CN113512115B - anti-Cry 2Aa idiotype single-domain antibody for broad-spectrum identification of plutella xylostella midgut receptor and application thereof - Google Patents
anti-Cry 2Aa idiotype single-domain antibody for broad-spectrum identification of plutella xylostella midgut receptor and application thereof Download PDFInfo
- Publication number
- CN113512115B CN113512115B CN202110909528.0A CN202110909528A CN113512115B CN 113512115 B CN113512115 B CN 113512115B CN 202110909528 A CN202110909528 A CN 202110909528A CN 113512115 B CN113512115 B CN 113512115B
- Authority
- CN
- China
- Prior art keywords
- domain antibody
- single domain
- plutella xylostella
- antibody
- cry
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- 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)
-
- 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
- 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
- C07K2317/50—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
- C07K2317/565—Complementarity determining region [CDR]
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Plant Pathology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Immunology (AREA)
- Agronomy & Crop Science (AREA)
- Pest Control & Pesticides (AREA)
- Biophysics (AREA)
- Engineering & Computer Science (AREA)
- Dentistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The application relates to an anti-Cry 2Aa idiotype single domain antibody for broad spectrum identification of plutella xylostella midgut receptor and application thereof, wherein the single domain antibody is an antigen binding fragment F (ab') obtained by pepsin digestion of Bt Cry2Aa toxin rabbit polyclonal antibody 2 And the midgut receptor protein of plutella xylostella is used as a coating antigen, biopanning is carried out on a phage display single-domain antibody library, and soluble expression is carried out in escherichia coli, wherein the amino acid sequence of the single-domain antibody is shown as SEQ ID NO.1, and the nucleotide sequence is shown as SEQ ID NO. 2; the single domain antibody has broad spectrum recognition capability on various receptors of the midgut of plutella xylostella, namely cadherin-like protein, aminopeptidase N2 and vacuole type ATP hydrolase A subunit, has competitive inhibition effect on the combination of Cry2Aa toxin and three receptors, and has good application potential in the aspects of excavation of Cry toxin substitution materials and biological pest control.
Description
Technical Field
The application relates to a genetic engineering antibody, in particular to an anti-Cry 2Aa idiotype single domain antibody for broad-spectrum recognition of a plutella xylostella midgut receptor and application thereof.
Background
The Bt Cry toxin is insecticidal protein produced by gram-positive bacillus thuringiensis (Bacillus thuringiensis, bt), has high specificity to target insects, has high safety to other environmental organisms, mammals and human beings, is widely used for constructing transgenic insect-resistant crops and developing microbial source pesticides, and is one of the most successful and wide biological pesticides applied in the world (Sanahuja G, banakar R, twyman RM, capell T, christou P (2011) Bacillus thuringiensis: a century of research, development and commercial applications.plant Biotechnology Journal, 9:283-300). However, cry toxins also suffer from a number of problems such as limited virulence, narrow insecticidal spectrum, and the like, and many pests are not currently controlled effectively by Cry toxins (Guo Yajie, li Ping, wu Zhongling (2016) Cry toxins have been developed for the modification of pesticidally active molecules. Biotechnology has progressed 61:19-24). In addition, the long-term and wide-range use has led to an increasing risk of insect resistance, and at present, there are already diamond back moth and spodoptera frugiperda populations worldwide that develop field actual resistance to Bt formulations (furlmj, wright DJ, dosdall LM (2013) Diamondback moth ecology and management: progles, progress, and processes.Annual Review of Entomology,58:517-541.Janmaat AF,Myers J (2003) Rapid evolution and the cost of resistance to Bacillus thuringiensis in greenhouse populations of cabbage loopers, tricholusia ni.proceedings Biological Science, 270:2263-2270). Bt resistance management strategies widely adopted at present are mainly shelter strategies and gene aggregation strategies, however, long-term selection pressures of target pests can evolve resistance to adapt to the strategies (Jin L, zhang H, lu Y, yang Y, wu K, tabashnik BE, wu Y (2015) Large-scale test of the natural refuge strategy for delaying insect resistance to transgenic Bt crops. Nature Biotechnology, 33:169-174). Therefore, the excavation of new insect-resistant gene resources is particularly important.
The numerous target receptors for bacillus thuringiensis Cry toxins and the toxicological mechanisms are controversial (Pardo-Lopez l., sobaron m., bravo a. (2013) Bacillus thuringiensis insecticidal three-domain Cry toxins: mode of action, insect resistance and consequences for crop protection.fems Microbiology Reviews 37:3-22), making it difficult to achieve targeted screening and rational design for new resource development. Cry toxins that have been reported to date for crystal structures include Cry1Aa, cry1Ac, cry2Aa, cry3Ba, cry4Aa, cry4Ba, cry5B, cry8Ea (Xu c.c., wang b.c., yu z.n., et al (2014) Structural insights into Bacillus thuringiensis Cry, cyt and parasporin oxides.toxins, 6:2732-2770). Although these toxins differ greatly in amino acid sequence and specificity, all of these toxins have similar three domain characteristics. Through sequence alignment, the highest degree of amino acid sequence variation in each Domain was found for Cry toxin Domain II, particularly the 3 loop at the tip of the Cry toxin Domain II region, which plays an important role in receptor binding and insecticidal specificity, was highly variable in length, sequence and spatial conformation, much like the Complementarity Determining Regions (CDRs) of antibodies (Li J.D., carroll J., ellar D.J. (1991) Crystal structure of insecticidal delta-endotoxin from Bacillus thuringiensis at 2.5A resolution.Nature,353:815-821). According to the concept of anti-idiotype antibodies described by immunologists Niels Jerne in the network immunology, anti-idiotype antibodies have the potential to mimic the original antigen. The imitation ability is derived from the 'internal image' or similar molecular interaction of the antigen of the anti-idiotype antibody, and the anti-idiotype antibody capable of simulating Cry toxin activity key epitope is prepared by utilizing the molecular imitation function of the anti-idiotype antibody and combining with the Cry toxin receptor binding function, so that a new idea is provided for the excavation of novel insecticidal resources. The applicant previously constructed a phage display single chain antibody library based on mouse immunization ("construction of Cry2A toxin anti-idiotype single chain antibody library" Liu Yuan et al, jiangsu agricultural journal, 2018), but the literature only identified the single chain antibody library and did not screen it.
Single domain antibodies are a class of antibodies that lack the light chain and only the heavy chain variable region of the antibody, typically consisting of only 110-130 amino acids, with molecular weights of only 12-15kDa, also known as nanobodies. The single domain antibody has similar or higher specific antigen affinity than the conventional whole antibody, but has the advantages of small molecular weight, strong stability, easy recombinant expression and the like (Wang x.r., chen q., sun z.c., wang y.d., su b.c., zhang c.h., cao h.m., liu x (2020) Nanobody affinity improvement: directed evolution of the antiochratoxin A singledomain antibody, international Journal of Biological macromolecules.151:312-321. Kang Xiaozhen, cao Jiali, zhang Baohui, yuan Quan (2018), development of research and application of single domain antibodies, journal of bioengineering 34:1974-1984). The single domain antibody screening method mainly comprises single domain antibody screening based on phage surface display technology, single domain antibody screening based on yeast surface display technology and single domain antibody screening based on bacterial display technology.
At present, no reports are yet made on active effectors capable of replacing Cry2Aa toxins, in particular on anti-Cry 2Aa toxin single-domain antibodies and the like.
Disclosure of Invention
In view of the above, the present application relates to an anti-Cry 2Aa idiotype single domain antibody capable of broad-spectrum recognition of three receptors of the midgut of plutella xylostella (i.e., cadherin-like, aminopeptidase N2 and vacuolar ATP hydrolase A subunit), and its use in mimicking Cry2Aa toxins.
Specifically, the application is realized by the following technical scheme:
firstly, the application provides a single-domain antibody for broad-spectrum recognition of three receptors of plutella xylostella midgut, which consists of 119 amino acids with the amino acid sequence shown as SEQ ID NO.1, and the nucleotide sequence shown as SEQ ID NO. 2.
The application further provides application of the single-domain antibody with the amino acid sequence shown as SEQ ID NO.1 in simulating the combination of Cry2Aa toxin and plutella xylostella midgut receptor. After the single domain antibody gene is transferred into an expression vector pET26b (+), the single domain antibody gene is transformed into escherichia coli BL21 for soluble expression. The ELISA shows that the single domain antibody has good broad spectrum recognition capability on three types of receptors of cadherin, aminopeptidase N2 and vacuole ATP hydrolase A subunits, has competitive inhibition effect on the combination of Cry2Aa and the three types of receptors, has broad spectrum recognition capability on the receptors and competitive inhibition effect on the combination of toxins and the receptors, and has important guiding significance on the development of Cry toxin substitutes.
Thirdly, the application provides application of the single domain antibody with the amino acid sequence shown as SEQ ID NO.1 in killing plutella xylostella larvae. The single-domain antibody is displayed on the surface of phage through the rescue of auxiliary phage, and the biological assay of plutella xylostella is carried out through a feed coating method, so that the result shows that the single-domain antibody can poison plutella xylostella larvae.
According to the application, 1 kind of anti-Cry 2Aa toxin idiotype single domain antibody with three kinds of receptor binding activities of the plutella xylostella midgut is obtained through screening from a natural single domain antibody library for the first time, and the anti-Cry 2Aa toxin idiotype single domain antibody has the function of broad-spectrum recognition of three kinds of receptors of the plutella xylostella midgut and can simulate Cry2Aa toxin. The resource excavation way of the insect-resistant gene is widened, the resistance risk existing in the long-term use of the existing microorganism-source insect-resistant gene can be delayed, and the method has important scientific significance for the development of the field of prevention and control of agricultural insect pests. Meanwhile, the Cry2Aa toxin idiotype single domain antibody overcomes the defect of complicated process for preparing the anti-idiotype antibody by the traditional method, can be obtained without animal immunization, has short preparation period and small molecular weight, and can be applied to in-vitro large-scale production.
Drawings
FIG. 1 is a single domain antibody and F (ab') 2 Schematic representation of binding assay results for the receptor.
FIG. 2 is a schematic representation of the homologous sequence analysis of a single domain antibody to Cry2Aa and the alignment of the single domain antibody to germline gene sequences.
FIG. 3 is a single domain antibody pair Cry2Aa and F (ab') 2 And competitive inhibition of receptor binding.
FIG. 4 is a schematic representation of SDS-PAGE and Western Blot analysis of single domain antibodies.
FIG. 5 is a graphical representation of apparent affinity assays for Cry2Aa and single domain antibodies binding to receptors.
FIG. 6 is a three-dimensional structure of Cry2Aa toxin, single domain antibody, plutella xylostella APN2, V-ATPaseA, cad-TBR proteins.
FIG. 7 is a graph showing the prediction of hot spot residues of a single domain antibody in a docking complex with APN2, V-ATPaseA and Cad-TBR proteins of plutella xylostella respectively.
FIG. 8 is a schematic representation of the insecticidal results of phage display single domain antibodies against Plutella xylostella.
FIG. 9 is a photograph of a bioassay petri dish of example 6.
Detailed Description
The application is further described below with reference to the accompanying drawings.
Reagents, instrument sources involved in the examples:
F(ab’) 2 (Pierce TM F(ab′) 2 the Preparation Kit is obtained by treating Cry2Aa rabbit polyclonal antibody), a cadherin-like toxin binding region (Cad-TBR), aminopeptidase N2 (APN 2) and vacuole type ATP hydrolase A subunit (V-ATPase A) proteins are prepared and stored by agricultural product quality safety and nutrition institute of the academy of agricultural sciences of Jiangsu province. These materials are all conventional materials in the field, such as literature Wu Aihua, preparation and activity research of Bt Cry2Aa toxin genetic engineering antibodies, 2015, nanjing university of agriculture, disclose a preparation method of Cry2Aa rabbit polyclonal antibodies; literature "Gao m.j., dong s., hu x.d., zhang x, liu y, zhong j.f., lu l.n., wang y, chen l.m., liu x.j. (2019) Roles of midgut cadherin from two moths in different Bt action mechanisms: correlation among toxin binding, cellular toxicity and syngism.journal of Agricultural and Food Chemistry,67 (48): 13237-13246," methods of preparing Cad-TBR are disclosed; the preparation method of V-ATPaseA is disclosed in "Xie Y.J., xu C.X, gao M.J., zhang X, lu L.N., hu, X.D.Chen W., jurat-Funtes J.L., zhu Q., liu Y., lin M.M., zhong J.F, liu X.J. (2021) Docking-based generation of antibodies mimicking Cry A/1B protein binding sites as potential insecticidal agents against diamondback moth (Plutella xylostella) Pest Management Science, DOI 10.1002/ps.6499)", and the gene sequence of APN2 is disclosed in Genbank No. GU479677.1, and the present example is prepared by the same method as that for preparing V-ATPase A.
Humanized phage display single domain antibody libraries, E.coli TG1 and helper phage KM13 were purchased from UK Source BioScience.
Cry2Aa toxin standard was purchased from Shanghai Biotechnology Inc.
Pierce TM F(ab′) 2 Preparathe station Kit was purchased from Thermo Scientific.
Taq enzyme, T4 ligase, restriction endonucleases Nco I and Not I, plasmid extraction kit was purchased from NEB Inc. of America.
Transformation competent E.coli BL21 (DE 3) was purchased from Beijing full gold biotechnology Co.
HRP-labeled anti-M13 murine monoclonal antibody, HRP-labeled anti-HIS tag monoclonal antibody, HRP-labeled goat anti-rabbit IgG Fc antibody, HRP-labeled goat anti-rabbit IgG (H+L) antibody, HRP-labeled goat anti-mouse antibody, and HRP-labeled anti-streptavidin were purchased from Katsumadai Biotechnology Inc., beijing Soy Biotechnology Co., ltd, respectively.
96-well elisa plate, 6-well elisa plate was purchased from Corning, usa.
The pET26b (+) vector is preserved by agricultural product quality safety and nutrition research by the academy of agricultural sciences of Jiangsu province.
Glucose and skimmed milk powder were purchased from beijing solebone technologies.
Yeast extract and peptone are Oxoid products.
Kanamycin and ampicillin were purchased from michibased organisms.
Nickel ion affinity chromatography column (His Trap HP) was purchased from GE Healthcare.
12% of pre-gel, tris-MOPS protein electrophoresis buffer, transfer buffer and Western blot TMB color development solution (ChromoSensor) TM One-Solution TMB Substrate) was purchased from Nanjing Jinsri biotechnology Co.
3, 5-Tetramethylbenzidine (TMB), trypsin, isopropyl- β -D-thiogalactose (IPTG) were purchased from Invitrogen, U.S.A.
The other chemical reagents used in the examples below were all of the analytical purity of home-made.
Cradle (Shanghai know Chu instrument ZQZY-70 BF), small table-type refrigerated centrifuge (Eppendorf 5430R), refrigerated centrifuge (Beckman Allegra 25R), constant temperature incubator (Jing Hong), balance (Liangping instrument FA 2004), pH meter (Sartorius PB-10), autoclave (Sanyo MLS-3750), digital display constant temperature water bath (Hemsleyaku Instrument manufacturing Co., ltd.), ultra-low temperature refrigerator (sea), ultrasonic cell breaker (Nanj month Xuezhi biotechnology YX-1000H), vertical plate electrophoresis tank (Beijing jun's JY-SCZ2+), western blot transfer membrane electrophoresis apparatus (Beijing jun's JY-ZY 5), decolorization cradle (Scilogex SF-O180-E), plate washer (Thermo Wellwash), pure water apparatus (Millipore Direct-Q3 UV), enzyme-labeled apparatus (Thermo Multiskan GO). PCR instrument (Takara), metal bath (Hangzhou European instrument MIULAB), nucleic acid electrophoresis instrument (Beijing Liuyi DYY-6C).
Example 1 screening of anti-Cry 2Aa toxin idiotype Single Domain antibodies
The method comprises the following specific steps:
(1) Amplification of phage antibody library:
mu.L of stock was added to 50mL of pre-warmed 2 XTY-AG medium (2 XTY medium supplemented with 100. Mu.g/mL ampicillin and 1% glucose). Shake culturing at 37deg.C to OD 600 =0.4. Adding 2X 10 11 KM13 helper phage. The culture was allowed to stand in a water bath at 37℃for 30 minutes. 3,300g was centrifuged for 10min and resuspended in 100mL 2 XTY-AKG medium (2 XTY medium supplemented with 100. Mu.g/mL ampicillin, 50. Mu.g/mL kanamycin and 0.1% glucose). Shake-culturing overnight at 25deg.C. Overnight culture broth 3,300g was centrifuged for 30 min. 20mL of PEG/NaCl (20% polyethylene glycol 6000,2.5M NaCl) was added to 80mL of the supernatant, and the mixture was thoroughly mixed and allowed to stand on an ice bath for 1 hour. 3,300g centrifugal 30 minutes, pouring PEG/NaCl, again centrifugal, suction residual PEG/NaCl. The pellet was resuspended in 4mL Phosphate Buffer (PBS), and the residual bacterial debris was removed by centrifugation at 11,600g for 10 minutes using a microcentrifuge, and the supernatant was the phage antibody library prepared.
(2) Screening of phage antibody library:
f (ab') 2 1 well (1 well) coated in 6 well plate, 3% MPBS (3% nonfat milk powder in PBS buffer) coated 1 well (2 well), 4℃overnight. The next day, the coated wells were washed 3 times with PBST (PBS buffer plus 0.05% Tween 20), 3mL MPBS was added, and left to stand at 37℃for 2 hours for blocking. PBST is washed 3 times, 1mL of phage antibody library is taken and evenly mixed with 1mL of MPBS, and added into a No.2 hole, and the mixture is slowly shaken and incubated for 1h at room temperature, and then is kept stand and incubated for 1h. Adding the solution after the negative screen into a closed No.1 hole, and the same stripIncubate under the pieces for 1h and rest for 1h. PBST was washed 10 times, 500. Mu.L of trypsin-PBS (50. Mu.L of 10mg/mL trypsin stock plus 450. Mu.L PBS) was added and phages were eluted by shaking incubation for 15 minutes at room temperature. 5mL log phase E.coli TG1 (OD) 600 =0.4), phage obtained by elution was added, and the mixture was allowed to stand in a water bath at 37 ℃ for 30 minutes. Sucking 100 μL stock solution and 100 μL 1:10 2 Dilutions were plated on TYE-AG plates (TYE supplemented with 100. Mu.g/mL ampicillin and 1% glucose) and incubated overnight at 37℃to determine phage concentration. The remaining culture broth was centrifuged and resuspended and plated on TYE-AG plates overnight. Phage antibody libraries were prepared from phage produced in the first round according to step 1. The second and third rounds of screening had coating antigen concentrations of 45. Mu.g/mL and 20. Mu.g/mL, respectively. The number of times of the second and third PBST washes is 20, and the third phage elution mode is competition elution and trypsin elution, specifically 1mL of Cry2Aa toxin with 100 mug/mL is taken to be added into a hole to compete with phage for binding F (ab') 2 In order to elute phage, the eluent is added into logarithmic phase TG1, and trypsin elution is carried out after competitive elution, and the steps are the same as the first round of screening.
(3) Identification of positive clones:
single colonies from the third round were randomly picked and inoculated into 96-well plates containing 200. Mu.L/well of 2 XSY-AG medium and incubated overnight at 37 ℃. Transferring 2 μl overnight bacterial liquid to new 96 Kong Bankong, shaking at 37deg.C for 2 hr, adding 1×10 per well 10 The KM13 helper phage of (F) was allowed to stand at 37℃for 2h, centrifuged at 2000g for 10min, and the pellet was resuspended in 200. Mu.L of 2 XSY-AK medium and incubated overnight at 25 ℃. The overnight culture was centrifuged at 2000g for 30min and the supernatant was taken.
With 2. Mu.g/mL F (ab') 2 10. Mu.g/mL APN2, 10. Mu.g/mL V-ATPase A, 10. Mu.g/mL CAD-TBR coated 96-well ELISA plates, 10. Mu.g/mL MPBS as negative control for the coating, overnight at 4 ℃. The following day, PBST plates were washed 3 times, 3% MPBS was added, blocked at 37℃for 2h, 100. Mu.L of the supernatant prepared in the above steps was added after the plate was washed again, incubated at 37℃for 1h, PBST plates were washed 3 times, HRP-labeled anti-M13 murine monoclonal antibody diluted 1:5000 times with PBS was added to 100. Mu.L/well, and incubated at 37℃for 1h, PBST plates were washed 3 times. The now prepared substrate solution (10 mL CPBS buffer was added to 100. Mu.L dimethylsulfoxide)Dissolved 10mg/ml TMB and 25. Mu.L 0.65% H 2 O 2 ) 100 mu L/well of 96-well ELISA plate is added, and the mixture is kept stand and developed for 15min at 37 ℃. 50. Mu.L/well 2mol/L H are added 2 SO 4 The reaction was terminated and absorbance was read with a microplate reader at 450 nm.
ELISA results are shown in FIG. 1, and a strain of F (ab') 2 APN2, V-ATPase A, cad-TBR all have single domain antibodies with binding activity. The applicant has self-named this single domain antibody 7E. And (3) adding glycerol into the corresponding bacterial liquid of the single-domain antibody in a 96-well plate, and freezing and preserving the single-domain antibody in a refrigerator at the temperature of minus 80 ℃ for later use.
EXAMPLE 2 sequence analysis of a Single-Domain antibody for broad-Spectrum recognition of the midgut receptor of Plutella xylostella
The single domain antibody sequences obtained in example 1 were analyzed for homology to Cry2Aa sequences using the Lipman-Pearson method in DNASTAR Lasergene 7.1.1 software. The on-line tool of an IMGT database of an international immune genetic information system is utilized, (http:// www.imgt.org/IMGT_vquest/vquest) to analyze single-domain antibody sequence information, submit single-domain antibody nucleic acid sequences, set a species as human, be Immunoglobulin (IG), analyze the information such as alleles, framework region and CDR region labels of a single-domain antibody variable region coding gene fragment.
The Lipman-Pearson method is used for analyzing the homology between a single-domain antibody sequence and a Cry2Aa sequence, and is shown in figure 2, wherein A is the homology analysis result of the amino acid sequence of the single-domain antibody 7E and the Cry2Aa toxin; b is the result of amino acid sequence comparison and mutation site analysis of the single domain antibody and the germ line gene. As can be seen, the partial sequence of CDR1 of the single Domain antibody has 57.1% identity with Cry2Aa Domain II Q286-N292, and the fragment of CDR2 of the single Domain antibody has 46.2% identity with Cry2Aa Domain II A408-D420. IMGT database analysis shows that the single domain antibody consists of V, J, D gene fragments, wherein the allele with highest consistency with the V gene fragments is homcap IGHV3-23 x 01F or homcap IGHV3-23 x 05F or homcap IGHV3-23d x 01, and the consistency is 86.46. The allele with highest identity to the J gene fragment was Homsap IGHJ4 x 02F, the identity was 72.92%, and the allele with highest identity to the D gene fragment was Homsap IGHD3-10 x 01F. IMGT marks VH with 4 framework regions of amino acid length 25-17-38-11, respectively, and single domain antibodies with 3 CDR regions of amino acid length 8-8-14, respectively.
The single domain antibody gene was shown to have 11 and 2 amino acid mutations in the CDR region and the framework region, respectively, by alignment with the germline gene sequence of highest identity, wherein the CDR1 region had 6 amino acid mutations (F27 > D, T28> S, S30> T, S31> T, Y32> K, A33> N), the CDR2 region 3 (G53 > K, S54> R, G55> S), and the CDR3 region had 2 amino acid mutations (G106 > A, E107> K).
EXAMPLE 3 competitive substitution of the Plutella xylostella midgut receptor broad-spectrum recognition Single-Domain antibodies for Cry2Aa
mu.g/mL F (ab') 2 10. Mu.g/mL APN2, 10. Mu.g/mL V-ATPase A, 10. Mu.g/mL CAD-TBR coated 96-well ELISA plates overnight at 4 ℃. The following day, PBST wash plates 3 times, 3% MPBS was added, blocked at 37℃for 2h, PBST wash plates 3 times, 50. Mu.L of biotin-labeled Cry2Aa toxin and 50. Mu.L of gradient diluted single domain antibody (example 1) were added to each well after premixing, control was incubated with 50. Mu.LPBS instead of single domain antibody, 37℃for 1h, PBST wash plates 3 times, HRP-labeled streptavidin diluted 1:5000 fold with PBS, 100. Mu.L/well was added to the ELISA plates, and PBST wash plates 3 times at 37 ℃. The now prepared substrate solution (10 mL CPBS buffer was added to 100. Mu.L dimethylsulfoxide dissolved 10mg/mL TMB and 25. Mu.L 0.65% H) 2 O 2 ) 100 mu L/well of 96-well ELISA plate is added, and the mixture is kept stand and developed for 15min at 37 ℃. 50. Mu.L/well 2mol/L H are added 2 SO 4 The reaction was terminated and absorbance was read with a microplate reader at 450 nm.
As a result, FIG. 3 shows that the single domain antibody was specific for Cry2Aa toxin and F (ab') 2 And binding to all three receptors, wherein 2×10 has competitive inhibition 7 The inhibition rate of CFU/mL single domain antibody to the combination of Cry2Aa toxin and V-ATPase A reaches 35.59 percent.
Example 4 soluble expression and apparent affinity assay of the midgut receptor broad-Spectrum recognition Single-Domain antibodies of plutella xylostella
mu.L of the glycerol stock of the single-domain antibody of example 1 was frozen and incubated overnight with 5mL of 2 XSTY-AG at 37℃and 250 rpm. Extracting plasmid, nco I and Not I double enzyme cutting antibody genes and pET26b (+) vector, carrying out electrophoresis cutting gel recovery after enzyme cutting, connecting a single domain antibody gene to the pET26b (+) vector by T4 ligase, converting into escherichia coli competent BL21 (DE 3), randomly picking 5 clones, carrying out bacterial liquid PCR verification and sequencing, and successfully constructing an expression vector of the single domain antibody gene, wherein the amino acid sequence of the single domain antibody is shown as SEQ ID NO. 1. The nucleotide sequence is shown as SEQ ID NO. 2. In a specific embodiment, the single domain antibody may also be synthesized artificially based on the nucleotide sequence disclosed in SEQ ID NO. 2.
The 50. Mu.L single domain antibody BL21 expression strain with the amino acid sequence shown in SEQ ID NO.1 is taken, 5mL of 2 xTY-K (2 xTY-containing kanamycin with the final concentration of 50. Mu.g/mL) is added, and shake culture is carried out at 37 ℃ and 250rpm for overnight. The next day 2.5mL of overnight culture was transferred to 250mL of 2 XTY-K and shake cultured at 37℃at 250rpm until OD600 = 0.9 (about 3 h). IPTG with a final concentration of 0.5mM was added thereto, followed by shaking culture at 25℃and 250rpm for 18 hours. The next day the culture broth was centrifuged at 4℃and 10000g for 15min, the cells were collected, weighed and resuspended in phosphate buffer (PBS, pH 7.4) at a ratio of 100 mg/mL. Vortex mixing well, and ultrasonic crushing in ice water bath. The ultrasonic conditions are as follows: output power 40%, mode 06, working time 30min, ultrasonic crushing 2s, interval 3s. Centrifuging at 4deg.C and 10000g for 30min, collecting supernatant to obtain whole fungus crushed supernatant. The single domain antibody was purified using a HisTrap HP column, and specific procedures were referred to the GE company product instructions, and the purified single domain antibody was obtained for SDS-PAGE analysis and Western Blot analysis, and the purified protein concentration was determined using the Bradford method. The results are shown in FIG. 4, in which FIG. 4, A is SDS-PAGE analysis of purified single domain antibodies, and lane 3 is purified single domain antibodies; b is a single-domain antibody purified by Western Blot analysis, and the HRP-labeled anti-HIS tag antibody detects the purified single-domain antibody. As can be seen from FIG. 4, the single domain antibody was successfully expressed and purified at a concentration of 0.8mg/mL.
The 96-well ELISA plate was coated with 10. Mu.g/mL APN2, 10. Mu.g/mL V-ATPase A, and 10. Mu.g/mL CAD-TBR, respectively, at 4℃overnight. The next day, PBST wash plates 3 times, 200. Mu.L 3% MPBS was added to each well, blocked at 37℃for 2h, PBST wash plates 3 times, and the apparent affinity of single domain antibodies to the receptor was determined as follows: 100. Mu.L of single domain antibody protein (diluted from a starting gradient of 20. Mu.g/mL to 0.3125. Mu.g/mL) was added per well, PBS as a control. Incubate at 37℃for 1h and wash the plates 3 times with PBST.HRP-labeled Staphylococcus aureus protein A100. Mu.L/well diluted with 1:5000 times PBS was added to the ELISA plate, incubated at 37℃for 1h, and the PBST plates were washed 3 times. The now prepared substrate solution (10 mL CPBS buffer was added to 100. Mu.L dimethylsulfoxide dissolved 10mg/mL TMB and 25. Mu.L 0.65% H) 2 O 2 ) 100. Mu.L/well of an ELISA plate was added thereto, and the mixture was allowed to stand at 37℃for 15 minutes for color development. Will 2mol/L H 2 SO 4 The absorbance was read at 450nm by rapid addition to the plate at 50. Mu.L/well. Apparent affinity analysis was performed using the Scatchard module of Graphpad Prism software.
Apparent affinity assay procedure for Cry2Aa toxin to receptor is as follows: 100. Mu.L Cry2Aa toxin protein (diluted from a starting gradient of 4. Mu.g/mL to 0.0625. Mu.g/mL) was added per well, PBS serving as a control. Incubate at 37℃for 1h and wash the plates 3 times with PBST. HRP-labeled streptavidin diluted 1:5000 times with PBS was added to the ELISA plate at 100. Mu.L/well, and the subsequent steps were identical to the single domain antibody and receptor binding assay.
As a result, as shown in FIG. 5, the affinity of the toxin to receptor APN2 was 16.4.+ -. 2.3nmol/L, the affinity to V-ATPase A was 11.7.+ -. 3.2nmol/L, the affinity to Cad-TBR was 11.7.+ -. 2.3nmol/L (as shown in FIG. 5 for D, E, F, respectively), and the affinity of the single domain antibody to receptor was 77.9.+ -. 13.1nmol/L, 70.8.+ -. 7.6nmol/L, and 114.0.+ -. 7.8nmol/L (as shown in FIG. 5 for A, B, C, respectively) lower than the affinity of the toxin to receptor. The affinity of the single domain antibody to the three receptors was consistent with the competitive inhibition results described above, wherein the affinity of the single domain antibody to V-ATPase A in the three receptors was highest, the competitive inhibition to Cry2Aa and V-ATPase A was also highest, APN2 times, the affinity of the single domain antibody to Cad-TBR was lowest in the three receptors, and the competitive inhibition to Cry2Aa and Cad-TBR was also lowest. This example demonstrates that the single domain antibody can broad-spectrum recognize three classes of plutella xylostella middleintestine receptors, cadherin-like, aminopeptidase N2, and vacuole ATP hydrolase a subunits, and can mimic Cry2Aa toxin application.
EXAMPLE 5 midgut receptor broad-spectrum recognition Single-Domain antibody homology modeling and molecular docking of plutella xylostella
The single domain antibody amino acid sequence, APN2 amino acid sequence, V-ATPase A amino acid sequence, cad-TBR amino acid sequence were submitted to SWISS-MODEL (https:// swissmodel. Expasy/tdsourcetag = s_pcqq_aiomsg) for homology modeling and evaluation, respectively. The three-dimensional structure of Cry2Aa was obtained from the PDB database (PDB ID:1i5 p). The model is docked by ZDOCK SERVER (http:// ZDOCK. Umassmed. Edu /), and the docking compound uses KFC Server (https:// mitchell-web. Ornl. Gov/KFC_Server/index. Php) to conduct hot spot prediction.
In FIG. 6, A-E are three-dimensional schematic diagrams of Cry2Aa toxin, single domain antibody (7E), plutella xylostella APN2, V-ATPase A and Cad-TBR proteins respectively. In FIG. 7, A-C are a single domain antibody (7E) APN2 docking complex and hotspot prediction map, a single domain antibody (7E) and V-ATPase A docking complex and hotspot prediction map, and a single domain antibody (7E) and Cad-TBR docking complex and hotspot prediction map, respectively. It can be seen that the hot spot of the 7E-APN2 docking complex in which 7E participates in binding is mainly located in the CDR3 region, the hot spot of the 7E-V-ATPase A docking complex in which 7E participates in binding is mainly located in the CDR2 region, and the hot spot of the 7E-Cad-TBR docking complex in which 7E participates in binding is mainly located in the CDR3 region.
EXAMPLE 6 midgut receptor broad-Spectrum recognition Single-Domain antibody bioassays for Plutella xylostella
Bioassays were performed using the feed graph method, 1mL of phage display single domain antibody was added to a petri dish (diameter 5.8 cm) with conventional feed for plutella xylostella, a Cry2Aa toxin standard was diluted to 10 μg/mL with PBS as a positive control to the petri dish, PBS and phage display independent antibodies (anti-BSA-scfv, see literature "liuy"), lin m.m., zhang x.f., hu x.d., lin J.R, hao j, he d, zhang x, xu c.x, zhong j.f., xie y.j., zhang c.z., liu x.j., and (2017) Development of competitive ELISA for the detection of bovine serum albumin using single-chain variable fragments, analytical Biochemistry,525:89-91 ") were added as negative controls to the petri dish, and 20 head 2-year plutella xylostella was naturally inoculated to each petri dish, and 3 samples were dried for each replicate and each replicate was averaged. Mortality was recorded after 5 days and death or death was considered as death for no 4 years.
The result of the bioassay is shown in FIG. 8, 2X 10 7 The insecticidal rate of the phage display single domain antibody of CFU/mL to plutella xylostella is 46.48 +/-6.57%, and Cry2Aa toxin standard substance of positive control 10 mug/mLThe insecticidal rate of (2) was 80.00.+ -. 1.92%, and the insecticidal rate of the negative control independent antibody was 3.11.+ -. 1.44%.
The photographs of the culture dishes of PBS and single-domain antibodies for killing plutella xylostella larvae in one bioassay experiment are shown in fig. 9, wherein in fig. 9, A is a PBS control group, and B is a single-domain antibody experiment group.
The key point of toxin disinsection is that the apparent binding force of the single domain antibody and 3 receptors is lower than the apparent binding force of the toxin and the receptors, one of the reasons possibly causing the disinsection effect to be weaker than that of the toxin is that the toxin is a three-domain, has an alpha helix perforation structure, and the single domain antibody only simulates the domain II or the domain III of the toxin and lacks the perforation structure. However, the single-domain antibody has the advantages of being derived from animals and not obtained through animal immunization, being more superior in safety, short in preparation period and suitable for in-vitro large-scale production. In addition, under the situation that the current new insecticidal gene resource is increasingly difficult to excavate, the anti-idiotype single domain antibody provides a new way for creating the insecticidal gene resource.
Sequence listing
<110> academy of agricultural sciences in Jiangsu province
<120> an anti-Cry 2Aa idiotype single domain antibody for broad-spectrum recognition of plutella xylostella midgut receptor and application thereof
<141> 2021-08-09
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 119
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 1
Met Ala Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Pro Gly
1 5 10 15
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Ser Phe Thr Thr
20 25 30
Lys Asn Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Val Ser Ala Ile Ser Lys Arg Ser Gly Ser Thr Tyr Tyr Ala Asp Ser
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
65 70 75 80
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
85 90 95
Cys Ala Gly Leu Thr Gln Arg His Gly His Ala Lys Leu Lys Tyr Trp
100 105 110
Gly Gln Gly Thr Leu Val Thr
115
<210> 2
<211> 359
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
atggcccagg tgcagctgtt ggagtctggg ggaggcttgg tatagcctgg ggggtccctg 60
cgtctctcct gtgcagcctc cggagatagc tttaccacta agaatatggc ctgggtccgc 120
caggctccag ggaagggtct agagtgggta tcagccattt ctaagagaag cggtagcaca 180
tactacgcag actccgtgaa gggccggttc accatctccc gtgacaattc caagaacacg 240
ctgtatctgc aaatgaacag cctgcgtgcc gaggacaccg cggtatatta ttgcgcgggt 300
ttgacgcaaa ggcatgggca cgcgaagttg aagtattggg gtcagggaac cctggtcac 359
Claims (2)
1. An anti-Cry 2Aa idiotype single domain antibody for broad spectrum recognition of plutella xylostella midgut receptor, which is characterized in that the amino acid sequence of the single domain antibody is shown as SEQ ID NO. 1.
2. The use of the single domain antibody of claim 1 for killing plutella xylostella.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110909528.0A CN113512115B (en) | 2021-08-09 | 2021-08-09 | anti-Cry 2Aa idiotype single-domain antibody for broad-spectrum identification of plutella xylostella midgut receptor and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110909528.0A CN113512115B (en) | 2021-08-09 | 2021-08-09 | anti-Cry 2Aa idiotype single-domain antibody for broad-spectrum identification of plutella xylostella midgut receptor and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113512115A CN113512115A (en) | 2021-10-19 |
| CN113512115B true CN113512115B (en) | 2023-09-15 |
Family
ID=78068001
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110909528.0A Active CN113512115B (en) | 2021-08-09 | 2021-08-09 | anti-Cry 2Aa idiotype single-domain antibody for broad-spectrum identification of plutella xylostella midgut receptor and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113512115B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP6933379B2 (en) * | 2015-09-24 | 2021-09-08 | ザ ユニバーシティ オブ ノース カロライナ アット チャペル ヒル | Methods and compositions for reducing metastasis |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104498501A (en) * | 2014-12-05 | 2015-04-08 | 江苏省农业科学院 | Human insecticidal gene and coding insecticidal peptide and application thereof |
| CN113150161A (en) * | 2021-05-26 | 2021-07-23 | 江苏省农业科学院 | Anti-idiotype monoclonal antibody of bacillus thuringiensis Cry2Aa toxin and application |
-
2021
- 2021-08-09 CN CN202110909528.0A patent/CN113512115B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104498501A (en) * | 2014-12-05 | 2015-04-08 | 江苏省农业科学院 | Human insecticidal gene and coding insecticidal peptide and application thereof |
| CN113150161A (en) * | 2021-05-26 | 2021-07-23 | 江苏省农业科学院 | Anti-idiotype monoclonal antibody of bacillus thuringiensis Cry2Aa toxin and application |
Non-Patent Citations (2)
| Title |
|---|
| Anti-idiotypic single-chain variable fragment antibody partially mimic the functionally spatial structure of Cry2Aa toxin;Manman Lin等;《Analytical Biochemistry》;20210428;第625卷;第1-10页 * |
| 磁珠筛选抗Cry2Aa人源化单链抗体及检测方法的;武爱华等;《江苏农业学报》;20171231;第33卷(第4期);第945-950页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113512115A (en) | 2021-10-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Gao et al. | A method for the generation of combinatorial antibody libraries using pIX phage display | |
| US9770038B2 (en) | Human-derived insect-resistant gene and anti-CRY1B toxin idiotype single-chain antibody encoded thereby and application thereof | |
| Xu et al. | Construction of an immunized rabbit phage display library for selecting high activity against Bacillus thuringiensis Cry1F toxin single-chain antibodies | |
| Zhao et al. | Development of a biotinylated broad-specificity single-chain variable fragment antibody and a sensitive immunoassay for detection of organophosphorus pesticides | |
| JP2017505109A (en) | Human insect resistance gene and anti-Cry1Ab toxin idiotype single chain antibody encoded thereby and uses | |
| Miyazaki et al. | Isolation and characterization of antigen-specific alpaca (Lama pacos) VHH antibodies by biopanning followed by high-throughput sequencing | |
| EP3067423B1 (en) | Human insecticidal gene and insecticidal peptide encoded thereby, and use thereof | |
| CN113512115B (en) | anti-Cry 2Aa idiotype single-domain antibody for broad-spectrum identification of plutella xylostella midgut receptor and application thereof | |
| CN114292820B (en) | Insect-resistant protein Cry3Bb hybridoma cell strain, antibody produced by same and application thereof | |
| JP6211703B2 (en) | Human insect resistance gene and anti-Cry1C toxin idiotype single chain antibody encoded thereby and uses | |
| Xu et al. | Microcystin-LR heterologous genetically engineered antibody recombinant and its binding activity improvement and application in immunoassay | |
| Xu et al. | Selection and application of broad-specificity human domain antibody for simultaneous detection of Bt Cry toxins | |
| AU2020101140A4 (en) | ELISA kit for quantitative detection of content of insect cadherin and use method thereof | |
| WO2021153129A1 (en) | Method for producing heavy chain variable domain of heavy-chain antibody | |
| Dong et al. | Screening of anti-idiotypic domain antibody from phage library for development of Bt Cry1A simulants | |
| Lin et al. | Anti-idiotypic single-chain variable fragment antibody partially mimic the functionally spatial structure of Cry2Aa toxin | |
| CN108997494B (en) | Improved humanized Bt Cry toxin protein functional effector (GGCC) and design method and application thereof | |
| Njeru et al. | Nanobodies: their potential for applications in biotechnology, diagnosis and antiviral properties in Africa; focus on application in agriculture | |
| Jiang et al. | A rapid live-cell ELISA for characterizing antibodies against cell surface antigens of Chlamydomonas reinhardtii and its use in isolating algae from natural environments with related cell wall components | |
| US11161914B2 (en) | Human-derived insecticidal bt cry toxin mimetic, and design method and application thereof | |
| CN113754762B (en) | anti-Cry 3Bb protein single-domain heavy chain antibody and application thereof | |
| CN105884892B (en) | A kind of detection of Bt Cry toxin wide spectrum albumen and its encoding gene and application | |
| Muraoka et al. | Selection and characterization of human serum albumin-specific porcine scFv antibodies using a phage display library | |
| CN111548414A (en) | Bivalent antibody for broad-spectrum detection of Bt Cry1 toxoid as well as gene sequence and application thereof | |
| CN108642017B (en) | Monoclonal antibody cell strain capable of stably secreting anti-conotoxin and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |