CN111647564A

CN111647564A - Monoclonal antibody of anti-EB virus LMP1, cell strain and application thereof

Info

Publication number: CN111647564A
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: 2020-09-11
Anticipated expiration: 2040-05-18
Also published as: CN111647564B

Abstract

The invention discloses an anti-EB virus LMP1 monoclonal antibody and a cell strain and application thereof, the invention uses a sequence of SEQ ID NO.2 as an antigen and adopts cell fusion to obtain the cell strain, the cell strain is selected by three rounds of subclones and combined with indirect ELISA and FACS detection technology, hybridoma cell strains which can stably secrete positive antibodies are finally screened out, the hybridoma cell strains are expanded and cultured, and the cell supernatants are all single subtypes after subtype identification; the cells 7B63B10 with better FACS detection result were used to prepare ascites and purify 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 values.

Description

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

Technical Field

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

Background

Epstein-Barr virus (EBV) is a DNA virus, belongs to human herpes virus type 4 (HHV-4) and is the first virus directly associated with human tumors. Statistically, more than 90% of all people worldwide are infected with the virus and can be carried for life after primary infection, thereby causing various diseases: burkitt's Lymphoma (BL), nasopharyngeal carcinoma (NPC), Hodgkin's Lymphoma (HL), post-transplantation lymphoproliferative disease (PTLD), EBV-associated gastric cancer (EBVaGC), and the like.

LMP1(Laten membrane protein 1, LMP1, Latent membrane protein 1) is one of the key proteins responsible for B cell immortalization. It is an EBV encoded membrane protein, the molecular weight is 66KDa, and the EBV encoded membrane protein consists of 386 amino acids (aa), and comprises a hydrophilic cytoplasmic N end (1-23aa), 6 water delivery transmembrane regions (24-186aa) and a C end (187-386aa) consisting of 200 amino acids; the C-terminus contains three important C-terminal activating domains (CTARs): CTAR1, CTAR2, and CTAR 3. These regions provide anchoring sites for aptamer proteins, including tumor necrosis factor receptor-associated factors (TRAFs), tumor necrosis factor receptor-associated death domain proteins (TRADD), receptor interacting protein kinases (RIP), BS69, JAK-3 proteins, and the like, which mediate cell immortalization, inhibit cell differentiation and apoptosis, promote tumor metastasis, escape from host immune responses, and the like, by transducing signals via NF-. kappa. B, JNK/p38-SAPK, PI3-K/Akt, ERK-MAPK, and JAK/STAT pathways.

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

Disclosure of Invention

The primary object of the present invention is to provide a cell strain secreting monoclonal antibodies against LMP 1.

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

Still another object of the present invention is to provide the use of the above cell strain and monoclonal antibody.

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

the invention provides a hybridoma cell strain 7B63B10 secreting an anti-LMP 1 monoclonal antibody, which is preserved in the China center for type culture Collection in 2019, 7 and 19 months, Wuhan university, with the preservation number of CCTCC NO: C2019160.

in the second aspect of the invention, the invention provides an anti-LMP 1 monoclonal antibody, wherein the heavy chain variable region amino acid sequence of the monoclonal antibody is shown as SEQ ID NO.5, and the light chain variable region amino acid sequence 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 present invention, the monoclonal antibody has high specificity for specifically recognizing LMP 1.

According to an embodiment of the invention, the Ig-type of the monoclonal antibody is IgG2 b.

The monoclonal antibody is secreted and produced by the hybridoma cell line 7B63B 10.

In a third aspect of the invention, a kit for detecting the LMP1 of the EB virus is provided, and the kit contains the anti-LMP 1 monoclonal antibody.

The fourth aspect of the invention provides application of hybridoma cell strain 7B63B10 in preparation of an LMP1 monoclonal antibody. According to embodiments of the invention, the monoclonal antibody can be used to specifically recognize LMP 1; the heavy chain variable region of the monoclonal antibody has an amino acid sequence shown by SEQ ID NO.5 in a sequence table; the light chain variable region has an amino acid sequence shown as SEQ ID NO.7 in the sequence table.

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

In a sixth aspect of the invention, an epstein barr virus LMP1 detection system is provided, which comprises a sampling device and a detection device, wherein the detection system adopts flow cytometry and/or cytofluorescence immunoassay, and the flow cytometry and/or cytofluorescence immunoassay uses any one of the monoclonal antibodies.

In the seventh aspect of the invention, the application of the monoclonal antibody in preparing a reagent for detecting LMP1 is provided.

The eighth aspect of the invention provides a preparation method of a hybridoma cell strain secreting the LMP1 monoclonal antibody, which comprises the following steps:

according to LMP1 sequence information published by NCBI, carrying out Escherichia coli codon optimization and carrying out gene synthesis;

constructing an escherichia coli expression plasmid, transforming BL21 host bacteria, and testing small-amount expression;

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

immunizing Balb/c mice with recombinant protein expressed by antigen LMP1 Escherichia coli;

collecting splenocytes from the immunized mice, fusing with Sp2/0 myeloma cells, and selectively culturing the fused hybridoma cells in HAT medium;

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 immunizing comprises: the mice are female Balb/c mice with the age of 6-8 weeks, purified LMP1 recombinant protein and an isovolumetric Freund complete adjuvant are fully mixed and emulsified, then an antigen is emulsified by the Freund incomplete adjuvant and fully mixed to a water-in-oil state for subcutaneous multipoint immunization, and 2-3 times of boosting immunization.

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

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

According to an embodiment of the invention, the detecting comprises: liquid and semi-solid fusion plate assays.

According to an embodiment of the invention, the liquid fusion plate detection comprises: and (3) starting detection when the liquid changing cells of the fusion plate grow to more than 1 ten thousand cells with medium size, and selecting the positive hole for subcloning after the indirect ELISA quality control is qualified.

According to an embodiment of the invention, the semi-solid fusion plate assay comprises: after conventional cell fusion, resuspending the cell pellet in a methylcellulose semisolid culture medium, resuspending in 20ml of semisolid culture medium with 2% of methylcellulose concentration, and culturing in a cell culture dish; and (3) observing the size and the number of cell clones in the culture dish after 7-10 days, picking out the cells in an HT culture medium of 20% fetal calf serum under a body type microscope, culturing for 2-3 days, and performing titer detection to obtain subclones after the quality control of ELISA (enzyme-linked immunosorbent assay) is qualified.

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

In a ninth aspect of the present invention, there is provided a method for preparing 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 the pretreatment.

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

The invention has the beneficial effects that:

the hybridoma cell strain 7B63B10 prepared by the invention can stably secrete anti-LMP 1 monoclonal antibody. The cell strain for specifically recognizing the LMP1 monoclonal antibody adopts a semi-solid high-throughput fusion technology, obviously shortens the subcloning period, increases the stock base number of positive clones, and combines indirect ELISA and FACS detection technologies in the screening process to obtain the high-affinity monoclonal antibody capable of specifically recognizing LMP 1.

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

Drawings

FIG. 1 is an SDS-PAGE electrophoresis image after protein purification, wherein lanes 1-7 are LMP1 sample loading detection before purification, LMP1 column penetration detection after column hanging, Marker, LMP1 detection under NTA20 buffer elution, LMP1 detection under NTA60 buffer elution, LMP1 detection under NTA200 buffer elution, and LMP1 detection under NTA500 buffer elution, respectively.

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

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

FIG. 4 is a FACS flow cytometric assay of monoclonal antibodies of the invention.

FIG. 5 is an immunofluorescence of IF cells with monoclonal antibodies of the invention, A being MDA-MB-231 and B being Raji.

Detailed Description

The technical solution of the present invention is clearly and completely illustrated below with reference to the following examples, but is not limited thereto.

The hybridoma cell strain 7B63B10 secreting the anti-LMP 1 monoclonal antibody is preserved in the China Center for Type Culture Collection (CCTCC) in 2019, 7 months and 19 days, and the preservation number is CCTCC NO: C2019160.

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

The LMP1 amino acid sequence is as follows:

MERDLERGPPGPPRPPLGPPLSSSIGLALLLLLLALLFWLYIVMSNWTGGALLVLYSFALMLIIIILIIFIFRRDLLCPLGGLGLLLLMSKYYTLCPPPPFPYASFSNALSPLSPVTLLLIALWNLHGQALYLGIVLFIFGCLLVLGLWIYFLEILWRLGATIWQLLAFILAFFLAIILLIIALYLQQNWWTLLVDLLWLLLFMAILIWMYYHGPRHTDEHHHDDSLPHPQQATDDSSHESDSNSNDGRHHLLVSGAGDGPPLCSQNLGAPGGGPDNGPQDPDNTDDNGPQDPDNTDDNGPQDPDNTDDNGPQDPDNTDDNGPQDPDNTDDNGPHDPLPHNPSDSAGNDGGPPNLTEEVENKGGDRDPPSMTDGGGGDPHLPTLLLGTSGSGGDDDDPHGPVQLSYYD(SEQ ID NO.1)

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

The selected antigen sequences were as follows:

HGPRHTDEHHHDDSLPHPQQATDDSSHESDSNSNDGRHHLLVSGAGDGPPLCSQNLGAPGGGPDNGPQDPDNTDDNGPQDPDNTDDNGPQDPDNTDDNGPQDPDNTDDNGPQDPDNTDDNGPHDPLPHNPSDSAGNDGGPPNLTEEVENKGGDRDPPSMTDGGGGDPHLPTLLLGTSGSGGDDDDPHGPVQLSYYD(SEQ ID NO.2)

the nucleotide sequence corresponding to the selected antigen sequence:

CATGGTCCGCGTCATACCGATGAACATCATCACGATGATAGCCTGCCGCATCCGCAGCAGGCAACCGATGATAGTAGCCATGAAAGCGATAGCAATAGCAATGATGGTCGTCATCATCTGCTGGTTAGCGGTGCCGGTGATGGTCCGCCTCTGTGTAGCCAGAATCTGGGTGCACC TGGTGGTGGTCCGGATAATGGTCCGCAGGATCCGGATAACACAGATGATAATGGCCCTCAAGATCCTGACAATACCGACGATAACGGACCTCAGGACCCAGATAATACGGATGACAACGGTCCACAAGACCCTGATAACACTGACGACAATGGACCGCAAGACCCCGACAACACGGACGATAACGGTCCGCATGATCCTCTGCCGCATAATCCGAGCGATAGCGCAGGTAATGATGGTGGTCCTCCGAATCTGACCGAAGAGGTTGAAAATAAAGGTGGTGATCGTGATCCGCCTAGCATGACCGATGGTGGTGGCGGTGATCCGCATCTGCCGACACTGCTGCTGGGCACCAGCGGTAGCGGTGGTGATGACGATGATCCTCATGGTCCGGTTCAGCTGAGCTATTATGAT(SEQ IDNO.3)

preparation and purification of antigens

Artificially synthesizing gene segments according to the corresponding coding sequence of the antigen, connecting the gene segments into recombinant plasmids, constructing recombinant expression plasmids, carrying out protein expression, bacterium breaking detection and purification, and preparing the monoclonal antibody as the antigen.

As a result: FIG. 1 illustrates LMP1 protein eluted by different buffers, but LMP1 eluted by NTA60 and NTA200 buffers is the best effect in the invention and can be used for the subsequent preparation of monoclonal antibody.

Preparation of LMP1 monoclonal antibody

After a mouse is immunized by the antigen prepared by the method, lymphocytes in the spleen of the mouse are separated and fused with SP2/0 myeloma cells, the indirect ELISA method is used for detection, after the ELISA quality control is qualified (namely negative control is less than 0.2, and positive control is more than 1.0), a monoclonal antibody with a high positive value is selected for limited dilution, and the second time is repeated until a monoclonal cell strain capable of stably secreting the positive antibody is finally screened for amplification culture.

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

TABLE 1 first subcloning indirect ELISA results

TABLE 2 first subclone supernatant flow cytometry (FACS) results

TABLE 3 second subcloning indirect ELISA results

TABLE 4 second subclone supernatant flow cytometry (FACS) results

Serial number	Name of Gene	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

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

Cell line determination and subtype identification

And performing amplification culture on the cell strains which are screened in the subcloning stage and stably secrete the positive antibodies, collecting the supernatant for antigen detection, verifying the stability by adopting ELISA gradient dilution and Western blot, collecting the cells, performing amplification culture, collecting the supernatant again, and detecting the subtype of the antibodies in the supernatant.

As can be seen from Table 5, the subtype identification results show that all the hybridoma cells are single subtypes, the hybridoma cell line with the best detection result in the invention is 7B63B10, and ascites is prepared using the cell line, and the Ig type of the secreted monoclonal antibody is IgG 2B.

TABLE 5 Stable hybridoma cell line subtype determination

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

Monoclonal antibody heavy chain variable region gene sequence:

GAGGTGAAGCTGGTGGAATCTGGGGGAGGCTTCGTGCAGCCTGGAGGGTCCCGGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTTTGGAATGCACTGGGTTCGTCAGGCTCCAGAGAAGGGGCTGGAGTGGGTCGCATACATTAGTAGTGGCAGTAGTACCATCTACTATGCAGACACAGTGAAGGGCCGATTCACCATCTCCAGAGACAATCCCAAGAACACCCTGTTCCTGCAAATGACCAGTCTAAGGTCTGAGGACACGGCCATGTATTACTGTGCAAGAGACTGGGTTCTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA(SEQ ID NO.4)；

heavy chain variable region amino acid sequence of monoclonal antibody:

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 antibody is prepared in large quantities.

The monoclonal antibody of the present invention is prepared by a conventional in vivo induction method and purified. As a result, it was found that: as can be seen from FIG. 2, the purity of the monoclonal antibody LMP 17B 63B10 prepared by the present invention was as high as 90% or more at a concentration of about 10mg/ml as determined by SDS-PAGE purity detection.

The monoclonal antibody is subjected to an application test.

1. Monoclonal antibody affinity constant determination

1) Coating: coating according to the conventional coating concentration of 2ug/ml, coating a 96-well plate with 50 ul of the coating per well, and incubating at 37 ℃ for 2 hours or reacting at 4 ℃ overnight;

2) and (3) sealing: 2% BSA or 5% skim milk blocking solution 200 u l/hole, 37 ℃ temperature 1 h incubation or 4 ℃ reaction overnight, TBST washing 4 times;

3) a first 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 and 50 mu l/well, avoiding foaming as much as possible, uniformly adding, oscillating on a micro oscillator to enable liquid phases to be more uniformly mixed, and incubating for 1 hour at 37 ℃;

4) secondary antibody: washing the primary antibody for 4 times after incubation by TBST, adding an enzyme-labeled secondary antibody into the mixture, performing incubation for 1 hour at the temperature of 37 ℃ in a 100 mu l/hole manner; color development: washing the incubated secondary antibody for 4 times by TBST, adding 100 mu l/hole of the substrate solution, and placing the mixture in a constant temperature box at 37 ℃ for 5-10 min;

5) terminating reaction and carrying out color comparison: after adding 30. mu.l/well of the stop solution and turning yellow in color, the absorbance at 450nm was measured using a microplate reader.

A non-competitive ELISA method established by Beatty and statistical software SPSS15.0 are adopted, the concentration of mAb is taken as an X axis, the OD value is taken as a Y axis (table 6), and a combined reaction Curve model and an equation thereof are fitted through a Curve Estimation process in Curve regression analysis. And substituting the OD50 value into a curve equation to calculate the corresponding antibody concentration.

As a result: formula derived from Beatty, K ═ n-1/2 (n [ Ab ]]’T-[Ab]T), calculate affinity constant the affinity constant of the anti-LMP 1 monoclonal antibody calculated according to the power curve model, according to the formula derived from Beatty and fig. 3, is K9 × 10⁹M^-1The present invention pertains to high affinity antibodies.

TABLE 67 OD values of anti-LMP 1 monoclonal antibody prepared from B63B10 cell line

First antibody concentration (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 rates

1) The purified antibody 7B63B10 of the cell line 7B63B10 of the present invention was verified by flow cytometry using the EB virus positive cell line Akata-EBV. In these, the Akata-EBV cell line induced by goat anti-human IgG for 6 hours started to express EB virus in a large amount, and the expression level of LMP1 was increased.

2) 2 × 10 per tube of cells⁵Each was resuspended in 100ul FACS buffer (2% FBS in pre-chilled PBS), using a Hu FcR binding Inhibitor (eBioscience 14-9161-73) 20. mu.l/tube, blocked on ice for 20min, centrifuged at 250g for 4min and the supernatant discarded.

3) A first antibody: resuspending the cells with the supernatant in an amount of 500ul, ice-cooling for 30min, centrifuging for 4min at 250g, and discarding the supernatant; cells were resuspended using 1ml of FACS Buffer, washed once and repeated once.

4) After dilution of the secondary antibody (Abcam ab150119)1:1000 with FACS buffer, 200. mu.l of each tube were resuspended and protected from light on ice for 30 min. The cells were washed twice with FACS buffer (same procedure as above) and resuspended in 400. mu.l FACS buffer before detection on the machine.

As a result: as shown in FIG. 4, the antibody secreted by the cell line 7B63B10 of the present invention can be applied to flow cytometry, and the positive rate of flow cytometry is 85.4%, which meets the detection requirement.

3. Cellular immunofluorescence assay

1) The confocal dish (Nest 801002) was coated with Fibro Nectin (Corning 354008), at 0.05ug/ul100ul, overnight at 4 ℃.

2) On the next day, after the coating solution was aspirated, the cells were washed three times with PBS, and Raji cells were added until the cells adhered to the wall.

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

4) And (3) sealing: preparing sealing liquid: 9ml PBS, 1ml goat serum and 10ul Tween-20; aspirate 500 ul/dish for 60 min at room temperature. PBS was washed three times.

5) A first antibody: antibody dilution: diluting the antibody prepared by the invention to 2ug/ml and 500 ul/dish by using a diluent, wherein the diluent comprises 10ml of PBS, 0.1g of BSA and 10ul of Tween-20; a negative control (antibody dilution only) was also set, and PBS was washed three times after overnight at 4 ℃.

6) Secondary antibody: antibody dilutions 1:1000 dilution (secondary antibody Abcam ab150119), 1 hour at room temperature, PBS wash three times.

7) And (3) cell nucleus staining: DAPI dye (Solibao C0060) was diluted with PBS at a volume ratio of 1:100, 300 ul/dish, at room temperature for 10 minutes. PBS was washed three times.

8) After addition of the anti-quenching mounting solution (Sigma F4680), confocal detection was carried out.

As a result: the cells stained with the antibody of the present invention were observed to be fluorescent, and as shown in FIG. 5, the EBV positive cells Raji could be bound by the antibody, while the EBV negative cells MDA-MB-231 showed negative results. It is demonstrated that the antibodies prepared by the present invention can recognize LMP 1.

In summary, (1) the hybridoma cell strain 7B63B10 of the invention can produce an anti-LMP 1 monoclonal antibody, the antibody has high specificity, is used for specifically recognizing LMP1, and has no detectable cross reactivity with LMP1 family protein or other high homology protein. In immunoassays for determining the presence or amount of LMP1 in a test sample, erroneous results are avoided.

(2) The monoclonal antibody against LMP1 generated by the hybridoma cell strain 7B63B10 prepared by the invention has high affinity, the common monoclonal antibody is only in the 8 th power level of 10, and the monoclonal antibody against LMP1 has the anti-affinity constant of K ═ 9 × 10⁹M^-1. Belong to high affinity antibodies.

(3) When hybridoma cell strains which stably secrete monoclonal antibodies are obtained through screening, two methods of liquid and semisolid fusion plate detection are combined for screening, so that the fusion cloning quantity is improved to the maximum probability; compared with the conventional screening mode which only uses one method, the capacity of the obtained antibody library is larger.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

SEQUENCE LISTING

<110> Lixin, Chongtuo Tuo, Longyufei

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

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catggtccgc gtcataccga tgaacatcat cacgatgata gcctgccgca tccgcagcag 60

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 9095

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 LeuGln 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. A hybridoma cell strain 7B63B10 secreting anti-LMP 1 monoclonal antibody is preserved in China center for type culture Collection in 2019, 7 months and 19 days, and the preservation number is CCTCC NO: C2019160.

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

3. The monoclonal antibody according to claim 2, wherein the gene sequence of the heavy chain variable region of the monoclonal antibody is represented by SEQ ID No.4, and the gene sequence of the light chain variable region is represented by SEQ ID No. 6.

4. The monoclonal antibody of claim 2, wherein the Ig-type of the monoclonal antibody is IgG2 b.

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

6. A kit for detecting EB virus LMP1, wherein the kit contains the monoclonal antibody of any one of claims 2-4.

7. An EB virus LMP1 detection system, which is characterized by comprising a sampling device and a detection device, wherein the detection system adopts flow cytometry and/or a cell fluorescence immunoassay method, and the flow cytometry and/or the cell fluorescence immunoassay method uses the monoclonal antibody of any one of claims 2-4.

8. Use of the monoclonal antibody of any one of claims 2-4 in the preparation of a reagent for detecting EB virus LMP 1.