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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 construct Adenovirus-mediated Her2 gene RNAi vector. Methods: The Her2-pSuppressor siRNA plasmid containing Her2 gene was constructed. The target gene was linked to the shuttle vector pshuttle by specific enzyme digestion, and the target fragment was linked to the adenovirus DNA by specific restriction sites. Identification method for screening and identification, access to recombinant adenovirus DNA. After linearization with Pac I, HEK293 cells containing adenovirus E1 were transfected. The titer of the recombinant adenovirus was calculated by the number of plaques on the soft agar plate.Results: The target fragment of 304bp was obtained by double enzyme digestion with Her2a-RNAi / pSuttle and Her2b-RNAi / pshuttle by XbaⅠ + MluⅠ, and the recombinant adenovirus Vectors Adeno-Her2a-RNAi and Adeno-Her2b-RNAi were amplified by PCR, the same size fragments were linearized and then transfected into 293 cells by liposome method, the cytopathic effect was observed. Virus titer of 1.2 × 10 ~ 8PFu / mL. Conclusion: Adeno-Her2-RNAi was successfully constructed, which laid a good foundation for the gene therapy of tumors.