Bacillus velezensis derived from sheep nasal cavity and application thereof

A technology based on Bacillus sp. and source Belles, which is applied in the fields of microbiology, molecular biology, and biological control. Effect

Active Publication Date: 2022-01-18
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have established a mucosal explant model of human cytomegalovirus (HCMV) infection to explore the characteristics of HCMV in the early infection stage, and this model is useful for evaluating new interventions for HCMV horizontal transmission, but the culture time of these studies shorter, less reliable
The influence of pathogenic microorganisms or drugs on explants cannot be observed in vitro for a long time

Method used

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  • Bacillus velezensis derived from sheep nasal cavity and application thereof
  • Bacillus velezensis derived from sheep nasal cavity and application thereof
  • Bacillus velezensis derived from sheep nasal cavity and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1: Isolation and identification of Bacillus velei NSV2 bacterial strains

[0039] 1.1 Sample collection

[0040] Select healthy flocks from a vineyard in Nanjing City, Jiangsu Province, insert a sterile cotton swab into the nasal cavity for more than 10 cm, and rotate it repeatedly to collect nasal mucus. After the collection is completed, quickly put it into a sterile sampling tube, mark it, and bring it back to the laboratory at 4°C.

[0041] Refer to GB / T 1.1-2009 to isolate Bacillus in pig nasal cavity swabs. The specific steps are as follows: 1) Aseptically open the centrifuge tubes containing nasal swabs in the ultra-clean bench, add 500 μL of PBS solution to each tube, and then shake and centrifuge repeatedly. tube three times, after oscillating for 15s each time, put it in a 4°C refrigerator and let it stand for 15 minutes; 2) Open the centrifuge tube, use tweezers to squeeze the leachate remaining from the swab into the centrifuge tube, and heat it ...

Embodiment 2

[0054] Embodiment 2: Bacillus velei NSV2 antiviral effect

[0055] 2.1 Cytotoxicity evaluation of Bacillus Velez NSV2 and its secreted products

[0056] Inoculate 100 μL of 10 8 / mL of fresh Bacillus velei NSV2 bacteria liquid into liquid 5mL LB medium, cultured at 37°C 220rpm for 12-16h. Centrifuge at 300g for 10min to collect the supernatant and bacterial pellet respectively. Bacteria were washed three times with PBS, counted and diluted to 10 with culture medium 4 , 10 5 , 10 6 , 10 7 and 10 8,spare. The supernatant was centrifuged again at high speed (10000g, 10min), and the supernatant was collected again and filtered with a 0.22 μM filter membrane, and diluted to 100%, 50%, 25%, 5% and 1% in proportion, and set aside. The above-mentioned diluted test substances were added respectively, and cultured on the VERO cells in a 96-well plate covering 90% of the monolayer, with 5 replicates for each concentration, and blank wells and control wells were set at the same ti...

Embodiment 3

[0074] Embodiment 3: Belaisia ​​bacillus NSV2 suppresses the action characteristic of PRV infection

[0075] For the experimental treatment method, refer to Example 2, the pretreatment group in step 2.2. Protein and RNA samples were collected at 0h, 2h, 4h, 6h, 12h and 24h after pretreatment of PK15 cells with Bacillus beilesiform NSV2. For detection steps, refer to 2.2 of Example 2. After Western blot and fluorescence quantitative detection, PRV began to decrease significantly at 6 hours after pretreatment with Bacillus beilesiformis NSV2 (P Figure 6 A, B and C).

[0076] further as Figure 6 As shown in D, after the cells were pretreated with Bacillus beilesiformis NSV2 for 12 hours, the bacterial liquid was aspirated, and the empty medium was added to shake gently, discarded, and the residual beileizia bacillus NSV2 was washed away three times. After incubating the cells and virus at 4°C for 1.5 h, they were washed away directly, and the RNA samples were collected. Acco...

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Abstract

The invention discloses Bacillus velezensis derived from sheep nasal cavity and application thereof. The Bacillus velezensis provided by the invention belongs to Bacillus and is named as NSV2, and the preservation number is CGMCC No.20345. The strain not only can inhibit the growth of pathogenic bacteria, but also can inhibit the infection of pseudorabies virus (PRV), and the inhibition rate reaches 50% or above; metabolite surfactin of the Bacillus velezensis NSV2 is separated and identified by researching the antiviral mechanism of the Bacillus velezensis NSV2, and the surfactin secreted by the Bacillus velezensis NSV2 can inhibit PRV infection and also can effectively inhibit PEDV infection; finally, by constructing a sheep Bacillus velezensis-sheep nasal mucosa explant immunostimulation model, it is found that the strain can induce expression of innate immune factors, and the innate immunocompetence of the nasal cavity is improved. In conclusion, the Bacillus velezensis strain NSV2 is expected to be developed into a respiratory tract probiotic preparation for enhancing congenital immunity of nasal mucosa of sheep and resisting invasion of pathogenic microorganisms.

Description

technical field [0001] The invention relates to the fields of microbiology and molecular biology, belongs to the technical field of biological prevention and control, and in particular relates to the application of a strain of Bacillus velesi derived from sheep nasal cavity in resisting virus infection and improving the innate immunity of sheep nasal cavity mucosa. Background technique [0002] 1. Current status of research on nasal infectious diseases in sheep [0003] The respiratory tract of sheep is an important infection channel for diseases. At present, the main viruses that can be transmitted through the respiratory tract in sheep include pseudorabies virus (Pseudorabies virus, PRV), foot-and-mouth disease virus (foot-and-mouth disease virus, FMDV), and Peste des petits ruminants. Peste des Petits Ruminants virus (PPR), Rabies virus (RV), Sheep pulmonary adenomatosis virus (SPAS), caprine parainfluenza virus type 3 (CPIV3), goat Poxvirus (Goatcapripoxvirus, GPV), cap...

Claims

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Application Information

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IPC IPC(8): C12N1/20A61K35/742A61P31/04A61P31/14C12R1/07
CPCC12N1/20A61K35/742A61P31/04A61P31/14Y02A50/30
Inventor 杨倩郑健李昱辰
Owner NANJING AGRICULTURAL UNIVERSITY
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