CN117551621B

CN117551621B - Hybridoma cell strain L008, monoclonal antibody and application thereof

Info

Publication number: CN117551621B
Application number: CN202311545626.6A
Authority: CN
Inventors: 侯经远; 邓巧亭; 钟志雄; 钟炜; 高慧敏
Original assignee: Meizhou Peoples Hospital
Current assignee: Meizhou Peoples Hospital
Priority date: 2023-11-20
Filing date: 2023-11-20
Publication date: 2024-04-30
Anticipated expiration: 2043-11-20
Also published as: CN117551621A

Abstract

The invention belongs to the technical field of biology, and particularly relates to a hybridoma cell strain L008, a monoclonal antibody and application thereof. The hybridoma cell strain provided by the invention is classified and named L008, is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) on the 26 th month of 2023, and has a preservation address of number 3 of West Song No. 1 of North Star in the Korean region of Beijing city and a preservation number of CGMCC No.45723. The invention also provides a fetuin B monoclonal antibody secreted by the antibody, polynucleotide for encoding the antibody, and a kit containing the hybridoma cell strain or the fetuin B monoclonal antibody or the polynucleotide. The antibody of the invention can be well specifically combined with natural fetuin B and recombinant fetuin B antigen in blood plasma or tissues, can be used for preparing kits for detecting the fetuin B or other immunological detection products, or preparing tumor diagnostic reagents and tumor treatment medicines, and has practical application value.

Description

Hybridoma cell strain L008, monoclonal antibody and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a hybridoma cell strain L008, a monoclonal antibody and application thereof.

Background

Fetuin B is one of the members of the cysteine protease inhibitor superfamily, and was first discovered by Olivier et al through the study of localization of rat liver inflammatory factor mRNA. The study showed that fetuin B is an acidic glycoprotein consisting of 382 amino acids with a relative molecular weight of about 60kD, synthesized mainly by the liver and secreted into the blood. Fetuin B contains 1623 nucleotides in the human coding cDNA sequence, localizes to human chromosome 3 (3q27.3), and consists of 8 exons where the genetic locus is thought to be closely related to the development and progression of tumors, suggesting a potentially important role in tumor disease.

Studies have shown that fetuin B molecules can be abnormally expressed in different types of tumor tissue, including ovarian cancer, breast cancer, skin cancer, kidney cancer, liver cancer, lung cancer, esophageal adenocarcinoma, cholangiocarcinoma, colorectal cancer, prostate cancer, melanoma, and the like. Researchers have used genistein-treated female rats, and found that the treated rats had a 67% increase in fetuin B content in the mammary glands of the 50 th day mammary specimen, suggesting a potential role in inhibiting breast cancer. Researchers have used a model of nude mouse skin cancer to find that fetuin B appears to be overexpressed and has an inhibitory effect on cutaneous squamous cell carcinoma, the mechanism of which may be associated with anti-angiogenesis. It has also been reported that the mRNA level of fetuin B in hepatocellular carcinoma of hepatitis virus-infected mice is reduced by at least 50 times compared with the peripheral liver tissue, whereas the mRNA expression level of fetuin B in hepatocellular carcinoma is reduced by at least 10 times compared with the normal liver tissue in patients with liver cancer caused by hepatitis B virus infection. Researchers have found that fetuin B can clearly distinguish between lung adenocarcinoma in smokers and non-smokers. In addition, there are studies reporting upregulation of fetuin B expression in hamster models of cholangiocarcinoma. It has been reported that the expression level of fetuin B in serum of patients with esophageal adenocarcinoma is 2.41 times that of healthy controls. Thus, the fetuin B is expected to become a new target for researching the tumorigenesis mechanism and developing relevant diagnostic reagents.

Therefore, there is a need in the art to develop monoclonal antibodies that specifically recognize and bind to fetuin B, and that can be practically used in enzyme-linked immunosorbent assay (ELISA), western Blot, cellular immunofluorescence, immunohistochemistry, etc., and that will help to the localization of fetuin B, tissue expression information, cell function research, and research on the molecular mechanism of fetuin B in tumor diseases, while providing a key antibody raw material for the development of various immunodetection reagents of fetuin B, and that has a wide practical application value.

At present, monoclonal antibodies against fetuin B have not been reported.

Disclosure of Invention

In order to overcome the above-mentioned drawbacks and disadvantages of the prior art, a primary object of the present invention is to provide a hybridoma cell line L008.

The invention also aims to provide the fetuin B monoclonal antibody secreted by the hybridoma cell line L008 or a passage cell line thereof. The fetuin B monoclonal antibody can be specifically combined with fetuin B, and can be used as an immunodetection reagent for preparing an in-vitro diagnostic kit.

It is still another object of the present invention to provide the use of the above-described fetuin B monoclonal antibody in the preparation of a kit.

The aim of the invention is achieved by the following scheme:

A hybridoma cell strain is classified and named L008, is preserved in China general microbiological culture Collection center (CGMCC) of China general microbiological culture Collection center (CGMCC) at the 09 month 26 of 2023, and has a preservation address of the Ganyang region North Star of Beijing city, the West Lu No. 1, no. 3 and a preservation number of CGMCC No.45723.

The invention also provides a fetuin B monoclonal antibody named Ab008, which is secreted by the hybridoma cell strain L008 or a passage cell strain thereof.

Further, the fetuin B monoclonal antibody of the present invention is secreted from the hybridoma cell line L008.

Further, the fetuin B monoclonal antibody is an IgG1 subtype monoclonal antibody.

Further, the light chain of the fetuin B monoclonal antibody is a kappa chain.

Further, the fetuin B monoclonal antibody is capable of specifically recognizing human fetuin B.

Further, the fetuin B monoclonal antibody comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region has an amino acid sequence shown as SEQ ID NO. 3, and the heavy chain variable region has an amino acid sequence shown as SEQ ID NO. 5.

Further, a polynucleotide is encoded as the fetuin B monoclonal antibody, wherein the light chain variable region of the polynucleotide has a nucleotide sequence shown in SEQ ID NO. 4, and the heavy chain variable region has a nucleotide sequence shown in SEQ ID NO. 6.

The fetuin B monoclonal antibody can be specifically combined with fetuin B, and can be used as an immunodetection reagent for preparing an in-vitro diagnostic kit.

The invention also provides application of the hybridoma cell strain in preparation of a kit.

The invention also provides application of the hybridoma cell strain in preparation of a kit for detecting fetuin B.

The invention also provides application of the fetuin B monoclonal antibody in preparation of a kit.

The invention also provides application of the fetuin B monoclonal antibody in preparation of a kit for detecting fetuin B.

The invention also provides application of the polynucleotide in preparation of a kit.

The invention also provides application of the polynucleotide in preparation of a kit for detecting fetuin B.

The invention also provides a kit, which comprises the hybridoma cell strain or the fetuin B monoclonal antibody or the polynucleotide.

Further, the kit is a kit based on immunodetection.

Further, the immunodetection comprises enzyme-linked immunodetection, western immunoblotting detection, immunofluorescence detection, immunohistochemical detection and the like.

Further, the kit can be a colloidal gold immunoassay kit, a chemiluminescent kit, a radioimmunoassay kit, an enzyme-linked immunoassay kit or a fluorescent immunoassay kit.

Furthermore, the kit is an enzyme-linked immunosorbent assay kit.

The invention provides application of the hybridoma cell strain, the fetuin B monoclonal antibody or the polynucleotide in preparing tumor diagnosis reagents, tumor treatment medicaments, immune detection reagents and fetuin B protein detection reagents.

The tumor comprises ovarian cancer, breast cancer, skin cancer, kidney cancer, liver cancer, lung cancer, esophageal adenocarcinoma, bile duct cancer, colorectal cancer, prostatic cancer, melanoma and the like.

Compared with the prior art, the invention has the following advantages:

The invention provides a hybridoma cell strain and a fetuin B monoclonal antibody secreted by the hybridoma cell strain, which can be well specifically combined with natural fetuin B and recombinant fetuin B antigen in blood plasma or tissues, have wide application, can be used for preparing a kit for detecting the fetuin B, or can be used for preparing an immunology detection product for Western blot, cell immunofluorescence and immunohistochemistry detection of the expression of the fetuin B, and have practical application value.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a SDS-PAGE diagram after purification of recombinant proteins; lanes: m is a 180kDa protein marker;1 is fetuin B (100 mM imidazole); 2 is fetuin B (200 mM imidazole); 3 is fetuin B (300 mM imidazole).

FIG. 2 is a subtype identification of monoclonal antibodies.

FIG. 3 is a Western blot identification of monoclonal antibodies. Wherein A: a plasma sample; b: recombinant fetuin B.

FIG. 4 is an indirect immunofluorescent assay of monoclonal antibodies.

FIG. 5 is a graph showing the results of immunohistochemical detection of colorectal cancer. Wherein A is antibody Ab008 secreted by L008, and B is commercial antibody (Abcam).

FIG. 6 is a graph showing the results of immunohistochemical detection of monoclonal antibody Ab008 against each tumor tissue. Wherein A is liver cancer tissue, B is breast cancer tissue, C is ovarian cancer tissue, and D is prostate cancer tissue.

Detailed Description

The present invention will be described in further detail with reference to examples, but embodiments of the present invention are not limited thereto. The materials referred to in the examples below are available commercially unless otherwise specified. The method is conventional unless otherwise specified.

Example 1: preparation of recombinant fetuin B immunogen

(1) Construction of recombinant expression plasmids

The full-length amino acid sequence of fetuin B, numbered Q9UGM5, was selected from the UniProt database (https:// www.uniprot.org) as the standard sequence:

MGLLLPLALCILVLCCGAMSPPQLALNPSALLSRGCNDSDVLAVAGFALRDINKDRKDGYVLRLNRVNDAQEYRRGGLGSLFYLTLDVLETDCHVLRKKAWQDCGMRIFFESVYGQCKAIFYMNNPSRVLYLAAYNCTLRPVSKKKIYMTCPDCPSSIPTDSSNHQVLEAATESLAKYNNENTSKQYSLFKVTRASSQWVVGPSYFVEYLIKESPCTKSQASSCSLQSSDSVPVGLCKGSLTRTHWEKFVSVTCDFFESQAPATGSENSAVNQKPTNLPKVEESQQKNTPPTDSPSKAGPRGSVQYLPDLDDKNSQEKGPQEAFPVHLDLTTNPQGETLDISFLFLEPMEEKLVVLPFPKEKARTAECPGPAQNASPLVLPP(SEQ ID NO:1).

in total 382 amino acids, a plasmid vector expressing fetuin B was constructed by eukaryotic expression in order to obtain a specific immunogen. After optimizing human codon of fetuin B gene, the cloning vector is synthesized by Jin Weizhi (su state) biotechnology limited company, and the cloning vector is inserted into eukaryotic expression vector pcDNA3.1 in homologous recombination mode, and the accuracy of constructing eukaryotic expression plasmid is finally confirmed through enzyme digestion and sequencing.

The nucleotide sequence of the fetuin B gene for human codon optimization is as follows:

ATGGGCCTGCTCCTGCCCCTGGCCCTGTGCATTCTCGTGCTCTGCTGCGGGGCTATGAGCCCCCCTCAGCTGGCCCTGAACCCTAGCGCCCTGCTGAGCAGAGGCTGCAACGACAGCGACGTGCTGGCCGTGGCCGGCTTCGCCCTGAGAGACATCAACAAGGACAGAAAGGACGGCTACGTGCTGAGACTGAACAGAGTGAACGACGCCCAAGAGTACAGAAGAGGCGGCCTGGGCAGCCTGTTCTACCTGACCCTGGACGTGCTGGAGACCGACTGCCACGTGCTGAGAAAGAAGGCCTGGCAAGACTGCGGCATGAGAATCTTCTTCGAGTCCGTCTACGGGCAGTGCAAGGCCATCTTCTACATGAACAACCCTAGCAGAGTGCTGTACCTGGCCGCCTACAACTGCACCCTGAGACCCGTGAGCAAGAAAAAGATCTACATGACCTGCCCCGACTGCCCTAGCAGCATCCCCACCGACTCCTCCAACCACCAAGTGCTGGAGGCCGCCACCGAGAGCCTGGCCAAGTACAACAACGAGAACACAAGCAAGCAGTACAGCCTGTTCAAGGTGACAAGAGCTAGCAGCCAATGGGTGGTGGGCCCTAGCTACTTCGTGGAGTACCTGATCAAGGAGAGCCCCTGCACCAAGAGCCAAGCTAGCAGCTGCAGCCTGCAGAGCAGCGACAGCGTGCCCGTGGGCCTGTGCAAGGGCAGCCTGACAAGAACCCACTGGGAGAAGTTCGTGAGCGTGACCTGCGACTTCTTCGAGAGCCAAGCCCCCGCCACCGGCAGCGAGAACAGCGCCGTGAATCAGAAGCCCACCAACCTGCCCAAGGTGGAGGAGAGCCAACAGAAAAACACCCCTCCCACCGACAGCCCCTCCAAGGCCGGGCCTAGAGGCAGCGTGCAGTACCTGCCCGACCTGGACGACAAGAACAGCCAAGAGAAGGGCCCCCAAGAGGCCTTCCCCGTGCACCTGGACCTGACCACCAACCCCCAAGGCGAGACCCTGGACATCAGCTTCCTGTTCCTGGAGCCCATGGAGGAGAAGCTGGTGGTGCTGCCCTTTCCCAAGGAGAAGGCTAGAACAGCCGAGTGCCCCGGCCCCGCTCAGAACGCTAGCCCCCTGGTGCTGCCCCCC(SEQ ID NO:2)

(2) Recombinant fetuin B expression and purification

HEK293F cells in good growth state were inoculated into a new culture flask at a cell density of 3.0-4.0X10 ⁶ cells/mL, and further cultured by adding 130mL of serum-free medium (Beijing Yiqiao Shenzhou). Taking 250 mL sterile centrifuge tubes marked as a tube A and a tube B, adding 20mL serum-free culture medium respectively, then adding constructed eukaryotic expression plasmid and EZ Trans transfection reagent into the tube A and the tube B respectively, gently reversing and uniformly mixing, standing for 15min, mixing the two, standing for 30min, and finally adding the mixture system into cultured cells. The following morning after transfection, the cell status was observed, and after 5 days of continuous cell expression, the cell culture supernatant was collected by centrifugation at 6000rpm for 10 min.

Protein purification is carried out by connecting a His-nickel column with a Bio-Rad biological LP chromatography system, imidazole gradient elution of target proteins with different concentrations is carried out, 2 mug of target proteins are sampled and then the purification result of the proteins is detected by SDS-PAGE (figure 1), recombinant fetuin B with higher purity is obtained, SDS-PAGE protein gel is carried out for detection, a clear band is arranged at about 55-58kDa, and the purity is more than or equal to 90%. Collecting single strip sample, sub-packaging, and storing at-20deg.C.

Example 2: preparation of hybridoma cell strain

(1) Immunization of mice with fetuin B

3 BALB/c (6-8 week old) SPF-grade female mice (purchased from animal technology development Co., ltd. Of southern medical science, guangzhou) were selected and immunized with the recombinant fetuin B antigen purified in example 1. In the primary immunization, the immunogen fetuin B and an equal volume of Freund's complete adjuvant (Sigma company) are stirred and emulsified fully, and each mouse is subjected to subcutaneous multipoint injection immunization; the amount of fetuin B immunogen used was 100. Mu.g/g. Stirring and emulsifying fetuin B and equal volume of Freund's incomplete adjuvant (Sigma company) at intervals of 3 weeks after primary immunization, and performing subcutaneous multipoint injection immunization on each mouse; the amount of fetuin B immunogen used was 100. Mu.g/g. On day 7 after the third immunization, mice were bled by tail-breaking and serum titers were determined by an indirect enzyme-linked immunosorbent assay (ELISA).

(2) The indirect ELISA comprises the following specific steps: recombinant fetuin B was diluted to 3. Mu.g/mL with 0.05M carbonate buffer (pH 9.6) and added at 100. Mu.L per well to 96-well reaction plates of polystyrene plates, and coated overnight at 4 ℃. Removing solution in holes, adding 300 mu L of blocking solution into each hole, incubating at 37 ℃ for 2 hours, washing with PBST for 4 times, beating, adding diluted mouse serum, incubating at 37 ℃ for 1 hour, washing with PBST for 4 times, adding 100 mu L of HRP-labeled goat anti-mouse IgG diluted according to 1:10000 into each hole, incubating at 37 ℃ for 1 hour, washing with PBST for 5 times, and mixing ECL color development liquid with solution B according to the solution A and the solution B according to the ratio of 1:1 were mixed in a ratio of 100. Mu.L per well, incubated at 37℃for 10min, then 2M H ₂SO₄ was added to terminate the reaction, and the absorbance at a wavelength of 450nm (OD ₄₅₀) was read. Meanwhile, the serum of the non-immunized mice (diluted 1:1000) was used as a negative control, and a blank control well was set. The test results are shown in Table 1, and the titer of the antiserum reaches more than 64000, which shows that the prepared recombinant fetuin B has good immunogenicity, and also shows that the mice are immunized successfully. 5 days before cell fusion, mice were boosted once with fetuin B immunogen diluted in normal saline (without any adjuvant), injected intraperitoneally, at 100. Mu.g/mouse fetuin B, and finally immunized BALB/c mice were obtained.

TABLE 1 serum titers after mice immunization test results

(3) Cell fusion

After 5 days of booster immunization, cell fusion was performed according to the conventional PEG method (polyethylene glycol, molecular weight 1450), as follows:

① After the mice are killed by removing eyeballs and taking blood and cervical dislocation, the mice are soaked in 75% alcohol for about 5min, the spleens of the mice are taken out through aseptic operation, the mice are rinsed with a serum-free 1640 culture medium, placed on a 200-mesh cell screen, fully ground by a rubber head of an aseptic syringe, the spleen cells are washed into a 50mL aseptic centrifuge tube through the serum-free 1640 culture medium, centrifuged at 1200rpm for 8min, and finally resuspended with the serum-free 1640 culture medium and counted.

② Collecting SP2/0 cells from myeloma cells SP2/0 in logarithmic growth phase, suspending in RPMI-1640 basic culture solution, and performing cell count;

③ Myeloma cells SP2/0 and mouse immune splenocytes were thoroughly mixed at a ratio of 1:5, centrifuged at 1200rpm for 5min, the supernatant was discarded, 1mL of 50% PEG which had been warmed up was slowly added over 1min while shaking, and after addition, the fusion was stopped with 30mL of warmed up serum-free 1640 medium. The fused cells were centrifuged at 900rpm for 8min, the supernatant was discarded, and the pellet was well suspended in complete RPMI 1640 medium containing 20% of HAT neonatal bovine serum. Then evenly split into 96-well cell culture plates with 200 mu L of each well, and placed in a 37 ℃ and 5% CO ₂ incubator for culture.

④ Hybridoma cell selection and subcloning.

After 3 days of culture after fusion, the growth of the cells was observed, and after 6 days, 1/2 of the medium was replaced with HAT medium. After 7-10 days the HAT medium was swapped out with HT medium. After 10 days of fusion culture, hybridoma cell fine culture supernatant was detected by the indirect ELISA method described above. The positive hybridoma cell strain detected by the indirect ELISA method is inoculated to an HT incomplete culture medium, a cell pore limiting dilution method with strong positive reaction is selected to conduct cloning culture on the positive cloning hybridoma cell, and the monoclonal hybridoma cell strain with the positive rate reaching 100% and stable secretion series is obtained. And (5) freezing and storing in liquid nitrogen after amplification culture. One of the hybridoma cell lines is named L008, and is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) at the year 09 of 2023 and the preservation address is number 3 of West Song No. 1, the Korean region North Star of Beijing, and the preservation number is CGMCC No.45723.

Example 3: expression and purification of fetuin B monoclonal antibodies

After obtaining a stable hybridoma cell line, monoclonal antibodies are mainly obtained by an in vitro culture method, and the specific steps are as follows:

Hybridoma cells L008 cultured to the logarithmic growth phase were inoculated at 5.0X10 ⁵ cells/mL into a CDM4 medium (Hyclone Co., USA) containing L-glutamine, 250mL shaking flasks were placed in a shaking table at 37℃and 5% CO ₂ at 100rpm to be suspension-cultured, the initial culture volume was 65mL, and 3mL of Cell Boost ^TM 5 feed (Hyclone Co., USA) was added every day from the next day. When cultured until the counted cell viability was below 50%, the cell culture broth was harvested, the supernatant was collected by centrifugation at 6000rpm, filtered into a clean centrifuge tube using a disposable sterile syringe and a 0.22 μm filter membrane, and the antibody was purified using rProtein ASepharose Fast Flow (GE) affinity chromatography column: ① Filling a column, namely filling a proper amount of purchased ProteinA packing into a gravity chromatographic column, balancing by using a balancing buffer solution (0.01M PBS, pH 7.0) with 5 times of column volume, and controlling the flow rate to be 2mL/min; ② Loading, namely adding the cell supernatant filtered by a 0.22 mu m filter membrane into a chromatographic column filled with a filler, and controlling the flow rate to be 0.8mL/min; ③ Balancing, namely balancing by using a balancing buffer solution (0.01M PBS, pH 7.0) with a volume which is 10 times that of the column after loading the sample solution, and controlling the flow rate to be 1.5mL/min; ④ Eluting, adding an eluting buffer (0.1M glycine-HCl solution, pH 2.7) to wash the column, and collecting the eluent, wherein the flow rate is controlled to be 1.5mL/min; ⑤ Preserving, adding the eluted and collected antibody into a neutralization buffer solution (1.0M Tris-HCl solution, pH 8.8), regulating the pH to be neutral, finally, identifying the purity of the antibody by adopting an SDS-PAGE method, measuring the concentration of the antibody by an ultraviolet micro-spectrophotometry method, and finally, the concentration of the antibody is 2.3mg/mL. The monoclonal antibody secreted by the hybridoma cell strain L008 is named Ab008, and the purified antibody is split-packed and preserved at-20 ℃ for standby.

Example 4: subtype identification of fetuin B monoclonal antibodies

Referring to the operation of a mouse monoclonal antibody subtype identification kit (Proteintech company), the monoclonal antibody obtained by hybridoma cell culture purification is subjected to immunoglobulin subtype identification, and the specific method is as follows: taking the 96-well ELISA plate out of the refrigerator, balancing to room temperature, diluting the culture supernatant of the hybridoma cells, and adding the diluted culture supernatant into a lath sample hole, wherein the volume of the sample hole is 50 mu L/hole; adding diluted goat anti-mouse IgA+IgM+IgG-HRP into the sample hole, and mixing uniformly at a concentration of 50 mu L/hole; covering a sealing plate membrane, incubating for 1h at room temperature, discarding liquid in the hole, washing the plate with PBST for 4 times, and beating on absorbent paper; adding the prepared color development liquid into the hole, and developing at room temperature for 10-20min in a dark place at 100 mu L/hole; the reaction was stopped by adding 100. Mu.L/well of stop solution to each well, and the OD at 450nm was measured by a microplate reader within 10-12 min. The results are shown in FIG. 2, in which the monoclonal antibody of the present invention is an IgG1 class immunoglobulin and the light chain class is kappa.

Example 5: fetuin B monoclonal antibody sequencing

The hybridoma cell line L008 obtained was sent to Nanjing Ming Biotech Co., ltd for sequencing. The total RNA extracted from the splitting of hybridoma cell strain L008 is used as a template, cDNA is synthesized by reverse transcription of RNA through a cDNA terminal rapid amplification technology (RACE), genes of heavy chain and light chain variable regions are amplified through Polymerase Chain Reaction (PCR), PCR products are connected to cloning vectors, a successfully constructed plasmid is transformed into DH5 alpha competent cells by a heat shock method, and positive strains are screened by a resistance plate and then sequenced.

The fetuin B monoclonal antibody comprises a light chain variable region and a heavy chain variable region, and the sequencing result is specifically as follows:

(1) Sequencing results of the light chain variable region

The amino acid sequence of the light chain variable region is shown in SEQ ID NO. 3:

DIVMTQSPSSLSVSVGEKVTMNCKSSQSLLNSGHQKNYLAWYQQKPGQPPKLLIYG ASTRKSGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDHRYPLTFGAGTKLELK(SEQ ID NO:3)

a polynucleotide encoding said fetuin B monoclonal antibody, wherein the light chain variable region of said polynucleotide has the nucleotide sequence shown in SEQ ID No. 4:

GACATTGTGATGACACAGTCTCCATCCTCCCTGAGTGTGTCAGTAGGAGAGAAGGTCACTATGAACTGCAAGTCCAGTCAGAGTCTGTTAAACAGTGGACATCAAAAGAACTATTTGGCCTGGTACCAGCAGAAACCAGGGCAGCCTCCTAAACTGTTGATCTACGGGGCATCCACTAGGAAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGAACCGATTTCACTCTTACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAGAATGATCATCGTTATCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA(SEQ ID NO:4)

(2) Heavy chain variable region sequencing results

The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 5:

EVQLQQFGAELVKPGASVKISCKASGYTFTDYNMDWVKQSHGRSLEWIGDINPNY DSTRYNQKFKGKATLTVDKSSSTAYMELRSLTSEDTAVYYCARDGLYAMDYWGQGTSVT VSS(SEQ ID NO:5)

a polynucleotide encoding said fetuin B monoclonal antibody, wherein the heavy chain variable region of said polynucleotide has the nucleotide sequence shown in SEQ ID No. 6:

GAGGTCCAGCTGCAACAGTTTGGAGCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGCTACACATTCACTGACTACAACATGGACTGGGTGAAGCAGAGCCATGGAAGGAGCCTTGAGTGGATTGGAGATATTAATCCTAACTATGATAGTACTAGGTACAACCAGAAGTTCAAGGGAAAGGCCACATTGACTGTAGACAAGTCCTCCAGCACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGACACTGCAGTCTATTACTGTGCAAGAGATGGCCTCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA(SEQ ID NO:6)

Example 6: western immunoblotting detection of fetuin B monoclonal antibody

Recombinant fetuin B and human plasma samples were loaded separately and antibody specificity was detected using Western Blot. The specific experimental steps are as follows: preparing SDS-PAGE protein gel with the concentration of 10%, electrophoresing a sample (plasma sample and commercially available recombinant fetoprotein B) through SDS-PAGE proteins, then electrically transferring the sample to a PVDF membrane by using a Bio-Rad transfer device, blocking overnight at 4 ℃ by using a blocking solution containing 5% skimmed milk diluted by TBST, washing the membrane for 3 times by using TBST, adding purified monoclonal antibody according to the ratio of 1:1500 for 12min each time, incubating for 1h at room temperature, washing the membrane for 3 times by using TBST, adding goat anti-mouse IgG polyclonal antibody (Sigma) diluted by 1:5000 for 2min each time, incubating for 1h at room temperature for 3 times by using TBST, preparing ECL color development liquid according to the ratio of 1:1 of A/B liquid, and performing imaging color development in a Chemidoc MP omnipotent imaging system (BIO-RAD). As shown in FIG. 3, the anti-human fetuin B monoclonal antibody secreted by the prepared hybridoma L008 can well identify fetuin B (FIG. 3A) and recombinant fetuin B antigen (FIG. 3B) in a human natural plasma sample, and meanwhile, no other impurity bands are found, so that the prepared monoclonal antibody has the capability of specifically binding to human fetuin B, and a Western blot experiment can be applied.

Example 7: cytoimmunofluorescence detection of fetuin B monoclonal antibodies

The hepatoma cell line HepG2 was spread 24h in advance into a cell culture plate, cultured to logarithmic growth phase, and cells were washed 3 times with PBS. 4% paraformaldehyde was added for fixation, incubated at room temperature for 30min, and cells were washed 3 times with PBS. The cell rupture fluid was added and incubated at room temperature for 15min, and the cells were washed 3 times with PBS. Blocking with blocking solution containing 5% BSA was performed for 30min at room temperature. The blocking solution was discarded, diluted monoclonal antibody Ab008 (5. Mu.g/mL) was added to the well plate and incubated at room temperature for 2h, while wells incubated with 5% BSA served as negative controls. Cells were washed 3 times with PBS, and Dylight 594-labeled goat anti-mouse IgG fluorescent secondary antibody (2. Mu.g/mL) was added and incubated for 1h at room temperature. Cells were washed 5 times with PBS, DAPI diluted 1:500 was added, and incubated for 5min at room temperature. Cells were washed 3 times with PBS, observed under an inverted fluorescence microscope and photographed, and the results are shown in fig. 4. The results show that: the Ab008 antibody prepared by the invention can be clearly combined with target protein (red fluorescence) of HepG2 cells, the red fluorescence is mainly distributed in cytoplasm, DAPI (DAPI) staining cell nucleus shows blue fluorescence, and the monoclonal antibody Ab008 prepared by the invention can be proved to be capable of recognizing natural fetuin B and can be used for immunofluorescence staining of cells.

Example 8: immunohistochemical detection of fetuin B monoclonal antibody

Paraffin sections of human colorectal cancer tissues are taken, placed in an oven at 60 ℃ for 3 hours, and dewaxed with conventional xylene for 2 times, each for 15 minutes. The sections were gradient hydrated in 100%, 95%, 85%, 75% ethanol for 5min each, and washed with tap water for 5min. Thermal antigen retrieval was performed using citrate buffer and washed 3 times with PBS. 3% H ₂O₂ is dripped into the sliced tissue, incubated for 20min at room temperature, and endogenous catalase is inactivated. Washing with PBS for 3 times, dripping 5% BSA, blocking at 37deg.C for 30min, and drying to remove excessive liquid. Monoclonal antibody Ab008 (2 μg/mL) was formulated as primary antibody, fetuin B antibody (Ab 191569) from Abcam as control antibody, and each was added drop wise to tissue, incubated overnight at 4 ℃, and washed 3 times with PBS. The secondary antibodies of the goat anti-mouse IgG antibody and the goat anti-rabbit IgG antibody marked by the general HRP are respectively dripped, and incubated for 30min at 37 ℃. Washing with PBS for 3 times, and adding freshly prepared DAB color development liquid for 5min. Rinsing with tap water for 10min, and lightly counterstaining with hematoxylin for 1min. After washing with tap water for 5min, PBS was soaked for 5min to return to blue. Sections were dehydrated sequentially with 75%, 85%, 95%, 100% alcohol gradient for 3min each, and finally xylene was transparent for 3min, gel-sealed with neutral resin, observed under microscope and photographed. The results show that: the antibody (figure 5A) and the commercial antibody (figure 5B) of the invention synchronously detect colorectal cancer tissue sections, the staining positioning is accurate, the staining is clear, no nonspecific staining is caused, the background is clean, the detection result is consistent, the specificity of the antibody in colorectal cancer tissues is equivalent to that of the commercial antibody, and the antibody can be used for immunohistochemical detection.

Example 9: immunohistochemical detection of fetuin B monoclonal antibodies in different tumor tissues

Paraffin sections of human different tumor (liver cancer, breast cancer, ovarian cancer, prostate cancer) tissues were taken, and the experimental procedure was the same as in example 8. The experimental results are shown in FIG. 6. The histochemical results show that the antibody has good expression effect in different tumors, has accurate staining and positioning in each tumor tissue section, clear staining and clean background, and shows that the fetuin B monoclonal antibody (Ab 008) can be applied to immunohistochemical detection, can well detect the expression quantity and the expression position of the fetuin B in tumor tissues, and has better effect and specificity of recognizing the fetuin B antigen.

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.

Claims

1. A hybridoma cell strain is characterized by being classified and named L008, and is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) on the 26 th month of 2023, wherein the preservation address is the number 3 of West Song No. 1 of the Korean area North Star of Beijing, and the preservation number is CGMCC No. 45723.

2. A fetuin B monoclonal antibody, designated Ab008, secreted by the hybridoma cell line L008 or a passaged cell line thereof according to claim 1.

3. The fetuin B monoclonal antibody according to claim 2, wherein: the fetuin B monoclonal antibody comprises a light chain variable region and a heavy chain variable region, wherein the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 3, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 5.

4. A polynucleotide encoding the fetuin B monoclonal antibody of any one of claims 2 to 3, wherein the light chain variable region of the polynucleotide has a nucleotide sequence as shown in SEQ ID No. 4 and the heavy chain variable region has a nucleotide sequence as shown in SEQ ID No. 6.

5. Use of the hybridoma cell line of claim 1, the fetuin B monoclonal antibody of any one of claims 2-3, or the polynucleotide of claim 4 in the preparation of a kit.

6. Use of the hybridoma cell line of claim 1, the fetuin B monoclonal antibody of any one of claims 2-3, or the polynucleotide of claim 4 for the preparation of a kit for detecting fetuin B.

7. A kit, characterized in that: the kit comprises the hybridoma cell line of claim 1 or the fetuin B monoclonal antibody of any one of claims 2 to 3, or the polynucleotide of claim 4.

8. The kit of claim 7, wherein the kit is an immunoassay-based kit.

9. Use of the hybridoma cell line of claim 1, the fetuin B monoclonal antibody of any one of claims 2-3, or the polynucleotide of claim 4 for the preparation of a tumor diagnostic reagent, a reagent for detecting fetuin B protein; the tumor is at least one selected from ovarian cancer, breast cancer, liver cancer, colorectal cancer and prostate cancer.