Nucleic acids, polypeptides, methods of expression, and immunogenic compositions associated with sars corona virus spike protein

Abstract

Nucleic acid molecules, polypeptides, immunogenic compositions, vaccines, and methods of making and using the nucleotides and encoded polypeptides associated with the Spike protein of SARS Corona Virus (SARS CoV) are disclosed.

Description

[0001] related application

[0002] This application claims the benefit of U.S. Patent Application No. 10 / 860,641, filed June 4, 2004, and U.S. Provisional Patent Application No. 60 / 578,348, filed June 10, 2004, which are hereby incorporated by reference The above application is incorporated into this application. A request to amend US Patent Application No. 10 / 860,641 as a provisional application was filed on May 23, 2005. technical field

[0003] The present invention relates to purified and isolated nucleic acids, polypeptides, purified and isolated polypeptides, nucleic acids encoding such polypeptides, methods of producing recombinant forms of such polypeptides, antibodies raised against these polypeptides, and such nucleic acids and polypeptides Use in diagnostic methods, kits, immunogenic compositions, vaccines or antiviral therapy. Background technique

[0004] In 2002, a new infectious disease called Severe Acute Respiratory Syndrome (SARS) emerged in Guangdong Pr...

Examples

Embodiment 1

[0111] A spike gene (Spike gene) from a patient infected with SARS CoV was obtained, called Spike-Pasteur (SEQ ID NO: 1). The Spike-Pasteur was cloned into pcDNA eukaryotic expression vector and transfected into 293T cells. Cells transfected with pcDNA-Spike-Pasteur did not express the Spike-Pasteur polypeptide, as no detectable levels of the Spike protein were observed by either FACS or Western blotting.

[0112] Spike-Pasteur was then expressed in a SFV viral expression vector capable of efficient expression in transfected BHK cells. However, the yield of SFV virions was low due to the presence of 2 Spe I sites in the spike gene. Spe I is commonly used to linearize the plasmid at the end of the SFV coding sequence. Since Spe I could not be used, PSFV-Spike-Pasteur was linearized using Sph I, which generated an additional 3' RNA sequence of >2000 bases of the carrier RNA. Representative expression levels from cells infected with SFV-Spike-Pasteur were weak.

[0113] To en...

Embodiment 2

[0115] Further bioinformatics analysis was performed on the Spike-Pasteur sequence. The cDNA for Spike-Pasteur contains many cis-acting sites that can negatively affect expression. To further enhance expression, 32 of the 33 negative cis-acting signals identified were eliminated from Spike-Pasteur and an additional signal that stimulated gene expression was added, resulting in Spike-HKU-PRC (SEQ ID NO: 3). Spike-HKU-PRC was cloned into pSC, pcDNA, and pSFV vectors.

[0116] As shown in Table 1, all 19 AU-rich RNA instability motifs of Spike-Pasteur were eliminated to generate Spike-HKU-PRC. In addition, 11 of 12 putative splice donor and acceptor sites, as well as internal poly(A) and repeat and secondary sequence stretches, were removed.

[0117] Spike-Pasteur

Spike-HKU-PRC

AU-rich RNA instability motif

19

0

Repeats and secondary sequence segments

1

0

splice donor and acceptor sites

12

1

interna...

Embodiment 3

[0123]The Spike protein expression of pcDNA-Spike-Pasteur and pcDNA-Spike-HKU-PRC was compared by transfecting the plasmid into 293T cells using the calcium phosphate method. Spike protein was not observed in cells transfected with pcDNA-Spike-Pasteur. In contrast, high levels of Spike protein were detected in 293T cells transfected with pcDNA-Spike-HKU-PRC (Fig. 1). The migration and oligomerization patterns of the spike protein were consistent with previous results, suggesting that this plasmid can express the full-length, native conformation of the SARS CoV protein. These results demonstrate that codon optimization of the Spike coding sequence resulted in dramatic improvements in expression outcomes.

[0001] related application

[0002] This application claims the benefit of U.S. Patent Application No. 10 / 860,641, filed June 4, 2004, and U.S. Provisional Patent Application No. 60 / 578,348, filed June 10, 2004, which are hereby incorporated by reference The above application is incorporated into this application. A request to amend US Patent Application No. 10 / 860,641 as a provisional application was filed on May 23, 2005. technical field

[0003] The present invention relates to purified and isolated nucleic acids, polypeptides, purified and isolated polypeptides, nucleic acids encoding such polypeptides, methods of producing recombinant forms of such polypeptides, antibodies raised against these polypeptides, and such nucleic acids and polypeptides Use in diagnostic methods, kits, immunogenic compositions, vaccines or antiviral therapy. Background technique

[0004] In 2002, a new infectious disease called Severe Acute Respiratory Syndrome (SARS) emerged in Guangdong Pr...

Examples

Embodiment 1

[0111] A spike gene (Spike gene) from a patient infected with SARS CoV was obtained, called Spike-Pasteur (SEQ ID NO: 1). The Spike-Pasteur was cloned into pcDNA eukaryotic expression vector and transfected into 293T cells. Cells transfected with pcDNA-Spike-Pasteur did not express the Spike-Pasteur polypeptide, as no detectable levels of the Spike protein were observed by either FACS or Western blotting.

[0112] Spike-Pasteur was then expressed in a SFV viral expression vector capable of efficient expression in transfected BHK cells. However, the yield of SFV virions was low due to the presence of 2 Spe I sites in the spike gene. Spe I is commonly used to linearize the plasmid at the end of the SFV coding sequence. Since Spe I could not be used, PSFV-Spike-Pasteur was linearized using Sph I, which generated an additional 3' RNA sequence of >2000 bases of the carrier RNA. Representative expression levels from cells infected with SFV-Spike-Pasteur were weak.

[0113] To en...

Embodiment 2

[0115] Further bioinformatics analysis was performed on the Spike-Pasteur sequence. The cDNA for Spike-Pasteur contains many cis-acting sites that can negatively affect expression. To further enhance expression, 32 of the 33 negative cis-acting signals identified were eliminated from Spike-Pasteur and an additional signal that stimulated gene expression was added, resulting in Spike-HKU-PRC (SEQ ID NO: 3). Spike-HKU-PRC was cloned into pSC, pcDNA, and pSFV vectors.

[0116] As shown in Table 1, all 19 AU-rich RNA instability motifs of Spike-Pasteur were eliminated to generate Spike-HKU-PRC. In addition, 11 of 12 putative splice donor and acceptor sites, as well as internal poly(A) and repeat and secondary sequence stretches, were removed.

[0117] Spike-Pasteur

Spike-HKU-PRC

AU-rich RNA instability motif

19

0

Repeats and secondary sequence segments

1

0

splice donor and acceptor sites

12

1

interna...

Embodiment 3

[0123]The Spike protein expression of pcDNA-Spike-Pasteur and pcDNA-Spike-HKU-PRC was compared by transfecting the plasmid into 293T cells using the calcium phosphate method. Spike protein was not observed in cells transfected with pcDNA-Spike-Pasteur. In contrast, high levels of Spike protein were detected in 293T cells transfected with pcDNA-Spike-HKU-PRC (Fig. 1). The migration and oligomerization patterns of the spike protein were consistent with previous results, suggesting that this plasmid can express the full-length, native conformation of the SARS CoV protein. These results demonstrate that codon optimization of the Spike coding sequence resulted in dramatic improvements in expression outcomes.

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