细胞因子midkine基因的克隆及其转化甲醇酵母Pichia pastoris

Cloning of midkine gene and its transformation of Pichia pastoris system

目的克隆细胞因子midkine(MK)基因,并转化甲醇酵母Pichia pastoris。方法利用RT-PCR方法从人胃癌MGC803细胞总RNA中扩增MK cDNA,构建分泌表达载体pPic9k-MK,然后转入宿主菌GS115中,提取GS115的基因组DNA,用PCR法扩增鉴定阳性克隆。结果成功构建了分泌表达载体pPic9k-MK,并转入GS115中,筛选到抗4.0 mg/mlG418的重组菌株,鉴定出5个阳性克隆。结论MK基因可以转入甲醇酵母Pichia pastoris中,为今后在Pichia pastoris中表达有活性的MK蛋白,并进一步研究其生物学功能和作用机制奠定了实验基础。

Objective To clone midkine （MK） gene and transfer it into Pichia pastoris GSllS. Methods MK gene was amplified by RT-PCR from total RNAof MGC803 cells of human gastric cancer cell line and subcloned it into Pichia pastoris GSllS. Genomic DNA in GS115 was extracted. The transfected MK gene in positive clones was confirmed by PCR. Results A secretory expressive vector （pPic9k-MK） had been constructed, which had been transfected into GS115. Five strains of transfectants resisting to 4.0 mg/ml G418 were selectively identified to show multi-copy target genes. Conclusion MK gene could be introduced into Pichia pastoris, which will help to express the active protein encoded by MK in Pichia pastoris. It is significant for further studying its biological functions and roles.