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Anti-spermidine monoclonal antibody hybridoma cell strain 4E4 and monoclonal antibody and application thereof

A hybridoma cell line, monoclonal antibody technology, applied in the direction of antibodies, antibacterial drugs, chemical instruments and methods, etc., to achieve the effect of reducing spermidine levels, reducing serum spermidine content, and improving survival rate

Active Publication Date: 2018-09-14
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are no relevant reports on hybridoma cell lines that can secrete monoclonal antibodies that specifically recognize spermidine in the prior art

Method used

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  • Anti-spermidine monoclonal antibody hybridoma cell strain 4E4 and monoclonal antibody and application thereof
  • Anti-spermidine monoclonal antibody hybridoma cell strain 4E4 and monoclonal antibody and application thereof
  • Anti-spermidine monoclonal antibody hybridoma cell strain 4E4 and monoclonal antibody and application thereof

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 with a spermidine-protein (HSA or OVA) conjugate 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 mass fra...

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 Mab4E4. 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 approximately 1.0 was reached, the activity of T3SS beta-galactosidase encoded by the lacZ promoter under the control of the exsCEBA promoter was determined. The experiment was repeated at least three times, and the data shown is the averag...

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Abstract

The invention belongs to the technical field of cellular immunity, and specifically discloses anti-spermidine monoclonal antibody hybridoma cell strain 4E4 and a monoclonal antibody and application thereof. The hybridoma cell strain 4E4 is deposited at the China Center for Type Culture Collection (CCTCC) on March 2, 2018 under the accession number of CCTCC NO: C201841. The anti-spermidine monoclonal antibody 4E4 secreted by the hybridoma cell strain 4E4 can specifically recognize spermidine and spermine, and protects A549 cells against Pseudomonas aeruginosa infection by blocking of polyamineabsorption and down-regulating of T3SS gene expression. The anti-spermidine monoclonal antibody 4E4 can reduce the level of the spermidine in serum, reduces the damage of the lung of mice, thereby significantly improving the survival rate of the mice, can be used for preparing a drug or preparation for treating the Pseudomonas aeruginosa infection, and has good promotion and application prospects.

Description

technical field [0001] The present invention relates to the technical field of cellular immunity, in particular to 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 severe 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 activate...

Claims

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Application Information

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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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