表达HPV-16 E6基因siRNA真核载体的构建及体外转染效率鉴定

Construction of HPV16 E6 siRNA expressing recombinants and identification of transfection efficiency in vitro

目的:采用RNA干扰技术选择性的沉默宫颈癌Ca Ski细胞株中HPV-16 E6基因的表达。为观察HPV-16 E6基因的小干扰片段能否在Ca Ski细胞特异性的抑制E6基因的表达,构建能在哺乳动物细胞中表达E6小干扰RNA的真核表达质粒,并观察其转染效率。方法:根据HPV-16 E6基因序列,设计特异性的小干扰片段,并将其定向克隆到带有卡那霉素抗性和增强绿色荧光蛋白的真核表达载体pGenesil-1上,将其转染至Ca Ski细胞中,观察荧光表达,鉴定其转染效率。结果:通过酶切鉴定和序列测定,成功的构建二条表达小干扰RNA的质粒及其阴性对照质粒,并成功的转染到Ca Ski细胞株中,转染效率达到50%以上。结论:siRNA真核表达载体的构建及体外转染的成功为下一步研究奠定了良好的基础。

Objective： To silence the expression of HPV16 E6 gene in Ca Ski cell line with RNA interference technique. In order to understand if the HPV16 E6 siRNA could inhibit the expression of E6 gene specifically, recomhinant eukaryotic vectors were constructed and transfected into Ca Ski cell line. The transfection efficiency was observed. Methods： According to the sequence of HPV16 E6 gene, the E6 siRNA was designed and cloned into the EGFP reporter plasmid pGenesil-1, and then transfected into CaSki cell line. The fluorescence expression was observed and the transfection efficiency was identified. Results： Two siRNA expressing recombinants and the corresponding negative control vector were constructed and transfected into Ca Ski cell successfully, and the transfection efficiency achieved about 50%. Conclusion： The construction of recombinant E6 siRNA eukaryotic vectors and transfection of them into Ca Ski cell line successfully established a favourable foundation for further study.