EB病毒LMP1基因靶向shRNA表达载体构建方法的比较研究

目的:探讨两种构建EB病毒LMP1 基因的shRNA表达载体(pshLMP1)方法的不同,寻找更加稳定方便的靶向shRNA表达载体的构建方式.方法:设计针对EB病毒LMP1基因的特异siRNA 编码序列,分别应用传统的双链退火法和新颖的双链PCR法构建pshLMP1,比较两种方法在设计原则、构建方法和鉴定结果上的不同.结果:两种方法均能得到pshLMP1重组质粒,但是双链PCR法构建效率高且不易引起碱基的缺失和突变.结论:双链PCR法构建EB病毒pshLMP1表达载体比传统双链退火法更加稳定,构建成功率较双链退火法高.

Comparative study between two construction methods of short hairpin RNA expression vector targeting Epstein-Barr virus LMP1 gene

AIM: To explore the differences between two construction methods of short hairpin RNA expression vector targeting Epstein-Barr virus(EBV) latent membrane protein-1(pshLMP1), so as to look for a more stable and convenient way of constructing the special shRNA vector. METHODS: The siRNA coding sequences targeting EBV-LMP1 were designed; pshLMP1 expression vector was achieved by the conventional annealing method or PCR method, and the differences of constructive strategies, construction processes and sequencing results between the two methods were analyzed. RESULTS: pshLMP1 expression vector was obtained by both methods but better cloning efficiency was achieved by PCR method with rare base deletion and mutation. CONCLUSION: PCR method is a more efficient way to construct the more stable pshLMP1 expression vector.