JP6968467B2 - Use of Lactobacillus plantarum ZN-3 in the preparation of medicines for the prevention or treatment of porcine epidemic diarrhea virus infection - Google Patents

Description

The present invention belongs to the art of antiviral microecologic formulations for livestock and poultry, and particularly relates to the use of lactobacillus plantarum ZN-3 in the preparation of pharmaceuticals for the prevention or treatment of porcine epidemic diarrhea virus infection.

Intestinal disease is a problem that is universally present in the modern pig farming industry, and diarrheal diseases including bacterial diarrhea, viral diarrhea, nutritional diarrhea, and diarrhea caused by parasites are more likely to occur in piglets. Porcine epidemic diarrhea virus (PEDV), which has continued to occur from 2010 to the present, has a high incidence of viral diarrhea, rapid spread of infection, high mortality, and unclear therapeutic effect. I focused on the prevention of diarrhea. Epidemic diarrhea is an acute infectious disease with clinical symptoms such as diarrhea, vomiting, and dehydration as the main signs. Mortality in some large pig farms has reached more than 80% and is an important cause of enteritis and mortality in newborn pigs, causing serious economic losses to the pig farming industry.

For diseases, the preventive effect is generally superior to the therapeutic effect, and PEDV, which is a viral disease, is no exception. PEDV has strict cell tactics and host specificity. However, genetic variation of the virus affects the protection of effective vaccines; antiviral drugs also usually have serious side effects, limited in dose and efficacy. Therefore, there is a need to continue to seek effective ways to control viral diseases, and probiotics are non-harmful, non-drug resistant, non-persistent, antibacterial, antiviral, growth-promoting, and environmentally friendly. Due to its advantage, it has received widespread attention.

Probiotic lactic acid bacteria are widely used to prevent and treat gastrointestinal disorders in humans and animals. Lactic acid bacteria are anaerobic or facultative anaerobes that can withstand the pH environment of the body's gastrointestinal tract and can colonize intestinal epithelial cells. Lactic acid bacteria also produce lactic acid, lower the pH in the intestine, and block the invasion of harmful bacteria such as Salmonella and Escherichia coli. Giang et al. (2010) added a composite microecologic of Enterococcus faecalis, Lactobacillus acidophilus, Pediococca spent Saceus, and Lactobacillus plantarum to the diet of wean piglets, and a blank control without addition. The incidence of piglet diarrhea was reduced when compared to the group. The effect of oral administration of lactic acid bacteria vaccine has been demonstrated by several research results, and existing data show that lactic acid bacteria have not only antibacterial activity but also antiviral activity. Mucosal immunity plays an important role in preventing infection because the mucosa is the most important route for many bacteria and viruses to invade and infect the body. Oral vaccines via the mucosal pathway can effectively induce the body's potential humoral and mucosal immunity and are an effective strategy for combating the disease. According to a research report (Van Niel et al. 2002; Zhang et al. 2008), Lactobacillus acidophilus can also enhance the immunity of the vaccine when used as an oral adjuvant for the rotavirus vaccine. Kritas and Morrison (2007) also reported that oral lactic acid bacteria could improve the vaccine's defense efficiency against PRRSV virus infection and increase the weight of PRRSV-infected pigs daily. Wang XN et al. (2017) found that Lactobacillus casei effectively induced mucosal immunity, humoral immunity, and Th2 type cell-mediated immunity in the body as a result of using Lactobacillus casei as an oral vaccine carrier for anti-PEDV. It has been shown that PEDV infection and onset can be prevented. In addition, probiotics as an oral vaccine have the advantages of being convenient, safe, immune-free, and economically beneficial. These data even demonstrate that probiotics can be used as formulations to promote intestinal mucosal immunity and prevent and treat intestinal diseases. However, research on the direct antiviral activity of probiotics has only just begun, and there are few reports on the antiviral activity of non-recombinant wild strains, and currently on the antiviral activity of Lactobacillus plantarum against PEDV. There is no research. An object of the present invention is to obtain probiotics having antiviral activity against porcine epidemic diarrhea virus (PEDV) through research.

It is an object of the present invention to propose the use of lactobacillus plantarum ZN-3 in the preparation of pharmaceuticals for the prevention or treatment of porcine epidemic diarrhea virus infection, wherein lactobacillus plantarum ZN-3 is a gastrointestinal tract. It can enter the intestine from the intestine, inhibit the invasion and proliferation of the enterovirus PEDV, and effectively prevent and treat the diseases caused by the epidemic diarrhea virus.

In the present invention, the following technical measures have been taken in order to achieve the above object.
The use of lactobacillus plantarum ZN-3 in the preparation of medicines for the prevention or treatment of porcine epidemic diarrhea virus infection utilizes lactobacillus plantarum ZN-3 as an inhibitor of epidemic diarrhea virus (PEDV). Including preparation, or preparation as a therapeutic or prophylactic agent for epidemic diarrhea virus (PEDV) infection.
The Lactobacillus plantarum ZN-3 was deposited under accession number CCTCC M 2017286.

In the above use, Lactobacillus plantarum ZN-3 may be a fermentation broth or fermentation supernatant of Lactobacillus plantarum ZN-3.

In the present invention, probiotics Lactobacillus plantarum ZN-3 is used to inhibit cell infection from porcine epidemic diarrhea virus (PEDV), and Lactobacillus plantarum is conventionally used as a potential antiviral microecologic agent. It has the following advantages over the antiviral chemotherapeutic agent of.

1. No harmful side effects, no persistence, antibacterial and antiviral, growth promotion and environmentally friendly.

2. It has the advantages of convenience, safety, no immune stress, and high economic benefit in use.

3. It enters the intestine through the gastrointestinal tract, inhibits the invasion and proliferation of enterovirus PEDV cells, and effectively prevents and treats diseases caused by epidemic diarrhea virus. In the present invention, all three treatment methods of the cell suspension of Lactobacillus plantarum ZN-3 have a direct inhibitory effect on the virus, and the inhibition rates are 28.30% and 20.75%, respectively. 22.64%; For Lactobacillus plantarum ZN-3 fermentation supernatant, first add the fermentation supernatant, then add the PEDV diluent, and add the fermentation broth and PEDV diluent at the same time 2 Both methods also had a direct inhibitory effect on the virus, and the inhibition rates were 32.08% and 13.21%, respectively.

4. The cells of lactobacillus plantarum or the fermentation supernatant of pH 7.0 in the present invention have no harmful side effects on the cells, slightly reduce the infectious activity of PEDV virus to vero cells, and cause cell invasion of PEDV virus. It was able to inhibit infection and prevent and treat porcine epidemic diarrhea.

5. The application of probiotics has already been applied for more than 10 years, and the therapeutic effect on gastrointestinal diseases has been clinically demonstrated to some extent, and the protection and enhancement of immune function and the antiviral effect are also attracting more and more attention. The mechanism of action is not yet clear. The present invention describes the inhibitory effect of probiotic lactobacillus plantarum on epidemic diarrhea virus (PEDV) from the cellular level.

It is a schematic diagram of the proliferation and lesions of PEDV virus in VERO cells (left figure: cell control not infected with PEDV; right figure: lesion formed by PEDV infection). Schematic diagram of PCR detection of PEDV virus amplification solution (lane 1: positive control; lanes 2, 3, 4: PEDV virus growth sample with different pancreatin concentrations (0, 5, 10 μg / ml); lane 5: negative control; lane 6, 7, 8: Overlap of lanes 2, 3, and 4 samples; lane 9: marker) . It is a schematic diagram (left figure: control cell; right figure: lesion cell) of PEDV lesion cell and control cell in TCID50 measurement of a 96-well plate . Schematic diagram of the inhibitory effect of Lactobacillus plantarum on PEDV virus (upper left figure: normal control cells; upper right figure: group to which ZN-3 was first added and then PEDV diluted solution; lower left figure: PEDV diluted solution first The group to which ZN-3 was added after the addition of the above; lower right figure: virus control group).

Unless otherwise stated explicitly, the test methods and conditions in the examples of the present invention are conventional methods. These examples are used only to illustrate the invention and the scope of protection of the invention is not limited by these examples. The technical means of the invention are conventional solutions in the art unless expressly stated, and all reagents or materials are commercially available unless otherwise stated. It shall be.

(Example 1)
(Use of Lactobacillus plantarum ZN-3 in the preparation of medicines for the prevention or treatment of porcine epidemic diarrhea virus infection)
(1) Cell culture VERO cells are subcultured in DMEM containing 10% FBS for about 48 hours.
Cell passage: At the time of passage, it is digested with 0.25% pancreatin and can be digested for about 10 minutes. The specific steps are as follows.
Remove the old culture from the adherent cell culture vessel with a dropper or pipette and rinse the remaining old medium with PBS solution;
Add 1-2 ml of digestive juice (0.25% pancreatin) to the flask and gently shake the culture flask to flush the digestive juice onto all cell surfaces;
Observe under an inverted microscope and if the cells are rounded with the protrusions removed, or if the cell gaps are widening, digestion is terminated immediately (suck the digestive juices and discard, a small amount containing serum. Add fresh culture to finish digestion);
The culture medium in the flask is sucked up with a pipette, and the digested cells are repeatedly pipetted to destroy the cell wall and disperse them to form a cell suspension;
Count, dispense into new culture flasks, add additional volume of fresh medium containing serum;
Cap the flask, tighten it appropriately so that CO2 gas can easily enter, and then return it slightly, and return the culture flask to the CO2 incubator.

(2) PEDV virus proliferation The digested Vero cells are inoculated into a T75 cell culture flask and placed in a 5% CO2 constant temperature incubator at 37 ° C. until a single layer of cells is formed;
0.1 ml of PEDV virus solution (CV777) stored at −80 ° C. was inoculated into a culture flask having a single layer of Vero cells, and incubated at 37 ° C. for 1 hour in a% CO2 constant temperature incubator;
A small amount of the virus incubation solution is aspirated with a pipette, 10 ml of DMEM cell maintenance solution containing 2% FBS is added, and different amounts of pancreatin having a final concentration of 0, 5, 10 ug / ml are added, and 37 ° C., 5%. Incubate in a CO2 constant temperature incubator for 48-72 hours;
Observe the cell condition daily and if more than 80% of the cells have lesions, collect viral cell proliferation fluid;
The virus cell proliferation solution is frozen and thawed 3 times repeatedly, centrifuged at 4000 rpm for 10 minutes, and the supernatant is collected and stored at −80 ° C. for later use.

RT-PCR detection of viral growth products: The collected viral fluid amplifies the M gene fragment of PEDV to verify the viral growth products. PEDV virus belongs to RNA virus and requires reverse transcription PCR.

The reverse transcription system (20ul) is as follows.
System volume (ul)
5 x AMV buffer 5
AMV enzyme 1
dNTP 2
Random primer 1.5
RNase inhibitor 0.5
RNA template 10
Reverse transcription reaction procedure: 42 ° C for 60 minutes, 94 ° C for 5 minutes, 4 ° C for 10 minutes.

The gene amplification reaction system (25ul) is as follows.
System volume P1 1
P2 1
10 x Buffer 5
dNTP 1
Taq enzyme 0.5
ddH ₂ 0 11.5
Reverse transcription template 5
Reaction procedure: 94 ° C. 3 minutes; 94 ° C. 45 seconds, 53 ° C. 45 seconds, 72 ° C. 1 minute 30 seconds for 30 cycles; 72 ° C. 10 minutes; 16 ° C. 10 minutes.

Measurement of PDEV virus titers: TCID50 is used to measure virus titers (TCID50 / 0.1 ml) in a grown PEDV virus solution. The specific steps are as follows:
Digested Vero cells are inoculated into 96-well plates and placed in a 5% CO2 constant temperature incubator at 37 ° C. until a single layer of cells is formed;
PEDV virus solution is continuously diluted 10-fold to ^10-6 using DMEM in a 2 ml sterile centrifuge tube;
The diluted virus solution was inoculated into 96-well plates on which a single layer of Vero cells was formed, each dilution step was inoculated into a total of 8 holes in tandem, and each hole was inoculated with 100 ul of virus, 7th and 8th. The column is inoculated with virus-free DMEM medium as a control and incubated at 37 ° C. in a 5% CO2 constant temperature incubator for 1 hour;
A small amount of virus incubation solution and DMEM culture medium is aspirated with a pipette, 100 ul of DMEM cell maintenance solution containing 2% FBS is added, and the cells are cultured in a constant temperature incubator at 37 ° C. and 5% CO2 for 120 hours;
The status of cytopathic effect (CPE) was observed and recorded daily and the TCID50 of the PEDV virus solution was calculated according to the Red-Muerch method.

From the measurement results of TCID50, the concentration of pancreatin has a certain effect on the proliferation of PEDV in VERO cells, and the maximum value of virus titer is ^{10-4.43 /} 0.1 ml under the condition of pancreatin concentration of 5 ug / ml. It turns out that.

(3) Preparation of bacterial solution of probiotics (Lactobacillus plantarum ZN-3 accession number CCTCC M 2017286) and its metabolites Lactobacillus plantarum ZN-3 activated on a flat plate was inoculated into 150 ml of MRS liquid medium. , Incubated at 37 ° C. for 24 hours, centrifuged at 2400 rpm for 10 minutes, the supernatant was taken, the pH value of the supernatant was detected and stored at 4 ° C. for later use. The cell pellet after centrifugation and resuspended in sterile PBS, the concentration of the bacterial suspension was adjusted to 10 9 ^CFU / ml, and stored at 4 ° C. for later use.

(4) Cytotoxicity test of probiotics and metabolites A 96-well plate is inoculated with a cell suspension and cultured until a single layer of cells is formed;
Take the bacterial suspension and metabolites of lactobacillus plantarum ZN-3, dilute the bacterial suspension on a 10-fold gradient, 10 ⁷ CFU / ml, 10 ⁸ CFU / ml, 10 ⁹ CFU / ml. Select the gradient, take the metabolites from the fermentation supernatant stock solution and adjust the fermentation supernatant to pH 7.0 with sterile NaOH;
Create columns of different treatments, add 100 ul of different treatments to each hole and _{incubate in a 5% CO 2} incubator at 37 ° C. for 90 minutes;
After incubation, divide into two treatment methods,
One is to discard the incubation solution and then add the cell maintenance solution directly to continue the culture;
The other method is to discard the incubation solution, wash it once with PBS, add a cell maintenance solution, continue the culture, and set a blank cell control and a virus target;
The state of cell degeneration is observed daily, and when the CPE of the virus control group cells reaches 80% or more, the cell activity is measured by the MTT method.
Cytotoxicity = (control group OD ₄₉₀ -treatment group OD ₄₉₀ ) / control group OD ₄₉₀ x 100%

As a result of indirectly measuring the toxic effect of probiotics ZN-3 or its metabolite on VERO cells by the MTT method, bacteria having three gradients of ^{10 7} CFU / ml, 10 ⁸ CFU / ml, and 10 ^{9 CFU / ml.} The suspension has no toxic effect on VERO cells, and after incubating for 90 minutes, discarding the incubation solution and adding the maintenance solution directly without washing with PBS results in some (12% -14%) cell growth. It can be promoted, and after incubating for 90 minutes, the incubation solution is discarded and washed with PBS, and then ^{the three gradients of 10 7} CFU / ml, 10 ⁸ CFU / ml, and 10 ⁹ CFU / ml also have a certain degree of growth promoting effect ( There are 2% -4%); for the treatment group in which the pH of the fermentation supernatant is not adjusted, the cell activity is extremely reduced (about 93%) regardless of the presence or absence of washing, and the pH is lowered to 7. In the case of the fermentation supernatant adjusted to 0, the activity of the unwashed group was not affected, indicating that the activity of the washed group increased by 4.37%. Based on this, in subsequent studies, it was measured viral inhibitory effects of probiotics and its metabolites 10 8 ^CFU / ml and pH of the fermentation supernatant was adjusted to 7.0 by selecting respectively.

(5) Indirect detection of probiotic inhibition rate against PEDV virus by MTT method: The test is divided into 8 groups.
Groups 1-2 are pretreated with probiotics or their metabolites and then infected with the virus: cells are first infused with probiotics or metabolites, co-cultured for 90 minutes, washed and then 100 TCID50 on the cells. /0.1 ml PEDV was infected, placed in an incubator and adsorbed for 90 minutes, washed, and then a cell maintenance solution was added to continue culturing.

Groups 3-4 simultaneously inoculate cells with probiotics or metabolites thereof: probiotics or metabolites thereof are mixed with an equal amount of 100 TCID50 / 0.1 ml PEDV into a monolayered 96-well culture plate. In addition, the cells were co-cultured for 90 minutes, the mixed solution was discarded, the cells were washed with PBS, and the cell maintenance solution was added to continue the culture.

Groups 5-6 each first add virus-infected cells and then infect probiotics or their metabolites: cells are infected with 100 TCIC 50 / 0.1 ml PEDV, adsorbed in incubator for 90 minutes and then washed. Then, probiotics or a metabolite thereof was added, and the cells were placed in a constant temperature incubator and cultured for 90 minutes, washed with PBS, and a cell maintenance solution was added to continue the culture.

Group 7 is a virus control: VERO cells were infected with 100 TCID50 / 0.1 ml PEDV, placed in a constant temperature incubator, adsorbed for 90 minutes, washed with PBS, and cell maintenance solution was added to continue culturing.

Group 8 was a normal cell control.

Virus inhibition rate = (probiotic treatment group OD ₄₉₀ -virus control group OD ₄₉₀ ) / (cell control group OD ₄₉₀ -virus control group OD ₄₉₀ ) × 100%

(Results of probiotics and their metabolite inhibitory effects on PEDV virus at the cellular level.)
The inhibitory effects of probiotics and their metabolites on PEDV virus were measured by different treatment methods, and from the experimental results, in the case of ZN-3 bacterial suspension, all three treatment methods have inhibitory effects on the virus. It can be seen that the inhibition rates were 28.30%, 20.75% and 22.64%, respectively. In the case of fermentation supernatant, the two methods of adding the fermentation supernatant first and then adding the PEDV diluted solution, and adding the fermentation broth and the PEDV diluted solution at the same time have an inhibitory effect on the virus, and the inhibition rate is high. It was 32.08% and 13.21%, respectively; those in which the PEDV diluted solution was first added and then the fermentation supernatant was added had no inhibitory effect.

(Additional note)
(Appendix 1)
Use of Lactobacillus plantarum ZN-3 deposited under accession number CCTCC M 2017286 in the preparation of medicines for the prevention or treatment of porcine epidemic diarrhea virus infection.

(Appendix 2)
Use of Lactobacillus plantarum ZN-3 in the preparation of porcine epidemic diarrhea virus inhibitors.

(Appendix 3)
The use according to Appendix 1, wherein the Lactobacillus plantarum ZN-3 is a fermentation broth or fermentation supernatant of Lactobacillus plantarum ZN-3.

Claims (2)

Use of Lactobacillus plantarum ZN-3 deposited under accession number CCTCC M 2017286 in the preparation of a porcine epidemic diarrhea virus inhibitor to inhibit the invasion and proliferation of porcine epidemic diarrhea virus into cells. And,
The Lactobacillus plantarum ZN-3 is a fermentation broth or fermentation supernatant of Lactobacillus plantarum ZN-3.
The pH of the fermentation supernatant is 7.0.
Use . The use according to claim 1, wherein the porcine epidemic diarrhea virus inhibitor is a drug for preventing or treating a porcine epidemic diarrhea virus infection.

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