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Protein adhered to surface layers of bacteria and application thereof

A cell adhesion and protein technology, applied in the direction of antibacterial immunoglobulin, bacterial peptide, serum immunoglobulin, etc.

Active Publication Date: 2010-06-30
SHANGHAI SIXTH PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Affected by this, there are few reports at home and abroad on the target proteins that play a role in the adhesion of bacteria such as Lactobacillus to the intestinal epithelium.

Method used

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  • Protein adhered to surface layers of bacteria and application thereof
  • Protein adhered to surface layers of bacteria and application thereof
  • Protein adhered to surface layers of bacteria and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0128] Embodiment 1. Identification of Lactobacillus cell surface adhesion protein

[0129] The surface protein obtained by guanidine hydrochloride combined with ultracentrifugation was subjected to SDS-PAGE electrophoresis and western blotting, and the results showed that the control Marker had multiple bands at positions such as 100-130KD, 55KD, 35-40KD, and 30-33KD ( See figure 2 a). There is a strong positive band at 30KD of the PVDF membrane, and the adhesion protein is located in this band, see figure 2 b.

Embodiment 2

[0130] Example 2. LC-MS / MS and data analysis

[0131] Get the corresponding band at the 30KD place of the PAGE gel for LC-MS / MS and use SEQUEST software (Thermo Fisher Inc.) to carry out database retrieval analysis and ProteinProphet algorithm (Nesvizhskii, A.I., etc., A statistical model for identifying proteins by tandem mass spectrometry.Analytical Chemistry 2003;75:4646-4658) for result filtering. The results showed that the coincidence rate of 5 proteins with the protein library of plant lactic acid bacteria WCFS reached 90%, as shown in Table 2.

[0132] Table 2. Expressed proteins

[0133] Numbering

[0134] Numbering

[0135] Among them, the integral membrane protein is divided into three protein fragments, which are respectively called IMP1 (sequence 32-100 in SEQ ID NO: 1), IMP2 (sequence 455-755 in SEQ ID NO: 1), and IMP3 (sequence 455-755 in SEQ ID NO: 1). ID NO: 693-993 sequence in 1).

Embodiment 3

[0136] Example 3. Cloning, expression and purification of proteins

[0137] The target gene of L. plantarum CGMCC No.1258 was amplified by PCR. After electrophoresis, the PCR product showed a single band at the position of 1500bp-500bp in 1% agarose gel, and the result was as expected, see image 3 a.

[0138]The PCR products were separated and purified by agarose gel electrophoresis and recombined into PET-16B vectors. The plasmid was transformed into competent DH10B and sequenced. The results showed that the amplified gene of L.plantarum CGMCC No.1258 was consistent with the data of Lplantarum WSFC in Genbank (GenBank accession number: NP_785773.1). Then the plasmid containing the target gene was transfected into E.coli BL21. IPTG induced 3hr. Cells were lysed by ultrasound, and the target protein was extracted and purified with His-tag Fusion Protein Purification Beads. After protein elution, 10% SDS-PAGE electrophoresis separation ( image 3 B) Simultaneously use HR...

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Abstract

The invention relates to a protein adhered to the surface layers of bacteria and an application thereof. The invention discloses an application of an integration membrane protein STY in preparing a reagent for promoting the adhesion of cells. The invention also discloses a method for changing the adhesion property of the cells by utilizing the integration membrane protein STY. The invention also discloses a specific antibody of the integration membrane protein STY.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically, the invention relates to a bacterial surface adhesion protein and its application. Background technique [0002] Adhesion is the first step in the interaction between bacteria and intestinal epithelial cells (IEC). There are 500-1000 different microorganisms in the human intestine, especially the number of bacteria in the colon reaches 10 12 / g feces, faced the highest bacterial load. More and more evidence shows that compared with other bacteria in feces, only bacteria that can tightly connect and adhere to intestinal mucosa can affect human health. As a non-invasive bacterium, Lactobacillus adheres and colonizes in the intestinal tract to inhibit the colonization, translocation and infection of pathogenic bacteria and maintain the integrity of the intestinal mucosal barrier. [0003] At present, it is believed that Lactobacillus can recognize specific receptors on the host ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/195C12N15/31C12N15/63C12N5/10C07K16/06C07K16/12
Inventor 沈通一秦环龙
Owner SHANGHAI SIXTH PEOPLES HOSPITAL
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