【摘要】 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。更多还原

【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. METHOD AND RESULT: 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 (BamHⅠand SalⅠrestriction 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 prolifera-tion of the hepatoma cell lines SMMC-7721 and HepG2. CONCLUSION: 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.更多还原