WO2020215727A1 - 一种克百威农药的纳米抗体及其制备方法和应用 - Google Patents

一种克百威农药的纳米抗体及其制备方法和应用 Download PDF

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WO2020215727A1
WO2020215727A1 PCT/CN2019/122580 CN2019122580W WO2020215727A1 WO 2020215727 A1 WO2020215727 A1 WO 2020215727A1 CN 2019122580 W CN2019122580 W CN 2019122580W WO 2020215727 A1 WO2020215727 A1 WO 2020215727A1
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nanobody
carbofuran
detection
recombinant
pesticide
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PCT/CN2019/122580
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French (fr)
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王弘
张瑾如
杨金易
徐振林
孙远明
沈玉栋
肖治理
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华南农业大学
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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
    • 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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • 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
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • 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
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • 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
    • 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
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present invention relates to the field of biotechnology, and more specifically, to a nano antibody of carbofuran pesticide and its preparation method and application.
  • Carbamate compounds are used as insecticides, acaricides, herbicides and fungicides in pesticides. They have formed a large category of pesticides with many varieties, good efficacy and low toxicity. Carbamate pesticides are the main cause of acute pesticide poisoning, and they are also the key test species for pesticide residues in vegetables nowadays.
  • Carbofuran trade name carbofuran, is a carbamate broad-spectrum systemic insecticide, acaricide, and nematicide. It can be used for a variety of crops to control more than 300 pests and nematodes in the soil and on the ground. And it has the function of shortening the growth period of crops and promoting the growth and development of crops, thereby effectively increasing crop yields. It is widely used in pest control of vegetables, fruits and food crops.
  • carbofuran is highly toxic to humans and animals, and it is not easily degraded and easily causes environmental pollution. my country has stipulated the maximum residue limit of carbofuran in food, but there are still many violations of use. Therefore, it is necessary to strengthen the detection of carbofuran pesticide residues.
  • carbofuran residue analysis generally uses gas chromatography (GC for short), high performance liquid chromatography (HPLC), and chromatographic mass spectrometry.
  • GC gas chromatography
  • HPLC high performance liquid chromatography
  • chromatographic mass spectrometry chromatographic mass spectrometry
  • the purpose of the present invention is to overcome the shortcomings of the prior art and provide a nanobody of carbofuran pesticide and its preparation method and application.
  • the first objective of the present invention is to provide a Nanobody that specifically recognizes carbofuran.
  • the second object of the present invention is to provide a gene encoding a nanobody that specifically recognizes Carbofuran.
  • the third object of the present invention is to provide a recombinant vector.
  • the fourth object of the present invention is to provide a recombinant cell.
  • the fifth objective of the present invention is to provide one or more of the Nanobody, the gene, the recombinant vector, or the recombinant cell in the detection kit for carbofuran or the preparation of carbofuran Applications.
  • the sixth objective of the present invention is to provide a method for preparing the Nanobody.
  • the seventh object of the present invention is to provide a detection method for carbofuran.
  • the eighth object of the present invention is to provide a kit for detecting carbofuran.
  • Nanobodies have good stability and high affinity, which overcomes the shortcomings of small-molecule functional antibodies. At the same time, they have the advantages of small molecular weight, weak immunogenicity, and strong tissue penetration that monoclonal antibodies and polyclonal antibodies do not possess.
  • the present invention uses phage display technology to screen out nanoantibodies that can specifically bind to the target molecule (carbofuran complete antigen) from the camel-derived immune single domain heavy chain antibody library, and establish a rapid, sensitive and stable detection of vegetables , The method of carbofuran residue in tea.
  • a Nanobody that specifically recognizes Carbofuran, and its amino acid sequence is shown in SEQ ID NO.1.
  • a recombinant vector is a vector linked to the gene.
  • the vector is an expression vector.
  • the expression vector is pComb3xss.
  • a recombinant cell a cell carrying the recombinant vector, or a cell capable of expressing the Nanobody.
  • the cell is Escherichia coli BL21 (DE3).
  • nanobody Application of one or more of the nanobody, the gene, the recombinant vector, or the recombinant cell in the detection of carbofuran or the preparation of a detection kit for carbofuran.
  • the preparation method of the Nanobody includes the following steps:
  • step S3 the recombinant cells are cultured to a logarithmic phase with an OD600 value of 0.6 to 0.8 to induce the expression of the Nanobody.
  • step S3 0.1-1 mM IPTG is used to induce expression at 28-37°C for 12-16h.
  • step S3 1mM IPTG is used to induce expression at 37°C for 12h.
  • step S4 the periplasmic cavity protein is extracted by the sucrose osmotic pressure method, and the soluble nanobody in the periplasmic cavity is recovered after a step of Ni column purification.
  • a detection method of carbofuran using the nanobody A detection method of carbofuran using the nanobody.
  • the detection is based on the indirect ELISA method, using the complete antigen of carbofuran obtained by coupling the carbofuran hapten of formula (I) with a carrier protein as the coating source, and using the nanobody as the detection antibody,
  • the carrier protein is ovalbumin.
  • the detection method includes the following steps: coating the original coated enzyme-labeled plate; adding carbofuran standard or the sample to be tested, and then adding the detection antibody; adding the enzyme-labeled secondary antibody and incubating; adding the color developing solution , Incubate; add stop solution and determine; take the log10 of carbofuran standard concentration as the abscissa, and use the ratio of the absorbance value of each carbofuran standard concentration to the absorbance value of the zero standard hole as the ordinate to establish a standard curve, Then the content of carbofuran in the sample to be tested is calculated according to the absorbance value of the sample to be tested.
  • the complete antigen of carbofuran obtained by coupling the carbofuran hapten described in formula (I) with ovalbumin is used as the coating source and diluted to? ? ? , Add 100 ⁇ L per well, and incubate overnight at 37°C; wash the plate twice with a plate washer, dry the liquid in the well, add 120 ⁇ L of blocking solution, incubate at 37°C for 2h, dry the liquid in the well, invert it, 37°C Dry for 1h; add 50 ⁇ L of gram Budweiser standard or sample to be tested to each well, then add 50 ⁇ L of the nanoantibody, incubate at 37°C for 1h, wash the plate with a plate washer 5 times, spin dry the liquid in the well; add 100 ⁇ L to each well Anti-HA-HRP antibody (diluted 20000 times), incubate at 37°C for 40min, wash the plate 5 times with a plate washer, and spin dry the liquid in the well; add 100 ⁇ L of TMB color developing solution to each well,
  • a kit for detecting carbofuran contains the nanobody.
  • the detection is performed based on the indirect ELISA method, and the complete antigen of carbofuran obtained by coupling the carbofuran hapten of formula (I) with a carrier protein is used as the coating source, and the nanobody is used as the detection antibody,
  • the carrier protein is ovalbumin.
  • it also contains an enzyme-labeled secondary antibody, a color developing solution and a stop solution.
  • the present invention has the following beneficial effects:
  • the present invention obtains a nanobody against carbofuran pesticide.
  • the nanobody can detect carbofuran pesticide with accurate detection result, good effect, good stability, and good detection under high temperature and organic solvent conditions. effect.
  • the antibody can not only be widely used in the detection of carbofuran pesticide residues in agricultural products; it can also be used as a precursor to be modified by random or site-directed mutagenesis techniques to obtain better properties (affinity, specificity, stability, etc.)
  • the mutants can be further used in food, medicine, agriculture and other fields, and have great application and promotion value.
  • Figure 1 shows the results of phage monoclonal indirect competition ELISA.
  • Figure 2 shows the standard curve of Nanobody for detecting carbofuran pesticide.
  • Figure 3 shows the binding ability of Nanobodies at high temperature for 5 minutes.
  • Figure 4 shows the binding activity of Nanobodies at 95°C.
  • Figure 5 shows the binding ability of methanol as a diluent antibody to antigen.
  • Figure 6 shows the binding ability of acetonitrile as a diluent antibody to antigen.
  • test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used, unless otherwise specified, are commercially available reagents and materials.
  • Carbofuran pesticide hapten BFNB is coupled with ovalbumin OVA (albumin) and keyhole limpet heocyanin (KLH) to prepare complete antigens BFNB-OVA and BFNB-KLH.
  • OVA ovalbumin
  • KLH keyhole limpet heocyanin
  • the chemical formula of the carbofuran pesticide hapten BFNB is:
  • BFNB-KLH Take 500 ⁇ g of BFNB-KLH and the same volume of Freund's complete adjuvant to emulsify, and then carry out multi-point immunization injection into the neck of Bactrian camel.
  • the serum titer was determined by indirect competitive ELISA method.
  • RNA extraction Take the blood sample with the best serum inhibition for lymphocyte separation and RNA extraction.
  • the extraction of NA was carried out according to the Trizol reagent method of Invitrogen.
  • RNA as a template, the first strand of cDNA was synthesized according to the instructions of TARAKA's first strand reverse transcription kit.
  • One-step PCR uses primers P1 and P2 and primers P1 and P3 to amplify the target fragments respectively.
  • the reaction conditions are 94°C, 5min, 94°C, 30s, 61°C, 30s, 72°C, 1min, 30 cycles, Extension at 72°C for 10 min.
  • the PCR products were respectively subjected to agarose gel electrophoresis to recover fragments of about 500 bp, and the target fragments were recovered through the DNA cutting gel recovery kit, combined to obtain nanobody gene fragments, quantified and stored at -20°C for future use;
  • the first step PCR uses primers Q1 and Q2 to amplify, the reaction conditions are 94°C, 5min, 94°C, 30s, 55°C, 30s, 72°C, 1min, 30 cycles, 72 Extend at °C for 10min.
  • the PCR product of the first step is subjected to agarose gel electrophoresis to recover fragments of about 500 bp, and the target fragments are recovered through the DNA gel recovery kit.
  • the second step primers Q3 and Q4 are used for amplification, and the PCR reaction conditions are the same as the first step.
  • the nanobody gene fragments are recovered by further cutting the gel, quantified and stored at -20°C for later use.
  • the phagemid vector pComb3xss and the two nanobody gene fragments obtained in the previous step were subjected to sfiI double digestion, and pComb3xss and two nanobody gene fragments were obtained through gel-cutting recovery and PCR purification. Then, mix pComb3xss and the target fragment at a molar ratio of 1:3 at 16°C, and use T4 ligase to ligate overnight.
  • the above-mentioned ligation product was recovered by precipitation with PCR purification kit, and dissolved in 35 ⁇ l of sterile water.
  • the one-step and two-step ligation products were mixed 1:1 and electrotransformed into competent cells ER2738 in 13 times.
  • the transformed bacteria were cultured in a shaker at 200 rpm and 37°C for 1 hour to recover and grow. Dilute the transformed bacteria solution in a gradient, spread on an LB culture plate containing ampicillin and tetracycline, and cultivate overnight at 37°C.
  • the next day, the single clones coated on the plate were randomly picked and sent to the company for sequencing to identify the diversity of the antibody library.
  • the storage capacity is calculated based on the number and diversity of clones.
  • BFNB-OVA BFNB-OVA
  • 100 ⁇ l each well of the microtiter plate incubate overnight at 37°C in a water bath.
  • the coating concentration gradient is 10, 5, 1, 0.2 ⁇ g/ml.
  • wash the plate twice with PBST add 120 ⁇ l of 1% BSA to the wells of 10 and 1 ⁇ g/ml, and add 120 ⁇ l of 1% fish collagen to the wells of 5 and 0.2 ⁇ g/ml, seal for 3h, 37°C, dry for later use .
  • Nb309 (393bp), Nb316 (393bp), Nb328 (375bp), Nb391 (393bp), Nb393 (372bp), Nb415 (372bp), Nb438 (372bp), Nb480 (384bp), Nb489 (366bp) antibodies.
  • Nb316 has the best inhibitory effect, and its amino acid sequence is shown in SEQ ID NO. 1; the nucleotide sequence is shown in SEQ ID NO. 2.
  • the Nb316-pComb3xss plasmid was extracted with an extraction kit, and then introduced into competent E. coli BL21 (DE3) by chemical transformation method. Take a single clone for PCR identification and sequencing to determine the inserted fragment as the target fragment.
  • the BL21(DE3) colony containing the target fragment of the Nanobody was cultured to a logarithmic phase with an OD 600 value of 0.6-0.8, IPTG was added at a final concentration of 1 mM, and the expression was induced at 37°C for 12 hours. The next day, centrifuge to get the bacteria. Then the periplasmic cavity protein was extracted by the sucrose osmotic pressure method, and the soluble nanoantibody Nb316 in the periplasmic cavity was recovered after a step of Ni column purification.
  • BFNB-OVA BFNB-OVA
  • dilute to a working concentration of 1 ⁇ g/mL add to a 96-well microtiter plate, 100 ⁇ L per well, and incubate overnight at 37°C. The next day, the plate was washed twice with a plate washer, dried the liquid in the well, added 120 ⁇ L of blocking solution, incubated at 37°C for 2h, dried the liquid in the well, placed it in an oven at 37°C for 1h, and removed it for use.
  • Add 50 ⁇ L of serially diluted drug and 50 ⁇ L of Nanobody to each well, and do three replicates for each concentration.
  • the indirect competitive ELISA method was used to evaluate the specificity and sensitivity of carbamate pesticides.
  • the specific steps are as follows: add 50 ⁇ L of gradient dilution drug and 50 ⁇ L of Nanobody to each well, and do three replicates for each concentration. Incubate at 37°C for 1 hour, wash the plate with a plate washer 5 times, spin-dry the liquid in the wells, add 100 ⁇ L of anti-HA-HRP antibody (diluted 20000 times) to each well, incubate at 37°C for 40 minutes, wash the plate 5 times with a plate washer, and spin dry the wells Internal liquid. Add 100 ⁇ L of TMB color developing solution to each well, incubate at 37°C for 10 min, then add 50 ⁇ L of 10% H 2 SO 4 to stop the color reaction, and measure the A 450 nm absorbance value with a microplate reader.
  • CR (%) 100 ⁇ IC 50 (Carbofuran)/IC 50 (Carbofuran analog).
  • Nanobody Nb316 Dilute Nanobody Nb316 to working concentration, divide into seven equal parts, and heat them in a water bath at 20, 35, 50, 65, 80, and 95°C for 5 minutes.
  • the binding ability of the antibody to the antigen is measured by ic-ELISA, and the binding ability of the unheated antibody to the antigen is taken as 100%, and the stability of the nanobody at different temperatures is evaluated.
  • Example 4 Dilute the Nanobody Nb316 to working concentration, divide into 7 equal parts, place them in a water bath at 95°C, and heat them for 10, 20, 30, 40, 50, and 60 minutes respectively. After the antibody returned to room temperature, the ic-ELISA of Example 4 was used to determine the binding ability of the antibody to the antigen. The binding ability of the unheated antibody to the antigen was taken as 100%, and the stability of the Nanobody was evaluated over time under high temperature. .
  • the Nanobody Nb316 was diluted to the same working concentration, and the ic-ELISA of Example 4 was used to determine the relationship between antibody and antigen. Binding capacity, the binding capacity of the antibody and antigen without dilution with the organic solvent diluent is taken as 100% to evaluate the tolerance of the Nanobody to different organic solvents and different concentrations of the same organic solvent.

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Abstract

一种克百威农药的纳米抗体及其制备方法和应用，该纳米抗体，其氨基酸序列如SEQ ID NO.1所示；编码其的基因，其核苷酸序列如SEQ ID NO.2所示。一种针对克百威农药的纳米抗体，该纳米抗体能够检测克百威农药，且检测结果准确、效果好、稳定性好，在高温和有机溶剂的条件下还有很好的检测效果。该抗体不仅可以广泛的应用于农产品中克百威农药残留的检测；还可以作为前体，通过随机或定点突变技术进行改造，能够获得性质(亲和性、特异性、稳定性等)更好的突变体，用来发展进一步用于食品、医药、农业等领域，有很大的应用推广价值。

Description

一种克百威农药的纳米抗体及其制备方法和应用 技术领域
本发明涉及生物技术领域，更具体地，涉及一种克百威农药的纳米抗体及其制备方法和应用。
背景技术
氨基甲酸酯类化合物在农药上用作杀虫剂、杀螨剂、除草剂和杀菌剂，已形成农药的一大类别，品种多、药效好、低毒。氨基甲酸酯类农药是农药急性中毒的主要原因，也是时下蔬菜中农药残留的重点检测品种。
克百威商品名呋喃丹，是氨基甲酸酯类广谱内吸杀虫杀螨杀线虫剂，可用于多种作物防治土壤内及地面上的300多种害虫和线虫。并有缩短作物生长期、促进作物生长发育从而有效提高作物产量的作用。广泛应用于蔬菜、水果和粮食作物等的害虫防治中。但是，克百威对人和动物有很高的毒性，并且不易降解，容易造成环境污染。我国已规定食品中克百威的最大残留限量，但违规使用现象仍然较多，因此加强对克百威农药残留的检测十分必要。
目前，克百威残留分析一般使用气相色谱法(简称GC)、高效液相色谱法(简称HPLC)及色谱质谱联用技术，这些方法灵敏、准确，可以同时测定多种药物，但样品前处理复杂、繁琐费时，且需要昂贵的仪器设备和专业的操作人员，检测成本高，难以满足样品现场、批量、快速检测的需要。因此，开发一种简单快速、适用于农药残留现场监控的分析方法具有重要意义。而基于抗体建立的免疫分析方法，虽然具有快速、灵敏、高通量的优势，但是在极端条件下抗体往往稳定性差，容易失活。
因此目前缺乏一种稳定性好的克百威抗体。
发明内容
本发明的目的是为了克服现有技术的不足，提供一种克百威农药的纳米抗体及其制备方法和应用。
本发明的第一个目的是提供一种特异性识别克百威的纳米抗体。
本发明的第二个目的是提供一种编码特异性识别克百威纳米抗体的基因。
本发明的第三个目的是提供一种重组载体。
本发明的第四个目的是提供一种重组细胞。
本发明的第五个目的是提供所述纳米抗体、所述基因、所述重组载体、或所述重组细胞中的一种或几种在检测克百威或制备克百威的检测试剂盒中的应用。
本发明的第六个目的是提供所述纳米抗体的制备方法。
本发明的第七个目的是提供一种克百威的检测方法。
本发明的第八个目的是提供一种检测克百威的试剂盒。
为了实现上述目的，本发明是通过以下技术方案予以实现的：
纳米抗体的稳定性好、亲和力较高，克服了小分子功能抗体的缺点，同时又具有分子质量小、免疫原性弱、组织穿透力强等单克隆抗体、多克隆抗体不具备的优点。本发明通过噬菌体展示技术，从驼源免疫单域重链抗体库中筛选出能与靶分子(克百威完全抗原)特异性结合的纳米抗体，建立了一种快速、灵敏且稳定的检测蔬菜、茶叶中克百威残留的方法。
因此本发明要求保护以下内容：
一种特异性识别克百威的纳米抗体，其氨基酸序列如SEQ ID NO.1所示。
编码特异性识别克百威纳米抗体的基因，其核苷酸序列如SEQ ID NO.2所示。
一个重组载体，为连接有所述基因的载体。
优选地，所述载体为表达载体。
优选地，所述表达载体为pComb3xss。
一个重组细胞，携带有所述重组载体的细胞，或能够表达所述纳米抗体的细胞。
优选地，所述细胞为大肠杆菌BL21(DE3)。
所述纳米抗体、所述基因、所述重组载体、或所述重组细胞中的一种或几种在检测克百威或制备克百威的检测试剂盒中的应用。
所述纳米抗体的制备方法，包括以下步骤：
S1.将所述纳米抗体的编码基因连接到表达载体上，获得重组载体；
S2.将重组载体转入受体细胞，得到重组细胞；
S3.培养重组细胞，诱导表达纳米抗体；
S4.分离纯化得到纳米抗体。
优选地，步骤S3中，将重组细胞培养至对数期OD600值为0.6～0.8，诱导 表达纳米抗体。
优选地，步骤S3中，使用0.1～1mM的IPTG，28～37℃诱导表达12～16h。
更优选地，步骤S3中，使用1mM的IPTG，37℃诱导表达12h。
优选地，步骤S4中，通过蔗糖渗透压法提取周质腔蛋白，经一步Ni柱纯化后回收周质腔中的可溶性纳米抗体。
一种克百威的检测方法，利用所述纳米抗体。
优选地，基于间接ELISA方法进行检测，用式(I)所述克百威半抗原与载体蛋白偶联得到的克百威完全抗原做包被原，用所述纳米抗体作为检测抗体，
Figure PCTCN2019122580-appb-000001
优选地，所述载体蛋白为卵清白蛋白。
更优选地，所述检测方法包括以下步骤：包被原包被酶标版；加入克百威标准品或待测样品，再加入检测抗体；加入酶标记的二抗，孵育；加入显色液，孵育；加入终止液并测定；以克百威标准品浓度的log10值为横坐标，以各克百威标准品浓度的吸光值与零标准孔吸光值的比值为纵坐标，建立标准曲线，进而根据待测样品的吸光值来计算待测样品中的克百威药的含量。
进一步更优选地，用式(I)所述克百威半抗原与卵清白蛋白偶联得到的克百威完全抗原做包被原，稀释至？？？，加入酶标板，每孔100μL，37℃孵育过夜；洗板机洗板2次，甩干孔内液体，加入120μL的封闭液，37℃孵育2h，甩干孔内液体，倒置，37℃烘干1h；每孔加入50μL克百威标准品或待测样品，再加入50μL的所述纳米抗体，37℃孵育1h，洗板机洗板5次，甩干孔内液体；每孔加入100μL抗HA-HRP抗体(20000倍稀释)，37℃孵育40min，洗板机洗板5次，甩干孔内液体；每孔加入100μL的TMB显色液，37℃孵育10min；再加入50μL的10％H 2SO 4终止显色反应；酶标仪测定A450nm吸光值；以克百威药物浓度的log10值为横坐标，吸光值比值B/B0为纵坐标，用Origin9.0的四参数拟合模块拟合标准曲线。
一种检测克百威的试剂盒，含有所述纳米抗体。
优选地，基于间接ELISA方法进行检测，还含有式(I)所述克百威半抗原与载体蛋白偶联得到的克百威完全抗原做包被原，用所述纳米抗体作为检测抗体，
Figure PCTCN2019122580-appb-000002
优选地，所述载体蛋白为卵清白蛋白。
优选地，还含有酶标记的二抗、显色液和终止液。
与现有技术相比，本发明具有如下有益效果：
本发明得到一种针对克百威农药的纳米抗体，该纳米抗体能够检测克百威农药，且检测结果准确、效果好、稳定性好，在高温和有机溶剂的条件下还有很好的检测效果。该抗体不仅可以广泛的应用于农产品中克百威农药残留的检测；还可以作为前体，通过随机或定点突变技术进行改造，能够获得性质(亲和性、特异性、稳定性等)更好的突变体，用来发展进一步用于食品、医药、农业等领域，有很大的应用推广价值。
附图说明
图1为噬菌体单克隆间接竞争ELISA的检测结果。
图2为纳米抗体检测克百威农药标准曲线。
图3为纳米抗体在高温下5min的结合能力。
图4为在95℃下纳米抗体的结合活性。
图5为甲醇作为稀释液抗体与抗原的结合能力。
图6为乙腈作为稀释液抗体与抗原的结合能力。
具体实施方式
下面结合说明书附图和具体实施例对本发明作出进一步地详细阐述，所述实施例只用于解释本发明，并非用于限定本发明的范围。下述实施例中所使用的试验方法如无特殊说明，均为常规方法；所使用的材料、试剂等，如无特殊说明，为可从商业途径得到的试剂和材料。
实施例1 抗克百威农药纳米抗体免疫文库的构建
一、实验方法
1、完全抗原BFNB-OVA和BFNB-KLH的制备
把克百威农药半抗原BFNB与卵清白蛋白OVA(albumin)和匙孔血蓝蛋白KLH(keyhole limpet heocyanin)通过偶联制备完全抗原BFNB-OVA和BFNB-KLH。
所述克百威农药半抗原BFNB的化学式为：
Figure PCTCN2019122580-appb-000003
2、免疫双峰驼
取500μg的BFNB-KLH与等量体积的弗氏完全佐剂乳化，对双峰驼颈部皮下进行多点免疫注射。每隔2周加强免疫一次，每次500μg的BFNB-KLH和等量体积的弗氏不完全佐剂乳化后进行免疫，每次免疫一周后静脉采血。采用间接竞争ELISA方法测定血清效价。
3、RNA的提取和cDNA的合成
取血清抑制最好的血液样品进行淋巴细胞分离以及RNA的提取。NA的提取依据Invitrogen公司的Trizol试剂方法进行。以RNA为模板，参照TARAKA公司第一链反转录试剂盒说明书进行cDNA第一链的合成。
4、纳米抗体基因片段的获得
利用Taq Mix DNA聚合酶，经PCR技术扩增获得骆驼重链抗体的可变区编码基因(引物如下表1)。
表1 扩增VHH基因的引物序列：
Figure PCTCN2019122580-appb-000004
Figure PCTCN2019122580-appb-000005
将上一步获得的cDNA第一链分别进行一步法PCR和两步法PCR
(1)一步法PCR采用引物P1、P2和引物P1、P3分别扩增目的片段，反应条件为，94℃，5min，94℃，30s，61℃，30s，72℃，1min，30个循环，72℃延伸10min。PCR产物分别经琼脂糖凝胶电泳，回收500bp左右的片段，通过DNA切胶回收试剂盒回收目的片段，合并，得到纳米抗体基因片段，定量并置于-20℃保存备用；
(2)两步法PCR扩增第一步PCR采用引物Q1和Q2扩增，反应条件为，94℃，5min，94℃，30s，55℃，30s，72℃，1min，30个循环，72℃延伸10min。
把第一步PCR产物经琼脂糖凝胶电泳，回收500bp左右的片段，通过DNA切胶回收试剂盒回收目的片段。以回收的目的片段为模板，利用第二步引物Q3和Q4进行扩增，PCR反应条件同第一步。通过进一步切胶回收得到纳米抗体基因片段，定量并置于-20℃保存备用。
5、纳米抗体基因库的制备
将噬菌粒载体pComb3xss及上一步获得的两种纳米抗体基因片段进行sfiI双酶切，通过切胶回收和PCR纯化回收获得pComb3xss和两种纳米抗体基因片段。然后在16℃下，以pComb3xss和目的片段1：3的摩尔比混合，利用T4连接酶过夜连接反应。
上述连接产物经PCR纯化试剂盒进行沉淀回收，溶于35μl的无菌水。将一步法和两步法连接产物1：1混合，分13次电转化入感受态细胞ER2738中，转化菌液在200rpm，37℃摇床培养1小时复苏生长。梯度稀释转化菌液，涂布含氨苄青霉素和四环素的LB培养板，37℃过夜培养。次日，随机挑取涂布平板的单克隆，送公司测序，鉴定抗体库的多样性。库容依据克隆数目和多样性来计算。
用培养基将细胞刮洗下来，加入终浓度20％的无菌甘油，-80℃保存，即为克百威农药纳米抗体基因库。
6、纳米抗体噬菌体库的制备
取冻存的纳米抗体库1mL接种至200mL的LB(Amp，Tet)培养基，37℃，250rpm振荡培养至对数期(OD600＝0.6～0.8)。加入1mL辅助噬菌体(1×1012pfu/mL)，37℃静置培养30min，250rpm震荡培养2h，然后加卡那霉素(helper phage抗性)到70μg/mL，250rpm震荡培养过夜。将培养液4℃，12000rpm离心20min，上清液转移至新的离心瓶，加入50mL 5xPEG/NaCl混匀，冰上孵育4h。4℃，12000rpm离心15min，弃上清，加入1mL TBS重悬噬菌体沉淀，0.22μm滤膜过滤，加20％灭菌甘油，-20℃保存，即为纳米抗体噬菌体库。
实施例2 抗克百威农药纳米抗体免疫文库的构建
一、实验方法
1、以BFNB-OVA为包被抗原，酶标板每孔各100μl，37℃水浴孵育过夜。包被浓度梯度为10，5，1，0.2μg/ml。12h后，PBST洗板2次，10和1μg/ml的孔加入120μl的1％BSA，5和0.2μg/ml的孔加入120μl的1％鱼胶原蛋白，封闭3h，37℃，烘干待用。
2、取100μl噬菌体库(约10 11pfu)，分别加入2％的BSA、OVA和KLH载体蛋白，37℃孵育1h，除去非特异性吸附载体带白的抗体。然后转移至包被抗原孔内，室温震荡1小时，吸出未结合的噬菌体，用PBST洗板5次，10次，15次，15次。以100μl洗脱液(2，1，0.5，0.1μg/ml克百威)洗脱吸附于板孔内的噬菌体抗体。取出10μl用于滴度的测定，其余洗脱产物经辅助噬菌体救援扩增后用于下一轮的淘选。共进行4轮淘筛(见表2)。随机挑取第三轮和四轮的噬菌体单克隆进行间接竞争ELISA。
表2 淘筛策略表：
Figure PCTCN2019122580-appb-000006
Figure PCTCN2019122580-appb-000007
Recovery＝output phage/input phage
Enrichment＝after round/previous round
二、实验结果
第三轮和四轮的噬菌体单克隆进行间接竞争ELISA结果如图1所示，获得九种不同序列的纳米抗体：Nb309(393bp)、Nb316(393bp)、Nb328(375bp)、Nb391(393bp)、Nb393(372bp)、Nb415(372bp)、Nb438(372bp)、Nb480(384bp)、Nb489(366bp)抗体。经过筛选，Nb316的抑制效果最好，其氨基酸序列如SEQ ID NO.1所示；核苷酸序列如SEQ ID NO.2所示。
实施例3 克百威农药纳米抗体的制备与间接竞争ELISA方法的建立
一、实验方法
1、将Nb316-pComb3xss质粒通过提取试剂盒抽提，然后通过化学转化方法导入感受态大肠杆菌BL21(DE3)。取单克隆进行PCR鉴定和测序，确定插入片段为目的片段。将含有纳米抗体目的片段的BL21(DE3)菌落培养至对数期OD 600值为0.6～0.8，加入终浓度为1mM的IPTG，37℃诱导表达12h。次日，离心得菌体。然后通过蔗糖渗透压法提取周质腔蛋白，经一步Ni柱纯化后回收周质腔中的可溶性纳米抗体Nb316。
2、以BFNB-OVA为包被原，稀释至工作浓度1μg/mL，加入96孔酶标板，每孔100μL，37℃孵育过夜。次日，洗板机洗板2次，甩干孔内液体，加入120μL的封闭液，37℃孵育2h，甩干孔内液体，倒置于37℃烘箱烘1h后取出备用。每孔加入50μL梯度稀释的药物和50μL的纳米抗体，每个浓度做三个重复。37℃孵育1h，洗板机洗板5次，甩干孔内液体，每孔加入100μL抗HA-HRP抗体(20000倍稀释)，37℃孵育40min，洗板机洗板5次，甩干孔内液体。每孔加入100μL的TMB显色液，37℃孵育10min，再加入50μL的10％H 2SO 4终止显色反应，酶标仪测定A 450nm吸光值。以克百威药物浓度为横坐标，吸光值比值B/B 0为纵坐标，用Origin9.0的四参数拟合模块拟合标准曲线。
二、实验结果
结果显示，基于纳米抗体Nb2检测克百威的间接竞争ELISA标准曲线(如图2)，IC 50为7.27±0.88ng/ml，线性范围为1.44～30.40ng/ml。
实施例4 间接竞争ELISA检测多种氨基甲酸酯类农药
一、实验方法
1、以BFNB-OVA为包被原，Nb316为检测抗体，采用间接竞争ELISA方法对氨基甲酸酯类农药的特异性及灵敏度进行评价。
具体步骤如下：每孔加入50μL梯度稀释的药物和50μL的纳米抗体，每个浓度做三个重复。37℃孵育1h，洗板机洗板5次，甩干孔内液体，每孔加入100μL抗HA-HRP抗体(20000倍稀释)，37℃孵育40min，洗板机洗板5次，甩干孔内液体。每孔加入100μL的TMB显色液，37℃孵育10min，再加入50μL的10％H 2SO 4终止显色反应，酶标仪测定A 450nm吸光值。以克百威药物浓度为横坐标，吸光值比值B/B 0为纵坐标，用Origin9.0的四参数拟合模块拟合标准曲线，得出各自的IC 50值。用下式计算各药物与抗克百威纳米抗体的交叉反应率：
CR(％)＝100×IC 50(克百威)/IC 50(克百威类似物)。
2、结果显示，纳米抗体Nb316与丙硫克百威、仲丁威、丁硫克百威、3-羟基克百威和异丙威的交叉反应率分别为5.05％、3.52％、2.56％、1.97％和0.35％，与其余7种农药交叉反应率低于0.1％(见表3)。
表3 纳米抗体Nb316的ic-ELISA方法检测农药的灵敏度和特异性：
Figure PCTCN2019122580-appb-000008
Figure PCTCN2019122580-appb-000009
实施例5 克百威农药纳米抗体的热稳定性分析
一、实验方法
1、将纳米抗体Nb316稀释至工作浓度，分成七等份，分别置于20、35、50、65、80和95℃水浴锅中加热5min。待抗体恢复至室温，采用ic-ELISA测定抗体与抗原的结合能力，未加热的抗体与抗原结合的能力作为100％，评价纳米抗体不同温度下的稳定性。
2、将纳米抗体Nb316稀释至工作浓度，分成7等份，置于95℃水浴锅中，分别加热10、20、30、40、50、60min。待抗体恢复至室温，采用实施例4的ic-ELISA测定抗体与抗原的结合能力，未加热的抗体与抗原结合的能力作为100％，评价纳米抗体在高温下随着时间的增加稳定性的变化。
二、实验结果
结果显示：纳米抗体Nb 316在95℃下5min仍能保持100％以上的活性，因此，纳米抗体在高温下仍具有较强的结合能力(图3)。在95℃下，0min到60min，纳米抗体始终保持在约100％的结合活性(图4)。
实施例6 克百威农药纳米抗体的热稳定性分析
一、实验方法
用不同浓度(10％，20％，40％，60％和80％)的甲醇和乙腈作为稀释液，将纳米抗体Nb316稀释至同一工作浓度，采用实施例4的ic-ELISA测定抗体与抗原的结合能力，未加有机溶剂稀释液稀释的抗体与抗原结合能力作为100％，评价纳米抗体对不同有机溶剂、同一有机溶剂不同浓度时的耐受能力。
二、实验结果
结果显示：甲醇浓度为25％时，纳米抗体Nb316仍可保持约100％的结合活性，甲醇浓度为50％时，结合活性仍有50％(图5)。而在乙腈环境下，乙腈浓度低于20％时纳米抗体的结合活性不会降低，30％时纳米抗体仍具有50％的结合活性(图6)。因此，纳米抗体具有优异的有机溶剂耐受性，在实际样品检测的前处理过程中，不会受到残留有机溶剂的影响。

Claims (10)

  1. 一种特异性识别克百威的纳米抗体，其特征在于，其氨基酸序列如SEQ ID NO.1所示。
  2. 一种编码特异性识别克百威纳米抗体的基因，其特征在于，其核苷酸序列如SEQ ID NO.2所示。
  3. 一种重组载体，其特征在于，为连接有权利要求2所述基因的载体。
  4. 一种重组细胞，其特征在于，携带有权利要求3所述重组载体的细胞，或能够表达权利要求1所述纳米抗体的细胞。
  5. 权利要求1所述纳米抗体、权利要求2所述基因、权利要求3所述重组载体、或权利要求4所述重组细胞中的一种或几种在检测克百威或制备克百威的检测试剂盒中的应用。
  6. 权利要求1所述纳米抗体的制备方法，其特征在于，包括以下步骤：
    S1.将权利要求1所述纳米抗体的编码基因连接到表达载体上，获得重组载体；
    S2.将重组载体转入受体细胞，得到重组细胞；
    S3.培养重组细胞，诱导表达纳米抗体；
    S4.分离纯化得到纳米抗体。
  7. 一种克百威的检测方法，其特征在于，利用权利要求1所述纳米抗体。
  8. 根据权利要7所述检测方法，其特征在于，基于间接ELISA方法进行检测，用式(I)所述克百威半抗原与载体蛋白偶联得到的克百威完全抗原做包被原，用权利要求1所述纳米抗体作为检测抗体，
    Figure PCTCN2019122580-appb-100001
  9. 一种检测克百威的试剂盒，其特征在于，含有权利要求1所述纳米抗体。
  10. 根据权利要求试剂盒，其特征在于，基于间接ELISA方法进行检测，还含有式(I)所述克百威半抗原与载体蛋白偶联得到的克百威完全抗原做包被原，用权利要求1所述纳米抗体作为检测抗体，
    Figure PCTCN2019122580-appb-100002
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