Construction Of RhoC Gene Eucaryotic Expressive Vector And Its Control CyclinD1 And Cathepsin B Expression

Objective To construct sense and antisense expression vector of humanRhoC and observe the relationship between RhoC gene and expression of QBC939 cell'scyclinD1 and Cathepsin B mRNA Methods Genomic RNA was extracted from freshQBC939 cells .Primers was designed according to the sequence of RhoC gene, the RhoCgene was amplified by means of retrotranscription polymerase chain reaction(RT-PCR). The purified RT-PCR products was cloned into the pcDNA3.1/Hyg(+) plasmidto reconstruct sense and antisense eukaryotic expression vector containingHygromycin B selection marker.the combinant expression plasmid was transferredinto E.coli TOP10,positive clones were screened and identified by endonuclease.Sequence determination analysis was performed to identify the sense and antisensecloned gene,Sense ,Antisence eukaryotic expression plasmid of RhoC andeukaryotic expression plasmid were transfected into QBC939 by Lipofectamine methodand the positive clones were selected by Hygromycin B .RT-PCR was used to examinethe expression of Hygromycin B ,cyclinD1 and Cathepsin B,the changes ofproliferation were checked by growth analysis in vitro .Results The positive recombinant plasmid pcDNA3.1/Hygro(+)was screened anddigested by Kpnâ… + BamHâ… . Sequence determination analysis showed that the clonedgene was gene fragment coding RhoC gene.The results showed that all transfecters had expressed Hygromycin B, The differenceof cyclinD1 and Cathepsin B expression in QBC939-AS group ,QBC939-S group andin control group was statistically significant ( P < 0. 05) ,Compared with thecontrol QBC939 cells and QBC939-V cells , QBC939-AS grew more slowly ,but QBC939-Sgrew more quickly .Conclusion Sense and antisense pcDNA3.1/Hygro(+) eukaryotic expression vectorcontaining RhoC gene was constructed successfull.RhoC could control QBC939 cells'cyclinD1 and Cathepsin B transcription.