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Genic engineering strain of expression recombinant prawn peptide Pen24 and its use

A genetically engineered strain and genetic engineering technology are applied to the genetically engineered strain expressing the recombinant prawn peptide Pen24 and its application field, which can solve the problems of high cost, complicated chemical synthesis steps, and low yield.

Inactive Publication Date: 2007-01-17
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the chemical synthesis method has complex steps, low yield and high cost

Method used

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  • Genic engineering strain of expression recombinant prawn peptide Pen24 and its use
  • Genic engineering strain of expression recombinant prawn peptide Pen24 and its use
  • Genic engineering strain of expression recombinant prawn peptide Pen24 and its use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0115] Example 1 Extraction of total RNA of Penaeus vannamei

[0116] The vannamei were reared in an oxygenated water tank at 22°C for use. Select healthy shrimp during the moulting period, rinse with DEPC-treated sterilized water, collect 750 μL of hemolymph from the abdominal sinus of the shrimp with a 2.5 mL disposable syringe, add an equal volume of anticoagulant (PH7.0), and examine under the microscope Counting, take the cell content as 1×10 7 The hemolymph was centrifuged at 4°C, 800 g for 15 min, the supernatant was removed, and the blood cells were collected. Total RNA was extracted according to the product manual of Qiagen's RNeasyMini Kit. Store the extracted RNA solution at -80°C for future use.

[0117] Agarose gel electrophoresis of the extracted total RNA of Penaeus vannamei showed that two bands of 28S and 18S were clearly visible, and the color intensity of the two bands was approximately 2:1, indicating that the extracted total RNA was basicall...

Embodiment 2

[0118] Embodiment 2 Synthesis of the first strand of reverse transcription cDNA of Penaeus vannamei

[0119] Using Oligo(dT)15 as a primer, the first strand of cDNA was synthesized according to the instructions of Promega's Reverse Transcription Reaction kit. The specific operation steps are as follows: Add the following reagents into the PCR reaction tube soaked in DEPC and sterilized: 25mM MgCl 2 , 4 μL; 10× reverse transcription buffer, 2 μL; 10 mM dNTP mix, 2 μL; recombinant RNasin  RNase inhibitor, 0.5 μL; 24 U / μL MV reverse transcriptase, 0.8 μL; 0.5 μg / μL Oligo(dT)15 primer, 1 μL; total RNA, 3 μL; nuclease-free water, 2.7 μL. The reaction conditions are: 42°C, 1 hour; 95°C, 5 minutes; 3°C, 5 minutes.

[0120] Store the synthesized first-strand cDNA at -80°C for future use.

Embodiment 3

[0121] Example 3 Amplification of the Penaeidin-2 gene of Penaeus vannamei

[0122] 1. Synthesis of amplification primers

[0123] Design primers, upstream primer P1: 5' GAATTC TACAGGGGCGGTTACACA 3' (EcoR I site is underlined), downstream primer P2: 3'GTGAATCATTTTCCTATT TTCGAA 5' (Hind III site is underlined). Primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd. and purified by PAGE.

[0124] 2. PCR amplification of the target gene

[0125] According to the operation manual of the Reverse Transcription Reaction kit, the target gene Penaeidin-2 was amplified by PCR using the first strand of cDNA synthesized by reverse transcription as a template. PCR reaction system: 10×reactionbuffer, 5μL; 1.5mM MgCl2, 3μL; 0.2mM dNTP, 1μL; 20pmol upstream primer P1, 1μL; 20pmol downstream primer P2, 1μL; 5U / μL Taq DNA polymerase 1μL; template 6μL; Bacterial water to a final volume of 50 μL. PCR reaction conditions: 95°C, 5min; 94°C, 30s; 53°C, 45s; 72°C, 30s (35...

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Abstract

Gene engineering strain of expression recombinant prawn peptide Pen24 and its use are disclosed. It has excellent antibacterial activity for G+ bacterium, negative bacterium, colicine of antiampicillin and strong pathogenic bacterium, excellent antiviral activity. It can be used to prevent and treat animal and plant virus and as antiseptic agent in use of cosmetics, food and animal forage.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, more specifically to a genetically engineered strain expressing a recombinant prawn peptide Pen24, and more specifically relates to a genetically engineered strain E.coli OrigamiB(DE3)pLysS(pET-pen) containing a prawn peptide gene, which is related to prawn peptide The recombinant prawn peptide Pen24 has obvious antibiological activity against Gram-positive bacteria, Gram-negative bacteria, fungi, and viruses. It also relates to the preparation method of genetically engineered bacterial strains, and at the same time, also relates to the application of recombinant genetically engineered protein Pen24 in the prevention and treatment of bacteria, fungi and viral diseases of animals and plants, and its application as a preservative in the fields of cosmetics, food and feed, etc. use. Background technique [0002] Antimicrobial peptides are an important part of th...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N1/21C12N15/09A61K38/17A61P31/04A61P31/12A61K8/64A23L3/3463A23K1/00A23K20/147
Inventor 徐进平孟小林王健鲁伟付百玲
Owner WUHAN UNIV
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