恒河猴MyD88基因沉默siRNA腺病毒载体的构建与鉴定

目的 构建恒河猴MyD88基因沉默siRNA腺病毒载体, 观察MyD88siRNA对树突状细胞中MyD88蛋白表达的影响。方法 将合成的MyD88RNAi片段与质粒载体连接, 转化进化学感受态的大肠杆菌, 然后进行菌落PCR鉴定、阳性克隆测序及质粒的抽提；然后转染HEK293细胞, 将获得的重组腺病毒进行两轮扩增后进行纯化；用终点稀释法测定腺病毒滴度；Western blot检测MyD88siRNA对树突状细胞中MyD88蛋白表达的作用。结果 阳性克隆PCR鉴定与理论条带一致, 测序鉴定通过, 腺病毒载体转染树突状细胞, Western blot检测显示干扰组MyD88蛋白表达降低。结论 成功构建恒河猴MyD88基因沉默siRNA腺病毒载体, MyD88siRNA对恒河猴树突状细胞中的MyD88蛋白表达具有抑制作用。

Construction and identification of adenovirus vector of Mucaca mulatta myD88 gene silencing

Objective To observe the effect of MyD88 siRNA on the expression of MyD88 protein in dendritic cells.Methods The recombinant MyD88 RNAi fragment was ligated into plasmid vector and transformed into chemically competent Escherichia coli, then subjected to colony PCR identification, positive cloning and plasmid extraction. HEK293 cells were transfected and the recombinant adenovirus was transfected into two groups. The effect of MyD88siRNA on the expression of MyD88 protein in dendritic cells was detected by Western blot. The effect of MyD88 siRNA on the expression of MyD88 protein in dendritic cells was detected by Western blot. Results MyD88 siRNA could inhibit the expression of MyD88 protein in mucaca mulatta dendritic cells, and the expression of MyD88 protein was inhibited by Western blot. The positive clones were identified by PCR and sequenced. The dendritic cells were transfected by adenovirus vector. Western blot showed that the expression of MyD88 protein in the interference group was decreased.Conclusion The construction of mucaca mulatta MyD88 gene silencing siRNA adenovirus vector, MyD88siRNA in dendritic cells could inhibit the expression of MyD88 protein.