siNgR重组质粒载体构建及效应检测

目的 构建具有特异阻断大鼠NgR基因功能的siRNA表达系统,为视神经损伤基因治疗提供新的方法 .方法 实验研究.根据Genbank提供的NgR基因mRNA序列,应用设计软件设计特异性的短链寡核苷酸,化学合成2对编码短发夹RNA的寡核苷酸序列,退火、克隆到经BglⅡ、Hind Ⅲ酶切处理的pSUPER-EGFP质粒,获得重组siNgR质粒,用EcoR Ⅰ和HindⅢ双酶切和序列测定对重组体进行鉴定,最后将构建的表达载体转染Wistar大鼠体内视网膜神经节细胞,免疫印迹法观察对NgR蛋白表达的影响.结果 重组siNgR表达载体的酶切鉴定结果 和测序结果 表明重组载体构建成功.视网膜冰冻切片后,经荧光显微镜观察,视网膜神经节细胞层和视神经纤维可见绿色荧光蛋白表达.免疫印迹法分析表明,siNgR-1和siNgR-2可抑制RGCs NgR蛋白的表达,而siNgR-c和pSUPER-EGFP对照对NgR蛋白的表达无明显影响.结论 成功构建了siRNA表达载体,此载体具有阻断NgR基因的表达的功能,为进一步研究视神经损伤基因治疗打下基础.(中华眼科杂志,2008,44:244-247)

Construction of siRNA expression vector of NgR and its inhibition on the expression of NgR

OBJECTIVE: To construct the expressing vector of siRNA in order to inhibit NgR expression, which provides a new gene therapy approach to the optic nerve impairment. METHODS: It was a experimental study. Specific short chain oligonucletides was designed by using the siRNA software according to the mRNA sequence provided by genebank, the double chain DNA sequence was gained through annealing after chemosynthesis and was inserted to the pSUPER-EGFP vector linearized with Bgl II and Hind II enzyme, the recombinant expression vector was evaluated by using EcoR I and Hind II enzyme cutting and sequencing. At last, the constructed vectors were transfected into wistar rat retinal ganglion cells and the expression level of NgR was observed. RESULTS: Identification by enzyme cutting and sequencing showed that the expression vector was constructed successfully, and it was proved that the NgR expression level was effectively inhibited. CONCLUSION: The constructed siRNA expression vectors can block the NgR gene expression, it will be the foundation for the research of gene thrapy to optic nerve regeneration.