小鼠野生型和截短型睫状神经营养因子基因的克隆及真核表达

Cloning and eukaryotic expression of wild type and truncated mouse ciliary neurotrophic factor gene

目的构建野生型和截短型小鼠睫状神经营养因子(ciliary neurotrophic factor,CNTF)基因的真核表达载体,并在ARPE-19细胞系表达目的蛋白。方法通过逆转录聚和酶链反应(RT-PCR)扩增小鼠CNTF野生型全长编码序列,体外定点突变获取截短型CNTF的互补DNA编码序列,将上述两序列分别克隆至pTracer-CMV真核表达载体,转染ARPE-19细胞,免疫印迹检测2种CNTF的表达。结果PCR成功扩增了野生型和截短型CNTF基因,DNA序列分析证实2种真核表达载体中的CNTF序列与Gene Bank中目的序列一致。2种重组真核表达质粒转染ARPE-19细胞后,免疫印迹证实CNTF在ARPE-19细胞培养上清中有表达。结论野生型和截短型CNTF基因在ARPE-19细胞系的真核表达为视网膜色素变性基因治疗研究奠定了基础。

Objective To construct the eukaryotic expression vectors of wild type and truncated mouse ciliary neurotrophic factor （CNTF） gene and to observe the interest proteins in ARPE-19 cell line. Methods RT-PCR was used to amplify the cDNA of CNTF gene, and truncated CNTF cDNA was obtained by site-directed mutagenesis. The two types of CNTF gene were cloned into plasmid pTracer-CMV and transfected to ARPE-19 cells. Dot blotting was used to detect the expression of CNTF. Results Wild type and truncated CNTF gene were amplified by RT-PCR, and their eukaryotic expression plasraids were successfully constructed. After ARPE-19 cells transfected with two types of recombinant plasmids, the CNTF had been detected in the cells culturing supernatant. Conclusion Expression of two types of CNTF in ARPE-19 cells gets prepared for gene therapy research of retinitis pigmentosa.