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目的:从鲨鱼肝脏中克隆表达一种新基因psl12,纯化该基因的表达产物,研究其对肝肿瘤细胞的抑制作用。方法与结果:我们前面已报道发现了鲨肝细胞再生刺激因子(sHRSF),根据sHRSF的N端氨基酸序列设计了引物,并利用RT-PCR方法从鲨鱼再生肝组织的总RNA中扩增到大小约为350bp的cDNA片段,序列分析表明350bp的片段含有一个开放阅读框(ORF),含有333个碱基,编码111个氨基酸残基,其编码的N端氨基酸序列与天然sHRSF的前7个氨基酸一致。该肽被命名为PSL12(来源于鲨肝的分子量约为12kD的肽)。该cDNA被连接到质粒pGEM-T-Easy,获得重组质粒pGEM-T-psl12。根据cDNA序列分析和质粒pET-32a的多克隆位点(BamHⅠ和SalⅠ)的序列,设计并合成了psl12基因的特异性引物,质粒pGEM-T-psl12作为模板,进行了PCR扩增。将此PCR产物插入pET-32a得到表达质粒pET-32a-psl12,并将它转化入E.coli的BL21菌株。经IPTG诱导,带有组氨酸标签的融合蛋白的表达量约为40%。用金属螯合层析纯化融合蛋白后,再用FXa切割融合蛋白,经Resource Q和Mono Q柱层析得到PSL12纯品,它可抑制肝肿瘤细胞株SMMC-7721和HepG2的增殖。结论:从鲨鱼肝脏中获得了一种新基因psl12。该基因的表达产物PSL12能显著抑制体外培养的肝肿瘤细胞的增殖。
OBJECTIVE: To clone and express a novel gene psl12 from shark liver and purify its expression product to study its inhibitory effect on hepatic tumor cells. Methods and Results We have previously reported the discovery of shark hepatocyte stimulating factor (sHRSF), primers designed based on the N-terminal amino acid sequence of sHRSF, and amplified from total RNA from shark liver tissue by RT-PCR A 350bp cDNA fragment showed that the 350bp fragment contained an open reading frame (ORF) containing 333 bases and encoded 111 amino acid residues encoding the first 7 amino acids of the native sHRSF Consistent. This peptide was named PSL12 (a peptide of about 12 kD in molecular weight derived from shark liver). The cDNA was ligated into the plasmid pGEM-T-Easy to obtain the recombinant plasmid pGEM-T-psl12. According to the cDNA sequence analysis and the sequence of multiple cloning sites (BamHⅠ and SalⅠ) of plasmid pET-32a, a specific primer of psl12 gene was designed and synthesized. The plasmid pGEM-T-psl12 was used as a template for PCR amplification. This PCR product was inserted into pET-32a to obtain expression plasmid pET-32a-psl12 and transformed into E. coli BL21 strain. After induction with IPTG, the expression of histidine tagged fusion protein was about 40%. After purification of the fusion protein by metal chelation chromatography, the fusion protein was cleaved with FXa and purified by Resource Q and Mono Q column to obtain pure PSL12, which can inhibit the proliferation of hepatoma cell lines SMMC-7721 and HepG2. Conclusion: A novel gene psl12 was obtained from shark liver. The gene expression product PSL12 can significantly inhibit the proliferation of cultured liver tumor cells in vitro.