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Antibacterial peptide gene of Chinese prawn and its colon technique

A Chinese prawn and antimicrobial peptide technology, applied in the field of molecular biology, can solve the problem that the research on antimicrobial peptides has not yet been reported.

Inactive Publication Date: 2004-10-20
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are many researches on insect antimicrobial peptides in China. The expression of limulus antifungal peptides in Limulus has also been reported, and gratifying results have been achieved in antimicrobial peptide transgenic plants, but the research on antimicrobial peptides from crustaceans has not been reported yet.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0094] Embodiment 1. A cloned Chinese prawn antimicrobial peptide gene has the following sequence:

[0095] sequence listing

[0096] Homologous gene one:

[0097] (1) Information of SEQ ID N0 1

[0098] (a) Sequence features:

[0099] *Length: 600 bp

[0100] *Type: nucleic acid

[0101] * Chain type: double chain

[0102] *Topology: Linear

[0103] (b) Molecular type: cDNA

[0104] (c) Assumption: No

[0105] (d) Antisense: no

[0106] (e) Original source: Prawns (Penaeus chinensis)

[0107] (f) Sequence description: SEQ ID NO.1

[0108]ATGCGCCTCGTGGTCTGCCTGGTCTTCTTGGCCTCCTTCGCCCTGGTCTGCCAAGGGCAA 60

[0109] AAGGGTGGTTACACACGCCCGATATCCAGACCACCCTATGGGGGAGGATATGGCAATGTT 120

[0110] TGCACTTCATGCCACGTTTCTTACCACCTCACAAGCTCGTTCTTGCTGCAGTCGGTTTGGA 180

[0111] CGTTGCTGTGTGCCAAGAAGAGGATACAGTGGTTGATGAAGAAGACAACGAAAACCTGAC 240

[0112] TTCACAACGTATTCATTATTGTGAAGAGACTGCAACCCTGATTTTGAACTGTATTTTCT 300

[...

Embodiment 2

[0130] Embodiment 2. Cloning method of Chinese prawn antimicrobial peptide gene

[0131] 1. Extraction of total RNA: extract hemolymph from the abdominal sinus of the base of the first abdominal segment of Chinese prawns over 10 cm in length, add 1 / 10 volume of anticoagulant (10% sodium citrate, pH7) and 200 μM phenyl Thiourea (as melanization inhibitor). 30 ml of hemolymph was centrifuged at 700g for 15 minutes at 4°C to collect blood cells. The collected blood cells were extracted with Nippon Takara Biotechnology RNA extraction kit Catrimox TM Extract total RNA.

[0132] 2. cDNA first-strand synthesis: about 30 micrograms of total RNA, plus 20 microliters of reaction solution (50 millimoles potassium chloride, 3 millimoles magnesium chloride, 10 millimoles tris-hydrochloric acid buffer (Tris-HCl ) pH 8.3, 1 millimolar dithiothreitol (DTT), 5 micromolar oligodeoxythymonucleotide (Oligod(T)17), 500 micromolar deoxyribonucleotide mixture (dNTP), 25 units of RNA Enzyme inhib...

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PUM

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Abstract

An antibacterial peptide gene of Chinese prawn and its cloning process are disclosed. Its gene sequence features 600 base pairs for length, nucleic acid type, dual chains, and linear topology structure. The said antibacterial peptide gene is cloned from Chinese prawn by RT-PCR or Nested-PCR reaction, 3' RACE method, and smart cDNA method, and can be used for recombinant expression of the antibacterial peptide gene and its transfer, and so for preventing and treating diseases of prawn.

Description

(1) Technical field [0001] The invention relates to a Chinese prawn antimicrobial peptide gene and cloning technology, belonging to the technical field of molecular biology. (2) Background technology [0002] The immune response of insects / arthropods is a very sensitive system and thus can effectively resist the invasion of external microorganisms. Their immune response mainly depends on three reactions: proteolytic cascade reaction, that is, the rapid immune response caused by animal injury stimulation, including blood agglutination reaction, phenoloxidase cascade reaction and opsonization; blood cell phagocytosis of invading microorganisms and enveloping effect; after being stimulated, a large number of antimicrobial peptides or polypeptides are rapidly and instantaneously synthesized, and these molecules are mainly synthesized by fat body or blood cells, and are rapidly released into the hemolymph. Since the discovery of the first antimicrobial peptide in an insect, to d...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C12P19/34
Inventor 王金星赵小凡相建海
Owner SHANDONG UNIV