Cloning and Expression of a Chitinase Gene from Serratia marcescens Strain C8-8
摘要
A 1692 bp long chitinase-encoding chiA gene was cloned from the genomic DNA of Serratia marcescens strain C8-8 by PCR, which was speculated to encode a 563 aa long polypeptide chain with molecular weight of about 60.9 kD.Homology analysis showed that the chiA gene sequence cloned from C8-8 shared the highest similarity with chiA sequences from Serratia marcescens strains 141 (DQ 990373.1) and 14041 (DQ 493896.1), which reached 99%.Domain analysis showed that N-terminal (23 aa) of the chiA gene cloned from C8-8 harbored typical signal peptide sequence, while C-terminal harbored the other two domains, including the PKD region (73 aa) and chitinase catalytic region (387 aa).The PCR fragment was digested with restriction endonucleases and cloned into plasmid pET28a.The recombinant plasmid pET28a-chiA was firstly transformed into Escherichia coli DH5, and then transformed into expression host E.coli DH3 to express chiA gene.The recombinant strain DH3 chiA could produce transparent hydrolysis circles on the colloidal chitin plate induced by isopropyl-1-thiogalactopyranoside (IPTG).SDS-PAGE electrophoresis analysis showed that, a protein with relative molecular weight of about 60 kD was expressed by the recombinant strain DH3 chiA, which was consistent with the except molecular weight.After initial purification, biological activity test showed that the recombinant expression product could hydrolyze chitin, which produced transparent hydrolysis circles on the colloidal chitin plates.Results indicated that chiA gene from Serratia marcescens strain C8-8 had biological functions and could be utilized as a potential biological control factor.