CN113311166B - Protein biomarker for diagnosing early pregnancy of sheep and method for detecting early pregnancy of sheep - Google Patents
Protein biomarker for diagnosing early pregnancy of sheep and method for detecting early pregnancy of sheep Download PDFInfo
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Abstract
The invention discloses a protein biomarker which is CHI3L1 protein, and the sequence number is <210> -1; PSMB4 protein, the sequence number of which 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 detecting early pregnancy of sheep, which is characterized by comprising the following steps: the method comprises the following specific steps: (1) and blood sample collection: collecting peripheral blood of sheep 14 days after hybridization; (2) and blood sample treatment: separating serum or plasma from the blood sample obtained in the step (1): (3) and detecting the kit: and (3) detecting the blood sample treated in the step (2) by using an ELISA kit, and detecting the contents of the CHI3L1 protein, the PSMB4 protein and the LGALS3BP protein.
Description
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 detecting the early pregnancy of the 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 by an effective and accurate detection 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 detection methods such as an examination method, an external observation method, an ultrasonic diagnosis method, a vaginal examination method and the like have respective defects, the detection time is long, the symptom diagnosis is not clear, the detection is influenced by the experience degree of a user, and the like, and misdiagnosis and missed diagnosis exist in different degrees. In view of the defects of the conventional pregnancy detection method, a more accurate and simple early pregnancy detection method applied to production management is urgently needed.
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 detecting early pregnancy of sheep.
The protein is 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, TMT (tandem mass tags), is used for marking peptide fragments after protein enzymolysis and accurately identifying and quantifying the peptide fragments by using the tandem mass spectrometry method. 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 an early stage of pregnancy. 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 detecting 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 with sequence number <210> -1; PSMB4, 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 detecting early pregnancy of sheep, which is characterized by comprising the following steps: the method comprises the following specific steps:
(1) and blood sample collection: collecting peripheral blood of sheep 14 days after hybridization;
(2) and blood sample treatment: separating serum or plasma from the blood sample obtained in the step (1):
(3) and detecting by using a 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 kit for detecting the soluble galectin 3 binding protein of the sheep is used for detecting the LGALS3BP protein;
the sheep proteasome B subunit 4 type detection kit 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. Separating protein, screening proteins differentially expressed in early pregnancy by adopting a bioinformatics analysis method of a TMT technology combined system, finally obtaining 3 proteins, namely CHI3L1, PSMB4 and LGALS3BP, in peripheral blood closely related to early pregnancy of sheep, wherein the expression levels of the peripheral blood samples of pregnant sheep and non-pregnant sheep at 14 days after hybridization are remarkably different and are verified by ELISA, and finally proving that the differentially expressed proteins provided by the invention, namely CHI3L1, PSMB4 and LGALS3BP are highly related to the early pregnancy of sheep and can be used as biomarkers for rapidly diagnosing the early pregnancy of 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 detecting early pregnancy of the sheep.
Furthermore, the invention provides an application of any one of CHI3L1, PSMB4 and LGALS3BP protein biomarkers in early pregnancy detection of sheep, wherein a peripheral blood sample is collected 14 days after sheep hybridization, an ELISA enzyme-linked immunosorbent assay technology is adopted to detect the expression levels of the CHI3L1, PSMB4 and LGALS3BP proteins in the blood, if LGALS3BP is detected to be significantly low expressed and CHI3L1 and PSMB4 are significantly 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 a sheep at the early stage of pregnancy are remarkably changed relative to that of a mating non-pregnant ewe, so that the invention mainly analyzes the expression conditions of the CHI3L1, PSMB4 and LGALS3BP proteins in the peripheral blood of the sheep at the early stage of pregnancy, and finds that the CHI3L1, PSMB4 and LGALS3BP proteins can be independently used as molecular markers for rapidly detecting the early-stage 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 early pregnancy detection of sheep, 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 be subjected to 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 pregnant sheep and a non-pregnant sheep control group in 14-day hybridization is obviously different; the expression level of PSMB4 in the pregnant sheep and the non-pregnant sheep control group at the 14 th mating day is obviously different, and the expression level of LGALS3BP in the pregnant sheep and the non-pregnant sheep control group at the 14 th mating day 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 mating 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 in 14-day-old and non-pregnant sheep was 0.844 and exhibited significance at the level of 0.017 (p =0.017 and woven fabric of 0.05), which means that 3 protein biomarkers can be used for early pregnancy detection of sheep individually 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 a graphic showing 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 shows ROC curve analysis of PSMB4 protein expression in blood 14 days after mating.
FIG. 4 is a ROC curve analysis of the expression of LGALS3BP protein 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 some examples of the present invention, and not all 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 new centrifuge tube using a ProteoMiner TM Protein enzyme Small-Capacity Kit (Bio-rad) instructions remove high-abundance proteins. A5 μ L protein sample was taken and protein concentration was determined using the BCA kit.
SDS-PAGE electrophoresis
(1) Sample preparation: according to the results of protein concentration measurement, an equal amount of protein was taken from each sample and added to a centrifuge tube, 5. Mu.L of 4 × Loading buffer, and then 2% SDS was added to make the volume 20. Mu.L;
(2) Sampling: sequentially Loading 1 mu L of pre-dyed protein marker and 20 mu L of protein sample, and sealing the blank Kong Shangyang adjacent to the sample by 20 mu L of 1 × Loading buffer;
(3) Electrophoresis: concentrating the gel at 15mA/gel to obtain a single line, wherein the concentration time is about 15min; separating gel 35 mA-dye electrophoresis to the bottom of the gel;
(4) Dyeing and decoloring: taking out the glue, 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.
TMT quantitative proteome sequencing screening of differential proteins
(1) Cleavage with Trypsin
The equal amount of each sample protein is subjected to 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, centrifuge for 5min, discard the supernatant, wash the precipitate with pre-cooled acetone 2 times. After air-drying the precipitate, TEAB was added to a final concentration of 200mM, the precipitate was broken up by sonication, and trypsin was added at a ratio of 1. Dithiothreitol (DTT) was added to give a final concentration of 5mM, and the mixture was reduced at 56 ℃ for 30min. 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 15min.
(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 Agilent 300Extend C18 (5 μm size, 4.6mm inner diameter, 250mm length) as column. The operation is as follows: the peptide fragment gradient is 8-32% acetonitrile, pH is 9, 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% of B;56-60min,80% B, the flow rate was maintained at 600.00nL/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 fixed starting point of the secondary mass spectrum scanning range is 100m/z, and the secondary scanning resolution is set to 35000.00. The data acquisition mode uses a data-dependent scanning (DDA) program, i.e., after the primary scanning, the first 20.00 peptide parent ions with the highest signal intensity are selected to enter the HCD collision cell in sequence and are fragmented by 30% of fragmentation energy, and the secondary mass spectrometry is also performed in sequence. In order to improve the effective utilization rate of the mass spectrum, the Automatic Gain Control (AGC) is set to be 5E4, the signal threshold is set to be 3.8E4ions/s, the maximum injection time is set to be 50ms, and the dynamic exclusion time of the tandem mass spectrum scanning is set to be 30s so as to avoid repeated scanning of 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 fragment is set to be 7 amino acid residues; the maximum modification number of the peptide fragment is set as 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 0.02Da. Cysteine alkylated Carbamidomethyl (C) was set as a fixed modification, variable modifications were [ 'ethyl (Protein N-term)', 'Oxidation (M)', or '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
(1) Functional annotation analysis: proteins can be annotated with 3-way classification at 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 test FDR and P < 0.05.
(3) Enrichment analysis and enrichment clustering of differential proteins: enrichment analysis and enrichment clustering were 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. And (3) representing a filtered P-value matrix by x = -log10 (P-value), and performing z transformation on each functional class by using the x value, wherein the z value is used for single-side clustering analysis (Euclidean distance and average distance clustering). 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, wherein the differential proteins are CHI3L1, PSMB4 and LGALS3BP respectively.
In the embodiment, peripheral blood samples of pregnant sheep and non-pregnant sheep 14 days after hybridization are collected, so that differentially expressed proteins 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 detection 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 (CHI 3L 1), sheep protease subunit B4 (PSMB 4) and sheep soluble galectin 3 (LGALS 3 BP) were purchased from Shanghai Hengyuan Biotech, inc. The ELISA enzyme-linked immunosorbent assay test is carried out according to the kit specification, and the specific operation flow is as follows:
(1) and (3) diluting the standard: 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 |
| No. 4 standard substance | 150 μ l of No. 5 standard substance was added to 150 μ l of the standard substance dilution |
| No. 3 standard product | 150 mul of No. 4 standard substance is added into 150 mul of 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 |
(2) Sample adding: blank holes (the blank reference holes are not added with samples and enzyme labeled reagents, 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) And (3) incubation: the plates were sealed with a sealing plate and incubated at 37 ℃ for 30 minutes.
(4) Preparing liquid: and diluting the 30 times of concentrated washing liquid by 30 times of distilled water for later use.
(5) 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.
(6) Adding an enzyme: 50 μ l of enzyme-labeled reagent was added to each well, except for blank wells.
(7) And (3) incubation: the operation is the same as (3).
(8) Washing: the operation is the same as (5).
(9) Color development: 50 mul of color developing agent A is added into each hole, 50 mul of color developing agent B is added into each hole, the mixture is evenly mixed by gentle shaking, and color development is carried out for 10 minutes at 37 ℃ in a dark place.
R terminates: the reaction was stopped by adding 50. Mu.l of stop solution to each well (blue color immediately turned yellow).
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 (CHI 3L 1), sheep protease subunit B4 (PSMB 4) and sheep soluble galectin 3 (LGALS 3 BP) were purchased from Shanghai Hengyuan Biotech, inc.
1. Sample information acquisition
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 blood samples are collected, B ultrasonic pregnancy detection is carried out on all the breeding sheep at the 25 th day after the breeding, then B ultrasonic retest is carried out on all the breeding sheep at the 35 th day after the breeding again, the pregnancy state of all the breeding ewes is determined and is divided into a pregnant group and a non-pregnant group, and all samples are tested and detected. The sample information detection result is 25 cases of pregnancy and 5 cases of non-pregnancy.
The ELISA test is carried out according to the kit specification, and the specific operation flow is as follows:
(1) dilution of the standard: the kit provides one original standard, diluted in a small tube as per the table below.
| No. 5 standard substance | Adding 150 μ l of original standard sample into 150 μ l of standard sample 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 substance | 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 |
(2) 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 hole of the enzyme label plate, keeping the sample from touching the hole wall as much as possible, and gently shaking and mixing the sample and the hole wall.
(3) And (3) incubation: the plates were sealed with a sealing plate and incubated at 37 ℃ for 30 minutes.
(4) Preparing liquid: and diluting the 30 times of concentrated washing liquid by 30 times of distilled water for later use.
(5) 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.
(6) Adding an enzyme: 50 μ l of enzyme-labeled reagent was added to each well, except for blank wells.
(7) And (3) incubation: the operation is the same as (3).
(8) Washing: the operation is the same as (5).
(9) Color development: adding 50 mul of color developing agent A into each hole, then adding 50 mul of color developing agent B, lightly shaking and mixing evenly, and developing for 10 minutes in a dark place at 37 ℃.
End in r: the reaction was stopped by adding 50. Mu.l of stop solution to each well (blue color immediately turned yellow).
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 is 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 exhibited at a 0.001 level (p = 0.001-stra 0.05) means that day 14-CHI 3L1 is of very high value for use in pregnancy tests;
the AUC value for day 14-PSMB 4 was 0.976, and the significance of presenting a 0.001 level (p = 0.001-stra 0.05) means that day 14-PSMB 4 is of very high value for pregnancy tests;
the AUC value for day 14-LGALS 3BP was 0.884, and the significance of the 0.017 level exhibited (p =0.017 felt-0.05) means that the value of day 14-LGALS 3BP for pregnancy tests was very high;
the AUC value for day 14-PSMB 4-CHI3L1 was 1.000, and the significance at a 0.001 level was exhibited (p = 0.001-stra 0.05) meaning that the combination of day 14-PSMB 4, day 14-CHI 3L1 for pregnancy tests was of very high value;
the AUC value corresponding to day 14-PSMB 4-LGALS3BP was 0.976, and the significance of the 0.001 level was exhibited (p = 0.001-straw 0.05) meaning that the combination of day 14-PSMB 4, day 14-LGALS 3BP for pregnancy tests was of very high value.
According to the results, the results of the 3 protein biomarker detection and B-ultrasonic diagnosis are further analyzed as follows:
TABLE 2 detection results of three protein biomarkers and B-ultrasonic diagnosis results
And (3) annotation: CHI3L1 and PSMB4 are down-regulated proteins, and are judged to be non-pregnant when being larger than the optimal threshold value and are judged to be pregnant when being smaller than the optimal threshold value; LGALS3BP is up-regulated protein, and is more than the optimal threshold value to judge pregnancy, and less than the optimal threshold value to judge non-pregnancy.
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-CHI3L1 and PSMB4-LGALS3BP are higher, respectively being 96%, 100% and 96%, and the LGALS3BP being 68%; when CHI3L1, LGALS3BP, PSMB4-CHI3L1 and PSMB4-LGALS3BP are judged to be 0 in false positive rate (the non-pregnant ewe is detected to be in a pregnant state), PSMB4 is 25 percent; the true negative rates (pregnancy status detected by non-pregnant ewes) CHI3L1, PSMB4, LGALS3BP, PSMB4-CHI3L1 and PSMB4-LGALS3BP are respectively 100%, 80%, 100% and 100%; the false negative rate (pregnancy status detected by non-pregnant ewes) CHI3L1 was 4%, PSMB4 was 0, LGALS3BP was 32%, PSMB4-CHI3L1 was 0, and PSMB4-LGALS3BP was 0. The accuracy rates of CHI3L1, PSMB4, LGALS3BP, PSMB4-CHI3L1 and PSMB4-LGALS3BP in detecting the pregnancy status of ewes are 96.67%, 73.33%, 100% and 96.67% respectively.
In conclusion, the combination of the different 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 hybridization is suitable for being used as the biomarker for detecting the early pregnancy of the sheep, and has better 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.
Performing ELISA sorbent assay according to kit instructions, wherein the specific operation flow is as follows:
(1) dilution of the standard: 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 mul of No. 5 standard substance is added into 150 mul of 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 to 150 μ l of standard substance diluentLiquid medicine |
(2) 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 hole of the enzyme label plate, keeping the sample from touching the hole wall as much as possible, and gently shaking and mixing the sample and the hole wall.
(3) And (3) incubation: the plates were sealed with sealing plates and incubated at 37 ℃ for 30 minutes.
(4) Preparing liquid: and diluting the 30 times of concentrated washing liquid by 30 times of distilled water for later use.
(5) Washing: carefully uncovering the sealing plate film, discarding the liquid, drying by spin, filling washing liquid into each hole, standing for 30 seconds, discarding, repeating the steps for 5 times, and patting dry.
(6) Adding an enzyme: 50 μ l of enzyme-labeled reagent was added to each well, except for blank wells.
(7) Incubation: the operation is the same as (3).
(8) Washing: the operation is the same as (5).
(9) Color development: 50 mul of color developing agent A is added into each hole, 50 mul of color developing agent B is added into each hole, the mixture is evenly mixed by gentle shaking, and color development is carried out for 10 minutes at 37 ℃ in a dark place.
End in r: the reaction was stopped by adding 50. Mu.l of stop solution to each well (blue color immediately turned yellow).
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 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 point protein concentration for detecting the CHI3L1 protein is 302.54ng/ml; the optimal threshold point protein concentration for PSMB4 protein detection is 2956.06ng/ml; the optimal threshold point protein concentration for detecting the LGALS3BP protein is 111.02ng/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 302.54ng/ml.
LGALS3BP and PSMB4-LGALS3BP, greater than the best threshold to judge as pregnant, less than the best threshold to judge as not pregnant; when the values of CHI3L1, PSMB4 and PSMB4-CHI3L1 are less than the optimal threshold, pregnancy is judged, and when the values are greater than the optimal threshold, non-pregnancy is judged.
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 the CHI3L1, PSMB4 and LGALS3BP proteins in the blood, if it is possible to detect significantly low expression of LGALS3BP, and detect significantly low expression of CHI3L1 and PSMB4, the ewe should be mated next time in time, so as to shorten the fetal time interval, further improve the 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 the CHI3L1, PSMB4 and LGALS3BP proteins have obvious difference in 14 th-day gestation and non-gestation sheep peripheral blood, so that when any one or combination of the 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 detect and determine whether sheep is pregnant or not in 14 th-day after breeding.
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 (1)
1. The application of a protein biomarker in preparing a product for diagnosing early pregnancy of sheep is characterized in that: the protein biomarker comprises one or more of the following proteins; CHI3L1 protein with sequence number <210> -1; PSMB4 protein, the sequence number of which is <210> < 2; LGALS3BP protein, with sequence number <210> < 3.
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