Human-derived insect-resistant gene and anti-CRY1B toxin idiotype single-chain antibody encoded thereby and application thereof

Abstract

Provided are a human-derived insect-resistant gene having a nucleotide sequence represented by SEQ ID NO.1, and an anti-Cry1B toxin idiotype single-chain antibody encoded by said human-derived insect-resistant gene and having an amino acid sequence represented by SEQ ID NO.2. The idiotype single-chain antibody is a -type and has insecticidal activity, and after expression by the prokaryotic system, the primary culture thereof has binding activity to Cnaphalocrocis medinalis midgut peritrophic membrane specific receptor BBMV.

Claims

1. A single-chain antibody comprising the amino acid sequence of SEQ ID NO.2.

2. An insecticide comprising a single-chain antibody, wherein the single-chain antibody comprises the amino add sequence of represented by SEQ ID NO.2.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a schematic of C7 ELISA detection result.

(2) FIG. 2 is a schematic of C7 biological determination result.

(3) FIG. 3 is a schematic showing the death condition of Cnaphalocrocis medinalis third instar larvae after they were fed with paddy leaves soaked with C7, CK+ and CK respectively.

(4) FIG. 4 is a schematic showing the death condition of Plutella xylostella third instar larvae after they were fed with cabbage leaves soaked with C7, CK+ and CK respectively.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Embodiment 1: Screen Human-Derived Insect-Resistant Gene

(5) Reagents and medium formulae involved in the embodiment:

(6) (1) 2 TY Fluid Medium: Add 16 g of tryptone, 10 g of yeast extract and 5 g of NaCl in 900 mL of double distilled water, mix them well, set the volume to 1 L by double distilled water, put the liquid in an autoclave, sterilize it at 121 C. for 20 minutes, cool it and store it at 4 C. for future use.

(7) (2) 2TY-AG Fluid Medium: Add ampicillin with final concentration of 100 g/ml and glucose with a mass ratio of 1% to 2TY culture medium.

(8) (3) 2TY-AK Fluid Medium: Add ampicillin with final concentration of 100 g/ml and kanamycin with final concentration of 50 g/ml to 2TY culture medium.

(9) (4) 2TY-AKG Fluid Medium: Add ampicillin with final concentration of 100 g/ml, kanamycin with final concentration of 50 g/ml and glucose with a mass ratio of 1% to 2TY culture medium.

(10) (5) TYE Solid Medium: Add 15.0 g of agarose, 8 g of NaCl, 10 g of tryptone and 5 g of yeast extract to 900 ml of double distilled water, set the volume to 1 L by double distilled water, put the liquid in an autoclave, sterilize it at 121 C. for 20 minutes, cool it and store it at 4 C. for future use.

(11) (6) TYE-AG Solid Medium: Add ampicillin with final concentration of 100 g/ml and glucose with a mass ratio of 1% to TYE Solid Medium.

(12) (7) PBS Solution Weigh 8.0 g of NaCl, 0.2 g of KCl, 2.9 g of Na.sub.2HPO.sub.4.12H.sub.2O and 0.2 g of KH.sub.2PO.sub.4, add them in distilled water respectively, dissolve them thoroughly and set the volume to 1 L.

(13) (8) PBST Solution Add Tween-20 with a volume ratio of 0.05% to PBS solution.

(14) (9) PEG/NaCl Solution: Weigh 20 g of PEG 8000 and 14.61 g of NaCl, add 80 ml of deionized water, set the volume to 100 ml, put the solution in an autoclave, sterilize it at 121 C. for 20 minutes, cool it and store it at 4 C. for future use.

(15) (10) Citrate Buffer Solution (CPBS, pH=5.5): Weigh 21 g of C.sub.6H.sub.7O.sub.8 (citric acid) and 71.6 g of Na.sub.2HPO.sub.4.12H.sub.2O, add them to distilled water respectively, dissolve them thoroughly and set the volume to 1 L.

(16) (11) Tetramethyl Benaidine (TMB) Solution: Weigh 10 mg of TMB, dissolve it in 1 ml of dimethyl sulfoxide, keep the solution in a dark place and store it at 4 C. for future use.

(17) (12) Substrate Chromogenic Solution: Composition of 10 ml formula: 9.875 ml of CPBS, 100 l of TMB solution and 25 l H.sub.2O.sub.2 at volume ratio of 20%.

(18) Sources of the Materials in Involved the Embodiment:

(19) Anti-Cry1B polyclonal antibody, BBMV, irrelevant Anti-Id single-chain antibody, non--type Anti-Id ScFv, cabbage leaves and Plutella xylostella third instar larvae were provided by the Key Laboratory for Agricultural Product Quality and Safety Control Technology and Standard of the Ministry of Agriculture, Jiangsu Academy of Agricultural Sciences;

(20) Humanized phage antibody library, TG1 bacteria and helper phage KM13 were purchased from British Source BioScience;

(21) HRP-goat-anti-M13-IgG was purchased from Wuhan Boster Biological Technology Co., Ltd.;

(22) Cry1B toxin and Cry1Ab toxin were purchased from Shanghai Youlong Biotech Co., Ltd.;

(23) Paddy leaves and Cnaphalocrocis medinalis third instar larvae were provided by Yangzhou Luyuan Bio-Chemical Co., Ltd.

Embodiment 1: Screen Anti-Cry1B Toxin Idiotype Single-Chain Antibody

(24) (1) Add 20 l of humanized phage antibody library bacterium liquid to 200 ml of 2TY-AG fluid medium, cultivate it at constant temperature 37 C. till OD.sub.600 is 0.4, measure 50 ml of the bacterium liquid, add 110.sup.12 pfu of helper phage KM13 for superinfection, incubate the liquid at 37 C. for 30 minutes, then centrifuge it at 3300 g for 10 minutes, discard the supernate, use 100 ml of 2TY-AKG fluid medium to resuspend the precipitate and cultivate it at 30 C. overnight; centrifuge it at 3300 g for 30 minutes next day, collect the supernate, add 20 ml of PEG/NaCl solution, keep it in ice bath for 1 h, then centrifuge it at 3300 g for 30 minutes and resuspend the precipitate by 4 ml of PBS; centrifuge the resuspension solution at 11600 g for 10 minutes, and the supernate is amplified phage antibody library; (2) Use the amplified phage antibody library obtained in step 1 for four rounds of Panning: in the first round of Panning, coat 4 ml of 100 g/ml anti-Cry1B polyclonal antibody to the bottom of a cell culture flask, keep it at 4 C. overnight, wash the cell culture flask with 1 ml of PBS for 3 times next day, then add 1 ml of thoroughly mixed amplified phage antibody library and 4 ml of 3% MPBS solution, put the flask on a shaking table, slowly shake it at room temperature for 1 h, let it rest for 1 h, remove the liquid in the culture flask, wash the flask with 1 ml of PBST solution for 20 times and add 1 ml of 10 mg/ml trypsin to elute the specifically bound phage antibody. The eluent is phage antibody obtained in the first round of Panning. The concentrations of the coated anti-Cry1B polyclonal antibody panned in the second, third and fourth rounds are 50 g/ml, 25 g/ml and 10 g/ml respectively. The used phage antibody is the phage antibody obtained from the previous round of panning. The panning method is same as adopted in the first round. 10 l of the phage antibody panned in the fourth round is used to infect 1 ml of TG1 bacteria in a logarithmic phase. After it is incubated at 37 C. for 1 h, it is coated on TYE-AG solid medium and cultivated at 37 C. overnight; next day, single colonies are picked randomly, incubated on a 96-well plate containing 100 W/well of 2TY-AG fluid medium and cultivated at 37 C. overnight; next day, 41 of bacterium liquid is sucked from the well plate, transferred to a new 96-well plate and incubated at 37 C. for 2 h. 25 l of helper phage KM13 with titer of 10.sup.12 is added to every well, incubated at 30 C. for 2 h, centrifuged at 1800 g for 10 minutes, the precipitate is resuspended with 150 W of 2TY-AK fluid medium and then cultivated at 30 C. overnight. Next day, it is centrifuged at 1800 g for 30 minutes. The supernate is collected; (3) 4 g/ml anti-Cry1B polyclonal antibody is measured and added to a 96-well plate, 100 l/well, and stored at 4 C. overnight. Next day, 100 l of the supernate obtained in step 2 is added to every well. 100 l of 2TY-AK fluid medium is added to the negative control. They are kept in 37 C. water bath for 2 h. After the plate is washed with 250 l/well of PBST, 100 l of 1:5000 diluted HRP-goat-anti-M13-IgG is added to each well and incubated at 37 C. for 2 h. 100 l of substrate chromogenic solution is added to each well and takes reaction at room temperature for 10 to 20 minutes till blue appears. Lastly 50 l of 2 mol/L H.sub.2SO.sub.4 is added to each well to quickly terminate the reaction. OD.sub.450 is determined by ELIASA. If OD.sub.450 of the solution/OD.sub.450 of negative control is greater than 2.1, it will be considered positive. The supernate in step 2 corresponding to this solution is the screened supernate containing anti-Cry1B toxin Idiotype single-chain antibody.

(25) The nucleotide sequence of the screened anti-Cry1B toxin idiotype single-chain antibody determined by Sanger sequencing method is SEQ ID NO.1, as shown below:

(26) TABLE-US-00001 attgtctgcggccccgtgatggtgatgatgatgtgcggccgcccgtttgatttccacctt 60 ggtcccttggccgaacgtagaaggataagcagcagcctgttgacagtagtaagttgcaaa 120 atcttcaggttgcagactgctgatggtgagagtgaaatctgtcccagatccactgccact 180 gaaccttgatgggaccccactttgcaaagaggatgcactatagatcaggagcttaggggc 240 tttccctggtttctgctgataccaatttaaatagctgctaatgctctgacttgcccggca 300 agtgatggtgactctgtctcctacagatgcagacagggaggatggagactgggtcatctg 360 gatgtccgtcgacccgccaccgccgctgccacctccgcctgaaccgcctccaccgctcga 420 gacggtgaccagggttccctggccccagtagtcaaaataagcaccagatttcgcacagta 480 atatacggccgtgtcctcggctctcaggctgttcatttgcagatacagcgtgttcttgga 540 attgtctctggagatggtgaaccggcccttcacggagtctgcgtaacctgtagcaccacc 600 attattagcaatagttgagacccactccagccccttccctggagcctggcggacccagct 660 catggcatagctgctaaaggtgaatccagaggctgcacaggagagtctcagggacccccc 720 aggctgtaccaagcctcccccagactccaacagctgcacctcggccatggccggctgggc 780 cgcgagtaataacaatccagcggctgccgtaggcaataggtatttcattatgactgtctc 840 ctgaaatagaattgt 855

(27) After nucleotide translation, the amino acid sequence of screened anti-Cry1B toxin idiotype single-chain antibody determined by Sanger sequencing method is SEQ ID NO.2, as shown below:

(28) TABLE-US-00002 H-CDR1 MKYLLPTAAAGLLLLAAQPAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVR 60 H-CDR2 QAPGKGLEWVSTIANNGGATGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK 120 H-CDR1-----Link----- SGAYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRAS 180 L-CDR1 L-CDR2 QSISSYLNWYQQKPGKAPKLLIYSASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATY 240 L-CDR3His-tag YCQQAAAYPSTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLNGAASTP 291

(29) The applicant names this anti-Cry1B toxin idiotype single-chain antibody as C7.

Embodiment 2: Prepare Primary Culture of C7

(30) The supernate obtained through screening in Embodiment 1 and containing anti-Cry1B toxin idiotype single-chain antibody is transferred to 10 ml of 2TY-AG fluid medium at a volume ratio of 1:100 and incubated at 37 C. for 2 h. 100 l of helper phage KM13 with titer of 10.sup.12 is added for rescue, incubated at 30 C. for 2 h and centrifuged at 1800 g for 10 minutes. The supernate is removed. 2TY-AK fluid medium is used to resuspend the precipitated bacteria. It is cultivated while being shaken at 30 C. 250 rpm overnight. Next day it is centrifuged at 1800 g for 30 minutes. Its supernate is supernate containing C7 primary culture.

Embodiment 3: Subtype Identification of C7

(31) (1) ELISA Detection Experiment of Competitive Inhibition

(32) The experiment adopts 6 experimental groups and corresponding control groups. Solutions are prepared based on Table 1.

(33) TABLE-US-00003 TABLE 1 Preparation of solutions for ELISA detection experiment Irrelevant Anti-Id single-chain Group C7 antibody 2 TY fluid medium Experimental group 1 5 l 45 l Control group 1 5 l 45 l Experimental group 2 10 l 40 l Control group 2 10 l 40 l Experimental group 3 20 l 30 l Control group 3 20 l 30 l Experimental group 4 30 l 20 l Control group 4 30 l 20 l Experimental group 5 40 l 10 l Control group 5 40 l 10 l Experimental group 6 50 l Control group 6 50 l

(34) In Table 1, C7 is the supernate obtained in Embodiment 2 and containing C7 primary culture;

(35) Add 50 l of 10 g/ml anti-Cry1B polyclonal antibody to the solutions prepared in Table 1 respectively, incubate them at 37 C. for 2 h, add them to a 96-well plate coated with 2 g/ml Cry1B toxin respectively (the 96-well plate coated with 2 g/ml Cry1B toxin is obtained by adding 2 g/ml Cry1B toxin to a 96-well plate on the previous day, 100 l/well and keeping it at 4 C. overnight), take reaction for 2 h; wash the plate with 250 l/well of PBST for 3 times, add 100 l/well of 1:5000 diluted HRP-goat anti-rabbit IgG incubate it at room temperature for 1 h; wash the plate with 250 l/well of PBST for 3 times, add 100 l/well of substrate chromogenic solution, take reaction at room temperature for 10 to 20 minutes till blue appears and in the end add 50 l/well of 2 mol/L H.sub.2SO.sub.4 to quickly terminate the reaction; determine OD.sub.450 by ELIASA.

(36) The experimental results are as shown in FIG. 1. The inhibition ratio increases with the increase of C7 content. The control groups do not have the phenomenon of competitive inhibition, suggesting C7 is -type Anti-Id single-chain antibody and can simulate Cry1B toxin to competitively bind with anti-Cry1B toxin polyclonal antibody.

(37) (2) Biological Determination Experiment

(38) The experiment has experimental group 1, experimental group 2, experimental group 3, positive control group, negative control group 1, negative control group 2 and negative control group 3; the experimental procedure is as follows: (a) Blocking: Coat 100 l/well of 5 g/ml BBMV in a 96-well plate, keep it at 4 C. overnight, wash the plate with 250 l/well of PBST for 3 times next day, add 200 l of BAS with a mass ratio of 3% respectively, incubate it at room temperature for 2 h, and carry out blocking; (b) Sample addition: Wash the 96-well plate blocked in step 1 with 250 l/well of PBST for 3 times, and add samples to the 96-well plate according to Table 2:

(39) TABLE-US-00004 TABLE 2 Preparation of solutions for biological determination experiment of C7 Non-- 2 TY- 2 g/ml type AG Cry1B Anti-Id fluid Group toxin C7 ScFv medium CPBS Experimental group 1 50 l 10 l 40 l Experimental group 2 50 l 30 l 20 l Experimental group 3 50 l 50 l Positive control 50 l 50 l group Negative control 50 l 10 l 40 l group 1 Negative control 50 l 30 l 20 l group 2 Negative control 50 l 50 l group 3

(40) In Table 2, C7 is the supernate obtained in Embodiment 2 and containing C7 primary culture; (c) Incubate the 96-well plate added with sample in step b at room temperature for 2 h, wash the plate with 250 l/well of PBST for 3 times, add 100 l/well of 10 g/ml anti-Cry1B polyclonal antibody, then wash the plate with 250 l/well of PBST for 3 times, add 100 l/well of 1:5000 diluted HRP-goat anti-rabbit IgG and incubate it at room temperature for 1 h; wash the plate with 250 l/well of PBST for 3 times, add 100 l/well of substrate chromogenic solution per well, take reaction at room temperature for 10 to 20 minutes till blue appears and in the end add 50 l/well of 2 mol/L H.sub.2SO.sub.4 to quickly terminate the reaction, and determine OD.sub.450 by ELIASA.

(41) The experimental result is as shown in FIG. 2. Compared with positive control, anti-Cry1B toxin idiotype single-chain antibody C7 (experimental groups 1, 2 and 3) can inhibit the binding between Cry1B toxin and its receptor BBMV; non--type negative control does not have the phenomenon of inhibition, which further proves that C7 is type.

Embodiment 4: Verify Insecticidal Activity of Anti-Cry1B Toxin Idiotype Single-Chain Antibody

(42) The experiment has experimental groups and control groups:

(43) The experimental groups use the supernate (C7) obtained in Embodiment 2 and containing C7 primary culture;

(44) The positive control groups adopt 0.2 g/L Cry1Ab toxin (CK+);

(45) The negative control groups adopt non- type Anti-Id ScFvs (CK);

(46) Experimental Procedure:

(47) Measure experimental groups, positive control groups and negative control groups each 10 ml, put them in sterilized culture dishes, add 6 paddy leaves and 6 cabbage leaves respectively, soak them for 30 minutes, take them out and dry them in the air; feed Cnaphalocrocis medinalis third instar larvae and Plutella xylostella third instar larvae with dried leaves.

(48) The experimental result is as shown in FIG. 3 and FIG. 4. FIG. 3 shows the death condition of Cnaphalocrocis medinalis third instar larvae respectively fed with paddy leaves, which have been soaked with C7, Cry1Ab toxin (CK+) and non--type Anti-Id ScFvs (CK). FIG. 4 shows the death condition of Plutella xylostella third instar larvae respectively fed with cabbage leaves, which have been soaked with C7, Cry1Ab toxin (CK+) and non--type Anti-Id ScFvs (CK). It can be seen that C7 has a good insecticidal effect.