CN-122012560-A - Bacterial secretion system for engineering efficient protein delivery and application thereof

CN122012560ACN 122012560 ACN122012560 ACN 122012560ACN-122012560-A

Abstract

The invention belongs to the technical field of microbial genetic engineering and protein delivery, and particularly relates to an engineering bacterial secretion system for efficiently delivering protein and application thereof. Specifically, the invention constructs a bacterial secretion system capable of efficiently and simultaneously delivering a plurality of proteins by engineering an effector protein toxic structural domain on the basis of analyzing a delivery mechanism of the Bacteroides fragilis GS086 type double-effect protein T6 SS. The system is modified into natural human intestinal symbiotic bacteria, so that the problems of high immunogenicity, poor stability and the like are effectively avoided, and the protein can be delivered in situ in the intestinal tract to realize the accurate regulation and control of the intestinal flora structure and the secretion of specific target proteins, so that the system has good practical application value.

Inventors

GAO XIANG
LI WEIXUN
Zheng Shuaining
ZHAO XUEZHENG
Jiao Xuyao

Assignees

山东大学

Dates

Publication Date: 20260512
Application Date: 20250930

Claims (10)

1. A bacterial secretion system for engineering high-efficiency protein delivery is characterized in that the bacterial secretion system takes a six-type secretion system T6SS of bacteroides fragilis as a main body, the T6SS comprises a core protein VgrG, an auxiliary protein and a plurality of effector proteins, wherein each effector protein has a conserved delivery domain and a replaceable toxic domain, and a target protein to be secreted is fused and expressed with the conserved delivery domain when in use, so that the toxic domain is replaced.
2. The bacterial secretion system of claim 1, wherein the core protein VgrG has: a1 Amino acid sequence shown as SEQ ID NO. 1; a2A protein with the same function, wherein the amino acid sequence shown as SEQ ID NO.1 is substituted and/or deleted and/or added by one or more amino acid residues; The accessory protein has: b1 Amino acid sequence shown as SEQ ID NO. 2; b2 Amino acid sequence shown as SEQ ID NO.2 is replaced and/or deleted and/or added by one or more amino acid residues and has the same function.
3. The bacterial secretion system of claim 1, wherein the plurality of effector proteins is two, a first effector protein and a second effector protein, the first effector protein having a delivery domain comprising: c1 Amino acid sequence shown as SEQ ID NO. 3; c2 A protein with the same function, wherein the amino acid sequence shown as SEQ ID NO.3 is subjected to substitution and/or deletion and/or addition of one or more amino acid residues; The amino acid sequence of the toxic structural domain of the first effector protein is shown as SEQ ID NO. 4; the second effector protein has a delivery system comprising: d1 Amino acid sequence shown as SEQ ID NO. 5; d2 A protein with the same function, wherein the amino acid sequence shown as SEQ ID NO.5 is substituted and/or deleted and/or added by one or more amino acid residues; the amino acid sequence of the toxic structural domain of the second effector protein is shown in SEQ ID NO. 6.
4. The bacterial secretion system of claim 1, wherein said protein of interest is an enzyme, an antibody, a toxin, or a signaling molecule, and further wherein said protein of interest is at least two, a first protein of interest replaces a toxic domain of said first effector protein and a second protein of interest replaces a toxic domain of said second effector protein.
5. A nucleic acid molecule encoding the bacterial secretion system of any of claims 1-4 engineered to deliver a protein with high efficiency.
6. An engineered bacterium comprising the bacterial secretion system of the engineered high efficiency delivery protein of any one of claims 1-4 and/or a nucleic acid molecule encoding the bacterial secretion system of the engineered high efficiency delivery protein, thereby secretory expression of a protein of interest.
7. The bacterium of claim 6, wherein said engineered bacterium further comprises an inducible expression system, whereby modulation of expression of a protein of interest by said bacterial secretion system is achieved by modulating the concentration of an inducer.
8. The bacterium of claim 7, wherein said inducible expression system is a rhamnose protein inducible expression system.
9. Use of the bacterial secretion system of any one of claims 1-4, the nucleic acid molecule of claim 2, the engineered bacterium of any one of claims 6-8 in protein delivery.
10. The use according to claim 9, wherein the protein is an enzyme, an antibody, a toxin or a signaling molecule; Further, the application is specifically the application of the bacterial secretion system, the nucleic acid molecules and the engineered bacteria in the preparation of protein delivery products, wherein the products are common experimental reagents for pharmaceutical or non-pharmaceutical use.

Description

Bacterial secretion system for engineering efficient protein delivery and application thereof Technical Field The invention belongs to the technical field of microbial genetic engineering and protein delivery, and particularly relates to an engineering bacterial secretion system for efficiently delivering protein and application thereof. Background The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art. The intestinal flora of human body is a complex microbial community, is closely related to host health, and the bacteroides are used as gram negative bacteria with highest abundance in intestinal tracts, and realize long-term stable colonization by virtue of interaction mechanisms among various bacteria. The six-type secretion system (Type Six Secretion System, T6 SS) is an important protein secretion machine in bacteroides, and can not only inject effector proteins into sensitive strains in a direct contact manner to play a killing role, but also secrete the effector proteins into extracellular supernatants. Bacteroides fragilis (Bacteroides fragilis) is a key component of human intestinal symbiotic bacteria, and exists mainly in the distal colon. Through bioinformatic analysis of the genome of bacteroides fragilis disclosed in NCBI, more than 86% of bacteroides fragilis code for T6SS, which shows that T6SS is the main protein secretory pathway in bacteroides fragilis, providing natural advantages for constructing a protein delivery system based on the bacteria. The traditional protein delivery system has many limitations that the virus vector delivery system has high immunogenicity and potential safety hazard, the chemical delivery methods such as liposome embedding and the like have poor stability, are easy to be degraded by lysosomes and have poor targeting, and the biological macromolecules such as cell penetrating peptide, antibody conjugate and the like have poor tissue specificity in a delivery mode mediated by biomacromolecules, and can enter normal cells in a non-selective way or trigger host immune response, so that the delivered protein is cleared. In recent years, researchers have begun to develop biological protein delivery systems. For example, the Massachusetts institute Zhang Feng team engineered bacterial T6SS delivered proteins into Cre recombinase by engineering bacterial extracellular injection systems (eCISs), base editors, bacterial toxins, etc., and the Canadian university of Calgari Dong Tao team 2021. However, the modification of these bacterial secretion systems still has the problems of poor targeting, low delivery efficiency, only one effector protein at a time, and the like, which limits the application range thereof. Disclosure of Invention In order to overcome the defects in the prior art, the inventor provides an engineering bacterial secretion system for efficiently delivering protein and application thereof through long-term technical and practical exploration. Specifically, the invention constructs a bacterial secretion system capable of efficiently and simultaneously delivering a plurality of proteins by engineering an effector protein toxic structural domain on the basis of analyzing a delivery mechanism of the Bacteroides fragilis GS086 type double-effect protein T6 SS. The system is modified into natural human intestinal symbiotic bacteria, so that the problems of high immunogenicity, poor stability and the like are effectively avoided, and the protein can be delivered in situ in the intestinal tract to realize the accurate regulation and control of the intestinal flora structure and the secretion of specific target proteins. Based on the above results, the present invention has been completed. In order to achieve the technical purpose, the invention adopts the following technical scheme: In a first aspect, the invention provides a bacterial secretion system for engineering high-efficiency protein delivery, which takes a six-type secretion system (T6 SS) of bacteroides fragilis as a main body, wherein the T6SS comprises a core protein VgrG, an auxiliary protein and a plurality of effector proteins, wherein each effector protein has a conserved delivery domain and a replaceable toxic domain, and a target protein to be secreted is fused and expressed with the conserved delivery domain when in use, so that the toxic domain is replaced. Wherein the core protein VgrG has: a1 Amino acid sequence shown as SEQ ID NO. 1; a2 Amino acid sequence shown as SEQ ID NO.1 is replaced and/or deleted and/or added by one or more amino acid residues and has the same function. The accessory protein has: b1 Amino acid sequence shown as SEQ ID NO. 2; b2 Amino acid sequence shown as SEQ ID NO.2 is replaced and/or deleted and/or added by one or mo