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

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Problems solved by technology

However, there are no relevant reports on hybridoma cell lines that can secret

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

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

[0046] A hybridoma cell line 4E4 that secretes an anti-spermidine monoclonal antibody. The hybridoma cell line 4E4 was deposited at the China Center for Type Culture Collection (CCTCC) on March 2, 2018, and the deposit number is CCTCC NO: C201841 .

[0047] The 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 by using spermidine-protein (HSA or OVA) coupling as the immunogen.

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

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

[0050] Human serum albumin (HSA) and ovalbumin (OVA) were dissolved in buffer solution (10mg / mL dissolved in 0.1M carbonate buffer; 0.9% NaCl solution, pH=9; mass frac...

Example Embodiment

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

[0063] 1. Western blot analysis of protein separation

[0064] Western blot was used to analyze the interaction of monoclonal antibody 4E4 with spermidine and spermine. When performing SDS-PAGE electrophoresis, 10μg OVA-spermidine, OVA-spermine, OVA, OVA-glutaraldehyde were loaded. Western blot analysis was performed using purified Mab4E4 according to standard protocols. For Western blot analysis of T3SS gene expression, bacteria cultured overnight were inoculated in fresh NTA-containing LB medium at a ratio of 1:200, and monoclonal antibody 4E4 or control IgG was performed according to the above steps. After culturing for 4 hours at 37°C, the bacterial culture was cooled on ice for 10 minutes. For each bacterial culture sample, take 10 mL and centrifuge. The supernatant and bacterial pellet are used to prepare extracellular protein and total cell protein, respectively. The supernatant wa...

Example Embodiment

[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 ligating the lacZ gene to the exsCEBA promoter) was grown overnight in LB liquid medium containing NTA 7.5 mM, the next day Dilute the bacterial cells 1:200 with fresh MINIS medium, and add IgG, monoclonal antibody 4E4, IgG+spermidine, monoclonal antibody 4E4+spermidine, respectively, the final concentration of spermidine is 10μM. Bacteria grow with shaking at 37℃ for 4h, when OD 600 When it reached about 1.0, the activity of the β-galactosidase encoded by lacZ promoter of T3SS under the control of the exsCEBA promoter was measured. Repeat the experiment at least three times. The data shown is the average of the three repeated experiments. A blank control is...

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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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