摘要
产于我国黄海的黄盖鲽(Pseudopleuronectes yokohamae)体内含有能阻止血液冰冻的抗冻肽(Antifreeze peptide AFP)。在对该蛋白进行纯化及对其特性的一系列研究的基础上,我们合成了一段抗冻肽基因的寡核苷酸片段。以此为引物,与黄盖鲽的mRNA进行杂交,从而特异性地反转录出抗冻肽基因cDNA片段。该片段经EcoRI Linker连接法克隆至大肠杆菌(E.coli)质粒载体pUC19上。抗冻肽cDNA插入片段经杂交证实后,对其进行了酶谱分析,核酸序列测定。重组克隆还在大肠杆菌JM83中得到了表达。
Pseudopleuronectes yokohamae, existing in the Yellow Sea of China, contains antifreeze peptide (AFP). This protein could depress the serum freezing point. On the basis of purification and characterization of this protein, we synthesized a segment of Oligo-nucleotides of antifreeze protein gene as a primer and hybridized with the mRNA of Pseudopleuronectes yokoh-amae. The cDNA of antifreeze protein gene has specifically been reversly transcripted. This segment has been cloned onto pUC19 with Ecor I linker method. After confirmation of the insert segment of the cDNA of antifreeze protein gene, the zymogram was detected and the sequence of the nucleotides was determined. This recombinated clone was transformed into E. coli JM83 and expressed well.
基金
农牧渔业部水产局支持课题
关键词
鱼类
抗冻蛋白
黄盖鲽
CDNA
克隆
Pseudofleuronectes yokohamae, cDNA clone, Antifreeze peptide, Expres-sion in prokaryotes
