人干燥综合征B抗原在巴斯德毕赤酵母中的高效分泌表达研究  

HIGH-LEVEL SECRETED EXPRESSION OF RECOMBINANT BUMAN SJOGREN'S SYNDROME ANTIGEN B IN METHYLOTROPHIC YEAST PICHIA PASTORIS

作　　者：杨湘越[1] 兰小鹏[1] 吴文冰[1] 冯福英[1] 廖剑[1] 朱忠勇[1] 

机构地区：[1]福州总医院全军检验医学研究所

出　　处：《福州总医院学报》2007年第3期153-155,共3页Journal of Fuzhou General Hospital

摘　　要：目的:通过基因克隆在巴斯德毕赤酵母中表达人干燥综合征B抗原(SS-B)。方法:PCR扩增SS-B基因,与酵母表达载体pPIC9k重组,构建表达质粒pPIC9k-SS-B。用电穿孔法转化酵母菌SMD1168,在MD平板上筛选重组克隆,用G418快速筛选高拷贝转化子,阳性克隆经甲醇诱导表达后,培养上清用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和免疫酶斑点法(immunodot)鉴定。结果:PCR产物约为1200 bp,与预期1224 bp接近,pPIC9k-SS-B重组阳性克隆测序结果与GenBank核酸数据库报道完全一致,双酶切鉴定正确。表达产物SS-B的分子量约48 000,高拷贝毕赤酵母转化菌的表达水平明显高于低拷贝的,表达量占分泌总蛋白60%以上,产物浓度为800- 900mg/L。免疫酶斑点法证实表达产物具有天然SS-B分子的免疫原性。阴性对照菌未见目的表达条带。结论:SS-B在巴斯德毕赤酵母中获得高效分泌表达,为下一步研究打下了基础。Objective： To clone and express human Sjogren＇ s syndrome antigen B （SS- B） in methylotrophic yeast Pichia Pastoris. Method： The gene SS- B was cloned by PCR.The PCR product was inserted into the vector pPICgk.The recombinant plasmid pPICgk - SS- B was transformed into yeast SMDl168 by eleetroporation. The positive clones were screened in MD plates. The high copy number transformants were rapidly selected by using G418 and were induced by methanol. Supernatants after induction were analyzed by SDS - PAGE and immunodot. Results： The PCR product was about 1 200 bp in size which was in accordance with predicted 1 224 bp. The pPICgk - SS- B showed the same seqencing result with GenBank＇s report and restriction enzyme analysis confirmed our prediction. The pPIC9k- SS - B positive done produced a 48 000 protein which had natural immunogenicity of human autoantlgen SS - B by SDS - PAGE and immunodot. The expression product of high copy number transformants amounted to 800 - 900 rag/llter, over 60% of the total secreted protein. Conclusion： Successfully cloning and high - level expression of human autoantigen SS - B in methylotrophic yeast Pichia Pastoris laid a foundation for further research work.

关 键 词：人干燥综合征B抗原 克隆表达 巴斯德毕赤酵母 

分 类 号：R373.21[医药卫生—病原生物学]