Optimized Zika virus envelope gene and expression thereof
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Publication Number
US-11434261-B2
Publication Date
2022-09-06
Expiration Date
Abstract
The present invention is directed to the expression and secretion the Zika virus envelope protein. Elements of the pre-membrane and envelope sequence have been modified to enhance the expression of the envelope protein as a secreted product in the culture medium of transformed insect cell lines. The expressed and purified product is suitable as a vaccine antigen.
Core Innovation
The invention relates to optimizing Zika virus prM-E (envelope) gene sequences to enable high-level expression and secretion of a soluble ZIKV envelope protein. The construct combines codon-optimized prM-E DNA, a synthetic optimized secretion signal for E processing at the prM-E junction, and an extended C-terminus of the E protein to stabilize the secreted envelope protein. The resulting purified E antigen is described as a vaccine immunogen.
The optimized expression construct is expressed in transformed Drosophila melanogaster S2 cells, and protein expression and secretion are compared among variants including wild-type, codon-optimized, secretion-signal optimized, C-terminal-extended, and combined improvements. The described prM-E junction processing is guided by a SignalP program to support cleavage for E processing, and protein stabilization is described in terms of interactions between domain III and domain I.
The document further describes optimized constructs that include specific sequence elements identified as SEQ ID NO:1 through SEQ ID NO:7, including a contiguous prM-E sequence, an optimized secretion signal element, and an E C-terminal extension element. It also reports downstream immunogenicity testing in mice, including measurements such as ELISA titers and PRNT titers, and it includes vaccine formulations with adjuvants for the purified E antigen.
Claims Coverage
The provided claims include three independent claims. Each independent claim centers on an expression vector that produces secretion of a soluble Zika virus envelope protein, with claim-specific emphasis on the SEQ ID NO:1 DNA sequence, an expression cassette comprising SEQ ID NO:7, or expression and secretion of heterologous proteins in cultured insect cells using an expression cassette shown in SEQ ID NO:7. Across the independent claims, the inventive features are primarily defined by the DNA sequence/cassette content and the stated secretion/host context.
Soluble Zika virus envelope protein secretion from SEQ ID NO:1 encoding prM-E
An expression vector comprising a DNA sequence encoding Zika virus pre-membrane and envelope protein, wherein expression results in secretion of a soluble envelope protein in the culture medium, and wherein the DNA sequence comprises SEQ ID NO:1.
Expression vector with SEQ ID NO:7 expression cassette enabling soluble prM-E envelope secretion
An expression vector comprising a DNA sequence encoding Zika virus pre-membrane and envelope protein, wherein expression results in secretion of a soluble envelope protein in the culture medium, and an expression cassette that comprises SEQ ID NO:7.
Insect-cell expression and secretion using an expression cassette shown in SEQ ID NO:7
An expression vector for expression and secretion of heterologous proteins in cultured insect cells, wherein the expression vector comprises the expression cassette shown in SEQ ID NO:7.
The claim set defines expression vectors by their encoded prM-E/envelope content and by the inclusion of specific sequence elements, particularly SEQ ID NO:1 and an expression cassette comprising SEQ ID NO:7. Secretion of a soluble envelope protein in culture medium is a core requirement for the prM-E-specific claims, while the heterologous protein claim extends the cassette framework to cultured insect cells.
Stated Advantages
Provides secretion of a soluble Zika virus envelope protein into the culture medium when the DNA sequence is expressed.
Provides an expression system for expression and secretion of heterologous proteins in cultured insect cells.
Documented Applications
Vaccine immunogen use of a purified ZIKV E antigen derived from the optimized constructs, with vaccine formulations that include adjuvants.
Expression and secretion in transformed Drosophila melanogaster S2 cells for producing the soluble envelope protein.
Immunogenicity testing in mice using measurements including ELISA titers and PRNT titers.
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