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A kind of human insecticidal protein and its preparation method and application

A technology of insecticidal protein and insecticide, applied in the field of genetic engineering, achieves high success rate, improved prediction accuracy, and less harm to human body

Active Publication Date: 2018-02-09
JIANGSU ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It pointed out the direction for further modification of the binding region of antibody molecules, and the method of screening anti-Cry1B toxin idiotype single-chain antibodies by combining site-directed mutagenesis has not been reported yet

Method used

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  • A kind of human insecticidal protein and its preparation method and application
  • A kind of human insecticidal protein and its preparation method and application
  • A kind of human insecticidal protein and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1 Homology modeling and molecular docking of anti-CrylB toxin idiotype single chain antibody C7, Cnaphalocrocismedinalis Guenee APN (CmAPN)

[0066] (1) Homologous modeling

[0067] The amino acid sequence of the anti-CrylB toxin idiotype single-chain antibody C7 (hereinafter referred to as C7) has been disclosed by the Chinese patent publication number CN103773775A, and its amino acid structure is as follows: figure 1 Shown: The C7 sequence consists of 291 amino acids, and the heavy and light chains respectively contain 3 CDR variable regions, respectively CDR-H1: 48-57, CDR-H2: 72-86, CDR-H3: 121-127, CDR-L1: 178, -188, CDR-L2: 204-210, CDR-L3: 243-251 (H: heavy chain, L: light chain), there are 15 amino acid residues between the heavy and light chains (GGGS )3 linker to connect, with a His tag consisting of 6 Hs at the end of the sequence;

[0068] The amino acid sequence of CmAPN was obtained from the NCBI protein database, and the gene accession number i...

Embodiment 2

[0081] Example 2 Construction of a Saturation Mutant Antibody Library for Site-Directed Mutagenesis

[0082] According to the molecular docking prediction results in Example 1, the heavy chain 3 region A123, Y124 and the light chain 2 region S204, S207 of the C7 sequence were selected as the mutated regions.

[0083] Primer sequences involved in introducing saturation mutation sites:

[0084] SEQ ID No.1(LMB3-F): CAGGAAACAGCTATGAC;

[0085] SEQ ID No.2 (pHENseq-R): CTATGCGGCCCCATTCA;

[0086] SEQ ID No.3(H-M): 5'CCCCAGTAGTCAAA NNKNNK ACCAGATTTCGC 3';

[0087] SEQ ID No.4(L-M): 5' CTCCTGATCTAT NNK GCATCC NNK TTGCAAAGTGG 3';

[0088] Among them, LMB3-F and pHENseq-R are the upstream and downstream primers for amplifying the full length of the fragment; H-M are the mutation primers for introducing heavy chain 3 region A123 and Y124 (the underlined part " NNKNNK " is the codon corresponding to the saturation mutation site, the same below); L-M is the mutation primer int...

Embodiment 3

[0112] Example 3 Screening of insecticidal proteins

[0113] (1) Preparation of BBMV from the midgut of the rice leaf roller

[0114] Referring to Wolfersberger's experimental method (Wolfersberger, 1987), the Mg-EGTA sedimentation method was used to prepare the midgut BBMV of the rice leaf roller. Wash in 0.15 M NaCl, add 3 mL of homogenization buffer for every 500 midguts; after 3 times of repeated homogenization in ice bath, take an appropriate amount and add 24 mMMgCl 2 Then vortex and mix, ice bath and centrifuge, then transfer the supernatant to a new high-speed centrifuge tube and then centrifuge; discard the supernatant, invert the centrifuge tube until the liquid is exhausted, resuspend the pellet in HEPES buffer, and separate After packaging, store at -80°C for use; BBMV protein concentration was determined by the Bradford method.

[0115] (2) Enrichment and screening of saturated mutant antibody library using BBMV

[0116] Using the solid-phase screening method o...

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Abstract

The invention discloses a human insecticidal protein, whose coding nucleotide sequence is shown in SEQ ID NO.5, and its amino acid sequence is shown in SEQ ID NO.6; the human insecticidal protein is composed of anti-Cry1B toxin idiotype The single-chain antibody C7 was screened by phage display technology after directional mutation to form a saturated mutation library; its affinity to insect BBMV was significantly higher than that of the antibody C7 before mutation, and it can effectively replace the Cry1B toxin for biological control of insect pests, and it is the same as C7 It is a human antibody, and when used as a biological pesticide, it is less harmful to the human body.

Description

technical field [0001] The invention belongs to the field of genetic engineering, in particular to a human insecticidal protein and its preparation method and application. Background technique [0002] Bacillus thuringiensis ( Bacillus thuringiensis , Bt) is an entomopathogenic bacterium, and its main insecticidal active substance is endotoxin parasporal crystal protein, which has specific poisonous effect on various agricultural pests (Bravo and Soberon, 2008); Cry1B is a kind of Bt toxin, Its target receptors for lepidopteran insects mainly include aminopeptidase N (APN) and cadherin (Bravo et al. al., 2004; Pigott and Ellar, 2007), APN belongs to the zinc-binding metalloprotease family protein, and its carbon-terminal binding site is rich in N-acetylgalactosamine (GalNAc), these regions are similar to Cry1B Toxin binding site (Knight et al., 1994; Knight et al., 2004). Anti-idiotype antibody scFvs (Anti-IdscFvs) can mimic the structure and function of Cry1B toxin, so I...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/12C12N15/13A01N47/44A01P7/04
Inventor 刘贤金仲建锋徐重新张霄刘媛张存政何鑫武爱华
Owner JIANGSU ACAD OF AGRI SCI
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