Whole peptidoglycan of lactobacillus cell wall and application in improving IEC (intestinal epithelial cell) antimicrobial peptide expression level thereof

A technology of Lactobacillus cells and Lactobacillus, applied in animal cells, vertebrate cells, artificial cell constructs, etc., can solve the problem of unclear mechanism of action of probiotics

Inactive Publication Date: 2012-10-03
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the research on the relationship between probiotics and the expression of antimicrobial peptides is still blank, and the relationship between the two and the mechanism of action of probiotics are still unclear.

Method used

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  • Whole peptidoglycan of lactobacillus cell wall and application in improving IEC (intestinal epithelial cell) antimicrobial peptide expression level thereof
  • Whole peptidoglycan of lactobacillus cell wall and application in improving IEC (intestinal epithelial cell) antimicrobial peptide expression level thereof
  • Whole peptidoglycan of lactobacillus cell wall and application in improving IEC (intestinal epithelial cell) antimicrobial peptide expression level thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Isolation and cultivation of small intestinal epithelial cells

[0051] Wipe the 18-day-old SPF chicken embryo with alcohol cotton, and place the big head up on the egg stand, gently break the big head of the egg with a sterilized metal utensil, use tweezers to clip the broken eggshell, and bend the tweezers to tear off the eggs. Remove the air chamber membrane, gently clamp the neck of the chicken embryo with tweezers, drag it out and put it into a clean and sterile Petri dish for later use. Take out the whole small intestine under aseptic conditions, carefully remove the mesentery, wash 3 times with D-Hank’s solution, and cut the small intestine to less than 1mm 3 Add D-Hank's solution to wash the tissue block, add double antibody (penicillin-streptomycin) with a concentration of about 200U / ml to the cleaning solution, let stand for 1min, and wash repeatedly 3 times until the supernatant is clear. Add 5 times the volume of collagenase I (1mg / ml), transfer t...

Embodiment 2

[0052] Example 2 Effect of Lactobacillus on the Expression of Chicken Small Intestinal Epithelial Cell Antimicrobial Peptide AvBD9 Gene

[0053] 2.1 Test materials: 8 strains of Lactobacillus, respectively Lactobacillus acidophilus La standard strain (Lactobacillus acidophilus ATCC 4356, derived from human intestinal tract, purchased from the American Type Culture Collection), porcine Lactobacillus salivarius X7 (Lactobacillus salivarius X7, isolated from the duodenal mucosa of healthy piglets, donated by Associate Professor Zhang Jinhua, Department of Preventive Veterinary Medicine, Jiangxi Agricultural University), Lactobacillus delbrueckii C21 (Lactobacillus delbrueckii C21, isolated from the digestive tract of healthy piglets, donated by Professor Zhu Weiyun, Nanjing Agricultural University) , Lactobacillus fermentum F6 (Lactobacillus fermentum F6, isolated from traditional dairy products in Inner Mongolia, donated by Professor Zhang Heping, Key Laboratory of Dairy Biotechn...

Embodiment 3

[0099] Example 3 Extraction of Lactobacillus rhamnosus MLGa (Lactobacillus rhamnosus MLGa) Active Components

[0100] 3.1 Extraction of peptidoglycan

[0101] Extracted according to the method of Kazunori Sekine et al. Specific steps are as follows;

[0102] Add 15g of heat-treated bacteria into 50ml of 0.5% Trilon x-100 solution, and put it in a water bath at 80-85°C for 1h while continuously stirring the solution. Then, the solution was immediately cooled and washed thoroughly with double distilled water. Then wash with methanol: water (2:1 V / V), methanol, and acetone successively to remove the detergent in the insoluble residue. Add 40ml containing 10mmol MgCl to the residue 2 Trypsin (1mg / ml), DNase (100μg / ml), RNase (150μg / ml), pH7.2 TrisHcl buffer, incubate at 37°C for 14h, centrifuge at 2000×g 4°C for 40min to separate the insoluble residue, and use trypsin-containing (0.5mg / ml), α-chymotrypsin (0.5mg / ml), Tris Hcl buffer 40ml, digested at 37°C for 14h. The residu...

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Abstract

The invention discloses a whole peptidoglycan of a lactobacillus cell wall and an application in improving IEC (intestinal epithelial cell) antimicrobial peptide expression level thereof. The invention detects the enhancement on the expression of IEC antimicrobial peptide AvBD9 by using eight lactobacilluses, screens out lactobacillus rhamnosus MLGa (Lactobacillus rhamnosus) with the best stimulation effect, extracts related components of cell wall whole peptidoglycan (WPG) of the MLGa and lipoteinchoic acid (LTA), and screens out the constituent cell wall whole peptidoglycan (WPG) with the best stimulation effect. Compared with blank control group, the lactobacillus MLGa with the best stimulation effect and the constituent cell wall whole peptidoglycan (WPG) thereof screened out by the invention can significantly improve the IEC antimicrobial peptide expression level. The difference of the two effects can reach more than 34 times. The lactobacillus rhamnosus MLGa and the constituent cell wall whole peptidoglycan thereof play an important role in enhancing the innate immunity of the body.

Description

technical field [0001] The invention relates to a cell wall intact peptidoglycan and its application, in particular to a cell wall intact peptidoglycan extracted from Lactobacillus rhamnosus MLGa (Lactobacillus rhamnosus MLGa) and its application in improving the expression level of IEC antimicrobial peptides . The invention belongs to the field of biotechnology. Background technique [0002] Main Functions of Chicken Intestinal Epithelial Cells (IEC) [0003] IEC is the main functional cell for digestion and absorption in mammals, and it is also the fastest-renewing type of cell in the body. It participates in the digestion and absorption of intestinal food, and is closely related to the internal and external secretion functions of the intestinal tract. The mucosal epithelium contains a large number of immune cells and immune molecules, which regulate various immune responses of the mucosa through different stimulation signals to the mucosal immune cells, constitute an im...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C08B37/00C12N5/071C12Q1/68
Inventor 刘思国黎观红贾永杰洪智敏司微于申业
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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