| Introduction and ObjectiveSince the report was first announced by Fire in 1998,RNAi has been playing an important role in the fields of functional gene group etc.Compared with the traditional therapeutic method such as gene substitution and anteiso-necleic acidetc,RNAi has more advantages.RNAi refers to the process in which interfering RNA from the living creature gives rise to the special degeneration with its isogenic RNAi then inhibits the expression of counterpart genes.Moreover,because of its function in successfully down regulating the expression level of a series of genes in cells of mammals and the improved method of cell introduction,RNAi has been widely applied to the studies on tumors,such as in the aspects of nosogenesis,infestation and aversion,signal conduction,control of cell cycle,apoptosis and healing.Survivin is the one of the strongest apoptosis inhibiting agents that has ever been found.It inhibits apoptosis through two ways:one is the classic method,Caspase;the other is through promoting cell division.As far as second way is concerned,survivin, as a dominant chromosome 'passing by' protein,participates in the modulation of cell division in many aspects.Kishi etc detected the level of survivin mRNA in prostatic carcinoma tissues and found out that the expression of survivin in the prostatic cancer group was obviously higher than in the normal prostatic tissues group.If the expression of survivin in PCa group is high,then the situations such as Staging,Gleason grading, aversion of absorbent gland,blood vessel invasion,positive rate of incision,are all obviously rare.Meanwhile,the expression level of survivin is in inverse ratio to the apoptosis of PCa cells.So far,RNAi aiming directly at survivin has been applied to induce the apoptosis of tumor cells such as gastric cancer,cervical cancer,ovarian cancer and large intestine cancer etc.successfully. RNAi was adopted in this experiment.Taking suivivin as the target gene,two expression vectors which could express siRNA aiming directly at survivin within cells were designed and constructed.These findings form a solid foundation for prostatic carcinoma's clinical gene therapy.Material and methodVector pBAsi-mU6-pur,E.coli DH5α,BamH1,HindⅢ,λ-HindⅢdigest,DL-2000(TaKaRa china).E.Z.N.A(R)(Omega USA)Two DNA sequences containing small hairpin structure were designed and synthesized.The complememt form was obtained by annealing and being cloned into vector pBAsi-mU6-pur and the recombinant plasmid was transformed into strain E.coli DH5α.The plasmid identified by restriction enzyme was used for sequecing.ResultsRNAi was adopted in this experiment.Taking suivivin as the target gene,two expression vectors which could express siRNA aiming directly at survivin within cells were designed and constructed.The recombinant plasmid was cloned and the aimed sequence was obtained.These findings form a solid foundation for prostatic carcinoma's clinical gene therapy.Conclusionsthe recombinant plasmid was cloned and the aimed sequence was obtained. Successful cloning of the recombinant plasmid help to find a new gene therapy for tumors. |
