Rice rubisco large subunit epitope, its antibody and its application
An antigenic epitope and rice technology, applied in the fields of molecular biology and immunology, can solve the problem that antibody specificity cannot be guaranteed, and achieve the effect of strong affinity, low preparation cost and good specificity
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Embodiment 1
[0023] Example 1: Prediction of Candidate Antigen Epitopes and Artificial Synthetic Antigens
[0024] The ID of the amino acid sequence corresponding to the rice rbcL used in GenBank is P0C510, and the full-length sequence is shown as SEQ ID NO: 3. After analyzing the hydrophilicity, exposure and flexibility of the amino acid sequence, a segment was selected at the N-terminal of the protein The amino acid sequence is KLTYYTPEYETKDTD (as shown in SEQ ID NO: 1, the sequence is located at positions 21-35 of SEQ ID NO: 3 in the sequence table). After Blast comparison with the rice protein database, it was confirmed that this polypeptide can uniquely identify rbcL.
[0025] In order to couple the sequence with the carrier protein, a cysteine residue was added at the C-terminus of the sequence, so the amino acid sequence synthesized was KLTYYTPEYETKDTDC (as shown in SEQIDNO: 2), which was carried out by Baiyixin Biotechnology Co., Ltd. It is synthesized in solid phase and coupled...
Embodiment 2
[0026] Example 2: Serum preparation and purification of polyclonal antibody anti-rbcL
[0027] Animals were immunized with the above-mentioned synthetic antigens, and healthy New Zealand white rabbits aged 3 months and weighing 2 kg were selected for the immunized animals. The specific steps are as follows:
[0028]Firstly, the rabbits were induced before immunization, and 0.5 mL of Freund's complete adjuvant was subcutaneously injected into the limbs, underarms and back to stimulate the local immune response, and immunization was carried out 1 week later. Before the first immunization, blood was taken from the ear vein as a negative control. The antigen (calculated as carrier protein) was washed with PBS buffer (137mMNaCl, 2.7mMKCl, 10mMNa 2 HPO 4 , 2mMKH 2 PO 4 , pH7.4) diluted to 1mg·mL -1 . Take 500 μL antigen diluent, add 300 μL PBS buffer and 800 μL Freund’s complete adjuvant (first immunization) or Freund’s incomplete adjuvant (second to fourth immunization), mix ...
Embodiment 3
[0032] Embodiment 3: the specificity test of polyclonal antibody anti-rbcL
[0033] 0.1 g of rice leaves were thoroughly ground with liquid nitrogen, and 0.5 mL of pre-cooled protein extraction buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 15 mM β-mercaptoethanol, 1% [ w / v] PVP) for 30min. Centrifuge at 15000g for 30min at 4°C, and the supernatant is Rubisco crude enzyme solution. The soluble protein in Rubisco crude enzyme solution was quantified by Bradford method and stored at -80°C.
[0034] Mix equal volumes of Rubisco crude enzyme solution (2.5 μg protein) and loading buffer for SDS-PAGE electrophoresis separation. Transfer the PAGE glue quickly (high electric field strength) to the nitrocellulose membrane through the tank transfer system, and use blocking solution (20mM Tris-HCl, pH7. Block the nitrocellulose membrane for 1 h. Using the polyclonal antibody anti-rbcL prepared in Example 2, diluted with blocking solution containing 5% skimmed milk powder at a ratio of 1:...
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