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Constructing method and application of transgenic chlamydomonas reinhardtii for expressing epinephelus antibacterial peptide piscidin

A grouper antibacterial peptide, Rhine coat technology, applied in application, genetic engineering, plant genetic improvement and other directions, can solve the problems of low yield of antibacterial peptide piscidin, complicated separation and purification process, inactive antibacterial peptide and other problems

Active Publication Date: 2013-10-02
SHENZHEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] In order to solve the problem of low yield, high cost and complex production process of the antimicrobial peptide piscidin, especially the antimicrobial peptide expressed in the process of gene engineering recombinant expression antimicrobial peptide has no activity or low activity, and the antimicrobial peptide molecule is easily hydrolyzed during the expression process, and the subsequent separation Purification process is complicated and other problems, the present invention is based on the technology of the applicant's previous patent No. The method of effectively expressing the antimicrobial peptide piscidin has achieved success, laying the foundation for industrialized large-scale production of the antimicrobial peptide piscidin

Method used

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  • Constructing method and application of transgenic chlamydomonas reinhardtii for expressing epinephelus antibacterial peptide piscidin
  • Constructing method and application of transgenic chlamydomonas reinhardtii for expressing epinephelus antibacterial peptide piscidin
  • Constructing method and application of transgenic chlamydomonas reinhardtii for expressing epinephelus antibacterial peptide piscidin

Examples

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Effect test

Embodiment 1

[0037] [Example 1] Optimization and synthesis of grouper antimicrobial peptide piscidin gene sequence

[0038] The inventors conducted experiments using the partial gene sequence of the piscidin mature peptide that was not optimized according to the codon preference of Chlamydomonas reinhardtii, and found that the unoptimized partial gene sequence of the mature piscidin peptide could not be effectively expressed in Chlamydomonas reinhardtii, the results of western blot showed , the corresponding target protein could not be detected in the transgenic Chlamydomonas reinhardtii integrated with unoptimized piscidin mature peptide partial gene. In addition, the antibacterial activity experiment was carried out with the total protein of transgenic Chlamydomonas reinhardtii integrated with the unoptimized piscidin mature peptide partial gene. The results showed that the transgenic Chlamydomonas reinhardtii total protein integrated with the unoptimized piscidin mature peptide partial g...

Embodiment 2

[0040] [Example 2] Selection and cultivation of transgenic recipient algal strains

[0041] When carrying out the genetic transformation of the exogenous gene of Chlamydomonas reinhardtii, different genetic transformation methods can select different strains of Chlamydomonas reinhardtii for genetic transformation. For example, "gene gun" or "electric shock transformation" is used for genetic transformation to select cell wall-deficient or non-cell wall-defective strains of Chlamydomonas reinhardtii. If the "bead milling method" is used for genetic transformation, the cell wall-defective Chlamydomonas reinhardtii can be selected, which helps to improve the genetic transformation efficiency of the exogenous gene. In the selection of selection markers for transgenic recipient algae strains, the above several genetic transformation methods can select auxotrophic algae strains or antibiotic selection markers.

[0042] The transgenic recipient strain selected in the present inventi...

Embodiment 3

[0043] [Example 3] Construction of the Chlamydomonas reinhardtii nucleus genetic expression vector of the antimicrobial peptide piscidin gene optimized by codons

[0044] The plasmid piscidin-pUC57 containing the piscidin gene optimized through Chlamydomonas reinhardtii codon preference can be obtained from Example 1, and due to the addition of appropriate restriction enzyme sites while optimizing the sequence, it can be passed through the restriction The codon-optimized piscidin gene was obtained by double digestion with endonucleases NheI and PmaCI.

[0045] What the present invention uses is Chlamydomonas reinhardtii nuclei genetic transformation plasmid pH124, first digests plasmid pH105 with EcoRI (Wang Chaogang et al., Hsp70A-RBCS2 integrated promoter regulates the expression of PHB synthase gene in Chlamydomonas reinhardtii, Chinese Science, Series C: Life Science, 2009, Volume 39, Issue 8, 778-782) Obtained about 731bp of the part containing the HSP70A-RBCS2 promoter a...

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Abstract

The invention provides a constructing method and application of transgenic chlamydomonas reinhardtii for expressing epinephelus antibacterial peptide piscidin, provides an epinephelus antibacterial peptide piscidin gene with an optimized chlamydomonas reinhardtii codon, a carrier with the gene and a transformant and also provides an application of the epinephelus antibacterial peptide piscidin gene with the optimized chlamydomonas reinhardtii codon in the preparation of the transgenic chlamydomonas reinhardtii.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the construction and application of a transgenic Chlamydomonas reinhardtii expressing grouper antibacterial peptides. Background technique [0002] Antimicrobial peptides are a class of small molecular polypeptides with broad-spectrum antimicrobial activity, widely exist in the biological world, and are important non-specific immune factors in the innate immune system. Antimicrobial peptides have the characteristics of small molecular weight, heat stability and good water solubility, and their mechanism of action is different from traditional antibiotics. After use, they will not produce drug resistance to bacteria, and antimicrobial peptides will be metabolized very quickly after entering the body, and will not be produced in the body. Harmful residue. Because antimicrobial peptides have broad-spectrum antibacterial activity, they have a strong killing effect on bacteri...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/63C12N1/13A23L1/29C12N15/79A01P1/00A01P3/00A23L33/00
Inventor 邓利何凡胡章立
Owner SHENZHEN UNIV
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