TW201527536A - Hybridoma cell lines used for preparation of cartilage oligomeric matrix protein monoclonal antibody and monoclonal antibody thereof - Google Patents

Abstract

Provided is a hybridoma cell lines used for preparation of cartilage oligomeric matrix protein monoclonal antibody and monoclonal antibody thereof. The hybridoma cell lines are deposited in Food Industry Research and Development Institute in which the storage number is BCRC 960472. The hybridomacell lines are fused together by a parental cell and a myeloma cell by which the parental cell is derived and prepared from the spleen cell of the body in a mouse. This cartilage oligomeric matrix protein fragements exhibited in procaryotic cells of a mouse is used as an antigen immune, and the monoclonal antibody prepared by the hybridoma cell lines can be specifically used for identifying cartilage oligomeric matrix protein antigen in human urine.

Description

Fusion tumor cell strain for producing cartilage oligomeric matrix protein monoclonal antibody and monoclonal antibody thereof

The present invention relates to a fusion tumor cell line, in particular to a fusion tumor cell strain for producing a cartilage oligomeric matrix protein monoclonal antibody and a monoclonal antibody thereof.

Osteoarthritis (OA) is a common joint degenerative disease. According to statistics, 70-80% of people over the age of 65 have diseases of articular cartilage degeneration.

At present, the degree of articular cartilage degradation is not easy to detect. Cartilage degradation is usually the most commonly used X-ray illumination to detect the size of the space between the articular cartilage to estimate the degree of cartilage degradation. However, the clinical detection and the development of such X-ray irradiation, or other physical detection such as MRI (magnetic resonance imaging) detection can not be observed for early joint deterioration, when the X-ray irradiation development or MRI detection has a difference, the articular cartilage is degraded. The degree of reversal is no longer recoverable, so X-ray detection or MRI does not immediately detect the condition in which articular cartilage begins to degenerate. However, in addition to the diagnosis by X-ray irradiation, the general preventive test requires invasive extraction of cartilage tissue fluid, such as LC/MS/MS (Liquid Chromatography/Mass Spectrometry/Mass Spectrometry); or extraction of joint fluid Or the serum is analyzed by the ELISA (Enzyme-Linked Immunosorbent Assay) method to determine the condition of cartilage degradation, and the amount of decomposed cartilage is analyzed to determine the condition of cartilage damage.

In order to improve the defects such as the sample which can only be used for invasive sampling, the US Patent No. 6642007 "Method for detecting type II collagen fragments of urine samples" discloses a method for detecting degenerative arthritis. The detection method is to prepare a composite antibody for detecting degenerative arthritis with a urine sample, and the composite anti-system is designed for two antigenic epitopes (Type II collagen neoepitope, TIINE for short) on the type II collagen fragment. a capture antibody and a detection antibody, and the type II collagen fragment is identified by the composite antibody; wherein the compound antibody must be subjected to a sandwich enzyme-linked immunosorbent assay ( Sandwich Enzyme-Linked ImmunoSorbet Assay) can effectively improve the detection limit of the composite antibody for urine samples, and can not achieve the purpose of detecting type II collagen fragments by only one antibody; therefore, in order to maintain proper detection sensitivity And potency, must spend more manpower, resources and time to prepare more than two kinds of resistance , Resulting in cost burden.

It is an object of the present invention to provide a fusion tumor cell strain for producing a cartilage oligomeric matrix protein monoclonal antibody and a monoclonal antibody thereof.

In order to achieve the above object, the technical content of the present invention includes the following:

A fusion tumor cell line for producing a monoclonal antibody against cartilage oligomeric matrix protein, which is deposited in the Food Industry Development Research Institute of the consortium, registered as BCRC 960472, using Escherichia coli (BL21(DE3)plysS E. coli) To express cartilage oligomeric matrix protein (COMP), and to fuse a parental cell with a myeloma cell (SP/o-Ag14) to form a fusion tumor cell line.

It uses a polymerase chain reaction (PCR) to amplify gene fragments derived from human cartilage oligomeric matrix proteins; continues to use pRSET B as a vector system for colonization, and uses prokaryotic bacteria for protein expression. Then, it is prepared by immunizing the mouse with the antigen expressed by prokaryotic bacteria as an antigen, and taking out the spleen cells in the mouse.

In addition, the present invention also provides a cartilage oligomeric matrix protein monoclonal antibody, which is produced by using a fusion cell strain harboring the accession number BCRC 960472, and can be specifically combined with a cartilage oligomeric matrix protein antigen to provide degradation. Rapid detection of the use of arthritis.

As apparent from the above description, the present invention is characterized in that the cartilage oligomeric matrix protein monoclonal antibody is isolated by animal immunosuppression by cartilage oligomeric matrix protein as an antigen, and the high sensitivity of the monoclonal antibody of the oligocartilage matrix protein is obtained. As well as high-priced properties, it is possible to detect cartilage oligomeric matrix protein antigens in urine samples in a single antibody manner, thereby saving process costs and further developing a rapid detection system for degenerative arthritis.

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Fig. 1A is a graph showing the results of a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) experiment of the purified cartilage oligomeric matrix protein.

Fig. 1B is a graph showing the results of detecting the cartilage oligomeric matrix protein purified by the western blot method of the monoclonal antibody of the present invention.

Fig. 2 is a graph showing the results of detection of different samples of the cartilage oligomeric matrix protein monoclonal antibody of the present invention by Western blotting.

Fig. 3 is a graph showing the results of an experiment for the enzyme-linked adsorption immunoassay of the present invention.

The detailed description and technical contents of the present invention are as follows:

The present invention relates to a fusion tumor cell strain for producing a cartilage oligomeric matrix protein monoclonal antibody, and a cartilage oligomeric matrix protein monoclonal antibody produced by using the fusion tumor cell strain, which is deposited in a corporation. The Food Industry Development Institute, the accession number is BCRC 960472; to prepare the fusion tumor cell line and the monoclonal antibody of the present invention, the following experimental steps are illustrated:

"Cartilage Oligomeric Matrix Protein Replication and Colonization"

S1: Primer design: Design a primer pair for the hydrophilicity of the human cartilage oligomeric matrix protein (equivalent to the amino acid region Nos. 335 to 500 of GenBank accession No. AB086984.1) The gene is amplified. The sequence of the forward primer of the primer is 5'-ATAGATCTCGACCAGCGCAACAC-3', which contains the cleavage site of the BglII restriction enzyme; the sequence of the reverse primer of the primer pair is 5'-CTGAATTCCACGCCGTCCCTG-3', which contains the cleavage site of the EcoRI restriction enzyme.

S2: PCR preheating denaturation step: the reaction mixture of the primer pair in step S1 and a DNA template was heated at 94 ° C for 5 minutes.

S3: PCR thermal cycle step: The following thermal cycle steps were repeated 30 times: Densification was carried out by heating at 94 ° C for 1 minute, followed by bonding at 55 ° C for 30 seconds, and then at 72 ° C for 1 time. Minute extension (Extension).

S4: PCR final extension step: The reaction mixture after the PCR thermal cycle step was extended at 72 ° C for 5 minutes.

S5: PCR product analysis: The product of the PCR step was analyzed by electrophoresis on a 0.8% agar colloid to determine its size.

S6: cartilage oligomeric matrix protein selection: the product was first treated with an appropriate restriction enzyme, and the restriction enzymes used in the present case were BglII and EcoRI, and then the positions of BamHI and EcoRI of the pRSET B vector were bonded to obtain A recombinant plasmid of pRCOMP166 was transformed into E. coli, and the Escherichia coli was selected from E. coli DH5α.

"Carbonogenic oligomeric matrix protein expression and purification"

S7: cartilage oligomeric matrix protein expression: the above-mentioned pRCOMP166 recombinant plastid containing the cartilage oligomeric matrix protein fragment in DH5α E. coli was extracted and transformed into another protein expressing cell, wherein the protein expressing cell was selected from BL21 (DE3) plysS E. coli. The protein expression cells were cultured in 2 ml of LB (Luria-Broth) medium containing 50 μg/ml of (μg/ml) amphibilin (Ampicillin) and 150 μg/ml of chloramphenicol ( Chloramphenicol), and cultured vigorously at 37 ° C for 16 hours to obtain a protein-expressing colony; continued to inject the protein expression colony into 100 ml of LB medium, and continue to shake vigorously at 37 ° C, and continue to measure The absorbance at a wavelength of 600 nm (OD600) is up to 0.4-0.6.

S8: induction of cartilage oligomeric matrix protein: an inducer was added to the LB medium to a final concentration of 1 millimolar (mM), and further cultured for 4 hours to induce the expression of the cartilage oligomeric matrix protein, wherein the inducer was selected. Isopropyl-β-thiogalactopyranoside (IPTG).

S9: Purification of cartilage oligomeric matrix protein: The E. coli colonies containing the cartilage oligomeric matrix protein were collected by centrifugation, dissolved in 10 ml of ultrasonic shock buffer solvent and vortexed by ultrasonic wave until the solution became clear. a clear solution; the supernatant is continuously centrifuged to obtain an upper clear liquid containing the cartilage oligomeric matrix protein, and the supernatant liquid is purified by using a His-Tag affinity chromatography column. A purified cartilage oligomeric matrix protein is obtained which can be used as a parental cell for subsequent generation of a fusion tumor cell line.

S10: analysis of cartilage oligomeric matrix protein purified: the purified cartilage oligomeric matrix protein product was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). To analyze the molecular weight, as shown in Fig. 1A, the molecular weight of the purified cartilage oligomeric matrix protein was about 29 kilodaltons (kDa). In the figure, the M row is the protein molecular weight symbol, and the P row is the cartilage. The molecular weight of the purified polystromal protein.

"An animal immunoassay using cartilage oligomeric matrix protein purified material"

S11: Animal immunization test: firstly, 50-75 μg of the purified cartilage oligomeric matrix protein was used as an antigen and mixed with a complete Freund's adjuvant to prepare a concentration of about 50 μg / 250 μl of the first injection was administered to the peritoneal cavity of a mouse by subcutaneous injection to complete the first immunization, wherein the mouse was a 6-week-old BALB/c line maternal experimental mouse; The matrix protein purified as an antigen is mixed with a Freund's complete adjuvant, and the supernatant containing the cartilage oligomeric matrix protein antigen is taken as a second injection, and after the first immunization, the third, fifth, and seventh At 9 weeks, the vaccination of the experimental mice was also performed by intraperitoneal injection, and the rats were administered intravenously for the last 4 days before the preparation of the fusion tumor cell line, and the mice were collected by tail vein blood collection. The blood, and the serum obtained from the blood test of the test mouse, confirms that the serum carries the antibody of the cartilage oligomeric matrix protein, and the application time and the dose of the application are shown in Table 1; Adsorption immunity Determination precipitation method (indirect Enzyme-Linked Immunosorbent Assay, indirect ELISA) serum antibody titers, and finally the antibody titer reached 100,000, the mice to subsequent further use of the antibody production.

Table 1: Application time and dosage of animal immunoassay

[0031] "Preparation method of fusion tumor cell strain for producing cartilage oligomeric matrix protein monoclonal antibody"

S12: preparing a fusion tumor cell strain: collecting the spleen cells and myeloma cells of the mouse subjected to the above experiment, and treating with a 50% PEG-8000 (polyethylene glycol-8000) cell fusion agent to form a fusion tumor cell; The medium is cultured in a suitable medium, which is a 4×10 ^-5 M HAT medium containing 15% fetal bovine serum, and cultured until the number of the fusion tumor cells reaches 10 ⁶ cells per well (cell/well). In this culture, only the cells in which the myeloma and the spleen cells are fused can survive, and the successfully fused cells can be screened; after 10 days of culture, the antibody-producing cell strain can be screened.

"Preparation of the antibody of the fusion tumor cell of the present invention and the antibody of the cartilage oligomeric matrix protein"

S13: Screening of fusion tumor cell lines: using Bovine Serum Albumin (BSA), untreated cartilage oligomeric matrix protein, purified cartilage oligomeric matrix protein purified, and concentrated in human urine The other proteins were screened by the Western blot method, and the fusion cell strains capable of clearly identifying the cartilage oligomeric matrix protein were selected, and then subjected to a single dilution by a limiting dilution method. In the step, a single antibody strain of the fusion tumor cell is obtained.

S14: preparation of cartilage oligomeric matrix protein antibody: selecting a BALB/c strain of a male experimental pig of about 12 weeks old, first injecting 0.5 ml of Pristane, and continuously injecting the above-mentioned selected fusion tumor cell antibody strain into the abdominal cavity; After 2 to 3 weeks, the ascites was taken out and centrifuged for 1 hour at 4 ° C. The supernatant was removed and the high concentration of cartilage oligomeric matrix protein antibody was used. The antibody purification kit Econo-Pac Protein was used. A kit (Bio-Rad Laboratories, CA, USA) Purification of individual antibodies was carried out to complete the preparation of the antibody to the cartilage oligomeric matrix protein.

In order to confirm the characteristics of the antibody of the cartilage oligomeric matrix protein, the present invention was subjected to the following experiments:

"The fusion tumor monoclonal antibody recognizes the specificity of the cartilage oligomeric matrix protein"

T1: Western blot: The present invention confirms that the cartilage oligomeric matrix protein monoclonal antibody specifically binds to the cartilage oligomeric matrix protein by Western blotting. The experimental method is as follows: After SDS-PAGE electrophoresis, it was transferred to polyvinylidene fluoride (PVDF) membrane. The protein sample contained commercially available cartilage oligomeric matrix protein (R&D system, MN, USA) and pRCOMP166 recombinant plasmid. The cartilage oligomeric matrix fragment protein and the protein obtained after concentration in human urine. The protein-containing PVDF membrane was cultured in 10% skim milk for 4 hours, followed by dilution of 50,00 times of the cartilage oligomeric matrix protein monoclonal antibody (original concentration 0.5 mg/ml) and 5% skim milk. Primary antibody, cultured at 4 ° C for 4 hours; followed by PBS-Tween 20 detergent for 3 times every 10 minutes to remove skim milk, and diluted 10,000 times anti-mouse HRP-IgG secondary antibody React for about 1 hour at room temperature, then wash 3 times with PBS-Tween20 detergent for 5-10 minutes each time; continue to add HRP-substrate (0.1 ml/cm ² ) in commercial kit. The reaction was carried out for 10 minutes in a warm and dark environment, and then the PVDF membrane was taken out and visualized by a chemical fluorescence imaging system (ChemiDocTM XRS+ System, Bio-Rad) to confirm the prepared cartilage oligomeric matrix protein. The antibody was detected to develop color only by binding to the cartilage oligomeric matrix protein. The results are shown in Fig. 1B and Fig. 2, and Fig. 1B shows that the antibody of the cartilage oligomeric matrix protein was detected by Western blotting. The test result of the purified cartilage oligomeric matrix protein, the cartilage oligomeric matrix protein single plant The body can be clearly detected for the purified cartilage oligomeric matrix protein; "Fig. 2" is a test result of the detection of different protein samples by the western blot method of the monoclonal antibody of the cartilage oligomeric matrix protein, wherein U1 to U6 are Urine samples of patients with different degenerative arthritis, C is a commercially available cartilage oligomeric matrix protein, and P is a cartilage oligomeric matrix fragment protein expressed by pRCOMP166 recombinant plastid. From the test results, the cartilage provided by the present invention is known. Oligomeric matrix protein monoclonal antibodies do detect cartilage oligomeric matrix proteins in urine samples from patients with degenerative arthritis.

"The sensitivity of the chimeric oligomeric matrix protein was identified by the monoclonal antibody of the fusion tumor"

T2: Enzyme-linked adsorption immunoassay: The present invention utilizes an enzyme-linked immunosorbent assay to confirm the minimum concentration of the antibody of the cartilage oligomeric matrix protein, as follows: The cartilage oligomeric matrix protein antigen is diluted to one. The antigen dilution solution, wherein the antigen dilution solution is used in the present invention at concentrations of 50, 100, 200, 300, 400, and 500 ng/ml (ng/ml), and 100 microliters of the antigen dilution solution is added to the microplate. After standing for 8 to 10 hours in each well of (Microplate), the antigen was fixed to a microplate, and then washed with a 0.1% PBS-Tween 20 detergent for 3 times every 5 minutes; the concentration was 10 ng/ 100 μl of ml of cartilage oligomeric matrix protein antibody was added to the wells of a microplate and allowed to stand at room temperature for 1 hour; again, washed with 0.1% PBS-Tween 20 detergent for 3 times every 5 minutes. Add 100 μl of anti-mouse HRP-IgG secondary antibody, also incubated for 1 hour at room temperature, and also wash 3 times with 0.1% PBS-Tween20 detergent for 5 minutes each time; Microliter of developer reaction for 2 minutes Color development, wherein the color developer selected for the present invention is SureBlue, and the absorbance of the wavelength of 450 nm is detected by an instrument. The experimental result is shown in FIG. 3, and the concentration detection limit of the antibody of the cartilage oligomeric matrix protein is single. Less than 50 ng/ml, and as the concentration of cartilage oligomeric matrix protein antigen increases, the absorbance also increases.

In summary, the present invention has the following features

1. The monoclonal antibody of cartilage oligomeric matrix protein produced by the provided fusion cell strain is specifically identified with the cartilage oligomeric matrix protein antigen in human samples, and can be further utilized for the development of degenerative arthritis. Rapid detection system.

Second, the Western blot method and enzyme-linked adsorption immunoassay for the detection of the characteristics of the cartilage oligomeric matrix protein monoclonal antibody can further confirm the specificity and sensitivity of the cartilage oligomeric matrix protein monoclonal antibody.

Domestic deposit information

Depository institution: Food Industry Development Research Institute

Date: October 1, 102

Number: BCRC 960472

<110> Guo Wei <120> <160> 1 <210> 1 <211> 498 <212> dna <213> homo sapiens <220> <223> A fragment obtained by polymerase chain reaction from an increase in human cartilage oligomeric matrix protein, equivalent to aminobank accession no. ab086984.1 No. 335 to 500 amino acid region <400> 1 Cggtcgtgcg tgtgtcgcgt tggctgggcc ggcaacggga tcctctgtgg 50 Tcgcgacact gacctagacg gcttcccgga cgagaagctg cgctgcccgg 100 Agccgcagtg ccgtaaggac aactgcgtga ctgtgcccaa ctcagggcag 150 Gaggatgtgg accgcgatgg catcggagac gcctgcgatc cggatgccga 200 Cggggacggg gtccccaatg aaaaggacaa ctgcccgctg gtgcggaacc 250 Cagaccagcg caacacggac gaggacaagt ggggcgatgc gtgcgacaac 300 Tgccggtccc agaagaacga cgaccaaaag gacacagacc aggacggccg 350 Gggcgatgcg tgcgacgacg acatcgacgg cgaccggatc cgcaaccagg 400 Ccgacaactg ccctagggta cccaactcag accagaagga cagtgatggc 450 Gatggtatag gggatgcctg tgacaactgt ccccagaaga gcaacccg 498

Claims (6)

[Item 1]
A fusion tumor cell line for producing a monoclonal antibody against a cartilage oligomeric matrix protein, which is deposited in the Food Industry Development Research Institute of the consortium, the accession number is BCRC 960472, and the fusion tumor cell line is composed of a parental cell and a bone marrow. The tumor cells are fused, and the parental cell line is obtained by removing one spleen cell in a mouse, and the mouse is immunized with a cartilage oligomeric matrix protein fragment represented by a prokaryotic cell as an antigen, and the fusion tumor The monoclonal antibody produced by the cell strain can specifically recognize the cartilage oligomeric matrix protein antigen in human urine, and the cartilage oligomeric matrix protein gene comprises the nucleic acid sequence shown in SEQ ID NO: 1. [Item 2]
A fusion tumor cell strain for producing a cartilage oligomeric matrix protein monoclonal antibody according to claim 1, wherein the mouse is a BALB/c strain adult mouse. [Item 3]
A fusion tumor cell strain for producing a cartilage oligomeric matrix protein monoclonal antibody according to claim 1, wherein the prokaryotic cell is an Escherichia coli. [Item 4]
A cartilage oligomeric matrix protein monoclonal antibody produced by using a fusion tumor cell strain for producing a cartilage oligomeric matrix protein monoclonal antibody according to the first aspect of the invention, which is characterized in that it can be combined with a cartilage oligomeric matrix The protein antigen specifically binds, and the cartilage oligomeric matrix protein gene comprises the nucleic acid sequence shown in SEQ ID NO: 1. [Item 5]
A cartilage oligomeric matrix protein monoclonal antibody according to claim 4, wherein the cartilage oligomeric matrix protein monoclonal antibody system is secreted from the fusion tumor cell line. [Item 6]
The cartilage oligomeric matrix protein monoclonal antibody according to the fourth aspect of the invention, wherein the cartilage oligomeric matrix protein monoclonal antibody system is obtained by intraperitoneal injection of the fusion tumor cell line into the ascites of the experimental mouse, and collecting ascites. .