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Fusant with bacteriostatic activity and preparation method thereof

A technology of antibacterial activity and protoplast fusion, which is applied in the biological field to achieve the effect of ensuring stability and genetic shape stability

Inactive Publication Date: 2017-10-17
GUANGDONG PHARMA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the research on the protoplast fusion of Serratia marcescens and Rhodotorula gluei has not been reported, therefore, it has very important research significance

Method used

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  • Fusant with bacteriostatic activity and preparation method thereof
  • Fusant with bacteriostatic activity and preparation method thereof
  • Fusant with bacteriostatic activity and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1, the culture medium that the present invention relates to

[0048] (1) Preparation of LB medium

[0049] Take 10g of peptone, 5g of yeast extract, and 10g of NaCl respectively, mix the above-mentioned peptone, yeast extract, and NaCl evenly, and dilute to 1L with distilled water.

[0050] (2) Preparation of PDA medium

[0051] Take 200g of potatoes, peel them, cut them into pieces, add water and boil for 30 minutes to obtain boiled potatoes, filter the boiled potatoes with double-layer gauze, then add 20g of glucose, stir to dissolve, add water to 1L, and get ready.

[0052] (3) Preparation of regeneration medium

[0053] NaCl was added to LB medium and PDA medium respectively until the final concentration of NaCl was 0.6mol / L to prepare the corresponding regeneration medium.

[0054] (4) Preparation of ZR medium

[0055] Take 0.1g of sucrose, 1.23g of beef extract powder, 0.3g of NaCl, MgSO 4 0.12g, CaCl 2 0.10g, mix the above components evenly and...

Embodiment 2

[0057] Embodiment 2, the main reagent involved in the present invention

[0058] (1) Preparation of PBS buffer

[0059] Take 8.34 g of potassium dihydrogen phosphate and 0.87 g of dipotassium hydrogen phosphate trihydrate, mix the above-mentioned potassium dihydrogen phosphate and dipotassium hydrogen phosphate trihydrate evenly, and dilute to 1 L with distilled water to obtain PBS buffer solution. Buffer pH = 5.8.

[0060] (2) Preparation of Protoplast Stable Solution

[0061] Add NaCl to the above PBS buffer solution until the final concentration of NaCl is 0.6 mol / L.

[0062] (3) Preparation of protoplast fusion agent

[0063] The ratio of the protoplast fusion agent is: PEG6000 with a mass fraction of 25%, 0.05mol / L CaCl 2 , 0.05mol / L glycine.

Embodiment 3

[0064] Embodiment 3, the fusion son with antibacterial activity

[0065] The preparation method of the fusion son with antibacterial activity is:

[0066] S1: Preparation and inactivation of protoplasts

[0067] (1) Preparation of Serratia marcescens protoplasts

[0068] ① Activate the glycerol strain of Serratia marcescens overnight, insert 5% inoculum into 30mL LB medium, culture at 30°C, 200r / min for 4h, and prepare bacterial solution A;

[0069] ②Take 8mL of the bacterial solution A prepared in step ①, centrifuge at 6000r / min for 8min, wash 3 times with PBS buffer and resuspend in 6mL of PBS buffer to obtain suspension B;

[0070] ⑧Take 50 μL of the bacterial suspension B prepared in step ②, add 6 μL of lysozyme with a mass fraction of 0.2% prepared in PBS buffer, bathe in 35°C water for 55 minutes, centrifuge at 4000r / min for 10 minutes, discard the supernatant to obtain the bacterial solution C;

[0071] ④ Wash the bacterial liquid C obtained in step ⑧ twice with PBS...

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Abstract

The invention belongs to the biotechnical field, and particularly relates to a fusant with bacteriostatic activity and a preparation method thereof. The fusant with bacteriostatic activity is named as Rhodotorula mucilaginosa SGM, preserved at Guangdong province microbiological culture collection center (address: fifth floor, building 59, No.100 courtyard, martyr middle road, Guangzhou city) on January 16th, 2017, with the preservation number of GDMCCNo:60141. The preparation method of the fusant with bacteriostatic activity comprises the following steps: preparing and deactivating protoplast, fusing and regenerating the protoplast, screening fusants, and screening the fusant with bacteriostatic activity. The fusant with bacteriostatic activity has potential application values in the fields of biopesticide, antibacterial agent screening, genetics and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a fusion son with antibacterial activity and a preparation method thereof. Background technique [0002] Commonly used bacteriostatic agents are some antibiotics, which can reversibly inhibit the reproduction of bacteria, but do not directly kill bacteria. In recent years, as antibiotics have been abused by people, some bacteria have developed drug resistance, thereby weakening or disappearing the antibacterial effect of antibiotics. Therefore, the development of biological antibacterial agents with antibacterial activity to replace antibiotics has become a worldwide research. hotspot. [0003] Highly N-methylated linear peptides produced by an atypical sponge-derived Acremonium sp [J Nat Prod, 2006, 69(9): 83-92] discloses a peptide compound isolated and purified from the metabolites of sponge Acremonium , the compound has a strong antibacterial effect, and the antibac...

Claims

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Application Information

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IPC IPC(8): C12N1/16C12N15/08C12R1/645
CPCC12N15/02C12N1/145C12R2001/645
Inventor 周林周子雄朱爽吴继宏
Owner GUANGDONG PHARMA UNIV
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