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Hyphantria cunea cecropin A gene as well as expression protein and application thereof

A technology of American white moth and cecropin, applied in the field of genetic engineering, can solve the problem of not being cloned, and achieve the effect of strong antibacterial activity and wide application prospect.

Inactive Publication Date: 2015-02-18
NANJING FORESTRY UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] According to the search results of the three major international nucleic acid sequence databases of GenBank, EMBL and DDBJ, the cDNAs of Cecropin A and Cecropin A have been cloned, and their sequence numbers have been applied for respectively in the GenBank database. The cDNA of the American white moth has not been cloned yet, and the research on the Cecropin A gene of the American white moth is still completely blank at home and abroad

Method used

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  • Hyphantria cunea cecropin A gene as well as expression protein and application thereof
  • Hyphantria cunea cecropin A gene as well as expression protein and application thereof

Examples

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Embodiment 1

[0017] Example 1 Cloning of Cecropin A cDNA

[0018] 1. Extract the total RNA of white moth.

[0019] Use RNA extraction reagent (TIANGEN) to extract the total RNA of fat body of white moth pupae according to its operation manual, and identify its purity by formaldehyde-denatured agarose gel electrophoresis, which is greater than 95%, and its concentration is 720 ng as determined by ultraviolet spectrophotometer / μL, to meet the needs of use.

[0020] 2. Use Transgen cDNA reverse transcription kit to transcribe into first-strand cDNA.

[0021] 1) Primer design: According to the sequence alignment of Cecropin A in NCBI, Bombyx mori and Drosophila, degenerate primers were designed:

[0022] Cec-A1: 5'-ATGAATTTCNCAANAATNNNTNTNCTTCGT-3',

[0023] Cec-A2: 5'-NTATTTNCNAANGNTTTTTNCNGTACCCAG-3'.

[0024] 2) Reverse transcription: Add 3 μL of total RNA extract, Oligo d(T) to DEPC-treated 1.5mL Eppendorf tubes sequentially 18 (0.5 μg / μL) 1 μL, 2×TS Reaction Mix 10 μL, DEPC water 5 ...

Embodiment 2

[0027] According to the cecropin A protein sequence obtained in Example 1, the polypeptide sequence (N-terminal-C-terminal) of the cecropin A antimicrobial peptide of the American white worm was synthesized in vitro: RWKIFKKIERVGQNVRDGIIKAGPAIQVLGTAKALGK, the main specific process is as follows:

[0028] First, an amino acid whose amino group is protected by a blocking group is covalently linked to a solid support. Under the action of trifluoroacetic acid, the protective group of the amino group is removed, so that the first amino acid is connected to the solid phase carrier. Then the carboxyl group of the second amino acid whose amino group is blocked is activated by N,N'-dicyclohexylcarbodiimide (DCC, Dicyclohexylcarbodiimide), and the carboxyl group is activated by DCC. Amino groups of two amino acids react to form peptide bonds, thus generating a dipeptide with a protecting group on the solid support.

[0029] Repeat the above peptide bond formation reaction to make the p...

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Abstract

The invention discloses a hyphantria cunea cecropin A gene as well as expression protein and application thereof. The hyphantria cunea cecropin A gene has a DNA (deoxyribonucleic acid) sequence as shown in SEQ ID NO.1 and an amino acid sequence as shown in SEQ ID NO.2. According to the hyphantria cunea cecropin A gene, the full-length cDNA (complementary DNA) sequence and the expression protein of the cecropin A gene cloned from hyphantria cunea for the first time are obtained, hyphantria cunea cecropin A antibacterial peptide with amidated C end is synthesized for the first time by a chemical method according to the protein sequence, and the antibacterial activity of synthetic hyphantria cunea cecropin A antibacterial peptide on escherichia coli K12D31 (E.coli K12D31) and agrobacterium EHA105 is tested by an agarose hole diffusion method; the hyphantria cunea cecropin A gene is very high in antibacterial activity and has a wide application prospect in bacterial inhibition.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a cecropin A (Cecropin A) gene of the American white moth, its expressed protein and its application. Background technique [0002] Antimicrobial peptides are a class of polypeptide substances with broad-spectrum and high-efficiency bactericidal activity, and are an essential part of the non-specific immune system in nature, especially insects. In 1972, Swedish scientist Boman et al. first discovered the antimicrobial peptide cecropins from silkworm chrysalis, and then isolated antimicrobial peptides from various animals (mammals, amphibians, etc.) and plants. In addition, some bacteria can also produce antimicrobial peptides. With the deepening of research, it is found that some antimicrobial peptides also have a significant killing effect on certain pathogenic fungi, pathogens, viruses and even tumor cells, and are a class of active substances with impor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C07K14/435A61P31/04
CPCY02A50/30
Inventor 诸葛强张嘉鑫武小龙王晓立潘惠新尹佟明
Owner NANJING FORESTRY UNIV
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