Application of milk exosome in preparation of medicine for resisting porcine epidemic diarrhea virus and medicine

Abstract

The invention discloses application of a milk exosome in preparation of a medicine for resisting porcine epidemic diarrhea virus, and belongs to the technical field of biological medicine, and the milk exosome is prepared by adopting a citric acid dissolved protein method and an ultrafiltration concentration method. The invention further discloses a medicine for resisting the porcine epidemic diarrhea virus, and the medicine contains the milk exosome in the application. According to the present invention, the porcine viral diarrhea virus resistance of the milk exosome milk-exo is researched, the results show that the milk-exo can significantly inhibit the replication ability of the PEDV virus, and can reduce the damage of the PEDV virus to PK1 cells and Vero cells, such that the application effect of the milk-exo in the preparation of the porcine epidemic diarrhea virus resistance drug is proved. The preparation method of the exosome from milk is simple, the raw materials are easy to obtain, and the exosome has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an application of milk exosomes in the preparation of a drug against porcine epidemic diarrhea virus and a drug against porcine epidemic diarrhea virus. Background Art

[0002] Animal husbandry is an important industry related to the national economy and people's livelihood, and it is also a strategic industry to ensure food safety and residents' lives. Pigs are one of the most important livestock resources. They not only provide livestock products for humans, but also provide experimental materials for related medical research because of their high similarity with humans in physiological and biochemical characteristics. With the expansion of the scale of pig farming in my country, farmers continue to use vaccines and antibiotics, which leads to a gradual decrease in the disease resistance and environmental adaptability of pigs, making them more susceptible to external pathogens, bringing new challenges to the prevention and control of pig diseases.

[0003] Porcine epidemic diarrhea virus (PEDV) is an enveloped positive-strand RNA virus of the Coronaviridae family and the genus Alphacoronavirus. Porcine epidemic diarrhea (PED) is an acute, highly contagious enteric disease caused by PEDV. Studies have found that PEDV can also be transmitted through aerosols and infected through the respiratory tract, which makes the prevention and control of PEDV more difficult. PEDV can infect pigs of all ages, with clinical symptoms such as anorexia, vomiting, intestinal villous atrophy, watery diarrhea, malignant dehydration, and weight loss. It is most harmful to piglets, causing huge economic losses to the global pig industry. At present, commercial PED vaccines are still mainly traditional vaccines, but with the high variation of the viral genome, the protective efficiency of the vaccine is difficult to guarantee. Some pig farms that have been vaccinated have experienced outbreaks of porcine epidemic diarrhea again, so there is an urgent need to find more effective measures to prevent and control the disease.

[0004] Exosomes (exo) are natural extracellular nanovesicles with a double-layer plasma membrane structure. They are widely present in various body fluids and participate in intercellular communication and regulate physiological functions by carrying bioactive substances such as proteins, miRNA, mRNA, DNA and other molecules. Exosomes from different sources have different functions. Milk is the only biological fluid containing exosomes that can be purchased on the market. Researchers have demonstrated the potential of milk exosomes as drug carriers under different pathological conditions. However, its antiviral properties remain to be explored. The use of milk-derived exosomes in the preparation of drugs against porcine epidemic diarrhea virus has not been reported, and there is currently no good antiviral drug for porcine epidemic diarrhea virus. Summary of the invention

[0005] In response to one or more of the above defects or improvement needs in the prior art, the present invention provides an application of milk exosomes in the preparation of a drug against porcine epidemic diarrhea virus and a drug.

[0006] To achieve the above objectives, according to one aspect of the present invention, there is provided a use of milk exosomes in the preparation of a drug against porcine epidemic diarrhea virus.

[0007] As a further improvement of the present invention, the milk exosomes are prepared by citric acid protein dissolution method and ultrafiltration concentration method.

[0008] As a further improvement of the present invention, the method for preparing milk exosomes comprises the following steps:

[0009] (1) adding a set amount of citric acid to milk, shaking and letting stand for 15 to 30 minutes; the volume mass ratio of the milk to the citric acid is (90 to 225):1;

[0010] (2) Centrifuge at 10,000 rpm for 20 min and collect the supernatant;

[0011] (3) first filtering the supernatant using a 0.45 μm filter, and then further filtering using a 0.22 μm filter to collect the filtrate;

[0012] (4) The filtrate is ultrafiltered and concentrated to obtain the milk exosomes.

[0013] As a further improvement of the present invention, the drug contains no less than 200 μg / mL of exosomes.

[0014] As a further improvement of the present invention, the particle size of the milk exosomes is 130 to 150 nm.

[0015] As a further improvement of the present invention, in step (1), the milk is pasteurized milk.

[0016] As a further improvement of the present invention, in step (4), the milk exosomes are packaged and stored at -80°C.

[0017] According to another aspect of the present invention, a drug for resisting porcine epidemic diarrhea virus is provided, comprising the milk exosomes used in the application.

[0018] As a further improvement of the present invention, the concentration of the milk exosomes is not less than 200 μg / mL.

[0019] As a further improvement of the present invention, the particle size of the milk exosomes is 130 to 150 nm.

[0020] The present invention uses CCK-8 kit to detect the effect of exosomes on the proliferation of pig kidney cells. The results show that exosomes have no significant effect on cell proliferation. In addition, through q-PCR detection, it was found that the addition of exosomes after PEDV infection significantly reduced the replication ability of PEDV virus and the expression of PEDV-M gene, indicating that milk exosomes can effectively inhibit the replication ability of PEDV in cells; and different concentrations of milk-exo can reduce the damage of PEDV virus to PK1 cells and Vero cells to varying degrees.

[0021] In general, the above technical solution conceived by the present invention has the following beneficial effects compared with the prior art:

[0022] (1) The present invention studies the anti-porcine viral diarrhea virus effect of milk exosomes (milk-exo) and finds that milk exosomes (milk-exo) can significantly inhibit the replication ability of PEDV virus and reduce the damage of PEDV virus to PK1 cells and Vero cells, which proves the application effect of milk exosomes (milk-exo) in the preparation of drugs against porcine epidemic diarrhea virus.

[0023] (2) The present invention adopts citric acid protein dissolution method and ultrafiltration concentration method to prepare milk exosomes, and can collect milk exosomes with high purity. In addition, the preparation method of milk-derived exosomes is simple, the raw materials are easy to obtain, and it has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The particle size distribution diagram and potential value of milk exosomes in Example 1 of the present invention;

[0025] Figure 2 This is a TEM image of milk exosomes in Example 1 of the present invention;

[0026] Figure 3 This is a graph showing the expression results of milk exosome protein markers in Example 1 of the present invention;

[0027] Figure 4 This is a graph showing the uptake of milk exosomes by pig kidney cells in Example 2 of the present invention;

[0028] Figure 5 This is a graph showing the effect of 0, 50, 100, 200, 400, and 600 μg / mL milk exosomes on the proliferation of pig kidney cells after 24 hours of culture in Example 3 of the present invention;

[0029] Figure 6 This is a graph showing the effect of 0, 50, 100, 200, 400, and 600 μg / mL milk exosomes on the proliferation of pig kidney cells after 48 hours of culture in Example 3 of the present invention;

[0030] Figure 7 This is a comparison diagram of different concentrations of milk exosomes reducing the damage of PEDV to pig kidney cells PK1 in Example 4 of the present invention;

[0031] Figure 8 This is a comparison chart of different concentrations of milk exosomes reducing the damage of PEDV to Vero cells in Example 4 of the present invention;

[0032] Fig. 9 This is a graph showing the results of milk exosomes reducing the expression level of the PEDV-M gene in Example 4 of the present invention. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0034] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0035] Example 1 Isolation, extraction and identification of milk exosomes

[0036] A certain amount of citric acid (45 mL milk + 0.25 g citric acid) was added to the milk, shaken well and allowed to stand for 30 minutes, shaken once every 10 minutes; centrifuged at 10,000 rpm for 20 minutes, took the supernatant, and repeated twice; the supernatant was first filtered through a 0.45 μm filter, and then filtered through a 0.22 μm filter; finally, it was concentrated using an ultrafiltration tube to obtain milk exosomes, and the concentrated milk exosomes were stored at -80°C.

[0037] The milk-exo protein concentration was determined using a BCA protein concentration assay kit. The milk-exo protein concentration determined by the BCA method was 11.36 μg / mL.

[0038] Nanoparticle tracking analysis was used to measure the size distribution and particle size of exosomes. Figure 1 As shown in the results, the particle size results show that the main peak is around 143.4nm. The morphology of exosomes was observed using transmission electron microscopy, and the results are as follows Figure 2 As shown, transmission electron microscopy observed milk-exo of uneven sizes, showing a typical saucer shape, and the morphology of exosomes was relatively complete.

[0039] Western blot was used to detect the expression of milk-exo marker proteins Alix, TSG101, CD63, and HSP70. The specific method of Western blot was as follows: the protein concentration was determined by the BCA method, and the protein was denatured with 5×SDS-PAGE Loading Buffer and then SDS-PAGE electrophoresis was performed. After transfer, the membrane was rinsed on a shaker with 1×TBS solution for 5 minutes. After blocking with 5% skim milk powder for 2 hours, the membrane was rinsed on a shaker with 1×TBS solution for 10 minutes. The primary antibody was incubated overnight in a 4°C refrigerator. The membrane was rinsed on a shaker with 1×TBST solution for 3 times, each for 10 minutes. After incubating the secondary antibody on a shaker at room temperature for 1 hour in the dark, the membrane was rinsed on a shaker with 1×TBST solution for 3 times, each for 10 minutes. The ECL chemiluminescent substrate kit was used for color development, and the chemiluminescent imager was used for imaging.

[0040] The results of Western blotting to detect the expression of protein markers in milk exosomes are as follows Figure 3 As shown, it can be seen that milk-exo highly expresses TSG101, Alix, HSP70, and CD63 surface protein markers. It can be seen that exo with higher purity can be collected through the citric acid protein dissolution method and concentration method, and used in subsequent experiments.

[0041] Example 2 Study on the uptake of milk-exo by PK1 cells

[0042] (1) Cell culture

[0043] The porcine kidney cell (PK1) cell line was provided by the Genetic Breeding and Precision Farming Laboratory of the College of Animal Science and Nutritional Engineering of Wuhan University of Technology. PK1 cells were cultured in a 37°C, 5% CO2 constant temperature incubator using high-glucose DMEM containing 10% FBS.

[0044] (2) Uptake of milk-exo by PK1 cells

[0045] To determine the uptake of milk-exo by PK1 cells, milk-exo was treated using the PKH67 green membrane staining kit; milk-exo with fluorescent markers (concentrations of 0, 50, 100, 200, and 400 μg / mL) was transfected into PK1 cells and cultured for 24 hours, and an immunofluorescence experiment was performed to observe the uptake of milk-exo by PK1 cells.

[0046] The specific method of the immunofluorescence experiment is as follows: remove the culture medium, wash 3 times with PBS, each time for 5 minutes; add 4% paraformaldehyde to fix for 30 minutes, wash 3 times with PBS, each time for 5 minutes; add Hochest staining solution, incubate in the dark for 5 minutes, wash 3 times with PBS, each time for 5 minutes; take pictures under a fluorescence microscope to observe the uptake of exosomes by PK1 cells.

[0047] Figure 4 This is the result of using the PKH67 green membrane staining kit to detect the uptake of exosomes by pig kidney cells. The results show that PK1 cells have better uptake when the exosome concentration is 400μg / mL.

[0048] Example 3 Effect of milk-exo on cell proliferation

[0049] (1) Cell culture

[0050] The porcine kidney cell (PK1) cell line was provided by the Genetic Breeding and Precision Farming Laboratory of the College of Animal Science and Nutritional Engineering of Wuhan University of Technology. PK1 cells were cultured in a 37°C, 5% CO2 constant temperature incubator using high-glucose DMEM containing 10% FBS.

[0051] (2) Effect of milk-exo on cell proliferation

[0052] To determine whether milk-exo would affect the proliferation of PK1 cells, the proliferation activity of PK1 cells was measured using a CCK-8 kit after culturing in milk-exo (concentrations of 0, 50, 100, 200, 400, and 600 μg / mL) for 24 h and 48 h, respectively.

[0053] Figure 5 and Figure 6 This is a graph showing the effect of exosomes on the proliferation of pig kidney cells detected using the CCK-8 kit. The results show that after 24h and 48h of culture, 50, 100, 200, 400, and 600 μg / mL of exosomes had no significant effect on cell proliferation.

[0054] Example 4 Study on the effect of milk-exo against porcine viral diarrhea virus

[0055] (1) milk-exo reduces the effect of PEDV virus on porcine kidney cells (PK1) and African green monkey kidney cells (Vero);

[0056] PK1 (Vero) cells were cultured in 2 groups and infected with PEDV when the cells were fully confluent:

[0057] ①Control group, cultured in serum-free DMEM medium containing 5 μg / mL trypsin;

[0058] ② PEDV-EXO group: After PEDV adsorption for 1 hour, the cells were washed twice with PBS and cultured in serum-free DMEM medium containing (0, 50, 100, 200, 400 μg / mL) milk-exo and 5 μg / mL trypsin. The degree of PK1 (Vero) cell pathology was observed.

[0059] The results are as follows Figure 7 and Figure 8 As shown, the results showed that different concentrations of milk-exo could reduce the damage of PEDV virus to PK1 cells and Vero cells to varying degrees, among which 200 μg / mL and 400 μg / mL exosomes could significantly reduce the damage of PEDV virus to PK1 cells.

[0060] In order to determine the effective concentration of exosomes against PEDV, the above examples added different concentrations of exosomes to PK1 cells after PEDV infection and observed the degree of cell pathology. The results showed that 200μg / mL and 400μg / mL of exosomes can significantly reduce the damage of PEDV virus to PK1 cells and Vero cells compared with 50, 100, and 200μg / mL. Therefore, the concentration of exosomes in the drug is preferably not less than 200μg / mL. In the following examples, exosomes with a concentration of 400μg / mL were selected to conduct experiments on the effect on the replication ability of PEDV.

[0061] (2) The effect of exosomes on the replication ability of PEDV;

[0062] PK1 cells were cultured in groups of 4 and infected with PEDV when the cells were fully confluent:

[0063] ①Control group, cultured in serum-free DMEM medium with 5 μg / mL trypsin;

[0064] ②EXO group, cultured in serum-free DMEM medium with 400 μg / mL milk-exo and 5 μg / mL trypsin;

[0065] ③ PEDV group: After PEDV adsorption for 1 h, the cells were washed twice with PBS and cultured in serum-free DMEM medium containing 5 μg / mL trypsin;

[0066] ④ PEDV-EXO group: After PEDV adsorption for 1 h, the cells were washed twice with PBS and cultured in serum-free DMEM medium containing 400 μg / mL milk-exo and 5 μg / mL trypsin.

[0067] After 24 hours of culture, cell RNA was extracted and the expression of PEDV-M gene was detected by real-time fluorescence quantitative PCR. The specific method of real-time fluorescence quantitative PCR is as follows: GAPDH is used as the internal reference gene, and the qRT-PCR method is used to detect the expression of PEDV-M gene 24 hours after PEDV infection. q-PCR amplification system (total volume of 10 μL): TBgreen 5 μL, upstream and downstream primers 0.2 μL each, ddH2O 3.6 μL, cDNA template 1 μL.

[0068] The results are as follows Fig. 9As shown, through q-PCR detection, it was found that 24 hours after the addition of 400 μg / mL of exosomes after PEDV infection, the PEDV virus replication ability was significantly reduced and the expression level of the PEDV-M gene was significantly reduced.

[0069] It should be noted that the present invention uses SPSS20.0 software, and uses Student's t test and one-way ANOVA to process the data. P<0.05 is considered to be statistically significant ("*" means P<0.05, "**" means P<0.01, "***" means P<0.001, and "****" means P<0.0001). Graphpad Pism9.5 software is used to draw the graph.

[0070] It will be easily understood by those skilled in the art that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of milk exosomes in the preparation of drugs against porcine epidemic diarrhea virus.

2. The use of milk exosomes according to claim 1 in the preparation of a drug against porcine epidemic diarrhea virus, characterized in that: The milk exosomes are prepared by adopting a citric acid protein dissolution method and an ultrafiltration concentration method.

3. The use of milk exosomes according to claim 1 in the preparation of a drug against porcine epidemic diarrhea virus, characterized in that: The method for preparing milk exosomes comprises the following steps: (1) adding a set amount of citric acid to milk, shaking and letting stand for 15 to 30 minutes; the volume mass ratio of the milk to the citric acid is (90 to 225):1; (2) Centrifuge at 10,000 rpm for 20 min and collect the supernatant; (3) first filtering the supernatant using a 0.45 μm filter, and then further filtering using a 0.22 μm filter to collect the filtrate; (4) The filtrate is ultrafiltered and concentrated to obtain the milk exosomes.

4. The use of milk exosomes according to any one of claims 1 to 3 in the preparation of a drug against porcine epidemic diarrhea virus, characterized in that: The medicine contains no less than 200 μg / mL of milk exosomes.

5. Use of the milk exosomes according to any one of claims 1 to 3 in the preparation of a drug against porcine epidemic diarrhea virus, characterized in that: The particle size of the milk exosomes is 130 to 150 nm.

6. The use of milk exosomes according to claim 3 in the preparation of a drug against porcine epidemic diarrhea virus, characterized in that: In step (1), the milk is pasteurized milk.

7. Use of milk exosomes according to claim 3 or 6 in the preparation of a drug against porcine epidemic diarrhea virus, characterized in that: In step (4), the milk exosomes are packaged and stored at -80°C.

8. A drug for resisting porcine epidemic diarrhea virus, comprising the milk exosomes used in any one of claims 1 to 7.

9. The drug against porcine epidemic diarrhea virus according to claim 8, characterized in that: The concentration of the milk exosomes is not less than 200 μg / mL.

10. The drug against porcine epidemic diarrhea virus according to claim 8 or 9, characterized in that: The particle size of the milk exosomes is 130 to 150 nm.

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