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Recombinant serine protease and fungicide containing the same

A technology of serine protease and fungi, which is applied in the direction of nematicides, biocides, fungi, etc., can solve the problems of non-fusion, etc., and achieve the effect of improving virulence and speed

Inactive Publication Date: 2006-04-26
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, no chitin-binding domains have been found in the serine proteases cloned from insect fungi, and no serine proteases derived from insect fungi have been fused with chitin-binding domains of insect chitinases. report

Method used

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  • Recombinant serine protease and fungicide containing the same
  • Recombinant serine protease and fungicide containing the same
  • Recombinant serine protease and fungicide containing the same

Examples

Experimental program
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Effect test

Embodiment 1

[0038] 1. Cloning of chitin binding domain

[0039] The amplified template is cDNA reverse-transcribed from the mRNA of the fourth-instar silkworm (provided by the Sericulture College of Southwest Agricultural University) during the molting period (the reverse transcription kit is a product of TaKaRa Company) as a template.

[0040] Primers were designed according to the sequence of silkworm chitinase (GenBank accession number: AB052914):

[0041] P1: 5'-ACT AGT CAC AAC CAC CAC CAC CGT G-3'

[0042] P2: 5'-GCG GCC GCC CGG GTT ACG AAC ATF CCG GTC TGT-3'

[0043] High-fidelity pfu DNA polymerase (product of Dingguo Company) was used for PCR amplification, and the PCR amplification conditions were as follows: 94°C for 5min; 94°C for 30sec, 58°C for 30sec, 72°C for 1min, 30 cycles; adding 0.7 units of Taq DNA polymerase was extended for another 20 min. A binding domain gene (sequence shown in SEQ ID NO.4) of about 340 bp was obtained by PCR amplification, and corresponding enzy...

Embodiment 2

[0065] 1. Construction of CDEP-1 yeast expression vector

[0066] pMD-CDEP was digested with EcoRI / NotI, and the foreign fragment of about 1 kb was recovered, and cloned into the pPIC9K (Invitrogen Company) vector digested with the same enzyme. It was verified by EcoRI / NotI digestion, and the positive clone was named pPIC9K-CDEP, and its plasmid structure is shown in figure 1 A (The amino acid sequence of the expression product is shown in SEQ ID NO.9).

[0067] 2. Construction of CDEP-SwChBD yeast expression vector

[0068] Digest pMD-hCDEP with EcoRI / XbaI, recover an exogenous fragment of about 1kb, digest pMD-SWChBD with SpeI / NotI, and recover a 340bp exogenous fragment, and clone the two fragments recovered above into EcoRI / NotI enzyme at the same time Cut pPIC9K (Invitrogen) vector. The positive clone identified by EcoRI / NotI digestion was named pPIC9K-CDEP-SwChBD, and its plasmid structure is shown in figure 1 B, the amino acid sequence of the expression product is s...

Embodiment 3

[0079] 1. Construction and transformation of CDEP-1 transformation vector of Beauveria bassiana

[0080] Digest pMD-spCDEP with EcoRI / SmaI, recover a 1140bp fragment, digest pUC-bar::gfp with SmaI / HindIII, recover the 750bp Trp terminator, and digest pUC-bar::gfp with EcoRI / HindIII , Recover the vector sequence of 10000bp. The three recovered fragments were ligated and transformed into Escherichia coli Dh5α. Validation was performed with EcoRI / HindIII. The positive clone identified by enzyme digestion was named pUC-bar::gfp-CDEP, and its plasmid structure is shown in figure 2 a. The plasmid of the positive clone was extracted and transformed into Beauveria bassiana Bb0062 strain after linearization with HindIII (Beauveria bassiana (Beauveriabassiana) Bb0062 was isolated from the cabbage caterpillar (Pieris rape) infected, preserved in the Southwest Agricultural University Biotechnology Center, It can also be obtained from known sources, such as CCTCCAF93297 or isolated fr...

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Abstract

The present invention relates to one kind of recombinant serine proteinase. The recombinant serine proteinase contains one kind of fusion protein comprising serine proteinase from fungus and chitin binding domain from insect chitinase fused together and contains one gene coding serine protein from fungus and one gene coding chitin binding domain from insect chitinase with separated nucleotide sequences. The recombinant serine proteinase of the present invention may be enriched in insect body wall to raise insect body wall degrading speed and raise pesticidal fungal virulence.

Description

technical field [0001] The present invention relates to a novel fusion protein, in particular to a recombinant protein containing chitin binding domain of insect chitinase and fungal serine protease sequence. [0002] The present invention also relates to the nucleotide sequence and amino acid sequence encoding the above-mentioned recombinant protein. [0003] The present invention relates to an expression vector for expressing the above-mentioned recombinant protein. [0004] The present invention also relates to an insecticide and fungicide containing the above-mentioned recombinant protein. Background technique [0005] Insect populations are kept in check in natural environments, largely due to the widespread prevalence of insecticidal fungal diseases (Chanley, The Mycota IV Environmental and Microbial Relationships, 1997, pp 185-201). Insecticidal fungi have been widely used as biocontrol agents in my country, Europe, America and Africa (Feng Mingguang, "Prospects for...

Claims

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Application Information

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IPC IPC(8): C12N9/50A01N63/04A01P5/00C07K19/00C12N1/15C12N15/62C12N15/81
Inventor 裴炎范艳华张永军方卫国肖月华
Owner SOUTHWEST UNIVERSITY
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