A mutagenesis library was constructed using GPS-LS system to insert a random 5 aa into the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoded by aroA gene. Active EPSPS pro- teins were identified by the abili...A mutagenesis library was constructed using GPS-LS system to insert a random 5 aa into the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoded by aroA gene. Active EPSPS pro- teins were identified by the ability to rescue growth of aroA-deleted mutant ER2799 on M9 minimal media. 12 unique sites, which can tolerate a 5-aa insertion, were identified. In all of the 12 sites, only F295/T296 site was found to split the G2-EPSPS properly by co-transformation of plasmids into E. coli ER2799. The G2-EPSPS gene was then divided into N-termi- nal and C-terminal from F295/T296 site which were fused to the N-terminal and C-terminal of Ssp.DnaE intein, respectively, creating two plasmids pMEPS- N295IN and pKEPSc296Ic. Co-transformation of plasmids, pMEPSN295IN and pKEPSc296Ic, rescu- ed growth of ER2799 in M9 minimal media, indicating that the intein splicing domains were bringing the EPSPS fragments together to generate activity. Re- consituted activity of splitted G2-EPSPS enzyme was 4.48 U/mg.展开更多
目的观察精神分裂症断裂基因1(dierupted in schizophrenia 1,DISC1)基因多态性与帕利哌酮治疗精神分裂症疗效及微小RNA-134-5p(miR-134-5p)的关联性分析。方法选取2018年3月至2019年3月绵阳市第三人民医院精神分裂症病人109例为疾病组...目的观察精神分裂症断裂基因1(dierupted in schizophrenia 1,DISC1)基因多态性与帕利哌酮治疗精神分裂症疗效及微小RNA-134-5p(miR-134-5p)的关联性分析。方法选取2018年3月至2019年3月绵阳市第三人民医院精神分裂症病人109例为疾病组,采用帕利哌酮单药治疗,选取同期健康体检者109例为健康组,检测并比较两组DISC1基因单核苷酸多态性(SNPs)位点间差异。比较疾病组不同基因型病人帕利哌酮治疗前与治疗6个月后阳性和阴性症状量表(PANSS)、精神分裂症认知功能成套测验共识版(MCCB)评分以及血清miR-134-5p水平。结果 DISC1 SNPs位点比较中,疾病组rs821616位点与健康组差异有统计学意义(P<0.05)。疾病组不同基因型病人帕利哌酮治疗后PANSS评分均较治疗前降低,MCCB评分较治疗前升高(P<0.05)。rs821616基因型病人中TT型病人PANSS评分(46.3±7.1)分低于AA型病人(59.4±5.8)分,TT型病人MCCB评分(154.7±32.3)分高于AA型病人(131.3±32.1)分,AT型(0.32±0.07)和TT型(0.41±0.09)病人miR-134-5p水平高于AA型病人(0.30±0.11)。rs11122319基因型病人中GG型、AG型病人PANSS评分低于AA型病人,GG型病人MCCB评分和miR-134-5p水平高于AA型和AG型病人(P<0.05)。rs1417584基因型病人中GG型、AG型病人PANSS评分低于AA型病人,GG型病人MCCB评分高于AA型和AG型,GG型病人miR-134-5p水平高于AA型病人(P<0.05)。结论帕利哌酮可以改善精神分裂症病人症状,改善病人认知功能,提高miR-134-5p水平,不同DISC1基因型病人间治疗效果存在差异。展开更多
目的探索精神分裂断裂基因1(DISC1)对APP/PS1转基因阿尔茨海默病(Alzheimer′s disease,AD)小鼠突触可塑性及学习记忆能力的影响。方法 Western blot比较正常人和AD患者脑组织中DISC1的表达情况;体外培养C57胎鼠神经元,分为GFP组、GFP+...目的探索精神分裂断裂基因1(DISC1)对APP/PS1转基因阿尔茨海默病(Alzheimer′s disease,AD)小鼠突触可塑性及学习记忆能力的影响。方法 Western blot比较正常人和AD患者脑组织中DISC1的表达情况;体外培养C57胎鼠神经元,分为GFP组、GFP+Aβ组、DISC1+Aβ组,激光共聚焦显微镜观察神经元树突棘。取2月龄的C57小鼠,海马注射对照病毒和DISC1过表达病毒,切脑片后Aβ处理,膜片钳技术检测长时程增强(LTP),评价突触可塑性。将C57小鼠分为WT+GFP组、TG+GFP组、TG+DISC1组三组,免疫荧光染色方法检测细胞中小鼠脑中Aβ斑块沉积;Morris水迷宫检测各组小鼠学习、记忆能力。结果与正常人比较,AD患者大脑中DISC1的表达量减少(P <0.05);与GFP组比较,GFP+Aβ组树突棘数量减少,而DISC1+Aβ组较GFP+Aβ组树突棘数量增多;Aβ处理后,小鼠脑切片的LTP减弱,而过表达DISC1后再行Aβ处理,LTP较只用Aβ处理增强;TG+DISC1组较TG+GFP组小鼠海马中的Aβ斑块减少(P <0.05),且寻找平台的时间更短、穿越平台的次数更多(P <0.05)。结论过表达DISC1对APP/PS1转基因AD小鼠突触可塑性具有保护作用并能够改善其学习记忆能力。展开更多
文摘A mutagenesis library was constructed using GPS-LS system to insert a random 5 aa into the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoded by aroA gene. Active EPSPS pro- teins were identified by the ability to rescue growth of aroA-deleted mutant ER2799 on M9 minimal media. 12 unique sites, which can tolerate a 5-aa insertion, were identified. In all of the 12 sites, only F295/T296 site was found to split the G2-EPSPS properly by co-transformation of plasmids into E. coli ER2799. The G2-EPSPS gene was then divided into N-termi- nal and C-terminal from F295/T296 site which were fused to the N-terminal and C-terminal of Ssp.DnaE intein, respectively, creating two plasmids pMEPS- N295IN and pKEPSc296Ic. Co-transformation of plasmids, pMEPSN295IN and pKEPSc296Ic, rescu- ed growth of ER2799 in M9 minimal media, indicating that the intein splicing domains were bringing the EPSPS fragments together to generate activity. Re- consituted activity of splitted G2-EPSPS enzyme was 4.48 U/mg.
