Gene Cloning,Prokaryotic Expression And Activity Analysis Of Caspase-3 And Caspase-6 Proteins From Cotton Leafworm,Spodoptera Litura

Apoptosis is an autonomous,programmed death process under normal physiological or pathological state,tightly regulated by a series of genes and proteins.Caspase,a family of cysteine proteases,which plays an irreplaceable role in apoptosis.So far,there are over fourteen caspases identitied in mammals and seven caspases identitied in Drosophila.In recent years,Caspases of Lepidoptera insects have been gradually identified and analyzed.There are some reports about apoptosis of Spodoptera litura,an insect in Lepidoptera family.However,few researches about caspase of S.litura were reported.In this study,we cloned and expressed the Sl-caspase-3,Sl-caspase-6 of S.litura,and their activities were analysed.This study provided useful information and data for further parsing molecular mechanism of cell apoptosis.1.The cloning,prokaryotic expression and activity analysis of Sl-caspase-3We cloned Sl-caspase-3,which ORF is 846bp in length,encoding 281 amino acids,predicted molecular weight of the protein is 31.8 kDa,the isoelectric point is 6.55.A five-peptide conservative domain(QACRG)of Caspase family but not the DED or CARD domain of effector caspase existed in Sl-caspase-3.The identity between Sl-caspase-3 and Bm ICE-2 is 56%.The cDNA of Sl-caspase-3 gene was amplified by PCR and inserted into the plamid pET22b and a prokaryotic expression vector was obtained to transform Rosetta cells.The recombinant caspase-3 was expressed and identified by SDS-PAGE and Western blotting.The proteins were purified and used for enzyme activity analysis.Active Sl-caspase-3 protein could cleave mutated S.litura caspase-1-C178A and caspase-5-C310A.These results suggested that caspase-3 was associated with other caspases of S.litura,but the specific function mechanism in apoptosis is not clear,which needs to be further investigated.2.The cloning,prokaryotic expression and activity analysis of Sl-caspase-6In this study,we successful cloned the Caspase-6 gene of S.litura.Its ORF is 1569 bp,encoding 522 amino acids with predicted molecular weight of 60.3 k Da and pI 6.71.The protein sequence analysis showed that Sl-caspase-6 consisted of DED domain,suggesting that it belongs to initiator caspase.SDS-PAGE and westernbloting analysis showed that the recombinant Sl-caspase-6 proteins were aut ocleaved.In order to get the active Sl-caspase-6,we constructed the plasmid pET-22b-caspase-6-N224 and expressed recombinant caspase-6-N224 protein.SDS-PAGE analysis showed that Sl-caspase-6-N224 could be activated by self-cleavage.The molecular mass of cleaved products were about 23.1kDa and 12.2kDa.The proteina se activity of purified Sl-caspase-6-N224 cleaved Ac-IEVD-AFC,an specific substr ate for human caspase-8.These results showed that Sl-caspase-6 have similar substr ate-cleaving specificity with mammalian caspase-8.This shows that Sl-caspase-6 and mammalian caspase-8 have high similarity in structure and function,suggestin g that Sl-caspase-6 is an initiator caspase,and probably mediates apoptotic death receptor pathway in S.litura.