Crassostrea gigas DM9-domain-containing protein CgDM9CP-2, preparation method and application
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A domain protein, long oyster technology, applied in the field of molecular biology, to achieve a strong inhibitory effect
Active Publication Date: 2018-05-08
DALIAN OCEAN UNIV
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[0003] However, so far, there are no relevant reports about the DM9 domain-containing protein CgDM9CP-2 of oyst
Method used
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experiment example 1
[0035] Experimental Example 1: Detection of the binding activity of the long oyster DM9 domain-containing protein CgDM9CP-2 of the present invention to Mannose, LPS, and PGN
[0036] Proceed as follows:
[0037] 1. Dissolve 10 μg of various PAMPs in 50 mM sodium carbonate-sodium bicarbonate buffer, 100 μl per well to coat the microtiter plate, 4°C, overnight.
[0038] 2. Wash with PBS-T 3 times, 5 min each time, add 200 μL 3% BSA to each well, and block at 37 °C for 1 h.
[0039] 3. Wash with PBS-T 3 times, 5 min each time, add 100 μL 2-fold serially diluted DM9 domain-containing protein CgDM9CP-2 of the present invention to each well, and incubate at 18°C for 3 h.
[0040] 4. Wash with PBS-T 3 times, 5 min each time, add 100 μL of diluted polyclonal antibody against the target protein (1:1000) to each well, and incubate at 37 °C for 1 h.
[0041] 5. Wash with PBS-T 3 times, 5 min each time, add 100 μL alkaline phosphatase-labeled goat anti-rat IgG (1:4000) to each well, a...
experiment example 2
[0044] Experimental example 2: Antibacterial activity detection of the long oyster protein CgDM9CP-2 containing DM9 domain of the present invention
[0045] Specific steps are as follows:
[0046] 1. Culture and preparation of microorganisms
[0047] Pichia pastoris (a laboratory-preserved strain) was cultured in YPD 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 so that the number of colonies per milliliter of bacteria liquid is about 1×10 3 CFU;
[0048] 2. Determination of antibacterial activity of recombinant protein CgDM9CP-2
[0049] Pichia pastoris in the logarithmic growth phase were collected by centrifugation and washed with TBS (50 mM Tris-Hcl, 150 mM NaCl) and resuspended (10 4 CFU). 50 μL of the DM9 domain-containing protein CgDM9CP-2 of the oyster of the present invention was incubated with an equal volume of Pichia pastoris for 2 h at room temperature. Take 20 μL of the ...
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Abstract
The invention discloses crassostrea gigas DM9-domain-containing protein CgDM9CP-2. The amino acid sequence of the protein is shown as SEQ ID NO.1. The preparation method includes the sequential stepsthat PCR amplification is performed on crassostrea gigas CgDM9CP-2 gene coding sequence fragments with specific primers P1 and P2, wherein the DNA sequence of the primer P1 is shown as SEQ ID NO.2, and the DNA sequence of the primer P2 is shown as SEQ ID NO.3; a PCR amplification product and a pET-30 a carrier are subjected to Nde1 and Xho1 digestion and then connected through ligase to be transformed, and sequencing authentication of a recombinant is performed; the recombinant is transferred into an escherichia coli Transetta (DE3) expression strain for induced culture, then, purification andrenaturation are performed, and the recombinant protein with the amino acid sequence shown in the sequence table SEQ ID NO.1 is obtained. The rassostrea gigas DM9-domain-containing protein CgDM9CP-2can be applied to preparation of pichia pastoris inhibiting medicine.
Description
technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a protein CgDM9CP-2 containing a DM9 domain of oyster, a preparation method and an 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...
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