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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 74000), but these products are mainly based on the test strip method, and are expensive and difficult to promote and use. No similar product production and sales

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

Embodiment 1

[0023] Example 1 Preparation of recombinant Cry1A protein

[0024] 1. Gene cloning

[0025] According to the published gene coding sequence of CP4 EPSPS (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, and NcoI and BamHI restriction sites were added to the 5' and 3' ends of the fusion protein gene by PCR. The PCR product was 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, recovered by electrophoresis again, and ligated with T4 DNA ligase. The ligation product was transformed into Escherichia coli competent cell BL21, the clones on the plate were picked and inoculated, the plasmid DNA was extracted and identified by PCR. PCR showed that the fusion protein gene positive clones were seque...

Embodiment 2

[0028] The establishment of embodiment 2 hybridoma cell lines

[0029] 1. Immunity

[0030] The cross-linked polypeptide in Example 1 was emulsified with complete Freund's adjuvant (Sigma Company), immunized 4-6 weeks old female Balb / c mice (provided by the Academy of Military Medical Sciences), and injected subcutaneously into each mouse at 6 o'clock in the abdomen. , the dose is 60μg / only. Immunization was boosted every 14 days, and the antigen was 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 polyantibody titer of the anti-immunogen in the mouse serum. 50μg / only.

[0031] 2. Cell Fusion

[0032] The mouse splenocyte suspension meeting the immune standard was aseptically prepared, mixed with mouse myeloma cell sp2 / 0 (ATCC) at a ratio of 5:1, and centrifuged at 1500 rpm for 5 min. After the supernatant was discarded, the centr...

Embodiment 3

[0037] Example 3 Preparation of Monoclonal Antibody by Ascites Induction Method

[0038] 1. Ascites preparation

[0039] Cells in the logarithmic growth phase were washed with serum-free medium and suspended, counted to 5×10 5 , 1ml. The suspended cells were injected intraperitoneally into mice previously 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 4°C or -20°C.

[0040] 2. Purification of monoclonal antibodies

[0041] 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.

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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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