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Cyanovirin-n Gene, Recombinant Protein and Application of I.

A gene and forestry technology, applied in the preparation of genes and recombinant proteins, in the field of Cyanovirin-N gene, can solve the problem of staying at the macro level, and achieve broad-spectrum bactericidal efficacy and pharmacological activity, stable properties, and yield. and high purity

Active Publication Date: 2021-06-18
TAIJI GROUP +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Yang Junyuan and other scholars have isolated a variety of valuable compounds from the cultured mycelia of I. adenocarpus, but their research is still at the macroscopic level.

Method used

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  • Cyanovirin-n Gene, Recombinant Protein and Application of I.
  • Cyanovirin-n Gene, Recombinant Protein and Application of I.
  • Cyanovirin-n Gene, Recombinant Protein and Application of I.

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] 1. Genomic DNA extraction

[0061] Genomic DNA extraction is extracted by the mean plant genome extraction kit and extracted in accordance with the instruction step.

[0062] 2. Genomic RNA extraction

[0063] The genomic RNA extracts the use of the mean plant genomic extract kit and extracted as described below.

[0064] 3. Trace fast PCR verification

[0065] Take a microphylfilate PCR amplification: sterile toothpick picking a small amount of powder bar to the EP tube, after liquid nitrogen quick-frozen grinding, add 50 μl of Lysis buffer, mix uniform, 95 ° C, 10 min; sterilized mitochronized liquid 2 μl Conventional PCR amplification.

[0066] 4. Biological information analysis

[0067] Use routine biological information analysis software.

[0068] 5. Biological information analysis results

[0069] IFCVN cDNA ORF is 369bp, encoded 122 amino acids ( figure 1 ), The molecular weight is approximately 12.9 kDa, the equidistant point is 5.99, the average hydrophobic is -0.2...

Embodiment 2

[0071] Full length of gene cDNA ORF:

[0072] Find the Powder Stamping Corpontotomy sequence to obtain a predicted IFCVN gene full length sequence, and design primers IFCVN F / R according to the sequence. The total RNA of the Brass Brass, reverse CDNA was extracted, and PCR amplification was performed, and the IFCVN gene cDNA ORF is fully long. Take 5 μl of PCR amplification product for electrophoresis verification.

[0073] Experimental results:

[0074] Amplifies a fragment of the total length of 369 bp, and the PCR amplification results are Figure 6 Indicated.

Embodiment 3

[0076] 1. Construction of IFCVN gene knockout box

[0077] (1) IFCVN gene, downstream and straw phosphine resistant gene fragment cloning

[0078] With wild-type strains FTRSY-2 genome, the IFCVN gene upstream sequence is amplified using primer IFCVNQCup F and IFCVNQCUP R, and the product is named IFCVNU; use primer IFCVNQCDOWN F and IFCVnqcdown R amplified IFCVN gene downstream sequence, the product is named IFCVND; PcambageGFP plasmids are templates, using primer BAR5 / 6 amplified lactitis resistant genes, and the product is named B. After electrophoresis test, the rubber recovery was used.

[0079] (2) Plasmid pCambiamx9-IFCVNU construction

[0080] The extracted plasmid pcambiamx9 and the recovered IFCVN gene upstream recovery fragment IFCVNu were subjected to SACI and Hind III bisase, and after electrophoresis, the recovered linearized plasmid PCAMBIAMX9 and IFCVN gene upstream fragment IFCVNU were connected, and connected The product transformed E. coli DH5α, and the primer...

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Abstract

The invention belongs to the field of molecular biology, and in particular relates to a Cyanovirin-N gene, a gene and a recombinant protein, a recombinant protein and an application thereof. The present invention starts from a strain of I. clavium FTRSY-2 bacterial strain with strong insecticidal activity, clones the I. cyrenia CVN gene from the I. cyrenia genome for the first time, and names it Ifcvn. Taking this gene as a starting point, through The means of molecular biology were used to construct recombinant vectors, engineering bacteria, recombinant CVN protein, etc., and the function of the gene was verified by constructing Ifcvn gene knockout and recovery strains. The recombinant CVN protein obtained by the invention is stable in property, high in yield and purity, has broad-spectrum bactericidal efficacy and pharmacological activity, and has the potential to be developed into biological control preparations, antibacterial drugs, polypeptide vaccines, health care products and the like.

Description

Technical field [0001] The present invention belongs to the field of molecular biology, and more particularly to the preparation method, recombinant protein and applications of powder lasee Cyanovirin-N gene, gene and recombinant protein. Background technique [0002] Pink Balloria Farinosa, is a Mito Fungal Bunch Mode, is a major insect-pathogenic fungi, and it is also a fungus that is very developing value and commercial utilization value. Specific performance At: 1) Existing studies have shown that the caids of powder bar have a good insecticidal effect on multiple types of pests; 2) Powder Bunch as a small fungus as a bat moth larvae, and the pharmaceutically acceptability is more and more It has been studied and found; 3) Separated from the strapless spore culture mycelium, including polysaccharide, insectic acid, alkaloid, pyridone, natural benzol, quinazolinone, cyclic peptide, anthracene A variety of new compounds such as compounds; 4) Studies to demonstrate a substance s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/31C12N15/10C12N15/70C12N1/21C07K14/37C07K16/14A61K38/16A61P31/04A61P31/18A23L33/17A01N61/00A01P7/04C12R1/19
CPCA01N61/00A23V2002/00A61K38/00A23L33/17A61P31/04A61P31/18C07K14/37C07K16/14C12N15/10C12N15/70A23V2200/324A23V2250/54
Inventor 秦少容胡军华卿玉玲陈仕江王帆陈若霓
Owner TAIJI GROUP