Macadamia plant defensin and its application

A technology of plant defensins and nuts, which is applied in the field of biotechnology and genetic engineering to achieve the effects of promoting expression, increasing the survival rate of animals challenged with viruses, and inhibiting growth

Active Publication Date: 2020-11-06
四川百可馨生物制药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Reports on the cloning and expression of plant defensin genes and other related genes are gradually increasing, but there are few reports on the optimization of plant defensin short peptide genes through codon degeneracy and high expression in Saccharomyces cerevisiae

Method used

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  • Macadamia plant defensin and its application
  • Macadamia plant defensin and its application
  • Macadamia plant defensin and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Cloning of Example 1 Macadamia Nut Plant Defensin Protein Gene

[0062] The sequence of macadamia plant defensin protein gene is:

[0063] ATTCAGGGGTTTGTGCTTAAGCCACGGAACTGTGCTAATGTGTGTCGGACTGAGGGTTTTCCCGGCGGTACTTGCAAAGGGTTCCGGCGTCGTTGCTTCTGTGAGAAGAATTGTCAT

[0064] The amino acid sequence of the encoded protein macadamia plant defensin is:

[0065] IQGFVLKPRNCANVCRTEGFPGGTCKGFRRRCFCEKNCH (shown in SEQ ID No.1)

[0066] There are deficiencies in the application of yeast expression system to express the target protein of exogenous gene, such as internal degradation, heterogeneous product, multimer formation, incomplete addition of signal peptide, etc., resulting in significant differences in the structure of the expressed protein. At the same time, excessive glycosylation of the expressed product will reduce the activity of the protein. The study found that the gene could not be expressed in yeast. Therefore, using bioinformatics analysis tools, the expression efficien...

Embodiment 2

[0081] Construction and identification of embodiment 2pYCT2-a recombinant expression vector

[0082] The cloning product of AZJG-DEFENSIN gene was double-digested with XhoI and XbaI to digest the plasmid, and the target fragment was recovered and ligated with the same double-digested secreted expression vector pYCa under the action of IN-FUSION ligase, and transformed into competent cells of INVSC1 Saccharomyces cerevisiae by electric shock , obtained pYCa-AZJG-DEFENSIN positive recombinant yeast, named AZJG-DEFENSIN recombinant yeast, and obtained positive recombinant transformants by screening on yeast selection medium SD-U plate; cultured colonies were extracted for plasmid PCR and XhoI and XbaI double enzyme digestion identification ( figure 1 ), gel electrophoresis showed that a single band appeared at all sizes, and the size was in line with expectations, proving that the recombinant pYCa plasmid was successfully constructed ( figure 2 ); Sequencing confirmed the corre...

Embodiment 3

[0083] Example 3 Expression Detection of Target Gene in RNA Level in Recombinant Saccharomyces

[0084] AZJG-DEFENSIN recombinant yeast was inoculated in uracil auxotrophic medium at 28°C, cultured overnight at 200 rpm, and centrifuged to obtain AZJG-DEFENSIN recombinant yeast cell. Extract the total RNA of the bacteria, and use the primer pair (F: 5'-ATGTATAAGATGCAGCTCTTGT-3' (SEQ ID No.6) and R: 5'-CTAATGGTGATGGTGATGAT-3' (SEQ ID No.7)) to detect the AZJG-DEFENSIN recombinant yeast The expression level of the target gene. The results showed that the size of the target band (122bp) in AZJG-DEFENSIN recombinant yeast was consistent with that of the marker band near the 100-150 interval. It indicated that the AZJG-DEFENSIN gene in the AZJG-DEFENSIN recombinant yeast was expressed. see image 3 .

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Abstract

The invention discloses macadimia nut plant defensin and an application thereof. The amino acid sequence of the macadimia nut plant defensin is as shown in SEQ ID No.1. The invention also discloses nucleotide coding a protein according to claim 1, and the nucleotide sequence of the nucleotide is as shown in SEQ ID No.2 or a degenerate sequence which codes the same protein as the SEQ ID No.2. The macadimia nut plant defensin can promote proliferation of lymphocytes and leukocytes, maintain stability of erythrocytes, inhibit growth of pathogenic microorganisms, enhance humoral immunity (IgG, IgG1, IgG2 and a specific antibody) and cellular immunity (CD4<+>, CD8<+> cells) and improve the immunity of human and animals and antimicrobial resistance.

Description

technical field [0001] The invention belongs to the field of biotechnology and genetic engineering, and specifically relates to the optimization of macadamia plant defensin gene, the construction of Saccharomyces cerevisiae expression vector, gene expression technology and its application methods for human and animal disease resistance health care, wound repair promotion and anti-inflammation. Background technique [0002] Defensins are a class of cationic antibacterial active peptides discovered in recent years and widely exist in animals and plants. They are usually composed of 28 to 54 amino acid residues, and the molecules are rich in arginine and intramolecular disulfide bonds formed by cysteine. They have broad-spectrum and high-efficiency bactericidal activity, and can effectively kill Gram-positive bacteria, Gram-negative bacteria, certain fungi, spirochetes, enveloped viruses and other microorganisms, so their research has become one of the current international aca...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/81C12N1/19A61K38/17A61P37/04A61P17/02A61P31/00C12R1/865
CPCA61K38/00A61P17/02A61P31/00A61P37/04C07K14/415C12N15/81Y02A50/30
Inventor 万小平魏于清杨玉玺陈兰方孙皓天付诗雨
Owner 四川百可馨生物制药有限公司
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