Recombinant Dermatophagoides pteronyssinus type 2 allergen protein and its preparation method and application
Inventors
MA, Bruce Yong • Fan, Yu • WANG, Anliang • Wang, Jun
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-11319353-B2
Publication Date
2022-05-03
Expiration Date
Abstract
A DNA sequence encoding Der p2 protein having a particular base sequence is codon-optimized for the Pichia pastoris expression system, which is conducive to expressing Der p2 in Pichia pastoris. After gene optimization and adding an activating element to increase the expression of Der p2 in molecular level, it was found that Der p2 is expressed at a higher level as compared with the prior art and has biological activity similar to the natural protein.
Core Innovation
The disclosure describes a recombinant Der p2 allergen, specifically a DNA encoding a Der p2 protein having a base sequence as shown in SEQ ID NO: 1, and the production of the Der p2 protein. The Der p2 is expressed in Pichia pastoris, and recombinant Der p2 is obtained and analyzed for identity and biological activity.
The Der p2 coding gene is codon-optimized and expressed in Pichia pastoris under the control of the AOX promoter. Expression constructions include insertion and integration into the Pichia pastoris chromosome, with a defined interval relative to the AOX1 ATG and an added Kozak sequence. Expression is confirmed by SDS-PAGE and western blot.
The recombinant Der p2 is purified by sequential ion-exchange and hydrophobic chromatography and is verified by N-terminal sequence identity to natural Der p2, including the first five residues DQVDV. Biological activity is demonstrated by dot-immunoblotting with patient sera, showing IgE reactivity comparable to natural Der p2.
Claims Coverage
The provided content identifies one independent claim centered on a Der p2 DNA sequence as shown in SEQ ID NO: 1. The covered features also include vectors, Pichia pastoris host cells, and construct features with a 242 bp interval to AOX1 ATG and a Kozak sequence.
Der p2 DNA sequence as shown in SEQ ID NO: 1
A DNA encoding a Der p2 protein, having a base sequence as shown in SEQ ID NO: 1.
Der p2 DNA carried by selected expression vectors
A vector comprising the DNA of claim 1, wherein the vector is pAO815, pPIC9, pPIC9K, pPIC3.5, pPIC3.5K, pPICZα A, B, C or pGAPZ A, B, C.
Pichia pastoris host cell for the Der p2 vector
A host cell comprising the vector of claim 2, wherein the host cell is a cell of Pichia pastoris strain SMD1168, GS115, KM71, X33 or KM71H.
Construct spacing and Kozak sequence relative to AOX1 ATG
The vector of claim 2, wherein there is a 242 bp interval between the base sequence encoding the Der p2 protein and ATG of AOX1 of Pichia pastoris; and the base sequence encoding the Der p2 protein is preceded by Kozak sequence GCCACCATGG (SEQ ID NO: 10).
Overall, the claim content centers on a Der p2 DNA defined by SEQ ID NO: 1, with further coverage for vectors containing that DNA, selected Pichia pastoris strains as host cells, and specific construct-context features defined by a 242 bp interval to AOX1 ATG and a preceding Kozak sequence (SEQ ID NO: 10).
Stated Advantages
Recombinant Der p2 shows similar biological activity to natural Der p2, demonstrated by dot-immunoblot with patient sera.
N-terminal sequence identity to natural Der p2 is confirmed, including first five residues DQVDV.
Purified recombinant Der p2 is produced with a reported yield of ≥ 200 mg/L.
Documented Applications
Use in medicament preparation for dust mite allergic disease, including allergic rhinitis and allergic asthma.
Interested in licensing this patent?