Expression and Purification of West Nile Virus prM Candidate Antigen
Dr. Ken Cornell
West Nile Virus (WNV) is a member of the mosquito borne flaviviruses that has caused almost 30,000 total cases of infection and 1,000 deaths since it emerged in the US in 1999. In 2006, Idaho was the leading state in the country for WNV cases. No vaccine is available for humans. An oral or intranasal vaccine would be ideal for ease of delivery. Since the virus is a member of the flaviviruses it has the unique property of having a structured pre-membrane protein (prM) around the nucleocapsid that contains the ssRNA genome. PrM has been shown to be a target for a protective antibody based immune response. For this reason the gene for prM was amplified from viral cDNA and expressed and purified from E. coli and Kluyveromyces systems for use in oral vaccines. For E. coli expression, the prM gene was cloned and expression in the pET200 plasmid vector that confers kanamycin resistance and yields a recombinant protein with a hexahistidine fusion suitable for Nickel chelate chromatography. To express prM in yeast, the gene was shuttled pKLAC2 plasmid, linearized and transformed into K. lactis. Positive transformants were selected based on co-expression of acetamidase that allows utilization of acetamide as the sole nitrogen source. Recombinant proteins expressed in each of these systems will be used to generate an oral vaccine.
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