【作者】 陈璇；

【导师】 马纪；

【作者基本信息】 新疆大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 抗冻蛋白(AFPs),也称热滞蛋白,具有降低溶液的冰点,抑制重结晶的能力。近些年,抗冻蛋白的研究主要集中在生物体的低温保护方面,且发展较快。由于天然抗冻蛋白纯化困难,成本高,使得其应用受到了限制,通过生物工程的办法获得大量生产抗冻蛋白,显得尤为必要。本文采用分子生物学技术,将新疆荒漠昆虫Blaps kashgarensis抗冻蛋白基因Bkafp972插入含AOX 1启动子和α分泌信号肽序列的巴斯德毕赤酵母(Pichia pastorris)表达载体pPIC9K中,构建重组表达质粒pPIC9K- Bkafp972,转化大肠杆菌E.coli Top10 F’,序列经分析后,取阳性转化子转化Pichia pastorris GS115,筛选多拷贝整合转化子。在0.5 %甲醇诱导条件下小量摇瓶发酵。Tricine SDS-PAGE、Western blotting和HPLC等技术被用于鉴定目的蛋白的表达,通过低温菌落保护试验,及重组蛋白对溶液冰晶形态修饰的观察检测目的蛋白的生物活性。结果显示,目的基因片断正确的插入穿梭质粒pPIC9K,目的基因没有发生突变,DNA测序结果说明了目的基因插入的正确性,成功的构建了重组表达载体pPIC9K- Bkafp972。Western blotting分析重组蛋白已成功表达,且产物具有良好的抗原性,这与Tricine SDS-PAGE和HPLC的结果相一致。表达粗提产物具有较强的细菌低温保护活性,且具有类似天然抗冻蛋白的溶液冰晶修饰能力。将新疆荒漠昆虫戈壁琵琶甲(Blaps kashgarensis)AFP基因Bkafp972原核表达,制备了特异性的抗血清,测量了原核表达产物的热滞活性。然后使用一种全新的静脉发送方法,将抗冻蛋白应用到生物器官的低温保护研究中。肝脏组织切片的结果表明,这种方法可以有效的保护小鼠肝脏在低温条件下不受冰冻的伤害。更多还原

【Abstract】 Antifreeze proteins (AFPs), known as thermal hysteresis proteins have the ability to inhibit ice nucleators and ice recrystallization. In recent years, researches on antifreeze proteins are mainly concentrated on the cryoprotection of AFP to organisms. However, the purification of enough amounts of natural antifreeze proteins is difficulty, which leads to the restriction of the application of AFP. To obtain mass production of antifreeze proteins, the employment of bioengineering technique might be essential.The antifreeze protein gene Bkafp972 from beetle Blaps kashgarensis was inserted into the Pichia pastorris expression vector pPIC9K, which containing AOX 1 promotor andαsecreting signal peptide. The recombinant expression plasmid pPIC9K- Bkafp972 was constructed and transformed into E. coli Top10 F’. The recombinant plasmid was transformed in to Pichia pastorris GS115. The transformants with multiple copies of insert were screened by DNA sequence analysis. The selected transformants were fermented and induced by 0.5% methanol in flasks. The protein expression was detected by TricineSDS-PAGE、Western blotting and HPLC. The biological activity of the recombinant protein was confirmed via low temperature bacteria protection experiment, and the morphology of ice crystals in the AFP solution.The results of all above showed that Bkafp972 was inserted into pPIC9K. Western blotting indicated that the recombinan protein was expressed and the product had antigenicity. The product could prevent the bacteria from freezing damage, as well as decorated the ice crystal shape when freezing occurred.In addition, Bkafp972 were prokaryotic expressed, and its antiserum was prepared. A novel strategy, vein blood transmission, for delivering AFP to protect mouse liver at -20°C was test, Microscopic cross-section showed that this method could protect the organ effectively against the low temperature freeze injury.更多还原