CLONING AND EXPRESSION OF THE CRIMEAN-CONGO HEMORRHAGIC FEVERVIRUS GLYCOPROTEINS

Abstract: Crimean-Congo Hemorrhagic Fever (CCHF) is a worldwide tick-borne disease that originally belongs to the Bunyaviridae family, the genus Nairovirus. In addition to infection from ticks, humans become infected if any contact with infected blood or tissue material occurs. To study the disease, several methods such as real-time Polymerase Chain Reaction, enzyme-linked immunosorbent assay and Immunofluorescence assay are used for detection of the virus. All viruses in Bunyaviridae consists of three single stranded RNA sequences, the small, the medium and the large segment, that encode for the nucleocapsid protein, the glycoproteins, GN and GC, and the RNA-dependent RNA polymerase, respectively. The main purpose of this study was to express the M RNA segment´s glycoproteins, GN and GC. By using the reverse transcription reaction, the cDNA was synthesized from vRNA and the M RNA sequence was amplified using Phusion DNA-polymerase. In the storage vector, pcDNA3.1/V5-His-TOPO, the insert was ligatured followed by transformation into Escherichia coli. Restriction digestion was made with specific enzymes that cut out the insert. In the second ligation and transformation two different expression vectors (pTM1/pI.18) was used. After observation of the gel analysis from the test-PCR, an insert in the expression vector was shown.