通用型10-23脱氧核酶表达载体的构建及其在巨噬细胞中的表达  

CONSTRUCTION AND IDENTIFICATION OF A GENERAL 10-23 DEOXYRIBOZYME EXPRESSION VECTOR

作　　者：李俊明[1] 王娜[1] 罗清[1] 万腊根[1] 

机构地区：[1]南昌大学第一附属医院检验科,南昌330006

出　　处：《现代预防医学》2010年第13期2498-2502,共5页Modern Preventive Medicine

基　　金：国家自然科学基金项目(30600528)

摘　　要：[目的]构建一种新型10-23脱氧核酶(Deoxyribozyme,DRz)的真核表达载体,并鉴定其在细胞内表达10-23DRz的能力。[方法]设计一条包含莫洛尼鼠白血病病毒逆转录酶(MLV-RT)引物结合序列、多克隆酶切位点及逆转录终止信号序列的寡脱氧核糖核苷酸ODN-PSL,并将其插入框架质粒pBUDCE4.1中PCMV的下游,获取重组质粒pBUD-PSL。通过二步亚克隆将MLV-RT的编码基因克隆至pBUD-PSL中另一启动子PEF-1α的下游,获取重组质粒pS-DE01。将pSDE01转染入巨噬细胞RAW264.7中,RT-PCR法鉴定MLV-RT的表达及逆转录酶活性。设计一taco基因特异性的10-23DRz—DZ1,并将其表达序列克隆入pSDE01中,获取重组表达载体pSDE01-DZ1.将pSDE01-DZ1转染入巨噬细胞RAW264.7中,分别经PCR和斑点杂交法检测DZ1在细胞内的表达。[结果]限制性内切酶酶切及测序结果显示pSDE01构建成功,将其转染入巨噬细胞RAW264.7后检测到了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,经PCR和点杂交方法均检测到了DZ1的表达。[结论]成功构建了一种操作简便的通用型10-23DRz表达载体,该载体可在真核细胞内有效表达特异性10-23DRz。[Objective] To construct a general 10-23DRz expression vector and identify its capacity of expressing specific 10-23 DRz in mammalian cells.[Methods] An oligonucleotide which contained the primer binding sequence of mouse Moloney leukemia viral reverse transcriptase（MLV-RT）,a multiple cloning sites（MCS） and a stem-loop structure for the termination of the reverse transcription was designed and inserted into one MCS of pBUDCE4.1,downstream the cytomegalovirus promoter（PCMV）.The gene fragment encoding MLV-RT was inserted into another MCS of pBUDCE4.1,downstream the elongation factor 1α promoter（PEF-1α）.The resulted plasmid was named pSDE01.pSDE01 was then transfected into RAW264.7 cell.The expression of MLV-RT and the reverse transcriptase activity of expression product was identified by RT-PCR.A 10-23DRz targeting the TACO mRNA of macrophage was designed.The expression sequence of the designed 10-23DRz,DZ1,was synthesized and inserted into the MCS of ODN-PSL in pSDE01.The result plasmid,pSDE01-DZ1,was transfected into RAW264.7 cell and the expression of DZ1 was identified by PCR and dot-blot respectively.[Results] pSDE01 could express active MLV-RT in RAW264.7 cells.pSDE01-DZ1 could express TACO mRNA specific 10-23 DRz in RAW264.7 cells.[Conclusion] pSDE01 is a general 10-23DRz expression vector.To express a desired 10-23DRz in cell,we need just insert the expression sequence into the MCS of ODN-PSL in pSDE01 through a simple clone procedure.

关 键 词：10-23脱氧核酶 表达载体 巨噬细胞 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]