雪蚤抗冻蛋白HhAFP在毕赤酵母中的表达及其抗冻作用  

Expression of Snow Flea(Hypogastrura harveyi)Antifreeze Protein HhAFP in Pichia pastoris and Its Antifreeze Effect

作　　者：钟文倩 颜倩倩 胡瑞芹 陈良标[1,2] ZHONGWen-Qian;YAN Qian-Qian;HU Rui-Qin;CHEN Liang-Biao(College of Fisheries and Life Science,Shanghai Ocean University,Shanghai 201306,China;Center for Aquacultural Breeding Research/Key Laboratory of Exploration and Utilization of Aquatic Resources,Ministry of Education/International Research Center for Marine Biological Sciences,Shanghai Ocean University,Shanghai 201306,China)

机构地区：[1]上海海洋大学水产与生命学院,上海201306 [2]上海海洋大学水产生物育种研究中心/水产种质资源发掘与利用教育部重点实验室/海洋生物科学国际联合研究中心,上海201306

出　　处：《农业生物技术学报》2024年第12期2848-2858,共11页Journal of Agricultural Biotechnology

基　　金：上海市教委研创新计划项目(2017-01-07-00-10-E00060)。

摘　　要：抗冻蛋白(antifreeze proteins,AFPs)是一类大分子蛋白质,可以与冰晶表面结合,以抑制冰晶的生长。毕赤酵母(Pichia pastoris)是一种可以表达外源蛋白的系统,可利用甲醇作为碳源进行表达获得目的蛋白。为获得高产量的具有活性的抗冻蛋白,选取昆虫雪蚤(Hypogastrura harveyi)抗冻蛋白(HhAFP),通过PCR在目的基因的5'端和3'端添加XhoⅠ、XbaⅠ酶切位点,并且在目的基因3'端添加6×His标签,将目的基因通过转化的方式添加至适用于毕赤酵母表达系统的表达载体pPICZαA中,成功构建重组表达载体pPICZαA-HhAFP。将pPICZαA-HhAFP转入毕赤酵母表达菌株X-33中,于29℃培养箱培养,通过博来霉素(zeocin)和甲醇筛选出2株阳性酵母转化子。将这2株酵母菌进行诱导表达,在29℃、250 r/min、pH 6.0的条件下,使用含1%甲醇的BMM培养基培养72 h。获得的酵母上清液进行亲和层析纯化和透析浓缩,通过基质辅助激光解吸电离飞行时间质谱(matrix-assisted laser desorption/ionization time of flight mass spectrometry,MALDI-TOF/TOF)分析,验证该蛋白为重组蛋白HhAFP。将重组蛋白HhAFP加入细胞冻存液检测其抗冻活性,结果表明,HhAFP可以显著提高冻存细胞复苏后的存活率,减少细胞损伤。通过进一步优化HhAFP的表达条件,确定培养温度为28℃,甲醇浓度为1.5%,表达培养72 h获得的重组蛋白HhAFP可达527 mg/L。该研究为通过毕赤酵母高产量表达具有活性的抗冻蛋白、实现抗冻蛋白的产业化提供了技术途径。Antifreeze proteins(AFPs)are a type of macromolecular proteins that can bind to the surface of ice crystals to inhibit the growth of ice crystals.Pichia pastoris is a system that can express foreign proteins.It can use methanol as a carbon source to express the target protein.In order to obtain high-yield active antifreeze protein,the insect snow flea(Hypogastrura harveyi)antifreeze protein(HhAFP)was selected.XhoⅠand XbaⅠrestriction sites were added to the 5'end and 3'end of the target gene through PCR.6×His tag was added to the 3'end of the target gene that could be used for identification and screening.The target gene was added to the expression vector pPICZαA suitable for the P.pastoris expression system through transformation,and the recombinant expression vector pPICZαA-HhAFP was successfully constructed.pPICZαA-HhAFP was transformed into P.pastoris expression strain X-33 by electroporation,and cultured in a 29℃incubator until positive yeast transformants grew.Two positive yeast transformants were then screened out using bleomycin(zeocin)and methanol.The expression of these 2 yeast strains was induced and cultured for 72 h using BMM medium containing 1%methanol at 29℃,250 r/min,and pH 6.0.The yeast supernatant was purified using affinity chromatography,and the obtained purified product was dialyzed and concentrated.It was analyzed by matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF/TOF)to verify that the protein was the recombinant protein HhAFP.The recombinant protein HhAFP was added to the cell cryopreservation solution to test its antifreeze activity.The results proved that HhAFP could significantly improve the survival rate of cells after cryopreservation and reduce cell damage.By further optimizing the expression conditions of HhAFP,the culture temperature was determined to be 28℃and the methanol concentration was 1.5%.The recombinant protein HhAFP obtained after 72 h of expression and culture could reach 527 mg/L.This study provides a technical approa

关 键 词：抗冻蛋白 冻存 毕赤酵母 重组表达 雪蚤抗冻蛋白(HhAFP) 发酵优化 

分 类 号：S188[农业科学—农业基础科学]