CN116769030B

CN116769030B - Panda relaxin 3 monoclonal antibody, hybridoma cell strain and application thereof

Info

Publication number: CN116769030B
Application number: CN202311050115.7A
Authority: CN
Inventors: 蔡开来; 李非平; 王神飞; 张梦诗; 胡贤彪; 王涓; 刘玉良; 侯蓉; 李明喜; 兰景超; 黄河
Original assignee: CHENGDU RESEARCH BASE OF GIANT PANDA BREEDING
Current assignee: CHENGDU RESEARCH BASE OF GIANT PANDA BREEDING
Priority date: 2023-08-21
Filing date: 2023-08-21
Publication date: 2023-10-20
Anticipated expiration: 2043-08-21
Also published as: CN116769030A

Abstract

The application provides a panda relaxin 3 monoclonal antibody, a hybridoma cell strain and application thereof, and belongs to the technical field of biological immunology. The amino acid sequence of the heavy chain variable region of the panda relaxin 3 monoclonal antibody is shown as SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 2. The application constructs a hybridoma cell strain RLN-1 secreting the RLN3 monoclonal antibody, collects and purifies cell strain culture supernatant to obtain the high-specificity RLN3 monoclonal antibody, and then applies the obtained high-specificity RLN3 monoclonal antibody to Western Blot protein detection. Provides new data and thinking for the localization and tissue expression information of relaxin protein, the research on the receptor action of relaxin and related peptide thereof, and further discussing the function of relaxin and revealing the reproduction physiological law.

Description

Panda relaxin 3 monoclonal antibody, hybridoma cell strain and application thereof

Technical Field

The application belongs to the technical field of biological immunology, and particularly discloses a panda relaxin 3 monoclonal antibody, a hybridoma cell strain thereof and application thereof.

Background

Relaxin belongs to a water-soluble polypeptide hormone and consists of two polypeptide chains alpha (also called A) and beta (B), wherein the alpha chain has intra-chain disulfide bonds, and the two chains are connected by two disulfide bonds. Relaxin precursor is a polypeptide chain comprising four structures of signal peptide, beta chain, linker peptide and alpha chain, and mature relaxin is obtained by removing signal peptide S and linker peptide C.

During vertebrate studies, we found that there are 7 family members of the relaxin family, respectively: RLN1, RLN2, RLN3, INSL4, INSL5 and INSL6, which have important roles in many physiological activities in mammals, for example, can inhibit uterine muscle contraction during pregnancy in women to facilitate pregnancy, improve cardiovascular, osmotic pressure regulation and promote reproductive tissue growth and development.

Relaxin 3 (RLN 3) is a novel member of the relaxin family, and in mammals, relaxin 3 is involved in regulating physiological activities such as memory, appetite, stress, cognitive ability, osmotic pressure, anxiety in the brain, and the like in mammals. Early studies found that relaxin was primarily found in reproduction-related tissues of gestational mammals, with corpus luteum and placenta being the major sources of relaxin in mammals. Furthermore, relaxin expression is also detected in tissues other than those of mammalian reproduction, such as porcine pituitary gland, rat brain, heart, kidney, human heart, etc. However, due to the species difference, specific antibodies are lacking in pandas, and the tissue expression of relaxin 3 (RLN 3) in pandas has not been reported.

Disclosure of Invention

The first aim of the application is to provide a panda relaxin 3 monoclonal antibody hybridoma cell strain, which can solve the technical problem that panda RLN3 specific antibodies cannot be obtained at present.

The second objective of the present application is to provide a panda relaxin 3 monoclonal antibody, which solves the problem that there is no specific anti-panda RLN3 specific antibody in the prior art.

In order to achieve the above purpose, the technical scheme adopted by the application for solving the technical problems is as follows:

a panda relaxin 3 monoclonal antibody has the amino acid sequence of a heavy chain variable region shown as SEQ ID NO.1 and the amino acid sequence of a light chain variable region shown as SEQ ID NO. 2.

Further, its subtype is IgG1.

An expression vector comprising the panda relaxin 3 monoclonal antibody described above.

Hybridoma cell strain RLN-1 secreting panda relaxin 3 monoclonal antibody, wherein the preservation number of the hybridoma cell strain RLN-1 is CCTCC NO: c202128, 1 month 7 of 2021, was deposited with the China center for type culture Collection, university of Wuhan, china.

The panda relaxin 3 monoclonal antibody is applied to panda relaxin 3 detection.

Further, western Blot was used for detection.

A kit for detecting panda relaxin 3, comprising the panda relaxin 3 monoclonal antibody.

Further, the kit is a kit for Western Blot detection.

Compared with the prior art, the application has at least the following advantages and positive effects:

the application provides a panda relaxin 3 (RLN 3) monoclonal antibody and a hybridoma cell strain thereof, wherein the high-specificity RLN3 monoclonal antibody is obtained by constructing the hybridoma cell strain secreting the RLN3 monoclonal antibody, collecting and purifying cell strain culture supernatant, and then the obtained high-specificity RLN3 monoclonal antibody is applied to Western Blot protein detection. Provides new data and thinking for the localization and tissue expression information of relaxin protein, the research on the receptor action of relaxin and related peptide thereof, and further discussing the function of relaxin and revealing the reproduction physiological law.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, 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 application 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 graph showing the standard curve of the concentration of cellular proteins in example 2 of the present application;

FIG. 2 is a graph showing the Western-blot detection result in example 2 of the present application.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions of the embodiments of the present application will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

It should be noted that, without conflict, the embodiments of the present application and features of the embodiments may be combined with each other. The present application will be described in detail with reference to specific examples.

Example 1

1. Preparation of anti-panda RLN3 monoclonal antibody:

1.1 design and Synthesis of specific polypeptide fragments: according to the NCBI database panda RLN3 amino acid sequence, accession number: XP_002921067.1, sequence: MAKHPLLLLLTVWVLAGELWLRTEARASPFGVKLCGREFIRAVIFTCGGSRWRRADVLAPEATGDPFPDADSDTDSELDEAVASSELLAMTKYPLASYGGRPGWQGTPGTLRGGRDVVAGLSSNCCKWGCSKSEISSLC (SEQ ID NO. 3), with reference to other species sequence analysis, the panda RLN3 specific sequence was designed: the sequence was RASPFGVKLCGREFIRAVIFTCGGSRW (SEQ ID NO. 4) (RLN 3-1) and was synthesized by man and conjugated to KLH (keyhole limpet hemocyanin) as an immunogen for the production of specific antibodies (RLN 3-1-KLH).

For the synthesis of polypeptide fragments, the process is mature, and the polypeptide fragment RLN3-1 and the immunogen RLN3-1-KLH can be produced by the sequences disclosed by the application and manufactured by the biological engineering (Shanghai) Co., ltd.

1.2 immunization of antigen mice: adjuvant is used: antigen=1:1 (antigen amount is not enough to be mixed with sodium chloride before emulsification with adjuvant). Freund's complete adjuvant was used first and Freund's incomplete adjuvant was used later for immunization. A syringe, a three-way tube, and a disposable syringe ready for sterilization prior to immunization. Firstly sucking the antigen and NaCl into the syringe (400 mu L in total) by using a sterilized syringe, and then sucking the adjuvant into the syringe (400 mu L in total) by using the sterilized syringe; finally, the sterilized injector is connected in a three-way pipe for emulsification (the emulsification is carried out for about 10 minutes or more until the oil is in a water-in-oil state), and finally, the fused mixed solution is transferred into a disposable injector for injection.

Day 1: intraperitoneal injection, 200. Mu.L of one. (antigen amount was 100. Mu.g/min)

Day 14: intraperitoneal injection, 200. Mu.L of one. (after which the antigen was 50. Mu.g/dose)

Day 21: intraperitoneal injection, 200. Mu.L of one.

Day 27: intraperitoneal injection, 200. Mu.L of one.

The tail of the mice can be sampled 3 to 4 days after the third immunization, 12000 rpm,8 min, the titer of the serum is measured, RLN3-1 is taken as an antigen, the ELISA method is used for detecting the serum titer, the fusion can be prepared after the titer reaches the standard, and the immunization is continued until the titer reaches the standard when the titer is not high enough. Immune serum titers are shown in table 1:

TABLE 1 serum titers after immunization

The results show that the mice reach the immune requirement, and cell fusion experiments can be carried out.

1.3 cell fusion:

1.3.1 Resuscitation of myeloma cells (SP 2): firstly, taking out the frozen cells from liquid nitrogen, and rapidly putting the frozen cells into a water bath kettle at 37 ℃ to be melted, so that the cells are loose; placing the cells into a centrifuge tube of 15 mL, mixing with PBS of about 5 mL, centrifuging at 1000 rpm for 5min, discarding supernatant, repeating washing for two times to obtain SP2 cells, culturing the cells in culture flask, marking, and placing the culture flask into 37 deg.C and 5% CO ₂ Culturing in an incubator.

1.3.2 Passage of SP2 cells: when the cells in the culture flask grow to about 80% of the bottom of the flask, the cells can be passaged. Blowing off cells by using a gun head, sucking out the culture solution into a 15 mL centrifuge tube for 1000 r/min, and centrifuging for 5 minutes; the supernatant was discarded, PBS 5 mL was added, and the mixture was blown and mixed well, the supernatant was discarded by centrifugation again, and the PBS washing step was repeated 2 times. After washing, adding 2 mL of 10% complete culture solution to resuspend the cells, taking a proper amount of cells into a culture flask, and putting the culture flask into a carbon dioxide incubator for culturing.

1.3.3 preparation of trophoblast macrophages (which can be prepared the day before fusion):

the mice are killed by cervical vertebra removal, the pressure on the abdominal cavity is reduced as much as possible when cervical vertebra is broken, and the blood vessels in the abdominal cavity are prevented from being damaged, so that a large number of blood cells are prevented from being contained in feeder cells. The mice were soaked in 75% alcohol for 5 minutes, then the tails of the mice were held by hand and rinsed in alcohol several times up and down. Placed in a sterile dish. The skin was cut from the back abdomen with sterile scissors, the skin on both sides was torn by hand, the abdomen was exposed, and care was taken not to damage the peritoneum. The peritoneum was rubbed with an alcohol cotton ball. The incomplete culture medium containing the double antibody of 7 mL is sucked by a syringe and injected into the abdominal cavity (double antibody: incomplete culture solution=1:100), and the peritoneum is lifted by forceps during injection, so that the needle is prevented from piercing abdominal organs such as intestinal tracts. The abdomen was gently massaged with a cotton ball for one minute, and the injected culture solution was aspirated and transferred into a centrifuge tube. 1000 Centrifuging at r/min for 5min, and discarding the supernatant. Washed four more times with PBS. Cells were resuspended in 10% complete medium.

Adding the above cell suspension into 96-well plate, adding 100 μl of each well, and adding CO into 96-well plate ₂ Is cultured in an incubator. (macrophages are not prone to excessive, and a portion of the collected cells can be discarded as appropriate.)

1.3.4 preparation of immune splenocytes:

(1) Spleen of mouse was taken

Mice meeting the immunization requirement were taken. Firstly, taking care of aseptic operation to prevent cell pollution, after killing mice, soaking the mice in 75% alcohol for about five minutes, placing the mice in an aseptic plate, placing the mice in a position (in an ultra clean bench) which is beneficial to self operation, and dissecting. Firstly, cutting a small opening at the tail of a mouse by using scissors, then, manually cutting the fur layer, lightly wiping the cut part by using an alcohol cotton ball, then, picking up the semitransparent film layer wrapping viscera by using forceps, cutting the semitransparent film layer, exposing the spleen, gently taking out the spleen, removing adipose tissues above the spleen as much as possible, and washing the taken-out spleen in PBS.

(2) Spleen cell suspension preparation

Washing spleen with PBS for about 3 times, placing spleen in a dish, shearing spleen as much as possible with scissors, washing with PBS, filtering, collecting separated spleen cell suspension without tissue cells, centrifuging for 5min, and discarding supernatant; washing with 5 mL PBS, 1000 r/min, and centrifuging for 5 min; repeating for three times, adding 2 mL incomplete culture solution (DMEM) to resuspend the cells after washing, diluting the cell suspension 100 times or 1000 times for cell counting, and placing the rest cells in a water bath kettle at 37 ℃ for standby.

(3) Preparation of SP2 cell suspension: sucking the cell liquid by using a rubber head suction pipe, blowing a film at the bottom of a culture bottle (cells are suspended or slightly adhered to grow), transferring the cell liquid into a 15 mL centrifuge tube by using a sample adding gun for 1000 r/min, and centrifuging for 5 minutes; discarding the supernatant, adding PBS 5 mL, mixing, centrifuging for 5min at 1000 r/min, washing twice repeatedly, adding 2 mL incomplete culture solution (DMEM) after washing, diluting the cell suspension 100 times or 1000 times for cell counting, and placing the rest cells in a water bath at 37 ℃ for standby.

1.3.5 Cell fusion under PEG

SP2 cell suspension was mixed with splenocytes at 1:4 (between 1:10 and 1:4), centrifuging at 600 rpm for 3 min; the supernatant was discarded. The bottom of the centrifugal tube is lightly flicked to loosen the cell sediment slightly. 0.6 mL of 50% PEG solution preheated at 37℃was slowly added over 1 min with gentle shaking and tapping. And standing for 1 min after the addition is completed. The incompletely cultured liquid 10 mL preheated at 37 ℃ is used for stopping PEG action, the centrifuge tube is tapped and rotated while dripping, the incompletely cultured liquid is added at a constant speed, and the culture liquid is stood for 2 min after the addition is finished. Centrifuging at 800 rpm for 5min, and discarding supernatant; the PEG was removed by washing 2 times with PBS or incomplete broth.

After washing, the supernatant was discarded and the cells were resuspended in 10 mL of HAT selection medium. The cells were added to a 96-well plate (using a macrophage plate prepared previously, the liquid in the well was not sucked out, and then washed once with the incomplete culture solution, and sucked out) of the existing feeder cell layer, and 100. Mu.L of the liquid was added to each well; placing the culture plate in CO ₂ Culturing in an incubator. After 4 hours, add complete HAT-containing broth (19.6 mL 10% complete broth+0.4 mL HAT) to wells with 100. Mu.L per well; placing the culture plate in CO ₂ Culturing in an incubator.

1.3.6 selection culture

(1) The HAT culture solution is changed by a half-liquid method on the fourth day of fusion culture: 100. Mu.L of the supernatant from the 96-well plate was pipetted off and fresh 100. Mu.L of HAT medium (HAT medium was 10% complete medium: HAT=1:50) was added to each well using a loading gun, and 200. Mu.L of 50 XHAT was added to each plate in 10 mL complete medium.

(2) The HT culture solution is replaced by a half-liquid method on the seventh day of fusion culture: 100. Mu.L of the supernatant from the 96-well plate was pipetted off and fresh 100. Mu.L of HT medium (HT medium prepared as 10% complete medium: HT=1:50) was added to each well using a loading gun, and 200. Mu.L of 50 XHT was added to each plate in 10 mL complete medium.

1.3.7 Positive clone screening

Obvious cloned cells in the wells can be seen after about 7 days of culture, and when the cloned cells grow sufficiently (about day 12), the culture solution can be sucked to detect the presence or absence of the secreted antibody.

(1) The corresponding ELISA (coated RLN3-1, coated concentration of 2. Mu.g/mL) plate coated previously was taken out from the refrigerator to allow to return to room temperature, 100. Mu.L of culture solution to be detected was added to the plate for each well, and both wells were used as a negative and positive control, 10% of culture solution was used for the negative control, and 10000-fold diluted serum (serum collected from orbit of mice before fusion) was used for the positive control.

(2) Incubation: the plates were then sealed and incubated in an incubator at 37℃for 1 hour.

(3) Washing: carefully removing the sealing plate film, washing for 4 times, and finally draining the water as much as possible.

(4) Adding double antibody: double antibody was diluted 10000-fold, 50 μl per well.

(5) Incubation: incubate with a plate membrane seal at 37℃for 30 minutes.

(6) Washing: carefully removing the sealing plate film, washing for 4 times, and finally draining the water as much as possible.

(7) Color development: 100 mu L of color reagent TMB is added into each hole, the mixture is gently mixed by shaking, and the color of the incubator is developed for about 10 min.

(8) Colorimetric determination: 50 μl of stop solution (stop solution=21.5/mL concentrated sulfuric acid to 200 mL) was added to each well, mixed by gentle shaking, and the microplate reader wavelength was set to 450nm, and the values of each well were measured.

The positive wells were selected as positive cloning wells with higher positive results (at least 4-fold compared to the negative control).

1.3.7 limiting dilution method for screening hybridoma cell lines of anti-panda RLN3 monoclonal antibody

(1) Count of positive well cells: limiting dilution is carried out on the positive clone holes obtained by screening. Firstly, transferring cells in the holes into a centrifuge tube of 15 mL (rotating while blowing to suspend the cells), and then adding 10% of complete culture solution to 2 mL; the cells were counted by a counter plate, and after counting, the cell fluid containing only 1000 cells was taken for the next experiment (about 100 cells were needed for one plate for 96-well plates, and 1000 cells were needed for 10 plates).

(2) The cell sap was added to 200 mL complete culture broth, mixed well, and applied in 96 well plates at 200 μl/well, ten 96 well plates total.

(3) Finally placing the culture plate into CO ₂ Culturing in an incubator.

(4) After 5 days of culture, small cell clones were visible on an inverted microscope, the growth of the cells was observed, and wells were recorded in which individual cells grew together.

(5) On day 5 of culture, wells were changed with wells recording growth aggregates of individual cells, and 100. Mu.L/well of 10% complete culture was added.

(6) On day 8, cell clones were seen with naked eyes, antibody detection was performed in time, and culture solution was detected (ELSIA detection) from wells in which single cells grew and were well grown, and wells with strong positivity were the panda RLN 3-resistant monoclonal antibody hybridoma cell strain. The application finally screens and obtains 2 anti-panda RLN3 monoclonal antibody hybridoma cell strains named RLN-1 and RLN-2.

1.3.8 subtype identification

Subtype identification was performed using the Pierce Rapid ELISA Mouse mAb Isotyping Kit 37503 kit.

Preparation: the TBS in the kit is dissolved in 500 mL double distilled water for diluting the sample, 870 mL double distilled water is mixed with 30 mL 30 xWash Buffer uniformly for washing the plate, the number of plates is determined according to the amount of the sample, the rest of plates are put back into a refrigerator at 4 ℃ for preservation, 450 mu L of sample diluent is prepared, 20 mu L of cell culture solution is sucked and added into 980 mu L of TBS for uniform mixing.

The experimental steps are as follows: balancing the plate to room temperature, adding a sample to be detected into each hole, adding 8 holes, namely one hole, into each sample, adding 50 mu L/hole of Goat Anti-Mouse IgG+IgA+IgM HRP, mixing the gently rocked plate uniformly, covering a sealing plate film, incubating for 4 times at room temperature, draining water, adding 75 mu L/hole of TMB color development liquid for developing color, changing the liquid in the holes into blue color, adding 75 mu L/hole of termination liquid for stopping reaction after developing color for 10 min, and changing the liquid from blue color into yellow color. The antibody subtypes secreted by panda RLN3 monoclonal antibody cell strains RLN-1 and RLN-2 obtained by identification and screening are of IgG1 type, and the results are shown in Table 2.

TABLE 2 hybridoma cell strain subtypes

Subtype type	RLN-1	RLN-2
			IgG1	0.9202	1.0583
IgG2a	0.0717	0.075
			IgG2b	0.0611	0.0555
IgG3	0.0493	0.0514
			IgGA	0.0486	0.0476
IgGM	0.0434	0.0427
			Kappa	1.4748	1.5181
Lamba	0.0428	0.0442

1.3.9 amplified culturing and purification of monoclonal antibody, and concentration.

(1) Batch culture: the subtype identification was performed, and the cell wells as a result of the monoclonal antibodies were transferred to a 24-well plate (blown while rotating, suspending the cells, and performing complete transfer) for culture, and 600. Mu.L of 10% complete culture solution was added for culture.

(2) After observing the growth of cells, the titer was measured after growing more cells, and the cells with high titers were transferred to a small flask for culture (cells in a 24-well plate were first blown to suspend the cells, and then transferred to the flask with a sample gun, and 10% of complete culture solution 7 mL was added).

(3) Observing the growth condition of the cells, transferring the cells to a large culture flask for culture after the cells grow well. One small flask was transferred to two large flasks for culture (cell passage), respectively.

(4) Several culture flasks can be used for culturing, a part of cells are frozen, and then the culture solution is taken for antibody column purification. The column is filled with Pierce Protein G Agarose, and the purified anti-panda RLN3 monoclonal antibody is concentrated by 10000 kDa ultrafiltration tube and stored at-20deg.C for use.

1.3.10 cryopreservation of monoclonal antibody cell lines

After the identified 2 anti-panda RLN3 monoclonal antibody cell lines were cultured stably, the cells in the flask were blown to suspend the cells in the culture medium (cells were typically suspended in the culture medium or grown on an adherent surface) and transferred to a centrifuge tube of 15 mL. Centrifuge, 1000 rpm/5 min. Wash twice with PBS: the supernatant in the centrifuge tube is sucked out, discarded, added with PBS, mixed evenly, centrifuged for 1000 revolutions per 5 minutes, and finally repeated once. Finally, the supernatant is sucked out by a sample gun, and a proper amount of frozen stock solution (frozen stock solution=5 mL serum+ 4 mL DMEM+1 mL DMSO) is added into the cells, and the mixture is inverted and mixed uniformly and filtered for later use, and then the mixture is uniformly mixed. Finally, the cells were added to the frozen tube, 1 mL cell fluid per tube. Placing into a freezing box, firstly placing overnight at-80 ℃, and then placing the cell strain into liquid nitrogen for long-term storage for standby.

Example 2

1. The application of the anti-panda RLN3 monoclonal antibody in the western blot technology.

(1) Extraction of total protein of target tissue

Protein extraction kit was used: c510004-0020, supplied by Shanghai, inc., and extracted from the company of Biotechnology, and the total protein concentration of the bone marrow mesenchymal stem cells of the panda is determined by BCA method.

(2) And (3) glue preparation: polyacrylamide gel was prepared, gel was concentrated 5% and gel was separated 12%.

(3) Sample preparation: the protein loading was 80. Mu.g.

(4) Electrophoresis: concentrating the gel at 80V for 30 min; the gel was separated for 120V and 90 min.

(5) Transferring: 250 mA,40 min.

(6) Closing: 5% skim milk, slowly shaking at 37℃for 2h.

(7) Incubating primary antibodies: the hybridoma cell strains RLN-1, RLN-2 antibody and beta-actin reference antibody are diluted respectively at a ratio of 1:1000, slowly shaken overnight at 4 ℃, and rewarmed at 37 ℃ for 1h the next day.

(8) Incubating a secondary antibody: dilution 1:8000, incubation 1h at 37 ℃.

(9) ECL exposure.

Western blot results show that only monoclonal antibodies secreted by the cell strain RLN-1 have specific target bands, and can be applied to the Western blot technology, and then the obtained hybridoma cell strain RLN-1 is preserved in China center for type culture collection (CCTCC NO) of university of Wuhan in China at 1 month 7 of 2021: C202128.

2. application results:

(1) Cellular protein concentration

TABLE 3 Standard Curve Table

The standard curve is shown in figure 1. Absorbance value of BSA (. Mu.g/. Mu.L)

TABLE 4 protein concentration meter

(2) The Western-blot detection result is shown in FIG. 2

As shown in the results of FIGS. 1 and 2, the anti-panda RLN3 monoclonal antibody developed in the present application can be prepared to recognize the target protein RLN3.

2. Determination of anti-panda RLN3 monoclonal antibody titer and sequence determination

The monoclonal antibody titer of the culture solution supernatant purified RLN-1 cell strain is detected by using an indirect ELISA, and the specific steps are as follows:

(1) The polypeptide fragment RLN3-1 2. Mu.g/mL, 50. Mu.L/well coated ELISA plate of example 1 was incubated overnight at 4 ℃.

(2) The coated plate was washed, 200. Mu.L/well of PBS containing 2% BSA was added thereto, and the plate was blocked at 37℃for 2 hours.

(3) After plate washing, anti-RLN 3 monoclonal antibodies (1000-fold, 2000-fold, 4000-fold, 8000-fold, 16000-fold, 32000-fold, 64000-fold, 128000-fold, 256000-fold, 512000-fold, respectively) and PBS wells were added as controls and incubated at 37℃for 1h.

(4) After washing the plates, add 1:5000 times of goat anti-mouse antibody marked by horseradish peroxidase is incubated for 1h at 37 ℃.

(5) TMB chromogenic substrate 100. Mu.L/well was added and reacted for 5min.

(6) The reaction was stopped by adding 50. Mu.L of stop solution.

(7) The OD value of each hole is read at 450nm of the enzyme label instrument, and the detection result is shown in Table 5.

As shown in Table 5, the antibody titer of RLN3 purified from the culture supernatant was 10 ⁶ Above mentionedIndicating high antibody titers.

TABLE 5 antibody titers

The obtained anti-panda RLN3 monoclonal antibody cell line (RLN-1) was sent to general biosystems (anhui) and sequenced, and the heavy chain variable region sequence of the monoclonal antibody was as follows:

EVQLEESGPQLLRPGASVKISCKASGYSFTRYWIHWVKQRPGQGLEWIGMIDPSDSESRLNQKFKDKATLTEDKSSSTAYMQLSSPTSEDSAVYYCVRRYFDYWGHGTTLTVSS（SEQ ID NO.1）

the light chain variable region sequence is as follows:

DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDVATYYCQQSNEDPLTFGAGTKLELK（SEQ ID NO.2）

in conclusion, the application constructs a hybridoma cell strain secreting the RLN3 monoclonal antibody, collects and purifies cell strain culture supernatant to obtain the high-specificity RLN3 monoclonal antibody, and then applies the obtained high-specificity RLN3 monoclonal antibody to Western Blot protein detection. Provides new data and thinking for the localization and tissue expression information of relaxin protein, the research on the receptor action of relaxin and related peptide thereof, and further discussing the function of relaxin and revealing the reproduction physiological law.

Finally, it should be noted that the above-described embodiments are some, but not all, embodiments of the application. The detailed description of the embodiments of the application is not intended to limit the scope of the application, as claimed, but is merely representative of selected embodiments of the application. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.

Claims

1. A panda relaxin 3 monoclonal antibody is characterized in that the amino acid sequence of a heavy chain variable region is shown as SEQ ID NO.1, and the amino acid sequence of a light chain variable region is shown as SEQ ID NO. 2.

2. The panda relaxin 3 monoclonal antibody according to claim 1, wherein the subtype thereof is IgG1.

3. An expression vector for expressing the panda relaxin 3 monoclonal antibody according to claim 1 or 2.

4. A hybridoma cell line RLN-1 secreting the panda relaxin 3 monoclonal antibody according to claim 1 or 2, wherein the hybridoma cell line RLN-1 has a preservation number of CCTCC NO: c202128, 1 month 7 days 2021, was deposited with the China center for type culture Collection, university of Wuhan, hubei, china.

5. Use of the panda relaxin 3 monoclonal antibody as claimed in claim 1 or 2 for the preparation of a kit for detecting panda relaxin 3.

6. A kit for detecting panda relaxin 3, comprising the monoclonal antibody against panda relaxin 3 according to claim 1 or 2.

7. The kit according to claim 6, wherein the kit is a kit for Western Blot detection.