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目的构建并鉴定Her-2特异性siRNA逆转录病毒载体,将其导入SKOV3卵巢癌细胞中,并初步筛选有效抑制Her-2表达的细胞株。方法体外合成含针对Her-2特异基因序列的寡核苷酸并定向克隆入逆转录病毒载体RNAi-ReadypSIREN-RetroQ,构建的载体通过测序、酶切鉴定后,脂质体介导下转染到包装病毒细胞株PT67中,嘌呤霉素筛选并挑选细胞克隆,收获病毒上清,将病毒上清转染SKOV3中,经过嘌呤霉素筛选得到稳定抑制Her-2表达的SKOV3细胞株,并分别通过RT-PCR和免疫组化方法鉴定抑制Her-2表达的效果。结果成功构建了重组逆转录病毒载体,转染包装病毒细胞株获得的病毒上清成功转染了SKOV3,并且抑制了Her-2的表达。结论抑制Her-2表达的SKOV3细胞株的建立,为观察转染siRNA的逆转录病毒表达载体的肿瘤细胞恶性生物学行为变化奠定了实验基础。
Objective To construct and identify Her-2 specific siRNA retroviral vector and introduce it into SKOV3 ovarian cancer cells and screen the cell lines that can effectively inhibit Her-2 expression. Methods The oligonucleotides containing Her-2 specific gene sequence were synthesized in vitro and cloned into the RNAi-ReadypSIREN-RetroQ retroviral vector. The constructed vector was identified by restriction endonuclease digestion and transfected into The packaging virus cell line PT67, puromycin screening and selection of cell clones harvested virus supernatant, the virus supernatant transfected SKOV3, after puromycin screening to obtain a stable inhibition of Her-2 expression SKOV3 cell lines, and respectively by RT-PCR and immunohistochemistry to identify the effect of inhibiting Her-2 expression. Results The recombinant retroviral vector was successfully constructed. The virus supernatant transfected by the packaged virus cell line was successfully transfected into SKOV3 and inhibited the expression of Her-2. Conclusion The establishment of SKOV3 cell line that inhibits the expression of Her-2 has laid the foundation for the observation of the malignant behavior of tumor cells transfected with siRNA retroviral vector.