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Deep sea shrimp C-type lectin and application thereof

A technology of lectin and deep-sea shrimp, applied in the field of molecular biology, can solve the problems of unclear function of lectin and lack of research on lectin

Inactive Publication Date: 2017-04-26
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, research on lectins in deep-sea invertebrates is lacking, and the functional roles of related lectins are unclear

Method used

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  • Deep sea shrimp C-type lectin and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] The C-type lectin (named P208) in the present invention is the amino acid sequence in SEQ ID No.1 in the sequence table.

[0014] The sequence listing SEQ ID No.1 is:

[0015] MTFGKTNFSLFLGGMLMILLALFTLPTGTVADKLQETLIEDDARIIGVDVIDHDNKYQRNKKGGAKFENTVQILNTEPECPPPYFRLMSECFYVHVHHHLPWEVARSFCKDMGGDLAEPSYYGALLAVVSDKYSDGPQHFWLGASDLEEEEGHFQWLSGGNHSVQSGWRTGQFPDDDGGIEDCAPSKHDCNNY

[0016] (a) Sequence features:

[0017] ●Length: 208

[0018] ●Type: amino acid sequence

[0019] ●Chain type: single chain

[0020] ●Topological structure: linear

[0021] (b) Molecule type: protein

[0022] (c) Assumption: No

[0023] (d) Antisense: no

[0024] (e) Original Source: Alvin Shrimp

[0025] Structural features: The protein is expected to contain a signal peptide (amino acids 1-31) and a C-type lectin domain (amino acids 91-205).

[0026] MTFGKTNFSLFLGGMLMILLALFTLPTGTVADKLQETLIEDDARIIGVDVIDHDNKYQRNKKGGAKFENTVQILNTEPECPPPYFRLMSECFYVHVHHHLPWEVARSFCKDMGGDLAEPSYYGALLAVVSDKYSDGPQHFWLGASDL...

Embodiment 2

[0028] Preparation of Recombinant Protein of Lectin P208

[0029] 1) Construction of plasmid pEtP208 expressing lectin P208 recombinant protein:

[0030] The construction of pEtP208 is as follows: take the deep-sea (1880m) Alvin shrimp cDNA as a template, and perform PCR amplification with primers F1 and R1. PCR conditions were: 940C 60s pre-denatured template DNA, then 940C 40s, 500C 60s, 720C 60s, 5 cycles, then changed to 940C 40s, 600C 60s, 720C 60s, 30 cycles. After the PCR product was purified with the Tiangen DNA Product Purification Kit, it was ligated with the PCR cloning vector pBS-T (purchased from "Tiangen Biochemical Technology Co., Ltd.", Beijing) at room temperature for 2-4 hours, and the ligation mixture was transformed into Escherichia coli DH5α After containing 100 μg / ml anka penicillin, 40 μg / ml 5-bromo-4-chloro-3-indole-β-D-lactoside and 24 μg / ml isopropyl-β-D-thiogalactoside Culture on LB solid medium for 18-24 hours, pick out the white transformant, ext...

Embodiment 3

[0037] Application of recombinant lectins

[0038] Step 1) Bacterial suspension preparation

[0039] Cultivate Vibrio anguillarum C312 and Vibrio harveyi T4 to an OD600 of 0.8 in LB medium, then centrifuge (5000g, 40C, 10min), collect the thalline, and suspend it in PBS to a final concentration of 10 cfu / ml, namely Suspensions of Vibrio anguillarum and Vibrio harveyi.

[0040] The Vibrio anguillarum C312 is preserved in CGMCC, General Microbiology Center of China Committee for Culture Collection of Microorganisms, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, postal code, 100101. The preservation number is: CGMCC No.6250, the preservation date is 2012.6.21, and the classification name is Vibrio anguillarum.

[0041] The Vibrio harveyi T4 is preserved in CGMCC, General Microorganism Center of China Microbiological Culture Collection Management Committee, with the same address as above, with the preservation number: CGMCC No.1985, and the preservation ...

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Abstract

The invention relates to the field of molecular biology, and concretely relates to a deep sea shrimp C-type lectin and an application thereof. The deep sea shrimp C-type lectin is amino acids in a sequence table represented by SEQ ID No.1. The agglutinin can be applied to the production of a bacterium agglutination preparation. The agglutinin is a recombinant protein expressed by agglutinin in Escherichia coli. The recombinant agglutinin is used for combining and agglutinating pathogenic bacteria.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a deep-sea shrimp C-type lectin and its application. Background technique [0002] Lectin is a kind of carbohydrate-binding protein, and C-type lectin is one of the major categories. It is a secreted protein or a transmembrane protein. Its structure contains a conserved carbohydrate-recognition domain (carbohydrate-recognition domain) of about 120 amino acid sequences. , can specifically recognize and bind exopolysaccharides. Some C-type lectins can recognize sugar molecules on the surface of bacteria and bind to bacteria, so they are classified as a pattern recognition receptor. After binding to bacteria, they can trigger a series of immune responses, such as enhanced phagocytosis, Complement activation, etc., thereby helping the body to eliminate pathogenic bacteria. Therefore, C-type lectins play an important role in the host innate immune response. At present, there is a l...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C12N15/70A61K38/17A61P31/04
Inventor 孙黎陈晨张健
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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