CN117050190A - Preparation of chicken angiotensin converting enzyme 2 (ACE 2) monoclonal antibody - Google Patents

Abstract

The invention relates to the technical field of biology, in particular to a monoclonal antibody. The monoclonal antibody can bind to angiotensin converting enzyme 2 (ACE 2) protein. The antibody titer is 1: 512000, and the specific binding of the chicken ACE2 protein can be successfully realized by using a mouse anti-chicken ACE2 monoclonal antibody. The antigen and the monoclonal antibody can lay a foundation for further developing a detection kit. ACE2 is used as a negative regulating molecule of an RAS system, and plays the roles of resisting injury, resisting fibrosis and the like through targeted degradation of AngII; as the main functional receptor of acute severe respiratory syndrome coronavirus (SARS-CoV), has the function of protecting lung injury; ACE2 plays an important role in cooperation with amino acid transport carrier transport, amino acid malabsorption, intestinal microecological balance and gastrointestinal tract inflammation. The invention is of great significance both in basic research and clinical use.

Description

Preparation of chicken angiotensin converting enzyme 2 (ACE 2) monoclonal antibody

Technical Field

The invention belongs to the technical field of biology, and particularly relates to preparation of a monoclonal antibody of chicken angiotensin converting enzyme 2 (Angiotensin converting enzyme 2, ACE 2).

Background

Angiotensin converting enzyme 2 (Angiotensin converting enzyme, ACE 2) is an important member of the Renin Angiotensin System (RAS) found in 2000, and the action of Angiotensin converting enzyme (Angiotensin converting enzyme, ACE) is counteracted by degrading Ang ii and producing Angiotensin1-7 (Ang 1-7 ), creating a protective axis for the RAS (i.e., ACE2-Ang1-7-Mas axis). This axis has been shown to be effective in producing beneficial effects on metabolic, immune and cardiovascular dysfunction by blocking apoptosis, fibrosis, oxidative stress and inflammation, thereby ameliorating metabolic dysfunction and the like. The level of ACE2 expression is significantly linked to the maintenance of homeostasis.

At present, detection methods of ACE2 mainly focus on methods such as real-time fluorescence quantitative PCR, westem blot and ELISA. Real-time fluorescence quantitative PCR and Westem blot, the experimental steps are complicated, a lot of time is required, and the method is not suitable for rapid detection of a large number of samples. The ELISA detection method has the characteristics of high selectivity, high sensitivity, high speed and the like, and is widely applied to the quantitative detection field of ACE 2. Commercial ACE2 primary antibodies applied to ELISA methods are prepared by antigen fragments, fragments selected by different companies are different, and the prepared antibodies have certain species specificity according to whether the selected fragments are conserved or not. In addition, most commercial primary antibodies to ACE2 are applied to animals such as humans and mice, but lack of primary antibodies to ACE2 applied to birds restricts the functional research of ACE2 in birds. Therefore, it is important to prepare an ACE2 primary antibody applied to poultry and establish a chicken ACE2 quantitative detection method based on the primary antibody.

In the research of the invention, a prokaryotic expression system is utilized to express pET32a-ACE2 recombinant protein, and a mouse and chicken ACE2 monoclonal antibody is prepared by purifying the pET32a-ACE2 recombinant protein. The research of the invention provides a method and a detection basis for the functional research of ACE2 on birds such as chickens.

In view of this, the present invention has been made.

Disclosure of Invention

The invention aims to play the roles of protecting the organism such as injury resistance, fibrosis resistance and the like by targeting degradation of AngII according to ACE2 as a negative regulating molecule of an RAS system; as the main functional receptor of acute severe respiratory syndrome coronavirus (SARS-CoV), has the function of protecting lung injury; the method can cooperate with amino acid transport carrier transport, plays key roles in amino acid malabsorption, intestinal microecology balance and gastrointestinal tract inflammation, lacks ACE2 antibodies applied to birds so far, can prepare corresponding monoclonal antibodies by prokaryotic expression of ACE2 protein, and can provide methods and detection basis for functional research of ACE2 in birds such as chickens.

In order to achieve the above purpose, the invention adopts the following technical scheme:

a method for preparing chicken ACE2 monoclonal antibody, which comprises the following steps:

s1, transferring an ACE2 prokaryotic expression vector of a chicken into BL21 competent cells, performing IPTG induced expression to obtain pET32a-ACE2 recombinant protein, then performing KCl dyeing, cutting off a silver-white target strip, adding PBS, shaking and mixing uniformly, and repeatedly freezing and thawing for 3 times at-20 ℃ to obtain the purified ACE2 recombinant protein.

S2, 5 SPF-class BALB/c female mice in the experimental group are subjected to tail-breaking blood collection (serving as negative control) respectively, and then are immunized. Primary immunization: freund's complete adjuvant was thoroughly mixed with 250ug of antigen. Each experimental group mice were immunized subcutaneously at 4 sites, each site 450uL. The immunization period was 56 days, for a total of 4 immunizations. Titers were determined by indirect ELISA at 7d after four immunizations.

S3, taking spleens of mice in the experimental group, separating spleen cells, mixing the spleens with mouse myeloma cells SP2/0 in a cell number ratio of 10:1, adding fusion agent PEG4000 into cell precipitates after centrifugation for fusion, transferring the cell precipitates into a 96-well plate for screening by using a selective medium containing 1% HAT, and then carrying out ELISA detection screening on cell culture supernatants after half-changing every 2 days and taking 10-14 d.

S4, for screening the titer of the mouse anti-ACE 2 monoclonal antibody, adding an ELISA plate coated with an antigen ACE2 protein into a cell culture supernatant, incubating for 1 hour at 37 ℃, washing PBST for 5 times, adding a horseradish peroxidase-labeled goat anti-mouse IgG secondary antibody, incubating for 1 hour at 37 ℃, washing PBST for 5 times, adding a TMB substrate for color development, and adding 2M H ₂ SO ₄ Termination of the reaction, enzymeThe reader reads OD450. A hybridoma cell with strong positive binding to ACE2 protein is obtained. And then, carrying out monoclonalization on the hybridoma cells by adopting a limiting dilution method, and freezing and preserving the seeds after the hybridoma cells are subjected to expansion culture.

S5, injecting Pristane into the abdominal cavity of the mouse 5-7 days before injecting the hybridoma cells, taking the hybridoma cells for intraperitoneal injection, collecting ascites of the mouse by intraperitoneal puncture after 10-14 days, and standing at 37 ℃ for half an hour and then standing at 4 ℃ overnight. The next day the blood clot was removed by centrifugation, 3 volumes of phosphate buffer was added to the ascites, centrifuged and the supernatant was filtered through a filter membrane and purified by protein A column.

S6, the monoclonal antibody detected by Western blot and immunofluorescence is well combined with ACE2 protein, and has good immunoreactivity.

The invention has the beneficial effects that:

the mouse anti-chicken ACE2 monoclonal antibody prepared by the invention provides a method and a detection basis for functional research of ACE2 on poultry such as chicken and the like. But ACE2 has the functions of anti-inflammation, anti-injury and the like, and can provide a new thought for treating intestinal inflammation of chickens, thereby laying a foundation for sustainable development of poultry industry.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 shows KCL staining results;

FIG. 2 shows hybridoma cell morphology after 11d cell fusion;

FIG. 3 shows the morphology of hybridoma cells after a third subcloning;

FIG. 4 is a graph showing the result of immunoblotting for detecting ACE2 monoclonal antibody of the present invention;

FIG. 5 is a graph showing immunofluorescence results of detection of an ACE2 monoclonal antibody of the present invention.

Detailed Description

The following description of the embodiments of the present invention will be made apparent and fully in view of the accompanying drawings, in which some, but not all embodiments of the invention are shown. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any method or material similar or equivalent to those described may be used in the present invention.

The present invention will be further described with reference to specific examples and comparative examples.

Example 1 expression and purification experiments of the antigen ACE2 protein

IPTG is selected to carry out large-scale induction expression of pET32a-ACE2 recombinant protein, bacterial liquid is collected after induction is carried out for 10 hours at 28 ℃, centrifugation is carried out for 15 minutes at 8000rpm/min, bacterial liquid sediment is collected, PBS is used for washing for 2 times, ultrasonic wave is utilized to crack thalli for 5 seconds, interval is 10 seconds, then SDS-PAGE loading buffer solution is added to boil for 10 minutes at 100 ℃.

SDS-PAGE is carried out according to a conventional method, after electrophoresis, the unloaded gel is placed into a clean large plate, 0.25mol/L KCl is dyed on a shaking table for 10min, after dyeing is finished, a target silver-white band is cut off by using a surgical blade, after washing for 5 times by distilled water, the gel is transferred into a clean small bag, 500uLPBS is added for shaking and mixing, and repeated freeze thawing is carried out for 3 times at-20 ℃ to obtain the purified ACE2 recombinant protein. KCL staining results as in fig. 1, the ace2 protein was stained silvery white.

EXAMPLE 2 immunization of BALB/c mice

10 SPF-class BALB/c female mice were randomly divided into 5 control groups and 5 experimental groups, which were then immunized after tail-cutting blood collection (as negative control). Primary immunization: 1.5mL of Freund's complete adjuvant was thoroughly mixed with 1.5mL of antigen (980 ug/mL) in preparation. Each experimental group mice were immunized subcutaneously at 4 sites, each site 450uL. Control group, directly immunized with 450uL Freund's complete adjuvant. The immunization period was 56 days, for a total of 4 immunizations. On day 7 after four immunizations, the eyebox of the mice was sampled, the collected blood was inactivated at 37 ℃ for 2 hours, and finally, the blood was allowed to coagulate at 4 ℃ overnight to release serum, the coagulated blood was centrifuged, and the supernatant was collected to obtain serum.

EXAMPLE 3 serum potency detection

Serum titers were determined using an indirect ELISA method. Antigen ACE2 coating: the white feather broiler pET32a-ACE2 purified recombinant protein is diluted to be 0.1, 0.2, 0.4, 0.8, 1.6 and 3.2ug/mL sequentially by antigen coating liquid, 100uL is dripped into each hole of a 96-well plate, and the mixture is coated overnight at 4 ℃. The antigen solution was poured off, 100uL of blocking solution was added to each well and incubated for 1h at room temperature. Pouring the sealing liquid, beating on the absorbent paper, and washing the plate with the washing liquid for 3 times. The antiserum to be tested was added to well 1:6400, and each subsequent well was diluted in a multiple of 1: 819200 on this basis, and incubated at 37℃for 1h. Pouring out the liquid, washing the plate with the washing liquid for 3 times, and beating to dry. 100uL of HRP-labeled mouse anti-rabbit IgG (1:5000 dilution) was added to each well and incubated for 1h at 37 ℃. Pouring out the liquid, washing the plate with the washing liquid for 3 times, and beating to dry. 100uL of TMB developer was added to each well and incubated at 37℃for 15min. 100uL of stop solution was added to each well, and the absorbance at 450nm was read on a microplate reader. The serum titers of the experimental groups are shown in Table 1, the antibodies of 5 mice in the experimental groups are positive, the titers of the antibodies are higher than 1:51200, and the P/N values are higher than 9.9. The highest antibody titers of experimental groups 3 and 4 were 1:409600.

Table 1 test group serum potency assay

EXAMPLE 4 cell fusion

1. Preparation of feeder cells

The day prior to fusion, non-immunized BALB/c mice were selected for the experiment, and the mice were enucleated and bled to death. The mice were sterilized in 75% ethanol solution for 10min with the left abdomen facing upward. A small opening was cut at the midline between the legs of the mice, torn along the small opening, and the peritoneum of the mice was fully exposed. By usingThe syringe infuses 5mL of RPMI-1640 medium into the peritoneum. The abdomen is continuously massaged by using the alcohol cotton ball, and the liquid in the abdominal cavity is pumped back into the 15mL centrifuge tube. The intraperitoneal macrophages were rinsed thoroughly by repeating 3 times. Centrifuging the cell suspension in the centrifuge tube at 800rpm for 5min, discarding the supernatant after the centrifugation is finished, repeating for 1 time, and washing macrophages in the abdominal cavity. Resuspension was performed with 20% RPMI-1640 medium (RPMI-1640+fetal bovine serum+1% HAT medium+penicillin and streptomycin double antibody), and the suspension was spread evenly in the middle of 96-well plates, with one round of sterile PBS. 96-well plates were placed at 37℃with 5% CO ₂ Culturing in a cell culture incubator.

2. Collection of myeloma SP2/0 cells

On the day of fusion, 2 bottles of SP2/0 cells in log phase and round and bright are decanted, about 3mL of RPMI-1640 medium is aspirated, the cells on the cell walls are quickly and gently blown off with a Pasteur pipette, the solution is transferred to a 15mL centrifuge tube, and after centrifugation to discard the supernatant, 5mL of RPMI-1640 is added for resuspension. The cell suspension was taken and counted under a microscope using a blood cell counting plate. After counting the cell suspension was placed at 37℃with 5% CO ₂ The cells are kept in an incubator for later use.

3. Preparation of spleen lymphocyte B cells

The mice with highest indirect ELISA detection antibody titers after 4 times of immunization are selected for intraperitoneal injection of 200ug of chicken ACE2 recombinant protein for impact immunization, and fusion is carried out after 3 days. The mice were sacrificed by cervical scission, sterilized in 75% ethanol solution for 10min, transferred to an ultra clean bench, and left abdomen placed up. Cutting off the outer epidermis of the abdomen, tearing off the outer epidermis, exposing the abdomen of the mouse, tearing off the inner epidermis of the mouse, and determining the position of the spleen. The spleens of the mice were removed and placed in RPMI-1640 dishes to remove fat and connective tissue from the spleens. A70 um nylon cell filter screen was placed over a 50mL EP tube, the spleen on the screen was ground with a syringe white push rod and constantly rinsed with RPMI-1640, allowing the spleen cells to be released into the RPMI-1640, and the above procedure was repeated until only connective tissue was left. Spleen lymphocytes were counted using the method described above.

4. Cell fusion

According to splenic lymphocyte B cells: mixing SP2/0 cells at a ratio of 10:1, adding RPMI-1640, centrifuging and discarding the supernatant. The EP tube was then rotated to loosen the cells. 1mL of PEG4000 preheated at 37℃was added to the tube wall over 1min, and the tube was water-bath at 37℃for 1min. 10mL of RPMI-1640 was added over 5min and the addition was run as above, from slow to fast. Fusion was then terminated by a single addition of 30 mLRPMI-1640. Slowly invert and centrifuge the supernatant. Water bath at 37deg.C for 10min, then add 20% RPMI-1640, mix gently, and add to feeder cells prepared the day before. Carefully put in 37℃and 5% CO ₂ Culturing in an incubator. The cell morphology was observed to see if there was a grape-like cell mass.

Example 5 selection of hybridoma cells and subcloning

As shown in FIG. 2, the cells fused with 11d were observed under a microscope, and a decrease in the number of living cells and an increase in cell debris were observed, and a vigorous growth of a cluster-like hybridoma was observed. 360 wells were fused together, 209 wells were successfully obtained, and the fusion rate was 58%. Taking the supernatant of the hybridoma cells which are successfully fused as the supernatant to be detected, taking the SP2/0 cell supernatant as a negative control, and determining the titer of the supernatant of the hybridoma cells which are successfully fused by the indirect ELISA. The results of potency assays are shown in Table 2, and 1D10, 3G9, 4B7, 4B9, 4C3, 4C8, 4D5, 4E3, 4E5, 4G4, 4G9, 4G10 with higher OD450 values were selected for subsequent subcloning in a total of 12 wells.

TABLE 2 determination of cell titers of fusion hybridomas OD450 s

Subcloning of positive wells was performed using limiting dilution as follows: and sucking up the cell culture solution to be subcloned, sucking up the new cell in the re-suspension hole of the culture solution, and then carrying out double-ratio dilution to ensure that the dilution multiple has a cell hole with only one cell mass. Then uniformly dripping into a new 96-well cell plate (feeder layer is added in advance), and placing at 37 ℃ and 5% CO ₂ Culturing in an incubator. Then, the cell state was observed, and when the cells grew to 1/2 of the wells, the indirect ELISA assay titers were performed again. HAT content and HT content in the medium at the second subcloning 1:1, at the thirdWhen subcloned, 1% HT was used throughout. When 100% of the cell plates are positive, the culture can be expanded. The results of titer detection are shown in Table 3, and positive monoclonal hybridoma cells are selected for mass expansion culture and frozen storage by judging that the OD450 value is more than 0.5. And (3) uniformly blowing and mixing cells in the 96-hole cell plate, transferring the cells to a 48-hole cell plate for culture, transferring the cells to a 24-hole plate and a 6-hole plate, and transferring the cells to a cell bottle for culture. The enlarged culture state of the hybridoma cells is shown in fig. 3, and the cells are round and bright and grow vigorously.

TABLE 3 determination of subclone hybridoma cell titers OD450 s

EXAMPLE 6 preparation and purification of ascites monoclonal antibodies

1. Preparation of ascites monoclonal antibody

4 female BALB/c mice of 6 weeks old were ascites injected with 1mL liquid paraffin to sensitize the body. Hybridoma cells with better growth status were collected after 7d and counted. Each mouse was intraperitoneally injected with 1 x 106 hybridoma cells. After 2 weeks, the mice are Mao Pengluan, the abdomen is expanded and difficult to exercise, when no reaction is counteracted after the mice are lifted, the mice are penetrated from the outer side areas of two nipples of the abdomen by a syringe needle, the ascites after the penetration flows out along the needle, blood cells can be separated after the collected ascites is centrifuged, and clear upper liquid is taken after the centrifugation, namely the ascites is obtained, and the mice are preserved at the temperature of minus 20 ℃.

Protein A affinity chromatography purification

Protein A was mixed up and down and added to 10mL of Protein A to PD-10 column. The murine anti-chicken ACE2 monoclonal antibody was diluted with PBS binding buffer and filtered through a 0.45um filter. The flow rate was set to 0.5mL/min and the UV monitor value was set to 0.05. Protein a column was run with 10 volumes of PBS binding buffer. 12mL of sample was added, the column was washed with PBS binding buffer, and unbound protein was removed. The filtrate was collected by passing 5 volumes of citric acid eluent through the column and stopping the collection when the UV detection was below 0.05. Mixing the collected liquid with glycerol at a ratio of-80 deg.C for preservation. The titer of the purified ACE2 monoclonal antibody is detected by the indirect ELISA method, and the result of the titer is shown in table 4, wherein the titer of the monoclonal antibody ascites can reach more than 1: 512000, which indicates that the prepared monoclonal antibody ascites has high titer.

TABLE 4 determination of ascites titers

EXAMPLE 7 identification of monoclonal antibodies

1. Westem blot identification of monoclonal antibodies

After electrophoresis of ACE2 recombinant proteins with different concentrations by polyacrylamide gel electrophoresis (SDS-PAGE), PVDF membrane transfer was performed by wet transfer (100V, 90 min). PVDF membrane was blocked for 3h at 5% BSA room temperature, then blocked overnight at 4℃with the addition of 1D10ACE2 monoclonal antibody (1:10000 dilution). HRP-labeled IgG (1:10000 dilution) was incubated at room temperature after washing. After the ECL luminescence liquid is developed, the photo is observed and photographed in a full-automatic chemiluminescence image analysis system. The results are shown in FIG. 4, in which ACE2 monoclonal antibodies specifically react with ACE2 recombinant proteins.

2. Immunofluorescence identification of monoclonal antibodies

Immunofluorescence identification of monoclonal antibodies was performed with DF-1 cells, DF-1 cells were spread evenly in 24 well plates with cell climbing plates, first cell climbing plates in 12 well plates were fixed with 4% paraformaldehyde at room temperature, 0.5% TritonX-100 permeabilized, and blocked with 3% BSA. A1:1000 dilution of the 1D10 murine anti-chicken ACE2 monoclonal antibody was then added, incubated overnight at 4℃and incubated with FITC-labeled IgG (1:1000 dilution) in the dark. And adding DAPI, incubating in a dark place, sealing with a sealing liquid sealing sheet containing an anti-fluorescence quenching agent, and observing the result under a laser confocal microscope. The results are shown in FIG. 5, which shows that the monoclonal antibody can recognize DF-1 cells and generate specific green fluorescence.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims (10)

1. A monoclonal antibody specifically binding to chicken ACE2 protein is prepared from BALB/c mice.

2. A hybridoma cell line producing monoclonal antibodies at a titer of 1: 512000.

3. The monoclonal antibody of claim 1, wherein white-feather broiler pET32a-ACE2 recombinant protein is used as an antigen.

4. The monoclonal antibody of claim 1 purified by affinity chromatography.

5. The monoclonal antibody of claim 1 prepared from ascites.

6. The monoclonal antibody of claim 1, wherein the titer is determined by indirect ELISA.

7. The monoclonal antibody of claim 2, wherein the use of the expression of chicken ACE2 protein is detected.

8. The use according to claim 7, wherein the detection is Western blot and immunofluorescence.

9. A test kit comprising the monoclonal antibody of claim 1.

10. Use of the monoclonal antibody of claim 1 in a detection tool for detecting chicken ACE2 protein.