CN112630449B - Blood exosome marker for judging buffalo birth time and application thereof - Google Patents
Blood exosome marker for judging buffalo birth time and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of biology, in particular to a blood exosome marker for judging buffalo birth time and application thereof. The invention specifically researches the newborn calf, mainly adopts a whole blood sample to research exosomes in the sample and performs a long-term research on the establishment process of a buffalo immune system, finds that exosomes of the newborn calf have great difference in protein content and type along with the establishment of the immune system in the research process, and can divide the birth time of the calf into the following parts according to the change condition of the exosomes: within 2h of birth, 3h-11d of birth and more than 12d of birth, the birth time of the calf is effectively judged by detecting the positivity and positivity of Calnexin, TSG101, CD9 and CD81 proteins according to a western blotting method aiming at the birth time, and the kit plays a good indicating role in later immune research, management, feeding and the like.
Description
[ technical field ] A method for producing a semiconductor device
The invention relates to the technical field of biology, in particular to a blood exosome marker for judging buffalo birth time and application thereof.
[ background of the invention ]
Exosomes (also called multivesicular bodies (MVBs) are a class of microvesicles secreted by cells, with a diameter of 30 to 150nm, having a monolayer membrane structure, and are widely present in various body fluids of organisms, such as plasma, urine, saliva, ascites, amniotic fluid, and the like. In recent years, with the progress of research, the structural size and shape of exosomes are clearly understood, but the molecular mechanism involved in physiological processes and the functional study of exosome contents are still in the beginning stage, and the species involved in exosome research are still limited.
At present, exosomes contain various active substances such as proteins, DNA, mRNA and some non-coding RNAs, and the structural size, shape and density of exosomes are mainly determined by the specific protein, lipid, enzyme and mineral contents of exosomes. Exosomes are formed by the invagination of intracellular lysosomal microparticles, contain a plurality of integral membrane signaling proteins, including Epidermal Growth Factor Receptors (EGFR), cytokine receptors, G-protein coupled receptors (GPCR), and the like, participate in the transmission of intercellular information, act on target cells, and further regulate the expression of target cell proteins and various signaling pathways. According to the research, the exosome has various different functions according to different cells from which the exosome comes, including participation in cell migration, angiogenesis promotion, tumor development and metastasis, tumor resistance, establishment of an immune system, immune regulation and other physiological processes.
The calf of a buffalo gradually establishes an own immune system after birth, the establishment of a passive immune system mainly depends on the supply of breast milk, and the maturation of the autogenous immune system is a complex process. The research on the formation process of the autogenous immune system of calves is still in a primary stage, and a plurality of problems to be solved are still needed. It has been reported that exosomes may participate in immune response reactions in the role of promoting factors, and may also play a role in processes such as antigen presentation, angiogenesis, and inflammatory responses. Therefore, it is necessary to study the morphology of exosomes of nascent buffalo and analyze the molecular biological characteristics of the isolated exosomes; can effectively analyze the process of establishing the immune system of the newborn buffalo, can also analyze the growth time of the newborn buffalo from the aspect of the content of exosome protein simultaneously, can judge the growth period where the newborn calf is located, can effectively utilize and should judge that better to raise and manage the newborn calf.
[ summary of the invention ]
In view of the above, it is necessary to study the morphology of exosomes of nascent buffalo and to analyze the molecular biological characteristics of the isolated exosomes; can effectively analyze the process of establishing the immune system of the newborn buffalo, can also analyze the growth time of the newborn buffalo from the aspect of the content of exosome protein simultaneously, can judge the growth period where the newborn calf is located, can effectively utilize and should judge that better to raise and manage the newborn calf.
In order to achieve the purpose, the invention comprises a blood exosome marker for judging the birth time of buffalo, wherein the exosome marker is marker proteins Calnexin, TSG101, CD9 and CD81.
Further, the kit comprises Calnexin, TSG101, CD9 and CD81 antibodies.
The invention also comprises an application of the blood exosome marker and the kit in judging the birth time of buffalo, which comprises the following steps:
(1) Collecting a whole blood sample of a buffalo calf born, and extracting exosomes in buffalo blood by adopting a centrifugal method;
(2) Carrying out Western blot detection on the exosomes extracted in the step (1), and when detecting that Calnexin and TSG101 proteins are positive, explaining that: the buffalo calf is born for 0-2h; when the above four proteins are not detected, it is stated that: buffalo calves are born for 3h-11d; when positive TSG101 and CD81 proteins were detected, it was indicated that: buffalo calves are born for more than 12 days.
Further, the centrifugation method in the step (1) is a differential centrifugation method, namely: taking a serum sample, adding phosphate buffer solution into the serum sample, and centrifuging for 15min at 300g and 4 ℃; centrifuging, collecting supernatant, and centrifuging at 12000g and 4 deg.C for 40min; centrifuging, collecting supernatant, and filtering with filter membrane; centrifuging the filtrate at 12000g and 4 deg.C for 1.5 hr for three times, discarding the supernatant and retaining the precipitate; beating the precipitate with phosphate buffer solution to wash the precipitate, and centrifuging for 1 hr at 100000g and 4 deg.c to obtain precipitate as exosome.
Further, the western blot detection method in step (2) comprises the following steps: adding 5 xlodang buffer into an exosome suspension, uniformly mixing, adding a sample, performing electrophoresis in 10% acrylamide gel for 2 hours, transferring proteins onto a PVDF membrane by using an electrotransfer instrument, putting the PVDF membrane after membrane transfer into 5% skimmed milk powder confining liquid, incubating for 2 hours by using a shaker, eluting by using 1xTBST buffer solution, cutting the membrane according to the molecular mass of the proteins, incubating Calnexin, TSG101, CD9 and CD81 for one-time resistance overnight at the temperature of 4 ℃, taking out the PVDF membrane, then performing elution again, adding a corresponding secondary antibody reagent, hybridizing for 1 hour at room temperature, then washing the PVDF membrane by using the 1xTBST buffer solution, and finally performing exposure imaging in a protein imaging system to obtain the recombinant hemoglobin variant exosome.
The invention has the following beneficial effects:
the application is specific to newborn calf study, adopt the whole blood sample to study through the exosome to in the sample mainly, judge the immune system of buffalo newborn calf and establish the process, for buffalo immune system's the process of establishing carries out the long-standing research, discover at research in-process, the exosome of newborn calf, along with immune system's the content of establishing its protein, the kind all has very big difference, according to the situation of change of exosome, can divide the birth time of calf into: within 2h of birth, 3h to 11d of birth and more than 12d of birth, the research aiming at the exosome protein in the time period discovers that: when Calnexin and TSG101 protein are detected to be positive, the following is stated: the buffalo calf is born for 0-2h; when the above four proteins are not detected, it is stated that: the buffalo calf is born for 3h-11d; when positive TSG101 and CD81 proteins were detected, it was indicated that: the buffalo calf is born for more than 12 days; by adopting the western blot detection method, the birth time of the newborn calf can be effectively divided, and the method plays a good indication role in later-stage immune research, management, feeding and the like.
[ description of the drawings ]
FIG. 1 is a morphological diagram of blood exosomes of buffalo calves born for 2h under an electron microscope;
FIG. 2 is a morphology of blood exosomes of buffalo calves born for 5d under an electron microscope;
FIG. 3 is a morphological diagram of blood exosomes of buffalo calf born 12d under an electron microscope
FIG. 4 is a graph showing exosome diameter results for samples of calves at different birth stages;
FIG. 5 is a Western Blot detection result of calf exosomes at different birth stages.
[ detailed description ] embodiments
The invention is further illustrated below with reference to the figures and examples and tests.
Example 1:
1. reagents and instruments:
EDTA anticoagulant blood collection tubes purchased from American BD company; l-100XP low temperature ultracentrifuge available from Beckman, USA; total Exosome Isolation (00520032) was purchased from Invitrogen; transmission Electron microscopy (H7650) was purchased from Hitachi, japan; a nanosized and zeta-potentiometer (Nano ZS 90) purchased from malvern, uk; PBS buffer was purchased from Biological Industries, israel; calnexin antibody (AC 018-1), TSG101 antibody (AF 8259), CD9 antibody (AF 1192), and CD81 antibody (AF 2428) were purchased from shanghai bi yunnan biotechnology limited.
2. Time-sharing sampling:
selecting 9 delivered Mora buffalo calves, sampling every 2 hours within 24h of birth, and sampling once a day at birth 2d till birth 14d; the collection process is as follows:
collecting 3 calf whole blood samples of buffalo by using EDTA (ethylene diamine tetraacetic acid) anticoagulant blood collection tubes respectively, wherein the blood sample collected by each buffalo is not less than 10ml, turning the blood collection tubes upside down after collection and uniformly mixing 8-10, centrifuging the collected whole blood, balancing the blood collection tubes with other centrifuge tubes by using a horizontal rotor centrifuge, 1900g (3000 rpm), and centrifuging at 4 ℃ for 10min. The supernatant plasma was pipetted into a fresh conical centrifuge tube (approximately 4-5mL plasma), 3000g, and centrifuged at 4 ℃ for 15min. And sucking the supernatant into a new 15mL centrifuge tube, reversing, uniformly mixing, subpackaging into 1.5mL or 2mL centrifuge tubes, and storing in an environment at-20 ℃.
3. Exosome extraction:
exosomes in buffalo blood were extracted using differential centrifugation. A2 ml serum sample was taken, and 13ml of Phosphate Buffered Saline (PBS) was added thereto, followed by centrifugation at 300g and 4 ℃ for 15min. After the initial centrifugation, the supernatant was collected, and centrifuged at 12000g and 4 ℃ for 40min. After centrifugation, the supernatant was taken and filtered through a filter. The filtrate was ultracentrifuged at 12000g and 4 ℃ for 1.5h, and the supernatant was discarded and the pellet was retained. Beating with PBS to wash the precipitate, centrifuging at 100000g and 4 deg.C for 1 hr to obtain precipitate as exosome.
Performing electron microscope microscopic examination on exosomes, analyzing the particle size of exosomes and performing Western Blot Western Blot detection on specific protein markers aiming at the samples; in the experiments of the applicant, it was found that: the exosome protein in the blood of buffalo calf born varies, and the experimental sample can be divided into 3 periods according to the experimental result: that is, the buffalo calf is born for 0-2h, the buffalo calf is born for 3-11 d, and the buffalo calf is born for more than 12 d; the applicant selects 3 most representative exosome protein stages for model experiments, which are respectively: buffalo A: buffalo calves are at birth for 2h, buffalo B: buffalo calf birth 5d, buffalo C: buffalo calves are at birth 12d.
The test and experimental procedures are as follows:
1. electron microscopy identification of exosomes:
the sample was centrifuged, 10. Mu.l of the sample was pipetted onto a copper mesh, and after standing for several minutes, the excess supernatant of the sample was pipetted using filter paper. Dripping 10 μ l of phosphotungstic acid on a copper net, dyeing at room temperature for 1min, absorbing the floating liquid by using filter paper, drying for several minutes, and then imaging at 80-120 kv under a transmission electron microscope to identify the physical characteristics of vesicles in a sample, including the shape, the diameter and the membrane structure, wherein the identification result is shown in figures 1-3; fig. 1 to 3 are respectively: the exosome forms of 2h, 5d and 12d of buffalo calf are shown in the figure, and the exosome forms of the buffalo calf are different in size in 3 periods, are similar to a circle or an ellipse, and have large difference of about 30-150 nm in diameter.
2. Exosome particle size analysis
And (3) sucking 10 mu l of extracted exosome sample, diluting the sample by using 1 XPBS (phosphate buffer solution), analyzing the particle size of the exosome by using a nanometer particle size and ZETA (Zeolite associated technologies) potentiometer, measuring the particle size of the exosome in the sample, and further identifying the distribution condition of the exosome. The temperature was maintained at 23 ℃ to 37 ℃ during the experiment. The results are shown in fig. 4, where fig. 4 shows the diameters of the exosomes of the sample, and buffalo a, buffalo B and buffalo C are shown from left to right in the middle; from fig. 4 it is shown that: the peak value of the diameter of an exosome of a buffalo A blood sample is 132.2nm, and the particle with the diameter concentrated between (132.2 +/-90.8) nm accounts for 96.5 percent; the peak value of the diameter of the exosome of the buffalo B blood sample is 123.1nm, and 98.2 percent of particles with the diameters concentrated between (123.1 +/-79.6) nm are contained; the peak exosome diameter of buffalo C blood samples was 112.6nm, with particles centered between (112.6 ± 58.3) nm in diameter accounting for 95.3%. All three samples were consistent with exosome diameter size and as seen by the peak, the exosomes of buffalo a, buffalo B and buffalo C had large differences in diameter peak, suggesting that exosome proteins in animal blood may not be consistent at different stages after parturition.
Observations made for both of the above experiments found that: the exosome protein in the blood of buffalo is inconsistent at different stages of birth, so the protein is detected by a marker, and the method and the result are as follows:
first, the protein concentration in the sample was measured using the BCA method. Then, an appropriate amount of 5xlodaing buffer is added to the exosome suspension and mixed evenly. After electrophoresis in a 10% acrylamide gel for 2h, the proteins were transferred to PVDF membrane using an electrotransfer apparatus (100V voltage, 60 min). And putting the PVDF membrane after membrane conversion into 5% skimmed milk powder sealing liquid, and incubating for 2h in a shaking table. After elution with 1xTBST buffer, the membrane is cut according to the molecular mass of the protein, and Calnexin, TSG101, CD9 and CD81 are incubated at 4 ℃ for overnight. And taking out the PVDF membrane, then eluting again, adding a corresponding secondary antibody reagent, hybridizing at room temperature for 1h, and then washing the PVDF membrane by using a 1xTBST buffer solution. Finally, exposure imaging is carried out in a protein imaging system; the results are shown in table 1 and fig. 5:
TABLE 1 protein concentration of exosome samples
| Sample name | Buffalo A | Buffalo B | Buffalo C |
| OD560nm | 0.160 | 0.186 | 0.398 |
| c ug/ul | 0.50 | 0.71 | 2.35 |
| m ug | 27.77 | 38.86 | 129.30 |
The results in Table 1 show that the protein concentrations in the three samples were 0.50, 0.71, 2.35ug/ul, respectively.
Western Blot is adopted to detect the exosome surface marker proteins of three samples, and the result is shown in figure 5, calnexin and TSG101 proteins on the exosome membrane of the buffalo A (which is a blood sample of 2h born by calves) are positive; no four proteins were detected on the exosome membrane of buffalo B (a blood sample from calves born for 5 d); buffalo C (a blood sample from 12d born calf) is positive for TSG101 and CD81 proteins on exosome membranes; thus, for the purposes of this application, proteins in exosomes are detected by western blot: the types of Calnexin, TSG101, CD9 and CD81 can well realize the typing of the buffalo birth time.
In summary, exosomes are important components of the extracellular environment, which can transmit signals and molecules to neighboring cells, participating in important biological processes. The research firstly concentrates on the separation of exosomes in buffalo blood cells of different growth periods of the newborn calf, identifies the molecular biological characteristics of the newborn calf, finds 3 characteristic periods, and types the newborn calf by a western blot method according to the characteristic periods, thereby providing a more accurate period typing basis for the immune function, exosome function, feeding and management of the newborn calf in the future.
During the research, applications found that: compared with other exosome extraction methods, the differential centrifugation method is higher in exosome purity. In addition, research shows that the exosome extracted by the differential centrifugation method has rich microRNA content. The morphological structure of the buffalo blood exosomes obtained in the research is well reserved, round or oval micro vesicles can be seen under a transmission electron microscope, and the particle size analysis result shows that the particle size of the buffalo blood exosomes is about 100nm and is consistent with the reported exosome morphology.
Various types of inclusion exist in exosome, and through research on exosome inclusion of newborn calves, the applicant finds that establishment of a passive immune system of a newborn calf depends on colostrum which is supplied in time after birth, and establishment and maturation of an autogenous immune system of the calf are relatively complex processes. The cell secretion is shown to participate in the immune regulation process of cells in the past research, so that the change condition of cell exosome small RNA of calves after birth and colostrum is deeply explored, the regulation and control target genes of the calves are facilitated to be predicted, and a basis is provided for analyzing the construction of the autogenous immune system of the calves from the molecular level.
Meanwhile, according to the protein production period, the method is beneficial to the research of the immune system establishment process of the buffalo, provides more scientific and accurate basis for the feeding and management of the buffalo in the future, and provides important molecular basis for the research of the immune system of the calf of the buffalo.
The above description is for the purpose of illustrating the preferred embodiments of the present invention, but the present invention is not limited thereto, and all changes and modifications that can be made within the spirit of the present invention should be included in the scope of the present invention.
Claims (2)
1. The method for judging the birth time of the buffalo by applying the blood exosome marker is characterized by comprising the following steps of:
(1) Collecting a whole blood sample of a buffalo calf born, and extracting exosomes in buffalo blood by adopting a centrifugal method;
(2) Carrying out Western blot detection on the exosomes extracted in the step (1), and when detecting that Calnexin and TSG101 proteins are positive, explaining that: the buffalo calf is born for 0-2h; when the four Calnexin, TSG101, CD9 and CD81 proteins are not detected, the following description is given: the buffalo calf is born for 3h-11d; when positive TSG101 and CD81 proteins were detected, it was indicated that: the buffalo calf is born for more than 12 d;
the exosome marker is Calnexin, TSG101, CD9 or CD81 protein.
2. The method according to claim 1, wherein the centrifugation of step (1) is a differential centrifugation, i.e.: taking a serum sample, adding phosphate buffer solution into the serum sample, and centrifuging for 15min at 300g and 4 ℃; centrifuging, collecting supernatant, and centrifuging at 12000g and 4 deg.C for 40min; centrifuging, collecting supernatant, and filtering with filter membrane; centrifuging the filtrate at 12000g and 4 deg.C for 1.5 hr for three times, discarding the supernatant and retaining the precipitate; beating the precipitate with phosphate buffer solution to wash the precipitate, and centrifuging for 1 hr at 100000g and 4 deg.C to obtain precipitate as exosome.
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Application publication date: 20210409 Assignee: Cenxi Qiliao Agricultural Development Co.,Ltd. Assignor: GUANGXI ZHUANG AUTONOMOUS REGION BUFFALO INSTITUTE Contract record no.: X2023980045747 Denomination of invention: Blood Exosomes Markers for Judging the Birth Time of Buffalos and Their Applications Granted publication date: 20221018 License type: Common License Record date: 20231106 |