竞争抑制0610009E02Rik长链非编码RNA重组腺病毒穿梭载体的构建与鉴定  

Construction and identification of competitive 0610009E02Rik long noncoding RNA recombinant adenovirus shuttle vector

作　　者：杨娜[1] 陈翀[1,2] 秘红岭 李小莉[1] 陈朝[1] 褚培培[1] 夏园[1] 姚海娜 徐开林[1,2] 曾令宇[2] 

机构地区：[1]徐州医学院附属医院血液科,221002 [2]徐州医学院移植免疫实验室

出　　处：《国际输血及血液学杂志》2015年第3期192-199,共8页International Journal of Blood Transfusion and Hematology

基　　金：国家自然科学基金资助项目（81370602）

摘　　要：目的 构建可竞争抑制肝细胞内0610009E02Rik长链非编码RNA（lncRNA）的重组腺病毒穿梭载体,为探索0610009E02Rik/Notch1在肝静脉闭塞病（HVOD）肝细胞损伤修复中的作用提供有效实验方法.方法 采用基因组DNA提取试剂盒对截取的C57BL/6小鼠尾进行基因组DNA的提取,通过PCR扩增出0610009E02Rik与Notch1基因第34个外显子重叠的1 000 bp基因序列,并连接入经BamHⅠ/Nhe Ⅰ双酶切后的腺病毒穿梭载体GV359;竞争抑制0610009E02Rik lncRNA重组腺病毒穿梭载体与辅助包装质粒pBHG共转染HEK293细胞,并对竞争抑制0610009E02Rik lncRNA重组腺病毒进行包装、扩增,采用氯化铯（CsCl）不连续密度梯度离心与连续密度梯度离心2个步骤对重组腺病毒进行浓缩纯化,并对重组腺病毒滴度进行测定;测定不同感染复数（MOI）竞争抑制0610009E02Rik lncRNA重组腺病毒感染肝细胞株H2.35的感染效率,并采用实时聚合酶链反应（RT-PCR）与Western blotting对竞争抑制0610009E02Rik lncRNA重组腺病毒感染肝细胞株H2.35与同步培养的对照肝细胞中竞争抑制0610009E02Rik lncRNA片段、Notch1 mRNA及0610009E02Rik lncRNA相对表达水平及其蛋白表达水平,并进行统计学分析.结果 ①经基因序列比对分析发现0610009E02Rik与Notch1的第34个外显子存在分子量为1 000 bp的重叠序列,依据0610009E02Rik与Notch1基因序列设计含BamH Ⅰ/Nhe Ⅰ酶切位点特异性引物,通过PCR扩增出片段长度为1 000 bp的0610009E02Rik与Notch1基因第34个外显子重叠序列的竞争抑制0610009E02Rik lncRNA片段,DNA测序结果显示竞争抑制0610009E02Rik lncRNA重组腺病毒穿梭载体与理论预期序列构建成功.②将竞争抑制0610009E02Rik lncRNA重组腺病毒穿梭载体转染HEK293细胞后第12天,约90％细胞出现细胞病变（CPE）,收集细胞获取病毒液.浓缩纯化病毒液经梯度稀释在感染HEK293细胞后第10天,于显微镜下观察稀释梯度1∶ （10～1010）均出现Objective To construct a competitive adenovirus vector against 0610009E02Rik,a long noncoding RNA （lncRNA） for investigating its role in the repairment of injured hepatocytes during hepatic veno-occlusive disease （HVOD）.Methods Firstly,genome DNA was extracted from the tail of C57BL/6 mice,and amplified the overlapping region of the gene 0610009E02Rik and the 34th exon of Notch1 by polymerase chain reaction（RT-PCR）,then it was linked into the adenovirus shuttle plasmid GV359 which was digested by BamH Ⅰ/Nhe Ⅰ restriction enzyme.Secondly,the constructed shuttle plasmid and packaging plasmid pBHG were co-transfected into HEK293 cells for assembly of recombination adenovirus,and subsequent amplification,followed by cesium chloride （CsCl） gradient ultracentrifugation for purification and measurement of the viral titer in HEK 293 cells.Finally,the recombination adenovirus was transfected into hepatocyte cell line H2.35 with different multiplicity of infection （MOI）,and transfection efficiency was evaluated by fluorescence microscope.Meanwhile,real-time polymerase chain reaction （RT-PCR） was used to detect the expression of anti-lncRNA,Notch1 and 0610009E02Rik.Western blotting was used for the detection of the expression level of Notch1.Results ① It was discovered that the gene 0610009E02Rik and the 34th exon of Notch1 have the overlapping region （its fragment length is about 1 000 bp）,and then it was obtained by PCR with the primer which has the BamH Ⅰ/Nhe Ⅰ digestion sites,and DNA sequencing result confirmed that the competitive adenovirus shuttle plasmid against 0610009E02Rik was successfully constructed.② At the 12th day after transfection,about 90％ cells presented cytopathic effect （CPE）,then all the transfected cells were obtained a large amount of virus extract.Then HEK293 cells were transfected by the condensed and purified virus with a series of dilution,and cells transfected with dilution fold of 1 ∶ （10～1010） were all had CPE,and this is verifi the virus

关 键 词：RNA 长链非编码 受体 Notch1 腺病毒 人 DNA 重组 肝细胞 

分 类 号：R575[医药卫生—消化系统]