应用外部引导序列切割人巨细胞病毒UL148 RNA的研究

Effective inhibition of human cytomegalovirus UL148 gene expression by external guide sequences in vitro

目的 研究人巨细胞病毒（Human cytomegalovirus,HCMV）临床病毒株UL/b＆#39;区域UL148基因功能及抗HCMV治疗新策略,应用DNA性质外部引导序列（External guide sequences,EGS）在体外抑制UL148 RNA的表达.方法 应用RNA structure生物软件模拟UL148 RNA二级结构,选择二级结构相对简单的区域设计EGS,并合成EGS核苷酸序列.应用PCR方法分别扩增UL148及M1RNA基因,克隆至T7启动子下游,在T7 RNA聚合酶的作用下,体外转录UL148 RNA及M1RNA,其中UL148以32p标记并进行体外转录.混合EGS、M1RNA及UL148 RNA于切割反应液中,反应1h后,进行尿素变性聚丙烯酰胺凝胶电泳分离,Typhoon扫描仪观察结果.结果 根据UL148 RNA二级结构选取第58 ～72位碱基作为EGS结合区域,其切割位点位于57位,针对此区域设计EGS57,并在体外进行EGS57与UL148 RNA结合的二级结构的模拟,可形成M1RNA天然作用底物类似的茎环结构.在T7 RNA聚合酶的作用下,分别在体外转录M1RNA及UL148 RNA,其大小均与预期相符.经切割反应,可获得与预期切割位点相符的切割条带.结论 EGS57可切割UL148RNA,其切割位点准确,与预期相符.该研究为进一步探讨UL148基因功能提供有效基因沉默工具.

Objective To investigate the UL148 gene function of human cytomegalovirus （HCMV） low passage clinic isolate and new strategies for anti-HCMV treatment,the DNA-based external guide sequences （EGSs） were designed to inhibit UL148 RNA expression.Methods UL148 RNA secondary structure was analyzed by RNA structure technique,an appropriate region was chosen for DNA-based EGS57 synthesis,targeted the UL148 RNA.The M1RNA and UL148 RNA were generated by PCR for transcription in vitro.The UL148 RNA and M1RNA were transcribed in vitro under the function of T7 RNA polymerase.The UL148 was labelled by 32p.The cleavage reactions were carried out by mixing up EGS,M1RNA with UL148 RNA for 1 h.The products were separated by urea denaturing polyacrylamide gel electrophoresis and detected with Typhoon Phosphor Imager.Results UL148 RNA ranged from 58 to 72 sites was the binding position,and 57 was a cleavage site.EGS57 was designed and synthesized.EGS57 was combined with UL148 RNA to form the natural substrate of M1RNA.UL148 RNA and M1RNA were synthesized through T7 RNA polymerase catalyzing,and the products were conformed.After cleaving reactions,DNA-based EGS57 was shown to be able to cleave UL148 RNA efficiently in vitro by a complex with M1RNA.Conclusions UL148 RNA was cleaved efficiently by EGS57,and the cleaving site was conformed as expectation.It will provide the gene silent tool effectively for further study the function of UL148 gene.