人补体受体1型活性片段SCR118在毕赤酵母中的表达纯化及活性.DOC

人补体受体1型活性片段SCR15-18在毕赤酵母中的表达、纯化及活性鉴定 刘高科 何莉 杨永涛 汪正清(（第三军医大学基础部微生物教研室，重庆400038） 摘要：目的 实现人补体受体1型（CR1）活性片段SCR15-18在毕赤酵母中的分泌表达，为大规模工业发酵奠定基础。方法 用PCR从原核表达质粒pET32a-sCR1- SCR15-18 上扩增CR1-SCR15-18的编码基因，并克隆到毕赤酵母分泌表达质粒pPIC9，构建重组质粒pPIC9-CR1-SCR15-18，鉴定后测序；重组质粒电转化整合到毕赤酵母GS115基因组中，菌落PCR技术筛选阳性转化株；经摇瓶发酵和甲醇诱导，SDS和Western blot鉴定目的蛋白的表达；Ni2+-NTA agarose亲和层析CR1-SCR15-18蛋白，目的蛋白能够明显抑制补体的体外溶血。结论 在毕赤酵母中成功实现了CR1-SCR15-18蛋白的分泌表达，具有较高的抑制补体溶血的生物活性。 关键词 补体受体1型 毕赤酵母 分泌表达 补体溶血 Expression，Purification and Activity Identification of SCR15-18 domain of Human CR1 in Pichia PastorisDepartment of microbiology , Third Military Medical University ,Chongqing, 400038，China） Abstract ：Objective xpress SCR15-18 domain of human CR1 in Pichia pastoris as a secreted protein for making a foundation of large-scale industry fementation．Methods The gene of CR1SCR15-18 was amplified by PCR from pET32a-sCR1-SCR15-18．The obtained sequence was cloned into Pichia pastoris secretory expression vector pPIC9；identification the recombinant plasmid pPIC9-CR1-SCR15-18 was electrotransformed into Pichia Pastoris；clones w identified using colony PCR；Positive recombinants were fermented in shake flaskes and induced with methanol；the recombinant proteins were identified with SDSand Western blot；the biological activityrecombinant protein was detected by inhibiting complement hemolysis in vitro after purification by Ni-NTA agarose metal chelate affinity chromatography；Results　he recombinant protein was successful secreted into culture supernatant and inhibited complement hemolysis． Conclusion　CR1SCR15-18 was successful expressed in Pichia Pastoris with high activity of inhibiting complement hemolysis．Keywords：CR1 　Pichia Pastoris yeast 　secretory expression complement hemolysis补体系统是先天和获得性免疫的重要组成部分，也是炎症反应因子。体内补体级联的活化受到一系列膜蛋白和血浆蛋白的抑制调节，补体异常过度活化则可以引起缺血再灌注损伤、超敏反应、移植反应和肾小球肾炎等多种严重的疾病[1]。补体受体1 型(complement receptor type 1,CR1)含有个SCR(short consensus repeat)，其中SCR15-18为CR1的活性位点之一，结合C3b抑制C3 /C5转化酶，辅助I因子裂解C3b/C4b，进而抑制C5a及攻膜复合的产生