Kong Xin-Juan, Song Yu-Hu, Lin Ju-Sheng, Huang Huan-Jun, Wang Nan-Xia, Liu Nan-Zhi, Li Bin, Jin You-Xin
Institute of Liver Disease, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, China.
World J Gastroenterol. 2003 Jul;9(7):1571-5. doi: 10.3748/wjg.v9.i7.1571.
To evaluate the specific inhibition of maxizyme directing against mutant-type p53 gene (mtp53) at codon 249 in exon 7 (AGG-AGT) in vitro.
Two different monomers of anti-mtp53 maxizyme (maxizyme right MzR, maxizyme left MzL) and control mutant maxizyme (G(5)-A(5)) were designed by computer and cloned into vector pBSKU6 (pBSKU6MzR, pBSKU6MzL). After being sequenced, the restrictive endonuclease site in pBSKU6MzR was changed by PCR and then U6MzR was inserted into pBSKU6MzL, the recombinant vector was named pU6Mz and pU6asMz (mutant maxizyme). Mtp53 and wild-type p53 (wtp53) gene fragments were cloned into pGEM-T vector under the T7 promoter control. The (32)p-labeled mtp53 transcript was the target mRNA. Cold maxizyme transcripts were incubated with (32)p-labeled target RNA in vitro and radioautographed after denaturing polyacrylamide gel electrophoresis.
In cell-free systems, pU6Mz showed a specific cleavage activity against target mRNA at 37 degrees and 25 mM MgCL(2). The cleavage efficiency of pU6Mz was 42 %, while pU6asMz had no inhibitory effect. Wtp53 was not cleaved by pU6Mz either.
pU6Mz had a specific catalytic activity against mtp53 in cell-free system. These lay a good foundation for studying the effects of anti-mtp53 maxizyme in HCC cell lines. The results suggest that maxizyme may be a promising alternative approach for treating hepatocellular carcinoma containing mtp53.
在体外评估针对第7外显子密码子249（AGG-AGT）处突变型p53基因（mtp53）的maxizyme的特异性抑制作用。
通过计算机设计两种不同的抗mtp53 maxizyme单体（maxizyme右侧MzR，maxizyme左侧MzL）和对照突变型maxizyme（G(5)-A(5)），并克隆到载体pBSKU6（pBSKU6MzR，pBSKU6MzL）中。测序后，通过PCR改变pBSKU6MzR中的限制性内切酶位点，然后将U6MzR插入pBSKU6MzL，重组载体命名为pU6Mz和pU6asMz（突变型maxizyme）。将mtp53和野生型p53（wtp53）基因片段克隆到T7启动子控制下的pGEM-T载体中。(32)p标记的mtp53转录本为靶mRNA。将冷的maxizyme转录本与(32)p标记的靶RNA在体外孵育，变性聚丙烯酰胺凝胶电泳后进行放射自显影。
在无细胞系统中，pU6Mz在37℃和25 mM MgCL(2)条件下对靶mRNA表现出特异性切割活性。pU6Mz的切割效率为42%，而pU6asMz无抑制作用。pU6Mz也不能切割wtp53。
pU6Mz在无细胞系统中对mtp53具有特异性催化活性。这些为研究抗mtp53 maxizyme在肝癌细胞系中的作用奠定了良好基础。结果表明，maxizyme可能是治疗含有mtp53的肝细胞癌的一种有前景的替代方法。
