自身抗原组氨酰转移核糖核酸合成酶基因在巴斯德毕赤酵母中的分泌表达研究

目的:通过基因克隆在巴斯德毕赤酵母中表达人自身抗原组氨酰转移核糖核酸合成酶(HRS或Jo-1)。方法:PCR扩增Jo-1基因,与酵母表达载体pPIC9k重组,构建表达质粒pPIC9k-Jo-1。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SDS-PAGE和免疫酶斑点法鉴定。结果:PCR产物长约1500bp,与预期1526bp接近;pPIC9k-Jo-1重组阳性克隆测序结果与GenBank核酸数据库的报道完全一致,双酶切鉴定正确,表达产物Jo-1的相对分子质量约55000,免疫酶斑点法证实表达产物具有天然Jo-1分子的免疫原性,阴性对照菌未见目的表达条带。结论:Jo-1在巴斯德毕赤酵母中分泌表达成功,为后续研究打下了基础。

Secreted Expression of Recombinant Human Autoantigen Histidyl-tRNA Synthetase in Methylotrophic Yeast Pichia pastoris

Objective: To clone and express human autoantibody histidyl-tRNA synthetase(HRS or Jo-1) in methylotrophic yeast Pichia pastoris. Methods: The gene Jo-1 was cloned by PCR. The PCR product was inserted into the vector pPIC9k. The recombinant plasmid pPIC9k-Jo-1 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. Results: The PCR product was about 1 500 bp in size which was in accordance with predicted 1 526 bp. The pPIC9k-Jo-1 showed the same seqencing result with GenBank's report and restriction enzyme analysis confirmed our prediction. The pPIC9k-Jo-1 positive clone produced a 55 000 protein which had natural immunogenicity of human autoantigen Jo-1 by SDS-PAGE and immunodot. Conclusion: Successfully cloning and expression of human autoantigen Jo-1 in methylotrophic yeast P.pastoris laid a foundation for further research work.