Recombinant Lactobacillus CRAMP Expression for Intestinal Immunomodulation
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Solution Overview
Problem
The existing technologies face challenges in delivering CRAMP effectively to the intestinal tract due to degradation by digestive enzymes, leading to inadequate local immunoregulatory effects and poor intestinal targeting.
Innovation Solution
A recombinant Lactobacillus expressing the CRAMP protein, utilizing a gene encoding CRAMP fused with a Usp45 signal peptide and a linker sequence, is introduced, which is carried by vectors like pMG36e or pNZ8148, to enhance secretion and expression of CRAMP in the intestinal tract.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If CRAMP is delivered orally to the intestinal tract, then it can exert immunoregulatory effects, but it is easily degraded by digestive tract enzymes resulting in insufficient expression
Solution Approach 1:
The patent uses lactic acid bacteria as an intermediary carrier to deliver CRAMP to the intestinal tract. The bacteria protect CRAMP from digestive enzyme degradation during transit and facilitate its targeted delivery to the intestine, where the bacteria colonize and continuously express CRAMP locally, bypassing the degradation issue of direct oral administration
Solution Approach 2:
The lactic acid bacteria express CRAMP autonomously within the intestinal tract, continuously producing the peptide locally without requiring external supplementation. This self-service mechanism ensures sustained immunoregulatory effects while the bacteria's natural resistance to digestive enzymes protects the expressed CRAMP
2Productivity
If a foreign gene is expressed in lactic acid bacteria, then CRAMP can be produced and secreted, but the complexity of genetic engineering increases
Solution Approach 1:
The patent merges the CRAMP gene with a signal peptide sequence to create a fusion gene construct. This combination enables the CRAMP peptide to be naturally secreted by the lactic acid bacteria through the bacterial secretion pathway, eliminating the need for complex purification systems while achieving high-level expression and secretion
Solution Approach 2:
The patent uses a universal prokaryotic expression system in lactic acid bacteria that can handle multiple functions: gene expression, protein folding, post-translational modification, and secretion. This multi-functional system simplifies the overall process by combining production and delivery mechanisms in a single organism
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The approach results in significant expression levels of CRAMP, up to 40 ng/μL in L. lactis and 20 ng/μL in L. plantarum, facilitating effective immunomodulation and intestinal flora regulation, with potential applications in treating colitis and maintaining intestinal health.
Implementation Method 1
a gene encoding CRAMP fused with a Usp45 signal peptide and a linker sequence, is introduced, which is carried by vectors like pMG36e or pNZ8148, to enhance secretion and expression of CRAMP in the intestinal tract
Implementation Method 2
Due to high adhesion of surface molecules, Lactococcus. lactis and Lactobacillus. plantarum can successfully colonize the intestinal tract of animal bodies and become the dominant flora in the intestinal tract
Data Source
AI summary
The disclosure discloses cathelicidin-expressing lactic acid bacteria, and belongs to the technical field of genetic engineering. The disclosure constructs Lactococcus. lactis and Lactobacillus. plantarum expressing a CRAMP protein by optimizing the nucleotide sequence of the CRAMP protein. The Lactobacillus constructed in the disclosure can be used to prepare a vaccine for regulation of intestinal flora disorder, and has advantages in regulation of intestinal flora and intestinal immune response and maintenance. An anaculture can be directly taken as an oral vaccine to stimulate mice and cause a strong cellular immune response. The recombinant L. lactis can be used as a new oral vaccine product with good industrial prospects, plays a positive role in reducing intestinal inflammation, and has important practical significance for promoting health development of the intestinal tract.