WO2021218053A1 - 一种特异性抗噻虫胺抗体的可变区序列及其重组完整抗体的制备与应用 - Google Patents

一种特异性抗噻虫胺抗体的可变区序列及其重组完整抗体的制备与应用 Download PDF

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WO2021218053A1
WO2021218053A1 PCT/CN2020/121649 CN2020121649W WO2021218053A1 WO 2021218053 A1 WO2021218053 A1 WO 2021218053A1 CN 2020121649 W CN2020121649 W CN 2020121649W WO 2021218053 A1 WO2021218053 A1 WO 2021218053A1
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clothianidin
antibody
variable region
recombinant
light chain
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WO2021218053A9 (zh
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郭逸蓉
常云云
赵颖
刘鹏琰
柳颖
焦沙沙
张天宜
朱国念
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浙江大学
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Priority to US17/921,355 priority Critical patent/US11884743B2/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • 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
    • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
    • 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
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2430/00—Assays, e.g. immunoassays or enzyme assays, involving synthetic organic compounds as analytes
    • G01N2430/10—Insecticides
    • 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
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the technical field of genetic engineering, and in particular relates to the preparation and application of a specific variable region sequence of an anti- clothianidin antibody and its recombinant complete antibody.
  • Clothianidin is a new type of nicotine insecticide with thiazole ring. Because of its unique structure and better performance than traditional nicotine insecticides, it is widely used to prevent and control the effects of various pests on crops. harm. Clothianidin has excellent systemic and osmotic effects, so it is distributed in various parts of crops. Because of its strong water solubility and long half-life, it can easily enter surface water and soil through rainfall and irrigation. Studies have shown that there are high levels of clothianidin residues in surface water, sediments and soils. These pesticide residues pose a huge threat to ecological health and non-target pollinators and aquatic invertebrates. The European Union has now banned the use of clothianidin in outdoor environments.
  • the maximum residue limits (MRLs) of clothianidin in different foods are 0.01-1.5mg/mg.
  • MRLs of clothianidin are set It is 0.01-2mg/kg.
  • the monitoring of clothianidin in the environment and agricultural products is of great significance.
  • the detection methods for clothianidin mainly include instrument-based detection methods and immunoassay-based detection methods.
  • Instrumental analysis methods include HPLC, GC and MS. These instrumental analysis methods have high precision, accuracy and sensitivity, and can meet the precise qualitative and quantitative detection requirements of clothianidin.
  • the instrumental analysis methods require expensive analytical instruments, Professional laboratory personnel and complex sample pre-processing procedures make it difficult to achieve large-scale on-site rapid analysis, and rapid immunoassay detection methods can meet the needs of pesticide residues on-site detection and large-scale sample screening.
  • Dinotefuran is a new tobacco pesticide with a very different chemical structure from the existing neonicotinoid pesticides. Because of its broad insecticidal spectrum, it is very safe for crops, humans, animals, and the environment. The actual amount of clothianidin used in production is not less than that of clothianidin, and its physical and chemical properties are similar to other neonicotinoid pesticides, so dinotefuran residues often exist in the environment.
  • the anti-clothianidin monoclonal antibody that can cross-recognize dinotefuran is used for rapid immunoassay detection. In actual field testing, it is impossible to quickly distinguish between clothianidin and dinotefuran. Therefore, there is an urgent need to develop highly specific and sensitive antibodies. Clothianidin antibody.
  • Pesticide immunoassay rapid detection technology is based on the specific combination of pesticide antigens and antibodies, so the preparation of antibodies is the core key of pesticide immunoassay rapid detection.
  • Traditional antibodies include polyclonal antibodies and monoclonal antibodies, both of which are prepared by immunizing animals, and the latter is widely used for rapid detection of pesticide immunoassays because of only one antigen-recognizing epitope and strong specificity.
  • the production of monoclonal antibodies based on hybridoma cells is costly, time-consuming, and requires long-term cryopreservation and regular domestication of hybridoma cells. However, in these processes, hybridoma cells will lose or mutate effective genes due to improper storage conditions. It cannot produce effective antibodies.
  • recombinant antibodies with clear sequences have gradually become research hotspots, including single-chain antibodies, Fab antibodies, and recombinant complete antibodies.
  • mammalian cell expression systems can guide the correct folding of recombinant antibodies, provide accurate multiple post-translational processing and modification functions, and therefore can stably produce recombinant antibodies with the same activity as the parent antibody, which not only confirms
  • the accuracy of the variable region sequence of the parent antibody can also solve many problems faced by conventional antibody preparation, such as the batch-to-batch difference of traditional antibodies and the easy loss of effective genes during the passage and preservation of cell lines, causing cell lines to fail to produce effective antibodies.
  • a large number of recombinant antibodies can be prepared in vitro without sacrificing mice to prepare ascites antibody.
  • the operation is simple and the preparation cycle is short, which is conducive to the large-scale production of antibodies, and contributes to the development and application of sensitive and reliable rapid detection techniques for pesticide residues.
  • the present invention provides a method for preparing and stable production of an anti- clothianidin recombinant complete antibody with high sensitivity and strong specificity, as well as the application of the antibody.
  • the technical solution adopted by the present invention to solve its technical problems is: a specific variable region sequence of an anti- clothianidin antibody, and the amino acid sequence of the gene encoding the heavy chain variable region is shown in SEQ ID NO: 2.
  • amino acid sequence of the gene encoding the light chain variable region is shown in SEQ ID NO: 4.
  • nucleotide sequence of the gene encoding the light chain variable region is shown in SEQ ID NO: 3.
  • the present invention also discloses an anti- clothianidin recombinant complete antibody, including a heavy chain constant region, a heavy chain variable region, a light chain constant region and a light chain variable region, and the heavy chain variable region encodes the amino acid sequence of the gene As shown in SEQ ID NO: 2.
  • nucleotide sequence of the gene encoding the heavy chain variable region is as shown in SEQ ID NO:1.
  • amino acid sequence of the gene encoding the light chain variable region is shown in SEQ ID NO: 4.
  • the present invention also discloses an antibody expression plasmid, which contains the nucleotide sequence of the heavy chain variable region and the mouse heavy chain IgG1 constant region described in any one of the above, and can express the heavy chain of the anti- clothianidin recombinant complete antibody Protein; or, containing the light chain variable region of any one of claims 2-3 and the nucleotide sequence of the mouse light chain Kappa constant region, which can express the light chain protein of the anti- clothianidin recombinant intact antibody .
  • the present invention further discloses an immune test strip for rapid detection of clothianidin residue, which contains the above-mentioned recombinant intact anti- clothianidin antibody.
  • the present invention successfully amplified, sequenced and synthesized the heavy chain and light chain variable region genes of a hybridoma cell line capable of stably secreting a highly specific and sensitive monoclonal antibody against clothianidin, and respectively constructed by using homologous recombination technology
  • the heavy chain and light chain expression vectors were co-transfected into mammalian cells HEK293F, and finally a recombinant complete antibody against clothianidin was obtained.
  • SPR surface plasmon resonance
  • the clothianidin gold-labeled test strip prepared by the recombinant intact antibody is used to construct an immunoassay method for the rapid detection of clothianidin residues in the environment and agricultural products, and the detection limit is as low as 2.5ng/mL .
  • the present invention has the following advantages: the heavy chain variable region and light chain variable region sequences of the anti-clothianidin recombinant complete antibody disclosed in the present invention are derived from mice after multiple immunizations, cell fusion and screening A highly specific monoclonal cell line resistant to clothianidin was obtained.
  • This sequence gene was respectively connected to the expression vector containing the heavy chain constant region gene and the light chain constant region gene, and the recombinant complete antibody was obtained by using the mammalian cell expression of the double plasmid system to ensure the recognition activity of the mouse parent antibody, and The anti- clothianidin antibody can be produced stably in large quantities, and it provides reliable core reagents for various immunoassay methods for detection of clothianidin residues.
  • the immunochromatographic test strip using the anti-clothianidin recombinant intact antibody of the present invention has high sensitivity and high specificity. advantage.
  • Figure 1 shows the results of PCR amplification of agarose gel electrophoresis of cDNA obtained by reverse transcription using 5'race technology as a template, and clothianidin antibody heavy chain variable region genes and light chain variable region genes.
  • Figure 2 shows the SDS-PAGE of the present invention to verify the expression of intact antibodies in mammalian cells HEK293F.
  • Figure 3 is the ELISA competition curve of clothianidin against clothianidin by the recombinant intact anti-clothianidin antibody and mouse monoclonal antibody of the present invention (concentrations from low to high are 0.01, 0.20, 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, 100ng/mL).
  • Figure 4 is a gold-labeled test strip based on recombinant intact antibodies to detect clothianidin standard solution (concentrations from low to high are CK, 0.63, 1.25, 2.5, 5, 10, 20, 40ng/mL).
  • the recombinant anti- clothianidin antibody of the present invention includes a heavy chain constant region, a heavy chain variable region, a light chain constant region and a light chain variable region, and its heavy chain constant region sequence is that of mouse IgG1
  • the light chain constant region sequence of the antibody is the mouse kappa antibody light chain constant region sequence.
  • RNA was extracted by the Trizol method. The integrity of RNA identified by agar gel electrophoresis can meet the requirements of this experiment.
  • 5'Race technology SMARTer RACE 5/3'Kit reverse transcription to synthesize specific cDNA.
  • the functional heavy chain variable region has a full length of 363 bases.
  • the structural domain starts from base 1 and encodes 121 amino acids.
  • the functional heavy chain belongs to IGHV1-14*01, and the matching rate of the V region is 94.9 %, the J area is 97.9%.

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Abstract

提供了一种特异性抗噻虫胺的抗体，该抗体重链可变区氨基酸序列如SEQ ID NO:2所示，轻链可变区氨基酸如SEQ ID NO:4所示，可用于噻虫胺的检测。还提供了一种含有该抗体的快速检测噻虫胺残留的免疫试纸条。

Description

一种特异性抗噻虫胺抗体的可变区序列及其重组完整抗体的制备与应用 技术领域
本发明属于基因工程技术领域，尤其是涉及一种特异性抗噻虫胺抗体的可变区序列及其重组完整抗体的制备与应用。
背景技术
噻虫胺是一种具有噻唑环的新型烟碱类杀虫剂，因其结构独特，性能与传统烟碱类杀虫剂相比更为优异，而被广泛应用于防治各种害虫对农作物的危害。噻虫胺有卓越的内吸和渗透作用，因此会分布在作物各个部位，且因其具有水溶性强和半衰期长等特点，很容易通过降雨和灌溉等途径进入到地表水和土壤中。研究表明，地表水、沉积物和土壤中存在着较高水平的噻虫胺残留，这些农药残留对生态健康和非目标传粉动物及水生无脊椎动物造成巨大威胁。欧盟现已禁止噻虫胺在室外环境下使用，在欧盟标准中，不同的食品中噻虫胺的最大残留限量(MRLs)为0.01-1.5mg/mg，在中国，噻虫胺的MRLs设定为0.01-2mg/kg。为防止消费者和有益生物受到噻虫胺残留的危害，对环境和农产品中的噻虫胺的监测具有重要的意义。
目前，针对噻虫胺的检测方法主要有基于仪器的检测方法和基于免疫分析的检测方法。仪器分析方法有HPLC、GC和MS等，这些仪器分析方法均具有很高精密度、准确度和灵敏度，能够满足噻虫胺的精准定性定量的检测要求，但仪器分析方法需要昂贵的分析仪器、专业的实验人员和复杂的样品前处理程序，因此很难实现大规模的现场快速分析，而免疫分析快速检测方法能满足农药残留现场检测以及大批量样本筛查的需求。
国内外虽然已经有抗噻虫胺单抗的相关报道，但是这些抗体均与呋虫胺有一定的交叉反应。李明等报道了抗噻虫胺单抗的制备和应用，其抗体灵敏度(抑制中浓度IC 50)为26.6ng/mL，与呋虫胺的交叉反应率为47.8％(Detecting clothianidin residues in environmental and agricultural samples using rapid,sensitive enzyme-linked immunosorbent assay and gold immunochromatographic assay.SCI TOTAL ENVIRON 2014,499:1-6)。Uchigashima等报道的抗噻虫胺抗体的IC 50为4.4ng/mL，与呋虫胺的交叉反应率为64％(Development of Immunoassay Based on Monoclonal Antibody Reacted with the Neonicotinoid Insecticides Clothianidin and Dinotefuran.SENSORS-BASEL 2012,12(11):15858-15872)。
呋虫胺是一种与现有的新烟碱类农药的化学结构有着很大差别的新烟类农药，因其具有很广的杀虫谱，并对作物、人畜和环境又十分安全，在实际生产中使用量不低于噻虫胺，其理化性质和其他新烟碱类农药相似，因此环境中也经常存在着呋虫胺残留。将与能交叉识别 呋虫胺的抗噻虫胺单抗用于免疫分析快速检测，在实际现场检测工作中，无法快速区分噻虫胺和呋虫胺，因此急需研制高特异、高灵敏的抗噻虫胺抗体。
农药免疫分析快速检测技术是基于农药抗原和抗体的特异性结合而实现的，故抗体的制备是农药免疫快速检测的核心关键。传统的抗体有多克隆抗体和单克隆抗体，均是通过免疫动物而制备的，且后者因只有一个抗原识别表位特异性强而被广泛应用于农药免疫分析快速检测。依赖杂交瘤细胞制备单克隆抗体这一技术生产成本高、耗时，且需要杂交瘤细胞的长期低温保存和定期驯化，但在这些过程中杂交瘤细胞会因保存条件不当，有效基因丢失或突变而无法产生有效的抗体。随着基因工程技术的发展，序列明确的重组抗体逐渐变成研究热点，包括单链抗体、Fab抗体、重组完整抗体等。在众多重组抗体表达系统中，哺乳动物细胞表达系统能够指导重组抗体的正确折叠，提供准确的多种翻译后的加工和修饰功能，因此可以稳定产生与亲本抗体活性一致的重组抗体，不仅证实了亲本抗体可变区序列的准确性，还可以解决常规抗体制备面临的许多问题，如传统抗体的批次间差异、细胞株传代保存过程中容易丢失有效基因而导致细胞株无法产生有效抗体。只要获得正确的抗体序列基因，就可以在体外大量制备多种形式的重组抗体(包括全长IgG完整抗体、单链抗体scfv、抗原识别片段Fab等)，而无需牺牲小鼠来制备腹水抗体，操作简便，制备周期短，有利于大规模产生抗体，并有助于灵敏、可靠的农药残留快速检测技术的发展和应用。
发明内容
为了克服现有技术的不足，本发明提供一种灵敏度高、特异性强的抗噻虫胺重组完整抗体的制备方法和稳定生产，以及该抗体的应用。
本发明解决其技术问题所采用的技术方案是：一种特异性抗噻虫胺抗体的可变区序列，重链可变区编码基因的氨基酸序列为SEQ ID NO:2所示。
作为优选，重链可变区编码基因的核苷酸序列如SEQ ID NO:1所示。
作为优选，轻链可变区编码基因的氨基酸序列为SEQ ID NO:4所示。
作为优选，轻链可变区编码基因的核苷酸序列如SEQ ID NO:3所示。
本发明还公开了一种抗噻虫胺重组完整抗体，包括重链恒定区、重链可变区、轻链恒定区和轻链可变区，所述重链可变区编码基因的氨基酸序列为如SEQ ID NO:2所示。
作为优选，所述重链可变区编码基因的核苷酸序列为如SEQ ID NO:1所示。
作为优选，所述轻链可变区编码基因的氨基酸序列如SEQ ID NO:4所示。
作为优选，所述轻链可变区编码基因的核苷酸序列如SEQ ID NO:3所示。
本发明又公开了一种抗体表达质粒，含有上述任一项所述的重链可变区和小鼠重链 IgG1恒定区的核苷酸序列，可以表达抗噻虫胺重组完整抗体的重链蛋白；或者，含有如权利要求2-3中任一项所述的轻链可变区和小鼠轻链Kappa恒定区的核苷酸序列，可以表达抗噻虫胺重组完整抗体的轻链蛋白。
本发明再公开了一种快速检测噻虫胺残留的免疫试纸条，含有如上述所述的抗噻虫胺重组完整抗体。
本发明将一株能稳定分泌抗噻虫胺高特异、高灵敏单抗的杂交瘤细胞株的重链和轻链可变区基因成功扩增出来、测序和合成，采用同源重组技术分别构建了重链和轻链表达载体，并共转染至哺乳动物细胞HEK293F中，最终获得抗噻虫胺的重组完整抗体。经ELISA和表面等离子共振(SPR)技术验证，该重组完整抗体与传统的抗噻虫胺小鼠腹水单抗相比，有一致的高灵敏度和高选择性，可以代替腹水单抗，应用于噻虫胺农药残留检测。
利用该重组完整抗体制备的噻虫胺金标试纸条，利用该重组完整抗体构建免疫分析方法，用于环境和农产品中噻虫胺残留的快速检测，其检出限低至2.5ng/mL。
与现有技术相比，本发明具有以下优点：本发明公开的抗噻虫胺重组完整抗体的重链可变区、轻链可变区序列来源于经多次免疫小鼠、细胞融合及筛选得到的一株高特异性的抗噻虫胺的单克隆细胞株。将此序列基因分别连接到包含了重链恒定区基因和轻链恒定区基因的表达载体，采用双质粒系统的哺乳动物细胞表达获得了重组完整抗体，保证了鼠源亲本抗体的识别活性，并使得抗噻虫胺抗体可大批量稳定生产，为噻虫胺残留检测的各种免疫分析方法提供了可靠的核心试剂。
在制备了抗噻虫胺重组完整抗体的基础上，将其应用于噻虫胺的快速检测，使用本发明的抗噻虫胺重组完整抗体的免疫层析试纸条具有灵敏度高、特异性高等优点。
附图说明
图1为本发明以5’race技术反转录获得的cDNA为模板，噻虫胺抗体重链可变区基因、轻链可变区基因PCR扩增琼脂糖凝胶电泳结果。
图2为本发明SDS-PAGE验证完整抗体在哺乳动物细胞HEK293F中的表达。
图3为本发明抗噻虫胺重组完整抗体和小鼠单克隆抗体对噻虫胺的ELISA竞争曲线(浓度从低到高依次为0.01、0.20、0.39、0.78、1.56、3.13、6.25、12.5、25、50、100ng/mL)。
图4为基于重组完整抗体的金标试纸条检测噻虫胺标准溶液(浓度从低到高依次为CK、0.63、1.25、2.5、5、10、20、40ng/mL)。
具体实施方式
为了使本技术领域的人员更好地理解本发明方案，下面将结合本发明实施例中的附图，对发明实施例中的技术方案进行清楚、完整的描述，显然，所描述的实施例仅仅是本发明的一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都应当属于本发明保护的范围，如无特殊说明，实施例中的实验方法均为常规方法。
本发明涉及的抗噻虫胺重组完整抗体包括重链恒定区、重链可变区、轻链恒定区和轻链可变区，其重链恒定区序列为小鼠IgG1的重链恒定区序列，该抗体的轻链恒定区序列为小鼠κ抗体轻链恒定区序列。
小鼠抗噻虫胺重组完整抗体的制备
1)抗噻虫胺单克隆抗体可变区基因扩增
经小鼠免疫、细胞融合和杂交瘤细胞筛选，制备了一株能分泌高特异性的抗噻虫胺抗体的杂交瘤细胞株B2，亚型IgG1/kappa，采用Trizol法提取B2的总RNA，经琼脂凝胶电泳鉴定RNA完整性较好可满足本实验的要求。以RNA为模板，利用5’Race技术(SMARTer RACE 5/3’Kit)反转录合成特异性cDNA。其中，重链和轻链上游引物为试剂盒中自带的接头引物，重链下游特异性引物为CTCAATTTTCTTGTCCACCTTGGT，轻链下游特异性引物为C T C A T T C C T G T T G A A G C T C T T G A C A A T G G G和C T C A T T C C T G T T G A A G C T C T T G A C G A C G G G。
PCR扩增的程序为：
Figure PCTCN2020121649-appb-000001
PCR扩增产物琼脂糖凝胶电泳结果参见图1，扩增出含有VH和VL基因片段的条带。用凝胶提取试剂盒纯化，将纯化后的产物克隆到pEASY-Blunt载体上，转化、测序，序列经NCBI数据库Blast比对后，鉴定出序列完整、亚型相符和正确表达框的VH和VL基因。
抗噻虫胺单克隆抗体B2重链可变区序列为：
Figure PCTCN2020121649-appb-000002
Figure PCTCN2020121649-appb-000003
功能性重链可变区全长为363个碱基，结构域从第1位碱基开始，编码121个氨基酸，该有功能的重链均属于IGHV1-14*01，V区匹配率为94.9％，J区为97.9％。
以IMGT法定义结构域，具体结构域划分为：
结构域 FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4
碱基序列 1-75 76-99 100-150 151-174 175-288 289-330 331-363
抗噻虫胺单克隆抗体B2重链可变区氨基酸序列
Figure PCTCN2020121649-appb-000004
抗噻虫胺单克隆抗体B2轻链可变区序列为
Figure PCTCN2020121649-appb-000005
功能性轻链可变区全长为个336个碱基，结构域从第1位碱基开始，编码112个氨基酸，该有功能的轻链属于IGKV1-117*01，V区匹配率为97.0％，J区为96.0％。
以IMGT法定义结构域，具体结构域划分为：
结构域 FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4
碱基序列 1-78 79-111 112-162 163-171 172-279 280-306 307-336
抗噻虫胺单克隆抗体B2轻链可变区氨基酸序列
Figure PCTCN2020121649-appb-000006
2)表达质粒的构建
在鉴定出正确的噻虫胺B2的重链和轻链可变区基因后，合成正确的基因序列。采用同源重组法分别将重链和轻链可变区基因连接到线性化的表达载体pCDNA3.4-Mouse-IgG1和 pCDNA3.4-Mouse-Kappa上。其中，pCDNA3.4-Mouse-IgG1上面含有小鼠IgG1重链恒定区域基因，pCDNA3.4-Mouse-Kappa上面含有小鼠Kappa轻链恒定区域基因，故构建的重/轻链重组质粒上面分别包含了重链可变区基因和恒定区基因/轻链可变区基因和恒定区基因。分别将重链/轻链表达载体转化至T1感受态、摇菌培养并测序。
3)重组完整抗体的表达
将测序正确的质粒对应的菌液继续大体积培养扩增，提取质粒，待用。复苏HEK293F细胞,120rpm，8％CO 2，37℃悬浮培养3代。转染前，将细胞接种至新的培养瓶，接种密度为1.5×10 6个/mL，悬浮培养2h后，将重链质粒、轻链质粒和转染试剂按照一定的比例静置孵育15min后，转染HEK293F细胞。转染后的细胞悬浮培养至活率低于70％后，4000rpm离心5min后，收集上清培养液，采用Protein A亲和层析柱纯化细胞上清中重组完整抗体，纯化过程中液体流速均为1mL/min。纯化后的抗体用0.01M PBS溶液进行透析过夜，透析结束后，测定抗体浓度为3mg/mL，采用SDS-PAGE(图2)进行了验证。
重组完整抗体的活性评价
1)抗噻虫胺重组完整抗体和抗噻虫胺天然小鼠腹水单抗用ELISA方法进行对比
在前期制备抗噻虫胺小鼠单抗时，比较了同源间接竞争ELISA(包被原为噻虫胺-OVA)和异源间接ELISA(包被原为氯噻啉-OVA)方法的灵敏度差异，研究结果发现，当使用氯噻啉-OVA作为异源包被时，方法灵敏度提高了。故本发明中采用异源间接竞争ELISA评价抗噻虫胺重组完整抗体的灵敏度和特异性。将氯噻啉-OVA(混合酸酐法制备)作为异源包被原包被96孔板，然后用兔抗鼠IgG-HRP作为检测抗体，TMB作为反应底物，噻虫胺和其他新烟碱类农药标准品作为检测物，进行ELISA检测。结果(图3)如下:
a)检测范围
抗噻虫胺重组完整抗体用于ELISA法检测范围(IC 20-IC 80)为：0.92-23.04ng/mL。
抗噻虫胺小鼠腹水单抗用于ELISA法检测范围(IC 20-IC 80)为：1.45-20.01ng/mL。
b)灵敏度(IC 50)
抗噻虫胺重组完整抗体IC 50为：4.62ng/mL。
抗噻虫胺小鼠单克隆抗体IC 50为：5.39ng/mL。
c)ELISA法特异性
本发明中通过检测与其他七种常见的新烟碱类杀虫剂的交叉反应来评价抗体的特异性。ELISA数据显示抗噻虫胺的鼠单抗和重组完整抗体均与其他七种新烟碱类杀虫剂无交叉反 应(交叉反应率均<0.1％)，可特异性识别噻虫胺，结果说明重组完整抗体与亲本单抗具有高度一致的选择性。
2)抗噻虫胺重组完整抗体动力学表面等离子共振(SPR)共振表征
本发明中用到的芯片为CM7芯片，两种抗体的最适标记pH均为5.0，最终芯片上偶联的抗体信号值分别为38663RU和39854RU。动力学实验确定了小鼠腹水亲本单抗和重组完整抗体在一系列分析物浓度(50、25、12.5、6.25、3.12、1.623和0.78nM)下对噻虫胺的亲和力。腹水单抗和重组完整抗体对噻虫胺的KD分别为5.05×10 -9M和3.24×10 -9M，表示重组完整抗体对噻虫胺的亲和力和腹水抗体接近。
实施例3：重组完整抗体在金标试纸条上的应用
经ELISA和SPR法验证，通过该方法制备的抗噻虫胺重组完整抗体代替腹水抗体，因此为了实现噻虫胺的快速检测，本发明制备一种噻虫胺残留检测的竞争免疫层析试纸条。
一、金标抗体制备
预先确定该方法中标记的最适缓冲液为0.01M PBS，最适标记pH为6.5，最适抗体标记量为75μg/mL。将适量的抗体滴加至10mL的胶体金溶液中，搅拌混匀。室温孵化1h后，金标抗体溶液被10％BSA和1％PEG 20000封闭(最终浓度分别为1％和0.1％)，然后再室温孵化1h后，13500g，4℃离心30分钟，沉淀用缓冲液(含1％BSA和0.1％PEG20000 pH6.5)复溶，在上述条件下再次离心，去除未结合抗体。最后，用1mL缓冲液(含5％蔗糖和1％BSA)重悬沉淀物，待用。
二、试纸条的组装
免疫层析试纸由塑料衬板、吸水垫、NC膜、样品垫(1％BSA、0.1％PEG20000、0.25％pvp处理)、金标结合垫、检测线(0.15mg/mL；氯噻啉-OVA)和控制线(0.09mg/mL；山羊抗小鼠IgG)组成按照常规试纸条组装方式进行组装。
三、试纸条灵敏度实验
用纯甲醇配置噻虫胺母液100μg/mL，用0.01M PBS缓冲液配置一系列标准工作液0.63，1.25，2.5，5，10，20，和40ng/mL，测试同一批次的试纸条，结果如图4所示。当噻虫胺浓度>2.5ng/mL时T线显色明显减弱，当噻虫胺浓度>10ng/mL，肉眼判断T线消线。同时用其他7种新烟碱类农药溶液进行试纸条特异性分析测试，将待测标准液用0.01M PBS稀释至1000ng/mL，进行测试，15分钟后，将测试后的试纸条T线的显色情况与噻虫胺结果进行对比，根据T线颜色的深浅来判断方法的特异性。结果表明7种噻虫胺结构类似物的测试试纸条的T线与不加任何农药的CK比较，显色颜色肉眼判断无明显减弱，故检测结 果阴性。
因此，采用本发明的抗噻虫胺重组抗体所制备的金标试纸法特异性强、灵敏度高，对噻虫胺的肉眼检出限为2.5ng/mL，可用于环境和农产品中噻虫胺残留的快速检测。
上述具体实施方式用来解释说明本发明，而不是对本发明进行限制，在本发明的精神和权利要求的保护范围内，对本发明作出的任何修改和改变，都落入本发明的保护范围。

Claims (10)

  1. 一种特异性抗噻虫胺抗体的可变区序列，其特征在于：重链可变区编码基因的氨基酸序列为SEQ ID NO:2所示。
  2. 根据权利要求1所述的特异性抗噻虫胺抗体的可变区序列，其特征在于：重链可变区编码基因的核苷酸序列如SEQ ID NO:1所示。
  3. 根据权利要求1或2所述的特异性抗噻虫胺抗体的可变区序列，其特征在于：轻链可变区编码基因的氨基酸序列为SEQ ID NO:4所示。
  4. 根据权利要求3所述的特异性抗噻虫胺抗体的可变区序列，其特征在于：轻链可变区编码基因的核苷酸序列如SEQ ID NO:3所示。
  5. 一种抗噻虫胺重组完整抗体，包括重链恒定区、重链可变区、轻链恒定区和轻链可变区，其特征在于：所述重链可变区编码基因的氨基酸序列为如SEQ ID NO:2所示。
  6. 根据权利要求5所述的抗噻虫胺重组完整抗体，其特征在于：所述重链可变区编码基因的核苷酸序列为如SEQ ID NO:1所示。
  7. 根据权利要求6所述的抗噻虫胺重组完整抗体，其特征在于：所述轻链可变区编码基因的氨基酸序列如SEQ ID NO:4所示。
  8. 根据权利要求7所述的抗噻虫胺重组完整抗体，其特征在于：所述轻链可变区编码基因的核苷酸序列如SEQ ID NO:3所示。
  9. 一种抗体表达质粒，其特征在于：含有如权利要求1-2中任一项所述的重链可变区和小鼠重链IgG1恒定区的核苷酸序列，可以表达抗噻虫胺重组完整抗体的重链蛋白；或者，含有如权利要求2-3中任一项所述的轻链可变区和小鼠轻链Kappa恒定区的核苷酸序列，可以表达抗噻虫胺重组完整抗体的轻链蛋白。
  10. 一种快速检测噻虫胺残留的免疫试纸条，其特征在于：含有如权利要求5-8中任一项所述的抗噻虫胺重组完整抗体。
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102875671A (zh) * 2012-09-27 2013-01-16 南京农业大学 噻虫胺抗原、抗体及其应用
CN105400742A (zh) * 2015-11-25 2016-03-16 浙江大学 杂交瘤细胞株imi-g12及其产生的新烟碱类农药通用单克隆抗体和应用
CN108948188A (zh) * 2018-08-16 2018-12-07 江南大学 一株分泌噻虫胺单克隆抗体的杂交瘤细胞株j6及其应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4841856B2 (ja) * 2005-03-31 2011-12-21 株式会社堀場製作所 クロチアニジンおよびジノテフランのハプテン化合物、抗体、ハイブリドーマおよびその測定手段、測定キットまたは測定方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102875671A (zh) * 2012-09-27 2013-01-16 南京农业大学 噻虫胺抗原、抗体及其应用
CN105400742A (zh) * 2015-11-25 2016-03-16 浙江大学 杂交瘤细胞株imi-g12及其产生的新烟碱类农药通用单克隆抗体和应用
CN108948188A (zh) * 2018-08-16 2018-12-07 江南大学 一株分泌噻虫胺单克隆抗体的杂交瘤细胞株j6及其应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LI MING, SHENG ENZE, CONG LUJING, WANG MINGHUA: "Development of Immunoassays for Detecting Clothianidin Residue in Agricultural Products", JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY, vol. 61, no. 15, 25 March 2013 (2013-03-25), US, pages 3619 - 3623, XP055862544, ISSN: 0021-8561, DOI: 10.1021/jf400055s *

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