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Hybridoma cell strain AntiTput-13 and monoclonal antibody AntiTput-DP10 secreted thereby

A hybridoma cell line and monoclonal antibody technology, applied in the field of genetic engineering related to dust mite allergens

Active Publication Date: 2019-07-05
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, Der p10 has received extensive attention, but Der p10 as a tropomyosin of dust mites has not been reported in detail

Method used

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  • Hybridoma cell strain AntiTput-13 and monoclonal antibody AntiTput-DP10 secreted thereby
  • Hybridoma cell strain AntiTput-13 and monoclonal antibody AntiTput-DP10 secreted thereby
  • Hybridoma cell strain AntiTput-13 and monoclonal antibody AntiTput-DP10 secreted thereby

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030]Example 1 Prokaryotic expression and purification of Der p10 protein

[0031] 1. Primer Design

[0032] According to the tropomyosin (tropomyosin) gene sequence (accession number: AAT40866.1) of tyrophagous mite registered in Genbank, design PCR amplification primers, the sequence is as follows: upstream primer 5'-ATGAATCACAAAGTGCATCATCATCATCAT CATCATGGC -3', downstream primer 5'-CCG TCTAGA TTAATAACCGGTCAGTTCGGCAAA-3', the underline is the restriction site.

[0033] 2. Extraction and reverse transcription of total RNA from Tyrophagous mite

[0034] Total RNA was extracted from Tyrophagous mite by Trizol method as a template, and cDNA was synthesized by reverse transcription with random primers.

[0035] Trizol method for RNA extraction: pick 30 adults of tyrophagous mite under a microscope, add 0.5mL Trizol to mix, let stand at room temperature for 5min, add 0.1mL chloroform, shake vigorously for 15s, incubate at room temperature for 2-3min, 12000g, 4℃ Centrifuge f...

Embodiment 2

[0063] The preparation of embodiment 2 monoclonal antibody

[0064] 1. Mice Immunization

[0065] Four 6-week-old female SPF BALB / c female mice were immunized with affinity-purified prokaryotic recombinant Der p10 protein for 3 times with a two-week interval between each immunization. immunity.

[0066] One week after the second and third immunizations, blood was collected from the eyes of the mice, and the serum was separated (4°C, 10,000rpm, 20min). The antibody level was detected by indirect ELISA. The results are shown in Table 1. No. 3 mouse had the best titer and cell fusion Select mouse No. 3. Three days before the cell fusion, the SPF BALB / c mice No. 3 with good immune effect were boosted again, and each mouse was intraperitoneally injected with 50 μg immune antigen.

[0067]

[0068] Indirect ELISA method: coated with purified recombinant Der p10 protein antigen, 2ug / ml, 100uL / well, 4°C overnight; then washed 3 times with washing solution; blocked with 2% skim m...

Embodiment 3

[0079] Example 3 Identification of monoclonal antibodies

[0080] 1. Subclass identification of monoclonal antibodies

[0081] According to SBA Clonotyping TM System-HRP (Southern Biotech) Antibody Subclass Identification Kit Operating Instructions The monoclonal antibody obtained in Example 2 was used for subclass identification.

[0082] The results show that the heavy chain of the monoclonal antibody AntiTput-DP10 of the present invention is IgG1, and the light chain is Kappa chain. The identification results are shown in Table 3.

[0083] Table 3 Identification of monoclonal cell subtypes

[0084]

[0085] 2.Western blot identification test

[0086] 5 adult mite bodies of tyrophagous mite were crushed with a pestle, centrifuged and sedimented, and then subjected to SDS-PAGE electrophoresis, and then the protein was transferred to a nitrocellulose membrane by electrotransfer. The electrotransfer condition was 18V for 30min, and blocked with 5% skimmed milk powder Bl...

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Abstract

The invention relates to the field of genetic engineering associated with dust mite allergens, and discloses a hybridoma cell strain AntiTput-13, which can stably secrete a monoclonal antibody againstTyrophagus putrescentiae allergen Der p10. The hybridoma cell strain is collected in the China General Microbiological Culture Collection Center at Beichen West Road, Chaoyang District, Beijing withthe number of CGMCC No. 16298. Tests prove that the monoclonal antibody antiTPUT-DP10 secreted by the hybridoma cell strain can specifically react with Tyrophagus putrescentiae allergen Der p10. Basedon the monoclonal antibody secreted by the hybridoma, a double sandwich ELISA method for detecting tyrophagus putrescentiae is established, and basis is laid for diagnostic methods of accurately detecting Dermatophagoides farinae, such as tyrophagus putrescentiae, in houses, warehouses, fields and other environments.

Description

technical field [0001] The invention belongs to the field of genetic engineering related to dust mite allergens, and in particular relates to a hybridoma cell line and a monoclonal antibody secreted by it against the tyrophagous allergen Der p10 protein. Background technique [0002] Tyrophagus putrescentiae (Tyrophagus putrescentiae) belongs to Arachnida (Arachinida), Acari (Acari), Acariformes (Acaridida), and is a worldwide distribution of storage pests with a wide range of hosts. It also widely exists in the human living and working environment. Its mite body, metabolites, excreta, etc. all have strong allergenicity and can cause type I allergic diseases such as allergic asthma, dermatitis, and allergic rhinitis. It is one of the members of dust mite and house dust mite. Studies have shown that more than 50% of patients with type I hypersensitivity diseases are caused by dust mites, and 10-20% of the global population are allergic to dust mites. Since Voorhort et al. f...

Claims

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Application Information

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IPC IPC(8): C07K16/18C12N5/20G01N33/577G01N33/68
CPCC07K16/18G01N33/577G01N33/68
Inventor 曲绍轩李辉平马林骆昕蒋宁
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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