阅读说明：本技术 MrgD免疫抗原片段、MrgD抗体及其制备方法和应用 (MrgD immune antigen fragment, MrgD antibody, preparation method and application thereof ) 是由 李鹏 吴晓光 赵锟 毛宇康 孔祥清 于 2020-11-12 设计创作，主要内容包括：本发明涉及抗体制备及其应用领域,具体是涉及单克隆抗体的制备及其应用于免疫荧光方面。此种抗体可与大鼠Mas相关性G蛋白耦联受体D(MrgD)结合,且具有良好的特异性,结合免疫荧光法,可快速检测心脏病理生理条件下MrgD表达水平的变化,对病理的发生发展过程提供可靠的依据。(The invention relates to the field of antibody preparation and application thereof, in particular to the preparation of a monoclonal antibody and the application thereof in immunofluorescence. The antibody can be combined with a rat Mas-related G protein coupled receptor D (MrgD), has good specificity, can be combined with an immunofluorescence method, can quickly detect the change of the MrgD expression level under the heart pathophysiology condition, and provides a reliable basis for the generation and development process of pathology.)

1. An MrgD immune antigen fragment having the sequence: ser Ser Pro Ala Pro Gly Leu ThrIle Ser Pro Thr Met Asp Cys.

2. An MrgD antibody produced by injecting the rat Mas-associated G protein-coupled receptor D immune fragment of claim 1 into an animal.

3. The MrgD antibody of claim 2, characterized in that: the antibody is a rabbit anti-mouse MrgD antibody.

4. A method of making an MrgD antibody comprising the steps of:

(1) synthesizing a polypeptide fragment Ser Ser Pro Ala Pro Gly Leu ThrIle Ser Pro Thr Met Asp Cys;

(2) injecting the polypeptide fragment synthesized in the step (1) into an animal body for immunization,

(3) taking the spleen of the immunized animal, preparing splenocytes, and culturing after the splenocytes and myeloma cells are fused;

(4) taking fusion culture cells, and screening cell strains which have the highest titer and are monoclonal by using an indirect ELISA method coated with MrgD protein for amplification culture;

(5) injecting the hybrid hybridoma cells subjected to expanded culture into the same animal body, collecting ascites, centrifuging and taking supernate;

(6) and (4) allowing the supernatant of the abdominal water to pass through a protein G microsphere column, eluting and purifying to obtain the MrgD antibody.

5. Use of the MrgD antibody of claim 2 or 3 for immunofluorescence detection of MrgD in vivo profiles.

6. The use of claim 5, comprising the steps of:

(1) extracting rat primary myocardial cells, and culturing in a culture medium;

(2) discarding the culture medium, washing, fixing and blocking the cardiac muscle cells, and adding the antibody of claim 2 or 3 overnight;

(3) and after the PBS is cleaned, adding a fluorescent secondary antibody for incubation, eluting the fluorescent secondary antibody, adding an anti-fluorescence quenching agent, and then sealing and photographing.

Technical Field

The invention relates to an MrgD immune antigen fragment, an MrgD antibody, and a preparation method and application thereof, and belongs to the field of biomedicine.

Background

Hypertension has increased year by year, and its harm is mainly target organ injury, including cardiovascular remodeling, cerebral apoplexy, renal failure, eye diseases, etc. The regulation of hypertension is mainly hormonal regulation and neural regulation, the former being mainly the renin-angiotensin system (RAS), the latter being sympathetic parasympathetic regulation. The research finds that Mas-related G protein coupled receptor D (MrgD) is a main receptor regulated by a renin-angiotensin system and plays an important role in the regulation of the renin-angiotensin system. However, the distribution and action mechanism of MrgD in vivo are not clear, so if a method can be invented, the distribution of MrgD in vivo can be rapidly determined, and the method has important significance for the regulation and control of hypertension.

Disclosure of Invention

The invention aims to provide a rat Mas-related G protein-coupled receptor D (MrgD) immune antigen fragment which can be used for preparing an MrgD antibody, and the MrgD antibody can be used for MrgD immunofluorescence tracing.

The technical scheme adopted by the invention is as follows: an MrgD immune antigen fragment having the sequence: ser Ser Pro Ala Pro Gly Leu Thr Ile Ser Pro Thr Met Asp Cys are provided.

The invention also discloses an MrgD antibody, which is an antibody generated after the MrgD immune fragment is injected into an animal body.

Preferably, the antibody is a rabbit anti-MrgD antibody.

The invention also discloses a preparation method of the MrgD antibody, which comprises the following steps:

(1) synthetic polypeptide fragment Ser Ser Pro Ala Pro Gly Leu Thr Ile Ser Pro Thr Met Asp Cys;

(2) injecting the polypeptide fragment synthesized in the step (1) into an animal body for immunization,

(3) taking the spleen of the immunized animal, preparing splenocytes, and culturing after the splenocytes and myeloma cells are fused;

(4) taking fusion culture cells, and screening cell strains which have the highest titer and are monoclonal by using an indirect ELISA method coated with MrgD protein for amplification culture;

(5) injecting the hybrid hybridoma cells subjected to expanded culture into the same animal body, collecting ascites, centrifuging and taking supernate;

(6) and (4) allowing the supernatant of the abdominal water to pass through a protein G microsphere column, eluting and purifying to obtain the MrgD antibody.

The invention also discloses application of the MrgD antibody in detecting the in-vivo distribution condition of MrgD by an immunofluorescence method.

Preferably, the steps include:

(1) extracting rat primary myocardial cells, and culturing in a culture medium;

(2) discarding the culture medium, cleaning, fixing and sealing the cardiac muscle cells, and adding the MrgD antibody overnight;

(3) after washing with PBS, adding a fluorescent secondary antibody cy3 for incubation, eluting the fluorescent secondary antibody, adding an anti-fluorescence quencher, and sealing and photographing.

The invention provides a specific immune antigen fragment of MrgD, which can be injected into an animal body to generate an MrgD antibody, the MrgD antibody can be combined with MrgD protein, has good specificity, can quickly detect the change of the MrgD expression level under heart pathology by combining an immunofluorescence method, and provides a theoretical basis for the generation and development of hypertension pathology.

Drawings

FIG. 1: schematic representation of MrgD distribution in normal rat cardiomyocytes in example 2;

FIG. 2: example 3 schematic representation of MrgD distribution in rat cardiomyocytes with angiotensin II induced myocardial hypertrophy.

Detailed Description

To further illustrate the technical means and effects of the present invention adopted to achieve the predetermined objects, the following detailed description of the embodiments, structures, features and effects according to the present invention will be made with reference to the accompanying drawings and preferred embodiments.

Example 1

1. Polypeptide antigen synthesis

The following fragment was selected from the rat MrgD sequence for synthesis, SSPAPGLTISPTMDC, as an immunizing antigen.

2. Animal immunization

The synthesized polypeptide antigen is used for immunizing a plurality of New Zealand rabbits of 2-3 kg.

Primary immunization: 100ug of antigen per mouse, complete Freund's adjuvant (antigen and adjuvant mixed in equal volume, ground into water-in-oil chyle) was added and injected subcutaneously into the back at multiple points in a volume of 1.5m l.

And (3) second immunization: the second immunization is carried out at an interval of 2 weeks after the primary immunization, the dosage route is the same as that of the primary immunization, and Freund incomplete adjuvant is added.

And (3) third immunization: carrying out the third immunization at an interval of 2 weeks after the second immunization, wherein the dose is the same as the above, no adjuvant is added, intraperitoneal injection is carried out, and tail tip blood sampling is carried out after 10 days to measure the titer and the immunization titer. The selection titer is 10⁵The mice were boosted 3 days before the fusion by intraperitoneal injection of 100ug of antigen.

3. Cell fusion and monoclonal antibody screening

Myeloma cell preparation: myeloma cells were expanded 2 days before fusion. On the day of fusion, cells were blown off the vial wall and collected in 50ml centrifuge tubes. Centrifuging at 1000r/min for 5-10 min, and discarding the supernatant. 30ml of incomplete medium RPMI-1640 was added and washed once by centrifugation. The cells were then resuspended in 10ml of RPMI-1640 incomplete medium.

Preparation of splenocytes: immunized New Zealand rabbits were sacrificed, immersed in 75% alcohol for 5 minutes, the spleen was removed from the clean bench and placed in a petri dish containing 10ml of incomplete RPMI-1640 medium, blood was rinsed off, and the surrounding connective tissue was carefully stripped off. A stainless steel mesh was placed on the dish, and the cell suspension was ground with a syringe needle and counted. The cell suspension in the plate was pipetted into a 50ml centrifuge tube and gently pipetted several times with a Pasteur pipette to make a single cell suspension. Usually 1X 10 per rabbit⁸-2.5×10⁸And (4) spleen cells.

Cell fusion: mixing splenocytes and myeloma cells at a ratio of 10:1 in a centrifuge tube, adding 1ml of 50% polyethylene glycol (PEG4000) for fusion, resuspending the cells in RPMI-1640 complete medium, and adding to a 96-well cell culture plate (about 10 cells per well)⁵Cells) per well, 100. mu.l, the plate was incubated at 37 ℃ in a 5% CO2 incubator, and on day 2 of confluency, 1% HAT medium was added at 100. mu.l per well, 5% at 37%Culturing in a CO2 incubator; after 5-7 days of culture, the HAT culture medium is changed once.

4. Screening hybridoma cells:

after fusion culture for 4-5 days, the culture solution is aspirated and screened by indirect ELISA method for coating MrgD protein.

5. Hybridoma cell cloning

After the optimal hybridoma cells are screened out by an indirect ELISA method, adding an HT culture medium containing 16% serum, uniformly mixing, paving in a 96-well plate, paving feeder cells before paving, culturing in a 37 ℃ and 5% CO2 incubator after paving, subcloning the hybridoma cells, performing subcloning screening after subcloning for about one week, sampling and screening when the clonal growth vigor is good, taking 100ul of supernatant for detection, selecting the wells with high positive reaction, selecting the wells with single clone as much as possible, transferring the cells into a 24-well plate when the cells grow to 1/4-1/3 which occupies about the bottom area of the wells after the cell growth vigor is good, paving feeder cells in the 24-well plate before transferring, performing titer determination after transferring the wells for 24h-72h, selecting the wells with high titer for subcloning again until 100% of the wells are positive, selecting a plurality of cell strains with high titer and are monoclonal, performing expanded culture, finally, the best cell is selected for ascites preparation.

6. Production of monoclonal antibodies

Injecting hybridoma cells into abdominal cavity of New Zealand rabbit, swelling the rabbit belly after 5-8 days, taking ascites, inserting 20ml syringe needle into the rabbit belly, placing 50ml centrifuge tube below, making the ascites flow into the centrifuge tube, taking about 3ml each time, generally taking 3-5 times until the ascites is taken out, centrifuging the taken ascites (2000r/min for 5 min), removing cell components and other precipitates, collecting supernatant, determining antibody titer, subpackaging, and storing at-80 ℃.

7. Purification of monoclonal antibodies

The pH of the ascites fluid containing the antibody was adjusted to 8.0 by adding 1/10 ascites volume of 1.0mol/L Tris (pH8.0) hydrochloric acid. The antibody solution was passed through a protein G microspherical column (abbimate). Each ml of microspheres can bind about 10-20 ml of antibody (1 protein G microsphere molecule binds 2 molecules of antibody). Record the approximate volume of the packed microspheres. ③ washing the microspheres with 100mmol/L Tris (pH8.0) hydrochloric acid in 10 times of the volume of the microsphere column, then washing the microspheres with 10mmol/L Tris (pH8.0) hydrochloric acid in 10 times of the volume of the microsphere column, and then eluting the microsphere column with 50mmol/L glycine (pH3.0), adding 1/2 buffer solution in each time in the volume of the microsphere column, and adding the buffer solution in portions. The eluate was collected in test tubes containing 1/10 microsphere column volumes of 1mol/L Tris (pH8.0) hydrochloric acid, and the tubes were gently shaken to return the pH to neutral. Sixthly, measuring the OD value of 280nm of each collecting pipe to obtain the protein content (10D is about equal to 0.75mg/ml immunoglobulin), and performing titer measurement on the antibody after purification.

Example 2

Extracting rat primary myocardial cells, culturing in a culture medium DMEM + 10% FBS for 48 hours, removing the culture medium, washing with PBS for 2 times, fixing with 4% paraformaldehyde for 30 minutes, blocking with 5% BSA for 30 minutes, adding the MrgD antibody obtained in example 1, standing overnight at 4 ℃, washing with PBS for 3 times, adding a fluorescent secondary antibody (cy3), incubating at normal temperature for 2 hours, eluting the secondary antibody, adding an anti-fluorescence quencher, and sealing and taking pictures. As shown in fig. 1, it can be seen that the MrgD content in normal cardiomyocytes is low.

Example 3

Extracting rat primary myocardial cells, adding angiotensin II (10 concentration)^-6mol/L) of the cells were cultured in DMEM + 10% FBS for 24 hours, the medium was discarded, PBS was washed 2 times, 4% paraformaldehyde was fixed for 30 minutes, 5% BSA was blocked for 30 minutes, MrgD antibody was added for 4 degrees overnight, then PBS was washed 3 times, secondary antibody was added and incubated at 37 ℃ for 1 hour, PBS was washed 3 times, and mounting was photographed. As shown in fig. 2, it can be seen that the angiotensin II-induced MrgD expression level (highlight) was higher in the hypertrophic cardiomyocytes.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.