光滑鳖甲抗冻蛋白结构与功能的关系及其低温保护分子机理的研究

Antifreeze proteins (AFPs) can be found in many organisms to protect against cold damage by specific binding and inhibiting the growth of ice crystal. AFPs can noncolligatively reduce the freezing point with no change of the melting point, resulting in the difference between the both known as the thermal hysteresis activity (THA). This study use the antifreeze protein gene Apafp914 from the desert beetle Anatolica polita as object, and produce the mutants of ApAFP914 with variant hydrophobicity and charge by site mutation of the conserved amino acid residues. The activity of the purified ApAFP914 and its mutants is analyzed by THA and in vitro antifreeze activity assay on the frozen bacteria. The acid and basic stability, modification to the ice crystals will be observed; their secondary structure composition is detected by circular dichroism and 3-dimension structure will be predicted. On this basis, the conserved amino acid of the ice-binding site of the ApAFP914s will be deduced and the relationship between the structure and function of the insect antifreeze proteins is further to clarify by comparative analysis of ApAFP914 and its mutants. The cryoprotective effects of ApAFP914 and its mutants on the mouse oocyte and liposomes with different composition of lipids will be detected for further elucidation of its molecular mechanism for cryopreservation and provide some foundation for the ApAFP's application study.

抗冻蛋白（AFPs）是生物体产生的能够特异性地与冰晶表面结合，保护机体避免低温损伤的生物活性多肽。AFPs可非依数性地降低溶液冰点但不改变熔点，引起二者之间的差值称之为热滞活性（THA）。本研究以新疆荒漠昆虫光滑鳖甲抗冻蛋白基因Apafp914为蓝本，采用定点突变获得具有不同疏水性和电荷的光滑鳖甲抗冻蛋白ApAFP914突变体。检测ApAFP914及其突变体的热滞活性、酸碱及热稳定性和菌体体外低温保护活性；观察对冰晶的修饰作用；分析二级结构组成建立三级结构预测模型；从而确定ApAFP与冰晶结合的关键位点，并通过热滞活性及结构的比较进一步探讨昆虫抗冻蛋白结构与功能之间的关系。研究ApAFP914及其突变体对小鼠卵母细胞以及不同脂质体的低温保护活性，初步阐明其低温保护作用的分子机理，并为光滑鳖甲抗冻蛋白的应用研究提供理论依据。

抗冻蛋白（AFPs）是生物体产生的能够特异性地与冰晶表面结合，保护机体避免低温损伤的生物活性多肽。AFPs可非依数性地降低溶液冰点但不改变熔点，引起二者之间的差值称之为热滞活性（THA）。本研究以新疆荒漠昆虫光滑鳖甲抗冻蛋白基因Apafp914为蓝本，采用定点突变以及原核表达体系获得具有不同疏水性和电荷的光滑鳖甲抗冻蛋白ApAFP914突变体。检测ApAFP914及其突变体的热滞活性、酸碱及热稳定性和菌体体外低温保护活性；观察对冰晶的修饰作用；分析二级结构组成建立三级结构预测模型；从而确定Thr33,Thr45是ApAFP与冰晶结合的关键位点，而ApAFP914-A19T突变蛋白热滞活性的增加则与其增加的并结合表面积有关。ApAFP914对小鼠红细胞，卵母细胞以及脂质体的低温保护活性研究，初步阐明其低温下对细胞膜的保护效应，为光滑鳖甲抗冻蛋白的应用研究提供理论依据。