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c-erbB-2原癌基因的产物是跨膜生长因子受体的一种,具有内在酪氨激酶活性,在肿瘤细胞生长信号传递中起着重要的作用.已发现该基因的扩增或蛋白的过度表达与许多腺癌的发生、发展、转移及预后密切相关.核酶(Ribozyme)因可直接切割靶RNA并能够反复利用,而在阻断有害基因的表达方面比反义核酸更有效.本文在计算机辅助下设计了C-erbB-2特异性核酶并观察了它对靶基因体外转录产物的切割作用.主要内容如下:1.以人癌基因c-erbB-2 mRNA为靶RNA,用计算机分析显示c-erbB-2 mRNA第2428～2776位和第2820～3004位之间的二级结构相对稳定,可成为核酶攻击理想的区域.该区第2438、2477、2751位是核酶的最佳切割位点;2.在计算机辅助下,根据锤头装结构设计了针对这三个位点相应的三个核酶,分别称为RZ1、RZ2、RZ3.合成了核酸RZ1基因,测序证实合成的核酶基因序列正确;3.构建并鉴定了RZ1的体外转录载体pGEM3Z-RZ1、靶基因体外转录
The product of the c-erbB-2 protooncogene is a transmembrane growth factor receptor, has intrinsic tyrosine kinase activity, and plays an important role in the signal transmission of tumor cell growth. The amplification or protein of the gene has been found Overexpression is closely related to the occurrence, development, metastasis and prognosis of many adenocarcinomas. Ribozyme can cut target RNA directly and can be used repeatedly, but it is more effective than antisense in blocking the expression of harmful genes. In this paper, C-erbB-2 specific ribozymes were designed and computer-assisted, and its cleavage of target gene in vitro transcription products was observed. The main contents are as follows:1. The human oncogene c-erbB-2 mRNA was used as target RNA. Computer analysis showed that the secondary structure of c-erbB-2 mRNA between positions 2428 to 2776 and 2820 to 3004 was relatively stable and could be an ideal region for ribozyme attack. Nucleotides 2438, 2477, and 2751 in the region were nuclear. The optimal cleavage site of the enzyme; 2. With computer-assisted design, three ribozymes corresponding to these three sites were designed according to the structure of the hammer head, called RZ1, RZ2, RZ3. The nucleic acid RZ1 gene was synthesized. Sequencing confirmed that the synthesized ribozyme gene sequence was correct; 3. Constructed and identified RZ1 In vitro transcription vector pGEM3Z-RZ1, target gene in vitro transcription