Gene complete sequence of siganus oramin L-amino acid oxidase and application thereof

A technology of gene sequence of the yellow-spotted bluefish, which is applied in the field of the complete sequence of the gene of the yellow-spotted bluefish L-amino acid oxidase, can solve problems such as food safety threats

Inactive Publication Date: 2011-07-13
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The extensive use of antibiotics and illegal drugs poses a serious

Method used

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  • Gene complete sequence of siganus oramin L-amino acid oxidase and application thereof
  • Gene complete sequence of siganus oramin L-amino acid oxidase and application thereof
  • Gene complete sequence of siganus oramin L-amino acid oxidase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0011] Example 1: The complete sequence of the L-amino acid oxidase gene of the yellow-spotted bluefish

[0012] The inventors obtained the active protein by separating and purifying the active protein from the serum of the yellow-spotted bluefish, determined the sequence of 15 amino acids at the N-terminal of the active protein, and carried out mass spectrometry sequencing analysis at the same time. The partial sequence of the protein gene, according to the obtained partial sequence of the active protein, the full-length sequence of the active protein gene was obtained by using RACE technology, and the active protein was confirmed to be L-amino acid oxidase by Blast analysis.

[0013] The full length of the L-amino acid oxidase gene sequence of the macula macula is 1725bp, the length of the open reading frame (ORF) is 1584bp, its specific position is in the 43-1626 region, and it encodes a protein of 527 amino acids. figure 1 .

Embodiment 2

[0014] Example 2: Verification of insecticidal and bactericidal functions of L-amino acid oxidase and determination of minimum action concentration

[0015] The present inventor adopts ammonium sulfate precipitation, cation exchange chromatography, reversed-phase high-performance liquid chromatography, secondary reverse-phase high-performance liquid chromatography, has obtained the L-amino acid oxidase of macula macula, the L-amino acid oxidase of this purification is to Stimulating Cryptocaryon has killing effect, has bactericidal effect to representative Gram-positive bacteria (Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli), and the present inventor has to purifying macular bluefish L-amino acid Oxidases were tested for insecticidal and bactericidal minimum protein concentrations.

[0016] 1. Verification of the insecticidal function of L-amino acid oxidase and determination of the minimum protein concentration for insecticide

[0017] The killing effe...

Embodiment 3

[0022] Example 3 High-fidelity cloning of the gene sequence encoding the L-amino acid oxidase of the yellow-spotted bluefish

[0023] The inventors designed a pair of specific primers, and in the optimized high-fidelity enzyme PCR reaction system, using the total cDNA of the spleen of A. Gene sequence for subsequent genetic engineering.

[0024] 1. Specific primers for amplifying the gene sequence encoding L-amino acid oxidase

[0025] Sense Primer: 5′-GGCTGAAGAAGGCAAAGAAGAAC-3′

[0026] Anti-sense Primer: 5′-AACAACATTGTGCTGACAGTGGC-3′

[0027] 2. High fidelity enzyme PrimeSTAR TM HS DNA Polymerase Reaction System

[0028] 5 x Prime STAR TM Buffer (Mg 2+ plus) 10μL

[0029] dNTP Mixture (2.5mM) 4μL

[0030] Sense Primer 1 μL

[0031] Anti-sense Primer 1μL

[0032] Spleen total cDNA 1 μL

[0033] Prime STAR TM HS DNA Polymerase 0.5μL

[0034] h 2 O 32.5 μL

[0035] 50 μL total

[0036] The reaction conditions are: 98°C for 1min; 30 cycles of 98°C for 10s, 68°...

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Abstract

The invention discloses a gene complete sequence of siganus oramin L-amino acid oxidase and application thereof. The gene complete sequence of the siganus oramin L-amino acid oxidase is 1725bp, the length of an open reading frame is 1584bp, and the gene complete sequence is specifically in a 43-1626 region and is used for coding 527 amino acids. The siganus oramin L-amino acid oxidase is obtained from siganus oramin serum through separation and purification, and has a function of killing cryptocaryon irritans brown and a bactericidal activity on typical gram-positive bacteria (staphylococcus aureus) and gram-negative bacteria (escherichia coli). In an optimized high-definition enzyme PCR (Polymerase Chain Reaction) system, by using total siganus oramin spleen cDNA as a template and adopting a pair of designed specific primers, the gene sequence coding the protein can be rapidly accurately cloned and used for the subsequent genetic engineering.

Description

technical field [0001] The invention belongs to the technical field of biological control, and in particular relates to a complete sequence of the gene of L-amino acid oxidase of macula macula and its use Background technique [0002] With the gradual improvement of the degree of intensive culture in seawater fishery, the density of culture increases, and the environment of culture gradually deteriorates, resulting in increasingly serious diseases. The extensive use of antibiotics and illegal drugs poses a serious threat to food safety, so the development of new drugs is imminent. [0003] Siganus oramin belongs to the order Perciformes, the suborder Acanthuroidei, the family Siganidae, and the genus Siganus. It is a small warm-water fish in the offshore. It has a wide range of water temperature and salinity suitable for growth, and it is abundant in the southeast coast of my country. [0004] The invention separates and purifies an active protein with resistance and killi...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/02C12N15/10A61K38/44A61P33/02
CPCY02A50/30
Inventor 李安兴黎睿君王方华
Owner SUN YAT SEN UNIV
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