CN114957464B

CN114957464B - Monoclonal antibody of anti-human mitochondrial outer membrane protein OMP25 and application thereof

Info

Publication number: CN114957464B
Application number: CN202210522277.5A
Authority: CN
Inventors: 高传杰; 赵明红
Original assignee: Hangzhou Heavy Chain Technology Co ltd
Current assignee: Hangzhou Heavy Chain Technology Co ltd
Priority date: 2022-05-13
Filing date: 2022-05-13
Publication date: 2023-07-07
Anticipated expiration: 2042-05-13
Also published as: CN114957464A

Abstract

The invention discloses a monoclonal antibody of an anti-human mitochondrial outer membrane protein OMP25 and application thereof. The monoclonal antibody is HC01-R001, has subtype of IgG1 and kappa type, and can be specifically combined with the outer membrane region of human mitochondrial outer membrane protein OMP 25. The hybridoma producing the antibody is obtained by fusing, screening, cloning and passaging the immunized BALB/C mouse spleen lymphocytes and mouse myeloma cells SP2/0, and can stably secrete HC01-R001. The protein has the advantages of simple structure and function, small influence on mitochondria, high sequence specificity and high conservation degree among different species, is an ideal mitochondrial outer membrane recognition target, has wide application, and can be used for detection of ELISA, western Blotting, immunofluorescence, immunohistochemistry, flow cytometry and the like of human mitochondrial outer membrane proteins and immunoprecipitation experiments.

Description

Monoclonal antibody of anti-human mitochondrial outer membrane protein OMP25 and application thereof

Technical Field

The invention belongs to the field of biotechnology and immunology, and relates to an anti-human mitochondrial envelope protein OMP25 monoclonal antibody and application thereof, wherein the monoclonal antibody is HC01-R001.

Background

Mitochondria are an organelle in eukaryotic cells that is coated with two membranes, and are the structures that produce energy in the cell, the primary sites for aerobic respiration by the cell. The mitochondria shape is different depending on the biological species and physiological state, and is generally in the shape of a short rod or sphere, and can also be in the shape of a ring, a line, a dumbbell, a fork, a flat disk or other shapes. Mitochondria possess their own genetic material and genetic system, but their genome size is limited, a semi-autonomous organelle.

Mitochondria can be divided into four functional regions from outside to inside, namely, the outer mitochondrial membrane, the mitochondrial membrane gap, the inner mitochondrial membrane and the mitochondrial matrix. Mitochondria are sites where eukaryotes undergo oxidative metabolism, and are the sites where sugars, fats, and amino acids ultimately oxidize and release energy. The common pathway responsible for the final oxidation by mitochondria is tricarboxylic acid cycle and oxidative phosphorylation. In addition to the primary functions of ATP synthesis that provide energy to cells, mitochondria also assume a number of other physiological functions, such as the storage of calcium ions, participation in processes such as cell differentiation, cellular information transfer and apoptosis, and the ability to regulate cell growth and cell cycle, cholesterol synthesis and certain heme.

Abnormalities in mitochondrial structure, morphology, function, etc. are associated with the development and progression of many diseases. Active oxygen radicals generated by mitochondria during the process of producing energy by using oxygen gas damage organisms and cause oxygen toxicity, which are the first cause of aging of the organisms. Human mitochondria are problematic in causing mitochondrial diseases, which are a large class of inherited metabolic diseases, and are mainly accumulated on tissues with high energy demands, including mitochondrial myopathy, multisystem diseases, cardiomyopathy, progressive extraocular muscle paralysis, leer hereditary optic neuropathy, mitochondrial myopathy, diabetes, deafness, ataxia chorea and other diseases. In recent years, mitochondria have been found to be involved in cancer cell metastasis, and when the function of mitochondria in tumor cells is altered, cell migration is promoted, ultimately leading to successful tumor metastasis. Furthermore, mitochondrial mediated autophagy plays an important role in many physiological and pathological processes.

Many studies are currently directed to the detection of mitochondrial morphology and function, and antibodies specific for mitochondrial outer membrane proteins play an important role in these studies and can be used in detection techniques such as mitochondrial isolation, immunofluorescence, immunohistochemistry, flow cytometry, and the like. Since the outer mitochondrial membrane proteins have important biological functions and only a part of the structural domain is located outside the mitochondria, selecting which outer mitochondrial membrane protein as a target not only affects the effects of mitochondrial separation and detection, but also affects the function of mitochondria. The structure and the function of the mitochondrial outer membrane protein OMP25 are simpler, the sequence specificity is high, the conservation degree among different species is higher, and the mitochondrial outer membrane protein OMP25 is an ideal mitochondrial outer membrane recognition target.

Disclosure of Invention

The invention aims to provide a monoclonal antibody of an anti-mitochondrial outer membrane protein OMP25, which is HC01-R001, has subtype of IgG1 and kappa type and can be specifically combined with the outer membrane region of the human mitochondrial outer membrane protein OMP 25; the hybridoma cell for producing the monoclonal antibody is obtained by fusing, screening, cloning and passaging the immunized BALB/C mouse spleen lymphocytes and mouse myeloma cell SP2/0, and can stably secrete HC01-R001. The heavy chain amino acid sequence of the monoclonal antibody is shown as SEQ ID No.1, and the light chain amino acid sequence is shown as SEQ ID No. 2; the nucleotide sequence of the heavy chain coding nucleic acid molecule is shown as SEQ ID NO.3, and the nucleotide sequence of the light chain coding nucleic acid molecule is shown as SEQ ID NO. 4.

A method for preparing the monoclonal antibody HC01-R001 comprises the following steps:

(1) Immunization of mice: selecting a male BALB/C mouse with 6 weeks of age, and immunizing the mouse with human mitochondrial outer membrane protein OMP25 whole protein; total immunization was 3 times,

day

1, 21, 35, 100 μg antigen plus an equal volume of adjuvant mixture injected each time inside the thigh of the mice; mice were treated on day 38 for fusion experiments;

(2) Culture of mouse myeloma cells: culturing the mouse myeloma cell SP2/0 and keeping a good growth state for hybridoma cell fusion;

(3) Cell fusion: taking BALB/C mouse peritoneal macrophages as feeder cells, inoculating the feeder cells into a 96-well plate, culturing the feeder cells for 1 day by using HAT culture medium containing 20% FCS, taking the mouse spleen lymphocytes in the step (1), mixing and centrifuging the spleen lymphocytes with myeloma cells in the step (2), mediating cell fusion by polyethylene glycol (PEG 6000), and inoculating the fused cells into a cell culture plate for culturing;

(4) Selection of hybridoma cells: selectively culturing the cultured cells in a HAT culture medium, sucking the supernatant to perform antibody identification when the cell colony grows to a certain size, and screening positive clones;

(5) Cloning and culturing hybridoma cells: diluting positive clones to 96-well plates by a limiting dilution method, enabling at most 1 hybridoma cell to grow in each well, sucking supernatant after cell colonies are formed for antibody identification, performing expansion culture on the positive clones, and performing physical and chemical property analysis on the antibodies;

(6) Ascites induction in mice: 1 week before hybridoma cell inoculation, BALB/C mice were intraperitoneally injected with 0.5 mL/mouse of pristane, followed by 5X 10 inoculation ⁶ Positive hybridoma cells, ascites were collected after 10 days, centrifuged, antibody titers were determined, and monoclonal antibodies were purified using Protein G affinity columns.

It is another object of the present invention to provide the use of the monoclonal antibody HC01-R001 according to the above, wherein the use of the monoclonal antibody HC01-R001 is used in performing experiments such as ELISA, western Blotting, immunofluorescence, immunohistochemistry, flow cytometry detection and the like of human mitochondrial outer membrane proteins.

The invention has the beneficial effects that: HC01-R001 specifically recognizes mitochondrial outer membrane protein OMP25, the protein has simpler structure and function, less influence on mitochondria, high sequence specificity and higher conservation degree among different species, and is an ideal mitochondrial outer membrane recognition target. The monoclonal antibody has wide application, and can be applied to detection such as ELISA, western Blotting, immunofluorescence, immunohistochemistry, flow cytometry and the like of human mitochondrial outer membrane proteins, and immunoprecipitation experiments.

Drawings

FIG. 1 purification of monoclonal antibodies HC01-R001.

FIG. 2 immunoglobulin subtype of monoclonal antibody HC01-R001.

FIG. 3 shows ascites fluid and culture supernatant titers of monoclonal antibodies HC01-R001.

FIG. 4 monoclonal antibody HC01-R001 ELISA for detection of human mitochondrial outer membrane proteins.

FIG. 5 monoclonal antibody HC01-R001 flow cytometry detected human mitochondria.

FIG. 6 shows immunofluorescence detection of human mitochondria by monoclonal antibody HC01-R001.

FIG. 7 shows the detection of human mitochondrial outer membrane proteins by monoclonal antibodies HC01-R001 Western Blotting.

FIG. 8 monoclonal antibodies HC01-R001 immunohistochemical detection of human mitochondrial outer membrane protein.

Detailed Description

For a further understanding of the present invention, preferred embodiments of the invention are described below with reference to the drawings and examples, but it should be understood that these descriptions are intended only to illustrate the invention further and are not limiting to the claims of the invention.

EXAMPLE 1 preparation of anti-human mitochondrial envelope protein OMP25 monoclonal antibody HC01-R001

(1) Antigen preparation: selecting a complete sequence of human mitochondrial outer membrane protein OMP25, adding a 6His tag at the C end, inserting a pet21a vector, taking escherichia coli BL21 as an expression host cell, expressing OMP25 protein, and purifying the protein by using the His tag protein.

(2) Immunization of mice: 6 male BALB/C mice of 6 weeks of age were selected and immunized for the first time, purified OMP25 protein with adjuvant at 1:1 volume was mixed well and the total volume was 600. Mu.L. Each mouse was injected intramuscularly at 100. Mu.L (containing 100. Mu.g antigen) on the inner thigh. Immunization was boosted once on day 21 in the same manner. The titer of the tail vein blood sampling detection antibody reaches 1:100000 on the 35 th day, and then 100 mug antigen is injected into the tail vein for boosting immunization for 1 time. Cell fusion was performed on day 38.

(3) Culture of mouse myeloma cells: the day before fusion, SP2/0 myeloma cell lines from BALB/C mice are subcultured with DMEM medium containing 10% FBS, and a good growth state is maintained for hybridoma cell fusion;

(4) Cell fusion: BALB/C mice abdominal macrophages are used as feeder cells, inoculated in 96-well plates, cultured for 1 day by HAT medium containing 20% FCS, spleen of immunized mice is taken, spleen cells are separated by a pressure water injection method, and the mice are centrifugally washed for 2 times for standby. SP2/0 cells were collected, washed 2 times by centrifugation, and then washed 1.0X10-fold ⁸ Spleen cells and 1.0X10 ⁷ Mixing SP2/0 cells, centrifuging, removing supernatant, dropwise adding 0.8mL 50% PEG6000 at 37deg.C in 60-90 seconds, gently shaking the centrifuge tube, standing for 1min, adding 1mL serum-free RPMI1640 medium in 1min according to the principle of quick speed and slow speed, adding 2mL serum-free RPMI1640 medium in 2min, adding 7mL serum-free RPMI1640 medium in 3min, and adding 30-40mL serum-free RPMI1640 medium in 4 min. Centrifuging at low speed at 800 rpm for 5-10min, culturing in HAT medium containing 20% FCS, inoculating into 96-well plate containing feeder cells, and placing in 5% CO at 37deg.C ₂ Incubator culture.

(5) Selection of hybridoma cells: changing 1/2 culture solution (HAT) every 4 days, changing to HT-containing culture medium after 10 days, continuously culturing for 2 weeks, sucking culture supernatant, diluting, and screening positive clone by indirect ELISA method, wherein P/N is more than or equal to 2.1.

(6) Cloning of hybridoma cells: positive clones were selected for limiting dilution. After accurately counting the cells, diluting the cells into 10 cells/mL of cell suspension by using RPMI1640 complete culture medium, inoculating the cell suspension into a 96-well plate of the existing feeder cells, observing the growth condition of the cells after 0.1mL of each well for 7-10 days, detecting the antibody level of the supernatant, and selecting 5 monoclonal growth holes with the highest antibody titer for recloned culture. This method can be repeated several times until the monoclonal hole antibody detection positive rate is 100%.

(7) Ascites induction in mice: 1 week before hybridoma cell inoculation, BALB/C mice were intraperitoneally injected with 0.5 mL/mouse of pristane, followed by 5X 10 inoculation ⁶ Positive hybridoma cells, ascites were collected after 10 days and centrifuged to determine antibody titer.

(8) Purification of monoclonal antibodies: the monoclonal antibody in the ascites of the mice is purified by adopting an affinity purification method. Pre-loading Protein G cross-linked Sepharose affinity purification column, diluting ascites with buffer solution, passing through the column once again, washing 3 times with buffer solution, and eluting with eluent. The eluted antibody concentration was determined and dialyzed overnight in 100 volumes of PBS. The dialyzed antibodies were identified by SDS-PAGE gel electrophoresis, as shown in FIG. 1, and the purified monoclonal antibodies were of higher purity.

(9) Subtype identification of monoclonal antibodies: the mouse monoclonal antibody immunoglobulin typing kit of Serotec company was used for analysis, and the procedure was performed according to the instructions. The results showed that monoclonal antibodies HC01-R001 were IgG1, kappa type (FIG. 2). In addition, the monoclonal antibody secreted by the hybridoma cell HC01-R021 of 1 strain is also IgG1 and kappa type.

(10) The HC01-R001 hybridoma cell line was cloned 3 times, and cultured for 6 months, and the secreted antibody was stable. The cell line grew well after resuscitation by freezing in liquid nitrogen, and antibody secretion did not decline (fig. 3). Sequencing, wherein the HC01-R001 heavy chain amino acid sequence is shown as SEQ ID No.1, and the light chain amino acid sequence is shown as SEQ ID No. 2; the nucleotide sequence of the heavy chain coding nucleic acid molecule is shown as SEQ ID NO.3, and the nucleotide sequence of the light chain coding nucleic acid molecule is shown as SEQ ID NO. 4.

EXAMPLE 2 application of monoclonal antibodies HC01-R001 and HC01-R021 in combination to ELISA detection of human mitochondrial outer membrane protein OMP25

The human mitochondrial outer membrane protein OMP25 is detected by a double antibody sandwich method, and the experiment is realized by the following ways:

(1) Monoclonal antibody HC01-R021 was diluted in 10mM carbonate buffer, pH 9.6, at a concentration of 1. Mu.g/mL, added to a 96-well ELISA plate at 0.1 mL/well, and coated overnight at 4 ℃.

(2) Plates were washed 3 times with 10mM pH 7.4PBS-Tween 20.

(3) 2%BSA 10mM pH 7.4PBS is closed for 2h.

(4) And washing the plate.

(5) Setting a standard substance hole 10 holes on an enzyme-labeled coating plate, respectively adding 100 mu L of standard substance into the first hole and the second hole, then adding 50 mu L of standard substance diluent into the first hole and the second hole, and uniformly mixing; then 100 mu L of each of the first hole and the second hole is added into a third hole and a fourth hole respectively, 50 mu L of standard substance diluent is added into the third hole and the fourth hole respectively, and the mixture is uniformly mixed; then 50 mu L of each of the third hole and the fourth hole is discarded, 50 mu L of each of the third hole and the fourth hole is added into the fifth hole and the sixth hole respectively, 50 mu L of standard substance diluent is added into the fifth hole and the sixth hole respectively, and the mixture is uniformly mixed; mixing, adding 50 μL of each of the fifth and sixth holes into the seventh and eighth holes, adding 50 μL of each of the standard substance diluents into the seventh and eighth holes, mixing, adding 50 μL of each of the seventh and eighth holes into the ninth and tenth holes, adding 50 μL of each of the standard substance diluents into the ninth and tenth holes, mixing, and discarding 50 μL of each of the ninth and tenth holes. (50. Mu.L of each well after dilution, and concentrations of 24. Mu.g/L, 16. Mu.g/L, 8. Mu.g/L, 4. Mu.g/L, and 2. Mu.g/L, respectively).

(6) Blank holes (blank control holes are not added with samples and enzyme-labeled reagents, and the rest steps are the same) and sample holes to be tested are respectively arranged. And adding 40 mu L of sample diluent into a sample hole to be detected on the enzyme-labeled coated plate, and then adding 10 mu L of sample to be detected. And (3) adding a sample to the bottom of the ELISA plate hole, so as not to touch the hole wall as much as possible, and slightly shaking and uniformly mixing.

(7) The plates were then covered with a plate membrane and incubated at 37℃for 30 minutes.

(8) And washing the plate.

(9) Monoclonal antibody HC01-R001 was diluted into the sample dilution to a final concentration of 100ng/mL, 100. Mu.L was added to each well, and incubated for 30 minutes at 37℃after membrane sealing with a sealing plate.

(10) And washing the plate.

(11) HRP-labeled goat anti-mouse secondary antibody 1:10000 dilutions were made into sample dilutions, 100 μl was added per well, and incubated for 30min at 37℃with a plate membrane seal.

(12) And washing the plate.

(13) 50 mu L/well of substrate (TMB) was added and the reaction was carried out at room temperature in the dark for 5-10min.

(12) The OD was measured at 450nm by adding 50. Mu.L/well of stop solution.

(13) A standard curve (FIG. 4) is drawn, and the OD value has a better corresponding curve relationship with the OMP25 protein concentration.

EXAMPLE 3 application of monoclonal antibodies HC01-R001 to human mitochondrial flow cytometry detection

293T cells were selected and mitochondrial in the cells were detected by flow cytometry using the mitotracker red as a tracer for the mitochondria and monoclonal antibodies HC01-R001, comprising the steps of:

(1) 293T cells were cultured to logarithmic growth phase and incubated with the addition of a mitotracker red for 10-15min, following the instructions.

(2) Cells were digested with pancreatin, collected, and washed 3 times with PBS.

(3) Cells were fixed with 80% acetone solution at 4℃for 30min and then washed 2 times with 1mL of PBS.

(4) 200. Mu.L of PBS blocking solution containing 3% BSA was added to each well, and after blocking for 30min at room temperature, the wells were washed 2 times with 1mL PBS.

(5) Monoclonal antibody HC01-R001 was diluted into PBS blocking solution, the final concentration of antibody was 500ng/mL, 200. Mu.L was added to each well, and incubated at 4℃for 2 hours.

(6) The primary antibody was blotted off and washed 2 times with 1mL of PBS.

(7) FITC-labeled goat anti-rabbit secondary antibody was diluted 1500-fold, 200. Mu.L was added to each well, and incubated at room temperature in the dark for 1h.

(8) The secondary antibody was blotted off and 500 μl of PBS containing 10% fbs was added to each well and washed once again with PBS.

(9) The results of the flow cytometry detection are shown in FIG. 5, and HC01-R001 is consistent with the result of Mitotracker staining, which suggests that HC01-R001 can be used for flow cytometry.

EXAMPLE 4 application of monoclonal antibody HC01-R001 to human mitochondrial immunofluorescence detection

(2) Cells were washed 3 times with PBS.

(6) The primary antibody was blotted off and washed 2 times with 1mL of PBS.

(8) After washing the secondary antibody, PBS 1: diluting DAPI by 500, adding 200 mu L of DAPI into each hole, and incubating for 5min at room temperature in dark place;

(9) Sucking DAPI dye liquor, and washing with PBS for 2 times;

(10) As shown in FIG. 6, when the mitochondria in the cells are observed by a fluorescence microscope, the 293T cells are stained by HC01-R001 (green), mitotracker (red) and DAPI (blue), and the HC01-R001 is highly coincident with the staining range of the mitochondrial marking kit, so that the HC01-R001 can be used for mitochondrial immunofluorescence marking.

EXAMPLE 5 application of monoclonal antibodies HC01-R001 to Western Blotting detection of human mitochondrial outer membrane protein OMP25

The supernatant was aspirated from the cultured 293T cells, washed 2 times with pre-chilled PBS, and then cell lysates were added and incubated on ice for 10min.

Scraping cells with a cell scraper, centrifuging at high speed, collecting supernatant, adding protein loading buffer solution, and boiling for 10min.

SDS-PAGE was performed and transferred to PDVF membrane, and blocked with TBST in which 5% skimmed milk powder was dissolved for 20min.

After washing 3 times with TBST, HC01-R001 solution (1. Mu.g/mL concentration) prepared by TBST was added and incubated overnight at 4 ℃.

After 3 times of TBST washing, the goat anti-mouse secondary antibody prepared by TBST dissolved with 5% skimmed milk powder is incubated for 1h at room temperature.

TBST was washed 3 times for post exposure.

As a result, as shown in FIG. 7, the cell samples were free of bands, indicating that HC01-R001 can specifically bind to OMP25 antigen, and as low as 2ng of antigen can be detected.

EXAMPLE 6 application of monoclonal antibodies HC01-R001 to human mitochondrial outer membrane protein OMP25 immunohistochemical detection

(1) Human liver tissue sections were dewaxed to water.

(2) 0.3% h2o2 methanol: endogenous peroxidase (198 ml methanol or water+2 ml 30% h2o 2) was blocked for 20min at room temperature. Washing with tap water.

(3) Antigen retrieval: the pH7.2 TBS buffer was washed three times and trypsin digested at 37℃for 30 minutes.

(4) The pH7.2 PBS buffer was washed three times.

(5) Normal serum was added dropwise at 37 ℃ for 20 minutes.

(6) Excess serum was removed by shaking, HC01-R001 was added thereto at 37℃for 30 minutes.

(7) The pH7.2 TBS buffer was washed three times.

(8) HRP-labeled secondary antibody was added dropwise at 37 ℃ for 30 min.

(9) The pH7.2 TBS buffer was washed three times.

(10) DAB color development is carried out for 3-10 minutes.

(11) And (5) washing with water.

(12) Hematoxylin light dyeing, bluing, dehydrating, transparent and sealing.

The results are shown in fig. 8, where all cellular mitochondria showed good results.

Sequence listing

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<120> monoclonal antibody against human mitochondrial outer membrane protein OMP25 and use thereof

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cccatcgtgc agtccttcaa ccggggcgac tgc 693

Claims

1. A monoclonal antibody of anti-human mitochondrial outer membrane protein OMP25, which is characterized in that the monoclonal antibody is HC01-R001, the subtype of the monoclonal antibody is IgG1 and kappa type, and can be specifically combined with the outer membrane region of human mitochondrial outer membrane protein OMP 25; the hybridoma cell for producing the monoclonal antibody is obtained by fusing, screening, cloning and passaging process screening of immunized BALB/C mouse spleen lymphocytes and mouse myeloma cells SP2/0, and can stably secrete HC01-R001, wherein the heavy chain amino acid sequence of the monoclonal antibody is shown as SEQ ID No.1, and the light chain amino acid sequence is shown as SEQ ID No. 2; the nucleotide sequence of the heavy chain coding nucleic acid molecule is shown as SEQ ID NO.3, and the nucleotide sequence of the light chain coding nucleic acid molecule is shown as SEQ ID NO. 4.

2. The use of monoclonal antibody HC01-R001 according to claim 1, in the preparation of an ELISA, western Blotting, immunofluorescence, immunohistochemistry, flow cytometry detection reagent for detecting human mitochondrial outer membrane proteins.