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Anti-spermidine monoclonal antibody hybridoma cell line 4e4 and its monoclonal antibody and application

A hybridoma cell line, monoclonal antibody technology, applied in the direction of antibodies, antibacterial drugs, chemical instruments and methods, etc., to achieve the effects of strong specificity, reduced cytotoxicity, and high sensitivity

Active Publication Date: 2021-06-15
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the prior art, there is no relevant report on a hybridoma cell line that can secrete a monoclonal antibody that specifically recognizes spermidine

Method used

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  • Anti-spermidine monoclonal antibody hybridoma cell line 4e4 and its monoclonal antibody and application
  • Anti-spermidine monoclonal antibody hybridoma cell line 4e4 and its monoclonal antibody and application
  • Anti-spermidine monoclonal antibody hybridoma cell line 4e4 and its monoclonal antibody and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Preparation and identification of hybridoma cell line 4E4 and its secreted monoclonal antibody 4E4

[0046] A hybridoma cell line 4E4 secreting anti-spermidine monoclonal antibody, the hybridoma cell line 4E4 was preserved in China Center for Type Culture Collection (CCTCC) on March 2, 2018, and the preservation number is CCTCC NO: C201841 .

[0047] Monoclonal antibody 4E4 secreted by the hybridoma cell line 4E4 as described above. The monoclonal antibody 4E4 is a high-quality mouse monoclonal antibody (mAb 4E4, IgG1 subtype) prepared from a spermidine-protein (HSA or OVA) coupling compound as an immunogen.

[0048] The preparation and identification of the hybridoma cell line 4E4 and the secreted monoclonal antibody 4E4 thereof are as follows:

[0049] 1. Preparation of HSA-spermidine and OVA-spermidine

[0050] Human serum albumin (HSA) and ovalbumin (OVA) were dissolved in buffer respectively (10mg / mL was dissolved in 0.1M carbonate buffer solution; the...

Embodiment 2

[0062] Example 2 Interaction of monoclonal antibody 4E4 with spermidine and spermine

[0063] 1. Protein separation and western blot analysis

[0064] The interaction of monoclonal antibody 4E4 with spermidine and spermine was analyzed by Western blot. When performing SDS-PAGE electrophoresis, 10 μg of OVA-spermidine, OVA-spermine, OVA, and OVA-glutaraldehyde were used to load the samples. Western blot analysis was performed according to standard protocols using purified Mab 4E4. For Western blot analysis of T3SS gene expression, bacteria cultured overnight were inoculated at 1:200 in fresh LB medium containing NTA, monoclonal antibody 4E4 or control IgG as described above. After 4 h incubation at 37°C, the bacterial culture was cooled on ice for 10 min. For each bacterial culture sample, 10 mL was taken and centrifuged, and the supernatant and bacterial pellet were used to prepare extracellular protein and total cellular protein, respectively. The supernatant was filtered...

Embodiment 3

[0069] Example 3 Effect of Monoclonal Antibody 4E4 on T3SS Gene Expression

[0070] 1. Exogenous monoclonal antibody 4E4 inhibits the expression of T3SS induced by spermidine

[0071] In order to determine the inhibitory effect of monoclonal antibody 4E4 on T3SS gene expression, the T3SS reporter strain PAO1pClacZ (obtained by linking the lacZ gene to the exsCEBA promoter) was grown overnight in LB liquid medium containing NTA 7.5mM, and the next day Bacterial cells were diluted 1:200 with fresh MINIS medium, and IgG, monoclonal antibody 4E4, IgG+spermidine, monoclonal antibody 4E4+spermidine were added respectively, and the final concentration of spermidine was 10 μM. Bacteria were grown with shaking at 37°C for 4 hours, when OD 600 When it reaches about 1.0, start to measure the activity of T3SS beta-galactosidase encoded by lacZ promoter under the control of exsCEBA promoter. The experiment was repeated at least three times, the data shown is the average value of the thre...

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Abstract

The invention belongs to the technical field of cellular immunity, and specifically discloses a hybridoma cell line 4E4 of anti-spermidine monoclonal antibody and its monoclonal antibody and application. The hybridoma cell line 4E4 was deposited in the China Center for Type Culture Collection (CCTCC) on March 2, 2018, and the deposit number is CCTCC NO: C201841. The anti-spermidine monoclonal antibody 4E4 secreted by the hybridoma cell line 4E4 can specifically recognize spermidine and spermine, and block the absorption and downregulation of polyamines T3SS Gene expression to protect A549 cells against Pseudomonas aeruginosa infection. The anti-spermidine monoclonal antibody 4E4 can reduce the level of spermidine in serum, reduce the damage of mouse lungs and significantly improve the survival rate of mice, and can be used to prepare medicines or preparations for treating Pseudomonas aeruginosa infection, and has good Promote application prospects.

Description

technical field [0001] The invention relates to the technical field of cellular immunity, in particular to the anti-spermidine monoclonal antibody hybridoma cell line 4E4 and its monoclonal antibody and applications. Background technique [0002] Pseudomonas aeruginosa (P. aeruginosa) is an important bacterial pathogen that can cause serious infections in cystic fibrosis patients and immunocompromised individuals, and infection with this bacterial pathogen is often associated with high mortality. Pseudomonas aeruginosa can cause epithelial damage and evade the host's innate immune response, mainly because it can directly transport its expressed toxic effector proteins into eukaryotic cells through the type III secretion system (T3SS). Many studies have demonstrated that T3SS is a key virulence determinant of Pseudomonas aeruginosa, which plays an important role in manipulating the acute infection response of eukaryotic host cells. [0003] The T3SS system can be activated b...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/44C12N5/20A61K39/40A61P31/04G01N33/577
CPCA61K2039/505A61P31/04C07K16/44G01N33/577
Inventor 王建和张炼辉
Owner SOUTH CHINA AGRI UNIV
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