腺病毒介导的Her2基因RNAi载体的构建和鉴定  

Identification and construction of a RNAi vector containing Her 2 gene mediated by adenovirus

作　　者：高洋[1] 杨红[1] 闫岩[2] 刘艳丽[2] 张芳琳[2] 吴兴安[2] 辛晓燕[1] 

机构地区：[1]第四军医大学西京医院妇产科,西安710032 [2]第四军医大学基础医学部微生物学教研室,西安710032

出　　处：《现代生物医学进展》2008年第8期1409-1411,1408,共4页Progress in Modern Biomedicine

基　　金：国家自然科学基金(30772305)

摘　　要：目的：构建腺病毒介导的Her2基因RNAi载体。方法：构建含有Her2基因片断的siRNA质粒Her2-pSuppressor，通过特异性酶切将目的基因与穿梭载体pshuttle相连，再通过特异性酶切位点将目的片断与腺病毒DNA相连，并用PCR和酶切鉴定方法进行筛选和鉴定，获得重组腺病毒DNA。以PacⅠ酶切线性化后转染包装含有腺病毒E1的HEK293细胞。以软琼脂平板上的空斑数量计算重组腺病毒的的滴度.结果：XbaⅠ＋MluⅠ双酶切鉴定Her2a-RNAi/pShuttle和Her2b-RNAi/pshuttle阳性重组子获得304bp的目的片段，重组腺病毒载体Adeno-Her2a-RNAi和Adeno-Her2b-RNAi经PCR扩增出同样大小片断，经线性化后用脂质体法转染293细胞，观察到细胞病变效应。病毒滴度达1.2×10^8PFu/mL。结论：成功构建了Adeno-Her2-RNAi，为肿瘤的基因治疗奠定了良好的基础。Objective：To express the gene containing Her2 RNAi fragment in adenovirs expression system.Methods：The recombinant plasmid Her2-pSuppressor was constructed and digested to construct the recombinant adenovirus transfer vector Her2-pshuttle,the target gene fragment in which was obtained and then inserted into adenovirus DNA.Positive clones were selected after digestion with XhoⅠand PCR.After digestion with PacⅠ,the plasmids was transfected into packing cells-293 cell line.Then the titer of recombinant adnovirus DNA was detected.Results：Identified by XbaⅠand MluⅠa gene fragment with the length of 304bp was obtained from Her2-RNAi/pShuttle,and a gene fragment with the same length was amplified from Adeno-Her2-RNAi constructed in this article.After linearization,the Adeno-Her2-RNAi was transfected into 293 cells and cytopathic effects were observed subsequently.Lastly,the titer of adeno-Her2 was detected for 1.6×10~8 PFU/mL.Conclusion：The recombinant adnovirus Adneo-Her2 RNAi was constructed successfully, which supply a potential agent for gene therapy in tumors in the future.

关 键 词：Her2 RNA干扰 基因 腺病毒 重组 

分 类 号：R-332[医药卫生] R322.61