Scylla paramamosain double-stranded specific deoxyribonuclease as well as preparation method and application thereof

A technology for deoxyribonucleic acid and Scylla simulans, which is applied in the field of Scylla simulatum double-strand specific deoxyribonuclease and its preparation, and can solve the problems of low enzyme cleavage activity and the like

Inactive Publication Date: 2013-08-21
JIMEI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, DSN has high enzyme-cleaving activity for short fragments of double-stranded DNA (8-12bp) that are completely complementary

Method used

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  • Scylla paramamosain double-stranded specific deoxyribonuclease as well as preparation method and application thereof
  • Scylla paramamosain double-stranded specific deoxyribonuclease as well as preparation method and application thereof
  • Scylla paramamosain double-stranded specific deoxyribonuclease as well as preparation method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0107] The extraction of the total RNA of embodiment 1 mud crab

[0108] Extraction of total RNA and synthesis of cDNA: Isolate the hepatopancreas of Scylla pseudocavena, extract the total RNA of the hepatopancreas by RDP method, extract with phenol / chloroform / isoamyl alcohol to remove impurities such as proteins, and obtain the total RNA of the hepatopancreas with good quality (see figure 1 ), the bands of 18s rRNA and 28s rRNA were clear; the OD of total RNA was measured by UV spectrophotometer 260 / OD 280 =2.0, the total RNA can be used for follow-up experiments.

Embodiment 2c

[0109] Synthesis of the first strand of embodiment 2cDNA

[0110] Take 3 μg of RNA and treat it with DNase I first, then add 1 μL of 3’CDS primer to the 3’RACE template; add 1 μL of 5’CDS primer (10 μmol / L) and 1 μL of SMART II Oligo (10 μmol / L) to the 5’RACE template ) for reverse transcription to synthesize the first strand of cDNA.

Embodiment 3

[0111] Embodiment 3SMART II RACE technology obtains result

[0112] The first strand of 3'RACE cDNA and the first strand of 5'RACE cDNA were used as templates, and 3'RACE outer and UPM, 5'RACE outer and UPM were used as primers at both ends to carry out the first round of SMART II RACE amplification. Then use the 50-fold dilution of the first round of RACE-PCR product as a template, and use 3'RACE inner and UPM, 5'RACE inner and UPM as primers at both ends to carry out the second round of SMART II RACE amplification to obtain 3'RACE And 5'RACE results, found that 3'RACE has a specific band at 907bp, and 5'RACE has a specific band at 698bp (see figure 2 and 3 ), recover the two bands, clone and sequence the recovered target bands.

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Abstract

The invention discloses a scylla paramamosain double-stranded specific deoxyribonuclease as well as a preparation method and an application thereof and relates to double-stranded specific deoxyribonucleases. The preparation method comprises the steps of extracting total RNA (Ribose Nucleic Acid) of hepatopancreas of scylla paramamosain, carrying out reverse transcription on the total RNA by using an RT-PCR (Reverse Transcription-Polymerase Chain Reaction) technology to obtain cDNA (complementary Deoxyribose Nucleic Acid), obtaining a new DSN (Duplex-Specific Nuclease) gene by utilizing a SMART II-RACE method, constructing a prokaryotic expression recombinant plasmid of the scylla paramamosain by utilizing a genetic recombination method and carrying out high-efficiency expression and purification in escherichia coli to obtain recombinant scylla paramamosain DSN with biological activity. The invention provides a recombinant vector, comprising the gene sequence of the scylla paramamosain double-stranded specific deoxyribonuclease. The invention provides a biological agent which contains the scylla paramamosain double-stranded specific deoxyribonuclease and is applied to degrading double-stranded DNA.

Description

technical field [0001] The invention relates to a double-strand specific deoxyribonuclease, in particular to a mud crab double-strand specific deoxyribonuclease and a preparation method and application thereof. Background technique [0002] Scylla paramamosain belongs to Arthropoda, Crustacea, Decapoda, Brachyura, Portunidae, Scylla, It is an important cultured crab species in the tropical waters of the Indian Ocean and the Pacific Ocean. It is widely cultivated in Zhejiang, Fujian, Guangdong and Hainan provinces. [0003] Double-strand-specific deoxyribonuclease (Duplex-Specific Nuclease, hereinafter referred to as DSN) is a thermostable nuclease discovered from the hepatopancreas of Kamchatka stone crab (Paralithodes camtschaticus, common name: king crab) in 2004. Enzymes are capable of degrading DNA in double-stranded DNA and DNA-RNA hybrids while having little effect on single-stranded nucleic acid molecules. In addition, DSN has a high enzyme-cleaving activity for sho...

Claims

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Application Information

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IPC IPC(8): C12N9/22C12N15/55C12N15/70C12N1/21C12R1/19
Inventor 王艺磊张子平兰旺仁
Owner JIMEI UNIV
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