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Human-derived insecticidal protein and preparation method and application thereof

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

Active Publication Date: 2015-09-23
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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  • Human-derived insecticidal protein and preparation method and application thereof
  • Human-derived insecticidal protein and preparation method and application thereof
  • Human-derived insecticidal protein and preparation method and application thereof

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, Cnaphalocrocis medinalis 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 accessi...

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 prim...

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 mM MgCl 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 ...

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Abstract

The invention discloses human-derived insecticidal protein. The coded nucleotide sequence of the human-derived insecticidal protein is as shown in SEQ ID NO.5, and the amino acid sequence of the human-derived insecticidal protein as shown in SEQ ID NO.6. Orthomutation is conducted on anti-Cry1B toxin idiotype single-chain antibody C7 to form a saturation mutagenesis library, then the saturation mutagenesis library is screened through a phage display technology, and the human-derived insecticidal protein is obtained; the affinity with insects BBMV of the human-derived insecticidal protein is remarkably higher than that of antibody C7 without mutation, the human-derived insecticidal protein can replace Cry1B toxin effectively to be used for biological control over pests, the human-derived insecticidal protein and C7 belong to a humanized antibody, and the human-derived insecticidal protein does less harm to the human body when used as biological pesticide.

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 poisoning effect on a variety of agricultural pests (Bravo and Soberon, 2008); Cry1B is a kind of Bt toxin , and its target receptors for Lepidoptera insects mainly include aminopeptidase N (APN) and cadherin (Bravo et 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 areas are the same as Cry1B Toxoid binding site (Knight et al., 1994; Knight et al., 2004). Anti-Cry1B toxin idiotype single-chain antibody (Anti-idiotype antibody scFvs, Anti-Id scF...

Claims

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Application Information

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Patent Type & Authority Applications(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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