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Recombinant serine protease inhibitor, fungus expression vector and fungus insecticide of recombinant protein

A technology of serine protease and inhibitor, applied in the field of insecticide fungicides, can solve the problems of low infection efficiency of entomopathogenic fungi, achieve the effects of improving insecticidal effect, increasing proliferation speed, and improving characteristics

Active Publication Date: 2013-04-10
SOUTHWEST UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In response to the above-mentioned deficiencies in the prior art, an object of the present invention is to provide a recombinant serine protease inhibitor, which contains a secreted signal peptide derived from fungi and a serine protease inhibitor derived from insects, and has the ability to inhibit insect immune response and Enhanced virulence properties of insecticidal fungi to address the low efficiency of entomopathogenic fungal infection

Method used

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  • Recombinant serine protease inhibitor, fungus expression vector and fungus insecticide of recombinant protein
  • Recombinant serine protease inhibitor, fungus expression vector and fungus insecticide of recombinant protein
  • Recombinant serine protease inhibitor, fungus expression vector and fungus insecticide of recombinant protein

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Experimental program
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Effect test

Embodiment 1

[0052] 1. Cloning of Drosophila serpin gene:

[0053] In the serpin gene of Drosophila (NM_080112), there is an intron, and the intron is removed by PCR mutual overlap. Use P1 / P2, P3 / P4 as primers respectively. The primer sequences during vector construction are as follows:

[0054] P1: 5'- GAATTC ATGGCGAGCAAAGTCTCGATCCTTCTCCTGC-3' (the underlined sequence is the EcoRI site);

[0055] P2: 5'-GGGGTACGAACTTGGCATAGGAAGCTGCCGAGGCC-3';

[0056] P3: 5'-GGCCTCGGCAGCTTCCTATGCCAAGTTCGTACCCC-3';

[0057] P4: 5'- CCCGGG TTAGACGCTCATGGGCGTGGGAT-3' (the underlined sequence is the SmaI site),

[0058] P5: 5'- TCTAGA GAGCTCATCGCTTGGCAACG-3' (the underlined sequence is the XbaI site);

[0059] P6: 5’-AAAAAA GCGGCCGC ATGGCTCCTTTTCTTCAAACC-3' (the underlined sequence is the NotI site);

[0060] P7: 5’-GG ACTAGT GCCGGCTCGCGGCGCCAAGGG-3' (the underlined sequence is the SpeI site);

[0061] P8: 5'-TCCAATTGCTTCCGATCTGG-3'.

[0062] Drosophila genomic DNA was used as a template, an...

Embodiment 2

[0070] 0.05% sterile Tween-80 to collect the spores of wild-type Beauveria bassiana and Bbsp:serpin transformants, vortex, filter with four layers of lens paper, centrifuge, and adjust the final concentration of spores to 1×10 8 ~5×10 6 individual / mL. The prepared spore suspension was inoculated with Melonella mellonella by body surface inoculation, and 0.05% Tween-80 was used as the blank control. The results showed that compared with the wild-type strain, the semi-lethal concentration of the serpin transformant was reduced by 3.5 times at 96 hours after inoculation, that is, under this condition, the transformant only needed less than 1 / 3 of the concentration of the wild-type strain to reach Same insecticidal effect as wild type; at 5×10 6 The half-lethal time was shortened by 14% under the concentration of spores / mL. This result indicated that the insecticidal effect of the strain could be significantly improved by overexpressing serpin in insecticidal fungi.

[0071] ...

Embodiment 3

[0073] The spore suspension (1 × 10 7 individuals / ml), with S. mellonella mellonella as the test worm, each worm was injected with 4 μL of spore suspension and raised at 26°C. The hemolymph was collected every 12 hours to observe the number of parasites. The result is as Figure 5 As shown, compared with the wild-type strain, the transgenic strain (serpin strain) proliferated significantly in the hemolymph of insects. Insects are silkworms, A, inoculated with 0.85% NaCl; B, inoculated with 1×10 7 individuals / ml Beauveria bassiana wild-type strain; C, inoculated with 1×10 7 pcs / ml Bbsp:serpin transgenic Beauveria bassiana strain. The picture shows the observation results 36h after inoculation.

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Abstract

The invention discloses a recombinant serine protease inhibitor, a fungus expression vector and a fungus insecticide of recombinant protein. The invention relates to the recombinant serine protease inhibitor; a molecular biology technology is used to construct a recombinant nucleotide sequence; and the recombinant serine protease inhibitor comprises a signal peptide sequence of encoded coccidio beauveriabassiana chitinase and a serine protease inhibitor gene of encoded drosophila, and has a nucleotide sequence shown as SEQ ID NO. 1 and an amino acid sequence shown as SEQ ID NO. 2. The obtained recombinant serine protease inhibitor can inhibit immunoreaction of insects, increase multiplication speed of fungi in haemocoeles, and therefore improve characteristics of insecticide fungus toxicity.

Description

technical field [0001] The invention relates to a new serine protease inhibitor, in particular to a recombinant protein containing fungal secreted signal peptide and insect serine protease inhibitor serpin sequence. [0002] The present invention relates to a fungal expression vector expressing the above-mentioned recombinant protein. [0003] The present invention also relates to an insecticide and fungicide expressing the above-mentioned recombinant protein. [0004] Background technique [0005] Fungi are one of the main factors controlling insect populations in nature, and biopesticides with fungi as the main active ingredient have been widely used in my country, Europe, America, and Africa (Feng Mingguang, "Plant Protection in the 21st Century Outlook and the Third National Youth Plant Protection Science and Technology Workers Symposium Proceedings", 1998). However, biopesticides, including bacteria, viruses, and fungal insecticides, have disadvantages such as too lon...

Claims

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Application Information

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IPC IPC(8): C07K14/81C12N15/15C12N15/62C12N15/80C12N1/15
Inventor 范艳华裴炎杨琳芝张永军金丹罗志兵李先碧
Owner SOUTHWEST UNIVERSITY
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