An improved functional effector of human-derived mimetic bt Cry insecticidal protein and its design method and application

Abstract

The invention provides a human molecular modified insecticidal protein and its preparation method and application, belonging to the field of genetic engineering and biological control. The present invention provides a human molecularly modified insecticidal protein, the amino acid sequence of the insecticidal protein CCL-CCL_scFv is shown in SEQ ID No.1. The insecticidal protein CCL-CCl_scFv has a significantly higher affinity to the midgut BBMV of diamondback moth (Plutella xylostella) than Cry1Ab toxin, and competes with Cry1Ab and Cry1Ac toxin for binding to midgut BBMV of diamondback moth, and is a mimic of Cry1Ab and Cry1Ac toxin; The indoor insecticidal biological activity of Plutella xylostella was measured, and the 3d lethality rate can reach 55.35%, which can effectively replace Cry1Ac toxin for biological control of insect 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...

Examples

preparation example Construction

[0043] In the present invention, the preparation method of the human-derived molecularly modified insecticidal protein 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:

[0044] (1) Carry out double enzyme digestion and connection of the gene encoding the modified insecticidal protein of the human source molecule and the vector respectively, and obtain the recombinant vector;

[0045] (2) The recombinant vector is introduced into the expression system, induced and cultured, and the recombinantly expressed human-derived molecularly modified insecticidal protein is obtained.

[0046] 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 Escherichia coli BL2...

Embodiment 1

[0069] A method for designing and preparing a human-derived molecularly modified insecticidal protein, the specific steps are as follows:

[0070] (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.

[0071] (2) Splicing is designed according to the results, and the heavy chain region (VH) of the anti-Cry1Ab toxin idiotype single-chain antibody B12_scFv sequence is replaced with the light chain region (VL) to obtain the double-chain antibody CCH-CCH_scFv; or the light chain region ( VL) was replaced by the heavy chain region (VH) to obtain the double light chain antibody CCL-CCL_scFv, and then BLAST comparison analysis was performed.

[0072] (3) Anti-Cry1Ab toxin idiotype single-chain antibody B12_scFv, amino acid sequence of human molecularly e...

Embodiment 2

[0079] The amino acid sequences of the anti-CrylAb toxin idiotype single chain antibody B12_scFv, the modified CCL-CCL_scFv, CCH-CCH_scFv and Cry1A toxoids (Cry1Aa, Cry1Ab, Cry1Ac) were compared and analyzed by BLAST:

[0080] 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 left) 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 in the middle.

[0081] The amino acid sequences of Bt toxins Cry1Aa, Cry1Ab and Cry1Ac were obtained from the NCBI protein database with accession numbers: AFK79795.1, ALJ10947.1 and ALT07695.1.

[0082] The amino aci...

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...

Examples

preparation example Construction

[0043] In the present invention, the preparation method of the human-derived molecularly modified insecticidal protein 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:

[0044] (1) Carry out double enzyme digestion and connection of the gene encoding the modified insecticidal protein of the human source molecule and the vector respectively, and obtain the recombinant vector;

[0045] (2) The recombinant vector is introduced into the expression system, induced and cultured, and the recombinantly expressed human-derived molecularly modified insecticidal protein is obtained.

[0046] 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 Escherichia coli BL2...

Embodiment 1

[0069] A method for designing and preparing a human-derived molecularly modified insecticidal protein, the specific steps are as follows:

[0070] (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.

[0071] (2) Splicing is designed according to the results, and the heavy chain region (VH) of the anti-Cry1Ab toxin idiotype single-chain antibody B12_scFv sequence is replaced with the light chain region (VL) to obtain the double-chain antibody CCH-CCH_scFv; or the light chain region ( VL) was replaced by the heavy chain region (VH) to obtain the double light chain antibody CCL-CCL_scFv, and then BLAST comparison analysis was performed.

[0072] (3) Anti-Cry1Ab toxin idiotype single-chain antibody B12_scFv, amino acid sequence of human molecularly e...

Embodiment 2

[0079] The amino acid sequences of the anti-CrylAb toxin idiotype single chain antibody B12_scFv, the modified CCL-CCL_scFv, CCH-CCH_scFv and Cry1A toxoids (Cry1Aa, Cry1Ab, Cry1Ac) were compared and analyzed by BLAST:

[0080] 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 left) 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 in the middle.

[0081] The amino acid sequences of Bt toxins Cry1Aa, Cry1Ab and Cry1Ac were obtained from the NCBI protein database with accession numbers: AFK79795.1, ALJ10947.1 and ALT07695.1.

[0082] The amino aci...

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