Dermatophagoides farina allergen Der f9 gene coded protein and application thereof

A gene encoding, dust mite technology, applied in the field of molecular biology, can solve problems such as difficult standardization

Inactive Publication Date: 2016-09-21
崔玉宝
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

We know that, as an organism, the crude extract of dust mite contains many kinds of proteins and other macromolecular substances. The concentration of crude extract produced by different manufacturers is different, so it is difficult to implement standardization

Method used

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  • Dermatophagoides farina allergen Der f9 gene coded protein and application thereof
  • Dermatophagoides farina allergen Der f9 gene coded protein and application thereof
  • Dermatophagoides farina allergen Der f9 gene coded protein and application thereof

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Experimental program
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Embodiment 1

[0012] 1 Amplification of Der f9 gene and synonymous mutation of NcoI site

[0013] Two pairs of primers were analyzed, designed and synthesized for cloning the active part of Der f9 gene and the NcoI in the synonymous mutation sequence, named F 6 :5′-CT CCATGG ATCATAGTGAAGAACAAGCTATTC-3' (underlined is the Nco I restriction site); R 6 : 5′-GTTCATTGATCATCCAAGGAAAACG-3′; F 7 :

[0014] 5′-CGTTTTCCTTGGATGATCAATGAAC-3′; R 7 :5′-TC CTCGAG AACTGTA

[0015] TTCGAAATGATCCAATTACG-3' (Xho I restriction site is underlined). Using plasmid pCold TF-Der f9 as template, F 6 -R 6 Or F 7 -R 7 Perform PCR with primers, the total reaction volume is 50μL, the specific composition is: 5μL of 10×PyrobestPCRBuffer, 0.5μL ofTaKaRa DNA Polymerase, 8μL of dNTP, 1μL of F 6 or F 7 ,1μLof R 6 or R 7 ,0.5μL of pCold TF-Der f9,34μL of ddH 2 O. The reaction conditions are as follows: denaturation at 94°C for 2 minutes and then enters the cycle. The cycle parameters are 94°C for 30s, 56°C for 30s, 72°C for 30s...

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Abstract

The invention discloses dermatophagoides farina allergen Der f9 gene recombinant protein and application thereof. The amino acid sequence of the recombinant protein is shown in SEQ ID No:2. The invention further discloses a preparation method and application of the recombinant protein. The dermatophagoides farina allergen Der f9 gene recombinant protein and the preparation method and application thereof have the advantages that a dermatophagoides farina allergen Der f9 full-length gene sequence is obtained, the recombinant protein is prepared according to the gene sequence, and the coded recombinant protein is high in purity, good in specificity and high in yield. The dermatophagoides farina allergen Der f9 gene recombinant protein can be applied to diagnosing whether a patient suffers from allergic diseases caused by a dermatophagoides farina allergen ninth component.

Description

Technical field [0001] The invention belongs to the field of molecular biology, and specifically relates to a Der f9 gene recombinant protein and its application. Background technique [0002] The mites of the family Acaridae are the main species of dust mites in our country. These mites can cause allergic diseases such as allergic asthma and rhinitis. Zhong Nanshan et al. used dust mite crude extract to test 6,304 patients with rhinitis and / or asthma in 17 cities in my country, and the results showed that both dust mite (D. farinae) and house dust mite (D. pteronyssinus) can cause mite allergic diseases in patients High-level IgE generation. [0003] The International Union of Immunological Societies (IUIS) naming database (nomenclature database, http: / / www.allergen.org / ) has published 33 different dust mite allergens, according to the order of discovery Named Der f1, Der f2, Der f3..... However, the nucleic acid sequence, amino acid sequence, physicochemical characteristics, an...

Claims

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Application Information

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IPC IPC(8): C07K14/435C12N15/12C12N15/10C12N15/70G01N33/68
CPCC07K14/43531G01N33/6893G01N2333/43582G01N2800/24
Inventor 崔玉宝
Owner 崔玉宝
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