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Chitin binding protein Bt-CBP, and coding gene and preparation method and application thereof

A protein-binding and chitin-binding technology, applied in the field of genetic engineering, can solve problems such as poor ability to inhibit fungal diseases, and achieve good inhibitory effect, good biological control function, and enhanced inhibitory effect

Inactive Publication Date: 2017-08-29
NANKAI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The purpose of the present invention is to solve the problem that Bacillus thuringiensis has good insecticidal performance but poor ability to inhibit fungal diseases, and provides a chitin-binding compound derived from Bacillus thuringiensis with high-efficiency anti-pathogenic fungal activity and a simple preparation method. Protein Bt-BCP and its coding gene, preparation method and application

Method used

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  • Chitin binding protein Bt-CBP, and coding gene and preparation method and application thereof
  • Chitin binding protein Bt-CBP, and coding gene and preparation method and application thereof
  • Chitin binding protein Bt-CBP, and coding gene and preparation method and application thereof

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Embodiment 1

[0027] Embodiment 1, expression and purification of chitin-binding protein Bt-CBP

specific Embodiment approach

[0028] In the present invention, the genome of Bacillus thuringiensis ATCC-35866 is used as a template, Up1 and Dn1 are used as upstream and downstream primers, and the Bt-CBP gene sequence is amplified by full gold high-fidelity TransStartFastPfu DNAPolymerase. Then, using the full gold seamless ligation kit, the amplified fragment and the pET-28a(+) linear fragment digested by NcoI and XhoI were mixed in a PCR tube at a ratio of 5:1, a one-step method Construction of recombinant expression plasmids. The recombinant expression plasmid was transformed into Escherichia coli DH5ɑ, and after verification was correct, the recombinant expression plasmid pET28(+)Bt-CBP was extracted and transformed into Escherichia coli BL21 to obtain the recombinant expression strain E.coli Bt-CBP. After culturing, IPTG inducer, sonication, and centrifugation to collect the supernatant is the crude protein. The crude protein was purified by affinity chromatography on a nickel column to obtain chit...

Embodiment 2

[0049] Example 2, the synergistic effect of chitin-binding protein Bt-CBP on Bt chitinase ChiB

[0050] Preparation of Bt chitinase ChiB:

[0051] Using the genome of Bacillus thuringiensis ATCC-35646 as a template, using Up2 (SEQ ID No.6) and Dn2 (SEQ ID No.7) as upstream and downstream primers, the high-fidelity TransStart FastPfu DNAPolymerase of Quanshijin Company was used to amplify Bt- ChiB gene sequence, PCR amplification system and procedure are consistent with the amplification of Bt-CBP gene. A 2056bp Bt-ChiB nucleic acid fragment was obtained, the sequence of which is shown in (SEQ ID No.8). Subsequent one-step construction of E. coli expression plasmid pET28a(+)Bt-ChiB, construction of recombinant expression strain E.coli(Bt-ChiB) and induction, fragmentation and protein purification of recombinant expression strains are all related to chitin-binding protein Bt-CBP The preparation method of Bt-ChiB is the same, and the amino acid sequence of the obtained Bt-ChiB ...

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Abstract

The invention relates to a chitin binding protein Bt-CBP, and a coding gene and a preparation method and an application thereof and belongs to the technical field of gene engineering. The coding gene of the chitin binding protein is cloned from bacillus thuringiensis for the first time, and an expression product with high activity is obtained by constructing a prokaryotic expression vector and expressing the gene in escherichia coli. The chitin binding protein Bt-CBP has a good inhibiting effect on cucumber gray mold and fusarium oxysporum. In addition, the chitin binding protein Bt-CBP has a synergic effect on catalytic activity of chitin ChiB, can promote ChiB to efficiently degrade chitin, can remarkably enhance the inhibiting effect of ChiB on cucumber gray mold, has a good biological control function, provides a novel research direction for research of antifungal drugs in the agricultural field, has wide application value and generates a huge industrial benefit and economical value.

Description

technical field [0001] The invention relates to a chitin-binding protein derived from Bacillus thuringiensis and its coding gene, preparation method and application, and belongs to the technical field of genetic engineering. Background technique [0002] Chitin, commonly known as chitin and chitin, is a polymer of N-acetylglucosamine (GlcNAc). In nature, chitin is a biopolymer substance second only to cellulose in content. The degradation of chitin is mainly accomplished by microorganisms producing chitinase. [0003] The microbial chitin degradation system is mainly composed of endochitinase, exochitinase, acetylhexosaminidase and chitin-binding protein. Among them, chitin-binding proteins (chitin-binding proteins, CBP) disperse chitin sugar chains, and endochitinase randomly cuts polysaccharide chains to generate chitin oligosaccharides; exochitinase degrades polysaccharides along the sugar chain ends Butin, the generated chitobiose is converted into monosaccharide under...

Claims

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Application Information

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IPC IPC(8): C07K14/32C12N15/70A01N37/46A01P3/00C12Q1/34
CPCA01N37/46C07K14/32C12N15/70C12Q1/34
Inventor 蔡峻张娜李蓬飞陈月华
Owner NANKAI UNIV
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