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Preparation method of hybrid tumor of monoclonal antibody inhibiting common bacillus thuringiensis CryI, and application of monoclonal antibody thereof

A monoclonal antibody, hybridoma cell line technology, applied in biochemical equipment and methods, anti-bacterial immunoglobulins, instruments, etc., can solve the problems of unrecognized ability to recognize multiple toxin proteins, inability to recognize Cry1Ah and Cry1C, etc. , to achieve the effect of high use value, high specificity and sensitivity, strong specificity and sensitivity

Active Publication Date: 2012-12-12
BEIJING PROTEIN INNOVATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After sequence comparison, we found that this polypeptide sequence only appears in Cry1Ab and Cry1Ac, and there are sequence differences in Cry1Ah and Cry1C, and the antibody prepared based on this will not be able to recognize Cry1Ah and Cry1C
Other monoclonal antibodies prepared using the complete recombinant Cry1Ab protein as an immunogen were not designed to recognize a variety of toxin proteins in advance, nor were their antigenic epitopes confirmed, so their ability to recognize a variety of toxin proteins cannot be confirmed (Zhang Xiao, Ji Wei, Wang Yun, Wen Shuang, Liu Xianjin, Establishment of an indirect ELISA method for detection of Bacillus thuringiensis (Bt) Cry1Ab toxin antibody antibody, Nanjing Agricultural Journal, 2010, 26(4))

Method used

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  • Preparation method of hybrid tumor of monoclonal antibody inhibiting common bacillus thuringiensis CryI, and application of monoclonal antibody thereof
  • Preparation method of hybrid tumor of monoclonal antibody inhibiting common bacillus thuringiensis CryI, and application of monoclonal antibody thereof
  • Preparation method of hybrid tumor of monoclonal antibody inhibiting common bacillus thuringiensis CryI, and application of monoclonal antibody thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Preparation of immune antigen

[0028] Use software to analyze Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ah, Cry1C and Cry1A protein sequences, and combine their homology ( figure 1 ), antigenicity, hydrophilicity, surface accessibility and secondary structure, select a polypeptide composed of 12 amino acid residues (see SEQ ID No: 1 in the sequence list) that has both immunogenicity and sequence homology The fragments were synthesized by solid-phase synthesis and coupled with the carrier protein by the maleimide method. Specific steps are as follows:

[0029] 10mg / ml sulfo-GMBS (PIERCE company) and 10mg / ml mcKLH (Thermo-Fisher company) were mixed at a ratio of 1:5, placed on a shaker at room temperature and shaken slowly for 30min, and centrifuged at 12000rpm for 5min. Take the supernatant and pass it through sephadex TM G-25Fine (GE Company) separates and collects the activated carrier protein KLH-sulfo-GMBS, adds it dropwise to the 10mg / ml peptide solution at an equal ma...

Embodiment 2

[0030] Example 2 Preparation of recombinant Cry1A protein

[0031] 1. Gene cloning

[0032] After the gene sequence encoding Cry1A (GenBank: ACF32736.1) was artificially synthesized, the 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 T4DNA 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 for the fusion protein gene were sequenced and analyzed by PCR, and the clones with the correct sequence were used to express the recombinant Cry1A protein.

[0033] 2. Protein expression and purification ...

Embodiment 3

[0035] Example 3 Establishment of Hybridoma Cell Line

[0036] One, immunity

[0037] The polypeptide coupled in Example 1 was emulsified with Freund's complete adjuvant (Sigma) to immunize female Balb / c mice (provided by the Academy of Military Medical Sciences) at the age of 4-6 weeks. Each mouse was injected subcutaneously at 6 points in the abdomen , The dosage 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.

[0038] 2. Cell Fusion

[0039] The spleen cell suspension of mice up to the immune standard was aseptically prepared, mixed with mouse myeloma cell...

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Abstract

The invention relates to a broad-spectrum monoclonal antibody aiming at a bacillus thuringiensis (Bt) CryI protein common in gene transformation, and a preparation method thereof. The invention aims at providing a novel monoclonal antibody which can be used for detecting whether Bt protein exists in natural transgenic plants (paddy rice, cotton, corn, and the like). A special antigen for the monoclonal antibody is prepared through the coupling of a polypeptide designed according to an amino acid sequence of the common Bt CryI protein and carrier protein. A subtype of the monoclonal antibody is IgG2a, and an affinity constant reaches 3.84*10<9>. The monoclonal antibody can identify a plurality of proteins in a CryI family, and can be used in detections of various Bt CryI proteins. The invention also provides a hybrid tumor cell line 70647s-9 which secret the monoclonal antibody. The collection number of the cell line is CGMCC No.4856. The cell line can stably secret the monoclonal antibody with high titer.

Description

Technical field [0001] The invention relates to an antibody and its hybridoma cell line which can bind to the foreign gene expression protein in a transgenic plant. Specifically, the present invention provides a monoclonal antibody against the common CryI protein of Bacillus thuringiensis in transgenic plants, which can be used to prepare a kit for detecting the common CryI protein in transgenic plants, and belongs to the field of biological detection. Background technique [0002] Bacillus thuringiensis is abbreviated as Bt, which belongs to Gram-positive Agrobacterium, which is widely distributed in soil, dust, water, desert, vegetation, and insect carcass. During the sporulation process, Bacillus thuringiensis can produce a large number of parasporal crystals, which are composed of crystal proteins with highly specific insecticidal activity. This protein is commonly referred to as Insecticidal Crystal Protein (ICP) or delta-endotoxin. ICP usually exists in the form of protox...

Claims

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Application Information

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IPC IPC(8): C07K16/12G01N33/577C12N5/20
Inventor 尹长城刘国振吴琳刘斯奇郝育杰邓汉超韦汉福潘秦
Owner BEIJING PROTEIN INNOVATION
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