Application of SAL1 protein as marker for early pregnancy diagnosis of sows

Abstract

The invention belongs to the technical field of live pig feeding, and particularly relates to application of SAL1 protein as a diagnostic marker for early pregnancy of sows, and the amino acid sequence of the SAL1 protein is as shown in SEQ ID NO.1. Whether the SAL1 protein expression level is highly related to pregnancy of the sow or not is found for the first time, the SAL1 protein expression level is suitable for serving as a protein marker for early pregnancy of the sow, and the pregnancy state of the sow can be rapidly judged in the early stage by detecting the SAL1 protein content in vaginal secretion of the bred sow. The kit has the advantages of high sensitivity, good specificity, high accuracy, low misdiagnosis rate, rapid detection, safety and harmlessness to a maternal body and a fetus and the like.

Description

Technical Field

[0001] The invention relates to the technical field of pig breeding, and in particular to application of SAL1 protein as a sow's early pregnancy diagnosis marker. Background Art

[0002] After sows are mated, timely and accurate pregnancy diagnosis is an important technical measure to improve the reproductive efficiency of sows and the production efficiency of the pig industry. Early diagnosis of pregnancy can reduce the empty period or non-reproductive days of non-pregnant sows, and is also conducive to early treatment of sows with reproductive disorders and timely elimination of sows with repeated infertility. Pregnant sows should strengthen feeding management in all aspects, improve the refinement of pregnancy management, maintain the health of sows and fetuses, and avoid "ineffective feeding". Therefore, early pregnancy diagnosis of sows after mating can reduce the empty period of sows, reduce non-productive days, shorten their farrowing interval and reduce feeding costs, thereby improving the reproductive rate of sows and the production efficiency of the pig industry.

[0003] Common methods for diagnosing sow pregnancy include external observation, rectal palpation, hormone diagnosis and ultrasonic diagnosis. These diagnostic methods have their own advantages and disadvantages. External observation refers to judging whether a sow is pregnant by observing whether it returns to estrus. If a sow does not estrus after one estrus cycle after mating, it is preliminarily determined to be pregnant. This method is based on the estrus pattern of sows, and its judgment time limit is long. In addition, the estrus symptoms of some breeds of pigs are not obvious, which can easily lead to misjudgment. Rectal palpation refers to palpating the uterus, cervix and middle uterine artery through the rectum. This method has high requirements for technical personnel and is difficult. It is also easy to cause rectal injury and infection to the sow. In addition, the accuracy is not high in the early stages of sow mating, and its application in production is gradually decreasing. The hormone diagnosis method uses exogenous estrogen to inject sows about 21 days after mating. If the sow is empty-stomach, it will show estrus within 2-3 days; if there is no estrus 2-3 days after injection, it means that it is pregnant; this method must be combined with the boar test and appearance to judge whether it is pregnant, and the injection of hormones is a great stress for the sows after mating, which is easy to cause the uterus of the pregnant sows to contract, making it difficult for the fertilized egg to implant, and the accuracy is not high. The ultrasonic diagnosis method is to explore the sow's uterus through high-frequency sound waves and amplify its echo. This method is used to diagnose early pregnancy in sows. Generally, it has a higher detection rate after the 28th day of pregnancy. Therefore, the currently commonly used early pregnancy diagnosis method has problems such as unclear symptom diagnosis and easy to miss the best mating period for sows. It is urgent to find a more economical, simple and accurate early pregnancy diagnosis method.

[0004] With the rapid development of molecular biology, biomarker-based sow pregnancy diagnosis technology has been increasingly promoted and applied. Protein is the final product of gene expression, and changes in the level of protein markers usually reflect changes in the physiological or pathological state of the body. Therefore, the use of molecular diagnostic technology to sensitively and specifically detect specific protein markers in pregnant sows at an early stage is conducive to the rapid diagnosis of sow pregnancy. However, there are currently few studies on sow early pregnancy proteins, and there is a lack of accurate and reliable protein markers for the diagnosis of sow early pregnancy. Summary of the invention

[0005] In view of the above technical problems in the prior art, the present invention discovers for the first time that the expression level of SAL1 protein is highly correlated with whether the sow is pregnant, and is suitable as a protein marker for early pregnancy in sows. By detecting the SAL1 protein content in the vaginal secretions of sows after mating, the pregnancy status of the sow can be quickly judged at an early stage. The invention has the advantages of high sensitivity, good specificity, high accuracy, low misdiagnosis rate, rapid detection, and safety to the mother and fetus.

[0006] In a first aspect, the present invention provides the use of SAL1 protein as a diagnostic marker in the preparation of a sow early pregnancy diagnostic reagent or kit, wherein the amino acid sequence of the SAL1 protein is shown in SEQ ID NO.1.

[0007] The second aspect of the present invention provides the use of a reagent for detecting the expression level of SAL1 protein in the preparation of a sow early pregnancy diagnostic reagent or kit, wherein the amino acid sequence of the SAL1 protein is shown in SEQ ID NO.1.

[0008] Furthermore, the above-mentioned reagent or kit detects SAL1 protein in the vaginal secretions of the sow.

[0009] Furthermore, the vaginal secretions mentioned above are collected on the 15th day after mating of the sow.

[0010] Furthermore, the preparation method of the vaginal secretions described above includes: adding the collected vaginal secretions to 2 mL of PBS solution, standing for 30 minutes, vortexing for 2 minutes, and then centrifuging at 1000g for 15 minutes, and taking the supernatant as the detection object.

[0011] Furthermore, the sow mentioned above is a Landrace pig, a Yorkshire pig or a hybrid offspring thereof.

[0012] Furthermore, the above-mentioned reagent for detecting the expression level of the SAL1 protein is an antibody that specifically binds to the SAL1 protein.

[0013] Furthermore, the above-mentioned reagent or kit is an enzyme-linked immunosorbent assay reagent or kit.

[0014] The third aspect of the present invention provides a method for diagnosing early pregnancy in sows for non-diagnostic purposes, comprising the following steps:

[0015] Collecting vaginal secretions of the sow to be tested after mating, detecting the content of SAL1 protein in the vaginal secretions, and judging whether the sow to be tested is pregnant according to the content of SAL1 protein;

[0016] When the SAL1 protein is detected to be highly expressed compared to that of a non-pregnant sow, the sow to be detected is determined to be in a pregnant state; when the SAL1 protein is detected to be lowly expressed compared to that of a non-pregnant sow, the sow to be detected is determined to be in a non-pregnant state.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention discovers for the first time that the expression level of SAL1 protein is highly correlated with whether the sow is pregnant or not, and is suitable as a protein marker for early pregnancy in sows for diagnosis of early pregnancy in sows. The expression level of SAL1 protein in vaginal secretions of pregnant and non-pregnant sows is significantly different, and the expression is significantly low in the pregnant group. By detecting the SAL1 protein content in vaginal secretions of sows after mating, the pregnancy status of the sow can be quickly judged at an early stage. The protein has the advantages of high sensitivity, good specificity, high accuracy, low misdiagnosis rate, rapid detection, safety and harmlessness to the mother and fetus, etc., which provides a new direction and approach for diagnosis of early pregnancy in sows and has great practical significance for improving the economic benefits of pig breeding enterprises. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0020] Figure 1 This is a graph showing the expression level of SAL1 protein in vaginal secretions of pregnant and non-pregnant sows detected based on 4D Label-free technology in an embodiment of the present invention;

[0021] Figure 2 This is a diagram showing the expression level of SAL1 protein in vaginal secretions of pregnant and non-pregnant sows detected by enzyme-linked immunosorbent assay in an embodiment of the present invention;

[0022] Figure 3 The figure is a ROC curve diagram of the expression of the SAL1 protein in the vaginal secretions of sows on the 15th day after mating according to the embodiment of the present invention. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. The embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0024] Based on the information contained in the present invention, it is easy for those skilled in the art to make various changes to the precise description of the present invention without departing from the spirit and scope of the appended claims. It should be understood that the scope of the present invention is not limited to the defined processes, properties or components, because these embodiments and other descriptions are only for the purpose of illustrating specific aspects of the present invention. In fact, various changes that a person skilled in the art or related fields can obviously make to the embodiments of the present invention are covered within the scope of the appended claims.

[0025] In order to better understand the present invention but not to limit the scope of the present invention, all the numbers and other numerical values ​​used in the present invention to express the amount, percentage, etc. should be understood as modified by the word "about" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification and the appended claims are approximate values, which may be changed according to the different ideal properties to be obtained. Each numerical parameter should at least be regarded as obtained according to the reported significant figures and by conventional rounding methods.

[0026] In addition, it should be noted that, unless otherwise defined, in the context of the present invention, the scientific and technical terms used should have the meanings commonly understood by those of ordinary skill in the art.

[0027] The terms "include", "comprising", "containing", "having" and the like are not limiting in meaning, that is, other steps and other components that do not affect the results may be added.

[0028] The term "and / or" should be regarded as a specific disclosure of each of the two specified features or components with or without the other. For example, "A and / or B" is regarded as including the following situations: (i) A, (ii) B, and (iii) A and B.

[0029] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0030] In the preliminary test of the present invention, the group samples were divided into a pregnant group and a non-pregnant group by ultrasonic pregnancy detection of sows at 25 days of mating and re-testing at 35 days of pregnancy. Vaginal secretion samples of pregnant and non-pregnant sows at 15 days of mating were collected, and differentially expressed proteins in vaginal secretions of pregnant and non-pregnant sows were screened out by 4D label-free technology combined with systematic bioinformatics analysis. The expression level differences of different group proteins were further verified by enzyme-linked immunosorbent assay (ELISA) technology to obtain a protein marker for early pregnancy diagnosis of sows - SAL1 protein, whose protein expression level is highly correlated with whether the sow is pregnant. The expression level of SAL1 protein in vaginal secretions of pregnant sows 15 days after mating was significantly lower than that of the non-pregnant group. Afterwards, the prediction accuracy of SAL1 protein for the pregnancy status of sows was verified in a new group, and a receiver operating characteristic curve (ROC) was constructed. It was found that the area under the ROC curve (AUC) of SAL1 protein was 1, and it showed great significance (p=0<0.01), which means that the protein marker can be used as an independent predictor for early pregnancy diagnosis of sows, and has a high pregnancy diagnosis value. Finally, the content of SAL1 protein between groups was detected by ELISA technology to identify whether the sows were pregnant, and a comparative analysis was performed with the results of ultrasonic pregnancy detection, showing that the true positive rate of judging the pregnancy status by the content of SAL1 protein was 96.875%, the false negative rate was 3.125%, and the false positive rate was 0, and the true negative rate was 100%. It was confirmed that the SAL1 protein marker of the present invention has good sensitivity and specificity when actually performing pregnancy diagnosis, which is conducive to greatly improving the accuracy of pregnancy detection results and reducing the misdiagnosis rate.

[0031] Based on the above findings, an embodiment of the present invention provides the use of SAL1 protein as a diagnostic marker in the preparation of a sow early pregnancy diagnostic reagent or kit, wherein the amino acid sequence of the SAL1 protein is shown in SEQ ID NO.1.

[0032] The SAL1 protein provided by the present invention is suitable for being used as a protein marker for early pregnancy in sows for early pregnancy diagnosis in sows. Vaginal secretion samples are collected 15 days after sow mating to detect the content of SAL1 protein in vaginal secretions. When the SAL1 protein in the vaginal secretions is detected to be significantly low in expression, the sow is already pregnant. If significantly high in expression is detected, the sow is not pregnant and the next artificial insemination and mating should be performed in time. Compared with existing methods such as hormone diagnosis and ultrasonic diagnosis, the present invention determines whether the sow is pregnant by detecting the content of SAL1 protein in the vaginal secretions of sows after mating. The operation is simple in production, the trauma to pregnant sows is small, and the sows basically do not have a stress reaction. Moreover, the pregnancy state of the sow can be quickly and accurately determined on the 15th day after mating. The method has the advantages of high sensitivity, good specificity, high accuracy, low misdiagnosis rate, rapid detection, safety and harmlessness to the mother and fetus, and provides a new direction and approach for early pregnancy diagnosis in sows. Knowing earlier whether a sow is pregnant is beneficial for shortening the parity interval, reducing the number of non-productive days of the sow, and saving the cost of raising the sow. At the same time, corresponding feeding and management of normal pregnant sows is of great practical significance for improving the economic benefits of pig breeding enterprises.

[0033] In actual operation, the expression level of SAL1 protein is based on the general level of expression of the protein in pigs in the art. The high or low is relative, and is determined based on whether the expression level is high or low compared to the general level. For example, relative to the general expression level of the protein in the non-pregnant group, if it is lower than the expression level, it is determined to be pregnancy; or, relative to the general expression level of the protein in the pregnant group, if it is within the expression level, it is determined to be pregnancy.

[0034] The present invention adopts ELISA technology to detect the expression level of SAL1 protein in the F1 sow group bred by Landrace×Yorkshire, and obtains the optimal threshold concentration of SAL1 protein by ROC analysis, which is 208.19 ng / mL. SAL1 protein is a down-regulated protein. When the concentration is greater than the optimal threshold, it is judged as non-pregnancy, and when the concentration is less than the optimal threshold, it is judged as pregnancy.

[0035] Optionally, the reagent or kit is an enzyme-linked immunosorbent assay reagent or kit.

[0036] Optionally, the reagent or kit detects SAL1 protein in the vaginal secretions of the sow.

[0037] Optionally, the vaginal secretions are collected on the 15th day after the sow is mated.

[0038] Optionally, the sow is a Landrace pig, a Yorkshire pig or a hybrid thereof.

[0039] Based on the same inventive concept as above, another embodiment of the present invention provides the use of a reagent for detecting the expression level of SAL1 protein in preparing a sow early pregnancy diagnostic reagent or kit, wherein the amino acid sequence of the SAL1 protein is shown in SEQ ID NO.1.

[0040] The advantages of using the reagent for detecting the expression level of SAL1 protein in preparing a reagent or kit for diagnosing early pregnancy in sows are the same as the advantages of using SAL1 protein as a diagnostic marker in preparing a reagent or kit for diagnosing early pregnancy in sows compared with the prior art, and will not be repeated here.

[0041] Optionally, the reagent for detecting the expression level of the SAL1 protein is an antibody that specifically binds to the SAL1 protein, and the antibody is used to perform enzyme-linked immunosorbent assay.

[0042] Based on the same inventive concept as above, an embodiment of the present invention further provides a method for diagnosing early pregnancy in sows for non-diagnostic purposes, comprising the following steps:

[0043] Collecting vaginal secretions of the sow to be tested after mating, detecting the content of SAL1 protein in the vaginal secretions, and judging whether the sow to be tested is pregnant according to the content of SAL1 protein;

[0044] When the SAL1 protein is detected to be highly expressed compared to that of a non-pregnant sow, the sow to be detected is determined to be in a pregnant state; when the SAL1 protein is detected to be lowly expressed compared to that of a non-pregnant sow, the sow to be detected is determined to be in a non-pregnant state.

[0045] The present invention uses the expression level of SAL1 protein as an important reference for whether the sow is pregnant, which can greatly improve the sensitivity and specificity of early pregnancy diagnosis in sows, and is beneficial to improving detection accuracy and reducing misdiagnosis rate.

[0046] Optionally, the vaginal secretions are collected on the 15th day after mating of the sow to be tested.

[0047] Optionally, the method for preparing vaginal secretions comprises: adding the collected vaginal secretions into 2 mL of PBS solution, standing for 30 minutes, vortexing for 2 minutes, and then centrifuging at 1000 g for 15 minutes, and taking the supernatant for detecting the content of SAL1 protein.

[0048] The present invention is further described below in conjunction with specific examples. The experimental methods in the following examples where specific conditions are not specified are usually carried out under conventional conditions, such as the conditions described in the Molecular Cloning Laboratory Guide (Fourth Edition) published by Cold Spring Harbor Laboratory, or the conditions recommended by the manufacturer. In addition, the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0049] Main reagents: BSA standard protein for protein quantification was purchased from Beijing Polymer, catalog number 23CA1204; C18 desalting column for protein purification was purchased from Comma Biotechnology, catalog number MPHLB9650; ELISA kit for detecting SAL1 protein was purchased from Shanghai Hengyuan Biotechnology Co., Ltd.

[0050] Sow group: originated from the pig breeding base of Xinjiang Tiankang Animal Husbandry Technology Co., Ltd., and are F1 sows bred from Changbai × Yorkshire.

[0051] Protein marker: SAL1 (Salivary lipocalin) protein, whose UniPort sequence number is A0A8D0HYQ4; the amino acid (AA) sequence of the protein is as follows:

[0052] MKLLLLLCLGTLASSHKEAGQDVVTSNFDASKIAGEWYSILLASDAKENIEENGSMRVFVEHIRVLDNSSLAFKFQRKVNGECTDFYAVCDKVGDGVYTVAYYGENKFRLLEVNYSDYVILHLVNVNGDKTFQLMEFYGRKPDVEPKLKDKFVEICQQYGIIKENIIDLTKIDRCFQLRGSGGVQESSAE (see SEQ ID NO.1).

[0053] Example 1 Screening for differential proteins in vaginal secretions of pregnant and non-pregnant sows on day 15 after mating

[0054] In this example, vaginal secretions of pregnant and non-pregnant sows on the 15th day after mating were collected to separate total protein, and then 4D label-free technology and bioinformatics analysis were used to screen out proteins that were differentially expressed in early pregnancy. The specific steps are as follows:

[0055] 1. Test samples

[0056] A total of 103 Landrace × Yorkshire F1 sows were used in this embodiment, all of which were from the pig breeding base of Xinjiang Tiankang Animal Husbandry Technology Co., Ltd. The parities of the experimental sows were 2 (23), 3 (16), 4 (36) and 5 (12), respectively, and the parities of the other 16 sows were 6-8. Vaginal secretions of the whole group of sows were collected on the 15th day after mating. The collection time was after feeding in the morning. When the vaginal secretions were collected, the sows were in a standing state. Before collection, the vulva was cleaned with clean water, the feces were washed away, and the gynecological cotton swab was inserted into the sow's genitals at an angle of 45° upwards and placed for 10 minutes. After the collection was completed, all samples were immediately frozen in liquid nitrogen and stored at -80°C until use. Whether the sows were pregnant was detected by ultrasonic diagnosis on the 25th day, and re-examination was performed on 35 days. Among them, 8 sows were non-pregnant groups and the rest were pregnant groups.

[0057] 2. Total protein extraction and quantification

[0058] Take out the sample from the -80℃ refrigerator, add 2mL PBS solution, let it stand for 30min, and then vortex for 2min. Use a disposable syringe to absorb all the liquid, centrifuge at 1000g for 15min, take the supernatant and transfer it to a 1.5mL centrifuge tube, add an appropriate amount of DB protein dissolving solution (including 8M urea and 100mM triethylammonium bicarbonate (TEAB), pH=8.5), shake and mix, and fully lyse it in an ice water bath for 5min. Centrifuge at 4℃ and 12000g for 15min, take the supernatant and add an appropriate amount of 1M dithiothreitol (DTT) to react at 56℃ for 1h, then ice bath for 2min, then add sufficient iodoacetamide (IAM) and react at room temperature in the dark for 1h.

[0059] Use Bradford protein quantification kit, prepare BSA standard protein solution according to the instructions, and the concentration gradient range is 0-0.5μg / μL. Take BSA standard protein solution with different concentration gradients and sample solution with different dilution multiples and add them to 96-well plates, make up the volume to 20μL, and repeat each gradient 3 times. Quickly add 180μL G250 staining solution, leave at room temperature for 5min, and measure the absorbance at 595nm. Use the absorbance of standard protein solution to draw a standard curve and calculate the protein concentration of the sample to be tested. Take 20μg of protein sample to be tested for 12% SDS-PAGE gel electrophoresis, where the concentrated gel electrophoresis conditions are 80V, 20min, and the separation gel electrophoresis conditions are 120V, 90min. After the electrophoresis, Coomassie Brilliant Blue R-250 staining is performed and decolorization is performed until the band is clear.

[0060] 3. Screening of differentially expressed proteins using 4D label-free proteomics technology

[0061] Label Free is a protein non-labeling quantitative technology based on liquid chromatography tandem mass spectrometry commonly used in proteomics research. Its general process is to detect the unlabeled protein enzymatic peptides through tandem mass spectrometry, and then analyze the mass spectrometry data such as the signal peak intensity of peptide ions and fragment ions to achieve relative quantification of the protein corresponding to the peptide. 4D Label-free proteomics is based on the timsTOF Pro2 mass spectrometer. On the basis of traditional three-dimensional data (retention time, mass-to-charge ratio, ion intensity), it adds a fourth dimension, ion mobility information, which can achieve a more comprehensive analysis of the proteome.

[0062] (1) Proteolysis

[0063] Take a protein sample, add DB protein dissolution solution to make up the volume to 100μL, add 1μg / uL trypsin and 100mM TEAB buffer, mix well and digest at 37℃ for 4h, then add 1μg / uL trypsin for overnight digestion. Add formic acid to adjust the pH to less than 3, mix well and centrifuge at room temperature and 12000g for 5min, take the supernatant and slowly pass it through the C18 desalting column, then use the cleaning solution (including 0.1% (v / v) formic acid and 3% (v / v) acetonitrile aqueous solution) to wash continuously for 3 times, then add an appropriate amount of eluent (including 0.1% (v / v) formic acid and 70% (v / v) acetonitrile aqueous solution), collect the filtrate, and freeze-dry.

[0064] (2) Fraction separation (high-depth quantitative analysis)

[0065] Prepare mobile phase A (including 2% (v / v) acetonitrile and 98% (v / v) water, ammonia water to adjust pH = 10) and mobile phase B (including 98% (v / v) acetonitrile and 2% (v / v) water, ammonia water to adjust pH = 10). Dissolve the freeze-dried protein powder in A solution and centrifuge at 12000g for 10 minutes at room temperature. Use L-3000 HPLC system, the chromatographic column is Waters BEH C18 (4.6×250mm, 5μm), the column temperature is 45°C, and the elution gradient is shown in Table 1. Collect 1 tube per minute, merge into 10 fractions, freeze-dry and set aside.

[0066] Table 1 Liquid chromatography elution gradient for peptide fraction separation

[0067]

[0068]

[0069] (3) Liquid quality testing

[0070] Prepare mobile phase A (aqueous solution containing 0.1% (wt) formic acid) and B (acetonitrile solution containing 0.1% (wt) formic acid). Use 10 μL of A to dissolve the freeze-dried peptide fraction powder, centrifuge at 14000g for 20 min at 4°C, take 200 ng of the supernatant and inject it for liquid chromatography-mass spectrometry detection. Use Bruker Nanoscale UHPLC system, liquid chromatography elution conditions are shown in Table 2.

[0071] Table 2 nanoElute liquid chromatography elution gradient

[0072]

[0073] A tims TOF pro2 mass spectrometer and a Captive Spray ion source were used. The ion spray voltage was set to 1.5 kV, the full mass spectrometry scan range was m / z 100-1700, the Ramp time was 100 ms, the Lock Duty Cycle was 100%, PASEF settings were: 10 MS / MS scans (total cycle time was 1.17 sec), the ion intensity threshold was 2500, and the Scheduling target intensity was 20000; the mass spectrometry detection raw data (.d) were generated.

[0074] (4) Data analysis

[0075] ① Protein identification and quantification: All result spectra were searched using the search software Proteome Discoverer (PD, Thermo, HFX and 480) or MaxQuant (Bruker, Tims) according to the Sus_scrofa_uniprot_2023_10_18.fasta (333489 sequences) protein database. The Proteome Discoverer search parameters were set as follows: the mass tolerance of the precursor ion was 10 ppm, and the mass tolerance of the fragment ion was 0.02 Da. The fixed modification was alkylation of cysteine, the variable modification was methionine oxidation modification, the N-terminal methionine of the protein was lost, and a maximum of 2 missed cleavage sites were allowed. The MaxQuant search parameters were set as follows: the mass tolerance of the precursor ion was 20 ppm, and the mass tolerance of the fragment ion was 0.05 Da. The fixed modification was alkylation of cysteine, the variable modification was methionine oxidation modification, the N-terminus was acetylated, and a maximum of 2 missed cleavage sites were allowed.

[0076] In order to improve the quality of the analysis results, the search results were further filtered using Proteome Discoverer or MaxQuant software: Peptide Spectrum Matches (PSMs) with a credibility of more than 99% were credible PSMs, and proteins containing at least one unique peptide were credible proteins. Only credible peptides and proteins were retained, and FDR verification was performed, and peptides and proteins with FDR greater than 1% were removed. The protein quantification results were statistically analyzed using the T-test test to screen out differentially expressed proteins (DEPs) between the pregnancy group and the non-pregnancy group. Among them, the proteins with a significant test value between the groups p<0.05 and a quantitative ratio FC>1.2 or FC<0.83 of the two groups of samples were defined as DEPs.

[0077] ② Functional analysis of proteins and DEPs: Based on Rfam, PRINTS, PrODOm, SMART, RroSite and PANTHER databases, InterProScan software was used for Gene Ontology (GO) and InterPro (IPR) functional annotations, and functional protein family and pathway analysis of the identified proteins was performed based on Clusters of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Volcano plot analysis, cluster heat map analysis, and pathway enrichment analysis of Gene Ontology (GO), IPR and KEGG were performed for DEPs, and possible protein-protein interactions were predicted using STRINGDB software (access link: http: / / STRING.embl.de / ).

[0078] In this example, vaginal secretion samples of pregnant and non-pregnant sows 15 days after mating were collected, and differentially expressed proteins in the vaginal secretion samples of pregnant and non-pregnant sows were screened and detected. Through GO and KEGG enrichment analysis and differential protein interaction network analysis, the differentially expressed protein SAL1 was screened (protein sequence see SEQ ID NO.1 or UniPort sequence number: A0A8D0HYQ4).

[0079] The expression level of SAL1 protein in vaginal secretions of pregnant and non-pregnant sows was shown in Figure 1 It can be seen that the expression level of SAL1 protein in the non-pregnancy (NP) group was significantly higher than that in the pregnancy (P) group, that is, SAL1 protein is a down-regulated protein, and the expression level of this protein will be significantly reduced after the sow becomes pregnant.

[0080] 4. ELISA verification of the expression level of SAL1 protein in different groups

[0081] To further verify the protein expression results of 4D label-free detection, the expression of SAL1 protein was verified using enzyme-linked immunosorbent assay (ELISA). The ELISA enzyme-linked immunosorbent assay was performed according to the instructions of the SAL1 protein detection kit. The specific operation process is as follows: (1) Preparation of standard solution: The kit provides one original standard and one diluent, and standard solutions of 4000, 2000, 1000, 500, and 250 ng / L are prepared; (2) Sample addition: Accurately add 50 μL of sample to the standard well on the enzyme-labeled plate, first add 40 μL of sample diluent to the sample well to be tested, and then add 10 μL of the sample to be tested (the final dilution of the sample is 5 times), add the sample to the bottom of the well of the enzyme-labeled plate, try not to touch the wall of the well, and gently shake to mix. In addition, set up a blank control well, which is not added Samples and enzyme-labeled reagents, the rest of the steps are the same; (3) Incubation: Seal the plate with a sealing film and incubate at 37°C for 30 minutes; (4) Washing: Dilute the 30-fold concentrated washing solution with 30-fold distilled water for later use; Carefully peel off the sealing film, discard the liquid, shake dry, fill each well with washing solution, let it stand for 30 seconds and then discard it, repeat this 5 times, and pat dry; (5) Add enzyme: Add 50μL of enzyme-labeled reagent to each well, except for the blank well; (6) Incubation: Same as (3); (7) Washing: Same as (4); (8) Color development: First add 50μL of color developer A to each well, then add 50μL of color developer B, gently shake and mix, and color develop at 37°C in the dark for 10 minutes. (9) Stop: Add 50μL of stop solution to each well to stop the reaction (the blue color immediately turns yellow). (10) Determination: Zero the blank well and measure the absorbance (OD value) of each well in sequence at a wavelength of 450nm. The measurement should be carried out within 15 minutes after adding the stop solution.

[0082] The sample processing method includes: taking out the collected vaginal secretion sample from the liquid nitrogen tank, adding 2 mL of PBS solution, standing for 30 minutes and vortexing for 2 minutes; then using a disposable syringe to absorb all the liquid, centrifuging at 1000g for 15 minutes, taking the supernatant and transferring it to a 1.5 mL centrifuge tube and storing it at -20℃ or -80℃, but repeated freezing and thawing should be avoided.

[0083] The standard curve was drawn with the concentration of the standard as the horizontal axis and the OD value as the vertical axis. According to the OD value of the sample to be tested, the corresponding concentration was calculated from the standard curve, and then multiplied by the dilution factor to calculate the sample concentration. SPSS21.0 software was used to analyze the differential protein expression between the pregnancy and non-pregnancy groups. The data were expressed as mean + standard error (SD), and the differences between the samples were tested by t-test or variance analysis. p<0.05 indicated a significant difference, and p<0.01 indicated an extremely significant difference.

[0084] Figure 2 The average expression level of SAL1 protein in the pregnant group and the non-pregnant group detected by ELISA is shown. The expression of SAL1 protein in the vaginal secretions of pregnant and non-pregnant sows 15 days after mating is the same as the 4Dlabel-free detection result, and the difference between the groups is extremely significant, suggesting that SAL1 protein can be used as a pregnancy diagnostic marker. Example 2 Group verification of SAL1 protein expression in vaginal secretions of sows in different groups

[0085] 1. Detection of SAL1 protein expression levels in the pregnancy group and the non-pregnancy group

[0086] To further verify the protein expression results of 4D label-free detection in Example 1, the expression of SAL1 protein in the pregnancy group and the non-pregnancy group was detected using ELISA technology in a new large group.

[0087] Sample information collection: 40 vaginal secretion samples of Landrace × Yorkshire F1 sows were collected on the 15th day after mating, immediately frozen in liquid nitrogen, transported back to the laboratory as soon as possible and stored in an ultra-low temperature refrigerator for future use.

[0088] Sample information confirmation: After the vaginal secretion samples were collected, all sows were subjected to ultrasound diagnosis on the 25th day after mating, and re-examined on the 35th day after mating to determine the pregnancy status of all sows. The sample test results showed that 32 cases were pregnant and 8 cases were not pregnant.

[0089] Detection of SAL1 protein content: ELISA enzyme-linked immunosorbent assay of SAL1 protein was performed according to Example 1.

[0090] 2. ROC curve and area under the curve AUC data analysis of protein biomarkers

[0091] The receiver operating characteristic curve (ROC curve for short) is a line graph established with 1-specificity (also called false positive rate or false alarm rate) as the X-axis and true positive rate (also called sensitivity) as the Y-axis. It is a comprehensive indicator reflecting the continuous variables of sensitivity and specificity. The true positive rate (TPR) refers to the ratio of the number of samples that are actually positive and are correctly judged as positive to the number of samples that are actually positive. TPR is also called Sensitivity; the false positive rate (FPR) refers to the ratio of the number of samples that are actually negative and are incorrectly judged as positive to the number of samples that are actually negative. FPR is equal to 1-Specificity, also known as the false alarm rate. In this field, the area value below the ROC curve represents the accuracy of the prediction, which is called the AUC (Area Under Curve) value. The larger the AUC value, the higher the prediction accuracy, and vice versa. The AUC value is between 0 and 1. The judgment of the AUC value is as follows:

[0092] AUC < 0.5: does not conform to the actual situation, the predicted diagnosis is worse than random guessing, which should not occur in the actual situation;

[0093] AUC = 0.5: indicates that there is no predictive diagnostic value at all, and the prediction accuracy is the same as the guessing effect;

[0094] 0.5<AUC<0.7: The predictive diagnostic value is very low, which is relatively common;

[0095] 0.7≤AUC<0.9: has certain predictive diagnostic value and is of great practicality for actual diagnosis;

[0096] AUC ≥ 0.9: indicates high predictive diagnostic value, which is a good situation;

[0097] AUC=1 means perfect prediction without any flaws. In most cases, there is no perfect predictive diagnosis.

[0098] According to the results of the ELISA test, the ROC curve was constructed for the expression period and the pregnancy outcome corresponding to the differentially expressed protein SAL1, and its AUC area and confidence level were calculated to determine the diagnostic value of SAL1 protein for pregnancy outcomes. Figure 3 and Table 3. The optimal critical value (Cut-off value) in the table refers to the maximum value of the Youden Index (Youden Index = Sensitivity + Specificity – 1). The maximum Youden Index can be obtained at the Cut-off value.

[0099] The results showed that on the 15th day of mating, the AUC value corresponding to the SAL1 protein in the vaginal secretions of sows was 1, and was extremely significant (p=0<0.01), which means that at the 15th day of pregnancy, the diagnostic value of detecting the expression level of SAL1 protein for pregnancy is very high, and SAL1 protein is a reliable marker for the diagnosis of early pregnancy.

[0100] Table 3 Summary of ROC curve results

[0101]

[0102] According to the ROC curve results, the optimal threshold concentration of SAL1 protein is 208.19 ng / mL, which is a down-regulated protein. When the SAL1 protein concentration obtained by ELISA is greater than 208.19 ng / mL, it is judged as a non-pregnant state, and when it is lower than this concentration, it is judged as a pregnant state.

[0103] The pregnancy diagnosis results of the present invention using the SAL1 protein marker were compared with the ultrasound diagnosis results (32 cases of pregnancy and 8 cases of non-pregnancy). The results are shown in Table 4, where the true positive rate is the proportion of individuals in which the actual pregnant sows were detected as pregnant, the false positive rate is the proportion of individuals in which the actual non-pregnant sows were detected as pregnant, the true negative rate is the proportion of individuals in which the actual non-pregnant sows were detected as non-pregnant, and the false negative rate is the proportion of individuals in which the actual pregnant sows were detected as non-pregnant.

[0104] Table 4 Comparative analysis of pregnancy diagnosis results in the population based on SAL1 protein concentration and ultrasound diagnosis results

[0105]

[0106] It can be seen from the table that the true positive rate of SAL1 is 96.875%, and the false negative rate is 3.125%, indicating that it has a high accuracy in actual pregnancy diagnosis, and the false positive rate is 0%, and the true negative rate is 100%, indicating that the misdiagnosis rate of SAL1 for detecting non-pregnant sows is extremely low. It is confirmed that the SAL1 protein provided by the present invention is suitable as a diagnostic marker for early pregnancy in sows, and has good sensitivity and specificity for early pregnancy diagnosis, which is conducive to improving the accuracy of the diagnostic results and reducing the misdiagnosis rate.

[0107] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of SAL1 protein as a diagnostic marker in the preparation of diagnostic reagents or kits for early pregnancy in sows.

2. Use of the SAL1 protein according to claim 1 as a diagnostic marker in the preparation of a sow early pregnancy diagnostic reagent or kit, characterized in that: The amino acid sequence of the SAL1 protein is shown in SEQ ID NO.

1.

3. Use of the SAL1 protein according to claim 1 as a diagnostic marker in the preparation of a diagnostic reagent or kit for early pregnancy in sows, characterized in that: The reagent or kit detects the SAL1 protein in the vaginal secretions of the sow.

4. Use of the SAL1 protein according to claim 3 as a diagnostic marker in the preparation of a diagnostic reagent or kit for early pregnancy in sows, characterized in that: The vaginal secretions were collected on the 15th day after the sow was mated.

5. Use of the SAL1 protein according to claim 3 as a diagnostic marker in the preparation of a diagnostic reagent or kit for early pregnancy in sows, characterized in that: The preparation method of the vaginal secretions comprises: The collected vaginal secretions were added to 2 mL of PBS solution, allowed to stand for 30 minutes, vortexed for 2 minutes, and then centrifuged at 1000 g for 15 minutes, and the supernatant was taken as the detection object.

6. Use of the SAL1 protein according to claim 1 as a diagnostic marker in the preparation of a diagnostic reagent or kit for early pregnancy in sows, characterized in that: The sow is a Landrace pig, a Yorkshire pig or a hybrid offspring thereof.

7. Use of the SAL1 protein according to claim 1 as a diagnostic marker in the preparation of a diagnostic reagent or kit for early pregnancy in sows, characterized in that: The reagent or kit is an enzyme-linked immunosorbent assay reagent or kit.

8. Use of a reagent for detecting the expression level of SAL1 protein in the preparation of a diagnostic reagent or kit for early pregnancy in sows, characterized in that: The amino acid sequence of the SAL1 protein is shown in SEQ ID NO.

1.

9. Use of the reagent for detecting the expression level of SAL1 protein according to claim 8 in preparing a diagnostic reagent or kit for early pregnancy in sows, characterized in that: The reagent for detecting the expression level of the SAL1 protein is an antibody that specifically binds to the SAL1 protein.

10. A method for diagnosing early pregnancy in sows for non-diagnostic purposes, characterized in that: The following steps are involved: Collecting vaginal secretions of the sow to be tested after mating, detecting the content of SAL1 protein in the vaginal secretions, and judging whether the sow to be tested is pregnant according to the content of SAL1 protein; When the SAL1 protein is detected to be highly expressed compared to that of a non-pregnant sow, the sow to be detected is determined to be in a pregnant state; when the SAL1 protein is detected to be lowly expressed compared to that of a non-pregnant sow, the sow to be detected is determined to be in a non-pregnant state.

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