源于鲨鱼肝的一种新基因的克隆表达及其对肝肿瘤细胞的抑制作用

目的：从鲨鱼肝脏中克隆表达一种新基因psll2，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，6-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-psll2。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psll2基因的特异性引物，质粒pGEM—T-psll2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—psll2，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ResourceQ和MonoQ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psll2。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。

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

AIM： To clone and express a new gene psll2 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 sHRSE 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-psll2. Based on the sequence of psll2 gene and multiple cloning sites （BamH I and Sal I restriction sites） of expression plasmid pET-32a, the specific primers for PCR amplifying psll2 gene were designed and synthesized. The PCR was operated using plasmid pGEM-T-psll2 as template. The expression plasmid pET-32a-psll2 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 HepG2. CONCLUSION： A new gene psll2 was obtained from shark liver. The expression product PSL12 of the gene could inhibit proliferation of the hepatoma ceils cultured in vitro.