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A hybridoma cell line secreting anti-human il-37b monoclonal antibody, anti-human il-37b monoclonal antibody and application thereof

A hybridoma cell line and monoclonal antibody technology, applied in the field of genetic engineering, can solve the problems of limited application scope, insufficient to meet experimental research, few types of monoclonal antibodies, etc., and achieve the effect of high specificity and good passaging ability.

Active Publication Date: 2019-08-09
GUANGDONG MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It can be seen that there are few types of monoclonal antibodies against IL-37b at present, and the scope of application is limited, which is not enough to meet the needs of experimental research.

Method used

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  • A hybridoma cell line secreting anti-human il-37b monoclonal antibody, anti-human il-37b monoclonal antibody and application thereof
  • A hybridoma cell line secreting anti-human il-37b monoclonal antibody, anti-human il-37b monoclonal antibody and application thereof
  • A hybridoma cell line secreting anti-human il-37b monoclonal antibody, anti-human il-37b monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Preparation, identification and purification of antibodies

[0049] 1. Immunogen preparation and animal immunization

[0050] The plasmid containing the IL-37b gene fragment stored in the laboratory was used to transform into Escherichia coli, and IPTG induced Escherichia coli to express a large amount of the target protein. After the Escherichia coli was sonicated, it was purified using a nickel column to obtain soluble IL-37b protein. Two 6-week-old female BALB / c mice were prepared, and the mice were immunized with human IL-37b protein according to the scheme in Table 1. The mice were immunized for the first time, and the second immunization was carried out 14 days later. After the first immunization, each mouse received 150 μg of purified recombinant IL-37b emulsified with Freund's incomplete adjuvant three times by intraperitoneal route at weekly intervals. Seven days after the fourth immunization, blood was collected from the mice via the tail vein and ...

Embodiment 2

[0090] Example 2: Identification and preservation of hybridoma cells

[0091] The preservation information of the hybridoma cell line of the present invention is:

[0092] Deposit unit: China Center for Type Culture Collection; address: Wuhan University; date of deposit: May 2016; name: hybridoma cell line 1C6; deposit number CCTCCNO: C201683.

[0093] 1. Stability analysis of antibody secretion ability of different generations of hybridoma cell clones

[0094] Firstly, passage the 1C6 hybridoma cells, and take the first generation, second generation, fifth generation, seventh generation, and tenth generation hybridomas (the initial cell density is that the cells are fully covered at the bottom of the culture flask) after two days of culture The supernatant sample of the cell secretion was identified by ELISA, and the supernatant sample of each generation of cells was used as the primary antibody to obtain the IL-37b prokaryotic purified protein plate, and the ELISA was perfo...

Embodiment 3

[0097] Example 3: Application of anti-human IL-37b monoclonal antibody

[0098] 1. Mouse anti-human IL-37b monoclonal antibody applied to WB

[0099] (1) Reagents and preparation

[0100] 1. Electrode buffer (prepared with 1× electrode buffer):

[0101] Tris base 3.03g, glycine 18.8g, SDS 1.0g, double distilled water to adjust the volume to 1L, pH8.3;

[0102] 2. Transfer buffer: glycine 2.9g, Tris base 5.8g, SDS 0.37g, methanol 200ml, add ddH 2 O is fixed to 1000ml, pH8.3;

[0103] 3. TBST (Tris-Hcl, Nacl) buffer: 6g Tris base, 9g Nacl, 500ml double distilled water, Hcl pH7.5, constant volume 1L, add 1ml Tween-20;

[0104] 4. Blocking solution: 8% skimmed milk powder;

[0105] 5. 30% acrylamide storage solution: 29.2g acrylamide, 0.8g methylenebisacrylamide, add double distilled water to 100ml;

[0106] 6. Stacking gel buffer: 1mol / L Tris base, pH6.8;

[0107] 7. Separating gel buffer: 1mol / L Tris base, pH8.8;

[0108] All the above reagents were prepared with double ...

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Abstract

The present invention relates to the technical field of gene engineering, and in particular relates to a hybridoma cell strain secreting an anti human IL-37 monoclonal antibody and the anti human IL-37 monoclonal antibody and application thereof, and the anti human IL-37 monoclonal antibody with the accession number of CCTCCNO:C20168 is secreted by the hybridoma cell strain or a passage cell strain of the hybridoma cell strain. The anti human IL-37 monoclonal antibody has high specificity and high affinity, and can recognize human IL-37 Prokaryotic expression products, human IL-37 eukaryotic expression products and natural expression human IL-37 proteins, and by use of the anti human IL-37 monoclonal antibody, the expression level of the human IL-37 proteins can be simply, rapidly and accurately determined by IHC, WB, ELISA, FCM and other immunological methods.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a hybridoma cell line secreting anti-human IL-37b monoclonal antibody, anti-human IL-37b monoclonal antibody and application thereof. Background technique [0002] IL-37 is a member of the interleukin-1 (interleukin-1, IL-1) family. There are 11 members in the IL-1 family, all of which have a β-clover structure and can bind to immunoglobulin-like receptors. Most of the factors in the family are pro-inflammatory cytokines, and a small part belongs to receptor antagonists, which can be blocked by blocking Binding of the receptor to the ligand reduces the inflammatory response. In 2000, Kumar et al. discovered an immature precursor peptide IL-1H4, which was discovered by Eleanor in 2001 as the seventh cytokine of the IL-1 family, named IL-1F7, and was officially renamed as IL-1F7 in 2010 IL-37. [0003] The human IL-37 gene exists on chromosome 2, its molecular weight...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C07K16/24G01N33/68G01N33/577G01N33/543A61K39/395A61P1/18A61P21/00A61P9/14A61P7/06A61P37/02A61P5/16A61P1/16A61P1/00A61P19/08A61P17/06A61P19/02A61P17/00A61P37/06A61P35/00A61P13/12A61P43/00A61P11/06A61P3/10
CPCA61K39/00C07K16/244G01N33/543G01N33/6869Y02A50/30
Inventor 徐军发高宇驰贾岩王鑫张俊爱
Owner GUANGDONG MEDICAL UNIV
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