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Fish serum amyloid P component and use thereof

A serum starch, fish technology, applied in the field of molecular biology, can solve problems such as unknown functions, and achieve the effect of improving anti-bacterial and anti-virus capabilities

Inactive Publication Date: 2016-01-20
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

SAP in fish is currently poorly studied and its function is almost completely unknown

Method used

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  • Fish serum amyloid P component and use thereof
  • Fish serum amyloid P component and use thereof
  • Fish serum amyloid P component and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Preparation of recombinant protein of serum amyloid P component

[0019] 1) Construction of plasmid pCsSap expressing serum amyloid P component recombinant protein:

[0020] The amino acid sequence of the serum amyloid P component of the present invention has been published (GenBankaccessionnumberKT025856):

[0021] MMEKLFLFTALMISTCCAVTQDLSGKVFVFPKETNTDHVKLLTTRSTFYNVTVCLRFLTDLNRAYSLFSMSTPTVENAFLIYGEPANHVLHVYSGSGPTFHAVPFPQNTWHSLCSTWGHGSGVAQIWLNGKPYVKKFVTNQPIGGKPISILGQEQDSYGGGFDAAQSFVGMISDVHMWDQVLTPTEIKSYMNHRHVTPGNVFNWRSLQYEITGLVLVDDAPDDM

[0022] The serum amyloid P component consists of 224 amino acids and contains a PTX domain consisting of 21-218 amino acid residues. Serum amyloid P component gene was amplified by PCR with primers SapF1 and SapR1 using the cDNA of tongue sole as template. The PCR conditions were: pre-denatured template DNA at 94°C for 60s, followed by 30 cycles at 94°C for 40s, 58°C for 60s, and 72°C for 60s, and then extended at 72°C for 7-10 minut...

Embodiment 2

[0028] Application of Serum Amyloid P Component - Interaction of Serum Amyloid P Component with Bacteria

[0029]1) Bacterial suspension preparation. The gram-negative bacteria Pseudomonas fluorescens (Pseudomonas fluorescens) were cultured in LB medium (preserved at CGMCC, General Microbiology Center of China Committee for Culture Collection of Microorganisms; the address of the preservation unit is Datun Road, Chaoyang District, Beijing, Chinese Academy of Sciences Institute of Microbiology; the preservation date is January 9, 2008, and the number is CGMCCNo.2329), Edwardsiella tarda (preserved in CGMCC, General Microbiology Center of China Committee for the Collection of Microbial Cultures; the address of the preservation unit is Beijing Datun Road, Chaoyang District, Institute of Microbiology, Chinese Academy of Sciences; preservation date is January 9, 2008, number CGMCCNo.2330), Escherichia coli (DH5α, purchased from "Tiangen Biochemical Technology (Beijing) Co., Ltd." ...

Embodiment 3

[0034] Application of Serum Amyloid P Component-Antibacterial Effect of Serum Amyloid P Component

[0035] The rCsSAP purified in Example 1 above was diluted to 200 ug / ml in PBS, which was the rCsSAP diluent. Pseudomonas fluorescens was cultivated to OD according to the method in Example 2 600 0.8, then centrifuged (5000g, 4°C, 10min), collected the bacteria, suspended in PBS to a final concentration of 10 7 cfu / ml is the bacterial suspension. The bacterial suspension was mixed with equal volume (1:1) of rCsSAP dilution or PBS (control). Twenty tongue soles (about 13.8g in weight) were randomly divided into 2 groups, 10 in each group. These two groups are named A and B respectively. Each fish in group A was injected with 100 ul of bacteria+rCsSAP dilution, and each fish in group B (control group) was injected with 100 ul of bacteria+PBS. At 24 hours after injection, the kidney and spleen tissues of groups A and B were taken, homogenized in 2ml of PBS, and 100ul of the hom...

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Abstract

The invention relates to the field of molecular biology and concretely relates to a fish (cynoglossus semilaevis) serum amyloid P component and a use thereof. Through a cynoglossus semilaevis cDNA as a template and primers SapF1 and SapR1, a serum amyloid P component gene is subjected to PCR amplification, and the amplification product is subjected to inducible expression and purification so the cynoglossus semilaevis serum amyloid P component recombinant protein is obtained. The serum amyloid P component recombinant protein can be bonded to a plurality of bacteria and can substantially improve antibacterial and antiviral capability of fish.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a serum amyloid P component of fish (lingual sole) and its application. Background technique [0002] Serum amyloid P component (Serumamyloid P component, SAP), belonging to the pentraxin (Pentraxins, PTX) family, is a typical extracellular secreted acute phase protein with a molecular weight of about 25.2kDa and contains a PTX domain. SAP plays a role in mammalian non-specific immunity, and its ligands are diverse; SAP can bind lipopolysaccharides, phosphoethanolamine and polysaccharides on the surface of bacteria, as well as DNA, histones, chromatin and small ribonucleic acid proteins. It plays an important role in the process of identifying damaged cells and their intracellular components and clearing apoptotic and necrotic cells. In addition, SAP can interact with C1q to activate the classical pathway of complement, thereby regulating inflammatory responses and opsonization....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/46A61K38/17A61P31/04A61P31/12
CPCC07K14/461A61K38/00
Inventor 孙黎王婷
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI