摘要
目的 构建表达p38丝裂原活化蛋白激酶（p38MAPK）-β基因的小分子干扰RNA（siRNA）载体,并观察感染神经元后表达.方法 p38MAPK-β mRNA序列中选择1个特异性靶序列,体外合成对应发卡样DNA片段,经退火后,将其定向克隆导入siRNA载体,获得重组质粒p38MAPK-β-短发卡RNA （shRNA）,后者与慢病毒试剂共转染神经元细胞,同源重组产生p38MAPK-β-siRNA-Lentivirus.经聚合酶链反应（PCR）鉴定目的基因的表达并测定病毒滴度.结果 PCR结果表明p38 MAPK-β-siRNA-Lentivirus构建正确,病毒滴度为3.5×108 TU/ml.结论 成功构建和筛选出p38MAPK-β-siRNA特异性介导的重组慢病毒载体.
Objective To construct a p38 mitogen-activated protein kinases （p38MAPK）-β recombinant lentivirus vector carrying small interfering RNA （siRNA） and observe the expression in neuronal cells transfected with this vector.Methods A target-specific sequence from p38MAPK-β mRNA was used to synthesize the corresponding hairpin DNA fragments in vitro.Mter annealing,the DNA products were cloned into siRNA vector to obtain the recombinant siRNA vector plasmid p38MAPK-β-shRNA.The neuronal cells were co-transfected by the lentivirus vector and p38MAPK-β-shRNA,and the recombinant vector of p38MAPK-β-siRNA-lentivirus was obtained.The expression of the transfected genes was evaluated by polymerase chain reaction （PCR） and the titer of purified virus was determined.Results The identification of PCR showed that the recombinant p38MAPK-β-siRNA-lentivirus plasmid was successfully constructed,and the titer of virus was 3 × 10s TU/ml after purification.Conclusion The recombinant lentivirus vector with p38MAPK-β-targeted shRNA is successfully constructed and screened.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2013年第11期2388-2390,共3页
Chinese Journal of Experimental Surgery
基金
湖北省自然科学基金资助项目（2012FFB05602）
湖北省卫生厅科研项目（JX6B35）
