Screening, Expression And Identification Of Multi-copy Recombinant HaSNPV Chintinase Strain And Optimization Of Its Cultivating Conditions

Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus(HaSNPV)is H. armigera-concentrated pathogeny microorganism characterized in unique host scope, high-toxin on target,harmless,safety and no pest drug-resistance.The genome of this virus has been completely sequenced and a chitinase gene belonging to family 18 chitinases was identified.The chitinase gene was considered to be essential and in liquefying infected cadavers.Therefore,further study on this gene will play an important role in heredity improvement on virus pesticide and building new expression system.This research tries to use eukaryotic expression system in order to overcome the purification difficulty and inclusion body formation of HaSNPV chitinase in the prokaryotic expression system.First,the chitinase gene was cloned to pPIC9K eukaryotic expression plasmid,and then electroporate the pichia GS115 competent cells.After intensive ridding,Mut~+ strain of contradiction 4.0mg/ml G418 can be obtained.The recombinant strain is pre-expressed below 30℃and induced by methanol.Identified with SDS-PAGE,the expression target protein(96 hours)could get to 50%of the whole secretory protein.When the chitinase activity is measured according to Schales method,the enzyme activity reaches up to 66U(108 hours)per ml supernatant of fermentation culture.After experiments with 6 aspects related with protein expression and analyse the protein yield,the best cultivating condition of the recombinant strain was considered to be 29℃,pH 6.0,250rpm,108 hours ferment,with 1.0%methanol and 0.05%oleic acid.Meanwhile the culture medium has been optimized by the orthogonal design, which is 1.0%methanol,0.67%of YNB,1:1 of the volume between BMGY and BMMY,1.5%of yeast extract.Under these condition,the protein expression of multi-copy recombinant chitinase gene strain is 171.99mg/L,and enzyme activity is 96.50U/mL。This research have built a good foundation for further study on phenotype transformant Mut~s,the properties and functions about HaSNPV chitinase,and also for extending production of HaSNPV chitinase using multi-copy recombinant chitinase gene GS 115-pPIC9K-ChiA.