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Monoclonal antibody obtained by taking Cfr protein as immunogen and application of monoclonal antibody in detection of Cf4 protein

Active Publication Date: 2015-05-20
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

After in-depth research on the mechanism of drug resistance mediated by methylation at position A2503, it was found that the Cfr protein catalyzed the methylation of the 8th position of adenosine, which led to bacterial resistance to amido alcohols, lincosamides, oxazolidines, etc. Drug resistance of ketones, truncated pleurotus, streptavidin A and some sixteen-membered ring macrolides

Method used

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  • Monoclonal antibody obtained by taking Cfr protein as immunogen and application of monoclonal antibody in detection of Cf4 protein
  • Monoclonal antibody obtained by taking Cfr protein as immunogen and application of monoclonal antibody in detection of Cf4 protein
  • Monoclonal antibody obtained by taking Cfr protein as immunogen and application of monoclonal antibody in detection of Cf4 protein

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Embodiment 1, preparation Cfr protein

[0019] 1. Preparation of recombinant plasmids

[0020] 1. Synthesize the double-stranded DNA molecule shown in sequence 2 of the sequence listing.

[0021] 2. Using the double-stranded DNA molecule synthesized in step 2 as a template, a primer pair composed of F1 and R1 is used for PCR amplification to obtain a PCR amplification product.

[0022] F1: 5'-GGAATTC CATATG ATGAATTTTAATAATAAAAC-3';

[0023] R1: 5'-CG GAATTC CTATTGGCTATTTTGATAAT-3'.

[0024] 3. Digest the PCR amplified product in step 2 with restriction endonucleases Nde I and EcoR I, and recover the digested product.

[0025] 4. Digest the vector pET-28a(+) with restriction endonucleases Nde I and EcoR I to recover a vector backbone of about 5369 bp.

[0026] 5. Ligate the digested product of step 3 with the vector backbone of step 4 to obtain the recombinant plasmid pET-28a-cfr.

[0027] 2. Preparation of Cfr protein

[0028] 1. Introduce the recombinant pl...

Embodiment 2

[0035] Embodiment 2, the acquisition of hybridoma cells

[0036] Immune BALB / c mice with the Cfr protein solution prepared in Example 1: mix Freund's complete adjuvant with antigen 400 μl (50-100 μg) in equal volumes during the initial immunization, fully emulsify, and inject subcutaneously in points; one month later The mice were given booster immunizations with the same amount of antigen, injection volume and injection method, but Freund's incomplete adjuvant was used instead; thereafter, booster immunizations were given every 2 weeks, and there were 4 booster immunizations in total; serum was collected 7 days after the last immunization , mouse splenocytes were fused with SP2 / 0 myeloma cells, and were selectively cultured with HAT medium (mouse peritoneal macrophages were used as trophoblasts).

[0037] Use the indirect ELISA method to detect the culture supernatant of hybridoma cells, and the steps are as follows: (1) Coat the ELISA plate with the Cfr protein solution prep...

Embodiment 3

[0040] Example 3, Preparation, Identification and Application of Monoclonal Antibody

[0041] 1. Preparation and purification of monoclonal antibodies

[0042] 1. Incremental cultivation method

[0043] The preparation method of cell culture medium (7.4): add calf serum and sodium bicarbonate to DMEM high glucose medium, the final concentration of calf serum is 20% (mass percentage content), the final concentration of sodium bicarbonate is 0.2 % (mass percentage content).

[0044] The hybridoma cell Cfr-D9 was placed in the cell culture medium and cultured at 37°C for 2 days, and the obtained culture medium was purified by octanoic acid-saturated ammonium sulfate method to obtain a monoclonal antibody solution (stored at -20°C).

[0045] Protein concentration in monoclonal antibody (mg / ml) = 1.45 x OD 280 -0.74×OD 260 .

[0046] The protein concentration in the monoclonal antibody was calculated using the above formula, which was 16.5 mg / ml.

[0047] 2. Ascites preparati...

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Abstract

The invention discloses a monoclonal antibody obtained by taking a Cfr protein as an immunogen and an application of the monoclonal antibody in detection of the Cfr protein. The invention provides a hybridoma cell Cfr-D9 with the preservation number of CGMCC No.8415. The invention also discloses a monoclonal antibody secreted by the hybridoma cell Cfr-D9. The monoclonal antibody provided by the invention can be combined with and identify the Cfr protein and has good specificity and high titer; and Cfr protein mediated pathogenic bacteria have drug resistance to multiple drugs; therefore, the monoclonal antibody provided by the invention has a good popularization value.

Description

technical field [0001] The invention relates to a monoclonal antibody obtained by using Cfr protein as an immunogen and its application in detecting Cfr protein. Background technique [0002] The cfr gene was first discovered by Professor Stefan Schwarz in 2000 in Staphylococcus squirrel isolated from nasal swabs of calves with respiratory infections. Sensitivity experiments showed that the strain was resistant to tetracycline, erythromycin, kanamycin, chloramphenicol and florfenicol. A plasmid pSCFS1 that mediates multidrug resistance was found through plasmid extraction, plasmid transformation experiments and antibiotic susceptibility assays. The size of the plasmid is 17.1-kb. In addition to containing the ermC gene that mediates erythromycin resistance, a gene that mediates resistance to chloramphenicol (64 μg / mL) and florfenicol (32 μg / mL) was also found. The medicinal protein is the Cfr protein (composed of 349 amino acid residues). CFR is the abbreviation of Chlora...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/12G01N33/68G01N33/577G01N33/569
Inventor 吴聪明沈建忠汪洋钟洁陈乐然
Owner CHINA AGRI UNIV
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