鲨肝刺激物质类似物cDNA片段的克隆及序列分析

Cloning and Sequence Analysis of cDNA Fragment of Shark Hepatic Stimulating Substance's Similarity

目的 :从鲨鱼肝脏中分离纯化肝刺激物质 ,测定N 端氨基酸残基序列 ,根据序列分析结果 ,合成简并引物 ,获得鲨鱼肝刺激物质的cDNA序列 ,并对其进行序列分析 ,比较其与已报道序列的同源性。方法与结果 :通过离子交换和凝胶过滤层析方法 ,从鲨鱼再生肝组织中分离到一种可刺激肝癌细胞株SMMC 772 1增殖活性的多肽 ,命名为鲨鱼肝刺激物质 (sHSS)。进一步通过FPLCMonoQ柱纯化后 ,纯度可达 95 %以上 ,其得率为 4 0mg/kg肝脏 ,SDS PAGE分析表明 :sHSS的分子量约为 1 6 0 0 0Da。对sHSS的氨基酸组分及N 端氨基酸序列进行了分析 ,并根据其N 端氨基酸残基序列设计了一套简并引物 ,利用RT PCR方法从再生肝组织的总RNA中扩增到一大小为 5 0 4bp的cDNA片段 ,序列分析表明其与我们分离到的天然鲨肝刺激物质有一定的差异 ,而与已报道的肝再生增强因子 (ALR)差异较大 ,而与人、鼠以及果蝇中的一种未知功能的蛋白质在氨基酸水平上的同源性较高 ,分别为 84 % ,83%和 5 7% ,这类物质可能在生物机体中发挥重要的生理功能。结论 :通过RT PCR扩增 。

AIM:To purify the hepatic stimulating substance from regenerated shark hepatic tissues and clone its cDNA fragment. METHOD: Shark regenerated hepatic tissues were homogenized and centrifuged, and supernatant was purified by DEAE Sephadex A25, sephadex G75 and FPLC Mono Q. All components were detected by MTT method, a kind of protein which can improve the proliferation of SMMC7721 was found, and its yield was about 40 milligrams per kilogram liver. The molecular weight of this protein is about 16,000 Da, which was detected by SDS PAGE. The composition of amino acids and their 10 N terminal amino acid residues were also analyzed. A set of degenerated oligonucleotide primers were designed according to its N terminal amino acid residues, and partial cDNA sequence which is about 504 bp, was amplified from the total RNA extracted from shark regenerated hepatic tissues. RESULT:A kind of protein which called shark hepatic stimulate substance (sHSS) were found in regenerated shark hepatic tissues, and its cDNA was cloned and sequenced. CONCLUSION:Sequence analysis shows 84%, 83% and 57% homology with a kind of unknown function protein in human, rat and fruit fly, but has lower homology with the augmenter of liver regeneration (ALR) found in the liver of human or rat.