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

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。

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

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 plasmids 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.