CN111647564B - Anti-Epstein-Barr virus LMP1 monoclonal antibody and its cell line and application - Google Patents

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-Epstein-Barr virus LMP1 monoclonal antibody and its cell line and application

technical field

The invention relates to the field of biotechnology, in particular to a monoclonal antibody against Epstein-Barr virus LMP1, cell lines and applications thereof.

Background technique

Epstein-Barr virus (EBV) is a DNA virus that belongs to human herpesvirus type 4 (HHV-4), and is the first virus directly associated with human tumors. According to statistics, more than 90% of human beings in the world have been infected with this virus, which can be carried for life after primary infection, resulting in a variety of diseases: Burkitt lymphoma (BL), nasopharyngeal carcinoma (NPC), Hodgkin lymphoma EBV-associated gastric cancer (EBVaGC) and post-transplantation lymphoproliferative disease (PTLD).

LMP1 (Latent membrane protein 1, LMP1, latent membrane protein 1) is one of the key proteins leading to the immortalization of B cells. It is a membrane protein encoded by EBV, with a molecular weight of 66KDa, consisting of 386 amino acids (aa), including a hydrophilic cytoplasmic N-terminal (1-23aa), 6 transmembrane domains for water transport (24-186aa) and The C-terminal (187-386aa) consists of 200 amino acids; the C-terminal contains three important C-terminal activating regions (CTARs): CTAR1, CTAR2 and CTAR3. These regions provide anchor sites for aptamer proteins, including tumor necrosis factor receptor-associated factors (TNFR-associated factors, TRAFs), tumor necrosis factor receptor-associated death domain proteins (TNFR-associated death domain, TRADD), Receptor interacting protein kinase (RIP), BS69, JAK-3 protein, etc. transduce signals through NF-κB, JNK/p38-SAPK, PI3-K/Akt, ERK-MAPK and JAK/STAT pathways , mediated cell immortalization, inhibited cell differentiation and apoptosis, promoted tumor metastasis, evaded host immune response, etc.

Therefore, finding a cell line that can stably secrete and specifically bind anti-LMP1 monoclonal antibody has become an urgent problem to be solved, which is of great significance for the deep molecular mechanism of Epstein-Barr virus LMP1.

Contents of the invention

The primary purpose of the present invention is to provide a cell line secreting anti-LMP1 monoclonal antibody.

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

Another object of the present invention is to provide the application of the above cell lines and monoclonal antibodies.

For achieving the above object, the technical scheme that the present invention takes is:

In the first aspect of the present invention, a hybridoma cell line 7B63B10 secreting anti-LMP1 monoclonal antibody is proposed, which was deposited in the China Center for Type Culture Collection, Wuhan, China, Wuhan University on July 19, 2019, with the deposit number CCTCC NO: C2019160.

In the second aspect of the present invention, an anti-LMP1 monoclonal antibody is proposed. The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.5, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. .7 shown.

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

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

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

The aforementioned monoclonal antibody is secreted and produced by the aforementioned hybridoma cell line 7B63B10.

The third aspect of the present invention proposes a kit for detecting LMP1 of Epstein-Barr virus, which contains the aforementioned anti-LMP1 monoclonal antibody.

The fourth aspect of the present invention proposes the application of hybridoma cell line 7B63B10 in the preparation of anti-LMP1 monoclonal antibody. According to an embodiment of the present invention, the monoclonal antibody can be used to specifically recognize LMP1; the heavy chain variable region of the monoclonal antibody has the amino acid sequence shown in SEQ ID NO.5 in the sequence listing; the light chain variable region It has the amino acid sequence shown in SEQID NO.7 in the sequence listing.

In the fifth aspect of the present invention, a kit for detecting Epstein-Barr virus LMP1 is proposed, the kit contains any one of the aforementioned monoclonal antibodies.

A sixth aspect of the present invention provides a detection system for Epstein-Barr virus LMP1, including a sampling device and a detection device, the detection system adopts flow cytometry and/or cytofluorescence immunoassay, and the flow cytometry and/or Or cell fluorescence immunoassay using any one of the aforementioned monoclonal antibodies.

The seventh aspect of the present invention proposes the use of monoclonal antibodies in the preparation of reagents for detecting LMP1.

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

According to the LMP1 sequence information published by NCBI, the codon optimization of Escherichia coli was carried out, and the gene synthesis was carried out;

Construct the expression plasmid of Escherichia coli, and transform it into BL21 host bacteria, and carry out small-scale expression test;

Enlarge the culture volume of E. coli, collect the supernatant or precipitate for the purification and detection of the target protein;

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

Spleen cells from immunized mice were collected and fused with Sp2/0 myeloma cells, and the fused hybridoma cells were selectively cultured with HAT medium;

The antibody content in the hybridoma cell culture supernatant was detected by indirect ELISA method, and the hybridoma cell line stably secreting monoclonal antibody was screened.

According to an embodiment of the present invention, the immunization includes: the mouse is a 6-8 week-old female Balb/c mouse, thoroughly mixing and emulsifying the purified LMP1 recombinant protein with an equal volume of Freund's complete adjuvant, and then using Incomplete Freund's adjuvant emulsified antigen, fully mixed to the water-in-oil state for subcutaneous multi-point immunization, 2-3 booster immunizations.

According to an embodiment of the present invention, the fusion includes: taking the mouse spleen under aseptic conditions to make a cell suspension, under the mediation of PEG1450, fusing the mouse myeloma SP2/0 cells and splenocytes, adding DMEM to culture after fusion base terminated.

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

According to an embodiment of the present invention, the detection includes: liquid and semi-solid fusion plate detection.

According to an embodiment of the present invention, the detection of the liquid fusion plate includes: the medium-changed cells on the fusion plate grow to a medium size of about 10,000 cells and start detection, and the positive wells are selected as subclones after the quality control of the indirect method ELISA is qualified.

According to an embodiment of the present invention, the detection of the semi-solid fusion plate includes: after conventional cell fusion, resuspend the cell pellet with methylcellulose semi-solid medium, resuspend in 20ml of semi-solid medium with 2% methylcellulose concentration 7-10 days later, observe the size and number of cell clones in the culture dish, pick them out under a stereomicroscope and culture them in HT medium with 20% fetal bovine serum for 2-3 days before titer detection , used as subclones in indirect ELISA quality control.

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

In the ninth aspect of the present invention, a method for preparing an anti-LMP1 monoclonal antibody is proposed, comprising the steps of: injecting the aforementioned hybridoma cells into BALB/c mice previously treated with Freund's incomplete adjuvant, and collecting the The ascites of the above mice was purified after pretreatment.

According to an embodiment of the present invention, the purification includes: selecting a Protein G-Sepharose affinity chromatography column for purification.

The beneficial effects of the present invention are:

The invention prepares a hybridoma cell line 7B63B10, which can stably secrete anti-LMP1 monoclonal antibody. The invention specifically recognizes the cell line of LMP1 monoclonal antibody, adopts semi-solid high-throughput fusion technology, in addition to significantly shortening the subcloning cycle, but also increases the reserve base of positive clones, and combines indirect ELISA and FACS detection technology during the screening process, Obtain a high-affinity monoclonal antibody that specifically recognizes LMP1.

The invention uses the anti-LMP1 monoclonal antibody secreted by the hybridoma cell line 7B63B10 as the detection antibody, and the established indirect ELISA method has good detection specificity and sensitivity, indicating that the indirect ELISA kit of the present invention has good popularization and application value.

Description of drawings

Figure 1 is the SDS-PAGE electrophoresis image after protein purification, in which lanes 1 to 7 are sample loading detection before LMP1 purification, penetration detection after LMP1 hanging on the column, Marker, LMP1 detection under NTA20 buffer elution, and NTA60 buffer elution LMP1 detection under NTA200 buffer elution, LMP1 detection under NTA500 buffer elution.

Figure 2 is the SDS-PAGE electrophoresis image after antibody purification, wherein lane 1 is Marker, and lane 2 is the monoclonal antibody purified by LMP1.

Figure 3 is a power curve of the affinity constant of anti-LMP1 monoclonal antibody.

Fig. 4 is a FACS flow cytometric detection graph of the monoclonal antibody of the present invention.

Figure 5 is the IF cell immunofluorescence image of the monoclonal antibody of the present invention, A is MDA-MB-231, B is Raji.

Detailed ways

The technical solutions in the present invention are clearly and completely described below in conjunction with the embodiments, but are not limited thereto.

The hybridoma cell line 7B63B10 secreting anti-LMP1 monoclonal antibody of the present invention was deposited in China Center for Type Culture Collection, Wuhan, China, Wuhan University on July 19, 2019, and the preservation number is CCTCC NO: C2019160.

Example 1 Sequence Analysis of LMP1 Gene and Selection of Antigen Sequence

The amino acid sequence of LMP1 is as follows:

MERDLERGPPGPPRPPLGPPLSSSIGLALLLLLLALLFWLYIVMSNWTGGALLVLYSFALMLIIIILIIFIFRRDLLCPLGGLGLLLMSKYYTLCPPPPFPYASFSNALSPLSPVTLLLIALWNLHGQALYLGIVLFIFGCLLVLGLWIYFLEILWRLGATIWQLLAFILAFLAIILLIIALYLQQNWWTLLVDLL WLLLFMAILIWMYYHGPRHTDEHHHDDSLPPHQQATDDSSHESDSNSNDGRHHLLVSGAGDGPPLCSQNLGAPGGGPDNGPQDPDNTDDNGPQDPDNTDDNGPQDPDNTDDNGPQDPDNTDDNGPQDPDNTDDNGPHDPLPHNPSDSAGNDGGPPNLTEEVENKGGDRDPPSMTDGGGGDPHLPTLLLLGTSGSGGDDDDPHGPVQ LSYYD (SEQ ID NO.1)

The LMP1 gene encodes 408 amino acids, 6 transmembrane regions, and is hydrophilic. After 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 screened out and used as the antigen sequence to express in Escherichia coli to prepare the antigen.

The selected antigen sequences are as follows:

HGPRHTDEHHHDDSLPHPQQATDDSSHESDSNSNDGRHHLLVSGAGDGPPLCSQNLGAPGGGPDNGPQDPDNTDDNGPQDPDNTDDNGPQDPDNTDDNGPQDPDNTDDNGPQDPDNTDDNGPHDPLPHNPSDSAGNDGGPPNLTEEVENKGGDRDPPSMTDGGGGDPHLPTLLGTSGSGGDDDDPHGPVQLSYYD(SEQ ID NO.2)

The nucleotide sequence corresponding to the selected antigen sequence:

CATGGTCCGCGTCATACCGATGAACATCATCACGATGATAGCCTGCCGCATCCGCAGCAGGCAACCGATGATAGTAGCCATGAAAGCGATAGCAATAGCAATGATGGTCGTCATCATCTGCTGGTTAGCGGTGCCGGTGATGGTCCGCCTCTGTGTAGCCAGAATCTGGGTGCACC TGGTGGTGGTCCGGATAATGGTCCGCAGGATCCGGATA ACACAGATGATAATGGCCCTCAAGATCCTGACAATACCGACGATAACGGACCTCAGGACCCAGATAATACGGATGACAACGGTCCACAAAGACCCTGATAACACTGACGACAATGGACCGCAAGACCCCGACAACACGGACGATAACGGTCCGCATGATCCTCTGCCGCATAATCCGAGCGATAGCGCAGGTAATGATGGTGGTCCTCCGAATCTGAAGAGGT TGAAAATAAAGGTGGTGATCGTGATCCGCCTAGCATGACCGATGGTGGTGGCGGTGATCCGCATCTGCCGACACTGCTGCTGGGCACCAGCGGTAGCGGTGGTGATGACGATGATCCTCATGGTCCGGTTCAGCTGAGCTATTATGAT (SEQ ID NO. 3)

Antigen preparation and purification

According to the coding sequence corresponding to the antigen, the gene fragment is artificially synthesized, connected into the recombinant plasmid, and the recombinant expression plasmid is constructed, and the protein expression, bacteriostasis detection and purification are carried out, and the monoclonal antibody is prepared as the antigen.

Results: Figure 1 illustrates the LMP1 protein eluted with different buffers, but the present invention uses the LMP1 eluted with NTA60 and NTA200 buffers, which has the best effect and can be used for subsequent monoclonal antibody preparation.

Preparation of LMP1 monoclonal antibody

After immunizing the mice with the antigen prepared by the present invention, the lymphocytes in the spleen of the isolated mice were fused with SP2/0 myeloma cells, detected by indirect ELISA method, and the ELISA quality control was qualified (ie negative control<0.2, positive control>1.0) Finally, the monoclonal with high positive value was selected for limiting dilution, and repeated for the second time, until finally a monoclonal cell line capable of stably secreting positive antibodies was selected for expanded culture.

Results: Based on Table 1 to Table 4, after fusion detection, 108 positive clones with OD450nm between 1.0 and 2.0 were screened by indirect ELISA, and some clones were selected for FACS detection. Finally, 14 strains with the best results were selected for the first test. Subcloning, 12 strains were selected for the first subcloning, and finally 9 strains of hybridoma cell lines were screened out; and then a cell line with the highest affinity, the best FCS flow cytometry results, and stable positive antibody secretion was selected , for subsequent ascites preparation and antibody purification experiments.

Table 1 Indirect ELISA results of the first subcloning

Table 2 The results of flow cytometry (FACS) on the supernatant of the first subcloning

Table 3 Second subcloning indirect ELISA results

Table 4 Second subcloning supernatant flow cytometry (FACS) results

serial number gene name clone 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

Based on Table 1 to Table 4, after fusion detection, 108 positive clones with OD450nm between 1.0 and 2.0 were screened by indirect ELISA, and some clones were selected for FACS detection, and finally 14 strains with the best results were selected for the first subcloning 12 strains were selected for the first subcloning, and finally 9 strains of fixed hybridoma cell strains were screened out; and then a cell strain with the highest affinity, the best FCS flow cytometry results, and stable positive antibody secretion was selected for Subsequent ascites preparation and antibody purification experiments.

Cell line determination and subtype identification

The cell lines that stably secrete positive antibodies screened out in the subcloning stage were expanded and cultured to collect the supernatant for antigen detection, and their stability was verified by ELISA serial dilution and Western blot, the cells were collected and expanded for culture, and the supernatant was collected again to detect the antibodies subtype.

As can be seen in Table 5, the subtype identification results show that they are all of a single subtype, and the hybridoma cell line with the best detection result in the present invention is 7B63B10, and the cell line is used to prepare ascites, and the Ig type of the monoclonal antibody secreted by it is IgG2b.

Table 5 Determination of Stable Hybridoma Cell Line Subtype

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

Monoclonal antibody heavy chain variable region gene sequence:

GAGGTGAAGCTGGTGGAATCTGGGGGAGGCTTCGTGCAGCCTGGAGGGTCCCGGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTTTGGAATGCACTGGGTTCGTCAGGCTCCAGAGAAGGGGCTGGAGTGGGTCGCATACATTAGTAGTGGCAGTAGTACCATCTACTATGCAGACACAGTGAAGGGCCGATTCACC TCTCCAGAGACAATCCCAAGAACACCCTGTTCCTGCAAATGACCAGTCTAAGGTCTGAGGACACGGCCATGTATTACTGTGCAAGAGACTGGGTTCTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO. 4);

Amino acid sequence of heavy chain variable region of monoclonal antibody:

EVKLVESGGGFVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEKGLEWVAYISSGSSTI YYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCARDWVLDYWGQGTTLTVSS (SEQ ID NO. 5);

Monoclonal antibody light chain variable region gene sequence:

GATATTGTGATAACCCAAACTCCACTCTCCCTGCCTGTCAGCCTTGGAGATCAAGCCTCCGTCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACAT TGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGA TTTCACACTCAATATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTTATTTCTGCTCTCAAAGTACACATGTTCCGTTCACGTTCGGAGGGGGGACCAAGCTGGAGCTGAAACGG (SEQ ID NO. 6);

Monoclonal Antibody Light Chain Variable Region Amino Acid Sequence:

DIVITQTPLSLPVSLGDQASVSCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLNISRVEAEDLGVYFCSQSTHVPFTFGGGTKLEIKR (SEQ ID NO. 7).

Large quantities of the above-mentioned monoclonal antibodies were prepared.

The monoclonal antibody of the present invention is prepared by conventional in vivo induction method and purified. The results found that: Figure 2 shows that the SDS-PAGE purity test results show that the concentration of the monoclonal antibody LMP1 7B63B10 prepared by the present invention is about 10 mg/ml, and the purity is as high as 90%.

The above-mentioned monoclonal antibodies were subjected to application tests.

1. Determination of Monoclonal Antibody Affinity Constant

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

2) Blocking: 200 μl/well of 2% BSA or 5% skimmed milk blocking solution, incubate at 37°C for 1 hour or react overnight at 4°C, wash 4 times with TBST;

3) Primary antibody: Dilute the purified antibody according to a certain ratio and add it to a 96-well plate. At the same time, add the corresponding volume of PBS, 50 μl/well. Try not to make foam, and add it evenly. Mix the phases more evenly and incubate at 37°C for 1 hour;

4) Secondary antibody: wash 4 times with TBST after primary antibody incubation, add enzyme-labeled secondary antibody, 100 μl/well, incubate at 37°C for 1 hour; color development: wash 4 times with TBST after secondary antibody incubation, add substrate solution 100 μl/well , placed in a 37°C incubator for 5 to 10 minutes;

5) Stop reaction, colorimetry: add 30 μl/well stop solution, after the color turns yellow, use a microplate reader to measure the absorbance at 450 nm.

Adopt the non-competitive ELISA method established by Beatty, SPSS15.0 statistical software, take mAb concentration as X-axis, OD value is Y-axis (Table 6), through the Curve Estimation process in the curve regression analysis, fit the binding response curve model and its equation. Substitute the OD50 value into the curve equation to calculate the corresponding antibody concentration.

Result: the formula derived by Beatty: K=(n-1)/2(n[Ab]'T-[Ab]T), calculate the affinity constant. According to the formula derived by Beatty and Figure 3, the affinity constant of the anti-LMP1 monoclonal antibody calculated according to the power curve model is K=9×10 ⁹ M ^-1 , and the present invention belongs to high affinity antibodies.

Table 6 OD value of anti-LMP1 monoclonal antibody prepared by 7B63B10 cell line

Primary 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

2. FACS flow cytometry positive rate

1) Use the Epstein-Barr virus positive cell line Akata-EBV to conduct flow cytometry experiments to verify the antibody 7B63B10 purified from the cell line 7B63B10 of the present invention. Among them, the Akata-EBV cell line was induced with goat anti-human IgG for 6 hours and began to express Epstein-Barr virus in large quantities, which increased the expression of LMP1.

2) Use 2× ¹⁰⁵ cells per tube to resuspend in 100ul FACS buffer (pre-cooled PBS containing 2% FBS), use Hu FcR binding Inhibitor (eBioscience 14-9161-73) 20μl/tube, block on ice for 20min , centrifuged at 250g for 4min and discarded the supernatant.

3) Primary antibody: use 500ul supernatant to resuspend cells, place on ice for 30min, centrifuge at 250g for 4min, discard supernatant; use 1ml FACS Buffer to resuspend cells, wash once and repeat once.

4) After diluting the secondary antibody (Abcam ab150119) 1:1000 with FACS buffer, resuspend the cells in 200 μl per tube, and keep it on ice for 30 minutes in the dark. Wash twice with FACS buffer (same as above), resuspend with 400 μl FACS buffer, and test on the machine.

Results: As shown in Figure 4, the antibody secreted by the cell line 7B63B10 of the present invention can be applied to flow cytometry detection, and the positive rate of flow cytometry detection is 85.4%, meeting the detection requirements.

3. Immunofluorescence detection of cells

1) Coat the confocal dish (Nest 801002) with Fibro Nectin (Corning 354008), 0.05ug/ul100ul, overnight at 4°C.

2) On the second day, after absorbing the coating solution, wash with PBS three times, add Raji cells, and wait for the cells to adhere to the wall.

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

4) Blocking: Prepare blocking solution: 9ml PBS, 1ml goat serum and 10ul Tween-20; pipette 500ul/dish, room temperature for 60 minutes. Wash three times with PBS.

5) Primary antibody: Antibody diluent: 10mlPBS, 0.1g BSA and 10ul Tween-20, dilute the antibody prepared by the present invention to 2ug/ml, 500ul/dish with diluent; set up negative control at the same time (only add antibody diluent) , washed three times with PBS after overnight at 4°C.

6) Secondary antibody: Antibody diluent diluted 1:1000 (secondary antibody Abcam ab150119), room temperature for 1 hour, washed three times with PBS.

7) Nucleus staining: dilute DAPI dye (Soleibo C0060) with PBS at a volume ratio of 1:100, 300ul/dish, at room temperature for 10 minutes. Wash three times with PBS.

8) After adding anti-quenching mounting solution (Sigma F4680), confocal detection.

Results: Fluorescence was observed in the cells stained with the antibody of the present invention, as shown in Figure 5, the EBV-positive cell Raji could be bound by the antibody, while the EBV-negative cell MDA-MB-231 showed a negative result. It is proved that the antibody prepared by the present invention can recognize LMP1.

In summary, (1) the hybridoma cell line 7B63B10 of the present invention can produce anti-LMP1 monoclonal antibodies, which are highly specific and used to specifically recognize LMP1, and do not have high homology with LMP1 family proteins or other Detectable cross-reactivity of sex proteins. In the immunoassay used to determine the presence or amount of LMP1 in a test sample, erroneous results are avoided.

(2) The anti-LMP1 monoclonal antibody produced by the hybridoma cell line 7B63B10 prepared by the present invention has a high affinity, and the common monoclonal antibody is only at the 8th power level of 10, while the anti-affinity constant of the anti-LMP1 monoclonal antibody of the present invention is K =9×10 ⁹ M ^-1 . It is a high-affinity antibody.

(3) When the present invention screens and obtains the hybridoma cell line that stably secretes the monoclonal antibody, adopts liquid and semi-solid fusion plate to detect two kinds of methods to carry out screening comprehensively, has improved fusion clone amount most likely; A method of screening, the capacity of the obtained antibody library is larger.

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.

SEQUENCE LISTING

<110> Li Xin, Chong Tuotuo, Long Yufei

<120> Anti-Epstein-Barr virus LMP1 monoclonal antibody and its cell line and application

<130>

<160> 7

<170> PatentIn version 3.5

<210> 1

<211> 408

<212> PRT

<213> Artificial sequence

<400> 1

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

1 5 10 15

Leu Gly Pro Pro Leu Ser Ser Ser Ser Ile Gly Leu Ala Leu Leu Leu Leu

20 25 30

Leu Leu Ala Leu Leu Phe Trp Leu Tyr Ile Val Met Ser Asn Trp Thr

35 40 45

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

50 55 60

Ile Ile Leu Ile Ile Phe Ile Phe Arg Arg Asp Leu Leu Cys Pro Leu

65 70 75 80

Gly Gly Leu Gly Leu Leu Leu Leu Met Ser Lys Tyr Tyr Thr Leu Cys

85 90 95

Pro Pro Pro Pro Phe Pro Tyr Ala Ser Phe Ser Asn Ala Leu Ser Pro

100 105 110

Leu Ser Pro Val Thr Leu Leu Leu Ile Ala Leu Trp Asn Leu His Gly

115 120 125

Gln Ala Leu Tyr Leu Gly Ile Val Leu Phe Ile Phe Gly Cys Leu Leu

130 135 140

Val Leu Gly Leu Trp Ile Tyr Phe Leu Glu Ile Leu Trp Arg Leu Gly

145 150 155 160

Ala Thr Ile Trp Gln Leu Leu Ala Phe Ile Leu Ala Phe Phe Leu Ala

165 170 175

Ile Ile Leu Leu Ile Ile Ala Leu Tyr Leu Gln Gln Asn Trp Trp Thr

180 185 190

Leu Leu Val Asp Leu Leu Trp Leu Leu Leu Phe Met Ala Ile Leu Ile

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

Gly Gly Pro Asp Asn Gly Pro Gln Asp Pro Asp Asn Thr Asp Asp Asn

275 280 285

Gly Pro Gln Asp Pro Asp Asp Asn Thr Asp Asp Asn Gly Pro Gln Asp Pro

290 295 300

Asp Asn Thr Asp Asp Asn Gly Pro Gln Asp Pro Asp Asn Thr Asp Asp

305 310 315 320

Asn Gly Pro Gln Asp Pro Asp Asn Thr Asp Asp Asn Gly Pro His Asp

325 330 335

Pro Leu Pro His Asn Pro Ser Asp Ser Ala Gly Asn Asp Gly Gly Pro

340 345 350

Pro Asn Leu Thr Glu Glu Val Glu Asn Lys Gly Gly Asp Arg Asp Pro

355 360 365

Pro Ser Met Thr Asp Gly Gly Gly Gly Asp Pro His Leu Pro Thr Leu

370 375 380

Leu Leu Gly Thr Ser Gly Ser Gly Gly Asp Asp Asp Asp Pro His Gly

385 390 395 400

Pro Val Gln Leu Ser Tyr Tyr Asp

405

<210> 2

<211> 196

<212> PRT

<213> Artificial sequence

<400> 2

His Gly Pro Arg His Thr Asp Glu His His His Asp Asp Ser Leu Pro

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Asn Gly Pro Gln Asp Pro Asp Asn Thr Asp Asp Asn Gly Pro Gln Asp

65 70 75 80

Pro Asp Asn Thr Asp Asp Asn Gly Pro Gln Asp Pro Asp Asn Thr Asp

85 90 95

Asp Asn Gly Pro Gln Asp Pro Asp Asn Thr Asp Asp Asn Gly Pro Gln

100 105 110

Asp Pro Asp Asn Thr Asp Asp Asn Gly Pro His Asp Pro Leu Pro His

115 120 125

Asn Pro Ser Asp Ser Ala Gly Asn Asp Gly Gly Pro Pro Asn Leu Thr

130 135 140

Glu Glu Val Glu Asn Lys Gly Gly Asp Arg Asp Pro Pro Ser Met Thr

145 150 155 160

Asp Gly Gly Gly Gly Asp Pro His Leu Pro Thr Leu Leu Leu Leu Gly Thr

165 170 175

Ser Gly Ser Gly Gly Asp Asp Asp Asp Pro His Gly Pro Val Gln Leu

180 185 190

Ser Tyr Tyr Asp

195

<210> 3

<211> 588

<212>DNA

<213> Artificial sequence

<400> 3

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 attackgtgc 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 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 (7)

1. A hybridoma cell line 7B63B10 secreting an anti-LMP1 monoclonal antibody, which was preserved in the China Center for Type Culture Collection on July 19, 2019, with a preservation number of CCTCC NO: C2019160; the heavy chain of the monoclonal antibody can be The amino acid sequence of the variable region is shown in SEQ ID NO.5, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.7. 2. An anti-LMP1 monoclonal antibody, characterized in that, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is as shown in SEQ ID NO.5, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.7 Show. 3. The monoclonal antibody according to claim 2, wherein the gene sequence of the heavy chain variable region of the monoclonal antibody is as shown in SEQ ID NO.4, and the gene sequence of the light chain variable region is as shown in SEQ ID NO.4. ID NO.6 shown. 4. The monoclonal antibody according to claim 2, wherein the Ig type of the monoclonal antibody is IgG2b. 5. The monoclonal antibody according to any one of claims 2 to 4 is secreted by the hybridoma cell line 7B63B10 according to claim 1. 6. A kit for detecting Epstein-Barr virus LMP1, characterized in that the kit contains the monoclonal antibody according to any one of claims 2-4. 7. The application of the monoclonal antibody described in any one of claims 2 to 4 in the preparation and detection of Epstein-Barr virus LMP1 reagent.

Images