WO2015109951A1 - 一种人源抗虫基因及其编码的抗Cry1Ab毒素独特型单链抗体与应用 - Google Patents

一种人源抗虫基因及其编码的抗Cry1Ab毒素独特型单链抗体与应用 Download PDF

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WO2015109951A1
WO2015109951A1 PCT/CN2015/070420 CN2015070420W WO2015109951A1 WO 2015109951 A1 WO2015109951 A1 WO 2015109951A1 CN 2015070420 W CN2015070420 W CN 2015070420W WO 2015109951 A1 WO2015109951 A1 WO 2015109951A1
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chain antibody
cry1ab toxin
toxin
resistant gene
cry1ab
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PCT/CN2015/070420
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French (fr)
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WO2015109951A8 (zh
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刘贤金
徐重新
张霄
刘媛
谢雅晶
张存政
余向阳
王冬兰
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江苏省农业科学院
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Priority to JP2016535242A priority Critical patent/JP6236531B2/ja
Priority to EP15739827.2A priority patent/EP3098315B1/en
Priority to US15/104,150 priority patent/US9751952B2/en
Priority to BR112016016035-5A priority patent/BR112016016035B1/pt
Publication of WO2015109951A1 publication Critical patent/WO2015109951A1/zh
Publication of WO2015109951A8 publication Critical patent/WO2015109951A8/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
    • 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
    • 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
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • 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)

Definitions

  • the invention relates to the field of genetic engineering and biological control, in particular to a human-resistant insect-resistant gene and an encoded anti-Cry1Ab toxin unique type single-chain antibody and application thereof.
  • Bacillus thuringiensis Bacillus thuringiensis
  • Bacillus thuringiensis Bacillus thuringiensis is an insect pathogenic bacterium, and its Bt toxin has a specific poisoning effect on a variety of agricultural and forest pests.
  • Plant Genetic Systems of Belgium reported the success of transgenic Bt insect-resistant tobacco for the first time in the world.
  • Bt gene has been transferred to corn, rice, cotton, tomato, potato, tobacco, etc. Among the world's major crops.
  • Bt toxin protein In the liver and kidney, cell swelling and vacuolar-like degeneration can be observed, and Seeing the lesions of glomerular vascular epithelial cells, of course, this cannot be ruled out by the immune response. Long-term high-dose use of Bt toxin protein also resulted in a significant decrease in the total number of white blood cells and hemoglobin in animals, indicating that Bt toxin protein has significant immunosuppressive toxicity. The development of alternative biological effectors with Bt toxin bioactivity (eg anti-idiotypic antibodies) is a hot spot in the development of biopesticide research.
  • the humanized antibody gene has the advantage of being homologous to the human immune system gene because it is derived from human, and can avoid the damage to the human immune system caused by the residue in the food after its preparation spray or transgenic expression.
  • An anti-idiotype antibody (hereinafter referred to as Anti-Id) is a specific antibody produced by a unique type (Idiotype, Id) located in the variable region of an antibody molecule.
  • Id an anti-idiotype antibody
  • Bona et al. classify anti-idiotypic 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 can be used to construct an anti-idiotypic antibody with a similar effect of a target antigen by constructing a phage antibody library.
  • the process of screening for specific antibodies using phage display technology is called “Panning” and consists of four steps of binding, washing, elution and amplification.
  • Raats et al. used anti-cortisol monoclonal anti-coating as a solid phase antigen for direct screening, and negative screening with negative monoclonal antibodies of the same species before screening to avoid screening for recombinant antibody fragments that bind to the constant region of the antibody.
  • the anti-Id against cortisol was successfully screened. Goletz et al.
  • Anti-Id ScFvs chain antibodies
  • a human insect-resistant gene having a nucleotide sequence as shown in SEQ ID NO.
  • the anti-Cry1Ab toxin idiotype single-chain antibody encoded by SEQ ID NO. 1 has an amino acid sequence as shown in SEQ ID NO.
  • a prokaryotic expression vector comprising the human insect resistance gene SEQ ID NO.
  • a human insect resistance gene SEQ ID NO. 1 is used in the control of agricultural pests.
  • an insecticide comprising an anti-Cry1Ab toxin unique type single chain antibody having an amino acid sequence as shown in SEQ ID NO.
  • the present invention screens and obtains an insecticidal activity " ⁇ " type anti-Cry1Ab toxin unique type single chain antibody from the published human gene library, and the single chain antibody is expressed by a prokaryotic system, and the primary culture has a rice vertical volume.
  • the binding activity of the peritrophic membrane-specific receptor BBMV in the midgut of the leaf mites the invention obtains the anti-Cry1Ab toxin unique type single-chain antibody of the " ⁇ " type without animal immunization, has a short preparation cycle, small amino acid sequence, and is suitable for large-scale in vitro.
  • the present invention as a brand new new insect-resistant genetic resource, explores and expands new insect-resistant genetic 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. It is of great scientific and practical significance to prevent and control the use of pesticides in biological control of agricultural pests.
  • Figure 1 is a schematic diagram of the results of the B12 ELISA assay.
  • Figure 2 is a schematic diagram showing the results of B12 biological assay.
  • Fig. 3 is a schematic diagram showing the death of the third instar larvae of Cnaphalocrocis medinalis after being fed with B12, CK+ and CK-soaked rice leaves, respectively.
  • Fig. 4 is a schematic diagram showing the death of third instar larvae of Plutella xylostella after being fed with B12, CK+, CK-soaked cabbage tablets, respectively.
  • a final concentration of 100 ⁇ g/ml ampicillin and a mass ratio of 1% glucose were added to the 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.
  • Tween-20 in a volume ratio of 0.05% was added to the PBS solution.
  • Anti-Cry1Ab polyclonal antibody, BBMV, unrelated anti-idiotype single-chain antibody, non-" ⁇ " type Anti-Id ScFv, cabbage leaf, Plutella xylostella larvae are all agricultural products of the Ministry of Agriculture of Jiangsu Academy of Agricultural Sciences Provided by the Quality Safety Control Technology and Standards Key Laboratory;
  • 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;
  • Cry1Ab toxin was 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 eluate is the phage antibody in the first round of panning; 2, 3,
  • the concentration of the anti-Cry1Ab polyclonal antibody in the four rounds of panning was 50 ⁇ g/ml, 25 ⁇ g/ml, and 10 ⁇ g/ml, respectively.
  • the phage antibody used was the phage antibody obtained by the previous round of panning, the panning method and the first round.
  • amino acid sequence is SEQ ID NO. 2, as follows:
  • Example 1 The supernatant containing the anti-Cry1Ab toxin unique type single-chain antibody 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 was added.
  • helper phage KM13 with a titer of 10 12 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 at 30 ° C, shaking at 250 rpm overnight; The next day, centrifugation at 1800 g for 30 min, the supernatant was the supernatant containing the B12 primary culture.
  • the experiment was divided into 6 experimental groups and corresponding control groups, and the solutions were prepared according to Table 1.
  • B12 is the supernatant of the B12-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:
  • B12 is the supernatant of the B12-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-Cry1Ab polyclonal antibody per well, and wash with 250 ⁇ l of PBST per well. 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 to each well, and react at room temperature for 10-20 min. Blue appeared, and finally, 50 ⁇ l of H 2 SO 4 having a concentration of 2 mol/L was added to each well to rapidly terminate the reaction, and the OD 450 value was measured by a microplate reader.
  • the experiment was divided into experimental group and control group:
  • the experimental group obtained the supernatant containing the B12 primary culture obtained in Example 2 (B12);
  • 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;
  • Figures 3 and 4 The experimental results are shown in Figures 3 and 4, wherein Figure 3 is a rice vertical roll fed with rice leaves soaked with B12, Cry1Ab toxin (CK+) and non-" ⁇ " type Anti-Id ScFvs (CK-).
  • FIG. 4 shows the death of the third instar larvae of Plutella xylostella after feeding the B12, Cry1Ab toxin (CK+), non-" ⁇ " type Anti-Id ScFvs (CK-) soaked cabbage slices. It can be seen that B12 has a good insecticidal effect.

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Abstract

一种核苷酸序列如SEQ ID NO.1所示的人源抗虫基因，以及由该人源抗虫基因编码的氨基酸序列为SEQ ID NO.2的抗Cry1Ab毒素独特型单链抗体。

Description

一种人源抗虫基因及其编码的抗Cry1Ab毒素独特型单链抗体与应用 技术领域
本发明涉及基因工程和生物防治领域，特别是一种人源抗虫基因及其编码的抗Cry1Ab毒素独特型单链抗体与应用。
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)的Bt毒素基因(例如Cry1Ab、Cry1Ac、Cry1C、Cry1F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。到1987年比利时植物遗传系统公司(Plant Genetic Systems)就在世界上首次报道了转Bt基因抗虫烟草获得成功，到现在，Bt基因已经被转到玉米、水稻、棉花、番茄、马铃薯、烟草等世界主要农作物中。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备受社会关注。“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。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。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。开拓具有Bt毒素生物活性的替代生物效应物(例如：抗独特型抗体)是开发生物农药领域研究的热点。
人源化的抗体基因因其来源于人类，具有与人类免疫系统基因同源的优势，可以避免在其制剂喷洒或转基因表达后在食品中的残留而对人体免疫系统造成危害。
1974年，丹麦免疫学家Jerne在其“免疫网络学说(Immune Network Theory)”中首次提出抗独特型抗体的概念。抗独特型抗体(Anti-idiotype antibody，以下简称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)。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。
目前普遍认为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毒素活性效应物，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)等相关材料及产品还未见报道。
发明内容
针对目前转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，开拓具有Bt毒素生物活性的可替代生物效应物及其在生物防治害虫中的应用，本发明是这样实现的：
一种人源抗虫基因，其核苷酸序列如SEQ ID NO.1所示。
本发明中,由SEQ ID NO.1编码的抗Cry1Ab毒素独特型单链抗体，其氨基酸序列如SEQ ID NO.2所示。
本发明中，一种含有人源抗虫基因SEQ ID NO.1的原核表达载体。
本发明中，一种人源抗虫基因SEQ ID NO.1在农业害虫防治方面的应用。
本发明中，一种含氨基酸序列如SEQ ID NO.2所示的抗Cry1Ab毒素独特型单链抗体的杀虫剂。
本发明从公开的人类基因库中，筛选获得1种具有杀虫活性的“β”类型抗Cry1Ab毒素独特型单链抗体，该单链抗体经原核系统表达，其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，本发明不经动物免疫而获得“β”类型的抗Cry1Ab毒素独特型单链抗体，制备周期短，氨基酸序列小，适合体外大规模生产；同时，本发明作为全新的新抗虫基因资源，对探索拓展具有模拟Bt毒素生物活性的新型抗虫基因资源，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，减少杀虫剂的使用等具有重要的科学及现实意义。
附图说明
图1为B12ELISA检测结果示意图。
图2为B12生物学测定结果示意图。
图3为分别喂食经B12、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B12、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％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，KH2PO40.2g，分别加入到蒸馏水中，充分溶解后，定容到1L。
(8)PBST溶液
在PBS溶液中加入体积比为0.05％的吐温-20。
(9)PEG/NaCl溶液：
称取20g PEG 8000，14.61g 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,pH＝5.5)：
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。
实施例所涉及材料来源：
抗Cry1Ab多克隆抗体、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；
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；
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 筛选抗Cry1Ab毒素独特型单链抗体
(1)取人源化的噬菌体抗体库菌液20μl加入到200ml 2×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4ml PBS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：第1轮淘筛将4ml100μg/ml的抗Cry1Ab多克隆抗体包被于细胞培养瓶底部，4℃过夜，次日分别用1ml PBS清洗细胞培养瓶3次后加入混匀的1ml扩增的噬菌体抗体库与4ml3％的MPBS溶液，置于摇床于室温下缓慢摇动1h，再静置1h后倾去培养瓶中液体，分别用1ml PBST溶液洗瓶20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Cry1Ab多克隆抗体浓度分别为50μg/ml、25μg/ml、10μg/ml，所使用的噬菌体抗体为前一轮淘筛获得的噬菌体抗体，淘筛方法与第1轮相同；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Cry1Ab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100μl2×TY-AK液体培养基，37℃水浴2h，每孔用250μl PBST洗板后，每孔加入100μl1:5000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Cry1Ab毒素独特型单链抗体的上清液。
以Sanger测序法测定筛选到的抗Cry1Ab毒素独特型单链抗体核苷酸序列为SEQ ID NO.1如下：
Figure PCTCN2015070420-appb-000001
以Sanger测序法测定筛选到的抗Cry1Ab毒素独特型单链抗体核苷酸翻译后的 氨基酸序列为SEQ ID NO.2，如下：
Figure PCTCN2015070420-appb-000002
申请人将此抗Cry1Ab毒素独特型单链抗体自命名为B12。
实施例2 制备B12的初级培养物
(1)将实施例1筛选到的含有抗Cry1Ab毒素独特型单链抗体的上清液按体积比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B12初级培养物的上清液。
实施例3 B12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 PCTCN2015070420-appb-000003
Figure PCTCN2015070420-appb-000004
表1中，B12为实施例2获得的含B12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Cry1Ab多克隆抗体37℃孵育2h，后分别加入到包被有2μg/ml Cry1Ab毒素的96孔板中(所述包被有2μg/ml Cry1Ab毒素的96孔板为前一天取2μg/ml的Cry1Ab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B12含量的增加而增大，对照组则无竞争抑制现象，说明B12为β型抗独特型单链抗体，能够模拟Cry1Ab毒素与其竞争结合抗Cry1Ab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100μl/孔5μg/ml BBMV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 B12生物学测定实验溶液配制
Figure PCTCN2015070420-appb-000005
表2中，B12为实施例2获得的含B12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Cry1Ab多克隆抗体，再每孔用250μlPBST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，抗Cry1Ab毒素独特型单链抗体B12(实验组1、2、3)能够抑制Cry1Ab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B12为“β”类型。
实施例4 抗Cry1Ab毒素独特型单链抗体杀虫活性验证
实验分为实验组与对照组：
实验组用实施例2获得的含有B12初级培养物的上清液(B12)；
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B12、Cry1Ab毒素(CK+)、非“β”类型的Anti-Id ScFvs(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B12、Cry1Ab毒素(CK+)、非“β”类型的Anti-Id ScFvs(CK-)浸泡过的甘蓝片后的小菜蛾三龄幼虫死亡情况，可看出，B12具有较好的杀虫效果。

Claims (5)

  1. 一种人源抗虫基因，其核苷酸序列如SEQ ID NO.1所示。
  2. 一种由权利要求1所述人源抗虫基因编码的抗Cry1Ab毒素独特型单链抗体，其氨基酸序列如SEQ ID NO.2所示。
  3. 一种含有权利要求1所述人源抗虫基因的原核载体。
  4. 一种如权利要求1所述人源抗虫基因在农业害虫防治方面的应用。
  5. 一种含权利要求2所述抗Cry1Ab毒素独特型单链抗体的杀虫剂。
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CN105131123B (zh) * 2015-09-16 2018-11-27 江苏省农业科学院 一种具有杀虫活性的牛源化改型抗体及其制备方法与应用
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