Preparation of polyclonal antibody based on specific fragment of coat protein of chrysanthemum B virus

Abstract

The invention belongs to the field of biology, and particularly relates to a method for preparing a polyclonal antibody based on a chrysanthemum B virus coat protein specific fragment. The invention discloses an antigenic determinant CVB-CP-B and an acquisition method of the antigenic determinant CVB-CP-B, and further discloses an antigen CVB-CP-B prepared from the antigenic determinant, and an Ab-CVB-CP-B of an antibody prepared from the antigen CVB-CP-B. The invention also discloses application of the antibody in detection of the chrysanthemum virus B.

Description

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

[0002] The invention belongs to the biological field, and particularly relates to a method for preparing a polyclonal antibody based on a specific fragment of a chrysanthemum B virus coat protein. Background Art

[0003] Chrysanthemum (Chrysanthemum morifolium Ramat.) is a perennial herbaceous plant of the genus Chrysanthemum in the Asteraceae family and one of the famous "Eight Flavors of Zhejiang" in Zhejiang Province. In production, chrysanthemum seedlings are mainly propagated by asexual propagation methods such as cuttings and divisions, which causes plant viruses to accumulate with the increase of reproductive generations, seriously reducing the yield and quality of chrysanthemums. To date, there are about 20 viruses and viroids reported in chrysanthemums, among which chrysanthemum virus B (CVB) is one of the most harmful viruses. The virus was first discovered in the Netherlands (Noordam, 1952) and is widely found in chrysanthemum bases around the world, seriously hindering the healthy and sustainable development of the chrysanthemum industry (Trolinger et al., 2018; Sastry et al., 2019). Virus infection has a serious impact on chrysanthemum production. Healthy virus-free seedlings have a significant effect on improving efficiency and quality. Therefore, it is urgent to establish a rapid detection technology for chrysanthemum seedling virus diseases in production to help the large-scale application of high-quality seeds and seedlings.

[0004] Enzyme linked immunosorbent assay (ELISA) is a quantitative or qualitative test that uses the principle of specific binding between antigen and antibody and encapsulates immunoactive and soluble antigens or antibodies on a solid carrier. ELISA has become a popular immunoassay method in many biomedical, clinical and immunology laboratories. ELISA technology requires specific antigens as immunogens to obtain specific antibodies. However, due to the high requirements for CVB virus particle purification technology, it is not possible to use complete 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 a polyclonal antibody based on a specific fragment of the chrysanthemum B virus coat protein. The method can be used to prepare and obtain highly specific and highly sensitive antibodies, thereby realizing rapid detection of CVB in chrysanthemum seedlings.

[0006] In order to solve the above 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 antigenic determinant, comprising the following steps:

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

[0012] 2) According to the three-dimensional structure predicted in step 1), the exposed structural region sequence is selected as a candidate antigenic determinant;

[0013] 3) Performing a preference analysis of the hydrophilicity of the amino acids in the two-dimensional structure on the candidate antigenic determinant obtained in step 2) to verify whether the selected fragment in the three-dimensional structure meets the requirements of the antigenic determinant;

[0014] 4) After selection and analysis by the above method, antigenic determinant clusters CVB-CP-A and CVB-CP-B were obtained.

[0015] That is, based on the analysis results of the three-dimensional structure and the two-dimensional structure, a comprehensive evaluation was performed in combination with the requirements of the antigenic determinants such as hydrophilicity, flexibility, and being on the surface of the structure; after selection and analysis by the above method, the antigenic determinants CVB-CP-A and CVB-CP-B were obtained.

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

[0017] The present invention also provides antigen CVB-CP-A and antigen CVB-CP-B which are useful for the preparation of the above antigenic determinant clusters.

[0018] The present 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, connected to the expression vector pET-41a(+), transformed into E.coli BL21(DE3) strain, and purified fusion protein of about 38 kDa is obtained by IPTG induction and Ni-NTA gravity column chromatography to obtain antigen CVB-CP-A;

[0020] A cysteine ​​(C) was added to the end of CVB-CP-B and coupled with KLH to obtain the antigen CVB-CP-B.

[0021] The present invention also provides antibodies prepared by the above antigens: antibody Ab-CVB-CP-A prepared by using CVB-CP-A, and antibody Ab-CVB-CP-B prepared by using antigen CVB-CP-B.

[0022] The present invention also provides a method for preparing the antibody:

[0023] Antigen CVB-CP-A / CVB-CP-B was used as the immunogen to immunize New Zealand white rabbits to prepare antiserum. A total of 4 immunizations were performed. Whole blood was collected 7 days after the last immunization to separate the antiserum. After the antiserum was affinity purified with Protein A, the concentrated antibodies Ab-CVB-CP-A / Ab-CVB-CP-B were obtained.

[0024] The present invention also provides application of the antibody in detecting chrysanthemum B virus.

[0025] As an improvement of the application of the present invention: chrysanthemum (chrysanthemum diseased leaves suspected of being infected with CVB) is used as a test sample, and Dot-ELISA method and RT-PCR are used to detect CVB carried by chrysanthemum.

[0026] The present invention has the following technical advantages:

[0027] 1. The peptide segments selected in the present invention are specific, thus determining the specificity of the immunogen.

[0028] 2. The present invention optimizes DNA codons according to protein sequences and excludes rare codons, thereby improving protein expression levels.

[0029] 3. The present invention uses artificial antigens to immunize rabbits instead of using the method of immunizing rabbits with purified virus particles to prepare antiserum. Therefore, the present invention has the advantages of high efficiency and simple preparation of artificial antigens.

[0030] In order to immunize rabbits with existing purified virus particles, the virus strain must first be obtained, then an artificial model plant host is inoculated, and then the virus is isolated and purified. Only the purified virus particles can be used to immunize rabbits. This leads to many steps, a long cycle, and high technical requirements for the experiment.

[0031] 3. Since the polyclonal antiserum used in the present invention uses a single antigenic determinant as an immunogen, the obtained antibody has the specificity of monoclonal resistance, and the production time is shortened, and the production cost is also greatly reduced.

[0032] In summary, the preparation of polyclonal antibodies using the antigen cluster selected by the present invention has high sensitivity and low cost, which is conducive to popularization and application. The above-mentioned specific antiserum can be further used to establish immunological detection technology (such as immune colloidal gold test strips, etc.). BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The specific implementation modes of the present invention are further described in detail below with reference to the accompanying drawings.

[0034] Figure 1 The three-dimensional structure of CVB CP and selected antigenic determinants (circles);

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

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

[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 It is the sensitivity analysis of Dot-ELISA method;

[0039] Note: CK is healthy and non-toxic chrysanthemum, used as negative control, and the dilution multiple is 1:20.

[0040] Figure 4 This is the sequence alignment diagram before and after optimization of CVB-CP-A. DETAILED DESCRIPTION

[0041] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0042] Embodiment 1:

[0043] 1) Selection and synthesis of specific fragments of chrysanthemum B virus coat protein

[0044] The amino acid sequence of CVB coat protein (UniProt accession number A0A3G5AWF4) was input into SWISS-MODEL to predict the three-dimensional structure of CVB coat protein; based on the predicted three-dimensional structure, the exposed structural region sequence was selected as the candidate antigenic determinant; the antigenic determinant was subjected to the hydrophilicity preference analysis of the two-dimensional structure amino acids, namely, Chou Fasman BetaTurn prediction, Emini surface accessibility prediction, Karplus Schulz flexibility prediction, Kolaskar Tongaonkar antigenicity prediction, Parker hydrophilicity prediction and other analyses; based on the analysis results of the three-dimensional structure and the two-dimensional structure, a comprehensive evaluation was performed in combination with the requirements of the antigenic determinant such as hydrophilicity, flexibility and being on the surface of the structure; after the above method, the fragment with the sequence IGRPALQPPPNMRGDPTNMYSQVSTDFLWKIKPQRISNNMATSEDMVKIQVALEGLGVPT was selected and analyzed as the antigenic determinant, numbered: CVB-CP-A (see Figure 1 ); Calculating the surface accessibility, hydrophilicity and flexibility of each amino acid can roughly infer the antigenic determinant region, which should combine the above three characteristics. Professional measurement software (such as MacVestor, Protean, GCG, etc.) can be used for measurement.

[0045] 2) Preparation of Chrysanthemum B Virus Antibodies

[0046] The following can be done with reference to the conventional and publicly published "Cellular Immunology Experimental Research Methods" (Edited by Sun Lifei, P124, 2009):

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

[0048] Antiserum was prepared by immunizing New Zealand white rabbits with antigen CVB-CP-A as the immunogen, and 4 immunizations were performed in total. Whole blood was collected 7 days after the last immunization to separate the antiserum. The antiserum was affinity purified with Protein A to obtain the concentrated antibody Ab-CVB-CP-A.

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

[0050] ATAGGTAGGCCTGCACTCCAGCCCCCACCCAATATGCGAGGGGATCCAACTAATATGTAC

[0051] AGTCAGGTGTCTACCGATTTCCTGTGGAAGATTAAACCACAGAGGATTTCCAACAACAT

[0052] GGCGACATCTGAGGATATGGTGAAAATACAAGTGGCCCTTGAAGGCCTGGGGGTACCTA

[0053] CC

[0054] The optimization result is:

[0055] ATTGGTCGTCCGGCACTGCAGCCTCCGCCTAATATGCGTGGTGATCCGACCAATATGTAT

[0056] AGCCAGGTTAGCACCGATTTTCTGTGGAAAATCAAACCGCAGCGCATTAGCAATAATATG

[0057] GCAACCAGCGAAGATATGGTGAAAATTCAGGTTGCACTGGAAGGTCTGGGTGTTCCGAC

[0058] C

[0059] Sequence alignment Figure 4 shown.

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

[0061] Nineteen chrysanthemum samples (chrysanthemum diseased leaves suspected of CVB infection) collected from the Hangbai Chrysanthemum Base in Tongxiang, Zhejiang Province were tested for CVB in chrysanthemums using the Dot-ELISA method and RT-PCR established by Ab-CVB-CP-A. The results showed that the detection rates of CVB in the samples tested by Dot-ELISA and RT-PCR were both 100%, and the detection consistency reached 100% (see Figure 2 B).

[0062] The Dot-ELISA method established with Ab-CVB-CP-A is as follows:

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

[0064] 2) Divide the area on the NC membrane, take 2-3 μL of the crude extract and spot it on the NC membrane, and dry it in a 37℃ thermostat for 10 min;

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

[0066] 4) Dilute Ab-CVB-CP-A in blocking solution 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 completed, discard the primary antibody, pour in PBST buffer, and wash the membrane three times, each time for 3 minutes;

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

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

[0070] The result is as follows Figure 2 A: The infection rate of CVB in 19 chrysanthemum samples was 100%, while the control had no spots and was negative.

[0071] The RT-PCR method for detecting CVB in chrysanthemum is (conventional technology):

[0072] 1) Before the experiment, disinfect the fume hood with alcohol and precool the centrifuge at 4°C. Cool the sterilized mortar and pestle in liquid nitrogen. When the liquid nitrogen is about to evaporate completely, quickly put in 19 pieces of chrysanthemum diseased leaves suspected of CVB infection as samples, about 100 mg, grind them quickly 5 times, add 1 mL of RNA isolate, and then use a vortex to mix them thoroughly;

[0073] 2) Place the mixed sample on ice for 5 minutes and then place it in a centrifuge precooled to 4°C at 12,000 r / min. -1, 10min;

[0074] 3) After centrifugation, the sample is divided into two layers, the upper layer of the supernatant is pipetted into a new EP tube, and 1 / 5 of the same volume of chloroform as the supernatant is added. Use a vortex instrument to mix it thoroughly. After the solution is fully mixed, place it on ice and let it stand for 5 minutes. Then put it into a centrifuge at a parameter of 12,000 r / min. -1 , 15min;

[0075] 4) After centrifugation, the solution in the centrifuge tube is divided into three layers. Only the upper layer needs to be carefully transferred to a new EP tube, otherwise the quality of the extracted RNA will deteriorate. Then add 1 / 5 of the same volume of chloroform as the supernatant, vortex and shake, mix well, place on ice, let stand for 5 minutes, and then centrifuge at 4°C with a parameter of 12,000 r / min. -1 After centrifugation, transfer the supernatant in the centrifuge tube to a new EP tube, add the same volume of pre-cooled isopropanol, slowly invert it upside down to mix, let it stand at room temperature for 10 minutes, and then put it into the centrifuge at a parameter of 12,000 r / min. -1 , 10min;

[0076] 5) After centrifugation, observe whether there is a white precipitate at the bottom of the tube. The white precipitate is RNA. Pour out the supernatant, then slowly add 1 mL of freshly prepared 75% ethanol prepared with DEPC water along the tube wall. Gently invert the centrifuge tube to remove the remaining solution, and place it in a centrifuge at a parameter of 12,000 r / min. -1 , 5min, after centrifugation, repeat this operation and wash the RNA twice with ethanol;

[0077] 6) Slowly discard the ethanol, transfer the EP tube to a sterilized clean bench and dry it for several minutes until the alcohol evaporates completely, then add an appropriate amount of DEPC water to dissolve it;

[0078] 7) After RNA is dissolved, put it in ice immediately, use NanoDrop to measure the concentration of extracted RNA, and finally freeze RNA to -80℃ for reverse transcription. Add 3μL of 5×gDNA digester Mix, template RNA, and RNase free dd H2O to a total volume of 15μL in an RNase free centrifuge tube, incubate on a 42℃ heating table for 2 minutes, and then add ⅢSuperMix plus, slowly mix, reverse transcription program set to 25℃5min, 55℃15min, 85℃5min. The prepared cDNA can be used for the next experiment. 9) RT-PCR amplification, according to the reported primers, CVB-U: 5'-ACCGAATTCTTAGTCACAATGCCTCCC-3' and CVB-L: 5'-TCCGAGCTCATAGAGACGGCATACCTT-3', refer to the Green Taq Mix instructions of Novozymes for PCR amplification, annealing temperature is 52℃. Electrophoresis observation.

[0079] The result is as follows Figure 2 C: In the 19 suspected CVB infected samples, the CVB infection rate was 100%; while the control had no bands, which was negative;

[0080] Therefore, it was proved that the detection rates of CVB in samples by Dot-ELISA method and RT-PCR established with Ab-CVB-CP-A were both 100%, and the detection consistency reached 100%.

[0081] 4) Dot-ELISA to detect the sensitivity of antibodies to chrysanthemum B virus

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

[0083] Note: The chrysanthemums infected with CVB mentioned above showed symptoms such as thickened leaves, clear veins, mottled leaves, upward curling of leaf edges, dwarfed plants, weak growth, and small flowers. The RT-PCR method, which is recognized by the industry for its high detection accuracy, was used to confirm that they were indeed infected with CVB.

[0084] Embodiment 2:

[0085] 1) Selection and synthesis of specific fragments of chrysanthemum B virus coat protein

[0086] The amino acid sequence of CVB coat protein (UniProt accession number A0A3G5AWF4) was input into SWISS-MODEL to predict the three-dimensional structure of CVB coat protein; based on the predicted three-dimensional structure, sequences of different spatial positions and lengths were selected as candidate antigenic determinants; the antigenic determinants were subjected to a preference analysis of the hydrophilicity of the amino acids in the two-dimensional structure, i.e., ChouFasman Beta Turn prediction, Emini surface accessibility prediction, Karplus Schulz flexibility prediction, KolaskarTongaonkar antigenicity prediction, Parker hydrophilicity prediction, etc., and a comprehensive evaluation was performed based on the analysis results of the three-dimensional and two-dimensional structures, combined with the requirements of the antigenic determinant such as hydrophilicity, flexibility, and being on the surface of the structure; different spatial positions were selected by the above method, and a fragment with the sequence MHLQQTPPSDWSAMGFHPNVKYAAFD was selected as the antigenic determinant, numbered: CVB-CP-B (see Figure 1 );

[0087] 2) Preparation of Chrysanthemum B Virus Antibodies

[0088] The following can be done with reference to the conventional and publicly published "Cellular Immunology Experimental Research Methods" (Edited by Sun Lifei, P124, 2009):

[0089] A cysteine ​​(C) was added to the end of CVB-CP-B and coupled with KLH to obtain the antigen CVB-CP-B. The antigen CVB-CP-B was used as an immunogen to immunize New Zealand white rabbits to prepare antiserum. A total of 4 immunizations were performed. Whole blood was collected 7 days after the last immunization to separate the antiserum. The antiserum was affinity purified with Protein A to obtain the concentrated antibody Ab-CVB-CP-B.

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

[0091] 19 chrysanthemum samples collected from the Hangbai Chrysanthemum Base in Tongxiang, Zhejiang (same as in Example 1) were used to detect CVB in chrysanthemum using the Dot-ELISA method established with Ab-CVB-CP-B. Ab-CVB-CP-B was used in the detection method, and the rest was the same as in Example 1.

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

[0093] Therefore, it is proved that the detection rate of CVB in the Dot-ELISA method established by Ab-CVB-CP-B is 100%, and the detection consistency reaches 100% (see Figure 2 B).

[0094] 4) Dot-ELISA to detect the sensitivity of antibodies to Chrysanthemum B virus

[0095] Chrysanthemum diseased leaves infected with CVB were ground in a mortar and homogenized, and diluted with 0.01M PBS from 1:20 to 1:2560 (w / v, g / mL). The sensitivity of Dot-ELISA method for detecting CVB was analyzed. The results are shown in the figure. Figure 3 As shown: purple spots appeared at 1:20 to 1:640 dilution ratios, while no spots appeared at 1:1280 to 1:2560 dilution ratios; thus, the sensitivity of Ab-CVB-CP-B was 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 connected to the expression vector pET-41a(+), and the rest of the operations were performed according to the method described in Example 1. The results were as follows: no effective fusion protein could be obtained, no antibody preparation could be performed, and thus no Dot-ELISA method based on Ab-CVB-CP-C could be established for chrysanthemum CVB virus detection.

[0098] Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

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

2. Use of the antibody prepared using the antigen CVB-CP-B as claimed in claim 1 in detecting chrysanthemum B virus.

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

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