人肺表面活性相关蛋白A1在甲醇酵母的胞内表达

Intracellular Expression of Human Pulmonary Surfactant Associated Protein in Methyltrophic Yeast, Pichia Pastoris

摘要:

目的：研究人肺表面活性相关蛋白A1(SP-A1)在甲醇酵母中的胞内表达。方法：将编码正常人肺表面活性相关蛋白A1基因的cDNA克隆至甲醇酵母的胞内，表达载体pPIC3.5K，构建了重组质粒pPIC3.5K/SPA1，电穿孔方法转化至酵母宿主菌GS115和KM71，用PCR方法筛选阳性转化子，并用斑点印迹法、G418方法筛选多拷贝转化子，经甲醇诱导表达，SDS-PAGE和Western印迹法鉴定表达产物。结果：SP-A1基因在甲醇酵母中成功的表达了蛋白质，SDS-PAGE分析证实目的蛋白分子量为32 kD，可被SP-A多克隆抗体特异识别。结论：人组织特异SP-A1蛋白可在甲醇酵母中高效表达，表达产物具有免疫源性。

Abstract:

OBJECTIVE: To study the expression of pulmonary sufactant associated protein A1 (SP A1) in Methyltrophic Yeast, Pichia pastoris. METHODS: The cDNA encoding SP A1 derived from the healthy adult's lungs was cloned into the pPIC3.5K, an intracellular expression vector of Pichia pastoris, and then transformed into Pichia pastoris by electroporation. The PCR fast detection method and G418 resistance method were used to screen the positive transformants and the multiple copy transformants, respectively. SP A1 intracellular expression was induced by methanol. SDS PAGE and Westernblotting were applied to detect the characteristics of the product. RESULTS: SP A1 gene was highly expressed in Pichia pastoris. The molecular weight of the protein was 32 kD by SDS PAGE analysis. Anti SP A polyclonal antibodies could specifically recognize SP A1 protein. CONCLUSIONS:The human tissue specific SP A1 protein can be highly expressed in Methyltrophic Yeast, Pichia pastoris and the products can show antigenicity.