Fermentative preparation method of an insecticidal fusion protein and insecticidal composition of the fermented product

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

[0001] Technical field:

[0002] 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.

[0003] Background technique:

[0004] 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.

[0005] 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 ...

Examples

Embodiment 1

[0051] The present invention: adopt the method described in this patent to ferment the fusion protein product, the specific method is as follows:

[0052] Fermentation medium pre-cultivation: add 20% (V / V) BSM medium to the fermenter, the pH regulator is 30% ammonia water, the parameters are set to air pressure 0.03MPa, temperature 23°C, pH 3, speed 200rpm , run for 55 hours, and complete the pre-cultivation of the medium, wherein, the BSM medium is a minimally buffered methanol medium.

[0053] Preparation of fusion protein seed solution: Pick a single colony of fusion protein containing the fusion protein gene fragment and inoculate it in non-resistant YPG liquid medium, and culture it on a shaker at 28°C and 180rpm for 48h, wherein, YPG liquid medium Yeast extract, peptone, sucrose medium.

[0054] 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 ferm...

Embodiment 2

[0072] The present invention: adopt the method described in this patent to ferment the fusion protein product, the specific method is as follows:

[0073] Fermentation medium pre-cultivation: add 35% (V / V) BSM medium to the fermenter, the pH regulator is 60% ammonia water, the parameters are set to air pressure 0.06MPa, temperature 32°C, pH 4.5, speed 300rpm , run for 75 hours, and complete the pre-cultivation of the medium, wherein, the BSM medium is a minimally buffered methanol medium.

[0074] Preparation of fusion protein seed solution: Pick a single colony of fusion protein containing the fusion protein gene fragment and inoculate it in non-resistant YPG liquid medium, and culture it on a shaker at 32°C and 250rpm for 75h, wherein, YPG liquid medium Yeast extract, peptone, sucrose medium.

[0075] 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 fermen...

Embodiment 3

[0094] Insecticidal composition 1: The following components are divided according to the mass ratio of the insecticidal composition: 35% of the fusion protein fermentation product obtained according to the fermentation preparation method of the present invention in Example 1, 45% of the solvent phosphate buffer, and the protective agent Ethylene glycol 1%, emulsifier alkylphenol polyoxyethylene ether 0.05%, castor oil polyoxyethylene ether 0.05%, dispersant silicone polyoxyethylene ether 0.1%, humectant xanthan gum 5%, the rest is water, The pesticidal composition 1 is formed.

[0095]Treatment A: Insecticidal composition 2, in addition to no solvent phosphate buffer, other components according to the mass ratio of the insecticidal composition are: fusion protein fermentation product 35%, protective agent ethylene glycol 1%, emulsifier 0.05% of alkylphenol polyoxyethylene ether, 0.05% of castor oil polyoxyethylene ether, 0.1% of silicone polyoxyethylene ether of dispersant, 5%...

[0001] Technical field:

[0002] 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.

[0003] Background technique:

[0004] 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.

[0005] 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 ...

Examples

Embodiment 1

[0051] The present invention: adopt the method described in this patent to ferment the fusion protein product, the specific method is as follows:

[0052] Fermentation medium pre-cultivation: add 20% (V / V) BSM medium to the fermenter, the pH regulator is 30% ammonia water, the parameters are set to air pressure 0.03MPa, temperature 23°C, pH 3, speed 200rpm , run for 55 hours, and complete the pre-cultivation of the medium, wherein, the BSM medium is a minimally buffered methanol medium.

[0053] Preparation of fusion protein seed solution: Pick a single colony of fusion protein containing the fusion protein gene fragment and inoculate it in non-resistant YPG liquid medium, and culture it on a shaker at 28°C and 180rpm for 48h, wherein, YPG liquid medium Yeast extract, peptone, sucrose medium.

[0054] 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 ferm...

Embodiment 2

[0072] The present invention: adopt the method described in this patent to ferment the fusion protein product, the specific method is as follows:

[0073] Fermentation medium pre-cultivation: add 35% (V / V) BSM medium to the fermenter, the pH regulator is 60% ammonia water, the parameters are set to air pressure 0.06MPa, temperature 32°C, pH 4.5, speed 300rpm , run for 75 hours, and complete the pre-cultivation of the medium, wherein, the BSM medium is a minimally buffered methanol medium.

[0074] Preparation of fusion protein seed solution: Pick a single colony of fusion protein containing the fusion protein gene fragment and inoculate it in non-resistant YPG liquid medium, and culture it on a shaker at 32°C and 250rpm for 75h, wherein, YPG liquid medium Yeast extract, peptone, sucrose medium.

[0075] 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 fermen...

Embodiment 3

[0094] Insecticidal composition 1: The following components are divided according to the mass ratio of the insecticidal composition: 35% of the fusion protein fermentation product obtained according to the fermentation preparation method of the present invention in Example 1, 45% of the solvent phosphate buffer, and the protective agent Ethylene glycol 1%, emulsifier alkylphenol polyoxyethylene ether 0.05%, castor oil polyoxyethylene ether 0.05%, dispersant silicone polyoxyethylene ether 0.1%, humectant xanthan gum 5%, the rest is water, The pesticidal composition 1 is formed.

[0095]Treatment A: Insecticidal composition 2, in addition to no solvent phosphate buffer, other components according to the mass ratio of the insecticidal composition are: fusion protein fermentation product 35%, protective agent ethylene glycol 1%, emulsifier 0.05% of alkylphenol polyoxyethylene ether, 0.05% of castor oil polyoxyethylene ether, 0.1% of silicone polyoxyethylene ether of dispersant, 5%...