Patent Document

CROSS REFERENCE TO RELATED APPLICATIONS 
     This application is a national stage filing under section 371 of International Application No. PCT/CN2015/070420, filed on Jan. 9, 2015, and published in Chinese on Jul. 30, 2015, as WO 2015/109951 A1 and claims priority of Chinese application no. 201410037000.9 filed on Jan. 26, 2014, the entire disclosure of these applications being hereby incorporated herein by reference. 
     SEQUENCE LISTING 
     This application contains a Sequence Listing in Computer Readable Form; the file, in ASCII format, is designated H0714991.txt, which is 4.2 kilobytes in size and was created on Oct. 7, 2015. The sequence listing file is hereby incorporated by reference in its entirety into the application. 
     FIELD OF THE INVENTION 
     The present invention relates to genetic engineering and biological control field, particularly to a human-derived insect-resistant gene and anti-Cry1Ab toxin idiotype single-chain antibody encoded by the same and application thereof. 
     BACKGROUND OF THE INVENTION 
     Currently, the insecticidal gene widely used in the world for biological control of pests is Bt toxin gene of  Bacillus thuringiensis  (Bt) (such as: Cry1Ab, Cry1Ac, Cry1C and Cry1F et al.).  Bacillus thuringiensis  is insect pathogenic bacterium. The Bt toxin generated by it has a specific killing effect to many species of agricultural and forestry pests. Since Belgian Plant Genetic Systems first reported the success of transgenic Bt insect-resistant tobacco in 1987 till today, Bt gene has been transferred to main crops in the world, such as: maize, paddy, cotton, tomato, potato and tobacco. According to the statistics of International Service for the Acquisition of Agri-biotech Applications (ISAAA) in 2012, the area of transgenic Bt cotton grown in China has exceeded 3.9 million hectares, accounting for 71.5% of the total area of the cotton grown in China. However, following the application and generalization of transgenic Bt crops, its possible potential hazards in gene escape, change of microbial ecological structure of soil, drug resistance of species and harm to normal immune system have gradually aroused the attention of the society. “Diversity of Rhizospheric Microorganisms and Bacterial Physiological Groups of Transgenic Bt Maize” (Wang Min et al, Chinese Journal of Ecology, Issue 03 of 2010) and “Influence of Transgenic Bt Maize on Bacterial Quantity and Diversity of Soil” (Liu Ling et al, Journal of Ecology and Rural Environment, Issue 03 of 2011) analyzed the bacterial quantity and diversity of the soil in which transgenic Bt maize is grown indoors and outdoors respectively. The results all show significant difference between the transgenic Bt maize growing group and the blank control group. 
     “Cry1Ac protoxin from  Bacillus thuringiensis  sp. kurstaki HD73 binds to surface proteins in the mouse small intestine” (Vazquez-Padron et al., Biochem Biophys Res Commun, Issue 01, 2000) discovered that when intrinsic toxic protein of Bt and extrinsic toxic protein of Bt taken in by a mouse reached 10 mg/kg and 100 mg/kg, T cell ANAE positive rate, spleen index and macrophage phagocytosis of the mouse all were inhibited obviously during animal experiment. The more the intake is, the more obvious the inhibiting effect will be. This experiment also discovered that when the cumulative coefficient of Bt toxin protein in animal body was greater than 6.24, it might result in injury of liver, kidney and gastrointestinal tract and in liver and kidney, anomalies of cellular swelling and vacuolar degeneration could be observed and glomerular vascular epithelial lesion could be seen. Of course, it can&#39;t be excluded that they were caused by immunoreactions. Meanwhile, long-term use of Bt toxin protein at a large dose may also result in significant decrease of total white blood cells (WBC) and hemoglobin (HGB) of animals. This also indicates Bt toxin protein has obvious toxicity of immunosuppression. Therefore, developing substitute biological effectors with Bt toxin bioactivity (such as: Anti-idiotype antibody) is a research hotspot in biological pest development field. 
     As humanized antibody gene is derived from human, it has the advantage of gene homology with human immune system. It may avoid harm of its residue in food to human immune system after spray of its preparation or transgenic expression. 
     In 1974, Danish immunologist Jerne introduced the concept of Anti-idiotype antibody in his “Immune Network Theory”. Anti-idiotype antibody (hereinafter referred to as “Anti-Id”) refers to the specific antibody generated to address the idiotype (hereinafter referred to as “Id”) in the variable regions of antibody molecules. Bona, et al classified Anti-Id into four types (α, β, γ and δ) based on serological reaction between Id and Anti-Id as well as the function of AId. β-type Anti-Id has the effect of “internal image”, i.e.: has antigenic determinant same as (haptin) antigen, so it may have the function and bioactivity of antigen. 
     Currently, it is universally believed that Anti-Id with an effect similar to target antigen may be obtained by phage display technology through establishment of a phage antibody library, and specific screening. The process of screening specific antibody by phage display technology is called “Panning” and mainly includes four steps: binding, washing, eluting and amplification. Raats et al. adopted anti-cortisol monoclonal antibody coating as solid-phase antigen for direct screening. Before screening, a same species of negative monoclonal antibody is negatively screened to avoid screening recombinant antibody fragments bound to the constant region of antibody and successfully screen Anti-Id against cortisol. Goletz et al. also applied phage antibody display system and researched and compared the influence of different elution methods on Anti-Id fragment screening results. Of the eventually screened 96 clones, 28 were positive clones with Anti-Id characteristics. So far, no materials and products specific to substitutable Bt active effector, particularly Anti-Bt toxin type Anti-Id single-chain antibody (hereinafter referred to as “Anti-Id ScFvs”), have been reported. 
     CONTENT OF THE INVENTION 
     To address the potential safety hazard from the extensive application of transgenic Bt toxin crops and toxin preparations thereof, hypersensitivity and other problems at present, the present invention is realized through the development of a substitutable biological effector with Bt toxin bioactivity, and its application in biological control of pests: 
     A human-derived insect-resistant gene, having a nucleotide sequence represented by SEQ ID NO.1; 
     In the present invention, an anti-Cry1Ab toxin idiotype single-chain antibody encoded by SEQ ID NO.1, having an amino acid sequence represented by SEQ ID NO.2; 
     In the present invention, a prokaryotic expression vector containing human-derived insect-resistant gene SEQ ID NO.1; 
     In the present invention, an application of human-derived insect-resistant gene SEQ ID NO.1 in control of agricultural pests; 
     In the present invention, an insecticide containing anti-Cry1Ab toxin idiotype single-chain antibody with an amino acid sequence as represented by SEQ ID NO.2. 
     The present invention screened and obtained from disclosed human gene bank a “β”-type anti-Cry1Ab toxin idiotype single-chain antibody with insecticidal activity. After expression by the prokaryotic system, the primary culture of this single-chain antibody has binding activity to  Cnaphalocrocis medinalis  midgut peritrophic membrane specific receptor BBMV. The present invention obtains “β”-type anti-Cry1Ab toxin idiotype single-chain antibody without animal immunization. The preparation cycle is short. The amino acid sequence is small. It is suitable for in vitro mass production. Meanwhile, the present invention as a new insect-resistant gene resource has important scientific and practical significance to exploring and developing new-type insect-resistant gene resources simulating Bt toxin bioactivity to lower the safety risks from the wide use of existing Bt toxins and even substitute Bt in the future in biological control of agricultural pests and reduce the use of pesticides. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a schematic of B12 ELISA detection result. 
         FIG. 2  is a schematic of B12 biological determination result. 
         FIG. 3  is a schematic showing the death condition of  Cnaphalocrocis medinalis  third instar larvae after they were fed with paddy leaves soaked with B12, CK+ and CK− respectively. 
         FIG. 4  is a schematic showing the death condition of  Plutella xylostella  third instar larvae after they were fed with cabbage leaves soaked with B12, CK+ and CK− respectively. 
     
    
    
     DETAILED DESCRIPTION OF THE EMBODIMENTS 
     Reagents and Medium Formulae Involved in the Embodiment: 
     (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.
 
(2) 2×TY-AG Fluid Medium:
 
             Add ampicillin with final concentration of 100 μg/ml and glucose with a mass ratio of 1% to 2×TY culture medium.
 
(3) 2×TY-AK Fluid Medium:
 
             Add ampicillin with final concentration of 100 μg/ml and kanamycin with final concentration of 50 μg/ml to 2×TY culture medium.
 
(4) 2×TY-AKG Fluid 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.
 
(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 121° C. for 20 minutes, cool it and store it at 4° C. for future use.
 
(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.
 
(7) PBS Solution
 
             Weigh 8.0 g of NaCl, 0.2 g of KCl, 2.9 g of Na 2 HPO 4 .12H 2 O and 0.2 g of KH 2 PO 4 , add them into distilled water respectively, dissolve them thoroughly and set the volume to 1 L.
 
(8) PBST Solution
 
             Add Tween-20 with a volume ratio of 0.05% to PBS solution.
 
(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.
 
(10) Citrate Buffer Solution (CPBS, pH=5.5):
 
             Weigh 21 g of C 6 H 7 O 8  (citric acid) and 71.6 g of Na 2 HPO 4 .12H 2 O, add them to distilled water respectively, dissolve them thoroughly and set the volume to 1 L.
 
(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.
 
(12) Substrate Chromogenic Solution:
 
             Composition of 10 ml formula: 9.875 ml of CPBS, 100 μl of TMB solution and 25 μl H 2 O 2  with volume ratio of 20%. 
           
         
       
    
     Sources of the Materials Involved in the Embodiment: 
     Anti-Cry1Ab 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; 
     Humanized phage antibody library, TGl bacteria and helper phage KM13 were purchased from British Source BioScience; 
     HRP-goat-anti-M13-IgG was purchased from Wuhan Boster Biological Technology Co., Ltd.; 
     Cry1Ab toxin was purchased from Shanghai Youlong Biotech Co., Ltd.; 
     Paddy leaves and  Cnaphalocrocis medinalis  third instar larvae were provided by Yangzhou Luyuan Bio-Chemical Co., Ltd. 
     Embodiment 1: Screen Anti-Cry1Ab Toxin Idiotype Single-Chain Antibody 
     
         
         (1) Add 20 μl of humanized phage antibody library bacterium liquid to 200 ml of 2×TY-AG fluid medium, cultivate it at constant temperature 37° C. till OD 600  is 0.4, measure 50 ml of the bacterium liquid, add 1×10 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 2×TY-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 μg/ml anti-Cry1Ab 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-Cry1Ab 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 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 fluid 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 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 μl of 2×TY-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 μg/ml anti-Cry1Ab polyclonal antibody is measured and added to a 96-well plate, 100 μl/well, and stored at 4° C. overnight. Next day, 100 μl of the supernate obtained in step 2 is added to every well. 100 μl of 2×TY-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 μl/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 minutes till blue appears. Lastly 50 μl of 2 mol/L H 2 SO 4  is added to each well to quickly terminate the reaction. OD 450  is determined by ELIASA. If OD 450  of the solution/OD 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-Cry1Ab toxin idiotype single-chain antibody. 
       
    
     The nucleotide sequence of the screened anti-Cry1Ab toxin idiotype single-chain antibody determined by Sanger sequencing method is SEQ ID NO.1, as shown below: 
     
       
         
               
               
               
             
           
               
                 caggaaacag ctatgaccat gattacgcca agcttgcatg caaattctat ttcaaggaga 
                  60 
                   
               
               
                   
               
               
                 cagtcataat gaaataccta ttgcctacgg cagccgctgg attgttatta ctcgcggccc 
                 120 
               
               
                   
               
               
                 agccggccat ggccgaggtg cagctgttgg agtctggggg aggcttgtac agcctggggg 
                 180 
               
               
                   
               
               
                 ggtccctgag actctcctgt gcagcctctg gattcacctt tagcagctat gccatgagct 
                 240 
               
               
                   
               
               
                 gggtccgcca ggctccaggg aaggggctgg agtgggtctc atatatttcg aatgagggta 
                 300 
               
               
                   
               
               
                 cgacgacaat gtacgcagac tccgtgaagg gccggttcac catctccaga gacaattcca 
                 360 
               
               
                   
               
               
                 agaacacgct gtatctgcaa atgaacagcc tgagagccga ggacacggcc gtatattact 
                 420 
               
               
                   
               
               
                 gtgcgaaacc ttggtcggcg tttgactact ggggccaggg aaccctggtc accgtctcga 
                 480 
               
               
                   
               
               
                 gcggtggagg cggttcaggc ggaggtggca gcggcggtgg cgggtcgacg gacatccaga 
                 540 
               
               
                   
               
               
                 tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc atcacttgcc 
                 600 
               
               
                   
               
               
                 gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca gggaaagccc 
                 660 
               
               
                   
               
               
                 ctaagctcct gatctatgct gcatcccggt tgcaaagtgg ggtcccatca aggttcagtg 
                 720 
               
               
                   
               
               
                 gcagtggatc ctgggacaga tttcactctc accatcagca gtctgcaacc tgaagatttt 
                 780 
               
               
                   
               
               
                 gaacttacta ctgtcaacag acttctcatc ctcctctgac gttcggccaa gggaccaagg 
                 840 
               
               
                   
               
               
                 tggaaatcaa acgggcggcc gcacatcatc atcaccatca cggggccgca gaacaaaaac 
                 900 
               
               
                   
               
               
                 tcatctcaga agaggatctg aatggggccg catag 
                 935 
               
             
          
         
       
     
     After nucleotide translation, the amino acid sequence of screened anti-Cry1Ab toxin idiotype single-chain antibody determined by Sanger sequencing method is SEQ ID NO.2, as shown below: 
     
       
         
               
               
               
             
           
               
                                                                H-CDR1 
                   
                   
               
               
                 MKYLLPTAAAGLLLLAAQPAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVR 
                  60 
               
               
                   
               
               
                            H-CDR2 
                   
               
               
                 QAPGKGLEWVS RIHTSGDSTQ YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK 
                 120 
               
               
                   
               
               
                 H-CDR3            _____Link_____                     ______ 
                   
               
               
                 DYRWFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRAS 
                 180 
               
               
                   
               
               
                 L-CDRI               L-CDR2 
                   
               
               
                 QSISSYLNWYQQKPGKAPKLLIYGASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATY 
                 240 
               
               
                   
               
               
                     L-CDR3               His-tag 
                   
               
               
                 YCQQASEAPSTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLNGAASTP 
                 291 
               
             
          
         
       
     
     The applicant names this anti-Cry1Ab toxin idiotype single-chain antibody as B12. 
     Embodiment 2: Prepare Primary Culture of B12 
     
         
         (1) The supernate obtained through screening in Embodiment 1 and containing anti-Cry1Ab toxin idiotype single-chain antibody is transferred to 10 ml of 2×TY-AG fluid 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 12  is added for rescue, incubated at 30° C. for 2 h and centrifuged at 1800 g for 10 minutes. The supernate is removed. 2×TY-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 the supernate containing B12 primary culture. 
       
    
     Embodiment 3: Subtype Identification of B12 
     (1) ELISA Detection Experiment of Competitive Inhibition 
     The experiment adopts 6 experimental groups and corresponding control groups. Solutions are prepared based on Table 1. 
     
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Preparation of solutions for competitive 
               
               
                 inhibition ELISA detection experiment 
               
             
          
           
               
                   
                   
                 Irrelevant Anti-Id 
                 2 × TY 
               
               
                 Group 
                 B12 
                 single-chain antibody 
                 fluid 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 
               
               
                   
               
             
          
         
       
     
     In Table 1, B12 is the supernate obtained in Embodiment 2 and containing B12 primary culture; 
     Add 50 μl of 10 μg/ml anti-Cry1Ab 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 μg/ml Cry1Ab toxin respectively (the 96-well plate coated with 2 μg/ml Cry1Ab toxin is obtained by adding 2 μg/ml Cry1Ab toxin to a 96-well plate on the previous day, 100 μl/well and keeping it at 4° C. overnight), take reaction for 2 h; 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, 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 10-20 minutes till blue appears and in the end add 50 μl/well of 2 mol/L H 2 SO 4  to quickly terminate the reaction; determine OD 450  by ELIASA. 
     The experimental results are as shown in  FIG. 1 . The inhibition ratio increases with the increase of B12 content. The control groups do not have the phenomenon of competitive inhibition, suggesting B12 is β-type Anti-Id single-chain antibody and can simulate Cry1Ab toxin to competitively bind with anti-Cry1Ab toxin polyclonal antibody. 
     (2) Biological Determination Experiment 
     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 μl/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 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:   

     
       
         
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Preparation of solutions for biological 
               
               
                 determination experiment of B12 
               
             
          
           
               
                   
                 2 μg/ml 
                   
                 Non-“β”- 
                 2 × TY-AG 
                   
               
               
                   
                 Cry1Ab 
                   
                 type 
                 fluid 
               
               
                 Group 
                 toxin 
                 B12 
                 Anti-Id ScFv 
                 medium 
                 CPBS 
               
               
                   
               
               
                 Experimental 
                 50 μl 
                 10 μl 
                   
                 40 μl 
                   
               
               
                 group 1 
               
               
                 Experimental 
                 50 μl 
                 30 μl 
                   
                 20 μl 
               
               
                 group 2 
               
               
                 Experimental 
                 50 μl 
                 50 μl 
               
               
                 group 3 
               
               
                 positive Control 
                 50 μl 
                   
                   
                   
                 50 μl 
               
               
                 group 
               
               
                 negative Control 
                 50 μl 
                   
                 10 μl 
                 40 μl 
               
               
                 group 1 
               
               
                 negative Control 
                 50 μl 
                   
                 30 μl 
                 20 μl 
               
               
                 group 2 
               
               
                 negative Control 
                 50 μl 
                   
                 50 μl 
               
               
                 group 3 
               
               
                   
               
             
          
         
       
     
     In Table 2, B12 is the supernate obtained in Embodiment 2 and containing B12 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 μl/well of PBST for 3 times, add 100 μl/well of 10 μg/ml anti-Cry1Ab 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 per well, take reaction at room temperature for 10-20 minutes till blue appears and in the end add 50 μl/well of 2 mol/L H 2 SO 4  to quickly terminate the reaction, and determine OD 450  by ELIASA.   

     The experimental result is as shown in  FIG. 2 . Compared with positive control, anti-Cry1Ab toxin idiotype single-chain antibody B12 (experimental groups 1, 2 and 3) can inhibit the binding between Cry1Ab toxin and its receptor BBMV; non-“β”-type negative control does not have the phenomenon of inhibition, which further proves that B12 is “β” type. 
     Embodiment 4: Verify Insecticidal Activity of Anti-Cry1Ab Toxin Idiotype Single-Chain Antibody 
     The experiment has experimental groups and control groups: 
     The experimental groups use the supernate (B12) obtained in Embodiment 2 and containing B12 primary culture; 
     The positive control groups adopt 0.2 g/L Cry1Ab toxin (CK+); 
     The negative control groups adopt non-“β” type Anti-Id ScFvs (CK−); 
     Experimental Procedure: 
     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. 
     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 B12, 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 B12, Cry1Ab toxin (CK+) and non-“β”-type Anti-Id ScFvs (CK−). It can be seen that B12 has a good insecticidal effect.

Technology Category: 1