Anthropogenic insect-resistant gene and Cry1C toxin idiotype single-chain antibody encoded thereby and application thereof

Abstract

An anthropogenic insect-resistant gene having a nucleotide sequence represented by SEQ ID NO.1, and a Cry1C toxin idiotype single-chain antibody encoded by said anthropogenic insect-resistant gene and having an amino acid sequence represented by SEQ ID NO.2; the antibody is a β-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; the β-type Cry1C toxin idiotype single-chain antibody of the present invention is obtained without animal immunization, has a short preparation period and small amino acid sequence, and is suitable for large-scale in vitro production. The present invention is an entirely new insect-resistant gene resource, and has significant implications for decreasing the various safety risks associated with the widescale use of existing Bt toxins, substituting Bt toxins in the biocontrol of agricultural pests, and reducing the use of pesticides.

Claims

1. An insect-resistant gene, comprising the nucleotide sequence of SEQ ID NO: 1.

2. An anti-Cry1C toxin idiotype single-chain antibody encoded by the insect-resistant gene according to claim 1, wherein the antibody comprises the amino acid sequence of SEQ ID NO: 2.

3. A prokaryotic vector containing the insect-resistant gene according to claim 1.

4. An insecticide composition comprising the anti-Cry1C toxin idiotype single-chain antibody according to claim 2.

5. A method for controlling agriculture pests wherein said method comprises contacting the pests with a pesticidally effective amount of the anti-Cry1C toxin idiotype single-chain antibody according to claim 2.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a schematic of E8 ELISA detection result.

(2) FIG. 2 is a schematic of E8 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 E8, 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 E8, CK+ and CK− respectively.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Embodiment 1: Screening Human-Derived Inset-Resistant Gene

(5) Reagents and Medium Formulae Involved in the Embodiment

(6) (1) 2×TY liquid 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 min, cool it and store it at 4° C. for future use.

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

(8) (3) 2×TY-AK liquid medium: Add ampicillin with final concentration of 100 μg/ml and kanamycin with final concentration of 50 μg/ml to 2×Ty culture medium.

(9) (4) 2×TY-AKG liquid medium: Add ampicillin with final concentration of 100 μg/ml, kanamycin with final concentration of 50 μg/ml and glucose with a mass ratio of 1% to 2×TY 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 in 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 12° C. for 20 min, cool it and store it at 4° C. for future use.

(11) (6) TYE-AG solid medium: Add ampicillin with final concentration of 100 μg/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 them to 80 ml of deionized water, set the volume to 100 ml, put the solution in an autoclave, sterilize it at 121° C. for 20 min, cool it and store it at 4° C. for future use.

(15) (10) Citrate buffer solution (CPBS, substrate buffer solution, 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: Components of 10 ml formula: 9.875 ml of CPBS, 100 μl of TMB solution and 25 μl of H.sub.2O.sub.2 with volume ratio of 20%.

(18) Sources of the materials involved in the embodiment:

(19) Anti-Cry1C polyclonal antibody, BBMV, irrelevant Anti-Id single-chain antibody, non-“β”-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) Human-derived 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 Co., Ltd.;

(22) Cry1C 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-Cry1C Toxin Idiotype Single-Chain Antibody

(24) (1) Add 20 μl of human-derived phage antibody library bacterium liquid to 200 ml of 2×TY-AG liquid medium, cultivate it at constant temperature 37° C. till OD.sub.600 reaches 0.4, measure 50 ml of the bacterium liquid, add 1×10.sup.12 pfu of helper phage KM13 for superinfection, incubate the obtained solution at 37° C. for 30 min, then centrifuge it at 3300 g for 10 min, discard the supernate, use 100 ml of 2×TY-AKG liquid medium to resuspend and precipitate it and cultivate it at 30° C. overnight; centrifuge it at 3300 g for 30 min 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 min and resuspend and precipitate it by 4 ml of PBS; centrifuge the resuspension solution at 11600 g for 10 min. 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 μg/ml anti-Cry1C 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 shaker, 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-Cry1C polyclonal antibody panned in the second, third and fourth rounds are 50 μg/ml, 25 μg/ml and 10 μg/ml respectively. The used phage antibody is the phage antibody obtained from the previous round of panning. The panning method is same as that adopted in the first round. 10 μl 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 μl/well of 2×TY-AG liquid medium and cultivated at 37° C. overnight; next day, 2 μl 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 μl of helper phage KM13 with titer of 10.sup.12 is added to each well, incubated at 30° C. for 2 h, centrifuged at 1800 g for 10 min, resuspended and precipitated with 150 μl of 2×TY-AK liquid medium and then cultivated at 30° C. overnight. Next day, it is centrifuged at 1800 g for 30 min. The supernate is collected; (3) 4 μg/ml anti-Cry1C polyclonal antibody is taken and added to a 96-well plate at a concentration of 100 μl/well, and stored at 4° C. overnight. Next day, 100 μl of the supernate obtained in step 2 is added to each well. 100 μl of 2×TY-AK liquid 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 μ/well of PBST, 100 μl of 1:5000 diluted HRP-goat-anti-M13-IgG is added to each well and incubated at 37° C. for 2 h. 100 μl of substrate chromogenic solution is added to each well and takes reaction at room temperature for 10 to 20 min till blue appears. Lastly 500 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 judged as positive. The supernate in step 2 corresponding to this solution is the screened supernate containing anti-Cry1C toxin Idiotype single-chain antibody.

(25) The nucleotide sequence of the screened anti-Cry1C toxin idiotype single-chain antibody determined by Sanger sequencing method is SEQ ID NO.1, as shown below:

(26) TABLE-US-00001 tctatttcaa ggagacagtc ataatgaaat acctattgcc tacggcagcc gctggattgt  60 tattactcgc ggcccagccg gccatggccg aggtgcagct gttggagtct gggggaggct 120 tggtacagcc tggggggtcc ctgagactct cctgtgcagc ctctggattc acctttagca 180 gctatgccat gagctgggtc cgccaggctc cagggaaggg gctggagtgg gtctcatcga 240 ttagtaagca tggtagtagg acaacttacg cagactccgt gaagggccgg ttcaccatct 300 ccagagacaa ttccaagaac acgctgtatc tgcaaatgaa cagcctgaga gccgaggaca 360 cggccgtata ttactgtgcg aaacggagta ggctgtttga ctactggggc cagggaaccc 420 tggtcaccgt ctcgagcggt ggaggcggtt caggcggagg tggcagcggc ggtggcgggt 480 cgacggacat ccagatgacc cagtctccat cctccctgtc tgcatctgta ggagacagag 540 tcaccatcac ttgccgggca agtcagagca ttagcagcta tttaaattgg tatcagcaga 600 aaccagggaa agcccctaag ctcctgatct atcatgcatc ccacttgcaa agtggggtcc 660 catcaaggtt cagtggcagt ggatctggga cagatttcac tctcaccatc agcagtctgc 720 aacctgaaga ttttgcaact tactactgtc aacageggca tcagcggcct cggacgttcg 780 gccaagggac caaggtggaa atcaaacggg cggccgcaca tcatcatcac catcacgggg 840 ccg 843

(27) After nucleotide translation, the amino acid sequence of screened anti-Cry1C 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 QAPGKGLEWVSSISKHGSRTTYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK 120 H-CDR1                 -----Link----- RSRLFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRAS 180 L-CDR1                    L-CDR2 QSISSYLNWYQQKPGKAPKLLIYHASHLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATY 240     L-CDR3                     His-tag YCQQRHQRPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLNGAASTP 291

(29) The applicant names this anti-Cry1C toxin idiotype single-chain antibody as E8.

Embodiment 2: Prepare Primary Culture of E8

(30) The supernate obtained containing anti-Cry1C toxin idiotype single-chain antibody screened in Embodiment 1 is transferred to 10 ml of 2×TY-AG liquid medium at a volume ratio of 1:100 and incubated at 37° C. for 2 h. 100 μl 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 min. The supernate is removed. 2×TY-AK liquid medium is used to resuspend and precipitate the bacteria. It is cultivated while being shaken at 30° C., 250 rpm overnight. Next day it is centrifuged at 1800 g for 30 min. Its supernate is supernate containing E8 primary culture.

Embodiment 3: Subtype Identification of E8

(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 competitive inhibition ELISA detection experiment Irrelevant Anti-Id Group E8 single-chain antibody 2 × TY liquid medium Experimental group 1  5 μl 45 μl Control group 1  5 μl 45 μl Experimental group 2 10 μl 40 μl Control group 2 10 μl 40 μl Experimental group 3 20 μl 30 μl Control group 3 20 μl 30 μl Experimental group 4 30 μl 20 μl Control group 4 30 μl 20 μl Experimental group 5 40 μl 10 μl Control group 5 40 μl 10 μl Experimental group 6 50 μl Control group 6 50 μl

(34) In Table 1, E8 is the supernate containing E8 primary culture obtained in Embodiment 2.

(35) Add 50 μl of 10 μg/ml anti-Cry1C polyclonal antibody to the solutions prepared according to Table 1 respectively, incubate them at 37° C. for 2 h, add them to a 96-well plate coated with 2 μg/ml Cry1C toxin respectively (the 96-well plate coated with 2 μg/ml Cry1C toxin is obtained by adding 2 μg/ml Cry1C toxin to a 96-well plate on the previous day at a concentration of 100 μl/well and keeping it at 4° C. overnight), take reaction for 2 h; wash the plate with 250 μg/well of PBST for 3 times, add 100 μl/well of 1:5000 diluted HRP-goat anti-rabbit IgG, incubate it at room temperature for 1 h; wash the plate with 250 μl/well of PBST for 3 times, add 100 μg/well of substrate chromogenic solution, take reaction at room temperature for 10 to 20 min till blue appears and in the end add 50 μl/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 shown in FIG. 1. The inhibition ratio increases with the increase of E8 content. The control groups do not have the phenomenon of competitive inhibition, suggesting E8 is β-type Anti-Id single-chain antibody and can simulate Cry1C toxin to competitively bind with anti-Cry1C 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 μg/well of 5 μg/ml BBMV in a 96-well plate, keep it at 4° C. overnight, wash the plate with 250 μl/well of PBST for 3 times next day, add 200 μl/well 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 μl/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 E8 Non-“β”- 2 × TY- 2 μg/ ml type AG CrylC Anti-Id liquid Group toxin E8 ScFv medium CPBS Experimental group 1 50 μl 10 μl 40 μl Experimental group 2 50 μl 30 μl 20 μl Experimental group 3 50 μl 50 μl Positive control group 50 μl 50 μl Negative control group 1 50 μl 10 μl 40 μl Negative control group 2 50 μl 30 μl 20 μl Negative control group 3 50 μl 50 μl

(40) In Table 2, E8 is the supernate containing E8 primary culture obtained in Embodiment 2. (c) Incubate the 96-well plate added with sample in step b at room temperature for 2 h, wash the plate with 250 μl/well of PBST for 3 times, add 100 μl/well of 10 μg/ml anti-Cry1C polyclonal antibody, then wash the plate with 250 μl/well of PBST for 3 times, add 100 μl/well of 1:5000 diluted HRP-goat anti-rabbit IgG and incubate it at room temperature for 1 h; wash the plate with 250 μl/well of PBST for 3 times, add 100 μl/well of substrate chromogenic solution, take reaction at room temperature for 10 to 20 min till blue appears and in the end add 50 μl/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 shown in FIG. 2. Compared with positive control, anti-Cry1C toxin idiotype single-chain antibody E8 (experimental groups 1, 2 and 3) can inhibit the binding between Cry1C toxin and its receptor BBMV; non-“β”-type negative control does not have the phenomenon of inhibition, which further proves that E8 is “β” type.

Embodiment 4: Verify Insecticidal Activity of Anti-Cry1C Toxin Idiotype Single-Chain Antibody

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

(43) The experimental groups use the supernate (E8) containing E8 primary culture obtained in Embodiment 2.

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

(45) The negative control groups adopt non-“β” type Anti-Id ScFvs (CK−).

(46) Experimental Procedure:

(47) Take 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 min, 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 results are 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 E8, Cry1Ab toxin (CK+) and non-“β”-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 E8, Cry1Ab toxin (CK+) and non-“β”-type Anti-Id ScFvs (CK−). It can be seen that E8 has a good insecticidal effect.