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Hybridoma cell strain 5A4 secreting anti-CLas membrane protein Cmp1 monoclonal antibody, monoclonal antibody and application

A technology of hybridoma cell line and monoclonal antibody, which is applied in the direction of anti-bacterial immunoglobulin, immunoglobulin, analytical materials, etc., and achieves the effect of good promotion and application prospects

Active Publication Date: 2020-02-07
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are many kinds of outer membrane proteins of citrus huanglongbing. It has not been reported which outer membrane protein is used as an antigen to make a monoclonal antibody to detect citrus huanglongbing. ) Research report on monoclonal antibody of membrane protein Cmp1 (WP_015452405.1)

Method used

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  • Hybridoma cell strain 5A4 secreting anti-CLas membrane protein Cmp1 monoclonal antibody, monoclonal antibody and application
  • Hybridoma cell strain 5A4 secreting anti-CLas membrane protein Cmp1 monoclonal antibody, monoclonal antibody and application
  • Hybridoma cell strain 5A4 secreting anti-CLas membrane protein Cmp1 monoclonal antibody, monoclonal antibody and application

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Example 1 Preparation and Identification of a Hybridoma Cell Line 5A4 Secreting Anti-CLas Membrane Protein Cmp1 Monoclonal Antibody and Its Secreted Monoclonal Antibody mAb 5A4

[0037]A hybridoma cell line 5A4 secreting anti-CLas membrane protein Cmp1 monoclonal antibody, the hybridoma cell line 5A4 was deposited in the China Center for Type Culture Collection (CCTCC) on October 3, 2019, and the preservation number is CCTCC NO: C2019219.

[0038] Monoclonal antibody mAb 5A4 secreted by hybridoma cell line 5A4 as described above. The monoclonal antibody mAb5A4 is a high-quality mouse monoclonal antibody (mAb 5A4, IgG 1 Subtype).

[0039] The amino acid sequence of Cmp1p is FRREKATISLSAHDKEGSKHTMN.

[0040] The preparation and identification of the hybridoma cell line 5A4 and its secreted monoclonal antibody mAb 5A4 are as follows:

[0041] 1. Preparation of BSA-Cmp1p and OVA-Cmp1p

[0042] Bovine serum albumin (BSA) and ovalbumin (OVA) were dissolved in buffer resp...

Embodiment 2

[0049] Example 2 Detection of Cmp1p and Cmp1 by Monoclonal Antibody 5A4

[0050] 1. Western blot analysis

[0051] When performing SDS-PAGE electrophoresis, use 10 μg of equal mass of OVA-Cmp1p chelate and OVA to load the sample, after polyacrylamide gel electrophoresis, transfer to NC membrane at 400mA, 60min, remove the NC membrane with 5% skimmed milk powder Block with PBST for 60 min. After blocking, take an appropriate amount of monoclonal hybridoma culture medium supernatant and incubate the NC membrane for 1 h, then wash with PBST three times, 10 min each time, add HRP-labeled secondary antibody at an appropriate concentration (1:2500-1:50000) Incubate for 1 hour, then wash with PBST three times, each time for 10 minutes, add appropriate amount of luminescent liquid ECL and observe.

[0052] The results showed that mAb 5A4 could specifically recognize Cmp1p ( Figure 4 ), but the OVA cannot.

[0053] 2. Prokaryotic expression protein Cmp1 and western blot analysis

...

Embodiment 3

[0059] Example 3 Detection application of monoclonal antibody mAb 5A4 to CLas protein

[0060] 1. Plant tissue DNA extraction and TaqMAN qPCR

[0061] According to the method of AxyPrepTM Multisource Genomic DNA Miniprep Kit, DNA was extracted from leaf tissues of healthy citrus trees, sub-healthy citrus trees and diseased citrus trees. Weighed 300mg of fresh plant tissues, cut them into small pieces and put them in a mortar, then added liquid nitrogen Grind to powder, grind and add 350 μL PBS, and then extract DNA according to the kit instructions. Take 2 μL of the extract, synthesize primers and probes, and use a Real-Time PCR instrument to measure its quantitative TaqMAN qPCR.

[0062] 2. Western blot of plant tissue

[0063] Take 200-300mg leaves of healthy citrus trees, sub-healthy citrus trees and susceptible citrus trees, add liquid nitrogen to grind, add 1mL lysis buffer (containing 1μL protease inhibitor, 10μL 1M DTT and 10μg 100mM PMSF), Incubate at 4°C for 30 min...

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Abstract

The invention discloses a hybridoma cell strain 5A4 secreting an anti-CLas membrane protein Cmp1 monoclonal antibody, a monoclonal antibody and an application. The hybridoma cell strain 5A4 is collected in China Center for Type Culture Collection (CCTCC) on October 3, 2019, with a collection number of CCTCC NO: C2019219. The anti-CLas membrane protein Cmp1 monoclonal antibody mAb 5A4 secreted by the hybridoma cell strain 5A4 can specifically identify candidatus liberibacter membrane protein Cmp1; and an immunoblotting reaction proves that the monoclonal antibody mAb 5A4 can specifically identify the membrane protein Cmp1 of a pathogenic bacterium CLas in a susceptible plant leaf tissue. A preparation, a kit or a test strip for detecting liberobacter asiaticum can be prepared from the monoclonal antibody mAb 5A4, so that the field detection of the liberobacter asiaticum is achieved quickly and conveniently; and the monoclonal antibody mAb 5A4 has very good popularization and applicationprospects.

Description

technical field [0001] The invention relates to the technical field of cellular immunity, in particular to a hybridoma cell line 5A4 secreting monoclonal antibody against CLas membrane protein Cmp1, the monoclonal antibody and its application. Background technique [0002] Citrus huanglongbing is a devastating disease of citrus, causing huge economic losses worldwide. After the citrus plants are infected with Huanglongbing, the leaves shrink and fall, the seed setting rate decreases, the fruit is small, and the color is yellow and green, which eventually leads to the death of the plants. Moreover, the citrus Huanglongbing disease is highly contagious and can spread to the entire orchard in a short period of time. The pathogenic bacteria of citrus huanglongbing is a Gram-negative bacterium, which has three pathogenic varieties, namely Candidatus Liberibacter asiaticus (CLas), Candidatus Liberibacter africanus (CLaf) and Candidatus Liberibacter americanus (Clam). At present, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/12G01N33/577G01N33/569G01N33/558
CPCC07K16/1203G01N33/577G01N33/56911G01N33/558G01N2469/10
Inventor 王建和张炼辉吴晓妍周筱帆陈群一
Owner SOUTH CHINA AGRI UNIV
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