Scylla paramamosain sensitizing protein Scy p 1 derivative and application thereof

A technology of simulating Scylla sinensis and derivatives is applied in the field of bioengineering, which can solve the problems such as the transformation of allergenic proteins to be studied, and achieve the effects of improving the safety index and reducing side effects.

Active Publication Date: 2021-02-12
JIMEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the modification of allergenic proteins in crab remains to be investigated

Method used

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  • Scylla paramamosain sensitizing protein Scy p 1 derivative and application thereof
  • Scylla paramamosain sensitizing protein Scy p 1 derivative and application thereof
  • Scylla paramamosain sensitizing protein Scy p 1 derivative and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Molecular cloning of allergenic protein Scy p 1 derivatives

[0031] (1) Design and synthesis of primers

[0032] According to the amino acid sequence (SEQ ID NO: 1) of the derivative of Scy p 1, the gene sequence of Scy p 1 obtained from NCBI was deleted, and the upper part of the derivative of Scy p 1 was designed by Primer 5.0 software. Downstream primers (F, R). At the same time, Nde I and EcoR I restriction endonuclease sites were added on the basis of the upstream and downstream primers. In addition, the forward and reverse primers (F mutants , R mutants ), the primers used in the experiment were synthesized by Bosun Biotechnology Co., Ltd.

[0033] f 1 : 5'-CATATGATGAAGCTGGAGAAGGACA-3'; (SEQ ID NO: 3)

[0034] R 1 : 5'-TACGCTCGTCCTCCTCGAGGAGCTG-3'; (SEQ ID NO: 4)

[0035] f mutants : 5'-GAGGACGAGCGTATGCGTAAGGTGC-3'; (SEQ ID NO: 5)

[0036] R mutants : 5'-TACGCTCGTCCTCCTCGAGGAGCTG-3'. (SEQ ID NO: 6)

[0037] (2) Construction of pET-28a-Scy p...

Embodiment 2

[0052] Example 2 Preparation of wild-type Scy p 1 and Scy p 1 derivatives

[0053] (1) Induced expression of wild-type Scy p 1 and Scy p 1 derivatives

[0054] The strains of wild-type Scy p 1 and derivatives of Scy p 1 were cultured in 10 mL LB medium overnight. The next day, the above culture solution was transferred to 500 mL of LB culture solution at a ratio of 1:1000, and cultured at 37°C until OD 600 =0.6~0.8, add IPTG at a final concentration of 0.5mmol / L, continue induction at 37°C for 16h, then centrifuge (12000g, 15min) to collect the bacteria; resuspend in 20mL pre-cooled ultrasonic buffer (20mmol / L Tris-HCl, 0.2 mol / L NaCl, pH 8.0), the supernatant was collected after centrifugation (12000g, 30min), which was the crude enzyme solution.

[0055] (2) Purification and identification of wild-type Scy p 1 and Scy p 1 derivatives

[0056] Load the crude enzyme solution in (1) at a speed of 1mL / min on the pre-balanced Ni 2+ - NTA affinity chromatography column (1.0 cm...

Embodiment 3

[0057] Example 3 Mast cell model detection of degranulation efficiency of wild-type Scy p 1 and Scy p 1 derivatives on LAD2 cells

[0058] Human mast cell LAD2 cells were used to establish a degranulation model, and 25 μL of wild-type Scy p 1 and Scy p 1 derivatives (final concentration 50 μg / mL) were added to LAD2 cells that had been sensitized by the serum of patients with crustacean allergy. The cells were stimulated by incubating for 1.0 h in a cell culture box, centrifuged, the supernatant was collected and the cells were lysed, and the wild-type Scy p 1 and Scy p 1 derivatives stimulated the LAD2 cells to produce β-hexosaminidase.

[0059] The result is as image 3 Shown: PBS in the figure is the negative control group; CA8 / 80 is the positive control group. Wild-type Scy p 1 stimulated the release rate of β-hexosaminidase in LAD2 cells by 21%, while Scy p 1 derivatives could significantly reduce the ability of LAD2 cells to release β-hexosaminidase (LAD2 cells β-hexosam...

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Abstract

The invention provides a scylla paramamosain sensitizing protein Scy p 1 derivative and an application thereof. The amino acid sequence of the derivative is shown as SEQ ID NO:1. According to the scylla paramamosain sensitizing protein Scy p 1 derivative, an N terminal, a C terminal and two key linear epitope regions of a wild type Scy p 1 amino acid sequence are deleted. Amplifying is carried outthrough an overlap extension PCR technology to obtain an Scy p 1 derivative gene sequence; connecting and transforming into a BL21 host cell are carried out, and inducible expression is carried out;the expressed derivative is purified by a Ni column, and the influence of Scy p 1 on mast cell degranulation before and after transformation is analyzed. Compared with wild-type Scy p 1, the Scy p 1 derivative can significantly reduce release of beta-aminohexosidase in LAD2 cells, is expected to be used for immunotherapy or immunological prevention, and reduces side effects in immunotherapy so asto improve the safety index of treatment.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a derivative of the allergenic protein Scy p 1 of the mud crab and its application. Background technique [0002] Food allergy is an immediate hypersensitivity reaction mediated by IgE. At present, the morbidity and mortality of food allergy in the world are increasing year by year, and it has become a global public health problem. The United Nations Food and Agriculture Organization and the World Health Organization pointed out that more than 90% of food allergies are caused by the eight major foods of milk, eggs, fish, beans, peanuts, wheat, nuts and crustacean aquatic products. [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 aqua...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C12N15/12C12N15/70A61K38/17A61P37/08C12R1/19
CPCC07K14/43509C12N15/70A61P37/08A61K38/00Y02A40/81
Inventor 刘光明李梦思夏菲陈一瑜云肖
Owner JIMEI UNIV
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