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Chlamys Farreri H2A gene clone and N terminal expression technology

A technology of scallops and genes, applied in the production of feed additives and preservatives and preservatives, cloned the complete DNA sequence and amino acid sequence of the histone gene H2A of scallops, in the field of drug production, can solve the problem of cultured scallops Mass deaths, heavy economic losses, etc.

Inactive Publication Date: 2009-06-17
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since 1994, farmed shellfish began to die on a large scale, and the economic loss was very heavy

Method used

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  • Chlamys Farreri H2A gene clone and N terminal expression technology
  • Chlamys Farreri H2A gene clone and N terminal expression technology
  • Chlamys Farreri H2A gene clone and N terminal expression technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Implementation example 1. A cloned Chlamys farreri H2A gene has the following sequence:

[0057] sequence listing

[0058] (1) Information on SEQ ID NO 1

[0059] sequence features

[0060] Length: 606 base pairs

[0061] Type: nucleic acid

[0062] Chain type: double chain

[0063] Topology: Linear

[0064] Source: Chlamysfarreri

[0065] Sequence description:

[0066]

[0067] (2) Information on SEQ ID NO 2

[0068] (a) Sequence features:

[0069] Length: 125 amino acids

[0070] Type: amino acid

[0071] Number of chains: single chain

[0072] Geometry: Linear

[0073] (b) Molecule type: protein

[0074] (c) Source: Chlamys farreri

[0075] (d) Sequence description:

[0076]

[0077]

[0078] The method for cloning the H2A gene and its N-terminal in vitro recombination from Chlamys farreri of the present invention comprises:

[0079] 1. (1) Extraction and purification of Chlamys farreri genomic DNA,

[0080] The DNA extraction and purificati...

Embodiment 2

[0089] Implementation example 2. Cloning and expression method of the gene of Chlamys farreri H2A

[0090] 1. Extraction and purification of Chlamys scallop genomic DNA: The total DNA was extracted from Chlamys scallop muscle column using the DNA extraction and purification Kit of Beijing Saibaisheng Technology Co., Ltd., and the quality of the extracted DNA was determined by Bechman730 spectrophotometer (Li Taiwu, Li Chenghua et al. 2003).

[0091] 2 Construction of the genomic library of Chlamys farreri: it is characterized in that, the construction of the genomic library utilizes the Universal GenomeWalker of Clontech Company TM Kit to build.

[0092] 3. Cloning of the full-length sequence of gene cluster DNA containing H2A in Chlamys farreri: According to the obtained amino acid sequence of H2A, use multiple sequence alignment software to find out its conserved amino acid positions, and design corresponding merger primers GSPF: 5 '-AAYGAYGARGARYTRAAYAA-3' and specific li...

Embodiment 3

[0106] Example 3: Inhibitory activity of recombinant H2A N-terminal products on bacteria in vitro

[0107] Antimicrobial peptides have antibacterial activity in vitro, so we studied the inhibitory activity of the recombinant H2A N-terminus in Examples 4-5 on Gram-positive and Gram-negative bacteria in vitro. Using the agar well diffusion method, Micrococcus luteus and Vibrio luteus were used as test strains to test the activity of the expression product, and cultured in SOB liquid medium until O.D. 600 0.6, take 400μl of bacterial suspension and mix with 100mL of solid medium, and spread it in a sterile petri dish. After solidification, make a small hole with a diameter of 2mm in the petri dish, and drop 10μl and 20μl into the hole respectively, 50 μl of the supernatant of the blank control group and the supernatant of the test group were cultured overnight at 37°C to observe the antibacterial effect. The results showed that the recombinant H2A N-terminal product showed obvio...

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Abstract

The invention relates to a scallop protein H2A gene clone and the monoclonal expression technique of N end antibiotic polypeptide in the field of molecular biology technique. It uses isogenesis clone technique to obtain the scallop group protein H2A gene group total length; the gene DNA has a 375bp open reading frame with the coding 125 amino acids, 3' non-coding area comprises two ending signals which are stem ring structure and polyadenylic acid rear signal. It uses pPIC9K expressing carrier to express the protein N end 30aa in Pichia pastoris, the expressing product has bacterial inhibition to the Micrococcus luteus and the Vibrio splendidus.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and relates to the cloning and N-terminal expression technology of Chlamys farreri H2A gene, specifically clones the full DNA sequence and amino acid sequence of Chlamys farreri histone gene H2A from Chlamys farreri genome, and also The invention relates to the application of the gene and its N terminal in the production of medicine, feed additive, antiseptic and fresh-keeping agent. Background technique [0002] Antimicrobial peptides are a class of amphipathic small molecule basic polypeptides that widely exist in the entire biological world, and are key factors in the body's innate immunity. After Steiner first discovered in celestial silkworm, so far, more than 800 kinds of antimicrobial peptides have been found in various organisms. According to the sequence, secondary structure and antibacterial properties of antimicrobial peptides, the discovered antimicrobial peptides are divid...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C07K14/435C12N15/81C12N15/10A61K38/16A23K1/16A23L3/34C12P19/34A61P31/04A23K20/142A23K20/153
Inventor 宋林生李成华赵建民相建海
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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