大黄鱼类Ⅳ型抗冻蛋白基因的克隆、表达及功能研究

Cloning, Expression and Characterization of Antifreeze Protein Ⅳ-like Gene from Large Yellow Croaker (Pseudosciaena Crocea)

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SSHcDNA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664bp,包含372bp开放阅读框,编码124个氨基酸,经SignalP3.0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BL21(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。

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 eDNA library. The complete eDNA 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 eDNA contained 664 base pairs, including a 372 bp open reading frame which codes for a 124-residue large yellow croaker AFP IV-like protein precursor. The 20-amino acid sequence of N terminal protein could be a signal peptide with SignalP 3.0 server. The deduced amino acid sequence was highly homologous to longhorn sculpin AFP IV-like gene with 52 % identity. RT-PCR revealed that the mRNA was expressed only in the liver of large yellow croaker. The gene encoding product was also successfully expressed with pGEX-4T-Lycafp vector in E.coli BL21（DE3） and purified by the affinity chromatography. Tests on low temperature protection to bacteria showed that GST fusion protein had no remarkable antifreeze activity. Conclusion The large yellow croaker AFP IV-like gene encoding product has no distinct antifreeze activity, which is consistent to the fact that large yellow croaker can not survive in low temperature.