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Portuguese oyster allergic protein and application thereof

A Portuguese oyster, allergenic protein technology, applied in application, anti-animal/human immunoglobulin, introduction of foreign genetic material using vectors, etc., can solve problems such as hindering the exploration of sensitization, lack of information, etc.

Inactive Publication Date: 2020-01-14
JIMEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, sarcoplasmic calcium binding protein (SCP) is mainly involved in the muscle contraction response of invertebrates, and its related reports as an allergen are mainly concentrated in shrimp and crabs. However, this largely hinders the exploration of its sensitization, making the prevention, control and treatment of allergic diseases targeting the oyster allergenic protein SCP limited.

Method used

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  • Portuguese oyster allergic protein and application thereof
  • Portuguese oyster allergic protein and application thereof
  • Portuguese oyster allergic protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Purification, mass spectrometric identification and antibody preparation of Portuguese oyster allergenic protein SCP

[0039] 1. Take fresh Portuguese oysters to remove viscera, gonads, etc., keep the muscle part, add 10 times the volume of Buffer A (10mM PBS, 0.15M NaCl, pH 7.5) buffer solution, use a tissue masher to mash the tissue, and use refrigerated centrifugation machine for centrifugation (12000g, 10min, 4°C); centrifuge supernatant through silk cloth, slowly add 70% ammonium sulfate for salting out, stir in an ice bath for 30min, stand at 4°C for 2h, and centrifuge (12000g, 10min, 4°C); Centrifuge the supernatant through silk cloth, slowly add 90% ammonium sulfate for salting out, stir in ice bath for 30min, stand at 4°C for 2h, centrifuge (12000g, 10min, 4°C); dialyze the centrifugal supernatant into 20mM TrisHCl (pH 7.5) , dialyzed overnight at 4°C, the obtained sample was concentrated in a 3kDa ultrafiltration tube, and then loaded on a Q-Sepharos...

Embodiment 2

[0042] Example 2 Cloning, expression, purification and identification of recombinant Portuguese oyster allergen protein rSCP

[0043] 1. Extraction of total RNA: extraction of total RNA from Portuguese oyster Super total RNA extraction kit manual method, the extracted RNA was stored at -70°C after testing its concentration and purity.

[0044] 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.

[0045] Table 1 Mix-1 and mix-2 preparation table

[0046]

...

Embodiment 3

[0084] Example 3 Application of Portuguese oyster allergenic protein in clinical diagnosis

[0085] Nitrocellulose membrane cutting: Cut the nitrocellulose membrane into a rectangle with a length of 7.7 cm and a width of 4.2 cm, and draw squares of 0.7×0.7 cm each on the membrane, a total of 66 grids.

[0086] Allergenic protein spotting: the Portuguese oyster allergenic protein SCP purified in Example 1 and the allergenic protein rSCP in Example 2 were both adjusted to a protein concentration of 1 mg / mL, and 2 μL of each grid was directly spotted on the nitrocellulose membrane. and let it 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, each time for 5 minutes, and the squares corresponding to the serum in the membrane were cut out, and serum from 5 shellfish allergy ...

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Abstract

The invention provides a Portuguese oyster allergic protein and application thereof. An amino acid sequence of the allergic protein is shown as SEQ ID NO: 1, and a nucleotide sequence of a coding geneof the allergic protein is shown as SEQ ID NO: 2. According to the coding gene of the Portuguese oyster allergic protein, the coding gene is cloned in a prokaryotic expression vector to construct a recombinant expression plasmid, then the recombinant expression plasmid is transformed into a host bacterium and is induced to be expressed, and an rSCP as a recombinant Portuguese oyster allergic protein can be obtained; and determination of IgE combining capacity is performed by utilizing the rSCP and serum of a patient with allergy to shellfish, so that a reference basis can be provided for clinical diagnosis and allergen detailed screening of a allergic symptom, and prevention and treatment on allergic diseases for the SCP are timely and accurate.

Description

technical field [0001] The invention belongs to the technical field of food detection, and in particular relates to a Portuguese oyster allergenic protein and an application thereof. Background technique [0002] Allergic diseases have always been an important health problem at home and abroad. Food allergy is a typical allergic disease, which belongs to type I allergic reaction mediated by immunoglobulin IgE. Crustaceous aquatic products are one of the eight categories of allergic foods announced by the Food and Agriculture Organization of the United Nations, so research on seafood allergens is mainly directed at crustacean allergens, but molluscs in seafood and aquatic products can also cause allergic reactions. It has been reported that crustaceans and molluscs have cross-allergens, but the research on mollusc allergens is far less than that of crustaceans. [0003] Oysters, the most popular food in the mollusk category. Portuguese oysters are mainly distributed in the ...

Claims

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Application Information

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IPC IPC(8): C07K14/435C07K16/18C12N15/12C12N15/70G01N33/68
CPCC07K14/43504C07K16/18C12N15/70G01N33/68G01N2333/43504
Inventor 刘光明韩天娇杨阳胡梦君刘红李梦思
Owner JIMEI UNIV
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