Cloning and Expression of a New Gene from Shark Liver and its Inhibitory Effects on Hepatoma Cells

Abstract

Aim To clone and express a new gene psl12 from shark liver, purify the expression product, and study its inhibitory effects on hepatoma cells. Methods and Results We have previously reported the discovery of Hepatocyte regenerational stimulatory factor from shark liver (sHRSF). According to the sequence of the N-terminal amino acid residues of sHRSF and using RT-PCR method, we obtained 350 bp cDNA fragment from the total RNA extracted from regenerated hepatic tissues. The cDNA has an ORF of 333 nucleotides and encodes a peptide of 111 amino acid residues. The front 7 N-terminal amino acid residues agree with those of sHRSF. We named this peptide PSL12 (peptide from shark liver with a molecular mass of about 12 kDa). The cDNA was ligated into plasmid pGEM-T-Easy and obtained recombinant plasmid pGEM-T-psl12. Based on the sequence of psl12 gene and multiple cloning sites ( BamH I and Sal I restriction sites) of expression plasmid pET-32a, the specific primers for PCR amplifying psl12 gene were designed and synthesized. The PCR was operated using plasmid pGEM-T- psl12 as template. The expression plasmid pET-32a- psl12 was constructed by inserting the PCR product into pET-32a and was transformed into an E. coli host cell BL21. After inducing with IPTG, the fusion protein with his-tag was expressed to about 40% and was purified using metal chelation chromatography on His-Bind resin. After the fusion protein was cleaved with FXa, PSL12 was purified using Resource Q and Mono Q column chromatography. The purified PSL12 could inhibit proliferation of the hepatoma cell lines SMMC-7721 and HepG 2. Conclusions A new gene psl12 was obtained from shark liver. The expression product PSL12 of the gene could inhibit proliferation of the hepatoma cells cultured in vitro.