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Coding gene of scylla paramamosain allergenic protein and application thereof

A technology of imitating blue crabs and encoding genes, which is applied in the fields of application, genetic engineering, and plant gene improvement, and can solve problems such as large molecular weight, hindering the exploration of allergenicity, and increasing the difficulty of cloning

Active Publication Date: 2019-12-31
厦门市波生生物技术有限公司
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

Among them, compared with the more systematic research on other allergens of Scylla syringae, there is no report on the gene encoding Scy p 9 in aquatic animals; and the large molecular weight of Scy p 9 increases the difficulty of its gene cloning, which is very difficult. It largely hinders the exploration of its sensitization, making the prevention, control and treatment of Scy p 9 allergic diseases limited

Method used

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  • Coding gene of scylla paramamosain allergenic protein and application thereof
  • Coding gene of scylla paramamosain allergenic protein and application thereof
  • Coding gene of scylla paramamosain allergenic protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Cloning and expression of the allergenic protein Scy p 9 of Scylla pseudocarpus

[0053] 1. Extraction of total RNA: The extraction of total RNA from Scylla pseudocavea is as follows: Super total RNA extraction kit manual method, the extracted RNA was stored at -70°C after testing its concentration and purity.

[0054] 2. Synthesis of cDNA: The first-strand cDNA was synthesized according to PrimeScript TM II 1st Strand cDNA Synthesis Kit Instructions Method: After adjusting the RNA to an appropriate concentration, prepare mix-1 according to the system in Table 1, mix well, heat at 65°C for 5 minutes, and immediately ice-bath after heating. When mix-1 is heated, mix-2 is prepared. After the heated mix-1 and mix-2 are mixed gently, the first-strand cDNA is synthesized according to the reaction conditions in Table 2. The synthesized first-strand cDNA was stored at -20°C for future use.

[0055] Table 1 Mix-1 and mix-2 preparation table

[0056]

[0057] ...

Embodiment 2

[0081] Example 2 Purification and identification of the allergenic protein rScy p 9 from Scylla pseudocarpus

[0082] Protein purification of the allergenic protein rScy p 9 of Scylla maculae: The supernatant of Example 1 was applied to the pre-equilibrated Ni 2+ - NTA affinity chromatography column (1.0 cm x 5.0 cm). After washing with 100mL eluent (200mmol / L Tris-HCl, 200mmol / L NaCl, 10mmol / L imidazole, pH 8.0) to remove impurities, use 100mL eluent (200mmol / L Tris-HCl, 200mmol / L NaCl , 100mmol / L imidazole, pH 8.0) for linear elution of the target protein, through A 280 and SDS-PAGE analysis, and the purified fractions were collected.

[0083] Mass spectrometric identification of the allergenic protein rScy p 9 in Scylla pseudosyrpa: excised figure 2 The rScy p 9 lane in the middle corresponds to the gel strip of rScy p 9 protein, with a molecular weight of about 90 kDa, which was sent to Micronavi Biotechnology Co., Ltd. (Shenzhen, Guangdong, China) for mass spectrometr...

Embodiment 3

[0084] Example 3 The application of the allergenic protein rScy p 9 of Scylla pseudocarpus in clinical diagnosis

[0085] Nitrocellulose membrane cutting: Cut the nitrocellulose membrane into a rectangle with a length of 28.8 cm and a width of 3.2 cm, and draw squares of 0.8×0.8 cm each on the membrane, a total of 36 grids.

[0086] Allergenic protein spotting: adjust the allergenic protein rScy p 9 purified in Example 2 to a protein concentration of 1 mg / mL, take 2 μL of each cell and directly spot on the nitrocellulose membrane, and let it stand to dry.

[0087] Nitrocellulose membrane sealing: Soak the dried nitrocellulose membrane in 5% skimmed milk and incubate on a shaker at room temperature for 1.5 h.

[0088] Serum incubation: After sealing, the nitrocellulose membrane was washed 3 times with TBST for 5 minutes each time, and the corresponding serum of 16 crustacean allergic patients diluted 1:5 with TBST was cut out from the grid corresponding to the serum in the memb...

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Abstract

The invention provides a coding gene of scylla paramamosain allergenic protein and an application thereof. The coding gene of the allergenic protein is composed of a nucleotide sequence shown in SEQ ID NO: 1. The invention also discloses an expression vector and a recombinant strain containing the gene, a method for preparing the allergenic protein and the application of the allergenic protein. The coding gene of the scylla paramamosain allergenic protein is cloned into a prokaryotic expression vector, recombinant expression plasmid is constructed, and transferred into host bacteria and inducing expression is carried out; the scylla paramamosain allergenic protein rScy p 9 can be obtained, the IgE binding capacity of the scylla paramamosain allergenic protein rScy p 9 and serum of a crustacean allergic patient is measured, a reference basis can be provided for clinical diagnosis of allergic symptoms and detailed screening of allergens, and prevention, control and treatment of allergicdiseases of the Scy p 9 are timely and accurate.

Description

technical field [0001] The invention belongs to the technical field of food detection, and in particular relates to a coding gene of an allergenic protein of Scylla pseudomaculata and an application thereof. Background technique [0002] As a hot issue of food safety, food allergy has attracted widespread attention around the world. Crustaceous aquatic products are one of the eight types of allergic foods announced by the Food and Agriculture Organization of the United Nations. Scylla paramamosain belongs to Crustacea and Scylla genus, and is widely distributed throughout the Indian Ocean and the Pacific Ocean. This species has a long history of breeding in the southeast coast of my country; it is a commercially important crab resource for fisheries and aquaculture. But the increase in its production and consumption has been accompanied by more and more food allergy problems. [0003] At present, the sarcoplasmic protein of Scylla cryptae can be recognized by specific immun...

Claims

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Application Information

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IPC IPC(8): C12N15/12C07K14/435C12N15/70G01N33/68
CPCC07K14/43509C12N15/70G01N33/6854G01N2333/43508
Inventor 刘光明何欣蓉杨阳张永霞刘红曹敏杰
Owner 厦门市波生生物技术有限公司
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