细胞周期蛋白D1特异性核酶表达载体的构建及其活性

Construction of Specific Hammerhead Ribozyme Targeting Cyclin D1 mRNA and Its Activity in Vitro and Hepatec Stellate Cells

应用mfold程序对锤头状核酶(ribozyme,Rz)和大鼠细胞周期蛋白(cyclin)D1基因的二级结构进行分析,设计合成锤头状Rz基因,通过RT-PCR扩增获得大鼠细胞周期蛋白D1目的基因,将Rz基因和细胞周期蛋白D1基因分别克隆入载体pGEM-3Zf(+)中,体外转录Rz基因和靶基因并进行切割实验;将Rz基因与逆转录病毒载体pLXSN重组得到Rz真核表达载体pLXSN-Rz,将其转染入HSC-T6细胞,G418筛选出阳性细胞克隆,用RT-PCR检测细胞周期蛋白D1基因的表达。结果显示:针对目的基因的832位点设计合成了Rz832,成功获得Rz832基因、细胞周期蛋白D1mRNA的体外转录载体pGEM3Zf-Rz832和pGEM3Zf-cD1,经体外转录出Rz832(105nt)及细胞周期蛋白D1mRNA(1079nt)。体外切割实验证实Rz832能够特异性切割细胞周期蛋白D1mRNA,产生1014nt和65nt的切割产物,切割效率为80%。所构建的pLXSN-Rz832经酶切电泳、PCR鉴定显示,插入的Rz832序列大小约为57bp,与预期结果相同,经测序证实Rz832序列正确。转染pLXSN-Rz832的肝星状细胞(hepaticstellatecells,HSCs)细胞周期蛋白D1mRNA的表达受到明显抑制,仅为对照组的42.22%(t=-193.443,P<0.01),结果表明:Rz832能够在体外特异性切割细胞周期蛋白D1mRNA、并在HSC-T6细胞内有效抑制细胞周期蛋白D1基因的表达。

The secondary structures of hammerhead ribozyme （Rz） and rat cyclin D1 were analyzed and simulated by mfold software. Hammerhead ribozyme DNA sequence was synthesized. Cyclin D 1 DNA sequence was obtained by reverse transcription PCR. Ribozyme and cyclin D1 DNA sequences were separately cloned into pGEM-3Zf（＋） vectors. Ribozyme and cyclin D1 mRNA were obtained in vitro transcription, and ribozyme cleavage experiment was made in vitro, pLXSN-Rz vector was constructed by inserting ribozyme gene into retroviral vector and transfected into rat-derived HSC-T6 cell line with lipofectin. The expression of cyclin D1 gene was detected by RT-PCR. The results showed that a ribozyme directing to 832 site of cyclin D1 mRNA coding domain was selected by mfold software, and its DNA sequence was synthesized. The transcription vectors of Rz832 and cyclin D1 mRNA were constructed successfully. Ribozyme and cyclin D1 mRNA were expressed in vitro transcription. Cleavage experiments in vitro showed that Rz832 cleaving cyclin D1 mRNA produced 1 014 nt and 65 nt fragments. The cleavage efficiency was 80%. Restriction analysis showed that Rz832 DNA was correctly inserted into plasmid pLXSN as expected. Sequencing showed that there were no mutation in the ribozyme gene. The expression of cyclin D1 at mRNA level was effectively down-regulated in Rz832-transfected hepatic stellate cells （HSCs） by 42.22% of that in control cells （t=-193.443, P〈0.01）. The results indicated that Rz832 can specifically cleave cyclin D1 mRNA in vitro and significantly inhibit cyclin D1 gene expression in HSCs.