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AIM:To evaluate the specific inhibition of maxizyme directingagainst mutant-type p53 gene (mtp53) at codon 249 in exon7 (AGG→AGT) in vitro.METHODS:Two different monomers of anti-mtp53maxizyme (maxizyme right MzR,maxizyme left MzL) andcontrol mutant maxizyme (G~5→A~5) were designed bycomputer and cloned into vector pBSKU6 (pBSKU6MzR,pBSKU6MzL).After being sequenced,the restrictiveendonuclease site in pBSKU6MzR was changed by PCR andthen U6MzR was inserted into pBSKU6MzL,the recombinantvector was named pU6Mz and pU6asMz (mutant maxizyme).Mtp53 and wild-type p53 (wtp53) gene fragments werecloned into pGEM-T vector under the T7 promoter control.The ~(32)p-labeled mtp53 transcript was the target mRNA.Coldmaxizyme transcripts were incubated with ~(32)p-labeled targetRNA in vitro and radioautographed after denaturingpolyacrylamide gel electrophoresis.RESULTS:In cell-free systems,pU6Mz showed a specificcleavage activity against target mRNA at 37℃ and 25 mMMgCL_2.The cleavage efficiency of pU6Mz was 42%,whilepU6asMz had no inhibitory effect.Wtp53 was not cleavedby pU6Mz either.CONCLUSION:pU6Mz had a specific catalytic activity againstmtp53 in cell-free system.These lay a good fundation forstudying the effects of anti-mtp53 maxizyme in HCC celllines.The results suggest that maxizyme may be a promisingalternative approach for treating hepatocellular carcinomacontaining mtp53.
AIM: To evaluate the specific inhibition of maxizyme directingagainst mutant-type p53 gene (mtp53) at codon 249 in exon7 (AGG → AGT) in vitro. METHODS: Two different monomers of anti-mtp53maxizyme (maxizyme right MzR, maxizyme left MzL) andcontrol 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. Coldmaxizyme transcripts were incubated with ~ (32) p-labeled targetRNA in vitro and radioautographed after denaturing polyacrylamide gel electrophoresis .RESULTS: In cell-free systems, pU6Mz showed a specific cleavage activity against target mRNA at 37 ° C and 25 mM MgCl_2. The cleavage efficiency o fpU6Mz was 42%, whilepU6asMz had no inhibitory effect. Wtp53 was not cleavedby pU6Mz either.CONCLUSION: pU6Mz had a specific catalytic activity againstmtp53 in cell-free system. The release lay a good fund forstudying the effects of anti-mtp53 maxizyme in HCC celllines . The results suggest that maxizyme may be a promising alternative approach for treating hepatocellular carcinoma con- taining mtp53.