Pacific oyster DM9-containing structural domain protein CgDM9CP-4 as well as preparation method and application

A domain protein, long oyster technology, applied in the field of molecular biology, to achieve a strong inhibitory effect

Active Publication Date: 2018-04-20
DALIAN OCEAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] However, so far there are no related reports about the long oyster DM9 domain-containing protein CgDM9CP-4, its preparation method and its application in the preparation of drugs for inhibiting Pichia pastoris and Vibrio anguillarum

Method used

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  • Pacific oyster DM9-containing structural domain protein CgDM9CP-4 as well as preparation method and application
  • Pacific oyster DM9-containing structural domain protein CgDM9CP-4 as well as preparation method and application

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experiment example

[0035] Experimental example: detection of antibacterial activity of long oyster protein CgDM9CP-4 containing DM9 domain of the present invention

[0036] Specific steps are as follows:

[0037] 1. Culture and preparation of microorganisms

[0038] Pichia( Pichia pastoris GS115 , purchased from Invitrogen) with YPD medium at 28°C, 220rpm, Vibrio anguillarum ( Vibrio anguillarum , purchased from Beijing Microorganism Culture Collection Center) with 2216E medium at 28°C, 220 rpm, and when it reached the logarithmic growth phase, Tris-HCl (50 mmol L -1 , pH = 8.0) to dilute the bacteria respectively, so that the number of colonies per milliliter of bacteria liquid is about 1×10 3 CFU.

[0039] 2. Determination of antibacterial activity of recombinant protein CgDM9CP-4

[0040] Pichia pastoris and Vibrio anguillarum in the logarithmic growth phase were collected by centrifugation and washed with TBS (50mM Tris-Hcl, 150 mMNaCl) and resuspended (1×10 4CFU). 50 μL of the long...

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Abstract

The invention discloses a pacific oyster DM9-containing structural domain protein CgDM9CP-4 of which the amino sequence is as shown in SEQ ID NO.1. A preparation method comprises the following steps:performing PCR (Polymerase Chain Reaction) amplification on a pacific oyster CgDM9CP-4 gene coding region segment by using specific primers P1 and P2, wherein the DNA (Deoxyribonucleic Acid) sequenceof the primer P1 is as shown in SEQ ID NO.2, and the DNA sequence of the primer P2 is as shown in SEQ ID NO.3; performing Nde I and Xho I digestion on the PCR amplification product and a pET-30a vector, connecting through a connection enzyme, converting, and sequencing and identifying recons; transferring the recons into an escherichia coli Transetta (DE3) expression strain, performing induced culture, and further performing purification and renaturation, thereby obtaining a recombinant protein with amino sequences of a sequence table SEQ ID NO.1. The pacific oyster DM9-containing structural domain protein CgDM9CP-2 can be adopted to prepare medicines for inhibiting pichia pastoris and vibrio anguillarum.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a protein CgDM9CP-4 containing DM9 domain of long oyster, a preparation method and application. Background technique [0002] The long oyster is an important marine cultured shellfish. Because the long oyster lacks an adaptive immune defense system and mainly relies on the innate immune system to resist the infection of exogenous pathogenic microorganisms, various diseases caused by bacteria, fungi and viruses continue to break out in the long oyster farming population, causing huge economic losses. loss. Proteins containing the DM9 domain were first identified in Drosophila. Later, Magalhaes et al. found some toxins with DM9 domains in fish and named them "natterin", which contain N-terminal DM9 domains and C-terminal toxins / bacterial toxins (ETX / MTX2) domain. Natterin can produce cytotoxicity in human tissues, resulting in cell necrosis, edema, local p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C12N15/70A61K38/17A61P31/04A61P31/10
CPCA61K38/00C07K14/43504
Inventor 宋林生刘宇刘兆群宋小瑞王玲玲
Owner DALIAN OCEAN UNIV
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