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Anti-FtsH2 protein monoclonal antibody and application thereof

A monoclonal antibody, antibody technology, applied in the field of agricultural biology

Inactive Publication Date: 2017-08-29
INST OF CEREAL & OIL CROPS HEBEI ACAD OF AGRI & FORESTRY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Recently, many studies have reported that members of this protein family are related to the tolerance of environmental stress in different plants, such as Arabidopsis, rice, maize and soybean. These stresses include salt stress, drought stress and high temperature stress; but there is no resistance Related reports on FtsH protein monoclonal antibody, the development of related antibodies, and the use of them to screen plant materials with high stress resistance have become a development trend

Method used

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  • Anti-FtsH2 protein monoclonal antibody and application thereof
  • Anti-FtsH2 protein monoclonal antibody and application thereof
  • Anti-FtsH2 protein monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Cloning of embodiment 1 wheat ftsh2 gene and preparation of recombinant FtsH2 antigenic protein

[0028] 1) Take plant samples

[0029] Potted Jimai 418 (Institute of Grain and Oil Crops, Hebei Academy of Agriculture and Forestry Sciences) was subjected to salt stress treatment from heading to flowering stage, about 0.1 g of flag leaf leaves three days after treatment were taken, ground into powder with liquid nitrogen, and 0.5 mL of total plant RNA was added Extraction reagent (Tiangen Biochemical Technology (Beijing) Co., Ltd.), shake until thoroughly mixed. Place at room temperature for 5 minutes, centrifuge at 12,000 rpm for 2 minutes at 4°C, and transfer the supernatant to a new RNase-free centrifuge tube. Add 0.1mL 5M NaCl (prepared with DEPC), mix gently, then add 0.3mL chloroform, and mix evenly by inversion. Centrifuge at 12,000 rpm for 10 min at 4°C, transfer the upper aqueous phase to a new RNase-free centrifuge tube, add isopropanol of an equal volume to...

Embodiment 2

[0036] The establishment of embodiment 2 hybridoma cell lines and clone screening

[0037] 1) Animal immunity

[0038] The recombinant protein expressed and purified in Example 1 was emulsified with complete Freund's adjuvant (Sigma Company), immunized 4-6 weeks old female Balb / c mice (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.), and injected subcutaneously in the abdomen 6 points per mouse, the dose is 60 μg / mouse. Immunization was boosted once every 14 days, and emulsified with Freund's incomplete adjuvant (Sigma Company) at a dose of 30 μg per mouse. 7 days after the third booster immunization, indirect ELISA (wavelength 450nm) was used to detect the multi-antibody titer of the anti-immunogen in the mouse serum. The mouse with the highest titer was immunized by intraperitoneal injection. 50 μg / only to obtain mice that reached the standard of immunity.

[0039] 2) cell fusion

[0040] Aseptically prepare the mouse splenocyte suspension that reaches ...

Embodiment 3

[0043] Example 3 Preparation of Anti-FtsH2 Protein Monoclonal Antibody by Ascites Induction Method and Characterization of Monoclonal Antibody

[0044] 1) Ascites preparation

[0045] The 30749-22 hybridoma cell line was cultured to the logarithmic growth phase, washed with serum-free medium and suspended, and counted to 5×10 5 , 1ml. The suspended two kinds of cells were intraperitoneally injected into mice sensitized with paraffin oil. Ascites collection was started 7 days later. The removed ascites was centrifuged at 4000rpm for 10min at 4°C. Carefully suck out the ascites in the middle and collect in a centrifuge tube, and store at -20°C. Antibody was purified from ascitic fluid by HiTrap rProtein A FF (GE Company) affinity chromatography according to the instructions. The purity was identified by SDS-PAGE gel, and the concentration was determined by Bradford method. Purified antibodies were stored at -20°C.

[0046] 2) Subclass identification

[0047] Dilute coa...

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Abstract

The invention provides an anti-FtsH2 protein monoclonal antibody and an application thereof. The antibody is secreted from a mouse hybridoma cell line 30749-22 and can be used for preparing a kit for screening a plant material with a stress resistance character. According to the anti-FtsH2 protein monoclonal antibody, a recombinant FtsH2 protein and a natural FtsH2 protein expressed in wheat leaves can be specifically combined and identified. The antibody is an IgG1 antibody, and has extremely high specificity and sensitivity when combined with FtsH2 protein; the monoclonal antibody is capable of carrying out qualitative and semi-quantitative analysis on FtsH2 protein in plant leaves through a Western blotting method, and is used for identifying genetic modified plants, thereby screening a plant material with high stress resistance.

Description

technical field [0001] The invention belongs to the field of agricultural biotechnology, in particular to a monoclonal antibody, in particular to an anti-FtsH2 protein monoclonal antibody and application thereof. Background technique [0002] FtsH (filamentation temperature-sensitive H) belongs to the AAA protease family, which is a kind of ATP and Zn encoded by the FtsH gene 2+ Dependent part-time protein, with ATPase activity, proteolytic activity and molecular chaperone activity, this gene is widely distributed in biological genomes. [0003] In eukaryotes, the known FtsHs are located on the chloroplast membrane or mitochondrial membrane. FtsH is responsible for the degradation of unassembled proteins on the bacterial plasma membrane, mitochondrial membrane, and chloroplast membrane, and avoids its possible harmful accumulation by timely degrading free subunits in the form of non-complexes. FtsH not only participates in the normal metabolic regulation process in organis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/40C12N15/70C12N1/21G01N33/577G01N33/573C12R1/91C12R1/19
CPCC07K16/40C07K2317/56C12N15/70G01N33/573G01N33/577G01N2333/95
Inventor 刘茜孙丽静李辉张颖君胡梦芸王培楠李倩影刘富爽
Owner INST OF CEREAL & OIL CROPS HEBEI ACAD OF AGRI & FORESTRY SCI
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