人SmD1抗原的酵母真核表达及其初步应用

Eukaryotic Expression and Primarily Application of Human Smith D1 Antigen in Methylotrophic Yeast Pichia pastoris

摘要:

通过PCR扩增Sm D1基因, 与酵母表达载体pPIC9k重组, 构建表达质粒pPIC9k-Sm D1。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。结果显示PCR产物约为360 bp, 符合预期, pPIC9k-Sm D1重组阳性克隆双酶切鉴定正确,测序结果与GenBank核酸数据库报道完全一致。表达产物Sm D1的分子量约16 kD, 高拷贝毕赤酵母转化菌的表达水平明显高于低拷贝的。immunodot证实表达产物具有天然Sm D1分子的免疫原性。阴性对照菌未见目的表达条带。Immunodot的敏感性为96%, 特异性为100%, 与免疫印迹法(immunoblot, IBT)的符合率为98%。Sm D1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。

Abstract:

To clone, express and primarily use human autoantigen Sm D1 in methylotrophic yeast Pichia Pastoris. The gene Sm D1 was cloned by PCR.The PCR product was inserted into the vector pPIC9k. The recombinant plasmid pPIC9k- Sm D1 was transformed into yeast SMD1168 by electroporation. 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. The PCR product was showed about 360 bp in size which was in accordance with predicted. The pPIC9k-Sm D1 showed the same seqencing result with GenBank’s report and restriction enzyme analysis confirmed our prediction. The pPIC9k-Sm D1 positive clone produced an about 16 kD protein which had natural immunogenicity of human autoantigen Sm D1 by SDS-PAGE and immunodot. The sensitivity and specificity of immunodot were 96% and 100%, respectively. The agreement between immunodot and immunoblot was 98%. Successfully cloning and high-level expression of human autoantigen Sm D1 in methylotrophic yeast Pichia pastoris laid a foundation for further research work.