Mutant of scylla paramamosain allergic protein Scy p 4 and application of mutant

A technology for imitating blue crabs and allergens, which is applied in the field of bioengineering, can solve the problems that the modification of allergens needs to be studied, and achieve the effect of improving the safety index and reducing side effects

Active Publication Date: 2019-12-31
JIMEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Therefore, the modification of the allergenic protein in crab still needs to be studied

Method used

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  • Mutant of scylla paramamosain allergic protein Scy p 4 and application of mutant
  • Mutant of scylla paramamosain allergic protein Scy p 4 and application of mutant
  • Mutant of scylla paramamosain allergic protein Scy p 4 and application of mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Cloning of Mutant D70A of Scylla allergenic protein Scy p 4

[0032] (1) Design and synthesis of primers

[0033] According to the gene sequence of Scyp 4 in GenBank, the primers were designed and synthesized by Primer 5.0 software, and the wild-type Scy p 4 forward and reverse primers (F 1 , R 1 ) was used to amplify the 3' end and 5' end of the Scy p 4 gene, and Nde I and Sal I restriction sites were added to the upstream and downstream primers respectively. To determine the forward and reverse primers of the mutant (F mutants , R mutants ) was used for the first round of overlap extension PCR amplification, and the primers used in the experiment were synthesized by Bosun Biotechnology Co., Ltd.

[0034] f 1 : 5'-CATATGATGGCTTACTCTTGGGACA-3'; (SEQ ID NO: 3)

[0035] R 1 : 5'-GTCGACCTGCACCTCCTTCAGGGGT-3'; (SEQ ID NO: 4)

[0036] f mutants : 5'-GAGATCGCCGAACTGGCTGCATTCA-3'; (SEQ ID NO: 5)

[0037] R mutants : 5'-CTGACCGTCCTTGTTGAATGCAGCC-3'. (SEQ I...

Embodiment 2

[0055] Example 2 Preparation of wild-type Scy p 4 and D70A proteins

[0056] (1) Induced expression of wild-type Scy p 4 and its variants

[0057] Inoculate the correctly sequenced strains containing the Scy p 4 target gene and the strains containing the mutation site into 10 mL of LB culture medium containing 1 mmol / L Amp, and culture overnight. On the next day, transfer the above culture solution to 500 mL LB culture solution containing 1 mmol / L Amp at a ratio of 1:1000, and culture at 37°C until OD 600 =0.6~0.8, add the final concentration of 0.5mmol / LIPTG, centrifuge (12000g, 20min) after induction at 16°C for 14h, collect the bacteria; resuspend in 20mL ice-cooled ultrasonic buffer (20mmol / L Tris-HCl, / L NaCl, pH 8.0), sonicated, centrifuged (12000g, 20min), collected the supernatant, carried out SDS-PAGE and Western blot analysis, the results were as follows figure 1 and figure 2 shown. figure 1 Middle A-B, SDS-PAGE (A) and Western blot (B) analysis of wild-type Scy...

Embodiment 3

[0060] Example 3 Wild-type Scy p 4 and D70A protein and serum Dot blot analysis of patients with crustacean allergy

[0061] Take 2 μL of wild-type Scy p 4 protein, D70A protein and BSA with a protein concentration of 1 mg / mL, respectively, and spot them on nitrocellulose membranes (0.7×0.7 cm per square), and let them stand to dry. Soak the dried nitrocellulose membrane in 5% skimmed milk and incubate on a shaker at room temperature for 1.5 h. After blocking, the nitrocellulose membrane was washed 3 times with TBST (17g NaCl, 40mL 1M Tris-HCl, pH 8.0, 1mL Tween-20, dissolved in 2L distilled water), 5min each time. The nitrocellulose membranes were respectively placed in the sera of crustacean allergic patients who had a positive reaction to wild-type Scy p 4 diluted 1:1 with TBST, and incubated overnight at room temperature on a shaker. The next day, take out the nitrocellulose membrane soaked in serum diluent, wash 5 times with TBST, 5 min each time, and then use TBST to di...

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Abstract

The invention provides a mutant of scylla paramamosain allergic protein Scy p 4 and application thereof, wherein the mutant is obtained by mutating the 70th site of an amino acid sequence of the scylla paramamosain allergic protein Scy p 4 into alanine, and the amino acid sequence of scylla paramamosain allergic protein Scy p 4 is shown in SEQ ID NO: 1. According to the mutant of the scylla paramamosain allergic protein Scy p 4, a wild type Scy p 4 gene is subjected to site-directed mutagenesis by means of overlap extension PCR, the mutated gene is recombined with an expression vector and thentransferred into host cells, and the mutant of the Scy p 4 gene with low allergenicity is successfully constructed; and compared with the wild type allergen, the mutant has reduced IgE binding activity, and the mutant can be used for immunotherapy or immunoprophylaxis, and can reduce side effects in immunotherapy and improve safety index of treatment.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a mutant of Scyp 4 allergic protein Scy p 4 and an application thereof. Background technique [0002] At present, the morbidity and mortality of food allergy in the world are increasing year by year, and food allergy has become a global public health problem. Eggs, soybeans, tree nuts, wheat, peanuts, milk, fish, and crustacean aquatic products are the eight recognized allergens in the world. [0003] Among the crustacean aquatic products, Scylla Paramamosain, also known as blue crab, has important economic value and nutritional value, and is loved by consumers. However, many people are allergic to aquatic products every year. Unlike other food allergies, crustacean aquatic products are usually accompanied for life, seriously affecting the living conditions of susceptible people. At present, immunotherapy for food allergy usually includes: avoiding contact, t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C12N15/12C12N15/70A61K38/17A61P37/08
CPCC07K14/43509C12N15/70A61P37/08A61K38/00
Inventor 刘光明陈一瑜胡梦君李梦思韩欣宇
Owner JIMEI UNIV
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