Milk, wheat, peanut and soybean allergen fused protein and construction method and application thereof

A soybean allergen and fusion protein technology, which is applied in the field of wheat, milk, peanut and soybean allergen fusion protein and its construction, can solve the problems of single protein, missed detection and wrong detection, poor sensitivity and specificity, etc., to reduce pain , Reduce missed detection and false detection, and improve the detection rate

Pending Publication Date: 2021-01-15
四川携光生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the existing allergen detection usually uses a single protein detection, which has poor sensitivity and specificity, which easily leads to missed and false detections.

Method used

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  • Milk, wheat, peanut and soybean allergen fused protein and construction method and application thereof
  • Milk, wheat, peanut and soybean allergen fused protein and construction method and application thereof
  • Milk, wheat, peanut and soybean allergen fused protein and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Synthesis of fusion gene (target gene):

[0036] The amino acid sequence of milk protein is selected as follows:

[0037] EEIVNSVEQKHIQKEDVPSERYLGYLEQLLRLKKYKVPQLEIVNSAEERLGGGGSEI NQFYQKFPQYLQYLYGGGGSTEVFTKKTKLTEEEKNRLNFLKKISQRYQKFALPQYLKTV Y QHQKAMKPWI QPKT.

[0038] The amino acid sequence of wheat protein is selected as follows:

[0039] YSVRFVANHGGGGSVLKNKHREWESCFQKQ.

[0040] Peanut protein selects the following amino acid sequence:

[0041] SATHAKSSPYQKKTENPCAQRCLQSCQQEPDDLKQKACESRCTKLEYDPRCVYDPRGHTGTTNQRSPPGERTRGRQPGGGGGSRRYTARLKEGGGGGSLAAHASARQQWELQGDRRCQSQ LERANLRPCE QHLMQKIQRD.

[0042] The amino acid sequence of soybean protein is selected as follows:

[0043] MGVFTFEDEINSPVAPATLYKALVTDADNVIPKALDSFKSVENVEGNGGPGTIKKITFLEDGETKFVLHKIESIDEANLGYSYSVVGGAALPDTAEKITFDSKLVAGPNGGSAGKLTVKYETKGDAEPNQDELKTGKAKADALFKAIEAY LLahPDYN.

[0044] S1. Sequentially connect the nucleotide sequences of milk, wheat, peanut and soybean allergens to obtain the target gene. Accordi...

Embodiment 2

[0046] Construction of recombinant plasmids containing fusion genes:

[0047] 2.1 Combine the fully synthetic sequence obtained in Example 1 with the vector pFastBac double-digested with restriction endonucleases BamH I and Not I TM HT A to connect;

[0048] 2.2 Transform the ligation product into Escherichia coli DH5α competent cells. The volume of the ligation product should not exceed 10% of the competent cells. Gently swirl several times to mix the contents, put it in an ice bath for 30 minutes, put it in a 42°C water bath and heat it for 60 seconds at regular intervals. Shock, quickly transfer the tube to an ice bath for 120 seconds, add 400 μL LB medium to each tube, shake slowly at 37°C for 60 minutes, centrifuge at low speed for 2 minutes, remove the supernatant, leave 100 μL medium in the centrifuge tube, resuspend the bacteria, and Spread the bacterial solution evenly on the LB plate;

[0049] 2.3 Place the plate upside down in a constant temperature incubator at 3...

Embodiment 3

[0051] Expression of fusion protein

[0052]3.1 Transform the recombinant plasmid obtained in Example 2 into DH10 Bac competent cells, the volume should not exceed 5% of the competent cells, gently swirl several times to mix the contents, and put the tube in ice bath for 30 minutes; put the tube in 42°C Timed heat shock in water bath for 90 seconds; quickly transfer the tube to ice bath for 120 seconds; add 800 μL LB medium to each tube, 37 ° C, shake slowly for 4 hours; spread 30 μL bacterial solution on the resistance agar plate; put the plate upside down In a constant temperature incubator at 37°C, blue-white colonies may appear after 30-48 hours;

[0053] 3.2 Pick a single positive white spot colony and inoculate it into 5mL of resistant LB culture medium, shake slowly for 12-16h, take the bacterial solution for PCR identification, and the results show that the bacmid recombination is correct. The recombinant bacmid is transfected into insect cells with a transfection rea...

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Abstract

The invention discloses a milk, wheat, peanut and soybean allergen fused protein and a construction method and application thereof. The construction method comprises the following steps of: S1, sequentially connecting nucleotide sequences of milk, wheat, peanut and soybean allergens to obtain a target gene, and adding Linker sequences into the adjacent allergen nucleotide sequences; S2, adding aneukaryotic KOZAK sequence to the N terminal of the target gene, adding a restriction endonuclease site Bam I to the upstream, adding a Not I enzyme cutting site to the downstream, and adding a 6*His tag sequence to the C terminal to obtain a complete sequence gene; S3, performing double enzyme digestion on the complete sequence gene obtained in the step S2 and plasmids to obtain an enzyme digestion product; S4, connecting the enzyme digestion product obtained in the step S3 through a T4 ligase to obtain recombinant plasmids; and S5, expressing the recombinant plasmids in insect cells to obtainthe milk, wheat, peanut and soybean allergen fused protein. The fused protein constructed by the method provided by the invention can improve the sensitivity and specificity of a detection kit and reduce missing detection and error detection.

Description

technical field [0001] The invention relates to the technical field of in vitro diagnosis, in particular to a milk, wheat, peanut and soybean allergen fusion protein and its construction method and application. Background technique [0002] Allergic disease (anaphylactia), also known as allergic disease, occurs after a patient inhales, ingests, or injects substances containing allergens (called allergens or allergens, Allergen) such as house dust mites, plant pollen, animal fur, etc. Trigger the body's B cells to produce specific immunoglobulin E (Immunoglobulin E, IgE), thereby triggering allergic reactions (or allergic reactions, Allergy), leading to the occurrence of clinical diseases such as asthma, allergic rhinitis, and eczema. [0003] Among them, IgE is a class of Ig discovered in 1966, with a molecular weight of 188kD. Its content in serum is extremely low, accounting for only 0.002% of total serum Ig, and it is synthesized late in individual development. IgE is ma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/866G01N33/68
CPCC07K14/415C12N15/86G01N33/6893C07K2319/02C07K2319/21C12N2710/14043C12N2800/22G01N2800/24
Inventor 秦枫曹长春吴荣桂张小飞颜松周玥黄敬双万文琴张伟鲜静吴斌
Owner 四川携光生物技术有限公司
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