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PGBD-2 fusion defensin, and preparation method and application thereof

A protein and sequence technology, applied in the field of PGBD-2 fusion defensin and its preparation and application, can solve the problems of lack of research and restrictions on the promotion and application of biological feed

Inactive Publication Date: 2012-11-14
成都市金之源生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the research on the effect and safety evaluation system of biological feed is extremely lacking at home and abroad, which seriously restricts the promotion and application of biological feed

Method used

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  • PGBD-2 fusion defensin, and preparation method and application thereof
  • PGBD-2 fusion defensin, and preparation method and application thereof
  • PGBD-2 fusion defensin, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1, construction and cloning of novel porcine defensin fusion gene PGBD-2

[0053] Modification of porcine defensin-2 gene by molecular design and overlapping PCR technique. Remove part of the inactive groups, retain the key active parts, and add a flexible linking peptide sequence to the sequence, and introduce BamHI and XhoI sites and EcoRI and XbaI restriction sites at both ends, design a stop codon at the end, and clone PCR product, and constructed the pGPGBD-2 gene expression vector. After BamHI, EcoRI digestion and plasmid PCR identification, it was confirmed that the PGBD-2 gene had been cloned into the prokaryotic expression vector pGEX-4T-1, which laid the foundation for further research on its induced expression conditions and biological functions. Specific steps are as follows:

[0054] 1. Cloning of fusion gene PGBD-2

[0055] 1. Design and synthesis of PGBD-2 gene primers

[0056] Based on the cDNA sequences encoding the mature peptides of por...

Embodiment 2

[0091] Example 2, Prokaryotic expression and expression activity research of novel porcine defensin fusion gene (PGBD-2)

[0092] The constructed PGBD expression vector plasmid was transformed into Escherichia coli BL21(DE3), and the expression of the fusion protein was induced by IPTG. SDS-PAGE analysis showed that the fusion protein could be correctly transcribed in E. coli, with a molecular weight of about 37KD, which was consistent with the expected target band size. The protein expression effect was the best when induced for 5 hours at 28°C with a final concentration of IPTG of 1 mM. The expression product of the fusion protein exists in a soluble form. After the fusion protein was purified with Glutathione Sepharose 4B, the antibacterial effect was detected on Escherichia coli ATCC25922, Pseudomonas aeruginosa (Pseudomonas aeruginosa) ATCC10211, Staphylococcus aureus (Staphylococcus aureus) ATCC26112, and Streptococcus pneumoniae strains, and it was found that the fusion...

Embodiment 3

[0115] Example 3, Yeast expression and expression activity of porcine defensin fusion gene (PGBD-2)

[0116] The defensin fusion gene PGBD-2 was cloned into the baker's yeast secretion expression vector pYCa (pYES2 / CT was inserted into the a-factor secretion signal peptide), transformed into yCY3 baker's yeast, and high-efficiency expression strains were screened; the recombinant strains were induced to express the fusion defensin Finally, the expression supernatant was analyzed for antibacterial activity and purified protein electrophoresis, and it was found that the fusion defensin existed in the expression supernatant, and the molecular weight was approximately 11kDa; the secretory expression of the fusion defensin was realized; the produced fusion defensin was effective against Escherichia coli, Salmonella , Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus pneumoniae all had significant antibacterial activity. Specific steps are as follows:

[0117] 1. Amp...

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Abstract

The invention discloses a PGBD-2 fusion defensin, and a preparation method and application thereof. The fusion defensin is a protein shown in 1), 2) or 3): 1) a protein composed of an amino acid sequence shown in SEQ 2 in a sequence table; 2) a protein fused by a GST (glutathione S-transferase) label and the protein shown in 1); and 3) a protein which has an antibacterial activity and is derived from the protein shown in 1) or 2) through treating the residue sequences of the amino acids used for forming the protein shown in 1) or 2) by the replacement and / or deletion and / or addition of one ormore amino acid residues. The invention firstly alters the protein structure of porcine defensin-2 gene so as to build a protein with higher antibacterial activity than original porcine defensin. A novel defensin molecule is acquired through a gene engineering means, and the biological and immunological characteristics of the defensin molecule are further studied, thus favoring the building of novel multifunctional defensin active molecules, the adjusting of porcine immune response and the resisting of pathogen infection of bacteria, viruses, etc.

Description

technical field [0001] The present invention relates to PGBD-2 fusion defensin and its preparation method and application. Background technique [0002] The aquaculture industry (animal husbandry and aquatic products) is an important part of large-scale agriculture. It is one of the important foundations for my country's agricultural modernization, rural economic development, and social stability. The aquaculture industry in advanced countries in Europe and the United States is very developed, accounting for 50% of large-scale agriculture. % above the specific gravity. It provides human beings with meat, milk, eggs, fur and other high-grade foods and important textile raw materials. Animal products are the main source of high-quality protein, minerals, vitamins and other nutrients for people. Their safety is directly related to human health. In recent years, vicious events such as poisoning and disease transmission caused by livestock products have occurred continuously, suc...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N5/10C12N1/21C12N1/19C12P21/02A61K38/17A61P31/04A61P31/20A61P31/14A23K1/16C12R1/91C12R1/19A23K20/147
Inventor 高荣苏向东王海燕贾贤齐万小平杨发秀严俊勇白光明
Owner 成都市金之源生物技术有限公司
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