Preparation method and application of rice Cu/Zn-SOD polyclonal antibody

A polyclonal antibody and rice technology, applied in the field of genetic engineering, can solve the problems of low sensitivity and incomplete expression of enzyme activity, and achieve the effects of low preparation cost, strong affinity and high yield

Inactive Publication Date: 2019-05-28
HUNAN AGRICULTURAL UNIV
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Problems solved by technology

[0005] As the research on rice Cu/Zn-SOD is deepening, the change of Cu/Zn-SOD is an extremely sensitive indicator in the process of rice stress resistance. So far, the monitoring of rice Cu/Zn-SOD is mainly Based on the change of enzyme activity, copper zinc-superoxide dismutase, as a special functional protein, its activity is regulated by many factors, and the change of its activity can only indicate the change of the function of the protein. There are a series of complex processes such as modification, assembly, activation, etc., but changes in enzyme activity cannot fully reflect the changes in protein expression in the post-transcriptional regulation of genes; secondly, the

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  • Preparation method and application of rice Cu/Zn-SOD polyclonal antibody
  • Preparation method and application of rice Cu/Zn-SOD polyclonal antibody

Examples

Experimental program
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Embodiment 1

[0037] Example 1: Sequence Analysis and Analysis and Prediction of Candidate Epitopes

[0038] Rice Cu / Zn-SOD has three corresponding genes according to its cell localization.

[0039]The gene ID of Os SODCC1 (LOC4332846) located in the intercellular substance is: 4332846 in GenBank, the nucleotide sequence of the coding region is shown in SEQ ID NO: 5, and the full-length sequence of the encoded amino acid is shown in SEQ ID NO: 6, the gene is located on rice chromosome 3 and has 8 exons.

[0040] Os SODCC2 (LOC4344210) located in the cytoplasm, the gene ID in GenBank is: 4344210, the nucleotide sequence of the coding region is shown in SEQ ID NO: 7, and the full-length amino acid sequence encoded is shown in SEQ ID NO: 8 According to the results, the gene is located on rice chromosome 7 and has 8 exons.

[0041] Os SODCP (LOC4346329) located in chloroplast, the gene ID in GenBank is: 4346329, the nucleotide sequence of the coding region is shown in SEQ ID NO: 2, and the fu...

Embodiment 2

[0045] Embodiment 2: construct SODCP recombinant plasmid, the prepared SODCP recombinant protein is used as antigen

[0046] S1. Intercept the nucleotide sequence corresponding to the 58-211 peptide of SODCP protein from SEQ ID NO: 2, and optimize the codons of the E. coli expression system through software to obtain SEQ ID NO: 3;

[0047] S2. Perform gene synthesis of SEQ ID NO: 3, insert the synthesized base sequence of SEQ ID NO: 3 into a prokaryotic expression vector, construct a recombinant plasmid, sequence the recombinant plasmid, and extract the base sequence of the recombinant protein coding region The sequence was translated into an amino acid sequence, and the obtained fusion protein sequence was SEQ ID NO: 4;

[0048] S3. Introduce the recombinant plasmid in step S2 into Escherichia coli (BL21de3) for induced expression, use the histidine fusion tag on the expression vector, and perform multi-step separation and purification to obtain a SODCP recombinant protein wi...

Embodiment 3

[0049] Embodiment 3: Preparation of polyclonal antibody serum

[0050] For the immunized animals, select healthy New Zealand white rabbits aged about three months and weighing about 2.5 kg, and carry out animal immunization with the antigen prepared in Example 2. The specific steps are as follows:

[0051] Before the first immunization, blood was taken from the ear vein as a negative control; the antigen was mixed with PBS (137mM NaCl, 2.7mM KCl, 10mM Na 2 HPO 4 , 2mM KH 2 PO 4 , pH 7.4) diluted to 1mg·mL -1 (calculated as carrier protein), store at -20°C after aliquoting; take 500 μL of 1 mg·mL -1 Antigen (that is, 0.5mg, add 300μL PBS to dilute again, then add an equal volume of Freund's complete adjuvant (first immunization) or Freund's incomplete adjuvant (second to fourth immunization), and in a vortex instrument Mixing and emulsification; multiple subcutaneous injections on the limbs, underarms and back of the rabbits, the second immunization three weeks after the f...

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Abstract

The invention discloses a preparation method of a rice Cu/Zn-SOD polyclonal antibody and application of the rice Cu/Zn-SOD polyclonal antibody, and belongs to the technical field of genetic engineering. By conducting analytic prediction on epitope of rice chloroplast Cu/Zn-SOD protein, an SODCP protein sequence without transporting signal peptide is used for constructing recombinant plasmid; SODCPrecombinant protein is prepared, and is used as an antigen; through antigen immunoreaction, the rice Cu/Zn-SOD polyclonal antibody is prepared. The prepared polyclonal antibody is high in titer, highin affinity and good in specificity, the polyclonal antibody and the rice Cu/Zn-SOD protein can be subjected to specific binding reaction, moreover, the preparation cost is low, the yield is high, and the polyclonal antibody has feasibility of industrial production. By means of the prepared polyclonal antibody, analytical investigation can be conducted on expression situations of rice in different development stages under different growth conditions, and the polyclonal antibody can be applied in all metabolism mechanism studies, which are relative to rice Cu/Zn-SOD protein action, of rice, sothat detection and verification technical support is provided for studies of functions and action mechanisms of the rice Cu/Zn-SOD protein.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a preparation method and application of a rice Cu / Zn-SOD polyclonal antibody. Background technique [0002] Throughout their life cycle, plants must respond to various biotic and abiotic stresses from the external environment. Environmental stress increases the production of reactive oxygen species in plant cells, resulting in the formation of OH radicals (and their derivatives) that damage proteins, membrane lipids, DNA, and other cellular components. The antioxidant system evolved in plants can scavenge toxic free radicals, protect plant cells from oxygen poisoning, and combat the harmful effects of reactive oxygen species. The activity of antioxidant enzymes is often used to measure ROS-mediated oxidative stress. Superoxide dismutase (Superoxide Dismutase referred to as SOD) is an important antioxidant enzyme in organisms. It can resist and block the ...

Claims

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Application Information

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IPC IPC(8): C07K16/40C07K16/06C12N9/02C12N15/53C12N15/70G01N33/573
Inventor 汪启明熊丹饶力群陈彦超罗彪
Owner HUNAN AGRICULTURAL UNIV
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