Cnaphalocrocis medinalis V-ATP enzyme A subunit protein as well as coding gene and application thereof

Abstract

The invention discloses the cnaphalocrocis medinalis V-ATP enzyme A subunit protein and the coding gene thereof for the first time, the amino acid sequence and the nucleotide sequence of the cnaphalocrocis medinalis V-ATP enzyme A subunit protein are shown as SEQ IN NO.11 and SEQ IN NO.10 in sequence, and the cnaphalocrocis medinalis V-ATP enzyme A subunit CmV-ATPase sub A has a combination effect with Cry1Ab, Cry1Ac, Cry2Aa and Cry2Ab toxins respectively and can be applied to detection of Cry toxins in crops; according to the present invention, the CmV-ATPase sub A is adopted as the capture protein, the anti-Cry1Ac or Cry2Aa polyclonal antibody is adopted as the detection antibody to perform the binding protein-antibody sandwich ELISA detection, and the method can simultaneously detect the four Cry toxins such as Cry1Ab, Cry1Ac, Cry2Aa and Cry2Ab, has characteristics of wide linear range and high sensitivity, and has important application prospects in the detection of the Cry toxin protein residue in the fresh leaf of the agricultural product.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural product quality safety and immune detection, in particular to the application of V-ATPase A subunit protein and its encoding gene in Cry toxin detection. Background Art

[0002] Bacillus thuringiensis (Bt) is a Gram-positive bacterium that produces toxin proteins such as Cry, Cyt and Vip with specific insecticidal activity during its growth and metabolism stages, which are widely used in agricultural pest control. The receptor proteins of BtCry toxins on the insect midgut can bind to the corresponding toxins, thereby causing insect death through a complex process of action. Based on this theory, developing a Cry toxin detection method should be an effective approach. The mechanism of action of Bt Cry toxins has not yet been clarified, and there are two modes of action, including the "perforation model" and the "signal transduction model". Both modes of action agree that the Cry protoxin is activated in the alkaline midgut environment of insects, and then the activated toxin binds to different receptor proteins on the brush border membrane vesicles (BBMV) of the midgut cells, which is a key step. However, due to the large number of Bt Cry families and the different types of receptors on the midgut cells of different insects, the mechanism of action of Cry toxins still needs to be further explored. The identified receptor proteins of Cry toxins mainly include cadherin (Cad), aminopeptidase, alkaline phosphatase (ALP), glycolipids, sodium solute symporter, lipocalin, ATP binding cassette (ABC) transporter proteins family ABCC2, ABCC3, ABCB1, ABCA2 and vacuolar ATPase (Vacuolar H + -ATP synthase, V-ATPase), etc. Therefore, the study on the Bt Cry toxin receptor protein in the insect midgut is of great significance for a deep understanding of the mechanism of action of Bt toxins and guiding the establishment of a Bt toxin detection system based on this.

[0003] V-ATPase, one of the insect midgut receptor proteins, is the target of Bt Cry toxin. V-ATPase consists of a conserved peripheral cytoplasmic catalytic domain (V1) and a variable transmembrane domain (V0). V1 is composed of 8 subunits from A to H, and V0 is composed of 6 subunits: a, c, c', c", d and e. V-ATPase is responsible for H by utilizing the energy released when ATP is hydrolyzed. +It is a proton pump that relies on ATP to transport proteins across the membrane and maintain the balance of the specific intracellular pH. Since the V-ATPase A subunit on the midgut BBMV of tobacco budworm (Heliothis virescens) was reported to bind to Cry1Ac (Krishnamoorthy, M.; Jurat-Fuentes, JL; McNall, RJ; Andacht, T.; Adang, MJ Identification of novel Cry1Ac binding proteins in midgut membranes from Heliothis virescens using proteomic analyses. Insect Biochem. Mol. Biol., 2007, 37: 189-201.), proteomics technology was used to identify Spodoptera exigua, Helicoverpa armigera, Aedes aegypti, Ostrinia furnacalis, and other organisms. furnacalis), Trichoplusiani, and Plutella xylostella, are binding proteins for Cry toxins (Xie, C.; Xiong, L.; Ye, M.; Shen, L.; Li, J.; Zhang, Z.; You, M.; You, S. Genome-wide analysis of V-ATPase genes in Plutella xylostella (L.) and the potential role of PxVHA-G1 in resistance to Bacillus thuringiensis Cry1Ac toxin. Int. J. Biol. Macromol., 2022, 194: 74-83.).Other studies have found that the A subunit of the V-ATPase of the Chilo suppressalis is a functional receptor for Cry1C and Cry2Aa (Qiu, L.; Sun, Y.; Jiang, Z.; Yang, P.; Liu, H.; Zhou, H.; Wang, X.; Zhang, W.; Lin, Y.; Ma, W. The midgut V-ATPase subunit A gene is associated with toxicity to crystal 2Aa and crystal 1Ca-expressing transgenic rice in Chilo suppressalis. Insect Mol. Biol., 2019, 28: 520-527.), and the E subunit of the V-ATPase of the cotton bollworm is a receptor for Cry2Ab (Zhao, Y.; Li, P.; Yao, X.; Li, Y.; Tian, ​​Y.; Xie, G.; Deng, Z.; Xu, S.; Wei, J.; Li, X.; An, SV-ATPase E mediates Cry2Ab binding and toxicity in Helicoverpa armigera. Pestic. Biochem. Physiol., 2024, 198.). The subunit of insect V-ATPase can bind to Cry1 and 2 toxins, which lays a theoretical foundation for the detection of Cry toxins using the subunit protein of insect V-ATPase.

[0004] Insect V-ATPase A subunit is an important receptor binding protein in understanding the insecticidal effect and binding mechanism of Cry toxins, which is of great significance for studying the mechanism of action between Cry toxins and V-ATPase A subunit and designing binding proteins for detecting Cry toxins. At present, there is no report on the V-ATPase A subunit gene of rice leaf folder, especially the V-ATPase A subunit protein encoded by this gene for detecting Cry toxins. Summary of the invention

[0005] In response to the above problems, the present application first cloned a full-length sequence of a V-ATPase A subunit gene cDNA from the larvae of the rice leaf folder, and then determined its nucleotide sequence and amino acid sequence, and on this basis, the rice leaf folder V-ATPase A subunit (CmV-ATPase sub A) was expressed in prokaryotes to obtain the CmV-ATPase sub A protein. This protein can be used as a universal capture protein, combined with anti-Cry1Ac or Cry2Aa polyclonal antibodies as detection antibodies, and a detection method combining protein-antibody sandwich ELISA was established, which can simultaneously detect the four Cry toxins Cry1Ab, Cry1Ac, Cry2Aa, and Cry2Ab, and further developed its application in the field of agricultural detection.

[0006] Specifically, this application is implemented through the following technical solutions:

[0007] First, the present application provides a rice leaf folder V-ATPase A subunit protein, whose amino acid sequence is shown in SEQ ID NO.11.

[0008] Secondly, the present application provides the above-mentioned rice leaf roller V-ATPase A subunit protein encoding gene, the nucleotide sequence of which is shown in SEQ ID NO.10

[0009] The present application provides the above-mentioned rice leaf folder V-ATPase A subunit protein (CmV-ATPase sub A protein) as a universal capture protein, and anti-Cry1Ac polyclonal antibody or Cry2Aa polyclonal antibody as a detection antibody, and its application in detecting the content of Cry toxins by a combined protein-antibody sandwich ELISA method; the above-mentioned Cry toxins include at least one of Cry1Ab, Cry1Ac, Cry2Aa, and Cry2Ab, and when detecting Cry1Ab or Cry1Ac toxins, anti-Cry1Ac polyclonal antibody is used as the detection antibody; when detecting Cry2Aa or Cry2Ab toxins, anti-Cry2Aa polyclonal antibody is used as the detection antibody.

[0010] Further, the above-mentioned ELISA detection refers to the establishment of a binding protein-antibody sandwich ELISA detection by using the CmV-ATPase sub A protein with an amino acid sequence as shown in SEQ ID NO.11 as a capture protein and anti-Cry1Ac polyclonal antibody or anti-Cry2Aa polyclonal antibody as a detection antibody. The linear working range of the standard curve of Cry1Ab determined by the sandwich ELISA method is 17.41-376.31 ng / mL, and the detection limit LOD is 17.41 ng / mL. The linear working range of the standard curve of Cry1Ac is 43.16-804.95 ng / mL, and the detection limit LOD is 43.16 ng / mL. The linear working range of the standard curve of Cry2Aa is 39.70-733.03 ng / mL, and the detection limit LOD is 39.70 ng / mL. The linear working range of the standard curve of Cry2Ab is 31.85-1022.20 ng / mL, and the detection limit LOD is 31.85 ng / mL.

[0011] Third, the present application provides a sandwich ELISA detection method for Cry toxins, and the specific steps are as follows:

[0012] 1) Cut the sample (fresh leaves of crops) of known Cry toxin species into pieces, immerse in liquid nitrogen, and grind into powder with a pestle to obtain sample powder; take a 2ml centrifuge tube, add 0.1g sample powder and 1ml plant sample extract (PBS solution containing final concentrations of 0.1% BSA and 0.05% Tween-20, pH 7.4) to dissolve; place the centrifuge tube on a rotator, rotate at 4°C for 2h, and then centrifuge at 4°C, 10000g for 30min. The supernatant obtained is the plant sample test solution. At the same time, set up a control group without adding samples to obtain the control group test solution for standby use;

[0013] The above-mentioned crops are preferably rice or corn;

[0014] The Cry toxin type is any one of Cry1Ab, Cry1Ac, Cry2Aa or Cry2Ab toxin;

[0015] 2) CmV-ATPase sub A protein with the amino acid sequence shown in SEQ ID NO.11 was diluted to a concentration of 10 ppm using CBS solution, 100 μl / well was coated on a 96-well ELISA plate, and the plate was incubated at 4°C overnight;

[0016] 3) The next day, wash the ELISA plate 4 times with 300 μl / well PBST, then add 300 μl / well 5% MPBS, and block at 37°C for 2 h; then wash the ELISA plate 4 times with PBST, add the plant sample test solution, and add PBS to the control group test solution, 100 μl / well, repeat 3 times for each group, add 100 μl / well to the ELISA plate, incubate at 37°C for 1 h, and wash 4 times with PBST;

[0017] 4) Add anti-Cry1Ac polyclonal antibody or anti-Cry2Aa polyclonal antibody (volume ratio 1:2000, diluted in PBS) to 100 μl / well and incubate at 37°C for 1 h; wash 4 times with PBST, add horseradish peroxidase-labeled goat anti-rabbit IgG (volume ratio 1:7500, diluted in PBS) to 100 μl / well and incubate at 37°C for 1 h; wash 4 times with PBST, add color development solution to 100 μl / well, develop at 37°C in the dark for 15 min, add stop solution to 50 μl / well, and measure OD with an enzyme reader 450 The values ​​are substituted into the established four-parameter fitting equation:

[0018] When detecting Cry1Ab or Cry1Ac toxins, anti-Cry1Ac polyclonal antibodies are used as detection antibodies; when detecting Cry2Aa or Cry2Ab toxins, anti-Cry2Aa polyclonal antibodies are used as detection antibodies;

[0019] The four-parameter fitting equation is:

[0020] Cry1Ab: Y = 3.99 / [1+x / 0.10 (-1.45) ]+0.12;

[0021] Cry1Ac: Y = 4.15 / [1+x / 0.28 (-1.44) ]+0.13;

[0022] Cry2Aa: Y = 3.95 / [1+x / 0.24 (-1.55) ]+0.16;

[0023] Cry2Ab: Y = 3.27 / [1+x / 0.36 (-1.43) ]+0.16;

[0024] 5) The OD of the sample to be tested 450 By substituting the value (corresponding to Y) into the corresponding equation for calculation, the residual amounts of the four toxins (corresponding to X values) can be obtained respectively.

[0025] Under the premise that the types of toxins that may be contained in the sample are known, the CmV-ATPase sub A protein provided by the present application can be used as a universal capture protein for ELISA detection to detect the content of four toxins, Cry1Ab, Cry1Ac, Cry2Aa, and Cry2Ab, with high accuracy. In one embodiment of the present application, the detection method is used to add different concentrations of Cry1Ab, Cry1Ac, Cry2Aa, and Cry2Ab toxins to rice leaves and corn leaf samples, respectively. The results show that the average recovery rate is between 83.08% and 118.58%, and the relative standard deviation (RSD) is between 1.41% and 6.37%, indicating the accurate quantitative detection of the four Cry toxins in rice and corn leaves.

[0026] Compared with the prior art, this application firstly uses the rice leaf folder V-ATPase A subunit protein to construct the CmV-ATPase sub A recombinant protein and clone it into the pET26b(+) vector, expresses and purifies it in Escherichia coli BL21(DE3), and also prepares and purifies anti-Cry1Ac or Cry2Aa polyclonal antibodies. On this basis, the rice leaf folder V-ATPase A subunit is used as a universal capture protein for ELISA detection for the first time, and anti-Cry1Ac polyclonal antibodies (PAbs) or anti-Cry2Aa polyclonal antibodies are used as detection antibodies to establish a detection method of binding protein-antibody sandwich ELISA. This universal capture protein can simultaneously detect whether a sample contains these four Cry toxins (currently commercial kits require at least two tests to obtain the detected Cry toxin types); in addition, when the types of Cry toxins that a sample may contain are known, this universal capture protein / detection method can accurately quantitatively detect the four Cry toxins, Cry1Ab, Cry1Ac, Cry2Aa, and Cry2Ab, to overcome the problems of low yield, instability, high cost, and lack of broad spectrum of monoclonal antibodies, and further develop the application of this protein in the field of agricultural product testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1This is a detailed alignment of the amino acid sequences of the V-ATPase A subunits of Chilo suppressalis, Ostrinia furnacalis and Diatraea saccharalis in the family Pidralidae of the order Lepidoptera. The conserved regions of the alignment results are indicated by black dashed boxes and are used to design degenerate primer pairs for amplifying the V-ATPase A subunit of the rice leaf roller. The V-ATPase A subunit sequences used for analysis are from the NCBI database, and the accession numbers of the amino acid sequences are as follows: C. suppressalis (AXF48683.1), O. furnacalis (XP_028155919.1) and D. saccharalis (CAH0755505.1).

[0028] Figure 2 It is an amino acid sequence alignment and structural feature analysis of the A subunit of V-ATPase of rice leaf folder with other Lepidoptera insects. The diagram shows the N-terminal β-folded domain (ATP-synt ab N), the C-terminal domain (ATP-synt ab C), the nucleic acid binding domain (Nucleotide-binding domain) and the conserved regions of Walker A and Walker B. The V-ATPase A subunit amino acid sequences used included Cnaphalocrocismedinalis, C. suppressalis (AXF48683.1), O. furnacalis (XP_028155919.1), Conogethes pinicolalis (ASN77692.1), Pectinophoragossypiella (AKK21114.1), Helicoverpa armigera (AJQ81220.1), D. saccharalis (CAH0755505.1) and Plutella xylostella (CAH0755505.1).

[0029] Figure 3The purification and Western blotting analysis of the expressed CmV-ATPase sub A recombinant protein. A is the purification process of the CmV-ATPase sub A recombinant protein. B is the Western blotting detection of the expressed CmV-ATPase sub A recombinant protein. Lane M: protein molecular weight standard, lane 1: uninduced CmV-ATPase sub A recombinant protein, lane 2: recombinant protein after IPTG induction, lane 3: supernatant of ultrasonic fragmentation after induction, lane 4: inclusion body protein after IPTG induction, lane 5: CmV-ATPase sub A recombinant protein purified by His column, lane 6: pET26b empty load and HRP-labeled anti-His tag antibody as a control, lane 7: HRP-labeled anti-His tag antibody to detect the purified CmV-ATPase sub A recombinant protein.

[0030] Figure 4 This is a ligand blotting analysis of the expressed CmV-ATPase sub A recombinant protein binding to Cry1Ab, Cry1Ac, Cry2Aa, and Cry2Ab toxins. Among them, the lane Marker is a protein molecular weight standard, the lane EP is pET26b empty as a negative control for ligand blotting analysis; the lanes Cry1Ab, Cry1Ac, Cry2Aa, and Cry2Ab are biotin-labeled Cry1Ab, Cry1Ac, Cry2Aa, and Cry2Ab to detect the ligand blotting of CmV-ATPase sub A recombinant protein; and the pET26b empty is used as a negative control for ligand blotting analysis.

[0031] Figure 5 The results of the protein-antibody sandwich method for detecting four Cry toxins are shown in Figure 1. A is the standard curve for the Cry1Ab toxin, B is the standard curve for the Cry1Ac toxin, C is the standard curve for the Cry2Aa toxin, and D is the standard curve for the Cry2Ab toxin. DETAILED DESCRIPTION

[0032] To facilitate understanding of the technical solution of the present invention, the following is further described in conjunction with the accompanying drawings and specific embodiments. The following embodiments are only used for illustration and are not intended to limit the scope of the claims of the present invention. The experimental methods described in the following embodiments are conventional methods unless otherwise specified. The reagents and biological materials are obtained from commercial sources unless otherwise specified.

[0033] Example 1 Amplification of the full-length sequence of the V-ATPase A subunit gene of the rice leaf folder

[0034] 1.1 Amplification of the conserved region sequence of the V-ATPase A subunit gene of the rice leaf folder

[0035] Rice leaf folder was collected from the experimental base of Jiangsu Academy of Agricultural Sciences in Nanjing, Jiangsu Province (118°52'E, 32°01'N). After several generations of indoor rearing, the fourth instar larvae of the same age were selected and RNA was extracted using the Invitrogen Trizol kit and the Invitrogen SuperScript TM II Reverse Transcriptase Kit was used to reverse transcribe the total RNA to obtain the cDNA template. The rice leaf roller belongs to the family Pidralidae in the order Lepidoptera. The amino acid sequences of the V-ATPase A subunit of three insects in the same family (O. furnacalis, D. saccharalis and C. pressalis) were compared using Clustal W ( Figure 1 ). Based on the conserved region characteristics of the A subunits of V-ATPase of the three insects, a pair of degenerate primers (the nucleotide sequence of the upstream primer CmV-ATPase sub A_con-F:NGAYATHAAYGARCTNACNC as shown in SEQ ID No.1, and the nucleotide sequence of the downstream primer CmV-ATPase sub A_con-R:CYARYTGNACDATYTCYGANARR as shown in SEQ ID No.2) were designed to amplify the conserved region of the A subunit of V-ATPase of rice leaf folder; then, the cDNA was used as a template and I-5 TM 2×High Fidelity Master Mix (MCLAB, USA) was used for PCR amplification. In SEQ IN NO.1 and SEQ IN NO.2, the IUBcode degenerate primer nucleotide codes were: R=A / G; Y=C / T; H=A / C / T; N=A / C / G / T.

[0036] The PCR amplification system was as follows: 2× High Fidelity Master Mix 25 μl, 10 μM CmV-ATPase subA_con-F primer and CmV-ATPase sub A_con-R primer 2 μl each, cDNA template 2 μl, and ddH2O to make up to a total reaction system of 50 μl;

[0037] PCR amplification conditions: 94°C for 5 min; 94°C for 30 s, 56°C for 30 s, 72°C for 1 min, for a total of 30 cycles; extension at 72°C for 10 min, hold at 4°C.

[0038] After the PCR reaction was completed, the PCR product was detected by agarose gel electrophoresis as a single product of 1163 bp, and the corresponding PCR product was sent to General Bio (Anhui) Company for sequencing. The sequencing results were compared with the amino acid sequence, and the obtained conservative sequence was highly conservative with three insects of the Lepidoptera family, indicating that the required conservative amino acid region had been obtained. The nucleotide sequence of the conservative region is shown in SEQ IN NO.3, with a size of 1163 bp, and the corresponding 389 amino acid residue sequence is shown in SEQ IN NO.4.

[0039] Its amplification method has been disclosed by patent CN 202110693950.7.

[0040] 1.2 Obtaining the full-length sequence of the rice leaf folder V-ATPase A subunit gene

[0041] Using the RNA obtained in step 1.1 as a template, a RACE cDNA library was constructed using the Smart RACE cDNA amplification kit (Clontech), and the operation steps were carried out according to the instructions of the kit. The products obtained were 5'RACEReady cDNA and 3'RACEReady cDNA, which were used as templates for 5'RACE and 3'RACE reactions, respectively.

[0042] According to the conserved region nucleotide sequence (SEQ ID No.3) obtained in step 1.1, two specific primers (primer CmV-ATPase sub A-5'-R with a nucleotide sequence as shown in SEQ ID No.5, and primer CmV-ATPase sub A-3'-F with a nucleotide sequence as shown in SEQ ID No.6) were designed using primer 5.0 software (primer biological software company, USA). 5'RACE PCR reaction was performed using the universal primer Universal Primer A Mix (UPM, whose nucleotide sequence is shown in SEQ ID No.7) and primer CmV-ATPase sub A-5'-R that come with the Smart RACE kit. 3'RACE PCR amplification was performed using primer UPM and primer CmV-ATPase sub A-3'-F. PCR reaction conditions: 94℃ 30s, 72℃ 3min, 5 cycles; 94℃ 30s, 70℃ 3min, 5 cycles; for 5'RACE PCR, 94℃ 30s, 68℃ 30s, 72℃ 3min, 25 cycles. For 3'RACE PCR, the extension time for each step was 2 minutes, and the other steps were the same as 5'RACE. The obtained PCR products were sent to General Biotech (Anhui) Company for sequencing.

[0043] The obtained 5' and 3' nucleotide sequences were spliced ​​with the conserved region nucleotide sequence of step 1.1 to obtain the full-length cDNA sequence of the V-ATPase A subunit gene of the rice leaf folder. After obtaining the full-length gene cDNA sequence, a pair of primers (CmV-ATPase sub A-full-F with a nucleotide sequence as shown in SEQ IN NO.8, and CmV-ATPase sub A-full-R with a nucleotide sequence as shown in SEQ IN NO.9) were designed to verify the open reading frame (Open Reading Fram, ORF) of the rice leaf folder cadherin. Finally, a 2,143bp full-length sequence CmV-ATPase sub A cDNA (SEQ INNO.10) was obtained.

[0044] Sequence analysis showed that the cloned cDNA sequence contained an ORF of 1,848 bp, a 5' untranslated region (UTR) of 152 bp, and a 3'-UTR of 143 bp, including a 31 bp poly(A) tail. AATAAA, one of the classic polyadenylation signal sequences, was located 18 bp upstream of the poly(A) tail. CmV-ATPase sub A cDNA encodes 616 amino acids (SEQ INNO.11), with a predicted molecular weight of 67.86 kDa and an isoelectric point of 4.86.

[0045] All primer sequences involved in the amplification of the embodiment, except UPM which comes with the kit, were commissioned to be synthesized by General Biotechnology (Anhui) Co., Ltd.

[0046] Example 2 Structural Analysis of the CmV-ATPase sub A Sequence of Rice Leaf Folder

[0047] The amino acid sequence alignment of the V-ATPase A subunit of the rice leaf folder with that of other lepidopteran insects such as Chilo suppressalis, Ostriniafurnacalis, Conogethes pinicolalis, Pectinophora sypiella, Helicoverpa armigera, Diatraea saccharalis and Plutella xylostella showed that the sequence identity reached more than 96.46% ( Figure 2 ), which shows that the V-ATPaseA subunit is quite conserved in Lepidoptera.

[0048] The NCBI CDD (conserved domain database) was used to analyze the conserved regions of the amino acid sequence of the A subunit of V-ATPase of rice leaf folder. The amino acid sequence has three conserved regions commonly found in the A subunit of V-ATPase, namely the ATP-synt ab N domain, i.e. the N-terminal β-folded domain (V21-K84), the ATP-synt ab C domain, i.e. the C-terminal domain (L474-D616), and the intermediate nucleic acid binding domain (G229-K455). There are also two conserved motif sequences, i.e. the Walker A conserved region (GAFGCGKT) located at G249-T256 and the Walker B conserved region (SMMAD) located at S344-D348 ( Figure 2 ). It can be seen that the A subunit of V-ATPase of rice leaf folder has typical sequence characteristics of A subunit of V-ATPase.

[0049] Example 3 Construction, expression and purification of rice leaf folder V-ATPase A subunit protein

[0050] First, the pET-26b(+) prokaryotic expression vector plasmid was constructed, and the CmV-ATPase sub A gene was subcloned into the pET-26b(+) vector (Novagen) using the BamHI and NotI restriction enzyme sites, and the ligation product was transformed into Escherichia coli (E. coli) BL21(DE3) competent cells (Quanshijin Biotechnology Co., Ltd.). The recombinant plasmid and nucleotide sequence of the successful transformation were verified by sequencing (Anhui General Biotechnology Co., Ltd.).

[0051] The pET-26b-CmV-ATPase sub A recombinant protein confirmed by sequencing was cultured in 2×TY medium containing 50 μg / ml kanamycin (Solerbo) at 37°C and 250 rpm until the late logarithmic growth period (OD 600 =0.5~0.6), 1mM isopropyl-β-D-thiogalactopyranoside (IPTG) (Solaibo) was added, and then cultured for another 14h at 30°C and 220rpm to induce the expression of CmV-ATPase sub A. The induced cells were centrifuged at 8,000×g for 15min, the pellet was resuspended in PBS, pH 7.4, and lysed by ultrasonic disruption. The cell lysate was centrifuged at 12,000×g for 30min, and the inclusion bodies in the pellet were dissolved in 8M urea and slowly rotated at 4°C overnight on a horizontal rotator. The dissolved recombinant protein was stained with HiTrap Ni 2+The protein was purified on a column (GE Healthcare) and the protein concentration was determined by Bradford protein assay kit (Solarbio). The purification process of the recombinant protein was analyzed by SDS-PAGE electrophoresis, and the 10% SDS-PAGE precast gel was stained with Coomassie Brilliant Blue and imaged on the Bio-Rad ChemiDoc system (Bio-Rad).

[0052] Western blotting was used to detect the expression of CmV-ATPase sub A recombinant protein. 2.0 μg of purified protein was separated by 10% SDS-PAGE precast gel and then electrotransferred to polyvinylidene difluoride (PVDF) membrane (Millipore). After transfer, the PVDF membrane was blocked in blocking buffer (PBS containing 0.1% Tween-20 and 3% BSA, PBST) at room temperature for 1 h, and then incubated with anti-His tag antibody (1:7500 dilution) (Proteintech, USA) for 1 h. The blot was washed 5 times with washing buffer (PBS containing 0.1% Tween-20, PBST) for 10 minutes each time, and then displayed using chemiluminescent reagent SuperSignal West Pico (ThermoFisher Scientific, USA), and photographed using Bio-Rad ChemiDoc imaging system (Bio-Rad). The purified recombinant protein that has been tested correctly is then desalted by a GE pre-packed desalting column for subsequent ELISA detection method establishment. The above method is a conventional detection method in the art, as disclosed in patents CN 202410141337.8 and CN202110693950.7.

[0053] In this study, the CmV-ATPase sub A recombinant protein was constructed and cloned into the pET26b(+) vector, expressed in Escherichia coli BL21(DE3), and the amount of induced protein was determined by SDS-PAGE under IPTG induction ( Figure 3 As shown in Figure 1A, lanes 1-4). Since the expressed CmV-ATPase sub A recombinant protein carries a His tag, HiTrap Ni 2+ After column purification, the expressed protein was between 70 and 100 kDa ( Figure 3 Lane A in the middle: 5). Western blotting further detected that the pET26b empty vector did not bind to the anti-His tag antibody ( Figure 3Lane B: 6), the size of the purified recombinant protein CmV-ATPase sub A binding to the anti-His tag antibody is above 70 kDa ( Figure 3 Lane B: 7), which is consistent with the SDS-PAGE results. These results indicate that the band above 70 kDa is the expressed recombinant protein CmV-ATPase sub A, whose nucleotide sequence is shown in SEQ ID NO.10 and amino acid sequence is shown in SEQ ID NO.11.

[0054] Example 4 Binding of the A subunit of V-ATPase of rice leaf folder to Cry toxin

[0055] Cry1Ab, Cry1Ac, Cry2Aa and Cry2Ab toxins were purchased from Envirologix, USA, and dissolved in sodium carbonate buffer (CBS, pH 9.6), aliquoted and stored at -80 °C until use. Soluble Cry toxins were biotinylated using the EZ-Link Sulfo-NHS-SS-Biotinylation Kit (Thermo Fisher Scientific) and operated according to the instructions of the kit. The reaction was slowly rotated at room temperature for 2 hours, and then the excess biotin reagent was removed by a desalting column. The biotin labeling efficiency was detected by combining Western blotting with horseradish peroxidase (HRP)-coupled streptavidin (Thermo Fisher Scientific). The concentration of biotinylated Cry toxins was determined using the Bradford method. The above method is disclosed by patent CN 202110693950.7.

[0056] In order to detect the binding of Cry1Ab, Cry1Ac, Cry2Aa and Cry2Ab toxins and CmV-ATPase sub A, the ligand blotting method was used. The specific method is basically the same as the above-mentioned Western blotting detection, except that after blocking with the blocking buffer, the PVDF membrane is incubated with 20nM biotin-labeled Cry toxin at room temperature for 1h. After washing 5 times with PBST, streptavidin-HRP (1:7,500 dilution) was then used to bind to the PVDF membrane for 1h. The final color development method is the same as the aforementioned Western blotting detection. The above method is a conventional method in the art, as disclosed in patents CN 202410141337.8 and CN202110693950.7.

[0057] Test results such as Figure 4As shown in the figure, the results of ligand blotting analysis confirmed the binding of Cry1Ab, Cry1Ac, Cry2Aa and Cry2Ab toxins to the CmV-ATPase sub A recombinant protein. In other words, the V-ATPase A subunit is the binding protein of the four toxins Cry1Ab, Cry1Ac, Cry2Aa and Cry2Ab on the midgut of the rice leaf roller, and based on this, a new detection method can be designed to detect these four toxins.

[0058] Example 5 Establishment of a sandwich detection method based on the A subunit of V-ATPase of rice leaf folder

[0059] Referring to the working principle of the double-antibody sandwich ELISA, the A subunit of the V-ATPase of the rice leaf roller was used as the capture protein, and the anti-Cry1Ac polyclonal antibody or the anti-Cry2Aa polyclonal antibody was used as the detection antibody to establish the sandwich ELISA method. The optimal working concentrations of the capture protein and the detection antibody were screened by the chessboard titration method (disclosed by patent CN 202410141337.8). When the working concentration of the capture protein was 10 μg / ml and the working concentration of the detection antibody was 0.25 μg / ml, the detection effect was best, and it was determined to be the optimal working concentration. Use CBS to dilute the purified and desalted (the method is the same as that in Example 3) CmV-ATPase sub A protein to 10 μg / ml, 100 μl / well was coated on a 96-well ELISA plate, and left to stand overnight at 4°C. Wash 4 times with 300 μl / well PBST. Add 5% (mass percentage) MPBS to 300 μl / well and block at 37°C for 2 hours. Wash 4 times with PBST, dilute the toxins (Cry1Ab, Cry1Ac, Cry2Aa, Cry2Ab) with PBS to 400ng / mL, 200ng / mL, 100ng / mL, 50ng / mL, 25ng / mL, 6.25ng / mL, 3.125ng / mL, respectively, with PBS as the control group, repeat 3 times for each group, add 100μl / well to the ELISA plate, and incubate at 37℃ for 1h. Wash 4 times with PBST, add 100μl / well of polyclonal antibody (1:2000 volume ratio, diluted in PBS), and incubate at 37℃ for 1h. Wash 4 times with PBST, add 100μl / well of horseradish peroxidase (HRP) labeled goat anti-rabbit IgG (1:7500 volume ratio, diluted in PBS) (Proteintech, USA), and incubate at 37℃ for 1h. Wash 4 times with PBST, add 100 μl / well of colorimetric solution, develop at 37°C in the dark for 15 min, add 50 μl / well of ELISA stop solution, and measure OD with an enzyme reader 450 The four-parameter fitting equation was established.

[0060] The above anti-Cry1Ac polyclonal antibody or anti-Cry2Aa polyclonal antibody is prepared by conventional methods in the art. In this embodiment, the preparation method of Cry1Ac and Cry2Aa polyclonal antibodies disclosed in patent CN118108826A is used to obtain the antibodies. When detecting Cry1Ab or Cry1Ac toxin, anti-Cry1Ac rabbit polyclonal antibody is used as the detection antibody; when detecting Cry2Aa or Cry2Ab toxin, anti-Cry2Aa rabbit polyclonal antibody is used as the detection antibody. In specific applications, anti-Cry1Ac polyclonal antibody or anti-Cry2Aa polyclonal antibody prepared by other methods can also be used.

[0061] Results Figure 5 A is the standard curve of sandwich ELISA for determination of Cry1Ab, the equation is: Y = 3.99 / [1 + x / 0.10 (-1.45) ]+0.12, the linear working range was 17.41—376.31 ng / mL, and the detection limit LOD was 17.41 ng / mL.

[0062] Figure 5 B is the standard curve of sandwich ELISA for determination of Cry1Ac, the equation is: Y = 4.15 / [1 + x / 0.28 (-1.44) ]+0.13, the linear working range was 43.16—804.95 ng / mL, and the LOD was 43.16 ng / mL.

[0063] Figure 5 C is the standard curve of sandwich ELISA for determination of Cry2Aa, the equation is: Cry2Aa: Y = 3.95 / [1 + x / 0.24 (-1.55) ]+0.16, the linear working range was 39.70—733.03 ng / mL, and the LOD was 39.70.

[0064] Figure 5 D in the figure is the standard curve of sandwich ELISA for determination of Cry2Ab, the equation is: Cry2Ab: Y = 3.27 / [1 + x / 0.36 (-1.43) ]+0.16, the linear working range is 31.85—1022.20 ng / mL, and the LOD is 31.85 ng / mL.

[0065] The sandwich detection method established in the present application using CmV-ATPase sub A protein as the capture protein and Cry1Ac and Cry2Aa polyclonal antibodies as the detection antibodies has a sensitivity similar to that of the traditional monoclonal antibody ELISA method (as disclosed in patent CN202410141337.8), but can simultaneously detect four toxins, Cry1Ab, Cry1Ac, Cry2Aa, and Cry2Ab, and has a wider application value.

[0066] Example 6 Application of the sandwich detection method on rice and corn leaves

[0067] After establishing the binding protein-antibody sandwich ELISA method in Example 5, this example used this method to add different concentrations of Cry1Ab, Cry1Ac, Cry2Aa and Cry2Ab toxins (all purchased from Envirologix, USA) to rice leaf and corn leaf samples.

[0068] Cut the sample to be tested into 1 cm 2 The fragments of about 1000 μg were immersed in liquid nitrogen and ground into powder with a pestle to obtain sample powder. Weigh 0.1 g of sample powder and dissolve it in 1 ml of plant sample extract (PBS containing 0.1% BSA and 0.05% Tween-20, pH 7.4). Four toxins, Cry1Ab, Cry1Ac, Cry2Aa and Cry2Ab, were added to make their concentrations 100 ng / mL, 50 ng / mL and 5 ng / mL, respectively. No toxins were added to the control group. Place the centrifuge tube on a rotator and rotate at 4°C for 2 hours. Centrifuge at 4°C 10000g for 30 minutes. The supernatant is the test solution.

[0069] The toxin in Example 4 was replaced by the above test solution to perform binding protein-antibody sandwich ELISA detection. The results were calculated based on the OD of the non-sample group. 450 The standard linear equation was established by converting the OD of the sample to be tested into 450 The value (corresponding to Y) is substituted into the equation to calculate the toxin residue (corresponding to X). The ratio of the toxin residue in the sample to be tested to the theoretical toxin concentration is the added recovery rate. The above method is a conventional method in the art, as disclosed in patent CN 202410141337.8.

[0070] The results showed that the average recoveries ranged from 83.08% to 118.58%, and the relative standard deviations (RSDs) ranged from 1.41% to 6.37% (Table 1).

[0071] Table 1 Addition recovery test of four Cry toxins

[0072]

[0073]

[0074] Table 1 shows that the average spike recovery rate is between 70% and 120%, and RSD ≤ 20% meets the International Union of Pure and Applied Chemistry (IUPAC) standard. The spike recovery experiment shows the accurate quantitative detection of the four Cry toxins in the two crop samples, so it can be actually applied to the detection of the four Cry toxins in crop residues, which is efficient and accurate.

Claims

1. A rice leaf folder V-ATPase A subunit protein, the amino acid sequence of which is shown in SEQ ID NO.

11.

2. The gene encoding the A subunit protein of V-ATPase of rice leaf folder as claimed in claim 1, wherein the nucleotide sequence thereof is shown as SEQ ID NO.

10.

3. The use of the rice leaf folder V-ATPase A subunit protein as claimed in claim 1 as a universal capture protein in detecting the content of Cry toxins using the ELISA method; the Cry toxins include at least one of Cry1Ab, Cry1Ac, Cry2Aa, and Cry2Ab.

4. A binding protein-antibody sandwich ELISA detection method for Cry toxin, characterized in that: The specific steps are as follows: 1) Add 0.1g sample and 1ml plant sample extract to a centrifuge tube and rotate at 4℃ for 2h. Centrifuge at 4℃, 10000g for 30min. The supernatant obtained is the plant sample test solution. At the same time, set up a control group without adding sample for later use. The sample is a powder obtained by cutting and grinding fresh leaves of crops; The plant sample extract is prepared as follows: BSA and Tween-20 are added to a PBS solution to a final concentration of 0.1% and 0.05%, respectively; 2) CBS was used to dilute the rice leaf folder V-ATPase A subunit protein with the amino acid sequence shown in SEQ ID NO.11 to a concentration of 10 μg / ml, 100 μl / well was coated on a 96-well ELISA plate, and the plate was incubated at 4°C overnight; 3) The next day, after washing with PBST, 300 μl / well of 5% MPBS was added and the plates were blocked at 37°C for 2 h. After washing with PBST, the plant sample solution was added and the control group was added with PBS. Each group was repeated 3 times, 100 μl / well of the plate was added to the ELISA plate, incubated at 37°C for 1 h, and washed with PBST. 4) Add detection antibody at 100 μl / well and incubate at 37℃ for 1h; wash with PBST, add goat anti-rabbit IgG labeled with horseradish peroxidase at 100 μl / well and incubate at 37℃ for 1h; wash with PBST, add color development solution at 100 μl / well and develop at 37℃ in the dark for 15min; add stop solution at 50 μl / well and measure OD with an enzyme reader 450 The corresponding Cry toxin content in the sample can be obtained by inserting the test results into the corresponding four-parameter fitting equation; When detecting Cry1Ab or Cry1Ac toxins, anti-Cry1Ac polyclonal antibodies are used as detection antibodies; when detecting Cry2Aa or Cry2Ab toxins, anti-Cry2Aa polyclonal antibodies are used as detection antibodies; The four-parameter fitting equation is: Cry1Ab:Y=3.99 / [1+x / 0.10 (-1.45) ]+0.12 (1); Cry1Ac:Y=4.15 / [1+x / 0.28 (-1.44) ]+0.13 (2); Cry2Aa: Y=3.95 / [1+x / 0.24 (-1.55) ]+0.16 (3); Cry2Ab:Y=3.27 / [1+x / 0.36 (-1.43) ]+0.16 (4)。 5. The detection method according to claim 4, characterized in that: The crops are rice or corn.

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