CN113311166A - Protein biomarker for diagnosing early pregnancy of sheep and method for detecting early pregnancy of sheep - Google Patents

Abstract

The invention discloses a protein biomarker which is CHI3L1 protein, and the sequence number is <210> 1; PSMB4 protein, wherein the sequence number is <210> 2; LGALS3BP protein, with sequence number <210> 3. The protein biomarker is applied to diagnosis of early pregnancy of sheep. A method for diagnosing early pregnancy of sheep, which is characterized by comprising the following steps: the method comprises the following specific steps: firstly, blood sample collection: collecting peripheral blood of sheep 14 days after hybridization; processing a blood sample: separating serum or plasma from the blood sample in the step (i): thirdly, detecting by using the kit: and (3) detecting the blood sample treated in the step (2) by using an ELISA kit, and detecting the contents of CHI3L1 protein, PSMB4 protein and LGALS3BP protein.

Description

Protein biomarker for diagnosing early pregnancy of sheep and method for detecting early pregnancy of sheep

Technical Field

The invention belongs to the field of biotechnology diagnosis, and particularly relates to a protein biomarker for diagnosing early pregnancy of sheep and a method for diagnosing early pregnancy of sheep.

Background

The pregnancy diagnosis of sheep has been paid attention by enterprises all the time, because the accurate diagnosis of early pregnancy can not only reduce the economic loss caused by nonpregnant of ewes, but also facilitate timely grouping of pregnant ewes, reasonable feeding and management, and simultaneously is beneficial to improving the sheep breeding efficiency, promoting intensive management of livestock and improving the benefit. How to discover a non-pregnant ewe through an effective and accurate diagnosis method, shorten the interval of fetal times and reduce the number of non-productive days (NPD) always troubles the sheep raising industry.

The traditional pregnancy diagnosis methods such as the examination method, the external observation method, the ultrasonic diagnosis method, the vaginal examination method and the like have respective defects, the detection time is long, the symptom diagnosis is not clear, the diagnosis is influenced by the experience degree of a user, and the like, and the misdiagnosis and the missed diagnosis exist in different degrees. In view of the defects of the traditional pregnancy diagnosis method, a more accurate and simple early pregnancy diagnosis method applied to production management is urgently needed to be found.

Disclosure of Invention

The invention aims to solve the problem of finding out a protein marker capable of detecting early pregnancy of sheep, application thereof and a method for diagnosing early pregnancy of sheep.

The protein is used as the final product of gene expression and directly reflects the complexity and the variability of the life of the organism. The proteins are analyzed to better explain the cellular activity or the process by which they function. The biomarker refers to a biochemical index which can clearly identify the change of the structure or function of a system, an organ and the like, and DNA, RNA, protein and the like can be used as the biomarker for research. The development of modern biomarkers is generally divided into three phases: a discovery phase, a verification phase and a validation phase. The technical method of proteomics in the discovery stage has been widely applied to discovery and detection of the self-diagnostic markers. At present, a mainstream protein quantification technology based on a tandem mass spectrometry method is tmt (tandem mass tags), and the technology marks peptide fragments after protein enzymolysis and utilizes the tandem mass spectrometry method to accurately identify and quantify the peptide fragments. At present, the research on early pregnancy protein of sheep is less, and an accurate and reliable protein biomarker for early pregnancy diagnosis of sheep is still lacked, so that development of some early pregnancy diagnosis protein biomarkers of sheep with early diagnosis value is urgently needed. The ideal protein biomarker needs to be specifically and sensitively detected from peripheral blood in sheep at the early gestation stage. At present, no protein biomarkers for diagnosis of early pregnancy in sheep have been disclosed.

In order to achieve the above purpose, the present invention provides the following technical solutions:

one of the purposes of the invention is to provide a protein biomarker for detecting early pregnancy of sheep.

The second purpose of the invention is to provide a new method for detecting early pregnancy of sheep, which can judge whether the sheep is pregnant or not by detecting the expression level of the protein biomarker in the early pregnancy stage of the sheep.

The invention also aims to provide a method for diagnosing early pregnancy of sheep.

The technical scheme of the invention is as follows:

the invention provides a protein biomarker for detecting early pregnancy of sheep, which comprises the following components in percentage by weight: CHI3L1, SEQ ID NO <210> 1; PSMB4, with sequence number <210> 2; LGALS3BP with sequence number <210> 3; the protein biomarker is applied to diagnosis of early pregnancy of sheep.

A method for diagnosing early pregnancy of sheep, which is characterized by comprising the following steps: the method comprises the following specific steps:

firstly, blood sample collection: collecting peripheral blood of sheep 14 days after hybridization;

processing a blood sample: separating serum or plasma from the blood sample in the step (i):

thirdly, detecting by using the kit: and (3) detecting the blood sample treated in the step (2) by using an ELISA kit, and detecting the contents of CHI3L1 protein, PSMB4 protein and LGALS3BP protein.

The ELISA kit is one of the following types: a kit for detecting a sheep chitinase 3-like protein 1, a kit for detecting a sheep protein proteasome B subunit 4 and a kit for detecting a sheep soluble galectin 3 binding protein; namely:

the sheep soluble galectin 3 binding protein detection kit is used for detecting the LGALS3BP protein;

the kit for detecting the type 4 proteasome B subunit of sheep is used for detecting the PSMB4 protein;

the kit for detecting the sheep chitinase 3-like protein 1 is used for detecting the CHI3L1 protein.

Peripheral blood samples of sheep 14 days after mating are collected, and B-ultrasonic pregnancy detection is carried out 25 days after mating, and pregnancy recheck is carried out 35 days later, so that a pregnant group and a non-pregnant ewe group are distinguished. Protein is separated, and the proteins differentially expressed in the early pregnancy are screened by adopting a bioinformatics analysis method of a TMT technology combined system, so that 3 proteins closely related to the early pregnancy of sheep, namely CHI3L1, PSMB4 and LGALS3BP, are finally obtained, the expression levels of the peripheral blood samples of the pregnant sheep and the non-pregnant sheep which are 14 days after hybridization are obviously different and are verified by ELISA, and finally the differentially expressed proteins provided by the invention, namely CHI3L1, PSMB4 and LGALS3BP, are highly related to the early pregnancy of the sheep and can be used as biomarkers for rapidly diagnosing the early pregnancy of the sheep, and the protein biomarkers have good diagnostic index characteristics.

The expression level of the CHI3L1 and PSMB4 proteins in the peripheral blood of the sheep in the pregnant group 14 days after hybridization is obviously lower than that of the non-pregnant group, the expression level of the LGALS3BP protein in the peripheral blood of the sheep in the pregnant group 14 days after hybridization is obviously higher than that of the non-pregnant group, and the CHI3L1, PSMB4 and LGALS3BP proteins can be used as protein biomarkers of early pregnancy of the sheep for sheep early pregnancy detection.

Furthermore, the invention provides an application of any one of CHI3L1, PSMB4 and LGALS3BP protein biomarkers in early pregnancy detection of sheep, peripheral blood samples are collected 14 days after sheep hybridization, the expression levels of CHI3L1, PSMB4 and LGALS3BP proteins in blood are detected by adopting an ELISA (enzyme-linked immunosorbent assay) reaction technology, if LGALS3BP is detected to be obviously low expressed and CHI3L1 and PSMB4 are obviously high expressed, the ewe should be subjected to next artificial insemination hybridization in time to shorten the fetal time interval, so that the number of non-productive days of ewes is effectively reduced, and the economic benefit of sheep raising enterprises is improved.

The principle of the invention is as follows: the applicant finds that the expression levels of CHI3L1, PSMB4 and LGALS3BP proteins in the peripheral blood of an early pregnancy sheep are remarkably changed relative to that of a mating non-pregnant ewe, so that the invention mainly analyzes the expression conditions of CHI3L1, PSMB4 and LGALS3BP proteins in the peripheral blood of the early pregnancy sheep, and finds that the CHI3L1, PSMB4 and LGALS3BP proteins can be independently used as molecular markers for rapidly detecting the early pregnancy proteins of the sheep and are applied for the first time.

The invention has the following beneficial effects:

(1) the invention aims at sheep early pregnancy diagnosis, protein molecules are screened, and by collecting 14-day postspecies pregnancy and non-pregnancy sheep peripheral blood samples, proteins which are differentially expressed in pregnancy and non-pregnancy ewe samples are screened and detected, so that 3 proteins which are significantly different between pregnancy and non-pregnancy are obtained, and finally, differentially expressed proteins related to sheep early pregnancy are obtained, namely: CHI3L1, PSMB4 and LGALS3BP proteins, thereby proving that 3 proteins can be used for detecting early pregnancy of sheep.

(2) The invention provides an application of one of protein biomarkers CHI3L1, PSMB4 and LGALS3BP in sheep early pregnancy detection, peripheral blood is collected in 14 days of sheep hybridization, the expression levels of CHI3L1, PSMB4 and LGALS3BP proteins in the blood are detected by adopting an ELISA (enzyme-linked immunosorbent assay) reaction technology, if the detection result is obviously low expressed, the ewe should carry out next artificial insemination hybridization in time to shorten the fetal time interval, further effectively reduce the number of non-productive days of ewes and improve the economic benefit of sheep raising enterprises.

(3) Among the 3 protein biomarkers disclosed by the invention, the expression quantity of CHI3L1 in a 14-day-matched pregnant sheep and a non-pregnant sheep control group is remarkably different; the expression level of PSMB4 in the pregnant sheep of 14-day mating and the non-pregnant sheep control group is obviously different, and the expression level of LGALS3BP in the pregnant sheep of 14-day mating and the non-pregnant sheep control group is obviously different.

(4) Of the 3 protein biomarkers disclosed in the present invention, CHI3L1 had an area under the ROC curve (AUC) of 0.992 for 14 days of breeding of pregnant and non-pregnant sheep and exhibited significance at a level of 0.001 (p ═ 0.001< 0.05); PSMB4 had an area under the ROC curve (AUC) of 0.976 for 14-day-old and non-pregnant sheep and exhibited significance at a 0.001 level (p ═ 0.001< 0.05); the area under the ROC curve (AUC) of LGALS3BP between 14-day-old and non-pregnant sheep in breeding was 0.844, and showed significance at a level of 0.017 (p ═ 0.017<0.05), which means that 3 protein biomarkers can be used for early pregnancy detection of sheep alone, and have higher early pregnancy detection value.

The invention provides new protein biomarkers CHI3L1, PSMB4 and LGALS3BP for early pregnancy detection of sheep and provides a new direction for early pregnancy detection of sheep.

Drawings

FIG. 1 is an ELISA validation of differential protein expression for TMT detection.

FIG. 2 shows ROC curve analysis of CHI3L1 protein expression in blood 14 days after mating.

FIG. 3 is a ROC curve analysis of the expression of PSMB4 protein in blood 14 days after mating.

FIG. 4 shows ROC curve analysis of LGALS3BP protein expression in blood 14 days after mating.

FIG. 5 shows ROC curve analysis of the expression of PSMB4-CHI3L1 protein in blood 14 days after mating.

FIG. 6 shows ROC curve analysis of the expression of PSMB4-LGALS3BP protein in blood 14 days after mating.

Detailed Description

The present invention is described in further detail by the following examples, and it is apparent that the described examples are only a part of the examples of the present invention, and not all of the examples. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1: TMT technology for screening differential protein in peripheral blood of pregnant and non-pregnant sheep at 14 th day after mating

In this example, the sheep blood during pregnancy and non-pregnancy at 14 th day after mating is collected, protein is separated, and the protein differentially expressed during the early stage of pregnancy is analyzed and screened by TMT technology and bioinformatics method, and the specific steps are as follows:

1. test sample

8 normal Kazakh sheep were randomly selected and divided into a control group and a test group. According to the conventional production flow, the estrus is checked and the breeding is carried out, wherein the test group ewes are fed with normal semen, and the control group ewes are fed with dead semen (the normal semen is boiled at high temperature and observed under a microscope to ensure that the semen is dead). Collecting blood of pregnant ewes and ewes of control group on 14 days after mating, separating plasma, immediately freezing and storing in dry ice, and transporting to laboratory as soon as possible and storing in ultra-low temperature refrigerator for later use.

2. Total protein extraction and quantification

The sample was removed from-80 ℃, centrifuged at 12000g for 10 min at 4 ℃, cell debris removed, and the supernatant transferred to a fresh centrifuge tube using a ProteoMiner^TMProtein enzyme Small-Capacity Kit (Bio-rad) instructions remove high-abundance proteins. A5. mu.L sample of protein was taken and the protein concentration was determined using the BCA kit.

SDS-PAGE electrophoresis

(1) Sample preparation: according to the protein concentration determination result, the same amount of protein is taken from each sample and put into a centrifuge tube, 5 mu L of 4 Xloading buffer is added, and then 2% SDS is added to make the volume be 20 mu L;

(2) loading: sequentially Loading 1 mu L of pre-dyed protein marker and 20 mu L of protein sample, and Loading 20 mu L of 1 × Loading buffer on adjacent blank holes of the sample and sealing;

(3) electrophoresis: concentrating the gel at 15mA/gel to obtain a single line, wherein the concentration time is about 15 min; separating gel 35 mA-dye electrophoresis to the bottom of the gel;

(4) dyeing and decoloring: taking out the gel, dyeing in Coomassie brilliant blue R-250 dye solution for 2h at room temperature, and adding a decolorizing solution to decolorize until the background is colorless and the bands are clear.

Screening for differential proteins by TMT quantitative proteome sequencing

(1) Cleavage with Trypsin

The equal amount of each sample protein is taken for enzymolysis, a proper amount of standard protein is added, and the volume is adjusted to be consistent by using a lysate. Adding 1 time volume of precooled acetone, uniformly mixing by vortex, adding 4 times volume of precooled acetone, and precipitating for 2 hours at-20 ℃. 4500g, centrifugating for 5min, discarding supernatant, and washing precipitate with precooled acetone for 2 times. Air drying the precipitate, adding TEAB with final concentration of 200mM, ultrasonically breaking the precipitate, adding trypsin at a ratio of 1:50 (protease: protein, m/m), and performing enzymolysis overnight. Dithiothreitol (DTT) was added to give a final concentration of 5mM, and the mixture was reduced at 56 ℃ for 30 min. After that, Iodoacetamide (IAA) was added to give a final concentration of 11mM, and the mixture was incubated at room temperature in the dark for 15 min.

(2) TMT mark

The tryptic peptide fragments were desalted using StrataX C18(Phenomenex) and vacuum freeze-dried. The peptide fragments were solubilized at 0.5M TEAB and labeled according to the protocol of the TMT kit. The simple operation is as follows: thawing the labeled reagent, dissolving with acetonitrile, mixing with the peptide segment, incubating at room temperature for 2h, mixing the labeled peptide segment, desalting, and vacuum freeze drying. Sample marking information:

P1

P2

P3

P4

NP1

NP2

NP3

NP4

127N

127C

128N

128C

129N

129C

130N

130C

(3) HPLC fractionation

The peptide fragments were fractionated by high pH reverse phase HPLC using an Agilent 300 extended C18 column (5 μm size, 4.6mm inner diameter, 250mm length). The operation is as follows: the peptide fragment gradient is 8-32% acetonitrile, pH9, 60 components are separated in 60min, then the peptide fragments are combined into 14 components, and the combined components are subjected to vacuum freeze drying and then are subjected to subsequent operation.

(4) Liquid chromatography-mass spectrometry tandem analysis

The peptide fragment is dissolved by a mobile phase A of liquid chromatography and then is separated by an EASY-nLC 1000 ultra-performance liquid phase system. The mobile phase A is an aqueous solution containing 0.1 percent of formic acid and 2 percent of acetonitrile; mobile phase B was an aqueous solution containing 0.1% formic acid and 90% acetonitrile. Setting a liquid phase gradient: 0-38min, 8% -23% B; 38-52min, 23% -35% B; 52-56min, 35% -80% B; 56-60min, 80% B, flow rate maintained at 600.00 nL/min. The peptide fragments are separated by an ultra-high performance liquid phase system, injected into an NSI ion source for ionization and then analyzed by QE +1 mass spectrometry. The ion source voltage was set at 2.2kV and both the peptide fragment parent ion and its secondary fragment were detected and analyzed using the high resolution Orbitrap. The scanning range of the primary mass spectrum is set to be 400-1500m/z, and the scanning resolution is set to be 70000.00; the secondary mass spectral scan range is fixed at 100m/z starting point and the secondary scan resolution is set at 35000.00. The data acquisition mode uses a data-dependent scanning (DDA) program, namely, after the primary scanning, the first 20.00 peptide fragment parent ions with the highest signal intensity are selected to sequentially enter an HCD collision cell for fragmentation by using 30% of fragmentation energy, and the secondary mass spectrometry is also sequentially performed. To improve the effective utilization of the mass spectra, the Automatic Gain Control (AGC) was set to 5E4, the signal threshold was set to 3.8E4ions/s, the maximum injection time was set to 50ms, and the dynamic exclusion time of the tandem mass spectrometry scan was set to 30s to avoid repeated scans of the parent ions.

(5) Database search

Secondary mass spectral data were retrieved using maxquant 1.5.2.8. And (3) retrieval parameter setting: the database is Ovis _ aries _9940(23111 sequences), a reverse library is added to calculate the false positive rate (FDR) caused by random matching, and a common pollution library is added into the database and is used for eliminating the influence of pollution proteins in the identification result; the enzyme cutting mode is set as Trypsin/P; the number of missed cutting sites is set to 2; the minimum length of the peptide segment is set to be 7 amino acid residues; the maximum modification number of the peptide fragment is set to be 5; the First-level parent ion mass error tolerance of the First search and the Main search is set to be 10.0ppm and 5ppm respectively, and the mass error tolerance of the second-level fragment ions is 0.02 Da. Cysteine alkylated carbamidomethyl (c) was set as a fixed modification, variable modifications were [ 'Acetyl (Protein N-term)', 'oxidation (m)', 'deamidation (nq)' ]. The quantitative method is set as TMT-10plex, and the FDR of protein identification and PSM identification is set as 1%.

(6) Bioinformatics analysis method

Functional annotation analysis: proteins can be annotated with 3-way classification in Gene Ontology (GO): biochemical processes, cellular localization, molecular functions. The annotation information for GO comes mainly from the UniProt-GOA database (http:// www.ebi.ac.uk/GOA /). Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used to analyze protein pathways. The functional annotation of proteins in the KEGG database was first performed using the KEGG online tool KAAS and then the metabolic pathways of the annotated proteins were mapped using the KEGG online tool KEGG mapper.

② function enrichment: differential proteins enriched for the identified GO and KEGG pathways and domains were detected using the two-tailed Fisher assay. Corrected with multiple hypothesis testing of FDR and P < 0.05.

Enrichment analysis and enrichment clustering of differential proteins: enrichment analysis and enrichment clustering are used to explore the potential relationships of different proteins in participating pathways, such as the KEGG pathway. We first enriched protein function by analyzing the p-values of all proteins and then filtered out proteins with low enrichment. The filtered P-value matrix is represented by x-log 10(P-value), and z-transform is performed on each functional class by the x-value, and the z-value is used for single-side cluster analysis (euclidean distance, average distance cluster). And (5) carrying out clustering analysis on the differential protein by using a pheatmap packet in the R language.

5. Differential protein interaction network analysis

The differential Proteins were analyzed on an interaction network using STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) on-line software, a database that was specifically studied and collected to predict protein interactions against known individuals. Comparing the protein of sheep with the protein of corresponding species in the STRING database, and obtaining the gene registration number for expressing the protein to carry out STRING analysis.

6. The result of the detection

The difference in protein expression between the pregnant and non-pregnant samples was analyzed using the T test under the conditions of significance test P <0.05 and quantitative ratio FC >1.20 or FC <0.83 for both samples. In the sequencing result, 3 differential proteins related to the early pregnancy of the sheep are screened out through GO and KEGG enrichment analysis and differential protein interaction network analysis, namely CHI3L1, PSMB4 and LGALS3 BP.

In the embodiment, by collecting peripheral blood samples of pregnant sheep and non-pregnant sheep 14 days after mating, proteins differentially expressed in the peripheral blood samples of the pregnant sheep and the non-pregnant sheep are screened and detected, and 3 differentially expressed proteins, namely CHI3L1, PSMB4 and LGALS3BP, in early pregnancy and related to embryo implantation are obtained.

Verification of TMT test results by ELISA

To further verify the protein expression results of the TMT assay, we used ELISA to verify the expression of 7 differentially expressed proteins in the above samples.

ELISA kits for sheep chitinase-3-like protein-1 (CHI3L1), sheep protease subunit B4 (PSMB4) and sheep soluble galectin 3(LGALS3BP) were purchased from Shanghai Hengyuan Biotech, Inc. The ELISA enzyme-linked immunosorbent assay is operated according to the kit specification, and the specific operation flow is as follows:

firstly, diluting a standard product: the kit provides one stock multiple standard, diluted in a small tube as per the table below.

Standard substance No. 5	Adding 150 μ l of original standard substance into 150 μ l of standard substance diluent
		Standard substance No. 4	150 μ l of No. 5 standard substance was added to 150 μ l of the standard substance dilution
No. 3 standard product	150 μ l of No. 4 standard substance was added to 150 μ l of the standard substance dilution
		Standard article No. 2	150 μ l of No. 3 standard substance is added into 150 μ l of standard substance diluent
Standard article No. 1	150 μ l of No. 2 standard substance is added with 150 μ l of standard substance diluent

Sample adding: blank holes (the blank reference holes are not added with the sample and the enzyme labeling reagent, and the rest steps are operated in the same way) and sample holes to be detected are respectively arranged. The standard hole on the enzyme labeling coated plate is accurately loaded with 50 mul, the sample hole to be detected is loaded with 40 mul of sample diluent, and then 10 mul of sample to be detected is loaded (the final dilution of the sample is 5 times). Adding sample to the bottom of the plate hole of the enzyme label, keeping the sample from touching the hole wall as much as possible, and gently shaking and mixing the sample and the hole wall.

③ incubation: the plates were sealed with a sealing plate and incubated at 37 ℃ for 30 minutes.

Fourthly, preparing the liquid: and diluting the 30 times of concentrated washing liquid by 30 times of distilled water for later use.

Washing: carefully uncovering the sealing plate film, discarding liquid, spin-drying, filling washing liquid into each hole, standing for 30 seconds, then discarding, repeating the steps for 5 times, and patting dry.

Sixthly, adding enzyme: 50 μ l of enzyme-labeled reagent was added to each well, except for blank wells.

And seventhly, incubation: the operation is the same as the third step.

And (b) washing: the operation is performed in the same fifth step.

Ninthly, color development: adding 50 μ l of color-developing agent A and 50 μ l of color-developing agent B into each well, shaking gently, mixing, and developing at 37 deg.C in dark for 10 min.

R terminates: the reaction was stopped by adding 50. mu.l of stop solution to each well (blue color immediately turned yellow).

11, measurement: the absorbance (OD value) of each well was measured sequentially at a wavelength of 450nm with the blank well being zeroed. The measurement should be performed within 15 minutes after the addition of the stop solution.

12ELISA results and data analysis

And drawing a standard curve on coordinate paper by taking the concentration of the standard substance as an abscissa and the OD value as an ordinate. Finding out the corresponding concentration from the standard curve according to the OD value of the sample; multiplying by the dilution times; or calculating a linear regression equation of the standard curve by using the concentration and OD value of the standard substance, substituting the OD value of the sample into the equation to calculate the concentration of the sample, and multiplying the concentration by the dilution factor to obtain the actual concentration of the sample.

The SPSS 21.0 software was used to analyze the amount of differential protein expression in the pregnant and non-pregnant groups. Data are expressed as mean + standard error and differences between test samples are analyzed by t-test or variance. P <0.05 indicates significant difference, and P <0.01 indicates very significant difference.

As shown in FIG. 1, the results showed that the expression of 7 differential proteins in peripheral blood of pregnant and non-pregnant sheep 14 days after mating was consistent with the TMT detection results.

Example 2: ELISA in the 14 days after mating in pregnancy and non-pregnant peripheral blood difference protein expression in the cases of further verification of TMT detection protein expression results, we used ELISA technology to sheep pregnancy early related 4 protein expression cases were carried out group verification.

ELISA kits for sheep chitinase-3-like protein-1 (CHI3L1), sheep protease subunit B4 (PSMB4) and sheep soluble galectin 3(LGALS3BP) were purchased from Shanghai Hengyuan Biotech, Inc.

1. Sample information collection

At day 14 post-mating, 30 sheep blood samples were collected, serum was isolated, immediately stored on dry ice for frozen storage, and transported back to the laboratory as soon as possible and stored in an ultra-low temperature freezer for future use.

2. Sample information validation

After the blood sample is collected, B ultrasonic pregnancy detection is carried out on all breeding sheep at the 25 th day after the breeding, then B ultrasonic retest is carried out on all breeding sheep at the 35 th day after the breeding again, the pregnancy state of all breeding ewes is determined and is divided into a pregnant group and a non-pregnant group, and all samples are subjected to test detection. The sample information detection result is 25 cases of pregnancy and 5 cases of non-pregnancy.

3, performing ELISA according to the kit specification, wherein the specific operation flow is as follows:

firstly, diluting a standard product: the kit provides one stock multiple standard, diluted in a small tube as per the table below.

Standard substance No. 5	Adding 150 μ l of original standard substance into 150 μ l of standard substance diluent
		Standard substance No. 4	150 μ l of No. 5 standard substance was added to 150 μ l of the standard substance dilution
No. 3 standard product	150 μ l of No. 4 standard substance was added to 150 μ l of the standard substance dilution
		Standard article No. 2	150 μ l of No. 3 standard substance is added into 150 μ l of standard substance diluent
Standard article No. 1	150 μ l of No. 2 standard substance is added with 150 μ l of standard substance diluent

Sample adding: blank holes (the blank reference holes are not added with the sample and the enzyme labeling reagent, and the rest steps are operated in the same way) and sample holes to be detected are respectively arranged. The standard hole on the enzyme labeling coated plate is accurately loaded with 50 mul, the sample hole to be detected is loaded with 40 mul of sample diluent, and then 10 mul of sample to be detected is loaded (the final dilution of the sample is 5 times). Adding sample to the bottom of the plate hole of the enzyme label, keeping the sample from touching the hole wall as much as possible, and gently shaking and mixing the sample and the hole wall.

③ incubation: the plates were sealed with a sealing plate and incubated at 37 ℃ for 30 minutes.

Fourthly, preparing the liquid: and diluting the 30 times of concentrated washing liquid by 30 times of distilled water for later use.

Washing: carefully uncovering the sealing plate film, discarding liquid, spin-drying, filling washing liquid into each hole, standing for 30 seconds, then discarding, repeating the steps for 5 times, and patting dry.

Sixthly, adding enzyme: 50 μ l of enzyme-labeled reagent was added to each well, except for blank wells.

And seventhly, incubation: the operation is the same as the third step.

And (b) washing: the operation is performed in the same fifth step.

Ninthly, color development: adding 50 μ l of color-developing agent A and 50 μ l of color-developing agent B into each well, shaking gently, mixing, and developing at 37 deg.C in dark for 10 min.

R terminates: the reaction was stopped by adding 50. mu.l of stop solution to each well (blue color immediately turned yellow).

11, measurement: the absorbance (OD value) of each well was measured sequentially at a wavelength of 450nm with the blank well being zeroed. The measurement should be performed within 15 minutes after the addition of the stop solution.

ELISA results and data analysis

And drawing a standard curve on coordinate paper by taking the concentration of the standard substance as an abscissa and the OD value as an ordinate. Finding out the corresponding concentration from the standard curve according to the OD value of the sample; multiplying by the dilution times; or calculating a linear regression equation of the standard curve by using the concentration and OD value of the standard substance, substituting the OD value of the sample into the equation to calculate the concentration of the sample, and multiplying the concentration by the dilution factor to obtain the actual concentration of the sample.

5.3 ROC Curve and area under Curve AUC data analysis of protein biomarkers

The ROC curve is a line graph created with 1-specificity, i.e., false alarm rate, as the X-axis and specificity (sensitivity) as the Y-axis. In the art, the area under the ROC curve represents the accuracy of the prediction, and is called AUC value. Obviously, the larger the AUC value, the higher the prediction accuracy, and vice versa, the lower the prediction accuracy. AUC values are between 0 and 1, and the judgments about AUC values are as follows:

AUC < 0.5: if the condition is not in accordance with the actual condition, the prediction diagnosis is worse than the random guess, and the condition should not appear in the actual condition;

AUC 0.5: the method has no prediction diagnosis value at all, and the prediction accuracy is the same as the guessing effect;

0.5 < AUC < 0.7: the predictive diagnostic value is low, and the situation is relatively common;

AUC more than or equal to 0.7 and less than 0.9: the method has certain predictive diagnosis value and greater practicability for actual diagnosis;

AUC is more than or equal to 0.9: it shows that the predictive diagnostic value is high, which is better;

AUC 1 is perfect prediction without flaws, and in most cases, no perfect predictive diagnosis exists.

According to the ELISA test results, ROC curve analysis is respectively carried out, the AUC area and the confidence coefficient of the test results are calculated, and the ROC curve analysis of 3 indexes is combined with binary logistic regression operation, as shown in figures 2 to 6 and table 1.

TABLE 1 summary of ROC curve results

From the above table, we constructed ROC curves for expression periods and corresponding pregnancy outcomes of proteins to determine their diagnostic value for pregnancy outcome, and based on the above results, we screened proteins and their expression periods that can be biomarkers with AUC values > 0.8 and P values <0.05 as thresholds:

the AUC value for day 14-CHI 3L1 was 0.992 and the significance of presenting a 0.001 level (p ═ 0.001<0.05) means that day 14-CHI 3L1 is of very high value for use in pregnancy tests;

AUC values for day 14-PSMB 4 were 0.976, and significance at the 0.001 level was exhibited (p ═ 0.001<0.05) meaning that day 14-PSMB 4 was of very high value for pregnancy testing;

the AUC value for day 14-LGALS 3BP was 0.884 and the significance of exhibiting a 0.017 level (p ═ 0.017<0.05) means that day 14-LGALS 3BP is of very high value for pregnancy tests;

the AUC value for day 14-PSMB 4-CHI3L1 was 1.000 and showed significance at a level of 0.001 (p 0.001<0.05) means that day 14-PSMB 4, day 14-CHI 3L1 in combination are of very high value for pregnancy tests;

the AUC value for day 14-PSMB 4-LGALS3BP was 0.976, and the significance of the 0.001 level (p 0.001<0.05) was shown to mean that day 14-PSMB 4, day 14-LGALS 3BP in combination for pregnancy tests were of very high value.

According to the results, the detection of 3 protein biomarkers and B-ultrasonic diagnosis results are further analyzed as follows:

TABLE 2 detection results of three protein biomarkers and B-ultrasonic diagnosis results

Note that: CHI3L1 and PSMB4 are down-regulated proteins, and are judged to be non-pregnant when the threshold value is larger than the optimal threshold value and are judged to be pregnant when the threshold value is smaller than the optimal threshold value; LGALS3BP is up-regulated protein, and is judged to be pregnant when it is greater than the optimal threshold, and non-pregnant when it is less than the optimal threshold.

TABLE 3 analysis of the results of the three protein biomarker detection

From the table, it can be seen that the true positive rates (pregnancy status detected by pregnant ewes) of CHI3L1, PSMB4, PSMB4-CHI3L1 and PSMB4-LGALS3BP have higher accuracy, respectively being 96%, 100% and 96%, and 68% for LGALS3 BP; CHI3L1, LGALS3BP, PSMB4-CHI3L1 and PSMB4-LGALS3BP are respectively 25% in the process of judging that the false positive rate (the non-pregnant ewe is detected to be in a pregnant state) is 0 and the PSMB4 is 25%; the true negative rates (non-pregnant ewes detected as pregnancy) of CHI3L1, PSMB4, LGALS3BP, PSMB4-CHI3L1 and PSMB4-LGALS3BP were 100%, 80%, 100% and 100%, respectively; the false negative rate (pregnancy status detected by non-pregnant ewes) was 4% for CHI3L1, 0 for PSMB4, 32% for LGALS3BP, 0 for PSMB4-CHI3L1, and 0 for PSMB4-LGALS3 BP. The accuracy of detecting the pregnancy status of ewes by CHI3L1, PSMB4, LGALS3BP, PSMB4-CHI3L1 and PSMB4-LGALS3BP is 96.67%, 73.33%, 100% and 96.67%, respectively.

In conclusion, the combination of the differential proteins CHI3L1, PSMB4, LGALS3BP, PSMB4-CHI3L1 and PSMB4-LGALS3BP in the peripheral blood of the pregnant sheep and the non-pregnant sheep 14 days after mating is suitable for being used as the biomarker for detecting early pregnancy of sheep, and has good accuracy and specificity.

Example 3: application of protein biomarker in diagnosis of early pregnancy of sheep

The embodiment provides application of a protein biomarker in diagnosis of early pregnancy of sheep. Whether the sheep is pregnant or not is judged by detecting the expression level of the protein biomarker, in the example, ELISA enzyme-linked adsorption reaction is taken as an example for detection, and an ELISA kit is purchased from Shanghai Hengyuan biological technology company Limited.

1. Type and collection of blood samples

Serum: placing the whole blood sample collected from the serum separation tube at room temperature for 2 hours or overnight at 4 ℃, then centrifuging at 1000 Xg for 20 minutes, and taking the supernatant, or storing the supernatant at-20 ℃ or-80 ℃, but avoiding repeated freezing and thawing.

Plasma: collecting sample with EDTA or heparin as anticoagulant, centrifuging the sample at 2-8 deg.C 1000 Xg for 15min within 30min, collecting supernatant, and detecting, or storing at-20 deg.C or-80 deg.C while avoiding repeated freeze thawing.

2, the ELISA enzyme-linked immunosorbent assay is operated according to the kit specification, and the specific operation flow is as follows:

firstly, diluting a standard product: the kit provides one stock multiple standard, diluted in a small tube as per the table below.

Standard substance No. 5	Adding 150 μ l of original standard substance into 150 μ l of standard substance diluent
		Standard substance No. 4	150 μ l of No. 5 standard substance was added to 150 μ l of the standard substance dilution
No. 3 standard product	150 μ l of No. 4 standard substance was added to 150 μ l of the standard substance dilution
		Standard article No. 2	150 μ l of No. 3 standard substance is added into 150 μ l of standard substance diluent
Standard article No. 1	150 μ l of No. 2 standard substance is added with 150 μ l of standard substance diluent

Sample adding: blank holes (the blank reference holes are not added with the sample and the enzyme labeling reagent, and the rest steps are operated in the same way) and sample holes to be detected are respectively arranged. The standard hole on the enzyme labeling coated plate is accurately loaded with 50 mul, the sample hole to be detected is loaded with 40 mul of sample diluent, and then 10 mul of sample to be detected is loaded (the final dilution of the sample is 5 times). Adding sample to the bottom of the plate hole of the enzyme label, keeping the sample from touching the hole wall as much as possible, and gently shaking and mixing the sample and the hole wall.

③ incubation: the plates were sealed with a sealing plate and incubated at 37 ℃ for 30 minutes.

Fourthly, preparing the liquid: and diluting the 30 times of concentrated washing liquid by 30 times of distilled water for later use.

Washing: carefully uncovering the sealing plate film, discarding liquid, spin-drying, filling washing liquid into each hole, standing for 30 seconds, then discarding, repeating the steps for 5 times, and patting dry.

Sixthly, adding enzyme: 50 μ l of enzyme-labeled reagent was added to each well, except for blank wells.

And seventhly, incubation: the operation is the same as the third step.

And (b) washing: the operation is performed in the same fifth step.

Ninthly, color development: adding 50 μ l of color-developing agent A and 50 μ l of color-developing agent B into each well, shaking gently, mixing, and developing at 37 deg.C in dark for 10 min.

R terminates: the reaction was stopped by adding 50. mu.l of stop solution to each well (blue color immediately turned yellow).

11, measurement: the absorbance (OD value) of each well was measured sequentially at a wavelength of 450nm with the blank well being zeroed. The measurement should be performed within 15 minutes after the addition of the stop solution.

3. And (4) calculating a result:

drawing a standard curve on coordinate paper by taking the concentration of the standard substance as an abscissa and the OD value as an ordinate, and finding out the corresponding concentration from the standard curve according to the OD value of the sample; multiplying by the dilution times; or calculating a linear regression equation of the standard curve by using the concentration and OD value of the standard substance, substituting the OD value of the sample into the equation to calculate the concentration of the sample, and multiplying the concentration by the dilution factor to obtain the actual concentration of the sample.

4. Pregnancy diagnosis based on protein concentration

The optimal threshold protein concentration for detecting the CHI3L1 protein is 302.54 ng/ml; the optimal threshold protein concentration for detecting the PSMB4 protein is 2956.06 ng/ml; the optimal threshold protein concentration of LGALS3BP protein detection is 111.02 ng/ml; the optimal threshold point protein concentration for detecting the PSMB4-CHI3L1 protein is-0.66 ng/ml; the optimal threshold point protein concentration for detecting the PSMB4-LGALS3BP protein is 302.54 ng/ml.

LGALS3BP and PSMB4-LGALS3BP, which are more than the optimal threshold value and judged as pregnant, and less than the optimal threshold value and judged as non-pregnant; CHI3L1, PSMB4 and PSMB4-CHI3L1, were judged to be pregnant when they were less than the optimal threshold and were judged to be non-pregnant when they were greater than the optimal threshold.

Preferably, this embodiment provides an application of any one of the CHI3L1, PSMB4 and LGALS3BP protein biomarkers in a sheep early pregnancy diagnosis test, a peripheral blood sample is collected 14 days after sheep mating, an ELISA enzyme-linked immunosorbent assay technology is used to detect the expression levels of CHI3L1, PSMB4 and LGALS3BP proteins in the blood, if LGALS3BP is significantly low expressed, and CHI3L1 and PSMB4 are significantly low expressed, the ewe should be timely mated next time, so as to shorten the fetal time interval, further improve sheep breeding efficiency, and improve the economic benefits of sheep raising enterprises.

In conclusion, CHI3L1, PSMB4 and LGALS3BP proteins are further verified in sheep peripheral blood in 14 th-day gestation and non-gestation sheep peripheral blood samples by adopting an ELISA method, and CHI3L1, PSMB4 and LGALS3BP proteins are significantly different from non-gestation sheep peripheral blood in 14 th-day gestation and non-gestation sheep peripheral blood, so that when any one or combination of CHI3L1, PSMB4 and LGALS3BP proteins is applied to the technical field of rapid detection of sheep early pregnancy diagnosis, the protein molecular marker can be used as a protein molecular marker for detecting sheep early pregnancy, has good detection index characteristics, and compared with the existing methods such as hormone determination, ultrasonic diagnosis and the like, the protein molecular marker has better specificity and sensitivity, and can be used for detecting and determining whether sheep is pregnant or not in 14 th-day after hybridization.

As noted above, while the present invention has been described in detail with reference to certain preferred embodiments, it is not to be construed as limited thereto. The present invention may take on many forms and details with some modifications or improvements in form and detail. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

<110> river university

<120> protein biomarker for sheep early pregnancy diagnosis and method for sheep early pregnancy detection

<141>

<160> 3

<210> 1

<211> 453

<212> amino acid sequence

<213> sheep

<220>

<221> CHI3L1

<223>

<400>

YKLICYYTSWSQYREGDGSCFPDAIDPFLCTHVIYSFANISNNEIDTWEWNDVTLYDTLN

TLKNRNPKLKTLLSVGGWNFGPERFSAIASKTQSRRTFIKSVPPFLRTHGFDGLDLAWLY

PGRRDKRHLTTLVKEMKAEFIREAQAGTEQLLLSAAVSAGKIAIDRGYDIAQISRHLDFI

SLLTYDFHGAWRQTVGHHSPLFAGNEDASSRFSNADYAVSYMLRLGAPANKLVMGIPTFG

RSFTLASSKTDVGAPVSGPGVPGRFTKEKGILAYYEICDFLHGATTHRFRDQQVPYATKG

NQWVAYDDQESVKNKARYLKNRQLAGAMVWALDLDDFRGTFCGQNLTFPLTSAVKDVLAE

V

<210> 2

<211> 264

<212> protein sequence

<213> sheep

<220>

<221> PSMB4

<223>

<400>MEALLESRSGLWAGGPAPGQFYRIPPTPGSSVDPASALYGAPITRTQNPMVTGTSVLGLK

FEGGVVIAADMLGSYGSLARFRNISRIMRVNNSTMLGASGDYADFQYLKQVLGQMVIDEE

LLGDGHSYSPKAIHSWLTRAMYSRRSKMNPLWNTMVIGGYADGDSFLGYVDMLGVAYEAP

SLATGYGAYLAQPLLREVLEKQPVLSQTEARELVERCMRVLYYRDARSYNRFQIATVTEK

GVEIEGPLSAETNWDIAHMISGFE

<210> 3

<211> 536

<212> amino acid sequence

<213> sheep

<220>

<221> LGALS3BP

<223>

<400> GVKDGDMRLADGGSANEGRVEIYYSGQWGTVCENMWDLTDASVVCRALGFRNATEALGGA

AFGPGHGPIMLDEVRCTGTEPSLANCSSLGWMRSNCRHDEDASVICTNETRGVYTLDLSG

ELPAALEQIFESQKGCDLFIRVKVREEDELAICAHKLILSTNPEAHGLWKEPGSRVTMEV

DAECVPTVKDFIRYLYSRRIDVSLSSVKCLHKLASAYQAEQLQSYCGHLFAILIPQDPSF

RTPLELYAYALATRDPVLEEICVQFLAWNFGALTQAEAWLSVPPGLLQDLLSRTELVVPS

ELVLLRAVDEWSRERSTSHKEVEGLVEKVRFPMMPPRDLFSLQFNLSLYWSHEALLQKKI

LQALEFHTVPFELLAQYWGLNLTEDAYQPRLYTSPTWSESVMSSSYNPYRSFQTPQHPSF

LFQASSVSWSLVYLPTLQSCWNYGFSCSSDDPPLLALSKSSYSSPTIGYENRALLHCEGS

FVADVIDFKGWKALIPSALGTNSSRSTSLFPCPAGLFSRFQVVIRPFYLTNSTGMD

Claims (4)

1. A protein biomarker for diagnosing early pregnancy in sheep, characterized by: the protein biomarker is one of the following protein markers:

CHI3L1 protein, wherein the sequence number is <210> 1;

PSMB4 protein, wherein the sequence number is <210> 2;

LGALS3BP protein, with sequence number <210> 3.

2. The application of a protein biomarker for diagnosing early pregnancy of sheep in sheep pregnancy detection is characterized in that: the use of the protein marker of claim 1 in the diagnosis of early pregnancy in sheep.

3. A method for diagnosing early pregnancy of sheep, which is characterized by comprising the following steps: the method comprises the following specific steps:

firstly, blood sample collection: collecting peripheral blood of sheep 14 days after hybridization;

processing a blood sample: separating serum or plasma from the blood sample in the step (i):

thirdly, detecting by using the kit: and (3) detecting the blood sample treated in the step (2) by using an ELISA kit, and detecting the contents of CHI3L1 protein, PSMB4 protein and LGALS3BP protein.

4. The method for diagnosing early pregnancy in sheep as set forth in claim 3, wherein: the ELISA kit is one of the following types: a kit for detecting a sheep chitinase 3-like protein 1, a kit for detecting a sheep protein proteasome B subunit 4 and a kit for detecting a sheep soluble galectin 3 binding protein; namely:

the sheep soluble galectin 3 binding protein detection kit is used for detecting the LGALS3BP protein;

the kit for detecting the type 4 proteasome B subunit of sheep is used for detecting the PSMB4 protein;

the kit for detecting the sheep chitinase 3-like protein 1 is used for detecting the CHI3L1 protein.

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