S.18. Cloning, expression, purification and homology modeling of envelope protein of Kyasanur forest disease virus (KFDV)
Abstract
Kyasanur Forest disease virus (KFDV) is a highly pathogenic member of family Flaviviridae producing a haemorrhagic disease in infected human beings. This virus is BSL-4 grade organism and listed under Class-A group of Bio-warfare agents. Despite this high pathogenicity and potential epidemiological importance, there have been relatively few detailed antigenic or molecular studies on KFD virus. Envelope protein of flaviviruses are of immense importance, as is known to be the principal target of host immune response and known to play critical role in invasion of the viruses to susceptible host cells. Envelope protein of KFDV shares 80% sequence homology with TBEV and 38–40% with dengue virus, JEV, WNV and YFV with positionally conserved cysteines. We did homology modeling of KFD virus glycoprotein E using TBEV and Dengue-2 virus envelope structure as template. It exhibited homologous sequence and structure of fusion peptide as that of TBEV indicating similar mechanism of virus membrane fusion. Homology modeling of Domain III of envelope protein indicated that the possible mechanism of receptor-ligand interaction involved in infection and pathogenesis of the KFDV may be the same as TBEV. Based on codon bias for E. coli, we designed a synthetic gene construct for envelope gene of KFDV in pDrive vector. The gene construct was sub-cloned to pQE-30 vector and expressed in E. coli SG13009. The protein is found to localize in intracellular soluble fractions and is ~60kDa in size. The recombinant KFDV envelope protein was purified to homogeneity by single step affinity chromatography. The purified protein reacted with penta-His monoclonal antibody and KFD vaccinated human sera in western blot analysis. The protein is suitable for development of rapid diagnostic kit for KFD and may have potential to be used as alternate vaccine candidate.
