Check patentability & draft patents in minutes with Patsnap Eureka AI!

Method for displaying lyase on cell surface and application thereof

A cell surface and lysing enzyme technology, applied in lysing enzymes, microbial-based methods, chemical instruments and methods, etc., can solve the problems of body toxicity risk, difficult screening, long cycle, etc., to promote intestinal health, good intestinal Microecology of the intestinal flora and the effect of maintaining the microecology of the intestinal flora

Active Publication Date: 2021-10-01
百葵锐(天津)生物科技有限公司
View PDF9 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the method of adding bacteriophage to the feed, because it is a virus and contains genetic material, has a risk of toxicity to the body, and the investment in product development is large, the screening is difficult, the cycle is long, and it is difficult to widely promote it in the market

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for displaying lyase on cell surface and application thereof
  • Method for displaying lyase on cell surface and application thereof
  • Method for displaying lyase on cell surface and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1 The lyase LysAB2 is displayed on the surface of Saccharomyces cerevisiae through a scaffold and the evaluation of its antibacterial effect on large intestine

[0062] 1. Use plasmid vector 1: pYD1

[0063] Insertion scaffold structure and sequence: CohesinI—CBD(t)—Cohesin II—CohesinIII, as shown in SEQ ID NO:12. Its structure is as figure 2 shown.

[0064] Insert it into the NheI and KpnI restriction sites of the pYD1 plasmid.

[0065] 2. Use plasmid vector 2: pESC-LEU

[0066] The insertion surface displays the sequence structure of the foreign protein: Yeast-secretion signal (α-factor)-LysAB2-GS linker-Dockerin (CelA), as shown in SEQ ID NO:13. Its structure is as image 3 shown.

[0067] Insert it into the BamHI and XhoI restriction sites behind the GAL1 promoter of the pESC-LEU plasmid.

[0068] 3. Transform the plasmid vector in the above steps 1 and 2 according to the following steps:

[0069] (1) Inoculate 10mL YPD with a single colony and cult...

Embodiment 2

[0084] 1. Use the second plasmid vector: pESC-LEU

[0085] The insertion surface displays the sequence structure of the foreign protein: Yeast-secretion signal (α-factor)-LL-37-SE1gp146-GSlinker-Dockerin (ScaA), as shown in SEQ ID NO:14. Its structure is as Figure 5 shown.

[0086] Insert it into the BamHI and XhoI restriction sites behind the GAL1 promoter of the pESC-LEU plasmid.

[0087] 2. Transform the plasmid vector in the above step 1 according to the following steps:

[0088] (1) Inoculate 10mL YPD with a single colony and culture overnight at 30°C

[0089] (2) Transfer to 50mLYPD, initial OD 600 0.2, cultured at 30°C for 4-5h

[0090] (3) Centrifuge at 2500rpm for 5min, then resuspend with 40mL 1×TE

[0091] (4) Centrifuge at 2500rpm for 5min, resuspend in 2mL of 1×LiAc / 0.5×TE, incubate at room temperature for 10min, aliquot 100ul / tube

[0092] (5) 100 μl of the above yeast suspension was mixed with 1 μg of plasmid DNA and 100 μg of denatured sheared salmon sp...

Embodiment 3

[0102] Example 3 Lyases LysAB2 and CF301 displayed on the combined surface of Saccharomyces cerevisiae through scaffolds and evaluation of their antibacterial effects on Escherichia coli and Staphylococcus aureus

[0103] 1. Use plasmid vector 1: pYD1

[0104] Insertion scaffold structure and sequence: CohesinI—CBD(t)—Cohesin II—CohesinIII, as shown in SEQ ID NO:12. Its structure is as figure 2 shown.

[0105] Insert it into the NheI and KpnI restriction sites of the pYD1 plasmid.

[0106] 2. Use plasmid vector 2: pESC-LEU

[0107] The insert surface displays the foreign protein sequence structure, including:

[0108] (1) Sequence structure one: Yeast-secretion signal (α-factor)-LysAB2-GS linker-Dockerin (CelA), as shown in SEQ ID NO:13. Its structure is as image 3 shown.

[0109] Insert it into the BamHI and XhoI restriction sites behind the GAL1 promoter of the pESC-LEU plasmid.

[0110] (2) Sequence structure 2: Yeast-secretion signal (α-factor)-CF301-GS linker-Do...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a method for displaying lyase on the surface of a cell, which is characterized in that a coding gene of the lyase and a coding gene of a surface binding protein are used as exogenous genes for production in an expression cell, and the lyase is bound on the surface of a display cell through the surface binding protein. The invention also discloses a display cell for displaying the lyase on the surface of the cell and application of the display cell. According to the scheme, lyase and probiotic cells are combined through a surface display method, the functions of related bacterial cells and bacteriophage lyase can be integrated, the display cells can kill or inhibit harmful bacteria, meanwhile, the lyase is more stable on a cell-based fixed carrier, and the cell-based fixed carrier is more stable. Pathogenic bacteria can be specifically killed in the intestinal environment; and one or more lyases are displayed on the surfaces of probiotic cells, so that a bactericidal effect is achieved aiming at one or more target spots of the same bacterium or aiming at various harmful bacteria, the intestinal health is promoted, and the good intestinal flora micro-ecology is maintained.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for displaying lyase on the cell surface and its application. Background technique [0002] Animal gut health plays a pivotal role in animal growth. Pathogenic bacteria such as Clostridium perfringens, Lawsonia ileum, Salmonella, Escherichia coli, etc. are the main factors that threaten the intestinal health of animals. However, due to the long-term abuse of antibiotics, the problem of antibiotic resistance has become one of the biggest health challenges in the world. In this context, my country has banned the addition of antibiotics to feed since July 1, 2020, and antibiotic substitutes have become the top priority of feed additives. At present, the existing alternative antibiotic products on the market include probiotics, prebiotics, enzymes and other microecological preparations, traditional Chinese medicine, and other products that improve the immunity of animals. [00...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12N9/88C12N15/62C12N15/74C12N15/75C12N15/81C12N1/21C12R1/225C12R1/01C12R1/07C12R1/46C12R1/645
CPCC12N9/88C12N15/74C12N15/75C12N15/81C07K2319/00Y02E50/10
Inventor 王晶张晓立李瑞琦李华珍章家泉
Owner 百葵锐(天津)生物科技有限公司
Features
  • R&D
  • Intellectual Property
  • Life Sciences
  • Materials
  • Tech Scout
Why Patsnap Eureka
  • Unparalleled Data Quality
  • Higher Quality Content
  • 60% Fewer Hallucinations
Social media
Patsnap Eureka Blog
Learn More

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2025 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com