Lactococcus lactis for expressing mouse antibacterial peptide gene

A technology of Lactococcus lactis and amino acids, which is applied in the field of genetic engineering, can solve the problems of decreased enzymes in the digestive tract and the inability to achieve intestinal targeted delivery, and achieve the effects of reducing intestinal inflammation, promoting the healthy development of the intestinal tract, and good industrial prospects

Active Publication Date: 2020-07-14
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology allows researchers to create small amounts of proteins called chlorella actinidase (CRAM) from natural sources like Bifdoba cerevisiae cells grown on special media containing specific nutrients such as yeast extract. These tiny particles are then added back into these cultures during fermentations. By controllably inducing gut floral rearrangement by adding certain signals derived from Crameric Aciduria virus type 1 enzymatic activity, we have developed methods for producing effective treatments targeted at preventing diseases associated with excessive gastrointestinal disturbances caused by various factors including stressors.

Problems solved by technology

This patented technical problem addressed in this patent relating to finding ways to prevent diseases caused by harmful organism such as helminthosis through their production of specific types of proteins known collectively as CRAMPs. These molecules were discovered during research into how they could protect tissues like gut walls without causing damage. However, these small polypeptides may still cause inflammatory responses when delivered alone. To address this issue, we developed different strategies based upon introducing them inside the enterocyte lining layer of the host defense mechanism.

Method used

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  • Lactococcus lactis for expressing mouse antibacterial peptide gene
  • Lactococcus lactis for expressing mouse antibacterial peptide gene
  • Lactococcus lactis for expressing mouse antibacterial peptide gene

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0060] Example 1 Construction of Recombinant Bacteria L.lactis NZ9000 / pMG36e-Usp45-CRAMP

[0061] 1. Construction of recombinant plasmid pMG36e-Usp45-CRAMP

[0062] (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, and the signal peptide sequence Usp45 added for the purpose of efficient secretion and expression, artificially synthesized The 228bp codon-optimized sequence of the Usp45-CRAMP gene was sent to the company for synthesis. Xbal-Usp45-CRAMP-F is the upstream primer that contains the restriction site Xbal fused with pMG36e and the initial sequence of the 5' end of the signal peptide Usp45-CRAMP, and Usp45-CRAMP-Sph1-R is the signal peptide Usp45-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 region of about 7...

Embodiment 2

[0067] Example 2 Construction of Recombinant Bacteria L.lactis NZ9000 / pNZ8148-Usp45-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 pNZ8148, as well as the signal peptide sequence Usp45 added for the purpose of efficient secretion and expression, artificially synthesized The 228bp codon-optimized sequence of the Usp45-CRAMP gene was sent to the company for synthesis. Sph1-Usp45-CRAMP-F is the upstream primer that contains the restriction site Xbal fused with pNZ8148 and the initial sequence of the 5' end of the signal peptide Usp45-CRAMP, and Usp45-CRAMP-Xbal-R is the signal peptide Usp45-CRAMP gene reverse primer. The optimized and synthesized Usp45-CRAMP sequence is shown in SEQ ID NO: 4; the optimized and synthesized Sph1-Usp45-CRAMP-F, Usp45-CRAMP-Xbal-R primer sequences are shown in SEQ ID NO: 7-8 respectively.

[0069] (2) PCR am...

Embodiment 3

[0073] Example 3 Induced expression of secretory recombinant Lactococcus lactis containing CRAMP gene in vitro

[0074] The recombinant bacteria L.lactis NZ9000 / pMG36e-Usp45-CRAMP recombinant expression bacteria constructed in Example 1 were respectively inoculated in GM17 liquid medium containing 5ug / mL erythromycin at a ratio of 1:100, and the recombinant bacteria constructed in Example 2 were Bacteria L.lactis NZ9000 / pNZ8148-Usp45-CRAMP was inoculated in GM17 liquid medium containing 5ug / mL chloramphenicol at a volume ratio of 1:100, and cultured overnight at 30°C; the overnight culture was inoculated at a ratio of 1:50 Inoculate in 10mL liquid medium containing corresponding antibiotics, and continue to cultivate for about 2.5h until the bacteria enter the logarithmic growth phase (OD 500 =0.4~0.6), add 40ng / mL nisin (nisin) to the L.lactis NZ9000 / pNZ8148-Usp45-CRAMP culture system to induce 4h, centrifuge at 10000rpm at 4°C for 5min, collect the culture supernatant, and a...

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Abstract

The invention discloses lactococcus lactis for expressing a mouse antibacterial peptide gene, and belongs to the technical field of gene engineering. The nucleotide sequence of a CRAMP protein is optimized, and the constructed lactococcus lactis for expressing the CRAMP protein is used for preparing vaccines for regulating intestinal flora disturbance, so that the lactobacillus lactis has advantages on regulation of intestinal flora and intestinal immune response and maintenance; the whole culture can be directly used as an oral vaccine to stimulate mice and cause strong cellular immune response, and the recombinant lactococcus lactis can be used as a novel oral vaccine product with a good industrial prospect, plays a positive role in relieving intestinal inflammation, and has important practical significance in promoting healthy development of intestinal tracts.

Description

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Claims

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Application Information

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Owner JIANGNAN UNIV
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