Expression Of HaSNPV Gene In E.coli And Pichia Pastoris GS115

Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HaSNPV) had been extensively used for the control of H. armigera since the first isolation of this pathogen in Hubei province of China. The genome of this virus has been completely sequenced, and then a chitinase gene belonging to family 18 chitinases was identified. The chitinase was found to be essential in liquefying infected cadavers.In order to express the HaSNPV chitinase gene in E.coli as extracellular and excretive fusion protein, the HaSNPV chitinase gene from pET28a-ChiA was subcloned into expression vector pMAL-p2X. The recombinant plasmid pMAL-p2X -ChiA was transformed into TB1 and induced by IPTG. Western blot analysis proved that fusion protein could be successfully expressed and was excretive and soluble partly. The fusion protein MBP- ChiA could cover about 14.7% of the total protein in E.coli TB1 after 4h of 1mM IPTG inducted. About 20% of the target fusion protein was soluble.Some questions occurred when the HaSNPV chitinase gene was expressed in E.coli. So the plasmid pUC19-ChiA was used as a template , and then DNA of ChiA was obtained by PCR and cloned into plasmid pPIC9K to construct expression plasmid pPIC9K-ChiA. After being linearized with restriction endonuclease SacI, the recombinant plasmid was transformed into Pichia pastoris GS115 by electroporation. The transformants with high level of G418 resistance on the YPD and fast growth on the MM plates were selected. After the selected transformants was grown in BMGY medium and induced by methanol, the culture supernatant was collected by centrifugation and analyzed by SDS-PAGE. The result showed that the recombinant ChiA molecular mass was about 70KDa and amounts 50% total soluble proteins by gel analysis.