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Gene expression cassette for coding secretory Mersicidin and preparation method thereof

A gene expression cassette and secretory technology, which is applied to the gene expression cassette encoding secreted Mersacidin and its preparation field, can solve the problems of low expression level and the like, and achieve the effects of short fermentation period, easy industrialization and good tolerance.

Pending Publication Date: 2022-05-10
CHONGQING ACAD OF ANIMAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Bacillus amyloliquefaciens FZB42 has been successfully used as a host to express Mersacidin exogenously, but the expression level is low

Method used

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  • Gene expression cassette for coding secretory Mersicidin and preparation method thereof
  • Gene expression cassette for coding secretory Mersicidin and preparation method thereof
  • Gene expression cassette for coding secretory Mersicidin and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1. Construction of recombinant vectors

[0052] 1) The vector pP43NMK (BioVector NTCC Inc.) was double digested with HindIII and PstI.

[0053] The digestion reaction system is as follows: 10 μL 10×FastDigest buffer, 10 μL carrier (100 ng / μL), 5 μL HindIII and 5 μL PstI, ddH 2 O 70 μL was mixed and placed in a water bath at 37°C for 60 min, and the linearized vector was recovered from the digested product.

[0054] 2) Using the Bacillus HIL Y-8554728 genome as a template, use G1 / G2, G3 / G4 and G5 / G6 as primers to amplify mrsK2R2FGE, mrsAR1D and mrsMT gene fragments, respectively. Primers G1 and G6 have homologous sequences with the vector, G2 and G3, G4 and G5 also have homologous sequences (such as figure 1 As shown, regions of the same color are homologous sequences).

[0055] The amplification system is 50 μL: 2.5U / μL high-fidelity enzyme, 1 μL; 2× reaction buffer, 25 μL; primers, 1 μL each; template, 1 μL; ddH 2 O, 21 μL; amplification conditions: denatur...

Embodiment 2

[0060] Example 2. Transformation of recombinant vectors

[0061] The vector pP43NMK-mersacidin was transformed into Bacillus subtilis SCK168 by electroporation, and the electroporation conditions were as follows: C=50 μF; PC=200 ohm; V=1.0 kV. The screened positive recombinants were named as MRS08 and MRS24.

[0062] The host bacteria include but not limited to Bacillus subtilis, other genetically engineered bacteria Bacillus licheniformis and Bacillus pumilus, which can also realize vector transformation and expression.

Embodiment 3

[0063] Example 3. The detection of fermentation supernatant to the antibacterial activity of Staphylococcus aureus

[0064] The MRS08 and MRS24 bacterial strains obtained by screening were cultured under the condition of 37°C and 200rpm / min in the culture medium (10g / L of soybean meal powder, 10g / L of yeast powder, 10g / L of peptone, 10g / L of glucose, and 5g / L of sodium chloride). After culturing for 16-22 hours, the antibacterial activity of the fermentation supernatant against Clostridium perfringens was measured at different time periods.

[0065] The method for measuring the antibacterial activity is as follows:

[0066] 1) Bacterial recovery: streak Clostridium perfringens ATCC13124 plate, culture at 37°C for 20 hours; pick a single colony in the corresponding liquid medium, place at 37°C, 200rpm / min and cultivate for about 12-16h. Dilute the bacterial solution to OD with normal saline 600 The absorbance value is 0.1 (about 10 8 CFU / mL), spare;

[0067] 2) Take 8000g o...

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Abstract

The invention belongs to the field of gene engineering, and particularly relates to a gene expression cassette for coding secretory Mersicidin and a preparation method of the gene expression cassette. The invention discloses a gene expression cassette for encoding secreting type Mersicidin. The nucleotide sequence of the gene expression cassette is shown as SEQ ID NO. 1. The method comprises the following steps: amplifying mrsK2R2FGE, mrsAR1D and mrsMT of a gene mrsK2R2FGEAR1DMT of a Merscidin gene cluster by utilizing a high-fidelity enzyme, connecting three sections of genes of the Merscidin gene cluster by utilizing a Gibson cloning technology to construct a Merscidin expression box, placing the expression box at the downstream of a strong promoter, and introducing the expression box into a genetically engineered bacterium Bacillus subtilis SCK168 to realize efficient secretory expression of the Merscidin. The invention provides a cheap biological antibacterial agent and preservative for feed additives, veterinary drugs, food and medicines.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a gene expression box for encoding secreted Mersacidin and a preparation method thereof. [0002] technical background [0003] Bacteriocins are a kind of polypeptides or precursor polypeptides with antibacterial activity synthesized by bacteria through ribosomes during the metabolic process. They have ideal probiotic properties and have the functions of effectively inhibiting pathogens, regulating inflammation, promoting wound healing and mucosal defense. , and not easy to produce drug resistance characteristics. [0004] Mersacidin is produced by Bacillus HIL Y-8554728, is 20aa in length, and belongs to class Ib bacteriocins. Mersacidin pure product is white amorphous powder, stable at ambient temperature, stable at pH 5.0-7.0, insoluble in water; metal salts are easily soluble in water, and can enhance its bactericidal activity. Mersacidin inhibits the transglyc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/75C12N15/52C12N1/21C12P21/00C12R1/125
CPCC12N15/75C12N15/52C12N1/20C12P21/00
Inventor 黄金秀苏国旗杨飞云
Owner CHONGQING ACAD OF ANIMAL SCI
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