鱼源Ⅲ型抗冻蛋白的生物学活性

Research on Biological Activity of Fish TypeⅢ Antifreeze Protein

为了通过细菌低温保护实验测定鱼源Ⅲ型抗冻蛋白(AFPⅢ)对大肠杆菌的低温保护效果,利用前期构建的原核表达质粒pET32a(+)-AFPⅢ转化大肠杆菌,目的蛋白以融合his标签的形式在大肠杆菌中表达,经NI柱亲和层析得到较高纯度的AFPⅢ-his融合蛋白。细菌抗冻实验的结果表明,外加一定浓度范围内的纯化的AFPⅢ融合蛋白对细菌有明显保护作用,但是抗冻蛋白的浓度对细菌的保护力不成一致的线性关系。鱼源Ⅲ型抗冻蛋白的浓度在50μg/mL时抗冻效果最好,并且随着温度的下降,抗冻蛋白对细菌的保护能力与Glycerol对细菌的保护能力的差距逐渐减少,在-80℃时基本可以代替甘油用于菌种保存。

In order to measure the protective effect of fish typeⅢ antifreeze protein on E. coli,a cryoprotective experiment was employed through counting the number of colonies after bacteria at low temperature for hours. Using pre-constructed prokaryotic expression plasmid pET32a （ ＋ ）-AFPⅢ transformed into E. coli. The target protein expressed in E. coli as a fusion form of his tags binding protein induced by IPTG. And AFP lll-his fusion protein was purified by affinity chromatography. The experimental results showed that type IU Antifreeze Protein can produce high protective effect on bacteria at a certain concentration. But the cryoprotective activity of typeⅢ antifreeze Protein increase,the concentration of bacteria on the contrary the protective effect reduce. Conclusion：The cryoprotective activity of typeⅢ antifreeze protein is highest when its concentration is 50μg/mL and following the temperature lower the disparity between AFPⅢ and 25% Glycerol reduced. It can replace Glycerol for the bacteria preservation at -80℃.