抗冻蛋白基因定向突变及其抗冻关系的研究

为探讨抗冻蛋白的抗冻机制，根据抗冻活力较高的肝型抗冻蛋白的氨基酸组成，改造皮肤型抗冻蛋白基因，使皮肤型抗冻蛋白基因的10位氨基酸－丙氨酸（A）改造成天冬氨酸（D），方法：合成一段含有突变位点的DNA片段，用其取代野生型DNA上相对应的片段，DNA测序法筛选阳性克隆并表达该蛋白，Western-blot法、氨基酸组成分析法、及质谱法鉴定表达蛋白，测定突变后抗冻蛋白的的活力并与野生型比较，以期揭示抗冻蛋白的抗冻机制。

Studies on the Relationship Between Activity and Site- directed Mutated Skin-type Antifreeze Protein

To investingate the mechanism of antifreeze protein, the gene of the skin type protein was mutated according to the amino acid sequence of liver type protein that has a higher activity than skin type. Therefore the residue alanine 10 of skin type antifreeze protein was coded and mutated into Aspartic acid. Methods: A fragment of DNA, which included the mutated amino acid site, was synthesized and then was used to replace the corresponding fragment from wild type gene. The mutated positive clone was picked up by DNA sequence assay and then was expressed. Western blot assay, amino acid analysis as well as mass spectroscopy were used to determinate the expressed mutant protein. After testing the antifreeze activity of mutant protein, the activities of both wild type and mutant proteins were compared.