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Monoclonal antibody capable of secreting anti-streptococcus suis 2 AK (Aspartokinase) protein and application thereof

A monoclonal antibody, Streptococcus suis technology, applied in the field of immunological detection of pathogenic microorganisms, to achieve the effect of high antibody titer

Inactive Publication Date: 2017-12-29
中国人民解放军南京军区军事医学研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, there is no report on the role of aspartokinase in the pathogenic mechanism of S. suis infection

Method used

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  • Monoclonal antibody capable of secreting anti-streptococcus suis 2 AK (Aspartokinase) protein and application thereof
  • Monoclonal antibody capable of secreting anti-streptococcus suis 2 AK (Aspartokinase) protein and application thereof
  • Monoclonal antibody capable of secreting anti-streptococcus suis 2 AK (Aspartokinase) protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Immunization and cell fusion, screening and cloning

[0029] 1. Use 6-week-old female Balb / c mice. Equal volumes of Freund's complete adjuvant and recombinant AK protein ("Prokaryotic expression and antigenicity detection of aspartokinase of Streptococcus suis type 2" Chinese Journal of Etiology, 2017, 12 (4)) were emulsified in equal volumes as immunogens. The amount of antigen immunization was injected subcutaneously in each mouse with a dose of 100 μg; additional immunization was given every 14 days, and emulsified with Freund’s incomplete adjuvant for a total of 3 times, and the amount of immunogen injection remained unchanged; 3 days before cell fusion, intraperitoneal injection For immunization, the immunization dose is still 100 μg / mouse.

[0030] 2. Cell fusion, screening and cloning

[0031] Spleen cells (1×10 7 ) and SP2 / 0 cells (2×10 6 ), then add 50% PEG1459 preheated to 37°C for fusion, the fused cells are suspended in HAT medium, and distrib...

Embodiment 2

[0032] Embodiment 2: Indirect ELASA method detects antibody

[0033] After screening and serial cloning, a hybridoma cell line stably secreting anti-AK monoclonal antibody was obtained, which was named 2E9. Inject the hybridoma cell 2E9 into the peritoneal cavity of the mouse (10 days before intraperitoneal injection of paraffin), 2×10 5 Ascites was collected after the abdominal distension of the mice. Antibodies were detected by indirect ELASA method.

[0034]Add 100 μl of 5 μg / ml recombinant AK protein to each well, coat overnight at 4°C; wash the plate three times with PBST, add 300 μl of 3% BSA blocking solution, and block for 2 hours at 37°C; discard the blocking solution, wash the plate three times with PBST, add 100 μl of hybridoma cells for culture Supernatant, incubated at 37°C for 1 hour; washed three times with PBST, added enzyme-labeled secondary antibody, incubated at 37°C for 1 hour; washed three times with PBST, added TMB for color development.

[0035] The r...

Embodiment 3

[0036] Embodiment 3: Western blot identification anti-AK monoclonal antibody

[0037] The recombinant AK protein was subjected to 10% SDS-PAGE, and then transferred to PVDF membrane after electrophoresis; 5% skimmed milk powder was blocked at 37°C for 1 hour, and washed three times with TBST; 2E9 ascites (1:5000) was incubated, overnight at 4°C, and washed three times with TBST; HRP- Incubate with goat anti-mouse (1:5000), at 37°C for 1 hour, wash three times with TBST; the bands are displayed by DAB color development.

[0038] Western blot results ( image 3 ) shows that there is a reactive band at 50KD between 2E9 ascites and recombinant AK protein. Monoclonal antibody can specifically react with AK protein.

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Abstract

The invention discloses a monoclonal antibody capable of secreting anti-streptococcus suis 2 AK (Aspartokinase) protein and application thereof. A hybridoma cell strain of the monoclonal antibody capable of secreting the anti-streptococcus suis 2 AK protein is named 2E9 and is preserved in CCTCC (China Center for Type Culture Collection), the preservation date is April 25, 2017, and the preservation number is CCTCC NO: C201763. The monoclonal antibody is secreted by the hybridoma cell strain of which the preservation number is CCTCC NO: C201763. The monoclonal antibody disclosed by the invention is very high in valence and has very good specificity.

Description

technical field [0001] The invention belongs to the technical field of immunological detection of pathogenic microorganisms, and relates to a monoclonal antibody secreting anti-type 2 Streptococcus suis AK protein and its application. Background technique [0002] Streptococcus suis (S. suis) is an important zoonotic pathogen, which can cause many diseases in humans or pigs, including meningitis, endocarditis, arthritis, pneumonia and sepsis. The pig breeding industry brings huge economic losses, and also poses a serious threat to human public health security. According to the type of S. suis capsular polysaccharide, it can be divided into 35 serotypes (including 1 / 2 type and 1-34 types), among which S. suis type 1, 1 / 2, 2, 7, Both types 9 and 14 are pathogenic, but S. suis 2 is the most widely distributed and most pathogenic. S. suis 2 is distributed globally, and outbreaks or epidemics of this pathogen have occurred many times in Canada, the United States, Denmark, the N...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/40G01N33/577G01N33/573G01N33/569
CPCC07K16/40
Inventor 潘秀珍高基民孙卫平王长军李先富沈玉洁张艳梅
Owner 中国人民解放军南京军区军事医学研究所
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