CN116338212A

CN116338212A - Anti-panda PRL monoclonal antibody, hybridoma cell strain and PRL enzyme-linked immunosorbent assay method

Info

Publication number: CN116338212A
Application number: CN202310182118.XA
Authority: CN
Inventors: 蔡开来; 张梦诗; 王东辉; 王神飞; 胡贤彪; 李非平; 王涓; 刘玉良; 侯蓉; 兰景超; 吴永胜
Original assignee: CHENGDU RESEARCH BASE OF GIANT PANDA BREEDING
Current assignee: CHENGDU RESEARCH BASE OF GIANT PANDA BREEDING
Priority date: 2022-04-29
Filing date: 2023-02-28
Publication date: 2023-06-27
Also published as: CN114624452A

Abstract

The invention discloses an anti-panda PRL monoclonal antibody, a hybridoma cell strain and a PRL enzyme-linked immunosorbent assay method. The method uses hybridoma cell strain PRL-1 and/or hybridoma cell strain PRL-5 to respectively produce anti-panda PRL monoclonal antibodies for detection; wherein, the preservation number of the hybridoma cell strain PRL-1 is CCTCC NO: C202183; the hybridoma cell strain PRL-5 has a preservation number of CCTCCNO: C202172, and is preserved in China center for type culture Collection of university of Wuhan in Wuhan, china, 10 months and 28 days in 2021. The invention develops a panda urine Prolactin (PRL) enzyme-linked immunosorbent assay method by taking the two prepared panda PRL antibodies as coating antibodies and labeled antibodies, and can monitor the gestation state of the panda by measuring the urine prolactin in the gestation period of the panda, thereby scientifically guiding the breeding production work of the panda.

Description

Anti-panda PRL monoclonal antibody, hybridoma cell strain and PRL enzyme-linked immunosorbent assay method

Technical Field

The invention belongs to the technical field of panda prolactin detection methods, and particularly relates to an anti-panda PRL monoclonal antibody, a hybridoma cell strain and a PRL enzyme-linked immunosorbent assay method.

Background

The confined pandas enter gestation period after effective hybridization. The gestation period of the containment pandas varies greatly from 83 days to 200 days. The research on panda gestation period, in particular pregnancy diagnosis, is an important key technology for panda containment breeding. It has been a difficult problem for many years. Little is known about the gestation mechanism of pandas: is mating effective? Is the panda put into gestation? At present, no method for accurate detection exists, and a certain difficulty is brought to feeding management. The diapause of panda embryo development causes great difficulty in pregnancy monitoring.

Embryo diapause is the physiological phenomenon that after embryo development reaches the blastula stage, the embryo is in a dormant state, cell proliferation is stopped, and when the internal and external environments are proper, the embryo can be activated to restart development and implantation. Embryo diapause separates mating from production from two important reproductive stages to ensure that both occur at the time when species survival is most beneficial, which is considered one of the evolutionary strategies to ensure successful animal reproduction and neonatal survival. It has now been found that more than 130 animals have a phenomenon of embryo diapause during embryo development, in which all of the existing known 8 felines including pandas have been identified.

Prolactin is a protein hormone secreted by the anterior pituitary gland eosinophils. The main functions are to promote the development and growth of mammary gland, stimulate and maintain lactation, stimulate the generation of follicle luteinizing hormone (luteinizing hormone, LH) receptor, start and delay implantation, etc. In obligate diapause animals, the photoperiod affects embryo diapause and subsequent restarting of embryo development by regulating the secretion of pituitary prolactin. Prolactin concentration plays an important role in stopping embryo diapause and embryo development in minks by inducing the expression of the ODC gene (ornithine decarboxylase). ODC proteins are rate limiting enzymes that produce polyamines (i.e., putrescine) and can induce reactivation of mink embryos. In facultative diapause, such as large-sized pouched animals, prolactin induces or inhibits the development and secretion of the corpus luteum by changes in concentration. Prolactin receptors are present on the surface of luteinizing cells and can terminate the dormant state by inhibiting prolactin secretion. The lactation stimulus promotes the release of prolactin through the nervous system. In curved-wing bats, an increase in prolactin concentration may induce the corpus luteum to end up resting and begin to implant, but progestin has no effect on corpus luteum function.

Current studies indicate that the process of reactivation of embryo diapause species, both when they enter diapause, during diapause and after embryo diapause, is dependent to varying degrees on the three hormones prolactin, estrogen and progestogen, but varies widely from species to species, resulting in different species characteristics. Resulting in different species characteristics. In species typified by mink, blastula enter diapause after mating due to high nocturnal melatonin levels, resulting in reduced prolactin levels. Reactivation of diapause is triggered by an increase in photoperiod after spring, an increase in prolactin followed by an increase in ovarian progestogen synthesis, and only prolactin from this species can terminate embryo diapause; for species such as tama Sha Daishu, if there is a young bag after mating, the blastula will enter diapause in the lactation period, maintained by high levels of prolactin, once prolactin inhibition is eliminated, followed by reactivation of the diapause blastula by progestogenic pulses; in mice, if mating occurs after parturition, the prolactin level increases, thereby inhibiting the rise in estrogen levels and causing the blastula to enter diapause, which is maintained by high levels of prolactin and progestin and can be terminated by high levels of estrogen. At present, a reliable detection method for estrogen and progestogen in panda urine is available, and a detection method for panda prolactin is lacked, so that the regulation mode of the prolactin in the gestation period of the panda is to be studied.

In summary, establishing a method for detecting prolactin in panda urine, and further grasping the change of the prolactin in the gestation period of the panda by the method is a problem to be solved urgently.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides an anti-panda PRL monoclonal antibody, a hybridoma cell strain and a PRL enzyme-linked immunosorbent assay method.

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

a panda prolactin ELISA detection method uses hybridoma cell strain PRL-1 and/or anti panda PRL monoclonal antibodies respectively generated by hybridoma cell strain PRL-5 to detect;

wherein, the preservation number of the hybridoma cell strain PRL-1 is CCTCC NO: C202183; the hybridoma cell strain PRL-5 has a preservation number of CCTCC NO: C202172, and is preserved in China center for type culture Collection of university of Wuhan in Wuhan, china, 10 months and 28 days in 2021.

Further, when detecting panda prolactin, anti-panda PRL monoclonal antibodies produced by hybridoma cell line PRL-1 and hybridoma cell line PRL-5 were used as primary antibodies or secondary antibodies, respectively.

Further, the anti-panda PRL monoclonal antibody takes a panda prolactin specific sequence with an amino acid sequence shown as SEQ ID NO.1 as an immunogen and is secreted by hybridoma cell lines PRL-1 and PRL-5.

Further, the preparation method of the panda PRL monoclonal antibody comprises the following steps:

(1) Designing a panda prolactin specific sequence, wherein the amino acid sequence of the panda prolactin specific sequence is shown as SEQ ID NO.1, and coupling the panda prolactin specific sequence with KLH to form an immunogen PRL-KLH;

(2) Immunizing a mouse with an immunogen PRL-KLH;

(3) Cell fusion to obtain hybridoma cell strains PRL-1 and PRL-5 of the anti-panda PRL monoclonal antibody;

(4) The anti-panda PRL monoclonal antibodies generated by the hybridoma cell strains PRL-1 and PRL-5 can be obtained by amplification culture.

Further, panda urine was used as a detection substrate.

Further, the method comprises the following steps:

(1) Adding panda urine into an ELISA plate coated by anti-panda PRL monoclonal antibodies, adding a PRL recombinant protein standard substance, and incubating for 30-35 min at 35-37 ℃;

(2) Washing the ELISA plate with washing liquid, adding anti-panda PRL monoclonal antibody-HRP complex, and incubating at 35-37 ℃ for 30-35 min; the anti-panda PRL monoclonal antibody used in this step is of a different source than the anti-panda PRL monoclonal antibody in step (1);

(3) Washing the ELISA plate with a washing solution, adding a substrate chromogenic reagent, incubating for 5-10 min at room temperature, and adding a reaction stopping solution to stop the reaction;

(4) And measuring and calculating the PRL content by using an enzyme-labeled instrument.

Further, the substrate chromogenic reagent is TMB chromogenic solution.

Further, the reaction termination solution was a sulfuric acid solution having a concentration of 1M.

The detection method or the application of the anti-panda PRL monoclonal antibody in embryo development monitoring of pandas gestation period.

A kit comprises the anti-panda PRL monoclonal antibodies respectively generated by the hybridoma cell strain PRL-1 and/or the hybridoma cell strain PRL-5.

A vector comprising the above anti-panda PRL monoclonal antibody.

The kit or the carrier is applied to detecting the panda prolactin or monitoring the embryo development of the panda gestation period.

The hybridoma cell strain is hybridoma cell strain PRL-1 and/or hybridoma cell strain PRL-5, wherein the preservation number of the hybridoma cell strain PRL-1 is CCTCC NO: C202183; the hybridoma cell strain PRL-5 has a preservation number of CCTCC NO: C202172, and is preserved in China center for type culture Collection of university of Wuhan in Wuhan, china, 10 months and 28 days in 2021.

The invention has the beneficial effects that:

1. the invention provides a preparation method of an anti-panda prolactin antibody, and the prepared two specific antibodies are applied to a detection method of panda urine prolactin, so that embryo development of a panda in gestation period can be monitored, and breeding production of the panda can be scientifically guided.

2. The invention establishes a sandwich type enzyme-linked immunosorbent assay method for the PRL metabolite in panda urine by utilizing panda PRL specific antibody, and the sensitivity of the assay method is high and can reach 1.2ng/mL; the detection method has high repeatability, and the errors in batch and between batches are less than 10%.

Drawings

FIG. 1 is a diagram showing the alignment of the sequencing result of the vector with the original target gene sequence;

FIG. 2 is a cleavage map after cleavage;

FIG. 3 is a SDS-PAGE analysis of fusion proteins;

FIG. 4 is a SDS-PAGE analysis of fusion protein purification;

FIG. 5 is a standard curve of the method for detecting giant panda prolactin of the present application;

FIG. 6 is a graph showing the trend of the variation of prolactin and progestogen in gestation period of pandas.

Detailed Description

The following description of the embodiments of the present invention is provided to facilitate understanding of the present invention by those skilled in the art, but it should be understood that the present invention is not limited to the scope of the embodiments, and all the inventions which make use of the inventive concept are protected by the spirit and scope of the present invention as defined and defined in the appended claims to those skilled in the art.

EXAMPLE 1 preparation of anti-panda PRL monoclonal antibody

1. Design and Synthesis of specific polypeptide fragments

According to the amino acid sequence of panda PRL of NCBI database, accession No.: np_001291863.1, sequence: MDKKGWSLKGSLLPLLLLVSDLLLCQSVASLPICPTGAVNCQVSLRDLFDRAVILSHYIHNLSSEMFNEFDKRYAQGRGFITKAINSCHTSSLSTPEDKEQAQQIHHEDLLNLILRVLRSWNDPLYHLVTEVRGMQEAPDSILSRAIEIEEQNRRLLEGMEKIVGQVHPGVRENEVYSVWSGLPSLQMADEDTRLFAFYNLLHCLRRDSHKIDNYLKLLKCRIVYDSNC and referring to other species sequence analysis, the panda PRL specific sequence DRAVILSHYIHNLSSEMFNEFDKRYAQGR (PRL) (SEQ ID NO. 1) was designed and was synthesized artificially and conjugated to KLH (keyhole limpet hemocyanin) to make immunogens (PRL-KLH) for specific antibodies. For the synthesis of polypeptide fragments, the process is mature, and the polypeptide fragments PRL and the immunogen PRL-KLH can be produced by the sequence disclosed in the application and by the division of biological engineering (Shanghai) Co., ltd

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.

…………………

(3 to 4 days after the third immunization, the tail of the mice can be sampled, 12000rmp is 8min, the serum is taken for measuring the titer, PRL-1 is taken as an antigen, the serum titer is detected by an ELISA method, fusion can be prepared after the titer reaches the standard, and the immunization is continued until the serum titer reaches the standard as shown in Table 1) table 1 after the fourth immunization

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

3. Cell fusion

(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 15mL centrifuge tube, taking approximately 5mL of PBS, uniformly mixing, 1000rpm for 5min, centrifuging, discarding the supernatant, repeatedly cleaning the SP2 cells twice, culturing the cells in a culture flask, marking, and finally culturing the culture flask in a 5% CO2 incubator at 37 ℃.

(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 15mL centrifuge tube for 1000r/min, and centrifuging for 5 minutes; the supernatant was discarded, 5mL of PBS was added, the mixture was blown and homogenized, the supernatant was discarded by centrifugation again, and the PBS washing step was repeated 2 times. After washing, 2mL of 10% complete culture solution is added to resuspend the cells, a proper amount of cells are taken into a culture flask, and the culture flask is placed into a carbon dioxide incubator for culture.

(3) Preparation of trophoblast macrophages (preparation 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. 6-8mL of incomplete culture medium containing the double antibody 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. Centrifuging at 1000r/min for 5min, and discarding 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.)

(4) Preparation of immune spleen cells

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 the spleen in a plate, shearing the spleen as much as possible with scissors, adding PBS for washing and filtering, collecting separated spleen cell suspension without tissue cells, centrifuging for 5 minutes at 1000r/min, and discarding supernatant; washing with 5mLPBS, centrifuging at 1000r/min for 5min; repeating for three times, adding 2mL of 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 suspensions

Sucking the cell liquid by using a rubber head suction pipe, blowing the film at the bottom of the culture bottle (the cells are suspended or slightly attached to the wall for growth), then transferring the cell liquid into a 15mL centrifuge tube by sample adding and rush transferring, and centrifuging for 5 minutes; discarding the supernatant, adding 5mL of PBS, uniformly mixing, centrifuging for 5 minutes at 1000r/min, repeatedly washing twice, adding 2mL of 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 kettle at 37 ℃ for standby.

(5) Cell fusion under PEG

SP2 and splenocytes were mixed at 1:4 (between 1:10 and 1:4), centrifuging at 600rpm for 3min; the supernatant was discarded. The bottom of the centrifugal tube is lightly flicked to loosen the cell sediment slightly. 0.6mL of 50% PEG solution preheated at 37℃was slowly added over 1min with gentle shaking and tapping. And standing for 1min after the addition is completed. 10mL of the preheated incomplete culture solution at 37 ℃ is used for stopping PEG action, the centrifuge tube is tapped and rotated while dripping, the incomplete culture solution is added at a constant speed, and the incomplete culture solution is kept stand for 2min after the addition is finished. Centrifuging at 800rpm for 5min, and discarding the 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 10mL of HAT selection medium. The cells were added to a 96-well plate of the existing feeder cell layer (using a previously prepared macrophage plate, the liquid in the well was not sucked out first, and then washed once with the incomplete culture solution, and the liquid was sucked out), 100. Mu.L 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.

(6) Selective culture

1) HAT culture solution is replaced by half-solution 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% of complete medium: HAT=1:50) was added to each well, and 200. Mu.L of 50 XHAT was added to 10mL of complete medium.

2) HT culture solution is exchanged by half liquid method on seventh day of fusion culture

The supernatant from the 96-well plate was pipetted 100. Mu.L per well using a loading gun, discarded, and fresh 100. Mu.LHT per well (HT medium was prepared as 10% complete medium: HT=1:50) was added, 200. Mu.L of 50 XHT per plate was added to 10mL of complete medium.

(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 PRL-1, coated concentration of 2. Mu.g/mL) plate previously coated 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 negative and positive controls, 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, buckling 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, buckling 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 10min.

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

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

(8) Limited dilution method for screening hybridoma cell strain of anti-panda PRL monoclonal antibody

1) Counting of positive well cells

The positive clone Kong Kongke obtained by screening was subjected to limiting dilution. Firstly, transferring cells in the holes into a 15mL centrifuge tube (rotating while blowing to suspend the cells), and then adding 10% of complete culture solution to 2mL; 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 200mL of complete culture broth, mixed well, applied to 96 well plates, 200 μl/well, ten 96 well plates total.

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

4) After 4-5 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 days 8-9, cell cloning is seen by naked eyes, antibody detection is carried out in time, culture solution detection (ELSIA detection) is carried out on holes which are aggregated by single cell growth and have good growth conditions, and the positive holes are the panda PRL monoclonal antibody hybridoma cell strain. The invention finally screens and obtains a strain of panda PRL monoclonal antibody hybridoma cell strain PRL-1 and PRL-5.

(9) Subtype identification

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

Preparation: the TBS in the kit is dissolved in 500mL double distilled water for diluting a sample, 870mL double distilled water is uniformly mixed with 30mL 30X Wash Buffer, the mixture is used for washing a plate, a plurality of plates are 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 per hole, namely one sample, adding 50 mu L per 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 per hole of TMB color development liquid for developing color, changing the liquid in the holes into blue color, adding 75 mu L per hole of termination liquid for terminating the reaction after developing color for 5-15min, and changing the liquid from blue color into yellow color. The antibody subtype secreted by the panda PRL monoclonal antibody hybridoma cell strains PRL-1 and PRL-5 obtained by identification and screening is IgG1 type. The results are shown in Table 2.

TABLE 2 hybridoma cell strain subtypes

Subtype type	PRL-1	PRL-5
			IgG1	0.767	0.799
IgG2a	0.077	0.066
			IgG2b	0.078	0.071
IgG3	0.086	0.091
			IgGA	0.076	0.074
IgGM	0.072	0.071
			Kappa	0.562	0.537
Lamba	0.066	0.07

(10) And (3) carrying out amplification culture and purification of the monoclonal antibody, and concentrating.

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.

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

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.

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 PRL monoclonal antibody is concentrated by 10000kda ultrafiltration tube and stored at-20deg.C for use.

(11) Freezing of monoclonal antibody cell lines

After the identified anti-panda PRL monoclonal antibody hybridoma cell lines PRL-1 and PRL-5 were cultured stably, the cells in the flask were blown to suspend the cells in the culture (cells were typically suspended in the culture or grown on an adherent) and transferred to a 15mL centrifuge tube. 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, sucking out the supernatant by using a sample gun, adding a proper amount of frozen stock solution (frozen stock solution=5 mL of serum+4mLDMEM+1 mLDMSO) into the cells, mixing the mixture upside down, and filtering the mixture for later use. Finally, the cells are added into the frozen storage tube, and each tube contains 1mL of cell fluid. Placing into a freezing box, firstly placing overnight at-80 ℃, and then placing cell strains PRL-1 and PRL-5 into liquid nitrogen for long-term storage for standby.

(12) Determination of anti-panda PRL monoclonal antibody titer

The monoclonal antibody titers of the culture solution supernatant purified PRL-1 and PRL-5 cell lines are detected by using an indirect ELISA, and the specific steps are as follows:

(1) The polypeptide fragment of example 1, 2. Mu.g/mL, 50. Mu.L/well, was coated onto an ELISA plate and 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-PRL monoclonal antibodies (1-fold, 2-fold, 4-fold, 8-fold, 16-fold, 32000-fold, 64000-fold, 128000-fold, 256000-fold, 512000-fold, respectively) and PBS wells at a double dilution concentration were added as controls and incubated at 37℃for 1 hour.

(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) Adding TMB chromogenic substrate 100 mu L/hole, reacting 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 3.

As shown in Table 3, the PRL monoclonal antibody titer of the culture supernatant was 10 ⁵ The above indicates high antibody titers.

TABLE 3 antibody titers

Antibody dilution fold	PRL-1	PRL-5
			1:10	2.068	2.258
1：100	2.026	1.934
			1：200	1.720	1.840
1：400	1.284	1.679
			1：800	0.670	1.403
1：1600	0.619	1.277
			1：3200	0.572	0.732
1：6400	0.517	0.535
			1：12800	0.391	0.386
1：25600	0.115	0.263
			Blank control	0.052	0.067

Example 2

The anti-panda prolactin antibody obtained in example 1 was HRP-labeled as follows:

1. dissolving HRP equivalent to antibody in 1mL pure water, adding 0.1mol/L NaIO ₄ 0.5mL, standing at 4deg.C for 30min;

2. 0.5mL of 2.5% ethylene glycol was added and the mixture was stirred slightly at room temperature for 1h;

3. adding an antibody to be marked, regulating the pH value of the reaction solution to 9.0, uniformly mixing, and standing at 4 ℃ overnight;

4. adding 0.1mL of sodium borohydride solution, uniformly mixing, and standing at 4 ℃ for 3 hours;

5. dropwise adding saturated ammonium sulfate solution with equal volume, standing at 4deg.C for 30min,

6. centrifuging at 4000rpm for 15min, removing supernatant, dissolving precipitate with appropriate amount of 0.01mol/l PBS with pH of 7.4, dialyzing at 4deg.C overnight, and changing liquid for 3 times to obtain HRP-labeled antibody.

Example 3

The preparation method of the panda prolactin recombinant protein comprises the following specific steps:

1. construction of panda PRL recombinant plasmid

According to the NCBI PRL gene sequence NM-001304934.1: ATGGACAAAAAAGGGTGGTCGCTGAAAGGGTCTCTCCTGCCCCTGCTGCTGCTGGTGTCTGACCTGCTCCTGTGCCAGAGCGTGGCCTCCCTGCCCATCTGTCCCACCGGGGCTGTCAACTGCCAGGTGTCCCTCCGAGACCTGTTTGACCGCGCGGTCATCCTGTCTCACTACATCCATAACCTCTCCTCGGAGATGTTCAACGAATTTGATAAAAGGTATGCCCAAGGCCGGGGGTTCATTACCAAGGCCATCAACAGCTGTCACACTTCCTCCCTCTCTACCCCTGAAGACAAGGAGCAAGCCCAACAGATCCATCATGAAGACCTTCTGAACCTGATACTCAGAGTGCTACGCTCGTGGAATGACCCCCTGTACCATCTAGTCACAGAAGTACGGGGTATGCAAGAAGCCCCAGATTCGATTCTGTCCAGAGCCATAGAGATTGAGGAACAAAACAGAAGACTTCTAGAGGGTATGGAGAAGATAGTTGGCCAGGTTCATCCTGGAGTCAGAGAAAATGAGGTCTACTCGGTCTGGTCAGGACTTCCATCCCTTCAGATGGCCGATGAAGACACTCGCCTTTTTGCTTTTTATAACCTGCTCCACTGCCTACGCAGGGATTCACATAAGATTGACAATTATCTCAAGCTCCTGAAGTGCCGCATCGTCTACGACAGCAACTGCTAA

The sequence is subjected to signal peptide excision and optimized, enzyme cutting sites Nde I and XhoI are added to carry out chemical synthesis on the final target sequence, and the final target sequence is constructed on a PET28b carrier in the forward direction. Sequencing is carried out, and enzyme digestion identification is carried out on the recombinant vector by using Nde I and XhoI enzymes. The sequencing result is compared with the original target gene sequence in FIG. 1 (both a and b in FIG. 1 are the gene sequence comparison), and the detection result after enzyme digestion is in FIG. 2. As shown in FIG. 1 and FIG. 2, the panda PRL recombinant plasmid successfully constructed in the application is shown to be successfully constructed and named pET28b-PRL.

2. Preparation of panda PRL recombinant protein

(1) Prokaryotic expression of recombinant proteins

1) Transformation

1. Mu.L of recombinant pET28b-PRL plasmid was used to transform Rosetta (DE 3) strain, which was heat-shocked at 42℃for 90s, allowed to stand on ice for 2min, plated (30. Mu.g/mL kanamycin and 34. Mu.g/mL chloramphenicol), and incubated overnight at 37 ℃.

2) The best induction conditions were selected for the pilot culture

Single colonies of the expression strain Rosetta (DE 3) were picked up and cultured in tubes (4 mL LB medium, 30. Mu.g/mL kanamycin and 34. Mu.g/mL chloramphenicol) at 37℃overnight at 220 rpm. The cultured bacterial solutions were inoculated into 4mL of LB medium at a ratio of 1:100, respectively, and 30. Mu.g/mL of kanamycin and 34. Mu.g/mL of chloramphenicol were added thereto for cultivation at 37℃and 220 rpm. When the OD value reaches about 0.6, IPTG with a final concentration of 0.5mM is added, and the mixture is induced at 220rpm and 20 ℃ for overnight; and 220rpm,37 ℃ for 5 hours, and not adding IPTG inducer as a negative control. The cells were collected and suspended in PBS buffer. SDS-PAGE electrophoresis detection, and selecting the optimal protein expression induction condition.

3) Massive induction of

The cultured bacterial liquid is inoculated into 4L of LB liquid medium according to the proportion of 1:100, 30 mug/mL kanamycin and 34 mug/mL chloramphenicol are added, the culture is carried out at 37 ℃ and 220rpm, when the OD value reaches about 0.6, 0.5mM IPTG with the final concentration is added, the temperature is 37 ℃,220rpm and 5h are carried out, and the cell bacterial body is collected by centrifugation.

4) Nickel agarose affinity chromatography

The collected bacterial cells were dissolved with a disruption Buffer (50mM Tris,300mM NaCl,0.1%Triton X-100, ph=8.0), and the cells were sonicated in an ice bath at 400w for 20min (sonicated for 3S, suspended for 5S as one cycle). After the completion of the ultrasonic treatment, the mixture was centrifuged at 12000rpm/min at 4℃for 20min. The pellet was dissolved with a dissolution Buffer (7M guanidine hydrochloride, 50mM Tris,300mM NaCl,0.1%Triton X-100, ph=8.0), sonicated in an ice bath to break the cells, power 400w,20min (sonication 3S, suspension 5S for one cycle). After completion of the sonication, the mixture was centrifuged at 12000rpm/min at 4℃for 20min, and the precipitate was discarded, and the supernatant was purified in the next step. The equilibrated column was washed with 10 bed volumes of Binding buffer at a flow rate of 60mL/h. The sample (supernatant of the solution) was applied to a column at a flow rate of 45mL/h and the permeate was collected. The column was washed with 10 bed volumes of Wash buffer at a flow rate of 60mL/h.

The Elute Buffer was eluted at a flow rate of 45mL/h and the eluate was collected. Samples of the above eluates were collected separately for SDS-PAGE analysis. And (3) injection: binding Buffer (8M urea, 50mM Tris,300mM NaCl,pH =8.0) Wash Buffer (8M urea, 50mM Tris,300mM NaCl,10/20/50mM imidazole, ph=8.0) protein SDS-PAGE detection after purification of the Binding Buffer (8M urea, 50mM Tris,300mM NaCl,500mM imidazole, ph=8.0), dialyzing 500mM imidazole eluate into 50mM Tris,300mM NaCl,0.1%SKL,PH =8.0 Buffer, dialyzing overnight at 4 ℃, filtering, sub-packaging, and preserving at-80 ℃.

3. Expression and localization of PRL recombinant proteins

SDS-PAGE analysis of the small samples indicated that specific bands of interest appeared at the corresponding positions (-27 KD) in the 37℃pellet, indicating that the fusion proteins were expressed in the form of primary inclusion bodies after induction, see FIG. 3.

4. Purification of PRL recombinant proteins

The fusion protein is purified by a nickel agarose affinity chromatography, and obvious bands appear at corresponding positions (-27 KD) by SDS-PAGE electrophoresis analysis, which shows that the fusion protein is successfully purified, and the panda PRL recombinant protein with high purity is prepared (see figure 4).

EXAMPLE 4 giant panda prolactin assay

The specific measurement method is as follows:

(1) For the assay, panda urine was added to the anti-PRL antibody coated elisa plate at 0.05mL per well. Meanwhile, sample diluent is added to prepare PRL recombinant protein standard products of 0 to 1250ng/mL, and the total number of the PRL recombinant protein standard products is 12. Incubate at 37 degrees for 30 minutes.

(2) The ELISA plate was washed 5 times with the washing solution.

(3) anti-PRL antibody-HRP complex 1:2000 in sample dilution was added, 0.05mL per well.

(4) Incubate at 37 degrees for 30 minutes.

(5) The ELISA plate was washed 5 times with the washing solution.

(6) TMB working solution was added at 0.1mL per well. Incubate at room temperature for 5 to 10 minutes.

(7) The color reaction was stopped with 1M H2SO4, 0.1mL per well.

(8) The standard optical density of the sample cell was read with a microplate reader at a wavelength of 450/630, and then the PRL content of the sample was calculated from the standard curve. The standard curve obtained is shown in FIG. 5.

Example 5

The invention establishes a sandwich type enzyme-linked immunosorbent assay method for the PRL metabolite in panda urine by using panda PRL specific antibodies.

The detection method of the panda luteinizing hormone provided by the invention is calculated by adding twice of the standard deviation to the average value of the 0-hole values of 10 repeated tests, and the detection method is high in sensitivity and can reach 1.2ng/mL.

To verify the reproducibility of the detection kit of the present invention, the results of table 1 were obtained by the test:

TABLE 1 repeatability results of panda LH detection kit

	Inter-batch error CV%	Within-batch error CV
			Standard curve (n=10)	9.2	5.37

As can be seen from Table 1, the average of the standard points was less than 10% for both the intra-and inter-batch errors. According to the standard known in the ELISA method, the error in the batch is lower than 10% and the error between batches is lower than 15%, so that the kit can be identified as having reliable repeatability (see Micillef J, ahsan R.Immunosaydevelopement. In: gosling JP, basso LV, editors.Immunoassay: laboratory analysisand clinical applications: london: butterworth-Heinemann;1994.P. 51-680). It can be seen from this: the panda PRL detection method provided by the invention has very reliable repeatability.

Example 6

The panda prolactin enzyme-linked immunosorbent assay method provided by the invention can realize monitoring of the panda gestation prolactin, indicates the implantation development time of panda embryos through the change of the gestation hormone, and effectively guides the breeding management of panda in the breeding period. Fig. 6 is a trend graph of 1 gestation progestin versus prolactin, and it can be seen from fig. 6 that high levels of prolactin occur before progestin rises rapidly, suggesting that high prolactin levels are required for initiation of embryonic implantation development in pandas.

Claims

1. The panda prolactin ELISA detection method is characterized in that anti panda PRL monoclonal antibodies respectively generated by hybridoma cell strain PRL-1 and/or hybridoma cell strain PRL-5 are used for detection;

wherein, the preservation number of the hybridoma cell strain PRL-1 is CCTCC NO: C202183; the hybridoma cell strain PRL-5 has a preservation number of CCTCC NO: C202172, and is preserved in China center for type culture Collection (China, with a preservation number of 10 months and 28 days of 2021).

2. The method for detecting panda prolactin according to claim 1, wherein the anti-panda PRL monoclonal antibodies produced by the hybridoma cell line PRL-1 and the hybridoma cell line PRL-5 are used as the primary antibody or the secondary antibody, respectively, in detecting panda prolactin.

3. The method for detecting panda prolactin enzyme-linked immunosorbent assay according to claim 1, wherein the anti-panda PRL monoclonal antibody is secreted by hybridoma cell lines PRL-1 and PRL-5 by using a panda prolactin specific sequence with an amino acid sequence shown as SEQ ID No.1 as an immunogen.

4. The method for detecting panda prolactin by enzyme-linked immunosorbent assay according to any one of claims 1 to 3, wherein panda urine is used as a detection substrate.

5. The method for detecting panda lactogenic enzyme-linked immunosorbent assay according to claim 4, comprising the steps of:

(2) Washing the ELISA plate with washing liquid, adding anti-panda PRL monoclonal antibody-HRP complex, and incubating at 35-37 ℃ for 30-35 min;

6. Use of the detection method according to any one of claims 1 to 5, or the anti-panda PRL monoclonal antibody according to any one of claims 1 to 3, for monitoring embryonic development in panda gestation.

7. A kit comprising the anti-panda PRL monoclonal antibody produced by the hybridoma cell line PRL-1 and/or the hybridoma cell line PRL-5 according to any one of claims 1 to 3, respectively.

8. A vector comprising the anti-panda PRL monoclonal antibody of any one of claims 1 to 3.

9. Use of the kit of claim 7, or the vector of claim 8, for detecting panda prolactin, or for monitoring embryonic development in gestation of pandas.

10. The panda PRL monoclonal antibody hybridoma cell strain is characterized in that the hybridoma cell strain is a hybridoma cell strain PRL-1 and/or a hybridoma cell strain PRL-5, and the preservation number of the hybridoma cell strain PRL-1 is CCTCC NO: C202183; the hybridoma cell strain PRL-5 has a preservation number of CCTCC NO: C202172, and is preserved in China center for type culture Collection (China, with a preservation number of 10 months and 28 days of 2021).