Patent Application
20160215066
The present invention relates to genetic engineering and biological control field, particularly to a human-derived insect-resistant gene and anti-Cry1C toxin idiotype single-chain antibody encoded thereby and application thereof.
Currently, the insecticidal gene widely used in the world for biological control of pests is Bt toxin gene of Bacillus thuringiensis (Bt) (such as: Cry1C, Cry1Ab, Cry1B and Cry1F et al.). Bacillus thuringiensis is an insect pathogenic bacterium. The Bt toxin generated by Bacillus thuringiensis 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 into 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 popularization 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 received the attention of the society. The document entitled “Diversity of Rhizospheric Microorganisms and Bacterial Physiological Groups of Transgenic Bt Maize” (Min Wang et al., Chinese Journal of Ecology, 2010(03)) and “Influence of Transgenic Bt Maize on Bacterial Quantity Diversity of Soil” (Ling Liu et al., Journal of Ecology and Rural Environment, 2011(03)) 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 group and the blank control group.
The document “Cry1Ac protoxin from Bacillus thuringiensis sp. kurstaki HD73 binds to surface proteins in the mouse small intestine” (Vázquez-Padrón et al., Biochem Biophys Res Commun, 2000(01)) disclosed that, in animal experiment, 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 respectively, T cell ANAE positive rate, spleen index and macrophage phagocytosis of the mouse all were inhibited obviously. The more doses are intaken, 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 etc. 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'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) number and hemoglobin (HGB) content of animals. This also indicates that 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 pesticide development field.
In 1974, Danish immunologist Jerne firstly proposed 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 directed to 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 ε) 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 envelope as solid-phase antigen for direct screening. Before screening, a same species of negative monoclonal antibody is used to perform negative screening to avoid screening recombinant antibody fragments bound to the constant region of antibody and successfully screen Anti-Id against cortisol. Goletz et al. also employed 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 related 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.
To address the potential safety hazard, hypersensitivity and other problems from the extensive application of transgenic Bt toxin crops and toxin preparations thereof at present, developing a substitutable biological effector with Bt toxin bioactivity, and its application in biological control of pests, the present invention is realized in the following way:
A human-derived insect-resistant gene, having a nucleotide sequence represented by SEQ ID NO.1;
In the present invention, an anti-Cry1C 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 of SEQ ID NO.1;
In the present invention, an application of human-derived insect-resistant gene of SEQ ID NO.1 in control of agricultural pests;
In the present invention, an insecticide containing anti-Cry1C toxin idiotype single-chain antibody with an amino acid sequence as represented by SEQ ID NO.2.
In the present invention, a “β”-type anti-Cry1C toxin idiotype single-chain antibody with insecticidal activity is screened and obtained from disclosed human gene banks. 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-Cry1C 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, as a new insect-resistant gene resource, the present invention 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.
(1) 2×TY liquid medium:
(2) 2×TY-AG liquid medium:
(3) 2×TY-AK liquid medium:
(4) 2×TY-AKG liquid medium:
(5) TYE solid medium:
(6) TYE-AG solid medium:
(7) PBS solution
(8) PBST solution
(9) PEG/NaCl solution:
(10) Citrate buffer solution (CPBS, substrate buffer solution, pH=5.5):
(11) Tetramethyl benaidine (TMB) solution:
(12) Substrate chromogenic solution:
Sources of the materials involved in the embodiment:
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;
Human-derived phage antibody library, TG1 bacteria and helper phage KM13 were purchased from British Source BioScience;
HRP-goat-anti-M13-IgG was purchased from Wuhan Boster Co., Ltd.;
Cry1C toxin and Cry1Ab toxin were purchased from Shanghai Youlong Biotech Co., Ltd.;
Paddy leaves and Cnaphalocrocis medinalis third instar larvae were provided by Yangzhou Luyuan Bio-Chemical Co., Ltd.
(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 OD600 reaches 0.4, measure 50 ml of the bacterium liquid, add 1×1012 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 1012 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 H2SO4 is added to each well to quickly terminate the reaction. OD450 is determined by ELIASA. If OD450 of the solution/OD450 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.
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:
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:
The applicant names this anti-Cry1C toxin idiotype single-chain antibody as E8.
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 1012 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.
(1) ELISA detection experiment of competitive inhibition
The experiment adopts 6 experimental groups and corresponding control groups. Solutions are prepared based on Table 1.
In Table 1, E8 is the supernate containing E8 primary culture obtained in Embodiment 2.
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 H2SO4 to quickly terminate the reaction; determine OD450 by ELIASA.
The experimental results are shown in
(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 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 2h, 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:
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 H2SO4 to quickly terminate the reaction, and determine OD450 by ELIASA.
The experimental result is shown in
The experiment has experimental groups and control groups.
The experimental groups use the supernate (E8) containing E8 primary culture obtained in Embodiment 2.
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:
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.
The experimental results are shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 201410037240.9 | Jan 2014 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2015/070422 | 1/9/2015 | WO | 00 |
