Pig small intestine tissue exosome and separation and purification method and application thereof

Abstract

The application discloses pig small intestine tissue exosomes and a separation and purification method and application thereof, wherein the pig small intestine tissue exosomes have an anti-swine epidemic diarrhea virus effect, and the separation and purification comprises the following steps: S1: washing the tissue with a PBS buffer, then taking out intestinal contents and mucosa, and cutting into tissue fragments; S2: immersing the tissue fragments into an HBSS buffer containing collagenase, then placing on ice to end enzymatic digestion and obtain an enzymolysis product; S3: homogenizing the enzymolysis product, then filtering into a fresh tube by using a 40 mu m filter, then performing centrifugation on the filtrate for three times; and S4: purifying supernatant obtained by the third centrifugation through an exosome collection and purification system, and obtaining high-purity exosomes. The pig small intestine tissue exosomes have the obvious anti-swine epidemic diarrhea virus effect, can be used for preparing medicines for preventing / treating the virus, and have important significance for preventing and controlling the swine epidemic diarrhea in the future.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a pig small intestine tissue exosome and a separation and purification method and application thereof. BACKGROUND

[0002] Porcine epidemic diarrhea (PEDV) is an acute infectious disease of newborn piglets caused by porcine epidemic diarrhea virus infection, and the diseased piglets have clinical manifestations of acute diarrhea, vomiting and dehydration, and has a high morbidity and mortality. At present, there is still a lack of effective vaccines and drugs for the prevention and control of the disease. Therefore, it is of great significance to find a drug or new substance that can play an anti-porcine epidemic diarrhea virus role for the research and disease prevention and control of the virus.

[0003] Exosomes are vesicles secreted by cells, widely exist in blood, urine, saliva, milk and other body fluids, and the particle size is 30-150nm. Exosomes were initially only regarded as the "excretion" of cell activity, but more and more studies have shown that they may be important substances for cell-to-cell communication and material exchange. Exosomes contain various biomolecules such as proteins, lipids, DNA, mRNA and miRNA, and these substance bases make them attract attention in biomedical applications. Exosomes realize cell-to-cell signal transduction through the biological active molecules contained therein, thereby inducing the occurrence of some physiological and pathological events. In addition, due to the endogenous nature of exosomes, exosomes containing various biological active substances are expected to be developed into safe and effective therapeutic drugs. However, there is no related research on exosomes against porcine epidemic diarrhea at present. SUMMARY

[0004] Based on this, the present application provides a pig small intestine tissue exosome and a separation and purification method and application thereof, which aims to break through the current research bottleneck of exosomes against porcine epidemic diarrhea virus.

[0005] In a first aspect of the present application, a pig small intestine tissue exosome is provided, which has an anti-porcine epidemic diarrhea virus effect.

[0006] The classification, purification and identification of the pig small intestine tissue exosome show that the exosome includes proteins, RNA and lipids, wherein the proteins include enzymes of metabolic type, ribosomal proteins, signal transduction factors, adhesion factors, cytoskeletal proteins and ubiquitin, and the exosome specifically further contains biomarker proteins such as tumor susceptibility gene 101 protein (TSG101), heat shock protein with a molecular weight of 70kD (HSP70), CD9 and β-actin, and the average particle size thereof is 87.23nm. It is found by transmission electron microscopy that the exosome has a cup-shaped structure.

[0007] In some experimental examples, Vero cells are cultured and subjected to exosome pretreatment, and then inoculated with PEDV viruses, and it is found that the higher the concentration of added exosomes, the weaker the cytopathic effect of Vero cells and the lower the degree of viral infection, thereby verifying the antiviral effect of the exosomes.

[0008] In a second aspect of the present application, a method for separating and purifying pig small intestine tissue exosomes is provided, comprising the following steps:

[0009] S1: washing the tissue with PBS buffer, then removing the intestinal contents and mucosa, and cutting into tissue fragments;

[0010] S2: immersing the tissue fragments in HBSS buffer containing collagenase, water bathing in a 37℃ water bath for 30min, and then placing on ice to end the enzymatic digestion and obtain the enzymatic hydrolysate;

[0011] S3: homogenizing the enzymatic hydrolysate, filtering into a fresh tube using a 40μm filter, then performing first centrifugation on the filtrate, removing the cell debris after the first centrifugation, transferring the supernatant and performing second centrifugation, filtering the supernatant obtained by the second centrifugation using a 0.22μm PVDF filter, and then performing third centrifugation on the filtrate;

[0012] S4: purifying the supernatant obtained by the third centrifugation through an exosome collection and purification system to obtain high-purity exosomes.

[0013] Further, in step S2, the collagenase is type I collagenase, and the concentration is 300U / mL.

[0014] Further, in step S3, the first centrifugation is performed at 1000×g for 10min.

[0015] Further, in step S3, the second centrifugation is performed at 4℃, and sequentially at 2000×g for 20min, 5000×g for 30min, and 15000×g for 1h.

[0016] Further, in step S3, the third centrifugation is performed at 4℃ at 120000×g for 2h.

[0017] In a third aspect of the present application, the pig small intestine tissue exosomes are used in the preparation of an anti-porcine epidemic diarrhea virus drug, and the drug dosage form is a tablet, a capsule, a powder, a pill, a granule or a solution.

[0018] Compared with the prior art, the present application has the following technical effects:

[0019] The present application separates and purifies high-purity exosomes from pig small intestinal tissues, and determines the antiviral ability of the pig small intestinal tissue exosomes through experiments, finds that the pig small intestinal tissue exosomes have obvious anti-porcine epidemic diarrhea virus effect, and can be used for preparing medicines for preventing / treating the virus, which has important significance for the prevention and control of future porcine epidemic diarrhea. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 is the separation and identification result diagram of the pig small intestinal tissue exosomes;

[0021] Figure 2 is the result diagram of the influence of the pig small intestinal tissue exosomes on the cytopathic effect of virus-infected Vero cells;

[0022] Figure 3 is the result diagram of the influence of the pig small intestinal tissue exosomes on the fluorescence specific to PEDV N protein;

[0023] Figure 4 is the determination result diagram of the influence of the pig small intestinal tissue exosomes on the PEDV N gene copy number.

[0024] Figure 1 In the figure, a is the result diagram of the Western blotting experiment, b is the result diagram of the pig small intestinal tissue average diameter and concentration determination, and c and d are the observation result diagrams of the pig small intestinal tissue exosomes at different multiples. DETAILED DESCRIPTION

[0025] In order to facilitate the understanding of the present application, the present application will be described more fully below with reference to the related drawings. The preferred embodiments of the present application are shown in the drawings. However, the present application can be realized in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the disclosure of the present application more thorough and comprehensive.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. The terms used herein in the specification of the present application are only for the purpose of describing the specific embodiments and are not intended to limit the present application.

[0027] In the present application, "first aspect", "second aspect", "third aspect", "fourth aspect" and the like are only for the purpose of description and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth" and the like only serve the purpose of non-exhaustive enumeration description, and should be understood as not constituting a closed limitation on the quantity.

[0028] In the present application, the technical features described in an open manner include both a closed technical solution consisting of the listed features and an open technical solution including the listed features.

[0029] In some embodiments of the present application, the Vero cells are from the Shanghai Veterinary Research Institute, the DMEM culture medium is from GIBCO Company, the trypsin digestion solution is from GIBCO Company, the porcine epidemic diarrhea virus is JS-2013 strain (NCBI accession number MH910099.1, provided by the Shanghai Veterinary Research Institute), and the HBSS buffer and collagenase type I are from Solabio Technology Co., Ltd.

[0030] Example 1: T25 culture flask culture of adherent Vero cells

[0031] The Vero cells are cultured in a conventional T25 culture flask, and when the cells in the culture flask reach a dense monolayer state, the cell density is about 6×10 6 cells / flask, which can be used for plating and inoculating viruses.

[0032] The culture solution is high-tension DMEM culture medium containing 10% fetal bovine serum, and the trypsin digestion solution is 0.25% trypsin. When the Vero cells in the T25 culture flask grow to 80%-90% of the density, they can be subcultured. The cells are repeatedly washed with sterile PBS buffer solution for three times, 1 mL of trypsin digestion solution is added, and the cells are digested in a cell culture box (37°C, 5% CO2) for 1-2 min. When the cells become round, the digestion is stopped by adding the culture solution, and the cells are uniformly blown and dispersed according to a certain proportion for subculture. 5 mL of culture solution is added to the T25 culture flask, and the culture is continued in the cell culture box.

[0033] Example 2: T25 culture flask inoculation of porcine epidemic diarrhea virus

[0034] The Vero cells in the T25 culture flask with a dense monolayer of cells are taken, the culture solution in the flask is discarded, 4 mL of PBS buffer solution is added, and the culture solution on the surface of the culture flask and the cells is washed by shaking. After the PBS buffer solution is discarded, the cells are repeatedly washed with PBS buffer solution for three times. In order to avoid the influence of serum on the effect of trypsin, the residual culture solution should be washed thoroughly. 1 mL of incubation solution is added, 50 μL of virus solution is added, and the mixture is incubated in a cell culture box (37°C, 5% CO2) for 1 h. During the incubation, the mixture is shaken every 20 min to prevent local drying and death of the cells. After the incubation of the cells is completed, 4 mL of maintenance solution is added, and the mixture is further cultured in the culture box. The mixture is observed every 8 h, and when the lesion reaches more than 80%, the virus solution is harvested together with the T25 culture flask and stored at -80°C.

[0035] The incubation solution is serum-free DMEM / F-12 medium with a final concentration of 10 μg / mL trypsin, and the maintenance solution is high-sugar DMEM medium containing trypsin with a final concentration of 10 μg / mL, and the PBS buffer has a pH of 7.2-7.4.

[0036] Example 3, Isolation and purification of pig small intestine tissue exosomes

[0037] (1) The tissue was washed thoroughly with ice-cold PBS buffer to remove intestinal contents and mucosa. Next, the tissue was placed on ice, spread longitudinally, and cut into tissue fragments of about 0.4 cm;

[0038] (2) The tissue fragments were immersed in HBSS buffer containing type I collagenase at a concentration of 300 units / mL, and incubated in a 37°C water bath for 30 min, with shaking every 5 min. After enzyme digestion, the mixture was placed on ice to stop the enzymatic digestion;

[0039] (3) After homogenization of the tissue sample, the sample was filtered through a sterile nylon mesh filter with a pore size of 40 μm into a fresh tube. The filtrate was centrifuged at 1000 x g for 10 min to remove cell debris, and then the supernatant was transferred to a new 15 mL tube;

[0040] (4) The centrifugation was repeated, with the temperature set to 4°C, and the centrifugation was performed in the following order: 2000 x g for 20 min, 5000 x g for 30 min, and 15,000 x g for 1 h. The supernatant was filtered through a 0.22 μm PVDF filter into a new 15 mL tube, with the temperature set to 4°C, and then centrifuged at 120,000 x g for 2 h;

[0041] (5) The supernatant obtained by centrifugation was collected and purified through an exosome collection and purification system to obtain high-purity exosomes.

[0042] a. The isolated exosomes were subjected to protein concentration determination using a BCA protein concentration determination kit (Bi Yun Tian), and the exosome biomarker proteins were identified by Western blotting, with a total protein loading amount of 30 μg per lane. The proteins were separated by 12% SDS-PAGE gel electrophoresis at 80 V for 30 min and at 100 V for 1-2 h. The PVDF membrane was transferred at 100 V for 1.5 h, and the primary antibodies against TSG101 (1:1000, Abeam), HSP70 (1:800, Abeam), β-actin (1:1000, Bi Yun Tian), and CD9 (1:1000, Abeam) were incubated at 4°C overnight.

[0043] b. After thorough washing with 0.1% TBST, the samples were incubated with anti-rabbit IgG-HRP secondary antibody (1:5000, Abcam) at room temperature for 1.5 h, followed by color development using a highly sensitive ECL chemiluminescence kit. Before starting the analysis, the exosome samples were diluted tenfold with PBS, and the mean diameter and concentration of the samples were automatically determined using a NanoFCM Flow Nanoanalyzer N30E (China).

[0044] Separation and identification results as follows Figure 1 As shown, a represents exosome biomarker proteins, tumor susceptibility gene 101 (TSG101), heat shock protein 70 (HSP70), and CD9. b represents isolated exosomes with a concentration of 3.53 × 10⁻⁶. 11 The particle count was 87.23 nm, with an average particle size of 87.23 nm. This indicates that the isolated exosomes exhibited a cup-shaped characteristic as observed by transmission electron microscopy.

[0045] Experiment Example 1: Test of the anti-porcine epidemic diarrhea virus activity of porcine small intestinal tissue exosomes

[0046] (1) Culture Vero cells in T25 cell culture flasks until the cells grow into a dense monolayer. The culture medium is DMEM medium containing 10% fetal bovine serum.

[0047] (2) After digesting the well-growing Vero cells in the T25 flask with trypsin, they were dispersed in DMEM containing 10% fetal bovine serum and then seeded into 24-well cell culture plates. They were cultured at 37°C in a 5% CO2 incubator for 24 hours. After 24 hours, the cell density reached 80-90%, and subsequent experiments were carried out.

[0048] (3) Prepare serum-free high-glucose DMEM containing pancreatic enzyme at a final concentration of 10 μg / mL as maintenance solution;

[0049] (4) Wash the cells three times with sterile PBS before adding exosomes to thoroughly remove the culture medium from the culture flask and the cell surface;

[0050] (5) Add 0, 0.05, 0.5, 5, 2.5 and 50 μg of total protein exosomes to cells in each well in sequence, so that the final concentrations are 0 μg / mL, 0.1 μg / mL, 1 μg / mL, 10 μg / mL, 50 μg / mL and 100 μg / mL in sequence. Incubate at 37℃ in a 5% CO2 incubator for 1 h, discard, and wash cells three times with sterile PBS;

[0051] (6) Add an appropriate amount of porcine epidemic diarrhea virus solution (to make the virus challenge concentration MOI = 0.001) and incubate in serum-free high-glucose DMEM medium containing 10 μg / mL trypsin for 1 h.

[0052] (7) Finally, add the trypsin-free high-sugar DMEM maintenance solution with a final concentration of 10 pg / mL.

[0053] (8) Observe the cells after 24 h.

[0054] Observe the cytopathic effect, and the results are shown in Table 1. Figure 2 The higher the exosome concentration, the weaker the cytopathic effect, indicating that the higher the exosome concentration, the stronger the antiviral effect. The PEDV N protein and Vero cells were specifically fluorescently dyed, and the PEDV N protein-specific fluorescence was observed. The results are shown in Table 2. Figure 3 The higher the exosome concentration, the weaker the PEDV N protein-specific fluorescence, i.e., the lower the degree of viral infection, again indicating that the higher the exosome concentration, the stronger the antiviral effect.

[0055] Experimental Example 2: Determination of the N gene copy number of porcine epidemic diarrhea virus

[0056] 1. Culture Vero cells in a T25 cell culture bottle, and use when the cells grow into a dense monolayer. The culture solution is DMEM medium containing 10% fetal bovine serum;

[0057] 2. After trypsin digestion of the well-grown T25 bottle of Vero cells, blow them with DMEM containing 10% fetal bovine serum, and inoculate them into a 24-well cell culture plate. Incubate at 37°C in a 5% CO2 incubator for 24 h. After 24 h, the cell density reaches 80-90%, and the subsequent experiment is performed;

[0058] 3. Prepare trypsin-free high-sugar DMEM containing a final concentration of 10 pg / mL as a maintenance solution;

[0059] 4. Before adding the exosomes, wash the cells with sterile PBS buffer three times to thoroughly remove the culture solution on the surface of the culture bottle and the cells;

[0060] 5. Add 0, 0.05, 0.5, 5, 2.5, and 50 pg of total protein of exosomes to each well of cells, respectively, so that the final concentration is 0 pg / mL, 0.1 pg / mL, 1 pg / mL, 10 pg / mL, 50 pg / mL, and 100 pg / mL, respectively. Incubate at 37°C in a 5% CO2 incubator for 1 h, and then discard and wash the cells with sterile PBS buffer three times;

[0061] 6. Add an appropriate amount of virus solution and trypsin-free high-sugar DMEM medium containing 10 pg / mL to incubate for 1 h;

[0062] 7. Finally, add the trypsin-free high-sugar DMEM maintenance solution with a final concentration of 10 pg / mL.

[0063] 8. After 24h post-virus infection, the cells were observed, and the cells were frozen and thawed, centrifuged to collect the supernatant, and stored at -80℃. The viral gene copy number was detected by absolute quantitative qPCR.

[0064] The detection results are shown in Table 1. Figure 4 As shown in Table 1, the higher the exosome addition concentration, the lower the PEDV N gene viral copy number, indicating that the higher the exosome addition concentration, the stronger the anti-virus proliferation effect.

[0065] The above-described embodiments only express several embodiments of the present application, which are described in detail and specifically, but should not be understood as a limitation on the scope of the patent. It should be noted that, for ordinary skilled in the art, without departing from the concept of the present application, several modifications and improvements can be made, which are all within the protection scope of the present application. Therefore, the protection scope of the present application patent should be subject to the appended claims.

Claims

1. The application of porcine small intestinal tissue exosomes in the preparation of drugs against porcine epidemic diarrhea virus, characterized in that, The method for isolating and purifying exosomes from porcine small intestinal tissue includes the following steps: S1: Wash the tissue with PBS buffer, then remove the intestinal contents and mucosa, and cut into tissue fragments; S2: Immerse the tissue fragments in HBSS buffer containing collagenase, incubate in a 37°C water bath for 30 min, and then place on ice to end the enzymatic digestion and obtain the enzymatic hydrolysis product. S3: Homogenize the enzymatic hydrolysate and filter it into a fresh tube using a 40μm filter. Then, centrifuge the filtrate for the first time. After the first centrifugation, remove cell debris, transfer the supernatant, and centrifuge a second time. Filter the supernatant obtained from the second centrifugation using a 0.22μm PVDF filter, and then centrifuge the filtrate a third time. The conditions for the first centrifugation are: 1000×g for 10 min; the temperature for the second centrifugation is 4℃, and the following conditions are repeated: 2000×g for 20 min, 5000×g for 30 min, and 15000×g for 1 h; the temperature for the third centrifugation is 4℃, and the centrifugation is 120000×g for 2 h. S4: The supernatant obtained from the third centrifugation was purified using an exosome collection and purification system to obtain high-purity exosomes.

2. The application according to claim 1, characterized in that, The drug dosage form is tablet, capsule, powder, pill, granule or solution.

3. The application according to claim 1, characterized in that, In step S2, the collagenase is type I collagenase with a concentration of 300 U / mL.

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