Preparation method, coding sequences and use of anti-CP4 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPS) monoclonal antibody

A monoclonal antibody and sequence technology, applied in anti-enzyme immunoglobulins, measuring devices, instruments, etc., can solve the problems of difficult to popularize and use, high price, etc., and achieve the effect of high use value, high specificity and sensitivity

Active Publication Date: 2014-06-11
BEIJING PROTEIN INNOVATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, a variety of products for detecting CP4 EPSPS have come out abroad (Agdia’s test strip product STX 74000 and sandwich ELISA product PSP 740

Method used

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  • Preparation method, coding sequences and use of anti-CP4 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPS) monoclonal antibody
  • Preparation method, coding sequences and use of anti-CP4 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPS) monoclonal antibody
  • Preparation method, coding sequences and use of anti-CP4 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPS) monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0023] Example 1 Preparation of recombinant Cry1A protein

[0024] 1. Gene cloning

[0025] According to the published CP4 EPSPS gene coding sequence (Genbank Accession No.AF464188), design specific upstream primer (SEQ ID No. 3) and downstream primer (SEQ ID No. 4) from Roundup Ready (RR) transgenic soybean DNA standard The CP4 EPSPS gene was amplified in the fusion protein gene, and NcoI and BamHI restriction sites were added to the 5'and 3'ends of the fusion protein gene by PCR. The PCR products were separated by agarose gel electrophoresis and recovered. The recovered fusion protein gene and the plasmid vector pET-BPI used for expression were digested with NcoI and BamHI respectively, and recovered by electrophoresis again, and ligated with T4 DNA ligase. The ligation product was transformed into E. coli competent cells BL21, the clones on the plate were picked and inoculated, the plasmid DNA was extracted, and PCR identification was performed. The clones that were positive f...

Example Embodiment

[0028] Example 2 Establishment of Hybridoma Cell Line

[0029] One, immunity

[0030] The cross-linked polypeptide in Example 1 was emulsified with Freund's complete adjuvant (Sigma) to immunize female Balb / c mice aged 4-6 weeks (provided by the Academy of Military Medical Sciences), and injected subcutaneously at 6 points per mouse in the abdomen , The dose is 60μg / only. The booster immunization was carried out every 14 days, and the antigen was emulsified with Freund's incomplete adjuvant (Sigma) at a dose of 30 μg per mouse. Seven days after the third booster immunization, the indirect ELISA (wavelength 450nm) was used to detect the polyclonal antibody titer of the anti-immunogen in the mouse serum. The mouse with the highest titer was injected into the tail vein and the antigen was mixed with normal saline. It is 50μg / only.

[0031] 2. Cell Fusion

[0032] The spleen cell suspension of mice up to the immune standard was aseptically prepared, mixed with mouse myeloma cells sp2 / 0...

Example Embodiment

[0037] Example 3 Preparation of monoclonal antibody by ascites induction method

[0038] 1. Ascites preparation

[0039] The logarithmic growth phase cells are washed and suspended in serum-free medium, and counted to 5×10 5 , 1ml. The suspended cells were injected intraperitoneally into mice sensitized with paraffin oil. After 7 days, ascites was collected. The removed ascites was centrifuged at 4000 rpm for 10 min at 4°C. Carefully aspirate the middle ascites, collect it in a centrifuge tube, and store at 4°C or -20°C.

[0040] 2. Purification of monoclonal antibodies

[0041] The antibody was purified from ascites fluid by HiTrap rProtein A FF (GE) affinity chromatography according to the instructions. SDS-PAGE gel identified the purity, and Bradford method was used to determine the concentration. The purified antibody is stored at -20°C.

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Abstract

The invention relates to an anti-glyphosate resistance protein 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPS) monoclonal antibody in commonly seen in transgenic crops and a preparation method thereof. The anti-CP4 EPSPS monoclonal antibody is a novel monoclonal antibody for detecting if a natural transgenic plant (such as soybean, corn, cotton or paddy rice) contains a CP4 EPSPS protein. A special-purpose antigen of the anti-CP4 EPSPS monoclonal antibody is obtained by recombination expression of a published CP4 EPSPS gene sequence in escherichia coli. The subtype of the anti-CP4 EPSPS monoclonal antibody is IgG1 and has affinity constant of 2.35*10<8>. The anti-CP4 EPSPS monoclonal antibody can identify a CP4 EPSPS protein in the transgenic plant and can be used for detection of a plant with the CP4 EPSPS gene. The invention also provides light chain and heavy chain variable-range coding sequences of the anti-CP4 EPSPS monoclonal antibody.

Description

technical field [0001] The invention relates to an antibody that can bind to proteins expressed by foreign genes in transgenic plants. Specifically, the present invention provides a common glyphosate resistance protein-5-enolpyruvyl-shikimate-3-phosphate synthase (5-enolpyruvyl-shikimate-3-phosphate synthase, EPSPS) monoclonal antibody can be used to prepare a test kit for detecting common CP4 EPSPS protein in transgenic plants, and belongs to the field of biological detection. Background technique [0002] Glyphosate (Glyphosate) is a widely used herbicide with the characteristics of high efficiency, low toxicity, broad spectrum and easy decomposition. my country is a big country in the production and use of glyphosate. Glyphosate-resistant transgenic plants are an important object of transgenic research. Currently, glyphosate-resistant transgenic plants are the most widely planted transgenic varieties in the world, and glyphosate N-acetyltransferase is the most used glyph...

Claims

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Application Information

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IPC IPC(8): C07K16/40G01N33/577
Inventor 尹长城刘国振吴琳郝育杰韦汉福潘秦韩宇宁奚文辉刘斯奇
Owner BEIJING PROTEIN INNOVATION
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