EXPRESSION OF MURINE POLYOMAVIRUS-LIKE PARTICLES WITH FIMBRIAL PROTEIN OF Pasteurella multocida.

Haemorrhagic septicaemia (HS) an acute septicaemic disease caused by the bacteria Pasteurella multocida, is an almost neglected disease although it had been significantly caused high mortality in infected buffaloes and cattles. A great effort has been made to search for vaccine candidates for HS using different approaches and methodologies, as the vaccine that is currently available only confer short-term protection with side effects. Thus, this study was conducted to construct a recombinant protein expression system expressing Murine polyomavirus-like particles and the fimbrial protein of Pasteurella multocida for future application as vaccine for HS in livestock. Virus-like particles (VLPs) are viral subunit that mimics the conformation of authentic viruses without requirement of containing infectious genetic material. In this study, polymerase chain reaction (PCR) was used to isolate and amplify fimbrial protein gene of P. multocida B:2 strain. To introduce restriction enzyme (RE) site at the fimbrial gene sequence, PCR was employed using the upstream primer and downstream primer with NotI and XhoI site. The ~435 bp of amplified fimbrial protein gene was cloned into an expression plasmid containing Murine polyomavirus major protein 1 (VP1), pGEX-4T-1 plasmid, between XhoI and NotI site and was then transformed into E. coli JM109 strain. The putative transformants were verified by RE digestion and sequencing. The plasmid (pGEX-VP1) and recombinant plasmid (pGEX-VP1-fimbrial) were transformed into BL21 (DE3) cells for protein expression. Control (GST-VP1) and recombinant (GST-VP1-fimbrial) protein was optimized under various IPTG concentrations (0.4 mM to 1.0 mM) in order to produce the highest protein yield. The highest expression level of control protein (GST-VP1) was observed at 0.8 mM IPTG (~66 kDa), while the IPTG concentration yielded the highest expression level for GST-VP1-fimbrial protein is 0.6 mM which showed the size ~82 kDa. Further analysis will be conducted in order to produce the ultimate protein product which is the purified VP1-fimbrial protein in order to elucidate the potential of the purified protein as a vaccine candidate. [ABSTRACT FROM AUTHOR]