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Antibacterial peptide gene of Chinese prawn containing single whey acidic protein structure domain and its coded antibacterial peptide and application

A technology of whey acidic protein, Chinese prawns, applied in the field of genetic engineering

Inactive Publication Date: 2006-08-02
SHANDONG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in many related reports, there is no report on the antimicrobial peptide gene containing a single whey acidic protein domain in Penaeus chinensis. The present invention clones a single whey acidic protein domain (peptide containing single whey acidic protein (WAP) domain) antimicrobial peptide gene, although similar genes have been found in Penaeus vannamei abroad, but so far there is no antimicrobial peptide gene recombinant expression and function of the Chinese shrimp containing a single whey acidic protein domain research report

Method used

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  • Antibacterial peptide gene of Chinese prawn containing single whey acidic protein structure domain and its coded antibacterial peptide and application
  • Antibacterial peptide gene of Chinese prawn containing single whey acidic protein structure domain and its coded antibacterial peptide and application

Examples

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Embodiment 1

[0067] Example 1: Cloning of antimicrobial peptide cDNA containing a single whey acidic protein domain in Penaeus chinensis

[0068] 1) Extraction of total RNA: Total RNA was extracted by one-step method using the prior art.

[0069] 2) Synthesis of the first strand of cDNA: 6 micrograms of total RNA, plus 20 microliters of reaction solution, the reaction solution is a mixture of 50 mmol potassium chloride and 3 mmol magnesium chloride, 10 mmol tris-hydrochloric acid buffer (Tris-HCl) pH8.3, 1 millimolar dithiothreitol (DTT), 5 micromolar oligodeoxythymonucleotide (Oligod(T) 17 ), 500 micromole deoxyribonucleotide mixture (dNTP), 25 units of RNase inhibitor, 8 units of AMV reverse transcriptase, reacted at 42° C. for 90 minutes, and terminated the reaction at 70° C. for 10 minutes.

[0070] 3) PCR reaction: chain polymerase reaction (PCR) reagents and conditions:

[0071] First mix the following reagents together:

[0072] 10xTaq DNA polymerase buffer 5 microliters (μ...

Embodiment 2

[0095] Example 2: Recombinant expression vector construction, expression and antibacterial function assay

[0096] (1) According to the sequence of the antimicrobial peptide gene containing a single whey acidic protein domain in Penaeus chinensis and the cloning site of the expression vector pPET30a (Novagen Company), design primers:

[0097] Wap F: CGC GAT GAA TTC ATG GTG AAC ATC AAG GAA G(EcoR I)

[0098] Wap R: TAG ATC CTC GAG TTT TCC GTA GGG AGA TCC CAC(XhoI)

[0099] The present invention selects the EcoR I and Xho I restriction sites of the pET30 a cloning site. Therefore, when designing the primers, the EcoR I restriction site is introduced into the upstream primer, and the Xho I restriction site is introduced into the downstream primer.

[0100] (2) Gene amplification, cloning and recombinant plasmid screening

[0101] Using pMD-18T-Wap as template, carry out PCR reaction with the above primers, the amplification conditions are: 94°C, 2min pre-denaturation; 94°...

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Abstract

The present invention discloses one kind of antibacterial peptide gene of Chinese prawn containing single whey acidic protein structure domain and its coded antibacterial peptide and application in preparing antibacterial active recombinant protein. The recombinant antibacterial peptide gene of Chinese prawn containing single whey acidic protein structure domain of the present invention may be applied in antibacterial feed additive, food preservation, animal and plant genetic conversion and medicine development.

Description

technical field [0001] The present invention relates to an antimicrobial peptide gene containing a single whey acidic protein domain and its encoded antibacterial polypeptide and its application, in particular to a Chinese prawn antimicrobial peptide gene containing a single whey acidic protein domain and its encoded Antibacterial polypeptide and its application; it belongs to the technical field of genetic engineering. Background technique [0002] Chinese prawn is an important marine species in my country, but since the 1990s, large-scale outbreaks of prawn diseases have caused serious losses to the marine aquaculture industry. Many domestic scholars have done a lot of work on the prevention and control of prawn diseases, and have made a lot of progress, and the production of prawns has also increased year by year; but so far, prawn diseases are still a problem that plagues the prawn farming industry. To solve this problem, we should start with controlling pathogens and i...

Claims

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Application Information

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IPC IPC(8): C12N15/12C07K14/435C12P21/02
Inventor 王金星赵小凡
Owner SHANDONG UNIV
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