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Ofoga gene of β-n-acetylglucosyl hydrolase from the Asian corn borer and its application

A technology of Asian corn borer and aminoacetyl, applied in the field of genetic engineering, can solve the problems of chemical pesticides without specific effects and time-consuming and laborious application methods

Inactive Publication Date: 2016-03-02
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no specific chemical pesticide, and due to its borer characteristics, the application method is time-consuming and labor-intensive

Method used

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  • Ofoga gene of β-n-acetylglucosyl hydrolase from the Asian corn borer and its application
  • Ofoga gene of β-n-acetylglucosyl hydrolase from the Asian corn borer and its application
  • Ofoga gene of β-n-acetylglucosyl hydrolase from the Asian corn borer and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Obtaining of the Ostrinia officinalis β-N-acetylglucosyl hydrolase (OfOGA) gene

[0026] 1. Extraction of totalRNA from the 5th instar larvae of Ostrinia officinalis

[0027] Total RNA was extracted from the 5th instar larvae of Ostrinia officinalis using RNAisoplus (Dalian Bao Biological Company, Code No. D9108A) to obtain the total RNA.

[0028] Take 1ul of the above-mentioned total RNA of Ostrinia officinalis and carry out 1% agarose gel electrophoresis, the result is as follows figure 1 , figure 1 Shown is the gel electrophoresis analysis of the total RNA extracted from the 5th instar larvae of Ostrinia officinalis. Two of the RNA samples have clear results and meet the quality requirements and are suitable for the next experiment.

[0029] 2. RT-PCR experiment

[0030] Using the total RNA of the 5th instar larvae of Ostrinia officinalis as a template, reverse transcription was carried out to synthesize cDNA.

[0031] The primers used for gene transfer...

Embodiment 2

[0143] Example 2 Expression of Ostrinia officinalis β-N-acetylglucosaminyl hydrolase (OfOGA)

[0144] 1. Escherichia coli expression system to obtain soluble expression products

[0145] The OfOGA gene was cloned into the NcoI and EcoRI sites of the vector pET-28a(+) (Novagen Company), and 6 His tags (CATCATCATCATCATCAT) were added to the C-terminus of the protein to construct the expression vector pET28a-OfOGA-6His.

[0146]

[0147] The detailed steps are:

[0148] 1. Design NcoI and EcoRI restriction sites on the primers of OfOGA. After the PCR reaction, use NcoI and EcoRI to digest; pET-28a(+) is digested with NcoI and EcoRI; use DNALigationKit (Dalian Bao Biological Company, Code No. D6020A) Solution I in the kit, respectively ligated the digested PCR product with the digested pET-28a(+).

[0149] 2. Transform E.coliDH5α, screen positive recombinants, identify by enzyme digestion and determine the DNA sequence correctly, then transfer into E.coliBL21(DE3), and identi...

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Abstract

The invention discloses a brand-new nucleotide sequence of an asiatic corn borer beta-N-amino acetyl glucosyl hydrolase (OfOGA) gene as well as an amino acid sequence of a protein coded by the nucleotide sequence. a pET-28a(+) recombinant expression vector of the OfOGA gene is constructed on the basis of the obtained target gene segment; the recombinant expression vector is converted to the OfOGA protein expressed in an escherichia coli host cell; the host cell converted by plasmid is cultured and a positive bacterial strain is obtained through screening; the bacterial strain is cultured and the protein expression is induced; the expressed protein is separated and purified so as to be applied to the green pesticide development field; moreover, a small molecule compound can be designed by the expressed protein so as to be applied to the medical treatment as well as the development of medicines for treating diseases (the Alzheimer disease and diabetes mellitus).

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to the gene of Ostrinia officinalis β-N-acetylglucosyl hydrolase (OfOGA), the construction of an expression vector containing the gene, the protein expression of the gene in prokaryotic host cells and its application. Background technique [0002] With the development of technology, more and more O-GlcNAc modified proteins have been discovered. Among them, the dbOGAP database (http: / / cbsb.lombardi.georgetown.edu / OGAP.html) provides O-GlcNAc-modified proteins and their modification sites from different species. This O-GlcNAc modification is only regulated by two enzymes: O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (OGA). The abnormality of O-GlcNAc modification of some important proteins in organisms can cause physiological function disorder and even cause body death. Previous studies have found that the reduction of the O-GlcNAc level of the Tau prot...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/56C12N9/24C12N15/63C12N15/70C12N1/21A01N61/00A01P7/04A61K45/00A61P3/10A61P25/28C12R1/19
Inventor 杨青周罡杨君姜秀萍刘田
Owner DALIAN UNIV OF TECH
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