A kind of Lactobacillus plantarum expressing antimicrobial peptide gene

A Lactobacillus plantarum, gene technology, applied in the field of genetic engineering, can solve problems such as the reduction of digestive tract enzymes and the inability to achieve intestinal targeted delivery, achieve strong cellular immune responses, promote healthy intestinal development, and help regulate Effect

Active Publication Date: 2021-06-25
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved by the present invention is to overcome the defects in the prior art that oral CRAMP is easily reduced by digestive tract enzymes, unable to achieve intestinal targeted delivery of CRAMP, and maximize its local immune regulation effect, and to provide a recombinant protein that secretes and expresses CRAMP Lactobacillus plantarum and its application

Method used

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  • A kind of Lactobacillus plantarum expressing antimicrobial peptide gene
  • A kind of Lactobacillus plantarum expressing antimicrobial peptide gene
  • A kind of Lactobacillus plantarum expressing antimicrobial peptide gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1 Construction of recombinant plasmid pMG36e-CRAMP

[0060] (1) Codon preference optimization and synthesis of the gene sequence: According to the sequence of the target gene CRAMP gene and the characteristics of the expression vector pMG36e, the 108bp codon-optimized sequence of the CRAMP gene was sent to the company for synthesis by artificial synthesis. Xbal-CRAMP-F is an upstream primer containing the restriction site Xbal (TCTAGA) fused with pMG36e and the initial sequence of the 5' end of the signal peptide CRAMP, and CRAMP-Sph1-R is an upstream primer with a restriction site Sph1 (GCATGC ) reverse primer for CRAMP gene. The optimized and synthesized CRAMP sequence is shown in SEQ ID NO: 1; the optimized and synthesized Xbal-CRAMP-F and CRAMP-Sph1-R primer sequences are shown in SEQ ID NO: 5-6, respectively.

[0061] (2) PCR amplification of CRAMP gene fragments: using the optimized synthesized CRAMP gene as a template, add high-fidelity DNA polymerase KO...

Embodiment 2

[0063] Example 2 Construction of recombinant plasmid pNZ8148-CRAMP

[0064](1) Codon preference optimization and synthesis of the gene sequence: According to the sequence of the target gene CRAMP gene and the characteristics of the expression vector pMG36e, the 108bp codon-optimized sequence of the CRAMP gene was sent to the company for synthesis by artificial synthesis. Sph1-CRAMP-F is an upstream primer containing the restriction site Sph1 (GCATGC) fused with pMG36e and the initial sequence of the 5' end of the signal peptide CRAMP, and CRAMP-Xbal-R is an upstream primer with restriction site Xbal (TCTAGA ) reverse primer for CRAMP gene. The optimized and synthesized CRAMP sequence is shown in SEQ ID NO: 1; the optimized and synthesized Sph1-CRAMP-F and CRAMP-Xbal-R primer sequences are shown in SEQ ID NO: 7-8, respectively.

[0065] (2) PCR amplification of CRAMP gene fragments: using the optimized synthesized CRAMP gene as a template, add high-fidelity DNA polymerase KOD-...

Embodiment 3

[0067] Example 3 Construction of recombinant plasmid pMG36e-Usp45-Linker-CRAMP

[0068] (1) Codon preference optimization and synthesis of the gene sequence: According to the sequence of the target gene CRAMP gene and the characteristics of the expression vector pMG36e, as well as the signal peptide sequence Usp45 added for the purpose of efficient secretion and expression, artificially synthesized The 243bp codon-optimized sequence of the Usp45-Linker-CRAMP gene was sent to the company for synthesis. Xbal-Usp45-Linker-CRAMP-F is an upstream primer containing the first sequence of the 5' end of the restriction site Xbal fused with pMG36e and the signal peptide Usp45-Linker-CRAMP, and Usp45-Linker-CRAMP-Sph1-R is Signal peptide Usp45-Linker-CRAMP gene reverse primer. At the same time, primers pNZ1 and pNZ2 for PCR detection and sequencing of recombinant plasmids were also designed, which were designed based on the 70-90bp upstream and downstream regions of the MCS of pMG36e em...

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Abstract

The invention provides a plant lactobacillus expressing an antimicrobial peptide gene, which belongs to the technical field of genetic engineering. The invention optimizes the nucleotide sequence of the CRAMP protein and can combine with the Usp45 signal peptide to promote the secretion and expression of the CRAMP gene, so that the lactic acid bacteria expression system becomes a food-grade expression system, which can be taken together with the bacterium. The recombinant Lactobacillus plantarum of the present invention can be used as a new type of oral vaccine product with good industrial prospects, which plays a positive role in reducing intestinal inflammation and has important practical significance in promoting the healthy development of the intestinal tract.

Description

technical field [0001] The invention relates to a plant lactobacillus expressing an antimicrobial peptide gene, which belongs to the technical field of genetic engineering. Background technique [0002] Antimicrobial peptides are major components of innate immunity and defenses in a variety of hosts including plants, invertebrates, and vertebrates, including humans. Cathelicidins are a major class of antimicrobial peptides characterized by a conserved anionic N-terminal precursor sequence called cathelin. The conservation of the cathelin sequence suggests that various members of the family evolved from duplication and modification of a common ancestral gene. CRAMP ( Cathelicidin-Related Anti-Microbial Peptide ) contains 34 amino acids (GLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPEQ) and has potent antibacterial activity against Gram-positive and Gram-negative bacteria, but no hemolytic activity against human erythrocytes. 1 mM CRAMP can directly lead to immediate permeabilization of ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/47C12N15/12C12N1/21C12N15/74A23L33/135A61K39/02A61P31/04A61P1/00C12R1/25
CPCC07K14/47C12N15/746A23L33/135A61K39/09A61P31/04A61P1/00A23V2002/00A61K2039/542A23V2400/169A23V2200/3204
Inventor 孙嘉潘礼龙张明陈卫张灏
Owner JIANGNAN UNIV
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