Monoclonal antibody for anti-XA21 protein and use thereof

A monoclonal antibody, XA21 technology, applied in the biological field, can solve the problems of not being able to understand whether XA21 protein is expressed or not and the temporal and spatial characteristics of expression, and achieve high use value, high specificity and sensitivity

Inactive Publication Date: 2015-09-30
INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the sequence of Xa21 is known, the presence or absence of Xa21 can be easily detected by PCR amplification, but this DNA-based amplification cannot understand the expression of the XA21 protein and the temporal and spatial characteristics of the expression, so the preparation of anti-XA21 Protein antibodies, the establishment of immunology-based methods has important practical significance and application value

Method used

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  • Monoclonal antibody for anti-XA21 protein and use thereof
  • Monoclonal antibody for anti-XA21 protein and use thereof
  • Monoclonal antibody for anti-XA21 protein and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1, the acquisition of mouse hybridoma cell line 60632-18

[0063] 1. Preparation of recombinant XA21 kinase domain protein (recombinant XA21K protein)

[0064] The amino acid sequence of the XA21K protein is shown in sequence 1 in the sequence listing, and its coding gene is shown in sequence 2 in the sequence listing.

[0065] 1. Construction of recombinant expression vector

[0066] Cloning XA21K into the expression vector pGTK to construct the expression clone pGST-XA21K, the recombinant expression vector pGST-XA21K and its specific process refer to the literature Liu, G.Z., Pi, L.-Y., Walker, J.C., Ronald, P.C., and Song, W.-Y.Biochemical characterization of the kinase domain of the rice disease resistance receptor-like kinase XA21.J.Biol.Chem.2002.277,20264–20269.

[0067] The recombinant expression vector pGST-XA21K carries the XA21K gene shown in sequence 2 in the sequence listing, and can express the XA21K protein shown in sequence 1 in the sequence li...

Embodiment 2

[0096] Example 2, Preparation and Identification of Monoclonal Antibodies

[0097] 1. Preparation of monoclonal antibodies

[0098] Wash and suspend the hybridoma cell line 60632-18CGMCC No.10416 in the logarithmic growth phase with serum-free medium, and count 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. Use the ELISA method to measure the titer of ascites. For specific operations, refer to "ELISA method for screening positive cells" in step 4 of Example 1, wherein step 6) is: measure the OD value of each well to be tested with a single wavelength of 450nm to compare with the negative control well. (The SP2 / 0 cell culture supernatant is used to replace the control of the sample to be teste...

Embodiment 3

[0140] Example 3, the application of the monoclonal antibody prepared by the present invention in immunological detection

[0141] Detect the application effect of the monoclonal antibody secreted by the hybridoma cell line 60632-18CGMCC No.10416 of the present invention in detecting whether the XA21 protein is expressed in the transgenic rice. The complete amino acid sequence of the XA21 protein is shown in sequence 7 in the sequence listing; the corresponding coding gene is shown in sequence 8 in the sequence listing.

[0142] Tested rice: rice varieties 4021 and CX6221b, which are known to express XA21 protein.

[0143] Rice varieties TP309 and MH86, which are known not to express the XA21 protein.

[0144] 1. Pre-treat the rice for testing to obtain samples to be tested

[0145] Rice samples (leaves) frozen at -70°C were fully ground into a fine powder with a high-throughput tissue grinder (Beijing Dinghaoyuan Technology Co., Ltd., TL2010) or manually, and protein extrac...

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Abstract

The invention discloses a monoclonal antibody for anti-XA21 protein and use thereof. The monoclonal antibody for anti-XA21 protein is secreted by a hybridoma cell line 60632-18, and the registered number of the hybridoma cell line 60632-18 in the China General Microbiological Culture Collection Center of China Committee for Culture Collection of Microorganisms is CGMCC No. 10416. The monoclonal antibody for the anti-XA21 protein is good in specificity and high in titer. The monoclonal antibody for the anti-XA21 protein can be used for developing immunology diagnostic reagents of the anti-XA21 protein, such as enzyme-linked diagnostic reagents, colloidal gold immunostrips, antibody chips and biosensors and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to an anti-XA21 protein monoclonal antibody and its application. Background technique [0002] The work on the rice bacterial blight resistance gene Xa21 can be traced back to 1977, when Indian breeder S.Devadath and others conducted an inoculation test on the long-drug wild rice (Oryza longistaminata) in Mali, Africa, and found that the wild rice was resistant to All bacterial blight (Xoo) races tested. Subsequently, the International Rice Research Institute Khush and others crossed the wild rice with the susceptible indica cultivar IR24, and after 12 years of breeding, they obtained a near-isogenic line of indica rice with IR24 as the genetic background in 1990, named IRBB21. Xoo inoculation identification showed that IRBB21 was resistant to all Xoo races in India and the Philippines. Genetic analysis showed that the resistance was determined by a single genetic locus, and Khush named...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C12N15/13C07K16/16C07K14/415G01N33/68G01N33/577C12R1/91
Inventor 翟文学江光怀刘鹏程尹长城刘国振郝育杰韦汉福韩宇宁史佳楠
Owner INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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