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Preparation method and use of insecticidal synergistic protein

A technology for synergistic proteins and uses, applied in botany equipment and methods, biochemical equipment and methods, pesticides, etc., can solve problems such as hazards and increased dosage of chemical preparations, and achieve environmental pollution reduction, easy operation, The effect of easy operation

Inactive Publication Date: 2012-05-23
孙修炼
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the shortcomings of chemical preparations are becoming more and more obvious: their toxicity not only acts on insects, but also acts on other animals, especially to humans and livestock.
Increased use of chemical agents along with increased pest resistance has serious consequences

Method used

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  • Preparation method and use of insecticidal synergistic protein
  • Preparation method and use of insecticidal synergistic protein
  • Preparation method and use of insecticidal synergistic protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Preparation of biological insecticidal synergistic protein by prokaryotic expression method

[0052] The sequence of CpGV orf13 was obtained from GenBank of NCBI (serial number: U53466), the full length of its coding region is 756bp, and it encodes a protein product with a size of 252 amino acids. After the present invention cuts off the signal peptide and related hydrophobic regions, the length of the coding region is 663bp ( Figure 1B ), encoding a protein product with a size of 221 amino acids.

[0053] A preparation method for insecticidal synergistic protein, the steps are:

[0054] 1) According to the sequence of codling moth granular virus (CpGV) orf13, starting from the 94th nucleotide, design a pair of primers, upstream primer P1-1: 5'-cgc atgccgttggcgagacagcg-3' (the italic part is the BamHI site); downstream primer P2-1: 5'-ccc ctacaaatcacttttcgtttgctt-3'(Italic part is HindIII site)

[0055] 2) The truncated fragment (663bp) was amplified by PCR using...

Embodiment 2

[0063] Purification of prokaryotic expression potentiators

[0064] 1) Collect the bacteria liquid (200ml) that induces expression, centrifuge at 5000×g for 10min at 4°C, discard the supernatant, blow up the precipitate with 10ml of PBS, centrifuge at 5000×g for 10min at 4°C, discard the supernatant;

[0065]2) The pellet was resuspended in 30-40ml PBS, then ultrasonically crushed for 20min, centrifuged at 8000×g for 10min at 4°C, washed 2-3 times with washing solution (2M urea, 50mM Tris, 1mM EDTA), and centrifuged at 8000×g for 15min;

[0066] 3) Collect the inclusion body precipitate, blow it away with Binding Buffer (5mM imidazole, 0.5M NaCl, 20mM Tris-HCl, 6M urea), treat it on ice for 1h, then centrifuge at 10000×g for 30min, take the supernatant and temporarily store it in a refrigerator at 4°C;

[0067] 4) NTA resin was loaded into the chromatography column, followed by adding 5ml Charge Buffer (50mM NiSO 4 ), 3mlMilli-Q water, 3ml Binding Buffer through the column; ...

Embodiment 3

[0072] A kind of insecticidal synergistic protein (Cp13) is improving the purposes in the activity of killing beet armyworm larvae of beet armyworm nucleopolyhedrosis virus (SeNPV), and its steps are:

[0073] Pick the end-2nd instar larvae of beet armyworm with uniform size, place them in a sterile 24-well plate, starve and culture them in a constant-temperature light incubator at 28°C for 24 hours, and the larvae molt and enter the 3rd instar by themselves.

[0074] The concentration of SeNPV was measured with a hemocytometer, and diluted to a final concentration of 1×10 3 , 1×10 4 , 3×10 4 , 1×10 5 , 3×10 5 Four gradients of OB / ml. The reagents used in the bioassay were divided into 5 different treatments, that is, SeNPV: only containing virus without adding any substances; SeNPV+Cp13: adding the induced expression of Cp13 to the above-mentioned virus suspensions of different concentrations, so that the final concentration of Cp13 30 μg / ml; SeNPV+Enhancin: Add AsGV Enh...

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Abstract

The invention discloses a preparation method and use of an insecticidal synergistic protein. The preparation method comprises the following steps of: A) designing a primer according to the sequence of CpGVorf13, and performing polymerase chain reaction (PCR) amplification on a truncated segment by taking the DNA of a CpGV genome as a template; B) identifying a PCR product by agarose gel electrophoresis, recycling the PCR product by using a PCR recycling purification kit, connecting the recycled PCR product with a pMD18-T vector; C) extracting plasmas, performing enzyme digestion, identifying positive clones, sequencing, recycling a target segment, suncloning until a vector pET-28a is expressed, and obtaining pET-28a-Cp13; D) transforming the pET-28a-Cp13 into colibacillus DH5alpha, extracting plasmas for identification, transforming the colibacillus BL21 by using the plasmas, and adding isopropyl thiogalactoside (IPTC) to induce expression; and E) analyzing and detecting an expression product by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to obtain Cp13 protein. The expressed Cp13 protein can be purified by His purification resin Ni-NTA. The Cp13 protein expressed by pronucleuses can obviously improve the insecticidal activity of a plurality of insect virus and Bt medicaments and destroy the integrality of the mesenteron funnel of insects. The synergistic protein is convenient to use, safe and environmentally-friendly and has no toxic and side effects.

Description

technical field [0001] The invention belongs to the technical field of green insecticide synergistic biological pesticides, more specifically relates to a method for preparing an insecticidal synergistic protein, and also relates to an application of an insecticidal synergistic protein, which is effective against insect viruses and thuringiensis The insecticidal activity of Bacillus has obvious synergistic effect. Background technique [0002] At present, a large number of reports have shown that from baculoviruses, especially the synergistic protein (Enhancin) of granuloviruses can not only increase the virulence of other baculoviruses, but also greatly improve the insecticidal activity of Bt (Xu Jian et al., East China Acta Entomological Sinica, 2005, 14(4): 343-347). No homologue of the enhancen gene was found in the genome sequence of CpGV, but it contained a spindle protein gene (orf13) homologous to the gp37 gene of baculovirus and the fusolin gene of entomopox virus,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/34C12N15/70C07K14/01A01N63/00A01N63/02A01P7/04
Inventor 孙修炼刘向阳
Owner 孙修炼
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