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An improved human-derived analog bt Cry toxin protein functional effector (GGCC) and its design method and application

A human-derived, insecticidal protein technology, applied in the field of genetic engineering and biological control, can solve problems such as unreported, and achieve the effect of less harm to human body

Active Publication Date: 2020-08-07
JIANGSU ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] With the deepening understanding of the relationship between antibody structure and function, antibody modification with the help of computer simulation technology can be purposefully designed within a limited range, and design and directional modification can be carried out based on the analysis of the amino acid site of antibody-antigen binding. Wong et al. (1995) based on the known three-dimensional structure of the antigen-antibody complex, after site-directed mutation of Phe at position 108 of the Fab heavy chain of anti-p-azophenylarsonate to Trp, relative to the wild antibody In other words, the affinity of the mutant is increased by 10 times; it has pointed out the direction for further modification of the binding region of the antibody molecule, but the current binding chain replacement molecular modification method to improve the affinity of the anti-Bt toxin anti-idiotypic single-chain antibody and through the Octet technology platform Screening methods have not been reported yet

Method used

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  • An improved human-derived analog bt Cry toxin protein functional effector (GGCC) and its design method and application
  • An improved human-derived analog bt Cry toxin protein functional effector (GGCC) and its design method and application
  • An improved human-derived analog bt Cry toxin protein functional effector (GGCC) and its design method and application

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preparation example Construction

[0042] In the present invention, the preparation method of the human-derived molecularly modified insecticidal protein GGCC_scFv is preferably prepared by a recombinant expression method. The method for preparing human-derived molecularly modified insecticidal proteins by the recombinant expression method preferably includes the following steps:

[0043] (1) Carry out double digestion and ligation of the gene encoding the modified insecticidal protein GGCC_scFv encoding the human molecule and the vector respectively, to obtain the recombinant vector;

[0044] (2) The recombinant vector is introduced into the expression system, induced and cultured, and the recombinantly expressed human-derived molecularly modified insecticidal protein GGCC_scFv is obtained.

[0045] In the present invention, the expression system is preferably a prokaryotic cell expression system. The prokaryotic expression system is preferably Escherichia coli. The strain of Escherichia coli is preferably E...

Embodiment 1

[0068] A method for designing and preparing a human-derived molecularly modified insecticidal protein, the specific steps are as follows:

[0069] (1) Perform BLAST comparison analysis on the amino acid sequences of anti-Cry1Ab toxin idiotype single-chain antibody B12_scFv and Cry1A toxoids (Cry1Aa, Cry1Ab, Cry1Ac), and analyze the similar sequences of B12 and Cry1A toxoids, respectively H-CDR 1, L -CDR 1, L-CDR 2 and GS-linker. The amino acid sequences of anti-Cry1Ab toxin idiotype monoclonal antibody G1_scFv and Cry1B toxoid were compared by BLAST analysis, and the similar sequences of G1 and Cry1B toxoid were analyzed, which were H-CDR1', H-CDR3', and L-CDR 2', respectively.

[0070] (2) Design splicing according to the results, and replace the heavy chain region (VH) of the anti-Cry1Ab toxin idiotype single chain antibody B12_scFv sequence with the heavy chain region (VH) of G1_scFv to obtain the recombinant antibody GGCC_scFv; or use the anti-Cry1Ab toxin idiotype The li...

Embodiment 2

[0077] Anti-CrylAb toxin idiotype single chain antibody B12_scFv, G1_scFv, modified GGCC_scFv, CCGG_scFv and Cry1A toxoids (Cry1Aa, Cry1Ab, Cry1Ac) were compared with the amino acid sequences of Cry1B toxin by BLAST analysis:

[0078] Anti-CrylAb toxin idiosyncratic single-chain antibody B12 (hereinafter referred to as B12_phage, derived from the phage vector pIT2, is a phage-displayed single-chain antibody (scFv), that is, a scFv co-expressed on the GIII protein of the phage coat, such as figure 1 The amino acid sequence shown on the upper left side) has been disclosed by the Chinese patent No. ZL201410037000.9. B12_scFv is the antibody sequence in B12_phage, which is recombined into the prokaryotic expression vector pET-26b for soluble expression of scFv, such as figure 1 Shown above middle.

[0079] Anti-CrylAb toxin idiosyncratic single-chain antibody G1 (hereinafter referred to as G1_phage, derived from the phage vector pIT2, is a phage-displayed single-chain antibody (sc...

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Abstract

The invention provides human molecule-modified insecticidal protein and a coding gene, design method and application thereof, and belongs to the fields of genetic engineering and biological control. The invention provides the human molecule-modified insecticidal protein GGCC_scFv, wherein the human molecule-modified insecticidal protein GGCC_scFv has an amino acid sequence shown in SEQ ID No. 1. The insecticidal protein GGCC_scFv has higher affinity with intestinal BBMV of Plutella xylostella than with Cry1Ab and Cry1B toxins, competes with the Cry1Ab, Cry1Ac and Cry1B toxins in combination with the intestinal BBMV of Plutella xylostella, and is a simulation of the Cry1Ab, Cry1Ac and Cry1B toxins; and it is shown through indoor insecticidal activity tests on Plutella xylostella, the fatality rate can reach 50% in 3 days, and can reach 95% in 7 days, and therefore the human molecule-modified insecticidal protein can effectively replace the Cry1A toxin, and is applied to biological control of pests.

Description

technical field [0001] The invention relates to the field of genetic engineering and biological control, in particular to a human-derived molecular transformation insecticidal protein, coding gene, design method and application thereof. Background technique [0002] Bacillus thuringiensis (Bt) is an insect pathogenic bacterium, and its main insecticidal active substance is endotoxin concomitant crystal protein, which has specific poisoning effect on various agricultural pests (Bravo and Soberon, 2008); Cry1Ac is A kind of Bt toxin, its target receptors to Lepidoptera insects mainly include alkaline phosphatase (alkaline phosphatase, ALP) and aminopeptidase ( aminopeptidase N, APN) and cadherin. ALP acts as a receptor for the Cry1A toxin, which facilitates membrane insertion and pore formation of the toxin. A number of ALPs isolated from Diptera and Lepidoptera insect species have been identified as receptors for the Cry1Ac toxin. Anti-Cry1Ac toxin idiotype single-chain an...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/12C12N15/13A01N47/44A01P7/04
CPCA01N47/44C07K16/1278C07K2317/24C07K2317/622C07K2317/76C07K2317/92
Inventor 刘贤金谢雅晶张霄徐重新何鑫高美静刘媛张存政
Owner JIANGSU ACAD OF AGRI SCI