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Colon bacillus outer membrane protein monoclonal antibody and preparation method and application thereof

A technology of monoclonal antibody and Escherichia coli, which is applied in the direction of anti-bacterial immunoglobulin, botany equipment and method, biochemical equipment and method, etc., can solve problems such as poor cross-immunity effect, difficulty in vaccine and drug prevention, etc., and achieve Extensive research and commercial use value, good neutralizing activity and protection efficiency, strong sensitivity effect

Active Publication Date: 2012-07-18
兆丰华生物科技(南京)有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are many serotypes of Escherichia coli, and the cross-immunity effect between each serotype is not good, which brings certain difficulties to the prevention of vaccines and drugs

Method used

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  • Colon bacillus outer membrane protein monoclonal antibody and preparation method and application thereof
  • Colon bacillus outer membrane protein monoclonal antibody and preparation method and application thereof
  • Colon bacillus outer membrane protein monoclonal antibody and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Avian Pathogenic Escherichia coli O 78 Preparation of immune antigen of outer membrane protein

[0029] (1) Cloning of outer membrane protein

[0030] Specific primers were designed according to the OMPA gene sequence of Escherichia coli K12W3110 strain (accession number AP009048) in Genebank, the upstream primer introduced the BamHI restriction site, and the downstream primer introduced the XholI restriction site. The upstream primer is: ATTGGATCCGCTCCGAAAGATAACA, and the downstream primer is: TTCTCGAGTTAAGCCTGCGGCTGAGTTA. avian pathogenic Escherichia coli O 78 The genome of the outer membrane protein OMPA was used as template for PCR amplification. The OMPA was connected to pMD18T for sequencing, and the correct OMPA identified by sequencing was double-digested with BamHI and Xhol, cloned into pET-28a(+), transformed into DH5α competent cells, and obtained by kanamycin resistance screening. The positive plasmid pET-28a(+)-OMPA was transformed into Escher...

Embodiment 2

[0035] Example 2 Avian pathogenic Escherichia coli O 78 Preparation of monoclonal antibody against outer membrane protein

[0036] (1) Immunization of BALB / c mice

[0037] Avian pathogenic Escherichia coli O prepared by intraperitoneal injection of 8-week-old BALB / c mice 78 Outer membrane protein Freund's complete adjuvant immunogen 0.5mL / rat; 7d and 14d after immunization, the immunogen in Freund's incomplete adjuvant was boosted with the same method and the same dose.

[0038] (2) Hybridoma cell fusion

[0039] On the third day after the last booster immunization, the splenic lymphocytes of the immunized mice were mixed with myeloma cells SP2 / 0 at a ratio of 4:1, and cell fusion was carried out by using the classic PEG method.

[0040] (3) Screening of hybridoma cells

[0041] After fusion, use HAT as a selective medium, add feeder cells at the same time, distribute in 96-well cell culture plates, and place at 37°C, 5% CO 2 Cultivate in an incubator, observe for 7-10 days...

Embodiment 38A4

[0050] Embodiment 38A4 strain monoclonal antibody specificity test

[0051] avian pathogenic Escherichia coli O 78 , O 1 , O 2 and the standard strain of Salmonella and the screened monoclonal antibody 8A4 strain were used for glass plate agglutination test, and the selected avian pathogenic E. coli O on a small amount of plate was dipped with an inoculation loop 78 , O 1 , O 2 and Salmonella colonies, mixed in normal saline, and then dripped onto the previously marked clean glass slides, and at the same time, drop the monoclonal antibody 8A4 strain to set the avian pathogenic Escherichia coli O 78 , O 1 , O 2 And the control group of Salmonella and saline and monoclonal antibody and saline. The results are shown in Table 1, the results show that the monoclonal antibody has good specificity, only with avian pathogenic Escherichia coli O 78 The reaction was positive, with avian pathogenic Escherichia coli O 1 , O 2 All types were negative, and there was no cross-react...

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Abstract

The invention belongs to the technical field of veterinary biology, and particularly relates to a colon bacillus outer membrane protein monoclonal antibody and a preparation method and an application thereof. The antibody is prepared by taking cloned, expressed and purified taking avian pathogenic escherichia coli 78 (APECO78) outer membrane protein as an antigen immune BALB / c mouse with a hybrid tumor technology. The antibody has high specificity and high sensitivity, and an experimental basis is laid for future rapid and specific detection of colon bacillus infection with an immunology method. Meanwhile, as proved by an experiment, the monoclonal antibody disclosed by the invention has high neutralizing activity and protecting efficiency on colon bacillus infection of the same type, and can be used for preventing colibacillosis.

Description

technical field [0001] The invention relates to an Escherichia coli outer membrane protein monoclonal antibody and a preparation method and application thereof, belonging to the field of biotechnology. Background technique [0002] Avian colibacillosis is caused by avian pathogenic Escherichia coli, and is one of the most serious diseases to the poultry industry. At present, vaccines and antibacterial drugs are mainly used to prevent colibacillosis. However, there are many serotypes of Escherichia coli, and the cross-immunity effect between each serotype is not good, which brings certain difficulties to the prevention of vaccines and drugs. [0003] Monoclonal antibody refers to the antibody against a single antigenic determinant produced by the progeny cells of a B cell differentiation and proliferation. Monoclonal antibody has strong pathogen specificity, does not affect normal tissues and cells, and is relatively safe. It is safe in bacteria and viruses. It plays an impo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/12C07K14/245C12N15/31C12N15/70G01N33/577G01N33/569A61K39/40A61P31/04
Inventor 杜金玲牟巍赵亚荣郭书豪卢会英王勇鹣
Owner 兆丰华生物科技(南京)有限公司
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