Protein marker for early pregnancy diagnosis of sows and application of protein marker

Abstract

The invention belongs to the technical field of live pig feeding, and particularly relates to a protein marker for early pregnancy diagnosis of sows and application of the protein marker. It is found for the first time that DEF6 and MUC5AC proteins can be independently or jointly used as the protein markers for rapidly detecting whether sows are pregnant or not, the pregnancy state of the sows can be rapidly and accurately judged in the early stage of hybridization by detecting the expression level of the protein markers, a new direction and approach are provided for diagnosis of early pregnancy of the sows, and the method is suitable for popularization and application. The method plays an important role in shortening the fetal interval of the sows, reducing the non-production days of the sows, improving the economic benefits of pig raising enterprises and the like.

Description

Technical Field

[0001] The invention relates to the technical field of pig breeding, and in particular to a protein marker for diagnosing early pregnancy in sows and an application thereof. Background Art

[0002] With the development of intensive pig farming, the role of early pregnancy diagnosis in improving the reproductive rate of sows and the economic benefits of pig farms is becoming more and more obvious. After the sow is mated, timely and accurate judgment can be made on whether the sow is pregnant. Re-breeding measures can be taken in time for empty-stomach sows to reduce their empty-stomach period and shorten the interval between parities, which is beneficial to improving the reproductive capacity of sows and reducing the feed costs increased by ineffective feeding; for pregnant sows, timely fetal preservation can be carried out and thoughtful pregnancy management can be provided. Therefore, early and accurate diagnosis of pregnancy can reduce the number of non-productive days of sows, improve the efficiency of sow utilization, increase the annual productivity of sows, reduce feeding costs, etc., which is very important for the effective management of economic pig farms.

[0003] Traditional methods for diagnosing early pregnancy in sows mainly include estrus cycle judgment method, hormone injection diagnosis method, and ultrasound diagnosis method. The estrus cycle judgment method observes whether the sow is in estrus after one estrus cycle (18-25 days) after mating. If the sow is no longer in estrus, has an increasing appetite, is docile, has a naturally drooping tail, walks with the tail tucked between legs when expelled, and has a vagina that shrinks into a line, then it is preliminarily determined that the sow is pregnant. The hormone injection diagnosis method injects estrogens such as diethylstilbestrol intramuscularly into sows 16-18 days after mating. If the sow is empty, it will show estrus within 2-3 days; if there is no estrus 2-3 days after injection, it means that the sow is pregnant. The ultrasound diagnosis method uses ultrasound image atlas to judge. This method is generally used for early pregnancy diagnosis of sows. The detection rate is generally higher on the 28th day of pregnancy. The aforementioned detection methods have problems such as long days, unclear symptom diagnosis, and easy to miss the best mating period for sows, and the reproductive efficiency of sows cannot be effectively improved.

[0004] With the rapid development of molecular biology, molecular diagnostic technology based on biomarkers has become an important guarantee for the early diagnosis and treatment of many diseases with its high sensitivity, high specificity and high efficiency. Biomarkers refer to biochemical indicators that can clearly identify changes in the structure or function of systems, organs, etc. Biological molecules such as DNA, RNA, and proteins can be used as biomarkers for research. Among them, protein, as the final product of gene expression, directly reflects the complexity and variability of the body's life. Protein markers play an irreplaceable role in the early prediction, diagnosis and treatment of diseases; changes in the level of protein markers usually reflect changes in the body's physiological or pathological state. The use of molecular diagnostic technology to quickly, sensitively and specifically detect specific protein markers in pregnant sows in the early stage is conducive to the early diagnosis of sow pregnancy.

[0005] However, there are few studies on early pregnancy proteins in sows, and there is still a lack of accurate and reliable protein biomarkers for the diagnosis of early pregnancy in sows. Therefore, the development of protein biomarkers with early diagnostic value for sows is of great significance for achieving cost reduction and efficiency improvement in pig farming. Summary of the invention

[0006] In view of the above technical problems in the prior art, the present invention discovers for the first time that DEF6 and MUC5AC proteins can be used alone or in combination as protein markers for quickly detecting whether a sow is pregnant. By detecting the expression level of the protein marker of the present invention, the pregnancy status of the sow can be quickly and accurately judged in the early stage of mating, which provides a new direction and approach for the early diagnosis of sow pregnancy, and plays an important role in shortening the parity interval of the sow, reducing the number of non-productive days of the sow, and improving the economic benefits of pig farming enterprises.

[0007] The first aspect of the present invention provides the use of a protein marker in preparing a sow early pregnancy diagnostic reagent or kit, wherein the protein marker is a DEF6 protein and / or a MUC5AC protein.

[0008] The second aspect of the present invention provides the use of a reagent for detecting the expression level of a protein marker in the preparation of a sow early pregnancy diagnostic reagent or kit, wherein the protein marker is a DEF6 protein and / or a MUC5AC protein.

[0009] Furthermore, the amino acid sequence of the DEF6 protein mentioned above is shown as SEQ ID NO.1, and the amino acid sequence of the MUC5AC protein mentioned above is shown as SEQ ID NO.2.

[0010] Furthermore, the protein marker mentioned above is the protein in the vaginal secretions of the sow.

[0011] Furthermore, the time for the above-mentioned early pregnancy diagnosis is the 15th day after the sow is mated.

[0012] Furthermore, the sow mentioned above is a Landrace pig, a Yorkshire pig or a hybrid offspring thereof.

[0013] Furthermore, the above-mentioned reagent for detecting the expression level of the protein marker is an antibody that specifically binds to the protein marker.

[0014] Furthermore, the above-mentioned reagent or kit is an enzyme-linked immunosorbent assay reagent or kit.

[0015] The third aspect of the present invention provides a method for diagnosing early pregnancy in sows for non-diagnostic purposes, comprising the following steps:

[0016] Collecting vaginal secretions of the sow to be tested after mating, detecting the expression level of DEF6 protein and / or MUC5AC protein in the vaginal secretions, and judging whether the sow to be tested is pregnant according to the expression level of the DEF6 protein and / or the MUC5AC protein;

[0017] When it is detected that the DEF6 protein and / or the MUC5AC protein are highly expressed compared to non-pregnant sows, the sow to be tested is determined to be in a pregnant state; when it is detected that the DEF6 protein and / or the MUC5AC protein are lowly expressed compared to non-pregnant sows, the sow to be tested is determined to be in a non-pregnant state.

[0018] Furthermore, the vaginal secretions are collected on the 15th day after the sow to be tested is mated.

[0019] Furthermore, the processing method of the collected vaginal secretions includes: adding the 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 for detecting the expression level of the DEF6 protein and / or the MUC5AC protein.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The expression level of the protein markers provided by the present invention, namely, DEF6 protein and / or MUC5AC protein, is closely related to whether the sow is pregnant. The expression levels of DEF6 protein and MUC5AC protein in the vaginal secretions of pregnant and non-pregnant sows are significantly different and are significantly highly expressed in the pregnant group, which is suitable as a biomarker for diagnosing early pregnancy in sows. It has good sensitivity and specificity, and a very low false alarm rate, and has high accuracy for early pregnancy diagnosis. By detecting the expression level of the protein markers of the present invention, it is possible to quickly and accurately judge the pregnancy status of sows in the early stage of mating, which provides a new direction and approach for the diagnosis of early pregnancy in sows, and is conducive to the early diagnosis of large-scale mating groups, and plays an important role in shortening the parity interval of sows, reducing the number of non-productive days of sows, and improving the economic benefits of pig farming enterprises. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] 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.

[0023] Figure 1This is a graph showing the expression levels of DEF6 and MUC5AC proteins in vaginal secretions of pregnant and non-pregnant sows detected using 4D Label-free technology in an embodiment of the present invention;

[0024] Figure 2 This is a diagram showing the expression levels of DEF6 and MUC5AC proteins in vaginal secretions of pregnant and non-pregnant sows detected by enzyme-linked immunosorbent assay in an embodiment of the present invention;

[0025] Figure 3 The ROC curve diagram of the expression of DEF6 protein in vaginal secretions of sows on the 15th day after mating according to the embodiment of the present invention;

[0026] Figure 4 The ROC curve diagram of the expression of MUC5AC protein in vaginal secretions of sows on the 15th day after mating according to the embodiment of the present invention;

[0027] Figure 5 This is a joint ROC curve analysis of the expression of DEF6 and MUC5AC proteins in the vaginal secretions of sows on the 15th day after mating according to the example of the present invention. DETAILED DESCRIPTION

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] In the preliminary test of the present invention, the sows that were mated for 25 days were detected by B-ultrasound pregnancy detection and re-examination at 35 days of pregnancy, and the group samples were divided into a pregnant group and a non-pregnant group. Vaginal secretion samples of pregnant and non-pregnant sows that were mated for 15 days were collected, and the differentially expressed proteins in the vaginal secretions of pregnant and non-pregnant sows were screened and detected by 4D label-free technology combined with a systematic bioinformatics analysis method, and two proteins DEF6 and MUC5AC proteins with significant differences in expression levels between the pregnant and non-pregnant groups were obtained, and both were significantly highly expressed in the vaginal secretions of pregnant sows. The expression levels of differentially expressed proteins DEF6 and MUC5AC in the pregnant group and non-pregnant group were further detected by enzyme-linked immunosorbent assay (ELISA) technology, and the above results were verified. It is proved that the expression levels of the differentially expressed proteins of the present invention, namely DEF6 and MUC5AC, are highly correlated with early pregnancy in sows, suggesting that DEF6 and MUC5AC proteins have the function of biomarkers and can be used as protein markers for diagnosing early pregnancy in sows.

[0036] The diagnostic value of DEF6 and MUC5AC protein markers for early pregnancy in sows was verified in a group. First, the prediction accuracy of judging the pregnancy status of sows according to protein markers was analyzed by receiver operating characteristic curve (ROC). Among the two protein biomarkers of the present invention, the areas under the ROC curves (AUCs) of DEF6 and MUC5AC proteins in pregnant sows and non-pregnant sows at 15 days after mating were both 0.844, and showed a significance level of 0.0026. Subsequently, the two proteins were jointly analyzed, and the area under the ROC curve (AUC) was 0.95, which means that these two protein biomarkers can be used alone or in combination for the diagnosis of early pregnancy in sows, and have a high diagnostic value for predicting early pregnancy. Then, the DEF6 and MUC5AC protein levels in different groups were detected by ELISA technology, and compared with the B-ultrasound diagnosis results. It was confirmed that the present invention has good sensitivity and specificity in judging the pregnancy status of sows by DEF6 and MUC5AC protein levels, with true positive rates of 71.88% and 59.58% respectively, and extremely low false alarm rates, true negative rates of 100%, and high accuracy of 77.5% and 67.5% respectively.

[0037] The above results show that DEF6 and MUC5AC proteins can be used as detection targets alone or in combination, and their protein expression levels can be used as an important reference for identifying whether the sow is pregnant. When DEF6 or MUC5AC protein is detected in early vaginal secretion samples compared with the non-pregnant group, the sow is already pregnant. If a significant low expression is detected, the sow should be artificially inseminated in time to shorten the parity interval, thereby effectively reducing the number of non-productive days of sows, improving the economic benefits of pig farming enterprises, and reducing feeding costs.

[0038] Based on the above findings, one embodiment of the present invention provides the use of a protein marker in the preparation of a sow early pregnancy diagnostic reagent or kit, wherein the protein marker is selected from at least one of the following a)-b):

[0039] a) DEF6 protein, the amino acid sequence of which is shown in SEQ ID NO.1;

[0040] b) MUC5AC protein, the amino acid sequence of which is shown in SEQ ID NO.2.

[0041] The present invention provides the application of any one or a combination of DEF6 and MUC5AC protein markers in the diagnosis of early pregnancy in sows. Vaginal secretion samples are collected 15 days after sow mating to detect the expression levels of DEF6 and MUC5AC proteins in the vaginal secretions. If significantly high expression of DEF6 and MUC5AC is detected, the sow is already pregnant. If significantly low expression is detected, the sow is not pregnant, and the sow should be timely artificially inseminated for the next mating. The protein markers of the present invention can quickly and accurately judge the pregnancy state of the sow in the early stage of mating, provide a new direction and approach for the diagnosis of early pregnancy in sows, and are conducive to the early diagnosis of large-scale mating groups, reduce identification costs and improve diagnostic efficiency, etc., which is of great significance to the cost and efficiency of pig farming.

[0042] In actual operation, the expression level of DEF6 and MUC5AC protein is based on the general level of expression of the protein in pigs in the field. The high and low are relative. According to the high or low expression level compared with the general level, for example, relative to the general expression level of the protein in the non-pregnant group, if it is higher than the expression level, it is judged as pregnancy; or, relative to the general expression level of the protein in the pregnant group, if it is within the expression level, it is judged as pregnancy.

[0043] The present invention uses ELISA enzyme-linked immunosorbent assay technology to detect the expression levels of DEF6 and MUC5AC proteins in the F1 sow group bred by Landrace × Yorkshire, and obtains the optimal threshold protein concentration by receiver operating curve analysis. Among them, the optimal threshold protein concentration of DEF6 protein detection is 310.25ng / mL, and the optimal threshold protein concentration of MUC5AC protein detection is 333.52ng / mL. DEF6 and MUC5AC are up-regulated proteins. Pregnancy is judged to be greater than the optimal threshold, and non-pregnancy is judged to be less than the optimal threshold.

[0044] Optionally, the reagent or kit is an enzyme-linked immunosorbent assay reagent or kit.

[0045] Optionally, the protein marker is protein in the vaginal secretions of the sow.

[0046] Optionally, the time for early pregnancy diagnosis is the 15th day after the sow is mated.

[0047] Optionally, the sow is a Landrace pig, a Yorkshire pig or a hybrid thereof.

[0048] 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 a protein marker in the preparation of a sow early pregnancy diagnostic reagent or kit, wherein the protein marker is selected from at least one of the following a)-b):

[0049] a) DEF6 protein, the amino acid sequence of which is shown in SEQ ID NO.1;

[0050] b) MUC5AC protein, the amino acid sequence of which is shown in SEQ ID NO.2.

[0051] The advantages of using the reagent for detecting protein markers in preparing a reagent or kit for diagnosing early pregnancy in sows are the same as the advantages of using protein markers in preparing a reagent or kit for diagnosing early pregnancy in sows, and will not be repeated here.

[0052] Optionally, the reagent for detecting the protein marker is an antibody that specifically binds to the protein marker, and the antibody is used to perform enzyme-linked immunosorbent assay. Therefore, the sow early pregnancy diagnostic reagent or kit is an enzyme-linked immunosorbent assay.

[0053] 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:

[0054] Vaginal secretions of the sow to be tested after mating are collected, the expression levels of DEF6 protein and / or MUC5AC protein in the vaginal secretions are detected, and whether the sow to be tested is pregnant is determined according to the expression levels of the DEF6 protein and / or the MUC5AC protein; when it is detected that the DEF6 protein and / or the MUC5AC protein are highly expressed compared with those of non-pregnant sows, it is determined that the sow to be tested is in a pregnant state; when it is detected that the DEF6 protein and / or the MUC5AC protein are lowly expressed compared with those of non-pregnant sows, it is determined that the sow to be tested is in a non-pregnant state.

[0055] The present invention uses DEF6 protein and / or MUC5AC protein as diagnostic markers for early pregnancy in sows, and changes in their expression levels are used to diagnose whether the sow is pregnant, which can greatly improve the sensitivity and specificity of pregnancy diagnosis and is conducive to improving detection accuracy.

[0056] Optionally, the vaginal secretions are collected on the 15th day after mating of the sow to be tested.

[0057] Optionally, the collected vaginal secretions are added to 2 mL of PBS solution, allowed to stand for 30 min, vortexed for 2 min, and then centrifuged at 1000 g for 15 min, and the supernatant is used to detect the expression level of DEF6 protein and / or MUC5AC protein.

[0058] 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.

[0059] Main reagents: BSA 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 mucin 5AC (MUC5AC) was purchased from Shanghai Hengyuan Biotechnology Co., Ltd., catalog number HB556-Pg; ELISA kit for detecting DEF6 guanine nucleotide exchange factor (DEF6) was purchased from Shanghai Hengyuan Biotechnology Co., Ltd., catalog number HB555-Pg.

[0060] Sow group: originated from the pig breeding base of Xinjiang Tiankang Animal Husbandry Technology Co., Ltd., and are F1 sows bred from Changbai × Yorkshire.

[0061] Protein markers: DEF6 protein, whose UniPort sequence number is F1RZ09; MUC5AC protein, whose UniPort sequence number is A0A8W4FM62. The specific protein sequences are shown in Table 1:

[0062] Table 1 Amino acid (AA) sequence information of protein markers of the present invention

[0063]

[0064]

[0065] Example 1 Screening for differential proteins in vaginal secretions of pregnant and non-pregnant sows on day 15 after mating

[0066] In this example, vaginal secretions of pregnant and non-pregnant sows on the 15th day after mating were collected, proteins were separated, and 4D label-free technology and bioinformatics methods were used to analyze and screen proteins that were differentially expressed in early pregnancy. The specific steps are as follows:

[0067] 1. Test samples

[0068] A total of 103 Changbai × Yorkshire F1 sows were used in this embodiment, all of which were sourced 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 (10min). After the collection was completed, all samples were immediately frozen in liquid nitrogen and stored at -80°C until use. Pregnancy diagnosis was performed by B-ultrasound on the 25th day, of which 8 sows were non-pregnant groups and the rest were pregnant groups.

[0069] 2. Total protein extraction and quantification

[0070] 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.

[0071] Prepare BSA standard protein solution with a concentration gradient range of 0-0.5μg / μL. Take BSA standard protein solutions with different concentration gradients and test sample solutions 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 the standard protein solution to draw a standard curve and calculate the protein concentration of the sample to be tested. Take 20μg of protein samples to be tested for 12% SDS-PAGE gel electrophoresis, where the conditions for concentrated gel electrophoresis are 80V, 20min, and the conditions for separation gel electrophoresis are 120V, 90min. After the electrophoresis, Coomassie Brilliant Blue R-250 staining is performed and decolorization is performed until the bands are clear.

[0072] 3. Screening of differentially expressed proteins using 4D label-free proteomics technology

[0073] 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.

[0074] (1) Proteolysis

[0075] Take a protein sample, add DB protein dissolution solution to make up the volume to 100μL, add 1μg / μL trypsin and 100mM TEAB buffer, mix well and digest at 37℃ for 4h, then add 1μg / μL 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.

[0076] (2) Fraction separation (high-depth quantitative analysis)

[0077] 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 BEHC18 (4.6×250mm, 5μm), the column temperature is 45°C, and the elution gradient is shown in Table 2. Collect 1 tube per minute, merge into 10 fractions, freeze-dry and set aside.

[0078] Table 2 Liquid chromatography elution gradient for peptide fraction separation

[0079]

[0080] (3) Liquid quality testing

[0081] 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 3.

[0082] Table 3 nanoElute liquid chromatography elution gradient

[0083]

[0084] A tims TOF pro2 mass spectrometer and a CaptiveSpray 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.

[0085] (4) Data analysis

[0086] 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.

[0087] 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.

[0088] ② Functional analysis of proteins and DEPs: Interproscam software was used for GO and InterPro (IPR) functional annotation (including Rfam, PRINTS, PrODOm, SMART, RroSite, PANTHER databases), and COG and KEGG were used for functional protein family and pathway analysis of the identified proteins. Volcano map analysis, cluster heat map analysis, and GO, IPR and KEGG pathway enrichment analysis were performed for DEPs, and STRINGDB software (access link: http: / / STRING.embl.de / ) was used to predict possible protein-protein interactions.

[0089] In this example, vaginal secretion samples of pregnant and non-pregnant sows were collected 15 days after mating, and differentially expressed proteins in vaginal secretion samples of pregnant and non-pregnant sows were screened and detected. Through GO and KEGG enrichment analysis, as well as differential protein interaction network analysis, a total of 2 differentially expressed proteins related to embryo implantation were screened, namely DEF6 and MUC5AC. The expression levels of differential proteins DEF6 and MUC5AC in vaginal secretions of pregnant and non-pregnant sows are shown in Figure 2. Figure 1 , it can be seen that in the pregnancy group, the expression levels of DEF6 and MUC5AC proteins were significantly higher than those in the non-pregnancy group.

[0090] 4. ELISA verification of differential protein expression levels

[0091] To further verify the protein expression results of 4D label-free detection, the expression of the two differentially expressed proteins screened above was verified using enzyme-linked immunosorbent assay (ELISA). The ELISA enzyme-linked immunosorbent assay was performed according to the instructions of the DEF6 and MUC5AC detection kits. 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.

[0092] 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.

[0093] 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.

[0094] Figure 2The average expression levels of DEF6 and MUC5AC proteins in the pregnant and non-pregnant groups detected by ELISA are shown. The results show that the expression of these two differential proteins in the vaginal secretions of pregnant and non-pregnant sows 15 days after mating is consistent with the results of 4Dlabel-free detection.

[0095] Example 2 Group verification of differential protein expression in vaginal secretions of pregnant and non-pregnant sows

[0096] 1. Detection of differential protein expression levels

[0097] To further verify the protein expression results of the 4D label-free detection of the population in Example 1, the expression of the above two proteins was verified in a new large population using ELISA technology.

[0098] 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.

[0099] Sample information confirmation: After the vaginal secretion samples were collected, all the sows were subjected to B-ultrasound pregnancy test on the 25th day after mating, and then all the sows were re-examined by B-ultrasound on the 35th day after mating to determine the pregnancy status of all the sows, which were divided into pregnancy and non-pregnancy groups, and all samples were tested. The sample information test results showed that 32 cases were pregnant and 8 cases were not pregnant.

[0100] The ELISA enzyme-linked immunosorbent assay was performed according to Example 1.

[0101] 2. ROC curve and area under the curve AUC data analysis of protein biomarkers

[0102] The receiver operating characteristic curve (ROC) is a line graph with 1-specificity (also known as false positive rate or false alarm rate) as the X-axis and true positive rate (also known as sensitivity) as the Y-axis. It is a comprehensive indicator that reflects 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:

[0103] 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;

[0104] AUC = 0.5: indicates that there is no predictive diagnostic value at all, and the prediction accuracy is the same as the guessing effect;

[0105] 0.5<AUC<0.7: The predictive diagnostic value is very low, which is relatively common;

[0106] 0.7≤AUC<0.9: has certain predictive diagnostic value and is of great practicality for actual diagnosis;

[0107] AUC ≥ 0.9: indicates high predictive diagnostic value, which is a good situation;

[0108] AUC=1 means perfect prediction without any flaws. In most cases, there is no perfect predictive diagnosis.

[0109] According to the ELISA test results, ROC curve analysis was performed and its AUC area and confidence level were calculated. The ROC curve results of DEF6 and MUC5AC proteins are shown in Figure 3-4 The calculation results of the corresponding parameters are summarized in Table 4. 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.

[0110] Table 4 Summary of ROC curve results

[0111]

[0112] ROC curves were constructed for the expression periods and pregnancy outcomes corresponding to the differentially expressed proteins to determine their diagnostic value for pregnancy outcomes. AUC values ​​> 0.8 and P values ​​< 0.05 were used as thresholds to screen proteins and their expression periods that could be used as biomarkers. Figure 3-4 It can be seen that the AUC value corresponding to the DEF6 protein on the 15th day of mating is 0.844, and it shows an extremely significant level of 0.0026 (p=0.0026<0.01). The AUC value corresponding to the MUC5AC protein on the 15th day of mating is 0.844, and it shows an extremely significant level of 0.0026, which means that on the 15th day of pregnancy, the expression levels of DEF6 and MUC5AC proteins have certain predictive diagnostic value for early pregnancy diagnosis and are of great practicality for actual diagnosis.

[0113] The two proteins were further analyzed for joint diagnosis, and the results are shown in Figure 5 . It can be seen that the AUC value is 0.95, and the predictive diagnostic value is higher.

[0114] According to the above results, the two protein biomarkers, DEF6 protein and MUC5AC protein, were up-regulated proteins in the pregnancy group, and the optimal threshold concentrations were 310.25 ng / mL and 333.52 ng / mL, respectively. The rules for early pregnancy diagnosis based on protein concentration are as follows: when the DEF6 protein concentration obtained by ELISA is greater than 310.25 ng / mL, it is judged as a pregnancy state, and when it is lower than this concentration, it is judged as a non-pregnancy state; when the MUC5AC protein concentration obtained by ELISA is greater than 333.52 ng / mL, it is judged as a pregnancy state, and when it is lower than this concentration, it is judged as a non-pregnancy state.

[0115] The pregnancy judgment results of the protein biomarkers of the present invention were compared and analyzed with the B-ultrasound diagnosis results (32 cases of pregnancy and 8 cases of non-pregnancy). The results are shown in Table 5, wherein 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.

[0116] Table 5 Results of pregnancy diagnosis in the population based on DEF6 protein and MUC5AC protein concentrations

[0117]

[0118]

[0119] As can be seen from Table 5, the true positive rates of DEF6 and MUC5AC were 71.88% and 59.58%, respectively, which were highly accurate in judging the pregnancy status of pregnant sows, and the misdiagnosis rate was 0, and the true negative rate was 100%. The false negative rate of DEF6 was 28.13%; the false negative rate of MUC5AC was 40.69%. The accuracy of DEF6 and MUC5AC in detecting the pregnancy status of sows was 77.5% and 67.5%, respectively.

[0120] In summary, the differentially expressed proteins DEF6 and MUC5AC in the vaginal secretions of pregnant and non-pregnant sows 15 days after mating provided by the present invention are suitable as biomarkers for diagnosing early pregnancy in sows, have good sensitivity and specificity, and have an extremely low false positive rate, and are highly accurate for early pregnancy diagnosis.

[0121] 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. The use of protein markers in the preparation of diagnostic reagents or kits for early pregnancy in sows, characterized in that: The protein markers are DEF6 protein and / or MUC5AC protein.

2. Use of the protein marker according to claim 1 in the preparation of a sow early pregnancy diagnostic reagent or kit, characterized in that: The amino acid sequence of the DEF6 protein is shown in SEQ ID NO.1, and the amino acid sequence of the MUC5AC protein is shown in SEQ ID NO.

2.

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

4. Use of the protein marker according to claim 1 in the preparation of a sow early pregnancy diagnostic reagent or kit, characterized in that: The time for early pregnancy diagnosis is the 15th day after the sow is mated.

5. Use of the protein marker according to claim 1 in preparing a sow early pregnancy diagnostic reagent or kit, characterized in that: The sow is a Landrace pig, a Yorkshire pig or a hybrid offspring thereof.

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

7. Use of a reagent for detecting the expression level of a protein marker in the preparation of a sow early pregnancy diagnostic reagent or kit, characterized in that: The protein markers are DEF6 protein and / or MUC5AC protein.

8. Use of the reagent for detecting the expression level of a protein marker according to claim 7 in the preparation of a sow early pregnancy diagnostic reagent or kit, characterized in that: The amino acid sequence of the DEF6 protein is shown in SEQ ID NO.1, and the amino acid sequence of the MUC5AC protein is shown in SEQ ID NO.

2.

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

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 expression level of DEF6 protein and / or MUC5AC protein in the vaginal secretions, and judging whether the sow to be tested is pregnant according to the expression level of the DEF6 protein and / or the MUC5AC protein; When it is detected that the DEF6 protein and / or the MUC5AC protein are highly expressed compared to non-pregnant sows, the sow to be tested is determined to be in a pregnant state; when it is detected that the DEF6 protein and / or the MUC5AC protein are lowly expressed compared to non-pregnant sows, the sow to be tested is determined to be in a non-pregnant state.

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