Vectors expressing SARS immunogens, compositions containing such vectors or expression products thereof, methods and assays for making and using

A recombinant baculovirus vector that comprises:

• an isolated nucleic acid comprising a nucleotide sequence GCCCTTGTAC AAATTGTTAA ACGTTTTGTG GTTGGTCGCC GTTTCTAACG CGAT (SEQ ID NO:
• 29) encoding a baculovirus signal peptide,
• wherein the recombinant baculovirus vector also expresses a SARS S protein,
• wherein expression of the SARS S protein is under control of a polyhedrin promoter.

A method for producing a biologically active, highly pure, recombinant SARS S protein, comprising:

• (a) infecting insect cells that grow in serum-free media with a recombinant baculovirus that comprises DNA coding for SARS S protein such that the SARS S protein is expressed recombinantly;
• (b) culturing the infected insect cells in serum-free media;
• and
• (c) purifying the recombinant SARS S protein to 95% or greater purity, wherein:
• the biologically active, highly pure, recombinant SARS S protein is produced;
• and
• is biologically active such that it agglutinates red blood cells.

A method for preparing a baculovirus transfer vector for preparing a recombinant baculovirus that expresses a heterologous protein and a baculovirus signal peptide, wherein DNA coding for the heterologous protein is seamlessly joined to DNA coding for the baculovirus signal peptide, without addition of a single nucleotide, comprising:

• (a) cutting an initial vector having DNA coding for a leader sequence and a restriction site at a distance from the restriction site by an enzyme that so cuts, whereby the restriction site is excised from the initial vector, and the initial vector has a unique sticky end;
• (b) performing in a separate reaction a nucleic acid amplification reaction of DNA coding for the SARS S protein to obtain an amplification product, whereby the restriction site is part of the amplification product;
• (c) cutting the amplification product with the enzyme, whereby the amplification product has a unique sticky end;
• and
• (d) ligating the initial vector having the unique sticky end and the amplification product having the unique sticky end such that the transfer vector is obtained and intervening nucleic acid molecules between the DNA coding for the leader sequence and the DNA encoding the SARS S protein are avoided.