摘要
目的在体外转录快速获得原核生物来源核酶P的核心催化亚基M1RNA。方法以大肠埃希菌DH5α菌液为模板,应用聚合酶链式反应的方法扩增M1RNA基因,并将该基因置于T7启动子下游,其聚合酶链式反应产物经电泳及测序鉴定。以M1RNA基因为模板,以32P标记,在T7 RNA聚合酶的作用下体外转录M1RNA。将产物以尿素变性聚丙烯酰胺凝胶分离并观察结果。结果应用聚合酶链式反应的方法从大肠埃希菌基因组扩增M1RNA基因,经凝胶电泳观察及测序鉴定,证实成功在体外扩增M1RNA基因,并置于T7启动子下游。在T7 RNA聚合酶的催化下,应用尿素变性聚丙烯酰胺凝胶电泳分离产物,结果显示377nt处可见与预期相符的条带,证实成功在体外获得M1RNA。结论成功在体外快速获得具有独立催化功能的M1RNA,基于此的基因沉默技术具有发展成新型反义核酸药物的良好前景。
Objective To prepare the core catalytic subunit M1RNA of prokaryotic source ribozyme P rapidly.Methods Using Escherichia coli DH5 as the template,the M1RNA gene was amplified by PCR and placed in the downstream of T7 promoter.The PCR products were identified by electrophoresis and sequencing.Using the M1RNA gene as the template and 32P labelling,M1RNA was obtained by the action of T7 RNA polymerase.The products were separated by urea denaturing polyacrylamide gel and the results were observed.Results PCR method was used to amplify the M1RNA gene from the Escherichia coli genome,and the M1RNA gene was successfully amplified in vitro and identified by gel electrophoresis and sequencing.The M1RNA was placed in the downstream of the T7 promoter.Under the action of T7RNA polymerase,the products were isolated by urea denaturing polyacrylamide gel,and the results showed that the band size was 377 nt,which is consistent with the expected size.These confirmed that M1RNA was successfully obtained in vitro.Conclusion The independent catalytic subunit of M1RNA has been successfully prepared rapidly in vitro,and the gene silencing technology based on this has good prospects for developing new antisense nucleic acid drugs.
作者
胡兢晶
伍苑宾
赵小琴
祁丹
谭琪琪
苏海浩
王波
Hu Jingjing;Wu Yuanbin;Zhao Xiaoqin;Qi Dan;Tan Qiqi;Su Haihao;Wang Bo(Guangdong Women and Children's Hospital,Guangzhou 511442,China)
出处
《中华临床医师杂志（电子版）》
CAS
2020年第1期48-51,共4页
Chinese Journal of Clinicians(Electronic Edition)
基金
国家自然科学基金(81070516)
广东省自然科学基金(2015A030313726、2016A030313788)
广东省医学科研基金(A2015133)。
