新型10-23脱氧核酶表达载体的构建及其效能研究  

Construction and efficacy study of a novel 10-23 deoxyribozyme expression vector

作　　者：李俊明[1] 万腊根[1] 张才成[1] 王娜[1] 罗清[1] 

机构地区：[1]南昌大学第一附属医院检验科,330006

出　　处：《中华医学杂志》2009年第38期2708-2712,共5页National Medical Journal of China

基　　金：国家自然科学基金（30600528）

摘　　要：目的构建一种新型10-23脱氧核酶（DZ）真核表达载体，并鉴定其在细胞内表达10—23DZ的能力以及10-23DZ抑制巨噬细胞TACO表达的效果。方法设计1条寡脱氧核糖核苷酸ODN—PSL，其序列包含鼠莫洛尼白血病病毒逆转录酶（MLV—RT）的引物结合序列、1个多克隆酶切位点（MCS）及1个逆转录终止信号序列。将ODN—PSL插入框架质粒pBUDCE4．1中启动子PCMV的下游，获取重组质粒pBUD—PSL。将MLV—RT的编码基因克隆至pBUD—PSL中另一启动子PEF-1a的下游，构建重组质粒pSDE01。将pSDE01转染入鼠巨噬细胞系RAW264．7细胞，采用RT—PCR方法鉴定细胞中MLV-RT的表达及其表达产物的逆转录酶活性。通过计算机软件模拟巨噬细胞TACOmRNA的二级结构，以此确定10-23DZ的作用靶位并设计一相应的10—23DZ——DZ1，将DZ1表达序列插入pSDE01中ODN—PSL的MCS中，获取重组表达载体pSDE01-DZ1。将pSDE01-DZ1转染入RAW264．7细胞，并设未转染对照组。转染后48h，采用PCR法和斑点杂交方法鉴定DZ1在细胞中的表达；并分别采用半定量RT-PCR和蛋白质印迹法检测TACO mPNA和蛋白的表达，各组实验均独立重复3次。结果限制性内切酶酶切及测序结果显示pSDE01构建成功，将其转染入RAW264．7细胞后检测到MLV-RT的表达，而且表达产物具有逆转录酶活性。pSDE01-DZ1转染入RAW264．7细胞后48h，PCR和斑点杂交方法均检测到DZ1的表达；半定量RT-PCR和蛋白质印迹检测结果显示，与未转染组相比，pSDE01-DZ1转染后48h巨噬细胞TACOmRNA表达水平下降了77．7％（0．193±0．008比0．864±0．005，P〈0．05），TACO蛋白表达量下降了73．3％（0．114±0．051比0．427±0．043，P〈0．05）。结论成功构建了一种操作简便的新型10—23DZ表达载体，所携带的特异性10-23DZ可在细胞内有效表达，并抑制相应靶基因的表达。Objective To construct a novel 10-23 deoxyribozyme （10-23DZ） expression vector, identify the intraeellular production of specific 10-23DZ and its inhibitory effect upon the expression of TACO gene in macrophage. Methods An oligonueleotide containing the primer binding sequence of mouse Moloney leukemia viral reverse transcriptase （MLV-RT） , a multiple cloning site （MCS） and a stem-loop structure for termination of reverse transcription was designed and inserted into one MCS of p]asmid pBUDCE4.1, downstream of the cytomegalovirus promoter （ PCMV ） The gene fragment encoding MLV-RT was inserted into another MCS of pBUDCFA. 1, downstream of elongation factor let promoter （PEF-1a）. The resulting plasmid was named pSDE01. Then pSDE01 was transfeeted into RAW204.7 cell and the expression of MLV-RT and the reverse transcriptase activity of expression product was identified by RT-PCR. To identify the cellular expression of 10-23DZ by pSDEO1, a 10-23DZ targeting the TACO mRNA of macrophage was designed according to the predicted secondary structure of TACO mRNA. The expression sequence of designed 10-23DZ, DZ1, was synthesized and inserted into the MCS of ODN-PSL in pSDE01. The resulting plasmid, pSDE01-DZ1, was transfected into RAW264.7 cell and the expression of DZ1 was identified by PCR and dot-blot respectively. At 48 h after transfection of pSDEO1 into RAW264.7 cells, total RNA and proteins were extracted and the TACO mRNA and protein expression level was assayed by RT- PCR and Western blotting respectively. Results Restrictive analysis and sequencing data showed that the 10-23DZ expression vector, pSDEO1, was successfully constructed and could express MLV-RT with reverse transcriptase activity in cell. When transfected into RAW264. 7 cells, pSDE01-DZ1 expressed DZ1 effectively and inhibited the expression of TACO gene. And TACO mRNA decreased by 77.7% （0. 193 ± 0. 008 vs 0.864 ± 0.005, P 〈 0.05） and TACO protein decreased by 73.3% （0.114 ± 0. 051 vs 0.427 ± 0. 043, P 〈 0.05 ）. Con

关 键 词：DNA 催化性 表达载体 微丝蛋白质类 巨噬细胞 

分 类 号：R686[医药卫生—骨科学]