小鼠睫状神经营养因子基因的克隆及真核表达载体的构建

Cloning and Eukaryotic Expression Vectors Construction of Wild Type and Truncated Mouse Ciliary Neurotrophic Factor Gene

目的:分别构建野生型和截短型小鼠睫状神经营养因子(CNTF)基因的真核表达载体。方法:通过逆转录聚合酶链反应(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(cDNA)编码序列,将上述两序列分别克隆至pGEM-TEasy载体,经限制性内切酶EcoRI和XbaI双酶切后,将野生型和截短型CNTF基因连入pTracer-CMV真核表达载体,DNA测序鉴定。结果:PCR成功扩增了野生型和截短型CNTF基因,DNA序列分析证实两种真核表达载体中的CNTF序列与GeneBank中目的序列一致。结论:野生型和截短型CNTF基因真核表达载体的成功构建为视网膜色素变性(RP)的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. Methods： RT-PCR was used to amplify wild type full-length coding sequence of CNTF gene, and the coding sequence of truncated CNTF cDNA was obtained by site-directed mutagenesis in vitro. The two types of CNTF gene sequence were cloned into pGEM-T Easy vector respectively. The cloning vectors were double-enzyme digested by restrictive enzyme EcoR I and Xba I followed by the wild and truncated CNTF gene cloned into eukaryotic expression vector pTracer-CMV. The reconbinant plasmids were verified by DNA sequence. Results： Wild type and truncated CNTF gene were amplified by PCR successfully, and unification of the CNTF gene in the constructed vectors and target sequence in gene loank were confirmed by DNA sequence analysis. Conclusion： The construction of the eukaryotic expression vector in wild type and truncated CNTF is basic preparation for gene therapy research of retinitis pigmentosa.