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Antibiotic peptide gene converted viscons bacillus engineering bacterium and its preparing method and use

A technology of bacillus and gene transformation, applied in the field of genetic engineering, can solve the problems of low titer of antimicrobial peptides, slow growth of yeast, application restrictions, etc., and achieve the effects of reducing production costs, promoting growth, and increasing body weight

Inactive Publication Date: 2007-04-11
WEIGUAN BIOLOGICAL SCI & TECH GUANGZHOU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the slow growth of yeast, the long period (72 hours), and the low titer of antimicrobial peptides (5000 units / mL), the application is limited, mainly used for livestock and poultry feed additives

Method used

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  • Antibiotic peptide gene converted viscons bacillus engineering bacterium and its preparing method and use
  • Antibiotic peptide gene converted viscons bacillus engineering bacterium and its preparing method and use
  • Antibiotic peptide gene converted viscons bacillus engineering bacterium and its preparing method and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] The preparation method of embodiment 1 antimicrobial peptide ADC gene transformation Bacillus marcescens

[0045] (1) Design and synthesis of antimicrobial peptide ADC gene

[0046] According to the bactericidal activity structure of natural antimicrobial peptides, design and synthesize antimicrobial peptide ADC genes, and synthesize F 1 (82bp) and F 2 (78bp) two fragments. There is 20 bp complementarity between the two fragments, and they serve as templates for PCR amplification.

[0047] f 1 : 5’GGATCCGT ATG AAA TGG AAG TTG TTC AAA AAG ATT GAA AAGGTT GGT CAA AGA GTT AGA GAC GCT GTC ATC TCT GCT GGT CC-3’

[0048] f 2 : 3’CGA CAG TAG AGA CGA CCA GGA CGA CAA CGG TGA CAA CGAGTT CGA TGA CGG AAC CGA TTC ATTATFCTTAAGCAGCTG-5’

[0049] Reaction conditions:

[0050] f 1 , F 2 Fragment (25mmol / L) 1.0μl each

[0051] dNTP (pmol / L) 2.5μl

[0052] 10×PCR buffer 5.0μl

[0053] Taq enzyme (5 units) 1.0μl

[0054] Double distilled water 39.5μl

[0055] Total 50μl

[00...

Embodiment 2

[0082] Example 2 Antimicrobial peptide gene transformation Bacillus marcescens engineering strain culture experiment

[0083] The present invention has carried out the culture medium optimization test on the engineering strain of Bacillus marcescens transformed with the antimicrobial peptide gene, see Table 1 for details.

[0084] Table 1. Orthogonal experiments for medium composition optimization

[0085]

[0086] 8

9

3

3

1.5

2.0

0.01

0.02

0.100

0.050

14

12

[0087] The best formula is peptone 3.0%, starch 1.5%, MgSO 4 ·7H 2 O 0.03%, K 2 HPO 4 ·3H 2 O O.1%, the fermentation bactericidal titer reaches the level of 10000 units / mL.

[0088] Optimization of fermentation conditions: under the formulation conditions of the above-mentioned optimized medium, find the best fermentation conditions, see Table 2 for details.

[0089] Table 2. Orthogonal experiments for optimization of ...

Embodiment 3

[0092] Example 3 Application of Antimicrobial Peptide ADC Gene Transformation of Bacillus marcescens

[0093] The product of Bacillus viscosus transfected with antimicrobial peptide ADC gene is added to the drinking water of chickens, and they can drink freely, which can prevent gastrointestinal diseases such as pullorum in chickens. Adding it to piglet feed can prevent bacterial diseases of piglets. The optimum doses to be used in chick drinking water are listed in Table 3. The effect of feeding chicks is shown in Table 4.

[0094]Table 3. The dosage of Bacillus marcescens fed to chickens with antimicrobial peptide ADC gene

[0095]

[0096] project

Antimicrobial peptide region

(7.5mL / feather)

Antimicrobial peptide region

(10.0mL / feather)

Chlortetracycline

(500mg / kg)

control

Number of feathers tested (feathers)

Starting weight (g / feather)

1 week (g / feather)

2 weeks (g / feather)

3 weeks (g / feather) ...

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Abstract

The present invention discloses antibiotic peptide gene converted engineering viscous bacillus and its preparation process and application. Insect antibiotic peptide has wide spectrum antibiotic effect. The present invention designs new antibiotic peptide gene, synthesizes its DNA sequence, converts to expressing viscous bacillus to form discloses antibiotic peptide gene converted engineering viscous bacillus based on the characteristic of amino acid sequence in natural antibiotic peptide. At the same time, the formula and the fermentation condition of the culture medium are optimized, and through fermentation in fermenting tank, the high density culture and high efficiency expression effects are reached. The fermented liquid is separated and purified to form the antibiotic peptide preparation. The antibiotic peptide preparation is used as veterinary medicine and externally applied medicine for human to replace antibiotic.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to an antimicrobial peptide gene transformation bacillus marcescens engineering bacteria and its preparation method and application. Background technique [0002] Natural antimicrobial peptides (Cecropin), which have been found to have a variety of A, B and D, have a broad-spectrum bactericidal effect. The application of antimicrobial peptides in medicine has achieved preliminary results. If bred into pupae powder, it can be used as the raw material of "Shenganning" medicine to treat nephritis and hepatitis with good effect. Silkworm chrysalis immune hemolymph is made into freeze-dried powder, and it is formulated into tussah capsules. As a second-class western medicine for the treatment of hepatitis B, it has entered the second phase of clinical observation. Antimicrobial peptides are being used as feed additives instead of antibiotics to feed chickens, piglets and prawns to pr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/31C12N15/75C12P21/02A61K38/16C12R1/07
Inventor 黄自然戴宏彬游锡元
Owner WEIGUAN BIOLOGICAL SCI & TECH GUANGZHOU