LMP1 in a test sample.

(2) The anti-LMP 1 monoclonal antibody generated by the hybridoma cell strain 7B63B10 has high affinity, the common monoclonal antibody is only at the level of 8 times of 10, and the anti-LMP 1 monoclonal antibody has an anti-affinity constant of K=9×10 ⁹ M ^-1 . Belongs to high affinity antibodies.

(3) When the hybridoma cell strain stably secreting the monoclonal antibody is obtained through screening, two methods of liquid and semisolid fusion plate detection are adopted to comprehensively screen, so that the fusion cloning quantity is improved to the greatest extent; the capacity of the resulting antibody library is greater than in conventional screening formats using only one method.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

SEQUENCE LISTING

<110> Li Xin, chong Tuo, long Yufei

<120> anti-EB virus LMP1 monoclonal antibody, cell strain and application thereof

<130>

<160> 7

<170> PatentIn version 3.5

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Met Glu Arg Asp Leu Glu Arg Gly Pro Pro Gly Pro Pro Arg Pro Pro

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Gly Gly Ala Leu Leu Val Leu Tyr Ser Phe Ala Leu Met Leu Ile Ile

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Trp Met Tyr Tyr His Gly Pro Arg His Thr Asp Glu His His His Asp

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Asp Ser Leu Pro His Pro Gln Gln Ala Thr Asp Asp Ser Ser His Glu

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Ser Asp Ser Asn Ser Asn Asp Gly Arg His His Leu Leu Val Ser Gly

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Ala Gly Asp Gly Pro Pro Leu Cys Ser Gln Asn Leu Gly Ala Pro Gly

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gcaaccgatg atagtagcca tgaaagcgat agcaatagca atgatggtcg tcatcatctg 120

ctggttagcg gtgccggtga tggtccgcct ctgtgtagcc agaatctggg tgcacctggt 180

ggtggtccgg ataatggtcc gcaggatccg gataacacag atgataatgg ccctcaagat 240

cctgacaata ccgacgataa cggacctcag gacccagata atacggatga caacggtcca 300

caagaccctg ataacactga cgacaatgga ccgcaagacc ccgacaacac ggacgataac 360

ggtccgcatg atcctctgcc gcataatccg agcgatagcg caggtaatga tggtggtcct 420

ccgaatctga ccgaagaggt tgaaaataaa ggtggtgatc gtgatccgcc tagcatgacc 480

gatggtggtg gcggtgatcc gcatctgccg acactgctgc tgggcaccag cggtagcggt 540

ggtgatgacg atgatcctca tggtccggtt cagctgagct attatgat 588

<210> 4

<211> 345

<212> DNA

<213> artificial sequence

<400> 4

gaggtgaagc tggtggaatc tgggggaggc ttcgtgcagc ctggagggtc ccggaaactc 60

tcctgtgcag cctctggatt cactttcagt agctttggaa tgcactgggt tcgtcaggct 120

ccagagaagg ggctggagtg ggtcgcatac attagtagtg gcagtagtac catctactat 180

gcagacacag tgaagggccg attcaccatc tccagagaca atcccaagaa caccctgttc 240

ctgcaaatga ccagtctaag gtctgaggac acggccatgt attactgtgc aagagactgg 300

gttcttgact actggggcca aggcaccact ctcacagtct cctca 345

<210> 5

<211> 115

<212> PRT

<213> artificial sequence

<400> 5

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Phe Val Gln Pro Gly Gly

1 5 10 15

Ser Arg Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ala Tyr Ile Ser Ser Gly Ser Ser Thr Ile Tyr Tyr Ala Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Pro Lys Asn Thr Leu Phe

65 70 75 80

Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Asp Trp Val Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr

100 105 110

Val Ser Ser

115

<210> 6

<211> 339

<212> DNA

<213> artificial sequence

<400> 6

gatattgtga taacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60

gtctcttgca gatctagtca gagccttgta cacagtaatg gaaacaccta tttacattgg 120

tacctgcaga agccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180

tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaatatc 240

agcagagtgg aggctgagga tctgggagtt tatttctgct ctcaaagtac acatgttccg 300

ttcacgttcg gaggggggac caagctggag ctgaaacgg 339

<210> 7

<211> 113

<212> PRT

<213> artificial sequence

<400> 7

Asp Ile Val Ile Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Ser Val Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser

85 90 95

Thr His Val Pro Phe Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg

Claims

1. Hybridoma cell line 7B63B10 secreting anti-LMP 1 monoclonal antibody is preserved in China Center for Type Culture Collection (CCTCC) in the 7 th month 19 of 2019, and the preservation number is CCTCC NO: c2019160; the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO.5, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 7.

2. An anti-LMP 1 monoclonal antibody is characterized in that the amino acid sequence of a heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO.5, and the amino acid sequence of a light chain variable region of the monoclonal antibody is shown as SEQ ID NO. 7.

3. The monoclonal antibody according to claim 2, wherein the heavy chain variable region of the monoclonal antibody has a gene sequence shown in SEQ ID No.4 and the light chain variable region has a gene sequence shown in SEQ ID No. 6.

4. The monoclonal antibody of claim 2, wherein the Ig type of the monoclonal antibody is IgG2b.

5. The monoclonal antibody of any one of claims 2 to 4 is secreted from the hybridoma cell line 7B63B10 of claim 1.

6. A kit for detecting epstein barr virus LMP1, characterized in that the kit comprises the monoclonal antibody of any one of claims 2-4.

7. The use of the monoclonal antibody of any one of claims 2-4 in preparing reagent for detecting epstein barr virus LMP1.