大黄鱼类IV型抗冻蛋白基因的克隆、表达及功能研究.pdfVIP

食品与药品 Food and Drug 2009 年第 11卷第09期 15 大黄鱼类IV型抗冻蛋白基因的克隆、表达及功能研究 * 张 晓，吴海珍 ，卫 玮，徐红艳，张元兴 （华东理工大学生物反应器工程国家重点实验室，上海 200237） 摘 要：目的 研究大黄鱼类IV型抗冻蛋白基因编码产物的功能。方法 从大黄鱼肝脏 SSH cDNA文库筛选到的类似极地 鱼类IV型抗冻蛋白的EST序列出发，用RACE方法克隆IV型抗冻蛋白基因全长cDNA ，RT-PCR分析此基因在大黄鱼不同组 织内的表达。构建原核表达载体pGEX-4T-Lycafp并表达蛋白质，亲和层析纯化GST融合蛋白，分析它的体外细菌抗冻保护 作用。结果 大黄鱼类IV型抗冻蛋白基因cDNA全长664 bp ，包含372 bp开放阅读框，编码124个氨基酸，经 Signal P 3.0软件 预测其N端前20位氨基酸为信号肽，氨基酸序列与长角杜夫鱼IV型抗冻蛋白同源性高达52 % ；RT-PCR分析表明其mRNA 仅在肝脏表达；重组蛋白在大肠杆菌BL2 1 （DE3 ）中成功表达，初步纯化后分析体外细菌抗冻保护作用表明，融合蛋白 抗冻活性并不明显。结论 大黄鱼类IV型抗冻蛋白不具有显著的抗冻活性，这与大黄鱼不能在低温下生存吻合。 关键词：鱼类IV型抗冻蛋白；RACE ；抗冻活性；大黄鱼 中图分类号：R973 文献标识码：A 文章编号：1672-979X （2009 ）09-0015-05 Cloning, Expression and Characterization of Antifreeze Protein IV-like Gene from Large Yellow Croaker (Pseudosciaena Crocea) * ZHANG Xiao, WU Hai-zhen , WEI Wei, XU Hong-yan, ZHANG Yuan-xing (State Key Lab of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China) Abstract: Objective To investigate the function of antifreeze protein (AFP) IV-like gene from large yellow croaker. Methods The EST fragment which was similar to the polar fish AFP IV gene was screened from the large yellow croaker liver SSH cDNA library. The complete cDNA of large yellow croaker AFP IV-like gene was cloned by the RACE method. The tissue expression pattern of the AFP IV-like gene was analyzed by RT-PCR. The prokaryotic expression vector pGEX-4T-Lycafp was expressed and GST fusion protein was purified by the affinity chromatography. The antifreeze protection of GST fusion protein to bacteria was detected in vitro. Results The complete cDNA contained 664 base pairs, including a 372 bp open reading frame which codes