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A kind of bacillus amyloliquefaciens wh3 and its preparation method and application

A technology for dissolving amyloid spores and bacilli, applied in the field of immunology, can solve problems such as toxic and side effects, and achieve the effects of easy preparation, high biological safety, and good immune enhancement effect.

Inactive Publication Date: 2011-12-21
武汉光谷世傲生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is an immunomodulator among biological response modifiers, and its non-specific immunostimulatory effect is the basic condition necessary for exerting its immune adjuvant effect and making the body produce a protective immune response, but if this effect is excessive or too strong, it will inevitably Toxic and side effects

Method used

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  • A kind of bacillus amyloliquefaciens wh3 and its preparation method and application
  • A kind of bacillus amyloliquefaciens wh3 and its preparation method and application
  • A kind of bacillus amyloliquefaciens wh3 and its preparation method and application

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Effect test

Embodiment 1

[0062] A preparation method of bacillus amyloliquefaciens WH3 bacterial strain, the steps are:

[0063] 1. Screening and isolation of Bacillus amyloliquefaciens WH3:

[0064] Rapeseed seedlings at the three-leaf stage were randomly selected from the rapeseed field of Hubei Academy of Agricultural Sciences in Wuhan City, Hubei Province, China, and the roots were separated after washing. Rinse the outer surface of the root of rapeseed with tap water, rinse it with sterile water for 5 times, then immerse it in 70% (V / V) ethanol for 30 seconds, take it out, rinse it with sterile water for 5 times, and cut it with sterile scissors. The rice roots were cut into pieces about 1 mm in size, and then placed on PDA medium (containing 200 g of potato boiled liquid, 20 g of glucose, 20 g of agar, and distilled water was added to 1 L) and cultured at 28°C for 48 hours. The isolated bacteria were cultured with PD liquid medium (containing 200g potato boiled liquid, 20g glucose, and distille...

Embodiment 2

[0068] Isolation, purification and identification of Safenour

[0069] Inoculate 10 μl of preserved Bacillus amyloliquefaciens WH3 strain into 10 ml sterilized PD medium, and cultivate overnight at 28° C. with shaking at 200 rpm. Then inoculate 2.5ml of fresh culture into a 1L Erlenmeyer flask containing 250ml of fresh PD medium, culture at 28°C or 37°C, and shake at 200rpm for 48h. Generally, 4 Erlenmeyer flasks are used to grow 1 L cultures at a time. Then the supernatant was collected by centrifugation at 8000g for 10min, and the antibacterial active substance was extracted by "two-step extraction method". After alcohol extraction, the n-butanol phase was collected by centrifugation at 8000 g for 20 min. The obtained n-butanol ester phase was evaporated to dryness at 65° C. with a vacuum rotary evaporator, and then dissolved with 10 ml of distilled water. After the solution obtained after dissolution was centrifuged to remove the precipitate, the supernatant was further ...

Embodiment 3

[0071] A kind of application of bacillus amyloliquefaciens WH3 bacterial strain in preparation vaccine and immune adjuvant, its steps are:

[0072] 1. Determination of immune effect of Safenour adjuvant injection:

[0073] The Kunming (KM) mice were randomly divided into four groups: Safenour adjuvant + antigen group, antigen group, Freund's adjuvant (FA) + antigen group, PBS control group, 6 mice in each group, and the dose of Safenour adjuvant was 100 μg / mouse Each time, the antigens were GST and OVA protein respectively, and the amount of antigen was 10 μg / animal / time. Two immunization methods were adopted: multi-point subcutaneous injection on the back and intraperitoneal injection, the amount of immunization was 0.2 mL / time, the immunization time was 0, 1, and 2 weeks, and blood was collected one week after the last immunization.

[0074] One week after the last immunization, the tail vein blood of the mice was collected, the serum was separated, and the serum anti-GST a...

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Abstract

The invention discloses a Bacillus amyloliquefaciens WH3 strain, and a preparation method and application thereof. The preparation method comprises the following steps: 1, separation and identification of bacteria: separating bacteria resistant to rape sclerotinia rot from rape seedlings, and carrying out 16SrDNA and morphological identification to determine that the WH3 strain obtained by separation is Bacillus amyloliquefaciens; 2, separation, purification and identification of an antifungal active substance Safenour: fermenting the WH3 strain, extracting the antifungal active substance, separating and purifying through a sephadex column, and carrying out MALDI-TOF (matrix-assisted laser desorption / ionization-time of flight ) mass spectrometry on the active antifungal substance to inferthat the substance is a ring type polypeptide; and 3, application of the strain in the preparation of vaccines and immunological adjuvants. After the Safenour used as the adjuvant is mixed with a protein antigen and mice are respectively immunized through oral administration and injection of the mixture, effective body fluid and cellular immune response can be activated, and a high-titer specificantibody can be detected in the blood serum. The Safenour has low production cost and high stability, does not need to be emulsified when mixed with the antigen, and has a better immunoenhancement effect in comparison with Freund adjuvants and cholera toxin B subunits.

Description

technical field [0001] The present invention belongs to the field of immunology, and more specifically relates to a Bacillus amyloliquefaciens WH3 strain, and also relates to a preparation method of a Bacillus amyloliquefaciens WH3 strain, and also relates to a method for preparing an immunization of a Bacillus amyloliquefaciens WH3 strain The application in adjuvants is also applicable to cyclic peptides with similar structures produced by other microorganisms. Background technique [0002] Immune adjuvants refer to substances that can enhance the immunogenicity of antigenic substances, enhance the immune response ability of the body against antigens, or change the type of immune response, but have no antigenicity when applied simultaneously or in advance with antigens. [0003] The mechanism of action of adjuvants is extremely complex, and the current research believes that they mainly include: (1) Antigen storage, mineral colloids, latex adjuvants, etc. can form an antige...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12P21/04A61K39/39C12R1/07
Inventor 祁高富苏纪宇李菁菁赵秀云
Owner 武汉光谷世傲生物科技有限公司
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