CN111893176B - miRNA diagnostic marker for indicating milk cow mammitis exosomes of Holstein - Google Patents
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Abstract
The invention discloses a miRNA diagnostic marker for indicating the milk source exosome of Holstein cow mastitis, which takes milk source exosome miR-223 and miR-200b as diagnostic markers for Holstein cow mastitis; successfully separating and identifying milk-derived exosomes of Holstein cows; the level of miR-223 in the in vitro secretion of Holstein cow mastitis individuals is obviously increased (P is less than 0.001) compared with the control group; the level of miR-200b is obviously reduced (P < 0.001) compared with the control group. As most protein components are removed in the extraction process of exosomes, the exosome miRNA can more accurately reflect whether dairy cow individuals suffer from mastitis or not compared with the milk miRNA.
Description
Technical Field
The invention relates to a diagnostic marker for predicting Holstein cow mastitis, and further provides an early diagnosis method for Holstein cow mastitis by taking milk source exosome miR-223/miR-200b as a diagnostic marker, belonging to the technical field of biological diagnosis of cow diseases.
Background
Cow mastitis is one of three diseases of cows accepted in the world, which causes temporary or permanent reduction of milk yield and milk quality, prolongation of postpartum oestrus period, loss of reproductive performance and the like of cows. For a long time, the economic loss caused by cow mastitis to the cow breeding industry is the first disease of various diseases, and is a disease with high cost in the dairy industry.
About one third of 2.2 hundred million cows in the global stock are reported to have mastitis and invisible mastitis, with high incidence and some difference in incidence of mastitis in cows in different countries and regions. Mastitis is an inflammation of breast tissue, which can be caused by various infectious agents, usually as a result of microbial infection by pathogens such as staphylococcus aureus, streptococcus uberis and escherichia coli, and is a chronic or acute disease which clinically manifests as localized changes in the breast and abnormal milk secretion, characterized by an elevated number of somatic cells, usually accompanied by physical, chemical or microbial changes in milk.
Along with the biological evolution process, both prokaryotes and eukaryotes have evolved a compact and efficient intercellular communication strategy. Classical cell biology suggests that there are two ways of intercellular communication, either through direct contact interactions or indirect effects of secretion of soluble factors such as hormones, growth factors, cytokines, etc. In recent years, the research finds that exosomes are used as a third newly discovered important intercellular communication carrier, can mediate the transportation of biological macromolecules such as proteins, lipids, nucleic acids and the like among cells, and widely influence the physiological and pathological processes of organisms. Exosomes refer to vesicle structures secreted by different types of cells and formed by phospholipid bilayer membranes wrapping proteins, lipids and nucleic acids, and are powerful carriers capable of transporting proteins, lipids and nucleic acids between cells and mediating communication between cells. mirnas are present in various body fluids and selectively packaged in exosomes, a membrane vesicle secreted by most cell types. These exosome mirnas can be actively delivered into recipient cells and can regulate target gene expression and recipient cell function. Exosomes naturally occur in various body fluids, such as blood, urine, saliva, cerebrospinal fluid and milk, providing a basis for their wide regulation of the physiological and pathological processes of the body.
Disclosure of Invention
The invention discloses a diagnosis marker for predicting Holstein cow mastitis, which is used for early diagnosis of Holstein cow mastitis by taking milk source exosomes miR-223 and miR-200b as diagnosis markers and solves the defect of detection of Holstein cow mastitis in the prior art.
The invention discloses a diagnostic marker for predicting Holstein cow mastitis, which is characterized in that the diagnostic marker is as follows: milk-derived exosomes miR-223 and miR-200b; wherein, the liquid crystal display device comprises a liquid crystal display device,
the miR-223 diagnosis marker sequence is shown as SEQ No. 1;
the miR-200b diagnosis marker sequence is shown as SEQ No. 2.
The invention relates to an early diagnosis method for indicating Holstein cow mastitis by taking milk source exosomes miR-223 and miR-200b as diagnosis markers, which comprises the following steps:
1. the milk sample is subjected to gradient low-speed centrifugation:
300 Xg/min, 10min; 3000 Xg/min, 20 min; centrifuging at 10000 Xg/min for 30 min to collect supernatant;
gradient ultracentrifugation was performed for 30000 Xg/min, 60 min;50000 Xg/min, 90min;70000 Xg/min, 90min; 95000 Xg/min 60 min;120000 Xg/min, 90min;120000 Xg/min, 90min;
wherein, the milk sample is centrifugated at 50000 Xg/min, and then the two-thirds supernatant is centrifugated at 70000 Xg/min after being filtered by a 3 μm filter membrane, and the two-thirds supernatant is filtered by a 1 μm filter membrane and a 0.45 μm filter membrane, and finally the two-thirds supernatant is centrifugated at 95000 Xg/min and then filtered by a 0.22 μm filter membrane, and then the two-thirds supernatant is centrifugated at 120000 Xg, and the milk source Exosomes are collected.
2. The two pairs of primer sequences designed according to the bovine miR-223 and miR-200b genes and promoter sequences are as follows:
miR-223：
F-primer TGCGGTGTCAGTGTCAAAT，
R-primer CAGTGCAGGGTCCGAGGT；
miR-200b：
F-primer TGCGGTAATACTGCCTGGTA，
R-primer CAGTGCAGGGTCCGAGGT。
3. extracting exosome total RNA from the milk source Exosomes of the milk cow separated in the step 1) by using a TRIzol method, reversely transcribing the Exosomes total RNA into cDNA by using a reverse transcription kit, and detecting the abundance levels of miR-223 and miR-200b among milk cow samples by using RT-qPCR;
the reaction system is miR-223 and miR-200b upstream primers designed in the step 2)0.5. Mu.L each, 0.5. Mu.L each of the downstream primer, 1. Mu.L of cDNA, ddH 2 O3. Mu.L, mix 5. Mu.L, total 10. Mu.L, 3 replicates per sample;
the reaction conditions are as follows: 50 ℃ for 2min;95 ℃ for 10min;95℃for 10s and 60℃for 30s (45 cycles); 95 ℃ for 15s; 15s at 55 ℃;95℃for 15s.
The invention analyzes the expression of the milk-specific miR-223 and miR-200b in the milk source exosomes of the Holstein cows, and discusses the possibility of using the milk-specific miR-223 and miR-200b as cow mastitis markers.
And (3) separating and extracting milk-derived exosomes by adopting an optimized ultracentrifugation method, and analyzing the expression and the difference of the milk-specific miR-223 and miR-200b in the milk-derived exosomes of the individuals suffering from the mastitis and the healthy individuals by adopting a real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) technology. The specificity and sensitivity of miR-223 and miR-200b are significantly higher than those of the existing mastitis detection technology.
The invention has the positive effects that:
discloses that milk source exosomes miR-223 and miR-200b are used as diagnosis markers of Holstein cow mastitis; successfully separating and identifying milk-derived exosomes of Holstein cows; the level of miR-223 in the in vitro secretion of Holstein cow mastitis individuals is obviously increased (P is less than 0.001) compared with the control group; the level of miR-200b is obviously reduced (P < 0.001) compared with the control group. Because most of protein components are removed in the extraction process of exosomes, exosome miRNA can reflect whether dairy cow individuals suffer from mastitis more accurately than milk miRNA.
Drawings
FIG. 1 shows the morphology and particle size distribution under a milk-derived Exosome transmission electron microscope and identification of the marker protein CD 81/TSG 101;
FIG. 2 is a graph showing the expression level of miR-223/miR-200b in milk-derived exosomes from healthy cows and mammitis cows.
Detailed Description
The present invention will be further described in detail with reference to the following examples and drawings for the purpose of enhancing the understanding of the present invention, which examples are provided for the purpose of illustrating the present invention only and are not to be construed as limiting the scope of the present invention.
Example 1
1. Milk sample collection:
1. the milk sample of Chinese Holstein cows comes from the great waste cattle farm in North Heilongjiang. 25 of Holstein cows with mastitis in the cattle farm 10 months in 2019 are selected as study groups, and 25 of healthy Holstein cows are selected as control groups.
2. Collecting and preserving milk samples: collecting milk sample with clean sterile collecting bottle, packaging into 50 mL centrifuge tube, centrifuging for 300×g10 min, 3000×g 20 min, and 10×g 30 min to obtain supernatant, removing impurities such as cell, large particle interference, fat, etc., and freezing at-80deg.C.
2. Separation of milk-derived exosomes:
1. thawing the pretreated milk sample stored at-80deg.C on ice, centrifuging the milk sample for 300×g10 min, 3000×g 20 min, and 10000×g 30 min to obtain supernatant, and removing impurities such as cell, protein, and fat. The samples were further subjected to the following steps, namely 30000 Xg 60 min, 50000 Xg 90min, 70000 Xg 90min, 95000 Xg 60 min, 120000 Xg 90min, 120000 Xg 90min, wherein 50 Xg was centrifuged to obtain two-thirds of the supernatant, and the two-thirds was filtered with a 3 μm filter, 70000 Xg was centrifuged to obtain two-thirds of the supernatant, and the two-thirds was filtered with a 1 μm and 0.45 μm filter, and 95 000Xg was centrifuged to obtain two-thirds of the supernatant, and the two-thirds was filtered with a 0.22 μm filter, and subjected to 120000 Xg ultracentrifugation.
2. Analysis of exosomes particle size and concentration: the number and size distribution of the particles were analyzed by Nanoparticle-tracking analysis (NTA). The pellet was resuspended in 30ul particle free PBS and diluted 1000-fold before testing.
3. The morphology of exosomes was observed by transmission electron microscopy: the exosome was resuspended in 200 mesh copper mesh for 5min, then blotted with filter paper, and after 1% phosphotungstic acid was counterstained for 2min, washed twice with PBS and observed with transmission electron microscopy. The morphology and size of the exosomes were visualized by transmission electron microscopy (JEM-2100 JEOL, tokyo, japan) (see FIG. 1).
4. Westenblot detection of exosome markers: exosome proteins were extracted using RIPA (Pierce, USA) lysate and BCA (Pierce, USA) was used to determine protein concentration. Proteins were separated by 12% SDS-PAGE and loaded at 40ug total protein per lane. Transferring the protein onto a PVDF film by a wet transfer method, sealing the protein for 1h at room temperature by 5% nonfat milk powder, respectively adding TSG101 and CD81 antibodies (1:1000), sealing overnight at 4 ℃, washing 3 times by TBST for 5 minutes each time, adding HRP-labeled secondary antibodies (1:10000), incubating for 1h at room temperature, washing for many times by 1 XTBST after incubation is finished, preparing developing solution (ECL, pierce, USA) according to the proportion of 1:1, slowly dripping the developing solution onto the PVDF film, spreading the whole film, and sucking off redundant developing solution so as to prevent too dark background caused by excessive developing solution. The exposure times were 5s,10s,15s,20s,30s.
Because casein interference in milk is serious, the method is optimized on the basis of traditional ultracentrifugation, fat layers, cells, casein and other proteins with larger molecular weight in the milk are removed by a low-speed differential centrifugation method, and finally exosomes in the milk are extracted by the ultracentrifugation method. The milk source exosomes obtained by the method are approximately circular, have concave parts, have the particle size of about 100nm, express marker molecules of exosomes such as TSG101, CD81 and the like, and have no obvious difference between a study group and a control group. Further NTA analysis shows that the particle size of the separated exosomes is not uniform and is mainly distributed near 100nm, which indicates that the milk-derived exosomes of the Holstein cows are successfully separated.
3. Milk-derived exosomes miR-223/miR-200b assay:
exosome RNA extraction and quantitative and real-time quantitative PCR: extraction of total exosome RNA 1mL TRIzol was added to each 200. Mu.L sample. The exosome samples were transferred to a 1.5mL EP tube and left at 15-30℃for 5 minutes. Chloroform (0.2×g, mL) was added, covered, and then vigorously shaken by hand for 15 seconds, left at 15-30℃for 2-3 minutes, and centrifuged for 12,000Xg, 15 minutes, and 2-8 ℃. After centrifugation, the mixture was separated into three layers, the lower red phase being phenol-chloroform phase, an intermediate layer and the one side being a colorless aqueous phase. RNA is only present in the aqueous phase. The aqueous phase represents 60% of the total TRIzol. The upper aqueous phase was transferred to another clean EP tube, 0.5mL of isopropanol was added, and 20. Mu.L of a trace RNA precipitation aid was added, left to stand for 10 minutes at 15-30℃and then centrifuged at 12,000Xg for 10 minutes at 2-8 ℃. Flocculent gum-like sediment, which is RNA sediment, can be seen on the side walls and bottom of the tube prior to centrifugation. The supernatant was removed, 1mL of 75% cold ethanol was added to wash the RNA pellet, mixed well with a shaker, centrifuged at 7,500 Xg for 5 minutes at 2-8deg.C. Removing supernatant, vacuum or air for 5-10 min, and drying RNA precipitate. Re-suspending RNA precipitate with RNase-free water, repeatedly blowing with gun head for several times, measuring concentration, and preserving at-20deg.C.
The extracted tissue RNA was reverse transcribed into cDNA samples using the TaKaLa reverse transcription kit. The reverse transcription conditions were: (1) denaturation of RNA: 5X gDNA Eraser Buffer. Mu.L, gDNA Erase 1. Mu.L, 1ug total RNA, and the RNA samples were heat denatured in a 42℃water bath for 2min, and then immediately cooled on ice. (2) preparation of a reaction solution: 5 xRT buffer 2. Mu.L; RT Enzyme mix 0.5. Mu.L; prime mix 0.5 μL; RNA 1. Mu.L, then using Nuclear-free ddH 2 O was made up to 10. Mu.L and was performed on ice. (3) reverse transcription reaction: 37. metal bath at c for 15 min. (4) enzyme inactivation: 85. metal bath at c, 5s. The obtained product is stored in a refrigerator at the temperature of-80 ℃ for standby. Reverse transcription into cDNA as a template, and dyeing with a fluorescent dye SYBR Green for real-time fluorescent quantitative PCR experiments, and detecting the expression levels of miR-223 and miR-200b in two groups of milk-derived exosomes, wherein each sample is repeatedly detected for 3 times.
The quantitative primer sequences of miR-223 and miR-200b are respectively as follows:
F-Primer TGCGGTGTCAGTGTCAAAT，
R-Primer CAGTGCAGGGTCCGAGGT；
F-Primer TGCGGTAATACTGCCTGGTA，
R-Primer CAGTGCAGGGTCCGAGGT；
the sequence of the primer of the reference gene U6 is as follows:
F-Primer CTCGCTTCGGCAGCACA，
R-Primer AACGCTTCACGAATTTGCGT。
RT-qPCR was performed using cDNA obtained by reverse transcription as a template. The reaction system was 5. Mu.L each for the upstream primer, 5. Mu.L each for the downstream primer, 1. Mu.L for cDNA, 8. Mu.L for Mix, 10. Mu.L for the total, and 3 replicates for each sample. The reaction conditions are as follows: 50 ℃ for 2min;95 ℃ for 10min;95℃for 10s and 60℃for 30s (45 cycles); 95 ℃ for 15s; 15s at 55 ℃;95℃for 15s. The resulting data was analyzed using SPSS after the reaction was completed.
Test example 1
Study group expression of miR-223/miR-200b in milk-derived exosomes:
the miR-223 and miR-200b in the milk-derived exosomes are detected by adopting Q-PCR (shown in figure 2), the expression abundance of miR-223 in the milk-derived exosomes of the individual suffering from mastitis in the study group is obviously increased (P < 0.001), and the expression abundance of miR-200b is obviously reduced (P < 0.001). Therefore, the miR-223 and miR-200b can be used as markers for diagnosis of the Holstein cow mastitis, and the specificity and sensitivity of the miR-223 and miR-200b disclosed by the invention are obviously higher than those of the existing mastitis detection technology.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Sequence listing
<110> Jilin university
<120> miRNA diagnostic marker for indicating mammitis of Holstein cows and exosomes
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 15
<212> DNA
<213> cow (Bos taurus)
<400> 1
gcaggcaaaa cccca 15
<210> 2
<211> 14
<212> DNA
<213> cow (Bos taurus)
<400> 2
aaacgccgga agag 14
<210> 3
<211> 19
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 3
tgcggtgtca gtgtcaaat 19
<210> 4
<211> 18
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 4
cagtgcaggg tccgaggt 18
<210> 5
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 5
tgcggtaata ctgcctggta 20
<210> 6
<211> 18
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 6
cagtgcaggg tccgaggt 18
Claims (1)
1. Use of milk-derived exosome miR-200b in the preparation of a diagnostic marker for the prediction of mammitis of a Holstein cow, characterized in that:
the miR-200b diagnosis marker sequence is shown as SEQ No. 2.
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Citations (3)
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| JP2010504350A (en) * | 2006-09-19 | 2010-02-12 | アシュラジェン インコーポレイテッド | Genes and pathways regulated by miR-200 as targets for therapeutic intervention |
| CN103961706A (en) * | 2013-01-30 | 2014-08-06 | 中国医学科学院医药生物技术研究所 | Application of microRNA or inhibitor thereof to lipid metabolism regulation |
| CN110496138A (en) * | 2019-06-11 | 2019-11-26 | 中国农业大学 | A kind of extracting method and its application of yak milk excretion body |
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| JP2010504350A (en) * | 2006-09-19 | 2010-02-12 | アシュラジェン インコーポレイテッド | Genes and pathways regulated by miR-200 as targets for therapeutic intervention |
| CN103961706A (en) * | 2013-01-30 | 2014-08-06 | 中国医学科学院医药生物技术研究所 | Application of microRNA or inhibitor thereof to lipid metabolism regulation |
| CN110496138A (en) * | 2019-06-11 | 2019-11-26 | 中国农业大学 | A kind of extracting method and its application of yak milk excretion body |
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