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Rice Rubisco large-subunit antigen epitope, large-subunit antibody and applications of antibody

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, good specificity and high yield

Inactive Publication Date: 2014-07-30
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there have been reports on the preparation of rbcL antibody synthesis at home and abroad, which are divided into three categories according to the source of the antigen: one is to extract the crude Rubisco enzyme solution from the plant, and use it as the antigen after ammonium sulfate fractional precipitation purification, which is the most common; the other is to use the purified The recombinant protein is used as an antigen to immunize animals; the third is to prepare the antigen through artificially synthesized polypeptides, and obtain antibodies by immunizing animals, which is rarely reported.
Although the first two methods purify the antigenic protein, the specificity of the prepared antibody cannot be guaranteed due to the existence of multiple potential antigenic determinants

Method used

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  • Rice Rubisco large-subunit antigen epitope, large-subunit antibody and applications of antibody
  • Rice Rubisco large-subunit antigen epitope, large-subunit antibody and applications of antibody
  • Rice Rubisco large-subunit antigen epitope, large-subunit antibody and applications of antibody

Examples

Experimental program
Comparison scheme
Effect test

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 in SEQ ID NO: 3. After analyzing the hydrophilicity, exposure and flexibility of the amino acid sequence, it was selected at the N-terminal of the protein An 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 this sequence with a carrier protein, a cysteine ​​residue is added at the C-terminal of this sequence, so the amino acid sequence synthesized is KLTYYTPEYETKDTDC (as shown in SEQ ID NO: 2), provided by Baiyixin Biotechnology Co., Ltd. The company conducts solid-phase synthesis, and couples succinimi...

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. Antigen (calculated as carrier protein) was washed with PBS buffer (137mM NaCl, 2.7mM KCl, 10mM NaCl 2 HPO 4 , 2mM KH 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 (2-4 immunization), mix and emulsif...

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 30 minutes. 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. The PAGE glue was quickly transferred (high electric field strength) to the nitrocellulose membrane through the tank transfer system, and after the transfer was completed, the blocking solution (20mM Tris-HCl, pH7. Block the nitrocellulose membrane at 37°C for 1h. Using the polyclonal antibody anti-rbcL prepared in Example 2, diluted with blocking soluti...

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Abstract

The invention rice photosynthesis key enzyme-ribulose-1,5-bisphosphate carboxylase / oxygenase (Rubisco) large-subunit antigen epitope, a polyclonal antibody resisting rice Rubisco large-subunit, and a preparation method and applications of the antibody. The amino acid sequence of the rice Rubisco large-subunit specific antigen epitope is as shown in SEQ ID NO:1. The preparation method comprises the following steps: synthesizing a polypeptide; coupling the polypeptide with carrier protein; preparing antiserum from an immune animal; and performing affinity chromatography for purification, thereby obtaining the polyclonal antibody resisting rice Rubisco large-subunit. The polyclonal antibody is high in titer, remarkable in affinity, excellent in specificity, low in preparation cost and high in yield, and can generate specific binding reaction with rice Rubisco large-subunit; an important tool is supplied for the fundamental research on the rice Rubisco large-subunit as well as the research on the relevant physiological functions of the rice Rubisco large-subunit.

Description

technical field [0001] The invention belongs to the field of molecular biology and immunology, and specifically relates to a large subunit epitope of a key enzyme of rice photosynthesis—ribulose-1,5-bisphosphate carboxylase / oxygenase (Rubisco), Its antibody and application. technical background [0002] Ribulose-1,5-bisphosphate carboxylase / oxygenase (Rubisco; EC4.1.1.39) is the most abundant protein in plant leaves, accounting for 12-35% of protein and 50% of soluble protein. Rubisco is a key enzyme of photosynthetic carbon assimilation with dual functions, on the one hand as CO in the photosynthetic Calvin cycle 2 Fixed key enzyme involved in catalyzing CO 2 React with ribulose-1,5-bisphosphate (RuBP) to form 2 molecules of 3-phosphoglycerate (3-PGA); at the same time, it participates in the photorespiration process of C3 plants, catalytic CO 2 React with RuBP to generate 1 molecule of 3-PGA and 1 molecule of 2-phosphoglycolic acid (2-phosphoglycolate, 2-PG). Therefo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C07K16/40C07K16/06G01N33/573
CPCC07K16/065C12N9/88C12Y401/01039G01N33/573G01N2333/988
Inventor 唐红玲何莹李海霞曾汉来
Owner HUAZHONG AGRI UNIV
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