Strawberry vein banding virus antibody, and antigen polypeptide, immunogen and application thereof
A technology of strawberry vein vein virus and antigenic polypeptides, which is applied in the direction of antiviral immunoglobulin, peptides, instruments, etc., can solve the problems of lack of rapid detection reagent development, etc., and achieve the effect of simple and rapid detection and low production cost
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Embodiment 1
[0025] Example 1 Preparation of Antibody to Strawberry Vein Disease Virus
[0026] (1) Antigen immunization
[0027] The sequence of the coat protein of Strawberry Vein Disease Virus was analyzed and studied, two antigenic dominant epitopes were selected, and two polypeptides were synthesized by solid-phase synthesis. The amino acid sequences were as follows:
[0028] Polypeptide A: SSRRERLEQLFEEDC; the first 14 amino acids of polypeptide A are the same as the N-terminal (2-15) amino acids of SVBV coat protein, in order to facilitate cross-linking with the carrier protein, a cysteine C is added at the end of the C-terminal of polypeptide A ;
[0029] Polypeptide B: CESSSDESDDSTDLE; the last 14 amino acids of polypeptide B are the same as the C-terminal (455-468) amino acids of SVBV coat protein. In order to facilitate cross-linking with the carrier protein, a cysteine C is added to the N-terminal of polypeptide B;
[0030] Polypeptide A and polypeptide B were cross-linke...
Embodiment 2
[0040] Example 2 Antibody Determination
[0041] The titer, reaction specificity and sensitivity of the antibody were determined by ELISA using the antigen polypeptide. At the same time, the strawberry plants that were positive by PCR were used as positive controls, the strawberry plants that were negative by PCR were used as negative controls, and 1 μg / mL BSA was used as blank control. Taking the polypeptide B+antibody B as an example, the specific detection method will be described in detail below.
[0043] 1) Take 1g sample (leaves of positive control and negative control plants), add sample extraction coating solution (0.05M carbonate buffer, pH 9.6) 10mL homogenate, filter with double gauze, and coat with coating buffer Dilute the homogenate to a concentration of 200 μL / mL; dilute polypeptide B to a concentration of 1 μg / mL for later use;
[0044] 2) Take 0.1 mL of each antigen dilution solution obtained in step 1), add it to the reaction we...
Embodiment 3
[0071] Example 3 Double Antibody Sandwich ELISA Test
[0072] The SVBV infection in the samples was detected by the double-antibody sandwich ELISA method, and the strawberry plants that were positive by PCR were used as the positive control, the strawberry plants that were negative by PCR were used as the negative control, and 1 μg / mL BSA was used as the blank control. Taking antibody A + enzyme-labeled antibody B as an example, the specific detection method will be described in detail below.
[0073] (1) Preparation of alkaline phosphatase (AP) labeled antibody
[0074] 1) Take Antibody B (2mg / mL) 1mL, add AP 5mg to dissolve;
[0075] 2) Put it into a dialysis bag, dialyze with 0.01mol / L, pH 7.2 PBS at 4°C for 18 hours, and change the solution 3 times;
[0076] 3) Add 20 μL of 2.5% glutaraldehyde, and place at room temperature (about 20°C) for 2 hours; dialyze with 0.01mol / L, pH 7.2 PBS at 4°C overnight, and change the medium 3 times;
[0077] 4) Transfer to 0.05mol / L, pH ...
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