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目的构建人基因组中16个组蛋白乙酰化酶的短发卡RNA(shRNA)慢病毒载体文库,为进一步研究表观遗传因子对胆囊癌的调控机制提供有利的工具。方法依据shRNA引物设计原则,分别针对每个基因设计4对shRNA引物,将引物退火形成粘性末端后连接至慢病毒载体;经菌落PCR、酶切验证正确后,进行转化及质粒抽提。结果构建了16个组蛋白乙酰化酶基因的shRNA慢病毒载体,共64个shRNA慢病毒载体克隆;并对胆囊癌细胞SGC996和GBC-SD进行了病毒感染的MOI值(multiplicity of infection)摸索,以确保可通过RNAinterference(RNAi)慢病毒干扰的方式对这两株细胞进行研究。结论成功构建了16个组蛋白乙酰化酶的shRNA慢病毒载体,从而为在SGC996和GBC-SD胆囊癌细胞中研究人组蛋白乙酰化酶奠定坚实的基础。
Objective To construct a short hairpin RNA (shRNA) lentiviral vector library of 16 histone deacetylases in the human genome, which provides a useful tool for further study on the regulatory mechanisms of epigenetic factors in gallbladder carcinomas. Methods According to the principles of shRNA primer design, 4 pairs of shRNA primers were designed for each gene respectively, and the primers were annealed to form a sticky end and then ligated to the lentiviral vector. After colony PCR and enzyme digestion were correct, transformation and plasmid extraction were carried out. Results 16 shRNA lentiviral vectors with histone acetylase gene were constructed, and 64 shRNA lentiviral vector clones were constructed. The multiplicity of infection of gallbladder cancer cells SGC996 and GBC-SD was explored. To ensure that both of these cells could be studied by means of RNA interference (RNAi) lentivirus. Conclusion 16 shRNA lentiviral vectors of histone acetylase were successfully constructed, which laid a solid foundation for the study of human histone acetylase in SGC996 and GBC-SD gallbladder carcinoma cells.