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Insecticidal fusion protein fermentation and preparation method and insecticidal composition of fermentation product thereof

A technology of insecticidal composition and fusion protein, which is applied in the fields of botanical equipment and methods, microorganism-based methods, biochemical equipment and methods, etc., to achieve the effects of high content, high-efficiency expression, and high expression level

Active Publication Date: 2016-03-23
北京依科曼生物技术股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003]However, in practice, there are uncontrollable protein expression, high acquisition cost, and protein A series of problems such as difficult purification, few active protein products, and easy loss of protein activity due to the influence of the application environment have caused limitations in the application of fusion proteins in the field of pest control.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Inoculation of fusion protein seed solution: add PTM1 to the pre-cultivated fermenter, the volume ratio of PTM1 and BSM medium is 3:1000; seed solution is inoculated in the fermenter, wherein the volume ratio of seed solution and BSM medium is 1: 20. After completion, carry out fermentation culture, the culture conditions are as follows: rotation speed 400rpm, pH 3.7, temperature 28°C.

[0052] Mass expression stage of fusion protein fermentation product:

[0053] Feeding process: feed 10h after inoculation, the flow rate of feeding liquid is 5mL / h, the feeding time is 12h, the speed is adjusted to 700rpm, the flow rate of feeding liquid is adjusted to 10mL / h, and the feeding time is 12h, After that, the feeding flow rate was increased step by step by 4mL / h on the basis of the previous feeding flow rate, and the single feeding time was 12h;

[0054] This stage lasts for 70 hours in total. In this stage, dissolved oxygen needs to be maintained at 25%, and an antifoaming...

Embodiment 2

[0072] Inoculation of fusion protein seed liquid: add PTM1 in the pre-cultivated fermenter, the volume ratio of PTM1 and BSM medium is 7:1000; inoculate the seed liquid in the fermenter, wherein the volume ratio of seed liquid and BSM medium is 3: 20. Carry out fermentation culture after completion, the culture conditions are: rotation speed 750rpm, pH 4.1, temperature 32°C.

[0073] Mass expression stage of fusion protein fermentation product:

[0074] Feed feeding process: feed feed 10h after inoculation, the flow rate of feed liquid is 5mLl / h, the feed time is 20h, the rotating speed is adjusted to 1000rpm, the flow rate of feed liquid is adjusted to 10mL / h, and the feed time is 15h, After that, the feeding flow rate was increased step by step by 8mL / h on the basis of the previous feeding flow rate, and the single feeding time was 15h;

[0075] This stage lasts for 90 hours in total. In this stage, dissolved oxygen needs to be maintained at 35%, and an antifoaming agent sh...

Embodiment 3

[0094] Use fixed-point and fixed-point investigation of drug efficacy, and 20 fixed-point investigations for each insecticidal composition. Investigate the population base of cotton bollworm before spraying, investigate the control effect 1, 3, 7, and 14 days after spraying, and record the number of cotton bollworm larvae in each plot respectively.

[0095] Decrease rate of insect population = (base number of insect population before treatment - number of insect population after treatment) × 100% / base number of insect population before treatment;

[0096] Control effect = (the reduction rate of the insect population in the treatment area - the reduction rate of the insect population in the control area) × 100% / (1-the reduction rate of the insect population in the control area).

[0097] Carry out different insecticidal compositions to field control effect experiment of cotton bollworm, specifically see the following table:

[0098] Table 5 of field control effects of differ...

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PUM

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Abstract

The invention relates to an insecticidal fusion protein fermentation and preparation method and an insecticidal composition of a fermentation product thereof, and belongs to the field of pest control. The fermentation and preparation method includes the steps of preparation of a fusion protein seed solution, inoculation of the fuse protein seed solution and the stage of increased expression of the fermentation product of fusion protein. The method is characterized in that a fermentation culture medium is pre-cultured needs to be completed before the fusion protein seed solution is inoculated, and a dynamically adjusted material supplement process exists in the stage of increased expression of the fermentation product of fusion protein. The preparation and fermentation method can achieve efficient and stable expression of protein. The insecticidal composition is formed by screening a solvent, a protecting agent, an emulsifier, a dispersing agent and a moisture retention agent which are matched with the expressed product. The insecticidal composition has the advantages of being good in insecticidal matter stability, good in dispersing effect, strong in field practicality and wide in insecticidal spectrum and has efficient killing effect on various pests.

Description

technical field : [0001] The invention belongs to the field of pest control, and in particular relates to a fermentation preparation method of an insecticidal fusion protein and an insecticidal composition of the fermentation product. Background technique: [0002] In the field of pest control, only the Bt and CpTI genes are relatively mature in the use of gene technology to achieve pest control. With the increasing demand for the use of biotechnology for pest control, the recombination of two or more gene sequences that have an inhibitory effect on pests to form a fusion protein to achieve pest control has attracted further attention. [0003] However, the technology of using toxin protein or gene sequence recombination with inhibitory effect on pests to form fusion protein has the disadvantages of uncontrollable protein expression, high acquisition cost, difficult protein purification, less active protein products, and protein activity easily affected by the application ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/00A01N63/04A01P7/04C12R1/84
CPCC12P21/00A01N63/30
Inventor 邓青陈育李永亮王广兴梁兴慧杜进平周震
Owner 北京依科曼生物技术股份有限公司
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