To Örebro University

Tick-borne encephalitis virus (TBEV) and Langat virus (LGTV) are both members of Flavivirus genus within the Flaviviridae family. TBEV is the main pathogenic arbovirus circulating in Europe, Russia, and China. Flaviviruses are characterized by a positive sense single-stranded RNA genome and an enveloped icosahedral virion structure. Previously, it has been observed that flavivirus envelope (E) protein and non-structural protein 1 (NS1) both play a critical role in the pathology of flavivirus. Therefore, in this study, we aim to investigate flavivirus E and NS1 protein as a good target for the development of a subunit vaccine with further potential as a putative diagnostic tool to distinguish between TBEV infected from TBEV vaccinated individuals. Thus, we have generated 4 different successful constructs with TBEV (E and NS1) and LGTV (E and NS1) in the pET SUMO vector. Restriction digestion and sequencing analysis confirmed successful clones of interest and their right orientation. Next, the right clones were transformed in BL21 (DE3) one shoot chemically competent E. coli and induce the expression with 0.5 mM IPTG in culture medium following 0-4h, and 24h incubation period. Next, bacterial cell pellets were collected and used for SDSPAGE/Western blot analysis. We used the champion™ pET SUMO expression system which may produce high levels of soluble protein in bacteria. It employs a small ubiquitin-related modifier (SUMO) fusion, belonging to the growing family of ubiquitin-related proteins, to enhance the solubility of expressed fusion proteins. We have stained with 6x-His tag antibody of interest (mouse monoclonal) for targeting both TBEV- E/NS1 and LGTV-E/NS1 proteins. Among them, the expression of TBEV-NS1 and LGTV-E proteins was verified and confirmed. Several attempts have also been made to obtain the TBEV-E and LGTV-NS1 protein in E. coli cells; however, these require further optimization with a suitable time and dose of IPTG induction. We have used the BL21(DE3) expression system, which could maximize the expression of soluble protein. After successful expression, the 13-kd SUMO moiety will be cleaved by the highly specific and active SUMO (ULP1) protease at the carboxyl terminal, producing a native protein. Furthermore, a protein purification assay (e-g., NI-NTA column/ÄKTA Protein Purification Systems) will be developed to obtain native recombinant protein. The purified proteins will be studied in combination with suitable adjuvants as putative TBE subunit vaccines. They will also be characterized with the potential to develop new tools for TBE diagnostics.