Polyclonal antibodies prepared based on specific fragments of the capsid protein of Chrysanthemum B virus.

Abstract

This invention belongs to the field of biology, specifically relating to a method for preparing polyclonal antibodies based on a specific fragment of the capsid protein of Chrysanthemum B virus. This invention discloses the antigenic determinant CVB-CP-B and its acquisition method, as well as the antigen CVB-CP-B obtained from the antigenic determinant, and Ab-CVB-CP-B, which facilitates the preparation of antibodies from antigen CVB-CP-B. This invention also discloses the application of the above-mentioned antibodies in the detection of Chrysanthemum B virus.

Description

[0001] This invention is a divisional application of the following invention: Application date: 2023-02-23; Application number: 202310194485.1; Invention title: "A method for preparing polyclonal antibodies based on specific fragments of chrysanthemum B virus coat protein". Technical Field

[0002] This invention belongs to the field of biology, specifically relating to a method for preparing polyclonal antibodies based on a specific fragment of the outer shell protein of the chrysanthemum B virus. Background Technology

[0003] Chrysanthemum (Chrysanthemum morifolium Ramat.) is a perennial herbaceous plant belonging to the genus Chrysanthemum in the family Asteraceae, and is one of the famous "Eight Delicacies of Zhejiang Province". In production, chrysanthemums are mainly propagated asexually through cuttings and division, leading to the accumulation of plant viruses with each generation, severely reducing chrysanthemum yield and quality. To date, approximately 20 types of viruses and viroids have been reported in chrysanthemums, among which Chrysanthemum Virus B (CVB) is one of the most damaging. First discovered in the Netherlands (Noordam, 1952), this virus has become widespread in chrysanthemum production bases worldwide, seriously hindering the healthy and sustainable development of the chrysanthemum industry (Trolinger et al., 2018; Sastry et al., 2019). Viral infection has a serious impact on chrysanthemum production, and healthy, virus-free seedlings have a significant effect on improving yield and quality. Therefore, there is an urgent need to establish rapid detection technology for viral diseases in Hangzhou white chrysanthemum seedlings to facilitate the large-scale application of high-quality seeds and seedlings.

[0004] Enzyme-linked immunosorbent assay (ELISA) involves coating immunologically active and soluble antigens or antibodies onto a solid carrier and using the principle of specific binding between antigens and antibodies for quantitative or qualitative detection. ELISA has become a popular immunoassay method in many biomedical, clinical, and immunological laboratories. ELISA technology requires specific antigens as immunogens to obtain specific antibodies. However, due to the high requirements of CVB virus particle purification technology, it is not possible to use intact CVB virus particles as immunogens. Therefore, it is particularly important to select highly specific viral capsid protein fragments as immunogens. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for preparing polyclonal antibodies based on specific fragments of the outer shell protein of chrysanthemum B virus. Using this method, highly specific and highly sensitive antibodies can be prepared and obtained, enabling rapid detection of CVB in Hangzhou white chrysanthemum seedlings.

[0006] To address the aforementioned technical problems, the present invention provides an antigenic determinant whose amino acid sequence is any of the following:

[0007] CVB-CP-A:

[0008] IGRPALQPPPNMRGDPTNMYSQVSTDFLWKIKPQRISNNMATSEDMVKIQVALEGLGVPT;

[0009] CVB-CP-B:MHLQQTPPSDWSAMGFHPNVKYAAFD.

[0010] The present invention also provides a method for obtaining the above-mentioned antigenic determinants, comprising the following steps:

[0011] 1) Input the amino acid sequence of the CVB shell protein (UniProt accession number A0A3G5AWF4) into SWISS-MODEL to predict the three-dimensional structure of the CVB shell protein;

[0012] 2) Based on the three-dimensional structure predicted in step 1), select exposed structural region sequences as candidate antigen determinants;

[0013] 3) Perform a two-dimensional structural amino acid hydrophilicity preference analysis on the candidate antigenic determinants obtained in step 2) to verify whether the selected fragments in the three-dimensional structure meet the requirements of the antigenic determinants.

[0014] 4) The antigenic determinants CVB-CP-A and CVB-CP-B were obtained through the above methods for selection and analysis.

[0015] That is, based on the analysis results of the three-dimensional and two-dimensional structures, and combined with the requirements of antigenic determinants such as hydrophilicity, flexibility, and location on the surface of the structure, a comprehensive evaluation is carried out; through the above methods, antigenic determinants CVB-CP-A and CVB-CP-B are obtained.

[0016] As an improvement to the method for obtaining antigenic determinants of the present invention: the hydrophilicity preference analysis of the two-dimensional amino acids in step 2) includes Chou Fasman Beta Turn prediction, Emini surface reachability prediction, Karplus Schulz flexibility prediction, Kolaskar Tongaonkar antigenicity prediction, and Parker hydrophilicity prediction analysis.

[0017] The present invention also provides antigens CVB-CP-A and CVB-CP-B, which are obtained by preparing the above-mentioned antigenic determinants.

[0018] This invention also provides a method for preparing the antigen:

[0019] The preparation method of antigen CVB-CP-A is as follows: the DNA sequence corresponding to CVB-CP-A is optimized according to the codon preference of Escherichia coli, ligated into expression vector pET-41a(+), transformed into E. coli BL21(DE3) strain, and purified fusion protein with a size of approximately 38 kDa is obtained by IPTG induction and Ni-NTA gravity column chromatography, thus obtaining antigen CVB-CP-A;

[0020] Adding a cysteine ​​residue (C) to the end of CVB-CP-B and coupling it with KLH yields the antigen CVB-CP-B.

[0021] The present invention also provides antibodies that are advantageous for the preparation of the above-mentioned antigens: antibody Ab-CVB-CP-A prepared using CVB-CP-A, and antibody Ab-CVB-CP-B prepared using antigen CVB-CP-B.

[0022] This invention also provides a method for preparing antibodies:

[0023] Antiserum was prepared by immunizing New Zealand white rabbits with antigens CVB-CP-A / CVB-CP-B. A total of 4 immunizations were performed. Whole blood was collected 7 days after the last immunization and antiserum was isolated. After affinity purification of the antiserum with Protein A, the concentrated antibodies Ab-CVB-CP-A / Ab-CVB-CP-B were obtained.

[0024] This invention also provides the application of antibodies in the detection of Chrysanthemum B virus.

[0025] As an improvement to the application of this invention: Chrysanthemum (chrysanthemum leaves suspected of being infected with CVB) was used as the test sample, and the Dot-ELISA method and RT-PCR were used to detect CVB in chrysanthemum.

[0026] This invention has the following technical advantages:

[0027] 1. The peptides selected in this invention are specific, thus determining the specificity of the immunogen.

[0028] 2. This invention optimizes DNA codons based on protein sequences, excluding rare codons, thereby improving protein expression levels.

[0029] 3. This invention uses artificial antigens to immunize rabbits, rather than using the method of immunizing rabbits with purified viral particles to prepare antiserum. Therefore, this invention has the advantages of high efficiency and simple preparation of artificial antigens.

[0030] The existing method of immunizing rabbits with purified virus particles requires obtaining the virus strain, inoculating it with an artificial model plant host, and then isolating and purifying the virus before it can be used to immunize rabbits. This method involves many steps, a long cycle, and requires high technical skills.

[0031] 3. The polyclonal antibody serum used in this invention employs a single antigenic determinant as an immunogen, resulting in antibodies with monoclonal resistance specificity, shortened production time, and significantly reduced production costs.

[0032] In summary, the preparation of polyclonal antibodies using the antigen clusters selected in this invention offers advantages such as high sensitivity and low cost, making it suitable for widespread application. Furthermore, the specific antiserum described above can be used to establish immunological detection techniques (such as immunochromatographic gold test strips). Attached Figure Description

[0033] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0034] Figure 1 The three-dimensional structure of CVB CP and the selection antigen determinants (circles);

[0035] Note: A: CVB-CP-A antigenic determinant, B: CVB-CP-B antigenic determinant.

[0036] Figure 2 Results of CVB infection detection in field chrysanthemum samples using Dot-ELISA method established with Ab-CVB-CP-A(A) and Ab-CVB-CP-B(B) and RT-PCR(C);

[0037] Note: 1-19 are 19 chrysanthemum samples suspected of being infected with the virus collected in the field. CK is a healthy, virus-free chrysanthemum as a negative control. M: DNA Marker.

[0038] Figure 3 Sensitivity analysis for the Dot-ELISA method;

[0039] Note: CK is a healthy, non-toxic chrysanthemum used as a negative control, diluted at a ratio of 1:20.

[0040] Figure 4 Sequence alignment diagrams before and after CVB-CP-A optimization. Detailed Implementation

[0041] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0042] Example 1:

[0043] 1) Selection and synthesis of specific fragments of the capsid protein of Chrysanthemum B virus

[0044] The amino acid sequence of the CVB shell protein (UniProt accession number A0A3G5AWF4) was input into SWISS-MODEL for three-dimensional structure prediction. Based on the predicted three-dimensional structure, sequences of exposed structural regions were selected as candidate antigenic determinants. Two-dimensional structural amino acid hydrophilicity preference analyses were performed on the antigenic determinants, including Chou Fasman BetaTurn prediction, Emini surface reachability prediction, Karplus Schulz flexibility prediction, Kolaskar Tongaonkar antigenicity prediction, and Parker hydrophilicity prediction. Based on the results of the three-dimensional and two-dimensional structure analyses, combined with the requirements of antigenic determinants such as hydrophilicity, flexibility, and location on structural surfaces, a comprehensive evaluation was conducted. After selection and analysis using the above methods, the fragment with the sequence IGRPATALQPPPNMRGDPTNMYSQVSTDFLWKIKPQRISNNMATSEDMVKIQVALEGLGVPT was selected as the antigenic determinant, numbered: CVB-CP-A (see [link to relevant documentation]). Figure 1 The antigenic determinant region can be roughly estimated by calculating the surface accessibility, hydrophilicity, and flexibility of each amino acid. The antigenic determinant region should combine all three characteristics. This can be determined using specialized software (such as MacVestor, Protean, GCG, etc.).

[0045] 2) Preparation of Chrysanthemum B Virus Antibody

[0046] The following procedures can be performed with reference to the conventional, publicly published "Experimental Research Methods in Cellular Immunology" (edited by Sun Lifei, p. 124, 2009):

[0047] The DNA sequence corresponding to CVB-CP-A was optimized according to the codon preference of E. coli, ligated into the expression vector pET-41a(+), transformed into E. coli BL21(DE3) strain, and purified into a fusion protein of approximately 38 kDa by IPTG induction and Ni-NTA gravity column chromatography, thus obtaining the antigen CVB-CP-A.

[0048] Antiserum was prepared by immunizing New Zealand white rabbits with antigen CVB-CP-A as the immunogen. A total of four immunizations were performed. Whole blood was collected seven days after the final immunization, and the antiserum was isolated. The antiserum was purified by affinity polymerization with Protein A to obtain concentrated antibody Ab-CVB-CP-A.

[0049] The above DNA sequence (i.e., the original sequence) is as follows:

[0050] ATAGGTAGGCCTGCACTCCAGCCCCCACCCAATATGCGAGGGGATCCAACTAATATGTAC

[0051] AGTCAGGTGTCTACCGATTTCCTGTGGAAGATTAAACCACAGAGGATTTCCAACAACAT

[0052] GGCGACATCTGAGGATATGGTGAAAATACAAGTGGCCCTTGAAGGCCTGGGGGTACCTA

[0053] CC

[0054] The result after optimization is:

[0055] ATTGGTCGTCCGGCACTGCAGCCTCCGCCTAATATGCGTGGTGATCCGACCAATATGTAT

[0056] AGCCAGGTTAGCACCGATTTTCTGTGGAAAATCAAACCGCAGCGCATTAGCAATAATATG

[0057] GCAACCAGCGAAGATATGGTGAAAATTCAGGTTGCACTGGAAGGTCTGGGTGTTCCGAC

[0058] C

[0059] Sequence alignment, such as Figure 4 As shown.

[0060] 3) Analysis of the accuracy of Ab-CVB-CP-A in CVB detection

[0061] Nineteen chrysanthemum samples (leaves suspected of being infected with CVB) collected from the Hangbai chrysanthemum base in Tongxiang, Zhejiang Province, were tested for CVB using a Dot-ELISA method established with Ab-CVB-CP-A and RT-PCR. The results showed that the detection rate of CVB in the samples was 100% for both Dot-ELISA and RT-PCR, with 100% consistency (see...). Figure 2 B).

[0062] The Dot-ELISA method established using Ab-CVB-CP-A for detecting CVB in chrysanthemums is as follows:

[0063] 1) Take the above 19 chrysanthemum samples as samples, grind them rapidly in liquid nitrogen, and serially dilute the tissue leaves with freshly prepared 0.01M PBS from 1:20 (w / v, g / mL) to 1:1280, at 8000 r·min. -1 Centrifuge for 5 minutes, and the supernatant is the crude extract of plant tissue.

[0064] 2) Divide the NC membrane into intervals, take 2-3 μL of crude extract and spot it onto the NC membrane, then dry it in a 37℃ constant temperature oven for 10 min;

[0065] 3) After drying, prepare the blocking solution, immerse the NC membrane in it, and block at room temperature for 1 hour. After blocking, discard the blocking solution, pour in PBST buffer, and wash the membrane thoroughly 3 times, 3 minutes each time.

[0066] 4) Dilute Ab-CVB-CP-A in blocking buffer at a ratio of 1:1000, then place the NC membrane in the prepared primary antibody and incubate at room temperature for 1 hour;

[0067] 5) After the primary antibody incubation is complete, discard the primary antibody, pour in PBST buffer, and wash the membrane thoroughly 3 times, 3 minutes each time;

[0068] 6) Dilute the AP enzyme-labeled goat anti-rabbit IgG secondary antibody in blocking buffer at a ratio of 1:5000. Then, place the NC membrane into the prepared secondary antibody and incubate at room temperature for 1 hour. Wash the membrane 6 times with PBST for 3 minutes each time.

[0069] 7) Color development: Blot the membrane dry with absorbent paper, add NBT and BCIP substrates for color development, observe the results with the naked eye, and take a picture and record the results when the positive control shows purple spots and the negative control does not change color.

[0070] The results are as follows Figure 2 According to A, the infection rate of CVB in all 19 chrysanthemum samples was 100%. The control group, however, showed no spots and was negative.

[0071] The RT-PCR method for detecting CVB in chrysanthemums is a standard technique:

[0072] 1) Before the experiment, disinfect the fume hood with alcohol and pre-cool the centrifuge to 4°C. Cool the sterilized mortar and pestle in liquid nitrogen until the liquid nitrogen is about to evaporate completely. Quickly add 19 samples of chrysanthemum leaves suspected of being infected with CVB, about 100mg each, grind them rapidly 5 times, add 1mL of RNA isolate, and then use a vortex mixer to mix them thoroughly.

[0073] 2) Let the well-mixed sample stand on ice for 5 minutes, then place it in a centrifuge pre-cooled to 4°C and set the parameters to 12000 r·min. -110 minutes;

[0074] 3) After centrifugation, the sample separates into two layers. Transfer the supernatant to a new EP tube, add 1 / 5 of the same volume of chloroform as the supernatant, and mix thoroughly using a vortex mixer. Once the solution is fully mixed, place it on ice and let it stand for 5 minutes. Then, centrifuge at 12,000 rpm. -1 15 minutes;

[0075] 4) After centrifugation, the solution in the centrifuge tube will separate into three layers. Carefully transfer only the upper layer to a new EP tube; otherwise, the quality of the extracted RNA will deteriorate. Then, suspend the tube in the air and add 1 / 5 of the same volume of chloroform as the supernatant. Vortex to mix, place on ice, and let stand for 5 minutes. Then, centrifuge at 4°C with parameters of 12,000 rpm. -1 Centrifuge for 15 minutes; after centrifugation, transfer the supernatant from the centrifuge tube to a new EP tube, add the same volume of pre-cooled isopropanol, gently invert to mix, let stand at room temperature for 10 minutes, then place in a centrifuge at 12,000 rpm. -1 10 minutes;

[0076] 5) After centrifugation, observe the bottom of the tube for a white precipitate, which is RNA. Discard the supernatant, then slowly add 1 mL of freshly prepared 75% ethanol (reconstituted with DEPC water) along the tube wall. Gently invert the centrifuge tube to remove any residual solution, and place it in a centrifuge at 12,000 rpm. -1 Centrifuge for 5 minutes. After centrifugation, repeat the operation and wash the RNA twice with ethanol.

[0077] 6) Slowly discard the ethanol, transfer the EP tube to the sterilized laminar flow hood and dry for several minutes until the alcohol has completely evaporated, then add an appropriate volume of DEPC water to dissolve it.

[0078] 7) After RNA dissolution, immediately place the sample on ice and use NanoDrop to determine the concentration of the extracted RNA. Finally, freeze the RNA to -80°C for subsequent reverse transcription. Add 3 μL of 5×gDNA digester mix, template RNA, and RNase-free ddH2O to an RNase-free centrifuge tube to a total volume of 15 μL. Incubate at 42°C for 2 min. After incubation, add [the remaining ingredients] to the reaction tube from step 1. III. SuperMix plus, mix slowly, and set the reverse transcription program to 25℃ for 5 min, 55℃ for 15 min, and 85℃ for 5 min. The prepared cDNA can be used for subsequent experiments. 9) RT-PCR amplification: According to the reported primers, CVB-U: 5'-ACCGAATTCTTAGTCACAATGCCTCCC-3' and CVB-L: 5'-TCCGAGCTCATAGAGACGGCATACCTT-3', PCR amplification was performed according to the Green Taq Mix instructions from Novizan. The annealing temperature was 52℃. Electrophoresis observation was performed.

[0079] The results are as follows Figure 2 According to C: In the 19 suspected CVB infection samples, the CVB infection rate was 100%; while the control group showed no bands and was negative.

[0080] Therefore, it is demonstrated that the detection rate of CVB in samples by the Dot-ELISA method and RT-PCR established with Ab-CVB-CP-A is 100%, and the detection consistency reaches 100%.

[0081] 4) Dot-ELISA detection of antibody sensitivity to Chrysanthemum B virus

[0082] Chrysanthemum leaves infected with CVB were ground into a homogenate in a mortar and serially diluted with 0.01M PBS (phosphate buffer) from 1:20 to 1:2560 (w / v, g / mL). The sensitivity of the Dot-ELISA method for detecting CVB was analyzed, and the results are as follows: Figure 3 As shown, purple spots were observed at dilutions of 1:20 to 1:1280, while no spots were observed at a dilution of 1:2560; thus proving that the sensitivity of Ab-CVB-CP-A is 1:1280.

[0083] Note: The chrysanthemums infected with CVB mentioned above exhibit symptoms such as thickened leaves, prominent veins, mottling, upward curling of leaf margins, stunted growth, weak growth, and small flower size. Furthermore, the infection was confirmed by testing using the industry-recognized, highly accurate RT-PCR method.

[0084] Example 2:

[0085] 1) Selection and synthesis of specific fragments of the capsid protein of Chrysanthemum B virus

[0086] The amino acid sequence of the CVB shell protein (UniProt accession number A0A3G5AWF4) was input into SWISS-MODEL for three-dimensional structure prediction of the CVB shell protein. Based on the predicted three-dimensional structure, sequences of different spatial locations and lengths were selected as candidate antigenic determinants. Two-dimensional structural amino acid hydrophilicity preference analyses were performed on these antigenic determinants, including ChouFasman Beta Turn prediction, Emini surface reachability prediction, Karplus Schulz flexibility prediction, KolaskarTongaonkar antigenicity prediction, and Parker hydrophilicity prediction. Based on the results of the three-dimensional and two-dimensional structure analyses, combined with the requirements of antigenic determinants such as hydrophilicity, flexibility, and location on structural surfaces, a comprehensive evaluation was conducted. Using the above methods, the sequence MHLQQTPPSDWSAMGFHPNVKYAAFD was selected as the antigenic determinant, numbered: CVB-CP-B (see [link to relevant documentation]). Figure 1 );

[0087] 2) Preparation of Chrysanthemum B Virus Antibody

[0088] The following procedures can be performed with reference to the conventional, publicly published "Experimental Research Methods in Cellular Immunology" (edited by Sun Lifei, p. 124, 2009):

[0089] A cysteine ​​residue (C) was added to the end of CVB-CP-B and conjugated with KLH to obtain the antigen CVB-CP-B. Using CVB-CP-B as an immunogen, antiserum was prepared by immunizing New Zealand white rabbits. A total of four immunizations were performed. Whole blood was collected seven days after the final immunization, and the antiserum was isolated. The antiserum was purified by affinity chromatography with Protein A to obtain the concentrated antibody Ab-CVB-CP-B.

[0090] 3) Analysis of the accuracy of Ab-CVB-CP-B in CVB detection

[0091] Nineteen chrysanthemum samples collected from the Hangbai chrysanthemum base in Tongxiang, Zhejiang (same as in Example 1) were used to detect CVB in chrysanthemums using the Dot-ELISA method established by Ab-CVB-CP-B. The detection method used Ab-CVB-CP-B, and the rest was the same as in Example 1.

[0092] The results of the Dot-ELISA method established using Ab-CVB-CP-B for detecting CVB in chrysanthemums are as follows: Figure 2 According to B, the infection rate of CVB in all 19 chrysanthemum samples was 100%. The control group, however, showed no spots and was negative.

[0093] Therefore, it is demonstrated that the Dot-ELISA method established using Ab-CVB-CP-B achieves a 100% detection rate for CVB and 100% consistency in sample detection (see...). Figure 2 B).

[0094] 4) Dot-ELISA detection of antibody sensitivity to Chrysanthemum B virus

[0095] Chrysanthemum leaves infected with CVB were ground into a homogenate in a mortar and serially diluted with 0.01M PBS from 1:20 to 1:2560 (w / v, g / mL). The sensitivity of the Dot-ELISA method for detecting CVB was analyzed, and the results are as follows: Figure 3 As shown, purple spots were observed at serial dilutions of 1:20 to 1:640, while no spots were observed at serial dilutions of 1:1280 to 1:2560; thus proving that the sensitivity of Ab-CVB-CP-B is 1:640.

[0096] Comparative Example 1

[0097] The optimization of step 2) in Example 1 was cancelled, that is, the DNA sequence corresponding to CVB-CP-A (the original sequence before optimization) was ligated into the expression vector pET-41a(+), and the rest was operated according to the method described in Example 1. The result was that an effective fusion protein could not be obtained, antibody preparation could not be carried out, and thus the Dot-ELISA method based on Ab-CVB-CP-C could not be established for the detection of Chrysanthemum CVB virus.

[0098] Finally, it should be noted that the above examples are merely some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of this invention should be considered within the scope of protection of this invention.

Claims

1. Antigen CVB-CP-B, characterized in that: The amino acid sequence of antigen CVB-CP-B is: MHLQQTPPSDWSAMGFHPNVKYAAFD.

2. The application of the polyclonal antibody prepared using the antigen CVB-CP-B as described in claim 1 in the detection of Chrysanthemum B virus.

3. The application according to claim 2, characterized in that: Chrysanthemum was used as the test sample, and the Dot-ELISA method was used to detect CVB in chrysanthemum.

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