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[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 eukaryotic expression vector of 10-23 deoxyribozyme (DRz) and identify its ability to express 10-23DRz in cells. [Method] An oligodeoxyribonucleotide ODN-PSL containing the MLV-RT primer binding sequence, polyclonal restriction sites and reverse transcription termination signal sequence was designed and compared Inserted downstream of PCMV in the framework plasmid pBUDCE4.1, the recombinant plasmid pBUD-PSL was obtained. The MLV-RT coding gene was cloned into the downstream of the other promoter PEF-1α in pBUD-PSL by two-step subcloning to obtain the recombinant plasmid pS-DE01. PSDE01 was transfected into RAW264.7 macrophages, and the expression of MLV-RT and the activity of reverse transcriptase were identified by RT-PCR. A taco gene-specific 10-23DRz-DZ1 was designed and cloned into pSDE01 to obtain the recombinant expression vector pSDE01-DZ1.The pSDE01-DZ1 was transfected into RAW264.7 macrophages, Dot blot hybridization was used to detect the expression of DZ1 in cells. [Result] Restriction endonuclease digestion and sequencing showed that pSDE01 was constructed successfully. After transfection into macrophage RAW264.7, the expression of MLV-RT was detected and the expression product had reverse transcriptase activity. After transfection of pSDE01-DZ1 into RAW264.7 cells, the expression of DZ1 was detected by PCR and dot blot hybridization. [Conclusion] The universal 10-23DRz expression vector was successfully constructed, which could efficiently express the specific 10-23DRz in eukaryotic cells.