A kind of recombinant Artemisia annua class 1 allergen protein and its application

A technology of allergen and Artemisia annua, applied in the direction of allergen antigen composition, application, recombinant DNA technology, etc., can solve the problems of difficult expression, diagnostic specificity, unsatisfactory curative effect, pollution, etc.

Active Publication Date: 2021-04-27
ZONHON BIOPHARMA INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the complex composition of the natural allergen extract, it is very difficult to define its components. It contains a large amount of impurities, pigments and inactive components. The diagnostic specificity and curative effect are not ideal, and it is easily contaminated by exogenous toxic substances and pathogenic microorganisms.
[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 class 1 allergen protein and its application
  • A kind of recombinant Artemisia annua class 1 allergen protein and its application
  • A kind of recombinant Artemisia annua class 1 allergen protein and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 rArta1 codon optimization

[0047] According to the EMBL-EBI published DNA sequence of Artemisia annua partial major pollenallergen Art V1-like protein (EMBL-EBI accession number: AHF71022.1), as shown in SEQ ID No: 1, the inventor optimized the codon of the gene Then the rArt a1 gene of the present invention is obtained, the nucleotide sequence is shown in SEQ ID No: 2, and the amino acid sequence is shown in SEQ ID No: 3. The following is a comparison of the parameters before and after rArt a1 codon optimization:

[0048] 1. Codon Adaptation Index (CAI)

[0049] Depend on Figure 2-a It can be seen that before codon optimization, the codon adaptation index (CAI) of rArt a1 original gene in mammalian cell CHO expression system was 0.75. Depend on Figure 2-b It can be seen that through codon optimization, the CAI index of rArt a1 gene in mammalian cell CHO expression system is 0.95. Usually, when CAI=1, it is considered that the gene is in the most ide...

Embodiment 2

[0054] Embodiment 2: Containing the expression plasmid construction of rArta1 gene

[0055] The codon-optimized rArt a1 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 (by Provided by Nanjing GenScript Technology Co., Ltd.), a long-term storage plasmid was obtained, which was designated as pUC57-rArt a1 plasmid.

[0056] The pUC57-rArt a1 plasmid was used as a template for PCR amplification, and the primer sequences used were as follows:

[0057] Upstream primer (SEQ ID No: 4):

[0058] M13 F: TGT AAA ACG ACG GCC AGT

[0059] Downstream primer (SEQ ID No: 5):

[0060] M13 R: CAG GAA ACA GCT ATG AC

[0061] 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 concentratio...

Embodiment 3

[0064] Example 3: Preparation of Mammalian Cell Line Containing Stable Expression of rArta1 Gene

[0065] 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.

[0066] The correctly sequenced pCHO-rArt a1 plasmid was linearized with Nr...

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Abstract

The invention relates to Art a1, the main allergenic protein of Artemisia annua expressed in mammalian cells, encoding gene, expression method, purification process, detection method and application of recombinant protein. The Artemisia annua allergen protein itself is a plant protein, and it is difficult to express it in the existing prokaryotic or eukaryotic expression systems, and it has not been disclosed in relevant literature; however, traditional extraction methods often have complex components, difficult quality control, Vulnerable to contamination by exogenous toxic substances, pathogenic microorganisms, etc. For this reason, the present invention provides the coding gene of the recombinant Art a1 shown in SEQ ID NO: 1, and expression, purification, recombinant protein detection method and application specially designed for this purpose, compared with the prior art, the present invention Recombinant Art a1 has a relatively high expression level and activity, and also establishes a foundation for cell line development, process optimization, preclinical and clinical research, and industrial production in 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 class 1 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 ha...

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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