球孢白僵菌丝氨酸蛋白酶基因CDEP-1在毕赤酵母中的表达  

Preparation of a serine protease CDEP-1 of Beauveria bassiana by pichia pastoris expression system

作　　者：范艳华[1] 张永军[1] 罗志兵[1] 冯静[1] 方卫国[1] 裴炎[1] 

机构地区：[1]农业部生物技术与作物品质改良重点实验室重庆市农业生物技术重点实验室西南大学生物技术中心,重庆400716

出　　处：《菌物学报》2006年第1期56-62,共7页Mycosystema

基　　金：国家重点基础研究发展计划(973)项目(2003CB114203);国家高技术研究发展计划(863)项目(2002AA245021)

摘　　要：我们从球孢白僵菌中克隆了丝氨酸蛋白酶Pr1类基因CDEP-1。为明确CDEP-1的功能、评价其在害虫生物防治中的潜力,需要大量制备具有生物活性的CDEP-1编码蛋白。由于大肠杆菌系统表达真核基因存在产物复性困难的问题,本文利用毕赤酵母系统来表达CDEP-1。结果表明,CDEP-1可在毕赤酵母中高效的分泌表达,而且产物活性高,甲醇诱导48h后上清液中的酶活即可达到38,266U/L。诱导表达的上清液经浓缩后进行凝胶过滤层析,得到了CDEP-1的初纯品,蛋白质含量为50mg/L。将纯化的蛋白酶CDEP-1免疫家兔,制备了CDEP-1的抗血清。Westernblotting分析表明,制备的抗血清可特异性地检测CDEP-1。CDEP-1, a serine protease, produced by entomopathogenic fungus Beauveria bassiana, is a potential virulence factor against insects. To meet the need of assessing the roles of CDEP-1 in the pest bio-control, large amount of active CDEP-1 was required. Escherichia coli is the microbial host widely used for the production of heterologous protein. However, gain of active proteins has remained a recalcitrant problem for the E. coli expression system due to being prone to form inclusion bodies. On the other hand, the methylotrophic yeast Pichia pastoris, which can perform the post-translational modifications of enkaryotic system, has turned out to be a suitable host organism for production of active protein. To produce large scale of active CDEP-1, the gene encoding CDEP-1 was cloned into yeast expression vector, pPIC9K, and then delivered into a Pichia pastoris strain, GSllS. The recombinant CDEP-1 can be secreted into the culture medium in the recombinant Pichia pastoris, and the total enzyme activity reached 38, 266U/L at 48^th h after inducing by methanol. The product of CDEP-1 was purified to homogeneity through gel filtration chromatography detected by SDS-PAGE analysis. Results demonstrated that the Pichia pastoris expression system is useful in producing recombinant active CDEP-1 （expression levels up to 50 mg/L）. Additionally, with purified product, the rabbit antiserum against CDEP-1 was prepared by immunization.

关 键 词：毒力因子 凝胶过滤层析 抗血清 

分 类 号：S476.12[农业科学—农业昆虫与害虫防治]