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A kind of recombinant Artemisia annua Type 3 allergen protein and its application

An allergen-like, Artemisia annua technology, applied in the field of recombinant Artemisia annua class 3 allergen proteins and their coding genes and expression purification, can solve the problems of cumbersome process, pollution, complex composition, etc., and achieve simple preparation and purification process, Reduce allergic reaction, good immunogenic effect

Active Publication Date: 2020-06-09
ZONHON BIOPHARMA INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because the composition of the natural allergen extract is very complex, it is very difficult to define its components, the extraction method is time-consuming, the process is cumbersome, the cost is high, and it is easy to be polluted by exogenous toxic substances and pathogenic microorganisms
In addition, the use of crude extracts for diagnosis cannot clearly determine the degree of response of the patient to the components of the allergen, which can easily lead to misdiagnosis
[0005] On the other hand, the Artemisia annua allergen protein itself is a plant protein, and it is difficult to express it in the existing commonly used prokaryotic or eukaryotic expression systems.

Method used

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  • A kind of recombinant Artemisia annua Type 3 allergen protein and its application
  • A kind of recombinant Artemisia annua Type 3 allergen protein and its application
  • A kind of recombinant Artemisia annua Type 3 allergen protein and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0045] Example 1 rArt a3 codon optimization

[0046] According to the mRNA sequence of Artemisia vulgaris clone1Art v3allergenprecursor (Art v3) published by NCBI (GenBank accession number: EU564846.1), the inventor obtained the cDNA sequence of Artemisia annua with high homology to Artv3 sequence but unknown gene function from NCBI EST ( NCBI accession number: GW332468.1), the sequence is shown in SEQ ID No: 2, the rArt a3 gene of the present invention is obtained after codon optimization of the gene, the nucleotide sequence is shown in SEQ ID No: 1, the amino acid sequence As shown in SEQ ID No:3. The following is a comparison of the parameters before and after rArt a3 codon optimization:

[0047] 1. Codon Adaptation Index (CAI)

[0048] Depend on Figure 2-a It can be seen that before codon optimization, the codon adaptation index (CAI) of rArt a3 original gene in mammalian cell CHO expression system was 0.72. Depend on Figure 2-b It can be seen that through codon o...

Embodiment 2

[0053] Embodiment 2: Containing the expression plasmid construction of rArt a3 gene

[0054] The codon-optimized rArt a3 gene was introduced into the AvrII restriction site sequence at the 5' end, and the BstZ17I restriction site sequence was introduced at the 3' end, and the whole gene synthesis was carried out, and the synthetic gene fragment was constructed into the pUC57 plasmid ( Provided by Nanjing GenScript Co., Ltd.), a long-term storage plasmid was obtained, which was designated as pUC57-rArt a3 plasmid.

[0055] The pUC57-rArt a3 plasmid was used as a template for PCR amplification, and the primer sequences used were as follows:

[0056] Upstream primer (SEQ ID No: 4):

[0057] M13F: TGT AAA ACG ACG GCC AGT

[0058] Downstream primer (SEQ ID No: 5):

[0059] M13R: CAG GAA ACA GCT ATG AC

[0060] The total reaction volume was 50 μL, in which 2.5 μL was added to each primer with a concentration of 10 μmol / L, and 1 μL was added to dNTP with a concentration of 10 m...

Embodiment 3

[0063] Example 3: Preparation of Mammalian Cell Line Containing Stable Expression of rArt a3 Gene

[0064] Puromycin is an aminoglycoside antibiotic that blocks protein synthesis in mammalian cells by interfering with ribosome function. The pac gene from Streptomyces has the effect of detoxifying Puromycin. The pCHO1.0 vector contains the pac gene, so Puromycin can be used as a screening antibiotic for pCHO1.0 as the expression vector. MTX is a folic acid antagonist, which can inhibit the activity of DHFR after conversion in cells, inhibit nucleic acid synthesis, and cause cytotoxicity. pCHO1.0 contains the DHFR gene, and MTX can be used as a screening reagent. The transfected cells contain Puromycin and MTX resistance genes, and the concentration of the screening reagent is continuously increased to increase the copy number of the target gene in the positive cells to increase the expression level.

[0065] The correctly sequenced pCHO-rArt a3 plasmid was linearized with N...

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Abstract

The invention relates to an Artemisia annua main allergenic protein Art a3 expressed by mammalian cells, a coding gene, an expression method, a purification process, a recombinant protein detection method and applications thereof. In the prior art, the Artemisia annua allergenic protein belongs to plant protein, is difficultly expressed in the existing commonly-used prokaryotic or eukaryotic expression systems, and is not disclosed in related literatures, and the traditional extraction methods have problems of complex components, difficult quality control, easy pollution by exogenous toxic substances and pathogenic microorganisms and the like. A purpose of the present invention is to solve the problems in the prior art. The present invention provides the Art a3 coding gene represented by SEQ ID NO:1, the expression method, the purification method, the recombinant protein detection method and the applications thereof. Compared to the allergenic protein in the prior art, the recombinantArt a3 of the present invention has advantages of high expression and high activity, and the foundation is established for the cell line development, the process optimization, the preclinical and clinical research and industrial production during the drug development process.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals, and in particular relates to a recombinant Artemisia annua Type 3 allergen protein, its coding gene and an expression and purification method. Background technique [0002] In recent years, the incidence of allergic hay fever has been increasing year by year, and the incidence in Europe has risen from 1% in the early 20th century to 20%, and it is expected to reach 35% in the next 20 years. There are more than 10 million hay fever patients in my country, the incidence rate of urban residents is about 0.9%, and it can reach 5% in endemic areas. With more and more varieties of trees and flowers in urban areas, the number of hay fever patients is increasing. [0003] Pollen is the male reproductive cell of higher plants. It is spread in the air under the action of wind or insects. Some people will have allergic reactions after inhaling pollen during breathing. The dispersal of pollen has...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C12N15/85C07K14/415C07K1/18A61K39/36A61P37/08
CPCA61K39/00C07K14/415C12N15/85C12N2800/22
Inventor 马永王安良王俊
Owner ZONHON BIOPHARMA INST
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