Variant recombinant dermatophagoides pteronyssinus type 1 allergen protein and its preparation method and application
Inventors
MA, Bruce Yong • Fan, Yu • WANG, Anliang • Wang, Jun
Assignees
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Publication Number
US-11359191-B2
Publication Date
2022-06-14
Expiration Date
Abstract
A DNA sequence encoding Der p1 protein having a particular base sequence is codon-optimized for the Pichia pastoris expression system, which is conducive to expressing Der p1 in Pichia pastoris. After gene optimization and adding an activating element to increase the expression of Der p1 in molecular level, it was found that Der p1 is expressed at a higher level as compared with the prior art and has biological activity similar to the natural protein.
Core Innovation
The invention describes a DNA sequence encoding a proDer p1 protein, wherein the DNA has the base sequence as shown in SEQ ID NO: 1. The proDer p1 protein is expressed in Pichia pastoris using an expression cassette integrated into the Pichia chromosome using the AOX promoter, with a defined positional relationship between the proDer p1 encoding region and the ATG of AOX1.
A codon optimization strategy is provided for producing proDer p1, including changes reported in terms of codon adaptation index (CAI), low-utilization codon frequency (POP), and GC content. Regulatory and signaling elements are specified for directing expression, including an alpha-factor signal peptide and a Kozak sequence GCCACCATGG associated with the proDer p1 encoding region.
The invention further describes a mature Der p1 exhibiting biological activity comparable to natural Der p1, together with inducible expression confirmation and IgE/serum reactivity in comparison with natural Der p1. The documented recombinant constructs include SEQ ID NO: 1 and multiple vectors and Pichia pastoris strains, together with supporting characterization of gene copy number and expression.
Claims Coverage
The document includes one independent claim and dependent claims that define the proDer p1-encoding DNA sequence (SEQ ID NO: 1) and narrow how it is carried and expressed, including host strain selection and specific positional/regulatory sequence features. The inventive features cover the defined proDer p1 DNA sequence, its use in specified vectors, its presence in particular Pichia pastoris strains, and a positional spacing and sequence context relative to AOX1 ATG with alpha-factor signal peptide and the Kozak sequence GCCACCATGG.
Defined proDer p1 DNA base sequence
A DNA sequence encoding a proDer p1 protein, said DNA having the base sequence as shown in SEQ ID NO: 1.
Carriage in selected expression vectors
The DNA sequence is contained in one of the specified vectors selected from pAO815, pPIC9, pPIC9K, pPIC3.5, pPIC3.5K, pPICZαA, pPICZαB, pPICZαC, pGAPZαA, pGAPZαB, and pGAPZαC.
Expression in selected Pichia pastoris strains
A DNA sequence in a vector, where the vector is present in Pichia pastoris strains selected from SMD1168, GS115, KM71, X33, and KM71H.
Defined spacing relative to AOX1 ATG with alpha-factor signal peptide and Kozak sequence
A DNA sequence for a proDer p1 protein in which a 242 bp interval separates the proDer p1 encoding region from the ATG of AOX1 on Pichia pastoris; and the proDer p1 encoding sequence is preceded by an alpha-factor signal peptide and a Kozak sequence GCCACCATGG.
Overall, the claims center on a defined SEQ ID NO: 1 DNA sequence encoding proDer p1, with coverage extended by incorporation into specific vectors, restriction to particular Pichia pastoris strains, and a defined 242 bp spacing and specified sequence context (alpha-factor signal peptide and Kozak GCCACCATGG) relative to the AOX1 ATG.
Stated Advantages
Mature Der p1 shows biological activity comparable to natural Der p1.
The recombinant proDer p1 strategy shows IgE/serum reactivity comparable to natural Der p1.
Documented Applications
A medicament for treating dust mite allergic diseases is described, including allergic rhinitis and allergic asthma.
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