Kit for improving pig frozen semen quality and conception rate and application thereof

Abstract

The invention provides a monoclonal antibody aiming at recombinant porcine relaxin, an ELISA (enzyme-linked immunosorbent assay) detection method and application of the monoclonal antibody in a kit for improving the quality and conception rate of frozen semen of pigs. The recombinant porcine relaxin protein is obtained after analysis and optimization by bioinformatics software, and the monoclonal antibody and the polyclonal antibody obtained by using the recombinant porcine relaxin protein as the antigen have the advantages of strong specificity, high binding force with the antigen and the like; and the established ELISA kit is higher in sensitivity during detection. According to the method disclosed by the invention, the relaxin in the seminal plasma is originally and quantitatively detected, the quality of the frozen seminal fluid can be more effectively improved, and various indexes are obviously improved.

Description

Technical Field

[0001] The present invention relates to the field of biological proteins, and more specifically to an antibody against relaxin in pig seminal plasma, quantitative detection of relaxin in pig seminal plasma, and application of the antibody in a kit for improving the quality and conception rate of pig frozen semen. Background Art

[0002] Semen consists of sperm and seminal plasma. Sperm is the main carrier of genetic material, and seminal plasma provides the necessary nutrition and environment for sperm. Among them, seminal plasma is an important component of semen, which is composed of secretions from various glands such as testicles, epididymis, prostate, seminal vesicle, and bulbourethral glands. About 70% of the liquid in seminal plasma is composed of seminal vesicle fluid, 20% is secretions from the prostate, and less than 10% is secretions from other glands. The pH of seminal plasma is maintained at 7.35-7.5, which has a buffering property and protects sperm from the acidic environment of the vagina during fertilization. Seminal plasma is rich in protein, with an average protein concentration of 35-55g / L. During the fertilization process, it mainly provides sperm with the nutrients and energy needed for survival. In addition, the expression of fucose and sialylated residues on certain glycoproteins in seminal plasma can prevent microorganisms and other antigens from penetrating the mucosal surface, thereby participating in innate and acquired immunity.

[0003] The technology of freezing and preserving pig semen has good application value in the protection of pig germplasm resources and pig breeding and production, but pig frozen semen has problems such as low sperm motility after thawing, changes in sperm physical and chemical properties, and low efficiency of artificial insemination. Existing reports have confirmed that adding seminal plasma from boars with good sperm freezing resistance to freezing diluent can significantly improve the quality of frozen semen. Some studies also believe that adding seminal plasma to the thawing solution is beneficial to improving the acrosome integrity and fertilization ability of thawed sperm. However, the sperm reproduction performance between boars of different breeds and even between boars of different individuals of the same breed is also different, which shows that the semen traits of boars are greatly affected by genetics. Moreover, directly using sperm as the diagnostic object often destroys the structure of sperm and reduces the storage of sperm, which is a huge challenge for scarce resources. Therefore, how to use indirect means to determine whether boars produce effective sperm and ensure the effect of artificial insemination requires in-depth research and determination.

[0004] Studies have confirmed that there is a direct correlation between the relaxin concentration in boar semen and sperm motility. The relaxin level in the semen of Yorkshire, Landrace, Duroc and Boar breeds has a high positive correlation with the sperm motility in the semen of boars of various breeds. However, there is no kit on the market for detecting the relaxin content in boar semen samples, and the relaxin detection reagents in the existing technology often have problems such as low detection sensitivity and low specificity. The lack of a convenient and accurate method to quickly quantify it makes it difficult to transform research results into real productivity. Summary of the invention

[0005] In order to solve the above problems, the present invention first provides a recombinant porcine relaxin protein, and further prepares polyclonal antibodies and monoclonal antibodies that specifically recognize the above recombinant protein, which are used as capture antibodies and detection antibodies in ELISA double antibody sandwich kits, respectively, and establishes a method for using a detection kit for relaxin content in porcine seminal plasma and its application.

[0006] Specifically, the present invention provides a recombinant porcine relaxin protein, the amino acid sequence of the recombinant porcine relaxin protein is shown in SEQ ID NO.1;

[0007] Furthermore, the present invention provides a monoclonal antibody 3B4 against the above recombinant relaxin protein, wherein the heavy chain amino acid sequence of the monoclonal antibody 3B4 is shown in SEQ ID NO: 2, and the light chain amino acid sequence is shown in SEQ ID NO: 3;

[0008] Furthermore, the present invention provides a polyclonal antibody, which is obtained by screening an animal using the above-mentioned recombinant relaxin protein and then by conventional means, and the effective titer of the polyclonal antibody is as high as 1:256000;

[0009] Furthermore, the present invention provides an ELISA kit for quantitatively detecting porcine relaxin protein, the ELISA kit comprising the above-mentioned monoclonal antibody 3B4, recombinant relaxin polyclonal antibody, ELISA plate, blocking solution, HRP-goat anti-mouse IgG secondary antibody, color development solution, stop solution, coating solution and washing solution. The recombinant relaxin polyclonal antibody is used as the capture antibody, and the monoclonal antibody 3B4 is used as the detection antibody.

[0010] Furthermore, the present invention provides a kit for improving the quality and conception rate of frozen semen of pigs, wherein the kit comprises the above-mentioned ELISA detection kit and a semen protection diluent; wherein the ingredients of the semen protection diluent are: 0.5% sodium dodecyl sulfate, 0.5% dodecylamide, 0.2% anhydrous sorbitan fatty acid ester, 0.3% fatty acid monoethanolamide, 0.5% lauric acid, 0.5% triethanolamine, 1% fatty acid monoethanolamide sulfosuccinate monoester salt, 0.2% EDTA, 0.2% epigallocatechin gallate, 10% citric acid buffer, and the balance is double distilled water.

[0011] Furthermore, the present invention provides the use of the above-mentioned kit in improving the quality and conception rate of frozen semen of pigs, wherein the use is:

[0012] 1) isothermally dilute the semen protection diluent with the original semen in a ratio of 1:1, and slowly cool down after packaging; centrifuge to obtain the supernatant of the seminal plasma and the sperm precipitate in the lower layer;

[0013] 2) Quantitatively detect relaxin in the seminal plasma supernatant using an ELISA detection kit, and select the seminal plasma sample with the highest content for use;

[0014] 3) The sperm precipitate is resuspended with a porcine sperm cryopreservation solution, wherein the porcine sperm cryopreservation solution is prepared by adding 20% ​​of the total volume of egg yolk and 2.5% of the total volume of a high relaxin seminal plasma sample to the above-mentioned protective diluent and filtering the mixture using a 0.22 μm sterilizing filter;

[0015] 4) The resuspended sperm solution is placed in a container and gradually cooled to 4°C, then sealed and frozen; after freezing, the semen in the tube is stored in liquid nitrogen.

[0016] Among them, the quantitative detection method of the ELISA detection kit is:

[0017] 1) Purified polyclonal antibody coating: dilute the above polyclonal antibody against porcine relaxin to 1:500 with coating solution, incubate at 4°C overnight, 100 μL per well, incubate at 4°C overnight;

[0018] 2) Blocking: Add 200 μL of 5% skim milk powder to each well and incubate at 37°C for 1.5 h;

[0019] 3) Add antigen: add 100 μL sample to each well and incubate at 37°C for 1 to 2 hours;

[0020] 4) Add purified monoclonal antibody: dilute monoclonal antibody 3B4 to 1:1000 with 5% skim milk powder, incubate at 37°C for 1-2 hours, 100 μL per well;

[0021] 5) Add enzyme-labeled secondary antibody: dilute HRP-goat anti-mouse IgG secondary antibody to 1:16000 with 5% skim milk powder, incubate at 37°C for 2h, 100 μL per well;

[0022] 6) Substrate color development: add 100 μL of color development solution to each well and color development for 10 min;

[0023] 7) Stop the reaction: add 50 μL / well of 2 mol / L concentrated sulfuric acid to each well, measure the OD450nm reaction value, and substitute it into the standard curve formula to calculate the relaxin content.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] 1) The recombinant porcine relaxin of the present invention is obtained after analysis and optimization based on the porcine relaxin protein data in the NCBI GenBank database using bioinformatics software. The monoclonal antibodies and polyclonal antibodies obtained using this as an antigen have the advantages of strong specificity and high antigen binding ability. The polyclonal antibody is used as the capture antibody and the monoclonal antibody is used as the detection antibody, so that the established ELISA kit has higher sensitivity during detection;

[0026] 2) The present invention innovatively performs quantitative detection on relaxin in seminal plasma, and uses the relaxin content as a standard to screen the source of seminal plasma added to the semen cryopreservation reagent, thereby avoiding the uncertainty and blindness of adding seminal plasma before, and can more effectively improve the quality of semen after freezing, with significant improvements in indicators such as sperm motility, acrosome integrity, catalase, glutathione peroxidase, and glutathione reductase activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Electrophoresis of PCR-amplified recombinant porcine relaxin, where M is maker and 1 is the amplified PCR product with a length of about 546 bp;

[0028] Figure 2 The SDS-PAGE electrophoresis results of the induced expression of recombinant porcine relaxin;

[0029] Figure 3 This is the western blot result after the target protein is expressed and purified, where M is the protein Maker, 1 is the negative control, and 2 is the recombinant protein;

[0030] Figure 4 Western blotting of recombinant porcine relaxin monoclonal antibody, M: protein molecular weight standard; 1: negative control; 2: recombinant porcine relaxin protein;

[0031] Figure 5 Standard curve of sandwich ELISA for porcine relaxin protein. Example

[0032] Although the present invention can be implemented in many different forms, what is disclosed here is its specific illustrative embodiment that verifies the principle of the present invention.It should be emphasized that the present invention is not limited to the specific embodiment illustrated.In addition, any section title used herein is only for organizational purposes and is not to be interpreted as limiting the subject matter described.

[0033] Generally, terms relating to cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein and their techniques are those well known and commonly used in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification.

[0034] Example 1. Preparation of relaxin monoclonal antibody

[0035] The amino acid and nucleotide sequences of porcine relaxin protein were downloaded from the NCBI GenBank database. The gene sequence was aligned and analyzed using DNAStar MegAlign software, and its conservative region was selected. The hydrophilicity was aligned and analyzed using DNAMAN, DNAstar and ExPASy software. The hydrophilic conservative region was selected and the amino acid difference sites were avoided to design synthetic peptides. The antigen polypeptide sequence was finally determined as follows: MPRLFSYLLGVWLLASQLPREIPGQSTNDFIKACGRELVRLWVEICGSVSWGRTALSLEEPQLETGPPAETMPSFINKDA EILKMMLEFVPNLPQELKLTLSEMQPALRELQQSALKDSNLNFEEFRKIILNRQNEAEDKSLLELKNLGLDKHSRKHRLF RMALSEKCCQVGCIRKDIARLC (SEQ ID NO.1). The above polypeptide was synthesized and prepared by Jier Biochemical (Shanghai) Biological Company.

[0036] According to the sequence provided by the biological company, the specific upstream primer RLX-F (5'-CCGGATGCATGACCGATGTCAT-3') and downstream primer RLX-R (5'-CTTGGGATCGCCAAACCGGGCACTTCTTTACTGC-3') were designed to amplify the recombinant porcine relaxin. The PCR product was recovered after separation by agarose gel electrophoresis ( Figure 1 ), the recovered fusion protein gene and the plasmid vector pMD20-T for expression were digested with HindIII and BamHI respectively, recovered again by electrophoresis, and connected with T4 DNA ligase. The ligation product was transformed into E. coli competent cells BL21, and the clones on the plate were picked and inoculated, and plasmid DNA was extracted and identified by PCR. The clones with positive fusion protein gene shown by PCR were sequenced and analyzed, and the clones with completely correct sequences were used.

[0037] Transfer the overnight bacteria cultured from a single colony to 100 ml LB medium at a ratio of 1:100, add kanamycin at a final concentration of 50 μg / ml, and culture at 37°C with shaking until the OD600 is 0.6-0.8. Add 0.1 mol / L IPTG, culture at 25°C with shaking for 8 hours, and then ultrasonically disrupt the bacteria after harvesting ( Figure 2 ). The recombinant protein has a histidine tag and is affinity purified using a nickel column. After elution with different concentrations of imidazole solution, each component and flow-through are loaded separately for SDS-PAGE separation and detection. The purity of the recombinant protein is above 90%, and the concentration is about 1-1.5 mg / mL, which can meet the requirements of immunized animals and antibody screening and identification ( Figure 3 ).

[0038] Recombinant porcine relaxin was used as an antigen to immunize 8 8-week-old SPF BALB / c female mice (purchased from the Experimental Animal Research Center of the Chinese Academy of Sciences). The antigen was mixed with an equal volume of complete Freund's adjuvant (first immunization) and incomplete Freund's adjuvant (boost immunization) and emulsified. The mixture was fully mixed to an oil-in-water state for subcutaneous multi-point immunization. 2-3 booster immunizations were performed, with an interval of 2 weeks between each immunization. After that, the titer was tested. If the titer was higher than >1:10000, the abdominal cavity was impacted within 1 week. The immunization dose of antigen was directly dissolved in 250 μL of PBS. The specific immunization times and immunization doses are shown in Table 1:

[0039] Table 1 Immunization schedule

[0040] Immunization times Immunization time (d) Immunization dose First immunization 1 200μg / head (equal amount of complete Freund's adjuvant emulsified) Second immunization 15 200μg / head (equal amount of incomplete Freund's adjuvant emulsified) The third immunization 30 200μg / head (equal amount of incomplete Freund's adjuvant emulsified) Fourth immunization 3 days before fusion 150 μg antigen

[0041] The serum collected before immunization was used as a negative serum control. Blood was collected from the tail vein of the immunized mice on the tenth day after each immunization. The antibody titer of the collected serum was tested by indirect ELISA. When the mouse serum titer reached 105 or above, the antigen without adjuvant was used for booster immunization, and cell fusion could be performed 3 days later.

[0042] Three days after the last shock, positive control blood was collected, spleen was taken, and single cell suspension was prepared; SP2 / 0 cells in the logarithmic phase were treated, mixed with spleen cells in a certain ratio (1:5-1:10), 50% PEG1450 was used for 1 min, and the suspension was terminated by dilution with basal medium DMEM. After low-speed centrifugation, the suspension was gently suspended and mixed with HAT medium containing 20% ​​fetal bovine serum, and spread on the pre-prepared feeder layer cell plate at 2×107 / plate, and cultured at 5% CO2 and 37°C.

[0043] After the cells on the fusion plate have grown to a medium size of about 10,000 cells or more, the test begins. 450 <0.2, positive control OD 450 >1.0) and then select the positive wells (generally OD 450 ≥0.5) for subcloning.

[0044] The wells with high positive values ​​(OD450>2.0) in the fusion plate were selected for limiting dilution, and 60% of the monoclonal wells on each plate were counted for subcloning. Each time, the monoclonal wells with higher positive values ​​were selected for limiting dilution. ELISA detection could be performed 5-7 days after each subcloning, until the monoclonal cell line that could stably secrete positive antibodies was finally screened out for expanded culture.

[0045] The cell lines that stably secrete positive antibodies screened out in the subcloning stage were expanded and cultured in 24-well plates. After expansion, the supernatant was collected for antigen detection. ELISA gradient dilution and western-blotting were used to verify its stability. The monoclonal antibody hybridoma cell line 3B4 (specific for porcine relaxin protein) was screened and obtained. Figure 4 ), and no positive reaction with relaxin from other species (horse, dog, rat, human).

[0046] First, pristane or liquid paraffin was injected into the mouse peritoneal cavity. One week later, the hybridoma cell line 3B4 was inoculated into the mouse peritoneal cavity. After the cells were established, 10% fetal bovine serum medium was used for expansion culture. When the cell density reached 1×10 6 -2×10 6 / mL, centrifuge at 800rpm, collect the precipitate, resuspend it with PBS, and inject it into the mouse (liquid paraffin) intraperitoneally. After 7-10 days, collect the ascites and purify it by Protein A-agarose affinity chromatography column after pretreatment, wherein the starting buffer is pH7.0, 20mM phosphate buffer; the elution buffer is pH2.70.1mM glycine hydrochloride. Use enough starting buffer (8-10mL) to balance the Protein A-agarose affinity chromatography column (HiTrap Protein A 1mL, Pharmacia Biotech). Take 15-25mL of the sample to be purified (10.2-21.1mg protein per milliliter of sample) and load it onto the column at a flow rate of 0.5mL / min, then wash it with 7-8mL of starting buffer, 6-7mL of elution buffer, and 5mL of starting buffer in sequence at the same flow rate, and collect 1mL of eluate in each tube. The purified antibody used recombinant porcine relaxin protein as antigen, and the titer of the purified monoclonal antibody was detected by indirect ELISA method. The titer of the purified 3B4 monoclonal antibody was 1:920000 as determined by ELISA.

[0047] Total RNA of hybridoma cell line 3B4 was extracted and reverse transcribed into cDNA using universal primers. The light and heavy chains of the antibody were amplified, separated, cloned into standard cloning vectors for expression, and identified by single colony PCR. 5-10 single colonies with correct light and heavy chain lengths were selected for sequencing. If the results of 5 sequencing runs were almost identical, it was considered to be the true sequence of the antibody (the sequencing process and large-scale antibody expression were completed by BGI (Beijing) Co., Ltd.).

[0048] Among them, the heavy chain amino acid sequence of 3B4 is:

[0049] EVQLEESGPQLLKPGASVKISCKADYVIHTRYWIHWVKQRPPGGSTWIGMIDPSASESLNQKYMQLSSTLTEDTAM QLSSPTSEDSAYCVRRYFGYWGQGGTTLTVSS(SEQ ID NO.2)

[0050] The light chain variable region sequence is as follows:

[0051] DIVLTQSPASLAVGLGQRASISCKASQSVDYDGDSYLQKPGAQKPGQPPKLLYAASGVPDRFSGSGTDFTLNIHPV EEEDVATHYCYQGSHVPLTFGAGSKLELK (SEQ ID NO. 3).

[0052] Example 2. Preparation of relaxin monoclonal antibody

[0053] The recombinant porcine relaxin prepared in Example 1 was used as an immunogen to immunize New Zealand white rabbits. An equal amount of complete Freund's adjuvant was added for emulsification during the first immunization, and an equal amount of incomplete Freund's adjuvant was added for the next three times. The emulsified immune complex was subcutaneously administered to 4-month-old male New Zealand white rabbits at multiple points on the neck and back. The immunization dose was 800 μL / rabbit. The second immunization was performed on the 14th day, the third immunization was performed on the 28th day, and the fourth immunization was performed on the 42nd day. Blood was collected from the neck vein on the 45th day, and the collected blood was stored at 4°C overnight. The blood stored overnight was centrifuged at 5000 r / min for 10 minutes to collect serum, and polyclonal antibodies in a portion of the serum were purified by the conventional caprylic acid-ammonium sulfate salting-out method in the art (see "Handbook of Molecular Cloning").

[0054] Indirect ELISA was used to detect the titer of polyclonal antibodies in another part of the collected serum. When the sample absorbance value / negative absorbance value>2.1, it was considered positive. The results are shown in Table 2. The results showed that the effective titer of the polyclonal antibody obtained by screening was as high as 1:256000, which also had good specificity.

[0055] Table 2 Indirect ELISA detection of polyclonal antibody titer

[0056]

[0057]

[0058] Example 3: Establishment of a sandwich ELISA method for porcine relaxin protein

[0059] Based on the applicant's previous research results, the basic reaction steps of establishing ELISA for relaxin in pig seminal plasma are as follows:

[0060] 1) Purified polyclonal antibody coating: dilute the above polyclonal antibody against porcine relaxin to 1:500 with coating solution, incubate at 4°C overnight, 100 μL per well, incubate at 4°C overnight;

[0061] 2) Blocking: Add 200 μL of 5% skim milk powder to each well and incubate at 37°C for 1.5 h;

[0062] 3) Add antigen: add 100 μL sample to each well and incubate at 37°C for 1 to 2 hours;

[0063] 4) Add purified monoclonal antibody: dilute monoclonal antibody 3B4 to 1:1000 with 5% skim milk powder, incubate at 37°C for 1-2 hours, 100 μL per well;

[0064] 5) Add enzyme-labeled secondary antibody: dilute HRP-goat anti-mouse IgG secondary antibody to 1:16000 with 5% skim milk powder, incubate at 37°C for 2h, 100 μL per well;

[0065] 6) Substrate color development: add 100 μL of color development solution to each well and color development for 10 min;

[0066] 7) Stop the reaction: add 2 mol / L concentrated sulfuric acid (50 μL / well) to each well and measure the OD450nm reaction value;

[0067] Wash the plate 3 to 5 times with washing solution between each step 1) to 5), each time for 8 minutes.

[0068] 8) Establishment of standard curve

[0069] The purified recombinant porcine relaxin was diluted to 300, 250, 125, 62.5, 31.25, 15.63, 7.82, 3.9, 1.95, and 0.975 pg / mL. Three replicates were performed for each concentration. The standard curve results are shown in Figure 5 .

[0070] It can be seen from the figure that when the concentration of recombinant porcine relaxin is between 1.95 and 300 pg / mL, the natural logarithm of the protein concentration has a good linear relationship with the OD value, and the linear regression equation is y=0.2304x-0.1507, R 2 =0.991, wherein x is the natural logarithm of the concentration of the recombinant porcine relaxin protein, the detection range of the kit is 1.95-300 pg / mL, and the detection limit is 1.95 pg / mL.

[0071] Example 4: Detection of pig frozen semen quality and conception rate

[0072] Based on the applicant's previous research results, a semen protection diluent was prepared and kept at 37° C. for standby use; the ingredients of the semen protection diluent were: 0.5% sodium dodecyl sulfate, 0.5% dodecylamide, 0.2% sorbitan fatty acid ester, 0.3% fatty acid monoethanolamide, 0.5% lauric acid, 0.5% triethanolamine, 1% fatty acid monoethanolamide sulfosuccinate, 0.2% EDTA, 0.2% epigallocatechin gallate, 10% citric acid buffer, and the balance was double distilled water.

[0073] Routine semen collection is carried out on the boars for semen collection. After being numbered, the fresh semen collected is subjected to routine semen examination. Normal semen is milky white in color, free of impurities, odor, and a slight bloody smell. The viability is above 90%, the vitality is above 80%, and the sperm density is weighed and measured.

[0074] The semen protection diluent was diluted with the original semen at an isothermal ratio of 1:1. The diluted semen was allowed to stand at room temperature for 1 hour, and then the semen was divided into 50mL centrifuge tubes. The centrifuge tubes were placed in a beaker filled with isothermal water, and then transferred to a 17℃ constant temperature box to slowly cool down. After about 2-3 hours, the semen was cooled to 17℃. After equilibrium, the centrifuge was idling to cool down to 17℃, and centrifuged at 800g for 12 minutes in a centrifuge at an ambient temperature of 17℃, and the sperm precipitate in the seminal plasma supernatant and the lower layer were taken respectively.

[0075] Among them, the seminal plasma supernatant was quantitatively analyzed using the sandwich ELISA method to screen out the two samples with the highest and lowest relaxin levels (S5 and S24), which were placed in a 17°C environment for later use.

[0076] The sperm precipitate in the lower layer was resuspended with pig sperm cryopreservation solution (6 times volume), wherein the pig sperm cryopreservation solution was prepared by adding 20% ​​egg yolk in the total volume to the above-mentioned protective diluent and filtering it with a 0.22 μm sterilizing filter.

[0077] At the same time, in order to verify the effect of adding seminal plasma, 2.5% (v / v) of S5 and S24 seminal plasma supernatants were added to the pig sperm cryopreservation solution, and the same volume of semen protection diluent was set up as a negative control.

[0078] The resuspended sperm solution is placed at 4°C for 2-3 hours, gradually cooled to 4°C at a rate of 5-8°C / h, and then filled and sealed with 0.5ml straw tubes; the filled straw tubes of semen are stacked on a rack and placed in a programmed freezer for freezing according to a set curve; after freezing, the straw tubes of semen are stored in liquid nitrogen.

[0079] Thawing procedure: Gently shake the cryovials stored in liquid nitrogen for 1 to 3 minutes in a water bath at 37°C until they are completely thawed, allowing them to revive and perform sperm motility testing.

[0080] The detection methods of sperm survival rate, acrosome integrity rate and seminal plasma antioxidant enzyme activity after freezing are as follows:

[0081] (1) Immediately after thawing, sperm motility test is performed at 35℃~37℃. During the assessment, a drop of semen is dipped into a glass rod and dropped onto a glass slide. A cover glass is added so that the space between the cover glass and the glass slide is filled with semen. The semen is then observed under a microscope at 400x~600x magnification. In the field of view of the microscope, the sperm motility is assessed by comprehensively observing the sperm motility in the upper, middle and lower layers of the semen.

[0082] (2) Take a drop of thawed semen and drop it on a clean glass slide. Use another glass slide at a 45-degree angle to spread the semen to the other side to make a smear. After natural air drying, fix it with 1-2 ml formalin phosphate buffer for 15 minutes, wash and dry it, and then stain it with Giemsa for 90 minutes. Then remove the stain on the smear, air dry it, and observe 300 sperm under a biological microscope at 1000 times. Record the total number of sperm and the number of sperm with intact acrosomes.

[0083] (3) The sperm tail bending rate was calculated using the hypo-osmotic swelling test (HOST) to determine the sperm plasma membrane integrity. Sperm plasma membrane integrity = number of sperm with bent tails / number of sperm counted × 100%.

[0084] (4) The activity of seminal plasma antioxidant enzymes was measured using relevant enzyme measurement kits (all enzyme measurement kits were from Nanjing Jiancheng Biotechnology Co., Ltd.) in strict accordance with the operating procedures.

[0085] The test results are shown in Table 3.

[0086] Table 3 Results of frozen semen sperm quality test

[0087]

[0088] From the above results, it can be seen that adding seminal plasma to the cryoprotectant can effectively protect sperm and reduce the physical and chemical damage to sperm during freezing and thawing. At the same time, compared with seminal plasma with low expression of relaxin screened by the above quantitative method, seminal plasma with high expression of relaxin can effectively improve the protective power of the diluent on frozen semen in terms of sperm motility, acrosome integrity, catalase, glutathione peroxidase, glutathione reductase activity and other indicators. This result is also consistent with the conclusions in the prior art, providing a direction for the next step of developing a more effective freezing diluent.

[0089] The above description of the embodiments is to facilitate the understanding and use of the present invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments. Improvements and modifications made by those skilled in the art based on the principles of the present invention without departing from the scope of the present invention should be within the protection scope of the present invention.

Claims

1. A recombinant porcine relaxin protein, characterized in that: The amino acid sequence of the recombinant porcine relaxin protein is shown in SEQ ID NO.

1.

2. A monoclonal antibody 3B4 that specifically recognizes porcine relaxin protein, wherein the heavy chain amino acid sequence of the monoclonal antibody 3B4 is shown in SEQ ID NO: 2, and the light chain amino acid sequence is shown in SEQ ID NO:

3.

3. An ELISA kit for quantitatively detecting porcine relaxin protein, the ELISA kit comprising the monoclonal antibody 3B4 according to claim 2, a recombinant relaxin polyclonal antibody, an ELISA plate, a blocking solution, an HRP-goat anti-mouse IgG secondary antibody, a color developing solution, a stop solution, a coating solution and a washing solution. The recombinant relaxin polyclonal antibody was used as the capture antibody, and the monoclonal antibody 3B4 was used as the detection antibody.

4. A kit for improving the quality and conception rate of frozen semen of pigs, the kit comprising the ELISA detection kit according to claim 3 and a semen protection diluent; wherein, The semen protection diluent comprises the following components: 0.5% sodium dodecyl sulfate, 0.5% dodecylamide, 0.2% dehydrated sorbitan fatty acid ester, 0.3% fatty acid monoethanolamide, 0.5% lauric acid, 0.5% triethanolamine, 1% fatty acid monoethanolamide sulfosuccinate monoester salt, 0.2% EDTA, 0.2% epigallocatechin gallate, 10% citric acid buffer, and the balance is double distilled water.

5. Use of the kit according to claim 4 in improving the quality and conception rate of frozen semen of pigs, wherein: 1) isothermally dilute the semen protection diluent with the original semen in a ratio of 1:1, and slowly cool down after packaging; centrifuge to obtain the supernatant of the seminal plasma and the sperm precipitate in the lower layer; 2) Quantitatively detect relaxin in the seminal plasma supernatant using an ELISA detection kit, and select the seminal plasma sample with the highest content for use; 3) The sperm precipitate is resuspended with a porcine sperm cryopreservation solution, wherein the porcine sperm cryopreservation solution is prepared by adding 20% ​​of the total volume of egg yolk and 2.5% of the total volume of a high relaxin seminal plasma sample to the above-mentioned protective diluent and filtering the mixture using a 0.22 μm sterilizing filter; 4) The resuspended sperm solution is placed in a container and gradually cooled to 4°C, then sealed and frozen; after freezing, the semen in the tube is stored in liquid nitrogen. Among them, the quantitative detection method of the ELISA detection kit is: 1) Purified polyclonal antibody coating: dilute the above polyclonal antibody against porcine relaxin to 1:500 with coating solution, incubate at 4°C overnight, 100 μL per well, incubate at 4°C overnight; 2) Blocking: Add 200 μL of 5% skim milk powder to each well and incubate at 37°C for 1.5 h; 3) Add antigen: add 100 μL sample to each well and incubate at 37°C for 1 to 2 hours; 4) Add purified monoclonal antibody: dilute monoclonal antibody 3B4 to 1:1000 with 5% skim milk powder, incubate at 37°C for 1-2 hours, 100 μL per well; 5) Add enzyme-labeled secondary antibody: dilute HRP-goat anti-mouse IgG secondary antibody to 1:16000 with 5% skim milk powder, incubate at 37°C for 2h, 100 μL per well; 6) Substrate color development: add 100 μL of color development solution to each well and color development for 10 min; 7) Stop the reaction: add 50 μL / well of 2 mol / L concentrated sulfuric acid to each well, measure the OD450nm reaction value, and substitute it into the standard curve formula to calculate the relaxin content.

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