WO2016086513A1 - 一种人源杀虫基因及其编码杀虫肽与应用 - Google Patents

一种人源杀虫基因及其编码杀虫肽与应用 Download PDF

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WO2016086513A1
WO2016086513A1 PCT/CN2015/070423 CN2015070423W WO2016086513A1 WO 2016086513 A1 WO2016086513 A1 WO 2016086513A1 CN 2015070423 W CN2015070423 W CN 2015070423W WO 2016086513 A1 WO2016086513 A1 WO 2016086513A1
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insecticidal
gene
human
toxin
solution
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French (fr)
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刘贤金
刘媛
谢雅晶
武爱华
张霄
徐重新
赵岩岩
仲建锋
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江苏省农业科学院
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Priority to JP2016535244A priority Critical patent/JP6211702B2/ja
Priority to EP15851632.8A priority patent/EP3067423B1/en
Priority to US15/103,515 priority patent/US9725521B2/en
Priority to BR112016014689-1A priority patent/BR112016014689B1/pt
Publication of WO2016086513A1 publication Critical patent/WO2016086513A1/zh

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/50—Isolated enzymes; Isolated proteins
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N47/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid
    • A01N47/40—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides
    • A01N47/42—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides containing —N=CX2 groups, e.g. isothiourea
    • A01N47/44—Guanidine; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
    • C07K16/1278—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Bacillus (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
    • C07K16/4208—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig
    • C07K16/4233—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-bacterial Ig
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the invention relates to the field of genetic engineering and biological control, in particular to a human insecticidal gene and an insecticidal peptide thereof and application thereof.
  • Bt toxin genes such as Cry1C, Cry1Ab, Cry1B, Cry1F, Cry2Aa, etc.
  • Bacillus thuringiensis is an insect pathogenic bacterium, and its Bt toxin has a specific poisoning effect on a variety of agricultural and forest pests. From 1987, Plant Genetic Systems of Belgium reported the success of transgenic Bt insecticide tobacco. Up to now, Bt gene has been transgenic into major crops such as corn, rice, cotton, tomato, potato and tobacco.
  • israeltaki HD73 binds to surface proteins in the mouse small intestine
  • Vázquez-Padrón et al Biochem Biophys Res Commun, 2000-01
  • T cells are positive for T cells in mice when the exotoxin reaches 10 mg/kg and 100 mg/kg.
  • the rate, spleen index and macrophage phagocytic function showed obvious inhibition reaction. With the increase of ingestion dose, this inhibition effect became more obvious.
  • the experiment also found that when the accumulation coefficient of Bt toxin protein in animals is greater than 6.24.
  • Bt toxin protein is also caused by the immune response, and the long-term high dose of Bt toxin protein also causes a significant decrease in the total white blood cell count and hemoglobin content of the animal, which indicates that the Bt toxin protein has obvious immunosuppressive toxicity. Therefore, the development of alternative biological effectors with biological activity of Bt toxin is a hot spot in the field of research and development of biological pesticides.
  • Insecticidal peptides are biologically active polypeptides induced by organisms. Some Bt insecticidal crystal proteins can be decomposed into insecticidal peptides in insects to achieve insecticidal effects, thus becoming a substitute for the biological activity of Bt toxins.
  • Anti-Id anti-idiotype antibody
  • Anti-Id is a kind of polypeptide with biological insecticidal effect. Anti-Id is a pointer to the variable region of antibody molecule. Specific antibodies produced by Idiotype (Id), Bona et al.
  • the classified anti-idiotype antibodies into four types ( ⁇ , ⁇ , ⁇ , and ⁇ ) based on the serological response of Id and Anti-Id and the function of AId.
  • the ⁇ -type anti-idiotypic antibody has an antigenic function and biological activity because it has an "inner image" effect, that is, has the same antigenic determinant as the (semi)antigen.
  • phage display technology is widely used.
  • a phage antibody library By constructing a phage antibody library, specific anti-idiotypic antibodies with similar effects of target antigens can be obtained by specific screening.
  • the process of screening specific antibodies by phage display technology is called “Panning”. It mainly includes four steps of binding, washing, elution and amplification.
  • Raats et al. use anti-cortisol monoclonal anti-coating as a solid phase antigen for direct screening. Before screening, negative monoclonal antibodies of the same species are used. Negative screening was performed to avoid screening for recombinant antibody fragments that bind to the constant region of the antibody, and finally the anti-Id against cortisol was successfully screened; Goletz et al.
  • Anti-Id ScFvs anti-Bt toxin type anti-idiotype single-chain antibodies
  • An insecticidal peptide encoded by a human insecticidal gene according to the present invention the amino acid sequence of which is shown in SEQ ID NO.
  • a prokaryotic vector containing the human insecticidal gene of the present invention is provided.
  • insecticidal peptide encoded by the human insecticidal gene for the control of crop pests.
  • An insecticide comprising an insecticidal peptide encoded by a human insecticidal gene according to the present invention.
  • the present invention screens and obtains a " ⁇ " type anti-Cry2Aa toxin unique type single-chain antibody (insecticidal peptide) having insecticidal activity from the disclosed human gene library, and performs amino acid and nucleotide sequencing on the insecticidal peptide.
  • the primary culture has the binding activity to the peritrophic membrane-specific receptor BBMV of the rice leaf roller, which is not immunized by the animal, has a short preparation cycle and a small amino acid sequence, and is suitable for large-scale production in vitro.
  • the insecticidal peptide explores and expands new insecticidal gene resources with simulated Bt toxin biological activity, reduces various safety risks existing in the widespread use of existing Bt toxins, and may replace Bt in the future.
  • the biological control of agricultural pests and the reduction of the use of pesticides have important scientific and practical significance.
  • Figure 1 is a schematic diagram showing the results of F2 ELISA.
  • Figure 2 is a schematic diagram showing the results of F2 biological assay.
  • Fig. 3 is a schematic diagram showing the death of the third instar larvae of Cnaphalocrocis medinalis after feeding F2, CK+ and CK-soaked rice leaves, respectively.
  • Fig. 4 is a schematic diagram showing the death of the third instar larvae of Plutella xylostella after being fed with F2, CK+, CK-soaked cabbage tablets, respectively.
  • Glucose having a final concentration of 100 ⁇ g/ml ampicillin and a mass ratio of 1% was added to a 2 ⁇ TY medium.
  • a final concentration of 100 ⁇ g/ml ampicillin and 50 ⁇ g/ml kanamycin was added to the 2 ⁇ TY medium.
  • a final concentration of 100 ⁇ g/ml ampicillin, 50 ⁇ g/ml kanamycin, and a mass ratio of 1% glucose were added to the 2 ⁇ TY medium.
  • a final concentration of 100 ⁇ g/ml ampicillin and a mass ratio of 1% glucose were added to the TYE solid medium.
  • 0.05% PBST was added to a PBS solution at a volume ratio of 0.05% Tween-20.
  • PBST 0.1% PBST was added to a PBS solution at a volume ratio of 0.1% Tween-20.
  • Citrate buffer (CPBS, substrate buffer, pH 5.5):
  • skim milk powder 3 g was weighed, dissolved in 80 ml of PBS solution, and made up to 100 ml with PBS solution.
  • the HRP-goat anti-rabbit IgG described in the examples was diluted in 3% MPBS solution.
  • BBMV irrelevant anti-idiotype single-chain antibody
  • non-" ⁇ " type Anti-Id ScFv cabbage leaf
  • third instar larvae of Plutella xylostella were provided by the Key Laboratory of Agricultural Product Quality Safety Control Technology and Standards, Ministry of Agriculture, Jiangsu Academy of Agricultural Sciences ;
  • Negative serum collected from 1.5-2.0 kg of pure male New Zealand white rabbits, the collection time is one week before immunization;
  • Anti-Cry2Aa polyclonal antibody Cry2A protein standard (Shanghai Youlong Biotechnology Co., Ltd.) was used as the immunogen. Three 1.5-2.0 kg purebred male New Zealand white rabbits were selected as test animals. The specific immunization procedure was as follows: Each 200 ⁇ g immunogen was mixed with an equal volume of Freund's complete adjuvant, emulsified into a water-in-oil structure and then subcutaneously injected into the back (about 40 points). After two weeks, the same dose of immunogen was used with an equal volume of incomplete The adjuvant was boosted and then boosted every two weeks. The last time the immunogen was diluted with an equal volume of normal saline, and the ear was injected directly into the ear.
  • the heart was bled, serum was prepared, and a final concentration of 0.01% thiomersal was added.
  • the serum was purified by the method described in "Contemporary Immunology Technology and Application” (Bad Year, Beijing Medical University, China Union Medical University, United Press, 1998. 309-322) to obtain an anti-Cry2Aa polyclonal antibody.
  • the humanized phage antibody library, TG1 bacteria and helper phage KM13 were purchased from Source BioScience, UK;
  • HRP-goat anti-M13-IgG was purchased from Wuhan Boster Company;
  • Cry2Aa toxin and Cry1Ab toxin were purchased from Shanghai Youlong Biotechnology Co., Ltd.;
  • the third instar larvae of rice leaves and leaf roller are provided by Yangzhou Luyuan Biochemical Co., Ltd.
  • the screening method adopts positive and negative screening, negative serum is used as negative screening, anti-Cry2Aa polyclonal antibody is used as positive screening, negative negative and negative cells first.
  • the culture flask is coated with negative serum, and the positive cell culture flask is coated with anti-Cry2Aa polyclonal antibody, and the elution method uses four rounds of competitive elution:
  • the anti-Cry2Aa polyclonal antibody and the negative serum concentration in the second, third and fourth rounds of panning were still 100 ⁇ g/ml, and the phage antibody used was the phage antibody obtained by the previous round of panning, and the panning method still used the first one.
  • the positive and negative screening strategy of the round the difference is that the second round of washing the bottle with 0.1% PBST solution 10 times, adding 1ml 10mg / ml trypsin (Trypsin) and competitive elution 1h, the third and fourth times are 0.1% Wash the PBST solution for 20 times, then add 500 ⁇ l of 100 ⁇ g/ml Cry2Aa polyclonal antibody instead. Trypsin competes for elution, the third round of competitive elution time is 1 h, and the fourth round of competitive elution time is 30 min.
  • 10 ⁇ l of the phage antibody in the fourth round of panning was infested with 1 ml of TG1 bacteria in logarithmic growth phase, incubated at 37 ° C for 1 h, then coated on TYE-AG solid medium, and cultured overnight at 37 ° C; the next day random pick list Colonies, inoculated into 96-well plates containing 100 ⁇ l/well 2 ⁇ TY-AG liquid medium, cultured overnight at 37° C.; 2 ⁇ l of bacterial solution was aspirated from the wells of the plate the next day and transferred to a new 96-well plate well at 37 ° C.
  • the supernatant in step 2 corresponding to the solution is the selected supernatant containing the anti-Cry2Aa toxin unique type single-chain antibody, i.e., insecticidal peptide.
  • the nucleotide sequence of the selected insecticidal peptide was determined by Sanger sequencing method as SEQ ID NO. 1 as follows:
  • amino acid sequence of the selected insecticidal peptide was determined by Sanger sequencing method as SEQ ID NO. 2, as follows:
  • the supernatant containing the insecticidal peptide screened in Example 1 was transferred to 10 ml of 2 ⁇ TY-AG liquid medium at a volume ratio of 1:100, incubated at 37 ° C for 2 h, and 100 ⁇ l of helper phage with a titer of 10 12 was added.
  • KM13 was rescued, incubated at 30 ° C for 2 h, centrifuged at 1800 g for 10 min, the supernatant was removed, and the precipitated strain was resuspended in 2 ⁇ TY-AK liquid medium, and cultured overnight at 30 ° C, 250 rpm; the next day, 1800 g was centrifuged for 30 min, on which The supernatant is the supernatant containing the F2 primary culture.
  • the experiment was divided into 6 experimental groups and corresponding control groups, and the solutions were prepared according to Table 1.
  • F1 in Table 1 is the supernatant of the F2-containing primary culture obtained in Example 2;
  • the experiment was divided into 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 steps were as follows:
  • F2 is the supernatant of the F2-containing primary culture obtained in Example 2;
  • step b Incubate the 96-well plate after step b for 2 h at room temperature, wash the plate 3 times with 250 ⁇ l PBST per well, then add 100 ⁇ l of 10 ⁇ g/ml anti-Cry2Aa polyclonal antibody per well, and use 250 ⁇ l PBST per well. Wash the plate 3 times, add 100 ⁇ l of 1:5000 diluted HRP-goat anti-rabbit IgG to each well for 1 h at room temperature; wash the plate 3 times with 250 ⁇ l PBST per well, add 100 ⁇ l of substrate color development solution per well, and react for 10-20 min at room temperature. To the appearance of blue, finally add 50 ⁇ l of 2 mol/L H 2 SO 4 per well to terminate the reaction, and then measure the OD 450 value with a microplate reader.
  • the experiment was divided into experimental group and control group:
  • the experimental group obtained the supernatant (F2) containing the F2 primary culture obtained in Example 2;
  • the positive control group used Cry1Ab toxin (CK+) at a concentration of 0.2 g/L;
  • the negative control group used anti-Id ScFvs (CK-) other than " ⁇ " type;
  • Figure 3 shows the rice leaf roller after feeding the rice leaves soaked in the experimental group (F2), the positive control group (CK+), and the negative control group (CK-).
  • the death of the third instar larvae Fig. 4 shows the death of the third instar larvae of Plutella xylostella after feeding the cabbage tablets soaked in the experimental group (F2), the positive control group (CK+) and the negative control group (CK-), respectively.
  • the experimental group insecticidal peptide F2 has a good insecticidal effect.

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Abstract

一种人源杀虫基因及其编码的杀虫肽与应用，所述人源杀虫基因核苷酸序列如SEQ ID NO.1所示，该基因编码的杀虫肽氨基酸序列如SEQ ID NO.2所示。

Description

一种人源杀虫基因及其编码杀虫肽与应用 技术领域
本发明涉及基因工程和生物防治领域，特别是一种人源杀虫基因及其编码杀虫肽与应用。
背景技术
目前全球广泛用于生物防治病虫害的杀虫基因是苏云金芽孢杆菌(Bacillus thuringiensis，Bt)产生的Bt毒素基因(如Cry1C、Cry1Ab、Cry1B、Cry1F、Cry2Aa等)。苏云金芽孢杆菌是一种昆虫性病原细菌，其产生的Bt毒素对多种农、林害虫都有具有特异性毒杀作用。从1987年比利时植物遗传系统公司(Plant Genetic Systems)首次报道了转Bt基因杀虫烟草获得成功，到现在，Bt基因已经被转基因到玉米、水稻、棉花、番茄、马铃薯、烟草等主要农作物中。据2012年国际生物技术应用服务组织统计，中国种植的转Bt棉面积就已经超过390万公顷，占棉花种植总面积的71.5％。然而，随着转Bt基因作物的应用和推广，其在基因逃逸、改变土壤微生物生态结构、物种的耐药性以及危害正常免疫系统等方面可能存在的潜在危害逐渐受到社会关注。在学术领域，“转Bt基因玉米根际微生物和细菌生理群多样性”(王敏等，生态学杂志，2010年03期)和“转Bt基因玉米对土壤细菌数量多样性的影响”(刘玲等，生态与农村环境学报，2011年03期)分别对室内、室外种植转Bt玉米的土壤进行了细菌数量和多样性分析，结果都发现种植转Bt玉米的与空白对照组相比出现显著差异；“Cry1Ac protoxin from Bacillus thuringiensis sp.kurstaki HD73binds to surface proteins in the mouse small intestine”(Vázquez-Padrón等，Biochem Biophys Res Commun，2000年01期)在动物试验发现，当小鼠摄取的Bt内、外毒蛋白达到10mg/kg和100mg/kg时，小鼠的T细胞ANAE阳性 率、脾脏指数及巨噬细胞的吞噬功能均出现了明显的抑制反应，随着摄取剂量的增加，这种抑制作用越发明显，该试验还发现，当Bt毒素蛋白在动物体内的蓄积系数大于6.24时，可以导致肝脏、肾脏及胃肠道等损伤，在肝脏和肾脏中可以观察到细胞肿胀和空泡样变性异象，并且能看见肾小球血管上皮细胞的病变，当然这也不能排除是由免疫反应造成的，同时长期大剂量使用Bt毒素蛋白，还会导致动物白细胞总数和血红蛋白含量显著性下降，这也说明Bt毒素蛋白具有明显的免疫抑制毒性。因此，开拓具有Bt毒素生物活性的替代生物效应物是开发生物农药领域研究的热点。
杀虫肽是生物体内经诱导产生的一类具有生物活性的多肽，一些Bt杀虫晶体蛋白可在昆虫体内被分解为杀虫肽，实现杀虫的效果，因而成为目前Bt毒素生物活性的替代生物效应物发展的新方向，抗独特型抗体(Anti-idiotype antibody，以下简称Anti-Id)即为这样一类具有生物杀虫效果的多肽，Anti-Id是指针对位于抗体分子可变区的独特型(Idiotype，Id)产生的特异性抗体，Bona等根据Id与Anti-Id的血清学反应以及AId的功能，将抗独特型抗体分为4种类型(α，β，γ和ε)。其中，β型抗独特型抗体由于具有“内影像”作用，即具有与(半)抗原相同的抗原决定簇，因而可以具有抗原的功能和生物活性。
目前普遍采用噬菌体展示技术，通过构建噬菌体抗体库，经特异性筛选，可以获得具有靶标抗原类似效应的抗独特型抗体，采用噬菌体展示技术筛选特异性抗体的过程称为“淘选(Panning)”，主要包括结合、洗涤、洗脱和扩增4个步骤，Raats等采用抗皮质醇单克隆抗包被做为固相抗原直接进行筛选，在进行筛选之前，用相同种属的阴性单克隆抗体进行负筛选以避免筛选到与抗体恒定区结合的重组抗体片段，最终成功筛选到针对皮质醇的Anti-Id；Goletz等同样利用噬菌体抗体展示系统并研究比较了不同洗脱方法对抗独特型抗体片段 筛选结果的影响，最终筛选到的96个克隆中有28个为具有抗独特型特性的阳性克隆。目前针对可替代Bt活性效应物，特别是抗Bt毒素类型的抗独特型单链抗体(以下简称Anti-Id ScFvs)小分子多肽等相关材料及产品还未见报道。
发明内容
针对目前目前转Bt毒素作物及其毒素制剂的广泛应用存在的安全隐患及超敏反应等问题，提供一种具有Bt毒素生物活性的可替代人源杀虫基因及其编码杀虫肽，本发明是这样实现的：
一种人源杀虫基因，其核苷酸序列如SEQ ID NO.1所示。
一种如本发明所述人源杀虫基因编码的杀虫肽，其氨基酸序列如SEQ ID NO.2所示。
一种含有本发明所述人源杀虫基因的原核载体。
如本发明所述人源杀虫基因编码的杀虫肽在农作物害虫防治方面的应用。
一种含有如本发明所述人源杀虫基因编码的杀虫肽的杀虫剂。
本发明从公开的人类基因库中，筛选获得具有杀虫活性的“β”类型抗Cry2Aa毒素独特型单链抗体(杀虫肽)，并对其进行氨基酸与核苷酸测序，该杀虫肽经原核系统表达，初级培养物具有与稻纵卷叶螟中肠围食膜特异性受体BBMV的结合活性，不经动物免疫获得，制备周期短，氨基酸序列小，适合体外大规模生产，此外，该杀虫肽作为全新的新杀虫基因资源，对探索拓展具有模拟Bt毒素生物活性的新型杀虫基因资源，降低现有Bt毒素广泛使用存在的各类安全风险乃至以后可能替代Bt用于农业害虫的生物防治，减少杀虫剂的使用具有重要的科学及现实意义。
附图说明
图1为F2ELISA检测结果示意图。
图2为F2生物学测定结果示意图。
图3为分别喂食经F2、CK+、CK-浸泡过的水稻叶片后的稻纵卷叶螟三龄幼虫死亡情况示意图。
图4为分别喂食经F2、CK+、CK-浸泡过的甘蓝片后的小菜蛾三龄幼虫死亡情况示意图。
具体实施方式
实施例所涉及的试剂和培养基配方：
(1)2×TY液体培养基：
在900mL双蒸水中加入16g胰蛋白胨，10g酵母提取物和5g NaCl，搅拌混匀，用双蒸水定容到1L，置于高压灭菌锅中，121℃，20min灭菌，冷却后，置于4℃保存备用。
(2)2×TY-AG液体培养基：
在2×TY的培养基中加入终浓度为100μg/ml氨苄青霉素和质量比为1％的葡萄糖。
(3)2×TY-AK液体培养基：
在2×TY的培养基中加入终浓度为100μg/ml氨苄青霉素、50μg/ml卡那霉素。
(4)2×TY-AKG液体培养基：
在2×TY的培养基中加入终浓度为100μg/ml氨苄青霉素、50μg/ml卡那霉素和质量比为1％葡萄糖。
(5)TYE固体培养基：
在900ml双蒸水中加入15.0g琼脂糖，8g NaCl，10g胰蛋白胨，5g酵母提取物，用双蒸水定容到1L，置于高压灭菌锅中，121℃，20min灭菌，冷却 后，置于4℃保存备用。
(6)TYE-AG固体培养基：
在TYE固体培养基中加入终浓度为100μg/ml氨苄青霉素和质量比为1％葡萄糖。
(7)PBS溶液
称取NaCl 8.0g，KCl 0.2g，Na2HPO4·12H2O 2.9g，KH2PO4 0.2g，分别加入到蒸馏水中，充分溶解后，定容到1L。
(8)PBST溶液
0.05％PBST是在PBS溶液中加入体积比为0.05％的吐温-20。
0.1％PBST是在PBS溶液中加入体积比为0.1％的吐温-20。
(9)PEG/NaCl溶液：
称取20g PEG 8 000，14.61g NaCl，加80ml去离子水，定容到100ml，置于高压灭菌锅中，121℃，20min灭菌，冷却后，置于4℃保存备用。
(10)柠檬酸盐缓冲液(CPBS,底物缓冲液，pH5.5)：
取C6H7O8(柠檬酸)21g，Na2HPO4·12H2O 71.6g，分别加入到蒸馏水中充分溶解后定容到1L。
(11)四甲基联苯胺(TMB)溶液：
称取10mg四甲基联苯胺溶于1ml二甲基亚砜中，避光，置于4℃保存备用。
(12)底物显色溶液：
10ml配方成分：9.875ml CPBS、100μl TMB溶液、25μl体积比为20％H2O2。
(13)3％的MPBS溶液：
称取3g脱脂奶粉，溶解于80mlPBS溶液中，加PBS溶液定容到100ml。
实施例中所述HRP-羊抗兔IgG是以3％的MPBS溶液稀释。
实施例所涉及材料来源：
BBMV、不相关的抗独特型单链抗体、非“β”类型的Anti-Id ScFv、甘蓝叶片、小菜蛾三龄幼虫均由江苏省农业科学院农业部农产品质量安全控制技术与标准重点实验室提供；
阴性血清：采集自1.5-2.0kg的纯种雄性新西兰大白兔，采集时间为免疫前一周；
抗Cry2Aa多克隆抗体：以Cry2A蛋白标准品(上海佑隆生物科技有限公司)作为免疫原，选取三只1.5-2.0kg的纯种雄性新西兰大白兔作为试验动物，具体免疫程序如下：首次免疫采用每只200微克免疫原与等体积弗氏完全佐剂混和，乳化成油包水结构后进行背部皮下多点注射(40点左右)，两周后用同样剂量的免疫原与等体积不完全弗氏佐剂进行加强，此后每隔两周加强一次，最后一次用等体积的生理盐水稀释免疫原，直接耳缘静脉注射，8天后心脏采血，制备血清，添加终浓度为0.01％的硫柳汞，然后将血清以“当代免疫学技术与应用”(巴德年，北京医科大学,中国协和医科大学联合出版社,1998.309-322)所记载的方法提纯，获得抗Cry2Aa多克隆抗体。
人源化的噬菌体抗体库、TG1细菌和辅助噬菌体KM13购于英国Source BioScience公司；
HRP-羊抗M13-IgG购于武汉博士德公司；
Cry2Aa毒素和Cry1Ab毒素购于上海佑隆生物科技有限公司；
水稻叶片和纵卷叶螟三龄幼虫由扬州绿源生物化工有限公司提供。
实施例1 筛选杀虫肽
(1)取人源化的噬菌体抗体库菌液20μl加入到200ml2×TY-AG液体培养基中，37℃恒温培养到OD600为0.4，取50ml菌液，加入1×1012pfu辅助噬菌体KM13 进行超感染，于37℃孵育30min后以3300g离心10min,弃上清液，用100ml2×TY-AKG液体培养基重悬沉淀，30℃培养过夜；次日3300g离心30min，收集上清液并加入20ml PEG/NaCl溶液，冰浴1h后再3300g离心30min，用4mlPBS重悬沉淀；重悬液以11600g离心10min，上清液即为扩增的噬菌体抗体库；
(2)取步骤1获得的扩增的噬菌体抗体库进行4轮淘筛：筛选方法采取正负筛选，以阴性血清作为负筛选，抗Cry2Aa多克隆抗体作为正筛选，先负后正，负细胞培养瓶中包被阴性血清，正细胞培养瓶瓶中包被抗Cry2Aa多克隆抗体，洗脱方法采用四轮竞争洗脱：
第一轮筛选先将4mL100μg/ml的阴性血清和4mL100μg/ml的抗Cry2Aa多克隆抗体分别包被于负细胞培养瓶与正细胞培养瓶底部，4℃过夜，次日先用PBS清洗负细胞培养瓶3次后加入步骤1获得的1ml扩增的噬菌体抗体库，4ml 3％的MPBS溶液，置于摇床于室温下缓慢摇动1h，再静置1h，用PBS清洗正细胞培养瓶，再将静置1h后的负细胞培养瓶中液体吸入正细胞培养瓶中，置于摇床于室温下缓慢摇动1h，再静置1h，之后弃去正细胞培养瓶中液体，用1ml 0.05％PBST溶液洗正瓶10次，加入1ml 10mg/ml的胰蛋白酶(Trypsin)洗脱特异性结合的噬菌体抗体，洗脱时间为30min，洗脱液即为第1轮淘筛的噬菌体抗体；
第2、3、4轮淘筛的包被抗Cry2Aa多克隆抗体和阴性血清浓度仍然为100μg/ml，所使用的噬菌体抗体为前一轮淘筛获得的噬菌体抗体，淘筛方法仍用第1轮的正负筛选策略，不同的是第二轮采用0.1％PBST溶液洗正瓶10次，加入1ml 10mg/ml的胰蛋白酶(Trypsin)后竞争洗脱1h，第三、四次均用0.1％PBST溶液洗正瓶20次，之后加入500μl浓度为100μg/mlCry2Aa多克隆抗体代替 胰蛋白酶竞争洗脱，第三轮竞争洗脱时间为1h，第四轮竞争洗脱时间为30min。
取第4轮淘筛的噬菌体抗体10μl侵染1ml处于对数生长期的TG1细菌，37℃孵育1h后，涂布于TYE-AG固体培养基上，37℃培养过夜；次日随机挑取单菌落，接种到含100μl/孔2×TY-AG液体培养基的96孔板中，37℃培养过夜；次日从板孔中吸出2μl菌液转接到新的96孔板孔中，37℃孵育2h，每孔加入25μl滴度为1012的辅助噬菌体KM13，30℃孵育2h，1800g离心10min，用150μl 2×TY-AK液体培养基重悬沉淀后30℃培养过夜，次日1800g离心30min，分别取上清液；
(3)取4μg/ml的抗Cry2Aa多克隆抗体加入到96孔板中，100μl/孔，4℃过夜，次日每孔分别加入100μl步骤2获得的上清液，阴性对照加100μl 2×TY-AK液体培养基，37℃水浴2h，每孔用250μlPBST洗板后，每孔加入100μl 1:5000稀释的HRP-羊抗M13-IgG，37℃孵育2h；每孔加入100μl底物显色溶液，室温下反应10～20min至出现蓝色，最后每孔加入50μl浓度为2mol/L的H2SO4快速终止反应后用酶标仪测定OD450值，其中溶液OD450值/阴性对照OD450值大于2.1的，判断为阳性，与该溶液对应的步骤2中的上清液即为筛选到的含有抗Cry2Aa毒素独特型单链抗体，即杀虫肽的上清液。
以Sanger测序法测定筛选到的杀虫肽核苷酸序列为SEQ ID NO.1如下：
Figure PCTCN2015070423-appb-000001
Figure PCTCN2015070423-appb-000002
以Sanger测序法测定筛选到的杀虫肽氨基酸序列为SEQ ID NO.2，如下：
Figure PCTCN2015070423-appb-000003
申请人将此杀虫肽自命名为F2。
实施例2 制备F2的初级培养物
将实施例1筛选到的含有杀虫肽的上清液按体积比1:100转接到10ml 2×TY-AG液体培养基中，37℃孵育2h，加入100μl滴度为1012的辅助噬菌体KM13进行救援，30℃孵育2h，1800g离心10min，去上清液，用2×TY-AK液体培养基重悬沉淀菌种，于30℃、250rpm振荡培养过夜；次日1800g离心30min，其上清液即为含有F2初级培养物的上清液。
实施例3 F2的亚型鉴定
(1)竞争抑制ELISA检测实验
实验分为6个实验组与相应的对照组，分别依表1配制溶液
表1 竞争抑制ELISA检测实验溶液配制
Figure PCTCN2015070423-appb-000004
Figure PCTCN2015070423-appb-000005
表1中F2为实施例2获得的含F2初级培养物的上清液；
分别向表1配制好的溶液中加入50μl 10μg/ml的抗Cry2Aa多克隆抗体37℃孵育2h，后分别加入到包被有2μg/ml Cry2Aa毒素的96孔板中(所述包被有2μg/ml Cry2Aa毒素的96孔板为前一天取2μg/ml的Cry2Aa毒素加入到96孔板中，100μl/孔，4℃过夜)反应2h；每孔用250μl PBST洗板3次后每孔分别加入100μl 1:5000稀释的HRP-羊抗兔IgG室温孵育1h；每孔用250μl PBST洗板3次后，每孔分别加入100μl底物显色溶液，室温下反应10～20min至出现蓝色，最后每孔分别加入50μl浓度为2mol/L的H2SO4快速终止反应；用酶标仪测定OD450值。
实验结果如图1所示，可见抑制率随着F2含量的增加而增大，对照组则无竞争抑制现象，说明F2为β型抗独特型单链抗体，能够模拟Cry2Aa毒素与其竞争结合抗Cry2Aa毒素多克隆抗体。
(2)生物学测定实验
实验分为实验组1、实验组2、实验组3、阳性对照组、阴性对照组1、阴性对照组2、阴性对照组3；实验步骤如下：
(a)封闭：将5μg/ml的BBMV以100μl/孔包被于96孔板中，4℃过夜，次日每孔用250μl PBST洗板3次后，分别加入200μl质量比为3％的BSA于室温孵育2h，进行封闭；
(b)加样：将步骤1封闭后的96孔板用250μl/孔的PBST洗板3次后，分别向96孔板按照表2进行加样：
表2 F2生物学测定实验溶液配制
Figure PCTCN2015070423-appb-000006
表2中，F2为实施例2获得的含F2初级培养物的上清液；
(c)将步骤b加样后的96孔板于室温下孵育2h，每孔用250μl PBST洗板3次后，然后每孔加入100μl 10μg/ml抗Cry2Aa多克隆抗体，再每孔用250μl PBST洗板3次，每孔加入100μl 1:5000稀释的HRP-羊抗兔IgG室温孵育1h；每孔用250μl PBST洗板3次，每孔加入100μl底物显色溶液，室温下反应10～20min至出现蓝色，最后每孔加入50μl浓度为2mol/L的H2SO4快速终止反应后，用酶标仪测定OD450值。
实验结果如图2所示，与阳性对照相比，杀虫肽F2(实验组1、2、3)能够抑制Cry2Aa毒素与其受体BBMV结合；而非“β”类型的阴性对照则无抑制现象，进一步证实了F2为“β”类型。
实施例4杀虫肽F2杀虫活性验证
实验分为实验组与对照组：
实验组用实施例2获得的含有F2初级培养物的上清液(F2)；
阳性对照组采用浓度为0.2g/L的Cry1Ab毒素(CK+)；
阴性对照组采用非“β”类型的Anti-Id ScFvs(CK-)；
实验步骤：
取实验组、阳性对照组、阴性对照组各10ml，置于灭菌后的培养皿中，分别放入6片水稻叶片和6片甘蓝叶片，浸泡30min,取出晾干；稻纵卷叶螟三龄幼虫和小菜蛾三龄幼虫以晾干后的叶片喂食。
实验结果如图3、4所示，其中，图3为分别喂食经实验组(F2)、阳性对照组(CK+)、阴性对照组(CK-)浸泡过的水稻叶片后的稻纵卷叶螟三龄幼虫死亡情况，图4为分别喂食经实验组(F2)、阳性对照组(CK+)、阴性对照组(CK-)浸泡过的甘蓝片后的小菜蛾三龄幼虫死亡情况，可看出，实验组杀虫肽F2具有较好的杀虫效果。

Claims (5)

  1. 一种人源杀虫基因，其核苷酸序列如SEQ ID NO.1所示。
  2. 如权利要求1所述人源杀虫基因编码的杀虫肽，其氨基酸序列如SEQ ID NO.2所示。
  3. 一种含有权利要求1所述人源杀虫基因的原核载体。
  4. 如权利要求2所述杀虫肽在农作物害虫防治方面的应用。
  5. 一种含有权利要2所述杀虫肽的杀虫剂。
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