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Small rhzomorph MccY and preparation method and application thereof

A microcin and preparation technology, applied in the field of microcin MccY and its preparation, can solve the problem of insufficient broad spectrum, and achieve the effect of simple operation, stable maintenance and significant bactericidal effect

Active Publication Date: 2021-06-01
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, for Salmonella Pullorum, Salmonella typhimurium, Salmonella kentucky, Salmonella infantis, Salmonella London, Shigella sonnei ( Shigella sonnei) and other common opportunistic pathogens, the use of MccJ25 alone is obviously not broad enough to inhibit bacteria

Method used

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  • Small rhzomorph MccY and preparation method and application thereof
  • Small rhzomorph MccY and preparation method and application thereof
  • Small rhzomorph MccY and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Mcc Y plasmid construction and expression

[0048] Synthesize the mccy gene fragment (Aiji Biosynthesis), the full length of the mccy gene is 4458bp, the sequence is shown in SEQ ID NO.6, including all 4 target genes mcyA (SEQ ID NO.2), mcyB (SEQ ID NO.3) , mcyC (SEQ ID NO.4), mcyD (SEQ ID NO.5) (by performing Blast retrieval on mcyA, mcyB, mcyC, and mcyD genes respectively, it was found that the amino acid sequences of mcjA, mcjB, mcjC, and mcjD genes of Escherichia coli MccJ25 The homology is respectively 51.9%, 54.8%, 52.3%, 70.4%. The homology of this sequence and MccJ25 is not high, but it possesses all the basic structural genes of type I microcin), using the restriction of NEB company Endonucleases (SalI, BamHI) were used to double-digest mccy gene fragments (enzyme digestion reaction system is shown in Table 2), and gel recovery and purification obtained mccy with open cohesive ends; at the same time, restriction endonuclease ( SalI, BamHI) carried ou...

Embodiment 2

[0054] Construction and expression of embodiment 2 empty plasmid engineering bacteria

[0055] The vector pET-28a (+) is introduced into BL21 (DE3) competent cells (the method is the same as in Example 1), and the BL21 (DE3) bacteria containing pET-28a (+) are obtained, and the pET-28a (+) containing BL21(DE3) bacteria were revived on LB plates with a kanamycin concentration of 30μg / mL, and a fresh bacterial lawn grew. Take a ring of bacteria and inoculate it into a 250ml Erlenmeyer flask containing 100mL of M9 basic salt medium, and add kanamycin Mycin, isopropyl-β-D-thiogalactopyranoside (IPTG) (the final concentration of kanamycin is 30 μg / mL, and the final concentration of IPTG is 0.1M), and the Erlenmeyer flask is placed in a shaker for cultivation (Culture conditions: 200r / min, 37°C, 14h); the expressed bacterial solution was collected, centrifuged at 5000r / min for 20min, and the centrifuged supernatant was collected and filtered with a 0.22μm filter membrane to obtain a...

Embodiment 3

[0056] Example 3 MccJ25 plasmid construction and expression

[0057] Synthesize the mccj25 gene fragment (Aiji Biosynthesis), the mccj25 gene full-length 4495bp, the sequence is shown in SEQ ID NO.9, use the restriction endonuclease (BgIII, HindIII) of NEB company to carry out double enzyme digestion to the mccj25 gene fragment ( The enzyme digestion reaction system is shown in Table 2), gel recovery and purification to obtain mccj25 with open cohesive ends; at the same time, the vector pET-28a (+) was double digested with restriction endonucleases (BgIII, HindIII) (enzyme The cleavage reaction system is shown in Table 2), and the linearized vector with open cohesive ends was obtained by the gel recovery and purification method of the DNA gel recovery kit of OMEGA; the pET-28a(+) linearized vector and the mccj25 was ligated by T4 DNA ligase (the ligation system is shown in Table 3, reaction conditions: 16°C for 3 h), the ligated product was transformed into Escherichia coli DH...

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Abstract

The invention discloses a small rhzomorph MccY and a preparation method and application thereof. The amino acid sequence of the small rhzomorph MccY is GGRGHIAEYFSGPITQVSFYG. Compared with MccJ25 which only has bactericidal activity on a small part of serotype salmonella such as enteritis and the like, the small rhzomorph MccY has a bacteriostatic / bactericidal effect on salmonella pullorum, salmonella typhimurium, salmonella kafenkii, salmonella infantis, salmonella London and shigella sonnei, and can kill other salmonella serotypes which cannot be killed by MccJ25; the small rhzomorph MccY has a remarkable bactericidal effect on salmonella typhimurium and salmonella pullorum which are common in livestock and poultry production, also has a bacteriostatic / bactericidal effect on shigella sonnei, overcomes the defect of narrow spectrum of small rhzomorph, has breakthrough significance, and has the potential of being used as an antibiotic substitute.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a microcin MccY and its preparation method and application. Background technique [0002] Microcin (Mcc) is a small molecule bacteriostatic peptide secreted by intestinal bacteria, with a molecular weight of about 10kDa, encoded by a bacterial plasmid or chromosome-related gene cluster, and some genes in this gene cluster are relatively conserved, with at least 4 Genes involved in encoding microcins: precursor genes, post-transcriptional modifier genes, secreted genes, autoimmune genes. Its structural gene mainly encodes the microcin precursor, which is the main structural component of microcin; the modification gene can process the microcin precursor to make it easier to be recognized by the corresponding receptors, and at the same time better play the microcin biology Activity; secretory genes encode secretion-related proteins; immune genes can protect bacteria from se...

Claims

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Application Information

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IPC IPC(8): C07K14/195C12N15/31C12N15/70C12N1/21A61K38/16A61P31/04A23K20/147C12R1/19
CPCC07K14/195C12N15/70A61K38/164A61P31/04A23K20/147A61K38/00A61K2300/00C12P21/02Y02A50/30C07K14/255C07K14/245C07K14/24
Inventor 曹伟胜李昱冯赛祥韩雨
Owner SOUTH CHINA AGRI UNIV
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